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

Volume 68 Number 1 - January/2013

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 Anna Sara Shafferman Levin 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

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

Emilia Inoue Sato 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

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

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

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

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

Bruno Zilberstein Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, 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

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 Walter Gomes Universidade Federal 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 2013 prices are as follows: fast track: US$ 1,500.00; original articles, review articles and rapid communications: US$ 1,200.00. 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 only need to 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 using 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. References must be restricted to directly relevant published works, papers, or abstracts. 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. Authors are responsible for the accuracy and completeness of their references and for correct text citation. Usually the total number of references should not exceed 35. For up to six authors, list all authors. For more than 6 authors, list first six authors followed by ‘‘et al.’’. Tables and Figures: The maximum number of tables and/or figures is six tables and/or figures. 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 or standard error) should be identified, and decimal places in tabular data should be restricted to those with mathematical and statistical significance. Figures: Photographs, illustrations, charts, drawings, line graphs, etc are all defined as figures. Number figures consecutively using Arabic numerals in order of appearance. Figure legend(s) should be descriptive and should allow examination of the figure without reference to text. Images must be of professional quality and uploaded as *.tiff files. 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.

FAST TRACK ARTICLES: Fast-track articles should follow the same format described above for original studies. 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, abstract and keywords. Authors should format rapid communications based on the subject at hand. 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. For keywords, 3-6 items from the Medical Subject Headings (MeSh) should be used.

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 and authors will be automatically notified of the submission. Progress of the manuscript through the Editorial Office’s procedures will be available to authors at all times.


ISSN-1807-5932

CLINICS CONTENTS Clinics 2013 68(1):1–126

CLINICAL SCIENCES

Post-thoracotomy wound separation (DEHISCENCE): A disturbing complication Aydin Nadir, Melih Kaptanoglu, Ekber Sahin, Hakan Sarzep . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1

Evaluation of the association between the JAK2 46/1 haplotype and chronic myeloproliferative neoplasms in a Brazilian population Sarah Pagliarini-e-Silva, Bruna Cunha Santos, Elizangela Mendes de Figueiredo Pereira, Mari Ellen Ferreira, Elaine Cristina Baraldi, Ana Maria Sell, Jeane Eliete Laguila Visentainer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

Dietary patterns in Brazilian patients with nonalcoholic fatty liver disease: a cross-sectional study Silvia Marinho Ferolla, Teresa Cristina Abreu Ferrari, Maria Luı´za Pereira Lima, Taˆmara Oliveira Reis, Wilson Campos Tavares-Jr., Osvaldo Fla´vio Melo Couto, Paula Vieira Texeira Vidigal, Maria Arlene Fausto, Cla´udia Alves Couto . . . 11

The relationship between hyperuricemia and the risk of contrast-induced acute kidney injury after percutaneous coronary intervention in patients with relatively normal serum creatinine Yong Liu, Ning Tan, Jiyan Chen, Yingling Zhou, Liling Chen, Shiqun Chen, Zhujun Chen, Liwen Li . . . . . . . . . . . . 19

RAPID COMMUNICATION

Use of an artificial neural network to predict persistent organ failure in patients with acute pancreatitis Wan-dong Hong, Xiang-rong Chen, Shu-qing Jin, Qing-ke Huang, Qi-huai Zhu, Jing-ye Pan . . . . . . . . . . . . . . . . . . . 27

CLINICAL SCIENCES

High levels of B-type natriuretic peptide predict weaning failure from mechanical ventilation in adult patients after cardiac surgery Thiago Martins Lara, Ludhmila Abrahao Hajjar, Juliano Pinheiro de Almeida, Julia Tizue Fukushima, Carmem Silvia Valente Barbas, Adriano Rogerio Baldacin Rodrigues, Emilia Nozawa, Maria Ignes Zanetti Feltrim, Elisangela Almeida, Vera Coimbra, Eduardo Osawa, Rafael de Moraes Ianotti, Alcino Costa Leme, Fabio Biscegli Jatene, Jose Otavio Costa Auler-Jr., Filomena Regina Barbosa Gomes Galas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Evaluation of renal function and immune system cells in elderly individuals from Sa˜o Paulo City Daniela Teixeira, Ieda Maria Longo-Maugeri, Yeda Aparecida Oliveira Duarte, Maria Lucia Lebra˜o, Valquiria Bueno . . . . 39

Evaluation of the correlation between dental occlusion and posture using a force platform Alberto Baldini, Alessandro Nota, Domenico Tripodi, Salvatore Longoni, Paola Cozza . . . . . . . . . . . . . . . . . . . . . . . 45


Rev 7.51n/W (Jan 20 2003)

Clinics

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Body size and longitudinal body weight changes do not increase mortality in incident peritoneal dialysis patients of the Brazilian peritoneal dialysis multicenter study Nata´lia Maria da Silva Fernandes, Marcus Gomes Bastos, Ma´rcia Regina Gianotti Franco, Alfredo Chaoubah, Maria da Glo´ria Lima, Jose´ Carolino Divino-Filho, Abdul Rashid Qureshi, on behalf of the Brazilian Peritoneal Dialysis Multicenter Study (BRAZPD) Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

BASIC RESEARCH

Evaluation of histological changes after tracheal occlusion at different gestational ages in a fetal rat model Rodrigo Melo Gallindo, Frances Lilian Lanhellas Gonc¸alves, Carolina Teixeira de Resende Barreto, Augusto Frederico Santos Schmidt, Luis Antonio Violin Dias Pereira, Lourenc¸o Sbragia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

CLINICAL SCIENCES

A new cultural adaptation of the University of Pennsylvania Smell Identification Test Marco Aure´lio Fornazieri, Richard L. Doty, Clayson Alan dos Santos, Fa´bio de Rezende Pinna, Thiago Freire Pinto Bezerra, Richard Louis Voegels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Prevalence of metabolic syndrome and related factors in bank employees according to different defining criteria, Vito´ria/ES, Brazil Luciane Bresciani Salaroli, Renata Aubin Dias Saliba, Eliana Zandonade, Maria del Carmen Bisi Molina, Nazare´ Souza Bissoli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Comparing the diagnostic values of circulating microRNAs and cardiac troponin T in patients with acute myocardial infarction Ying-Qing Li, Mei-Fen Zhang, Hong-Yan Wen, Chun-Lin Hu, Rong Liu, Hong-Yan Wei, Chen-Mu Ai, Gang Wang, XiaoXing Liao, Xin Li . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Hereditary angioedema: quality of life in Brazilian patients Maria Abadia Consuelo M. S. Gomide, Eliana Toledo, Solange Oliveira Rodrigues Valle, Regis A. Campos, Alfeu T. Franc¸a, Nieves Prior Gomez, Heitor Franco Andrade Jr., Teresa Caballero, Anete S. Grumach . . . . . . . . . . . . . . . . . 81

Factors associated with hyperglycemia and low insulin levels in children undergoing cardiac surgery with cardiopulmonary bypass who received a single high dose of methylprednisolone Ronaldo Arkader, Luiz Marcelo Malbouisson, Gilda Maria Barbaro Del Negro, Lidia Yamamoto, Thelma Suely Okay . . . 85

BASIC RESEARCHES

Fenitrothion induced oxidative stress and morphological alterations of sperm and testes in male spraguedawley rats Izatus Shima, Taib, Siti Balkis Budin, Ahmad Rohi Ghazali, Putri Ayu Jayusman, Santhana Raj Louis, Jamaludin Mohamed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

Tamoxifen decreases the myofibroblast count in the healing bile duct tissue of pigs ´ scoli, Antonio Cla´udio Orlando Hiroshi Kiono Siqueira, Benedito Herani Filho, Rafael Erthal de Paula, Fa´bio Otero A Lucas da No´brega, Angela Cristina Gouveˆa Carvalho, Andre´a Rodrigues Cordovil Pires, Nicolle Cavalcante Gaglionone, Karin Soares Gonc¸alves Cunha, Jose´ Mauro Granjeiro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Exercise training prevents skeletal muscle damage in an experimental sepsis model Carla Werlang Coelho, Paulo R. Jannig, Arlete B. de Souza, Hercilio Fronza Jr., Glauco A Westphal, Fabricia Petronilho, Larissa Constantino, Felipe Dal-Pizzol, Gabriela K. Ferreira, Emilio E. Streck, Eliezer Silva . . . . . . . . . . . . . . . . . . 107


Rev 7.51n/W (Jan 20 2003)

Clinics

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READER’S OPINIONS

The platelet volume in patients with cardiac syndrome X Ercan Varol, Mehmet Ozaydin, Abdullah Dogan, Dogan Erdogan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Authors’ comments on: The platelet volume in patients with cardiac syndrome X Sait Demirkol, Sevket Balta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Differences in the mechanisms that induce obesity and metabolic syndrome in experimental animal models and humans may cause treatment failure Mustafa Cakar, Sevket Balta, Sait Demırkol, Ugur Kucuk, Seref Demırbas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Prediction of hospital events based on the severity of illness Sevket Balta, Mustafa Cakar, Sait Demirkol, Zekeriya Arslan, Murat Unlu, Omer Kurt . . . . . . . . . . . . . . . . . . . . . 121

RAPID COMMUNICATION

Residual C-peptide in patients with Type 1 diabetes and multiethnic backgrounds Mirella Hansen de Almeida, Joana Rodrigues Dantas, Bianca Barone, Fabiano Marcel Serfaty, Rosane Kupfer, Marta Albernaz, Maria Rocio Bencke, Lenita Zajdenverg, Melanie Rodacki, Jose´ Egı´dio Paulo de Oliveira. . . . . . . . . . . . . 123


CLINICAL SCIENCE

Post-thoracotomy wound separation (DEHISCENCE): A disturbing complication Aydin Nadir, Melih Kaptanoglu, Ekber Sahin, Hakan Sarzep Cumhuriyet University, School of Medicine Sivas, Department of Thoracic Surgery, Sivas/Turkey.

OBJECTIVES: We described the treatment of dehiscence of thoracotomy incisions in patients who underwent thoracic surgery in the present study. METHODS: Twenty-four patients with either partial or complete dehiscence of their thoracotomy incisions were included in the study from 2005 to 2010. The patients were evaluated regarding their age, sex, indication for thoracotomy, and surgical approaches. We also described our method of re-closure. RESULTS: The male/female ratio was 17/7. The youngest and oldest patients were 15 and 75 years old, respectively, and the mean age was 43 years. Among the indications for thoracotomy, empyema was the most common reason (determined in eight [33%] patients), followed by vertebral surgery (determined in six [25%] patients). Bacterial growth was detected in the wound site cultures from 13 (54%) patients. For the patients with dehiscence of their thoracotomy incisions, an en block approximation technique with debridement was performed under general or local anesthesia in 16 (66%) and eight (33%) of the cases, respectively. Three patients exhibited an open thorax with dehiscence of the thoracotomy incision. Thoracoplasty was required in two patients. Using this method, successful closure was obtained in 91.7% (n = 22) of the patients with dehiscence of their thoracotomy incisions. CONCLUSION: Dehiscence of the thoracotomy incision is an important complication that causes concern in patients and their thoracic surgeons and strongly affects the success of the surgery. An en block approximation technique with significant debridement that enables removal of the necrotic tissues from the wound site can successfully be applied to patients with dehiscence of their thoracotomy incisions. KEYWORDS: Dehiscence; Failure; Suture; Thoracotomy; Wound. Nadir A, Kaptanoglu M, Sahin E, Sarzep H. Post-thoracotomy wound separation (DEHISCENCE): A disturbing complication. Clinics. 2013;68(1):14. Received for publication on April 27, 2012; First review completed on June 26, 2012; Accepted for publication on July 5, 2012 E-mail: anadir@ttmail.com Tel.: 00-90-346-2580211

manuscript that includes a series of dehiscence of thoracotomy incisions (DTI). The incidence of wound dehiscence after cardiac or general surgery is approximately 0.3-5% and 0.25-3%, respectively (1,7). However, the incidence is unclear for thoracic surgery. Our dehiscence rate was 6.6% (24/360 patients) over five years, which alerted us to investigate this relatively high incidence. Traditional surgical approaches or institution-based techniques at surgical clinics are preferred for addressing similar problems (8,9). The publication of these procedures adds to the essential knowledge base. Therefore, we aimed to draw attention to this disturbing complication and publish our experiences. The new term ‘‘thoracotomy dehiscence’’ may potentially be added to the thoracic surgical nomenclature upon recognition of this entity.

& INTRODUCTION Wound dehiscence is a surgical complication in which a wound fails to heal or opens along its incision line following surgery. The are many etiologies, including infection, weak tissue or muscle at the wound site, injury to the wound area, and other factors, which are commonly related to poor closure techniques (1). Articles on thoracotomy wound dehiscence are scarce in the literature (2-4). We could not find the term ‘‘thoracotomy dehiscence’’ in thoracic surgery textbooks or thoracic surgery articles. The commonly found papers are related to sternal separation after cardiac surgery and dehiscence of laparotomy incisions (5-7). Indeed, we did not find any

Copyright ß 2013 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.

& PATIENTS AND METHODS Twenty-four patients with either partial or complete DTIs were included in the study from 2005 to 2010. This study was approved by the local ethical committee at our university (No: 10/161). The patients were retrospectively

No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(01)OA01

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evaluated regarding age, sex, indication for thoracotomy, surgical approach, and re-closure method for the DTI. The separations occurred postoperatively within the first two weeks. The separation included the muscle tissue layers in the complete or partially separated wounds. The length of the separation was between eight and ten centimeters and predominantly located in the anterior section of the incision in the partially separated wounds. Specimens for cultures and antibiograms were obtained from each wound site. In coordination with the Infection Control Committee, the patients were given either sensitive antimicrobial treatment in the case of growth in their cultures or wide-spectrum antimicrobial treatment otherwise. The sutures were immediately removed in patients with surgical site drainage and separation. Superficial debridements combined with saline irrigation were conducted under local anesthesia. During the early years of the study, this procedure was performed at least once per day until the cultures became negative. However, in later years, the decision was made to close the incisions after sufficient vascularization of the wounds and before waiting for the cultures to become negative. These repairs were performed under general anesthesia. After surgical site disinfection, a large segment of skin, subcutaneous tissue, and muscle was debrided, removing the necrotic tissues (including the wound margins) until fresh bloody tissues were reached. The surgical site was cleansed by scrubbing with povidone-iodine and saline. The skin, subcutaneous tissue and muscular layers were approximated by polypropylene (no:1). To decrease the tension and increase the strength of the sutures, four or five U-shaped retention sutures were inserted based on the size of the wound. To prevent tissue damage, these sutures were supported by Foley catheters that were three centimeters in length (Figure 1A-C). The skin was closed separately by polypropylene sutures (2/0). Dressing of the wounds was performed daily, starting on the second day of the operation. The patients were discharged on the fifth or sixth day after the operation. The sutures were removed on approximately the 15th day. In patients with open thoraces, the pleural space was cleansed by saline and hydrogen peroxide (3%). After the thickened, necrotic pleural tissues were removed, the thorax was closed.

A

Vacuum-assisted closure (VAC) was applied in three cases because the wound was too large and suppurative. In eight of the patients who had partial dehiscence, the incision was closed using single polypropylene sutures (2/0) under local anesthesia after sufficient vascularization was observed, and debridement was performed every three to four days, once or twice a given day.

& RESULTS Seventy percent of the patients in the study were male. The youngest patient was 15 years old, and the oldest was 75 years old, with a mean age of 43 years. Empyema (n = 8) was the most frequent indication for thoracotomy, followed by vertebral fracture (n = 6). Ten (40%) of the patients with either empyema or vertebral fracture had a history of trauma (Table 1). Two of the patients with empyema had concurrent bronchopleural fistulas. Recurrent pneumothorax, hemothorax, lung abscess, solitary pulmonary nodule, mesothelioma, lung carcinoma, and congenital cystic adenomatous malformation were among the other thoracotomy indications. Bacterial growth was detected in wound cultures from 13 (54%) patients. Staphylococcus epidermidis was isolated from six patients, but we considered this organism to be contamination from the skin. In total, 25 microorganisms were isolated: one type in 14 patients, two types in four patients, and three types in one patient. Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, and Enterococcus cloacea were other responsible microorganisms that were isolated in four, three, two, two and two patients, respectively. The first result observed in the DTI patients was an adequate closure of the skin, except at the site of the separation. However, upon removing the sutures one by one and exploring the wound, we noticed that the subcutaneous tissue and muscular layers were all separated and that the exudate had spread throughout the incision (Figure 2). Almost all the patients exhibited surgical site infections. However, there were no serious purulent massive discharges in these wounds requiring dressings two-three times per day, despite the full-thickness involvement of the thoracotomy incisions—that is, the majority of our patients presented non-suppurative surgical site dehiscence and/or infection.

B

C

Figure 1 - A) The photomicrograph displays the retention sutures that were placed after debridement in a patient who had complete dehiscence. B) The drawing depicts the placement of sutures during our re-closure technique. C) Retention and skin sutures are shown in a patient during the early postoperative period.

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Dehiscence of the Thoracotomy Incision Nadir A et al.

Table 1 - Characteristics of the patients. NO

AGE

ETIOLOGY

1 2 3 4 5 6 7 8

43 63 38 26 44 28 24 38

Trauma Trauma Trauma Trauma Trauma Trauma Trauma Trauma

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

15 62 63 41 28 53 39 75 55 59 59 23 59 29 65 23

Trauma Trauma Mal de Pott Pneumonia Pneumonia Pneumonia Pneumonia BPF Pneumonia Tumor Tumor Pneumothorax Pneumothorax SPN Tumor CCAM

INDICATION

PROCEDURE

L1 fracture L1 corpectomy+stabilization T10 fracture T10 corpectomy+stabilization L1 fracture L1 corpectomy+stabilization T12 fracture T12 corpectomy+stabilization L1 fracture L1 corpectomy+stabilization T6 fracture T6 corpectomy+stabilization Hemothorax Decortication Diaphragmatic Primary repair rupture Empyema Decortication Empyema Decortication Empyema T10 corpectomy Empyema Decortication Empyema Decortication Empyema + Sepsis Thoracomyoplasty Empyema Decortication Empyema Thoracomyoplasty Lung Abscess Wedge Resection Pancoast Lobectomy+chest wall resection Carcinoid Pneumonectomy Pneumothorax Wedge Resection Pneumothorax Wedge Resection SPN Wedge Resection Mesothelioma Pleurectomy CCAM Lobectomy

DEHISCENCE

VAC

BG

ANESTHESIA

RESULT

C C C P P P C C

+ + -

+ + + + + + + +

G G G L L L G G

Success Success Success Success Success Success Success Success

C C OT C C OT C OT P C C C C P P P

+ -

+ + + + + + + + + + + -

G G G G G G G G L L G G G L L L

Success Success Ex Success Success Ex Success Ex * Success Success Success Success Success Success Success Success

BPF: Bronchopleural fistula, SPN: Solitary pulmonary nodule, CCAM: Congenital cystic adenomatous hyperplasia, L: Lumbar, T: Thoracic, C: Complete P: Partial, OT: Open thorax, BG: Bacterial growth, G: General, L: Local. (*): Re-closure was successful.

Wound site healing was not achieved in a patient with empyema and sepsis and in another patient who underwent spinal surgery for Pott’s disease; VAC was also applied to the latter patient. In another two patients with large wound defects that ruled out primary closure, VAC was applied, enabling primary closure in one patient and closure using a skin flap in the other patient. There were open thoraces in three patients with wound separation; thoracoplasty was required in two of these patients. Three patients in this series died. The first patient (75 years old), who exhibited empyema and a bronchopleural fistula, suffered an additional lung collapse. This subject had to receive mechanical ventilation due to respiratory failure that developed after thoracomyoplasty, although the thoracotomy incision was successfully closed. He died from cardiac arrest during follow-up in the intensive care unit. Another patient (53 years old) with poor wound healing was lost because of disseminated pneumonia and sepsis. The third patient (63 years old) had Pott’s disease. He had undergone vertebral surgery in another center and was admitted to our clinic with a diagnosis of open thorax and empyema. Despite repetitive debridements and irrigations, the treatment for empyema failed, and the patient expired due to the emerging sepsis.

An en block approximation technique combined with debridement was performed under general anesthesia in 16 (66%) of the DTI patients. In two patients, superficial general anesthesia was provided because these individuals did not appear to tolerate intratracheal general anesthesia because of their poor general health status. Debridement and suturation were performed in eight patients with local anesthesia. Pathogenic microorganisms were isolated from the cultures of six of these eight patients. The improvement was complete in the patients whose interventions were conducted under local anesthesia. Successful results were obtained in 14 (87.5%) of the 16 patients whose en block approximation was performed under general anesthesia.

& DISCUSSION Wound dehiscence is a mechanical failure of wound healing, which is a significant problem that can be affected by multiple factors. Reports reveal a significantly higher incidence of wound dehiscence after emergency surgery than after elective surgery (7). Trauma was the most common etiology (n = 10) in our patients. It is possible that contusion, edema, and extravasation into deeper layers due to the intensity of the trauma were responsible for the delayed wound healing. However, we experienced the same problem in our

Figure 2 - A patient’s necrotic and suppurative tissues, including the skin, subcutaneous tissue and muscle layers, were observed before debridement. Only the pericostal sutures were intact.

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non- traumatic cases. Notably, DTIs were observed in four consecutive patients during seasonal transition periods, from summer to winter or vice-versa. Thereupon, the ‘‘Infection Control Committee’’ of the hospital traced the etiology. Specimens for culture were obtained from the surgical team and the surgical environment, including the operating room, surgical sets, and suture materials. Unfortunately, this procedure did not reveal a responsible factor. Separation or dehiscence of thoracotomy incisions is rare, and the treatment is undetermined. Similar phenomena, such as post-thoracotomy empyema or open chest, are managed either with long-lasting dressings on an open chest window or with repeated scheduled mechanical debridements and packing of the pleural cavity with povidone-iodine-soaked dressings and temporarily closing the chest after each session until final closure (8,9). We preferred local dressings, followed by large-scale debridement and final closure in a single session. The quality and nature of the suture material is critical, and failure of the suture material is a risk factor. Published articles have compared various suture materials or suturing techniques; however, the various brands of materials have not been compared based on their tensile strength or structural integrity (10-11). We noticed that the structural material of the polyglactin sutures, which were used for closure of either subcutaneous or muscular layers, deformed prematurely and could not retain the ability to hold the tissue layers together. Although we returned the suture materials to the manufacturer, we have not received a response. We speculate that inexpensive suture materials with toxic or allergic side effects may be responsible for this complication. In fact, we have not encountered this problem since purchasing a different brand of sutures. The vacuum-assisted closure system was introduced into clinical practice in 1997 and has evolved as the standard of care in the treatment of sternal wound infections during recent years (12). Articles on the use of VAC in thoracotomy wounds first appeared in the year 2006 and increased thereafter (3,4). VAC application in patients with large defects enables the approximation of the wound margins and a decrease in the bacterial load at the infection site. This technique can be used either before approximation or to prepare for reconstruction of failed cases. Although it is a time-consuming (2-4 months) and costly procedure, VAC is a safe and effective alternative, especially in patients with large defects or associated comorbidities.

Surgical site infection is a serious complication that greatly affects the success and cost of the surgery. An immediate en block approximation technique, combined with a wide debridement of the infected and necrotic tissues, may be successfully performed in the majority of cases.

& AUTHOR CONTRIBUTIONS Nadir A and Kaptanoglu M organized and conducted the study and provided substantial scientific contributions. Nadir A also illustrated the picture of the surgical technique. Sahin E and Sarzep H contributed to the data collection.

& REFERENCES 1. Ridderstolpe L, Gill H, Granfeldt H, Ahlfeltdt H, Rutberg H. Superficial and deep sternal wound complications: incidence, risk factors and mortality. Eu J of Cardiothorac Surg. 2001;20(6):1168-75, http://dx.doi. org/10.1016/S1010-7940(01)00991-5. 2. Welvaart WN, Oosterhuis JWA, Paul MA. Negative pressure dressing for radiation- associated wound dehiscence after posterolateral thoracotomy. Interact Cardiovasc and Thorac Surg. 2009;8(5):558-60, http:// dx.doi.org/10.1510/icvts.2008.196485. 3. Varker KA, Ng T. Management of Empyema cavity with the vacuumassisted closure device. Ann Thorac Surg. 2006;81(2):723-5, http://dx. doi.org/10.1016/j.athoracsur.2004.10.040. 4. O’Toole MJ, Kolb JE, Lindblad WJ, Cohen IK, McKneally MF. Pneumothorax and wound dehiscence related to collagenase deregulation: treatment with diphenylhydantoin. Ann Thorac Surg. 1996; 61(6):1646-50, http://dx.doi.org/10.1016/0003-4975(96)00212-3. 5. Fawzy H, Alhodaib N, Mazer CD, Harrington A, Latter D, Bonneau D, et al. Sternal plating for primary and secondary sternal closure; can it improve sternal stability? J Cardiothorac Surg. 2009;4:19, http://dx.doi. org/10.1186/1749-8090-4-19. 6. Wynne R, Botti M, Stedman H, Holsworth L, Harinos M, Flavell O, et al. Effect of three wound dressings on infection, healing comfort, and cost in patients with sternotomy wounds. A randomized trial. Chest 2004;125(1):43-9. 7. Spiliotis J, Tsiveriotis K, Datsis AD, Vaxevanidou A, Zacharis G, Giafis K, et al. Wound dehiscence: is still a problem in the 21th century: a retrospective study. World J Emerg Surg. 2009;4:12. 8. Deschamps C, Allen MS, Miller DL, Nichols III FC, Pairolero PC. Management of postpneumonectomy empyema and bronchopleural fistula. Seminars Thorac Cardiovasc Surg. 2001;13(1):13-9. 9. Schneiter D, Grodzki T, Lardinois D, Kestenholz PB, Wojcik J, Kubisa B, et al. Accelerated treatment of postpneumonectomy empyema: A binational long-term study. J Thorac Cardiovasc Surg. 2008;136(1):17985, http://dx.doi.org/10.1016/j.jtcvs.2008.01.036. 10. Karabay O, Fermanci E, Silistireli E, Aykut K, Yurekli I, Catalyurek H, et al. Intracutaneous versus transcutaneous suture techniques. Tex Heart Ins J. 2005;32(3):277-82. 11. Durkaya S, Kaptanog˘lu M, Nadir A, Yılmaz S, C ¸ ınar Z, Dog˘an K. Do absorbable sutures exacerbate presternal scarring? Tex Heart Inst J. 2005;32(4):544-8. 12. Fleck T, Kickinger B, Moidl R, Waldenberger F, Wolner E, Grabenwoger M, et al. Management of open chest and delayed sternal closure with the vacuum assisted closure system: preliminary experience. Interac Cardiovasc and Thorac Surg. 2008;7(5):801-4.

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CLINICAL SCIENCE

Evaluation of the association between the JAK2 46/1 haplotype and chronic myeloproliferative neoplasms in a Brazilian population Sarah Pagliarini-e-Silva,I,II Bruna Cunha Santos,I Elizangela Mendes de Figueiredo Pereira,I Mari Ellen Ferreira,II Elaine Cristina Baraldi,III Ana Maria Sell,I Jeane Eliete Laguila VisentainerI I

Universidade Estadual de Maringa´ (UEM), Departamento de Cieˆncias Ba´sicas da Sau´de, Laborato´rio de Imunogene´tica, Maringa´/PR, Brazil. II Hospital do Caˆncer de Maringa´, Maringa´/PR, Brazil. III Instituto do Caˆncer de Londrina, Londrina/PR, Brazil.

OBJECTIVE: The JAK2 46/1 haplotype has recently been described as a major contributing factor to the development of myeloproliferative neoplasm, whether positive or negative for the JAK2 V617F mutation. The G allele, identified by a single-nucleotide polymorphism known as JAK2 rs10974944, is part of the JAK2 46/1 haplotype. The aim of this study was to verify the association between the presence of the G allele and the development of BCR-ABL-negative chronic myeloproliferative neoplasms in our population. METHODS: Blood and oral mucosa swab samples were obtained from 56 patients of two local Brazilian hospitals who had previously been diagnosed with BCR-ABL-negative chronic myeloproliferative neoplasms. Blood samples from 90 local blood donors were used as controls. The presence of the G allele was assessed using a PCR-RFLP assay after extracting DNA from the samples. RESULTS: The presence of the G allele was strongly associated with the presence of BCR-ABL-negative chronic myeloproliferative neoplasms (p = 0.0001; OR = 2.674; 95% CI = 1.63024.385) in the studied population. CONCLUSION: In agreement with previous reports, the JAK2 46/1 haplotype, represented in this study by the presence of the G allele, is an important predisposing factor in the oncogenetic development of these neoplasms in our population. KEYWORDS: Myeloproliferative Disorders; JAK2 Human; Haplotype. Pagliarini e Silva S, Santos BC, Pereira EM, Ferreira ME, Baraldi EC, Sell AM, et al. Evaluation of the association between the JAK2 46/1 haplotype and chronic myeloproliferative neoplasms in a Brazilian population. Clinics. 2013;68(1):5-9. Received for publication on June 15, 2012; First review completed on July 23, 2012; Accepted for publication on September 7, 2012 E-mail: jelvisentainer@gmail.com / jelvisentainer@uem.br Tel.: 55 44 3011-4864

screening became a cornerstone in the molecular diagnostic approach for cMPN (1,7). Even before identifying a common mutation associated with the development of cMPN, family clusters of patients sharing various types of these disorders had already been identified (8). Landgren et al. (9) studied the risk of PV, ET and MF development among the first-degree relatives of patients who had been previously diagnosed with cMPN. First-degree relatives were observed to have a five to seven times greater risk of developing cMPN, particularly in certain probands, suggesting that family members may share some of the oncogenes involved in myeloproliferative disorders. Pardanani et al. (10) studied the genetic factors that could contribute to the phenotypic diversity of cMPN. The authors observed that some JAK2 gene SNPs and haplotypes occurred more frequently in PV patients than in ET or MF patients. They suggested that individual genetic factors could contribute to the diverse phenotypic presentation of cMPN. Following the above findings, Jones et al. (11) analyzed several JAK2 SNPs. Considering uniparental disomy as the

& INTRODUCTION Chronic myeloproliferative neoplasms (cMPNs) are disorders that are characterized by the clonal proliferation of a single hematopoietic stem cell and result in increased peripheral blood counts of mature cells; they include chronic myeloid leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (MF) (1). The JAK2 V617F mutation was identified in more than 95% of PV patients and in approximately half of the patients with ET and MF but was not observed in healthy individuals (2-6). Consequently, JAK2 V617F mutation

Copyright ß 2013 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. DOI: 10.6061/clinics/2013(01)OA02

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Figure 1 - JAK2 V617F screening using the PCR-RFLP technique. After digestion, the JAK2 V617F-positive samples presented a 460-bp band relative to a 50-bp molecular weight ladder. (A) An ET JAK2 V617F negative patient. (B) A PV JAK2 V617F positive patient. (C) A JAK2 V617F positive control. (D) A JAK2 V617F negative control. (E) Blank.

main cause of JAK2 V617F mutation homozygosity, the authors concluded that the JAK2 V617F mutation does not occur randomly but rather in a specific JAK2 gene haplotype. A comparison of JAK2 gene SNPs led to the identification of 92 different haplotypes. Two of them, referenced together as the 46/1 haplotype, were more frequently observed in patients carrying the JAK2 V617F mutation than in the general population. Using genomic array techniques, Kilpivaara et al. (12) searched for SNPs that could predispose an individual to develop cMPN or that might act as phenotypic modifiers in these diseases. The JAK2 G allele, characterized by the presence of the rs10974944 SNP, occurred more frequently in PV than in ET patients. It was also more frequently observed in cMPN patients than in the general population. The C allele, characterized by the absence of the rs10974944 SNP, was considered to be the common allele that was most frequently observed in the general population. The G allele was also strongly associated with the JAK2 V617F mutation, with both occurring more frequently on the cis strand. The aim of this study is to analyze the possible association between the G allele in cMPN patients and to verify its role in the development of these diseases in a Brazilian population.

& METHODS The study was submitted to and approved by the local ethics committee. All of the participants signed an informed consent. Blood and oral swab samples were collected from 56 patients who received treatment at one of two regional oncologic services (Hospital do Caˆncer de Maringa´ and Instituto do Caˆncer de Londrina); these patients were previously diagnosed with BCR-ABL-negative cMPN according to the 2008 WHO diagnostic criteria (1). Blood samples were also collected from 90 healthy individuals (blood donors from the Hemocentro Regional de Maringa´) and used as controls, as the JAK2 V617F mutation is not found in healthy individuals (3-6,13,14). DNA was extracted from the biological specimens using a commercial kit according to the recommendations of the manufacturer (QIAampH DNA Blood Mini Kit, Qiagen). All of the blood samples obtained from the cMPN patients were genotyped for the JAK2 V617F mutation using a PCR-RFLP assay as previously described (14). To better visualize the post-digestion bands, we modified the described technique to include 2 U (1 mL) of BsaXI enzyme in each PCR amplicon digestion and used a 3% agarose gel to run the post-digestion electrophoresis. The JAK2 V617F-

Figure 2 - JAK2 rs10974944 SNP allele screening using the PCR-RFLP technique. The presence of the G allele is characterized by the observation of a 213-bp band, whereas the presence of the C allele is noted by the observation of a 176-bp band, relative to a 50-bp molecular weight ladder. Lanes A-D represent patient blood samples. (A) Homozygous C allele. (B) Heterozygous C/G alleles. (C) Homozygous G allele. (D) Heterozygous C/G alleles.

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JAK2 haplotype and myeloproliferation Pagliarini-e-Silva S et al.

the cMPN patients alone, 16 (94%) of the 17 PV patients, 14 (64%) of the 22 ET patients and 7 (58%) of the 12 MF patients were positive for the JAK2 V617F mutation. Five (10% of 56) patients could not be classified with one of the three disease variants and were therefore classified as myeloproliferative neoplasms, unclassifiable (MPNu) according to the 2008 WHO diagnostic criteria (1). Some of those patients lacked information in their medical records or clinical history, and this missing data could have compromised the accuracy of the classification. The JAK2 V617F mutation screening in those patients indicated 80% (four of the five MPNu patients) positive results, which suggested that they should be placed into the BCR-ABL-negative cMPN group. The distribution of JAK2 rs10974944 SNP genotypes and alleles is shown in Table 2, according to JAK2 V617F mutational status. When comparing the JAK2 rs10974944 SNP genotypes and allelic frequencies between the patients and controls (Table 3), higher frequencies of the GG genotype and G allele were observed in the cMPN patients (p = 0.0116; OR = 3.079; 95% CI = 1.34727.04 and p = 0.0001; OR = 2.674; 95% CI = 1.63024.385, respectively). The CC genotype and C allele were observed more frequently in the healthy controls (p = 0.0029; OR = 0.331; 95% CI = 0.16420.666 and p = 0.0001; OR = 0.374; 95% CI = 0.22820.614, respectively). A significant association was also observed between the JAK2 V617F mutation and the JAK2 rs10974944 SNP genotype or allele after comparing the patients who carried the mutation with the controls (Table 4). The GG genotype and G allele were more frequently observed in the patients who carried the mutation than in the healthy controls (p = 0.006; OR = 3.482; 95% CI = 1.45428.339 and p = 0.0001; OR = 3.222; 95% CI = 1.88425.510, respectively). A comparison between the JAK2 rs10974944 SNP genotype or allele frequencies in the JAK2 V617F-negative patients and in the controls revealed no significant difference (data not shown). In addition, a comparison of the JAK2 rs10974944 SNP genotype or allele frequencies between the JAK2 V617F positive and negative patients did not reveal a significant difference in contrast to what has been previously described (11,12,16). This distinction may be explained by the modest number of patients enrolled in this study.

Table 1 - General characteristics and JAK2 V617F mutational status of the cMPN patients. Age (median range, in years) Sex Male Female M:F Diagnosis PV JAK2 V617F-positive JAK2 V617F-negative ET JAK2 V617F-positive JAK2 V617F-negative MF JAK2 V617F-positive JAK2 V617F-negative MPNu JAK2 V617F-positive JAK2 V617F-negative TOTAL (cMPN patients)

22-83 (60.6)

25 (45%) 31 (55%) 0.8:1 17 (30%) 16 (94%) 1 (6%) 22 (39%) 14 (64%) 8 (36%) 12 (21%) 7 (58%) 5 (42%) 5 (10%) 4 (80%) 1 (20%) 56

cMPN: chronic myeloproliferative neoplasms; PV: polycythemia vera; ET: essential thrombocytosis; MF: primary myelofibrosis; MPNu: myeloproliferative neoplasms, unclassifiable

positive samples presented a 460-bp band (Figure 1). The JAK2 V617F-negative samples contained two bands: 241 bp and 189 bp. The absolute and relative frequencies of the JAK2 V617F mutation in the cMPN patients were calculated. Considering the possibility that a loss of heterozygosity occurred due to uniparental disomy, leading to a misinterpretation of the haplotype results, germline haplotypes (determined by oral swab) were compared to the results of the blood samples from the cMPN patients. Blood and oral swab samples obtained from cMPN patients and blood samples obtained from healthy subjects were submitted to JAK2 rs10974944 SNP screening using a PCR-RFLP assay developed by Trifa et al. (15,16). After digestion with the MboI enzyme, the presence of the G allele was detected by the observation of a 213-bp band, whereas the presence of the C allele is noted by the observation of a 176-bp band (Figure 2). The allelic and genotypic frequencies were calculated using direct counts. The allelic and genotypic frequencies for the patients and the controls were compared using a Chi-Square test with Yates’ correction, considering a 95% confidence interval (CI).

& DISCUSSION Recent reports have suggested that hereditary genetic factors, specifically the JAK2 haplotypes, can strongly contribute to the development of cMPN (11,12,16-18). These observations help explain previously known cMPN family clusters (8,9).

& RESULTS The overall characteristics of the cMPN patients enrolled in this study are shown in Table 1. After analyzing each of

Table 2 - JAK2 rs10974944 SNP genotype/allele frequencies in cMPN patients and controls. cMPN patients

JAK2rs10974944 genotype/alleles

Genotype CC CG GG Allele C G

Controls (n = 90)

JAK2 V617F-positive (n = 43)

JAK2 V617F-negative (n = 13)

Total (n = 56)

11 (26%) 17 (40%) 15 (34%)

7 (54%) 3 (23%) 3 (23%)

18 (32%) 20 (36%) 18 (32%)

53 (59%) 25 (28%) 12 (13%)

39 (45%) 47 (55%)

17 (65%) 9 (35%)

56 (50%) 56 (50%)

131 (73%) 49 (27%)

cMPN: chronic myeloproliferative neoplasms, SNP: single-nucleotide polymorphism

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Table 3 - Correlation between JAK2 rs10974944 SNP genotype/allele frequencies and the presence of cMPN (cMPN patients vs. control group). cMPN patients vs. control group

JAK2rs10974944 genotype/alleles

Genotype CC CG GG Allele C G

Patients (n = 56)

Controls (n = 90)

OR (95% CI)

p-value

18 (32%) 20 (36%) 18 (32%)

53 (59%) 25 (28%) 12 (13%)

0.331 (0.164-0.666) 1.44 (0.706-2.953) 3.079 (1.347-7.04)

0.0029 0.4 0.0116

56 (50%) 56 (50%)

131 (73%) 49 (27%)

0.374 (0.228-0.614) 2.674 (1.630-4.385)

0.0001 0.0001

cMPN: chronic myeloproliferative neoplasms, SNP: single nucleotide polymorphism.

The aim of this study was to analyze whether the genetic factors described above also play an important role in the development of cMPN in our population. The results of our study corroborate the hypothesis that JAK2 haplotypes are a major predisposing factor toward cMPN in the studied population. The GG genotype and G allele frequencies, representative of the JAK2 46/1 haplotype, were found to be significantly higher in our sample of patients. It was observed that the JAK2 V617F mutation could occur in a homozygous or heterozygous fashion in the affected hematopoietic cells (2-5). Homozygosity frequently occurs for a specific allele through duplication of the mutated allele and consequent loss of the non-mutated allele (2). This event is called uniparental disomy. To rule out acquired uniparental disomy as a misleading factor in the genotype and allelic frequencies estimation, we analyzed oral swabs (data not shown) and verified that these results were concordant with the blood sample results. The correlation between the JAK2 46/1 haplotype and the presence of the JAK2 V617F mutation has been well documented (11,12). Recent studies evaluating this correlation among other populations have also confirmed the JAK2 46/1 haplotype as a predisposing factor across various ethnic groups (19-20). It was suggested that the JAK2 46/1 haplotype brings genetic instability to the JAK2 gene, favoring the emergence of JAK2 acquired mutations, such as the JAK2 V617F and JAK2 exon 12 mutations (10-12,18). Our data reinforces these findings because the G allele and the GG genotype were more frequent in the patients who carried the JAK2 V617F mutation than in the healthy controls. As shown in Table 1, the characteristics of the studied patients are very similar to those observed in the literature (2-5), which indicates the comparability of our data and

supports the assertion that the presence of the G allele is a pivotal factor in the development of cMPN in our population. Various hypotheses have been proposed to explain the correlation between particular JAK2 haplotypes and the risk of cMPN. As mentioned above, one hypothesis is that a genetic instability brought by the presence of a specific inherited haplotype facilitated the emergence of an acquired JAK2 mutation (10-12,18). Another hypothesis suggests that the JAK2 46/1 haplotype could confer a proliferative or survival advantage to the neoplastic clone, which may explain the increased frequency of the JAK2 46/1 haplotype in different populations of cMPN patients (18). It is also possible that the various JAK2 haplotypes could produce different intensities of intracellular signaling, conferring the proliferative or survival advantage mentioned above (11,12). These hypotheses might coexist or even act synergistically in the process of cMPN oncogenesis. The JAK2 V617F mutation also seems to play a role in other myeloproliferative and myelodysplastic diseases, reinforcing the importance of intracellular signaling pathways in the progression of disease (21). The evaluation of genetic factors implicated in the development of chronic myeloproliferative disorders helps to identify acquired mutations, such as JAK2 V617F, and allows for the development of diagnostic assays. Recently, these molecular assays became a valuable tool in the evaluation of patients presenting with polycythemias, making diagnostic workup easier. In recently published data, inherited genetic factors were suggested to be important characters in the pathways of cMPN oncogenesis. More studies are needed to clarify the exact role these genetic factors play in modifying intracellular signaling and

Table 4 - Correlation between JAK2 rs10974944 SNP genotype/allele frequencies and the presence of the JAK2 V167F mutation (JAK2 V617F-positive patients vs. control group). JAK2 V617F-positive patients vs. control group

JAK2rs10974944 genotype/alleles

Genotype CC CG GG Allele C G

JAK2 V617F-positive patients (n = 43)

Controls (n = 90)

OR (95% CI)

p-value

11 (26%) 17 (40%) 15 (34%)

53 (59%) 25 (28%) 12 (13%)

0.24 (0.108-0.536) 1.7 (0.790-3.656) 3.482 (1.454-8.339)

0.0006 0.244 0.0078

39 (45%) 47 (55%)

131 (73%) 49 (27%)

0.311 (0.182-0.531) 3.222 (1.884-5.510)

0.0001 0.0001

cMPN: chronic myeloproliferative neoplasms, SNP: single-nucleotide polymorphism.

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JAK2 haplotype and myeloproliferation Pagliarini-e-Silva S et al.

perhaps to identify new targets for future diagnostic tools or even therapeutic agents.

9.

& ACKNOWLEDGMENTS 10.

The authors are thankful to all of the professionals of the Hemocentro Regional de Maringa´ for offering the control samples and to the professionals of the Laboratory of Molecular Diagnostics on Oncohematologic Diseases from Unicamp, especially Daiane de Almeida, who generously provided assistance with technical advice concerning the molecular techniques employed in this study.

11.

12.

& AUTHOR CONTRIBUTIONS 13.

Pagliarini e Silva S and Sell AM were responsible for the statistical analysis. Pagliarini e Silva S, Santos BC, and Pereira EM designed the study and performed the molecular biological analysis. Pagliarini e Silva S, Ferreira ME, and Baraldi EC were responsible for the evaluation and collection of the clinical data. Pagliarini e Silva S, Sell AM, and Visentainer, JE were responsible for the manuscript writing and critical review.

14.

15.

& REFERENCES 16.

1. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW (Ed.) WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon: International Agency for Research on Cancer: 2008. 439p. 2. Jones AV, Kreil S, Zoi K, Waghorn K, Curtis C, Zhang L, et al. Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders. Blood. 2005;106(6):2162-8. 3. Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. The Lancet. 2005;365(9464):1054-61. 4. Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg J. et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med. 2005;352(17):1779-90. 5. James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C. et al. A unique clonal JAK2 mutation leading to constitutive signaling causes polycithemia vera. Nature. 2005;434(7037):1144-8. 6. Rane SG, Reddy EP. Janus kinases: components of multiple signaling pathways. Oncogene. 2000;19(49):5662-79. 7. Barcelos MM, Santos-Silva MC. Molecular approach to diagnose BCR/ ABL negative chronic myeloproliferative neoplasms. Rev Bras Hematol Hemoter. 2011;33(4):290-6. 8. Bellane´-Chantelot C, Chaumarel I, Labopin M, Bellanger F, Barbu V, De Toma C, et al. Genetic and clinical implications of the Val617Phe JAK2

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mutation in 72 families with myeloproliferative disorders. Blood. 2006; 108(1):346-52. Landgren O, Goldin LR, Kristinsson SY, Helgadottir EA, Samuelsson J, Bjo¨rkholm M. Increasead risk of polycythemia vera, essential thrombocythemia, and myelofibrosis among 24577 first-degree relatives of 11039 patients with myeloproliferative neoplasms in Sweden. Blood. 2008;112(6):2199-04. Pardanani A, Fridley BL, Lasho TL, Gilliland DG, Tefferi A. Host genetic variation contributes to phenotypic diversity in myeloproliferative disorders. Blood. 2008;111(5):2785-9. Jones AV, Chase A, Silver RT, Oscier D, Zoi K, Wang YL, et al. JAK2 haplotype is a major risk factor for the development of myeloproliferative neoplasms. Nat Genet. 2009;41(4):446-9. Kilpivaara O, Mukherjee S, Schram AM, Wadleigh M, Mullally A, Ebert BL, et al. A germline JAK2 SNP is associated with predisposition to the development of JAK2 V617F-positive myeloproliferative neoplasms. Nat Genet. 2009;41(4):455-9. Monte-Mo´r BCR, Costa FF. A mutac¸a˜o JAK2 V617F e as sı´ndromes mieloproliferativas. Rev Bras Hematol Hemoter. 2008;30(3):241-8. Monte-Mo´r BCR, Cunha AF, Pagnano KBB, Saad ST, Lorand-Metze I, Costa FF. JAK2 V617F prevalence in Brazilian patients with polycythemia vera, idiopathic myelofibrosis and essential thrombocythemia. Genet Mol Biol. 2007;30(2):336-8. Trifa AP, Cucuianu A, Popp RA. Development of a reliable PCR-RFLP assay for investigation of the JAK2 rs10974944 SNP, which might predispose to the acquisition of somatic mutation JAK2 V617F. Acta Haematol. 2010;123(2):84-7. Trifa AP, Cucuianu A, Ljubomir P, Urian L, Militaru MS, Dima D, et al. The G allele of the JAK2 rs10974944 SNP, part of the JAK2 46/1 haplotype, is strongly associated with JAK2 V617F-positive myeloproliferative neoplasms. Ann Hematol. 2010;89(10):979-83. Olcaydu D, Skoda RC, Looser R, Li S, Cazzola M, Pietra D, et al. The ’GGCC’ haplotype of JAK2 confers susceptibility to JAK2 exon 12 mutation-positive polycythemia vera. Leukemia. 2009;23(10):1924-6. Goldin LR, Bjorkholm M, Kristinsson SY, Samuelsson J, Landgren O. Germline and somatic JAK2 mutations and susceptibility to chronic myeloproliferative neoplasms. Genome Medicine. 2009;1(5):55-5. Ohyashiki JH, Yoneta M, Hisatomi H, Iwabuchi T, Umezu T, Ohyashiki K. The C allele of JAK2 rs4495487 is an additional candidate locus that contributes to myeloproliferative neoplasm predisposition in the Japanese population. BMC Med Genet. 2012;17:13-6. Tian ZQ, Zhu P, Liu HX, Chen Y, Wang F, Zhang Y, Teng W, et al. Relationship between V617F mutation and 46/1 haplotype in JAK2 gene in patients with chronic myeloproliferative diseases and frequencies of 46/1 haplotype in different Chinese nationalities. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2012;20(2):362-7. Machado-Neto JA, Traina F, Lazarini M, Campos PM, Pagnano KB, Lorand-Metze I, Costa FF, Saad ST. Screening for hotspot mutations in PI3K, JAK2, FLT3 and NPM1 in patients with myelodysplastic syndromes. Clinics. 2011;66(5):793-9.


CLINICAL SCIENCE

Dietary patterns in Brazilian patients with nonalcoholic fatty liver disease: a cross-sectional study Silvia Marinho Ferolla,I Teresa Cristina Abreu Ferrari,II Maria Luı´za Pereira Lima,I Taˆmara Oliveira Reis,I Wilson Campos Tavares-Jr.,III Osvaldo Fla´vio Melo Couto,I Paula Vieira Texeira Vidigal,IV Maria Arlene Fausto,V Cla´udia Alves CoutoII I

Universidade Federal de Minas Gerais, University Hospital, Alfa Institute of Gastroenterology, Belo horizonte/MG, Brazil. II Universidade Federal de Minas Gerais, Department of Internal Medicine, School of Medicine, Belo Horizonte/MG, Brazil. III Universidade Federal de Minas Gerais, University Hospital, Division of Radiology, Belo horizonte/MG, Brazil. IV Universidade Federal de Minas Gerais, School of Medicine, Department of Pathology and Forensic Medicine, Belo Horizonte/MG, Brazil. V Universidade Federal de Ouro Preto, University Campus, School of Nutrition, Ouro Preto/MG, Brazil.

OBJECTIVE: Recent evidence suggests that non-alcoholic fatty liver disease is associated with diet. Our aim was to investigate the dietary patterns of a Brazilian population with this condition and compare them with the recommended diet. METHODS: A cross-sectional study was conducted on 96 non-alcoholic fatty liver disease patients before any dietetic counseling. All patients underwent abdominal ultrasound, biochemical tests, dietary evaluations, and anthropometric evaluations. Their food intake was assessed by a semi-quantitative food-frequency questionnaire and 24-hour food recall. RESULTS: The median patient age was 53 years, and 77% of the individuals were women. Most (67.7%) participants were obese, and a large waist circumference was observed in 80.2% subjects. Almost 70% of the participants had metabolic syndrome, and 62.3% presented evidence of either insulin resistance or overt diabetes. Most patients (51.5, 58.5, and 61.7%, respectively) exceeded the recommendations for energy intake, as well as total and saturated fat. All patients consumed less than the amount of recommended monounsaturated fatty acids, and 52.1 and 76.6% of them consumed less polyunsaturated fatty acids and fiber, respectively, than recommended. In most patients, the calcium, sodium, potassium, pyridoxine, and vitamin C intake did not meet the recommendations, and in 10.5-15.5% of individuals, the tolerable upper limit intake for sodium was exceeded. The patients presented a significantly high intake of meats, fats, sugars, legumes (beans), and vegetables and a low consumption of cereals, fruits, and dairy products compared with the recommendations. CONCLUSIONS: Although patients with non-alcoholic fatty liver disease exhibited high energy and lipid consumption, most of them had inadequate intake of some micronutrients. The possible role of nutrientdeficient intake in the development of non-alcoholic fatty liver disease warrants investigation. KEYWORDS: Non-Alcoholic Fatty Liver Disease; Diet; Food; Obesity; Metabolic Syndrome; Brazil. Ferolla SM, Ferrari TC, Lima ML, Reis TO, Tavares-Jr WC, Couto OF, et al. Dietary patterns in Brazilian patients with non-alcoholic fatty liver disease: a cross-sectional study. Clinics. 2013;68(1):11-17. Received for publication on July 23, 2012; First review completed on August 26, 2012; Accepted for publication on September 16, 2012 E-mail: tferrari@medicina.ufmg.br Tel.: 55 31 3409 9746

interrelated risk factors for cardiovascular disease. Obesity and diabetes are predictors of advanced liver fibrosis and cirrhosis in NAFLD patients (2). In the United States, the prevalence of obesity in 2007-2008 was 32.2% among adult men and 35.5% among adult women (3). In Brazil, 48% of adults were overweight in 2008-2009, and approximately 12.5% of men and 16.9% of women were obese (4). Because the prevalence of MS and obesity has increased in most countries, the burden of NAFLD is also expected to rise (5). Lifestyle interventions, including dietary changes and increases in physical activity, are the first-line treatment for NAFLD (6). It has been suggested that dietary composition plays a role in NAFLD pathogenesis; thus, changing dietary patterns may constitute a therapeutic resource even in the

& INTRODUCTION Non-alcoholic fatty liver disease (NAFLD) is currently recognized as one of the most common causes of chronic liver disease. It is usually associated with metabolic syndrome (MS) (1), which is characterized by numerous

Copyright ß 2013 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. DOI: 10.6061/clinics/2013(01)OA03

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The laboratory assessment included liver biochemistry, lipid profile, uric acid, TSH and fasting serum insulin and glucose analyses. Insulin sensitivity was calculated using the homeostasis model assessment (21), and insulin resistance was defined by homeostasis model assessment values $3 (22). Fatty liver was diagnosed by abdominal ultrasound using standardized criteria (17). The abdominal ultrasound was performed in all subjects using the same equipment and by the same operator, who was unaware of the clinical and laboratory results. Some patients underwent a liver biopsy according to clinical judgment, and the diagnosis of nonalcoholic steatohepatitis was based on the accepted clinicalpathological criteria (23).

absence of weight reduction (7). A few recent studies have been performed to address these questions, but their results are quite ambiguous (8-14). Some authors note a positive association of the increased consumption of cholesterol (8), saturated fatty acids (8), total fatty acids (12), and a high n6/n-3 fatty acid ratio (12) with the presence of the disease. On the other hand, others report an association between NAFLD and a lower intake of polyunsaturated fats (8) and total lipids (13). Although a positive association of carbohydrate intake with NAFLD (10,13) and MS (14) was previously demonstrated, there is no consensus regarding the relationship between the type of carbohydrate ingested and NAFLD (9,14). Clearly, this issue warrants further investigation. Considering the lack of knowledge regarding the diet of NAFLD patients in Brazil, we undertook this study to evaluate the dietary patterns of Brazilians with NAFLD and compare it with the Dietary Reference Intakes (15) and the Dietary Guide for Brazilians (16).

Dietary assessment Dietary intake was assessed by 24-hour food recall (24hFR) and a semi-quantitative food frequency questionnaire (FFQ) adapted to the Brazilian population, which served as a representation of the usual intake. The first method is a suitable tool with which to evaluate food and beverage intake within the previous 24 hours. It is easy to apply, is inexpensive, and does not depend on the respondent’s literacy. The semi-quantitative FFQ includes a list of more than 80 food items and provides information on long-term nutritional habits. It is the most commonly used method to assess dietary intake in epidemiological studies, and its reproducibility and validity have been investigated in the Brazilian adult population in different studies (24,25). For each food item, the participants indicated their average frequency of consumption over the past year in terms of number of specified meal sizes consumed per day/weekly/ biweekly/rarely/never. The selected frequency category for each food item was converted into a daily intake (9). The nutrient components of each food item were taken from the Brazilian Food Composition Table (26). The dietary reference values for nutrient intake by Americans and Canadians described in the Dietary Reference Intakes guide (15) were used to evaluate the nutrient intake adequacy, taking into account the estimated average intake and the adequate intake. The estimated average intake corresponds to the amount of daily ingestion of a nutrient estimated to satisfy the needs of half of the individuals in a group. The adequate intake values, which are calculated to cover or exceed the needs of all members of a group, were used for nutrients without an available estimated average intake. Both the estimated average intake and the adequate intake take into consideration the individual’s gender and age. The nutrient consumption over the tolerable upper intake limit was determined. The tolerable upper limit intake represents the maximum daily amount of a nutrient that appears to be safe for the majority of healthy people (15). The Dietary Guide for Brazilians, which consists of guidelines proposed by the Brazilian Ministry of Health according to the recommendations of the World Health Organization, was used to evaluate food intake adequacy (16). Groups of foods were classified according to the Dietary Guide for Brazilians as follows: 1) cereals, roots and tubers; 2) fruits and vegetables; 3) legumes (beans and other plant foods rich in protein); 4) milk and dairy products; 5) meat and eggs; and 6) fats and sugars (16).

& METHODS A cross-sectional study was performed that included 96 of the 114 consecutive patients diagnosed with NAFLD in the Hepatology Clinic, University Hospital, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, during a two-year period (2007-2009). This institution is a reference center of the Brazilian public health system. The inclusion criteria comprised a diagnosis of NAFLD according to the American Gastroenterological Association criteria (17) and a lack of previous nutritional counseling. The American Gastroenterological Association criteria for NAFLD include steatosis on an abdominal ultrasound and/ or liver biopsy (performed according to clinical judgment), the exclusion of other causes of liver disease (namely alcohol intake .20 g/day, markers of chronic B or C hepatitis virus infections, auto-immune hepatic disorders, Wilson disease, hemochromatosis, and alpha-1-antitripsin deficiency), no use of steatogenic medications within the past six months, no exposure to hepatotoxins, and no history of bariatric surgery (17). Alcohol use was addressed on at least three different occasions, namely by two doctors and by a dietician during a nutritional interview. Physical activity was evaluated according to the patient’s information. Considering the frequency, duration and type of physical activity (walking for recreation, moderate activity, or vigorous activity), our patients were classified as very active, active, irregularly active, or sedentary (18). MS was defined according to the National Cholesterol Education Program’s Adult Treatment Panel III (19). The study was approved by the local ethics committee (ETIC 280/07), and all patients signed an informed consent form at the time of screening for the study.

Clinical, laboratory, ultrasound and histological investigations All participants underwent anthropometric, laboratory and abdominal ultrasound investigations. The anthropometric data included height (m), weight (kg), body mass index (kg/m2), and waist circumference (cm). Being overweight was defined as having a body mass index .25 and ,30 kg/m2, and obesity was defined as having a body mass index $30 kg/m2 (20). A waist circumference $88 cm (women) or $102 cm (men) was defined as high (19).

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Diet in non-alcoholic fatty liver disease Ferolla SM et al.

and fiber intake. According to both the 24h-FR and the semiquantitative FFQ, all patients had a monounsaturated fat intake lower than the recommended amount, and more than 50% of them had a lower consumption of fiber and polyunsaturated fats than recommended. On the other hand, the intake of saturated fat and energy was higher than recommended in most patients. According to the semiquantitative FFQ evaluation, the carbohydrate intake was below and the total fat intake was above the recommended values.

Statistical analysis Statistical analyses were performed using the STATA software, version 9.0 (StataCorp LP, College Station, TX, USA). The data are presented as frequencies, proportions, means and standard deviations, and medians. The ShapiroWilk test was used to determine whether continuous variables were normally distributed. Continuous variables were compared using the t-test (normal distribution) or the MannWhitney U test (asymmetrical distribution), and proportions were compared using the chi-square test or Fisher’s exact test, where appropriate. To compare the nutrient intake with both the Dietary Reference Intakes and the Dietary Guide for Brazilians, the paired t-test or the Wilcoxon test was used for normal or asymmetrical distributions, respectively. For all tests, p-values ,0.05 were considered statistically significant.

Adequacy of vitamin and mineral intake The proportions of patients who met (or did not meet) the recommended amounts of vitamins and minerals are presented in Table 4. Both the 24h-FR and the semiquantitative FFQ evaluations demonstrated that almost all patients had a calcium intake lower than the recommended value. Most individuals had a low consumption of potassium, vitamin C, and pyridoxine. According to the 24h-FR evaluation, 10.6% of the subjects exceeded the tolerable upper limit intake of sodium, and according to the FFQ evaluations, the amount of sodium consumption was higher than recommended in 15.5% of the cases. Based on the 24h-FR results, more than 50% of the patients had a low intake of retinol, thiamin, magnesium, and manganese.

& RESULTS Characteristics of the study population The demographic, anthropometric, and key laboratory data of the patients are presented in Table 1. Of the 96 patients, 74 (77%) were women, and the median age was 53 years (25th and 75th percentiles = 48 and 60 years, respectively). None of the participants was undernourished, four (4.2%) had normal weight, 27 (28.1%) were overweight, and 65 (67.7%) were obese according to the body mass index classification. A high waist circumference was observed in 77 (80.2%) individuals, namely, 15 men (68.2%) and 62 women (83.8%) (p = 0.11). Seventy-seven (80.2%) patients were sedentary, and 19 (19.8%) were irregularly active. Six (6.2%) individuals were smokers. Almost 70% of the study population had MS, and 60 individuals (62.3%) presented evidence of either insulin resistance (homeostasis model assessment values $3) or overt diabetes. HDL cholesterol was low in 11 (52.4%) men and 53 (75.7%) women (p = 0.05). Other clinical features are shown in Table 2. Approximately 20% of the subjects underwent liver biopsy, and 13 (13.5%) of them had non-alcoholic steatohepatitis based on histological examination.

Food intake adequacy The dietary data concerning food group intake are summarized in Table 5. There were differences in the intake of all food groups compared with the guide. The average number of consumed meals that included cereals, fruits, and dairy products was lower than the recommended number. However, the number of meals consumed with legumes (beans), vegetables, meats, fats, and sugar was higher than recommended in the Dietary Guide for Brazilians.

& DISCUSSION Our NAFLD patients recalled a diet richer in lipids (specifically saturated fat and energy) and poorer in monounsaturated fat, polyunsaturated fat, fiber, calcium, potassium, vitamin C, and pyridoxine than the recommendations. A large proportion of this population also exceeded

Adequacy of energy, macronutrient, and fiber intake Table 3 shows the proportion of patients who met (or did not meet) the recommendations for energy, macronutrient,

Table 1 - Demographic, anthropometric, and laboratory data of NAFLD patients. Total

Male

Female

Parameters

Median/Mean (range)

Median/Mean (range)

Median/Mean (range)

Age (years) Weight (kg) BMI (kg/m2) Glucose (mg/dl) Insulin (mU/ml) HOMA Total cholesterol (mg/dl) Triglycerides (mg/dl) AST (x RV) ALT ( x RV) AP (x RV) GGT (x RV) Albumin (g/dl)

54.4 80.4 32.2 98.0 10.1 1.9 211.0 173.0 0.7 0.6 0.8 1.1 4.4

56.6 87.2 31.0 94.0 9.2 1.7 187.0 156.0 0.7 0.6 0.7 1.0 4.5

53.7 78.4 32.6 98.0 10.4 2.0 216.0 178.0 0.7 0.6 0.8 1.1 4.4

(29.0; 81.0) (53.5; 130.8) (23.4; 44.7) (64.0; 387.0) (2.0; 23.5) (0.5; 6.3) (120.0; 423.0) (61.0; 620.0) (0.3; 4.9) (0.1; 3.3) (0.2; 2.7) (0.3; 15.5) (3.7; 5.2)

(36.0; 70.0) (63.0; 96.4) (34.3; 31.1) (77.0; 275.0) (2.0; 15.8) (0.5; 3.7) (139.0; 291.0) (61.0; 618.0) (0.5; 1.8) (0.3; 2.4) (0.33; 1.1) (0.4; 7.8) (3.9; 5.0)

(29.0; 72.0) (53.5; 107.5) (24.4; 41.6) (64.0; 387.0) (3.9; 24.0) (0.8; 6.3) (120.0; 423.0) (75.0; 620.0) (0.3; 4.9) (0.1; 3.3) (0.2; 2.7) (0.3; 15.5) (3.7; 5.2)

p-value 0.23a 0.008a 0.16a 0.48 0.50a 0.60 0.03 0.60 1.00 0.50 0.30 0.51 0.61a

NAFLD, non-alcoholic fatty liver disease; BMI, body mass index; WC, waist circumference; HOMA, homeostasis model assessment; HDL, high-density lipoprotein; AST, aspartate aminotransferase; ALT, alanine aminotransferase; AP, alkaline phosphatase; GGT, gamma-glutamyltransferase; RV, reference value. Means were compared using the t-test (a), and medians were compared using the Mann-Whitney test.

13


Diet in non-alcoholic fatty liver disease Ferolla SM et al.

CLINICS 2013;68(1):11-17

suggests that the type of carbohydrate ingested may also influence NAFLD pathogenesis (9,14,27). Foods with a high glycemic index (e.g., sugar) increase de novo hepatic lipogenesis, hypertriglyceridemia, hepatic insulin resistance, and liver steatosis (27). Despite their high vegetable consumption, most of our NAFLD population reported low dietary fiber and vitamin C intake. These findings may be due to the low intake of fruits. Approximately 40% of our patients had a high protein intake, which is in agreement with the high ‘‘meat group’’ consumption. Similar results were described by Zelber-Sagi et al. (9). Although a direct association between NAFLD and protein intake has never been investigated, there are reports of an association between protein intake and both insulin resistance and diabetes (27). The higher energy consumption in relation to the estimated requirements, determined by both recent and long-term assessments, most likely reflects the increased intake of food from the ‘‘sugar group’’ and the ‘‘fat group’’. According to some authors, patients with non-alcoholic steatohepatitis have a higher energy intake compared with a control group (11-14). Even in healthy people, hypercaloric food intake could be related to alanine aminotransferase abnormalities (28). Calcium intake was below the recommended value in more than 94% of our population, most likely due to the reduced consumption of milk and dairy products. Recent studies have suggested that calcium deficiency is related to obesity by increasing lipogenesis and hyperinsulinemia and inhibiting lipolysis (29). Furthermore, prospective studies have also suggested that the intake of dairy products and calcium reduces the risk of obesity, abdominal obesity, diabetes, hypertension, and MS (29,30). The magnesium intake was also below the recommended value in most of our patients. As magnesium deficiency plays a role in increasing insulin resistance in diabetes and MS patients (31), we can speculate that this mineral may have some effect on the pathogenesis of NAFLD. Although the magnesium intake of 13.8% of our patients was above the recommended value, the effects of excessive magnesium consumption are known only for magnesium derived from a pharmacological agent, not when the source is food and water (15).

Table 2 - Clinical characteristics of NAFLD patients. Parameters

Total (%) Male (%) Female (%) p-value

Glucose intolerance or diabetes HOMA $3 (without diabetes) Insulin resistance Hypercholesterolemia Low HDL Hypertriglyceridemia Hypertension Hyperuricemia Hypothyroidism Metabolic syndrome

43.8

36.4

46.0

0.43

22.5 62.3 60.4 69.6 63.2 70.8 11.7 11.8 69.5

20.0 55.6 45.5 52.4 57.1 63.6 19.1 9.5 52.3

23.0 64.1 64.9 75.7 64.9 73.0 9.6 12.5 74.3

1.00a 0.51 0.10 0.05 0.52 0.40 0.26a 1.00a 0.05

NAFLD, non-alcoholic fatty liver disease. Proportions were compared using the chi-square test or Fisher’s exact test (a).

the tolerable upper limit for sodium intake. These findings correspond to a higher consumption of meats, fats, and sugar groups, with a lower intake of cereals, fruits, and dairy products. Interestingly, and in contrast to the results obtained by Cortez-Pinto et al. (12), all of our patients reported low monounsaturated fat consumption. Most of them did not consume any foods rich in monounsaturated fats, and less than 12% reported olive oil intake. The relationship between a high intake of monounsaturated fat and a good lipid profile (reduction of low-density lipoprotein (LDL) cholesterol and triglycerides) is well known, and the beneficial effects of a diet rich in monounsaturated fats, particularly a diet in which monounsaturated fats replace both saturated fat and high amounts of carbohydrates, have been extensively investigated (27). As previously reported (8), we also observed a significantly high consumption of total and saturated fat. The intake, corresponding to approximately five and two times the recommendations, respectively, of foods belonging to the ‘‘rich in fat group’’ and the ‘‘meat group’’, certainly contributed to these findings. Although our patients recalled recent low and adequate long-term carbohydrate consumption, their daily intake of meals containing the ‘‘sugar group’’ was approximately twice the recommended intake. It is well known that a high carbohydrate intake plays an important role in the pathogenesis of NAFLD. Furthermore, the available evidence

Table 3 - Distribution of NAFLD patients by category of adequacy / inadequacy of energy, macronutrient and fiber intake. 24-hour food recall

Parameters Recommendation

Male Energy Carbohydratea Protein Total fat Saturated fat MUFAa PUFAa Fiber

Female

Intake Amount Median/ Mean (range)

EER EER 2084.8 (1116.2; 4212.1) 50-60% TEI 270.5 (101.9; 485.7) 0.8-1.0 g/kg 75.4 (5.7; 178.2) 25-35% TEI 82.9 (32.4; 178.5) ,10% TEI 22.8 (7.6; 82.2) ,20% TEI 23.2 (7.3; 45.4) ,10% TEI 24.1 (1.8; 50.5) 20-30 g 18.2 (6.3; 56.5)

Food frequency questionnaire

Adequate

Lower

Over

Intake Amount

Adequate

Lower

Over

(%)

(%)

(%)

Median/ Mean (range)

%

%

%

0.0 28.7 22.3 36.2 0.0 0.0 0.0 2.1

48.9 53.2 34.0 5.3 38.3 100.0 52.1 76.6

51.1 18.1 43.7 58.5 61.7 0.0 47.9 21.3

0.0 38.1 22.6 44.1 0.0 0.0 0.0 0.0

32.1 34.5 32.1 14.3 33.3 100.0 54.8 53.6

67.9 27.4 45.2 41.7 66.7 0.0 45.3 46.4

2507.9 320.3 73.5 85.1 20.9 24.1 25.8 24.3

(473.9; 6575.3) (49.0; 1150.2) (21.3; 238.8) (17.7; 203.7) (4.5; 64.5) (6.2; 73.2) (4.2; 81.5) (8.0; 76.3)

NAFLD, non-alcoholic fatty liver disease; EER, energy expenditure requirements; TEI, total energy intake; MUFA, monounsaturated fat; PUFA, polyunsaturated fat. Mean (a).

14


65.5 67.9 100.0 64.3 44.1 4.8 60.0 60.7 61.5 61.9 63.1 35.7 65.4 4.8 5.9 349.7 (95.4; 2696.3) 2.4 (0.6; 9.6) 1003.6 (294.6;2791.2) 7.4 (2.9; 21.1) 1.3 (0.2; 3.7) 2.6 (1.0; 7.0) 1257.2 (380.9; 5442.5) 8.0 (2.4; 30.6) 267.4 (1.4; 5337.5) 1.1 (0.3; 4.0) 1.2 (0.2; 4.1) 0.9 (0.1; 4.7) 14.0 (3.4; 57.9) 79.2 (2.5; 620.6) 472.0 (108.6; 1734.4)

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.2 0.0 0.0 0.0 0.0

34.5 32.1 0.0 35.7 56.0 95.2 40.0 39.3 38.5 38.1 35.7 64.3 34.5 95.2 94.1

Sodium intake was most likely underestimated in our sample population, as we took into account only the amount of sodium naturally contained in food but not the addition of sodium. This limitation was due to the difficulties in measuring the addition of sodium to foods and preparations. Nevertheless, 10.5-15.5% of our patients exceeded the upper limit intake of sodium. It is well known that this dietary pattern increases the risks of hypertension, cerebrovascular disorders, and coronary heart disease (32,33). Our NAFLD patients reported a low consumption of antioxidant vitamins, specifically vitamins A and C. The low intake of retinol is at least partially due to the reduced consumption of milk (average, 0.4 meals per day) and eggs (average, 0.04 meals per day). The low intake of fruits, as mentioned previously, may have contributed to the low intake of vitamin C and beta-carotene, a precursor of vitamin A. These findings are consistent with the results of an Italian study, which demonstrated the low consumption of vitamin C by non-alcoholic steatohepatitis patients (8). Additionally, low serum levels of retinol in NAFLD individuals were observed in a Brazilian investigation (34). It is well known that antioxidant vitamins have a protective role against oxidative stress (34); therefore, the supplementation of these vitamins for treating NAFLD has been widely investigated (35). In this study, the high zinc, iron, copper, phosphorus, riboflavin, and niacin consumption above the estimated average intake and adequate intake values may be related to the high intake of the ‘‘meat group’’ (average, 0.7 meals/ 1,000 kcal). Moreover, the high consumption of beans, a typical food in Brazilian cuisine, may also explain the high iron and cooper intake. Contrary to our observation, Toshimitsu et al. (14) found low zinc consumption by NASH individuals. Additionally, in accordance with our findings, Alla and Bonkovsky (36) demonstrated a high intake of iron, especially heme-iron, which plays a role in NAFLD pathogenesis by increasing oxidative stress. In this context, some authors observed that the dietary restriction of iron, calories, and fat is associated with a decrease in serum aminotransferases and ferritin levels in NAFLD (37). The data from a recent study suggest that the dietary restriction of copper in rats may be involved in the development of hepatic steatosis and insulin resistance (38). In our study, in contrast, the intake of copper was above the recommended level. However, the association between NAFLD and copper intake has never been investigated in humans. To our knowledge, there are no published data relating phosphorus, niacin and riboflavin to NAFLD. Our study has some methodological limitations. As this study was a cross-sectional investigation, it is not possible to associate dietary patterns with the relative risk of NAFLD. However, the study provides the first description of the dietary patterns of Brazilian patients with NAFLD. In this context, a control group would have added more information regarding possible differences (or lack of differences) in relation to the dietary patterns of Brazilians without NALFD. We did not stratify our population according to age and socioeconomic level, which are factors that could influence dietary patterns. However, most of our subjects (66%) were aged between 40 and 59 years, and almost all of them (98%) were aged between 30 and 70 years; furthermore, it is well known that patients utilizing the public health system in Brazil have low purchasing power. It

60

6.8 500 0.9 0.9 #50 yr: 1.3 .50 yr: 1.5 11

19-50 yr: 8.1 .50 yr: 5.0

NAFLD, non-alcoholic fatty liver disease. Recommendations are expressed as the adequate intake (a) or as the estimated average intake.

13.8 31.9 100.0 66.7 31.9 2.1 60.6 70.2 8.4 31.9 53.2 23.2 68.1 23.4 0.0 86.2 68.1 0.0 33.3 66.0 97.9 39.4 29.8 91.6 68.1 46.8 76.6 31.9 76.6 100.0 0.0 0.0 0.0 0.0 2.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 183.8 (74.3; 534.3) 1.6 (0.2; 13.8) 858.1 (178.5;1995.3) 7.1 (2.4; 19.4) 1.2 (0.0; 3.8) 2.0 (0.9; 5.4) 654.1 (209; 3004.4) 8.8 (1.4; 25.4) 125.0 (0.0; 1275.2) 0.7 (0.1; 2.6) 1.0 (0.0; 3.7) 0.7 (0.1; 3.7) 14.7 (0.5; 37.7) 18.0 (0.0; 444.0) 360.0 (13.8; 1213.3) #30 yr: 255 .30 yr: 265 1.8

Magnesium (mg) Manganese (mg) Phosphorus (mg) Iron (mg) Sodium (g)a Potassium (g)a Copper (mg) Zinc (mg) Vitamin A (mg) Thiamine (mg) Riboflavin (mg) Pyridoxine (mg) Niacin (mg) Calcium (mg)a Vitamin C (mg)

a

Male

Diet in non-alcoholic fatty liver disease Ferolla SM et al.

#30 yr: 330 .30 yr: 350 2.3 34 6.0 #50 yr: 1.5 51-70 yr: 1.3 .70 yr: 1.2 4.7 700 9.4 625 1.0 1.1 #50 yr: 1.3 .50 yr: 1.7 12 #50 yr: 1000 .50 yr: 1200 75

(%) (%) (%) Mean/Median (range) (%) (%) (%) Female

Mean/Median (range)

Lower Adequate Intake Amount Over Lower Adequate Intake Amount

24-hour food recall Recommendations Parameters

Table 4 - Distribution of NAFLD individuals by category of adequacy / inadequacy of mineral and vitamin intake.

Food frequency questionnaire

Over

CLINICS 2013;68(1):11-17

15


Diet in non-alcoholic fatty liver disease Ferolla SM et al.

CLINICS 2013;68(1):11-17

Table 5 - Comparison between the number of meals consumed by NAFLD patients containing various food groups and the recommended numbers. Food groups Cereals, roots and tubers Legumes (beans and other plant foods rich in protein) Vegetables Fruits Milk and dairy products Meat and eggs Fats Sugars

Recommendation per 1000 kcal 0.3 0.5 1.5 1.5 1.5 0.5 0.5 0.5

meals/day meals/day meals/day meals/day meals/day meals/day meals/day meals/day

Median/Minimum (range) 2.1 0.6 1.6 0.9 0.4 0.6 2.4 1.0

(0.4; (0.0; (0.0; (0.0; (0.0; (0.0; (0.3; (0.0;

4.4) 3.9) 12.4) 7.4) 1.9) 2.1) 5.5) 4.5)

p-value ,0.00005a 0.0 a 0.01 ,0.00005 ,0.00005 0.003 ,0.00005a ,0.00005

NAFLD, non-alcoholic fatty liver disease. Non-normally distributed variables were compared using the Wilcoxon test, and normally distributed variables were compared using the paired t-test (a).

5. Mokdad AH, Bowman BA, Ford ES, Vinicor F, Marks JS, Koplan JP. The continuing epidemics of obesity and diabetes in the United States. JAMA : the journal of the American Medical Association. 2001;286(10):1195-200, http://dx.doi.org/10.1001/jama.286.10.1195. 6. Bellentani S, Dalle Grave R, Suppini A, Marchesini G. Behavior therapy for nonalcoholic fatty liver disease: The need for a multidisciplinary approach. Hepatology. 2008;47(2):746-54. 7. Clark J. Weight loss as a treatment for nonalcoholic fatty liver disease. J Clin Gastroenterol. 2006;40 Suppl 1:S39-43. 8. Musso G, Gambino R, De Michieli F, Cassader M, Rizzetto M, Durazzo M, et al. Dietary habits and their relations to insulin resistance and postprandial lipemia in nonalcoholic steatohepatitis. Hepatology. 2003;37(4):909-16, http://dx.doi.org/10.1053/jhep.2003.50132. 9. Zelber-Sagi S, Nitzan-Kaluski D, Goldsmith R, Webb M, Blendis L, Halpern Z, et al. Long term nutritional intake and the risk for nonalcoholic fatty liver disease (NAFLD): a population based study. J Hepatol. 2007;47(4):711-7, http://dx.doi.org/10.1016/j.jhep.2007.06. 020. 10. Solga S, Alkhuraishe AR, Clark JM, Torbenson M, Greenwald A, Diehl AM, et al. Dietary composition and nonalcoholic fatty liver disease. Digestive diseases and sciences. 2004;49(10):1578-83, http://dx.doi.org/ 10.1023/B:DDAS.0000043367.69470.b7. 11. Capristo E, Miele L, Forgione A, Vero V, Farnetti S, Mingrone G, et al. Nutritional aspects in patients with non-alcoholic steatohepatitis (NASH). European review for medical and pharmacological sciences. 2005;9(5):265-8. 12. Cortez-Pinto H, Jesus L, Barros H, Lopes C, Moura MC, Camilo ME. How different is the dietary pattern in non-alcoholic steatohepatitis patients? Clin Nutr. 2006;25(5):816-23, http://dx.doi.org/10.1016/j.clnu. 2006.01.027. 13. Kang H, Greenson JK, Omo JT, Chao C, Peterman D, Anderson L, et al. Metabolic syndrome is associated with greater histologic severity, higher carbohydrate, and lower fat diet in patients with NAFLD. Am J Gastroenterol. 2006;101:2247-53. 14. Toshimitsu K, Matsuura B, Ohkubo I, Niiya T, Furukawa S, Hiasa Y, et al. Dietary habits and nutrient intake in nonalcoholic steatohepatitis. Nutrition. 2007;23(1):46-52, http://dx.doi.org/10.1016/j.nut.2006.09.004. 15. Murphy SP, Poos MI. Dietary Reference Intakes: summary of applications in dietary assessment. Public Health Nutr. 2002;5(6A):843-9, http://dx.doi.org/10.1079/PHN2002389. 16. Brasil. Ministe´rio da Sau´de. Secretaria de Atenc¸a˜o a` Sau´de. Coordenac¸a˜oGeral da Polı´tica de Alimentac¸a˜o e Nutric¸a˜o. Guia alimentar para a populac¸a˜o brasileira: promovendo a alimentac¸a˜o sauda´vel/ Ministe´rio da Sau´de, Secretaria de Atenc¸a˜o a` Sau´de, Coordenac¸a˜o-Geral da Polı´tica de Alimentac¸a˜o e Nutric¸a˜o. Brası´lia, DF: Ministe´rio da Sau´de; 2005. 17. Sanyal AJ. American Gastroenterological Association. AGA Technical Review on Nonalcoholic Fatty Liver Disease. Gastroenterology. 2002;123(5):1705-25, http://dx.doi.org/10.1053/gast.2002.36572. 18. Matsudo SM, Matsudo VR, Arau´jo T, Andrade D, Andrade E, Oliveira L, et al. Nı´vel de atividade fı´sica da populac¸a˜o do Estado de Sa˜o Paulo: ana´lise de acordo com o geˆnero, idade e nı´vel so´cio-econoˆmico, distribuic¸a˜o geogra´fica e de conhecimento. Rev Bras Cien Mov. 2002;10(4):41-50. 19. Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults. Executive summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation and Treatment of High Cholesterol (Adult Treatment Panel III). JAMA. 2001;285(19):2486-97. 20. WHO: Physical Status. The use and interpretation of anthropometry: report of WHO expert committee. Geneva: World Health Organization; 1995.

would be better if the diagnosis of NAFLD had been established by liver biopsy in all patients. To minimize this limitation, we rigorously followed the American Gastroenterology Association criteria (17). Additionally, the possibility of both memory and reporting bias in dietary assessment should not be ruled out, especially when considering individuals, such as obese subjects, who have knowledge regarding ‘‘healthy diets". The omission of information on food consumption has been reported by several authors (39-42). Furthermore, all methods for dietary pattern evaluation are subject to sampling error. Thus, to minimize these potential errors, we used two methods to assess food intake. The FFQ questionnaire is one of the most accepted methods for measuring food consumption in epidemiological studies, as it is simple, fast and reliable. In conclusion, our NAFLD patients had excessive saturated fat, total lipid and energy intake, as well as some nutritional deficiencies, such as monounsaturated fat, polyunsaturated fat, calcium, vitamin C, and pyridoxine intake deficiencies. These dietetic features could be related to the poor long-term consumption of grains, fruits and dairy products, as well as to the consumption of too many meals containing legumes (beans), meats, fats and sugars, compared with the recommendations of the Dietary Guide for Brazilians. The possible role of each nutrient deficiency in the development of NAFLD needs further investigation.

& AUTHOR CONTRIBUTIONS Ferolla SM, Couto CA, Couto OF and Ferrari TC conceived and designed the study. Ferolla SM, Lima ML and Reis TO collected the data. TavaresJr WC performed the abdominal ultrasounds. Vidigal PV performed the pathological analysis and disease classification (non-alcoholic fatty liver disease or non-alcoholic steatohepatitis). Fausto MA performed the statistical analysis. Ferolla SM, Couto CA, and Ferrari TC prepared the manuscript. All authors have read and approved the final version of the manuscript.

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17


CLINICAL SCIENCE

The relationship between hyperuricemia and the risk of contrast-induced acute kidney injury after percutaneous coronary intervention in patients with relatively normal serum creatinine Yong Liu,I Ning Tan,I Jiyan Chen,I Yingling Zhou,I Liling Chen,III Shiqun Chen,II Zhujun Chen,I Liwen LiI I

Guangdong Academy of Medical Sciences, Guangdong General Hospital, Guangdong Cardiovascular Institute, Department of Cardiology, Guangzhou, China. II Department of Mathematics, Yibin University, Yibin, China. III Fujian Longyan No. 1 Hospital, Department of Cardiology, Longyan, China.

OBJECTIVES: Hyperuricemia is a risk factor for contrast-induced acute kidney injury in patients with chronic kidney disease. This study evaluated the value of hyperuricemia for predicting the risk of contrast-induced acute kidney injury in patients with relatively normal serum creatinine who were undergoing percutaneous coronary interventions. METHODS AND RESULTS: A total of 788 patients with relatively normal baseline serum creatinine (,1.5 mg/dL) undergoing percutaneous coronary intervention were prospectively enrolled and divided into a hyperuricemic group (n = 211) and a normouricemic group (n = 577). Hyperuricemia is defined as a serum uric acid level.7 mg/ dL in males and .6 mg/dL in females. The incidence of contrast-induced acute kidney injury was significantly higher in the hyperuricemic group than in the normouricemic group (8.1% vs. 1.4%, p,0.001). In-hospital mortality and the need for renal replacement therapy were significantly higher in the hyperuricemic group. According to a multivariate analysis (adjusting for potential confounding factors) the odds ratio for contrastinduced acute kidney injury in the hyperuricemic group was 5.38 (95% confidence interval, 1.99-14.58; p = 0.001) compared with the normouricemic group. The other risk factors for contrast-induced acute kidney injury included age .75 years, emergent percutaneous coronary intervention, diuretic usage and the need for an intra-aortic balloon pump. CONCLUSION: Hyperuricemia was significantly associated with the risk of contrast-induced acute kidney injury in patients with relatively normal serum creatinine after percutaneous coronary interventions. This observation will help to generate hypotheses for further prospective trials examining the effect of uric acid-lowering therapies for preventing contrast-induced acute kidney injury. KEYWORDS: Hyperuricemia; Contrast-Induced Acute Kidney Injury; Risk Factors; Percutaneous Coronary Intervention. Liu Y, Tan N, Chen J, Zhou Y, Chen L, Chen S, et al. The relationship between hyperuricemia and the risk of contrast-induced acute kidney injury after percutaneous coronary intervention in patients with relatively normal serum creatinine. Clinics. 2013;68(1):19-25. Received for publication on August 19, 2012; First review completed on September 23, 2012; Accepted for publication on September 23, 2012 E-mail: tanning100@126.com / chenjiyandr@126.com Tel.: +86-20-83819161

solution (3), few prophylactic strategies for CI-AKI are clearly effective. A key step in minimizing the risk of CI-AKI is identifying at-risk patients. Many risk factors, such as pre-existing renal dysfunction, exposure to a high volume of contrast medium, older age, hypovolemia, congestive heart failure, emergent PCI, and exposure to nephrotoxic drugs, have been associated with developing CI-AKI (4-7). Currently, the most widely recognized risk factor for developing CI-AKI is baseline renal impairment, which is conventionally defined as a serum creatinine (SCr) level$1.5 mg/dL (132 mmol/dL) (5,8). Few studies have investigated patients with normal renal function, although these patients constitute the majority of those undergoing PCI. CI-AKI prophylaxis, including hydration, is not typically administered to patients with normal baseline SCr (,1.5 mg/dL).

& INTRODUCTION Contrast-induced acute kidney injury (CI-AKI) has been recognized as a serious complication of percutaneous coronary intervention (PCI) and may cause an increase in morbidity and mortality (1,2). In addition to prophylactic intravenous volume expansion with isotonic crystalloid

Copyright Ă&#x; 2013 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. DOI: 10.6061/clinics/2013(01)OA04

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48-72 h after PCI (17,18). We obtained the blood samples used to measure preoperative serum uric acid, baseline serum creatinine (SCr) (prior to the pre-procedural hydration), blood urea nitrogen (BUN), random blood glucose, electrolytes before PCI, serum lipids, fasting blood glucose, albumin, hemoglobin, and other standard clinical parameters after an 8-h overnight fast. Follow-up SCr and BUN levels were measured one, two, and three days after the procedure (19% of the patients stayed in the hospital for three days and participated in the sampling on day 3). Baseline creatinine clearance was calculated with the CockCroft–Gault formula: (140-age)6weight (kg)/SCr (mg/ dL)726(0.85 for females) (19). The major in-hospital clinical event (e.g., death) was recorded at the same time.

The relationship between hyperuricemia (HUA) and CIAKI has not been extensively studied. It has been suggested that tubular obstruction by uric acid plays a role in the pathogenesis of CI-AKI (9,10). HUA is accompanied by enhanced synthesis of reactive oxygen species, activation of the renin–angiotensin–aldosterone system, an increase in endothelin-1, and inhibition of the nitric oxide system; all of these factors play a role in the pathogenesis of CI-AKI (11-13). Toprak et al. observed that HUA was a risk factor for CIAKI in patients undergoing coronary angiography with chronic kidney disease (SCr$1.2 mg/dL) (14). We aim to identify the risk predictors in this cohort and evaluate the value of HUA in predicting the risk of CI-AKI in patients with relatively normal SCr who are not receiving sufficient renal prophylaxis.

Statistical analysis SPSS software for Windows (version 13.0, SPSS Inc., Chicago, Illinois, USA) was used for the analyses. The demographics and traditional risk factors were compared between the hyperuricemic and normouricemic groups. All of the values are expressed as the mean¡SD or medians and interquartile ranges. The differences between the means were compared with an unpaired t-test for the variables that were normally distributed and with the Mann-Whitney Utest for the variables that were not normally distributed. Categorical variables were analyzed using the x2 test. We performed logistic regression analysis with CI-AKI as the dependent variable. The variables that were statistically significant according to the univariate analysis were included in the final multivariate model to identify the CIAKI predictors. A two-sided 95% confidence interval (CI) was constructed around the point estimate of the odds ratio (OR). All of the tests were two-sided, and a p-value,0.05 was considered statistically significant.

& METHODS Study population We conducted a prospective, controlled single-center observational study at Guangdong Cardiovascular Institute, Guangdong General Hospital, Guangdong Academy of Medical Sciences between February 2010 and January 2011. The exclusion criteria were pregnancy, lactation, sepsis, the intravascular administration of a contrast medium within the past seven days, nephroprotective drug treatment (e.g., N-acetylcysteine, theophylline, prostaglandin E1, sodium bicarbonate), nephrotoxic drug intake (e.g., non-steroidal anti-inflammatory drugs, metformin, aminoglycosides, amphotericin B, cisplatin) within the past seven days, a history of serious reactions to contrast media, renal transplantation, end-stage renal disease necessitating dialysis, and severe concomitant disease of other systems. We also excluded the patients who died during PCI or who had hemodynamic instability requiring an intra-aortic balloon pump (IABP) before PCI. The Ethics Committee of the Guangdong General Hospital approved the study. All of the eligible patients provided written informed consent.

& RESULTS A total of 788 patients were enrolled in the study. Based on their serum uric acid concentrations, 211 patients were assigned to the hyperuricemic group and 577 to the normouricemic group (mean serum uric acid 481¡72 and 310¡66 mmol/L, respectively, p,0.001); mean age (65¡12 and 62¡11, respectively, p,0.001); and proportion of males (70% and 82%, respectively, p,0.001). The following demographic, clinical, and angiographic characteristics were compared between the hyperuricemic and normouricemic groups (as shown in Tables 1 and 2): older age (21% vs. 11%, p,0.001), pre-existing renal dysfunction (48% vs. 29%, p,0.001), serum cholesterol levels (total cholesterol 4.77¡1.29 vs. 4.56¡1.11 mmol/L, p = 0.024; low-density lipoprotein cholesterol 3.07¡1.06 vs. 2.92¡0.95, p = 0.055), severely reduced LVEF (12% vs. 8%, p = 0.061), hypertension (70% vs. 54%, p,0.001), anemia (22% vs. 16%, p = 0.065), multivessel coronary disease (79% vs. 69%, p,0.001), the exposure time to contrast agents, the number of coronary lesions, and the ratio of smokers to non-smokers were significantly different between the two groups. There were no significant differences between the two groups in the incidence of diabetes mellitus (20% vs. 23%, p = 0.441), emergent PCI (23% vs. 26%, p = 0.404), contrast amount, coronary heart disease type, or other parameters. We determined the CI-AKI risk scores according to Scheme Model A developed by Mehran et al. (7). The mean and median CI-AKI risk scores in the hyperuricemic group

Study protocol We screened 960 patients who underwent PCI. A total of 788 patients met the inclusion criteria. The eligible patients were divided into two groups: hyperuricemic (n = 211) and normouricemic (n = 577). As in previous studies (15,16), HUA was defined as a serum uric acid level.7 mg/dL (417 mmol/L) in males and.6 mg/dL (357 mmol/L) in females. None of the patients in the hyperuricemic group had gout, documented uric acid stones, or any acute HUA complications (i.e., they had asymptomatic HUA). Left ventricular function was evaluated with echocardiography in all of the patients within 24 h of admission. The patients were hydrated with intravenous isotonic saline (0.9%) at a rate of 1 mL/kg body weight per h for 1-8 h before and 6-8 h after PCI. In the patients with left ventricular ejection fraction (LVEF) ,40% or overt heart failure, the hydration rate was reduced to 0.5 mL/kg per h. PCI was performed with a low-osmolar, non-ionic contrast medium (LOCM). The PCI operations and the use of an IABP, inotropic drugs, tirofiban, beta-blockers, angiotensinconverting-enzyme inhibitors, and diuretics were left to the discretion of the cardiologists. The primary endpoint of the study was the occurrence of CI-AKI, which was defined as an increase in serum creatinine of$0.5 mg/dL above the baseline value within

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Table 1 - Baseline demographics and clinical characteristics. Variables Age (y) Age.75 y (%) Males (%) Weight (kg) SBP (mmHg) Smokers (%) Hypertension (%) Diabetes (%) Unilateral/bilateral RAS$50 (%) Hyperlipidemia (%) Total cholesterol (mg/dl) LDL-cholesterol (mg/dl) Serum uric acid (mmol/L) Coronary heart disease type Acute coronary syndrome (%) Stable angina (%) Previous MI (%) CABG (%) LVEF (%) LVEF ,40% (%) Emergent PCI (%) ACEI/ARB usage (%) Diuretic usage Baseline SCr (mmol/L) CrCl, (mL/min) CrCl,60 mL/min, (%) Anemia (%) Hypoalbuminemia (%) HbAlc (%)

Hyperuricemic group (n = 211)

Normouricemic group (n = 577)

p-value

65¡12 44 (21) 147 (70) 65¡11 132¡23 87 (41) 148 (70) 42 (20) 7 (3) 40 (19) 4.77¡1.29 3.07¡1.06 481¡72

62¡11 63 (11) 472 (82) 64¡10 131¡21 267 (46) 311 (54) 130 (23) 17 (3) 96 (17) 4.56¡1.11 2.92¡0.95 310¡66

,0.001 ,0.001 ,0.001 0.240 0.940 0.208 ,0.001 0.430 0.788 0.445 0.024 0.055 ,0.001 0.347

193 (92) 18 (8) 24 (11) 4 (2) 57¡13 26 (12) 48 (23) 185 (88) 36 (17) 94¡19 65¡24 100 (48) 46 (22) 28 (13) 6.5¡1.1

539 (93) 38 (7) 73 (13) 4 (1) 58¡12 46 (8) 148 (26) 529 (92) 70 (12) 85¡18 75¡24 165 (29) 93 (16) 67 (12) 6.5¡1.3

0.629 0.220 0.469 0.061 0.404 0.088 0.073 ,0.001 ,0.001 ,0.001 0.064 0.527 0.876

SBP, systolic blood pressure; RAS, renal artery stenosis; LDL, low-density lipoprotein cholesterol; MI, myocardial infarction; CABG, coronary artery bypass grafting; LVEF, left ventricular ejection fraction; ACEI/ARB, angiotensin-converting enzyme inhibitors/angiotensin receptor blocker; CrCl, Creatinine clearance; HbAlc, glycated hemoglobin.

were significantly greater than the scores in the normouricemic group (mean 8.1¡4.8, median 7.4, interquartile range 5.8–10.8 vs. mean 6.2¡4.0, median 6.3, interquartile range 1.8–9.2, p,0.001) (Table 2). Overall, the incidence of CI-AKI was significantly different (p,0.001) between the two groups; CI-AKI occurred in 17 (8.1%) members of the hyperuricemic group and 8 (1.4%) members of the normouricemic group. The levels of uric acid were significantly higher in the CI-AKI group than in the group without CI-AKI (448¡131 vs. 353¡99 mmol/L, p = 0.001). In-hospital mortality was significantly higher in the hyperuricemic group than in the normouricemic group (2.4% vs. 0.3%, p = 0.007) (Table 3).

Hyperuricemia increased the rates of CI-AKI requiring renal replacement therapy (1.4% vs. 0%, p = 0.004), IABP therapy (6.2% vs. 2.8%, p = 0.025), and post-procedural hypotension (5.3% vs. 1.9%, p = 0.011). A multivariate analysis (adjusted for other potential confounding factors, gender, age.75 y, CrCl,60 ml/min, multivessel coronary disease, emergent PCI, LVEF,40%, hypertension, diabetes, ACEI/ARB, IABP, exceeding maximum contrast dose, anemia, diuretic usage) revealed that the odds ratio (OR) for CI-AKI in the hyperuricemic group compared to the normouricemic group was 5.38 (95% confidence interval [CI], 1.99-14.58, p = 0.001) (Table 4). Notably, age.75 years (OR: 3.55, 95% CI: 1.21-10.42,

Table 2 - Angiographic and procedural characteristics. Characteristic Number of diseased vessels Multivessel coronary disease (%) Number of stents used Total stent length (mm) Type of LOCM Iopamiron (%) Ultravist (%) Contrast amount (mL) Exceeding MCD (%) Procedure duration of (min) CIN score mean (SD) { CIN score median (IQR) {

Hyperuricemic group (n = 211)

Normouricemic group (n = 577)

2.3¡0.8 167 (79) 2.1¡1.3 52¡34

2.1¡0.9 398 (69) 2.0¡1.2 47¡32

109 (52) 102 (48) 156¡69 13 (6) 88¡37 8.1¡4.8 7.4 (5.8-10.8)

297 (52) 280 (48) 150¡60 22 (4) 83¡37 6.2¡4.0 6.3 (1.8-9.2)

Abbreviations: LOCM, low-osmolar contrast media. MCD: maximum contrast dose (mL) = (56body weight [kg]) divided by serum creatinine (mg/dL); { : Scheme (Model A) to define contrast-induced nephropathy (CIN) risk score by Mehran et al. (J Am Coll Cardiol Vol. 44, No. 7, 2004).

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p-value ,0.001 0.005 0.163 0.070 0.963

0.256 0.157 0.087 ,0.001 ,0.001


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Table 3 - Hyperuricemia and in-hospital clinical complications. Complication

Hyperuricemic group (n = 211)

Normouricemic group (n = 577)

p-value

17 (8.1) 5 (2.4)

8 (1.4) 2 (0.3)

,0.001 0.007 0.001

0 1 (0.5) 2 (0.9) 2 (0.9) 3 (1.4) 1 (0.5) 1 (0.5) 6 (2.8) 13 (6.2) 6 (2.8) 7 (3.3) 7 (3.3) 11 (5.3) 1 (0.5)

1 (0.2) 1 (0.2) 0 0 0 0 2 (0.3) 1 (1.0) 16 (2.8) 5 (0.9) 37 (6.4) 12 (2.1) 11 (1.9) 1 (0.2)

CI-AKI (%) Death (%) Cause of death Cardiogenic shock (%) Cardiac rupture (%) Arrhythmia (VT/VF) (%) Non-cardiac cause (%) Renal replacement therapy (%) 2nd myocardial infarction (%) Target revascularization (%) Acute heart failure (%) IABP (%) Mechanical ventilation (%) 2nd angina (%) Tachyarrhythmia (%) Hypotension (%) Cerebrovascular accident (%)

0.004 0.098 0.797 0.095 0.025 0.078 0.094 0.316 0.011 0.458

VT/VF, ventricular tachycardia/ventricular fibrillation; IABP, intra-aortic balloon pump.

p = 0.021), emergent PCI (OR: 3.66, 95% CI: 1.40-9.55, p = 0.008), diuretic usage (OR: 4.87, 95% CI: 1.87-12.69, p = 0.001) and IABP (OR: 7.29, 95% CI: 2.29-23.15, p = 0.001) were retained in the final model.

with normal SCr values, however, did not receive any prophylaxis, as they are typically thought to be at low risk. Chong et al. (22) observed that subgroups of patients with normal baseline SCr who were undergoing PCI were at risk of developing CI-AKI, which results in higher mortality. Therefore, despite normal baseline SCr values, the subgroups of patients undergoing PCI may be at higher risk of developing CI-AKI. In our study, we identified several independent risk predictors of CI-AKI in addition to baseline renal impairment. These risk predictors include older age (.75 years), emergent PCI, and IABP therapy. This is the first study in which HUA has been identified as an independent risk predictor of CI-AKI. Hence, the patients with normal baseline SCr values who have the risk factors discussed above should be considered for additional renal prophylaxis treatment. A few studies have shown an association between HUA and the progression of kidney disease (23,24). Only two small studies, which investigated the relationship between the use of contrast agents and uric acid, have observed that contrast agents had a uricosuric effect that appeared to be caused by an increase in the renal tubular secretion of uric acid (25,26). Little information, however, is available in the literature about the relationship between HUA and the progression of CI-AKI (9,27). Toprak et al. (14) conducted the most rigorous observational study of the value of HUA for predicting the risk of CI-AKI in patients. The subjects were patients with chronic kidney disease (SCr$1.2 mg/dL) who were considered at high risk of developing CI-AKI. They received daily prophylaxis using a different definition for CI-AKI (an increase of 25% in creatinine) than the definition that used in the present study. The patients presented with relatively normal baseline SCr levels. Our study could be regarded as an extension of the study by Toprak et al. The results of the present study were consistent with those obtained by Toprak and colleagues; the HUA patients were at risk of developing CI-AKI. Recently, Park et al. (28) also concluded that HUA was independently associated with an increased risk of in-hospital mortality and CI-AKI in patients treated with PCI, although they performed a retrospective analysis that used a different definition of CI-AKI (an increase in SCr$0.5 mg/dL or$50% over baseline within seven days of

& DISCUSSION The main finding of this study, which was the first to evaluate the value of HUA in predicting CI-AKI development in patients with normal baseline SCr after PCI, was that HUA was a significant and independent predictor of CI-AKI after PCI and resulted in significantly greater inhospital mortality and incidence of CI-AKI requiring renal replacement therapy after PCI. Pre-existing renal dysfunction has been known to be the most important risk factor for CI-AKI. At our institution, the patients with elevated baseline SCr received sufficient perioperative CI-AKI prophylaxis following the recent guidelines by the American College of Cardiology (20) and the European Society of Cardiology (21). The patients

Table 4 - Multivariate analysis of CI-AKI risk indicators. Variable HUA Gender (female) Age.75 y CrCl,60 ml/min Multivessel coronary disease Emergent PCI LVEF,40% Hypertension Diabetes ACEI/ARB IABP Exceeding MCD Anemia Diuretic usage

Odds ratio

95% confidence interval

p-value

5.38 0.96 3.55 0.21 1.10

1.99-15.58 0.33-2.81 1.21-10.42 0.15-1.50 0.34-3.54

0.001 0.942 0.021 0.207 0.875

3.66 1.86 1.89 0.61 0.34 7.29 1.84 1.16 4.87

1.40-9.55 0.50-6.87 0.64-5.59 0.18-2.02 1.00-1.20 2.29-23.15 0.31-11.10 0.36-3.72 1.87-12.69

0.008 0.351 0.250 0.416 0.094 0.001 0.504 0.800 0.001

CrCl, Creatinine clearance; LVEF, left ventricular ejection fraction; ACEI/ ARB, angiotensin-converting enzyme inhibitors/angiotensin receptor blocker; IABP, intra-aortic balloon pump; MCD, maximum contrast dose (mL) = (56body weight [kg]) divided by serum creatinine (mg/dL).

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PCI). Consequently, the incidence of CI-AKI in the present study was lower than the incidence in previous studies (14,28). An understanding of the potential pathogenic role of uric acid in acute renal failure (ARF) may be helpful for understanding the relationship between HUA and the progression of CI-AKI. Renal vasoconstriction is thought to play a pathogenic role in ARF. The mechanism for the uric acid–dependent decrease in renal blood flow seems to be the loss of nitric oxide (NO) because the vasoconstriction can be reversed with L-arginine (29). Furthermore, uric acid strongly inhibits the release of NO from endothelial cells (30,31). Uric acid increases the production of the chemotactic factor monocyte chemoattractant protein-1 (MCP-1) in vascular smooth muscle cells and enhances C-reactive protein (CRP) synthesis in human vascular endothelial and smooth muscle cells (31,32). Uric acid also inhibits endothelial cell proliferation and migration (28) and causes endothelial cell apoptosis (33). When HUA was identified as a potentially modifiable risk factor, we became interested in evaluating its connection with CI-AKI and in-hospital adverse outcomes. Pakfetrat et al. (34) found that serum uric acid levels did not differ significantly between those patients with and without CIAKI (34). The authors explained that they failed to find a difference because their study included a significant number of patients with normal kidney function. Chen et al. (35) found that HUA was an independent risk factor for mortality from all causes, total cardiovascular disease, and ischemic stroke in the general Taiwanese population, in high-risk groups and potentially in low-risk groups (35). In that study, HUA was significantly associated with increased in-hospital mortality, and both CI-AKI and the need for IABP therapy were significantly correlated with death. However, HUA was not a significant and independent predictor of in-hospital death. There were key differences between our study and these other studies; for example, we only recorded short-term outcomes (in-hospital vs. mean follow-up of 8.2 years), and we used a relatively small number of patients (788 vs. 146,900). In another study of stage 3–4 CKD, HUA appeared to be an independent risk factor for all-cause and cardiovascular disease-induced mortality but not for kidney failure (36). Kowalczyk et al. (37) evaluated the impact of HUA on outcomes in patients undergoing invasive treatment for impaired renal function and acute myocardial infarction (AMI). They found that HUA was an independent risk factor for death associated with an adjusted HR of 1.38 (95% CI, 1.23–1.53) in patients with baseline kidney dysfunction (p = 0.027), but there was no significant increase in risk for patients with contrast-induced nephropathy (p = 0.08). The exact pathophysiological mechanisms behind the relationship are unknown. Uric acid, a marker of xanthine oxidase (XO) activity, reflects the generation of reactive oxygen species. In experimental studies, increased XO activity leads to oxidative stress, which is associated with heart failure after AMI (38). Published data show that both elevated and decreased serum uric acid levels negatively impacted patient outcomes, most likely because of the loss of the plasma antioxidant properties of uric acid (39). In our study, although most of the subjects had not experienced AMI and had relatively normal renal function while undergoing PCI (and thus had a relatively low risk of CI-AKI or death), we also found a significant relationship between higher serum

uric acid levels and in-hospital death. However, the proportion of HUA among the patients was higher than the proportion in the study conducted by Kowalczyk et al. (37) (68% [17/25] vs. 35.1% [343/693]). In the multivariate analysis, after adjusting for potential confounding factors (baseline creatinine clearance, LVEF, contrast medium volume), age.75 years, emergent PCI, and IABP therapy were retained in the final model. Because acute kidney injury may result from hemodynamic instability rather than from CI-AKI, performing elective PCI of noninfarct-related arteries in patients with multivessel disease, especially in older patients with multivessel coronary disease, may be beneficial in protecting renal function. Our results suggested that HUA was an independent, modifiable risk factor for CI-AKI and that it significantly increased the in-hospital adverse outcomes among the patients who were undergoing coronary angiography. Measuring serum uric acid levels before PCI seems to be a useful method for assessing the risk of developing CI-AKI and short-term clinical outcomes. In addition, an increase in the serum uric acid level may serve as a disease biomarker during the perioperative period. Physicians should be encouraged to identify males whose uric acid levels approached or exceeded 7 mg/dL and females whose levels approached or exceeded 6 mg/dL, as they are the patients at high risk for CI-AKI, and prophylactic measures should be considered to preserve renal function. Another implication of this result is that uricosuric agents may be useful in treating CI-AKI in patients who have undergone PCI or other procedures. Setting a new marker for CI-AKI may motivate large-scale future studies, which may find that reducing the level of serum uric acid is an effective prophylactic strategy for treating CI-AKI. A recent study prospectively randomized 159 patients with SCr.1.1 mg/dL undergoing cardiac catheterization and interventions to receive allopurinol (300 mg, p.o.) 24 h before the administration of a radiocontrast agent and routine hydration (40). They found that CIN occurred in 6 of 80 patients (7.5%), and the median uric acid concentration decreased from 6.8 mg/dL [3.5-13.5 mg/dL] to 6.3 mg/dL [3.1-10.4 mg/dL] 48 h after administering contrast (p,0.0001) in the control group, but none of the subjects developed CIN in the allopurinol patients (p = 0.013), whose mean uric acid concentration decreased from 6.8 mg/dL [3.3-17.2] to 5.48 mg/dL [2.1-14.6 mg/dL]. Although the authors concluded that the reduction in the prevalence of CIN after administering allopurinol might have been caused by the antioxidant capacity of allopurinol, the results clearly showed a reduction in the uric acid concentration.

Study limitations The current study has four limitations. First, it was a prospective, observational study with a small sample size, and it was conducted in a single center over a short observation period. PCI operations and the decision to use IABP, inotropic dugs, tirofiban, beta-blockers, ACE-inhibitors, and diuretics were left to the discretion of cardiologists; therefore, the results were influenced by operator bias. In addition, to avoid introducing potential selection bias, we used serum creatinine,1.5 mg/dl as our inclusion criteria instead of creatinine clearance ,60 ml/min (using the Cock-Croft-Gault formula to better assess kidney function). Second, because of the variation in the measurement times, we may have missed the peak levels of creatinine

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Uric acid and the state of the intrarenal reninangiotensin system in humans. Kidney Int. 2004;66(4):1465-70, http://dx. doi.org/10.1111/j.1523-1755.2004.00909.x. 14. Toprak O, Cirit M, Esi E, Postaci N, Yesil M, Bayata S. Hyperuricemia as a risk factor for contrast-induced nephropathy in patients with chronic kidney disease. Catheter Cardiovasc Interv. 2006;67(2):227-35, http://dx. doi.org/10.1002/ccd.20598. 15. Chiou WK, Wang MH, Huang DH, Chiu HT, Lee YJ, Lin JD. The relationship between serum uric acid level and metabolic syndrome: differences by sex and age in Taiwanese. J Epidemiol. 2010;20(3):21924. 16. Sui X, Church TS, Meriwether RA, Lobelo F, Blair SN. Uric acid and the development of metabolic syndrome in women and men. Metabolism. 2008;57(6):845-52, http://dx.doi.org/10.1016/j.metabol.2008.01.030. 17. Marenzi G, Lauri G, Assanelli E, Campodonico J, De Metrio M, Marana I, Grazi M, et al. 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Task Force on Myocardial Revascularization of the European Society of Cardiology (ESC) and the European Association for Cardio-Thoracic Surgery (EACTS); European Association for Percutaneous Cardiovascular Interventions (EAPCI), Kolh P, Wijns W, Danchin N, Di Mario C, et al. Guidelines on myocardial revascularization. Eur J Cardiothorac Surg. 2010;38 Suppl :S1-S52, http://dx.doi.org/10.1016/j.ejcts.2010.08.019. 22. Chong E, Poh KK, Liang S, Tan HC. Risk factors and clinical outcomes for contrast-induced nephropathy after percutaneous coronary intervention in patients with normal serum creatinine. Ann Acad Med Singapore. 2010;39:374-80. 23. Chonchol M, Shlipak MG, Katz R, Sarnak MJ, Newman AB, Siscovick DS, et al. Relationship of uric acid with progression of kidney disease. Am J Kidney Dis. 2007;50(2):239-47. 24. Bo S, Cavallo-Perin P, Gentile L, Repetti E, Pagano G. Hypouricemia and hyperuricemia in type 2 diabetes: two different phenotypes. Eur J Clin Invest. 2001;31(4):318-21, http://dx.doi.org/10.1046/j.1365-2362.2001. 00812.x. 25. Postlethwaite AE, Kelley WN. Uricosuric effect of radiocontrast agents. A study in man of four commonly used preparations. Ann Intern Med. 1971;74(6):845-52. 26. Mudge GH. Uricosuric action of cholecystographic agents: possible nephrotoxicity. N Engl J Med. 1971;284(17):929-33. 27. Dudzinski PJ, Petrone AF, Persoff M, Callaghan EE. Acute renal failure following high dose excretory urography in dehydrated patients. J Urol.1971;106(5):619-21. 28. Park SH, Shin WY, Lee EY, Gil HW, Lee SW, Lee SJ, Jin DK, Hong SY. The impact of hyperuricemia on in-hospital mortality and incidence of acute kidney injury in patients undergoing percutaneous coronary intervention. Circ J. 2011;75(3):692-7, http://dx.doi.org/10.1253/circj.CJ10-0631. 29. Tapia E, Sanchez-Lozada LG, Sanchez-Gonzalez DJ, Medina-Campos ON, Soto V, Avila-Casado C, et al. Amelioration of inflammation and oxidative stress preserves microvascular structure, and decrease systemic and glomerular pressure, and proteinuria in albumin overload (OA) rats. J Am Soc Nephrol 2006;17:685A. 30. Khosla UM, Zharikov S, Finch JL, Nakagawa T, Roncal C, Mu W, et al. Hyperuricemia induces endothelial dysfunction. Kidney Int. 2005;67(5):1739-42, http://dx.doi.org/10.1111/j.1523-1755.2005.00273.x.

post-procedure. The variation and the failure to carry out systematic measurements at the optimal times for determining the peak creatinine concentrations post-procedure may have led us to underestimate the true incidence of nephropathy in the current study population. Third, information about the repeated measurements of serum uric acid levels and body mass index were not available in the original data set. Fourth, although some patients had relatively normal SCr levels and did not receive the same hydration strategy (i.e., the same duration and rate of hydration) as those with elevated SCr values, these patients may have actually exhibited some degree of renal damage, and thus, the incidence of CI-AKI may be underestimated. Our study found that CI-AKI might still develop even in patients with normal SCr levels. HUA is a significant and independent predictor of CI-AKI after PCI and results in a significant increase in in-hospital mortality and the incidence of CI-AKI requiring renal replacement therapy after PCI. Thus, despite having normal baseline SCr levels, patients with HUA, IABP, emergent PCI or older age should receive more comprehensive renal prophylaxis to reduce the occurrence of CI-AKI. The serum uric acid measurements that are taken before PCI seem to be a useful method for assessing the risk of developing CI-AKI and short-term clinical outcomes. This observation may generate hypotheses for future prospective trials that examine the effectiveness of uric acid-lowering therapies for preventing CI-AKI.

& ACKNOWLEDGMENTS Grants were received from the Guangdong Cardiovascular Institute and Science and Technology Planning Project of Guangdong Province (Number: 2008A030201002), China.

& AUTHOR CONTRIBUTIONS Liu Y, Tan N, Chen J, Zhou Y, Chen Z, Li L conceived and designed the study, analyzed and interpreted the data. Chen S drafted the manuscript and critically revised the manuscript for intellectual content. Chen L inspected and approved the final version of the manuscript. All of the authors were involved in the manuscript preparation.

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Hyperuricemia and Acute Kidney Injury Liu Y et al. 36. Madero M, Sarnak MJ, Wang X, Greene T, Beck GJ, Kusek JW, et al. Uric acid and long-term outcomes in CKD. Am J Kidney Dis. 2009;53(5):796803. 37. Kowalczyk J, Francuz P, Swoboda R, Lenarczyk R, Sredniawa B, Golda A, et al. Prognostic significance of hyperuricemia in patients with different types of renal dysfunction and acute myocardial infarction treated with percutaneous coronary intervention. Nephron Clin Pract. 2010;116(2):c114-22, http://dx.doi.org/10.1159/000314660. 38. Engberding N, Spiekermann S, Schaefer A, Heineke A, Wiencke A, Mu¨ller M, Fuchs M, Hilfiker-Kleiner D, Hornig B, Drexler H, Landmesser U: Allopurinol attenuates left ventricular remodeling and dysfunction after experimental myocardial infarction: a new action for an old drug? Circulation. 2004;110(15):2175-9, http://dx.doi.org/10.1161/ 01.CIR.0000144303.24894.1C. 39. Waring WS, Convery A, Mishra V, Shenkin A, Webb DJ, Maxwell SR. Uric acid reduces exercise-induced oxidative stress in healthy adults. Clin Sci. 2003;105(4):425-30, http://dx.doi.org/10.1042/CS20030149. 40. Erol T, Tekin A, Katircibasi MT, Sezgin N, Bilgi M, Tekin G, et al. Efficacy of allopurinol pretreatment for prevention of contrast-induced nephropathy: a randomized controlled trial. Int J Cardiol. 2012 May 8. [Epub ahead of print].

31. Kang D-H, Park SK, Lee IK, Johnson RJ. Uric acid induced C-reactive protein (CRP) expression: Implication on cell proliferation and nitric oxide production in human vascular cells. J Am Soc Nephrol. 2005;16(12):3553-62, http://dx.doi.org/10.1681/ASN.2005050572. 32. Kanellis J, Watanabe S, Li JH, Kang D-H, Li P, Nakagawa T, et al. Uric acid stimulates monocyte chemoattractant protein-1 production in vascular smooth muscle cells via mitogen-activated protein kinase and cyclooxygenase-2. Hypertension. 2003;41(6):1287-93, http://dx.doi.org/ 10.1161/01.HYP.0000072820.07472.3B. 33. Kang DH, Kim JH, Shin KS, Kim SJ, Johnson RJ. Uric acid induced apoptosis of HUVEC via ROS-dependent path-way, but not of HVSMC [Abstract]. J Am Soc Nephrol .2005;16:167A, http://dx.doi.org/10.1681/ ASN.2005050572. 34. Pakfetrat M, Nikoo MH, Malekmakan L, Tabande M, Roozbeh J, Reisjalali G, et al. Risk Factors for Contrast-related Acute Kidney Injury According to Risk, Injury, Failure, Loss, and End-stage Criteria in Patients with Coronary Interventions. Iran J Kidney Dis. 2010;4(2):116-22. 35. Chen JH, Chuang SY, Chen HJ, Yeh WT, Pan WH. Serum uric acid level as an independent risk factor for all-cause, cardiovascular, and ischemic stroke mortality: a Chinese cohort study. Arthritis Rheum. 2009;61(2): 225-32, http://dx.doi.org/10.1002/art.24164.

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RAPID COMMUNICATION

Use of an artificial neural network to predict persistent organ failure in patients with acute pancreatitis Wan-dong Hong,I Xiang-rong Chen,I Shu-qing Jin,I Qing-ke Huang,I Qi-huai Zhu,I Jing-ye PanII I The First Affiliated Hospital of Wenzhou Medical College, Department of Gastroenterology and Hepatology, Zhejiang Province, People’s Republic of China. II The First Affiliated Hospital of Wenzhou Medical College, Intensive Care Unit, Zhejiang Province, People’s Republic of China.

Email: panjingye011@163.com Tel.: 86 13566289666

Medical College within 72 hours of the onset of symptoms between Jan 2008 and Mar 2009 were eligible. Acute pancreatitis was defined as previously described (11). Organ failure was defined as a Marshall score $2 for at least one of the following organs involved (12): (i) cardiovascular failure – systolic pressure #90 mmHg, unresponsive to fluids; (ii) respiratory failure – PaO2/Fio2 ratio ,300 mmHg; or (iii) renal failure – creatinine .1.9 mg/dL. Duration of organ failure was defined as persistent if it exceeded 48 hours. Exclusion criteria included (3): prior surgery for pancreas, endoscopic retrograde cholangiopancreatography or trauma-induced pancreatitis, chronic pancreatitis, pancreatic cancer, pleural effusions preceding the development of acute pancreatitis, pleural effusions that resulted from concomitant diseases (e.g., pneumonia or chronic heart failure), chronic renal disease and cases with incomplete data. Age, gender, temperature, pulse, systolic blood pressure, and biochemical parameters were recorded within 12 hours of admission before the development of persistent organ failure. The systemic inflammatory response syndrome (SIRS) and APACHE II scores (11) were calculated at admission according to the laboratory and physiological data. Continuous values were expressed as the means ¡ SD or medians and interquartile ranges and compared using Student’s t-test or the Mann-Whitney non-parametric test. Categorical values were described by counts and proportions and compared using the x2 test. Variables that were significantly related to the presence of organ failure were selected as candidates for input into the final ANN model. Sensitivity analysis (also known as independent variable importance analysis) was performed to determine the optimum variables for construction of the final ANN model (9). For sensitivity analysis, an exploratory three-layer multiplayer perceptron (MLP) ANN model with a back propagation algorithm was constructed. In the exploratory ANN model, the data were randomly divided into a training sample (250 cases, 80%) and a test sample (62 cases, 20%). Sigmoid transfer functions were used in the hidden and output layers. Gradient descent was used to estimate the synaptic weights. The initial learning rate was 0.4, and the momentum was 0.9. According to the results of the univariate and sensitivity analyses, a final three-layer feed-forward ANN model with a back propagation algorithm was constructed for all 312 patients. The ANN model was trained with a maximum of

& INTRODUCTION One-third to one-half of the deaths that occur due to acute pancreatitis (AP) during the first week after the onset of AP symptoms are primarily due to progressive organ failure (OF). Deaths that occur more than one week after admission to the hospital are often associated with local complications, such as infected pancreatic necrosis, and these patients often present symptoms of sepsis and multiple organ dysfunction syndromes (1). A recent meta-analysis indicated that organ failure is the key determinant of severity, regardless of the presence or absence of local pancreatic complications (2). Early identification of patients who are likely to develop organ failure would assist physicians in selecting the patients who would benefit from close surveillance or aggressive intervention. Many prognostic models or single predictors have been developed to predict mortality or the severity of acute pancreatitis, which is primarily defined based on the Atlanta classification (3-6). However, information regarding the early prediction of persistent organ failure in patients with acute pancreatitis is not widely available (7). As one of the clinical prediction rules (8), an artificial neural network (ANN) is composed of a series of interconnecting parallel nonlinear processing elements (nodes) with limited numbers of inputs and outputs (9). A systematic review suggested that artificial neural network analysis is potentially more successful than conventional statistical techniques at predicting clinical outcomes when the relationship between the variables that determine the prognosis is complex, multidimensional and non-linear (10). The aim of this study was to develop an artificial neural network to predict persistent organ failure in patients with acute pancreatitis.

& MATERIALS AND METHODS A total of 312 patients with acute pancreatitis who were admitted to the First Affiliated Hospital of Wenzhou Copyright ß 2013 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. DOI: 10.6061/clinics/2013(01)RC01

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500 iterations and 10 tours. The overfit penalty was assigned as 0.001, and the convergence criterion was 0.00001 (9). Fivefold cross-validation was used (13). The output of the ANN model was transformed to range from 0-1. Organ failure was predicted if the output was greater than or equal to 0.5 (9). The sensitivity, specificity, negative predictive value, positive predictive value and diagnostic accuracy of the ANN model are reported herein. The clinical or patient-relevant utility of a diagnostic test is evaluated by a Fagan plot. The Fagan plot allows the reader to estimate the post-test probability of the target condition in an individual patient based on a selected pretest probability (14). Forward conditional step-wise logistic regression analysis was performed to develop a logistic regression function (LR) for comparison. The conditional probabilities for stepwise entry and removal of a factor were 0.05 and 0.06, respectively. The area under the receiver operating characteristic (ROC) curve (AUC) was used to evaluate the performance of the ANN model; LR model and APACHE II score (15). Differences were considered statistically significant if the two-tailed P value was less than 0.05. SPSS 16.0 (SPSS Inc., Chicago, IL, USA) and JMP 6.0 (SAS, Cary, North Carolina, USA) were used for ANN analysis.

Univariate and multivariate analysis Thirteen variables considered relevant to the presence of OF were tested using univariate and multivariate analyses. As shown in Table 2, univariate analysis suggested that age, SIRS, hematocrit, serum glucose, BUN and serum calcium were significantly associated with organ failure. Multivariate analysis by logistic regression identified the following three independent variables as predictive of persistent organ failure in acute pancreatitis: SIRS (p = 0.004), BUN (p,0.001) and serum calcium (p = 0.003). A logistic regression function (LR model) was developed to predict organ failure as follows: -0.38+0.12 BUN (mg/dl)1.94 calcium (mmol/l)+1.16 SIRS (1 vs. 0).

ANN analysis As shown in Figure 1, age, hematocrit, serum glucose, BUN and serum calcium were the most important predictors of persistent organ failure by sensitivity analysis (the exploratory ANN model constructed for the sensitivity analysis is not shown). The final three-layer 5-5-1-feed-forward back propagation ANN model with variables consisting of age, hematocrit, serum glucose, BUN and serum calcium was developed and trained in 312 patients (Figure 2). The sensitivity, specificity, positive likelihood ratio, negative likelihood ratio and diagnostic accuracy of the ANN were 81.3%, 98.9%, 71.5, 0.19 and 96.2%, respectively. Using the prevalence of organ failure in acute pancreatitis (15.4% in this study) as the pretest probability, the Fagan plot (Figure 3) shows that ANN can be clinically informative because it increases the probability of being classified as organ failure to 93% when positive and lowers the probability to 3% when negative.

& RESULTS The median age of the 312 patients included in the study was 53, and 192 patients (61.5%) were male. The demographic and clinical characteristics of the patients are shown in Table 1. Among all of the patients, the acute pancreatitis was most commonly biliary in nature (56.4%). The median APACHE II score at the time of hospital admission was 5. Of the 48 patients who developed persistent organ failure, multiple organ failure was noted in 16 (33.3%). Respiratory failure (70.8%) was the most frequent. Five patients with organ failure died from acute pancreatitis, and four died during the first week in the hospital. None of the patients died from acute pancreatitis without organ failure.

Comparison of the ANN model, the LR model and APACHE II scores The ROC curves for the ANN model and APACHE II scores for predicting organ failure in acute pancreatitis are shown in Figure 4. The AUC of the ANN model (AUC = 0.96ยก0.02) was statistically higher than the AUC of the LR model (AUC = 0.88ยก0.03, p,0.001) or the APACHE II scores (AUC = 0.83ยก0.03, p,0.001).

Table 1 - Demographic and Clinical Characteristics of 312 patients with acute pancreatitis. Characteristic Age (yr) Male (%) APACHE II score at admission Etiology, % Biliary Alcohol Other Idiopathic OF classification Single OF Multiple OF Organ affected by OF Cardiovascular Respiratory Renal

Table 2 - Univariate analysis of predictive factors of organ failure in AP.

Data 53 (42-65) 61.5 5 (2-7)

Variable Age (yr) Male (%) SIRS (%) Systolic blood pressure (mmHg) Hemoglobin (mg/dL) Hematocrit (%) Platelets (109/L) Serum glucose (mmol/L) BUN (mg/dl) Serum calcium (mmol/L)

56.4 32.1 2.2 9.3 32 (66.7%) 16 (33.3%) 8 (16.7%) 34 (70.8%) 22 (45.8%)

Data are shown as numbers of observations, percentages, medians and interquartile ranges.

OF (N = 48)

No-OF (N = 264)

p-value

63 (50.5-71.5) 64.6 19.3 139 (120-150)

51 (41-63) 61.0 58.3 130 (120-145)

0.001 * 0.637m ,0.001m 0.07 *

14.1 41 173 9.2

13.4 38 190 7.4

(12.3-16.2) (34-47) (108-233) (6.7-13.3)

23.9 (16.3-37.7) 1.92 (1.61-2.10)

(12.2-14.7 (35-42) (146-232) (5.8-9.9)

0.087* 0.045* 0.10* 0.001*

11.8 (8.4-15.4) 2.19 (2.0-2.3)

,0.001* ,0.001*

OF: Organ failure; *Mann-Whitney non-parametric test; mx2 test.

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ANN for prediction of organ failure in AP W Hong et al.

Figure 1 - Sensitivity analysis of the input variables. The value shown for each input variable is a measure of its relative importance.

& DISCUSSION The results of this study demonstrate the following: (i) an ANN model (Figure 2) with variables consisting of age, hematocrit, serum glucose, BUN and serum calcium achieved a sensitivity of 81.3% and a specificity of 98.9% and classified a total of 96.2% of patients correctly; (ii) as shown in the Fagan plot (Figure 3), if a patient’s ANN value was more than or equal to 0.5, the probability of developing persistent OF increased from 15.4% to 93%, and if the ANN value was less than 0.5, the probability decreased to 3%; (iii) Based on ROC analysis (Figure 4), the diagnostic performance of the ANN model was superior to both the LR model and the APACHE II score. Of the standard ANNs, the MLP is perhaps the most popular network architecture currently in use (16). An MLP model consists of an input layer, a hidden layer and an output layer. All of the artificial neurons are arranged in a layered feed–forward topology. Our ANN model was developed using the SPSS neural networks program and

Figure 2 - A neural network for the prediction of organ failure in patients with acute pancreatitis consisting of five input variables, a hidden layer with five nodes, and one output variable.

Figure 3 - Fagan plot.

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Figure 4 - Receiver operating characteristic curves for the ANN model, logistic regression function (LR model) and APACHE II score.

transient OF (2,19). Patients with persistent OF should be classified as having severe or critical acute pancreatitis (2,19). A previous study also showed that persistent OF during the first week after the onset of AP symptoms was a marker of a fatal outcome in cases of acute pancreatitis (1). To the best of our knowledge, this is the first investigation of the possible predictors of persistent organ failure in cases of acute pancreatitis using ANN analysis. Our study has several limitations. First, the data were collected retrospectively, which may introduce a population bias. Second, the data were all obtained from a tertiary care university hospital based on a 72-hour window of symptoms, which may not be applicable in a local clinic or community hospital setting. It will be necessary to analyze the performance of an ANN within a 48-hour window in future studies, which could generalize our results. Third, the sample size in this study was small. Finally, although we performed five-fold cross-validation and filtered out irrelevant input variables by univariate analysis and sensitivity analysis to avoid overfitting, testing the performance of the ANN model in an independent sample will be necessary in the future. In conclusion, an artificial neural network model with variables consisting of age, hematocrit, serum glucose, BUN and serum calcium may be useful for predicting the development of persistent organ failure in patients with acute pancreatitis.

JMP software, which can both run the MLP model (9,13). ANNs are nonlinear statistical data modeling tools. They can take into account outliers and nonlinear interactions among variables and can reveal previously unrecognized and/or weak relationships between given input variables and an outcome (13). Therefore, ANNs often include parameters that may not reach significance using conventional statistics, as evidenced by the fact that age, hematocrit, and serum glucose included in our ANN model were not significant in logistic regression analysis. Similar phenomena were also observed in a previous study (17). ANN models have been increasingly used in the clinical prediction of acute pancreatitis. Mofidi et al. (18) developed an ANN consisting of ten input values to predict severe acute pancreatitis that achieved a high sensitivity and specificity of 89% and 96%, respectively. However, several limitations to that study were noted by Andersson et al. (17), such as the lack of an ensemble approach and a priori variable selection. Andersson et al. (17) successfully constructed an ANN using six variables to predict severe acute pancreatitis. They enrolled 208 patients and reported that the performance of the ANN with an AUC of 0.92 was superior to a logistic regression model or APACHE II score. The limitations of the study by Andersson et al. (17) included a small sample size with missing data points and unclear time intervals between the onset of AP and data collection. In addition, aspartate aminotransferase, which was used as one of the variables in the ANN model, is not available in most emergency situations. These issues could limit the general applicability of the ANN described in the Andersson et al. study in different populations. The gold standard for severe acute pancreatitis used in both of the previously mentioned studies was based on the Atlanta classification, which has several limitations that have been highlighted in multiple publications. Revisions to the classification have been suggested (2,19), and two additional categories, including moderate and critical acute pancreatitis, have been introduced. Moderate acute pancreatitis is defined as patients with sterile pancreatic complications or

& ACKNOWLEDGMENTS This study was supported in part by the Wenzhou Municipal Science and Technology Bureau (No.Y20120014) and the Zhejiang Provincial Health Department (No. 2012ZB103). We are very thankful to the two anonymous reviewers for their thoughtful and precise input.

& AUTHOR CONTRIBUTIONS Hong WD, Chen XR, Zhu QH, and Pan JY designed the study. Hong WD, Jin SQ, and Huang QK participated in data collection. Hong WD performed the data analysis and drafted the manuscript. All of the authors read and approved the manuscript.

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CLINICAL SCIENCE

High levels of B-type natriuretic peptide predict weaning failure from mechanical ventilation in adult patients after cardiac surgery Thiago Martins Lara, Ludhmila Abrahao Hajjar, Juliano Pinheiro de Almeida, Julia Tizue Fukushima, Carmem Silvia Valente Barbas, Adriano Rogerio Baldacin Rodrigues, Emilia Nozawa, Maria Ignes Zanetti Feltrim, Elisangela Almeida, Vera Coimbra, Eduardo Osawa, Rafael de Moraes Ianotti, Alcino Costa Leme, Fabio Biscegli Jatene, Jose Otavio Costa Auler-Jr., Filomena Regina Barbosa Gomes Galas Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Heart Institute (InCor), Surgical Intensive Care Unit and Department of Anesthesiology, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: The failure to wean from mechanical ventilation is related to worse outcomes after cardiac surgery. The aim of this study was to evaluate whether the serum level of B-type natriuretic peptide is a predictor of weaning failure from mechanical ventilation after cardiac surgery. METHODS: We conducted a prospective, observational cohort study of 101 patients who underwent on-pump coronary artery bypass grafting. B-type natriuretic peptide was measured postoperatively after intensive care unit admission and at the end of a 60-min spontaneous breathing test. The demographic data, hemodynamic and respiratory parameters, fluid balance, need for vasopressor or inotropic support, and length of the intensive care unit and hospital stays were recorded. Weaning failure was considered as either the inability to sustain spontaneous breathing after 60 min or the need for reintubation within 48 h. RESULTS: Of the 101 patients studied, 12 patients failed the weaning trial. There were no differences between the groups in the baseline or intraoperative characteristics, including left ventricular function, EuroSCORE and lengths of the cardiac procedure and cardiopulmonary bypass. The B-type natriuretic peptide levels were significantly higher at intensive care unit admission and at the end of the breathing test in the patients with weaning failure compared with the patients who were successfully weaned. In a multivariate model, a high Btype natriuretic peptide level at the end of a spontaneous breathing trial was the only independent predictor of weaning failure from mechanical ventilation. CONCLUSIONS: A high B-type natriuretic peptide level is a predictive factor for the failure to wean from mechanical ventilation after cardiac surgery. These findings suggest that optimizing ventricular function should be a goal during the perioperative period. KEYWORDS: B-Type Natriuretic Peptide; Cardiac Surgery; Mechanical Ventilation; Weaning Failure. Lara TM, Hajjar LA, Almeida JP, Fukushima JT, Barbas CS, Rodrigues AR, et al. High levels of B-type natriuretic peptide predict weaning failure from mechanical ventilation in adult patients after cardiac surgery. Clinics. 2013;68(1):33-38. Received for publication on July 10, 2012; First review completed on July 26, 2012; Accepted for publication on September 4, 2012 E-mail: thiagomlara@hotmail.com Tel.: 55 11 3893-3267

coronary artery bypass grafting (CABG), Wong et al. (4) identified increased age, female gender, the postoperative use of an intra-aortic balloon pump (IABP), the use of inotropes, bleeding, and atrial arrhythmia as risk factors for weaning failure and prolonged mechanical ventilation. Perioperative ventricular dysfunction and cardiac failure are frequent causes of weaning failure from mechanical ventilation (4). Prolonged cardiopulmonary bypass (CPB), inadequate myocardial protection during surgery, perioperative myocardial ischemia and previous left ventricular dysfunction are associated with a higher incidence of perioperative heart failure (5,6). A diagnosis of low output syndrome after surgery is suggested by decreased central venous oxygen saturation (ScvO2), low urine output, low

& INTRODUCTION The failure to wean from mechanical ventilation after cardiac surgery is associated with worse outcomes, including increased length of hospital stays and higher costs (1-3). In a prospective study of 885 patients who underwent

Copyright ß 2013 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. DOI: 10.6061/clinics/2013(01)OA05

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the European system for cardiac operative risk evaluation score (EuroSCORE) (22), were obtained for all patients. The intraoperative data, including the cardiac procedure and CPB durations and the use of inotropes or vasopressors, were also recorded. After ICU admission, the following hemodynamic parameters were obtained and recorded: heart rate, mean arterial pressure (MAP), central venous pressure (CVP), pulmonary arterial pressures, pulmonary capillary wedge pressure, cardiac output, cardiac index and systemic vascular resistance (SVR). We also recorded the mechanical ventilation parameters, including the ventilation mode, plateau pressure and positive end-expiratory pressure (PEEP), respiratory rate in controlled and spontaneous mode, fraction of inspired oxygen (FiO2), oxygen saturation by pulse oximetry (SpO2), tidal volume and minute volume, rapid shallow breathing index or respiratory rate/tidal volume ratio and static respiratory system compliance (23,24). A blood gas analysis and measurement of hemoglobin levels were performed and recorded after ICU admission every 6 h during the first 24 h after cardiac surgery. The TriageH BNP Test (Biosite, San Diego, CA, USA) was used to determine the BNP levels in the plasma specimens through immunofluorescence, with EDTA as the anticoagulant. The BNP levels were measured immediately after ICU admission and at the end of the spontaneous breathing test (SBT). The fluid balance, need for vasopressor or inotropic support, and length of the ICU and hospital stays were also recorded.

cardiac index, high cardiac filling pressure and elevated levels of B-type natriuretic peptide (BNP) (7-10). BNP is produced by cardiac ventricular myocytes in response to volume or pressure overload. Active BNP and its inactive form, NT-proBNP, are derived from the cleavage of their precursor molecule, proBNP. BNP decreases systemic vascular resistance, improves myocardial relaxation, increases natriuresis and suppresses endothelin and the renin-angiotensin system (10). The BNP levels are increased in patients who have left ventricular dysfunction, right ventricular dysfunction or valvular dysfunction. Therefore, BNP is considered to be a quantitative biomarker of heart failure (10). BNP levels are also related to left ventricular dysfunction in the postoperative period, including after cardiac surgery (11-20). In a prospective study, Zapata et al. evaluated the value of the BNP level as a marker of failure to wean from mechanical ventilation in a mixed population of 100 medical and surgical patients (excluding cardiac surgery patients) who were treated with mechanical ventilation for over 48 h (21). BNP levels predicted failure to wean cases that were caused by ventricular dysfunction, and a DBNP of 48 ng/L identified heart failure as the cause of failed SBT, with 91.7% sensitivity and 88.5% specificity (21). We hypothesized that BNP levels could help in the early identification of patients who cannot be weaned from mechanical ventilation due to postoperative ventricular dysfunction after cardiac surgery.

& MATERIAL AND METHODS

Weaning protocol

The study was approved by the Ethics Committee, and written informed consent was obtained from all patients. We prospectively included all of the patients older than 18 years who underwent elective CABG surgery with CPB during a 1-year period from January 2009 and January 2010 at Heart Institute, University of Sao Paulo. The exclusion criteria were a history of pulmonary disease or chronic renal failure, the need for IABP and consent refusal. The patients were anesthetized according to our standard institutional protocol for CABG surgery. Preoperative medication consisted of midazolam (0.1 to 0.2 mg/kg given orally 30 minutes before surgery). Anesthesia was induced with fentanyl (3-5 mg/kg), midazolam (0.05 mg/kg), etomidate (0.2- 0.3 mg/kg), and pancuronium bromide (0.1 mg/ kg). Anesthesia was maintained with isoflurane in oxygen and fentanyl as needed. During CPB, additional doses of midazolam and pancuronium were administered as required to reach a bispectral index of approximately 4060. After the tracheal intubation, all of the patients received invasive mechanical ventilation with intermittent positive pressure with a tidal volume of 8 mL/kg, positive endexpiratory pressure of 5 to 8 cm H2O, and fraction of inspired oxygen (FiO2) of 0.6 to 1 to maintain the arterial oxygen saturation above 95%. During surgery, the patients were monitored with a central venous line and indwelling radial artery catheter. Forty-four patients also had a pulmonary artery catheter inserted if the surgical and anesthesiology teams believed that it was warranted. In these patients, the cardiac index was obtained from a continuous cardiac output monitor, Vigilance II (Edwards Lifesciences, Irvine, CA 92614 USA). Preoperative information, including demographic data, preoperative left ventricular ejection fraction (LVEF), and

After ICU admission, all of the patients were initially ventilated using the following parameters: synchronized intermittent mandatory ventilation (SIMV) using pressurecontrolled ventilation with an I/E ratio of 1:2 and enough support pressure to give a tidal volume of approximately 8 mL/kg, 5 cm H2O PEEP, respiratory rate of 12 breaths/ min, and FiO2 of 60% or greater if the SpO2 was less than 90%. The degree of support was reduced, if possible, by 2 to 4 cm H2O at least hourly. The first spontaneous breathing test was given if the patients were awake and hemodynamically stable, as defined by the absence of bleeding (chest tube drainage #100 ml per hour or #300 mL in one hour), ScvO2.65%, CI.2.2 L/min/m2 and MAP.65 mmHg with low-dose norepinephrine #0.2 mg/Kg/min) or no vasopressor agents. Additionally, SBT was started after correcting acid-base and electrolyte disorders. The SBT test lasted 60 min and was considered failed if the patient presented with one or more of the following signs at the end of the first SBT: respiratory rate ,35 breaths/min; heart rate ,140 beats/min; SpO2,90% or PaO2,60 mmHg; respiratory acidosis (pH,7.3 or PaCO2.50 mmHg); signs of respiratory distress, such as thoracoabdominal dyssynchrony, anxiety and diaphoresis, or reintubation within 48 h in patients who were successful in the first SBT.

Statistical analysis We compared the baseline characteristics, follow-up measures, and clinical outcomes between the groups. Continuous variables were tested for normal distribution using the Kolmogorov-Smirnov test and compared using Student’s t test or the Mann-Whitney U-test. The sample size was calculated based on a power of 80% and a 5% typeI error. Estimating an event incidence of 10%, 158 patients

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BNP and weaning failure in heart surgery Lara TM et al.

between the groups in the other hemodynamic variables or in the cumulative fluid balance. The BNP levels were significantly higher in the patients who failed to wean compared with those who weaned successfully, both at ICU admission (214 ng/mL [65-487] vs. 73 [28-127], p = 0.02) and after the SBT (416 ng/mL [311-561] vs. 140 [80-226], p,0.001) (Table 2). In the multivariate model, only BNP at the end of the SBT (BNP-2) was predictive of weaning failure from mechanical ventilation (odds ratio [OR], 1.006 per ng/mL [95% CI, 1.003-1.009]; p,0.001). A BNP concentration of 299 ng/L at the end of the SBT identified weaning failure with 92% sensitivity and 88% specificity, with an AUC of 0.91 (CI 95% [0.86 - 0.97], p,0.001) (Figure 1). Figure 2 shows the BNP concentrations for the individual patients.

were needed to complete the study. An interim analysis was scheduled after 101 patients had been enrolled in the study. The results are expressed as means with 95% confidence intervals (CIs) or medians with interquartile ranges (IQRs). A multiple logistic regression analysis was performed to assess the predictive factors for weaning failure from mechanical ventilation, and the significance level was set at p,0.10 in the univariate model (i.e., the patient age, body mass index [calculated as weight in kilograms divided by height in meters squared], comorbidity, left ventricular ejection fraction, EuroSCORE, surgery type, CPB duration, initial and final hemoglobin concentrations, lactate concentration, and ScvO2 and BNP levels). We built a multivariate Cox proportional hazard model in the overall population with weaning failure as the dependent factor using the variables above. To determine the best cut-off for BNP, we calculated the area under the receiver operating characteristic curve (AUC) and compared the BNP levels in the failed weaning group with those in the successful weaning group. A two-sided p-value,0.05 was considered statistically significant. The statistical analyses were performed using SPSS version 18.0 (SPSS Inc., Chicago, IL).

Clinical outcomes A total of 716 patients were assessed for eligibility during the study period. In total, 101 patients were enrolled, and the mortality rate was 5%. The patients who failed to wean from mechanical ventilation after the first SBT had higher ICU mortality rates than the patients who did not fail (3 [25%] vs. 2 [2.2%], p,0.0011). Five patients in the failure group were reintubated within 48 h after the SBT because of congestive heart failure. Two of the reintubated patients died from cardiogenic shock. Seven patients had failure to wean in the first SBT but were not reintubated in the first 48 h. One of these patients died from pneumonia, septic shock and multiple organ failure.

& RESULTS An interim analysis was performed when 101 patients had been included, and because a significant p-value was found, we interrupted the data collection. Of the 101 patients studied, 12 failed the weaning trial. Of those 12 patients, five required reintubation, and seven were weaned from mechanical ventilation after they failed to wean in the first SBT. There were no differences between the groups in their baseline or intraoperative characteristics, including the left ventricular function, EuroSCORE, and the durations of the cardiac procedure and CPB bypass (Table 1). The patients who failed weaning had longer ICU (9 (4-14) vs. 3 (2-5), p = 0.024) and hospital (15 (13-20) vs. 8 (7-13), p = 0.047) lengths of stay compared with successfully weaned patients. The patients who failed to wean from mechanical ventilation had higher CVP values at the end of the SBT than the patients who weaned successfully (11 (9-15) vs. 9 (510) mmHg, p = 0.023); they also had lower ScvO2 values (62 [58-65] vs. 69% [68-71], p = 0.002) at this time point and required higher doses of dobutamine (15 (11-18) vs. 12 (9-16) mg/Kg/min, p = 0.044) (Table 2). There were no differences

& DISCUSSION Our study shows that a high level of BNP is an independent risk factor for the failure to wean from mechanical ventilation after cardiac surgery. In clinical practice, BNP levels are widely used to diagnose and stratify risk in heart failure patients and as a predictor of outcomes, including re-hospitalization and death (10,25). An elevated BNP level is considered to be a biomarker of ventricular dysfunction and can identify early decompensated heart failure after cardiac surgery (11-14,16-20,25,26). Elevated postoperative BNP levels have also been associated with prolonged hospital stays and mortality in patients undergoing cardiac surgery (11,17,18). Underlying left ventricular dysfunction is an important cause of weaning failure in critically ill patients, particularly during the postoperative period after cardiac surgery, and

Table 1 - Baseline, intraoperative characteristics and clinical outcomes of patients. Variable Sex* Male Female Age (years)** LVEF (%)*** EuroSCORE*** Duration of procedure (min)*** Duration of CBP (min)*** Duration of mechanical ventilation (min) *** ICU length of stay (days) *** Hospital length of stay (days) *** Hospital Mortality

Total (101)

76 25 63 54 5 270 95 420 3 9 5

Successful weaning (n = 89)

(75.2%) (24.8%) (61-65) (40-64) (3-7) (240-330) (80-110) (360-613) (2-5) (7-14) (5%)

69 20 63 54 4 270 94 420 3 8 2

(77.5%) (22.5%) (61-65) (40-64) (3-7) (240-330) (80-108) (360-625) (2-5) (7-13) (2,2%)

Failure to wean (n = 12)

7 5 63 56 6 283 111 488 9 15 3

(58.3%) (41.7%) (55-72) (40-68) (4-8) (240-383) (91-120) (390-559) (4-14) (13-20) (25%)

p-value

0.165 0.289 0.560 0.246 0.281 0.433 0.868 0.024 0.047 0.011

*Chi-square test, **mean (95% confidence Interval), t-test, ***median (interquartile range), Mann-Whitney test. EuroSCORE: European System for Cardiac Operative Risk Evaluation; LVEF: left ventricular ejection fraction; CPB: cardiopulmonary bypass.

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Table 2 - Hemodynamic, respiratory and gas exchange variables. Variable Hemodynamic HR-1 (beats/min)* HR-2 (beats/min)* MAP-1 (mmHg)* MAP-2 (mmHg)* PAOP-1 (mmHg)*$ PAOP-2 (mmHg)*$ CVP-1 (mmHg)* CVP-2 (mmHg)** PCP-1 (%)*$ PCP-2 (%)*$ ScvO2-1 (%)** ScvO2-2 (%)* CI-1 (L/min/m2)$* CI-2 (L/min/m2)$* Lactate-1 (mmol/L)** Lactate-2 (mmol/L)** Hb-1 (g/dL)** Hb-2 (g/dL)** Dobutamine-1** Dobutamine-2** Respiratory PaO2-1 (mmHg)* PaO2-2 (mmHg)** PaCO2-1 (mmHg)** PaCO2-2 (mmHg)** pH-1** pH-2** Tobin-2** SC-1 (mL/cmH2O)** Natriopeptides (ng/L) BNP-1** BNP-2**

Successful weaning (n = 89)

Failure to wean (n = 12)

p-value

96 103 92 89 14 10 10 9 14 10 77 69 3.17 3.33 4.0 2.9 10.8 10.6 10 12

(92-100) (101-106) (89-96) (87-92) (13-15) (9-12) (9-11) (5-10) (13-15) (9-12) (70-81) (68-71) (2.83-3.50) (3.08-3.58) (2.9-6.1) (1.8-4.6) (9.8-11.8) (9.6-11.8) (8-17) (9-16)

93 96 93 85 14 12 10 11 14 12 77 62 2.75 2.76 2.3 1.9 9.8 10 14 15

(80-105) (89-103) (83-102) (78-93) (10-17) (9-15) (8-12) (9-15) (10-17) (9-15) (69-77) (58-65) (2.31-3.20) (2.21-3.30) (1.3-5.0) (1.4-3.6) (9.2-11.7) (8.9-10.2) (9-18) (11-18)

0.435 0.087 0.983 0.239 0.855 0.440 0.883 0.023 0.855 0.440 0.781 0.002 0.361 0.068 0.313 0.296 0.154 0.084 0.519 0.044

145 107 40 37 7.3 7.4 36 38

(136-154) (90-143) (36-44) (33-40) (7.3-7.4) (7.3-7.4) (26-45) (32-49)

142 117 39 37 7.4 7.4 45 39

(113-171) (88-152) (36-45) (34-41) (7.3-7.4) (7.3-7.4) (29-83) (33-51)

0.754 0.550 0.806 0.303 0.313 0.232 0.098 0.889

73 (28-127) 140 (80-226)

214 (65-487) 416 (311-561)

0.020 ,0.001

*mean (95% Confidence Interval), t-test, **median (Interquartile Range), Mann-Whitney test. $evaluated in the 44 patients who had a pulmonary artery catheter inserted – 36 in the successful weaning group and 8 in the failure to wean group. 1- at intensive care unit admission; 2- at the end of the spontaneous breathing test; HR: heart rate; MAP: mean arterial pressure; PAOP: pulmonary artery occlusion pressure; CVP: central venous pressure; ScvO2: central venous oxygen saturation; Hb: hemoglobin concentration; SC = Static compliance; CI: cardiac index.

can be the result of perioperative myocardial ischemia, prolonged CPB, or inadequate myocardial protection (2730). This condition may be difficult to recognize within the

first 24 h after cardiac surgery. Previous studies have reported that early indirect parameters of cardiac function, such as capnometric recirculation gas tonometry and gastric

Figure 1 - Area under receiving operating characteristic curve for BNP-2 (at the end of spontaneous breathing test) to predict weaning failure.

Figure 2 - Individual values of BNP concentration. A cut-off of 299 ng/L at the end of the SBT predicted failure to wean from mechanical ventilation after cardiac surgery with 92% sensitivity and 87% specificity. Red dots represent patients with weaning failure.

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patients, including effective myocardial protection during CPB and the judicious administration of inotropic agents and vasodilators to obtain adequate cardiac indices and ScvO2 levels (6). The careful evaluation of fluid status is also needed to prevent elevated filling pressures and pulmonary congestion. Our study has several limitations. First, it was an observational and single-center study with a small number of patients and relatively few failure events. Second, BNP levels can be altered in various conditions in patients undergoing cardiac surgery, and we are unable to identify the exact cause of the myocardial dysfunction that caused the weaning failure in our patients. Third, there are many other conditions in critically ill patients that may be associated with increased BNP levels. Therefore, these investigations should be extended to a larger patient population in a multicenter setting. In conclusion, our study demonstrated that a high BNP level is a predictor of weaning failure from mechanical ventilation in patients after cardiac surgery. Therefore, measuring BNP levels may help to guide and evaluate the effects of therapeutic strategies, such as optimizing ventricular function during cardiac surgery, prior to weaning from mechanical ventilation. This report describes human research. IRB contact information: CAPPesq - Comissa˜o de E´tica para Ana´lise de Projetos de Pesquisa (cappesq@hcnet.usp.br), do Hospital das Clinicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil.

intramucosal pH, were altered in patients who presented with failure to wean from mechanical ventilation. These findings can be explained by the likelihood that the increased respiratory workload during spontaneous test breathing can induce an intestinal mucosa hypoperfusion because of the adrenergic response and effort required to increase the blood flow to the respiratory muscles (31-33). Similarly, the BNP levels during the spontaneous breathing test might help physicians identify earlier those patients with postoperative heart failure and support decisions to discontinue mechanical ventilation after cardiac surgery, potentially decreasing the risk of weaning failure (6,18,21). All of the patients with weaning failure in the current study had higher BNP levels both at their ICU admissions and at the end of the SBT compared with the patients who had successful weaning. The highest BNP levels were found at the end of the SBT, suggesting that postoperative left ventricular dysfunction was involved in the weaning failure. In addition, these patients had higher CVP and lower ScvO2 levels and needed higher doses of dobutamine compared with the patients who were successfully weaned. Paulus et al. (34) reported that in patients with left ventricular dysfunction who had presented a failure in the first or additional spontaneous breathing tests after cardiac surgery, the use of enoximone, a phosphodiesterase III inhibitor, was associated with an improvement in hemodynamic parameters and successful withdrawal of mechanical ventilation. Although these hemodynamic parameters were not predictors of weaning failure in our study, they reinforce our hypothesis that underlying heart failure caused weaning failure in this subgroup of patients. A previous study showed that increased age, female gender, the postoperative use of an intra-aortic balloon pump (IABP), inotropes, bleeding, and atrial arrhythmia were risk factors of delayed extubation after cardiac surgery (4). Our data did not show any differences in gender. However, our findings suggested that poor ventricular function, expressed through high levels of BNP, is a predictor of weaning failure after cardiac surgery. In our study, the prevalence of weaning failure was 12%. This failure rate is higher than that reported in other studies (1), most likely because our study was performed in a cardiac surgery referral center and included severely ill patients, as shown by the high EuroSCOREs (22). The patients who had weaning failure in our study may have developed myocardial dysfunction secondary to CPB. A CPB duration of more than 120 minutes has been considered a predictive factor for weaning failure in patients after cardiac surgery in previous studies (35), but there was no significant difference in the duration of CPB between our groups. Moreover, none of the patients with weaning failure met the perioperative myocardial infarction criteria. Nevertheless, the etiology of myocardial dysfunction after CPB is multifactorial and can occur even without prolonged CPB or perioperative infarction (6). Using transesophageal echocardiography, Bernard et al. described a 30% rate of diastolic dysfunction after cardiac surgery with CPB (27). If BNP levels are predictors of weaning failure from mechanical ventilation in patients after cardiac surgery, as suggested by our results, and ventricular dysfunction is one cause of weaning failure, then optimizing ventricular function may result in better outcomes after cardiac surgery. A bundle of interventions could be used to optimize ventricular function during the perioperative period in such

& ACNOWLEDGMENTS This study was supported by the Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo (FAPESP), Brazil – Number 08553750.

& AUTHOR CONTRIBUTIONS Lara TM designed the study and wrote the manuscript. Hajjar LA designed the study and wrote the manuscript, reviewed the data analysis, approved the final version of the manuscript, and archived the study files. Almeida JP designed the study and wrote the manuscript, analyzed the original study data and approved the final version of the manuscript. Fukushima JT analyzed the data, reviewed the analysis of the data, and approved the final version of the manuscript. Barbas CS designed the study, wrote and approved the final version of the manuscript. Rodrigues AR, Coimbra V, Ianotti RM and Leme AC conducted the study and approved the final version of the manuscript. Almeida E and Osawa E reviewed the analysis of the data, wrote the manuscript and approved its final version. Nozawa E, Feltrim MI and Jatene FB designed the study, wrote and approved the final version of the manuscript. Auler Junior JO wrote, reviewed the analysis of the data and approved the final version of the manuscript. Galas FR helped design and write the study, reviewed the analysis of the data, and approved the final manuscript.

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18. Nozohoor S, Nilsson J, Algotsson L, Sjo¨gren J. Postoperative increase in B-type natriuretic peptide levels predicts adverse outcome after cardiac surgery. J Cardiothorac Vasc Anesth. 2011;25(3):469-75, http://dx.doi. org/10.1053/j.jvca.2010.07.002. 19. Jeong DS, Kim KH, Kim CY, Kim JS. Relationship between plasma Btype natriuretic peptide and ventricular function in adult cardiac surgery patients. J Int Med Res. 2008;36(1):31-9. 20. Schachner T, Wiedemann D, Fetz H, Laufer G, Kocher A, Bonaros N. Influence of preoperative serum N-terminal pro-brain type natriuretic peptide on the postoperative outcome and survival rates of coronary artery bypass patients. Clinics. 2010;65(12):1239-45, http://dx.doi.org/ 10.1590/S1807-59322010001200004. 21. Zapata L, Vera P, Roglan A, Gich I, Ordonez-Llanos J, Betbese´ AJ. B-type natriuretic peptides for prediction and diagnosis of weaning failure from cardiac origin. Intensive Care Med. 2011;37(3):477-85, http://dx.doi.org/ 10.1007/s00134-010-2101-4. 22. Nashef SA, Roques F, Michel P, Gauducheau E, Lemeshow S, Salamon R. European system for cardiac operative risk evaluation (EuroSCORE). Eur J Cardiothorac Surg. 1999;16(1):9-13, http://dx.doi.org/10.1016/ S1010-7940(99)00134-7. 23. Yang KL, Tobin MJ. A prospective study of indexes predicting the outcome of trials of weaning from mechanical ventilation. N Engl J Med. 1991;324(21):1445-50. 24. Rossi A, Gottfried SB, Zocchi L, Higgs BD, Lennox S, Calverley PM, Begin P, et al. Measurement of static compliance of the total respiratory system in patients with acute respiratory failure during mechanical ventilation. The effect of intrinsic positive end-expiratory pressure. Am Rev Respir Dis. 1985;131(5):672-7. 25. Jankowski M. B-type natriuretic peptide for diagnosis and therapy. Recent Pat Cardiovasc Drug Discov. 2008;3(2):77-83, http://dx.doi.org/ 10.2174/157489008784705395. 26. Eliasdottir SB, Klemenzson G, Torfason B, Valsson F. Brain natriuretic peptide is a good predictor for outcome in cardiac surgery. Acta Anaesthesiol Scand. 2008,52(2):182-7, http://dx.doi.org/10.1111/j.13996576.2007.01451.x. 27. Bernard F, Denault A, Babin D, Goyer C, Couture P, Couturier A, Buithieu J. Diastolic dysfunction is predictive of difficult weaning from cardiopulmonary bypass. Anesth Analg. 2001;92(2):291-8. 28. Demoule A, Lefort Y, Lopes ME, Lemaire F. Successful weaning from mechanical ventilation after coronary angioplasty. Br J Anaesth. 2004;93(2):295-7. 29. Rao V, Ivanov J, Weisel RD, Ikonomidis JS, Christakis GT, David TE. Predictors of low cardiac output syndrome after coronary artery bypass. J Thorac Cardiovasc Surg. 1996;112(1):38-51, http://dx.doi.org/10.1016/ S0022-5223(96)70176-9. 30. Nozawa E, Azeka E, Igneˆz Z M, Feltrim Z, Auler Ju´nior JO. Factors associated with failure of weaning from long-term mechanical ventilation after cardiac surgery. Int Heart J. 2005;46(5):819-31, http://dx.doi. org/10.1536/ihj.46.819. 31. Maldonado A, Bauer TT, Ferrer M, Hernandez C, Arancibia F, Rodriguez-Roisin R, et al. Capnometric recirculation gas tonometry and weaning from mechanical ventilation. Am J Respir Crit Care Med. 2000;161(1):171-6. 32. Bouachour G, Guiraud MP, Gouello JP, Roy PM, Alquier P. Gastric intramucosal pH: an indicator of weaning outcome from mechanical ventilation in COPD patients. Eur Respir J. 1996;9(9):1868-73, http://dx. doi.org/10.1183/09031936.96.09091868. 33. Mohsenifar Z, Hay A, Hay J, Lewis MI, Koerner SK. Gastric intramural pH as a predictor of success or failure in weaning patients from mechanical ventilation. Ann Intern Med. 1993;119(8):794-8. 34. Paulus S, Lehot JJ, Bastien O, Piriou V, George M, Estanove S. Enoximone and acute left ventricular failure during weaning from mechanical ventilation after cardiac surgery. Crit Care Med. 1994;22(1):7480. 35. Natarajan K, Patil S, Lesley N, Ninan B. Predictors of prolonged mechanical ventilation after on-pump coronary artery bypass grafting. Ann Card Anaesth. 2006;9(1):31-6.

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CLINICAL SCIENCE

Evaluation of renal function and immune system cells in elderly individuals from Sa˜o Paulo City Daniela Teixeira,I Ieda Maria Longo-Maugeri,I Yeda Aparecida Oliveira Duarte,II Maria Lucia Lebra˜o,II Valquiria BuenoI I Federal University of Sa˜o Paulo (UNIFESP), Immunology, Sa˜o Paulo/SP, Brazil. II University of Sa˜o Paulo - USP, Epidemiology Department, Sa˜o Paulo/SP, Brazil.

OBJECTIVES: Both renal function and immune system function decline with age. Although controversial, a significant number of studies have shown that the decline in kidney function is associated with the worsening of the immune system. These findings are reinforced by the increased susceptibility to infections and deficient immunization coverage after vaccination both in patients with chronic renal disease and in elderly individuals. Our objective was to evaluate a non-institutionalized elderly population from Sa˜o Paulo City and correlate the estimated glomerular filtration rate with the percentage of lymphocytes in circulation. METHODS: A random population of 237 individuals (107 men and 130 women), ranging in age from 60 to 101 years, who were enrolled in the Health, Well-Being and Aging Study was evaluated for renal function (Modification on Diet in Renal Disease formula) and lymphocyte percentage (flow cytometry). RESULTS: Aging was associated with a decrease in the estimated glomerular filtration rate in both male and female individuals. We did not identify a significant correlation between the estimated glomerular filtration rate and either the percentage of CD4, CD8, and B cells or CD4/CD8 ratio. The median percentage of CD8+ T cells was significantly lower in individuals with an estimated glomerular filtration rate $60 mL/min/1.73 m2. CONCLUSIONS: In this study, no statistical correlation was found between the estimated glomerular filtration rate and either the lymphocyte phenotype (CD4+, CD8+, and CD19+ cells) or the CD4/CD8 ratio in blood. KEYWORDS: Renal Function; Elderly; Immune System; Flow Cytometry. Teixeira D, Longo-Maugeri IM, Duarte YA, Lebra˜o ML, Bueno V. Evaluation of renal function and immune system cells in elderly individuals from Sa˜o Paulo City. Clinics. 2013;68(1):39-44. Received for publication on August 28, 2012; First review completed September 11, 2012; Accepted for publication on September 16, 2012 E-mail: valquiria@nefro.epm.br Tel.: 55 11 5549-6073

immune system. However, these studies are controversial, probably because of the different parameters used to measure kidney function and immune response (5). In healthy elderly individuals, the decreased cellularity in the peripheral blood has been attributed to thymus involution (6) rather than bone marrow insufficiency (7). However, in patients with chronic kidney disease, other factors, such as malnutrition, parathyroid hormone deficiencies, and trace element deficiencies, could contribute to the decrease in the cell numbers observed in the blood (8). Verkade et al. and Litiens et al. (9,10) showed that the progressive loss of kidney function is associated with poor generation of antigen-specific T cells after vaccination and increased susceptibility to infection. Betjes et al. (11) evaluated young (25-45 years old) individuals, either healthy or with chronic kidney disease, and observed that changes in renal function were associated with significant decreases in circulating naı¨ve CD4+, naı¨ve CD8+, and memory CD4+ cells and discrete changes in memory CD8+ T cells. The percentages of CD4+ and CD8+ T cells in young individuals with chronic kidney disease were similar to those observed in healthy elderly individuals (6080 years old), suggesting that the decrease in kidney

& INTRODUCTION Renal function declines with age (1) and corresponds to a high prevalence of chronic kidney disease in older individuals (2). Additionally, the percentage and function of immune cells have been reported to change in elderly individuals (3). Teixeira et al. (4) evaluated individuals ranging in age from 60 to 101 years and observed that the percentages of T (CD4+) and B (CD19+) cells were decreased, whereas there was an increase in the percentage of T CD8+ cells. The study also revealed a decrease in the CD4/CD8 ratio, and all changes were more significant in men than in women. A significant number of studies have shown that the decline in kidney function is associated with changes in the

Copyright ß 2013 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. DOI: 10.6061/clinics/2013(01)OA06

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Immune cell phenotyping: A total of 100 mL of blood was lysed with Tris-buffered solution for 10 minutes and centrifuged at 377 g for 5 minutes. The cells were washed in PBS twice and centrifuged at 377 g for 5 minutes. The cells were then incubated with monoclonal antibodies (CD3PerCP, CD4FITC, CD8Pe-tritest, and CD19Pe; BD Biosciences, San Jose, California) to determine the percentages of T and B lymphocytes using flow cytometry in a FACSCalibur cell counter (BD Biosciences). Estimated glomerular filtration rate (eGFR): SCr was used to calculate the eGFR according to the Modification of Diet in Renal Disease (MDRD) formula: eGFR (mL/min/ 1.73 m2) = 1866(Scr)-1.1546(age)-0.20860.742 (if female).

function is associated with premature immunological aging. Guo et al. (8) showed that the CD4/CD8 ratio was significantly lower in patients with chronic kidney disease compared with healthy individuals. Libetta et al. (12) observed that patients with chronic kidney disease exhibited significantly lower peripheral blood cell secretion of IFN-c and IL-12 compared with healthy individuals. In contrast, these patients secreted constitutively more IL-4 and IL-10 than healthy individuals. Thus, it was suggested that these patients exhibit a deviation in their immune system that appears to correspond to the existence of a cell phenotype resulting in strong chronic activation of the immune system that inhibits a further response to acute stimulation, i.e., vaccination and new infections. B cells, responsible for antibody production, are generated and maturated in bone marrow mostly by the action of IL-7 (13,14). A decrease in B cells has been shown in children (15) and adults (16) with chronic kidney disease and could be the reason for the defective humoral (antigenspecific antibody production) response to infections, vaccinations and recall antigens in these patients. The elderly Brazilian population is increasing, and there are few reports on the condition of their health. Therefore, our aim was to evaluate renal function in a random elderly (60 to 101 years old) population from Sa˜o Paulo City and to correlate renal function with the percentage of T (CD4+ and CD8+) and B (CD19+) cells in the blood.

Statistical analysis Variables were analyzed using Pearson’s correlation test, and p-values,0.05 were considered to indicate statistical significance. Individuals were evaluated according to whether they had an eGFR lower or higher than 60 mL/min/1.73 m2 using the Student’s t-test. The Minitab software version 16.1 was used for all statistical tests.

& RESULTS Table 1 shows the clinical characteristics of the 237 elderly individuals studied. In total, 144 individuals had a serum creatinine level higher than 0.9 mg/dL, 66 individuals had glucose levels $100 mg/dL, and one individual had an albumin level higher than 5.0 g/dL.

& METHODS

Renal function evaluation The use of serum creatinine as a method to evaluate renal function presents limitations that are even more pronounced in the elderly due to their decreased muscle mass, nutritional status, use of multiple drugs, and comorbidities. For example, a marked reduction in GFR can occur before the serum creatinine rises (it is possible for up to 50% of kidney function to be lost before the creatinine level changes). Several equations (the Modification of Diet in Renal Disease (MDRD), Cockroft-Gault (CG), and Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations) were developed to estimate the eGFR. However, because all of these equations use serum creatinine, they lack complete accuracy. To better evaluate renal function in our studied individuals, we calculated the eGFR using the Modification of Diet in Renal Disease (MDRD) equation. Figure 1A shows a negative statistical correlation between eGFR and age, indicating that kidney function (i.e., eGFR) decreases with age. When eGFR was evaluated according to gender, this correlation was still observed (Figure 1B). We recently published results showing that T and B cell levels change in elderly individuals, which could account for the decreased immune response observed in this

The present study is part of a larger epidemiologic survey called the Health, Well-Being and Aging Study, which was coordinated by the Pan-American Health Organization, Washington, and conducted in Brazil by the School of Public Health of the University of Sa˜o Paulo. From 2000 to 2001, the Health, Well-Being and Aging Study evaluated a sample of 2,143 non-institutionalized individuals, representing 836,204 aging people (60 years and older) living in the municipality of Sa˜o Paulo, who were selected through multi-stage sampling. In 2006, the School of Public Health continued the survey in Sa˜o Paulo and transformed it into a multi-cohort study with 1,115 individuals from the previous study who agreed to participate in the follow-up. Trained examiners visited the participants at home and gathered information regarding socioeconomic variables, general health and living conditions. They also evaluated anthropometric and physical parameters and collected blood. In the present study, the same inclusion/exclusion criteria were applied as the criteria cited above, and the individuals were enrolled as their biological samples were received. After obtaining written informed consent, the creatinine and blood lymphocyte levels of a total of 237 individuals (107 men and 130 women), between the ages of 60 and 101 years, were evaluated. The study was approved by the Ethics Committee of the University of Sa˜o Paulo (USP), School of Public Health, Protocol number 2044/10. A 3-mL sample of blood in EDTA was collected from each individual to evaluate serum creatinine and immune cell levels. Creatinine measurement: Serum creatinine (SCr) levels in the blood were measured with a Dimension RXL (Siemens Laboratory Diagnostics, Tarrytown, NY, USA) automatic analyzer.

Table 1 - Baseline clinical characteristics of the 237 elderly individuals. Gender Age Glucose (mg/dL) Albumin (g/dL) Creatinine (mg/dL)

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Female (n = 130) Male (n = 107) 79.1¡9.6 (60-101) 95.0¡34.4 (32-373) 3.79¡0.36 (2.8-5.7) 1.03¡0.35 (0.4-3.37)


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Elderly renal function and immune system Teixeira D et al.

Figure 1 - Aging was associated with a decrease in the estimated glomerular filtration rate (eGFR) in 237 elderly individuals (A). The statistically significant correlation remained when individuals were evaluated according to gender (B). Pearson correlation r = 20.398, p,0.0005.

evaluated the percentages of T and B cells according to eGFR values, where values ,60 mL/min/1.73 m2 and $60 mL/min/1.73 m2 represented chronic kidney disease and the absence of chronic kidney disease, respectively. In our study population, 148 individuals presented with an eGFR$60 mL/min/1.73 m2. In Figure 4, it can be observed that an eGFR$60 mL/ min/1.73 m2 was associated with a significantly lower median percentage of CD8+ T cells whereas no significant differences were found with respect to the CD4+ T cell percentage, B cell (CD19+) percentage or CD4/CD8 ratio between individuals with similar renal functions. However, we observed a tendency toward higher median percentages of CD4+ and CD19+ cells in individuals with an eGFR$60 mL/min/1.73 m2 compared with the percentages in individuals with an eGFR,60 mL/min/1.73 m2.

population (4). Also, it has been reported that patients with chronic kidney diseases present changes in lymphocytes (17). Therefore, in our study population, we investigated whether the confirmed decrease in kidney function (Figures 1A and 1B) was correlated with changes in the levels of CD4+ T, CD8+ T, and B cells. Figures 2 and 3 show that no significant correlations were observed between eGFR and either the percentage of CD4, CD8, and B cells or CD4/CD8 ratio. The American Kidney Foundation classification for chronic renal disease according to the estimated glomerular filtration rate is as follows: eGFR between 45 and 60 mL/ min/1.73 m2 = Stage 3A, eGFR between 30 and 45 mL/min/ 1.73 m2 = Stage 3B, eGFR between 15 and 30 mL/min/ 1.73 m2 = Stage 4 and eGFR,15 mL/min/1.73 m2 = Stage 5 (end-stage renal disease). Using this classification, we

Figure 2 - Estimated glomerular filtration rate (eGFR) versus percentage of CD4 T (A) and CD8 T (B) lymphocytes in 237 elderly individuals. Pearson correlation r = 0.068, p = 0.299 (A); Pearson correlation r = 20.099, p = 0.132 (B).

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Figure 3 - Estimated glomerular filtration rate (eGFR) versus the CD4/CD8 ratio (A) and percentage of CD19 B lymphocytes (B) in 237 elderly individuals. Pearson correlation r = 0.068, p = 0.301 (A); Pearson correlation r = 0.078, p = 0.258 (B).

Figure 4 - Median percentage of CD4+, CD8+, and CD19+ cells and the CD4/CD8 ratio in elderly individuals grouped according to whether they had an eGFR,60 mL/min/1.73 m2 or an eGFR$60 mL/min/1.73 m2.

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Additionally, the median CD4/CD8 ratio was higher in individuals with an eGFR$60 mL/min/1.73 m2.

known to be associated with cell function (proliferation after a stimulus, cytokine production, and apoptosis).

& DISCUSSION

& ACKNOWLEDGMENTS

Our results are in agreement with those of other studies showing that an increase in age is associated with a decrease in kidney function (1,2). Patients with chronic kidney disease have been reported to present with immune system changes, such as lower percentages of T and B cells. Chung et al. (18) observed that healthy individuals (creatinine: 0.9 mg/dL) presented with a higher percentage of naı¨ve CD4+ T cells (38.9%) than patients with chronic kidney disease (creatinine: dialysis = 9.7 mg/dL and pre-dialysis = 8.9 mg/dL; CD4+ T cells: dialysis = 31% and pre-dialysis = 25%). In the present study, no significant correlation was observed between the eGFR calculated using the MDRD equation based on creatinine levels and either CD4+, CD8+, and CD19+ cell percentages or the CD4/CD8 ratio. In elderly individuals, the decline in creatinine clearance and the greater individual variability associated with discrepancies in measured GFR and estimated GFR are factors that contribute to the inconsistency in results. Moreover, most of the individuals studied (n = 148) presented with an eGFR greater than 60 mL/min/1.73 m2, and, as reported by Van Pottelbergh et al. (19), individuals in this age range with an eGFR$60 mL/min/1.73 m2 have low probability of developing chronic renal disease. When we compared individuals with an eGFR indicating chronic renal disease (.60 mL/min/1.73 m2) and individuals with an eGFR not suggestive of renal disease (#60 mL/min/1.73 m2), a tendency toward higher median levels of CD4+ and CD19+ cells was observed in individuals with better renal function. In addition, the median number of CD8+ cells was significantly higher in individuals with worse renal function. In contrast to our findings, CD8+ T cell levels have been shown to be decreased in patients with chronic kidney disease (18,20,21), and, consequently, the CD4/CD8 ratio is also decreased (22,23). Compared with healthy controls, a decrease in the percentage of B cells (CD19+) has been observed in patients with chronic kidney disease (8,16). Considering the important role of B cell-producing antibodies, these findings should be considered in the development of new vaccines. For example, Yaghoubian et al. (23) showed that the mortality of patients 65 years or older admitted for general surgery with a serum creatinine level greater than 2.0 mg/ dL was 42% and was associated with infections. It should be noted that the changes observed in the immune system (immunosenescence) could have several causes, and the reason why some individuals are more protected than others in this process still needs to be clarified. Additionally, it has to be considered that this study has some deficiencies, such as the use of serum creatinine levels in elderly individuals, which could underestimate kidney function even when the results are adjusted for age and gender; the evaluation of immune cells in blood only instead of also investigating them in lymphoid organs (bone marrow, thymus, spleen, and lymph nodes); and the observed changes in the blood phenotype (CD4+, CD8+, and CD19+ cell percentages and the D4/CD8 ratio) that are

We thank Thais Cocarelli for performing the statistical analysis and FAPESP and CNPQ for providing financial support.

& AUTHOR CONTRIBUTIONS Teixeira D and Longo-Maugeri IM performed flow cytometry and contributed to the manuscript writing. Duarte YA and Lebra˜o ML designed and coordinated the study, collected blood and contributed to the manuscript writing. Bueno V performed flow cytometry, conducted the statistical analysis and contributed to the manuscript writing.

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21. Saurwein-Teissl M, Lung TL, Marx F, Gschosser C, Asch E, Blasko I. Lack of antibody production following immunization in old age: association with CD8(+) CD28(-) T cell clonal expansions and an imbalance in the production of Th1 and Th2 cytokines. J Immunol 2002;168(11):5893-9. 22. Yoon J-W, Gollapudi S, Pahl MV, Vaziri ND. Na覺穡ve and central memory T-cell lymphopenia in end-stage renal disease. Kidney Int. 2006;70(2):371-6, http://dx.doi.org/10.1038/sj.ki.5001550. 23. Yaghoubian A, Ge P, Tolan A, Saltmarsh G, Kaji AH, Neville AL, et al. Renal insufficiency predicts mortality in geriatric patients undergoing emergent general surgery. Am Surg. 2011;767(10):1322-5.

in patients aged $50 years. Nephrol Dial Transplant. 2011;27(6):2297303. 19. Litjens NH, de Wit EA, Betjes MG. Differential effects of age, cytomegalovirus-seropositivity and end-stage renal disease (ESRD) on circulating T lymphocyte subsets. Immun Ageing. 2011;8(1):2, http://dx. doi.org/10.1186/1742-4933-8-2. 20. Vacher-Coponat H, Brunet C, Lyonnet L, Bonnet E, Loundou A, Sampol J, et al. Natural killer cell alterations correlate with loss of renal function and dialysis duration in uraemic patients. Nephrol Dial Transplant. 2008;23(4):1406-14.

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CLINICAL SCIENCE

Evaluation of the correlation between dental occlusion and posture using a force platform Alberto Baldini,I Alessandro Nota,I Domenico Tripodi,II Salvatore Longoni,III Paola CozzaI I

University of Rome ‘‘Tor Vergata’’, Department of Orthodontics, Rome/Italy. II University of Chieti, Department of Oral and Biotechnological Medical Sciences, Chieti, Italy. III University of Milano-Bicocca, Department of Biosciences and Biomedical Technologies, Monza/Italy.

OBJECTIVES: Force platforms are widely used to evaluate the relationship between posture and dental occlusion. This study evaluated whether force platforms are able to detect eventual postural modifications resulting from dental occlusion. METHOD: A total of 44 healthy volunteers who were given no information on the aim of the study underwent six postural stabilometric exams under different mandibular and visual conditions. Four parameters were considered: sway area, sway velocity, X axis displacement of the center of the foot pressure and Y axis displacement of the center of the foot pressure. RESULTS: An analysis of variance (ANOVA) revealed the relative influence of each factor; specifically, the ocular afference significantly influenced the sway area and sway velocity parameters, and the mandibular position had only a weak influence on the sway area parameter. CONCLUSIONS: Vision was shown to influence body posture, and a weak correlation was observed between mandibular position and body posture in healthy subjects. However, the force platform is most likely not able to clearly detect this relationship. Gnathologists must use caution when using force platform analysis to modify a therapeutic plan. The sway area seems to be the most sensitive parameter for evaluating the effect of occlusion on body posture. KEYWORDS: Posture; Dental Occlusion; Occlusal Splint; Mandible. Baldini A, Nota A, Tripodi D, Longoni S, Cozza P. Evaluation of the correlation between dental occlusion and posture using a force platform. Clinics. 2013;68(1):45-49. Received for publication on July 19, 2012; First review completed on July 26, 2012; Accepted for publication on September 16, 2012 E-mail: studiomedicobaldini@gmail.com Tel.: 39 035/271935.

connections have been documented between the oral and cervical areas (3-6). Based on the available literature, it seems that in this system, the mandible represents a sort of balancing pole that is capable of affecting posture and of being influenced by the posture itself (7). In the majority of studies involving the hypothetical influence of dental occlusion on posture, as well as in clinical practice, the force platform is the principal instrument used to analyze these correlations, although the results are still contradictory (8). Many authors (9) do not consider the force plate to be a reliable instrument, although there are a few studies demonstrating the scientific reliability of the results obtained using the force plate and its clinical implications (8). Before using the force plate for the analysis of the correlation between dental occlusion and posture, the following factors should be confirmed:

& INTRODUCTION The human posture represents the position of the body and the spatial relationships between its anatomical segments that maintain balance under dynamic and static conditions (anti-gravity function of the muscles) according to the requirements of the environment and the motor goals. A dedicated ‘‘Tonic Postural System’’ regulates and adjusts postural balance based on visual, vestibular and somatosensory inputs (1), as well as (in some cases) respiration and mood states. In particular, the head and neck position can modify the postural pattern of each individual (2). In fact, the erect position of the head is maintained by a balanced tension between the craniocervical bones, myofacial structures and dental occlusion, and many neuroanatomical

Copyright ß 2013 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.

- The accuracy and precision of the test. - The reliability of the test. - Whether the force platform and its parameters are able to demonstrate an influence of dental occlusion on posture.

No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(01)OA07

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Only after having confirmed these elements is it possible to verify the existence and degree of the correlation between dental occlusion and posture. The accuracy and precision of the instrument are guaranteed by the manufacturer and by the maintenance and calibration of the device. Some authors (10-12) have studied the reliability of the force plate exam, concentrating on the intrasession variability, and they have obtained excellent results. Other authors have attempted to confirm whether there is also good intersession reliability (13). In this study, we sought to evaluate whether the force plate is capable of demonstrating a relationship between dental occlusion and posture in healthy subjects.

& MATERIALS AND METHODS Forty-four healthy volunteer subjects, including 30 males and 14 females (ages 17-35 years, mean age 23.75¡4.10 years), who were given no information about the aim of the study, were enrolled in the study, which was approved by the ethical committee, after signing an informed consent form. Based on a clinical and anamnestic analysis, subjects were included in the study if they met the following inclusion criteria:

Figure 1 - Force platform (DL Medica Spa Milano).

The subjects were required to remain as stable as possible, relaxed, with their arms hanging free beside their trunk, and facing the wall without concentrating on a precise point. Moreover, all of the subjects were asked to avoid alcohol, sports and conservative therapies during the 24 h before the clinical recordings. The placement of the subjects on the force plate was standardized; specifically, a hand was placed under the foot of the subject, lifting the foot until it met the following criteria using the markers painted on the surface of the platform (Figure 1):

- Good general health according to the medical history. - Absence of trauma or surgery, which can influence posture. - Absence of visual or vestibular problems. - Absence of any other disorder capable of influencing posture. - Absence of evident postural problems. - Presence of at least 28 teeth. - Dental overjet between 1 and 4 mm. - Absence of any type of crossbite, open bite or deep bite. - Absence of cast restoration and extensive occlusal restoration. - Absence of craniomandibular disorders.

- Foot angle of 30 ˚ following the principal red line. - Calcaneal tendon positioned along the length of the foot, expressed in French points and centered on the principal red line. - Malleolus positioned corresponding to the angled red line. - Second toe root projection corresponding to the principal red line. - Foot outline corresponding to the areas drawn on the surface of the platform.

The single-blind experimental protocol was carried out by performing posturographic and stabilometric analysis with a force platform, Postural Health Station (DL Medica SpA, Milano, Italy) (Figure 1). This platform is characterized by load cells with an internal circuit that changes electrical resistance when a force is applied. The participants underwent a force plate exam; each recording had a duration of 51.2 sec (in accordance with the guidelines of the French Posturology Association) and was performed under the following conditions: mandibular rest position, with eyes opened and closed; mandibular position of centric occlusion, with eyes opened and closed; and mandibular position, with cotton rolls and eyes opened and closed. To obtain the ‘‘cotton rolls’’ mandibular position, cotton rolls that were 8 mm thick and 37 mm long were positioned between the mandibular teeth distal to the canines. Quiet conditions were maintained during the exam, and disturbing elements were eliminated to avoid any influence on posture. A force plate was placed 150 cm from a wall such that the subjects were positioned perpendicular to the wall.

Based on the results obtained, four parameters were evaluated: sway area; sway velocity; COP X (position of the center of the foot pressure on the X axis compared with the theoretical ideal position); and COP Y (position of the center of foot pressure on the Y axis compared with the theoretical position) The statistical analysis of the data was performed using the software Minitab 15 (Minitab Inc. State College, Pennsylvania, USA). An analysis of variance (ANOVA) with statistical significance indicated by a p-value,0.1 was performed to evaluate the influence of each of the considered factors (visual condition and mandibular position) on the posture of healthy subjects.

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Dental occlusion and body posture Baldini A et al.

Table 1 - Mean and standard deviation of the results.

Table 2 - ANOVA results.

Parameter

Conditions

Mean

Standard dev.

Parameter

Sway area

Rest eyes open Rest eyes closed Centric eyes open Centric eyes closed Cotton eyes open Cotton eyes closed Rest eyes open Rest eyes closed Centric eyes open Centric eyes closed Cotton eyes open Cotton eyes closed Rest eyes open Rest eyes closed Centric eyes open Centric eyes closed Cotton eyes open Cotton eyes closed Rest eyes open Rest eyes closed Centric eyes open Centric eyes closed Cotton eyes open Cotton eyes closed

94.30 143.70 108.00 172.00 109.70 178.00 5.74 8.20 5.77 8.14 5.78 7.97 1.40 2.20 1.06 1.84 2.02 2.06 -8.12 -6.60 -7.00 -9.20 -8.00 -9.80

43.50 94.10 63.00 130.00 80.60 144.00 1.33 2.39 1.36 2.63 1.53 2.49 7.62 7.51 8.02 8.51 7.51 6.81 9.96 10.50 12.20 11.30 11.60 11.20

Sway area

Sway velocity

Cop x

Cop y

Sway velocity Cop x Cop y

Influence Vision Mandibular Vision Mandibular Vision Mandibular Vision Mandibular

pos. pos. pos. pos.

p-value 0.000 0.065 0.000 0.905 0.794 0.931 0.260 0.146

p-values of the ANOVA show the statistical relevance of the influence of vision and mandibular postitions on the postural parameters.

& DISCUSSION The results of the ANOVA in this study confirm the existence of an important and clear correlation between vision and postural control (14). The dynamic stabilometric parameters (area and velocity) seem to be influenced by vision, and we observed a loss of postural control when a subject closed his or her eyes, as demonstrated by an increase in the postural parameter values. This result can be understood if we assume that the vision represents a fundamental component of the tonic postural system and that the exclusion of vision prevents the superior system from controlling the posture. We observed that the position of the center of the foot pressure was not influenced by visual or occlusal components. It can be supposed that the coordinates of the center of the foot pressure are dependent on the patient’s positioning on the platform and are not related to the test itself and that the position of the center of the foot pressure on the X axis changes less than on the Y axis because the relationships between the anatomical body parts are more subject to anterior-posterior sway than to lateral sway. In this study, the mandibular position significantly influenced the sway area parameter, as some authors have previously reported (7,15-17), but it did not influence the sway velocity parameter. The influence of the mandibular position on sway area appears to be weak; however, this result is not completely reliable due to the abnormal statistical distribution of its values. In a clinical study conducted by Bracco et al. on a sample of 95 healthy subjects, posturometric and stabilometric analyses were performed with a force platform to investigate the influence of three mandibular positions on body posture. All subjects exhibited statistically significant variations of body posture with different mandibular positions according to the asymmetry index and the position of the COP on the X and Y axes (15); this result is in contrast to the results of the present study, which did not reveal an influence of the different mandibular positions on the COP values. A study by Perinetti et al. showed no significant differences in postural parameters values between the centric occlusion mandibular position and the resting position in a sample of 26 healthy subjects. The sway area, sway velocity and length of the force platform were significantly higher when the subjects had their eyes closed versus open for both mandibular positions. There were no differences between the mandibular rest position and dental occlusion under the different visual conditions. From a theoretical viewpoint, the absolute displacement of the COP was not correlated with the visual condition or mandibular position (9). This finding is

& RESULTS A preliminary analysis of the results showed that the values of the postural parameters tended to increase when the subjects’ eyes were closed. Results in Table 1. SWAY AREA: The mean values of the sway area obtained when the eyes were closed were between 143 and 178 mm2, which is more than 60 mm2 larger than the values obtained with the eyes open (mean values between 94 and 110 mm2), demonstrating that the area increased by approximately 39% when the eyes were closed. The variations between the areas recorded in different mandibular position were lower under the same visual conditions (15-30 mm2). The lowest areas were recorded in mandibular resting position. The mandibular position was able to increase the sway area by approximately 16%. SWAY VELOCITY: The mean values of the sway velocity when the eyes were closed were between 7.97 and 8.20 mm/s, which is approximately 2.21-2.42 mm/s greater than the velocity obtained when the eyes were open (mean values between 5.74 and 5.78 mm/s), demonstrating that the sway velocity increased by approximately 29% when the eyes were closed. In different mandibular positions under the same visual condition, there were variations of 0.04 mm when the eyes were open and 0.21 when the eyes were closed; thus, the mandibular position was able to increase the sway area by approximately 0.7-2.5%. The position of the center of the foot pressure on the X and Y axis is affected by wide variations. An ANOVA revealed that the sway area and sway velocity parameters were significantly influenced by vision (p,0.001). In contrast, the p-values for the COP X and COP Y parameters were particularly high, with values of p = 0.26 for the Y axis and p = 0.79 for the X axis. The mandibular position significantly influenced only the sway area (p = 0.065) without significantly affecting the other parameters (p.0.14).

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intraoral instrument (7,25). Contrary to the conclusions of other studies (9), the clinical influence of this relationship could be important in gnathopostural approaches to treating painful trunk muscle symptomatology. Future studies should focus on the development of new experimental protocols based, for example, on 3D analysis to clearly verify the correlation between dental occlusion and posture. These studies should evaluate the importance of this correlation for and its influence on each anatomical segment of the body.

compatible with our results, with the exception that we identified a weak influence of the mandibular position on the sway area. Finally, Sakaguchi et al. evaluated the effect of changing the mandibular position relative to the body posture and, reciprocally, changing the body posture relative to the mandibular position by using a force platform and performing a computerized analysis of dental occlusion loads in a sample of 45 asymptomatic subjects. Statistically significant differences were found in the sway length and sway area parameters between five different mandibular positions. Furthermore, the occlusal load values revealed by the T-Scan II (Tekscan, Inc., South Boston, MA) system showed a significant difference when a heel lift was positioned under the right foot. It was concluded that changing the mandibular position affected the body posture and vice versa (7,18). Considering these previous conclusions, it seems probable that, despite the existence of a correlation between dental occlusion and posture, our results are similar to Perinetti’s findings. Due to the presence of some significant differences between the results in the scientific literature and in accordance with the conclusion of Perinetti in his review article (19), the most reasonable interpretation of our results is that the force platform and its most widely used parameters, although commonly used in gnathopostural clinical practice, do not constitute the most ideal method for the analysis of the correlation between dental occlusion and posture because of the lack of sensitivity of the method, especially in healthy subjects. We can suppose that, because of the increased compliance among young, healthy subjects (especially those without craniomandibular disorders and without occlusal problems), the influence of dental occlusion on posture was not clearly observed in the lower legs, and the use of a force plate makes this difference even more difficult to detect. The sway area was the only parameter that demonstrated a weak ability to detect an influence of dental occlusion on posture. The force plate is commonly used by clinicians to analyze posture in healthy, pathological and elderly individuals and athletes undergoing postural changes. It is also frequently used by specialists of a variety of disciplines who are interested in posture (e.g., physiatrists, physiotherapists, orthopedists, neurologists, and sports doctors). However, the utility of this instrument for verifying the relationship between occlusion and posture in healthy subjects appears to be less reliable. Based on the results of this study, gnathologists should be careful in using force platform analysis to modify their therapeutic plans, especially in young patients and those without TMJ disorders, due to its low sensitivity for revealing occlusion-related postural alterations. Thus, the force platform does not seem to be the ideal instrument for use in gnathoposturology. In fact, despite the results of this study, a correlation between dental occlusion and body posture is suggested by gnathopostural results obtained in clinical practice (20-22), by clinical studies that have been conducted without the use of a force platform (23,24) and especially by the important finding of the existence of a reciprocal relationship between dental occlusion and body posture described by Sakaguchi et al. based on results obtained using a computerized

& AUTHOR CONTRIBUTIONS Baldini A, Nota A, and Tripodi D participated in the execution of the experiment. Longoni S conducted the statistical analysis. Cozza P coordinated the study.

& REFERENCES 1. Guez G. The posture. In: Kandel ER, Schwartz JH, Jessel TM, editors. Principles of Neural Science. Amsterdam: Elsevier; 1991.612-23. 2. Wada M, Sunaga N, Nagai M. Anxiety affects the postural sway of the antero-posterior axis in college students. Neurosci Lett. 2001;302(23):157-9, http://dx.doi.org/10.1016/S0304-3940(01)01662-7. 3. Michelotti A, Buonocore G, Manzo P, Pellegrino G, Farella M. Dental occlusion and posture: an overview. Prog Orthod. 2011;12(1):53-8. 4. Pinganaud G, Bourcier F, Buisseret-Delmas C, Buisseret P. Primary trigeminal afferents to the vestibular nuclei in the rat: existence of a collateral projection to the vestibulo-cerebellum. Neurosci Lett. 1999;264(1-3):133-6, http://dx.doi.org/10.1016/S0304-3940(99)00179-2. 5. Marfut CF, Rajchert DM. Trigeminal primary afferent projection to ‘‘non trigeminal’’ areas of the rat central nervous system. J Comp Neurol. 1991;303(3):489-511, http://dx.doi.org/10.1002/cne.903030313. 6. Buisseret-Delmas C, Compoint C, Delfini C, Buisseret P. Organization of reciprocal connections between trigeminal and vestibular nuclei in the rat. J Comp Neurol. 1999;409(1):153-68, http://dx.doi.org/10.1002/(SICI) 1096-9861(19990621)409:1,153::AID-CNE11.3.0.CO;2-#. 7. Sakaguchi K, Mehta NR, Abdallah EF, Forgione AG, Hirayama H, Kawasaki T, et al. Examination of the relationship between mandibular position and body posture. Cranio. 2007;25(4):237-49. 8. Baldini A, Cravino G, Dental occlusion and athletic performances. A review of literature Mondo Ortodontico. 2011;36(3):131-41, http://dx. doi.org/10.1016/j.mor.2010.09.003. 9. Perinetti G. Dental occlusion and body posture: no detectable correlation. Gait Posture. 2006;24(2):165-8, http://dx.doi.org/10.1016/j.gaitpost. 2005.07.012. 10. Lafond D, Corriveau H, He´bert R, Prince F. Intrasession reliability of center of pressure measures of postural steadiness in healthy elderly people. Arch Phys Med Rehabil. 2004;85(6):896-901, http://dx.doi.org/ 10.1016/j.apmr.2003.08.089. 11. Ruhe A, Fejer R, Walker B. The test-retest reliability of centre of pressure measures in bipedal static task conditions--a systematic review of the literature. Gait Posture. 2010;32(4):436-45, http://dx.doi.org/10.1016/j. gaitpost.2010.09.012. 12. Corriveau H, Hebert R, Prince F, Raıche M. Intrasession reliability of the ‘‘center of pressure minus center of mass’’ variable of postural control in the healthy elderly. Arch Phys Med Rehabil. 2000;81(1):45-8. 13. Baldini A, Nota A. Evaluation of the reliability of the force platform exam. Act of the XXIII International Congress of the Italian Society of Orthodontics; Nov. 10-12 2011; Rome. 14. Edwards AS. Body sway and vision. J Exp Psychol. 1946;36(6):526-35, http://dx.doi.org/10.1037/h0059909. 15. Bracco P, Deregibus A, Piscetta R. Effects of different jaw relations on postural stability in human subjects. Neurosci Lett 2004;356(3):228-30, http://dx.doi.org/10.1016/j.neulet.2003.11.055. 16. Gangloff P, Louis JP, Perrin PP. Dental occlusion modifies gaze and posture stabilization in human subjects. Neurosci Lett. 2000;293(3):203-6, http://dx.doi.org/10.1016/S0304-3940(00)01528-7. 17. Sforza C, Tartaglia GM, Solimene U, Morgun V, Kaspranskiy RR, Ferrario VF. Occlusion, sternocleidomastoid muscle activity, and body sway: a pilot study in male astronauts. Cranio. 2006;24(1):43-9. 18. Baldini A. Clinical and instrumental treatment of a patient with dysfunction of the stomatognathic system: a case report. Ann Stomatol (Roma). 2010;1(2):2-5. 19. Perinetti G, Contardo L. Posturography as a diagnostic aid in dentistry: a systematic review. J Oral Rehabil. 2009;36(12):922-36, http://dx.doi.org/ 10.1111/j.1365-2842.2009.02019.x. 20. Baldini A, Beraldi A, Nota A, Danelon F, Ballanti F, Longoni S. Gnathological postural treatment in a professional basketball player: a

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case report and an overview of the role of dental occlusion on performance. Ann Stomatol (Roma). 2012;3(2):51-8. 21. Baldini A, Tecco S, Cioffi D, Rinaldi A, Longoni S. Gnathological postural treatment in an air force pilot. Aviat Space Environ Med. 2012;83(5):5226, http://dx.doi.org/10.3357/ASEM.2852.2012. 22. Baldini A, Cravino G, Rinaldi A, Cioffi D Gnathological postural analysis and treatment in Air Force pilots: a case report Mondo Ortodontico. 2011;36(5):208-15.

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Body size and longitudinal body weight changes do not increase mortality in incident peritoneal dialysis patients of the Brazilian peritoneal dialysis multicenter study Nata´lia Maria da Silva Fernandes,I Marcus Gomes Bastos,I Ma´rcia Regina Gianotti Franco,I,II Alfredo Chaoubah,III Maria da Glo´ria Lima,II Jose´ Carolino Divino-Filho,IV Abdul Rashid Qureshi,IV on behalf of the Brazilian Peritoneal Dialysis Multicenter Study (BRAZPD) Group I

Federal University of Juiz de Fora, Interdisciplinary Program of Studies, Research, and Treatment in Nephrology, Juiz de Fora/MG, Brazil. II Clı´nica GAMEN, Rio de Janeiro/RJ, Brazil. III Federal University of Juiz de Fora, Department of Statistics, Juiz de Fora/MG, Brazil. IV Karolinska Institute, Division of Renal Medicine, CLINTEC, Stockholm, Sweden.

OBJECTIVES: To determine the roles of body size and longitudinal body weight changes in the survival of incident peritoneal dialysis patients. PATIENTS AND METHODS: Patients (n = 1911) older than 18 years of age recruited from 114 dialysis centers (Dec/ 2004-Oct/2007) and participating in the Brazilian Peritoneal Dialysis Multicenter Cohort Study were included. Clinical and laboratory data were collected monthly (except if the patient received a transplant, recovered renal function, was transferred to hemodialysis, or died). RESULTS: Survival analyses were performed using Kaplan-Meier survival curves and Cox proportional hazards. Total follow-up was 34 months. The mean age was 59 years (54% female). The weight category percentages were as follows: underweight: 8%; normal: 51%; overweight: 29%; and obese 12%. The multivariate model showed a higher risk of death for a body mass index ,18.5 kg/m2, a neutral risk between 25 and 29.9 kg/m2 and a protective effect for an index .30 kg/m2. Patients were divided into five categories according to quintiles of body weight changes during the first year of dialysis: ,23.1%, 23.1 to+0.12%, +0.12 to ,+3.1% (reference category), +3.1 to +7.1% and .+7.1%. Patients in the lowest quintile had significantly higher mortality, whereas no negative impact was observed in the other quintiles. CONCLUSION: These findings suggest that overweight/obesity and a positive body weight variation during the first year of peritoneal dialysis therapy do not increase mortality in incident dialysis patients in Brazil. KEYWORDS: Overweight; Obesity; Incident; Peritoneal Dialysis; Survival; Cohort Study. Fernandes NM, Bastos MG, Franco MR, Chaoubah A, Lima MG, Divino-Filho JC, et al. Body size and longitudinal body weight changes do not increase mortality in incident peritoneal dialysis patients of the Brazilian peritoneal dialysis multicenter study. Clinics. 2013;68(1):51-58. Received for publication on June 4, 2012; First review completed on July 23, 2012; Accepted for publication on September 23, 2012 E-mail: nataliafernandes02@gmail.com Tel.: 55 32 3231-6319

cardiovascular diseases, and certain types of cancer (2). In patients with chronic kidney disease on hemodialysis (HD), a high body mass index (BMI) is associated with better survival, whereas a low BMI and weight loss are associated with increased mortality (3-7). The role of BMI in the survival of patients undergoing peritoneal dialysis (PD) has yet to be established; available studies have provided conflicting results (8-10). Although most physicians do not consider PD to be the treatment of choice for obese patients, the percentage of incident patients with high BMIs has risen progressively in some countries since 1980 (11,12,13). McDonald et al. evaluated 9,440 PD patients in the ANZDATA registry and could not show a protective effect of BMI (11). However, in a prospective study with 45,982 PD patients, Snyder et al. reported an association between adjusted mortality and BMI changes over time, with better

& INTRODUCTION Over the last 20 years, the prevalence of overweight and obesity has increased dramatically in the Brazilian population, especially among those aged 20 years and over (1). Overweight and obesity are known risk factors for highly prevalent conditions such as type 2 diabetes, hypertension,

Copyright ß 2013 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. DOI: 10.6061/clinics/2013(01)OA08

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survival among overweight and obese patients until the third year of PD (14). The conflicting results regarding the effect of body size on PD outcomes may relate in part to the inability of BMI to differentiate between muscle mass and fat tissue (9). Ram Kumar et al. evaluated the body size and body composition of 10,140 PD patients (15); the authors concluded that both factors influence survival in incident patients receiving PD and that those patients with higher BMIs associated with normal or high muscle mass exhibited the best survival. In a recently published paper, Pellicano et al. (16) followed up 60 incident HD (n = 39) and PD (n = 21) patients longitudinally for 12 months. Using gold standard methods, they found that dialysis modalities affected changes in total body fat; extended hours of home HD were associated with the smallest increase, and PD was associated with the greatest increase in total body fat, with a significant increase in the ratio of visceral to subcutaneous fat. Obese patients experienced greater preservation of total body protein compared with normal and overweight patients, suggesting that energy storage as fat mass is of value in the dialysis patient population. The Brazilian Peritoneal Dialysis Multicenter Cohort Study (BRAZPD) is the first large observational PD cohort study performed in Brazil, and the percentage of obese PD patients (17) is similar to that of the general population (18). The aim of this observational study is to determine the impact of both body size and longitudinal body weight changes on survival in a cohort of incident PD patients in the BRAZPD study.

Davies comorbidity score was used to assess the severity of comorbid conditions (19). The data obtained from the patients’ charts included the following: sociodemographic information, chronic kidney disease etiology, hypertension, and comorbidities. During the follow-up period, body weight (BW), height, and BMI were evaluated monthly. BMI, defined as weight in kilograms divided by the square of the height in meters, was classified according to the World Health Organization (WHO): underweight (,18.5 kg/m2), normal (18.5 to 24.9 kg/m2), overweight (25 to 29.9 kg/m2) and obese (.30 kg/m2). Body weight was measured monthly without PD fluid in the abdominal cavity. Laboratory measurements were taken monthly, including creatinine, urea, potassium, calcium, phosphate, alanine amino-transferase (ALT), glucose, hemoglobin, albumin, total cholesterol, and triglycerides, and were determined using routine methods. The patients were followed until they received kidney transplants, recovered renal function, were transferred to HD, died, or ended their participation in the study.

Statistics The patients were divided into four groups according to BMI. The demographic, clinical, and laboratory data were evaluated for each group. Normally distributed variables were expressed as means¡SD (unless noted otherwise), and non-normally distributed variables were expressed as medians and ranges. Differences between the BMI groups were examined using the Kruskal-Wallis analysis of variance (ANOVA), followed by a post hoc Dunn’s test for nonparametric comparisons. A x2 test was used for categorical variables. Survival was analyzed with Kaplan-Meier survival curves and Cox proportional hazards models. The latter were used to examine survival differences after the analysis had been adjusted for potential confounding factors (age, gender, Davies score, and BMI) in incident patients. We used competing risk analysis to study the influence of BMI on survival in this cohort of incident PD patients. We also performed two sensitivity analyses. First, the main analysis with censoring of follow-up time for the patients (n = 1,615) who either reached the final follow-up time (n = 1,304) or ended PD therapy for any reason (drop out not related to death, n = 311). Second, we reclassified the patients who ended PD therapy for any reason. Patients who received a renal transplant, were transfer to HD or recovered renal function were excluded from the analysis. Survival was determined after a median follow-up of 13 months (range, 3 to 34 months). Moreover, survival for the entire follow-up period was analyzed according to the evolution of BW during the patients’ first year of therapy (n = 1,738). Patients were divided into five quintiles (,23.1%, 23.1 to +0.12%, +0.12 to ,+3.1% [reference category], +3.1 to +7.1% and .+7.1%) according to the BW evolution expressed as a percentage normalized to the ideal body weight calculated from the Broca formula (ideal BW = (height in cm 2 100) for males and ideal BW = (height in cm 2 104) for females). Restricted cubic splines were used to evaluate nonlinear relationships between BMI levels and mortality. This method has been suggested to offer adequately fit models and is a good compromise between flexibility and loss of precision caused by over-fitting a small sample. Statistical significance was set at p,0.05. Statistical analyses were performed using Stata software (Version 12.1; Stata Corp.,

& PATIENTS AND METHODS Setting and patients Incident PD patients recruited from 114 dialysis centers treating more than 10 PD patients each and reporting monthly to BRAZPD were included in this study. All of the patients were 18 years or older, remained on PD for at least 90 days and provided complete information on body weight and height. This study was conducted in accordance with the Declaration of Helsinki, and all participants provided written informed consent before enrollment. Details of the study design and characteristics of the cohort are described elsewhere (17). Of the 3,439 incident patients enrolled in the BRAZPD from December 2004 through October 2007, 1,911 were eligible for the study, and 1,528 patients were excluded (867 for not completing 90 days of therapy and 661 with more than 90 days of therapy but lacking either weight or height data). Out of the 1,528 excluded patients, 1,172 were still alive and on PD in October 2007, 210 died and 146 dropped out for reasons other than death as follows: 51 were transferred to HD, 13 received kidney transplantations, 4 recovered renal function, 13 were transferred to other clinics, 2 abandoned the study, 6 were lost for other reasons and 57 had no available data.

Data collection Data were collected monthly from December 2004 through October 2007. Sociodemographic and clinical data were evaluated at baseline. Each patient’s medical chart was thoroughly reviewed by nephrologists who extracted data pertaining to the underlying renal disease, history of cardiovascular disease and other comorbid conditions. The

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College Station, TX, USA) and SAS statistical software (Version 9.2 SAS Institute Inc., Cary, NC, USA). As p-values were not adjusted for multiple testing, they should be considered descriptive.

were normal, 27.2% were overweight, and 13% were obese. No significant differences in baseline characteristics were observed between the group of patients who had PD as their initial renal replacement therapy (RRT) (32%) and the group of patients who had HD as their first RRT (68%). Table 1 shows some demographic characteristics and clinical and laboratory data for the incident PD patients according to BMI. Overweight patients were older (60¡13 years old; p = 0.001), whereas obese patients were mostly female (64%; p = 0.001) and exhibited higher systolic (p = 0.001) and diastolic blood pressures (p = 0.003). The Davies score was higher than 2 in 18% of the overweight patients and 16% of the obese patients. Patients who were overweight or obese presented higher hemoglobin (p = 0.02), glucose (p = 0.001) and triglyceride (p = 0.007) levels than the other two groups (normal and underweight). There were no significant differences in the values of albumin, urea, potassium, calcium, phosphate, and cholesterol among the BMI groups. For creatinine, there was a trend towards a significant difference with higher levels in patients with higher BMIs. Figure 1 depicts the frequency distribution of BMI in the incident PD patients, with most patients being normal or overweight. Figure 2 shows an unadjusted Kaplan-Meier plot for incident patients according to BMI, with significant differences in the mortality curves among the different BMI groups (log rank x2, p,0.0001). The majority (34%) of patients died due to cardio-vascular causes in all groups. The most common cause of dropout was not related to the PD technique in all groups (67.5%). Figure 3A (univariate and multivariate analysis; conventional Cox method), Figure 3B (univariate and multivariate analysis; competing Cox method), and Figure 4 (Spline curve) show the Cox proportional hazard for the incident patients, according to BMI. In a conventional Cox multivariate analysis (Figure 3A), both BMI 25-29.9 kg/m2 (HR = 0.74, CI: 0.56 to 0.98, p,0.05) and BMI.30 kg/m2 (HR = 0.63, CI: 0.42 to 0.95, p,0.05) reflected protection from mortality risk, whereas BMI,18.5 kg/m2 reflected a higher mortality risk (HR = 1.72, CI: 1.15 to 2.55, p,0.001). Figure 3B indicates that in the multivariate Cox analysis with competing risks, BMI,18.5 kg/m2 (HR = 1.46 (0.98-2.1), p = 0.05) and BMI 25-29.9 kg/m2 (HR = 0.78 (0.591.04), p = 0.08) were not significant, whereas BMI.30 kg/m2

& RESULTS This study included 1,911 incident patients treated by either Automated PD (APD) or Continuous Ambulatory Peritoneal Dialysis (CAPD), who started PD between December 2004 and October 2007 and were followed up until October 2007. According to the WHO classification, the BMI distribution of the cohort was as follows: underweight 8%; normal (51%); overweight (29%) and obese (12%). The mean age of the patients was 59¡16 years old, 54% were female and 62% were Caucasian. The most common chronic kidney disease etiology was diabetic nephropathy (38%). A Davies score greater than 2 was present in 15% of patients, and the most frequent comorbidities were hypertension (76%) and diabetes (39%). CAPD was the PD modality for 51% of the patients. The majority of patients (68%) starting PD were transferred from HD, and the median time on HD before switching to PD was 7.3 (range, 1.3-60.2) months. Patients were mainly referred to nephrologists from internists (31%), and 18% of patients were referred from an emergency unit. In this cohort, 58% of patients had not received pre-dialysis care, 67% were illiterate or had ,4 years of schooling and 79% had a family income less than 5 times the national minimum wage (, USD$ 7.71/person/day) per month. Only glucose-based PD solutions were prescribed for all patients (Dianeal, Baxter Healthcare), and HomechoiceTM (Baxter, Healthcare) was the cycler used for APD. There were no significant differences in age, gender, socioeconomic status, and race distribution when comparing the 1,911 patients in this study and the 1,528 patients (867 for not completing 90 days of therapy and 661 with more than 90 days of therapy but lacking either weight or height data) excluded from the study. Moreover, there were no significant differences (p = 0.08) among the four BMI groups when analyzing the 867 patients excluded from the study for not having completed 90 days of therapy. In this group of excluded patients, 11.3% were underweight, 48.5%

Table 1 - Characteristics of incident patients with PD by underweight (,18.5 kg/m2), normal (18.5 to 24.9 kg/m2), overweight (25 to 30 kg/m2), and obese (.30 kg/m2).

Sex female (%) Age (years) Systolic BP (mm Hg) Diastolic BP (mm Hg) S-albumin (g/L) S-creatinine (mg/dL) S-urea (mg/dL) Hemoglobin (g/L) Phosphate (mg/dL) Calcium (mg/dL) Potassium (mEq/L) Glucose (mg/dL) Triglycerides (mg/dL) Tot cholesterol (mg/dL) Davies score: 0 1-2 .2

Under-weight N = 159

Normal N = 985

Over-weight N = 547

Obese N = 220

p-value

68% 55¡21 132¡23.5 81¡14 3.7¡1.4 6.8¡3.1 109.6¡47 10.4¡2.3 4.9¡1.9 8.5¡2.2 4.4¡1.1 106¡67 143¡86 190.8¡55

55% 57¡17 138.9¡24 83.2¡14.2 4.2¡1.2 7.5¡4.3 116.3¡48 10.4¡2.9 5.5¡1.8 8.4¡3.5 4.6¡1 115¡76 172¡101 190¡63

46% 60¡13 141.7¡25.5 85¡14 4.3¡2.4 7.7¡4.6 117¡48 10.8¡2.6 5.1¡1.8 8.3¡2.4 4.7¡2.4 122¡78 209¡166 184¡61

64% 59¡12 144.8¡26.6 85¡14 4.2¡1.2 8.2¡4.8 115.7¡44 10.5¡2.1 5.4¡3.3 8.38¡2.4 4.5¡0.9 127¡75 223.2¡97 199.9¡62

0.001 0.001 0.001 0.003 NS 0.07 NS 0.02 NS NS NS 0.001 0.007 NS 0.03

27% 62% 11%

22% 64% 14%

16% 65% 18%

16% 67% 16%

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Figure 1 - Histogram of BMI (kg/m2) for all PD patients at baseline.

reflected protection from mortality risk (HR = 0.67 (0.45-0.99), p = 0.04). Figure 4 shows the Spline Cox proportional hazard based on baseline BMI; the model is adjusted for age and gender. The curve depicts the relationship between log Hazard Ratios and BMI levels in incident PD patients, demonstrating that low BMI is associated with higher mortality and that high BMI is associated with lower mortality. Table 2A depicts the Cox proportional hazard for the evolution of body weight during the first year of PD in incident patients. The patients were divided into five categories according to the quintile distribution: ,23.1%, 23.1 to +0.12%, +0.12 to ,+3.1% (reference category), +3.1 to +7.1% and .+7.1%. There was significantly higher mortality (HR = 2.10, p,0.001) in the lowest quintile (-3.1%), whereas no impact was observed in the 3.1 to +0.12% quintile when compared to the reference category. In patients who gained +3.1 to +7.1% or .+7.1%, no impact was observed when

compared to the reference weight. The patients who gained weight (.+7.1%) had an HR = 0.81 with a narrow CI 0.541.24 in relation to the reference weight. In a multivariate Cox proportional hazard analysis (Table 2B), the lowest quintile (-3.1%) was associated with higher mortality (HR = 1.94, p,0.0001) when compared to the reference weight, whereas no impact was observed in the other quintiles. There were no differences in the frequencies of categories of BW evolution during the first year of therapy among the baseline BMI groups in 1,738 incident PD patients. BW gain during the first year was similarly distributed among the four groups (underweight, 42%; normal, 42%; overweight, 38%; and obese, 37%). Thirty-four percent of patients were more than 65 years of age. The younger and older (.65 years of age) patients presented similar underweight (7.8% vs. 8.3%), overweight (27.5% vs. 31.4%), and obesity (12% vs. 11.3%) distributions (p = 0.31).

Figure 2 - Unadjusted Kaplan-Meier survival curves for incident PD patients and all-cause mortality, according to BMI levels.

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Figure 3 - A) Univariate and multivariate Cox regressions for predictors of all-cause mortality in incident patients. B) Univariate and multivariate Cox regressions for predictors of all-cause mortality in incident patients with competing risk analysis. Hazard Ratios (HR) for all-cause mortality [and 95% confidence intervals (CI)] for patients with BMI ,18.5 kg/m2, BMI 25 to 29.9 kg/m2 and BMI .30 kg/m2. Patients with BMIs ranging from 18.5 to 24.9 kg/m2 were considered the reference. p-value, significance level ,0.05. The models are adjusted for potential confounders, i.e., baseline comorbidities (cardiovascular disease (CVD) and diabetes mellitus (DM)), automated peritoneal dialysis or continuous ambulatory peritoneal dialysis, renal replacement therapy, and calendar year (CY) (2005, 2006 and 2007).

In the last 20 years, the number of people with obesity has increased dramatically in Brazil (www.ibge.gov.br, accessed in November 2007) (18). The BRAZPD is the first large observational PD study performed in this country, and the percentage of PD patients with obesity is similar to that of the general population (18). In the general population, obesity is considered unhealthy; obesity is associated with a higher risk of cardiovascular disease (20), diabetes (21), cancer (22), kidney disease (23,24), and other comorbidities, and it correlates with mortality. It is important to note that while obesity has been shown to be a

& DISCUSSION In this large observational study of incident PD patients performed in Brazil, we demonstrated that a higher BMI (overweight and obese) is not associated with higher mortality when compared to normal (neutral risk) or underweight (higher risk of death). In a multivariate Cox proportional hazard analysis, the evolution of BW in the lowest quintile was associated with a higher mortality when compared to the reference, whereas no negative impact was observed in the other quintiles.

Figure 4 - In the left Y-axis, restricted spline curve showing the age, gender and comorbidities-adjusted Hazard ratios and 95% confidence intervals (CI) (dashed lines) for all-cause mortality associated with BMI in 1,911 incident PD patients. The model is plotted as restricted cubic splines with four knots. Log HR, Log transformed Hazard ratio; 95% CI, lower and upper 95% confidence intervals, respectively. P for linearity = 0.01.

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Table 2 - Univariate (A) and multivariate (B) Cox regressions for predictors of all causes of mortality in according to the evolution of body weight during the first year of PD treatment. A- Univariate analysis – all causes of mortality. Variable

Hazard Ratio

95% CI

,23.1%, vs 0.12 to ,3.1% 2.10 1.47-3.02 23.1 to ,0.12% vs 0.12 to ,3.1% 1.18 0.81-1.77 +3.1 to ,+7.1% vs 0.12 to ,3.1% 1.08 0.73- 1.61 .+7.1%. vs 0 to 0.12 to ,3.1% 0.85 0.56-1.29 B- Multivariate -all cause of mortality adjusted for age, gender, CVD, DM, APD/CAPD, RRT and calendar year ,23.1%, vs 0.12 to ,3.1% 1.94 1.35 - 2.8 23.1 to ,0.12% vs 0.12 to ,3.1% 1.29 0.86 - 1.95 +3.1 to ,+7.1% vs 0.12 to ,3.1% 1.10 0.74 - 1.63 .+7.1% vs 0 to 0.12 to ,3.1% 0.81 0.54 - 1.24

x2

p-value

4.06 0.84 0.41 0.74

,0.001 0.41 0.68 0.46

3.59 1.24 0.47 0.94

,0.001 0.21 0.63 0.34

Indicated are Hazard Ratios (HRs) for all-cause mortality [and 95% confidence intervals (CI)] for patients with weight evolution ,23.1%, 23.1 to ,0.12%, +3.1 to ,+7.1% and .+7.1%. The patients with weight evolution between 0.12 to ,3.1% were considered as reference. A p-value with significance level ,0.05. The models adjust for potential confounders i.e. baseline comorbidities (cardiovascular disease (CVD), and diabetes mellitus (DM)), automated peritoneal dialysis or continuous ambulatory peritoneal dialysis, renal replacement therapy, calendar year (CY) (2005, 2006, 2007).

a low BMI had a two-fold increased mortality risk. Our results also demonstrate higher mortality in patients either presenting lower BMI (,18.5 kg/m2) or a longitudinal weight loss of .-3.1 kg during the first year of therapy. A recent study involving the NECOSAD database examined the differences between the impact of BMI on mortality in HD and PD patients, stratified by age (.65 years and ,65 years). They found that in patients younger than 65 years of age, obesity was associated with higher mortality (37). This study presents relevant differences in relation to our study: first, the percentage of older patients was higher (47.3%) than in our study (34%); second, BMI was evaluated only at baseline, and there was no information about the variation of the BMI over time. Pellicano et al. (16), in a longitudinal study evaluating BMI, stratified the patients by age and did not observe differences between age groups. In our study, the prevalences of malnutrition and overweight/obesity in patients younger and older than 65 years were the same; like the majority of the studies, we adjusted for age, and no impact was observed. In this study, mortality curves adjusted for age, gender, and Davies score indicated that obesity does not increase mortality for incident PD patients (HR = 0.67, CI: 0.47 to 0.95, p = 0.02). Pellicano et al. have hypothesized that a higher BMI is protective because of increased energy storage as total body fat, which could lead to relative preservation of lean body mass (16). In their study, obese patients experienced greater preservation of total body protein when compared with normal and overweight patients. Recently, Kalantar-Zadeh et al. (7) raised the question, ‘‘what is better: fat or muscle?’’, and concluded that body fat is a protective factor for patients on hemodialysis. In our study, BW gain during the first year was similarly distributed among the four groups. However, a BW loss .3.1% (first quintile) during the first year of treatment was associated with significantly increased mortality when compared to the reference, whereas no impact was observed in the other quintiles. Our findings are in agreement with the study by Kalantar-Zadeh et al. (32), which examined a cohort of 54,535 MHD patients in the United States over two years. The authors found that for each BW loss category below -1%, there was a significant increase in patient death. Obesity, including morbid obesity, was associated with better survival and reduced cardiovascular death, even after accounting for changes in BMI and laboratory values. Over

risk factor for mortality in the general population with more than 10 years of follow-up (25), CVD mortality in the dialysis patient population, even after stratification, is 10 to 20 times higher than in the general population (26). Consequently, the follow-up time in studies evaluating dialysis patients and the general population should be different (27). However, obesity may be protective against mortality under several conditions, including in elderly and HD patients and in cases of chronic obstructive pulmonary disease, acquired immunodeficiency syndrome and rheumatoid arthritis (3,28). Obesity has also been reported to have a protective effect for patients with congestive heart failure and geriatric patients, i.e., populations with extraordinarily high mortality rates (28). Exploring the causes and consequences of this epidemiological observation for obesity in PD patients may enhance our insights into similar paradoxes observed for other conventional risk factors, such as blood pressure, serum cholesterol and homocysteine (23,29-33). It is important to note that in this study adverse clinical characteristics are readily observed in the epidemiological profile of the overweight and obese PD patients, i.e., higher age, increased hypertension, diabetes and higher Davies comorbidity scores. However, in this study, obesity is still not associated with higher mortality risk. Similar results have been observed by Chazot et al. (3) in a cohort of 5,592 HD patients; despite increased comorbidities, overweight and obese HD patients carried a significantly lower mortality risk than those HD patients with normal and lower BMI ranges. Agarwal et al. (34) evaluated the relationship between blood pressure (BP) control and BMI in HD patients. They observed that a lower BMI was associated with worse BP control and higher mortality during patients’ first two years on dialysis, justifying this higher mortality to the patients with greater severity. However, no time-dependent analysis of BP, body composition or inflammation was evaluated (35). These findings that higher mortality is associated with lower BMI are in agreement with our results. However, in our study, the overweight/obese patients did not have higher mortality, even though they presented with higher Davies comorbidity scores than the lower BMI group. De Mutsert et al. (36) evaluated incident PD patients selected from the NECOSAD study and followed them for five years; they found that obese incident PD patients did not have worse survival compared with normal BMI incident PD patients. In the same study, PD patients with

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that positive BW variation during the first year of therapy does not have a significant impact on mortality, allowing us to suggest that both overweight/obesity and BW gain during the first year of therapy do not increase mortality in incident PD patients in Brazil. The following centers participated in the Brazilian Peritoneal Dialysis multicentric study and contributed to the preparation of this paper: Ameneg, Associac¸a˜o Hospital Bauru, Biocor Hospital Doenc¸as Cardiolo´gicas, Casa de Sau´de e Mat. Nossa Sra. Perpe´tuo do Socorro, CDR Curitiba, CDR Goiania, CDR Imperatriz, CDR Sa˜o Jose´ Pinhais, CDTR - Centro Dia´lise Transplante Renal, Centro Nefrologia Tereso´polis, Centro Nefrolo´gico Minas Gerais, Centro Tratamento Doenc¸as Renais Joinville, Centro Tratamento Renal Zona Sul, Clinef Rio de Janeiro, Clinepa Clı´nica de Nefrologia Da Paraiba, Clines, Clinese, Clı´nica do Rim do Carpina, Clı´nica Evange´lico S/C Ltda., Clı´nica Nefrologia de Franca, Clı´nica Nefrologia Santa Rita, Clı´nica Nefrolo´gica Sa˜o Gonc¸alo, Clı´nica Paulista Nefrologia, Clı´nica Renal Manaus, Clı´nica Senhor do Bonfim, Clı´nica Senhor do Bonfim Ltda. Filial, Clı´nica Tratamento Renal, Cuiaba - Cenec, Clire Clı´nica Doenc¸as Renais, FAMESP Botucatu, Unicamp - Universidade Estadual de Campinas, Hospital Clı´nicas FMRPUSP, Fundac¸a˜o Civil Casa Mis Franca, Fundac¸a˜o Instituto Mineiro Est. Pesq. Nefrol, Gamen Rio de Janeiro, GDF Hospital de Base, Histocom Sociedade Civil Ltda, Hospital Universidade Prof. Edgard Santos, Hospital Beneficencia Portuguesa Pernambuco, Hospital Cidade Passo Fundo, Hospital Clı´nica Universidade Federal Goia´s, Hospital e Maternidade Angelina Caron, Hospital Evange´lico Vila Velha ES, Hospital Geral Bonsucesso, Hospital Geral de Goiania, Hospital Infantil Joana de Gusma˜o, Hospital Sa˜o Joa˜o Deus, Hospital Sa˜o Jorge, Hospital Sa˜o Jose do Avai, Hospital Sa˜o Vicente de Paula - Joa˜o Pessoa, Hospital Sa˜o Vicente De Paulo, Hospital Servidor do Estado Ipase, Hospital Universidade Presidente Dutra MA, Hospital Universita´rio Antoˆnio Pedro, Hospital Vita Volta Redonda S/A, IAMSPE Sa˜o Paulo, IMIP, Instituto Capixaba Doenc¸as Renais, Instituto Capixaba Doenc¸as Renais Cariacica, Instituto Capixaba Doenc¸as Renais Serra, Instituto do Rim de Fortaleza, Instituto do Rim de Marilia, Instituto do Rim do Parana S/C Ltda., Instituto do Rim Santo Antoˆnio da Platina, Instituto Hemodia´lise de Sorocaba, Instituto Medicina Nuclear Endocrina, Instituto Nefrologia de Mogi das Cruzes, Instituto Nefrologia de Suzano, Instituto Nefrologia Souza e Costa, Instituto Urologia e Nefrol Barra Mansa, Instituto Urologia e Nefrol Sa˜o Jose´ do Rio Preto, MEDSERVSP, Nefrocentro, Nefroclı´nica Caxias do Sul, Nefroclı´nica Foz do Iguac¸u, Nefroclı´nica Uberlandia, Nefron Clı´nica Natal, Nefron Contagem, Nephron Pelotas, Nephron Sa˜o Paulo, Nu´cleo Nefrologia Belo Horizonte, Pro Nephron, Prorim Campos dos Goitacazes, PUC Porto Alegre, Renalcare Servic¸os Me´dicos Ltda, Renalcor Angra dos Reis, Renalcor Rio de Janeiro, Renalvida, Rien Rio de Janeiro, Santa Casa de Adamantina, Santa Casa de Jau´ - UNEFRO, Santa Casa de Marı´lia, Santa Casa de Ourinhos, Santa Casa de Santo Amaro, Santa Casa de Sa˜o Jose´ dos Campos, Santa Casa de Votuporanga, Servic¸o de Nefrologia de Ribeira˜o Preto, UERJ Hospital das Clı´nicas da Universidade Estadual do Rio de Janeiro, Uni Rim Joa˜o Pessoa, Unidade Nefrologia Assis, Unirim Unidade de Doenc¸as Renais, UNIRIM Unidade Renal do Porta˜o, UNTR Unidade Nefrologia Transplante.

time, progressively worsening weight loss was associated with poor survival, whereas weight gain showed a tendency toward decreased cardiovascular death. Chazot et al. (3) have also demonstrated that BW variation during the first year of HD treatment is associated with patient survival, reinforcing the importance of nutrition in this setting. Our results, based on a large cohort of incident PD patients, corroborate the findings reported by Chazot et al. (3) and give support to the recently published results by Pellicano et al. (16) with both incident PD and HD patients. Our incident PD patients who gained weight did not present increased mortality when compared to the reference. However, a BW loss .3.1% (first quintile) during the first year of treatment was associated with significantly increased mortality, whereas the second quintile (23.1 to 0.12%) had no significant reduction in survival. As a matter of a fact, there are more review articles than original papers on the BMI and mortality theme, especially in the PD field. Moreover, these articles report on populations from North America, Europe, Asia and Australia-New Zealand but not on Latin American populations. Brazil was settled by the Portuguese, and the Brazilian ethnicity is primarily mixed. Therefore, some of the positive aspects of this paper are its originality, as it is characterized by Brazilian incident PD patients and a longitudinal approach to body changes. As far as we know, this is the second study in PD patients evaluating changes in BMI over time. Although this is an observational study, the large number of patients and statistically significant findings indicate that a BMI.30 kg/ m2 does not increase the mortality risk for incident patients on PD. Despite the negative selection (only 32% of patients actually chose PD as an RRT modality), as most patients were either transferred from HD or started PD as late referrals and showed a great deal of poor social indicators, the clinical outcomes of the BRAZPD cohort (38) are not different from those reported elsewhere (39,40). However, this observational study does not provide an explanation for the effect of BMI, and future prospective interventional studies may lead to a clarification of the ‘‘obesity paradox’’, adding substantially to the strength of our findings. Our study has some limitations. First, we relied on registry data, and the limitations of this study model are well known. Second, we lack data on residual renal function and D/P creatinine as potential modifiers of outcomes. Third, the fact that BMI does not measure body composition is an inherent limitation. Finally, the short median follow-up time and lack of adjustment for smoking are additional study limitations. It is important to note that studies evaluating body weight longitudinally in dialysis patients have reported body weight changes up to 12 months, as we did in our study. Our total follow-up time was 34 months. It is of utmost importance for us to stress that this study does not suggest that dialysis patients should become obese. Our findings reflect routine dialysis practice where overweight/obese incident PD patients seem to have a protective survival advantage. Therefore, there is a compelling need to design studies to understand the possible mechanisms that lead to a protective effect of higher BMI in dialysis patients. The study designed, performed and published by Pellicano et al. (16) with a small number of patients serves as timely support to our novel finding, as they suggest that energy storage as total body fat is of value in the dialysis population. A novel observation from the results of the large cohort (BRAZPD) with incident PD patients in this study is

& ACKNOWLEDGMENTS We would like to thank all of the dialysis centers for participating in the BRAZPD and the Brazilian Baxter Renal team for their important

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contributions to this project. This study was funded by Baxter Laboratories, Brazil.

20.

& AUTHOR CONTRIBUTIONS Fernandes NS, Bastos MG, Divino-Filho JC wrote the manuscript. Fernandes NS, Chaoubah A, Qureshi AR performed the statistical analysis. Franco MR, Lima MG and the BRAZPD group sent data and reviewed the manuscript.

21.

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BASIC RESEARCH

Evaluation of histological changes after tracheal occlusion at different gestational ages in a fetal rat model Rodrigo Melo Gallindo,I Frances Lilian Lanhellas Gonc¸alves,I Carolina Teixeira de Resende Barreto,II Augusto Frederico Santos Schmidt,III Luis Antonio Violin Dias Pereira,IV Lourenc¸o SbragiaI I

University of Sa˜o Paulo, School of Medicine of Ribeira˜o Preto, Division of Pediatric Surgery, Department of Surgery and Anatomy, Ribeira˜o Preto/SP, Brazil. II State University of Campinas (UNICAMP), School of Medical Sciences, Department of Obstetrics and Gynecology, Campinas/SP, Brazil. III Cincinnati Children’s Hospital Medical Center, Department of Pediatrics, Cincinnati/OH, USA. IV State University of Campinas, UNICAMP, Department of Histology and Embryology, Campinas/SP, Brazil.

OBJECTIVES: To evaluate the histological changes of tracheal cartilage and epithelium caused by tracheal occlusion at different gestational ages in a fetal rat model. METHODS: Rat fetuses were divided into two groups: a) External control, composed of non-operated rats, and b) Interventional group, composed of rats operated upon on gestational day 18.5 (term = 22 days), divided into triads: 1) Tracheal occlusion, 2) Internal control and 3) Sham (manipulated but not operated). Morphological data for body weight, total lung weight and total lung weight/body weight ratio were collected and measured on gestational days 19.5, 20.5 and 21.5. Tracheal samples were histologically processed, and epithelial, chondral and total tracheal thicknesses were measured on each gestational day. RESULTS: The tracheal occlusion group exhibited an increase in total lung weight/body weight ratio (p,0.001). Histologically, this group had a thicker epithelial thickness (p,0.05) and thinner chondral (p,0.05) and total tracheal thicknesses (p,0.001). These differences were more prominent on gestational days 20.5 and 21.5. CONCLUSION: Tracheal occlusion changed tracheal morphology, increased epithelial thickness and considerably decreased total tracheal thickness. These changes in the tracheal wall could explain the development of tracheomegaly, recently reported in some human fetuses subjected to tracheal occlusion. KEYWORDS: Therapeutic Occlusion; Trachea; Hernia; Diaphragmatic; Rats; Experimental Model. Gallindo RM, Gonc¸alves FL, Barreto CT, Schmidt AF, Pereira LA, Sbragia L. Evaluation of histological changes after tracheal occlusion at different gestational ages in a fetal rat model. Clinics. 2013;68(1):59-63. Received for publication on July 23, 2012; First review completed August 10, 2012; Accepted for publication on September 23, 2012 E-mail: sbragia@fmrp.usp.br Tel.: 55 16 3602-2593

Fetal tracheal occlusion (TO) via FETO (Fetoscopic Endoluminal Tracheal Occlusion) aims to promote in utero lung growth and decrease neonatal CDH mortality (3,4). TO prevents the amniotic fluid outflow from the lungs into the amniotic cavity; through mechanical action, it causes accelerated pulmonary growth, alveolarization and alveolar distension, decreasing the deleterious effects of pulmonary hypoplasia and hypertension (5-7). After TO removal, there is a large increase in the amount of intrapulmonary mucus. It is unclear whether this is due to the accumulation of mucus as a result of TO or the increased production of mucus by goblet cells in the tracheal epithelium (6). Yoshizawa et al. (8) concluded that TO stimulates cell cycle progression and type I lung cell differentiation in rat fetuses. However, TO has adverse effects on type II cells and induces a surfactant deficiency, which depends on pulmonary maturity and occlusion duration. It is possible that the effects of TO, both adverse and beneficial, are not only restricted to the lungs but may also extend to the trachea. It is known that the balloon causes slight changes in the

& INTRODUCTION Congenital diaphragmatic hernia (CDH) is an embryological defect of the diaphragm that occurs in approximately 1:2500 live births and accounts for 8% of the major congenital anomalies. It occurs because of a defect in the development of the pleuroperitoneal folds, leading to the passage of abdominal organs into the thorax. The presence of abdominal viscera in the thorax, usually the intestine and liver, occupies space, thus compressing the lung, impairing its normal development, and causing a defect in lung maturation and consequent pulmonary hypoplasia and hypertension (1,2).

Copyright ß 2013 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. DOI: 10.6061/clinics/2013(01)OA09

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trachea, such as local inflammatory changes and limited epithelial defects, such as a reduction in air contact surface (9). However, these changes are not well understood. Thus, using the experimental model of TO, we assessed the histological changes caused by the mechanical effects of TO on the trachea at different gestational ages.

Sample harvesting Each of the four groups was harvested on GD 19.5, 20.5 and 21.5, forming a total of twelve sub-groups. Each subgroup was composed of 12 fetuses, with a total of 144 fetuses. On harvest day, the rats were anesthetized and subjected to Caesarian section. Fetuses were removed from the uterus and weighed. After weighing, the fetuses were sacrificed by occipital puncture, the thorax and abdomen were opened, and the lungs and trachea were carefully removed and weighed. A proper TO was confirmed by observation of distended lungs. Tracheas were dissected under a 2.56 magnification loupe, and for TO fetuses, tracheal samples were cut one or two rings below the clip area. Samples were fixed in formaldehyde 10%.

& METHODS Animals This study was approved by the Ethics Committee on Animal Research of the School of Medicine of Ribeira˜o Preto of the University of Sa˜o Paulo – USP – (#043/2011) and followed the Council for International Organization of Medical Sciences (CIOMS) ethical code for animal experimentation and the principles of the Brazilian College on Animal Experimentation. Sprague-Dawley rats were kept under a controlled dark-night cycle with ad libitum food and water supply. The animals were mated during the night cycle, and a sperm-positive vaginal smear on the following day confirmed mating and designated gestational day (GD) 0 (term = 22 days).

Morphometrical evaluation Body weight (BW) and total lung weight (TLW) allowed for a statistical analysis of these variables in all groups. A TLW/BW ratio was calculated to exclude the variable BW from the TLW evaluation. Twelve fetuses were weighed in each sub-group.

Histological evaluation Samples were cut into 5-mm axial sections, starting below the cricoid cartilage and progressing towards the carina. Sections were stained with H/E for measurements and cell count evaluation. Histological sections were photographed under 406 magnification, and digital images were analyzed using Image Pro Plus 6.0 software for measurement of epithelial thickness (ET), chondral thickness (CT) and total tracheal thickness (TTT) in mm and epithelial cell count (ECC). Each section was divided into four quadrants, using six sections for each trachea. A mean of measurements for the four quadrants was used because of the irregular form of the trachea. The pars membranacea was not studied because it is too thin in rat fetuses. Using six slices per trachea, epithelial cells were manually counted in the whole slice, with each nucleus corresponding to a cell. Six fetuses were studied in each sub-group.

Experimental groups Pregnant rats were divided into two groups: external control (EC, n = 6) and interventional group (IT, n = 15). EC rats were not subjected to any treatment. IT rats were subjected to surgery on GD 18.5, forming triads: internal control (C), sham (S) and TO.

Surgical procedure for tracheal occlusion On GD 18.5, time-pregnant rats were anesthetized, and a median laparotomy was performed under sterile conditions. The uterine horn was exposed, and a purse string suture was placed in the uterine wall near the fetal head. A hysterotomy was performed, and the fetal head and neck were exposed. The neck was incised, and the trachea was isolated and occluded on its upper portion using a titanium microclip (Teleflex Medical Research Triangle Park, NC, USA) (Figure 1). The incision was not sutured. The fetus was then returned to the amniotic cavity, the amniotic fluid was replaced with saline 0.9% and the purse string was tightened. Sham fetuses were subjected to a similar procedure without cervical incision and tracheal occlusion; internal control fetuses were not manipulated. This sequence (TO, C and S) was repeated as many times as possible in each uterine horn. The uterine horn was then returned to the peritoneal cavity, and the abdominal wall was closed in two layers.

Statistical analysis Morphological and histological variables were described as the means ¡ standard deviations (SD) and analyzed using the ANOVA method followed by the Tukey-Kramer post-test in GraphPad Prism 3.02. A difference with a p,0.05 was considered significant.

& RESULTS Morphometrical results All fetuses subjected to surgery on GD 19.5 had a lower initial BW compared with EC fetuses (p,0.001) (Figure 2-A). TLW in TO fetuses progressively increased, differing from the normal curve observed in the EC, C and S groups (Figure 2-B). The TLW/BW ratio indicated the magnitude of this weight gain (Figure 2-C). This increase was so significant that on GD 21.5, the weight loss due to surgical intervention was reversed, and the BW of TO fetuses was similar to EC fetuses (p.0.05). Morphometrical results and differences are shown in Table 1.

Figure 1 - TO procedure in a rat fetus: A) exposition of the cephalic pole after opening the uterine cavity; B) after cervical incision, the titanium clip is affixed on the trachea in a way that occludes the organ without cutting it.

Histological results Despite the lower BW of operated fetuses, an initial increased ET was observed compared with EC fetuses, but

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Fetal rat tracheal changes after TO Gallindo RM et al.

Figure 2 - Morphological results: BW: Body weight (A), TLW: Total lung weight (B) and TLW/BW ratio (C).

the difference was not statistically significant. This difference was reversed in the following harvests, except for TO fetuses, where a thicker epithelium was observed on GD 21.5 compared with C and S fetuses (p,0.05) (Figure 3-A). It was also observed that the tracheal cartilage of the TO group was progressively thinner compared with EC (GD 20.5 and GD 21.5 = p,0.05) and C fetuses (GD 21.5 = p,0.05) (Figure 3-B). In general, it was observed that tracheal cartilage was responsible for 60-70% of TTT in this study. Additionally, similar results were noted in the CT and TTT analysis. TO fetuses had a thinner TTT compared with EC (p,0.001), C (p,0.05) and S fetuses (p,0.05) on GD 21.5 (Figure 3-C). TO led to an increase in ECC after the first post-operative day compared with other groups (p,0.001) (Figure 3-D). Histological sections from all groups are shown in Figure 4. Histological results and differences are shown in Table 2.

for ECC, where an increase was observed in the TO group compared with the other groups (p,0.001). This increase in ET and ECC could explain the increased mucus production reported by Harrison et al. (6). Tracheal cartilage thickness increased in groups EC, C and Sham over time, unlike the TO group, in which the cartilage initially increased after the placement of the clip and decreased significantly at the end of gestation (p,0.001). Thus, it can be inferred that the TTT reduction in the TO group occurs due to a reduction in cartilage and other components of the tracheal ring, such as the lamina propria and the submucosal layer; this reduction is even more important considering that the epithelium was thicker. This could be explained by mechanical stimulation from the accumulation in the airways of the fluid produced by the lung, which acted on the tracheal epithelium, but not over the whole trachea, because there was a reduction in TTT, with a thinner cartilage. Although there is a difference between the TO procedure in rats and humans, which is performed externally using a clip in this experimental model and performed by FETO using an intra-tracheal balloon in humans, our results support the idea that, as suggested by Yoshizawa et al. (8), both pulmonary and tracheal epithelial growth could be aligned. In premature animals, there is a decreased capacity of the cartilage rings and tracheal muscle to withstand pressure, leading to greater tracheal compliance (12,13). As described by Deoras et al., the acute distension of the trachea of extremely premature sheep neonates promotes a major distortion of the tracheal architecture, especially affecting the muscular posterior wall of the trachea (pars membranacea), leading to a decrease in tracheal cartilage thickness (14). Although the TO duration is short, these changes could be explained by Laplace’s law (c = P x r/h), where the constant

& DISCUSSION Temporary TO fosters clinical and experimental growth of hypoplastic lungs in CDH (4,5); the experimental rat model is effective for the study of lung and tracheal development (10). The TLW/BW ratio demonstrated that TO was effective and validated the mechanical action on the trachea, revealing the most significant increase between GD 20.5 and 21.5. TO acted as a barrier to fluid outflow produced by the lungs, leading to expansion and weight gain; similar results were observed by Kitano et al. (11). Except for the TO group, tracheal epithelium thickness was the thickest on GD 19.5, decreased during the course of pregnancy, and was considerably thinner at term. In the TO group, the epithelial thickness decreased between days 19.5 and 20.5 and increased on GD 21.5, which was different than the other groups (p,0.001). Similar results were found

Table 1 - Morphometrical results in the different groups, expressed as the means and SD.

BW (g)

TLW (g)

TLW/BW (g)

GD

EC

C

S

TO

p-value

19.5 20.5 21.5 19.5 20.5 21.5 19.5 20.5 21.5

4.027 (¡0.301) 5.403 (¡0.401) 5.769 (¡0.322) 0.134 (¡0.013) 0.137 (¡0.012) 0.177 (¡0.016) 0.033 (¡0.0020) 0.025 (¡0.0016) 0.031 (¡0.0020)

2.693 (¡0.169) 4.017 (¡0.286) 4.881 (¡0.444) 0.076 (¡0.009) 0.111 (¡0.021) 0.119 (¡0.011) 0.028 (¡0.0039) 0.028 (¡0.0010) 0.024 (¡0.0021)

2.617 (¡0.170) 3.996 (¡0.380) 4.908 (¡0.163) 0.068 (¡0.019) 0.109 (¡0.021) 0.115 (¡0.010) 0.026 (¡0.0100) 0.027 (¡0.0043) 0.023 (¡0.0017)

2.793 (¡0.245) 4.001 (¡0.477) 5.630 (¡0.799) 0.091 (¡0.024) 0.239 (¡0.084) 0.422 (¡0.123) 0.033 (¡0.0100) 0.060 (¡0.0200) 0.075 (¡0.0100)

p,0.001 A,B,C p,0.001 A,B,C p,0.05 E,F p,0.001 A,B p,0.001 A,B,C,F p,0.001 C,E,F p,0.001 C,E,F NS p,0.001 C,E,F p,0.05 A,B p,0.001 C,E,F

GD: Gestational day; EC: External control; C: Control; S: Sham; TO: Tracheal occlusion; BW: Body weight; TLW: Total lung weight; TLW/BW: TLW/BW ratio. NS: Non-significant, A: EC x C; B: EC x S; C: EC x TO; D: C x S; E: C x TO; F: S x TO.

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Figure 3 - Histological results: ET – Epithelial thickness (A), CT – Chondral thickness (B), TTT – Total trachea thickness (C), ECC – Epithelial cell count (D).

pressure of the amniotic fluid would exert a pressure on the tracheal tube, in an attempt to decrease the wall’s thickness. Deprest et al., when performing TO using a balloon or clip in a sheep model, did not observe changes in the tracheal cartilage thickness, which differed from our results.

However, in that study, the impact of balloon placement on the tracheal wall and its recovery after the TO removal were evaluated, emphasizing the inflammatory aspect. In addition, in the evaluation of the area below TO, an epithelial unfolding was observed (9). We have not assessed the

Figure 4 - Photomicrographs of tracheal sections from the four studied groups (EC, C, S and TO) on GD 21.5 showing increased epithelial thickness and decreased chondral thickness in the TO group. Bar = 250 mm.

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Fetal rat tracheal changes after TO Gallindo RM et al.

Table 2 - Histological results in the different groups, expressed as the means and SD.

ET (mm)

CT (mm)

TTT (mm)

ECC (cell number)

GD

EC

C

S

TO

p-value

19.5 20.5 21.5 19.5 20.5 21.5 19.5 20.5 21.5 19.5 20.5 21.5

16.17 (¡2.427) 14.00 (¡1.638) 12.95 (¡1.901) 80.32 (¡13.982) 98.13 (¡6.322) 99.40 (¡11.104) 147.93 (¡32.475) 138.62 (¡12.302) 140.62 (¡10.906) 287 (¡33.187) 291 (¡20.928) 295 (¡13.277)

16.88 (¡2.285) 14.76 (¡1.977) 11.57 (¡1.638) 71.49 (¡11.501) 94.55 (¡8.420) 96.47 (¡13.773) 118.40 (¡16.960) 142.44 (¡7.142) 136.67 (¡15.416) 311 (¡14.980) 314 (¡11.296) 323 (¡24.367)

16.73 (¡2.848) 14.41 (¡1.518) 11.41 (¡1.128) 68.86 (¡7.045) 86.95 (¡13.262) 90.40 (¡9.826) 113.44 (¡9.598) 132.68 (¡14.606) 127.61 (¡14.223) 312 (¡11.165) 318 (¡7.521) 325 (¡9.704)

17.01 (¡1.703) 14.00 (¡1.470) 14.45 (¡1.378) 67.35 (¡11.780) 78.12 (¡13.014) 72.19 (¡12.024) 111.56 (¡10.138) 119.12 (¡13.084) 102.12 (¡12.965) 382 (¡12.215) 398 (¡16.346) 401 (¡48.798)

NS NS p,0.05 E,F NS p,0.05 C p,0.05 C,E p,0.05 B,C p,0.05 E p,0.05 E,F, p,0.001 C p,0.001 C,E,F p,0.05 B, p,0.001 C,E,F p,0.001 C,E,F

GD: Gestational day; EC: External control; C: Control; S: Sham; TO: Tracheal occlusion; ET: Epithelial thickness; CT: Chondral thickness; TTT: Total tracheal thickness; ECC: Epithelial cell count. NS: Non-significant, A: EC x C; B: EC x S; C: EC x TO; D: C x S; E: C x TO; F: S x TO. 4. Deprest J, Jani J, Gratacos E, Vandecruys H, Naulaers G, Delgado J, et al. Fetal intervention for congenital diaphragmatic hernia: the European experience. Semin Perinatol. 2005;29(2):94-103, http://dx.doi.org/10. 1053/j.semperi.2005.04.006. 5. DiFiore JW, Fauza DO, Slavin R, Peters CA, Fackler JC, Wilson JM. Experimental fetal tracheal ligation reverses the structural and physiological effects of pulmonary hypoplasia in congenital diaphragmatic hernia. J Pediatr Surg. 1994;29(2):248-56; discussion 256-7, http://dx.doi. org/10.1016/0022-3468(94)90328-X. 6. Harrison MR, Adzick NS, Flake AW, VanderWall KJ, Bealer JF, Howell LJ, et al. Correction of congenital diaphragmatic hernia in utero VIII: Response of the hypoplastic lung to tracheal occlusion. J Pediatr Surg. 1996;31(10):1339-48, http://dx.doi.org/10.1016/S0022-3468(96)90824-6. 7. Nardo L, Hooper SB, Harding R. Stimulation of lung growth by tracheal obstruction in fetal sheep: relation to luminal pressure and lung liquid volume. Pediatr Res. 1998;43(2):184-90, http://dx.doi.org/10.1203/00006450199802000-00005. 8. Yoshizawa J, Chapin CJ, Sbragia L, Ertsey R, Gutierrez JA, Albanese CT, et al. Tracheal occlusion stimulates cell cycle progression and type I cell differentiation in lungs of fetal rats. Am J Physiol Lung Cell Mol Physiol. 2003;285(2):L344-53. 9. Deprest JA, Evrard VA, Verbeken EK, Perales AJ, Delaere PR, Lerut TE, et al. Tracheal side effects of endoscopic balloon tracheal occlusion in the fetal lamb model. Eur J Obstet Gynecol Reprod Biol. 2000;92(1):119-26, http://dx.doi.org/10.1016/S0301-2115(00)00435-8. 10. Sbragia L, Toelen J, Gijsbers R, Lewi P, Zeger D, Deprest J. Feasibility and efficacy of different tracheal occlusion methods in a fetal rat model. American Journal of Obstetrics & Gynecology. 2006;195(6):S189-S, http://dx.doi.org/10.1016/j.ajog.2006.10.676. 11. Kitano Y, Kanai M, Davies P, von Allmen D, Yang EY, Radu A, et al. BAPS prize-1999: Lung growth induced by prenatal tracheal occlusion and its modifying factors: a study in the rat model of congenital diaphragmatic hernia. Journal of pediatric surgery. 2001;36(2):251-9, http://dx.doi.org/10.1053/jpsu.2001.20683. 12. Bhutani VK, Shaffer TH. Time-dependent tracheal deformation in fetal, neonatal, and adult rabbits. Pediatr Res. 1982;16(10):830-3, http://dx.doi. org/10.1203/00006450-198210000-00006. 13. Penn RB, Wolfson MR, Shaffer TH. Developmental differences in tracheal cartilage mechanics. Pediatr Res. 1989;26(5):429-33, http://dx. doi.org/10.1203/00006450-198911000-00013. 14. Deoras KS, Wolfson MR, Bhutani VK, Shaffer TH. Structural changes in the tracheae of preterm lambs induced by ventilation. Pediatr Res. 1989;26(5):434-7, http://dx.doi.org/10.1203/00006450-198911000-00014. 15. Deprest J, Nicolaides K, Done’ E, Lewi P, Barki G, Largen E, et al. Technical aspects of fetal endoscopic tracheal occlusion for congenital diaphragmatic hernia. J Pediatr Surg. 2011;46(1):22-32, http://dx.doi. org/10.1016/j.jpedsurg.2010.10.008. 16. Ruano R, Yoshisaki CT, da Silva MM, Ceccon MEJ, Grasi MS, Tannuri U, et al. A randomized controlled trial of fetal endoscopic tracheal occlusion versus postnatal management of severe isolated congenital diaphragmatic hernia. Ultrasound Obstet Gynecol. 2012;39(1):20-7, http://dx.doi. org/10.1002/uog.10142. 17. Deprest J, Breysem L, Gratacos E, Nicolaides K, Claus F, Debeer A, et al. Tracheal side effects following fetal endoscopic tracheal occlusion for severe congenital diaphragmatic hernia. Pediatr Radiol. 2010;40(5):670-3, http://dx.doi.org/10.1007/s00247-010-1579-9. 18. Jani J, Valencia C, Cannie M, Vuckovic A, Sellars M, Nicolaides KH. Tracheal diameter at birth in severe congenital diaphragmatic hernia treated by fetal endoscopic tracheal occlusion. Prenat Diagn. 2011;31(7):699-704, http://dx.doi.org/10.1002/pd.2806.

epithelial folding degree, but we noted a thickening of that layer. Apparently, in the rat model, no epithelial folding was observed in either the TO or the other groups. The explanation for this difference may be intrinsic to histological conformations of these animals. Human FETO has increased survival in severe CDH cases (15,16). Recently, tracheomegaly was observed at later follow-up of children undergoing FETO (17,18). Given the limitations of our study that are related to the TO method and the use of a small animal, our results support the idea that after TO, there may be a thinning of the tracheal cartilage, which could explain tracheomegaly. The longterm follow-up of a larger number of patients subjected to FETO will tell us if these changes in tracheal diameter will persist. In addition, we observed epithelial thickening associated with increased cellularity, which could lead to increased mucus production. This difference in the amount of epithelial cells encourages further studies to distinguish which cell types are increased in the tracheal epithelium.

& ACKNOWLEDGMENTS FAPESP – Sa˜o Paulo Research Foundation – Research Grant #11/ 00794-1, Scholarships #08/52772-9 and #11/12587-0 and CAPES (Higher Education Consortia Program - Brazilian Ministry of Education).

& AUTHOR CONTRIBUTIONS Gallindo RM and Gonc¸alves FL contributed to the preparation of the manuscript and experimental surgery procedure. Barreto CT contributed to the animal care and experimental surgery procedure. Schmidt AF contributed to the animal care, statistical analysis and English correction. Pereira LA performed the analysis of histologic samples and prepared the manuscript. Sbragia L contributed to the experimental surgery procedure and prepared the manuscript.

& REFERENCES 1. Tonks A, Wyldes M, Somerset DA, Dent K, Abhyankar A, Bagchi I, et al. Congenital malformations of the diaphragm: findings of the West Midlands Congenital Anomaly Register 1995 to 2000. Prenat Diagn. 2004;24(8):596-604, http://dx.doi.org/10.1002/pd.908. 2. Doyle NM, Lally KP. The CDH Study Group and advances in the clinical care of the patient with congenital diaphragmatic hernia. Semin Perinatol. 2004;28(3):174-84, http://dx.doi.org/10.1053/j.semperi.2004.03.009. 3. Flageole H, Evrard VA, Vandenberghe K, Lerut TE, Deprest JA. Tracheoscopic endotracheal occlusion in the ovine model: technique and pulmonary effects. J Pediatr Surg. 1997;32(9):1328-31, http://dx.doi. org/10.1016/S0022-3468(97)90314-6.

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A new cultural adaptation of the University of Pennsylvania Smell Identification Test Marco Aure´lio Fornazieri, Richard L Doty, Clayson Alan dos Santos, Fa´bio de Rezende Pinna, Thiago Freire Pinto Bezerra, Richard Louis Voegels Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Otolaryngology, Sa˜o Paulo/SP, Brazil.

OBJECTIVES: The University of Pennsylvania Smell Identification Test, a test of olfactory function that is widely used by otolaryngologists, geriatricians, and neurologists, has been translated into more than a dozen languages. In some instances, cultural and socioeconomic factors have necessitated changes in the odorant items or the response alternatives to make the test scores congruent with North American norms. The objective of this study was to compare the performance of Brazilian subjects on a new Portuguese language version of the University of Pennsylvania Smell Identification Test with their performance on an earlier Portuguese language version of the test, as well as to assess the influences of gender, age, ethnicity, and economic status on the test scores. METHODS: Based on pilot data, several response alternatives of the earlier Portuguese language version of the test were altered in an effort to improve test performance. Forty-nine healthy Brazilian volunteers, who represented several economic classes, were tested. The test scores of the study cohort who received the newer version of the test were compared with those of a group of 25 subjects who received the earlier version of the test. RESULTS: The mean score for the new version [35 (2.1)] was significantly (p = 0.002) higher than that for the earlier version [32.5 (3.5)]. Although no apparent influence of socioeconomic status was observed, the female participants outperformed the male participants in the current subject cohort. CONCLUSION: The changes made in the new cultural adaptation of the Portuguese version of the University of Pennsylvania Smell Identification Test were effective in increasing the average test scores of the participants. Overall, the female subjects outperformed the male subjects on the test. KEYWORDS: Diagnostic Tests; Human; Olfactory Nerve (I); Olfaction; Olfaction Disorders; Smell. Fornazieri MA, Doty RL, Santos CA, Bezerra TF, Pinna FR, Voegels RL. A new cultural adaptation of the University of Pennsylvania Smell Identification Test. Clinics. 2013;68(1):65-68. Received for publication on June 25, 2012; First review completed on July 2, 2012; Accepted for publication on September 25, 2012 E-mail: marcofornazieri@gmail.com Tel.: 55 43 3029-1436

identification tests (8,9). In the case of the UPSIT, cultural and socioeconomic factors have necessitated changes in the odorant items or the response alternatives in a number of foreign-language versions to make the test scores more congruent with North American norms (2). For example, researchers in a pilot study in Taiwan made changes to several UPSIT odors and response alternatives, which markedly and effectively increased the test scores, although some items still caused the total test score to be lower than that observed in the U.S. population (10). In Australia, Mackay-Sim et al. (11), without changing any odors, substituted some alternatives and evaluated the performance of Australians on the test. Even with this adaptation, patients with normal smelling ability had average scores lower than those of the U.S. population. Therefore, those investigators suggested adding a correction factor of two points to the test scores if U.S. norms were to be used. The cultural adaptation of the UPSIT to another language or culture is not a simple task. In addition to performing the translation and replacing the odors and the response alternatives that are unfamiliar to the local population, the validation and collection of normative data are required.

& INTRODUCTION Originally published only in English, the University of Pennsylvania Smell Identification Test (UPSIT; commercially known as the Smell Identification TestTM, Sensonics, Inc., Haddon Hts., NJ) (1,2) has now been translated into over a dozen languages, including Portuguese. This widely used test, which is considered by many to be the gold standard to which other tests of olfactory function have been compared, is sensitive to the influences of a wide range of variables, including age (3), gender (4), environmental pollution (5), and numerous diseases (6). Interestingly, the American Academy of Neurology now recommends olfactory testing as an aid in diagnosing Parkinson’s disease (7). It is well established that cultural factors, which are sometimes quite subtle, can influence test scores on odor Copyright ß 2013 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. DOI: 10.6061/clinics/2013(01)OA10

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Moreover, numerous factors must be considered, including the gender, age, health, education, and socioeconomic level of the subjects. Importantly, even within a culture, alterations in the viability of a given odorant item can change over time. For example, synthetic motor oil does not have the same odor as traditional motor oil, suggesting that lack of knowledge of this UPSIT odor may influence some norms based upon younger cohorts. The present study examined the performance of healthy Brazilian subjects on a new Portuguese language version of the UPSIT. Specifically, scores from this new test were compared with those obtained in a pilot study using an earlier Portuguese version of the test (12). We examined whether individuals from different economic strata perform differently on the test. The ultimate goal of our ongoing program, of which this study is a part, is to develop a Brazilian Portuguese UPSIT with scores that can be directly classified on the basis of North American norms (2) and without a correction factor (11).

University of Pennsylvania Smell Identification Test The UPSIT consists of four booklets, each with 10 pages. Microencapsulated ‘‘scratch and sniff’’ odorant strips are positioned on brown strips that are located at the bottom of each page, resulting in a total of 40 odorants (4). The subject releases the odor by scratching the strip with a pencil tip in a standardized manner. He or she then indicates the smell that is perceived by choosing a name from a set of four odor descriptors located just above the odorized strip (1). The number of correctly identified odors serves as the test score. A response is required for each odor even if no smell is perceived (i.e., the test is a forced-choice test). This procedure enables the detection of malingering based on improbable responses and increases the likelihood that a subject will pay close attention to the released odorant. The UPSIT is strongly correlated with odor threshold tests, and the magnitude of these correlations is limited by the reliability of the threshold test that is being evaluated (2,5).

Experimental procedures In the earlier Portuguese test, we had replaced odors unfamiliar to certain people with odors more familiar to Brazilians. For example, the smell of root beer was replaced by the scent of a rubber tire. In the new version, we changed the response alternatives to words better known by the local population or to words that were less likely to be misconstrued as the target stimulus. For example, the correct answer for Item 22, namely, ‘‘popcorn,’’ was replaced by "rubber" because, to most of the subjects, the popcorn odor smelled more like rubber than popcorn. The other substitutions or changes that were made are shown in Table 2.

& MATERIALS AND METHODS Subjects The study population was composed of two groups of Brazilians. The first group, which was administered an earlier Portuguese version of the UPSIT, consisted of 25 volunteers [13 men; 12 women; mean (SD) age, 32.4 (11.5) years] (12). The second group was composed of 49 Brazilian volunteers who represented a broader spectrum of ages, economic classes, and professions than the first group [21 men; 28 women; mean (SD) age, 30.4 (8.9) years]. Individuals were excluded from participation if they had a current upper respiratory infection or any history of head trauma or neurological or psychiatric disease. All the subjects reported that they believed they had normal olfactory function. The volunteers in both groups were divided into three socioeconomic classes: individuals earning a monthly income greater than $3,000 (Class A), individuals earning a monthly income between $1,201 and $3,000 (Class B), and individuals earning $1,200 or less per month (Class C). All the subjects provided informed written consent. The study was reviewed and approved by the institution’s ethics committee under Protocol 0359/09. The two study groups did not differ significantly with respect to the demographic variables of age, gender, smoking behavior, ethnicity, and income (Table 1).

& RESULTS The changes in the response alternatives resulted in a higher overall mean UPSIT test score on the revised version of the test compared with the earlier version (respective means (SD), 35 (2.1) and 32.5 (3.5); t-test p = 0.002). The primary reason for this increased score was improved performance on the items whose response alternatives were changed (Table 2). Chi-square tests revealed that the percentage of correct answers was significantly higher for the test items that were modified, except for Items 32 and 34 (Table 3). To most of the subjects, Item 22 smelled more like rubber than popcorn; thus, providing a response alternative of ‘‘rubber’’ increased the rate of correct identification from 24 to 76%. No significant correlations between the UPSIT scores on the revised version and either the subject’s age or the time spent taking the test were present [respective Pearson r values (pvalues) = 0.06 (0.681) and 0.17 (0.25)], nor where there any differences between the UPSIT scores with respect to social class [respective mean (SD) values for A, B, and C = 34.3 (2.5), 35.5 (1.8), and 34.9 (1.8); p-values.0.35). However, the women outperformed the men (respective male and female means (SDs) = 35.7 (2.1) and 33.9 (1.7); p = 0.003).

Table 1 - Study group demographics. All the p-values reflect uncorrected chi-square p values except for the pvalue for age, which represents the p-value from an independent t-test. Variable

Gender Age (years) Smoking Race Income

Category

Male Female

White Nonwhite Class A Class B Class C

Group 1

Group 2

N (%) 13 (52%) 12 (48%) 32.4¡11.5 1 (4%) 13 (52%) 12 (48%) 6 (24%) 12 (48%) 7 (28%)

N (%) 21 (42.9%) 28 (57.1%) 30.4¡8.9 5 (10.2%) 32 (65.3%) 17 (34.7%) 12 (24.5%) 16 (32.7%) 21 (42.9%)

p-value

0.455 0.573 0.355

& DISCUSSION Compared with the previous version of the test, the revised Portuguese UPSIT described in this paper resulted in test scores that were closer to the North American norms. For the current version, the average test score was 35, which falls within the normal range, unlike the prior version’s

0.267

0.368

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CLINICS 2013;68(1):65-68

New cultural adaptation of the UPSIT Fornazieri MA et al.

Table 2 - Changes in the test alternatives in the earlier and newer UPSIT versions. Item No.

Odor

Alternative

Earlier Version (English)

Earlier Version (Portuguese)

New Version (English)

New Version (Portuguese)

4

Cherry

19

Chocolate (Honey Bread)

21

Flower (Perfume)

22

Rubber (Popcorn)

24

Tire

25

Pickles

32

Grass

34

Pine

37

Soap

A B C D A B C D A B C D A B C D A B C D A B C D A B C D A B C D A B C D

Beer Honey Vanilla Cherry Garlic Chocolate Tire Pepper Flower Spaghetti Coconut Beer Popcorn Soap Dog Spaghetti Tire Watermelon Banana Smoke Pineapple Cucumber Tire Pepper Peppermint Apple Grass Strawberry Wood Smoke Flower Orange Soap Pepper Baby Powder Peanut

Cerveja Mel Baunilha Cereja Alho Chocolate Pneu Pimenta Flor Espaguete Coco Cerveja Pipoca Saba˜o Cachorro Espaguete Pneu Melancia Banana Fumac¸a Abacaxi Pepino Pneu Pimenta Menta Mac¸a˜ Grama Morango Madeira Fumac¸a Flor Laranja Saba˜o Pimenta Talco de Bebeˆ Amendoim

Fish Lemon Garlic Cherry Garlic Honey Bread Tire Pepper Perfume Clove Gasoline Smoke Rubber Pineapple Pizza Mint Tire Watermelon Banana Honey Bread Pineapple Pickles Watermelon Flower Honey Bread Apple Grass Strawberry Wood Baby Powder Bubble Gum Grape Soap Pepper Orange Peanut

Peixe Lima˜o Alho Cereja Alho Pa˜o de Mel Pneu Pimenta Perfume Cravo Gasolina Fumac¸a Borracha Abacaxi Pizza Hortela˜ Pneu Melancia Banana Pa˜o de Mel Abacaxi Picles Melancia Flor Pa˜o de Mel Mac¸a˜ Grama Morango Madeira Talco de Bebeˆ Chiclete Uva Saba˜o Pimenta Laranja Amendoim

average score of 32. Nonetheless, the mean test score was below the average that was expected based on the North American normative data; the mean female score corresponded to the 16th percentile of scores of North American women in the same age group, and the mean male score corresponded to the 27th percentile of scores of men in the same age group (12). Assuming that the cohort of individuals tested in this study truly had normal olfactory function, additional modifications to the test appear to be necessary to increase the test scores to the levels observed in the North American normative data set. Alternatively, given the cultural changes that have occurred since the determination of the

North American norms during the early 1980s, revised norms may be required for the North American test to equate the test scores. Indeed, a revision of the North American UPSIT norms is presently underway. Although the present study demonstrated significant improvements in most of the modified test items (Table 2), certain test items in the modified group could be refined further (e.g., grass and wood). Changes in these and other items that yield a lower percentage of correct responses would most likely adjust the test scores to values closer to the current North American norms. One function of the UPSIT is to provide an olfactory diagnosis by comparing a patient’s scores with the normal score from normative tables, adjusted for gender and age (15). Such age- and gender-adjusted comparisons are necessary because women tend to outperform men and olfactory function declines with age, with a lesser decline in women (3,15). With a culturally adapted UPSIT in which the average scores of the Brazilian population is similar and comparable to the American population, the physician can determine, without calculation, the patient’s percentile rank relative to normal individuals of equivalent age and gender. As in our previous study, no statistically significant differences in UPSIT scores between the various economic classes appeared. In an earlier Portuguese UPSIT study, Silveira-Moriyama et al. (13) observed a poorer performance of subjects who had a lower socioeconomic status and

Table 3 - Comparison between the percentages of correct answers according to group. Item No.

Group 1 (n = 25)

Group 2 (n = 49)

4 19 21 22 24 25 32 34 37

% 60 52 36 24 68 36 76 72 52

% 92 82 90 76 100 76 65 71 88

Chi-Square p-value 0.002* 0.015* ,0.001* ,0.001* ,0.001* 0.002* 0.484 0.856 0.002*

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suggested that this finding may have been due to the subjects’ increased exposure to occupational dusts, their unfamiliarity with the test items, or their levels of education. The difference between this result and the results of the present study could be attributed to several factors. First, we used an improved version of the UPSIT, in which some of the items were presumably less difficult and did not require participants to make subtle distinctions. Second, our study group was significantly younger than the study group used in the study by Silveira-Moryama et al. (mean ages = 32.5 and 54.7 years, respectively). Third, our sample size was smaller than that of Silveira-Moriyama et al.’s study (n = 49 and 88, respectively); thus, it is possible that our statistical power to detect a socioeconomic effect was limited. Supporting this possibility, the regression analysis that was performed in the 1984 UPSIT development study, in which over 1,400 subjects were tested, revealed a significant, albeit weak, effect of education (1). Finally, the two studies’ samples may have differed in other ways, although the subjects of both studies came from the same catchment area of the same hospital. Regardless of the reasons for these differences, it is clear that the present version of the Portuguese UPSIT is greatly improved over the earlier versions and provides test scores that closely approximate the North American norms. The new cultural adaptation of the UPSIT in Portuguese was effective in increasing the average scores of volunteers compared with the average scores derived from an earlier Portuguese version of the test. Women outperformed men on this new version, as was the case in the earlier version. Revisions to the test are ongoing to ensure that Brazilian test scores fall within the UPSIT normative values that are obtained in North America.

& REFERENCES 1. Doty RL, Shaman P, Dann M. Development of the University of Pennsylvania Smell Identification Test: A standardized microencapsulated test of olfactory function. Physiol Behav. 1984;32(3):489-502, http:// dx.doi.org/10.1016/0031-9384(84)90269-5. 2. Doty RL. The Smell Identification TestTM Administration Manual, Sensonics, Inc., Haddon Heights, New Jersey 1995. 3. Doty RL, Shaman P, Applebaum SL, Giberson R, Siksorski L, Rosenberg L. Smell identification ability: changes with age. Science. 1984;226(4681):1441-3, http://dx.doi.org/10.1126/science.6505700. 4. Doty RL, Applebaum SL, Zusho H, Settle RG. Sex differences in odor identification ability: a cross-cultural analysis. Neuropsychologia 1985;23(5):667-72. 5. Calderon-Garciduenas L, Franco-Lira M, Henriquez-Roldan C, Osnaya N, Gonzalez-Maciel A, Reynoso-Robles R, et al. Urban air pollution: influences on olfactory function and pathology in exposed children and young adults. Experimental and toxicologic pathology : official journal of the Gesellschaft fur Toxikologische Pathologie. 2010;62(1):91-102, http:// dx.doi.org/10.1016/j.etp.2009.02.117. 6. Hawkes CH, Doty RL. The Neurology of Olfaction. Cambridge: Cambridge University Press. 2009, 244 pp. 7. Suchowersky O, Reich S, Perlmutter J, Zesiewicz T, Gronseth G, Weiner WJ, et al. Practice Parameter: Diagnosis and prognosis of new onset Parkinson disease (an evidence-based review) Report of the Quality Standards Subcommittee of the American Academy of Neurology. Neurology. 2006;66(7):968-75, http://dx.doi.org/10.1212/01.wnl.000021543 7.80053.d0. 8. Shu CH, Yuan BV, Lin SH, Lin SZ. Cross-Cultural application of the ‘‘Sniffin’ Sticks’’ odor identification test. Am J Rhinol. 2007;21(5):570-3. 9. Doty RL, Marcus A, Lee WW. Development of the 12-item CrossCultural Smell Identification Test. Laryngoscope. 1996;106(3 Pt 1):353-6, http://dx.doi.org/10.1097/00005537-199603000-00021. 10. Jiang RS, Su MC, Liang KL, Shiao JY, Wu SH, Hsin CH. A pilot study of a traditional Chinese version of the University of Pennsylvania Smell Identification Test for application in Taiwan. American Journal of Rhinology and Allergy. 2010;24(1):45-50, http://dx.doi.org/10.2500/ ajra.2010.24.3388. 11. Mackay-Sim A, Doty RL. The University of Pennsylvania Smell Identification Test: Normative adjustment for Australian subjects. Australian Journal of Oto-Laryngology. 2001;4:174-7. 12. Fornazieri MA, Pinna Fde R, Bezerra TF, Antunes MB, Voegels RL. Applicability of the University of Pennsylvania Smell Identification Test (SIT) in Brazilians: pilot study. Brazilian journal of otorhinolaryngology. 2010;76(6):695-9, http://dx.doi.org/10.1590/S1808-86942010000600004. 13. Silveira-Moriyama L, Azevedo AMS, Ranvaud, Barbosa ER, Doty RL, Lees AJ. Applying a new version of the Brazilian-Portuguese UPSIT smell test in Brazil. Arq Neuropsiquiatr. 2010;68(5):700-5, http://dx.doi. org/10.1590/S0004-282X2010000500005. 14. Doty RL. Studies of human olfaction from the University of Pennsylvania Smell and Taste Center. Chem Senses. 1997;22(5):565-86, http://dx.doi. org/10.1093/chemse/22.5.565. 15. Katotomichelakis M, Balatsouras D, Tripsianis G, Tsaroucha A, Homsioglou E, Danielides V. Normative Values of Olfactory Function Testing Using the ‘Sniffin Sticks’. Laryngoscope. 2007;117(1):114-20, http://dx.doi.org/10.1097/01.mlg.0000246518.79894.7e.

Conflicts of Interests: Professor Richard L. Doty is President and a major shareholder of Sensonics, Inc., the manufacturer of the UPSIT.

& ACKNOWLEDGMENTS This work was supported by Programa de Apoio a` Po´s-Graduc¸a˜o of Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES).

& AUTHOR CONTRIBUTIONS Fornazieri MA executed the research, Doty RL co-wrote the manuscript, Santos CA collected data, Bezerra TF and Pinna FR performed data analysis, and Voegels RL coordinated the study.

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CLINICAL SCIENCE

Prevalence of metabolic syndrome and related factors in bank employees according to different defining criteria, Vito´ria/ES, Brazil Luciane Bresciani Salaroli,I Renata Aubin Dias Saliba,I Eliana Zandonade,II Maria del Carmen Bisi Molina,II Nazare´ Souza BissoliI I

Federal University of Espı´rito Santo, Physiological Sciences, Postgraduate Program, Vito´ria/ES, Brazil. II Federal University of Espı´rito Santo, Public Health, Postgraduate Program, Vito´ria/ES, Brazil.

OBJECTIVE: To determine the prevalence of metabolic syndrome and related factors in bank employees in the city of Vitoria/ES, Brazil. METHODS: This was a cross-sectional study that included 521 working men and women $20 years of age. Sociodemographic, lifestyle, anthropometric, biochemical, and hemodynamic characteristics were collected. Metabolic syndrome was diagnosed using the criteria of the National Cholesterol Education Program-ATPIII and the International Diabetes Federation. A logistic regression model was used to calculate the crude and adjusted OR of the variables, and the statistical level of significance was set at 5.0%. RESULTS: We identified 86 (17.2%) and 113 (22.6%) subjects with metabolic syndrome according to the criteria of the National Cholesterol Education Program-ATPIII and the International Diabetes Federation, respectively. The risk of developing metabolic syndrome was higher in individuals with a high school education (OR 2.6 [CI95%, 1.1-6.1]). In overweight and obese subjects, the risks were also higher (OR 12.6 [CI95%, 4.8-33.2, p = 0.000] and OR 43.7% [CI95%, 16.1-118.9, p = 0.000], respectively). CONCLUSION: A large number of bank employees have metabolic syndrome, which can be associated with an increased risk of developing cardiovascular disease. Individuals who had college degrees had a higher prevalence of metabolic syndrome; this finding can be explained by the high rates of overweight and obesity found in subjects with college and graduate school educations. KEYWORDS: Metabolic Syndrome; Employees; Obesity; Insulin Resistance. Salaroli LB, Saliba RA, Zandonade E, Molina MC, Bissoli NS. Prevalence of metabolic syndrome and related factors in bank employees according to different defining criteria, Vito´ria/ES, Brazil. Clinics. 2013;68(1):69-74. Received for publication on June 13, 2012; First review completed on July 18, 2012; Accepted for publication on August 31, 2012 E-mail: luciane.bresciani@ufes.br Tel.: 55 27 3335-7223

and diet, are determinants of cardiovascular diseases (5), little attention has been paid to the risk factors inherent in work activities. The substantial increase of acute and chronic circulatory system disorders in the population warrants further research on these diseases. The worldwide prevalence of MS varies from 13.6% to 46% (6-9), depending on the diagnostic criteria being applied and the population being evaluated. MS research in workers has been rare in Brazil (10,11); to date, MS studies, specifically of bank employees in Brazil, have not been found in the literature. Cavagione et al. (2008) (10) studied 258 men who were professional long-haul drivers and found an MS prevalence of 24%, according to the NCEP-ATP III criteria. A study of administrative officials from the petroleum industry used the NCEP-ATP III criteria and found the MS prevalence to be 15% and determined that sex, age, and smoking were associated with the presence of MS in the study population (11). In this context, occupation might correlate with MS development (12). This study aimed to determine the prevalence of MS in bank

& INTRODUCTION Metabolic syndrome (MS) is a growing concern worldwide because it is associated with cardiovascular co-morbidities (1). MS is characterized by the presence of dyslipidemia, glucose intolerance, hypertension, overweight, abdominal obesity, and other abnormalities; the association of MS with type 2 diabetes mellitus and cardiovascular disease (CVD) is also important (2). According to multiple researchers, insulin resistance appears to link the alterations present in MS (3,4) and increased abdominal adiposity. Although individual lifestyle factors, such as a lack of regular exercise, smoking,

Copyright ß 2013 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. DOI: 10.6061/clinics/2013(01)OA11

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employees and identify the risk factors associated with developing MS in a large banking network in Vitoria, ES, Brazil.

laboratory using commercial kits (Roche Diagnostics Ltd., with the COBAS e601 analyzer, Roche, Rotkreuz, Switzerland) to measure the levels of glucose, total cholesterol, HDL cholesterol, VLDL cholesterol, triglycerides, uric acid, and ultra-sensitive CRP. The LDL cholesterol level was calculated using the Friedewald formula (18). The insulin dosage was determined using a commercial kit from Siemens with the IMMULITEH 2000 analyzer (Siemens Healthcare Diagnostics Inc., NY, USA). Five parameters were used to characterize MS: waist circumference, blood pressure, serum concentrations of fasting glucose, TG, and HDL-C. Moreover, the parameters were characterized according to two different sets of criteria. The first set of criteria, initially proposed by the NCEP-ATPIII (2001) (19), described the diagnosis of MS in the presence of at least three of the following criteria: waist circumference .88 cm for females or .102 cm for males; HDL-C ,50 mg/dL for females or ,40 mg/dL for males; TG.150 mg/dL; blood pressure values .130/85 mm Hg; and fasting glucose .110 mg/dL. The second set of criteria, proposed by the IDF (20), reduced the threshold values for fasting glucose (.100 mg/dL) and waist circumference (.80 cm for women and .90 cm for men); however, these parameters were established by South Asians and not for individuals from South or Central America. Statistical analyses were conducted using SPSS for Windows, version 15.0 (SPSS Inc., Chicago, USA). The mean values were compared using Student’s T-test for independent samples, and when the normality was not verified by the Kolmogorov-Smirnov test, we used the nonparametric Mann-Whitney U-test. To analyze the differences in proportions, we used the chi-squared test (X2). The level of significance for all tests was set at a = 0.05. After a bivariate analysis, a logistic regression analysis was performed using a dependent variable in the presence of MS according to the NCEP-ATP III criteria. Crude and adjusted OR were calculated from the variables that were entered into the logistic regression model. All variables with p,0.20 in the bivariate analysis were inserted into the regression model. In the final model, only those variables that retained p,0.05 were determined to be significant.

& METHODS We conducted an observational, cross-sectional study of employees, aged 20 to 64 years, from a state-owned banking network located in southeastern Brazil. Data were collected from August 2008 to August 2009. The study was approved by the Research Ethics Committee (no. 059/08) of the Center for Health Sciences, Federal University of Espirito Santo. The sample size was calculated to estimate the prevalence of MS in a population of 1,410 bank employees. We used simple random sampling for a prevalence of 20%, a 3% level of error and a 95% significance level. The quotas were calculated based on occupation (general direction and agency), sex, and age. Thus, the minimum sample size was 461 bank employees. Because of a possible low response rate, 525 bank employees were invited to participate. The data were collected at the workplace, and the employees were relieved of their duties during the data collection. Socioeconomic status was determined according to the Brazilian Economic Classification (13). Ethnicity was self-identified as black, brown, white, yellow, or indigenous, according to the IBGE (14). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) measurements were performed during the interviews, three times in each individual; the first SBP and DBP measurements were discarded. The digital OMRON 742H (OMRON Healthcare Inc, China) calibrated and validated by Inmetro, was used for blood pressure measurements. Prior to the measurements and after having been asked to empty their bladders, the participants remained seated and at rest for approximately five minutes. They refrained from consuming food, alcohol, coffee, or cigarettes during the previous 30 minutes. For the data analysis, the mean value of the two measurements was considered. Blood pressure was measured again if the difference between the readings was greater than 4 mm Hg. The blood pressure levels were measured according to the VII Joint National Committee criteria (15). Clamps suitable for obese patients were used when needed. All of the anthropometric measurements were performed by trained researchers. Body weight was obtained using a TANITAH electronic scale, which was accurate to within 0.1 kg, and the individuals were asked to be weighed with empty bladders and wearing only underwear. Height was measured in meters using a SannyH stadiometer, which was accurate to 0.1 cm, while the subjects were barefoot with their arms along their bodies and their eyes fixed on a spot on the horizon. The body mass indices were calculated (BMI = weight/height2) as recommended by the World Health Organization (16) to assess nutritional status. The following cutoff points were used to classify individuals according to BMI (kg/m2): underweight BMI ,18; normal $18.5 and ,25; pre-obese $25 and ,30; and obese $30. The types of obesity, including grades I to III, were pooled in the analysis to ensure that the groups would be more representative. Waist and hip measurements were obtained using a metal tape. To measure the waist circumference (WC), the tape was positioned at the natural waist or at the lower curvature located between the last rib and the iliac crest (17). Biochemical tests were performed in the reference

& RESULTS Of the 525 individuals who were invited to participate in the study, 521 attended the interviews; however, 20 did not submit to the biochemical tests and were excluded from the analysis. Therefore, the dataset included 501 subjects, 255 (50.9%) men and 246 (40.1%) women; the representative percentages were similar to the overall study population. Table 1 shows the prevalence of MS by sex, age, occupation, social status, education, race/ethnicity, and marital status, based on the NCEP-III and IDF criteria. We identified 86 (17.2% CI95%, 13.8-20.6) and 113 (22.6% CI95%, 18.8-26.3) individuals with MS based on the criteria from the NCEP and IDF, respectively. MS prevalence varied by sex according to the IDF criteria (males 27.8% vs. females 17.1%, p = 0.004). An increase in age correlated with an increase in MS using both sets of criteria (NCEP and IDF, p = 0.004 and p = 0.001, respectively). Individuals who worked in agencies or in customer service in banks showed a higher prevalence of MS compared with the general rate (p = 0.034), according to the NCEP criteria. No significant differences were observed in race or socioeconomic status, although

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Metabolic Syndrome in Bank Employees Salaroli LB et al.

Table 1 - Prevalence of MS according to the NCEP-III and IDF criteria by gender, sex, age, position, social status, education level, race/ethnicity and marital status of the study population. Variable Sex Total Age

Position Social status

Education*

Ethnicity

Marital status**

Category

Total (N = 501)

NCEP N (%)

p-value

IDF N (%)

p-value

Male Female

255 246

0.140

90 41 270 100 279 222 10 80 189 204 18 6 123 241 121 26 168 292 10 5 120 325 55

71(27.8) 42(17.1) 113(22.6) 8(8.9) 5(12.2) 68(25.2) 32(32.7) 56(20.1) 57(26.0) 1(11.1) 20(25.0) 44(23.7) 46(22.5) 2(11.1) 1(0.9) 36(32.5) 55(48.2) 21(18.4) 8(30.8) 32(19.0) 73(25.3) 15(12.5) 87(26.8) 11(20)

0.004

,30 years 31 to 40 years 41 to 50 years .50 years General direction Agencies A1+A2 B1 B2 C D Junior high High school College Graduate school Black Mixed White Yellow Indian Single Married/Live together Separated/Divorced/Widowed

50(19.6) 36(14.6) 86(17.2) 5(5.6) 6(14.6) 60(18.5) 25(25.0) 39(14.0) 47(21.2) 1(11.1) 16(20.0) 30(15.9) 38(18.6) 1(5.6) 31(36.0) 42(48.8) 13(15.1) 7(26.9) 28(16.7) 51(17.5) 9(10.5) 67(77.9) 10(11.6)

0.004

0.034 0.680

0.016

0.291

0.005

0.001

0.113 0.74

0.156

0.109

0.005

p,0.05, Chi-squared test * N = 491 ** N = 500. MS was identified according to the National Cholesterol Education Adult Treatment Panel Program’s-III (NCEP-ATP III) and International Diabetes Federation (IDF, 2005) criteria. Social Status A to E: based on the scholarship of the family head and items purchased.

respectively, more likely to develop MS compared with normal-weight individuals.

education level affected the distribution of MS according to the NCEP criteria (p = 0.016). Marital status had a significant influence on the prevalence of MS according to both sets of criteria (p = 0.005). The biochemical, anthropometric, and hemodynamic indicators for individuals with and without MS are shown in Table 2. Individuals with and without MS syndrome showed higher mean values of fasting plasma glucose according to both sets of criteria, whereas individuals diagnosed using the IDF criteria showed higher insulin levels. Those subjects with MS according to the NCEP criteria had the highest average total cholesterol as well as lower HDL levels. The CRP levels were significantly elevated in the individuals with MS, according to both sets of criteria. The anthropometric indicators were similar using either criteria; the average WC values were slightly higher in those subjects who were diagnosed with the IDF criteria; and higher levels of hemodynamic indicators were also identified in those subjects diagnosed using the IDF criteria. Figure 1 shows the relative contributions of the five criteria to the diagnosis of MS, according to the NCEPATPIII and the IDF. The parameters we assessed included high blood pressure, triglycerides, and HDL cholesterol, which yielded similar results. High fasting glucose levels and large waist circumferences were more prevalent in those subjects diagnosed using the IDF criteria, whereas large waists showed a significant difference (p = 0.001). Table 3 shows the crude and adjusted OR of the variables that were entered into the logistic regression model. The risk of MS, according to the NCEP ATP III criteria, was 2.6 times higher in the individuals who had only a high school education, compared to those with college degrees. Overweight and obese subjects were 12.6 and 43.7 times,

& DISCUSSION To the best of the authors’ knowledge, this was the first study of MS prevalence of in bank employees. The lack of studies is concerning because MS patients have clinical and laboratory results that strongly indicate other health problems, particularly cardiovascular issues. Additionally, the lack of unanimity of the MS diagnostic criteria should be highlighted because it complicates the comparisons between studies. To narrow this gap, two diagnostic criteria were used in the present investigation; these criteria were established by the NCEP-ATPIII (19) and the IDF (20); the number of cases of MS found using the NCEP-ATPIII and IDF criteria were 86 (17.2% CI95% 13.8-20.6) and 113 (22.6% CI95% 18.8-26.3), respectively. The prevalence of MS in this study was similar to that found by Felipe-de-Melo et al. (15%) (11) in administrative workers from the Brazilian oil industry but lower than that found in an urban population in the Brazilian capital (29.8%) from our previous study (9). Cavagione (2008) (10) assessed 258 long-haul drivers and observed an MS prevalence 24% using the NCEP-ATP III criteria. In other countries, we found diverse results for the prevalence of MS in other occupations, including 23.5% in the German chemical industry (21), 10% in different categories of the Spanish population (22), and 8.2% in Taiwanese high-tech workers (23). A Spanish study found that of ten active workers from an automobile company, only one had MS, and managers had a more favorable profile when compared with other workers (24). These

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CLINICS 2013;68(1):69-74

Table 2 - Biochemical, anthropometric and hemodynamic parameters in the study population, according to the diagnostic criteria and presence of MS. NCEP

Variable No (N = 415)

Glucose (md/dL) Insulin mcIU/mL Total cholesterol (mg/dL) HDL (mg/dL) LDL (mg/dL) VLDL (mg/dL) Triglycerides Hs CRP (mg/L) Mean blood pressure (mm Hg) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Height (m) Weight (kg) BMI (kg/m2) WC (cm)

IDF

Yes (N = 86)

p-value

Mean

SD

Median

Mean

SD

Median

84.4 5.2 190.2 51.1 120.5 21.3 107 2.7 92.9 123.6 78.3 1.7 70.2 24.9 85.8

9.0 3.7 36.1 13.2 55.8 10.8 62.9 5.0 12.9 16.5 10.9 0.1 14.4 4.0 11.9

83 4.1 189 48 117.2 19.2 94 1.4 92 122 77.5 1.7 69.1 24.4 85

106.4 10.8 201.2 38.9 121.3 37.7 216.8 4.4 105.0 139.7 88.1 1.7 87.4 30.7 102.1

44.1 8.6 39.4 7.4 34.3 15.7 136.6 4.7 12.3 17.1 11.0 0.1 14.5 4.6 9.6

92 9.4 198 38 120.8 35.6 180 2.7 103 138 86 1.7 85.8 29.7 103

0.000b 0.000b 0.012a 0.000b 0.364b 0.000b 0.000b 0.000b 0.000a 0.000a 0.000a 0.300a 0.000a 0.000b 0.000a

No (N = 388)

Yes (N = 113)

p-value

Mean

SD

Median

Mean

SD

Median

84.1 5.7 191.5 49.9 120.9 22.5 116.8 2.9 93.6 124.7 79.0 1.7 71.6 24.7 87.0

8.3 4.6 36.1 13.2 54.2 11.7 78.5 5.0 12.9 16.8 11.1 0.1 15.2 4.0 12.5

83 4.0 188 49 117.2 18.4 91.5 1.3 91.2 121 76.5 1.7 68.2 24.3 84

126.8 11.0 197.9 39.6 118.4 39.4 216.8 4.0 107.4 141.9 89.3 1.7 88.6 30.0 103.0

51.1 8.7 44.1 8.9 36.6 18.4 139.9 3.9 13.2 19.1 11.2 0.1 12.7 4.3 8.9

83 7.5 199 39 120.8 33 167.5 2.3 103.5 139.3 86.3 1.7 85.6 29.1 101

0.000b 0.000b 0.048a 0.000b 0.326b 0.000b 0.000b 0.000b 0.000a 0.000a 0.000a 0.000a 0.000a 0.000b 0.000a

The values are given as the means, medians and SDs (standard deviations). N = number of individuals. a - Student’s t-test. b - Mann-Whitney- U-test. p,0.05.

results were similar to those found in the present study, which showed a difference in MS rates between the general officers and agency employees using the NCEP-ATPIII criteria. The prevalence of MS was higher in individuals working in agencies, demonstrating that in bank employees, MS might be associated with the nature of their jobs. Using the IDF criteria, the results indicated that the prevalence of MS varied according to sex: the prevalence was higher in males than females. The IDF criteria for defining central obesity seemed to explain much of this difference: the limits of the NCEP waist circumference were 102 cm for men and 88 cm for women, reflecting different population percentiles compared with the limits set by the IDF criteria, which were 90 cm for males and 80 cm for

females. In particular, men tended to accumulate visceral fat deposits, a condition that increases with age and with increased BMI; this condition is associated with MS (5). Similarly, the rigidity of the cohort, particularly based on the IDF criteria, encompasses most individuals with risk factors for the syndrome. In this study, waist circumference and blood glucose changes appeared more frequently in individuals when the IDF criteria were used as the diagnostic criteria for SM. Similar results were observed by Gu¨ndogan (25) in the Mediterranean region of Turkey. Additionally, the distribution of body fat could have strongly influenced metabolic abnormalities in the lipid profiles and glucose levels (26). Our study showed that higher levels of insulin and triglycerides and lower HDL-C

Figure 1 - Student’s T-test * p = 0.001 (the difference between the criteria considered and high waist circumference).

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Metabolic Syndrome in Bank Employees Salaroli LB et al.

Table 3 - Logistic regression analysis, including the crude and adjusted OR for MS according to the NCEP-diagnosed employees of a state bank in Vito´ria/ES. Variable Age (years)

Education

Marital status

Position BMI

Category ,30 31 to 40 41 to 50 .50 High school College Graduate school Single Married/Living together Separated/ Divorced/Widowed Agency General direction Normal weight Overweight Obesity

p-value

NCEP OR Crude/CI

0.004

1 2.91(0.835-10.176) 3.86(1.490-10.018) 5.66(2.066-15.545) 2.79(1.383-5.664) 1.67(0.859-3.243) 1 1 3.20(1.543-6.650)

0.011

0.005

0.034 0.000

2.74(1.044-7.193) 1.65(1.036-2.693) 1 1 12.29(4.736-31.926) 43.33(16.235-115.681)

NCEP OR Adjusted/CI 1 1.97(0.478-8.129) 1.65(0.531-5.177) 2.52(0.747-8.562) 2.60(1.112-6.113) 2.18(1.019-4.696) 1 1 2.04(0.842-4.971) 1.84(0.564-6.062) 1.39(0.780-2.504) 1 1 12.63(4.802-33.265) 43.69(16.056-118.885)

The odds ratios were adjusted in a logistic regression analysis for other variables in the table, with 95% confidence intervals for the presence of MS according to the NCEP ATP III criteria. OR = odds ratio; CI = confidence interval.

levels were found in individuals with MS. Changes in the lipid metabolism of subjects with insulin resistance (IR) are triggered by excessive levels of circulating fatty acids that are derived from visceral adipose tissue in the liver, which increases the production of triglycerides and lowers HDL-C. These excessive levels of circulating fatty acids also affect insulin sensitivity in muscle tissue by inhibiting glucose uptake. Hyperglycemia and excessive fatty acids can both result in hyperinsulinemia (27). In our study, the high percentage of hypertensive subjects (32.5%) was remarkable. A population study in Vitoria-ES, Brazil, indicated a prevalence of hypertension 38% (28), which was higher than the 25% to 30% estimated prevalence in the adult Brazilian population (29). In the 1990s, a study of state bank employees in Rio de Janeiro, Brazil, estimated hypertension prevalence as 18.3% in the subjects. The subjects were the same ages as those in this study; the prevalence of hypertension was higher in males (22.3%) than females (12.6%) (p,0.001), and the prevalence increased with age (30). Chronic inflammation plasma markers are associated with risk for coronary artery disease; hsCRP has been the most studied marker (31), and in our study, it was higher in the individuals with MS. This result was similar to that of another study of various professional categories (10). Studies conducted in apparently healthy young adults have shown the potential of hsCRP in predicting future coronary events and its role as an important marker for this purpose (32-33), which demonstrates that individuals with MS have additional risk factors for CVD. Social determinants are strongly correlated with MS, but the contribution of these determinants has not been fully elucidated. For example, individuals with higher education levels and greater access to information have greater opportunities in the labor market and, consequently, higher purchasing power; these individuals usually have a lower prevalence of MS (34). However, in our study, the individuals who had college degrees had a higher prevalence of MS compared with those who had only completed high school. This finding can be explained by the high rates of overweight and obesity found in populations with higher education levels. Thus, the increased

adiposity in both low-income individuals and those with higher education levels supports the hypothesis that obesity could be a causative factor for MS in this population. Obesity has appeared to be a risk factor for MS because obese individuals have a 43.3 times higher risk of developing MS compared with normal-weight subjects. The population in this study showed a high prevalence of overweight (36.9%) and obesity (17.9%), which might be related to the physical inactivity that is also prevalent in this group, as well as the sedentary type of work performed by the subjects. A 1994 epidemiological study of bank employees in Rio de Janeiro has already demonstrated the rates of overweight and obesity in this population: 27.8% and 6.4%, respectively (35). Our results showed a large number of workers with MS and high levels of PCR; therefore, these subjects had increased risks of developing cardiovascular disease, although they did not have low education levels. In the studied population, obesity emerged as a risk factor for MS, and it should be the subject of further research. Furthermore, these findings support the need for programs to promote employee health, to facilitate the monitoring of the magnitude and temporal trends of these factors and to assess the actions that are directed toward this population group.

& ACKNOWLEDGMENTS The authors acknowledge the State Bank of Espirito Santo, Brazil, for its financial support in all phases of this study.

& AUTHOR CONTRIBUTIONS All of the authors participated appropriately in the study conception and design, data acquisition, analysis and interpretation, and the final approval of the published version.

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Detection, Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III). JAMA. 2001;285(19):2486-97. International Diabetes Federation (IDF). International Diabetes Federation consensus worldwide definition of the metabolic syndrome. 2008. Available at http://www.idf.org/webdata/docs/IDF_Metasyndrome_ definition.pdf. Accessed January 05, 2011. Oberlinner C, Humpert PM, Nawroth PP, Zober A, Morcos M. Metabolic syndrome in a large chemical company: prevalence in a screened worksite sample. Acta Diabetol. 2008;45(1):31-5, http://dx.doi.org/10. 1007/s00592-007-0015-6. Sanchez-Chaparro MA, Calvo-Bonacho E, Gonzalez-Quintela A, Fernandez-Labandera C, Cabrera M, Sainz JC, et al. Occupation-related differences in the prevalence of metabolic syndrome. Diabetes Care. 2008;31(9):1884-5, http://dx.doi.org/10.2337/dc08-0431. Tsai TY, Cheng JF, Lai YM. Prevalence of metabolic syndrome and related factors in Taiwanese high-tech industry workers. Clinics. 2011;66(9):1531-5, http://dx.doi.org/10.1590/S1807-59322011000900004. Alegrı´a E, Cordero A, Laclaustra M, Grima A, Leo´n M, Casasnovas JA, et al. Prevalence of metabolic syndrome in the Spanish working population: MESYAS Registry. Rev Esp Cardiol. 2005;58(7):797-806, http://dx.doi.org/10.1157/13077231. Gu¨ndogan K, Bayram F, Capak M, Tanriverdi F, Karaman A, Ozturk A, et al. Prevalence of Metabolic Syndrome in the Mediterranean Region of Turkey. Evaluation of Hypertension, Diabetes Mellitus, Obesity, and Dyslipidemis. Metab Syndr Relat Disord. 2009;7(5):427-33, http://dx.doi. org/10.1089/met.2008.0068. Palaniappan L, Carnethon M R, Wang Y, Hanley AJ, Fortmann SP, Haffner SM, et al. Predictors of the incident metabolic syndrome in adults: The Insulin Resistance Atherosclerosis Study. Diabetes Care. 2004;27(3):788-93, http://dx.doi.org/10.2337/diacare.27.3.788. Pajuelo J, Pando R, Leyva M, Herna´ndez K, Infantes R. Resistencia a la insulina en adolescentes con sobrepeso y obesidad. Anais da Faculdade Medicina de Lima. 2006;67(1):23-9. Mill JG, Molina MDC, Silva IO, Marquezine AL, Ferreira AVL, Cunha RS, et al. Epidemiologia da hipertensa˜o arterial na cidade de Vito´ria, Espı´rito Santo. Rev. Hipertensa˜o Arterial. 2004;7(3):109-16. LESSA I. Epidemiologia da hipertensa˜o arterial sisteˆmica e da insuficieˆncia cardı´aca no Brasil. Rev. Brasileira de Hipertensa˜o. 2001; 8(4):383-92. Chor D. Hipertensa˜o Arterial Entre Funciona´rios de Banco Estatal No Rio de Janeiro: Ha´bitos de Vida e Tratamento. Arq Bras Cardio. 1998;71(5):653-60. Yu H, Rifai N. High-sensitivity C-reactive protein and atherosclerosis: from theory to therapy. Clin Biochem. 2000;33(8):601-10, http://dx.doi. org/10.1016/S0009-9120(00)00186-7. Rifai N, Ridker PM. High-sensitivity C-reactive protein: a novel and promising marker of coronary heart disease. Clin Chem. 2001;47(3):40311. Ridker PM, Rifai N, Rose L, Buring JE, Cook NR. Comparison of C-reactive protein and low- density lipoprotein cholesterol levels in the prediction of first cardiovascular events. N Engl J Med. 2002;347(20):1557-65. Nayga RM Jr. A note on schooling and obesity. J Health Soc Policy. 2000;12(2):65-72, http://dx.doi.org/10.1300/J045v12n02_04. Ell E, Camacho LAB, Chor D, Bastos LA. Perfil antropome´trico de funciona´rios de banco estatal no Estado do Rio de Janeiro/Brasil: I ´ındice de massa corporal e fatores so´cio-demogra´ficos. Cad. Sau´de Pu´blica. 1999; 15(1):113-22, http://dx.doi.org/10.1590/S0102-311X1999000100012.


CLINICAL SCIENCE

Comparing the diagnostic values of circulating microRNAs and cardiac troponin T in patients with acute myocardial infarction Ying-Qing Li,I Mei-Fen Zhang,II Hong-Yan Wen,III Chun-Lin Hu,I Rong Liu,I,IV Hong-Yan Wei,I Chen-Mu Ai,II Gang Wang,II Xiao-Xing LiaoI*, Xin LiI* I

Sun Yat-Sen University, The First Affiliated Hospital, Department of Emergency, Guangzhou. II Sun Yat-sen University, School of Nursing, Guangzhou, People’s Republic of China. III NanHua University, The First Affiliated Hospital, Department of Cardiology, Hengyang, People’s Republic of China. IV Guangzhou Medical College, The First Affiliated Hospital, Department of Emergency, Guangzhou, People’s Republic of China.

OBJECTIVE: Recent studies have shown that circulating microRNAs might be useful, novel biomarkers for the diagnosis of acute myocardial infarction. The aims of this study were to evaluate the expression of cardiacspecific miRNAs (miR-1, -133a, -208b, and -499) in patients with acute myocardial infarction and to compare the diagnostic values of these miRNAs with that of cardiac troponin T. METHODS: Sixty-seven plasma samples obtained from patients with acute myocardial infarction and 32 plasma specimens collected from healthy volunteers were analyzed in this study. The levels of cardiac-specific miRNAs (miR-1, -133a, -208b, and -499) were measured by quantitative reverse transcription-polymerase chain reaction, and the concentrations of plasma cardiac troponin T were measured using electrochemiluminescence-based methods and an Elecsys 2010 Immunoassay Analyzer. RESULTS: The levels of plasma miR-1, -133a, -208b, and -499 were significantly higher in acute myocardial infarction patients (all p,0.001) than in healthy volunteers. The expression of the cardiac-specific miRNAs in acute myocardial infarction patients decreased to close to the baseline levels at the time of hospital discharge (all p.0.05). There were no correlations between the levels of the four circulating miRNAs and the clinical characteristics of the study population (all p.0.05). Furthermore, receiver operating characteristic curve analyses showed that the four plasma miRNAs were not superior to cardiac troponin T for the diagnosis of acute myocardial infarction (all p.0.05). CONCLUSION: Our results demonstrate that circulating miR-1, -133a, -208b, and -499 may be useful biomarkers in acute myocardial infarction patients but that these miRNAs are not superior to cardiac troponin T for the diagnosis of acute myocardial infarction. KEYWORDS: microRNAs; cardiac troponin T; Acute Myocardial Infarction; Circulating Biomarkers. Li YQ, Wen HY, Zhang MF, Hu CL, Liu R, Wei HY, et al. Comparing the diagnostic values of circulating microRNAs and cardiac troponin T in patients with acute myocardial infarction. Clinics. 2013;68(1):75-80. Received for publication on August 9, 2012; First review completed August 28, 2012; Accepted for publication September 12, 2012 E-mail: xlidoct@qq.com / liaowens@163.com *Corresponding author Tel.: +86-20-28823350

The circulating levels of cardiac troponins (cTns) are considered the ‘‘gold standard’’ for the early diagnosis of AMI because cTns are both highly specific and sensitive for cardiac injury (3). However, early studies have reported that cTn concentrations are also elevated in patients with endstage renal disease (ESRD) and that these molecules might serve as biomarkers for renal failure (4,5). Therefore, it is essential to explore new biomarkers with greater specificity for AMI. MicroRNAs (miRNAs) are a class of endogenous, noncoding single-stranded small RNAs of 19 to 25 nucleotides in length (6) that can regulate gene expression and play critical roles in various pathophysiological processes (7,8). miRNAs generally suppress gene expression by inhibiting mRNA translation or inducing mRNA degradation. Each miRNA can regulate several distinct target mRNAs, and

& INTRODUCTION Acute myocardial infarction (AMI) is a leading cause of morbidity and mortality worldwide, and approximately three to four million people are estimated to suffer from AMI each year (1). Early reperfusion can reduce the mortality rate of AMI (2); therefore, the timely diagnosis of AMI is critical.

Copyright ß 2013 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. DOI: 10.6061/clinics/2013(01)OA12

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were treated with thrombolysis. Thirty-two age- and sexmatched samples from healthy volunteers (no history of cardiovascular disease and normal electrocardiographic findings) were used as controls. The study protocol was approved by the Ethics Committees of Sun Yat-Sen University and NanHua University, and written informed consent was obtained from all participants.

conversely, single mRNAs can be targeted by several distinct miRNAs (9). Thus, the overall effects of miRNAassociated regulation can be very complex. To date, over 700 human miRNAs have been cloned or sequenced (10,11). Although the biological and pathological functions of miRNAs are not completely understood, it is clear that many miRNAs demonstrate tissue- or cell-specific distributions and play important roles in cell and/or tissue functions under various conditions (12,13). Furthermore, recent studies have demonstrated that miRNAs can be detected in the peripheral blood or plasma, and that the levels of circulating miRNAs are characteristically altered in individuals with various pathological conditions, indicating that circulating miRNAs may be useful diagnostic biomarkers for specific diseases (14,15). Interestingly, miRNAs are present in the blood and/or plasma in a strikingly stable form that withstands even repeated freeze/thaw cycles and is resistant to RNase degradation (16). Although the mechanisms by which miRNAs are released into the circulation are not clear, the levels of several miRNAs in the blood and/or plasma were observed to be altered during AMI. This result suggests that circulating myocardial-derived miRNAs, such as miR-1, 133a, -208b, and -499, might be good biomarkers for the diagnosis of AMI (17-25). However, the suitability of these four circulating miRNAs for the diagnosis of AMI in humans remains somewhat controversial. For example, Wang et al. found that circulating miR-208b was a good biomarker for the diagnosis of AMI, whereas circulating miR-1, -133a, and -499 were not good biomarkers because of their low sensitivities (17). Meanwhile, Alessandra et al. have reported that circulating miR-208b could not be detected in AMI patients and that circulating miR-1, -133a, and -499 are novel biomarkers for the diagnosis of AMI (25). Therefore, the objectives of the current work were to measure the levels of circulating miRNAs (miR-1, -133a, -208b, and -499) and the concentration of cardiac troponin T (cTnT) in AMI patients at two medical centers. Additionally, we analyzed the utility of these four miRNAs as novel biomarkers for the early diagnosis of AMI and compared their diagnostic values with that of cTnT.

Plasma collection and storage Venous blood samples (3 mL) were collected from AMI patients on Day 1 (n = 67) within 12 h of the onset of symptoms (4.6¡2.9 h) and on Day 14 (n = 18) and from healthy volunteers using K2-EDTA-coated tubes. Plasma was isolated by centrifugation at 1,000 g for 10 minutes at 4 ˚C, followed by centrifugation of the supernatant at 14,000 g for 15 minutes at 4 ˚C. The plasma was transferred into RNase/DNase-free tubes and stored at -80 ˚C until RNA extraction.

RNA isolation Total RNA was isolated from plasma using the TRIzol LSH reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and dissolved in 10 mL of diethylpyrocarbonate (DEPC)-treated water. The concentration and quality of the RNA samples were determined using a BioPhotometer (Eppendorf).

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) The Bulge-LoopTM miRNA qRT-PCR Primer Set (RiboBio Co, Guangzhou, China) was used to detect and quantify the expression of mature microRNAs (miR-1, -133a, -208b, and -499) according to the manufacturer’s instructions; a Caenorhabditis elegans microRNA (cel-miR-39) was used as the internal control (14). Total RNA (0.5 mg) from each sample was reverse transcribed using microRNA-specific primers and M-MLV reverse transcriptase (Promega, Madison, WI, USA). The reaction was carried out for 60 min at 42 ˚C, followed by 10 min at 70 ˚C, and the resulting cDNA was stored at -20 ˚C until use. The qRT-PCR reactions were performed on the PRISM 7900HT sequence detection system (Applied Biosystems, Carlsbad, CA, USA) in 20-mL reactions containing 2 mL of reverse transcription product, 2 mL of PCR forward primer (5 mM), 2 mL of Universal Adaptor PCR Primer (5 mM), 9 mL of Platinum SYBR Green qPCR SuperMix-UDG reagent (Invitrogen, Carlsbad, CA, USA), and 5 mL of ddH2O. The reactions were incubated at 95 ˚C for 2 min, followed by 40 cycles of 95 ˚C for 15 s, 60 ˚C for 30 s and 95 ˚C for 15 s. The samples were then heated from 60 ˚C to 95 ˚C to obtain melting curves. Reactions containing either no reverse transcriptase or no template were used as negative controls, and all assays were performed in triplicate. These procedures were performed by an investigator (L.L.) blinded to the clinical characteristics of the patients. The threshold cycle (Ct) was defined as the fractional cycle number at which the fluorescence exceeded the given threshold, and the relative expression values of the microRNAs were calculated using the 22DDCt method (27).

& MATERIALS AND METHODS Clinical specimens Sixty-seven patients diagnosed with AMI were enrolled in this study at the First Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China) and the First Affiliated Hospital of NanHua University (Hengyang, China) between October 2010 and March 2011. The inclusion criteria for patients with AMI were based on the redefined guidelines from the most recent standard definition of MI (26). Briefly, AMI was diagnosed in patients with increased cTnT or creatine kinase-MB (CK-MB) levels combined with chest pain lasting .30 minutes and electrocardiogram (ECG) findings such as new pathological Q waves or ST-segment elevation or depression. Patients were excluded if they had received intravenous thrombolytic or anticoagulant treatment before the initial blood samples were acquired. Fortyfour patients (66%) experienced ST-segment elevation myocardial infarction (STEMI), whereas 23 (34%) experienced non-ST-segment elevation myocardial infarction (NSTEMI). Thirty-nine patients (58%) were treated with percutaneous coronary intervention (PCI), whereas 28 (42%)

cTnT determination The levels of plasma cTnT were determined using electrochemiluminescence-based methods with the Elecsys 2010

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The diagnostic values of miRNAs and cTnT in AMI Li YQ et al.

We also measured the levels of the four miRNAs in plasma from AMI patients at the time of hospital discharge (n = 18, patients who lived more than 14 days and agreed to take part in this part of the study), and we found that the miRNA levels had decreased to baseline levels, i.e., they were not significantly different from the levels in the controls (p.0.05) (Figure 3).

Immunoassay Analyzer (Roche Diagnostics, Switzerland) according to the manufacturer’s protocol.

Statistical analysis The Statistical Package for Social Sciences, version 16.0 (SPSS, Inc., Chicago, IL, USA), was used for the statistical analysis. All data are presented as the mean¡standard deviation unless otherwise noted. The significance of the differences in clinical characteristics between AMI patients and controls was tested using Student’s t-test or Fisher’s exact test. The Mann-Whitney test was conducted to compare the expression of microRNAs between the AMI patients and controls. The microRNA levels measured in each AMI patient at the two time points were compared using the Wilcoxon signed rank test. Receiver operating characteristic (ROC) curves were established to discriminate between AMI patients and controls with Stata version 10.0 and were compared using the chi-square test. All p-values were two-tailed, and p,0.05 was considered statistically significant.

Circulating cardiac-associated microRNAs as potential predictors of AMI To evaluate the predictive power of circulating cardiacassociated microRNAs for AMI, we performed ROC analysis for 67 patients with AMI. As shown in Figure 4, the areas under the ROC curves (AUCs) for miR-1, miR-133a, miR208b, and miR-499 were 0.8265 (95% confidence interval, 0.7441-0.9088), 0.9468 (95% confidence interval, 0.9057-0.9879), 0.8899 (95% confidence interval, 0.8259-0.9540), and 0.8841 (95% confidence interval, 0.8187-0.9495), respectively. The AUC measured for cTnT was 0.9820 (95% confidence interval, 0.9289-0.9975). These results demonstrate that the four miRNAs had marked sensitivity and specificity for AMI. However, none of the four miRNAs tested was superior to cTnT for the diagnosis of AMI (all p.0.05).

& RESULTS Clinical characteristics of the study population In the present study, we enrolled 67 patients with AMI and 32 controls. The clinical characteristics of each group are listed in Table 1. The mean age of the AMI patients was 63.84¡11.17 years, and the mean age of the controls was 61.75¡9.58 years (p = 0.332). Both groups were predominantly male (52/67 in the AMI group and 22/32 in the control group; p = 0.343). The mean cTnT level at admission in AMI patients was 1.30¡1.06 ng/mL, which was significantly higher than the mean cTnT level in the control group (p,0.001). There were no significant differences in any other clinical characteristics between the AMI and control groups.

& DISCUSSION In our present study, we found that the levels of miRNA-1, 133a, -208b, and -499 were significantly increased in patients after AMI relative to the levels in healthy volunteers, and our data indicated that the elevated expression of these four miRNAs was coincident with the progression of the AMI. As we now know, in the early stages of AMI, pathological changes such as myocardial ischemia, hypoxia, edema, and necrosis occur rapidly, followed by the release of necrotic products, such as cardiac troponins (cTns), creatine kinase (CK) and brain natriuretic peptide (BNP), into the bloodstream. These established biomarkers can be useful for the early diagnosis of AMI. The results of the present work suggest that miRNA-1, -133a, -208b, and -499 might leak out of the necrotic myocardium and into the circulation during the early stages of AMI, and the levels of these miRNAs would thus become elevated as the AMI progresses; therefore, these four circulating miRNAs might be useful biomarkers for the diagnosis of AMI. AMI is a leading cause of death in both developed and developing countries, and the mortality rate of this disease can be reduced by early diagnosis. Established biomarkers such as cTnT, CK-MB, BNP and C-reactive protein (CRP) play critical roles in the diagnosis of AMI (3,28,29). According to the recent redefinition of myocardial infarction by the ESC/ACC (European Society of Cardiology/ American College of Cardiology) Consensus group, troponins are more sensitive and specific measures of myocyte necrosis with respect to AMI diagnosis than CK-MB, BNP, and CRP (30-32). Nevertheless, cTns have several weaknesses, including incomplete specificity for AMI. For example, some studies have reported that cTn levels were elevated in ESRD patients and that cTns might serve as prognostic biomarkers in RF patients (4,5). Therefore, it is important to continue the search for new biomarkers with higher sensitivity and specificity for the early diagnosis of AMI. Several recent studies have reported that miRNAs exhibit tissue- and cell-specific characteristics under various conditions;

Circulating cardiac-associated microRNA levels were elevated in AMI patients To determine whether the circulating cardiac-associated microRNAs were associated with the onset of AMI, we investigated the plasma levels of four microRNAs (miR-1, -133a, -208b, and -499) in AMI patients and controls. As shown in Figure 1, the plasma concentrations of miR-1, -133a, -208b, and -499 were all markedly elevated in AMI patients relative to controls (p,0.001). Furthermore, there were no significant differences in the levels of these four miRNAs between STEMI and NSTEMI patients (all p.0.05, Figure 2A), and there were also no significant differences between the PCI and thrombolysis groups (all p.0.05, Figure 2B). Table 1 - Clinical characteristics of the study population. Characteristics

Control (n = 32)

AMI (n = 67)

p-value

Age (years) 61.75¡9.58 63.84¡11.17 0.332 Male/Female (n/n) 22/10 52/15 0.343 Currently smoking, n (%) 13 (40.63) 32 (47.76) 0.505 Diabetes mellitus, n (%) 5 (15.63) 13 (19.40) 0.649 Hypertension, n (%) 15 (46.88) 38 (56.72) 0.358 Hyperlipidemia, n (%) 14 (43.75) 36 (53.75) 0.353 Systolic blood pressure (mmHg) 125.56¡12.52 129.66¡15.35 0.203 Diastolic blood pressure (mmHg) 79.75¡6.81 81.57¡9.76 0.210 Cardiac troponin T (ng/mL) 0.03¡0.02 1.30¡1.06 ,0.001

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Figure 1 - Levels of the four circulating miRNAs in AMI patients. The plasma levels of miR-1 (A), miR-133a (B), miR-208b (C), and miR-499 (D) were significantly increased in AMI patients (n = 67) relative to the controls (n = 32) (p,0.001).

additionally, miRNAs can be released into the circulation, where they persist in a stable form (12,16,17). Several consecutive reports have shown that the levels of circulating, cardiac-specific miRNAs were altered in conjunction with the progression of cardiovascular diseases; therefore, these miRNAs might be promising novel biomarkers for the diagnosis of AMI. The findings from our present study are consistent with those reported in recent articles, indicating that levels of the circulating miRNAs miRNA-1, -133a, -208b, and -499 were increased during AMI and were significantly higher than those in healthy volunteers. The levels of the four circulating miRNAs decreased to normal levels at the time of hospital discharge. We also analyzed the concentration of cTnT during AMI. cTns are established biomarkers for the diagnosis of AMI and reflect infarct size. Compared with cTnI, cTnT is more specific and sensitive for the diagnosis of AMI (33-35). Therefore, we selected cTnT as a benchmark for comparison with circulating miRNAs in the present study.

We performed ROC curve analyses to determine the diagnostic values of the four circulating miRNAs and to compare them with cTnT. The results of these analyses indicated that circulating miR-1, -133a, -208b, and -499 were specific and sensitive for the early diagnosis of AMI and are promising novel biomarkers for AMI. Although there were positive relationships between the four circulating miRNAs and cTnT within 12 hours of the onset of symptoms (Day 1), none of the four circulating miRNAs (miR-1, -133a, -208b, and -499) was superior to cTnT for the early diagnosis of AMI. According to these results, cTnT seems to be more sensitive and specific than any of the four circulating miRNAs in patients with AMI. We hypothesized that cTnT might be released from necrotic myocardium at the time of the AMI in patients prior to the onset of chest pain. Previous studies have demonstrated that cTnT is primarily bound to myofibrils and that miRNAs are primarily bound to cytosolic protein complexes. We hypothesized that these differences might affect the patterns of cTnT and miRNA release during the progression of myocardial necrosis.

Figure 2 - Stratified analysis of the levels of the four circulating miRNAs in AMI patients. (A) Circulating miR-1, -133a, -208b, and -499 levels in STEMI and NSTEMI patients; (B) circulating miR-1, -133a, -208b, and -499 levels in AMI patients treated with PCI or thrombolysis.

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Figure 3 - Alteration of the plasma miRNA levels in AMI patients. (A-D) The levels of miR-1, -133a, -208b, and -499 were markedly increased in plasma samples gathered within 12 hours of the onset of AMI (Day 1). At the time of hospital discharge (Day 14), the levels of these four miRNAs had returned to levels similar to those in the control subjects (n = 32 control subjects, n = 67 AMI patients at Day 1, n = 18 AMI patients at Day 14). The values shown represent the fold change of each miRNA in AMI patients relative to control subjects. The results are presented as the mean ยก standard deviation. *p,0.001 vs. control.

Our present study provides clinical evidence to support the use of circulating miR-1, -133a, -208b, and -499 as biomarkers for AMI. However, our work has some limitations, including the small sample size. Therefore, additional studies with

larger cohorts of healthy volunteers and patients are needed to definitively demonstrate the diagnostic value of the four circulating miRNAs as practical biomarkers in comparison with other markers and to reduce the rate of false-positive

Figure 4 - Comparisons of the sensitivity and specificity of the four plasma miRNAs and cTnT for the diagnosis of AMI. Receiver operating characteristic (ROC) curves were constructed to evaluate the diagnostic values of miR-1, -133a, -208b, and -499 for AMI patients in comparison with cTnT. AUC, area under the ROC curve.

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12. Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W, Tuschl T. Identification of tissue-specific microRNAs from mouse. Curr Biol. 2002;12(9):735-9. 13. Kloosterman WP, Plasterk RH. The diverse functions of microRNAs in animal development and disease. Dev Cell. 2006;11(4):441-50. 14. Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, PogosovaAgadjanyan EL. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A. 2008;105(30):10513-8. 15. Skog J, Wurdinger T, van Rijn S, Meijer DH, Gainche L, Sena-Esteves M, et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol. 2008;10(12):1470-6. 16. Gilad S, Meiri E, Yogev Y, Benjamin S, Lebanony D, Yerushalmi N, et al. Serum microRNAs are promising novel biomarkers. PLoS One. 2008;3(9):e3148. 17. Wang GK, Zhu JQ, Zhang JT, Li Q, Li Y, He J, et al. Circulating microRNA: a novel potential biomarker for early diagnosis of acute myocardial infarction in humans. Eur Heart J. 2010;31(6):659-66. 18. Adachi T, Nakanishi M, Otsuka Y, Nishimura K, Hirokawa G, Goto Y, et al. Plasma microrna 499 as a biomarker of acute myocardial infarction. Clin Chem. 2010;56(7):1183-5. 19. Ai J, Zhang R, Li Y, Pu J, Lu Y, Jiao J, et al. Circulating microRNA-1 as a potential novel biomarker for acute myocardial infarction. Biochem Biophys Res Commun. 2010;391(1):73-7. 20. Bostjancic E, Zidar N, Stajer D, Glavac D. MicroRNAs miR-1, miR-133a, miR-133b and miR-208 are dysregulated in human myocardial infarction. Cardiology. 2010;115(3):163-9. 21. Ji X, Takahashi R, Hiura Y, Hirokawa G, Fukushima Y, Iwai N. Plasma miR-208 as a biomarker of myocardial injury. Clin Chem. 2009;55(11): 1944-9. 22. Dong S, Cheng Y, Yang J, Li J, Liu X, Wang X, et al. MicroRNA expression signature and the role of microRNA-21 in the early phase of acute myocardial infarction. J Biol Chem. 2009;284(43):29514-25. 23. Tijsen AJ, Creemers EE, Moerland PD, de Windt LJ, van der Wal AC, Kok WE, et al. MiR423-5p as a circulating biomarker for heart failure. Circ Res. 2010;106(6):1035-9. 24. Corsten MF, Dennert R, Jochems S, Kuznetsova T, Devaux Y, Hofstra L, et al. Circulating microRNA-208b and microRNA-499 reflect myocardial damage in cardiovascular disease. Circ Cardiovasc Genet. 2010;3(6):499506. 25. D’Alessandra Y, Devanna P, Limana F, Straino S, Di Carlo A, Brambilla PG, et al. Circulating microRNAs are new and sensitive biomarkers of myocardial infarction. Eur Heart J. 2010;31(22):2765-73. 26. Thygesen K, Alpert JS, White HD. Universal definition of myocardial infarction. Eur Heart J. 2007;28(20):2525-38. 27. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) Method. Methods. 2001;25(4):402-8. 28. Ramos RB, Strunz CM, Avakian SD, Ramires JA, Mansur Ade P. B-type natriuretic peptide as a predictor of anterior wall location in patients with non-ST-elevation myocardial infarction. Clinics. 2011;66(3):437-41. 29. Martins OM, Fonseca VF, Borges I, Martins V, Portal VL, Pellanda LC. CReactive protein predicts acute myocardial infarction during high-risk noncardiac and vascular surgery. Clinics (Sao Paulo). 2011;66(5):773-6. 30. Myocardial infarction redefined—a consensus document of the Joint European Society of Cardiology/American College of Cardiology Committee for the redefinition of myocardial infarction. Eur Heart J. 2000;21(18):1502-13. 31. Alpert JS, Thygesen K, Antman E, Bassand JP. Myocardial infarction redefined--a consensus document of the Joint European Society of Cardiology/American College of Cardiology Committee for the redefinition of myocardial infarction. J Am Coll Cardiol. 2000;36(3):959-69. 32. White HD. Evolution of the definition of myocardial infarction: What are the implications of a new universal definition? Heart. 2008;94(6):679-84. 33. Panteghini M, Cuccia C, Bonetti G, Giubbini R, Pagani F, Bonini E. Single-point cardiac troponin T at coronary care unit discharge after myocardial infarction correlates with infarct size and ejection fraction. Clin Chem. 2002;48(9):1432-6. 34. Licka M, Zimmermann R, Zehelein J, Dengler TJ, Katus HA, Kubler W. Troponin T concentrations 72 hours after myocardial infarction as a serological estimate of infarct size. Heart. 2002;87(6):520-4. 35. Reiter M, Twerenbold R, Reichlin T, Haaf P, Peter F, Meissner J, et al. Early diagnosis of acute myocardial infarction in the elderly using more sensitive cardiac troponin assays. Eur Heart J. 2011;32(11):1379-89.

results. Additionally, the diagnostic values of the circulating miRNAs need to be investigated in further studies involving ESRD patients with AMI and in AMI patients without ESRD. In conclusion, this study verified the diagnostic value of four circulating miRNAs (miR-1, -133a, -208b, and -499) in the early stage of AMI and compared them with the established biomarker cTnT. We demonstrated that miR-1, -133a, -208b, and -499 might serve as novel biomarkers for the diagnosis of AMI; however, the diagnostic values of these circulating miRNAs were not superior to that of cTnT. Because the cTnT concentration is also increased in ESRD patients, we propose that cTnT and miRNA analyses could be combined to enhance the specificity of AMI diagnosis.

& ACKNOWLEDGMENTS This study was supported by grants from the National Natural Science Foundation of China (81071030), the Science and Technology Foundation of Guangdong Province, China (2010B031600089, 2011B080701006), and the Training Foundation for the Youth Scholars of Sun Yat-sen University (09ykpy31). The funders played no role in the study design, data collection, data analysis, decision to publish or preparation of the manuscript.

& AUTHOR CONTRIBUTIONS Li YQ, Zhang MF and Wen HY contributed equally to the study. Li YQ, Li X, and Zhang MF helped performing the research and data analysis and writing the manuscript. Hu CL, Wei HY, Ai CM, and Wang G contributed to the recruitment of patients and volunteers. Liu R helped performing the experiments. Corresponding authors (Li X and Liao XX) conceived and designed the study. All authors have approved the final manuscript.

& REFERENCES 1. White HD, Chew DP. Acute myocardial infarction. Lancet. 2008;372(9638):570-84. 2. Antman EM, Hand M, Armstrong PW, Bates ER, Green LA, Halasyamani LK, et al. 2007 Focused Update of the Acc/Aha 2004 guidelines for the management of patients with ST-elevation myocardial infarction: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2008;51(2):210-47. 3. Jaffe AS, Ravkilde J, Roberts R, Naslund U, Apple FS, Galvani M, et al. It’s time for a change to a troponin standard. Circulation. 2000;102(11):1216-20. 4. Collinson PO, Hadcocks L, Foo Y, Rosalki SB, Stubbs PJ, Morgan SH, et al. Cardiac troponins in patients with renal dysfunction. Ann Clin Biochem. 1998;35(Pt 3):380-6. 5. Khan NA, Hemmelgarn BR, Tonelli M, Thompson CR, Levin A. Prognostic value of troponin T and I among asymptomatic patients with end-stage renal disease: a meta-analysis. Circulation. 2005;112(20):3088-96. 6. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science. 2001; 294(5543):853-8. 7. Pheasant M, Mattick JS. Raising the estimate of functional human sequences. Genome Res. 2007;17(9):1245-53. 8. Soifer HS, Rossi JJ, Saetrom P. MicroRNAs in disease and potential therapeutic applications. Mol Ther. 2007;15(12):2070-9. 9. Van Rooij E, Liu N, Olson EN. MicroRNAs flex their muscles. Trends Genet. 2008;24(4):159-66. 10. Bentwich I, Avniel A, Karov Y, Aharonov R, Gilad S, Barad O, et al. Identification of hundreds of conserved and nonconserved human microRNAs. Nat Genet. 2005;37(7):766-70. 11. Berezikov E, Guryev V, van de Belt J, Wienholds E, Plasterk RH, Cuppen E, et al. Phylogenetic shadowing and computational identification of human microRNA genes. Cell. 2005;120(1):21-4.

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Hereditary angioedema: quality of life in Brazilian patients Maria Abadia Consuelo M. S. Gomide,I Eliana Toledo,II Solange Oliveira Rodrigues Valle,III Regis A. Campos,IV Alfeu T. Franc¸a,III Nieves Prior Gomez,V Heitor Franco Andrade Jr.,VI Teresa Caballero,V Anete S. GrumachI,VII I

Faculdade de Medicina da Universidade de Sa˜o Paulo, Department of Dermatology, Sa˜o Paulo/SP, Brazil. II University of Sa˜o Jose´ do Rio Preto, Faculty of Medicine, Brazil. III Federal University of Rio de Janeiro, Department of Immunology, Rio de Janeiro/RJ, Brazil. IV Federal University of Bahia, Department of Clinical Immunology, Salvador/BA, Brazil. V Hospital Universitario La Paz, Servicio de Alergia, Madrid, Spain. VI Faculdade de Medicina da Universidade de Sa˜o Paulo, Department of Pathology, Sa˜o Paulo/SP, Brazil. VII Faculty of Medicine ABC, Santo Andre´/SP, Brazil.

OBJECTIVE: Hereditary angioedema is a serious medical condition caused by a rare autosomal dominant genetic disorder and it is associated with deficient production or dysfunction of the C1 esterase inhibitor. In most cases, affected patients experience unexpected and recurrent crises of subcutaneous, gastrointestinal and laryngeal edema. The unpredictability, intensity and other factors associated with the disease impact the quality of life of hereditary angioedema patients. We evaluated the quality of life in Brazilian hereditary angioedema patients. METHODS: Patients older than 15 years with any severity of hereditary angioedema and laboratory confirmation of C1 inhibitor deficiency were included. Two questionnaires were used: a clinical questionnaire and the SF-36 (a generic questionnaire). This protocol was approved by the Ethics Committee of Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo. RESULTS: The SF-36 showed that 90.4% (mean) of all the patients had a score below 70 and 9.6% had scores equal to or higher than 70. The scores of the eight dimensions ranged from 51.03 to 75.95; vitality and social aspects were more affected than other arenas. The internal consistency of the evaluation was demonstrated by a Cronbach’s alpha value above 0.7 in seven of the eight domains. CONCLUSIONS: In this study, Brazilian patients demonstrated an impaired quality of life, as measured by the SF36. The most affected domains were those related to vitality and social characteristics. The generic SF-36 questionnaire was relevant to the evaluation of quality of life; however, there is a need for more specific instruments for better evaluation. KEYWORDS: Hereditary Angioedema; Quality of Life; Questionnaires; SF-36; Asphyxia. Gomide MA, Toledo E, Valle SO, Campos RA, Franc¸a AT, Gomez NP, et al. Hereditary angioedema: quality of life in Brazilian patients. Clinics. 2013;68(1):81-83. E-mail: asgrumach@gmail.com Tel.: 55 11 3284 5335

observed in the face, extremities, genitals, gastrointestinal system and upper airways. Edema affecting the skin is typically painless but may cause disfigurement or difficulty driving or operating machinery. Gastrointestinal edema is very painful and can lead to unnecessary laparotomy, while upper airway edema can be life threatening due to swelling of the tongue and larynx (3). Some patients are able to identify triggering factors, although in certain cases, no factors can be found. The most common triggering factors are emotional stress, local trauma, medical or dental procedures, infection, menses and oral contraceptives (4). These attacks are unpredictable, intense and vary in terms of the site of edema. There is a potential risk of dying due to asphyxiation. The efficacy of long-term prophylactic treatment is inconsistent and it can cause side effects. All these factors decrease the quality of life of this patient population. However, few publications have described the quality of life in patients with hereditary angioedema and no specific questionnaire is currently available.

& INTRODUCTION Hereditary angioedema (HAE) is a serious medical condition caused by a rare autosomal dominant genetic disorder that is characterized by deficient production or dysfunction of the C1 esterase inhibitor. The C1 inhibitor acts as a regulatory protein in the complement, contact, coagulation and fibrinolytic systems (1). The prevalence of HAE is estimated to be between 1:10,000 and 1:50,000 (2). Patients affected by HAE experience unexpected and recurrent ‘‘attacks’’ of bradykinin-mediated edema. Edema may occur in any part of the body but is most commonly

Copyright ß 2013 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. DOI: 10.6061/clinics/2013(01)OA13

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The aim of the study was to evaluate the quality of life in Brazilian patients with angioedema.

correlation. A Cronbach’s alpha value equal to or greater than 0.7 was considered acceptable.

& METHODS

& RESULTS

Ethical Aspects: The study protocol was approved by the Ethics Committee of the Clinics Hospital of the Faculty of Medicine of the University of Sa˜o Paulo. All patients signed the informed consent form before any study procedures took place. Patient Population: Patients older than 15 years with any severity of HAE and laboratory confirmation of C1 inhibitor deficiency were included. Data Collection: Two questionnaires were used: a clinical questionnaire and the SF-36. The SF-36, a generic tool, was previously culturally adapted and validated in Brazil (5). The clinical questionnaire consisted of personal questions and information about the disease; patients were asked about their history of angioedema attacks in the previous six months, treatment, side effects of medication, influence on their social life, whether they had missed days of work or school due to attacks, visits to emergency rooms and psychological disturbances due to angioedema. A severity score was calculated according to the data considered by Agostoni et al. (3): adverse events from long-term therapy, the frequency of attacks, absence from school and/or work due to clinical symptoms, depression and anxiety, specific treatment and need for hospitalization and/or ICU. The SF36 is a generic instrument that consists of 36 questions in eight domains, which are pooled into two domains: physical and mental. The physical domain includes questions regarding the following items: physical function (10 questions related to routine activities), general health (five questions related to the patient’s professional/educational life), pain (two questions) and physical aspects (four questions related to general well-being). The mental domain includes questions about mental health (five items), vitality (four items), social function (two items) and emotional well-being (three items). A question asking the patient to compare his/her current health to their health in the previous year is also included. Statistical Analysis: The data were evaluated with Microsoft Excel 2007, SPSS 17 and Statistica 7.0. Endpoints were considered statistically significant at p,0.05. The SF-36 internal reliability was tested using Cronbach’s alpha with a range of zero to one, with zero indicating the absence of correlation between items and one indicating perfect

Thirty-five patients were included in the study. The majority of patients were female (25 = 71.4%), from an urban area (85.7%) and had an average level of education (42.9%) and a medium-low socio-economic level (57.1%). The mean age was 40.7 years (DP¡16.6 years). In 54% of the patients, the disease was well controlled, while 32% had mild symptoms and 14% had a moderate degree of symptoms. The SF-36 showed that 90.4% (mean) of patients had a score below 70 and 9.6% had scores equal to or higher than 70. The scores of the eight dimensions applied in all patients varied from 51.03 to 75.95; vitality and social aspects were more affected. The internal consistency of the evaluation was demonstrated by a Cronbach’s alpha above 0.7 in seven of the eight domains (Table 1). There were no differences between the mean scores of the SF-36 in relation to gender, age, education level, or disease severity.

& DISCUSSION Although HAE is a rare disease, the patient burden is similar to other more common chronic diseases (6); the decrease in work productivity experienced by HAE patients is comparable with that experienced by patients with Crohn’s disease or severe asthma. In addition, HAE patients must contend with the uncertainties of their disease. In our case, it should be considered that Brazil has yet to invest in the identification of these patients or access to therapy for the attacks. Danazol, the only medication supported by the Brazilian government, is classified as a high-cost drug. Patients taking androgens have reported more depressive symptoms and reduced productivity levels compared to patients not taking androgens (6). Androgens are often prescribed to patients with higher attack frequencies and more severe disease profiles (2). Only a few studies have evaluated quality of life (QOL) in HAE patients (6-8). In this study, Brazilian patients exhibited an impaired QOL, as measured by the SF-36, which is a generic questionnaire. The areas of their lives that were most affected were those related to vitality and social arenas. The patients felt tired, and their physical and emotional conditions impacted their social and professional

Table 1 - Descriptive analysis of the SF-36 domains in 35 hereditary angioedema patients and Cronbach’s alpha values.

Physical functioning Lim. physical health Body pain General health perception Vitality Social functioning Lim. emotional problems Mental health

N

Minimum

Maximum

Mean

Median

Standard deviation

Cronbach’s alpha

35

15.00

100.00

75.57

85.00

22.61

0.90

35

0.00

80.00

60.57

70.00

22.02

0.94

35 34

0.00 20.00

100.00 90.00

58.10 59.26

52.00 40.00

30.15 18.71

0.90 0.84

34 35 35

15.00 25.00 0.00

65.00 87.50 100.00

51.03 54.29 75.95

60.00 50.00 83.30

12.42 13.87 25.14

0.78 0.66 0.90

34

25.00

91.67

65.20

64.58

16.85

0.86

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Hereditary Angioedema and Quality of Life Gomide MA et al.

activities. Poon et al. measured and compared Dermatology Life Quality Index (DLQI) scores in different dermatological conditions and included five patients with HAE (7). Bygum et al. addressed the improvement in the QOL of seven HAE patients related to medication used to ameliorate the edema (8). Most recently, Lumry et al. (6) evaluated the impact of HAE on QOL, applying one disease-specific survey and three standardized instruments; approximately 19% of the patients invited by the US HAE Association (n = 457) responded to the questionnaires. Patients with HAE reported decreased physical and mental health compared to a normal population (p,0.001) for all subscales and overall summary components. According to our data, the social effects of HAE had a larger impact on patient QOL than their physical condition. Several questionnaires have been developed to evaluate health-related quality of life. They provide information while evaluating or comparing the effects of clinical interventions or treatments, comparing differences between groups and evaluating the impact of side effects and potential problems demanding medical intervention. These questionnaires also help to decide resource allocation (9). The generic SF-36 questionnaire is important in the evaluation of quality of life in HAE patients; however, there is a need for studies using disease-specific surveys with specific instruments to serve as a point of comparison with generic questionnaires and evaluate the correlation with clinical severity (10). A specific HAE QOL questionnaire is currently being developed in Spain and will soon be translated and validated in other countries, including Brazil (11).

AT participated in the study design, data acquisition, analysis, and interpretation and manuscript draft, revision and final approval. Gomez NP and Caballero T participated in the conception of the study, data analysis and interpretation, and manuscript draft, revision and final approval. Andrade Jr HF participated in data analysis and interpretation, and manuscript draft, revision and final approval.

& REFERENCES 1. Davis AE 3rd, Medja P, Lu F. Biological activities of C1-Inhibitor. Mol Immunol. 2008;45(16):4057-63, http://dx.doi.org/10.1016/j.molimm. 2008.06.028. 2. Bowen T, Cicardi M, Bork K, Zuraw B, Frank M, Ritchie B, et al. Hereditary angioedema:a current state of the art review, VII: Canadian Hungarian 2007 International Consensus Algorithm for the Diagnosis, Therapy, and Management of Hereditary Angioedema. Ann Allergy Asthma Immunol. 2008;100(1 Suppl 2):S30-40, http://dx.doi.org/10. 1016/S1081-1206(10)60584-4. 3. Agostoni A, Aygoren-Pursun E, Binkley KE, Blanch A, Bork K, Bouillet L, et al. Hereditary and acquired angioedema: problems and progress: proceedings of the third C1 esterase inhibitor deficiency workshop and beyond. J Allergy Clin Immunol. 2004;114(3 Suppl):S51-131, http://dx. doi.org/10.1016/j.jaci.2004.06.047. 4. Bowen T, Cicardi M, Farkas H, Bork K, Longhurst HJ, Zuraw B, et al. 2010 International consensus algorithm for the diagnosis, therapy and management of hereditary angioedema. Allergy Asthma Clin Immunol. 2010;6(1):24-36, http://dx.doi.org/10.1186/1710-1492-6-24. 5. Ciconelli RM, Ferraz MB, Santos W, Meina˜o I, Quaresma MR. Traduc¸a˜o para a lı´ngua portuguesa e validac¸a˜o do questiona´rio gene´rico de avaliac¸a˜o da qualidade de vida SF-36. Rev. Bras. Reumatol. 1999;39(3):143-50. 6. Lumry WR, Castaldo AJ, Vernon MK, Blaustein MB, Wilson DA, Horn PT. The humanistic burden of hereditary angioedema: impact on healthrelated quality of life, productivity, and depression Allergy Asthma Proc. 2010;31(5):407-14. 7. Poon E, Seed PT, Greaves MW, Kobza-Black A. The extent and nature of disability in different urticarial conditions. Br J Dermatol. 1999;140(4):667-71. 8. Bygum A, Andersen KE, Mikkelsen CS. Self-administration of intravenous C1-inhibitor therapy for hereditary angioedema and associated quality of life benefits. Eur J Dermatol. 2009;19(2):147-51. 9. Bradlyn AS, Ritchey AK, Harris CV, Moore IM, O’Brien RT, Parsons SK, et al. Quality of life research in pediatric oncology. Research methods and barriers. Cancer. 1996;78(6):1333-9, http://dx.doi.org/10.1002/ (SICI)1097-0142(19960915)78:6,1333::AID-CNCR24.3.0.CO;2-0. 10. Both H, Essink-Bot ML, Busschbach J, Nijsten T. Critical review of generic and dermatology-specific health-related quality of life instruments. J Invest Dermatol. 2007;127(12):2726-39, http://dx.doi.org/10. 1038/sj.jid.5701142. 11. Prior N, Remor E, Go´mez-Traseira C, Lo´pez Serrano C, Caban˜as R, Contreras J, et al. Development of a disease-specific quality of life questionnaire for adult patients with hereditary angioedema due to C1 inhibitor deficiency (HAE-QOL): Spanish multi-centre research project. Health Qual Life Outcomes. 2012;10(1):82, http://dx.doi.org/10.1186/ 1477-7525-10-82.

& ACKNOWLEDGMENTS We are thankful to Mrs Roberta Trefiglio and Mrs Sara Davis for revising the manuscript and its English version, respectively. This work has been partially supported by a grant from FIS (Fondo de Investigaciones Sanitarias, Grant number 060843). Dr. Teresa Caballero is an investigator from the IdiPAZ program for promoting research activities (2009).

& AUTHOR CONTRIBUTIONS Gomide MA and Grumach AS participated in the conception and design of the study, data acquisition, analysis and interpretation, and manuscript draft, revision and approval. Toledo E, Valle SO, Campos RA and Franc¸a

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CLINICAL SCIENCE

Factors associated with hyperglycemia and low insulin levels in children undergoing cardiac surgery with cardiopulmonary bypass who received a single high dose of methylprednisolone Ronaldo Arkader,I Luiz Marcelo Malbouisson,II Gilda Maria Barbaro Del Negro,III Lidia Yamamoto,IV Thelma Suely OkayIV I Faculdade de Medicina da Universidade de Sa˜o Paulo, Postgraduate Programme, Sa˜o Paulo/SP, Brazil. II Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Anesthesiology Department, Sa˜o Paulo/SP, Brazil. III Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Instituto de Medicina Tropical de Sa˜o Paulo, Clinical Dermatological Division, Laboratory of Medical Mycology (LIM-53), Sa˜o Paulo/SP, Brazil. IV Universidade de Sa˜o Paulo, Instituto de Medicina Tropical de Sa˜o Paulo, Brazil, Laboratory of Seroepidemiology and Immunobiology, Sa˜o Paulo/SP, Brazil.

OBJECTIVES: Administering steroids before cardiopulmonary bypass in pediatric heart surgery modulates systemic inflammatory response syndrome and improves postoperative recovery. However, the use of steroids aggravates hyperglycemia, which is associated with a poor prognosis. Adult patients with systemic inflammatory response syndrome usually evolve with hyperglycemia and high insulin levels, whereas .90% of pediatric patients exhibit hyperglycemia and low insulin levels. This study aims to determine: A) the metabolic and inflammatory factors that are associated with hyperglycemia and low insulin levels in children who underwent cardiac surgery with cardiopulmonary bypass and who received a single high dose of methylprednisolone and B) the best predictors of insulin variation using a mathematical model. METHODS: This preliminary study recruited 20 children who underwent heart surgery with cardiopulmonary bypass and received methylprednisolone (30 mg/kg) immediately after anesthesia. Among the 20 patients initially recruited, one was excluded because of the absence of hyperglycemia and lower insulin levels after surgery. However, these abnormalities were confirmed in the remaining 19 children. The C-peptide, CRP, IL-6, and adrenomedullin levels were measured before surgery, immediately after cardiopulmonary bypass, and on the first, second, and third days after cardiac surgery. RESULTS: IL-6, CRP, and adrenomedullin increments were observed, whereas the C-peptide levels remained within reference intervals. CONCLUSION: The multiple regression model demonstrated that in addition to age and glycemia (two wellknown factors that are directly involved in glucose metabolism), adrenomedullin and IL-6 levels were independent factors associated with lower insulin concentrations. These four parameters were responsible for 64.7% of the observed insulin variances. In addition, the fact that C-peptide levels did not fall together with insulin could have grounded the medical decision not to administer insulin to patients. KEYWORDS: Cardiopulmonary Bypass; Adrenomedullin; Insulin; Hyperglycemia; Systemic Inflammatory Response Syndrome. Arkader R, Malbouisson LM, Del Negro GM, Yamamoto L, Okay TS. Factors associated with hyperglycemia and low insulin levels in children undergoing cardiac surgery with cardiopulmonary bypass who received a single high dose of methylprednisolone. Clinics. 2013;68(1):85-92. Received for publication on September 3, 2012; First review completed on October 3, 2012; Accepted for publication on November 23, 2012 E-mail: thelma.okay@usp.br Tel.: 55 11 3061-7280

& INTRODUCTION Metabolism and immunity are closely associated; therefore, a systemic inflammatory response syndrome (SIRS) produces and activates multiple proteins, including CRP, and inflammatory cascade-releasing cytokines, such as IL-6. Studies have demonstrated that IL-6 is increased earlier and returns to baseline levels more rapidly than CRP in SIRS. However, CRP levels are routinely measured in hospital laboratories because of their multiple clinical applications,

Copyright ß 2013 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. DOI: 10.6061/clinics/2013(01)OA14

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regulating insulin in the context of SIRS triggered by cardiac surgery with CPB and MP, which leads to hyperglycemia and low insulin levels (5,6,15,16). The development of ageappropriate immune modulatory interventions for preventing and treating SIRS abnormalities requires an investigation of the mechanisms that are responsible for the unique pediatric inflammatory response to injury. Therefore, we expanded our investigation of the clinical parameters that can be used to monitor children after cardiac surgery with CPB and MP. In addition to glycemia and CRP levels, which are typically monitored in these children, we evaluated insulin, IL-6, and adrenomedullin (ADM) levels. ADM is a novel powerful vasodilating peptide that acts as an immune modulator with bactericidal activity and as an inflammation marker during SIRS with or without infection (17). In addition, monitoring ADM levels is important due to its capacity to inhibit insulin release through a direct action on pancreatic b-cells (18,19). The ability of ADM to act specifically on insulin release supports its use as a marker in this type of pediatric patient. Adult and pediatric patients differ according to insulin levels after a hyperglycemic stimulus that is triggered by SIRS and aggravated by MP. Additionally, there is no consensus on the routine administration of exogenous insulin to these pediatric patients. Therefore, we investigated children who were subjected to cardiac surgery with CPB and MP and who presented with hyperglycemia and low insulin levels. We monitored blood glucose, insulin, Cpeptide, CRP, IL-6, and ADM levels to improve our understanding of the metabolic and inflammatory factors that are involved in glucose regulation in these children and determine the best predictors of insulin variance based on a mathematical model.

e.g., evaluating cardiac risk and detecting inflammation and infection, whereas IL-6 is currently used as a research tool (1). Cardiac surgery with cardiopulmonary bypass (CPB) triggers systemic inflammatory response syndrome (SIRS) and evolves with hyperglycemia and insulin resistance in adults, whereas .90% of children exhibit hyperglycemia accompanied by low insulin levels (2,3). Insulin is synthesized as preproinsulin and turned into proinsulin. Proinsulin is then converted to insulin and Cpeptide and stored in granules that await release on demand. C-peptide is secreted into the bloodstream in equimolar amounts with insulin in response to blood glucose levels (4). A single high dose of methylprednisolone (MP) is routinely used in the majority of pediatric heart surgery centers to blunt SIRS; however, MP aggravates hyperglycemia (5,6), which is associated with a poor prognosis (7,8). Verhoeven et al. (2011) studied 49 children undergoing cardiac surgery and analyzed their blood glucose, insulin, lactate, cortisol, ACTH, IL-6, and IL-10 levels before surgery, immediately after surgery, and 12 h and 24 h after surgery. Glucocorticoids were administered to 65% of the children, and this administration was the main factor associated with hyperglycemia, which was observed in 52% of the children after surgery. However, hyperglycemia disappeared spontaneously without insulin therapy after 24 h in 94% of children, which supported the results of other studies (9). Because the postoperative morbidity was low in the study group, the authors concluded that the positive effects of glucocorticoid administration outweighed the adverse effects of iatrogenic hyperglycemia. There is a consensus on the usefulness of intensive insulin therapy for glucose control to improve morbidity and mortality rates in adult cardiac surgical patients (10). In contrast, there have only been a few randomized controlled studies in critically ill children (8,11). Agus et al. enrolled 980 children up to 36 months of age who were undergoing cardiac surgery with CPB. The patients were randomly assigned to either undergo tight glycemic control with targeted blood glucose levels that ranged from 80-110 mg/ dL or receive standard care in the intensive care unit. Continuous glucose monitoring was used to detect hypoglycemia. Overall, 444 of the 490 children (91%) who were assigned to the tight glycemic control group received insulin compared with 9 of the 490 children (2%) who were assigned to receive standard care. Normoglycemia was achieved earlier with tight glycemic control than with standard care (6 h vs. 16 h, p,0.001) and was maintained in a greater proportion of those patients (50% vs. 33%, p,0.001); however, tight glycemic control was not associated with a significantly decreased rate of associated infections (8.6 vs. 9.9 per 1,000 patient days, p = 0.67). Secondary outcomes, such as morbidity, mortality, and multiple organ system failure, did not differ significantly between the groups; therefore, it was concluded that high-risk subgroups did not benefit from tight glycemic control. In addition, 3% of the patients who were assigned to undergo tight glycemic control had severe hypoglycemia (blood glucose ,40 mg per deciliter) (12). Considering the results of these studies, there is no consensus on insulin use in children despite the frequent association between hyperglycemia and cardiac surgery with CPB (7,8). Many studies have been conducted on hyperglycemia caused by SIRS in children with sepsis (5,6,13,14); however, little is known about the factors that are involved in

& MATERIALS AND METHODS After written informed consent was obtained from their legal guardians, 20 children from the Heart Institute in the School of Medicine at the University of SaËœo Paulo, Brazil were enrolled in the study. The inclusion criteria included the presence of ventricular or atrial defects that required surgical correction with CPB, aged over one year, the presence of hyperglycemia and a decrease in insulin levels after surgery. The exclusion criteria included the presence of an infection one month before surgery, pre-existing endocrine diseases, abnormal glucose and/or insulin values before the study, the need for insulin administration during the hospital stay or for corticosteroids up to 30 days before surgery, renal failure, pulmonary hypertension or congestion, the need for diuretics or oxygen therapy, overt cardiac failure, an inability to provide blood samples for all of the laboratory tests during the five study time points, and the refusal of parents or legal guardians to participate.

Anesthetic procedures, surgical procedures, methylprednisolone administration and postoperative care On the day of surgery, the children received midazolam (0.5 mg.kg-1) as premedication 30 minutes before surgery. Anesthesia was induced and maintained with 2.0% sevoflurane inhalation and a target-controlled infusion system of remifentanil (0.20 mg.kg-1.min-1). Then, an esophageal thermometer, an indwelling bladder catheter, a 22G-24G arterial catheter (right radial artery) and a central venous line (right

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Angeles, California, USA), and the normal range varied from 1.1-4.4 ng.mL-1. Serum CRP concentrations were determined using immunonephelometry (nephelometer-2, Dade-Behring, La De´fense, France); the reference values were #5.0 mg.L-1. Serum IL-6 levels were measured using a highly sensitive EIA kit (Human Interleukin-6 Quantikine HS ELISA, R&D Systems, Minneapolis, USA). The detection limit was 0.7 pg.mL-1. Serum ADM concentrations were measured using the EIA Kit from Phoenix Pharmaceuticals (Inc., Belmont, CA). The detection range was 0.01-100 ng.mL-1, and the reference interval was ,0.5 ng.mL-1.

internal jugular vein) were inserted. Arterial and central venous blood pressure, core temperature, heart rate, oxygen saturation and remifentanil dosing were continuously recorded. Intraoperative mechanical ventilation was set using pressure control ventilation with airway pressure (1510 cmH2O) to achieve a tidal volume of 8-10 mL.kg-1, a PEEP of 5 cmH2O, an inspiratory oxygen fraction of 0.6 and an inspiratory time of 33%. The respiratory rate was adjusted to maintain end-tidal CO2 at 35 cmH2O. All of the patients received 30 mg.kg-1 of MP and 30 mg.kg-1 of cefuroxime immediately after anesthesia was induced. After sternotomy and systemic intravenous anticoagulation with 400 U/kg of heparin, large bore aortic, superior and inferior vena cava cannulas were inserted. Non-pulsatile CPB was performed using a hollow fiber membrane oxygenator (Terumo CapioxC RX05 Cardiovascular Systems, Ann Arbor, MI, USA) with uncoated polyvinyl chloride bypass tubing and a non-occlusive roller pump. Hypothermia (28-32˚C) was induced in all of the patients. The CPB circuit was primed with normal saline, mannitol, sodium bicarbonate, calcium chloride, heparin and red blood cells to achieve a hematocrit of 20%. The circuit volumes were 450 mL for the patients ,10 kg, 800 mL for the patients 10-15 kg and 1.0-1.2 L for the patients .15 kg. The pump flow rates were 200 mL/kg for the children ,5 kg, 150 mL/kg for the children between 5 and 9 kg, 125 mL/kg for the children between 10 and 17 kg, and 100 mL.Kg-1 for the children .17 kg. After initiating CPB, hypothermia (28-32 ˚C) was induced in all of the patients. An initial dose of 30 mL.kg-1 was used to induce cardioplegia, followed by 10 mL.kg-1 every 10-20 minutes. At the end of CPB, the vascular cannulas were removed and anticoagulation was reverted with protamine sulfate. Vasoactive drugs were infused at the discretion of the attending anesthesiologist. The same surgical team performed all of the procedures. After surgery, the patients were transferred to the Pediatric Intensive Care Unit (PICU). The patients were weaned from the vasoactive drugs within the first 12 h after surgery. Fluids were restricted to 30 mL/kg/d during the first 24 h and increased to 60 mL/kg/d while the glucose infusion rate was maintained at 2 mg.kg -1.min.-1.

Statistical analysis The normal distribution of data was studied using the Skewness, Kurtosis and Shapiro-Wilk tests (20). For measurements over time, the Friedman analysis of variance test was used to establish the overall group differences. Multiple comparisons were performed using the Wilcoxon test. The variation in the insulin concentrations was tested for normality. Because the distribution was normal, a multiple regression analysis with the Bonferroni correction was used to assess the independent variables associated with low insulin levels. To evaluate the influence of glucose, C-peptide, CRP, IL-6, and ADM on insulin levels after cardiac surgery with CPB and MP, the concentration variations were calculated as the difference between the first postoperative day (POD1) levels and the baseline time (BT) levels (i.e., POD1-BT). All of the variables were initially tested, and a backward stepwise elimination of the variable with the largest p-value was performed at each step to enable the model to be refitted. In each subsequent step, the least significant variable was removed from the model until all of the remaining variables had individual p-values,0.05. The IL-6 and CRP concentrations both increased; therefore, we selected one inflammation marker (IL-6) to avoid artificial increments of R2. The variation in the glucose levels (glycemia) and the patients’ ages (in months) were arbitrarily included in the model irrespective of their p-values because of their central roles in pancreatic function and insulin regulation. The fitness in each step was assessed using the adjusted coefficient of determination (adjusted R2). The collinearity and multicollinearity among the independent variables were tested using the variance inflation factor (VIF). The beta coefficients (ß) of the variables were used to express the effect of the different variable sizes in the final model. The coefficients of semi-partial determination (spR2) were assessed to evaluate the relative importance of each variable with respect to the other variables. The limit of agreement between the observed and predicted insulin variations, which were obtained using the multiple regression equation, was performed according to the method proposed by Bland and Altman (21). The data were expressed as the mean¡standard deviation or the median [25th and 75th percentiles] according to the data distribution. In the boxplots, the bottom and top of the box were the 25th and 75th percentiles, respectively. The line within the box was the median, and the lower and upper whiskers were the 1.5 IQR of the lower quartile and the 1.5 IQR of the upper quartile, respectively (22). The

Blood sampling and laboratory tests An initial blood sample (basal time, BT) was collected from a peripheral vein to determine the pre-surgical glycemia, insulin, C-peptide, IL-6, CRP, and ADM values. Intraoperative and postoperative blood samples were taken using a catheter after CPB (ACPB) and on the first (POD1), second (POD2), and third postoperative days (POD3) to analyze the parameters. The dead space and the extension of the indwelling venous catheter were cleared before blood collection. Plasma glucose (glycemia) was analyzed using the hexokinase enzymatic method. Normal glucose levels ranged from 60-125 mg.dL-1 (5). Serum insulin levels were determined using a luminescence enzyme immunoassay (Auto DELFIA automatic immunoassay system, Perkin Elmer Life and Analytical Sciences, Shelton, CT, USA). The reference interval was 216 mU.L-1, and the detection limit was 3 mU.L-1. Serum C-peptide levels were determined using a luminescent immunoassay (Immulite; Diagnostic Products, Los

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used to predict insulin levels, including patient age expressed in months and the variations in the glycemia, ADM and IL-6 values. Taken together, these parameters explained 64.7% of the insulin reduction on POD1, F (4, 14) = 9.22, p,0.001. The following four tested variables reached statistical significance and were successfully correlated with insulin variation according to their semipartial R2: age (0.55, p,0.001), glycemia variation (0.17, p = 0.01), ADM variation (0.18, p = 0.009), and IL-6 variation (0.2, p = 0.007). The diagram displayed on the left side of Figure 2 shows the observed insulin values and the predicted values that were calculated using the multiple regression equation. The following final equation was used to predict insulin variation (mU.L-1):

statistical analyses were performed using the STATA 11.1 statistical package (StataCorp., College Station, Texas, USA).

& ETHICS This study was approved by the Institutional Ethics Committee (protocol CAPPesq number 675/05).

& RESULTS The study group consisted of 19 children, ten girls and nine boys, after excluding one child because of the absence of hyperglycemia and lower insulin levels after surgery. The study group was a convenience sample that was limited by the number of children who met the inclusion criteria of the study, whose parents or legal guardians agreed to participate and who underwent five successful blood samplings. We recruited children with congenital heart defects of moderate severity who were clinically stable to ensure that the five blood samplings would not damage their health. The homogeneity of the participant heart disease was essential to ensure the internal and external validity of the study and allow for the extrapolation of the data to populations of children with congenital heart defects of moderate severity who were subjected to cardiac surgery with CPB and MP in pediatric intensive care units. The mean age of the patients was 39¡16 months, and the mean weight was 14¡3.8 kg. Atrial and ventricular defects were corrected in 10 and 9 children, respectively. The mean CPB duration was 55¡14 min., and the mean aortic crossclamp length was 25¡6 min. None of the children needed inotropic support or experienced surgical complications. The patients were all discharged from the pediatric intensive care unit (PICU) within 72 hours. Many parents and legal guardians did not consent to participate, primarily because of the volume of blood samples that were needed for the five study time points and/or the fear of increased risks to their children. In addition, this study was limited by its budget because most of the exams are not routinely performed in hospital laboratories, and several exams needed to be tested in triplicate to comply with the manufacturers’ instructions, particularly the enzyme immunoassays that were used to evaluate IL-6 and ADM. Figure 1 shows the insulin, glycemia, C-peptide, ADM, IL-6 and CRP levels throughout the study period. Compared with the BT values (p,0.05), the insulin levels at the post-CPB time point and at the POD1 time point had decreased by 72% and 74%, respectively. On POD2 and POD3, the insulin levels increased by 47% with respect to the BT values (p = NS). Glycemia increased significantly from the BT time point to the POD1 time point (87 mg.dL-1 [68-88] to 172 mg.dL-1 [155-207], p,0.05) but returned to the BT levels on POD3. The C-peptide levels (Figure 1) did not significantly change. The ADM levels at the BT time point were slightly higher than normal, and 323% increments were observed on POD1 (p,0.05); however, the levels returned to BT levels on POD3. There was a significant increase in the IL-6 and CRP levels, which peaked on POD1 and returned to BT levels on POD3. During the fitting process, several variables were removed from the initial model (step-by-step) in the following order: 1) the variation in the C-peptide values, 2) the aortic cross-clamp duration, and 3) the CPB duration. Table 1 shows the final multiple regression model that was

 {16:3z0:14|agez0:03|glycemia mg:dL{1 z   0:29|ADM ng:mL{1 {0:17|IL{6 pg:mL{1 , where -16.3 was a negative constant, patient age was expressed in months and the glycemia, ADM and IL-6 variations were the difference between the POD1 and BT values. The diagram displayed on the right side of Figure 2 shows that the 95% limit of agreement for the observed and predicted insulin values according to the Bland and Altman analysis was within the interval -2.76 to+2.76 mU.L-1.

& DISCUSSION A better understanding of the complex interactions between metabolism and inflammation in critically ill children, such as children with congenital heart defects of moderate severity, will lead to appropriate general and metabolic support for these patients. In this study, all 19 children had hyperglycemia and low insulin levels caused by SIRS, which was induced by cardiac surgery with CPB and attenuated by MP. The insulin resistance state, which is characterized by decreased peripheral use of glucose in skeletal muscles and the liver (13), is more prone to occur in the context of prolonged hyperglycemia that is caused by multiple mechanisms, including the counter regulation of gluconeogenesis and glycogenolysis and the downregulation of glucose transporters (23). To our knowledge, this is the first study that has demonstrated variations in insulin concentrations and their association with inflammatory markers and other factors in pediatric cardiac surgery with CPB and MP, which leads to SIRS, hyperglycemia and low insulin levels. This study has several limitations, such as the lack of MP evaluation on the study time points, although it is well-known that MP effects are exerted during the first 12 h after surgery (24). In addition, most of the findings in this study occurred at the ACPB time point and persisted until the POD2 and POD3 time points, according to the parameter. Another limitation was the convenience sample, which was composed of only 19 patients who were evaluated over time (five study time points). However, age, glycemia, and the IL-6 and ADM levels of the patients reached strong statistical significance; therefore, the results indicate that these four parameters in the proposed mathematical model were consistently correlated with insulin variation in this study. In adult patients with SIRS, hyperglycemia is frequently associated with high insulin and C-peptide levels because of a

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Pediatric cardiac surgery Arkader R et al.

Figure 1 - Insulin, glycemia, C-peptide, ADM, IL-6, and CRP levels throughout the study period. The shaded area in each diagram represents the normal range of the test. *indicates a statistically significant difference between the value at the study time point and the baseline value (Wilcoxon).

peripheral resistance to insulin (2). In contrast, Vignewaran et al. described reduced insulin levels in children after cardiac surgery with CPB, which supported our findings; however, the authors did not monitor the other laboratory parameters (25). Bialkowski et al. reported hyperglycemia accompanied by insulin reduction during and after cardiac surgery with CPB in children; however, the authors did not use MP. In contrast to our findings, the C-peptide levels were reduced in the study along with insulin levels (3), most likely because of more pronounced and/or prolonged hyperglycemia.

As expected in pediatric patients after cardiac surgery with CPB and MP, glycemia levels increased and insulin levels decreased in all 19 patients and reached maximum and minimum levels at the POD1 time point, respectively. The glycemia and insulin levels returned to baseline levels at the POD3 and POD2 time points, respectively. These findings were in agreement with those of other studies (26). During this study, all of the patients had glycemia that exceeded 125 mg.dL-1, which was the threshold adopted in this study; however, these values were higher than 150 mg/

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Table 1 - Statistical analysis results show the coefficient (Coef), the standard error (Std err.), the p value, the 95% confidence interval, the variance inflation factor (VIF), the 1/VIF, the Ă&#x; coefficient, and the coefficients of the semipartial determination (spR2) of the four independent variables (age, glycemia, ADM and IL-6) that were significantly correlated with insulin variation.

Age (months) Glycemia variation (mg.dL-1) ADM variation (ng.mL-1) IL-6 variation (pg.mL-1) Intercept

Coef.

Std. Err.

p-value

0.14 0.03 0.29 -0.17 -16.34

0.03 0.01 0.1 0.05 1.59

,0.001 0.01 0.009 0.007 ,0.001

95% CI 0.08 0.01 0.09 -0.28 -19.74

dL-1. None of the patients received insulin, most likely because the hyperglycemia levels were not persistently high. Decisions related to patient care were made by the attending physicians at the Heart Institute without interference from the researchers. In this study, it is likely that the main factor associated with hyperglycemia was a single high dose of MP, which was administered in all of the children; this result supported previous data (5). The decreased insulin response after cardiac surgery with CPB in these children may be caused by the greater vulnerability to beta cell dysfunction that is observed in critically ill children compared with adults. Therefore, adults who are subjected to cardiac surgery with CPB evolve with hyperglycemia and resistance to insulin, whereas insulin concentrations are reduced in the majority of children. Preissig and Rigby (8) hypothesized that beta cells are extremely sensitive to rapid physiological changes; therefore, these cells may become dysfunctional if these changes occur quickly and exceed a certain threshold. Changes may be induced by multiple factors, such as hypothermia during CPB, vasopressors to maintain blood pressure, elevations of proinflammatory cytokines, including IL-6, and the use of glucocorticoids, such as MP (1,9,12,25). Excluding the use of vasopressors, all of these factors were present in this study. The acute and temporary nature of the physiological disturbance that is induced by SIRS may explain the low insulin levels and the unaltered Cpeptide levels. In this context, the C-peptide is a more stable marker of beta cell function than insulin because of its longer half-life, lower first-pass hepatic extraction and lack

-

0.19 0.05 0.5 -0.05 -12.94

VIF

1/VIF

Ă&#x; coef.

SP R2

1.23 1.18 1.09 1.03

0.81 0.84 0.92 0.97

0.82 0.43 0.43 -0.48

0.55 0.17 0.18 0.2

of adherence to the CPB circuit (4,27). Because of this stability and the temporal nature of the hyperglycemia that was triggered by SIRS and aggravated by MP in this study, the C-peptide levels were not as reduced as the insulin levels; therefore, this marker could not be used to predict insulin behavior. Nevertheless, the monitoring of C-peptide levels was useful and could have led to the medical decision not to administer insulin to patients. Regarding the CRP and IL-6 levels, there were similar variations in these two parameters, including a significant increment after surgery and a return to baseline levels at the POD3 time point. According to the multivariate analysis, the insulin reduction was directly proportional to patient age and glycemia, whereas the ADM and IL-6 increments followed the magnitude of inflammation (28). To elaborate the mathematical model, we arbitrarily included the IL-6 levels instead of the CRP levels. However, these markers demonstrated similar behavior. Because only CRP levels are routinely evaluated after cardiac surgery in pediatric intensive care units, this parameter could replace the IL-6 levels in the model. Several studies in adults have found that ADM concentrations increased during CPB (17,29). Komai et al. were the first to describe increased ADM levels in 14 children after cardiac surgery with CPB (29). This trend was later confirmed by Szekely et al., Takeuchi et al. and Sekine et al. (18). These authors found that ADM can inhibit insulin/C-peptide exocytosis via the activation of G proteins that are located on the insulin/C-peptide cellular receptor. We observed a slight elevation in the ADM concentrations at the BT time point; however, the children in our study were not healthy because of

Figure 2 - The diagram on the left shows the observed insulin values and the predicted values that were calculated using the multiple regression equation. The diagram on the right shows that the 95% limit of agreement for the observed and predicted insulin values was within an interval of -2.76 to+2.76 mU.L-1 (Bland and Altman).

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Pediatric cardiac surgery Arkader R et al. (adrenomedullin and interleukin). Okay TS conceived the study, oversaw the writing of the article, and was responsible for obtaining the funding from FAPESP and the doctoral thesis orientation.

previous heart conditions, and this finding was expected. In addition, a strong ADM increment occurred after CPB, which was accompanied by a decrement in insulin levels but not in C-peptide concentrations. The latter is secreted in equimolar amounts with insulin; therefore, ADM may have temporarily affected insulin release but did not inhibit C-peptide exocytosis or the inhibition was not strong enough to reduce the C-peptide concentrations in this study. We expected to observe C-peptide levels below the reference interval. The results of this study were based on a convenience sample of 19 children; however, the strict application of the study inclusion criteria and the homogeneity of the patient heart conditions enabled us to evaluate the role of all of the laboratory parameters during SIRS. Therefore, the semipartial R2 analysis indicated that patient age, glycemia, and the ADM and IL-6 levels were independent factors that were associated with insulin variation. These findings were confirmed in the multiple regression analysis with the Bonferroni correction, which demonstrated that these four parameters were responsible for 64.7% of the observed insulin variance. Although significant, this percentage indicates that childhood SIRS that is induced by cardiac surgery with CPB and MP and leads to hyperglycemia and low insulin levels is a complex process that involves more factors than those investigated in this study. In our patients, the insulin levels decreased, but there were no significant variations in the C-peptide levels during the study. Therefore, the insulin levels were normal in these children, which indicated that hyperglycemia did not persist for a long time period because glycemia returned to BT levels at the POD3 time point, and the insulin concentrations returned to BT levels one day earlier (POD2). Considering the potential hazards of iatrogenic hypoglycemia that is caused by exogenous insulin and the deleterious effects of hyperglycemia in these patients, insulin administration should be considered when a concomitant reduction in the insulin and C-peptide levels occurs in children with sustained hyperglycemia after cardiac surgery with CPB and MP. Therefore, the equation proposed in this study, modified or not by the replacement of IL-6 by CRP, and of ADM for another inflammation or metabolic marker could be useful to predict most of the insulin variation in pediatric intensive care units. In addition, the monitoring of insulin and C-peptide levels in parallel with glycemia is useful in the medical decision not to administer insulin to patients. Therefore, insulin and C-peptide exams are advised for these patients.

& REFERENCES 1. Oberhoffer M, Karzai W, Meier-Hellmann A, Bogel D, Fassbinder J, Reinhart K. Sensitivity and specificity of various markers of inflammation for the prediction of tumor necrosis factor-alpha and interleukin-6 in patients with sepsis. Crit Care Med. 1999;27(9):1814-8, http://dx.doi. org/10.1097/00003246-199909000-00018. 2. Rapp-Kesek D, Stridsberg M, Andersson LG, Berne C, Karlsson T. Insulin resistance after cardiopulmonary bypass in the elderly patient. Scand Cardiovasc J. 2007;41(2):102-8, http://dx.doi.org/10.1080/14017 430601050355. 3. Bialkowski J, Rubi J, Valino JM, Sanchez PA, Dominguez F, Alonso A. [Glucose metabolism in children undergoing extracorporeal circulation: its correlation with weight and the degree of hypothermia]. Rev Esp Cardiol. 1997;50(11):782-9. 4. Van Cauter E, Mestrez F, Sturis J, Polonsky KS. Estimation of insulin secretion rates from C-peptide levels. Comparison of individual and standard kinetic parameters for C-peptide clearance. Diabetes. 1992;41(3):368-77, http://dx.doi.org/10.2337/diabetes.41.3.368. 5. Scohy TV, Golab HD, Egal M, Takkenberg JJ, Bogers AJ. Intraoperative glycemic control without insulin infusion during pediatric cardiac surgery for congenital heart disease. Paediatr Anaesth. 2011;21(8):872-9, http://dx.doi.org/10.1111/j.1460-9592.2011.03571.x. 6. Clarizia NA, Manlhiot C, Schwartz SM, Sivarajan VB, Maratta R, Holtby HM, et al. Improved outcomes associated with intraoperative steroid use in high-risk pediatric cardiac surgery. Ann Thorac Surg. 2011;91(4):12227, http://dx.doi.org/10.1016/j.athoracsur.2010.11.005. 7. Vlasselaers D, Milants I, Desmet L, Wouters PJ, Vanhorebeek I, van den Heuvel I, et al. Intensive insulin therapy for patients in paediatric intensive care: a prospective, randomised controlled study. Lancet. 2009;373(9663):547-56, http://dx.doi.org/10.1016/S0140-6736(09)600441. 8. Preissig CM, Hansen I, Roerig PL, Rigby MR. A protocolized approach to identify and manage hyperglycemia in a pediatric critical care unit. Pediatr Crit Care Med. 2008;9(6):581-8, http://dx.doi.org/10.1097/PCC. 0b013e31818d36cb. 9. Verhoeven JJ, Hokken-Koelega AC, den Brinker M, Hop WC, van Thiel RJ, Bogers AJ, et al. Disturbance of glucose homeostasis after pediatric cardiac surgery. Pediatr Cardiol. 2011;32(2):131-8, http://dx.doi.org/10. 1007/s00246-010-9829-z. 10. Van den Berghe G. Coronary bypass surgery: protective effects of insulin or of prevention of hyperglycemia, or both? J Clin Endocrinol Metab. 2011;96(5):1272-5, http://dx.doi.org/10.1210/jc.2011-0683. 11. Van Herpe T, Gielen M, Vanhonsebrouck K, Wouters PJ, Van den Berghe G, De Moor B, et al. Assessment of blood glucose control in the pediatric intensive care unit: extension of the glycemic penalty index toward children and infants. J Diabetes Sci Technol. 2011;5(2):353-7. 12. Agus MS, Steil GM, Wypij D, Costello JM, Laussen PC, Langer M, et al. Tight glycemic control versus standard care after pediatric cardiac surgery. N Engl J Med. 2012;367(13):1208-19. 13. Dhar A, Castillo L. Insulin resistance in critical illness. Curr Opin Pediatr. 2011;23(3):269-74, http://dx.doi.org/10.1097/MOP.0b013e328 3464b3e. 14. Verhoeven JJ, den Brinker M, Hokken-Koelega AC, Hazelzet JA, Joosten KF. Pathophysiological aspects of hyperglycemia in children with meningococcal sepsis and septic shock: a prospective, observational cohort study. Crit Care. 2011;15(1):R44, http://dx.doi.org/10.1186/ cc10006. 15. Knapik P, Nadziakiewicz P, Urbanska E, Saucha W, Herdynska M, Zembala M. Cardiopulmonary bypass increases postoperative glycemia and insulin consumption after coronary surgery. Ann Thorac Surg. 2009;87(6):1859-65, http://dx.doi.org/10.1016/j.athoracsur.2009.02.066. 16. Rubens FD, Nathan H, Labow R, Williams KS, Wozny D, Karsh J, et al. Effects of methylprednisolone and a biocompatible copolymer circuit on blood activation during cardiopulmonary bypass. Ann Thorac Surg. 2005;79(2):655-65, http://dx.doi.org/10.1016/j.athoracsur.2004.07.044. 17. Nagata N, Kitamura K, Kato J, Naruo H, Eto T, Takasaki M. The effect of hypothermic cardiopulmonary bypass on plasma adrenomedullin in adult cardiac surgical patients. Anesth Analg. 1997;84(6):1193-7. 18. Sekine N, Takano K, Kimata-Hayashi N, Kadowaki T, Fujita T. Adrenomedullin inhibits insulin exocytosis via pertussis toxin-sensitive G protein-coupled mechanism. Am J Physiol Endocrinol Metab. 2006;291(1):E9-E14, http://dx.doi.org/10.1152/ajpendo.00213.2005. 19. Martinez A, Weaver C, Lopez J, Bhathena SJ, Elsasser TH, Miller MJ, et al. Regulation of insulin secretion and blood glucose metabolism by adrenomedullin. Endocrinology. 1996;137(6):2626-32, http://dx.doi. org/10.1210/en.137.6.2626.

& ACKNOWLEDGMENTS This study was supported by the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), grant number 2006/59214-6.

& AUTHOR CONTRIBUTIONS Arkader R co-wrote the article and was responsible for collecting all of the biological material during the five study days, conducting the follow-up of patients and collecting the medical records to query the results of the lab tests, which were performed at the INCOR. Malbouisson LM co-wrote the article and assisted in the description of this particular portion of the study and was our statistics expert. Del Negro GM was responsible for supervising the laboratory tests that were not included among the routine laboratory tests in the study, specifically the adrenomedullin and interleukin-6 tests. She oversaw the completion of routine tests, such as blood glucose and hormone, at the clinical laboratory of the Instituto da Crianc¸a. Yamamoto L conducted the non-routine laboratory tests

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20. Royston JP. A Simple Method for Evaluating the Shapiro-Francia W’ Test of Non-Normality. Journal of the Royal Statistical Society Series D (The Statistician). 1983;32(3):297-300. 21. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet. 1986;1(8476):30710, http://dx.doi.org/10.1016/S0140-6736(86)90837-8. 22. Frigge M, Hoaglin DC, Iglewicz B. Some Implementations of the Boxplot. The American Statistician. 1989;43(1):50-4. 23. Brealey D, Singer M. Hyperglycemia in critical illness: a review. J Diabetes Sci Technol. 2009;3(6):1250-60. 24. Kong AN, Jungbluth GL, Pasko MT, Beam TR, Jusko WJ. Pharmacokinetics of methylprednisolone sodium succinate and methylprednisolone in patients undergoing cardiopulmonary bypass. Pharmacotherapy. 1990;10(1):29-34. 25. Vigneswaran VT, Pollock JCS, Jamieson MPG, Torsney B, Beastal GH. Plasma levels of glucose, insulin and cortisol in children undergoing

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cardiac surgery: effects of pulsatile and nonpulsatile perfusion. Perfusion. 1989;4(1):33-9, http://dx.doi.org/10.1177/026765918900400 105. Moga MA, Manlhiot C, Marwali EM, McCrindle BW, Van Arsdell GS, Schwartz SM. Hyperglycemia after pediatric cardiac surgery: impact of age and residual lesions. Crit Care Med. 2011;39(2):266-72, http://dx.doi. org/10.1097/CCM.0b013e3181fee88e. Urban K, Redford D, Larson DF. Insulin binding to the cardiopulmonary bypass biomaterials. Perfusion. 2007;22(3):207-10, http://dx.doi.org/10. 1177/0267659107081632. Cheung BM, Ong KL, Tso AW, Leung RY, Cherny SS, Sham PC, et al. Plasma adrenomedullin level is related to a single nucleotide polymorphism in the adrenomedullin gene. Eur J Endocrinol. 2011;165(4):571-7. Komai H, Naito Y, Fujiwara K, Noguchi Y, Nishimura Y. Plasma adrenomedullin level after cardiopulmonary bypass. Perfusion. 1998;13(5):334-7.


BASIC RESEARCH

Fenitrothion induced oxidative stress and morphological alterations of sperm and testes in male spraguedawley rats Izatus Shima Taib,I Siti Balkis Budin,I Ahmad Rohi Ghazali,I Putri Ayu Jayusman,I Santhana Raj Louis,II Jamaludin MohamedI I

Programme of Biomedical Science, School of Diagnostic and Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia. II Unit of Electron Microscope, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia.

OBJECTIVE: Fenitrothion residue is found primarily in soil, water and food products and can lead to a variety of toxic effects on the immune, hepatobiliary and hematological systems. However, the effects of fenitrothion on the male reproductive system remain unclear. This study aimed to evaluate the effects of fenitrothion on the sperm and testes of male Sprague-Dawley rats. METHODS: A 20 mg/kg dose of fenitrothion was administered orally by gavages for 28 consecutive days. Blood sample was obtained by cardiac puncture and dissection of the testes and cauda epididymis was performed to obtain sperm. The effects of fenitrothion on the body and organ weight, biochemical and oxidative stress, sperm characteristics, histology and ultrastructural changes in the testes were evaluated. RESULTS: Fenitrothion significantly decreased the body weight gain and weight of the epididymis compared with the control group. Fenitrothion also decreased plasma cholinesterase activity compared with the control group. Fenitrothion altered the sperm characteristics, such as sperm concentration, sperm viability and normal sperm morphology, compared with the control group. Oxidative stress markers, such as malondialdehyde, protein carbonyl, total glutathione and glutathione S-transferase, were significantly increased and superoxide dismutase activity was significantly decreased in the fenitrothion-treated group compared with the control group. The histopathological and ultrastructural examination of the testes of the fenitrothion-treated group revealed alterations corresponding with the biochemical changes compared with the control group. CONCLUSION: A 20 mg/kg dose of fenitrothion caused deleterious effects on the sperm and testes of SpragueDawley rats. KEYWORDS: Morphology; Oxidative; Organophosphate; Sperm; Stress; Testes. Taib IS, Budin SB, Ghazali AR, Jayusman PA, Louis SR, Mohamed J. Fenitrothion induced oxidative stress and morphological alterations of sperm and testes in male sprague-dawley rats. Clinics. 2013;68(1):93-100. Received for publication on May 24, 2012; First review completed on June 26, 2012; Accepted for publication on August 31, 2012 E-mail: balkis6466@yahoo.com.my Tel.: 603-9289 7645

cholinesterase activity (1). It is employed in agriculture to control insects and mites that affect cereals, rice, fruits, vegetables, stored grains and cotton (2). FNT also acts against flies, mosquitoes and cockroaches and has been successfully used as a vector control agent for malaria in public health programs (1). The widespread use of FNT insecticides has also caused the release of the parent compound and its metabolites into natural water sources, such as rivers and ponds, thus categorizing it as a common river water pollutant (3). It has been classified on the ‘‘red list’’ by the United Kingdom Environment Agency as one of the most dangerous substances to the aquatic environment (4). The presence of FNT in the environment, such as in soil, water and food products, can also lead to toxic effects on non-target organisms (3). Humans are potentially exposed to FNT either directly through occupational exposure or indirectly via food

& INTRODUCTION Pesticides such as organophosphates have been widely used in agriculture to enhance the quality of food products. They have also been utilized in public health efforts to control vector diseases by eradicating unwanted insects. Fenitrothion [O,O-dimethyl-O-(3-methyl-4-nitrophenyl) phosphorothioate] (FNT) is a broad-spectrum organophosphate insecticide that distresses the nervous system by inhibiting acetyl

Copyright ß 2013 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. DOI: 10.6061/clinics/2013(01)OA15

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consumption. Previous research has found that various concentrations of FNT caused histopathological effects on the liver and kidneys of rats, cytotoxic effects on the lungs of rats and immunosuppressive effects in rats (5,6). Furthermore, after a seven-day exposure of FNT at doses of 15 and 30 mg/kg, the compound acted as an antiandrogen that could reduce the growth of androgendependent tissues in rats (7). However, the pathophysiological effect of FNT on sperm and testes remains unclear and underreported. These toxic effects likely occur through the generation of reactive oxygen species (ROS) that may be produced as a result of the FNT metabolism by cytochrome P450s or due to the high-energy consumption coupled with the inhibition of oxidative phosphorylation (8). The imbalance between the formation of ROS and mechanism of enzymatic and nonenzymatic antioxidants as a body defense system can lead to oxidative stress. Oxidative stress has been reported to be the primary mechanism of organophosphate toxicity after prolonged exposure (9). In addition, oxidative stress conditions may cause alterations in sperm cells due to the high levels of polyunsaturated fatty acid (PUFA) in their plasma membrane (10). FNT at a dose of 20 mg/kg body weight (bw) by oral administration caused significant damage to the liver and kidneys (5) and changes in the histology of lungs (6) in male albino rats. Therefore, this study aimed to assess the potential effects of FNT at a dose of 20 mg/kg bw on the reproductive parameters of male Sprague-Dawley rats to provide a potential extrapolation of the findings to humans. The purpose of this study was to determine the effects of FNT on sperm characteristics, sperm and testicular morphology, biochemical and oxidative stress markers in testes of male Sprague-Dawley rats.

Animal treatment schedule After the one-week acclimatization, the rats were divided randomly into control and treated groups, with ten rats per group. FNT (dissolved in corn oil) was administered at a dose of 20 mg/kg bw (1/3 LD50) using oral gavages for 28 consecutive days (five days/week), whereas the control group received only corn oil (1 mg/kg bw). Toxicity signs were observed and recorded daily. Body weight was recorded weekly throughout the study duration. At the end of the study, the rats were fasted overnight and anaesthetized with diethyl ether. Blood was collected in an EDTA tube via cardiac puncture. Plasma was obtained by centrifugation of blood samples at 3,500 rpm at 4 ˚C for 10 minutes. Plasma samples were maintained at -40 ˚C until further biochemical analysis. At the end of the experiment, the rats were sacrificed and dissected. The weights of the cauda epididymis, epididymis and testes were recorded. Left and right of cauda epididymis were immediately placed in 2 ml of Hank’s Buffered Salt Solution (HBSS) enriched with 0.5% BSA and pre-warmed at 37 ˚C to obtain the sperm. The cauda epididymis was cut into small pieces and centrifuged at 1,000 rpm at 4 ˚C for 3 minutes to allow the sperm to be released for further analysis. Testes were homogenized in 0.15 M KCl solution (1:2) at 4 ˚C and centrifuged at 10,000 X g for 20 minutes at 4 ˚C. The homogenated testes were maintained at -40 ˚C until further biochemical analysis.

Biochemical analysis Plasma cholinesterase (ChE) was assayed using the Ellman method (11). The cholinesterase in the samples hydrolyzed the propionythiocholine to form thiocholine, which was then reacted with 5,59-dithiobis-2-nitrobenzoic acid (DTNB) to form a yellow solution containing 5-thio-2nitrobenzoate. The formation of the yellow color was monitored at 405 nm and the cholinesterase activity was calculated in U/l. Protein and cholesterol levels in the testes were determined by the Bradford (12) and Franey and Amador (13) methods, respectively. Briefly, an aliquot of the homogenated testes was added to Bradford’s reagent and incubated for 5 minutes at room temperature. The protein reacted with Bradford’s reagent to produce colored complexes and the intensity of these complexes was measured at 595 nm using a microplate reader. Similarly, cholesterol detection was based on the reaction between cholesterol, ferric chloride and concentrated sulfuric acid. Briefly, 100 ml of homogenate testes was added to ethanol for cholesterol extraction purposes. The obtained supernatant was treated with ferric chloride and sulfuric acid. The concentration of cholesterol in each sample was determined at 560 nm, where it exhibited a color change from yellow to brown. The protein and cholesterol levels were calculated based on their standard curves in mg/dl and mg/ml, respectively. The homogenated testes were evaluated for malondialdehyde (MDA), protein carbonyl (PC), total glutathione (GSH), glutathione S-transferase (GST) and superoxide dismutase (SOD) to assess the redox processes. The production of MDA was measured for lipid peroxidation based on the Stocks and Dormandy methods (14). Briefly, an aliquot of (0.5 ml) of homogenated testes was added to a TCA/HCl solution, vortexed and incubated at room temperature for 15 minutes. The mixture was added to TBA/NaOH, vortexed and heated in a boiling water bath

& MATERIALS AND METHODS Animals Twenty adult male Sprague-Dawley rats weighing 230250 g were used in this study. All rats were obtained from the Laboratory Animal Resource Unit, Faculty of Medicine, Universiti Kebangsaan Malaysia, one week prior to the study for acclimatization purposes. The rats were kept in plastic cages, with two rats per cage under standard environmental conditions (12 h light/dark cycles, 25-28 ˚C) and maintained on a standard pellet and water ad libitum diet for the duration of the study. All of the animal handling protocols were approved by the Animal Ethics Committee of Laboratory Animal Resource Unit, Faculty of Medicine, Universiti Kebangsaan Malaysia.

Chemicals The chemicals and reagents used in this study were of high purity. FNT with a purity of 99.9% was purchased from SUPELCO Analytical, USA with lot number: LB75917. Other chemicals used in this study were purchased from Sigma Chemicals Co., St. Louis, Missouri, USA, with the exception of 2,4-nitrophenylhydrazine, which was purchased from Mallinckrodt Chemicals, Lenzing AG, Salzburg, Austria. Nitro blue tetrazolium, reduced glutathione and other reagents were purchased from Merck, Darmstadt, Bundesland of Hesse, Germany.

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(100 ˚C) for 30 minutes. The MDA in the sample reacted with the thiobarbituric acid to form a pink chromogen containing thiobarbituric acid reactive substances (TBARS). The level of TBARS in the supernatant was measured using a spectrophotometer at 532 nm. The MDA concentration was expressed as mM/mg of tissue protein. The PC content was measured for oxidative stress in the protein precipitate using the method described by Levine (15), with some modifications. Briefly, the homogenated testes were added to TCA (1:1) to precipitate the protein, incubated in ice for 15 minutes and then centrifuged at 15,000 X g for 5 minutes at 4 ˚C. The obtained pellet was reacted with 10 mM DNPH and incubated for 1 hour at room temperature in the dark. TCA was added to the mixture to precipitate the protein; the mixture was incubated in ice for 5 minutes and then centrifuged at 15,000 X g for 5 minutes at 4 ˚C. The obtained pellet was then washed twice using a mixture of ethanol and ethyl acetate and then diluted with 5 M urea. The derivatives obtained after centrifugation at 15,000 X g for 3 minutes at 4 ˚C were monitored between 375 and 380 nm. PC formation was expressed as nmol/mg of tissue protein. GSH content was measured in the homogenated testes based on the Ellman method (16), with some modification. Deproteinized homogenated testes were treated with a metaphosphoric acid solution and centrifuged at 3,000 X g for 10 minutes at 4 ˚C. The obtained supernatant was mixed with a reaction buffer at pH 8.0 and DTNB for 15 minutes and measured at 412 nm by using a microplate reader. The GSH content was expressed as mmol/mg protein. SOD and GST enzyme activity was determined using the Beyer and Fridovich (17) and Habig and Jacoby (18) methods, respectively. Briefly, an aliquot of (20 ml) of the homogenate was mixed with the substrate containing [PBS(EDTA); L-methionine; NBT.2HCl; Triton-X] and riboflavin. The mixture was incubated in an aluminum box under 20 watt lamp for 7 minutes. The SOD activity was measured spectrophotometrically by monitoring the inhibition of ferricytochrome reduction using a xanthine-xanthine oxidase as a source of peroxides. An SOD unit is defined as one unit of enzyme, which inhibits 50% of the nitro blue tetrazolium (NBT) reduction. SOD activity was calculated and expressed in U/mg protein. GST was assayed by measuring the rate that the enzyme catalyzed the conjugation of reduced glutathione with CDNB. Briefly, 50 ml of homogenated testes was added to 1 mM GSH and 75 mM of 1-chloro-2,4-dinitro benzene (CDNB). The rate of GST activity was measured at 340 nm and expressed as U mM/ min/mg protein.

Sperm density and morphology Sperm density was calculated by a Markler Counting Chamber based on a WHO method (19). Briefly, 10 ml of a sperm suspension was calculated for at least ten different microscopic fields under 40X magnification using a light microscope. Results are expressed as 106 cells/ml. A smear was conducted by using a drop of sperm suspension. After the smear had dried, the slide was fixed with absolute ethanol for 5 minutes. Then, the slide was immersed in Diff-Quik Stain I and II for 5 minutes each. A cover slip was applied and two hundred sperms were calculated per animal to measure the morphological abnormalities under oil immersion. The abnormal sperm heads were calculated in triplicate. Data are shown as a percentage of sperm head abnormalities.

Morphological analysis The testes in both groups were treated with 10% buffered formalin solution and a routine histological procedure was conducted. All testicular sections were stained with hematoxylin and eosin (H & E) stains and monitored for morphological changes under X10 and X40 magnifications. All changes were verified by a histopathological expert. A section of the testes was cut into small pieces (1 mm3), treated with 2.5% glutaraldehyde 0.1 N PBS at room temperature for one hour and post-fixed with osmium tetraoxide for another hour. Testes tissue was dehydrated in 70, 90 and 100% (twice) acetone for five minutes each, followed by 1:1 (acetone: resin) for five minutes and then embedded in epoxy resin. Ultrathin slices (90 nm) were observed using a transmission electron microscope, Tecnai G2 (FEI, USA), at 100 kV.

Statistical analysis All obtained data from the analysis were normally distributed. The differences between the treated and control groups were statistically evaluated using an independent Student’s t-test. All data are expressed as the mean values¡SD, with significant values at p,0.05.

& RESULTS Signs of toxicity Administration of a 20 mg/kg bw dose of FNT caused cholinergic signs, such as hypoactivity, lacrimation, piloerection and tremor. All of the toxicity signs occurred within one or two hours after FNT administration. The toxicity signs remained in the same animals for approximately 48 hours throughout the experimental period. Approximately 60% of treated rats showed signs of toxicity. No deaths were recorded in either experimental group.

Sperm viability

Body and organ weight

Sperm viability was determined by a routine gold standard method suggested by WHO (19) involving the determination of eosin, which penetrates the membranes of damaged cells. Approximately 100 ml of sperm suspension was mixed with two drops of eosin 1% and allowed to remain undisturbed for 30 seconds. Then, three drops 10% nigrosin were mixed with the solution and within 30 seconds, a thick smear was performed in triplicate. The results were determined by counting both motile and immotile sperm and are presented as a percentage (%).

The body and organ weight of the FNT and control groups are provided in Table 1. The treated and control groups showed increased body weight throughout the experimental period. However, the rats treated with FNT showed a significantly lower weight-gain compared with the control group (p,0.05). The absolute and relative epididymis weight decreased significantly for the FNT group compared with the control group, with p,0.05 and p,0.001, respectively. The relative weight of the testes was significantly higher in the FNT group than control group

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Table 1 - Body and reproductive organ weights of the treated and untreated groups. Data are reported as the mean values¡SD. Statistically significant differences compared with the control group are reported when p,0.05a and p,0.01b. Weight (g)

Initial Body Weight End of Body Weight Weight Gain Absolute Weight Testes Epididymis Cauda Epididymis Relative Weight Testes Epididymis Cauda Epididymis

Table 3 - Sperm analysis of the treated and untreated groups. Data are reported as the mean values¡SD. Statistically significant differences compared with the control group are reported when p,0.05a. Experimental Groups

Experimental Groups

Control

FNT (20 mg/kg bw)

243.1¡14.64 326.9¡26.01 83.30¡7.59

236.3¡13.40 295.9¡23.81a 59.60¡8.57a

2.54¡0.45 0.94¡0.35 0.40¡0.15

2.68¡0.23 0.53¡0.08a 0.39¡0.03

0.77¡0.14 0.34¡0.07 0.12¡0.05

0.91¡0.09a 0.18¡0.03b 0.13¡0.02

Sperm density (X106 cells) Sperm non-viability (%) Abnormal sperm morphology (%)

Control

FNT (20 mg/kg bw)

68.77¡3.69 7.24¡1.46 11.33¡0.99

35.71¡3.13a 38.61¡5.75a 57.33¡3.98a

Histological examination The normal histological structure of rat testes is illustrated in Figure 1 (A-C). The seminiferous tubule consists of normal somatic and spermatogenic cells and is surrounded by peritubular myoid cells (Figure 1B). Clusters of Leydig’s cells were observed in the intertubular space that is in close contact with blood vessels and lymphatic channels (Figure 1C). However, histopathological observations such as degeneration of germ cells, expansion of interstitial space, disarrangement of spermatogonia in seminiferous tubule, degeneration of Leydig cells and the presence of cellular debris throughout the lumen of the seminiferous tubule of rat testes were observed in the FNT group (Figure 1 D–F). The ultra-structure of the rat testis showed morphological alterations in the treated groups compared with the control group (Figure 2). Abnormalities included the presence of a lipid droplet, increased number of mitochondria, less chromatin condensation in the late spermatid and a vacuolated mitochondrial helix (Figure 2 D-F). However, histopathological and ultra-structure alterations were only observed in approximately 40% of the treated rat population.

(p,0.05). No statistically significant differences were noted in absolute and relative weight of cauda epididymis between the treated and untreated groups.

Biochemical analysis The results of the biochemical analysis are provided in Table 2. A marked reduction in the cholinesterase activity was found for the FNT group compared with the control group (p,0.05). However, testicular cholesterol and protein levels were not significantly different between the treated and untreated groups. FNT not only increased the formation of MDA (p,0.05) and PC (p,0.01) but also increased the content of GSH and activity of GST compared with the control group (p,0.05). However, SOD activity decreased in the FNT treated group compared with the control group (p,0.001).

& DISCUSSION Sperm parameters

The extensive use of organophosphate in the environment has affected many non-target organisms, including humans. The high lipid solubility of fenitrothion may lead to the bioaccumulation of the parent compound and its toxic metabolites in the adipose tissue. The release of this compound from the fat storage could be enhanced in stressful situations, such as during illness and diet changes (20). The current toxicity scenarios have attracted the attention of researchers studying the toxic effects of organophosphate on the male reproductive system. FNT has been found to cause some reproductive abnormalities in Japanese sprayers, with a reduction in the sperm quality (21). This study showed that the administration of FNT at a dose of 20 mg/kg bw for 28 days in male Sprague-Dawley rats induced significant adverse effects, including toxicity signs and symptoms, alterations in the biochemical status, reductions in sperm quality and histopathological and ultrastructural changes in the testes. FNT administration inhibits the acetyl cholinesterase enzymes that cause acetylcholine accumulation in cholinergic synapses. Therefore, cholinergic signs were observed, including hypoactivity, lacrimation, piloerection and tremor. Significant inhibition of acetyl cholinesterase enzyme by FNT directly supports the common acute toxicity mechanism of organophosphate poisoning (1). These findings are generally consistent with the studies by Turner (22), who reported that the muscle tremor was

The sperm concentration, sperm viability and sperm head abnormality data are provided in Table 3. A significant decrease in sperm concentration was found in the FNT group (p,0.05). Furthermore, FNT significantly decreased sperm viability and normal sperm morphology compared with the control group (p,0.05). Table 2 - Biochemical and oxidative stress analysis of the treated and untreated groups. Data are reported as the mean values¡SD. Statistically significant differences compared with the control group are reported when p,0.05a, p,0.01b and p,0.001c. Experimental Groups

Plasma cholinesterase (U/ml) Protein Testes (mg/ml) Cholesterol Testes (mg/ml) SOD (U/mg protein) GSH (mmol/mg protein) GST (mM/min/mg protein) MDA (mM/mg protein) PC (nmol/mg protein)

Control

FNT (20 mg/kg bw)

0.975¡0.39 34.81¡1.90 0.22¡0.03 1.23¡0.11 0.013¡0.003 6.47¡0.36 0.060¡0.008 0.035¡0.009

0.208¡0.13b 31.96¡0.98 0.24¡0.02 0.62¡0.06c 0.035¡0.007a 8.28¡0.52a 0.130¡0.020a 0.096¡0.017b

SOD: superoxide dismutase; GSH: total glutathione; GST: glutathione Stransferase; MDA: malondialdehyde; PC: protein carbonyl.

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Figure 1 - Histology of the testes of treated and untreated group (H&E staining). Figure 1 (A–C): Normal histological structure of rat’s ) and interstitial tissue (*)(Figure 1A). The seminiferous tubule consists of testis. Normal structure of seminiferous tubules ( normal somatic, sperm (S) and spermatogenic cells (G) surrounded with peritubularmyoid cells (My)(Figure 1B). Clusters of Leydig’s cells (L) were observed in the intertubular space that was in close contact with blood vessels and lymphatic channels (Figure 1C). Figure 1(D– F): Histology of FNT rat testis showing: degenerative of germ cells (DG) and Leydig cells (DL), expansion of interstitsial space, cellular debris (CD), vacuoles (v) and disarrangement of spermatogonia throughout the lumen of seminiferous tubule. (Magnification A and D,X10; Magnification B, C, E and F,X40).

observed in rats treated with FNT at doses of 20 and 25 mg/ kg/day. A significant decrease in body weight gain was observed in the FNT treated group; this might be associated with the toxic symptoms, such as cholinergic signs. The reduced weight gained could be attributed to systemic toxicity in male rats (23). FNT significantly decreased epididymis weight but increased testis weight. In this study, the reduction in the weight of reproductive organs, such as the epididymis, could be due to the reduction of androgen availability (23). The increase in the weight of the testes induced by chemicals can be correlated with testicular swelling, followed by atrophy (24). Under pathological conditions, high levels of ROS induce lipid peroxidation in male reproductive system (25). FNT increased MDA and PC production, which could be used as

important biomarkers of lipid and protein peroxidation. These findings support a previous study that found that most of the organophosphate insecticides altered MDA levels in experimental animals, especially in rats (5). PUFA peroxidation in the sperm head leads to a loss of membrane fluidity and decrease in the activity of the membrane enzyme and ion channels, which in turn increases the sperm permeability, causing sperm morphological abnormalities and decreased sperm function (26). ROS may also initiate a chain of reactions that ultimately leads to apoptosis. The process of apoptosis may also be accelerated by ROS-induced DNA damage and ultimately decrease the sperm count (26). Decrease in spermatogenesis can explain the occurrence of sperm with excessive residual cytoplasm in the mid-piece, thus altering sperm morphology. The abnormal morphology of sperm may occur in the

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Figure 2 - Ultra structure of testes of treated and untreated group. Figure 2 (A–C): The normal ultra structure of control rat testis showing: dense of chromatin (K) in late spermatid and normal structure of sperm tail. Figure 2 (D-F): The ultra structure of FNT rats testis showing: presence of lipid droplet (L), increased number of mitochondria (M), less chromatin (K) condensation in late spermatid and vacuolated of mitochondriae helix (red arrow) (Bar A & D,5000 nm; Bar B & E,2000 nm; Bar C & F,1000 nm).

in both enzymatic and non-enzymatic antioxidants. Several mechanisms of enzymatic and non-enzymatic antioxidants in humans and animals can counteract the harmful effects caused by ROS (9). In this study, SOD activities significantly decreased in testicular tissues of the FNT-treated group. These findings have also been reported in rat tissues induced by other organophosphate insecticides (30). Furthermore, the activity of melatonin as an antioxidant was also found to decrease in rats exposed to 2.45 GHz microwave radiation (31). However, phosphodiesterase type 5 increased total antioxidant levels, explaining the acute beneficial effect of this compound in patients with erectile dysfunction (32). This study also found that FNT increased GSH content and GST activity. Although GSH is an early consequence of oxidative stress, the increase in the GSH content of testes treated with FNT could be due to the early stage of the detoxification process by GST. GST is an enzymatic

epididymis and seminiferous tubules (27). The above condition might explain the alterations in the sperm morphology of the FNT-treated group. During the maturation process of sperm, cytoplasmic enzymes are extruded at the final stages of maturation. Therefore, mature sperm can attain their characteristic morphology and prevent oxidative stress, thus altering sperm functions, such as viability (27). In this study, FNT decreased the percentage of sperm viability. Decreases in sperm production and viability and increases in abnormal sperm morphology are common hazardous effects of organophosphate insecticides on sperm parameters (28). However, the supplementation of Eurycoma longifolia Jack increased the epididymal sperm quality, such as sperm count and motility, of estrogen-treated rats (29). Due to the lack of the cytoplasmic enzyme in mature sperm, the male genital tract, including the testes and semen, are rich

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antioxidant that is involved in the detoxification of organophosphate insecticides to form a non-toxic product (33). Organophosphate insecticides have been reported to alter the biochemical status in experimental animals depending on the various types of organophosphates, exposures durations and doses (34). There is a slight difference in the total protein and cholesterol content in testes of the FNT group. Changes in protein and cholesterol levels in the testes have been attributed to the inhibition of the androgen concentration (34). The above findings were supported by the histopathological changes in sperm and testis induced by FNT. FNT caused pathological changes in seminiferous tubules following a 28-day exposure. The apoptosis induced by ROS helped remove abnormal germ cells in the testes and prevent their overproduction by activating caspases (10). This explained the degenerative nature of germ and Leydig’s cells found in the seminiferous tubule and interstitial tissues of the FNT-treated group. FNT also allowed the crossing of the blood-testes barrier, thereby decreasing the spermatogenesis. These findings supported the study performed by Turner (22), who found that FNT inhibited the maturation and differentiation of germ cells. Toxicants can directly disturb the function of Sertoli cells, thereby causing the disorganization of germ cells in the seminiferous tubule of the FNT-treated group. In this study, FNT increased the formation of vacuoles and number of mitochondria in the testes. Vacuoles can be described as an early stage of damage induced by any toxicants (35). In addition, the increase in the number of mitochondria may be attributed to the high-energy consumption of the redox process in testes (9). FNT also caused fewer chromatins in late spermatids and vacuolated mitochondria of the sperm tail. A decrease in the chromatin in late spermatids may be due to genetic abnormalities that occurred during the differential process in spermatogenesis (28). In conclusion, FNT at a dose of 20 mg/kg bw has a detrimental effect on the sperm and testes of SpragueDawley rats.

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8. 9. 10. 11. 12.

13. 14. 15. 16. 17.

& ACKNOWLEDGMENTS 18.

The authors would like to thank the Ministry of Higher Education and Universiti Kebangsaan Malaysia for research funds (UKM-GUP-2011126). We also extend our gratitude to the Director of Institute for Medical Research (IMR), Malaysia, for the permission to use the electron microscopy facilities. The authors gratefully acknowledge the staff of Programme of Biomedical Sciences, School of Diagnostic and Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, and Electron Microscopy Unit, Institute for Medical Research (IMR), Malaysia, for providing the research facilities. We would like to acknowledge all of the lecturers, researchers and those who directly or indirectly supported this research.

20.

& AUTHOR CONTRIBUTIONS

23.

19.

21. 22.

Taib IS performed the majority of the experiments and drafted the manuscript. Budin SB was the project leader and was responsible for the project design. Ghazali AR and Mohamed J revised the manuscript for important intellectual content. Jayusman PA and Louis SR performed some of the experiments. All authors read and approved the final manuscript.

24. 25.

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BASIC RESEARCH

Tamoxifen decreases the myofibroblast count in the healing bile duct tissue of pigs Orlando Hiroshi Kiono Siqueira,I Benedito Herani Filho,II Rafael Erthal de Paula,III Fa´bio Otero A´scoli,IV Antonio Cla´udio Lucas da No´brega,IV Angela Cristina Gouveˆa Carvalho,V Andre´a Rodrigues Cordovil Pires,V Nicolle Cavalcante Gaglionone,VI Karin Soares Gonc¸alves Cunha,VII Jose´ Mauro GranjeiroVIII I

Fluminense Federal University, Department of General and Specialized Surgery, Nitero´i/RJ, Brazil. II Sa˜o Paulo Federal University, Surgical Gastroenterology, Sa˜o Paulo/SP, Brazil. III Fluminense Federal University, School of Medicine, Nitero´i/RJ, Brazil. IV Fluminense Federal University, Biomedical Institute, Nitero´i/RJ, Brazil. V Fluminense Federal University, Pathology, Nitero´i/RJ, Brazil. VI Fluminense Federal University, Clinical Research Unit, Nitero´i/RJ, Brazil. VII Federal Fluminense University, Postgraduate Program in Pathology, Nitero´i, RJ/Brazil. VIII Federal Fluminense University - Clinical Research Unit, Nitero´i/RJ, Brazil.

OBJECTIVE: The aim of this study was to evaluate the effect of oral tamoxifen treatment on the number of myofibroblasts present during the healing process after experimental bile duct injury. METHODS: The sample consisted of 16 pigs that were divided into two groups (the control and study groups). Incisions and suturing of the bile ducts were performed in the two groups. Tamoxifen (20 mg/day) was administered only to the study group. The animals were sacrificed after 30 days. Quantification of myofibroblasts in the biliary ducts was made through immunohistochemistry analysis using anti-alpha smooth muscle actin of the smooth muscle antibody. Immunohistochemical quantification was performed using a digital image system. RESULTS: In the animals treated with tamoxifen (20 mg/day), there was a significant reduction in immunostaining for alpha smooth muscle actin compared with the control group (0.1155 vs. 0.2021, p = 0.046). CONCLUSION: Tamoxifen reduced the expression of alpha smooth muscle actin in the healing tissue after bile duct injury, suggesting a decrease in myofibroblasts in the scarred area of the pig biliary tract. These data suggest that tamoxifen could be used in the prevention of biliary tract stenosis after bile duct surgeries. KEYWORDS: Tamoxifen; Myofibroblasts; Biliary Wound Healing; Bile Duct Stricture; Bile Duct Injury. Siqueira OH, Herani Filho B, Paula RE, A´scoli FO, No´brega AC, Carvalho AC, et al. Tamoxifen decreases the myofibroblast count in the healing bile duct tissue of pigs. Clinics. 2013;68(1):101-106. Received for publication on May 19, 2012; First review completed July 4, 2012; Received for publication on September 21, 2012 E-mail: ohksiqueira@gmail.com Tel.: 55 21 2629-9025

When the bile duct has lost continuity after injury from cholecystectomy or other biliary operations, surgical reconstruction is the only feasible treatment option (11,12). Nevertheless, the management of major BDIs is a surgical challenge (13), even for experienced hepatobiliary surgeons. Due to the small caliber of the main bile duct, anastomosis is difficult to perform and favors the occurrence of stenosis, which is usually secondary to the inflammatory process and fibrosis (14). The prevalence of bile duct stenosis varies from 0.2-0.5% (4) and can potentially progress to cholangitis, biliary cirrhosis, portal hypertension, end-stage liver disease and death (15,16). Myofibroblast differentiation and activation are critical events in the pathogenesis of human fibrotic diseases (17), as is post-operative biliary stenosis. Myofibroblasts are present in large numbers and represent the main cause of scar contracture and the occurrence of fibrosis (18,19). These cells exhibit features that are intermediate between fibroblasts and smooth muscle cells; specifically, they produce collagen, they express alpha smooth muscle actin (a-SMA) and their differentiation and activation are induced by

& INTRODUCTION Cholecystectomy is one of the most commonly performed general surgery procedures in the world (1). With the advent of video laparoscopy, nearly 90% of all cholecystectomies are currently performed laparoscopically, which has resulted in an increased incidence of bile duct injuries (BDIs). The incidence has increased from 0.1-0.2% (open cholecystectomy) to 0.4-0.6% (video laparoscopy) (2,3). Despite their low prevalence, iatrogenic BDIs are significant with regard to their absolute numbers (4) and are important in terms of health care costs (5,6); furthermore, they are among the leading causes of negligence claims against surgeons (7-10). Copyright ß 2013 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. DOI: 10.6061/clinics/2013(01)OA16

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transforming growth factor-beta 1 (TGF-b1) (17). Studies have indicated that the expression of TGF-b1 in stenotic bile ducts is significantly higher than that in normal bile ducts, suggesting that TGF-b1 is a key factor in the prolonged healing process of the bile duct and in the proliferation of cicatrix (20,21). Tamoxifen is a synthetic nonsteroidal antiestrogen agent that exhibits antifibrotic properties and has been shown to successfully treat many fibrotic diseases (e.g., hypertrophic scars (22), keloids (22), encapsulating peritoneal sclerosis (23), retroperitoneal fibrosis (24), fibrosing mediastinitis (25), sclerosing cervicitis (25) and recurrent desmoid tumors (26,27)). It is believed that the antifibrotic property of tamoxifen is mainly due to its downregulation of TGF-b1 (22,28). Considering that tamoxifen may inhibit the increase in myofibroblasts during wound healing, the aim of this study was to experimentally investigate the effect of oral tamoxifen treatment on the number of myofibroblasts in the healing tissue after BDI.

Figure 1 - Repair stiches in the choledoco duct.

duodenum. In all of the animals, two repair stitches were placed (on each side of the future incision) using 6.0 polypropylene sutures (Figure 1). A longitudinal 5-mm incision was made in the anterior wall of the bile duct. This incision was measured using digital calipers, starting from a distance of 1 cm from the superior border of the first portion of the duodenum and extending in the cranial direction (Figure 2). These wounds were closed using four noncontinuous stitches that were distributed in an equidistant manner and placed using 6.0 polydioxanone sutures (Figure 3). To close the abdominal cavity, the musculoaponeurotic layer was sutured using simple running stitches of 2.0 vicryl sutures. The skin was closed using continuous, simple running 3.0 nylon sutures.

& MATERIALS AND METHODS This study was conducted with approval from the Ethics Committee in Animal Experimentation of Fluminense Federal University, Rio de Janeiro, Brazil.

Animals Female pigs (Sus scrofa domesticus) of the Large White breed that weighed between 20 and 32 kg were used in the experiments. The animals were kept under standard conditions (12 h/12 h day/night cycle), were fed a standard diet and received water ad libitum. For the sample size calculation, a pilot study was carried out in which tamoxifen was administered to three of the six animals. Considering an effect size of 0.65, a significance level of 5% (a) and a statistical test power of 80% (1-b), it was estimated that a minimum sample size of nine animals would be needed for each experimental group. Eighteen pigs were included in this study and divided into two groups: a control group without treatment (Group A) and an experimental group treated with oral tamoxifen (Group B).

Tamoxifen administration Following the surgery, the group B animals received tamoxifen at a dose of 20 mg/day orally for 30 days.

Data sampling The animals were euthanized 30 days after the surgery. After withholding food overnight, the animals were sedated intramuscularly with ketamine (5.0 mg/kg), xylazine (1.0 mg/kg) and acepromazine (0.1 mg/kg). Thiopental sodium (5%) was administered intravenously to induce anesthesia. When an animal was deeply anesthetized,

Anesthesia and analgesia The pigs were intramuscularly (IM) pre-medicated with ketamine (5 mg/kg), midazolam (0.5 mg/kg) and acepromazine (0.05 mg/kg). Anesthesia was induced with propofol (4 mg/kg). After orotracheal intubation, an epidural block was performed with bupivacaine 0.125% (10 mL) and morphine (0.1 mg/kg). The isoflurane concentration was maintained at 1.5%. Tramadol was administered at a dose of 2 mg/kg to the animals that exhibited pain during the post-operative period and required rescue analgesic. Oxytetracycline (15 mg/kg IM) was administered before and 48 hours after each surgery.

Surgical procedures The peritoneal cavity was accessed using a right subcostal incision. Dissection, ligation and sectioning of the cystic artery, together with the cystic duct, were performed. The gallbladder was dissected and removed. The hepatoduodenal ligament was opened and the bile duct was measured using digital calipers from a location that was 1.5 cm away from the superior border of the first portion of the

Figure 2 - Longitudinal incision made in the anterior wall of the biliary duct.

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Effect of tamoxifen on biliary wound healing Siqueira OH et al. TM

in the ImageScope software (Aperio Technologies, Vista, CA, USA). The pen tool was used to select the scar areas and to exclude the immunopositive vessels.

Statistics Statistical analysis was performed using the software SASH System, version 6.11 (SAS Institute, USA). The results were expressed as the median and interquartile intervals (Q1 and Q3). The Shapiro-Wilk test was employed to examine the normality of the data distribution. Comparisons of the PI and surgical data between the two groups (control and study) were performed using the Mann-Whitney test. The level of significance was set at 5%.

& RESULTS One animal from the control group died on the sixth day after surgery from a biliary fistula and was excluded from the study. Another pig was excluded from the experimental group due to its characterization as an outlier, it once had considerably higher PI value than did the other pigs: in the interval between Q3+1.5*AIQ and Q3+3*AIQ; Q1 and Q3 are the 1st and 3rd quartiles and AIQ corresponds to the interquartilic amplitude (Q3-Q1). Therefore, the results were analyzed and presented based on a total of 16 animals (eight in each group). One representative immunohistochemical image for alpha smooth muscle actin is shown in Figure 4. Table 1 provides the surgical data of the control and study groups. There were no statistically significant differences in any of the parameters evaluated (p.0.05), demonstrating the homogeneity of the groups according to the analyzed parameters. Table 2 shows the descriptive data for the PI values of the control and study groups and the corresponding p values (Mann-Whitney test). The data are expressed as the median and interquartile intervals (Q1-Q3). The study group exhibited a significantly lower PI than the control group (p = 0.046), as illustrated in Figure 5.

Figure 3 - Biliary duct wound closing using separate stitches.

potassium chloride (10%) was administered intravenously to stop the heartbeat. The bile duct from each animal was resected through a median abdominal incision. The resected bile ducts were opened longitudinally along the posterior wall and immersed in 10% buffered formalin. The tissues were routinely processed. Five-micron-thick sections were cut from paraffin blocks, stained with hematoxylin and eosin and submitted for immunohistochemistry analysis.

Immunohistochemical staining After dewaxing, the presence of a-SMA protein was assessed using immunohistochemistry by employing the polymer method (Mach 4, Biocare Medical, Concord, CA, USA). Endogenous peroxidase activity was blocked by incubation in 1.3% H2O2 in methanol at room temperature for 15 min. Antigen retrieval was performed by incubating samples in Trilogy Antigen Retrieval Solution (Cell Mark, Hot Springs, CA, USA) at 96 ËšC for 40 min. Non-specific protein binding was blocked by incubation with Dako Blocking Solution (Dako, Carpinteria, CA, USA) for 15 min at 37 ËšC. The sections were incubated for 1 hour at room temperature with a 1:200 dilution of the primary monoclonal antibody anti-a-SMA (clone 1A4, Cell Mark, Hot Springs, CA, USA). Visualization was performed by incubation in diaminobenzidine for 5 min. The sections were counterstained with Harris hematoxylin for 30 seconds. Between each step, the sections were washed in phosphate-buffered saline (PBS buffer). All of the incubations were carried out in humidified chambers to prevent evaporation. Histological sections from the uterus were used as a positive control. The negative control was performed by omitting the primary antibody.

& DISCUSSION This study investigated the effect of oral tamoxifen on the myofibroblast count in the healing tissue after BDI in an experimental pig model. To our knowledge, this is the first study to investigate the possible use of oral tamoxifen as a therapeutic option for reducing stenosis after bile duct reconstruction. We observed that the oral treatment of pigs with tamoxifen reduced the a-SMA expression in the healing tissue after BDI, suggesting a reduction of the number of myofibroblasts through the inhibition of TGF-b1 production. Because the biliary tracts of pigs are anatomically similar to those of humans, pigs have been used in many studies to evaluate biliary tract processes (29-32). Therefore, the results obtained in this study approximate the results that can be expected in humans (9) because the greater the physiological and anatomical similarity, the more applicable the conclusions (33). The arterial supply of the common hepatic duct and the bile duct have a longitudinal anastomotic chain arranged in a ladder form in humans (34). Therefore, we performed a longitudinal incision to maintain the best blood supply in the scarring area. Myofibroblasts are the principle cause of biliary tract stenosis (35). In BDIs, the exposure of the underlying stroma to bile appears to serve as a nidus for the crystallization of biliary sludge and a stimulus for inflammation and the

Image analysis The immunohistochemical quantification of alpha smooth muscle actin was performed using a digital image system (Aperio Technologies, Vista, CA, USA). All of the glass slides were scanned at an objective magnification of 40x TM CS scanning system (Aperio using a ScanScope Technologies, Vista, CA, USA). The positivity index (PI; positive area/total area) was calculated for each image within the scar area using the positive pixel count algorithm

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Figure 4 - Example of the immunostaining quantification using the digital imaging system (Aperio Technologies; USA). A. Tissue section showing the scar area marked with a pen. B. Scar area showing immunopositivity for alpha smooth muscle actin. Blood vessels were manually excluded using the pen tool. C. Results of the analysis: blue = no staining, yellow = low-intensity staining, orange = mediumintensity staining and red = high-intensity staining.

activation of myofibroblasts (19). This process may lead to biliary strictures due to wound contraction and fibrosis (19). In addition to a-SMA, myofibroblasts also express smooth muscle myosin isoforms, which are responsible for the contraction and/or motility of smooth muscle (36). Myofibroblasts also produce matrix proteins and additional growth factors in response to proinflammatory cytokines (36). After repair or scar formation, myofibroblasts are eliminated by apoptosis (36). In dogs, myofibroblasts appear one week after BDI, peak after three weeks and remain for a long period (35). Therefore, we chose to sacrifice the animals 30 days after the surgery to ensure that the peak number of myofibroblasts had been achieved.

In this study, myofibroblasts were identified using immunohistochemistry with an anti-a-SMA antibody. It is important to note that we used computerized digital image analysis procedures to calculate the immunopositive areas. Many studies use conventional pathologist-based manual scoring of the immunohistochemical results (37-39), but this approach suffers from a greater risk of interobserver and intraobserver variability (40). In a model of nephropathy in rats, tamoxifen exhibited an antifibrotic effect that, according to the authors, could be explained by its ability to reduce myofibroblast proliferation, as there was also a reduction of a-SMA expression (41). In another study, tamoxifen inhibited keloid fibroblast

Table 1 - Surgical data for the control and study groups. Variable

Group

Median

Q1

Q3

Weight at surgery (kg)

control study control study control study control study control study

26.5 27.0 33.3 33.3 7.31 7.53 5.59 5.01 55.0 58.5

25.0 24.8 31.0 27.8 6.07 6.99 5.30 3.67 46.0 54.5

28.7 30.3 36.8 35.0 10.43 8.15 6.65 5.96 71.0 65.0

Weight at necropsy (kg) Caliber of the bile duct at surgery (mm) Caliber of the bile duct at necropsy (mm) Duration of the surgery (min)

Q1: 1st quartile; Q3: 3rd quartile,

a

Mann-Whitney test.

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p-value 0.83 0.71 0.79 0.17 0.95

a


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straightforward and provide novel evidence for the modulatory role of tamoxifen in myofibroblast proliferation in biliary wound healing. Biliary duct reconstruction is complex and although a large variety of suggested reconstruction techniques and materials exist in the literature, the BDI treatment results are still not satisfactory [49]. Therefore, other options must be investigated to improve reconstruction outcomes. In conclusion, our results demonstrate that oral tamoxifen is a promising therapeutic option for reducing stenosis after bile duct reconstruction because it reduces the number of myofibroblasts present in the healing tissue. More studies must be undertaken to validate the inclusion of oral tamoxifen in clinical practice.

Table 2 - Positivity indices of the control and study groups. Group

Median

Q1

Q3

Control Study

0.2021 0.1155

0.1249 0.0873

0.3259 0.1607

Q1: 1s t quartile; Q3: 3rd quartile.

a

p-value

a

0.046

Mann-Whitney test.

proliferation and decreased collagen production by decreasing the expression of TGF-b1 (42). It is known that biliary epithelial cells and periductal myofibroblasts are closed linked because damage to the former leads to activation and proliferation of the latter (19). Various experimental models in rats have previously shown that estrogen plays a key role in the biology of cholangiocytes (43,44) because the administration of tamoxifen or ovariectomy reduces biliary tree growth and induces cholangiocytes apoptosis (45,46). This concept is consistent with the findings in humans that estrogen receptors are over-expressed in the cholangiocytes of primary biliary cirrhosis patients (47). The present results should be interpreted in light of the potential limitations of the study. First, it is not possible to know whether the modulatory effect of tamoxifen would be present in male animals because we only examined females. However, this deliberate choice provided us with a more homogeneous sample and enhanced the translational impact of the results, considering that there is a known higher prevalence of biliary gallstone disease in women. Second, because we addressed the biliary wound healing process after mechanical injury at a single one-month follow-up time point, it is not known whether tamoxifen exerts a long-lasting action. Further studies are needed to describe the time course of this modulatory action of tamoxifen. The one-month time point was chosen because myofibroblast proliferation reaches its peak after three weeks (35). Third, because our aim was specifically to investigate the number of myofibroblasts present during the biliary wound healing process, we did not obtain an overview of the entire wound healing process, including the changes in collagen. Although a more comprehensive approach would be desirable, the present results are

& AUTHOR CONTRIBUTIONS Siqueira OH, Herani Filho B, de Paula RE and No´brega AC designed the ´ scoli FO, Carvalho AC, Cunha KS, Pires AR and study. Siqueira OH, A Gaglionone NC performed the research. Siqueira OH and Cunha KSG analyzed the data. Siqueira OH, No´brega AC, Cunha KS and Granjeiro JM wrote the paper.

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Figure 5 - Box plot showing the positivity indexes (PIs) of the control and study groups.

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BASIC RESEARCH

Exercise training prevents skeletal muscle damage in an experimental sepsis model Carla Werlang Coelho,I,II,III Paulo R. Jannig,IV Arlete B. de Souza,V Hercilio Fronza, Jr.,V Glauco A Westphal,II Fabricia Petronilho,VI Larissa Constantino,VI Felipe Dal-Pizzol,VI Gabriela K. Ferreira,VI Emilio E. Streck,VI Eliezer SilvaI,VII I

Faculdade de Medicina da Universidade de Sa˜o Paulo, Anesthesiology Strict Sensus Post Graduation Program of the Medical School, Sa˜o Paulo/SP, Brazil. Universidade da Regia˜o de Joinville (UNIVILLE), Physical Education and Medicine Department, Joinville/SC, Brazil. III Universidade do Estado de Santa Catarina (UDESC-Joinville), Chemistry Department, Joinville/SC, Brazil. IV Faculdade de Medicina da Universidade de Sa˜o Paulo, Experimental Pathophysiology Department, Sa˜o Paulo/SP, Brazil. V Centro de Diagno´stico Anatomo Patolo´gicos (CEDAP), Pathology Department, Joinville/SC, Brazil. VI Universidade do Extremo Sul Catarinense (UNESC), Experimental Pathophysiology Laboratory, Criciu´ma/SC, Brazil. VII Hospital Israelita Albert Einstein, Intensive Care Unit, Sa˜o Paulo/SP, Brazil. II

OBJECTIVE: Oxidative stress plays an important role in skeletal muscle damage in sepsis. Aerobic exercise can decrease oxidative stress and enhance antioxidant defenses. Therefore, it was hypothesized that aerobic exercise training before a sepsis stimulus could attenuate skeletal muscle damage by modulating oxidative stress. Thus, the aim of this study was to evaluate the effects of aerobic physical preconditioning on the different mechanisms that are involved in sepsis-induced myopathy. METHODS: Male Wistar rats were randomly assigned to either the untrained or trained group. The exercise training protocol consisted of an eight-week treadmill program. After the training protocol, the animals from both groups were randomly assigned to either a sham group or a cecal ligation and perforation surgery group. Thus, the groups were as follows: sham, cecal ligation and perforation, sham trained, and cecal ligation and perforation trained. Five days after surgery, the animals were euthanized and their soleus and plantaris muscles were harvested. Fiber cross-sectional area, creatine kinase, thiobarbituric acid reactive species, carbonyl, catalase and superoxide dismutase activities were measured. RESULTS: The fiber cross-sectional area was smaller, and the creatine kinase, thiobarbituric acid reactive species and carbonyl levels were higher in both muscles in the cecal ligation and perforation group than in the sham and cecal ligation and perforation trained groups. The muscle superoxide dismutase activity was higher in the cecal ligation and perforation trained group than in the sham and cecal ligation and perforation groups. The muscle catalase activity was lower in the cecal ligation and perforation group than in the sham group. CONCLUSION: In summary, aerobic physical preconditioning prevents atrophy, lipid peroxidation and protein oxidation and improves superoxide dismutase activity in the skeletal muscles of septic rats. KEYWORDS: CLP; Skeletal Muscle; Myopathy; Oxidative Stress; Antioxidant Enzymes; Aerobic Exercise. Coelho CW, Jannig PR, Souza AB, Fronza Jr H, Westphal GA, Petronilho F, et al. Exercise training prevents skeletal muscle damage in an experimental sepsis model. Clinics. 2013;68(1):107-114. Received for publication on August 1, 2012; First review completed on August 24, 2012; Accepted for publication on September 22, 2012 E-mail: silva.eliezer@einstein.br Tel.: 55 11 2151-1521

dysfunctions, severe myopathy plays an important role in prolonged intensive care unit stays and late sequelae (2-6). The main signs of sepsis-induced myopathy are generalized weakness, fatigue, atrophy (7), and a delay in weaning from mechanical ventilation (8). These symptoms are associated with a pronounced catabolic response in skeletal muscle that results from proteolytic stimulation and inhibited protein synthesis (9,10) (particularly in the myofibrillar proteins) and can negatively impact patient recovery (11). Oxidative stress plays an important role in skeletal muscle dysfunction in different clinical settings, including sepsis (12,13). Reactive oxygen species (ROS) production is exacerbated in sepsis disease mainly because of mitochondrial

& INTRODUCTION Severe sepsis has been associated with an uncontrolled systemic inflammatory response to infection that is associated with developing organ dysfunction (1). Among these

Copyright ß 2013 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. DOI: 10.6061/clinics/2013(01)OA17

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method used to detect exercise intolerance. Thus, exercise capacity was evaluated using a graded treadmill exercise protocol, as previously described (20).

dysfunction (13), which in turn promotes an imbalance in the redox status because the antioxidant defenses do not increase in the same proportion. This altered redox balance causes cell damage, particularly in proteins, lipids and DNA (14). Because aerobic exercise training induces higher oxygen consumption in parallel with increased ROS production (15), an antioxidant system is launched during such training to maintain an adequate redox balance (16). In addition, exercise training improves mitochondrial function and even increases the number of muscle mitochondria (17,18). The net effects of training are improved efficiency of the antioxidant defenses and oxygen consumption, which could be useful for scavenging ROS in sepsis (16). Therefore, aerobic exercise training enhances the antioxidant defenses in various tissues, particularly in the skeletal and cardiac muscles (19). As an ROS scavenger strategy, the impact of exercise training on sepsis-induced muscle damage has never been investigated (to the best of our knowledge). Therefore, it was hypothesized that aerobic exercise training before the presentation of a sepsis stimulus could attenuate skeletal muscle damage by modulating oxidative stress. Thus, the aim of this study was to evaluate the effects of aerobic physical preconditioning on the different mechanisms that are involved in sepsis-induced myopathy.

Aerobic exercise training protocol The aerobic exercise training protocol consisted of an eight-week program of running on a motorized treadmill (KT-4000 model INBRAMED, RS, Brazil), five days a week, for 60 minutes per sessions at 60% of the maximum running speed that was obtained in the graded treadmill test, which corresponded to the maximal lactate steady-state workload (MLSSw), as described elsewhere (20). All of the untrained rats were exposed to treadmill exercises (5 minutes) three times per week to become accustomed to the exercise protocol and handling.

Cecal ligation and perforation surgery The animals in both the untrained and trained groups were subjected to the surgical procedure 72 hours after the last treadmill exercise test, as previously described (21,22). For the CLP surgery, the rats were anesthetized with ketamine (80 mg/kg), which was administered intraperitoneally. Under aseptic conditions, a 3-cm midline laparotomy was performed to expose the cecum with the adjoining intestine. The cecum was tightly ligated with a 3.0-silk suture at its base (below the ileocecal valve) and was perforated once with a 14-gauge needle. The cecum was then gently squeezed to extrude a small amount of feces from the perforation site before being returned to the peritoneal cavity. The laparotomy was then closed with 3.0 silk sutures. All of the animals received isotonic saline solution (30 ml/kg subcutaneously) immediately after the CLP surgery, as well as antibiotics (ceftriaxone 30 mg/kg and clindamycin 25 mg/kg) starting 6 hr after the CLP surgery and again every 6 hr up to 24 hr after the CLP surgery. Afterward, the animals were returned to their cages with free access to food and water. In the sham group, the rats were submitted to all of the surgical procedures and received isotonic saline solution immediately after the surgery. They also received the same antibiotics as the CLP groups, but their ceca were not ligated or perforated. To minimize the possibility that the animals did not truly develop sepsis, the CLP procedure was always performed by the same investigator. In addition, the animals were observed after the CLP surgeries for signs of infection, such as piloerection, lethargy, tachypnea, and weight loss (21,22). The animals were weighed daily for five days after the surgery, and their soleus and plantaris muscles were harvested at the time of euthanization for later analysis. The rate of sepsis survival in this model was approximately 40%.

& MATERIALS AND METHODS Animals Adult male Wistar rats (70 days old) were obtained from the UNIVALI (Universidade do Vale de Itajai, Itajai, Brazil) breeding colony. The rats were maintained in a 12/12-hr light-dark cycle in a temperature-controlled (22˚C) environment with free access to standard laboratory chow (protein 20 kcal%, carbohydrates 70 kcal%, and lipids 10 kcal%, Nuvital Nutrientes, Curitiba, Parana´, Brazil) and tap water. Initially, the animals were randomly assigned to the untrained and trained groups. After completing the eight-week aerobic exercise training protocol, animals in the trained group were randomly assigned to either the sham or CLP (cecal ligation and perforation) surgery group. The untrained group was subjected to the same surgical procedures. At this phase, there were four groups: 1) sham trained (ShamT), 2) CLP trained (CLPT), 3) sham and 4) CLP. All of the rats were euthanized five days post-surgery. This study was conducted in accordance with the ethical principles in animal research adopted by the Brazilian College of Animal Experimentation (www.cobea.org.br) and was approved by the UNIVILLE (Universidade da Regia˜o de Joinville, Joinville, Brazil) Ethics Committee (Protocol No. 008/08 – COEA).

Graded treadmill exercise test Fiber CSA

Before the first exercise test, the rats were conditioned to exercise on a treadmill over a week-long period (10 min of exercise per session). During the graded treadmill exercise test, the rats were placed on a treadmill and allowed to acclimatize for at least 15 min. The exercise intensity was then increased by 3 m/min (6-33 m/min) every three minutes at a 0% grade until exhaustion (the point of maximum running speed). The graded treadmill exercise test was performed prior to the exercise training and then during the fourth and eighth weeks of exercise training. Exercise capacity was estimated by the total distance run and correlated with the skeletal muscle capacity, which is a

The fiber cross-sectional area (CSA) in the soleus and plantaris muscles was used as an indicator of muscle atrophy. Liquid nitrogen-frozen muscles were vertically mounted and serially sectioned. The muscle sections were then incubated in alkali (mATPase, pH 10.3) or acid (mATPase, pH 4.6), as described by Brooke & Kaiser (23), to assess myofibrillar ATPase activity. The myosin ATPase reaction was used to identify the muscle fiber type (Figure 1). Fiber CSA was evaluated in five areas at 2006 magnification and further analyzed using a digitizing unit connected to a computer (Image-Pro Plus; Media

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Figure 1 - Skeletal muscle histochemical staining for myosin ATPase. Fiber type I appears dark and type II is beige or white in the soleus (A) and plantaris (C) muscles at pH 4.6. The opposite is shown at pH 10.3 in the soleus (B) and plantaris (D) muscles.

Cybernetics, Silver Spring, MD). All of the analyses were conducted by a single observer (CWC) who was blinded to the rat group assignments.

Protein carbonyls The oxidative damage to the protein in the soleus and plantaris muscles was measured by determining levels of the carbonyl groups based on the reaction of the groups with dinitrophenylhydrazine (DNPH) (26). The proteins were precipitated by adding 20% trichloroacetic acid and reacted with DNPH. The samples were then re-dissolved in 6 M guanidine hydrochloride, and the carbonyl contents were determined by measuring the absorbance at 370 nm.

CK activity The creatine kinase (CK) activity was measured in the tissue homogenate of the soleus and plantaris muscles. The reaction mixture for the creatine kinase assay contained 100 mM Tris-HCl buffer (pH 7.5), 30 mM phosphocreatine, 20 mM glucose, 12 mM magnesium acetate, 10 mM diadenosine pentaphosphate, 15 mM sodium azide, 20 mM nacetylcysteine, 2 mM ADP, 5 mM AMP, 2 mM NADP, 3500 U/L hexokinase, 2000 U/L glucose-6-phosphate dehydrogenase and approximately 1.5 mg of protein in a final volume of 1200 mL. The creatine kinase activity was calculated based on reduced nicotinamide adenine dinucleotide (NADH) formation, which was monitored with a spectrophotometer at a wavelength of 340 nm at 37 ˚C (24).

CAT and SOD activity To determine catalase (CAT) activity, the soleus and plantaris muscles were sonicated in a 50-mM phosphate buffer, and the resulting suspension was centrifuged at 30006g for 10 minutes. The supernatant was used for the enzymatic assay. CAT activity was measured by the rate of decrease in hydrogen peroxide absorbency at 240 nm (27). Superoxide dismutase (SOD) activity was assayed by measuring the inhibition of adrenaline auto-oxidation, as previously described (28).

TBARS Lipid peroxidation in the soleus and plantaris muscles was measured by the formation of thiobarbituric acid reactive species (TBARS) during an acid-heating reaction, as previously described (25). Briefly, the samples (200 ml) were mixed with trichloroacetic acid (10%) (400 ml) and centrifuged for 10 minutes (40006g), and the supernatant was mixed with an equal volume of 0.67% thiobarbituric acid (TBA). The mixture was then heated in a boiling water bath for 15 minutes. The formation of TBARS was determined by measuring the absorbance at 535 nm.

Statistical analysis The data are presented as the means and standard errors of the means (mean¡SEM). A two-way ANOVA with Fisher’s post hoc test (Statistica software, StatSoft, Tulsa, OK) was used to compare the effects of training and surgery in all of the analyses except for the analyses of body weight and running distance, for which a repeated-measures two-way ANOVA with Fisher’s post hoc test and Student’s t-test, respectively, were used. Statistical significance was set at p,0.05.

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levels of both muscle types in the CLP group compared with the levels in the sham and CLPT groups (p,0.05). Regulating antioxidant enzymes is important for maintaining the balance between ROS generation and consumption; therefore, the SOD and CAT activity levels were evaluated in the soleus and plantaris muscles. As shown in Figures 6A and 6C, the CLP group exhibited no difference in SOD activity compared with the sham group for both muscle types, whereas the CLPT group exhibited a significant increase in SOD activity in both muscle types compared with the sham and CLP groups (p,0.05). However, there was a significant decrease in CAT activity in the CLP group for both muscle types compared with the sham group (p,0.05). In contrast, the CAT activity in the soleus muscles of the CLPT group was similar to that of the sham group, although it differed from that of the CLP group in the plantaris muscle (Figures 6B and 6D).

& RESULTS In the trained group, there was a significant increase in the running distance (375.3ยก37.9 m) compared with the untrained group (175.3ยก13.3 m) (p,0.05). The pre-training weights of the trained (333.9ยก5.8 g) and untrained (332.8ยก5.8 g) groups were similar. After eight weeks of aerobic exercise training, during the pre-surgery time interval, the trained groups (ShamT and CLPT) exhibited an 18.7-g increase in body weight compared with the untrained groups (Sham and CLP), but this increase was not statistically significant (p = 0.11). Seventy-two hours after surgery, the CLP group exhibited a significant decrease in body weight compared with their weight during the presurgery period, and the CLPT group exhibited a similar decrease after 96 hours (Figure 2). The CSA of muscle fibers was smaller in the CLP group than in the other groups. Myosin ATPase staining of the skeletal muscle sections showed that in the soleus, only the type I fibers from the CLP group were smaller than those from the other groups (p,0.05). In the plantaris muscle, only the type II fibers were smaller in the CLP group than in the other groups (p,0.05) (Figure 3). The soleus muscle CK activity was higher in the CLP group than in the sham and CLPT groups. However, the plantaris CK activity was higher in the CLP group than in the ShamT and CLPT groups (Figure 4). In the soleus and plantaris muscles, TBARS formation was evaluated as an index of lipid peroxidation and carbonyl group levels were evaluated as an index of protein oxidation. As shown in Figures 5-A and 5-C, the TBARS levels in the soleus and plantaris muscles of the rats in the CLP group were significantly increased compared with those of the sham and CLPT groups (p,0.05). Figures 5-B and 5-D demonstrate a greater increase in the carbonyl

& DISCUSSION The present study contributes valuable information regarding the effects of aerobic physical preconditioning on different mechanisms that are involved in sepsis-induced skeletal myopathy, including preventing atrophy, lowering lipid peroxidation and protein oxidation, and improving antioxidant defenses. Several molecular mechanisms of inflammation and cellular damage are involved in the pathogenesis of sepsis, including the excessive generation of ROS (29). These key mediators of cellular injury greatly contribute to the development of sepsis-induced multiple organ dysfunction (29-31). The proinflammatory properties of ROS include endothelial cell damage, chemotactic factor formation, lipid peroxidation and oxidation, neutrophil recruitment, DNA damage, the release of interleukin (IL) and tumor necrosis

Figure 2 - Time course of the body weight changes from pre-surgery until 5 days after surgery in the sham (n = 8), ShamT (n = 8), CLP (n = 6) and CLPT (n = 6) groups. The data are presented as the means ยก SEM. * p,0.05 vs. the same group pre-surgery.

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Figure 3 - Cross-sectional area (CSA) of type I and II fibers in the soleus (A and B) and plantaris (C and D) muscles at five days after surgery. The animals were divided into four groups: sham (n = 5), ShamT (n = 5), CLP (n = 5) and CLPT (n = 5). The data are presented as the means ยก SEM. *, p,0.05 vs. sham; #, p,0.05 vs. CLP; and 1, p,0.05 vs. ShamT.

(Figure 2). Importantly, 120 hours after sepsis induction, the skeletal muscle fiber CSA revealed atrophic conditions only in the soleus and plantaris muscles from the untrained CLP group (Figure 3); in addition, there was significant skeletal muscle injury, as observed by the increase in the TBARS and carbonyl activity (Figure 5). Additionally, there was a significant increase in skeletal muscle anaerobic metabolism in the soleus muscle and a trend toward this increase in the plantaris muscles from rats in the CLP group, as demonstrated by the increase in CK activity (Figure 4). However, in the rats submitted to the aerobic exercise protocol before sepsis induction (CLPT group), this early weight loss was prevented, and skeletal muscle fiber CSA and regular levels of CK, lipid peroxidation and protein oxidation were maintained. These results suggest that the training effects, such as maintaining muscle mass and improving the enzymatic antioxidant system, could be preserved even after 120 hours of sepsis. It has been shown that exercise training, particularly aerobic exercise, protects skeletal muscles against a variety of stressors, including oxidative stress (15,35). In this context, the elevation of several cytoprotective proteins occurs as a consequence of exercise. For instance, the antioxidant enzymes SOD, CAT and glutathione peroxidase (GPX) in skeletal muscles were shown to be up-regulated (19,36,37), and these enzymes were subsequently able to

factor (TNFa), and the formation of peroxynitrite (31). The results of the present study indicated that lipid peroxidation and protein oxidation increased in the oxidative and glycolytic skeletal muscles of rats in the untrained group (CLP) and that tissue damage could be attenuated through exercise training, as observed in the trained group (CLPT) (Figure 5). In agreement with the results of the present study, some authors have demonstrated that septic patients produce a large amount of ROS without any corresponding antioxidant defenses, which is accompanied by lipid peroxidation (32). Note that one of the mechanisms causing this oxidative stress is the reduction of altered tetravalent oxygen as a consequence of endotoxic, hypoxic, and acidic conditions, which occur in sepsis (14). Thus, in skeletal muscle tissue, acidosis increases the quantity of free iron that is released from myoglobin and hemoglobin (33), thereby activating leukocytes and resulting in superoxide generation (34). Thus, the outcome is a vicious cycle that increases cellular dysfunction, promotes muscle wasting, and is associated with a more serious prognosis (11). Septic patients frequently develop severe myopathy (2-7) that results in significant weight loss. In the present study, as expected, the CLP group exhibited significant decreases in body weight after 72 hours of sepsis, while the CLPT group exhibited this significant decrease only after 96 hours

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Figure 4 - The creatine kinase (CK) activity. (A) The soleus and (B) plantaris muscles five days after surgery. The animals were divided into four groups: sham (n = 8), ShamT (n = 8), CLP (n = 6) and CLPT (n = 6). The data are presented as the means ยก SEM. *, p,0.05 vs. sham; #, p,0.05 vs. CLP; and 1, p,0.05 vs. ShamT.

protect skeletal muscle against ROS-mediated damage (15,35). The impact of aerobic physical preconditioning on sepsis has not been fully evaluated. Some authors have suggested that 12-wk treadmill training maintains the integrity of the beta-adrenergic receptor adenylate cyclase system, which can be depressed by in vivo endotoxin administration (38). Moreover, Bagby et al. demonstrated that exercise to near exhaustion before intravenous LPS challenge in rats markedly suppressed the systemic TNF response that is normally observed in response to LPS challenge (39). Some studies have shown that appropriate exercise in humans

increases anti-inflammatory cytokine IL-6 plasma levels and suppresses the endotoxemia-induced elevation in TNFa (40). For instance, Chen et al. reported that four weeks of exercise training before a sepsis challenge attenuated sepsisinduced systemic hypotension and tachycardia, decreased the number of blood cells, increased the levels of proinflammatory cytokines (TNFa and IL-1b); and, also protected organs from sepsis damage (41). However, there are conflicting studies regarding the effects of transcutaneous electrical muscle stimulation (TEMS) on the quadriceps muscle in septic patients. Gerovasili et al. demonstrated that TEMS can preserve muscle mass; however, Poulsen et al.

Figure 5 - Oxidative stress parameters. (A, C) Thiobarbituric acid reactive substances (TBARS) and (B, D) carbonyl in the soleus and plantaris muscles at five days after surgery. The animals were divided into four groups: sham (n = 8), ShamT (n = 8), CLP (n = 6) and CLPT (n = 6). The data are presented as the means ยก SEM. *, p,0.05 vs. sham; #, p,0.05 vs. CLP; and 1 p,0.05 vs. ShamT.

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Figure 6 - Antioxidant enzymes. (A, C) Superoxide dismutase (SOD) and (B, D) catalase (CAT) in the soleus and plantaris muscles at five days after surgery. The animals were divided into four groups: sham (n = 8), ShamT (n = 8), CLP (n = 6) and CLPT (n = 6). The data are presented as the means ยก SEM. *, p,0.05 vs. sham; #, p,0.05 vs. CLP; and 1 p,0.05 vs. ShamT.

concluded that the loss of muscle mass was unaffected by TEMS (42,43). Remarkably, the present study demonstrated that aerobic exercise training was able to prevent atrophy, oxidative stress and muscle damage in skeletal muscle in a rat sepsis model (Figures 3 and 5). The exercise-induced increase in the antioxidant defense system of muscles is most likely the underlying mechanism responsible for protecting muscle cells against oxidative damage. Regarding the removal of ROS from muscle fibers, SOD activity is the first-line of defense against superoxide radicals (15). Subsequently, the present findings show that the ShamT and CLPT groups exhibited significant increases in SOD activity compared with the sham and CLP groups (Figure 6). Other researchers have shown that SOD expression can be induced in skeletal muscle by exercise, which can provide cellular protection against ROS (19,44). For instance, Pinho et al. investigated the effect of 12 weeks of treadmill exercise on the skeletal muscle of rats, and they found that aerobic exercise could increase the SOD/CAT ratio (45). The results of the present study suggest that exercise increased SOD activity and could be a contributory factor in preventing sepsis-related myocyte injury. CAT, another cellular antioxidant enzyme, is responsible for the catalytic reduction of hydrogen peroxide to water. In the present study, CAT activity levels did not decrease in the CLPT group, whereas a significant decrease in these levels was found in the CLP group compared with the sham group (Figure 6). The trained groups did not exhibit an increase in CAT activity levels, which could be associated with reduced hydrogen peroxide levels due to increased GPX enzyme activity. Moreover, in this study, the CAT activity data from the trained group was in accordance with the results of the

study by Powers et al., who showed that there was no change in oxidative and glycolytic muscle CAT activity in response to chronic exercise training (19). Thus, one limitation of the present study is that the exact mechanism that is involved in preventing lipid peroxidation and protein oxidation could not be clarified. In conclusion, aerobic physical preconditioning prevents atrophy, lipid peroxidation and protein oxidation and improves SOD activity in the skeletal muscle of septic rats. Furthermore, this study suggested that regular exercise may minimize the muscle damage that results from serious bacterial infections.

& ACKNOWLEDGMENTS We thank Hospital Israelita Albert Einstein for covering the publication fees.

& AUTHOR CONTRIBUTIONS Coelho CW, Westphal GA, Dal-Pizzol F, Streck EE, and Silva E conceived and designed the study, conducted the experiment, performed the data analysis, reviewed the drafts of the manuscript, and approved the final version of the manuscript. Coelho CW, Jannig PR, Souza AB, and Fronza Jr H conducted the experiment and muscle morphological analysis, reviewed the drafts of the manuscript, and approved the final version of the manuscript. Petronilho F, Constantino L, and Ferreira GK conducted the biochemical analyses, reviewed the drafts of the manuscript, and approved the final version of the manuscript.

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READER’S OPINION

The platelet volume in patients with cardiac syndrome X Ercan Varol, Mehmet Ozaydin, Abdullah Dogan, Dogan Erdogan Suleyman Demirel University Isparta, Faculty of Medicine, Department of Cardiology, Isparta, Turkey. Email: drercanvarol@yahoo.com Tel.: +90 5323468258

Demirkol et al. (1) compared the mean platelet volume (MPV) in the plasma of patients with cardiac syndrome X, patients with coronary artery disease and healthy controls. They observed that the MPV in patients with cardiac syndrome X and coronary artery disease was significantly higher than that observed in the control group. There were no significant differences in the MPV between the cardiac syndrome X and the coronary artery disease groups. Although this study is interesting, we would like to make a minor criticism based on its methodological aspects. Blood was collected in a Vacutainer tube containing ethylenediaminetetraacetic acid (EDTA) to measure the MPV. The authors analyzed the blood samples after two hours of venipuncture. However, a two-hour delay after blood sampling can cause abnormal MPV measurement results. This abnormality is important in tubes containing EDTA. As we know, platelets exhibit a time-dependent swelling when blood samples are anticoagulated with EDTA; however, this swelling does not occur in the presence of citrate (2). With impedance counting, the MPV increases over time as the platelets swell in EDTA. An increase of 7.9% within 30 min and an overall increase of 13.4% over 24 hours have been reported, although the majority of this increase occurs within the first 6 hours (3). The recommended optimal measuring time for MPV is 120 min after venipuncture (4). For reliable MPV measurements, the potential influence of the anticoagulant must be carefully controlled either by using an alternative anticoagulant, such as citrate, or by standardizing the time delay between sampling and analysis (less than 2 hours).

The pathophysiology of cardiac syndrome X is still being debated; however, endothelial dysfunction leading to a reduced coronary microvascular dilatory response and increased coronary resistance are thought to play important roles. We wish to add our experience with respect to this subject. In our previous study, we investigated whether MPV increased in patients with idiopathic dilated cardiomyopathy and whether increased MPV correlated with the degree of coronary microvascular dysfunction (5). We have found that MPV is an independent predictor of lower coronary flow reserve. Therefore, we believe that impaired coronary microvascular function as a result of endothelial dysfunction plays an important role in increasing the MPV.

& REFERENCES 1. Demirkol S, Balta S, Unlu M, Yuksel UC, Celik T, Arslan Z, et al. Evaluation of the mean platelet volume in patients with cardiac syndrome X. Clinics. 2012;67(9):1019-22, http://dx.doi.org/10.6061/ clinics/2012(09)06. 2. Bath PM, Butterworth RJ. Platelet size: measurement, physiology and vascular disease. Blood Coagul Fibrinolysis. 1996:7(2):157-61, http://dx. doi.org/10.1097/00001721-199603000-00011. 3. Bowles KM, Cooke LJ, Richards EM, Baglin T. Platelet size has diagnostic predictive value in patients with thrombocytopenia. Clin Lab Haematol. 2005;27(6):370-3, http://dx.doi.org/10.1111/j.1365-2257.2005.00726.x. 4. Lance´ MD, van Oerle R, Henskens YM, Marcus MA. Do we need time adjusted mean platelet volume measurements? Lab Hematol. 2010; 16(3):28-31, http://dx.doi.org/10.1532/LH96.10011. 5. Erdogan D, Tayyar S, Icli A, Uysal BA, Varol E, Ozaydin M, et al. Elevated mean platelet volume is associated with impaired coronary microvascular function in patients with idiopathic dilated cardiomyopathy. Platelets. 2012;23(3):177-83, http://dx.doi.org/10.3109/09537104. 2011.611273.

Copyright ß 2013 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. DOI: 10.6061/clinics/2013(01)LE01

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Authors’ comments on: The platelet volume in patients with cardiac syndrome X Sait Demirkol, Sevket Balta Gulhane School of Medicine, Department of Cardiology, Tevfik Saglam St., 06018 Etlik-Ankara, Turkey. Email: saitdemirkol@yahoo.com Tel.: 90-312-3044281

Dear Editor,

angiography. We then waited two hours to allow for the stabilization of platelet shape changes. Finally, the blood was collected in tripotassium EDTA tubes; all of the samples from all of the groups were analyzed immediately using an automatic blood counter.

We would like to thank Varol et al. (1) for their kind interest regarding our article. They indicated that a twohour delay after blood sampling can cause abnormal mean platelet volume measurement results and that platelets exhibit time-dependent swelling when blood samples are collected in ethylenediaminetetraacetic acid We are in agreement on these issues; however, we did not wait two hours before analyzing the MPV measurements. As mentioned in the Methods section of our study, we gained vascular access via venipuncture prior to coronary

& REFERENCES 1. Varol E, Ozaydin M, Dogan A, Erdogan D. The platelet volume in patients with cardiac syndrome X. Clinics. 2013;68(1):115, http://dx.doi. org/10.6061/clinics/2013(01)LE01.

Copyright ß 2013 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. DOI: 10.6061/clinics/2013(01)LE02

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READER’S OPINION

Differences in the mechanisms that induce obesity and metabolic syndrome in experimental animal models and humans may cause treatment failure Mustafa Cakar,I Sevket Balta,II Sait Demırkol,II Ugur Kucuk,II Seref DemırbasI I

Gulhane Medical Academy, Department of Internal Medicine, Ankara/Turkey. II Gulhane Medical Academy, Department of Cardiology, Ankara/Turkey.

Email: drsevketb@gmail.com Tel.: +90 312-3044281

Dear Editor,

glutamate may differ from any hereditary or acquired obesity and from MS in humans, i.e., that the enzymatic pathways utilized by and the physiologic effects resulting from these two entities may be different. These differences may affect treatment success, and we believe that the study results should be assessed with this perspective in mind.

We recently read the article ’’Monosodium glutamate neonatal treatment induces cardiovascular autonomic function changes in rodents’’ by Signora Peres Konrad et al. with great interest(1). These authors concluded that obesity induced by neonatal monosodium glutamate treatment impairs cardiac autonomic function and most likely contributes to increased arterial pressure and insulin resistance in rats. The authors are to be commended for contributing such a well-designed and successfully presented study. We believe that these findings will guide further study of obesity and metabolic syndrome (MS). Obesity is a growing public health problem, and metabolic syndrome is an important risk factor for cardiovascular diseases (2,3). Although it is correct that this study should be emphasized in terms of these factors, we believe that obesity and MS induced in rats with monosodium

& REFERENCES 1. Konrad SP, Farah V, Rodrigues B, Wichi RB, Machado UF, Lopes HF, et al. Monosodium Glutamate Neonatal Treatment I˙nduces Cardiovascular Autonomic Function Changes I˙n Rodents. Clinics. 2012;67(10):1209-14, doi: 10.6061/clinics/2012(10)14. 2. Bahia L, Aguiar LG, Villela N, Bottino D, Godoy-Matos AF, Geloneze B, et al. Relationship Between Adipokines, I˙nflammation, And Vascular Reactivity I˙n Lean Controls And Obese Subjects With Metabolic Syndrome. Clinics. 2006;61(5):433-40, doi: 10.1590/S1807-5932200600050 0010. 3. Tsai TY, Cheng JF, Lai YM. Prevalence of metabolic syndrome and related factors in Taiwanese high-tech industry workers. Clinics. 2011;66(9):1531-5, doi: 10.1590/S1807-59322011000900004.

Copyright ß 2013 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. DOI: 10.6061/clinics/2013(01)LE03

119


READER’S OPINION

Prediction of hospital events based on the severity of illness Sevket Balta,I Mustafa Cakar,II Sait Demırkol,I Zekeriya Arslan,I Murat Unlu,I Omer KurtII I

Gulhane Medical Academy, Department of Cardiology, Ankara/Turkey. II Gulhane Medical Academy,Department of Internal Medicine, Ankara/Turkey.

Email: drmustafacakar@gmail.com Tel.: +90-312-3044210

Dear Editor,

severity, cognitive impairment, comorbidity scores, polypharmacy, neoplastic diseases, immobility, congestive heart failure history, albumin levels ,3.3 g/dL, creatinine levels $1.3 mg/dL and advanced age are all highly correlated with mortality in older patients who are admitted to geriatric wards (4). The importance of this diagnosis as an indicator of poor prognosis and increased hospital stays indicates that geriatric patients who are agitated or have acutely impaired cognitive functions should be assessed for delirium. Implementing preventive measures and daily cognitive evaluations are crucial for diminishing the impact of delirium on patient outcomes. According to this study, patient mortality was significantly predicted by the Simplified Acute Physiology Score II score, poorer functional status and adverse medical events. Further studies are being conducted to investigate this issue. Adverse medical events should be monitored in all elderly hospitalized patients because there is no risk profile for sensitive patients, and the consequences of the adverse events are serious and may lead to longer hospital stays or even death.

We read with interest the article ‘‘Medical adverse events in elderly hospitalized patients: A prospective study,’’ by Szlejf et al. (1). Using predictive factors, the authors aimed to determine the frequency of adverse medical events in elderly patients who were admitted to an acute care geriatric unit and the correlation between adverse events and hospital mortality rates. They defined an adverse medical event as an unintentional injury or complication that resulted in disability and was caused by physician management rather than the patient’s underlying disease process. The patients with adverse medical events had significantly longer hospital stays and higher mortality rates than those patients without adverse events. Mortality was significantly predicted by the Simplified Acute Physiology Score II score, poorer functional status at admission and the occurrence of adverse medical events (1). We believe that these findings will serve as a guide for further studies about adverse events in elderly patients in acute care geriatric units. Delirium correlates with higher hospital mortality rates and greatly impacts the health of hospitalized older patients. Delirium is an acute psychiatric syndrome that is characterized by a transitory and important disorganization of cognitive function, which is caused by changes in brain metabolism. It is a frequent problem in hospitalized older patients and has a prevalence rate of 25%, depending on the study, and a hospital mortality rate of 30% (2). In a multicenter European study of patients who were at least 65 years old, death correlated with functional and cognitive impairment (3). One of the main contributions of this study was confirmation of the importance of delirium as a factor associated with death in elderly hospitalized patients. Patients who are affected by this disorder experience prolonged hospitalizations and functional decline. In addition to delirium, functional decline, disease

& REFERENCES 1. Szlejf C, Farfel IJM, Jose II, Curiati A, Euro II, Couto DB, et al. Medical adverse events in elderly hospitalized patients: A prospective study. Clinics. 2012;67(11):1247-52, http://dx.doi.org/10.6061/clinics/2012(11) 04. 2. Lima DP, Ochiai ME, Lima AB, Curiati JAE, Farfel JM, Filho WJ. Delirium in hospitalized elderly patients and post-discharge mortality. Clinics. 2010;65(3):251-5, http://dx.doi.org/10.1590/S1807-59322010000300003. 3. Campbell SE, Seymour DG, Primrose WR, Lynch JE, Dunstan E, Espallargues M, et al. A multi-centre European study of factors affecting the discharge destination of older people admitted to hospital: analysis of in-hospital data from the ACMEplus project. Age Ageing. 2005;34(5):467-75, http://dx.doi.org/10.1093/ageing/afi141. 4. Silva TJA, Jerussalmy CS, Farfel JM, Curiati JAE, Jacob-Filho W. Predictors of in-hospital mortality among older patients. Clinics. 2009;64(7):613-8, http://dx.doi.org/10.1590/S1807-59322009000700002.

Copyright ß 2013 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. DOI: 10.6061/clinics/2013(01)LE04

121


RAPID COMMUNICATION

Residual C-peptide in patients with Type 1 diabetes and multiethnic backgrounds Mirella Hansen de Almeida,I,III Joana Rodrigues Dantas,I,II Bianca Barone,I,II Fabiano Marcel Serfaty,I,II Rosane Kupfer,II Marta Albernaz,IV Maria Rocio Bencke,IV Lenita Zajdenverg,I Melanie Rodacki,I Jose´ Egı´dio Paulo de OliveiraI I

Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro (HUCFF), Nutrology Department, Rio de Janeiro/RJ, Brazil. II State Institute of Diabetes and Endocrinology Luiz Capriglione (IEDE), Rio de Janeiro/RJ, Brazil. III Air Force Central Hospital (HCA), Endocrinology Department, Rio de Janeiro/RJ, Brazil. IV Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro (HUCFF), Nuclear Medicine Department, Rio de Janeiro/RJ, Brazil.

OBJECTIVE: To evaluate serum C-peptide in 88 patients from a multiethnic population with Type-1 diabetes and variable disease durations. METHOD: Eighty-eight patients with a mean disease duration of 8.1¡7.6 years were included and underwent C-peptide measurement before and after glucagon stimulation. Chi-squared and Mann Whitney U-tests were used to compare the variables between groups (all two-tailed, a = 0.05). Spearmans correlation coefficient was used to test the association between the continuous variables. Logistic regression was used for the multivariate analysis. Twenty-eight (31.8%) individuals had significantly detectable C-peptide levels after stimuli, particularly those with a shorter disease duration (p,0.001). RESULTS: Patients with detectable C-peptide levels required lower insulin doses (p,0.009) and had similar HbA1C results (p = 0.182) and fewer chronic complications (p = 0.029). CONCLUSION: C-peptide detection was common in Type-1 diabetics, particularly shortly after being diagnosed. This result may have clinical implications. KEYWORDS: Diabetes Mellitus Type-1, C-Peptide, Insulin Secretion, Disease Duration, GADA, Autoimmune Disease. Almeida MH, Pereira JD, Barone B, Serfaty FM, Kupfer R, Albernaz M, et al. Residual C-peptide in patients with Type 1 diabetes and multiethnic backgrounds. Clinics. 2013;68(1):123-126. E-mail: joanardantasp@ig.com.br Tel.: 55 21 3251-3304

insulin secretion in individuals of other ethnicities with Type-1 diabetes. The aim of this study was to evaluate the bcell function in multiethnic Type-1 diabetes patients with variable disease durations using C-peptide measurements after glucagon stimulation.

& INTRODUCTION Type-1 diabetes mellitus is characterized by destroyed pancreatic b cells, insulin deficiency and hyperglycemia (1,2). The progression of this process is heterogeneous and may range from months to many years (3,4). For long periods after being diagnosed, some Type-1 diabetes patients maintain a residual b-cell function (5), which may be associated with reducing the risk of complications and hypoglycemia and improving metabolic control (6-10). Cpeptide is considered a reliable method for estimating the bcell residual function (5-7), particularly after glucagon stimulation or a mixed meal. Most studies that have evaluated the C-peptide secretion in Type-1 diabetes have included Caucasians or Asians. Little is known about

& RESEARCH DESIGN AND METHODS Volunteers who had Type-1 diabetes (11) with short (Group 1) and long (Group 2) disease durations (#5 years and .5 years, respectively) were included in this crosssectional study. A diabetes diagnosis was based on the American Diabetes Association criteria (11). The patients’ clinical and epidemiological data were obtained from questionnaires and medical records, and all participants provided informed written consent. Blood samples from each patient were measured for fasting glucose and stimulated C-peptide (6 minutes after intravenous injections of 1 mg of glucagon), glycosylated hemoglobin (HbA1C) and anti-glutamic acid decarboxylase antibody (GADA). The tests were performed if a patient’s fasting capillary glucose measured between 70 and 200 mg/dL. The participants were classified as whites and non-whites based

Copyright ß 2013 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. DOI: 10.6061/clinics/2013(01)RC02

123


C-peptide in patients with Type-1 diabetes Almeida MH et al.

CLINICS 2013;68(1):123-126

and the Type-1 diabetes duration (r = -0.342, p = 0.08). In group 1, the patients with detectable C-peptide levels had a shorter Type-1 diabetes duration (2.01¡1.4 vs. 3.04¡1.5 years, p = 0.034). The patient’s age at disease onset, ethnicity, gender, DKA at diagnosis and GADA status were not associated with the C-peptide detection. GADA was positive in 43.7% of patients. Patients with detectable Cpeptide levels had a lower mean age than the other patients (p = 0.001), but this link disappeared after the multivariate analysis. There was a correlation between overweight (BMI$25 kg/m2) and undetectable C-peptide levels (p = 0.06). Fasting glucose was not associated with C-peptide detection (p = 0.16). The patients with detectable C-peptide levels required lower insulin doses/kg than the other patients (0.61¡0.34 vs. 0.85¡0.28, p,0.009), but had similar HbA1c levels (8.49¡1.9 vs. 9.5¡2.6, p = 0.182). These results were observed in the sample as a whole and separately in each group. C-peptide detection was also linked to a lower frequency of chronic complications (3.7% vs. 21.7%, p = 0.029) but not to retinopathy or nephropathy individually. In group 2, other autoimmune diseases (usually thyroid diseases) occurred more frequently in the patients with detectable C-peptide levels (p = 0.018). This association was not observed in group 1 (p = 0.53). After the logistic regression analysis, the link between the detectable Cpeptide levels and the lower insulin doses (in use and with the Type-1 diabetes duration) remained significant.

on their phenotypes and family backgrounds. C-peptide was determined using chemiluminescence (Immulite DPC; UK). A C-peptide level $0.5 ng/dL was considered detectable (reference 1.1 to 5.0 ng/mL). The inter- and intra-assay coefficients of variation (CV) were 7.6% and 8.2%, respectively. GADA was determined using radioimmunoassay (RSR-Limited, Cardiff, UK). Values .1.0 U/ mL were considered positive. The inter- and intra-assay CVs were 4.9 to 7.0% and 3.6 to 3.7%, respectively. The statistical analyses were performed using SAS 6.1 version (SAS Institute Inc., Cary, NC). Chi-squared and Mann Whitney U-tests were used to compare the variables between the groups (all two-tailed, a = 0.05). Spearmans correlation coefficient was used to test the association between the continuous variables. Logistic regression was used for the multivariate analysis.

& RESULTS The study population comprised 88 individuals with Type-1 diabetes. The characteristics of the study group are shown in Table 1. Fasting C-peptide was detectable in 19 patients (21.6%), and these C-peptide levels were within the normal range in 7 (8%) patients. After the glucagon stimulus, C-peptide was detectable in 31.8% of the patients (n = 28, p = 0.004) and within the normal range in 14.8% of the patients (n = 13, p = 0.016). The mean C-peptide titers in the patients with detectable C-peptide levels were 1.3¡0.8 ng/ml (fasting) and 1.6¡1.2 ng/ml (after the stimulus). Given the superiority of C-peptide (after the stimulus) over the fasting measurement, the former was used for the following tests. Adverse events occurred in 35.2% of the patients (n = 30) after the glucagon infusion (e.g., transient mild nausea that was reversible within minutes in most cases, vomiting, paresthesia and malaise). There was an association between the detectable Cpeptide levels and the diabetes disease duration (detectable CP = 5¡7.5 vs. undetectable CP = 10¡6.99 years, p,0.001), but there was no correlation between the C-peptide titers

& DISCUSSION This study evaluated the frequency and clinical implications of C-peptide detection in Type-1 diabetes patients. This study population is particularly interesting because of the complex and mixed ethnic backgrounds of the patients. Most studies that have evaluated pancreatic function in Type-1 diabetes were performed in Caucasian and Asian patients. This study provides a unique opportunity to improve our knowledge of the progression of b-cell

Table 1 - Clinical characteristics of patients grouped by disease durations. Disease duration

Sex

Female n (%) Male n (%) Ethnicity White n (%) Non-white n (%) Mean actual age (years) Mean diagnostic age (years) Mean disease duration (years) DKA at diagnosis n (%) Yes No Mean BMI (kg/m2 ¡SD) HbA1C (mean ¡SD) Mean insulin dose/kg Detectable CP n (%) Fasting (CP1) Stimulated (CP2) Preserved CP n (%) Fasting (CP1) Stimulated (CP2) Other Auto-immune diseases n (%)* GADA Total number of patients

Group 1 (#5 years)

Group 2 (.5 years)

Total

p-value

14(35.9) 25(64.1) 19(48.7) 20(51.3) 18.6¡5.6 16.5¡5.7 2.51¡1.51 8(20.5) 31(79.5) 21.1¡3.5 8.97¡2.29 0.72¡0.34 16(41) 23(63.9) 6(15.4) 11(28.2) 6 (15) 24(62.5) 39

34(68.8) 15(31.2) 32(64.6) 17(35.4) 26.9¡9.1 13.3¡7.9 13.73¡6.72 21(41.7) 27(56.3) 23.6¡3.2 8.45¡1.62 0.87¡0.29 3(6.1) 5(10.2) 1(2.0) 2(4.1) 7(14.5) 14(29.8) 49

48(54.0) 40(46.0) 51(57.5) 37(42.5) 23.1¡8.8 14.7¡7.2 8.7¡ 7.6 29(33.0) 59(67.8) 22.4¡3.5 8.6¡1.95 0.8¡0.3 19(21.6) 28(31.8) 7(8) 13(14.8) 13(14.7) 38(43.7) 88

0.002 0.13 ,0.001 0.004 ,0.001 0.022 ,0.001 0.22 0.023 ,0.001 ,0.001 0.029 0.009 0.95 0.004

BMI – Body mass index, HBA1c – Glycohemoglobin, DKA- Diabetic ketoacidosis, CP – C-peptide. * Thyroid disease in 12 cases, Sjogren’s syndrome and rheumatoid arthritis in one case each.

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CLINICS 2013;68(1):123-126

C-peptide in patients with Type-1 diabetes Almeida MH et al.

comorbidities in patients with Type-1 diabetes and detectable C-peptide levels (23). It is known that short- and long-term glycemic control may influence b-cell function in patients with Type-1 diabetes. However, previous data have indicated that residual pancreatic function may influence glycemic control (8,24,25). In this study, we excluded patients with glycemia ,70 or .200 mg/dl to minimize any possible interference. Although the patients with Type-1 diabetes and detectable C-peptide levels had similar glycemic control, these patients used lower daily insulin doses, which indicated that maintaining endogenous insulin secretion might reduce the insulin requirements of patients with Type-1 diabetes. The association between chronic complications and residual C-peptide levels was also analyzed (25,26). We found a link between C-peptide and chronic complications as a whole but not with retinopathy or nephropathy separately. It is possible that the sample size and the small number of individuals with chronic complications included in this study might have influenced our results. To summarize, this study identified a significant number of individuals with Type-1 diabetes (of both short and long duration) with detectable C-peptide levels. It is possible that this residual b cell secretion is associated with a lower insulin requirement, a lower frequency of chronic complications and a higher frequency of other autoimmune diseases. However, prospective studies are still needed to resolve these questions.

secretion in patients with Type-1 diabetes in our population because it is still unclear whether the results that were obtained in previous studies can be extrapolated to other ethnic populations, such as ours. The tests were performed after the patients were stimulated with glucagon, which is a faster method for increasing b-cell secretion compared with the mixed meal test. However, nausea and flushing may occur more frequently as adverse events (5-7,12). This study found that adverse events occurred in 35.2% of patients. These adverse events included mild and transient nausea (in most cases), which suggested that C-peptide measurement after glucagon infusion can be well tolerated. At the beginning of this study, both tests were considered equivalent in the literature (7), and the mixed meal test was preferable (13). We have shown that a significant proportion of patients with Type-1 diabetes had detectable C-peptide levels, and these levels were higher than those observed in the Diabetes Control and Complications Trial Research Group DCCT (8,10). We also compared the serum C-peptide levels between individuals with short and long disease durations (#5 and .5 years, respectively) of Type-1 diabetes. As expected, detectable C-peptide levels occurred more frequently in the individuals with shorter disease durations, which indicated that the progression of the b cell destruction occurred primarily during the first years of the disease (8,13-16). However, a significant proportion of the patients with long-standing Type-1 diabetes had detectable levels of C-peptide. This finding was compatible with the anatomopathological data that described the presence of insulin cells in Type-1 diabetes patients with long disease durations (5). In a group of patients with extremely favorable clinical outcomes and fewer diabetic complications, Keenan et al. have reported a surprisingly large number (18%) of Type-1 diabetes patients with disease durations .50 years and detectable C-peptide levels (17). Recently, using an ultrasensitive assay, Wang et al. have reported residual Cpeptide levels in patients with long-standing Type-1 diabetes (18). However, the potential clinical implications of sustained b-cell function have been reported only for those patients with C-peptide levels above the approximate threshold that was used in our study (8). One could question whether the patients with Type-1 diabetes and detectable C-peptide levels actually have Type-1 diabetes rather than other forms of diabetes. However, most patients needed insulin shortly after their diagnoses, were not overweight and lacked a significant family history of diabetes in more than one generation; these histories make other diagnoses, such as Type-2 diabetes and Maturity Onset Diabetes of the Young (MODY), unlikely. However, we cannot exclude the possibility that atypical forms of diabetes could be present in populations with mixed ethnic backgrounds, particularly those with GADA (). Although the GADA prevalence was low in this study group (19), it is likely that its positivity has decreased over the years (20,21). We found an association between autoimmune comorbidities and detectable C-peptide levels after stimulating patients with long disease durations, particularly in patients with thyroid diseases. Although the sample size was limited, this association has not been reported in other studies (22). Vondra et al. reported the exact opposite association, with a lower frequency of autoimmune

& ACKNOWLEDGMENTS This study was performed at the Clementino Fraga Filho University Hospital (HUCFF) at the Federal University of Rio de Janeiro (UFRJ) and at the State Institute of diabetes and Endocrinology Luiz Capriglione (IEDE). Thanks to Novo Nordisk Pharmaceutical of Brazil for the GlucagonH kits. The study was supported by grants from CAPES e CNPQ.

& AUTHOR CONTRIBUTIONS Dantas JP and Almeida MH recruited patients, performed analyses, contributed to the statistical analyses and wrote the manuscript. Barone B recruited patients and analyzed samples. Sertafy FM recruited patients. Kupfer R contributed to the study design. Albernaz M and Bencke MR performed the radioimmunoassays. Zajdenverg L reviewed and edited the manuscript. Rodacki M contributed to the study design and interpretation of data and reviewed and edited the manuscript. Oliveira JE contributed to the discussion and reviewed the manuscript.

& REFERENCES 1. Atkinson MA, Eisenbarth GS. Type 1 Diabetes: new perspectives on disease pathogenesis and treatment. Lancet. 2001;358(9277):221-9, http://dx.doi.org/10.1016/S0140-6736(01)05415-0. 2. Daneman D. Type 1 Diabetes. Lancet. 2006;367(9513):847-58, http://dx. doi.org/10.1016/S0140-6736(06)68341-4. 3. Screenan S, Pick AJ, Levisettim, Baldwin AC, Pugh W, Polonsky KS. Increased beta-cell proliferation and reduced mass before diabetes onset in the nonobese diabetic mouse. Diabetes. 1999;48(5):989-96, http://dx. doi.org/10.2337/diabetes.48.5.989. 4. Marchetti P, Dotta F, Zhiong L, Lupi R, Del Guerra S, Santangelo C, et al. The function of pancreatic islets isolated from type 1 diabetic patient. Diabetes Care. 2000;23(5):701-3, http://dx.doi.org/10.2337/diacare.23.5. 701. 5. Tsai EB, Sherry NA, Palmer JP, Herold KC. The Rise and Fall of Insulin Secretion in type 1 diabetes mellitus. Diabetologia. 2006;49(2):261-70. 6. Palmer JP, Fleming GA, Greenbaum CJ, Herold KC, Jansa LD, Kolb H, et al. C-peptide is the appropriate outcome measure for type 1 diabetes clinical trials to preserve beta-cell function: report of an ADA workshop, 21-22 October 2001. Diabetes. 2004;53(1):250-64.

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7. Vendrame F, Zappaterreno A, Dotta F. Markers of beta cell function in type 1 diabetes mellitus. Minerva Med. 2004;95(2):79-84. 8. Steffes MW, Sibley S, Jackson M, Thomas W. b-cell Function and the development of diabetes-related complications in diabetes control and complication trial. Diabetes Care. 2003;26(3):832-6, http://dx.doi.org/10. 2337/diacare.26.3.832. 9. Sherry NA, Tsai EB, Palmer JP, Herold KC. Natural history of beta cell function in type 1 diabetes. Diabetes. 2005;54:Suppl 2:S32-9. 10. Effect of intensive therapy on residual beta-cell function in patients with type 1 diabetes in the diabetes control and complications trial. A randomized, controlled trial. The Diabetes Control and Complications Trial Research Group. Ann Intern Med. 1998;128(7):517-53. 11. American Diabetes Association (ADA). Diagnosis and classification of diabetes mellitus (position statements). Diabetes Care. 2012;35 Suppl1:S64-71. 12. Scheen AJ, Castillo MJ, Lefebvre PJ. Assessment of residual insulin secretion in diabetic patients using the intravenous glucagon stimulatory test: methodological aspects and clinical applications. Diabetes Metab. 1996;22(6):397-446. 13. Greenbaum Cj, Mandrup-Poulsen T, Mcgee Pf, Battelino T, Haastert B, Ludvigsson J, et al. Mixed-meal tolerance test versus glucagon stimulation test for the assessment of b-cell function in therapeutic trials in type 1 diabetes. Diabetes Care. 2008;31(10):1966-71, http://dx.doi.org/10. 2337/dc07-2451. 14. Wallensten M, Dahlquist G, Persson B, Landin-Olsson M, Lernmark A, Sundkvist G, et al. Factors influencing the magnitude, duration, and rate of fall of b-cell function in type 1 (insulin dependent) diabetic children followed for two years from their clinical diagnosis. Diabetologia. 1988;31(9):664-9, http://dx.doi.org/10.1007/BF00278749. 15. Effects of age, duration and treatment of insulin-dependent diabetes mellitus on residual beta-cell function: observations during eligibility testing for the Diabetes Control and Complications Trial (DCCT). The DCCT Research Group. J Clin Endocrinol Metab. 1987;65(1):30-6. 16. Steele C, Hagopian WA, Gitelman S, Masharani U, Cavaghan M, Rother KI, et al. Insulin Secretion In Type 1 Diabetes. Diabetes. 2004;53(2):426-33, http://dx.doi.org/10.2337/diabetes.53.2.426. 17. Keenan HA, Sun JK, Levine J, Doria A, Aiello LP, Eisenbarth G, et al. Residual insulin production and pancreatic cell turnover after 50 years of

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