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Volume 68 Number 12 - December/2013


CLINICS Editors Wagner Farid Gattaz, Edmund Chada Baracat 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

Joaquim Prado Moraes-Filho 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

Jose´ Luiz Gomes do Amaral Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Antonio Egidio Nardi Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil

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

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

Luı´z Eugeˆnio Garcez-Leme Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Berenice Bilharinho Mendonc¸a Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Luiz Fernando Onuchic 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

Lydia Masako Ferreira Universidade Federal 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

Marcos Intaglietta University of California, San Diego San Diego, CA, USA

Carmen Silvia Valente Barbas Faculdade de Medicina da Universidade 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

Claudia Regina Furquim de Andrade Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Mauricio Etchebehere Universidade Estadual de Campinas Campinas, SP, Brazil

Emilia Inoue Sato Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Michele Correale University of Foggia Foggia, Italy

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

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

Geraldo Busatto Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Gustavo Franco Carvalhal Faculdade de Medicina da Pontifı´cia Universidade Cato´lica do Rio Grande do Sul Porto Alegre, Rio Grande do Sul, 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

Heitor Franco de Andrade Jr. 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

Jesus Paula Carvalho 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

Patricia Rieken Macedo Rocco Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil Paulo Hoff Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Raul Coimbra University of California, San Diego La Jolla, CA, USA Renato Delascio Lopes Duke University Medical Center Durham, NC, USA Ricardo Bassil Lasmar Universidade Federal Fluminense Nitero´i, RJ, Brazil Rosa Maria Rodrigues Pereira Faculdade de Medicina da Universidade 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 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 Vale´ria Aoki Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

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

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

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

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

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

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

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

Ke-Seng Zhao Southern Medical University Guangzhou, China

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

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

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

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

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

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

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

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

Andrea Schmitt University of Goettingen Goettingen, Germany Arnaldo Valdir Zumiotti

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

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

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

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

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

Hiroyuki Hirasawa Chiba University School of Medicine Chiba, Japan

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

Irismar Reis de Oliveira Faculdade de Medicina da Universidade Federal da Bahia Salvador, BA, Brasil Irshad Chaudry University of Alabama Birmingham, AL, USA Ivan Cecconello

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


Michael Gregory Sarr Mayo Clinic Rochester, MN, USA

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

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

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

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

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

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

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

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

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

Noedir Antonio Groppo Stolf Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Roberto Chiesa San Raffaele Hospital

Milan, Italy Ronald A. Asherson Netcare Rosebank Hospital Rosebank, Johannesburg, South A´frica Samir Rasslan Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Tarcisio Eloy Pessoa de Barros Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Valentim Gentil Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Wagner Farid Gattaz Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Board of Governors Alberto Jose´ da Silva Duarte Aluisio Augusto Cotrim Segurado Ana Claudia Latronico Xavier Berenice Bilharinho de Mendonc¸a Carlos Roberto Ribeiro de Carvalho Clarice Tanaka Claudia Regina Furquim de Andrade Cyro Festa 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 Giovanni Guido Cerri 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 Marcus Castro Ferreira Maria Aparecida Shikanai Yasuda Miguel Srougi Milton de Arruda Martins Nelson de Luccia Olavo Pires de Camargo Paulo Andrade Lotufo Paulo Hila´rio Nascimento Saldiva Paulo Manuel Peˆgo Fernandes Paulo Marcelo Gehm Hoff

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

Paulo Rossi Menezes 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@hc.fm.usp.br 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$ 2,000.00; 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(12):1475–1560

CLINICAL SCIENCES

The impact of glucocorticoids and anti-cd20 therapy on cervical human papillomavirus infection risk in women with systemic lupus erythematosus Claudia Mendoza-Pinto, Mario Garcia-Carrasco, Veronica Vallejo-Ruiz, Alejandro Taboada-Cole, Margarita Mun˜oz-Guarneros, Juan Carlos Solis-Poblano, Elias Pezzat-Said, Adriana Aguilar-Lemarroy, Luis Felipe Jave-Suarez, Luis Vazquez de Lara, Gloria Ramos-Alvarez, Julio Reyes-Leyva, Aurelio Lopez-Colombo . . . . . . . . . . . . . . . . . . 1475

Ankle-brachial index as a predictor of coronary disease events in elderly patients submitted to coronary angiography Eduardo D. E. Papa, Izo Helber, Manes R. Ehrlichmann, Claudia Maria Rodrigues Alves, Marcia Makdisse, Livia N. Matos, Jairo Lins Borges, Renato D. Lopes, Edson Stefanini, Antonio Carlos Carvalho . . . . . . . . . . . . . . 1481

pH in exhaled breath condensate and nasal lavage as a biomarker of air pollution-related inflammation in street traffic-controllers and office-workers Thamires Marques de Lima, Cristiane Mayumi Kazama, Andreas Rembert Koczulla, Pieter S. Hiemstra, Mariangela Macchione, Ana Luisa Godoy Fernandes, Ubiratan de Paula Santos, Maria Lucia Bueno-Garcia, Dirce Maria Zanetta, Carmen Diva Saldiva de Andre´, Paulo Hilario Nascimento Saldiva, Naomi Kondo Nakagawa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1488

The impact of metabolic syndrome on metabolic, pro-inflammatory and prothrombotic markers according to the presence of high blood pressure criterion Juliana S. Gil, Luciano F. Drager, Grazia M. Guerra-Riccio, Cristiano Mostarda, Maria C. Irigoyen, Valeria Costa-Hong, Luiz A. Bortolotto, Brent M. Egan, Heno F. Lopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1495

The involvement of multiple thrombogenic and atherogenic markers in premature coronary artery disease Antonio P. Mansur, Julio Y. Takada, Ce´lia M. C. Strunz, Solange D. Avakian, Luiz Antonio M. Ce´sar, Jose´ A. F. Ramires. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1502

Reproducibility of ambulatory blood pressure changes from the initial values on two different days Garrett I. Ash, Timothy J. Walker, Kayla M. Olson, Jeffrey H. Stratton, Ana L. Go´mez, William J. Kraemer, Jeff S. Volek, Linda S. Pescatello . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1509

Predictors of in-hospital mortality in patients with ST-segment elevation myocardial infarction undergoing pharmacoinvasive treatment Felipe Jose´ de Andrade Falca˜o, Cla´udia Maria Rodrigues Alves, Adriano Henrique Pereira Barbosa, Adriano Caixeta, Jose´ Marconi Almeida Sousa, Jose´ Augusto Marcondes Souza, Amaury Amaral, Luiz Carlos Wilke, Fa´tima Cristina A. Perez, Iran Gonc¸alves Ju´nior, Edson Stefanini, Antoˆnio Carlos Carvalho. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1516

Posterior-only spinal fusion without rib head resection for treating type I neurofibromatosis with intracanal rib head dislocation Dong Sun, Fei Dai, Yao Yao Liu, Jian-Zhong Xu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1521


BASIC RESEARCHES

The putative role of ovary removal and progresterone when considering the effect of formaldehyde exposure on lung inflammation induced by ovalbumin Adriana Lino-dos-Santos-Franco, Renata Midori Amemiya, Ana Paula Ligeiro de Oliveira, Amı´lcar Sabino Damazo, Ana Cristina Breithaupt-Faloppa, Luana Beatriz Vitoretti, Beatriz Golega˜ Acceturi, Wothan Tavares-de-Lima . . . . 1528

Gender differences in microcirculation: Observation using the hamster cheek pouch Diogo Guarnieri Panazzolo, Lucia Henriques Alves da Silva, Fa´tima Zely Garcia de Almeida Cyrino, Fernando Lencastre Sicuro, Luiz Guilherme Kraemer-Aguiar, Eliete Bouskela . . . . . . . . . . . . . . . . . . . . . . . . . . . 1537

Biomechanical comparison of the four-strand cruciate and Strickland techniques in animal tendons Raquel Bernardelli Iamaguchi, William Villani, Marcelo Rosa Rezende, Teng Hsiang Wei, Alvaro B. Cho, Gustavo Bispo dos Santos, Rames Mattar Jr . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1543

Local and remote ischemic preconditioning protect against intestinal ischemic/reperfusion injury after supraceliac aortic clamping Nilon Erling Junior, Edna Frasson de Souza Montero, Paulina Sannomiya, Luiz Francisco Poli-de-Figueiredo (in memoriam) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1548

RAPID COMMUNICATION

Association between phase angle, anthropometric measurements, and lipid profile in HCV-infected patients Mariana de Souza Dorna, Nara Aline Costa, Erick Prado de Oliveira, Ligia Yukie Sassaki, Fernando Gomes Romeiro, Sergio Alberto Rupp de Paiva, Marcos Ferreira Minicucci, Giovanni Faria Silva . . . . . . . . . . . . . . . . . . . . . . . . . 1555

ERRATUM

Errata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1559


CLINICAL SCIENCE

The impact of glucocorticoids and anti-cd20 therapy on cervical human papillomavirus infection risk in women with systemic lupus erythematosus Claudia Mendoza-Pinto,I,II Mario Garcia-Carrasco,I,II,III Veronica Vallejo-Ruiz,II Alejandro Taboada-Cole,I Margarita Mun˜oz-Guarneros,IV Juan Carlos Solis-Poblano,V Elias Pezzat-Said,III Adriana Aguilar-Lemarroy,VI Luis Felipe Jave-Suarez,VI Luis Vazquez de Lara,IV Gloria Ramos-Alvarez,I Julio Reyes-Leyva,II Aurelio Lopez-ColomboVII I

Instituto Mexicano del Seguro Social (IMSS), Systemic Autoimmune Diseases Research Unit, HGR 36, Puebla, Mexico. II Instituto Mexicano del Seguro Social (IMSS), Centro de Investigacio´n Biome´dica de Oriente (CIBIOR), Molecular Biology and Virology Laboratory, Metepec, Puebla, Mexico. III Beneme´rita Universidad Auto´noma de Puebla, Medicine School, Department of Immunology and Rheumatology, Puebla, Mexico. IV Beneme´rita Universidad Auto´noma de Puebla, Medicine School, Research and Posgraduate, Studies Secretary, Puebla, Mexico. V Instituto Mexicano del Seguro Social (IMSS), Unidades Me´dicas de Alta Especialidad (UMAE), Department of Hematology, Puebla, Mexico. VI Instituto Mexicano del Seguro Social (IMSS), Centro de Investigacio´n Biome´dica de Occidente (CIBO), Immunology Department, Guadalajara, Jalisco, Mexico. VII Instituto Mexicano de Seguro Social, Puebla, Me´xico.

OBJECTIVE: To identify the prevalence and factors associated with cervical human papillomavirus infection in women with systemic lupus erythematosus METHODS: This cross-sectional study collected traditional and systemic lupus erythematosus-related disease risk factors, including conventional and biologic therapies. A gynecological evaluation and cervical cytology screen were performed. Human papillomavirus detection and genotyping were undertaken by PCR and linear array assay. RESULTS: A total of 148 patients were included, with a mean age and disease duration of 42.5¡11.8 years and 9.7¡5.3 years, respectively. The prevalence of squamous intraepithelial lesions was 6.8%. The prevalence of human papillomavirus infection was 29%, with human papillomavirus subtype 59 being the most frequent. Patients with human papillomavirus were younger than those without the infection (38.2¡11.2 vs. 44.2¡11.5 years, respectively; p = 0.05), and patients with the virus had higher daily prednisone doses (12.8¡6.8 vs. 9.7¡6.7 mg, respectively; p = 0.01) and cumulative glucocorticoid doses (14.2¡9.8 vs. 9.7¡7.3 g, respectively; p = 0.005) compared with patients without. Patients with human papillomavirus infection more frequently received rituximab than those without (20.9% vs. 8.5%, respectively; p = 0.03). In the multivariate analysis, only the cumulative glucocorticoid dose was associated with human papillomavirus infection. CONCLUSIONS: The cumulative glucocorticoid dose may increase the risk of human papillomavirus infection. Although rituximab administration was more frequent in patients with human papillomavirus infection, no association was found. Screening for human papillomavirus infection is recommended in women with systemic lupus erythematosus. KEYWORDS: Cervical Human Papillomavirus Infection; Systemic Lupus Erythematosus; Risk Factors; Rituximab. Mendoza-Pinto C, Garcia-Carrasco M, Vallejo-Ruiz V, Taboada-Cole A, Mun˜oz-Guarneros M, Solis-Poblano JC, et al. The impact of glucocorticoids and anti-cd20 therapy on cervical human papillomavirus infection risk in women with systemic lupus erythematosus. Clinics. 2013;68(12):1475-1480. Received for publication on April 30, 2013; First review completed on May 28, 2013; Accepted for publication on May 28, 2013 E-mail: mgc30591@yahoo.com Tel.: 52 22 2323-3498

& INTRODUCTION Systemic lupus erythematosus (SLE) is a multisystemic disease that mainly affects young women and is caused by autoantibodies to a variety of autoantigens. SLE has been associated with cervical dysplasia (1-3), for which some risk factors have also been identified, including a history of sexually transmitted disease, early onset of sexual activity, the number of sexual partners, and low educational levels (3). Immunosuppressive therapy can increase the risk of

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(12)01

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activity and damage, respectively. The drug history was determined by chart review. The daily prednisone dose at the study visit and the cumulative glucocorticoid (GC) dose were measured. The continuous use of immunosuppressive therapy (azathioprine, leflunomide, methotrexate, mycophenolic acid, and cyclophosphamide) and rituximab in the three years before study recruitment was recorded. Rituximab administration (261 g) was added to immunosuppressive treatment (azathioprine, leflunomide, methotrexate or mycophenolic acid) in patients with refractory disease. The genitalia were clinically examined by a single gynecologist, who examined the vulva, vagina, and cervix of each woman. Pap smears were collected using conventional techniques. Briefly, the cervix was visualized after insertion of a speculum into the vagina with rotation using gentle pressure. A Cytobrush was inserted two-thirds of the way into the endocervical canal and external cervix and was rotated 360 ˚; the material obtained was fixed on a microscope slide. A second Cytobrush was inserted into the endocervical canal, and the material obtained was deposited in a 15 ml tube containing 3 ml of phosphate-buffered saline (PBS) (137mM NaCl, 1.8mM KH2PO4, 2.7mM KCl, 10mM Na2HPO4). All Pap smears were evaluated by a cytopathologist who was blinded to the results of the gynecological examinations. Cervical cytology results were classified according to the 2001 Bethesda system as negative for intraepithelial lesion or malignancy; epithelial cell abnormalities that include atypical squamous cells; low-grade squamous intraepithelial lesion (LGSIL); high-grade squamous intraepithelial lesion (HGSIL); squamous cell carcinoma; atypical glandular cells; endocervical adenocarcinoma in situ; adenocarcinoma; or other (17).

viral infections, such as cervical human papillomavirus (HPV) infection, which is strongly associated with cervical dysplasia (4,5). Furthermore, a high risk of vulvar cancers has been found in patients with SLE, and one important factor is the possibility of altered clearance of viruses, particularly HPV, which is linked to this malignancy and cervical cancer (6). A recent analysis of a multicenter SLE cohort demonstrated that the standardized incidence ratio (SIR) for cervical cancer is consistent with increased risk (SIR 1.65, 95% CI 1.09-2.41) (7). There is also a relationship between immunosuppressive therapy and cervical abnormalities (8). However, some studies have found no association between cervical HPV infection and immunosuppressive therapy (3,9). In recent years, new, targeted therapies have been administered to SLE patients, and there has been some evidence for the efficacy and safety of B cell depletion by anti-CD20 therapy with rituximab. Although there was a case report of rituximab administration and JC papovavirus infection in a patient with non-Hodgkin lymphoma (10), the effect of this type of therapy on cervical HPV infection is unknown. In Mexico, the prevalence of HPV in cervical samples is estimated at 9.3% (11), and the prevalence of cervical cancer in women in the general population (aged $35 years old) ranges between 0.5% and 0.9% (12). Mexican patients with SLE have an elevated risk of major organ involvement (13). This type of patient usually requires immunosuppressive drugs and even biologic therapy, which may increase the risk of squamous intraepithelial lesions in patients with cervical HPV infection. The prevalence of cervical HPV infections in Mexican women with SLE was recently evaluated (5). The objective of this study was to identify the prevalence and factors associated with cervical HPV infection in women with SLE.

Determination of HPV in cervical smears All samples were labeled and stored at -20 ˚C until DNA extraction. Cervical smears were shaken vigorously and centrifuged for 10 min at 4000 rpm. The pellets were resuspended in 1 ml PBS, and a 0.5 ml aliquot was used for DNA extraction using the QIAamp DNA Mini (Qiagen, Hilden, Germany) kit, according to the manufacturer’s recommendations. Purified DNA was suspended in 100 ml of water and stored at -20 ˚C until use. DNA integrity was verified by 1% agarose gel electrophoresis.

& MATERIALS AND METHODS Patient selection and assessment In this cross-sectional study, consecutive female patients who presented at the Systemic Autoimmune Disease Research Unit of General Regional Hospital No. 36, Instituto Mexicano del Seguro Social, Puebla, Mexico, and fulfilled the 1997 American College of Rheumatology revised criteria for the classification of SLE (14) were recruited. Patients were eligible for the study if they were married or sexually active. Patients were excluded if they were pregnant or had had a hysterectomy, cervical cancer, or a previous diagnosis of papillomavirus infection. None of the patients had been immunized against any HPV subtypes. The local institutional ethics committee approved the study, and written informed consent was obtained from all participants. All women with abnormal Pap smears were referred for gynecologic follow-up. The study visit for each participant included a structured interview detailing demographic information and medical history, including sexual, gynecological, and obstetric histories, and a gynecological examination, including the collection of samples for a Pap test and HPV test. The Systemic Lupus Erythematosus Disease Activity Index, validated for the Mexican population (mexSLEDAI) (15), and the Systemic Lupus International Collaborating Clinics/American College of Rheumatology damage index (SLICC/ACR DI) for SLE (16) were used to assess disease

HPV detection by PCR assay HPV was detected using PGMY09/11 primers (18) which allow for the detection of more than 30 HPV genotypes. The final volume of the PCR reaction was 25 ml, including 10.5 ml of the DNA sample, 10 pmol of each PGMY09/11 primer, and the Taq PCR Master Mix kit (Promega, Madison, WI, USA). Amplification was performed in a thermal cycler (PTC 200, MJ Research, Watertown, MA, USA) using 40 cycles of 95 ˚C for 1 min and 55 ˚C for 1 min to allow for alignment and 72 ˚C for 1 min to allow for elongation. These cycles were followed by a final extension at 72 ˚C for 5 min. A fragment of the cyclophilin gene was amplified as a control for the samples. The amplification products were analyzed by 1.5% agarose gel electrophoresis. The gels were stained with ethidium bromide (1 mg/ ml), observed in an ultraviolet transilluminator, and photographed using a digital camera Canon (Melville, NY, USA) (18-20).

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Identification of viral genotypes

Table 1 - Sociodemographic, clinical, and treatment characteristics of patients with systemic lupus erythematosus.

HPV DNA genotyping was performed with the Linear Array (LA) HPV assay (Roche Diagnostic) in samples with a positive HPV PCR test. The LA assay uses the PGMY09/ PGMY11 primer set, which amplifies a 450-bp fragment of the L1 gene, and it can detect 37 HPV genotypes (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 56, 57, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39, and CP6108). On each strip, two different concentrations of bglobin probes are present, which serve as internal controls to assure adequate amplifiable DNA in each specimen. Beta-globin negative samples were considered inadequate and genotyping test was repeated. Genital HPV has traditionally been classified into low-risk (LR) and highrisk (HR) types. HR HVP types are associated with HGSIL and cervical cancer. The HPV types classified in the HR group include 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 (21). The assay was performed according to the manufacturer’s instructions. Briefly, DNA was amplified in a total volume of 100 ml, containing 50 ml of sample DNA and 50 ml of the master mixture provided by the manufacturer. The amplification protocol was as follows: 9 min of denaturation at 95 ˚C; 40 cycles of 30 s of denaturation at 95 ˚C, 1 min of annealing at 55 ˚C, and 1 min of elongation at 72 ˚C; and a final extension for 5 min at 72 ˚C. After amplification, the whole PCR product was denatured and hybridized with oligonucleotide probes immobilized on strips. After a stringent wash, the hybrids were detected by the addition of streptavidin-horseradish peroxidase conjugate, which binds to the biotinylated PCR primers, and a substrate (hydrogen peroxide and 3, 39, 5, 59-tetramethylbenzidine) that generates a purple precipitate at the probe line.

Variable Age, mean ¡ SD, years Formal education, mean ¡ SD, years Current smoker, n (%) Age at first intercourse, mean ¡ SD, years Pregnancies, n (%) 0 1-2 3 or more Number of sexual partners, n (%) 1 2 3 or more Oral contraceptive use, n (%) Disease duration, mean ¡ SD, years Previous medication Antimalarials, n (%) Daily prednisone (mg/d), mean ¡ SD Cumulative GC dose (g), mean ¡ SD Azathioprine, n (%) Methotrexate, n (%) Leflunomide, n (%) Mycophenolic acid, n (%) Cyclophosphamide, n (%) Cumulative cyclophosphamide (g), mean ¡ SD Rituximab, n (%)

N = 148 42.5¡11.8 11.0¡4.4 16 (10.6) 20.4¡3.7 133 (88.1) 18 (12.1) 62 (41.8) 68 (45.9) 105 (70.9) 24 (16.3) 19 (12.8) 1 (0.7) 9.7¡5.3 144 (95.4) 10.5¡6.8 11.0¡8.4 47 (31.3) 29 (19.2) 9 (5.9) 5 (3.3) 4 (2.7) 5.8¡3.2 18 (12.1)

SD: Standard deviation; GC: glucocorticoid.

leflunomide 20 mg per day, and mycophenolic acid 2 g per day. Most patients who had received intravenous cyclophosphamide had been administered the NHI protocol. Forty-three patients (29%) had HPV infections, of whom 31 (72%) had HR HPV infections, and 20 (13.5%) had $2 HPV infections. The most prevalent HR HVP types were 59 (34.8%), 18 (18.6%), 62 (16.2%), and 16 (13.9%). Cervical HPV (+) patients were younger than cervical HPV (-) patients (38.2¡11.2 years vs. 44.2¡11.5 years, respectively; p = 0.05) and had higher prednisone doses (12.8¡6.8 mg vs. 9.7¡6.7 mg, respectively; p = 0.01) and higher cumulative GC doses (14.2¡9.8 g vs. 9.7¡7.3 g, respectively; p = 0.005). The mexSLEDAI score and SLICC score did not significantly differ between the two groups. Although cervical HPV (+) patients were more likely to have received immunosuppressive therapy than cervical HPV (-) patients, the difference was not significant (58.1% vs. 54.1%, respectively; p = 0.7). However, mycophenolic acid therapy (9.3% vs. 0.9%, respectively, p = 0.02; OR: 10.6, 95% CI: 1.15-98.4) and prior rituximab therapy (20.9% vs. 8.5%, respectively, p = 0.03; OR: 2.8, 95% CI: 1.03-7.7) were more frequent in cervical HPV (+) patients than in HPV (-) patients. The cumulative cyclophosphamide dose was higher in HPV (+) patients than in HPV (-) patients (6.3¡3.4 g vs. 5.2¡3.0 g, respectively; p = 0.05). Table 2 shows the differences between patients with and without cervical HPV infections. The prevalence of cervical HVP infection did not differ significantly according to the response to rituximab therapy. In the logistic analysis, only the cumulative GC dose was associated with cervical HPV infection (OR: 1.03; 95% CI 1.01-1.11), with no association found with other traditional risk factors or immunosuppressive treatments. Similarly, there was no association with rituximab therapy.

Statistical analysis Quantitative variables are expressed as means ¡ standard deviations (SDs), and qualitative variables are expressed as frequencies (%). The means of two groups were compared using Student’s unpaired t-test. Proportions between groups were compared using the chi-square test or Fisher’s exact test. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to estimate the association between each possible risk factor and cervical HPV infection. Logistic regression was used to determine the independent risk factors for cervical HPV infection, and variables with a significance of p,0.05 in the univariate analysis were included in the multivariate analyses. The final models were assessed for interaction effects. All analyses were twotailed, and p-values #0.05 were considered significant. The statistical analysis was performed using SPSS for Windows, version 18.0 (SPSS, Chicago, IL, USA).

& RESULTS The sociodemographic, clinical, and treatment variables of the 148 SLE patients included are shown in Table 1. Sixtythree patients (42.5%) were postmenopausal. At the study visit, the mean mexSLEDAI and SLICC scores were 2.3¡2.1 and 1.3¡0.4, respectively. The mean mexSLEDAI score since disease onset was 3.54¡1.96. Eighteen patients had received rituximab, of whom three were non-responders (16.6%). The median daily doses of each immunosuppressive agent were azathioprine 100 mg per day, methotrexate 15 mg per week,

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Table 2 - Sociodemographic, clinical, and treatment characteristics of patients with systemic lupus erythematosus with and without cervical HPV infection.* Variable Age, mean ¡ SD, years Current smoker, n (%) Formal education, mean ¡ SD, years Pregnancies, n (%) 0 1-2 3 or more Age at first intercourse, mean ¡ SD, years Number of sexual partners, n (%) 1 2 3 or more Oral contraceptive use, n (%) Disease duration, mean ¡ SD years mexSLEDAI at study visit, mean ¡ SD, score mexSLEAI since diagnosis, mean ¡ SD, score SLICC/ACR DI, mean ¡ SD, score Previous medication Antimalarials, n (%) Daily prednisone dose, mean ¡ SD, mg Cumulative GC dose, mean ¡ SD, mg Azathioprine, n (%) Methotrexate, n (%) Leflunomide, n (%) Mycophenolic acid, n (%) Cyclophosphamide, n (%) Cumulative CYC (g), mean ¡ SD Rituximab, n (%) *

Patients with HPV (N = 43)

Patients without HPV (N = 105)

p-value

38.2¡11.2 4 (9.3) 10.7¡4

44.2¡11.5 12 (11.4) 11.2¡4.6

0.05 0.40 0.30

8 (18.6) 21 (48.8) 14 (32.6) 20.4¡3.4

10 (9.5) 43 (41) 52 (49.5) 20.5¡3.8

0.07 0.08 0.06 0.80

27 (62.7) 8 (18.6) 8 (18.6) 1 (2.3) 9.5¡6.2 2.3¡2.1 4.0¡2.0 1.2¡0.5

78 (74.2) 16 (15.2) 11 (10.4) 0 (0) 9.8¡6.1 2.1¡2.0 3.3¡1.9 1.3¡0.4

0.30 0.40 0.20 0.29 0.77 0.70 0.08 0.80

36 (83.7) 12.8¡6.8 14.2¡9.8 16 (37.2) 5 (11.6) 5 (11.6) 4 (9.3) 3 (6.9) 6.3¡3.4 9 (20.9)

81 (77.1) 9.7¡6.7 9.7¡7.3 30 (28.5) 25 (23.8) 6 (4.7) 1 (0.9) 1 (0.9) 5.2¡3.0 9 (8.5)

0.50 0.01 0.005 0.33 0.17 0.28 0.02 0.07 0.50 0.03

HPV: human papillomavirus; SD: standard deviation; GC: glucocorticoid; CYC: cyclophosphamide.

Six percent of patients had low-grade squamous intraepithelial lesions, and 0.6% had high-grade squamous intraepithelial lesions, according to the 2001 Bethesda system classification. None of the patients had squamous cell carcinoma or adenocarcinoma.

with SLE, possibly related to sexual activity. However, we found no associations between the variables measuring sexual activity and infection, possibly due to the crosssectional nature of the study. Disease activity may be associated with abnormal cervical cytology in juvenile-onset SLE (26). However, we found no association between cervical HPV infection and lupus activity measured by the mexSLEDAI at the study visit or the mean activity since disease onset. The cumulative GC dose was the only risk factor associated with cervical HPV infection in our SLE patients. The cumulative GC dose was recently associated with Tolllike-receptor (TLR) downregulation and the risk of HPV infection (27). Prednisolone suppressed the functions of TLR-stimulated human plasmacytoid dendritic cells, thereby reducing the ability to clear HPV infection (28). The recent addition of biologic therapies, such as rituximab, to the treatment of SLE led us to include this factor in our analysis to identify any possible association with the risk of cervical HVP infection. The possible mechanism by which anti-CD20 therapy might increase susceptibility to HPV infection is unclear. Rituximab administration results in profound depletion of normal B cells for several months, but immunoglobulin levels remain unaltered in most patients. These effects may occur because long-lived plasma cells do not express CD20. The lack of an effect on immunoglobulin levels suggests that rituximab administration could have a minimal effect on the occurrence of infections. Depletion of B cells would be expected to result in poor antibody responses to new antigens. Several studies have reported that patients receiving rituximab exhibit decreased to absent humoral responses to new

& DISCUSSION Several studies have investigated the prevalence of and risk factors for cervical HPV infection in SLE patients. Although immunosuppressive therapy has been studied as a risk factor for HPV infection in these patients with contradictory results (3-5,8), biologic therapy has not previously been analyzed as a possible risk factor. We found a prevalence of cervical HVP infection of 29%, which was higher than in some studies. Tam et al. (22) found that 12.5% of SLE patients had a persistently high risk of HPV, which increased to 25% three years after diagnosis. Rojo-Contreras et al. (5) recently found a prevalence of 14.7% in Mexican SLE patients. In contrast, a Brazilian study found a prevalence of 80.7% (9). These differences may be due to environmental, cultural, or genetic risk factors or geographical variations (23). Our study assessed a greater number of HPV types than previous studies (24). HR HPV type 59 was the most prevalent type in our cohort (34.8%). Tam et al. (3) found subtype 59 in only 1.2% of patients with SLE, in whom the most prevalent HR HPV subtype was 16 (4.7%). In addition, 13.5% of our patients had $2 HPV subtypes, which is higher than in some studies (3,5) but lower than the 21.7% found by Klumb et al. (24). The prevalence of cervical HPV infection decreases sharply in women after the age of 30 years (25). We found a higher prevalence of cervical HPV in younger patients

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antigens compared with recall antigens (29-31). Our results showed that patients treated with rituximab had a higher prevalence of cervical HPV infection than those treated with conventional immunosuppressive therapy. However, this possible association was lost after adjusting for others factors, such as GC. Similarly, Abud-Mendoza et al. (32) found no associations between HPV infection and rituximab in patients with SLE and rheumatoid arthritis. One possible reason why rituximab was not associated with cervical HPV infection might have been that other routes, such as the innate immune response, could have been affected in these patients. Innate immune abnormalities have been reported in patients with SLE and HPV (27). In the univariate analysis, mycophenolic acid was associated with cervical HPV infection, although the association disappeared in the multivariate analysis, possibly because few patients were analyzed. A very recent Mexican study detected low levels of B and NK cells and an enhanced risk of HPV infection in SLE patients receiving mycophenolate mofetil (32). We also found no association between HPV infection and other immunosuppressive therapies, such as azathioprine and methotrexate. RojoContreras et al. (5), who also studied Mexican SLE patients, found no association between cervical HPV and azathioprine but did find an association with methotrexate and a longer duration of prednisone therapy (5). These differences may be explained by the different methods used to assess the impact of these therapies and to geographical variations in the prevalence of HPV in Mexican women (23). A positive association has been found between cervical dysplasia and cyclophosphamide (33,34), and Klumb et al. (24) found higher cumulative cyclophosphamide doses in SLE patients with HPV infections. In our study, cyclophosphamide was not associated with cervical HPV infection. The importance of vaccination, including HPV immunization, in preventing and reducing infectious morbidity and mortality in SLE patients was recently suggested (35,36). However, the onset of some autoimmune diseases, particularly SLE, following HPV vaccination has also been reported (37). Mok et al. (38) evaluated the immunogenicity and safety of GARDASIL, a quadrivalent HPV vaccine, in patients with SLE, and they found that the vaccine was well tolerated and reasonably effective in patients with stable SLE and that it did not induce an increase in lupus activity or flares. Our study is not without limitations. First, there were the inherent difficulties of cross-sectional studies and the effect of sample size in establishing causal relationships and identifying incidence. Longitudinal studies with more patients who have received biologic therapy are necessary to establish associations. All the patients receiving rituximab had been refractory to conventional treatment, which was still continued during rituximab therapy, and the combination of therapies could have resulted in a greater risk of HPV infection than the administration of biologic therapy alone. Furthermore, although we measured the cumulative GC and cyclophosphamide doses, we did not measure the cumulative doses of other immunosuppressive agents, identifying only those drugs that had been administered and the median daily doses. In addition, we did not measure B cell counts to reflect depletion due to rituximab treatment or the role of other elements of the innate immune system.

SLE and HPV Mendoza-Pinto C et al.

In conclusion, women with SLE, and particularly younger patients, had an increased prevalence of cervical HPV infection. Our results suggest that the cumulative GC dose may increase the risk of HPV. Although rituximab administration was more frequent in patients with HPV infections, no associations were found. Screening for HPV infection is recommended in women with SLE, particularly those receiving high GC doses. Longitudinal studies are necessary to demonstrate these possible associations and to evaluate the natural history of cervical HPV infection in women with SLE who are receiving biologic therapies.

& ACKNOWLEDGMENTS This research was supported, in part, by PROMEP/Benemerita Universidad Autonoma de Puebla. We would like to thank David Buss for his valuable guidance and advice during this project.

& AUTHOR CONTRIBUTIONS Mendoza-Pinto C substantially contributed to conception and design, and drafted the manuscript. Garcia-Carrasco M substantially contributed to conception and design, obtained funding and administrative, technical, and material support. Vallejo-Ruiz V acquired and interpreted data, drafted the manuscript. Taboada-Cole A substantially contributed to conception and design. MunËœoz-Guarneros M, Lara LV and Reyes-Leyva J analyzed and interpreted data. Solis-Poblano JC provided study supervision. PezzatSaid E obtained funding and administrative, technical, and material support. Aguilar-Lemarroy A and Jave-Suarez LF acquired data. RamosAlvarez G provided administrative, technical, and material support. LopezColombo A contributed to study planning, and provided administrative and technical support. All authors approved the final drafting of the manuscript.

& REFERENCES 1. Dhar JP, Kmak D, Bhan R, Pishorodi L, Ager J, Sokol RJ. Abnormal cervicovaginal cytology in women with lupus: a retrospective cohort study. Gynecol Oncol. 2001;82(1):4-6, http://dx.doi.org/10.1006/gyno. 2001.6207. 2. Blumenfeld Z, Lorber M, Yoffe N, Scharf Y. Systemic lupus erythematosus: predisposition for uterine cervical dysplasia. Lupus. 1994;3(1):5961, http://dx.doi.org/10.1177/096120339400300112. 3. Tam LS, Chan AY, Chan PK, Chang AR, Li EK. Increased prevalence of squamous intraepithelial lesions in systemic lupus erythematosus: association with human papillomavirus infection. Arthritis Rheum. 2004;50(11):3619-25, http://dx.doi.org/10.1002/art.20616. 4. Nath R, Mant C, Luxton J, Hughes G, Raju KS, Shepherd P, et al. High risk of human papillomavirus type 16 infections and of development of cervical squamous intraepithelial lesions in systemic lupus erythematosus patients. Arthritis Rheum. 2007;57(4):619-25, http://dx.doi.org/10. 1002/art.22667. 5. Rojo-Contreras W, Olivar-Flores EM, Gamez-Nava JI, Montoya-Fuentes H, Trujillo-Hernandez B, Suarez-Rincon AE, et al. Cervical human papillomavirus infection in Mexican women with systemic lupus erythematosus or rheumatoid arthritis. Lupus. 2012;21(4):365-72, http://dx.doi.org/10.1177/0961203311425517. 6. Bernatsky S, Ramsey-Goldman R, Labrecque J, Joseph L, Boivin JF, Petri M, et al. Cancer risk in systemic lupus: An updated international multicentre cohort study. J Autoimmun. 2013;42:130-5, http://dx.doi.org/10. 1016/j.jaut.2012.12.009. 7. Bernatsky S, Clarke AE, Petri MA, Urowitz MB, Fortin PR, Gladman DD, et al. Further defining cancer risk in systemic lupus: updated results in an expanded international multi-centre cohort [abstract]. Arthritis Rheum. 2010;62(Suppl.):S731. 8. Santana IU, Gomes AD, Lyrio LD, Rios Grassi MF, Santiago MB. Systemic lupus erythematosus, human papillomavirus infection, cervical pre-malignant and malignant lesions: a systematic review. Clin Rheumatol. 2010;30(5):665-72. 9. Lyrio LD, Grassi MF, Santana IU, Olavarria VG, Gomes AD, Costapinto L, et al. Prevalence of cervical human papillomavirus infection in women with systemic lupus erythematosus. Rheumatol Int. 2013;33(2):631-6. 10. Matteucci P, Magni M, Di Nicola M, Carlo-Stella C, Uberti C, Gianni AM. Leukoencephalopathy and papovavirus infection after treatment with chemotherapy and anti-CD20 monoclonal antibody. Blood. 2002;100 (3):1104-5, http://dx.doi.org/10.1182/blood-2002-04-1271.

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11. Flores Y, Bishai D, Lazcano E, Shah K, Lo¨rincz A, Herna´ndez M, Salmero´n J. Improving cervical cancer screening in Mexico: results from the Morelos HPV Study. Salud Publica Mex. 2003;45:S3:388-98, http:// dx.doi.org/10.1590/S0036-36342003000900013. 12. Kuri-Morales P, Emberson J, Alegre-Dı´az J, Tapia-Conyer R, Collins R, Peto R, et al. The prevalence of chronic diseases and major disease risk factors at different ages among 150,000 men and women living in Mexico City: cross-sectional analyses of a prospective study. BMC Public Health. 2009;9:9, http://dx.doi.org/10.1186/1471-2458-9-9. 13. Zonana-Nacach A, Yan˜ez P, Jime´nez-Balderas FJ, Camargo-Coronel A. Disease activity, damage and survival in Mexican patients with acute severe systemic lupus erythematosus. Lupus. 2007;16(12):997-1000, http://dx.doi.org/10.1177/0961203307083175. 14. Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus (letter). Arthritis Rheum. 1997;40(9):1725, http://dx.doi.org/10.1002/art.1780400928. 15. Guzman J, Cardiel MH, Arce-Salinas A, Sanchez-Guerrero J, AlarconSegovia D. Measurement of disease activity in systemic lupus erythematosus. Prospective validation of 3 clinical indices. J Rheumatol. 1992;19(19):1551-8. 16. Galdman D, Ginzler E, Goldsmith C, Fortin P, Liang M, Urowitz M, et al. The development and initial validation of the Systemic Lupus International Collaborating Clinics/American College of Rheumatology damage index for systemic lupus erythematosus. Arthritis Rheum. 1996;39(3):363-9, http://dx.doi.org/10.1002/art.1780390303. 17. Solomon D, Davey D, Kurman R, Moriarty A, O’Connor D, Prey M, et al. The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA. 2002;287(6):2114-9, http://dx.doi.org/10.1001/jama. 287.16.2114. 18. Gravitt PE, Peyton CL, Alessi TQ, Wheeler CM, Coutle´e F, Hildesheim A, et al. Improved amplification of genital human papillomaviruses. J Clin Microbiol. 2000;38(1):357-61. 19. van den Brule AJ, Meijer CJ, Bakels V, Kenemans P, Walboomers JM. Rapid detection of human papillomavirus in cervical scrapes by combined general primer-mediated and type-specific polymerase chain reaction. J Clin Microbiol. 1990;28(12):2739-43. 20. Yasojima K, Kilgore KS, Washington RA, Lucchesi BR, McGeer PL. Complement gene expression by rabbit heart: upregulation by ischemia and reperfusion. Circ Res. 1998;82(11):1224-30, http://dx.doi.org/10. 1161/01.RES.82.11.1224. 21. Mun˜oz N, Bosch FX, de Sanjose´ S, Herrero R, Castellsague´ X, Shah KV, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med. 2003;348(6):518-27. 22. Tam LS, Chan PK, Ho SC, Yu MM, Yim SF, Cheung TH, et al. Natural history of cervical papilloma virus infection in systemic lupus erythematosus - a prospective cohort study. J Rheumatol. 2010;37(2): 330-40, http://dx.doi.org/10.3899/jrheum.090644. 23. Orozco-Colin A, Carrillo-Garcia A, Mendez-Tenorio A, Ponce-de-Leo´n S, Mohar A, Maldonado-Rodrı´guez R, et al. Geographical variation in human papillomavirus prevalence in Mexican women with normal cytology. Int J Infect Dis. 2010;14(12):1082-7. 24. Klumb EM, Pinto AC, Jesus GR, Araujo M Jr, Jascone L, Gayer CR, et al. Are women with lupus at higher risk of HPV infection? Lupus. 2010;19(13):1485-91, http://dx.doi.org/10.1177/0961203310372952.

25. Wright TC Jr, Schiffman M. Adding a test for human papillomavirus DNA to cervical-cancer screening. N Engl J Med. 2003;348(6):489-90. 26. Febronio MV, Pereira RM, Bonfa E, Takiuti AD, Pereyra EA, Silva CA. Inflammatory cervicovaginal cytology is associated with disease activity in juvenile systemic lupus erythematosus. Lupus. 2007;16(6):430-5, http://dx.doi.org/10.1177/0961203307079298. 27. Yu SL, Chan PK, Wong CK, Szeto CC, Ho SC, So K, et al. Antagonistmediated down-regulation of toll-like receptors increases the prevalence of human papillomavirus infection in systemic lupus erythematosus. Arthritis Res Ther. 2012;14(2):R80, http://dx.doi.org/10.1186/ar3803. 28. Boor PP, Metselaar HJ, Mancham S, Tilanus HW, Kusters JG, Kwekkeboom J. Prednisolone suppresses the function and promotes apoptosis of plasmacytoid dendritic cells. Am J Transplant. 2006;6 (10):2332-41. 29. Takata T, Suzumiya J, Ishikawa T, Takamatsu Y, Ikematsu H, Tamura K. Attenuated antibody reaction for the primary antigen but not for the recall antigen of influenza vaccination in patients with non-Hodgkin Bcell lymphoma after the administration of rituximab-CHOP. J Clin Exp Hematop. 2009;49(1):9-13, http://dx.doi.org/10.3960/jslrt.49.9. 30. Oren S, Mandelboim M, Braun-Moscovici Y, Paran D, Ablin J, Litinsky I, et al. Vaccination against influenza in patients with rheumatoid arthritis: the effect of rituximab on the humoral response. Ann Rheum Dis. 2008;67(7):937-41. 31. Gelinck LB, Teng YK, Rimmelzwaan GF, Van den Bemt BJ, Kroon FP, van Laar JM. Poor serological responses upon influenza vaccination in patients with rheumatoid arthritis treated with rituximab. Ann Rheum Dis. 2007;66(10):1402-3, http://dx.doi.org/10.1136/ard.2007.071878. 32. Abud-Mendoza C, Cuevas-Orta E, Santilla´n-Guerrero EN, Martı´nezMartı´nez MU, Herna´ndez-Castro B, Estrada-Capetillo L, et al. Decreased blood level of B lymphocytes and NK cells in patients with systemic lupus erythematosus (SLE) infected with papillomavirus (HPV). Arch Dermatol Res. 2013;305(2):117-23, http://dx.doi.org/10.1007/s00403012-1258-9. 33. Ognenovski VM, Marder W, Somers EC, Johnston CM, Farrehi JG, Selvaggi SM, et al. Increased incidence of cervical intraepithelial neoplasia in women with systemic lupus erythematosus treated with cyclophosphamide. J Rheumatol. 2004;31(9):1763-7. 34. Bateman H, Yazici Y, Leff L, Peterson M, Paget SA. Increased cervical dysplasia in intravenous cyclophosphamide-treated patients with SLE: a preliminary study. Lupus. 2000;9(7):542-4, http://dx.doi.org/10.1177/ 096120330000900711. 35. Shoenfeld Y. Infections, vaccines and autoimmunity. Lupus. 2009;18(13): 1127-8, http://dx.doi.org/10.1177/0961203309351081. 36. O’Connor MB, Bond U, Phelan MJ. The impact of human papillomavirus and human papillomavirus vaccination among rheumatology patients. J Clin Rheumatol. 2010;16(7):355. 37. Soldevilla HF, Briones SFR, Navarra SV. Systemic lupus erythematosus following HPV immunization or infection? Lupus. 2012;21(2):158-61, http://dx.doi.org/10.1177/0961203311429556. 38. Mok CC, Ho LY, Fong LS, To CH. Immunogenicity and safety of a quadrivalent human papillomavirus vaccine in patients with systemic lupus erythematosus: a case-control study. Ann Rheum Dis. 2013;72(5): 659-64, http://dx.doi.org/10.1136/annrheumdis-2012-201393.

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Ankle-brachial index as a predictor of coronary disease events in elderly patients submitted to coronary angiography Eduardo D. E. Papa,I Izo Helber,I Manes R. Ehrlichmann,II Claudia Maria Rodrigues Alves,I Marcia Makdisse,II Livia N. Matos,I Jairo Lins Borges,I Renato D. Lopes,III Edson Stefanini,I Antonio Carlos CarvalhoI I

Universidade Federal do Estado de Sa˜o Paulo, Departamento de Cardiologia, Sa˜o Paulo/SP, Brazil. II Hospital Israelita Albert Einstein, Departamento de Cardiologia, Sa˜o Paulo/SP, Brazil. III Duke University School of Medicine, Department of Medicine, Durham/North Carolina, NC, USA.

OBJECTIVES: To correlate the importance of the ankle-brachial index in terms of cardiovascular morbimortality and the extent of coronary arterial disease amongst elderly patients without clinical manifestations of lower limb peripheral arterial disease. METHODS: We analyzed prospective data from 100 patients over 65 years of age with coronary arterial disease, as confirmed by coronary angiography, and with over 70% stenosis of at least one sub-epicardial coronary artery. We measured the ankle-brachial index immediately after coronary angiography, and a value of ,0.9 was used to diagnose peripheral arterial disease. RESULTS: The patients’ average age was 77.4 years. The most prevalent risk factor was hypertension (96%), and the median late follow-up appointment was 28.9 months. The ankle-brachial index was ,0.9 in 47% of the patients, and a low index was more prevalent in patients with multiarterial coronary disease compared to patients with uniarterial disease in the same group. Using a bivariate analysis, only an ankle-brachial index of ,0.9 was a strong predictive factor for cardiovascular events, thereby increasing all-cause deaths and fatal and non-fatal acute myocardial infarctions two- to three-fold. CONCLUSION: In elderly patients with documented coronary disease, a low ankle-brachial index (,0.9) was associated with the severity and extent of coronary arterial disease, and in late follow-up appointments, a low index was correlated with an increase in the occurrence of major cardiovascular events. KEYWORDS: Peripheral Artery Disease; Prognosis; Coronary artery Disease; Ankle Brachial Index; Elderly. Papa ED, Helber I, Ehrlichmann MR, Alves CM, Makdisse M, Matos LN, et al. Ankle-brachial index as a predictor of coronary disease events in elderly patients submitted to coronary angiography. Clinics. 2013;68(12):1481-1487. Received for publication on March 1, 2013; First review completed on April 15, 2013; Accepted for publication on June 8, 2013 E-mail: eduardo.papa@yahoo.com.br Tel.: 55 11 99963-5141

to patients 40–70 years of age (14.5% and 4.3%, respectively) (2). The coexistence of PAD with Coronary Arterial Disease (CAD) and cerebrovascular disease (CVD) is well known. The REACH trial, which involved elderly patients with CAD, CVD and symptomatic or asymptomatic PAD with three or more atherothrombotic risk factors, demonstrated that 70% of patients with PAD have atherosclerotic disease in other vascular beds (3). According to Belch et al. (1), individuals with low ankle-brachial indices (ABIs) have twice the chance of presenting CAD compared to subjects with normal ABIs and have increased risks of fatal and nonfatal myocardial infarctions, stroke and cardiovascular mortality as well as increased overall mortality. The ABI is a simple, non-invasive method used to diagnose PAD. Compared to lower limb arterial angiography, an ABI ,0.9 has been shown to have a sensitivity of 90–97% and a specificity of 98–100% for detecting stenosis that affects the lumen in more than 50% of one or more leg arteries (4). Some studies have shown an association

& INTRODUCTION Peripheral Arterial Disease of the lower limbs (PAD) is a prevalent form of atherosclerotic disease. It has been estimated that in both North America and Europe, PAD affects approximately 27 million people, representing 16% of the North American population over 55 years of age (1).

Longevity is a risk factor for the development of PAD Data from the National Health and Nutrition Examination Survey (NHANES) indicate that PAD prevalence among people over 70 years of age is three times higher compared

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(12)02

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between the extent of CAD and PAD detected by ABI, regardless of the presence of symptoms (5), and it is known that the association between PAD and CAD increases allcause mortality and the cardiovascular risk of these patients two- to three-fold (6). Additionally, in some cases, isolated PAD has been responsible for increased mortality compared to patients with isolated CAD or CVD (3). Data from patients with suspected CAD who have been referred for coronary angiography indicated that PAD prevalence is high when evaluated by ABI (6,7). Therefore, although we have examined a high-risk group of patients, elderly patients with documented CAD and no PAD manifestations, this study aims to evaluate the impact of ABI as a marker of cardiovascular events in these patients and to evaluate the relationship between ABI and the extent of CAD as documented by coronary angiography.

Federal de Sa˜o Paulo in accordance with the 1975 Helsinki Declaration and was registered as CEP 1254/09. We excluded elderly patients with a clinical history of intermittent claudication, patients who had no significant coronary atherosclerotic disease after cardiac catheterization angiography, patients with an ABI $1.3 (non-compressible arteries), patients with decompensated heart failure and/or significant edema in their lower limbs, patients with amputation and patients who refused to participate in the study (11–13). The same physician (EDEP) performed all of the clinical evaluations and ABI measurements; at the inclusion of patients in the study, the physician did not know the specific results of the coronary angiography and the extent of coronary disease in each patient. However, the physician did know that all of the patients would have significant coronary disease.

& METHODS

Ankle-brachial index measurements The ABI was measured using a portable vascular Doppler scanning MEDPEJH (Sa˜o Paulo, Brazil) 10 Mhz DV 2001 model and a BICH aneroid sphygmomanometer (Sa˜o Paulo, Brazil) with appropriate cuff inflators for brachial circumference, in accordance with international standards (11). During physical examination, the right arm circumference was measured at the mid-point between the acromion and the olecranon, and an appropriate cuff was selected. After a least a 5-minute rest, the systolic pressures of the upper (brachial) and lower (tibial posterior and dorsalis pedis) limbs were measured in the supine position, initially taking measures of the right superior member and, in sequence, the left superior member, left inferior member and right inferior member. The ABI of each inferior member was calculated by dividing the highest systolic pressure at the ankles by the highest systolic pressure of the upper arms. The lowest value obtained was validated for analysis, and according to the ABI value, PAD was classified as: severe (#0.5), moderate (0.51–0.7), mild (0.71–0.9), and without PAD (greater than or equal to 0.9). A pathological ABI was considered to be a value ,0.9 (11,12). Patients with an ABI of 1.3 or more (noncompressible arteries) (13), an acute decompensated heart failure and/or significant lower limb edema and an inferior member amputation as well as those who refused to participate in the study were not analyzed.

During the study period, 425 coronary angiographies were performed in patients older than 65 years. Of these patients, 109 were evaluated, and 100 patients met the inclusion criteria. Therefore, we conducted a prospective observational cohort study of 100 consecutive patients aged $65 years who were asymptomatic for peripheral vascular disease and documented CAD by coronary angiography and who, immediately after hemodynamic investigation, were submitted to ABI determination and were followed for a mean period of 28.9¡6.6 months by either medical care or telephonic contact. Angiographic inclusion criteria were defined as a stenosis that was $70% of the epicardial coronary artery in at least one vessel and/or greater than 50% of the left coronary branch. The extent of CAD was evaluated by the number of vessels involved: uniarterial when there was an isolated lesion in one coronary artery and multiarterial when evidence of CAD was present in two or more vessels. Hypertension was defined according to the criteria of the IV Brazilian Guidelines on Hypertension (a systolic blood pressure greater than or equal to 140 mmHg and a diastolic blood pressure greater than or equal to 90 mmHg with an associated cardiovascular risk factor) and/or use of antihypertensive medication (8). Diabetes mellitus was diagnosed using the criteria of the American Diabetes Association: two fasting glucose measurements greater than or equal to 126 mg/dl, an oral glucose tolerance test with a post-load value within the 2 hours that was greater than or equal to 200 mg/dl or a casual plasma glucose greater than or equal to 200 mg/dl (9). Dyslipidemia was defined as having plasma cholesterol levels greater than 240 mg/dl, low-density lipoprotein (LDL) cholesterol levels between 160 and 189 mg/dl and high-density lipoprotein (HDL) cholesterol levels ,40 mg/ dl, according to the III Brazilian Guidelines on Dyslipidemia and Atherosclerosis Prevention (10). Smoking was assessed (current smoker or former) regardless of smoking history. We considered the following items as major cardiovascular events (MACE) to be prospectively studied: all-cause mortality, fatal and non-fatal myocardial infarction (MI) and stroke (transient or not). All patients included in the study were outpatients who were cared for in the Cardiogeriatrics Center at UNIFESP, and the patients who agreed to take part in the study signed an informed consent document. This study was approved by the ethics and research committee of the Universidade

Statistical analysis and descriptive statistics Quantitative data were described as the means and standard deviations (SD). The cutoff for the ABI was ,0.9 for PAD diagnosis, taking into consideration values in the literature as references (11,12). Taking into account the characteristics of our sample, we assumed that we would have a 6% yearly event rate in patients with ABIs ,0.9 and a 1% event rate in patients with normal ABIs.

ABI analysis We evaluated possible ABI predictors (as a dichotomized variable) with bivariate analysis using the chi-square test or Fisher test. The continuous variable (age) in relation to ABI was evaluated using Student’s t-test for independent samples and the Kolmogorov-Smirnov test when necessary to determine whether the age followed a normal distribution.

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multiarterial patients in the PAD group compared to uniarterial patients of the same group. However, there was a high prevalence of uniarterial patients without PAD (Table 1). Thus, we observed that the more severe the CAD, the lower the ABI with higher PAD prevalence. In fact, in multiarterial patients, the ABI medium value was significantly lower compared to uniarterial patients. (0.8¡0.2 and 1.0¡0.3, respectively, p = 0.047). Data analysis indicated that the presence of multiarterial coronary disease is a risk factor for PAD development, with a two-fold increase (RR = 2.19, CI 95% = 1.27–3.77). Over a mean period of 28.9 months (SD 6.6 months), we performed late follow-up assessments on all of the 100 patients who were part of the trial since the trial began. There were 17 major events with 11 deaths (11%), 9 by cardiovascular causes (8 by acute myocardial infarction [AMI], 1 by myocardial revascularization surgery complication), 1 by acute cholecystitis (sepsis) and 1 patient by an undetermined cause. Most deaths occurred in the PAD group. AMI was the main cause of death and was more frequent among patients with PAD, as demonstrated in Table 1. The average annual event rate was greater than 10%. Mortality in the PAD group was higher for both overall and cardiovascular causes. There was also a higher incidence of stroke in this group (6%) compared to the normal ABI group, where there was no stroke. For non-fatal events, there was no significant difference between patients with or without PAD. However, MACE incidence was higher in PAD patients (Table 1). An ABI,0.9 was the most important factor for all-cause mortality with a three-fold increased risk (RR 3.01; CI 95% 0.91–9.55). Additionally, a low ABI was a strong predictor for AMI and MACE, as shown in Table 2. Even correcting for the statistically significant difference between normal versus low ABI and the presence of multivessel coronary disease, the relationship between ABI and MACE remained. Thus, adjusting the analysis for single-vessel or multivessel disease did not significantly modify the RR (original RR 2.71 [95% CI 1.03 to 7.12] and corrected RR 2.90 [95% CI 1.11 to 7.62], as shown in Table 4). An event-free survival curve analysis (MACE and AMI – Figures 1 and 2, respectively) indicated that the time elapsed between PAD diagnosis and the occurrence of MACE and AMI was shorter in the PAD group, as shown in Table 3.

The strength of association was measured using the Relative Risk (RR) with a 95% confidence interval (CI). For the bivariate analysis, we considered a 10% significance level to be significant if more than one isolated tested variable was identified. A Poisson Regression model with an estimated RR was used to evaluate the combined occurrence of highest systolic blood pressure as a continuous variable and multiarterial disease events.

Analysis of clinical events We evaluated possible clinical event predictors (deaths, acute myocardial infarction [AMI], hospital admissions, MACE) with a bivariate analysis using the chi-square test or the Fisher Test. The continuous variable (age) was evaluated in relation to the ABI using Student’s t-test for independent samples and the Kolmogorov-Smirnov test when necessary. Time elapsed between the PAD diagnosis and occurrence of MACE, and AMI was estimated calculating the last patient contact date minus the PAD diagnosis date for patients with no events. For patients with events, we determined the time considering the event occurrence date and the PAD diagnosis date. When the event date was uncertain, we considered the last patient contact date as the event date. We used the time elapsed between the PAD diagnosis and the occurrence of MACE and AMI to obtain event-free survival curves using the Kaplan-Meier method. We compared the survival curves in relation to the ABI risk using the Log-Rank test.

& RESULTS There were no significant hematomas or systemic complications after cardiac catheterization that would have interfered with the ABI measurements. In our sample (n = 100) there was a predominance of women (57%) and the average age was 77.4 years old (SD 6.7 years), ranging from 65 to 93 years, and the risk factor with the highest prevalence was hypertension (96%). The medium value for the ABI was 0.88 (SD 0.26), and we diagnosed PAD in 47% of the patients. The demographic characteristics of the population studied are presented in Table 1. The mean systolic blood pressure in the PAD group was higher than in the group without PAD (p,0.001), and after applying two correlation coefficients, we noted a significant negative correlation between systolic blood pressure and ABI,0.9, which is expected because the calculation of ABI is always inversely proportional to the systolic blood pressure. The prevalence of hypertension was high in both groups, among both patients with PAD (100%) and those with a normal ABI (94.3%). The drugs that were used in this study are shown in Table 1. There was a higher proportion of patients without PAD using calcium blockers (32.2% and 13.3%, p = 0.024); Acetylsalicilic acid was the most frequently prescribed medication in both groups, followed by statins, beta blockers, ACE inhibitors, diuretics, oral hypoglycemics, ARBs, insulin, fibrates and antiplatelet agents, with no difference between groups with PAD and without PAD. Chronic renal failure, defined by a plasma creatinine level greater than 2.0, was not associated with reduced ABI, as shown in Table 1. The extent of CAD was evaluated according the number of coronary arteries involved and revealed a high prevalence of

& DISCUSSION In this study, which involved elderly patients consecutively selected accordingly to coronary cineangiography and with obstructive lesions greater than 70% in at least one epicardial vessel, we found that 47% of these patients had low ABIs. Similarly, a high prevalence of PAD measured by the ABI has been reported in studies focusing on both populations at high risk for PAD and primary care patients. Poredos and Jug (14) correlated 42% of PAD prevalence in elderly patients (with an average age of 63.7 years) with CAD or cerebrovascular disease. In a study regarding acute coronary syndrome, Nun˜ez et al. (5) reported that approximately 40% of the studied subjects (with an average age of 67.7 years) had an ABI#0.9. The high average age of the patients included in our study (77.4 years) was higher than the described series and may partially explain the high prevalence of PAD we detected using the ABI, as this is a

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Table 1 - Demographic and angiographic characteristics and the incidence of cardiovascular events in patients with and without PAD. PAD (ABI,0.9) Variables Age, years (mean ¡ standard deviation) Blood pressure, mmHg (mean ¡ standard deviation) Systolic blood pressure Diastolic blood pressure Women, n (%) Men, n (%) Hypertension, n (%) Diabetes Mellitus, n (%) Smoking (current or former) n (%) Chronic Renal failure (Creatinine . 2.0) CAD extent – uniarterial, n (%) CAD extent – multiarterial, n (%) Drugs n (%) Nitrate Calcium antagonists Statins Beta-blockers Angiotensin (IECA) Diuretics Acetylsalicylic acid Oral hypoglycemic Insulin ARBs Fibrates Antiplatelets Death, n (%) Acute myocardial infarction, n (%) Stroke, n (%) MACE n (%)

No PAD

p-value

(n = 47)

(n = 53)

77.8¡6.6

77.1¡8.1

0.524

158.34¡18.75 83.40¡7.15 29 (61.7%) 18 (38.3%) 47 (100%) 20 (37.7%) 15 (31.9%) 6 (12.8%) 8 (17%) 39 (83.0%) 17 (32.1%) 7 (13.3%) 44 (83.1%) 44 (83.1%) 37 (69.9%) 29 (54.8%) 49 (92.5%) 11 (20.8%) 4 (7.6%) 8 (15.1%) 2 (3.8%) 2 (3.8%) 8 (17%) 9 (19.1%) 3 (6.4%) 12 (25.5%)

143.96¡20.45 83.00¡5.66 28 (52.8%) 25 (47.2%) 50 (94.3%) 19 (40.4%) 15 (28.3%) 5 (9.4%) 23 (43.4%) 30 (56.6%) 16 (34.1%) 15 (32.2%) 44 (93.7%) 35 (74.5%) 32 (68.1%) 32 (68.1%) 44 (93.7%) 11 (23.5%) 6 (12.8%) 11 (23.5%) 3 (6.4%) 3 (6.4%) 3 (5.7%) 4 (7.5%) 0 5 (9.43%)

,0.001 0.753 0.371 0.245 0.783 0.694 0.595 0.004 0.835 0.024 0.104 0.295 0.852 0.171 1.000 0.750 0.509 0.290 0.664 0.664 0.070 0.085 0.100 0.032

Age (mean ¡ standard deviation) of patients with or without Peripheral Arterial Disease of the lower limbs (PAD). Major cardiovascular events (MACE); Coronary Arterial Disease (CAD). Student’s t-test was used for the variable age (years). The chi-square test was used for the variables gender, diabetes mellitus, smoking, CAD extent (uniarterial or multiarterial), death, acute myocardial infarction, stroke and MACE. The Fisher test was used to analyze hypertension. The chi-square test was used to analyze chronic renal failure. Drugs were analyzed by the chi-square test or Fisher’s exact test. Student’s ttest was used to analyze blood pressure.

well-known correlation both in the general population and in patients with documented PAD (2,3,5,20). Major cardiovascular risk factors for CAD are usually the same for PAD. Nonetheless, some authors suggest that there are more specific strong risk factors associated with atherosclerosis in certain vascular beds, such as smoking and PAD, hypertension and cerebrovascular disease as well as dyslipidemia associated with PAD (14). In our study, there was no difference between the prevalence of risk factors in PAD patients and patients with CAD only (Table 1). This observation could be partially explained by the fact that we studied a group of patients with a high risk for cardiovascular events. Additionally, at the time of inclusion, all patients were adequately medicated, and any risk factors, such as smoking, were well controlled. As such, only 20% of the patients were smokers at the beginning of our study. The evaluation of coronary cineangiography data from this study indicated that patients with a low ABI (,0.9) have a higher prevalence of multiarterial coronary disease compared to uniarterial patients. Additionally, an ABI,0.9 was independently related to the extent of CAD, as measured by the number of coronary arteries with obstructive CAD that were detected in the coronary angiography. Similarly, Sukhija et al. (7,16) analyzed patients with an average age of 71 years who were submitted to coronary angiography for suspicion of CAD and evaluated them for PAD using the ABI. Following their

Table 2 - Analysis of the incidence of death, fatal and non-fatal acute myocardial infarction and major cardiovascular events related to cardiovascular risk factors and the presence of peripheral arterial disease (PAD) evaluated by the ankle-brachial index. RR Death PAD (ABI,0.9) Gender (female) Diabetes mellitus Smoking Fatal and non-fatal AMI PAD (ABI,0.9) Gender (female) Diabetes mellitus Smoking MACE PAD (ABI,0.9) Gender (female) Diabetes mellitus Smoking

CI 95%

p-value

3.01 1.32 1.30 1.33

0.91–9.55 0.41–4.22 0.43–3.98 0.42–4.26

0.070 0.637 0.642 0.625

2.54 1.21 1.34 1.46

0.87–7.36 0.42–3.44 0.48–3.71 0.51–4.13

0.085 0.723 0.571 0.475

2.70 1.38 1.09 1.27

1.08–6.77 0.55–3.43 0.45–2.65 0.51–3.16

0.032 0.481 0.840 0.601

Relative risk values (RR), confidence interval (CI) and p-value. The chisquare test was used for the variables gender, diabetes mellitus and smoking. The presence of Peripheral Arterial Disease (PAD) was considered to have an ankle-brachial index of ,0.9. Bivariate analysis was performed using the chi-square test or Fisher’s exact test to evaluate possible ABI predictors.

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atherosclerosis (carotid and/or femoral) (15); they concluded that an ABI#0.9 was the only predictor of major cardiovascular events and revascularization procedures when adjusted for age, LDL and cholesterol levels, intimal-medial thickness and CAD extent. Diehn et al. (20) concluded that symptomatic or asymptomatic PAD (ABI#0.9) has been independently and significantly associated with mortality and major cardiovascular events, with an up to two-fold increase in their occurrences compared to elderly patients without PAD in primary care centers in Germany. Although we studied a group of patients with a high risk for cardiovascular events, that is, elderly patients with documented CAD, our conclusions were similar to those from the patients in primary care centers described in previous studies. In our study, during late follow-up visits, we detected the presence of stroke in patients with low ABIs (6%); this pathology did not occur in patients with normal ABI. Murabito et al. (21) evaluated patients from the Framingham study, with an average age of 80 years, who were tested for PAD by ABI; most were asymptomatic for the condition (82%). In these patients, the authors also observed a higher prevalence of stroke among persons with PAD compared to patients with normal ABI (13% and 5%, respectively), with a two-fold increased risk of stroke or transitory ischemic stroke compared to patients with normal ABI. In fact, elderly population-based studies revealed a positive correlation between reduced ABI and the presence of carotid atherosclerosis (carotid stenosis or medio-intimal thickness increase) in addition to an increased stiffness of the aorta and the carotid arteries, a common condition in elderly patients with PAD (22). This study had a few limitations, including the sample size and follow-up time. Although 100 elderly patients with PAD diagnosed by ABI and submitted to coronary angiography could not be regarded as a small casuistic, this sample size was not sufficient for more conclusive associations. In fact, the inclusion of patients after catheterization may have been a limiting factor for the evaluation of patients and may therefore have influenced the results. Furthermore, our follow-up time could have been longer; an increased follow-up time would contribute to an increased number of events and would strengthen our conclusions. However, we have not lost any patients to follow-up since their consecutive inclusion, which strengthens our sample power; additionally, we had more events than initially considered. Finally, the finding of an RR of 3.01 regarding the risk related to a low ABI was similar to findings reported in the literature, even though our starting point for patient selection was considerably different from previous studies. In elderly patients with asymptomatic PAD and with documented CAD by coronary cineangiography, an ABI,0.9 proved to be a predictor of global and vascular mortality, fatal and non-fatal AMI and stroke, with an up to

Table 3 - Time elapsed between the PAD diagnosis and the occurrence of major cardiovascular events (MACE) and acute myocardial infarction (AMI). Time (years)

CI 95%

p-value

3.05 2.73

(2.94–3.16) (2.51–2.94)

0.022

3.07 2.83

(2.97–3.17) (2.63–3.03)

0.082

MACE ABI$0.9 ABI,0.9 AMI ABI$0.9 ABI,0.9

Time (average, years); Major cardiovascular events (MACE); Ankle-brachial index (ABI); Confidence interval (CI); Acute myocardial infarction (AMI). Average time was estimated by the Kaplan-Meier method. The p-value was calculated by the Log-Rank test.

analysis, they reported a high prevalence of multiarterial patients (63%) in the PAD group and 11% of multiarterial patients without PAD (p,0.001). Among patients in this same population, it was noted that the lower the ABI measurements, the higher the prevalence of multiarterial and the lower the prevalence of uniarterial patients (84% and 5%, respectively, p,0.001). Papamichael et al. (15), in a study on asymptomatic PAD patients, with an average age of 60 years, evaluated by ABI and submitted to elective coronary angiography demonstrated that a low ABI (#0.9) was related to a greater extension of CAD, evaluated according to the number of coronary arteries with obstructive CAD (variance analysis, p = 0.04) and to the Gensini score (p = 0.01). A late follow-up analysis in our study revealed that only a low ABI (,0.9) was a strong predictor of all-cause death (RR 3.01; CI 95% = 0.91–9.95), AMI occurrence (RR 2.54; CI95% = 0.87–7.36) and MACE incidence (2.70; CI 95% = 1.08–6.77) when evaluated in relation to other risk factors. In fact, Criqui et al. (17) demonstrated, for the first time, a six-fold increase in CAD mortality in elderly patients (with an average age of 66 years) with reduced ABI compared to patients with normal ABI. Subsequently, Newman et al. (18) also demonstrated an up to three-fold increase in CAD mortality in elderly individuals (with an average age of 77 years) with reduced ABI. Similarly, data from ‘‘The Cardiovascular Health Study’’ (19), which involved 5,888 patients at over 65 years of age, demonstrated that, after a six-year follow up, patients with a low ABI and prevalent cardiovascular disease exhibited a 50% increase in all-cause mortality and up to a 61% increase in fatal and non-fatal AMI when gender and age were adjusted. Papamichael et al. also evaluated the ABI as a prognostic factor in elderly patients who were submitted to elective coronary angiography and diagnosed with extracoronary

Table 4 - MACE analysis adjusting for confounding factors (ABI and multiarterial coronary disease). Crude Analysis

Variable

CI (95%)

RR Multiarterial ABI

1.078 2.706

(0.416 1.029

2.797) 7.115

Multivariate Analysis p-value

RR

0.877 0.043

0.775 2.90

CI (95%) 0.302 1.11

p-value 1.983 7.62

0.594 0.030

Relative risk values (RR), confidence interval (CI) and p-value. The confounding effect was assessed by regression models considering the Poisson distribution and Robust estimation, considering the RR (relative risk) given the study design. The p-value was estimated using the Poisson regression model.

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Figure 1 - Event-free survival by ABI categories. Kaplan-Meier estimates showing MACE during the follow-up visit.

three-fold increase compared to individuals without PAD. We also demonstrated that, in this cohort of patients, the lower the ABI value, the more extensive the CAD. Therefore, ABI could be a useful tool not only for early detection of PAD, a condition frequently underdiagnosed

on several levels of elderly care but also for the purpose of risk stratification among CAD patients. Those patients with reduced ABIs should undergo more aggressive cardiovascular management because these patients are part of a highrisk group for cardiovascular events and all-cause mortality.

Figure 2 - Event-free survival by ABI categories. Kaplan-Meier estimates showing AMI during the follow-up visit.

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& AUTHOR CONTRIBUTIONS Papa EDE conceived and designed the study, acquired data, analyzed and interpreted data and drafted the manuscript. Helber I, Erlichmann MR conceived and designed the study and analyzed data. Alves CM conceived and designed the study, analyzed data and performed coronary cineangiography. Makdisse M analyzed data. Mattos LN analyzed and interpreted data. Borges JL, Stefanini E analyzed data. Lopes RD critically reviewed the manuscript. Carvalho AC conceived and designed the study, analyzed and interpreted data and critically reviewed the manuscript.

12.

& REFERENCES 13.

1. Belch JJF, Topol EJ, Agnelli G, Bertrand M, Califf RM, Clement DL, et al. Critical Issues in Peripheral Arterial Disease Detection and Management A call to action. Arch Intern Med. 2003;163(8):884-92, http://dx.doi.org/ 10.1001/archinte.163.8.884. 2. Selvin E, Erlinger TP. Prevalence of and risk factors for peripheral arterial disease in the United States: results from the National Health and Nutrition Examination Survey, 1999-2000. Circulation. 2004;110(6):73843, http://dx.doi.org/10.1161/01.CIR.0000137913.26087.F0. 3. Eagle KA, Hirsch AT, Califf RM, Alberts MJ, Steg PG, Cannon CP, et al. Cardiovascular ischemic event rates in outpatients with symptomatic atherothrombosis or risk factors in the united states: insights from the REACH Registry. Crit Pathw Cardiol. 2009;8(2):91-7. 4. Doobay AV, Anand SS. Sensitivity and specificity of the ankle-brachial index to predict future cardiovascular outcomes: a systematic review. Arterioscler Thromb Vasc Biol. 2005;25(7):1463-9, http://dx.doi.org/10. 1161/01.ATV.0000168911.78624.b7. 5. Nu´n˜ez D, Morillas P, Quiles J, Cordero A, Guindo J, Soria F, et al. Usefulness of an abnormal ankle-brachial index for detecting multivessel coronary disease in patients with acute coronary syndrome. Rev Esp Cardiol. 2010;63(1):54-9, http://dx.doi.org/10.1016/S0300-8932(10)70009-9. 6. Moussa ID, Jaff MR, Mehran R, Gray W, Dangas G, Lazic Z, et al. Prevalence and prediction of previously unrecognized peripheral arterial disease in patients with coronary artery disease: the Peripheral Arterial Disease in Interventional Patients Study. Catheter Cardiovasc Interv. 2009;73(6):719-24, http://dx.doi.org/10.1002/ccd.21969. 7. Sukhija R, Aronow WS, Yalamanchili K, Peterson SJ, Frishman WH, Babu S. Association of ankle-brachial index with severity of angiographic coronary artery disease in patients with peripheral arterial disease and coronary artery disease. Cardiology. 2005;103(3):158-60, http://dx.doi. org/10.1159/000084586. 8. Brazilian Society of Hypertension. Brazilian Society of Cardiology. Brazilian Society of Nephrology. IV Brazilian Guidelines on Hypertension Arq Bras Cardiol. 2004:82(supl 4):1-14. 9. Brazilian Society of Cardiolgy. III Brazilian Guidelines on Dyslipidemia and Atherosclerosis Prevention Guideline for The Department of Atherosclerosis of Brazilian Society of Cardiology. Arq Bras Cardiol. 2001;77(supl 3):1-48, http://dx.doi.org/10.1590/S0066-782X2001001500001. 10. Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Report of the expert committee on the diagnosis and classification of diabetes mellitus. Diabetes Care. 2003;26 Suppl 1:S5-20, http://dx.doi.org/10.2337/diacare.26.2007.S5. 11. Hirsch AT, Haskal ZJ, Hertzer NR, Bakal CW, Creager MA, Halperin JL, et al. ACC/AHA 2005 Practice Guidelines for the management of patients with peripheral arterial disease (lower extremity, renal,

14. 15.

16.

17.

18.

19.

20.

21.

22.

1487

mesenteric, and abdominal aortic): a collaborative report from the American Association for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (Writing Committee to Develop Guidelines for the Management of Patients With Peripheral Arterial Disease): endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation; National Heart, Lung, and Blood Institute; Society for Vascular Nursing; TransAtlantic Inter-Society Consensus; and Vascular Disease Foundation. Circulation. 2006;113(11):e463-654. Al-Qaisi M, Nott DM, King DH, Kaddoura S. Ankle brachial pressure index (ABPI): An update for practitioners. Vasc Health Risk Manag. 2009;5:833-41, http://dx.doi.org/10.2147/VHRM.S6759. Aboyans V, Lacroix P, Postil A, Guilloux J, Rolle´ F, Cornu E, et al. Subclinical peripheral arterial disease and incompressible ankle arteries are both long-term prognostic factors in patients undergoing coronary artery bypass grafting. J Am Coll Cardiol. 2005;46(5):815-20, http://dx. doi.org/10.1016/j.jacc.2005.05.066. Poredos P, Jug B. The prevalence of peripheral arterial disease in high risk subjects and coronary or cerebrovascular patients. Angiology. 2007;58(3):309-15, http://dx.doi.org/10.1177/0003319707302494. Papamichael CM, Lekakis JP, Stamatelopoulos KS, Papaioannou TG, Alevizaki MK, Cimponeriu AT, et al. Ankle-brachial index as a predictor of the extent of coronary atherosclerosis and cardiovascular events in patients with coronary artery disease. Am J Cardiol. 2000;86(6):615-8, http://dx.doi.org/10.1016/S0002-9149(00)01038-9. Sukhija R, Yalamanchili K, Aronow WS. Prevalence of left main coronary artery disease, of three- or four-vessel coronary artery disease, and of obstructive coronary artery disease in patients with and without peripheral arterial disease undergoing coronary angiography for suspected coronary artery disease. Am J Cardiol. 2003;92(3):304-5, http://dx.doi.org/10.1016/S0002-9149(03)00632-5. Criqui MH, Langer RD, Fronek A, Feigelson HS, Klauber MR, McCann TJ, et al. Mortality over a period of 10 years in patients with peripheral arterial disease. N Engl J Med. 1992;326(6):381-6. Newman AB, Sutton-Tyrrell K, Vogt MT, Kuller LH. Morbidity and mortality in hypertensive adults with a low ankle/arm blood pressure index. JAMA. 1993;270(4):487-9, http://dx.doi.org/10.1001/jama.1993. 03510040091035. Newman AB, Shemansky L, Manolio TA, Cushman M, Mittelmark M, Polak JF, et al. Ankle-arm index as a predictor of cardiovascular disease and mortality in the Cardiovascular Health Study. The Cardiovascular Health Study Group. Arterioscler Thromb Vasc Biol. 1999;19(3):538-45, http://dx.doi.org/10.1161/01.ATV.19.3.538. Diehm C, Allenberg JR, Pittrow D, Mahn M, Tepohl G, Haberl RL, et al. Mortality and vascular morbidity in older adults with asymptomatic versus symptomatic peripheral artery disease. Circulation. 2009;120(21):2053-61, http://dx.doi.org/10.1161/CIRCULATIONAHA. 109.865600. Murabito JM, Evans JC, Larson MG, Nieto K, Levy D, Wilson PW, et al. The ankle-brachial index in the elderly and risk of stroke, coronary disease, and death: the Framingham Study. Arch Intern Med. 2003;163(16):1939-42, http://dx.doi.org/10.1001/archinte.163.16.1939. van Popele NM, Grobbee DE, Bots ML, Asmar R, Topouchian J, Reneman RS, et al. Association between arterial stiffness and atherosclerosis: The Rotterdam Study. Stroke. 2001;32(2):454-60, http://dx.doi. org/10.1161/01.STR.32.2.454.


CLINICAL SCIENCE

pH in exhaled breath condensate and nasal lavage as a biomarker of air pollution-related inflammation in street traffic-controllers and office-workers Thamires Marques de Lima,I Cristiane Mayumi Kazama,I,II Andreas Rembert Koczulla,III Pieter S. Hiemstra,IV Mariangela Macchione,I Ana Luisa Godoy Fernandes,V Ubiratan de Paula Santos,VI Maria Lucia BuenoGarcia,VII Dirce Maria Zanetta,VIII Carmen Diva Saldiva de Andre´,IX Paulo Hilario Nascimento Saldiva,I Naomi Kondo NakagawaI,II I

Faculdade de Medicina da Universidade de Sa˜o Paulo, Department of Pathology, LIM 05, Sa˜o Paulo/SP, Brazil. II Faculdade de Medicina da Universidade de Sa˜o Paulo, Department of Physiotherapy, Communication Science and Disorders, and Occupational Therapy, LIM 34, Sa˜o Paulo/SP, Brazil. III Philipps University, Department of Pulmonology, Marburg/Germany. IV Leiden University Medical Center, Department of Pulmonology, Leiden, The Netherlands. V Universidade Federal de Sa˜o Paulo, School of Medicine, Department of Pneumology, Sa˜o Paulo/SP, Brazil. VI Faculdade de Medicina da Universidade de Sa˜o Paulo, Instituto do Corac¸a˜o (INCOR), Pneumology Division, Sa˜o Paulo/SP, Brazil. VII Faculdade de Medicina da Universidade de Sa˜o Paulo, Department of Internal Medicine, LIM 20, Sa˜o Paulo/SP, Brazil. VIII Universidade de Sa˜o Paulo, Public Health Faculty, Department of Epidemiology, Sa˜o Paulo/SP, Brazil. IX Universidade de Sa˜o Paulo, Institute of Mathematics and Statistics, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: To utilize low-cost and simple methods to assess airway and lung inflammation biomarkers related to air pollution. METHODS: A total of 87 male, non-smoking, healthy subjects working as street traffic-controllers or officeworkers were examined to determine carbon monoxide in exhaled breath and to measure the pH in nasal lavage fluid and exhaled breath condensate. Air pollution exposure was measured by particulate matter concentration, and data were obtained from fixed monitoring stations (8-h work intervals per day, during the 5 consecutive days prior to the study). RESULTS: Exhaled carbon monoxide was two-fold greater in traffic-controllers than in office-workers. The mean pH values were 8.12 in exhaled breath condensate and 7.99 in nasal lavage fluid in office-workers; these values were lower in traffic-controllers (7.80 and 7.30, respectively). Both groups presented similar cytokines concentrations in both substrates, however, IL-1b and IL-8 were elevated in nasal lavage fluid compared with exhaled breath condensate. The particulate matter concentration was greater at the workplace of trafficcontrollers compared with that of office-workers. CONCLUSION: The pH values of nasal lavage fluid and exhaled breath condensate are important, robust, easy to measure and reproducible biomarkers that can be used to monitor occupational exposure to air pollution. Additionally, traffic-controllers are at an increased risk of airway and lung inflammation during their occupational activities compared with office-workers. KEYWORDS: Exhaled Breath Condensate; Nasal Lavage Fluid; Air Pollution; Airway Inflammation. Lima TM, Kazama CM, Koczulla AR, Hiemstra PS, Macchione M, Fernandes AL, et al. pH in exhaled breath condensate and nasal lavage as a biomarker of air pollution-related inflammation in street traffic-controllers and office-workers. Clinics. 2013;68(12):1488-1494. Received for publication on March 28, 2013; First review completed on May 14, 2013; Accepted for publication on June 8, 2013 E-mail: naomi.kondo@usp.br Tel.: 55 11 3061-8520

pulmonary inflammation, infections, emergency room visits and hospital admissions (1–5). Among the pathophysiological mechanisms of these events, in vitro and in vivo studies have shown that air pollutants, particularly fine and ultrafine particles, induce the release of reactive oxygen species into airways and cause lung inflammation (6–8). In Sa˜o Paulo, which is one of the largest cities in the world, seven million vehicles circulate daily, resulting in traffic jam chaos in several areas of the city. Because of the high levels of vehicle-released pollutants in the atmosphere, outdoor activities in these areas may pose an increased health hazard, particularly to the respiratory system.

& INTRODUCTION There is epidemiological evidence that air pollution is directly associated with increases in respiratory symptoms,

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(12)03

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stored for a maximum of 4 weeks at 280 ˚C for the determination of cytokine levels.

Inflammation in the airways and lungs has an important role in the development and progression of several respiratory diseases. We examined the air pollution-related airway and lung inflammation in non-smoking, healthy street traffic-controllers and office-workers by measuring the pH and cytokines concentrations in exhaled breath condensate (EBC) and nasal lavage fluid (NLF) using low-cost and simple methods (9–16). Both EBC and NLF contain particles from the upper and the lower airway lining fluid and may be potential sources of air pollution-related inflammatory biomarkers.

Nasal lavage collection Subjects were asked to tilt their head back at a 45 ˚ angle and close the nasopharynx with the soft palate. Room temperature isotonic sodium chloride solution (0.9% NaCl, 5 ml) was instilled into each nostril. After 10 sec, the subject blew their nose forcefully into a sterile plastic container. The average recovery of fluid from NLF was approximately 70%. The lavage fluid was centrifuged (10 min, 300 g, 5 ˚C), and the supernatant was separated from the pellet and divided into five aliquots of 500 ml. One of these supernatant aliquots was immediately used for pH measurements. The remaining aliquots were coded (for blinding purposes) and stored at 280 ˚C for up to 4 weeks to determine the cytokine levels. The cell pellet was used for total and differential cell counts as previously described (15,16).

& MATERIALS AND METHODS Study population This cross-sectional study was approved by the local Ethics Committee of Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo (CAPPesq 0565/ 07). Non-smoking healthy male subjects aged 18 to 60 years were recruited from a list provided by the Engineering Traffic Company of Sa˜o Paulo City (street traffic-controllers) and from Faculdade de Medicina da Universidade de Sa˜o Paulo (office-workers). The subjects were enrolled in the study after providing informed consent. The exclusion criteria were as follows: the use of any chronic medications or the presence of any diagnosed acute or chronic disease. A healthy status was defined after a medical examination of each subject.

Total and differential cell counts in the NLF The cell pellet was resuspended in 1 ml of phosphate buffer saline solution (PBS). Thereafter, 20 ml of the mixed solution was added to a Neubauer chamber, and the cells were counted using a 400x light microscope (Olympus CH2, Olympus America Inc., Palo Alto, USA). For differential cell proportions (%), 100 ml of the mixed solution was centrifuged (96 g, 25 ˚C, 6 min) to obtain a slide with two areas of cells that were stained with hematoxylin and eosin. Differential cell counts were performed by two different observers with the aid of a 1000x light microscope (Olympus CH2, Olympus America Inc., Palo Alto, USA) (15,16).

Study design The subjects were assessed only once after one work week (five days of 8-h shifts) at the University Laboratory of Pulmonary Defense on a Saturday morning (from 8 A.M. until noon). The subjects were asked to sit in a chair in a quiet room. Clinical and job history, arterial blood pressure (mmHg), heart rate (bpm), pulse oximetry (%) and respiratory rate (rpm) were registered for each subject after 10 min of rest. The other variables were determined as described below.

pH measurements in EBC and NLF In a room maintained at a constant ambient temperature (23 ˚C) and relative humidity (65%), 500 ml of fluid (EBC or NLF) was de-aerated with a gentle 350 ml/min flow of ultrapure (99.9%) argon gas (Gama Gases Ltd., Sao Paulo, Brazil) for 15 min. The pH was determined with the aid of a microelectrode and a pH meter (827 pH Lab, Metrohm Ltd., Herisau, Switzerland). The pH meter was calibrated before each measurement using solutions with pH values of 4, 7 and 9. After pH determination, the EBC and NLF aliquots were discarded (9–11).

Exhaled carbon monoxide measurements The concentrations of exhaled carbon monoxide (CO) were determined (in ppm) with the aid of a Micro CO analyzer (Cardinal Health U.K., 232 Ltd., Chatham, UK). The subjects were asked to exhale slowly from their total lung capacity with a constant expiratory flow of 5–6 l min21 over 10 to 15 sec. The mean of two reproducible measurements with a variation of less than 5% was considered.

Measurement of cytokines in EBC and NLF The concentrations of tumor necrosis factor-alpha (TNFa), interleukin (IL)-1b, IL-8 and IL-10 in EBC and NLF samples were determined using high sensitivity enzymeimmunoassays (Quantikine HS, R&D Systems Inc., Minneapolis, USA). The limits of detection of the assays were as follows: (a) TNF-a: 0.106 pg/ml, with the standard curve fitted between 0 and 32 pg/ml; (b) IL-1b: 0.057 pg/ ml, with the standard curve fitted between 0 and 8 pg/ml; (c) IL-8: 3.50 pg/ml, with the standard curve fitted between 0 and 2,000 pg/ml; and (d) IL-10: 0.50 pg/ml, with the standard curve fitted between 0 and 50 pg/ml.

Exhaled breath condensate The EBC was obtained as previously described (10). At the start of EBC collection, all subjects rinsed their mouths with distilled water and were instructed to swallow saliva as necessary and to hold a slight head extension (approximately 15 ˚). The EBC sample was collected over 15 min of quiet and normal breathing (regular tidal volumes and respiratory rate) through a mouthpiece that was connected to a collector device with dry ice (220 ˚C). The total EBC (2.0–2.5 ml) was immediately divided and transferred to sterile 500 ml polypropylene tubes. One aliquot was immediately used for pH measurements. The remaining EBC sample aliquots were coded (for blinding purposes) and

Air pollutants The estimation of the exposure of each volunteer (trafficcontroller or office-worker) to 10 mm diameter particulate matter (PM10) was obtained during five consecutive days of an 8-h work shift from one of the seven fixed monitoring stations of the Sa˜o Paulo State Environmental Agency in Sa˜o

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Paulo City (Ibirapuera, Pinheiros, Cerqueira Cesar, Centro, Congonhas, Mooca and Parque Dom Pedro). The fixed monitoring stations that were geographically closest to the workplaces of the volunteers were chosen. None of the workplaces was .5 km distant from a fixed monitoring station.

it was correlated with BMI (r = 0.22 and p = 0.039). The NLF pH was correlated with both age (r = 20.60 and p,0.001) and BMI (r = 20.27 and p = 0.011). The pH measurements indicated that the EBC and NLF of traffic-controllers were more acidic than the EBC and NLF of office-workers (Figure 1). In the multiple linear regression model, there was a mean reduction in pH of 20.42 (SE = 0.13, p = 0.001), which was independent of the substrate (EBC or NLF, p = 0.608).

Statistical analysis Normality distribution was assessed using normal probability plots. The data are expressed as the mean and standard deviation (SD) when normally distributed or the median and interquartile range (IQR) when otherwise appropriate. Comparisons between groups (traffic-controllers and office-workers) were performed using the t-test or Mann-Whitney test. Pearson’s correlation or Spearman’s rank correlation coefficients were utilized to quantify the degree of association between variables. The estimated effect of traffic-controllers on the pH and the comparison between substrates (EBC and NLF) was determined using linear multiple regression adjusted for age and body mass index (BMI). Interactions between substrate and group and substrate and age were included in the model. The amount of variability in the response variable explained by the model was evaluated by the coefficient of determination, R2. To compare cytokines distributions between groups and between substrates, we created a factor with 4 levels (the combination between groups and substrates), and we used the Kruskal-Wallis test followed by Bonferroni’s method (when necessary) to localize the differences. Statistical analyses were carried out using the SPSS statistical package, version 15 (SPSS Inc., Chicago, IL, USA). No correction for the multiplicity of tests was performed; however, p-values are given explicitly wherever reasonable. Statistical significance was set at 5%.

Cytokine expression in EBC and NLF The concentrations of cytokines between EBC and NLF were not correlated. No significant differences in IL-10 were observed between the street traffic-controllers and the office-workers and between the substrates (EBC and NLF) (p = 0.455) (Table 2). However, significantly greater concentrations of IL-1 and IL-8 were observed in the NLF of trafficcontrollers and office-workers compared with the EBC.

Cellularity of NLF The number of total cells (median and IQR) in the NLF of the office-workers (126 and 170 cells/mm3) was greater than that of the traffic-controllers (42 and 77 cells/mm3), with an increase in lymphocytes (Figure 2). However, no other significant differences were observed in the NLF of officeworkers and traffic-controllers with respect to the percentages of neutrophils, eosinophils, macrophages and epithelial cells (ciliated and goblet cells).

Environmental exposure data The outdoor mean PM10 concentrations (SD) in the workplaces of the street traffic-controllers and office-workers were 26.4 (9.45) mg/m3 and 19.7 (1.71) mg/m3, respectively, and were significantly different (p = 0.006). No significant correlations were found between the PM10 concentrations and the EBC pH (r = 20.19 and p = 0.125), the NLF pH (r = 20.21 and p = 0.07), as either with exhaled CO (r = 0.21 and p = 0.876). However, among the officeworkers, the correlation coefficient between the NLF pH and PM10 was r = 20.46 (p = 0.154) as a result of the lower statistical power due to the reduced PM10 sample size (n = 11).

& RESULTS The demographic and clinical characteristics of the 87 adult male subjects who entered into the study (Table 1) showed differences in age and BMI between the street traffic-controllers and office-workers. However, the two occupational groups had similar vital signs (arterial blood pressure, heart rate, pulse oximetry and respiratory rate). Despite being within the normal range, the median levels of exhaled CO were two-fold higher in the street trafficcontrollers compared with the office-workers.

& DISCUSSION We conducted a cross-sectional study to assess the effects of two levels of air pollution exposure on human airways: a busy street with considerable traffic and the inside of an office. Because street traffic-controllers perform their occupational activities under direct exposure to outdoor vehiclerelated air pollution, they are at increased risk of airway

pH measurements in EBC and NLF The correlation between pH in the EBC and in the NLF was r = 0.12 and p = 0.38 in the traffic-controllers and r = 20.31 and p = 0.277 in the office-workers. The EBC pH was not correlated with age (r = 0.07 and p = 0.496); however,

Table 1 - Demographic and clinical data of the street traffic-controllers and office-workers. Traffic-controllers n = 73 Age, years, mean (SD) Body mass index, kg/m2, mean (SD) Systolic blood pressure, mmHg, mean (SD) Diastolic blood pressure, mmHg, mean (SD) Heart rate, bpm, mean (SD) Respiratory rate, rpm, mean (SD) Exhaled CO, ppm, median (IQR)

42 27.4 118 82 69 15 4.5

Abbreviations: CO, carbon monoxide; *, t test; {, Mann-Whitney test.

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(7) (3.7) (13) (8) (9) (3) (3)

Office-workers n = 14 30 24.5 118 78 70 14 2.5

(5) (3.0) (12) (11) (10) (2) (1.3)

p-value ,0.001* 0.005* 0.923* 0.261* 0.792* 0.186* ,0.001{


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Figure 1 - The pH of the exhaled breath condensate and nasal lavage fluid of the traffic-controllers and office-workers.

inflammation. Street traffic-controllers presented lower NLF and EBC pH values accompanied by increased amounts of exhaled CO compared with office-workers. However, the concentrations of IL-1b and IL-8 in NLF and EBC were similar in traffic-controllers and office-workers. The workplace of the street traffic-controllers was 25% more polluted than the workplace of the office-workers, although the PM10 concentrations were within the air quality limits advocated by international environmental agencies. Inflammation is a physiological response to a variety of stimuli comprising a complex series of events that involve macrophages, monocytes and neutrophils among others and their molecular products (i.e., cytokines and chemokines). Exposure to air pollution may pose a significant health risk to some outdoor professionals. The nasal mucosa is the first barrier of the respiratory system that protects against inhaled pollutants and other agents and plays an important role in the innate immune response to environmental stimuli (17). This response is often studied using nasal biopsies, which themselves induce inflammatory responses. We have used low-cost and simple methods to

study the response of the nasal mucosa and lower airways; these methods involve the collection of NLF and EBC. In the present study, we obtained sufficient amounts of sampling fluid, and both were effective sources of inflammatory biomarkers that provided complementary information. The acidification of the EBC suggests endogenous airway inflammation, which is implicated in the pathophysiology of several respiratory disorders. All the volunteers in this study were healthy non-smoking adults with no history of acute or chronic respiratory disease. The office-workers had a mean EBC pH of 8.12, which is within the normal values reported by others (10,18–21). However, the street trafficcontrollers showed significant reductions in the EBC pH values. The lower pH level was similar to that observed in mechanically ventilated patients experiencing respiratory failure in the intensive care unit (18). Experimental and human studies have shown that exposure to air pollutants may generate reactive oxygen species due to the presence of free radicals and oxidants on the particle surface (22) and may cause alterations in the transcription of inflammatory cytokines (7). Because cytokines may play important roles in the pathophysiology of airway and lung parenchyma diseases, interest has been focused on cytokines determinations (23). IL-1b plays an important protective role against pathogen invasion because it is a potent inflammatory mediator that stimulates chemokine production and leukocyte recruitment to the site of injury. Additionally, IL-1b is involved in a variety of cellular activities including cell proliferation, differentiation and apoptosis (24). IL-8 is responsible for the recruitment of inflammatory cells from the circulation to the airways in many respiratory diseases (25,26). Few studies on health effects related to air pollution have explored cytokines levels in EBC, primarily because low concentrations of cytokines have been reported (27,28). Indeed, in the present study, the IL-1b concentrations in EBC were between 0 and 1 pg/ml, similar to the findings of other reports in healthy subjects (27,29). In this study, the IL-8 concentrations in EBC were similar in the office-workers and the

Table 2 - Cytokines concentrations in the exhaled breath condensate and nasal lavage fluid of the trafficcontrollers and office-workers. The data were analyzed by the Kruskal-Wallis test and Bonferroni post-hoc test. The results are presented as median values (IQRs). Group TNF-a (pg/ml) Traffic-controllers Office-workers IL-1b (pg/ml) Traffic-controllers Office-workers IL-8 (pg/ml) Traffic-controllers Office-workers IL-10 (pg/ml) Traffic-controllers Office-workers

EBC 0.5 0.4 0 0 8.9 8.4 1.2 1.0

(0.3) (0.0) (0.7) (0.0) (1.8) (0.9) (1.6) (1.4)

NLF 0.4 0.3 8.4 8.4 257.0 293.6 1.2 1.5

(0.4) (0.2) (5.2) (5.4) (411.2) (589.6) (2.7) (2.2)

p-value 0.590 0.879 ,0.001 ,0.001 ,0.001 ,0.001 0.455 0.455

TNF-a, tumor necrosis factor-alpha; IL-1b, interleukin-1 beta; IL-8, interleukin-8; IL-10, interleukin-10.

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Figure 2 - The proportions of cells (%) in the nasal lavage fluid of the traffic-controllers and office-workers. The data were analyzed by the Kruskal-Wallis test.

street traffic-controllers (,10 pg/ml); however, these concentrations were four- to nine-fold higher than previous findings in healthy individuals (15,30,31) and similar to those observed young smokers (30,32). We can not exclude the possibility that these differences can be explained by the use of different standards in the assays. In NLF, we found an increased number of total cells in office-workers, which was primarily due to an increased number of lymphocytes. In contrast, traffic-workers presented a similar trend in the number of ciliated cells, although statistical significance was not achieved. We also found higher concentrations of IL-1b and IL-8 in NLF compared with EBC (eight-fold and three-fold, respectively). Data related to IL-1b levels in NLF are very scarce in the literature. Riechelmann et al. (25) reported IL-1b concentrations of 15ยก13 pg/ml in healthy non-smoking volunteers (aged 18-60 years); in the current study, we observed IL-1b concentrations that were 50% lower. However, the IL-8 concentrations in the NLF samples in the present study were ten-fold greater than those in volunteers with occupational rhinitis (mean IL-8 concentrations of ,27 pg/ml) (33). In agreement with this finding, other reports have shown direct associations between exposure to PM2,5 and nasal inflammation (34) as well as increases in IL-8 production in the airways of healthy individuals after acute exposure to diesel exhaust (35). In the present study, the levels of IL-1b and IL-8 were increased in NLF compared with EBC in the trafficcontrollers and office-workers, and these increases were not accompanied by changes in the percentage of neutrophils. This finding raised the possibility that the increase in these pro-inflammatory mediators (IL-1b and IL-8) may have resulted from epithelial cell production, as reported in in vitro studies (36) with human bronchial epithelial cells

exposed to PM10 and in studies of the nasal epithelial cells of asthmatic children (37). Human activity patterns and microenvironmental exposure can significantly affect exhaled CO levels; however, it has been suggested that regular monitoring of exhaled CO levels in healthy subjects has the potential to be used as a functional index of air pollution (38). We observed higher exhaled CO levels in traffic-controllers (two-fold) than in office-workers, although both levels were within the normal values for non-smokers (,10 ppm, according to the ATS guidelines). This study has limitations. There were some differences in demographic characteristics between our study groups. For example, the street traffic-controllers were older than the office-workers. However, previous studies have shown that aging has no effect on the pH of EBC (18), with the exception of individuals between 60 and 80 years old (20), which was not the age range of our study population. Additionally, the BMI of the traffic-controllers was higher than that of the office-workers. Obesity has been reported to be associated with systemic inflammation, particularly if coupled with sedentary behavior (39). However, the difference in BMI between the two groups was only approximately 10; thus, it may not have affected our results. In the present study, the outdoor air pollution data must be interpreted with caution. We used the PM10 concentration data provided by fixed monitoring stations that were located near the workplace of each street traffic-controller and office-worker (mean values of 8-hr intervals over 5 consecutive work days). We detected a 25% difference in exposure between the traffic-controllers and the officeworkers. However, this difference may still underestimate the PM10 exposure among traffic-controllers because considerably higher levels of pollutants are present at the

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Airway and lung inflammation by pH Lima TM et al. 8. Mukae H, English D, Anderson G, Terashima T, Hogg JC, van Eeden SF. Phagocytosis of PM10 by human alveolar macrophages stimulates the release of PMN from the bone marrow. Am J Respir Crit Care Med. 1999;159:A317. 9. Kostikas K, Papatheodorou G, Ganas K, Psathakis K, Panagou P, Loukides S. Ph in expired breath condensate of patients with inflammatory airway disease. Am J Respir Crit Care Med. 2002; 165:1364-70, http://dx.doi.org/10.1164/rccm.200111-068OC. 10. Koczulla R, Dragonieri S, Schot R, Bals R, Gauw SA, Vogelmeier C, et al. Comparison of exhaled breath condensate pH using two commercially available devices in healthy controls, asthma and COPD patients. Respir Res. 2009;10:78, http://dx.doi.org/10.1186/1465-9921-10-78. 11. Borrill Z, Starkey C, Vestbo J, Singh D. Reproducibility of exhaled breath condensate pH in chronic obstructive pulmonary disease. Eur Respir J. 2005;25(2):269-74, http://dx.doi.org/10.1183/09031936.05.00085804. 12. Maestrelli P, Canova C, Scapellato ML, Visentin A, Tessari R, Bartolucci GB, et al. Personal exposure to particulate matter is associated with worse health perception in adult asthma. J Investig Allergol Clin Immunol. 2011;21(2):120-8. 13. Quirce S, Lemie`re C, de Blay F, del Pozo V, Gerth Van Wijk R, Maestrelli P, et al. Noninvasive methods for assessment of airway inflammation in occupational settings. Allergy. 2010;65(4):445-58, http://dx.doi.org/10. 1111/j.1398-9995.2009.02274.x. 14. Barraza-Villarreal A, Sunyer J, Hernandez-Cadena L, Escamilla-Nun˜ez MC, Sienra-Monge JJ, Ramı´rez-Aguilar M, et al. Air pollution, airway inflammation, and lung function in a cohort study of Mexico City schoolchildren. Environ Health Perspect. 2008;116(6):832-8, http://dx. doi.org/10.1289/ehp.10926. 15. Naclerio RM, Meier HL, Kagey-Sobotka A, Adkinson NF Jr, Meyers DA, Norman PS, et al. Mediator release after nasal airway challenge with allergen. Am Rev Respir Dis. 1983;128:597-602. 16. Nakagawa NK, Nakao M, Goto DM, Saraiva-Romanholo BM. Air pollution and non-invasive respiratory assessments. In: Esquinas AM, ed. Applied Technologies in Pulmonary Medicine. Basel: Karger AG. 2011:223-30. 17. Auger F, Gendron MC, Chamot C, Marano F, Dazy AC. Responses of well-differentiated nasal epithelial cells exposed to particles: role of the epithelium in airway inflammation. Toxicol Appl Pharmacol. 2006; 215(3):285-94, http://dx.doi.org/10.1016/j.taap.2006.03.002. 18. Vaughan J, Ngamtrakulpanit L, Pajewski TN, Turner R, Nguyen TA, Smith A, et al. Exhaled breath condensate pH is a robust and reproducible assay of airway acidity. Eur Respir J. 2003;22(6):889-94, http://dx.doi.org/10.1183/09031936.03.00038803. 19. Wells K, Vaughan J, Pajewski TN, Hom S, Ngamtrakulpanit L, Smith A, et al. Exhaled breath condensate pH assays are not influenced by oral ammonia. Thorax. 2005;60(1):27-31, http://dx.doi.org/10.1136/thx.2003. 020602. 20. Cruz MJ, Sanchez-Vidaurre S, Romero PV, Morell F, Munoz X. Impact of age on pH, 8-Isoprostane, and nitrogen oxides in exhaled breath condensate. Chest. 2009;135(2):462-7, http://dx.doi.org/10.1378/chest. 08-1007. 21. Koczulla AR, Noeske S, Herr C, Jo¨rres RA, Ro¨mmelt H, Vogelmeier C, et al. Acute and chronic effects of smoking on inflammation markers in exhaled breath condensate in current smokers. Respiration. 2010; 79(1):61-7, http://dx.doi.org/10.1159/000245325. 22. Risom L, Moller P, Loft S. Oxidative stress-induced DNA damage by particulate air pollution. Mutat Res. 2005;592(1-2):119-37, http://dx.doi. org/10.1016/j.mrfmmm.2005.06.012. 23. Barnes PJ. The cytokine network in asthma and chronic obstructive pulmonary disease. J Clin Invest. 2008;118(11):3546-56, http://dx.doi. org/10.1172/JCI36130. 24. Dinarello CA. Immunological and inflammatory functions of the interleukin-1 family. Annu Rev Immunol. 2009;27:519-50, http://dx. doi.org/10.1146/annurev.immunol.021908.132612. 25. Riechelmann H, Deutschle T, Friemel E, Gross H-J, Bachem M. Biological markers in nasal secretions. Eur Respir J. 2003;21(4):600-605, http://dx. doi.org/10.1183/09031936.03.00072003. 26. Cyktor JC, Turner J. IL-10 and immunity against prokaryotic and eukaryotic intracellular pathogens. Infect Immun. 2011;79(8):2964-73, http://dx.doi.org/10.1128/IAI.00047-11. 27. Garey KW, Neuhauser MM, Robbins RA, Danziger LH, Rubinstein I. Markers of inflammation in exhaled breath condensate of young healthy smokers. Chest. 2004;125(1):22-26, http://dx.doi.org/10.1378/chest.125.1.22. 28. Jungraithmayr W, Frings C, Zissel G, Prasse A, Passlick B, Stoelben E. Inflammatory markers in exhaled breath condensate following lung resection for bronchial carcinoma. Respirology. 2008;13(7):1022-7. 29. Gessner C, Scheibe R, Wo¨tzel M, Hammerschmidt S, Kuhn H, Engelmann L, et al. Exhaled breath condensate cytokine patterns in chronic obstructive pulmonary disease. Respir Med. 2005;99(10):1229-40, http://dx.doi.org/10.1016/j.rmed.2005.02.041. 30. Gessner C, Kuhn H, Toepfer K, Hammerschmidt S, Schauer J, Wirtz H. Detection of p53 gene mutations in exhaled breath condensate of nonsmall cell lung cancer patients. Lung Cancer. 2004;43(2):215-22, http:// dx.doi.org/10.1016/j.lungcan.2003.08.034.

location where the traffic-controllers work compared with the city average, as we have recently demonstrated in vehicle corridors with high traffic (40). In addition, the PM10 results likely do not reflect the real difference in personal exposure (41). However, unpublished data from our laboratory show that the mean PM10 concentration measured by fixed monitoring stations is 50% lower than the personal exposure. Another issue is that the personal exposure of office-workers may be lower than the value reported by the fixed monitoring stations because these individuals work indoors. Several reports indicate that exposure to urban traffic is markedly attenuated in the indoor environment (41). Additionally, the present study was performed during a period of school vacation when there were 30–40% fewer vehicles circulating in the city. However, we found that the pH in NLF and EBC was an efficient biomarker that can be used to assess the inflammatory effects of air pollution on the airways and lungs. Inflammatory disturbances were markedly present in the nasal cavities of both groups. However, only trafficcontrollers showed extended inflammation in the airways based on acidification of their EBC. In conclusion, the pH values of NLF and EBC are important, robust, easy-to-measure and reproducible biomarkers that can be used to monitor occupational exposure to air pollution. Additionally, traffic-controllers are at an increased risk of airway and lung inflammation during their occupational activities compared with office-workers.

& ACKNOWLEDGMENTS The authors would like to thank Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP 07/51605-9 and 09/50056-7) and the National Council of Technological and Scientific Development (CNPq 555.223/06-0) for providing financial support for this study. We also thank Carolina Tieko Yoshida for revising tables, figures and references.

& AUTHOR CONTRIBUTIONS All the authors participated in the study design, results analysis, discussion and manuscript writing. Lima TM, Kazama CM, Santos UP, BuenoGarcia ML and Nakagawa NK also participated in the data collection.

& REFERENCES 1. Johns DO, Stanek LW, Walker K, Benromdhane S, Hubbell B, Ross M, et al. Practical advancement of multipollutant scientific and risk assessment approaches for ambient air pollution. Environ Health Perspect. 2012;120(9):1238-42, http://dx.doi.org/10.1289/ehp.1204939. 2. Ristovski ZD, Miljevic B, Surawski NC, Morawska L, Fong K, Goh F, et al. Respiratory health effects of diesel particulate matter. Respirology. 2012;17(2):201-2, http://dx.doi.org/10.1111/j.1440-1843.2011.02109.x. 3. Dockery DW, Brunekreef B. Longitudinal studies of air pollution effects on lung function. Am J Respir Crit Care Med. 1996;154(6):S250-6, http:// dx.doi.org/10.1164/ajrccm/154.6_Pt_2.S250. 4. Pope CA 3rd, Kanner RE. Acute effects of PM10 pollution on pulmonary function of smokers with mild to moderate chronic obstructive pulmonary disease. Am Rev Respir Dis. 1993;147(6):1336-40. 5. Olmo NRS, Saldiva PHN, Braga ALF, Lin CA, Santos UP, Pereira LAA. A review of low-level air pollution and adverse effects on human health: implications for epidemiological studies and public policy. Clinics. 2011;66(4):681-90, http://dx.doi.org/10.1590/S1807-59322011000400025. 6. Matsumoto G, Nakagawa NK, Vieira RP, Mauad T, da Silva LF, de Andre´ CD, et al. The time course of vasoconstriction and endothelin receptor A expression in pulmonary arterioles of mice continuously exposed to ambient urban levels of air pollution. Environ Res. 2010;110(3):237-43, http://dx.doi.org/10.1016/j.envres.2010.01.003. 7. Shukla A, Timblin C, BeruBe K, Gordon T, McKinney W, Driscoll K, et al. Inhaled particulate matter causes expression of nuclear factor (NF)kappa B-related genes and oxidant-dependent NF-kappa B activation in vitro. Am J Respir Cell Mol Biol. 2000;23(2):182-7.

1493


Airway and lung inflammation by pH Lima TM et al.

CLINICS 2013;68(12):1488-1494

31. Carpagnano GE, Resta O, De Pergola G, Sabato R, Foschino-Barbaro MP. The role of obstructive sleep apnea syndrome and obesity in determining leptin in the exhaled breath condensate. J Breath Res. 2010;4(3):036003, http://dx.doi.org/10.1088/1752-7155/4/3/036003. 32. Carpagnano GE, Kharitonov SA, Resta O, Foschino-Barbaro MP, Gramiccioni E, Barnes PJ. Increased 8-isoprostane and interleukin-6 in breath condensate of obstructive sleep apnea patients. Chest. 2002; 122(4):1162-7, http://dx.doi.org/10.1378/chest.122.4.1162. 33. Castano R, Maghni K, Castellanos L, Trudeau C, Malo J-L, Gaudrin D. Proinflammatory mediators in nasal lavage of subjects with occupational rhinitis. Otolaryngol Head Neck Surg. 2010;143(2):301-3, http://dx.doi. org/10.1016/j.otohns.2010.04.272. 34. Chen BY, Chan CC, Lee CT, Cheng TJ, Huang WC, Jhou JC, et al. The association of ambient air pollution with airway inflammation in schoolchildren. Am J Epidemiol. 2012;175(8):764-74. 35. Salvi SS, Nordenhall C, Blomberg A, Rudell B, Pourazar J, Kelly FJ, et al. Acute exposure to diesel exhaust increases interleukin-8 and GRO-alpha production in healthy human airways. Am J Respir Crit Care Med. 2000;161(2 Pt 1):550-7, http://dx.doi.org/10.1164/ajrccm.161.2.9905052. 36. Sakamoto N, Hayashi S, Gosselink J, Ishii H, Ishimatsu Y, Mukae H, et al. Calcium dependent and independent cytokine synthesis by air pollution

37.

38.

39. 40.

41.

1494

particle-exposed human bronchial epithelial cells. Toxicol Appl Pharmacol. 2007;225(2):134-41, http://dx.doi.org/10.1016/j.taap.2007. 07.006. Pringle EJ, Richardson HB, Miller D, Cornish DS, Devereux GS, Walsh GM, et al. Nasal and bronchial airway epithelial cell mediator release in children. Ped Pulmonol. 2012;47(12):1215-25, http://dx.doi.org/10.1002/ ppul.22672. Jones AY, Lam PK. End-expiratory carbon monoxide levels in healthy subjects living in a densely populated urban environment. Sci Total Environ. 2006;354(2-3):150-6, http://dx.doi.org/10.1016/j.scitotenv.2005. 02.018. Handschin C, Spiegelman BM. The role of exercise and PGC1 in inflammation and chronic disease. Nature. 2008;454(7200):24-5. Carneiro MFH, Ribeiro FQ, Fernandes-Filho FN, et al. Pollen abortion rates, nitrogen dioxide by passive diffusive tubes and bioaccumulation in tree barks are effective in the characterization of air pollution. Environ Exp Botany. 2011;72(2):272-7, http://dx.doi.org/10.1016/j.envexpbot.2011.04.001. Baxter LK, Wright RJ, Paciorek C, Laden F, Suh HH, Levy JI. Effects of exposure measurements error in the analysis of health effects from traffic-related air pollution. J Expo Sci Environ Epidemiol. 2010;20(1):10111, http://dx.doi.org/10.1038/jes.2009.5.


CLINICAL SCIENCE

The impact of metabolic syndrome on metabolic, proinflammatory and prothrombotic markers according to the presence of high blood pressure criterion Juliana S. Gil,I Luciano F. Drager,I Grazia M. Guerra-Riccio,I Cristiano Mostarda,I Maria C. Irigoyen,I Valeria Costa-Hong,I Luiz A. Bortolotto,I Brent M. Egan,II Heno F. LopesI,III I

Faculdade de Medicina da Universidade de Sa˜o Paulo, Heart Institute (InCor), Sa˜o Paulo/SP, Brazil. II Medical University of South Carolina, Charleston/SC, USA. III Universidade Nove de Julho – UNINOVE, Sa˜o Paulo/SP, Brazil.

OBJECTIVES: We explored whether high blood pressure is associated with metabolic, inflammatory and prothrombotic dysregulation in patients with metabolic syndrome. METHODS: We evaluated 135 consecutive overweight/obese patients. From this group, we selected 75 patients who were not under the regular use of medications for metabolic syndrome as defined by the current Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults criteria. The patients were divided into metabolic syndrome with and without high blood pressure criteria ($130/$85 mmHg). RESULTS: Compared to the 45 metabolic syndrome patients without high blood pressure, the 30 patients with metabolic syndrome and high blood pressure had significantly higher glucose, insulin, homeostasis model assessment insulin resistance index, total cholesterol, low-density lipoprotein-cholesterol, triglycerides, uric acid and creatinine values; in contrast, these patients had significantly lower high-density lipoprotein-cholesterol values. Metabolic syndrome patients with high blood pressure also had significantly higher levels of retinolbinding protein 4, plasminogen activator inhibitor 1, interleukin 6 and monocyte chemoattractant protein 1 and lower levels of adiponectin. Moreover, patients with metabolic syndrome and high blood pressure had increased surrogate markers of sympathetic activity and decreased baroreflex sensitivity. Logistic regression analysis showed that high-density lipoprotein, retinol-binding protein 4 and plasminogen activator inhibitor-1 levels were independently associated with metabolic syndrome patients with high blood pressure. There is a strong trend for an independent association between metabolic syndrome patients with high blood pressure and glucose levels. CONCLUSIONS: High blood pressure, which may be related to the autonomic dysfunction, is associated with metabolic, inflammatory and prothrombotic dysregulation in patients with metabolic syndrome. KEYWORDS: Hypertension; Sympathetic Activity; Insulin Resistance; Inflammation; Prothrombosis; Metabolic Syndrome. Gil JS, Drager LF, Guerra-Riccio GM, Mostarda C, Irigoyen MC, Costa-Hong V, et al. The impact of metabolic syndrome on metabolic, proinflammatory and prothrombotic markers according to the presence of high blood pressure criterion. Clinics. 2013;68(12):1495-1501. Received for publication on April 27, 2013; First review completed on May 7, 2013; Accepted for publication on June 11, 2013 E-mail: hipheno@incor.usp.br Tel.: 55 11 2661-5084

better define whether MetS predicts cardiovascular morbidity and mortality better than the sum of the individual components and whether one component is pivotal over the others. Most evidence points to visceral obesity and insulin resistance as central features of MetS. Little attention has been given to exploring other components of MetS, such as blood pressure and the potential factors that can influence not only blood pressure but also the metabolic dysregulation observed in MetS. Obesity-related sympathetic activation is an attractive explanation for several components of MetS. In particular, in 1994, the aggregation of cardiovascular risk factors and signs of a hyper sympathetic state was demonstrated in the Tecumseh population (2). Indeed, increased sympathetic activity (faster heart rate, higher cardiac output and plasma

& INTRODUCTION In 1988, the cardiovascular risk factor cluster, which includes obesity, increased blood pressure, high triglycerides and glucose and low HDL (high-density lipoprotein)cholesterol, was given the name ‘‘metabolic syndrome’’ (MetS) (1). Since then, the scientific community has tried to

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(12)04

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noradrenaline) correlated with higher levels of glucose, insulin, cholesterol, triglycerides, body weight and hematocrit and lower levels of HDL-cholesterol. In the Framingham Heart Study (3), more than 50% of hypertensive patients had 2 or more metabolic abnormalities. Only 19% of males and 17% of females had isolated hypertension. In view of this finding, the name hypertensive syndrome had been used in the past (4). However, it is not clear whether the increase in blood pressure is associated with exacerbations of the metabolic, proinflammatory, prothrombotic, vascular and autonomic dysfunctions in patients with MetS. Evidence suggests that autonomic dysregulation also contributes to elevated blood pressure and metabolic abnormalities. Moreover, a1-adrenoceptor antagonists lower blood pressure, improve insulin sensitivity and ameliorate dyslipidemia (5). However, it is unclear whether autonomic dysregulation in metabolic syndrome is associated not only with hemodynamic impairment but also with metabolic, inflammatory and other abnormalities associated with the syndrome. We hypothesized that high blood pressure, which may reflect increased sympathetic activity, is independently associated with metabolic dysregulation and inflammation in patients with MetS.

Figure 1 - Flow diagram used to screen patients.

stored at -80 ˚C. During the second visit, anthropometric measurements (body mass index – BMI; waist and hip circumferences; and bicipital, tricipital, subscapular and suprailiac skinfolds) and fat mass, lean mass, basal metabolism and total body water were measured using bioimpedance-measuring equipment (BIA 450, Bio dynamics, Seattle, USA) (7).

& METHODS Study population This research was performed at the Heart Institute (InCor) at the University of Sa˜o Paulo Medical School. Over a 1-year period, we initially evaluated 135 consecutive overweight or obese patients from the Sa˜o Paulo metropolitan area. From this group, we selected 75 patients with MetS, diagnosed according to ATP III (Third Report of the Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults criteria, and with at least three of the following criteria: high blood pressure - arterial blood pressure $130 and/or $85 mmHg for systolic and diastolic blood pressure, respectively; high glucose - fasting glucose $100 mg/dL; increased waist circumference - $102 cm in men and $88 in women; increased triglycerides - $150 mg/ dL; decreased HDL - ,40 mg/dL in men and ,50 mg/mg/ dL in women) (6). We excluded patients with morbid obesity (body mass index $40 kg/m2), severe hypertension (BP .180/110 mmHg), under regular physical activity, secondary forms of hypertension, diabetes, smokers, patients with any chronic disease and regularly using medications (including antihypertensives, see Figure 1). The patients were divided into 2 groups according to blood pressure status (patients with MetS without high blood pressure, i.e., ,130/,85 mmHg: MetS-BP) and (patients with MetS with high blood pressure, i.e., $130 systolic and/ or $85 mmHg diastolic: MetS+BP).

Biochemistry measurements Total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, uric acid, glucose and creatinine levels were measured using commercial kits.

Insulin resistance Insulin resistance was measured using the following 2 methods: the traditional Homeostasis Model Assessment Insulin Resistance method (HOMA-IR) and the RetinolBinding Protein 4 method (RBP4). The HOMA-IR index was calculated as [fasting serum glucose (mmol/L) x fasting serum insulin (UI/mL)]/22.5.

Cytokines Cytokines, including total adiponectin, resistin, leptin, insulin, interleukin 6 (IL-6), tumoral necrosis factor alpha (TNF-a), plasminogen activator inhibitor 1 (PAI-1) and monocyte chemoattractant protein 1 (MCP-1), were measured using a multiplex sandwich enzyme-linked immunosorbent assay (ELISA, Millipore, MA, USA) (8).

Power spectral analysis The data for spectral analysis were derived from a finger pulse contour recorded by beat-to-beat blood pressure measurements (FinometerH, FMS, Finapres Medical System BV, Holland) and from the electrocardiogram (ECG) for 10 minutes to characterize the RR interval series. Power spectral density was obtained by the fast Fourier transformation, using Welch’s method with a Hanning window of 512 points and 50% overlap as previously described (9). The entire time, series were previously re-sampled at 5.0 Hz to be equally spaced in time by a special signal editor developed for Matlab (MATLAB 6.0; Mathworks, Natick, Massachusetts, USA). Interpolated time series were decimated to be equally time-spaced. Two main components were considered for the RR interval variability: low

Clinical evaluation Patients who fulfilled the criteria for the study underwent a medical history evaluation. Blood pressure was measured in triplicate after 5 minutes of seated rest using a calibrated sphygmomanometer by the same investigator (J.S.G). The average from 3 blood pressure values was used to define the presence or absence of the ATP III, the criterion used to define elevated blood pressure ($130 or $85 mmHg for systolic and diastolic blood pressure levels, respectively). Fasting blood samples were drawn for biochemical tests and cytokine levels. Serum and plasma were obtained and then

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Logistic regression analysis (forward model) was used to identify the metabolic, pro-inflammatory and prothrombotic markers that were independently associated with the MetS+BP. The model was controlled for age and BMI. ANOVA was used to select variables for the logistic regression analysis. All variables with a p,0.1 in the univariate analysis were selected for the final model. P,0.05 was considered to be statistically significant.

frequency (LF; 0.04 to 0.15 Hz) and high frequency (HF; 0.15 to 0.4 Hz). The power density of each spectral component from the RR interval was calculated in absolute values and normalized units. The normalized units were obtained by calculating the percentages of LF variability (LFms2) and HF variability (HFms2) with respect to the total power after subtracting the power of very low frequency components (VLF; frequencies of ,0.04 Hz). The LF/HF ratio was also calculated to evaluate the sympathovagal balance. In addition, we also evaluated the power spectral analysis of systolic BP. Systolic BP spectral powers were quantified in the LF component (0.04–0.15 Hz) and were reported in absolute units. LF of systolic BP has been associated as a reliable marker of sympathetic activity (10).

Ethics statement The local ethics committee (Institutional Review Board – Heart Institute) approved the protocol, which was in accordance with the ethical standards and with the Helsinki Declaration of 1975, and all participants gave written informed consent.

Baroreflex sensitivity Beat-to-beat values of systolic BP and RR interval were used to estimate the cardiac baroreflex sensitivity (BRS) by spectral analysis, using the alpha index for the lowfrequency band (0.04 to 0.15 Hz). The coherence between the RR interval and the systolic BP signal variability was assessed by cross-spectral analysis. The alpha index was calculated only when the magnitude of the squared coherence between the RR and systolic BP signals exceeded 0.5 (range 0–1) in the LF band. After coherence calculation, the alpha index was obtained from the square root of the ratio between RR and systolic BP variability in the 2 major LF bands (10-13).

& RESULTS Figure 1 depicts derivation of the study sample of 75 subjects from the initial sample of 135 patients with MetS. The percentage of each MetS criterion is reported in the supplemental file. Baseline descriptive data on the MetS patients without (N = 45) and with (N = 30) high blood pressure are provided in Table 1. We observed a significant trend for increasing age in the MetS+BP group. There was no significant difference in gender, % of Caucasians and anthropometric data, including body mass index, waist circumference, hip circumference, waist-to-hip ratio, fat percentage as calculated by skinfold, fat mass, lean mass, metabolic tax and total body water estimated by bioelectrical impedance analysis (BIA) (Table 1). As expected, there were significant differences in the systolic and diastolic blood pressure measurements between groups. Pulse intervals during spectral analysis were significantly lower (heart rate faster) in the MetS+BP compared to the MetS-BP group (Table 2). The LF components for heart rate and blood pressure and the LF/HF ratio were significantly higher in the MetS+BP group than in the MetS-BP group. Baroreflex sensitivity, as evaluated by the alpha index, was impaired in the MetS+BP group compared with the MetS-BP group. No differences were detected between groups with regards to anxiety and depression (supplemental file).

Anxiety and depression evaluation We performed the Hamilton Scale to assess anxiety (1415) and the Beck questionnaire to assess depression (16).

Statistical analysis The SPSS software (SPSS 10.0, Chicago, IL) was used for statistical calculations. Data were expressed as the means¡standard deviations, medians or percentages, as indicated. The Kolmogorov-Smirnov test was used to assess the normality of distribution of each variable studied. Categorical variables (sex, race and percentage of MetS components) were compared using Fisher’s test. Numerical variables were compared using the unpaired Student’s t test.

Table 1 - Demographic, hemodynamic and anthropometric (clinical and bioimpedance) data (means¡sd) in metabolic syndrome patients without blood pressure criterion (metabolic syndrome - blood pressure) or with blood pressure (metabolic syndrome + blood pressure). Variables Age (years) Sex (F/M) Caucasians, n (%) Systolic BP (mmHg) Diastolic BP (mmHg) Heart Rate (bpm) Body mass index (kg/m2) Waist circumference (cm) Waist-to-hip ratio Fat (%) Fat mass (BIA), % Lean mass (BIA), % BMR (BIA), kcal TBW (BIA), %

MetS-BP (n = 45)

MetS+BP (n = 30)

p-value

38¡11 34/11 32 (71%) 121¡10 76¡8 72¡9 32.2¡4.0 108¡10 (men)/ 100¡11 (women) 0.93¡0.06 32¡6 28¡7 59¡10 1,829¡328 43¡8

45¡9 19/11 20 (67%) 144¡18 90¡13 77¡12 32.5¡4.0 111¡12 (men)/ 101¡11 (women) 0.95¡0.08 32¡7 28¡9 60¡13 1,842¡398 44¡10

0.061 0.843 0.789 ,0.0001 ,0.0001 0.081 0.767 0.6 (men) 0.7 (women) 0.265 0.817 0.878 0.534 0.743 0.73

BMR = basal metabolic rate; TBW = Total body water.

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cardiovascular risk, including metabolic, proinflammatory, prothrombotic and autonomic variables (17-19). We hypothesized that MetS patients with elevated blood pressure may have greater abnormalities in these same variables. By stratifying patients with similar obesity levels according to the presence or absence of high blood pressure, we found that those with high blood pressure (MetS+BP) had higher surrogate markers of sympathetic activity derived from spectral analysis and greater impairment in several components of MetS, including higher glucose and triglycerides and lower HDL-cholesterol. The MetS group with high blood pressure also had higher values for other risk factors, including total cholesterol, LDL-cholesterol, uric acid and insulin, along with HOMA index and RBP4, which are associated with insulin resistance (17,20,21). In addition, several markers of inflammatory and prothrombotic activity, including IL-6, PAI-1 and MCP-1, were higher, while adiponectin levels were lower in patients with MetS and high blood pressure. Finally, we found that HDL-cholesterol, RBP4 and PAI-1 were independently associated with MetS+BP. Taken together, our results suggest a significant heterogeneity of the MetS in promoting metabolic, pro-inflammatory, prothrombotic and autonomic impairments. MetS was defined based on the observation that a number of interrelated characteristics and diseases tended to cluster in the same individual and contributed to increased cardiovascular risk. However, MetS is not a well-defined pathophysiological entity, and the diagnostic criteria are variable and based on expert opinions (22). The literature has debated the existence of a major trigger for MetS and its components or whether the whole is really greater than the sum of the parts. Obesity emerges as an attractive candidate to explain all manifestations of MetS, but the best way to measure this parameter in the MetS scenario is also debatable. Some studies identified an association between abdominal obesity (waist circumference) and components of MetS (23,24), while others showed an association with body mass index or waist-to-hip ratio (25-28). Regardless of the obesity parameter, the importance of obesity in inducing high blood pressure and metabolic and inflammatory dysregulation is clear. Elevated sympathetic nervous system activity seems to be crucial to the development of obesity-related risk (29). In our study, patients with MetS and high blood pressure had higher surrogate markers of sympathetic

Table 2 - Power spectral analysis in metabolic syndrome patients without blood pressure criterion (metabolic syndrome - blood pressure) or with blood pressure criterion (metabolic syndrome + blood pressure). Variable PI (ms) VAR PI (ms2) LF (ms2) HF (ms2) LF (%) HF (%) LF/HF SBP (mmHg) VAR SBP (mmHg2) LF (mmHg2) Alpha index (ms/mmHg)

MetS-BP

MetS+BP

p-value

887¡120 2653¡1910 638¡607 687¡563 48¡15 52¡15 1.0¡0.7 126¡13 35¡25 6.7¡4.4 10.4¡5.2

823¡126 2578¡1529 671¡498 590¡549 56¡13 44¡13 1.5¡0.7 141¡21 50¡25 13.0¡9.2 7.7¡3.7

0.1 0.9 0.9 0.5 0.04 0.04 0.08 0.003 0.03 0.001 0.04

PI = Pulse interval; VAR PI = Variance of PI; LF = low frequency; HF = High frequency; SBP = Systolic blood pressure; VAR SBP = Variance of SBP.

Biochemistry data (glucose, total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides, uric acid and creatinine), along with insulin and the HOMA-IR index, were significantly higher in the MetS+BP group than in the MetSBP group (Table 3). RBP4 was significantly higher in the MetS+BP group than the MetS-BP group (Figure 2A). Leptin, resistin and TNF-a levels did not differ between the MetS+BP group (49.4¡41.5 pg/mL, 12.6¡4.6 pg/mL and 4.8¡1.7 ng/mL, respectively) and the MetS-BP group (41.4¡20.0 pg/mL, 12.9¡4.9 pg/mL and 4.4¡1.4 ng/mL, respectively). IL-6, PAI-1 and MCP-1, a marker for inflammatory activity, were significantly higher in the MetS+BP group (Figures 2B-2D). Adiponectin was lower in the MetS+BP group compared to the MetS-BP group (Figure 2E). The differences in RBP4, PAI-1, IL-6, MCP-1 and adiponectin levels between groups remained significant after adjusting for age and sex. Logistic regression analysis indicated that HDL, RBP-4 and PAI-1 levels were independently associated with MetS+BP. There was a strong trend for an independent association between MetS+BP and glucose levels (Table 4).

& DISCUSSION Previous studies have shown that patients with MetS have multiple abnormalities that contribute to increased

Table 3 - Biochemical data from metabolic syndrome patients without blood pressure criterion (metabolic syndrome blood pressure) or with blood pressure criterion (metabolic syndrome + blood pressure). Variables Glucose (mg/dL) Insulin (U/mL) HOMA-ir T-cholesterol (mg/dL) HDL-cholesterol (mg/dL)

Triglycerides (mg/dL) Uric Acid (mg/dL) Creatinine (mg/dL)

MetS-BP (n = 45)

MetS+BP (n = 30)

p-value

97¡8 10.5¡3.9 2.5¡1.0 194¡33 40.3¡5.9 (men)/ 51.5¡10.6 (women) 119¡27 150¡36 (men)/ 124¡54 (women) 4.6¡1.2 0.8¡0.2

102¡7 21.5¡20.0 5.4¡5.2 221¡43 38.3¡6.6 (men)/ 42.6¡7.6 (women) 145¡39 190¡78 (men)/ 167¡57 (women) 5.6¡1.3 1.0¡0.2

0.013 0.007 0.006 0.001 0.5 (men) 0.002 (women) 0.002 0.2 (men) 0.07 (women) 0.001 0.008

HDL = High-density lipoprotein; LDL = Low-density lipoprotein; HOMA-ir = homeostasis model assessment-estimated insulin resistance index (means¡sd). Triglycerides values are reported as the medians (interquartile ranges).

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Figure 2 - Retinol-binding protein 4 levels (2A), plasminogen activator inhibitor 1 levels (2B), interleukin 6 levels (2C), monocyte chemoattractant protein 1 levels (2D) and adiponectin levels (2E) in metabolic syndrome - blood pressure and metabolic syndrome + blood pressure subjects. The results are presents as the meansยกSD.

activity. Long-term sympathetic activation can lead to small artery vasoconstriction and remodeling, which increases the wall-to-lumen ratio and may act synergistically with large artery damage to raise blood pressure (30). Sympathetic activity is also related to baroreflex impairment, as observed in this study. However, sympathetic activity may not be related only to hemodynamic impairment but also to metabolic and inflammatory dysregulation. In addition to the hemodynamic effects, sympathetically mediated vasoconstriction may also antagonize insulinmediated glucose uptake (29). This theory is supported by longitudinal studies demonstrating that elevated indices of sympathetic activation precede the development of insulin resistance (31). Jamerson et al. showed that norepinephrine infusion, but not angiotensin, impaired insulin-stimulated glucose uptake (29-31). In line with the metabolic dysregulation observed in our study, Lichtand et al. found that autonomic nervous system dysregulation was strongly associated with MetS and its components in a large cohort of participants aged 18-65 years (35). However, these authors did not stratify patients with MetS according to blood pressure status and did not explore other risk factors not included in the MetS definition, such as proinflammatory and prothrombotic markers.

In our study, we also observed that adiponectin levels were lower and that markers of inflammation were higher in MetS patients with high blood pressure than in MetS patients without high blood pressure. Sympathetic activation reportedly reduces adiponectin release, whereas central-sympathetic blockade may increase adiponectin levels (36). Plasma adiponectin is inversely related to insulin resistance (35). Thus, the increased sympathetic activity in patients with MetS and high blood pressure may decrease adiponectin levels that, in turn, can also contribute to insulin resistance. Systemic inflammation may also be linked to sympathetic activity. Previous evidence found that indirect markers of sympathetic activity (increased heart rate and reduced heart-rate variability) were associated with subclinical inflammation in healthy middle-aged and elderly subjects. These results suggest that an autonomic imbalance in favor of the sympathetic system may interact with inflammatory processes to play a more important role in the process of vascular stiffness and atherosclerosis. A few limitations from this investigation should be addressed. First, due to the study design, our findings suggest an association rather than a cause-effect relationship between high blood pressure and metabolic, inflammatory, prothrombotic and autonomic impairments in patients with MetS. Although consistent with the cited literature, we cannot prove that sympathetic activity is the main obesityrelated trigger underlying not only the increased blood pressure but also impairments in the metabolic, inflammatory and prothrombotic profiles in this subgroup of patients with MetS. Second, direct measurements of peripheral sympathetic activity using microneurography were not performed. However, spectral analysis is an acceptable non-invasive method for estimating sympathovagal activity (13). Our findings consistently suggest a decrease in vagal activity and the activation of the sympathetic system in the spectral analyses of both heart rate and blood pressure. Third, these results cannot be extrapolated to MetS patients with diabetes. Finally, the precise reasons by which a subset

Table 4 - Logistic regression analysis for variables independently associated with metabolic syndrome with high blood pressure (metabolic syndrome + blood pressure) Variables

GLIC HDL RBP4 PAI1 Constant

B

0.118 -0.147 1.507 0.270 -20.148

Sig.

0.057 0.008 0.001 0.002 0.011

OR

1.125 0.863 4.511 1.311 0.000

95% C.I. for OR Lower

Upper

0.996 0.775 1.909 1.103

1.270 0.962 10.663 1.558

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6. Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) final report. Circulation. 2002;106(25):3143-421. 7. Pimentel GD, Bernhard AB, Frezza MR, Rinaldi AE, Burini RC. Bioelectric impedance overestimates the body fat in overweight and underestimates in Brazilian obese women: a comparison with Segal equation 1. Nutr Hosp. 2010;25(5):741-5. 8. Schipper HS, de Jager W, van Dijk ME, Meerding J, Zelissen PM, Adan RA, et al. Clin Chem. 2010;56(8):1320-8, http://dx.doi.org/10.1373/ clinchem.2010.146118. 9. Drager LF, Ueno LM, Lessa PS, Negra˜o CE, Lorenzi-Filho G, Krieger EM. Sleep-related changes in hemodynamic and autonomic regulation in human hypertension. J Hypertens. 2009;27(8):1655-63, http://dx.doi. org/10.1097/HJH.0b013e32832c6982. 10. Pagani M, Lombardi F, Guzzetti S, Rimoldi O, Furlan R, Pizzinelli P, et al. Power spectral analysis of heart rate and arterial pressure variabilities as a marker of sympatho-vagal interaction in man and conscious dog. Circ Res. 1986;59(2):178-93, http://dx.doi.org/10.1161/01.RES.59.2.178. 11. Heart rate variability: standards of measurement, physiological interpretation and clinical use. Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. Circulation. 1996;93(5):1043-65. 12. Lucini D, Cusumano G, Bellia A, Kozakova M, Difede G, Lauro R, et al. Is reduced baroreflex gain a component of the metabolic syndrome? Insights from the LINOSA study. J Hypertens. 2006;24(2):361-70, http:// dx.doi.org/10.1097/01.hjh.0000202817.02836.9c. 13. Pagani M, Somers V, Furlan R, Dell’Orto S, Conway J, Baselli G, et al. Changes in autonomic regulation induced by physical training in mild hypertension. Hypertension. 1988;12(6):600-10, http://dx.doi.org/10. 1161/01.HYP.12.6.600. 14. Hamilton M. The assessment of anxiety states by rating. Br J Med Psychol. 1959;32(1):50-5. 15. Maier W, Buller R, Philipp M, Heuser I. The Hamilton Anxiety Scale: reliability, validity and sensitivity to change in anxiety and depressive disorders. J Affect Disord. 1988;14(1):61-8, http://dx.doi.org/10.1016/ 0165-0327(88)90072-9. 16. Gomes-Oliveira MH, Gorenstein C, Lotufo Neto F, Andrade LH, Wang YP. Validation of the Brazilian Portuguese version of the Beck Depression Inventory-II in a community sample. Rev. Bras. Psiquiatr. 2012;34(4):389-94, http://dx.doi.org/10.1016/j.rbp.2012.03.005. 17. Graham TE, Yang Q, Blu¨her M, Hammarstedt A, Ciaraldi TP, Henry RR, et al. Retinol-binding protein 4 and insulin resistance in lean, obese, and diabetic subjects. N Engl J Med. 2006;354(24):2552-63. 18. Wallace TM, Levy JC, Matthews DR. Use and abuse of HOMA modeling. Diabetes Care. 2004;27(6):1487-95, http://dx.doi.org/10.2337/diacare.27. 6.1487. 19. Grundy SM, Cleeman JI, Daniels SR, Donato KA, Eckel RH, Franklin RA, et al. Diagnosis and management of the metabolic syndrome: an American Heart Association/National Heart, Lung, and Blood Institute Scientific Statement. Circulation. 2005;112(17):2735-52, http://dx.doi. org/10.1161/CIRCULATIONAHA.105.169404. 20. Janssen I, Katzmarzyk PT, Ross R. Waist circumference and not body mass index explains obesity-related health risk. Am J Clin Nutr. 2004;79(3):379-84. 21. Lean ME, Han TS, Morrison CE. Waist circumference as a measure for indicating need for weight management. BMJ. 1995;311(6998):158-61, http://dx.doi.org/10.1136/bmj.311.6998.158. 22. Costa GB, Horta N, Resende ZF, Souza G, Barreto LM, Correia LH, et al. Body mass index has a good correlation with proatherosclerotic profile in children and adolescents. Arq Bras Cardiol. 2009;93(3):261-7, http:// dx.doi.org/10.1590/S0066-782X2009000900010. 23. Katzmarzyk PT, Srinivasan SR, Chen W, Malina RM, Bouchard C, Berenson GS. Body mass index, waist circumference, and clustering of cardiovascular disease risk factors in a biracial sample of children and adolescents. Pediatrics. 2004;114(2):e198-205, http://dx.doi.org/10.1542/ peds.114.2.e198. 24. Schneider HJ, Glaesmer H, Klotsche J, Bo¨hler S, Lehnert H, Zeiher AM, et al. J Clin Endocrinol Metab. 2007;92(2):589-94. 25. Yusuf S, Hawken S, Ounpuu S, Bautista L, Franzosi MG, Commerford P, et al. Obesity and the risk of myocardial infarction in 27,000 participants from 52 countries: a case-control study. Lancet. 2005;366(9497):1640-9, http://dx.doi.org/10.1016/S0140-6736(05)67663-5. 26. Lambert EA, Lambert GW. Stress and its role in sympathetic nervous system activation in hypertension and the metabolic syndrome. Curr Hypertens Rep. 2011;13(3):244-8, http://dx.doi.org/10.1007/s11906-0110186-y. 27. Kotsis V, Stabouli S, Papakatsika S, Rizos Z, Parati G. Mechanisms of obesity-induced hypertension. Hypertens Res. 2010;33(5):386-93, http:// dx.doi.org/10.1038/hr.2010.9. 28. Flaa A, Aksnes TA, Kjeldsen SE, Eide I, Rostrup M. Increased sympathetic reactivity may predict insulin resistance: an 18-year follow-up study. Metabolism. 2008;57(10):1422-7, http://dx.doi.org/10. 1016/j.metabol.2008.05.012.

of patients with MetS had higher blood pressure and greater sympathetic activity despite similar indices of obesity are not clear. Recent studies reported the potential role of psychosocial stress in inducing sympathetic activity (29,37,38). Another potential candidate is obstructive sleep apnea. This clinical condition is characterized by sympathetic activation that is independent of obesity, and it is a well-established cause of high blood pressure and increased arterial stiffness (39-41). Recent evidence in consecutive patients with MetS suggests that obstructive sleep apnea is quite common and is independently associated with increased sympathetic activity (41), metabolic and inflammatory dysregulation (42) and increased markers of atherosclerosis (43). These potential confounders should be addressed in future investigations. In conclusion, this study provides several provocative new findings that suggest the importance of characterizing patients with MetS according to the presence or absence of high blood pressure. Although we cannot prove a causeeffect relationship, our results demonstrated that patients with MetS and high blood pressure have higher surrogate markers of sympathetic activity and significant metabolic, inflammatory and prothrombotic impairments. Thus, it is reasonable to speculate that blocking sympathetic activity is an attractive strategy for treating patients with MetS. A previous study showed that a combined a- and b-adrenergic blockade significantly reduced blood pressure in obese patients compared to lean patients with essential hypertension (44). Moreover, renal denervation, which reduces sympathetic activity, lowers blood pressure and reduces insulin resistance, may improve obstructive sleep apnea (45). Future studies will clarify whether pharmacological suppression of sympathetic activity in patients with MetS will improve not only blood pressure but also the metabolic, inflammatory and prothrombotic dysfunctions observed in these patients.

& ACKNOWLEDGMENTS This work was supported by grants from FAPESP (Fundac¸a˜o de Amparo a` Pesquisa de Sa˜o Paulo) and Zerbini Foundation.

& AUTHOR CONTRIBUTIONS Gil JS conceived, designed and performed the experiments; analysed the data and wrote the paper. Drager LF and Lopes HF conceived and designed the experiments, analysed the data and wrote the paper. Mostarda C performed the experiments and analysed the data. GuerraRiccio GM and Costa-Hong V performed the experiments. Irigoyen MC and Bortolotto LA analysed the data. Egan BM wrote the paper.

& REFERENCES 1. Reaven GM. Banting lecture 1988. Role of insulin resistance in human disease. Diabetes. 1988;37(12):1595-607, http://dx.doi.org/10.2337/diab. 37.12.1595. 2. Smith S, Julius S, Jamerson K, Amerena J, Schork N. Hematocrit levels and physiologic factors in relationship to cardiovascular risk in Tecumseh, Michigan. J Hypertens. 1994;12(4):455-62. 3. Kannel WB. Risk stratification in hypertension: new insights from the Framingham Study. Am J Hypertens. 2000;13(1Pt 2):3S-10S. 4. Houston MC. New insights and approaches to reduce end-organ damage in the treatment of hypertension: subsets of hypertension approach. Am Heart J. 1992;123(5):1337-67, http://dx.doi.org/10.1016/0002-8703(92) 91042-Y. 5. Chapman N, Chen CY, Fujita T, Hobbs FD, Kim SJ, Staessen JA, et al. Time to re-appraise the role of alpha-1 adrenoceptor antagonists in the management of hypertension? J Hypertens. 2010;28:1796-803. Erratum in: J Hypertens. 2010;28(11):2351.

1500


CLINICS 2013;68(12):1495-1501

Heterogeneity of the Metabolic Syndrome Gil JS et al.

29. Jamerson KA, Nesbitt SD, Amerena JV, Grant E, Julius S. Angiotensin mediates forearm glucose uptake by hemodynamic rather than direct effects. Hypertension. 1996;27(4):854-8, http://dx.doi.org/10.1161/01. HYP.27.4.854. 30. Jamerson KA, Smith SD, Amerena JV, Grant E, Julius S. Vasoconstriction with norepinephrine causes less forearm insulin resistance than a reflex sympathetic vasoconstriction. Hypertension. 1994;23(6 Pt 2):1006-11, http://dx.doi.org/10.1161/01.HYP.23.6.1006. 31. Pollare T, Lithell H, Selinus I, Berne C. Application of prazosin is associated with an increase of insulin sensitivity in obese patients with hypertension. Diabetologia. 1988;31(7):415-20, http://dx.doi.org/ 10.1007/BF00271585. 32. Licht CM, Vreeburg SA, van Reedt Dortland AK, Giltay EJ, Hoogendijk WJ, DeRijk RH, et al. Increased sympathetic and decreased parasympathetic activity rather than changes in hypothalamic-pituitary-adrenal axis activity is associated with metabolic abnormalities. J Clin Endocrinol Metab. 2010;95(5):2458-66, http://dx.doi.org/10.1210/jc.2009-2801. 33. Weyer C, Funahashi T, Tanaka S, Hotta K, Matsuzawa Y, Pratley RE, et al. Hypoadiponectinemia in obesity and type 2 diabetes: close association with insulin resistance and hyperinsulinemia. J Clin Endocrinol Metab. 2001;86(5):1930-5, http://dx.doi.org/10.1210/jc.86.5.1930. 34. Lambert E, Dawood T, Straznicky N, Sari C, Schlaich M, Esler M, et al. Association between the sympathetic firing pattern and anxiety level in patients with the metabolic syndrome and elevated blood pressure. J Hypertens. 2010;28(3):543-50, http://dx.doi.org/10.1097/ HJH.0b013e3283350ea4. 35. Wirtz PH, Ehlert U, Bartschi C, Redwine LS, von Kanel R. Changes in plasma lipids with psychosocial stress are related to hypertension status and the norepinephrine stress response. Metabolism. 2009;58(1):30-7, http://dx.doi.org/10.1016/j.metabol.2008.08.003. 36. Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL Jr, et al. Seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Hypertension. 2003;42(6):1206-52, http://dx.doi.org/10.1161/01.HYP. 0000107251.49515.c2. 37. Drager LF, Bortolotto LA, Figueiredo AC, Krieger EM, Lorenzi GF. Effects of continuous positive airway pressure on early signs of

38.

39.

40.

41.

42.

43.

44.

45.

1501

atherosclerosis in obstructive sleep apnea. Am J Respir Crit Care Med. 2007;176(7):706-12, http://dx.doi.org/10.1164/rccm.200703-500OC. Trombetta IC, Somers VK, Maki-Nunes C, Drager LF, Toschi-Dias E, Alves MJ, et al. Consequences of comorbid sleep apnea in the metabolic syndrome-implications for cardiovascular risk. Sleep. 2010;33(9):11939. Drager LF, Lopes HF, Maki-Nunes C, Trombetta IC, Toschi-Dias E, Alves MJ, et al. The impact of obstructive sleep apnea on metabolic and inflammatory markers in consecutive patients with metabolic syndrome. PLoS One. 2010;5(8):e12065, http://dx.doi.org/10.1371/journal.pone. 0012065. Drager LF, Bortolotto LA, Figueiredo AC, Silva BC, Krieger EM, LorenziFilho G. Obstructive sleep apnea, hypertension, and their interaction on arterial stiffness and heart remodeling. Chest. 2007;131(5):1379-86, http://dx.doi.org/10.1378/chest.06-2703. Wofford MR, Anderson DC, Jr., Brown CA, Jones DW, Miller ME, Hall JE. Antihypertensive effect of alpha- and beta-adrenergic blockade in obese and lean hypertensive subjects. Am J Hypertens. 2001;14(7 Pt 1):694-8. Witkowski A, Prejbisz A, Florczak E, Ka˛dziela J, S´liwin´ski P, Bielen P, et al. Effects of renal sympathetic denervation on blood pressure, sleep apnea course, and glycemic control in patients with resistant hypertension and sleep apnea. Hypertension. 2011;58(4):559-65. Drager LF, Bortolotto LA, Maki-Nunes C,Trombetta IC, Alves MJ, Fraga RF, et al. The incremental role of obstructive sleep apnoea on markers of atherosclerosis in patients with metabolic syndrome. Atherosclerosis. 2010;208(2):490-5, http://dx.doi.org/10.1016/j.atherosclerosis.2009.08. 016. Wofford MR, Anderson DC Jr., Brown CA, Jones DW, Miller ME, Hall JE. Antihypertensive effect of alpha- and beta-adrenergic blockade in obese and lean hypertensive subjects. Am J Hypertens. 2001;14(7 Pt 1):694-8. Witkowski A, Prejbisz A, Florczak E, Kadziela J, Sliwinski P, Bielen P, et al. Effects of renal sympathetic denervation on blood pressure, sleep apnea course, and glycemic control in patients with resistant hypertension and sleep apnea. Hypertension. 2011;58(4):559-65, http://dx.doi. org/10.1161/HYPERTENSIONAHA.111.173799.


CLINICAL SCIENCE

The involvement of multiple thrombogenic and atherogenic markers in premature coronary artery disease Antonio P. Mansur, Julio Y. Takada, Ce´lia M. C. Strunz, Solange D. Avakian, Luiz Antonio M. Ce´sar, Jose´ A. F. Ramires Faculdade de Medicina da Universidade de Sa˜o Paulo, Heart Institute (InCor), Sa˜o Paulo/SP, Brazil.

OBJECTIVE: To examine the association of atherogenic and thrombogenic markers and lymphotoxin-alfa gene mutations with the risk of premature coronary disease. METHODS: This cross-sectional, case-control, age-adjusted study was conducted in 336 patients with premature coronary disease (,50 years old) and 189 healthy controls. The control subjects had normal clinical, resting, and exercise stress electrocardiographic assessments. The coronary disease group patients had either angiographically documented disease (.50% luminal reduction) or a previous myocardial infarction. The laboratory data evaluated included thrombogenic factors (fibrinogen, protein C, protein S, and antithrombin III), atherogenic factors (glucose and lipid profiles, lipoprotein(a), and apolipoproteins AI and B), and lymphotoxin-alfa mutations. Genetic variability of lymphotoxin-alfa was determined by polymerase chain reaction analysis. RESULTS: Coronary disease patients exhibited lower concentrations of HDL-cholesterol and higher levels of glucose, lipoprotein(a), and protein S. The frequencies of AA, AG, and GG lymphotoxin-alfa mutation genotypes were 55.0%, 37.6%, and 7.4% for controls and 42.7%, 46.0%, and 11.3% for coronary disease patients (p = 0.02), respectively. Smoking, dyslipidemia, family history, and lipoprotein(a) and lymphotoxin-alfa mutations in men were independent variables associated with coronary disease. The area under the curve (Cstatistic) increased from 0.779 to 0.802 (p,0.05) with the inclusion of lipoprotein(a) and lymphotoxin-alfa mutations in the set of conventional risk factors. CONCLUSIONS: The inclusion of lipoprotein(a) and lymphotoxin-alfa mutations in the set of conventional risk factors showed an additive but small increase in the risk prediction of premature coronary disease. KEYWORDS: Coronary Artery Disease; Risk Factors; Thrombosis; Inflammation; Lipids; Lipoprotein(a); Risk Prediction; Lymphotoxin-Alfa. Mansur AP, Takada JY, Strunz CM, Avakian SD, Ce´sar LA, Ramires JA. The involvement of multiple thrombogenic and atherogenic markers in premature coronary artery disease. Clinics. 2013;68(12):1502-1508. Received for publication on May 15, 2013; First review completed on June 4, 2013; Accepted for publication on June 15, 2013 E-mail: apmansur@usp.br Tel.: 55 11 2661-5387

metabolism and inflammation have been identified as main biological pathways in the pathogenesis of CAD. Arterial inflammation is a key component of plaque progression and plaque rupture, with atherosclerotic lesions established as active sites of inflammation (4). Biomarkers, coagulation factors, and proteins appear to coordinate the development of atherosclerosis and lead to the formation of complex atherosclerotic plaques and thrombus formation (5-7). Several biomarkers and cytokines likely play a role in determining the degree of inflammation and contribute to the promotion or retardation of atherothrombosis development. Despite the improved understanding of atherothrombosis pathophysiology, the conventional CAD risk factors do not fully account for the overall CAD risk (8,9). Moreover, risk factors associated with premature CAD are poorly recognized. Studies also show conflicting results with respect to some atherogenic and thrombogenic biomarkers of CAD (10-12). Genetic influence is often

& INTRODUCTION Coronary atherosclerosis and its complications constitute a complex and polygenic disorder resulting from the combined effects of multiple environmental and genetic factors (1). Several traditional risk factors increase the risk of coronary heart disease (CHD) and heart attack (2-4). Coronary artery disease (CAD) results from the progression of atherosclerotic plaque. Studies show that almost half of an individual’s susceptibility to CAD is heritable (3). Lipid

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(12)05

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Biomarkers in premature coronary disease Mansur AP et al.

replacement therapy. Thrombogenic factors (fibrinogen, assessed by the Clauss method; protein C, protein S, and antithrombin III, assessed by chromogenic methods); and atherogenic factors (fasting glucose and lipid profiles, assessed by standard protocols; and lipoprotein (a) and apolipoprotein AI and B fractions, assessed using the immunoturbidimetric method) were determined.

suggested as an important factor associated with the risk of premature CAD (13). The proinflammatory cytokine lymphotoxin-alfa (LTA) is a key mediator of the initiation of local vascular inflammatory responses. The actions of LTA are characterized by the stimulation of adhesion molecule production, thrombogenesis, smooth muscle proliferation, platelet activation, release of vasoactive agents (14-17), and control of nitric oxide production (8). Functional single nucleotide polymorphisms (SNPs) in the LTA gene have been found to be associated with MI in the Japanese population (17,18). However, other researchers failed to provide evidence of the relationship between LTA and CAD (19-21). The mutant allele results in significantly increased production of LTA in mononuclear cells stimulated in vitro, which is related to increased gene transcription (17). The aim of this study was to investigate whether traditional risk factors, thrombogenic and atherogenic biomarkers, and the mutation A252G in the LTA gene may be related to an increased predisposition to premature CAD and its interaction with traditional risk factor modulation.

Genotyping Genomic DNA was extracted from peripheral blood leukocytes by the standard salting-out procedure (10). We studied SNP 252A.G (rs909253) in the LTA gene, which encodes LTA on Chromosome 6p21 in intron 1 (11). The genetic variability of LTA was determined by amplification of the genomic DNA using polymerase chain reaction (PCR) followed by the restriction fragment length polymorphism (RFLP) technique. A 30-cycle PCR was performed in a PTCDNA Engine Tetrad2 (MJ Research, Waltham, Massachusetts, USA) using a 10-mL reactive solution containing 10 mM TrisHCl (pH 9.0), 50 mM KCl, 2.5 mM MgCl2, 100 mM of each dNTP, 0.3 U of Easy Taq DNA Polymerase, 5 pmol of each primer, and 1 mL of genomic DNA template. PCR products were digested with 1 U of NcoI restriction enzyme and visualized using 2.5% agarose gel electrophoresis. The presence of the 782-pb fragments indicated homozygosis of the wild allele (AA), while the presence of the 586- and 196-pb products indicated homozygosis of the mutant allele (GG) and the presence of 782-, 586-, and 196-pb products indicated heterozygosis (AG genotype). Genotype distribution was in Hardy-Weinberg equilibrium in all study samples (p.0.001).

& MATERIALS AND METHODS Study population A cross-sectional, case-control, age-matched study was conducted in 525 consecutive subjects ,50 years of age. Clinical characteristics and laboratory data were analyzed in 189 healthy control individuals and in 336 patients with documented premature stable CAD (,50 years of age) selected from the outpatient clinic of the Heart Institute (InCor), Faculdade de Medicina da Universidade de Sa˜o Paulo, Brazil. We hypothesized that young adult patients exhibit a larger proportion of CAD driven by genetic risk factors compared with older patients with CAD. The control group with no CAD included healthy subjects with normal clinical histories, physical examinations, and resting and exercise stress electrocardiographic assessments. The CAD group included outpatients from the Heart Institute (InCor) with angiographically documented disease (.50% luminal reduction) and patients with a previous myocardial infarct episode. The local institutional review boards approved the study protocol, and written informed consent was obtained from each study participant.

Statistical methods The Hardy Weinberg equilibrium for the distribution of genotypes was estimated by the chi-square test in the groups studied (no-CAD and CAD). Chi-square tests, t tests, and analysis of variance with Tukey’s correction for multiple comparisons were used for baseline comparisons. The odds ratios for different association models were calculated with a 95% confidence interval (CI) by multiple logistic regressions with confounders determined by a backward conditional elimination method for a significance level below 0.05. Logistic regression was used to estimate the cross-sectional association of the traditional risk factors for CAD with the following independent variables: gender, smoking status, family history of premature CAD, diabetes, dyslipidemia, history of arterial hypertension, Lp(a), and LTA polymorphisms. Lp(a) was dichotomized into normal (,30 mg/dL) and high ($30 mg/dL) values. CAD was the dependent variable. To evaluate the model’s performance, a receiver operating characteristic (ROC) curve was developed, and the area under the ROC curve (AUC or Cstatistic) was used to measure the discriminative power. The C-statistic for the clinical model and the biomarker information added to the clinical model were compared. The significance level adopted for the statistical tests was 5% (p,0.05). Statistical analyses were performed using the SAS for Windows (Statistical Analysis System) program, version 9.2 (SAS Institute Inc., 1989-1996, Cary, NC, USA).

Data collection The clinical data obtained included age, gender, body mass index (weight [kg]/height [m2]), smoking history, arterial hypertension, diabetes, dyslipidemia, previous myocardial infarction, and family history of premature CAD. Smokers who had quit more than 1 year prior to the study were considered ex-smokers. Current and former smokers were considered as one group and were compared with the patients who had never smoked. Hypertension was defined as a systolic blood pressure $140 mm Hg, diastolic blood pressure $90 mm Hg, or both measured at study entry or following antihypertensive treatment. A family history of CAD was defined as obstructive CAD occurring in parents (before age 55 for men and 65 for women) and siblings. Dyslipidemia was considered as the presence of hypercholesterolemia (total cholesterol .6.2 mmol/L) or hypertriglyceridemia (triglycerides .2.87 mmol/L) with medication use (e.g., fibrates and statins). Diabetes was diagnosed when a patient was taking hypoglycemic drugs or exhibited fasting glucose blood levels .7.0 mmol/L. No patients used contraceptives or hormone

& RESULTS Baseline characteristics The demographic and laboratory characteristics of the study population are shown in Table 1.

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Table 1 - Demographic and laboratory characteristics of the study population.

Age (years) Women (%) Hypertension Smokers Diabetes Dyslipidemia Family history of CAD Body mass index (kg/m2) Leukocytes (mm3) Platelets (mm3) Cholesterol (mg/dL) HDL-cholesterol (mg/dL) LDL-cholesterol (mg/dL) Triglycerides (mg/dL) Glucose (mg/dL) Creatinine (mg/dL) Uric acid (mg/dL) Apolipoprotein A1 (mg/dL) Apolipoprotein B (mg/dL) C-reactive protein (mg/dL) Fibrinogen (g/L) Lipoprotein (a) (mg/dL) Protein-S % Protein-C % Antithrombin III % Factor V activity %

All patients N = 525

Controls N = 189 (36%)

Coronary artery disease patients N = 336 (64%)

p-value

40.4¡5.3 271 (51.6) 150 (28.6) 56 (10.7) 87 (16.6) 298 (56.9) 255 (48.8) 25.4¡5.8 6647¡1747 181,730¡112,077 115.5¡64.2 42.1¡13.3 69.3¡34.4 127.5¡213.0 96.1¡32.3 0.74¡0.21 5.4¡2.1 0.43¡0.26 0.65¡0.28 6.54¡17.7 291.9¡144.5 39.0¡41.2 72.1¡22.0 66.3¡26.6 80.1¡18.5 49.9¡37.5

40.7¡5.9 69 (36.5) 33 (17.5) 7 (3.7) 23 (12.2) 83 (43.9) 33 (17.5) 24.9¡5.4 6467¡1632 178,784¡102,551 114.0¡49.4 45.7¡13.0 65.7¡27.4 106.6¡122.4 91.0¡11.2 0.74¡0.16 5.2¡2.6 0.42¡0.22 0.61¡0.28 4.80¡13.7 297¡134.1 31.4¡32.1 63.4¡23.0 63.2¡29.6 80.4¡26.4 49.7¡38.3

40.2¡5.0 202 (60.1) 117 (34.8) 49 (14.6) 64 (19.2) 215 (64.2) 222 (66.5) 25.6¡6.0 6753¡1806 183378¡117185 116.4¡71.3 40.0¡13.0 71.5¡37.8 138.8¡248 99.2¡39.7 0.74¡0.24 5.5¡1.8 0.44¡0.27 0.67¡0.25 7.5¡19.5 289¡150.1 43.3¡45.0 73.0¡21.7 66.6¡26.2 80.1¡17.8 49.9¡37.0

0.275 ,0.001 ,0.001 ,0.001 0.039 ,0.001 ,0.001 0.235 0.080 0.641 0.656 ,0.001 0.098 0.078 0.005 0.688 0.109 0.544 0.037 0.064 0551 ,0.001 0.038 0.537 0.966 0.953

Females, smokers, and individuals with hypertension, diabetes, dyslipidemia, and a family history of CAD were significantly more prevalent in the CAD group. Compared with the controls, the CAD patients exhibited significantly

lower plasma concentrations of HDL-cholesterol (p,0.001) and higher levels of fasting glucose (p = 0.005), lipoprotein (a) (p,0.001), and protein S (p = 0.038). The genotypic frequency distributions of the LTA A252G polymorphism

Table 2 - Clinical and biochemical data of all subjects according to the genotype of the LTA 252A.G polymorphism. LTA 252A.G

AA N = 247(47.1)

AG N = 225(42.9)

GG N = 52(10)

p-value

Age (years) Women (%) Hypertension Smokers Diabetes Dyslipidemia Family history of CAD Coronary artery disease Body mass index (kg/m2) Leukocytes (mm3) Platelets (mm3) Cholesterol (mg/dL) HDL-cholesterol (mg/dL) LDL-cholesterol (mg/dL) Triglycerides (mg/dL) Glucose (mg/dL) Creatinine (mg/dL) Uric acid (mg/dL) Apolipoprotein A1 (mg/dL) Apolipoprotein B (mg/dL) C-reactive protein (mg/dL) Fibrinogen (g/L) Lipoprotein(a) (mg/dL) Protein-S % Protein-C % Antithrombin III % Factor V activity %

40.4¡5.3 120(48.6) 58(23.5) 100(40.5) 37(15.1) 135(54.7) 116(47.3) 143(57.9) 25.8¡6.2 6723¡1711 186,313¡118,890 114.9¡66.7 43.7¡13.3 67.2¡34.3 137.0¡289.5 96.0¡32.2 0.76¡0.18 5.5¡2.3 0.43¡0.25 0.66¡0.27 5.5¡15.0 292.2¡143.4 40.2¡39.8 70.8¡21.7 66.8¡28.5 79.8¡20.0 52.8¡37.6

40.8¡5.4 127(56.4) 74(32.9) 96(42.7) 39(17.3) 131(58.5) 107(47.8) 154(68.4) 25.4¡5.7 6524¡1767 176,638¡103,955 116.6¡61.7 40.8¡12.5* 72.3¡34.8 113.5¡108.0 95.7¡30.4 0.72¡0.24* 5.4¡1.9 0.44¡0.26 0.63¡0.26 5.8¡12.4 283.6¡145.2 37.8¡39.8 73.3¡21.5 67.8¡24.4 81.3¡16.5 45.5¡37.3*

40.1¡5.1 24(44.2) 17(32.7) 19(37.2) 11(21.1) 32(61.5) 32(61.5) 38(73.1) 24.0¡4.4 6863¡1811 182,634¡114,614 115.6¡63.6 40.2¡15.8 66.7¡33.4 150.1¡151.2 98.4¡40.6 0.73¡0.20 5.1¡1.9 0.42¡0.27 0.64¡0.23 12.0¡32.0** 323.1¡144.8 38.9¡52.9 72.2¡24.9 56.9¡26.5 77.1¡19.6 53.8¡36.3

0.274 0.126 0.060 0.749 0.533 0.551 0.159 0.021 0.181 0.329 0.646 0.960 0.038 0.379 0.446 0.913 0.133 0.519 0.793 0.575 0.030 0.211 0.816 0.683 0.172 0.517 0.107

Values in parenthesis are percentages. *HDL (AA vs. AG), p = 0.020; creatinine (AA vs. AG), p = 0.046; FV (AA vs. AG), p = 0.048; ** C-reactive protein (AA vs. GG), p = 0.010, and (AG vs. GG), p = 0.014.

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for A/A homozygotes, A/G heterozygotes, and G/G homozygotes were 47.1%, 42.9, and 10% for all subjects; 55.0%, 37.6%, and 7.4% for the control group; and 42.7%, 46.0%, and 11.3% for the patient group, respectively (p = 0.021). The frequencies of the A and G alleles of the LTA gene were 68.6% and 31.4% among the 524 individuals in the entire population; 73.8% and 26.2% in the controls; and 65.7% and 34.3% in the CAD group, respectively. Genotyping distributions in both the control and CAD groups were consistent with Hardy-Weinberg equilibrium. No association was observed between LTA A252G and traditional risk factors. HDL-cholesterol and C-reactive protein plasma levels were higher in the AA (p = 0.038) and GG genotype subjects (p = 0.030), respectively. A multivariate logistic regression analysis showed that smoking (OR 4.81; 95% CI 1.98 – 11.7, p = 0.001), dyslipidemia (OR 2.35; 95% CI 1.10 – 5.05, p = 0.028), family history of CAD (OR 8.20; 95% CI 4.692 - 12.103, p,0.001), and Lp(a) (OR 1.29; 95% CI 1.01 1.65, p = 0.042) and LTA (OR 3.38; 95% CI 1.03 – 11.10, p = 0.007) mutations in men, but not in women (OR 1.34; 95% CI 0.49 – 3.63, p = 0.567), were independent factors associated with CAD (Figure 1). The performances of the predictive risk models are shown in the ROC curves (Figure 2). Model1 included the clinical variables gender, hypertension, smoking, diabetes, family history of CAD, dyslipidemia, and body mass index. The AUC (C-statistic) of this model for the prediction of CAD in patients ,50 years old was 0.779. There was a progressive increase in the AUC with the inclusion of the Lp(a) and Lp(a) + LTA mutation variables of 0.796 (p = 0.047, for comparisons between model1 and model1+Lp(a)) and 0.802 (p = 0.024, for comparisons between model1 and model1+Lp(a)+LTA mutation).

Biomarkers in premature coronary disease Mansur AP et al.

& DISCUSSION Our study shows that Lp(a) and LTA mutations in men are associated with premature CAD. Lp(a) and LTA mutations exhibit an additive effect on the C-statistic AUC for the traditional risk factors. Several studies have shown conflicting results regarding the association between specific biomarkers and the prediction of CAD (22-27).

Lipoprotein (a) and risk prediction for CAD In a nested case-control study, Kim et al. (28) analyzed 18 biomarkers previously associated with CAD in 321 patients with CAD and 743 control postmenopausal women. Five (vWF, factor VIII, homocysteine, IL-6, and D-dimer) of the 18 biomarkers tested were associated with CAD, but only Ddimer improved the C-statistic compared with traditional risk factors. Contrary to our results, Lp(a) was not associated with CAD, but the previous study included only women who were older (aged between 50 and 79 years) than our patients. Blakenberg et al. (29) analyzed the risk prediction of CAD associated with 30 biomarkers in 2 middle-aged European populations. The study analyzed Lp(a) and other biomarkers and showed that no biomarker improved risk estimation in either population. The structure of lipoprotein (a) is similar to the structure of LDL cholesterol, which includes one additional plasminogenlike glycoprotein, namely apolipoprotein (a). Due to these characteristics, this particle may contribute to the processes of atherogenesis and thrombogenesis. Lp(a) studies showed conflicting results regarding the risk prediction of CAD (3034). Bennet et al. (31) showed that the odds ratios for CAD were progressively higher with increasing Lp(a) levels in a

Figure 1 - Predicted probabilities of coronary artery disease based on a multivariate-adjusted model. LTA+ means LTA genotype GG.

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family history. Paynter et al. (37) calculated a genetic risk score based on 101 SNPs in 19,313 healthy Caucasian women who were followed up over a median of 12.3 years in a prospective study. After adjustment for traditional cardiovascular risk factors, the genetic risk score was not associated with cardiovascular disease risk. The LTA gene mutation was not included in the genetic risk score of the two previous studies. Thanassoulis et al. (38) showed a marginal increase in the risk prediction of cardiovascular events with the genetic risk score in addition to standard cardiovascular risk factors and a high risk of coronary artery calcium. However, future studies evaluating the utility of coronary artery calcium and genetic risk scores in predicting lifetime risk are needed. Genome-wide single nucleotide polymorphism association studies have identified several SNPs that are significantly associated with CAD and with traditional risk factors for CAD (39-42). However, the results of all these studies still showed inconsistencies with regard to the presence of a causal relationship between biomarkers, with or without the inclusion of genetic markers, and CAD and traditional risk factors. A mechanistic basis for the association between SNPs and coronary heart disease was observed, although the atherosclerotic process was unknown (43). However, none of the previously mentioned studies analyzed multiple biomarkers for the prediction of CAD in young adults aged ,50 years. In this study, we analyzed several biomarkers related to thrombogenic and atherogenic processes, and we included the LTA mutation as a candidate gene approach to risk assessment. The LTA mutation was frequently associated with CAD. In our study, the presence of the LTA gene mutant allele provided incremental information about CAD risk prediction. Previous case-control and cross-sectional studies examined the association between LTA gene polymorphisms and cardiovascular disease, but the results were inconsistent. The study by Ozaki et al. (17) described significant associations between LTA gene polymorphisms and myocardial infarction; however, the authors did not adjust for relevant covariates, including gender and age, and the genotype distributions among the control subjects were not in Hardy-Weinberg equilibrium. The association between LTA gene polymorphisms and CHD was confirmed in another Japanese population and in the familybased European PROCARDIS (precocious coronary artery disease) study (44). A significant association was observed between the LTA C804A genotype and the extent of coronary atherosclerosis in Caucasian patients with angiographically confirmed coronary atherosclerosis (45). However, several other studies did not detect an association between LTA gene polymorphisms and myocardial infarction (10,14,15). A meta-analysis performed by Clarke et al. (46) also showed no relationship between LTA gene polymorphisms and CHD. However, they did not study the LTA gene polymorphism (rs909253) evaluated in the Japanese study and our study. The most straightforward explanation for the findings of our study may be associated with the genetic differences of studied populations. Among populations with similar genetic backgrounds, the differences in allele frequency make it difficult to extrapolate genetic findings from one population to another. In our population, we demonstrated an association between LTA gene polymorphisms and increased risk of well-documented stable CAD in younger patients aged ,50 years. Genetic diversity in different populations may be especially relevant

Figure 2 - Receiver operating characteristic (ROC) curves. Model1 included the following variables: gender, hypertension, smoking, diabetes, family history of CAD, dyslipidemia, and body mass index. Model1 vs. Model1+Lp(a) (p = 0.047); Model1 vs. Model1+Lp(a)+LTA (p = 0.024).

large, prospective, population-based cohort that was adjusted for several established risk factors. Virani et al. (33) also showed that Lp(a) levels are associated with an increased risk of cardiovascular diseases in both African American and Caucasian male and female subjects. Sawage et al. (34) demonstrated an association between low Lp(a) levels and all-cause or cancer deaths and similar outcomes for low and high Lp(a) levels for cardiovascular deaths.

Use of genetic markers in risk prediction for CAD Few studies have utilized genetic markers of CAD in risk prediction. Rossouw et al. (35) analyzed 23 inflammatory, lipid, and thrombotic biomarkers and 8 genetic polymorphisms in postmenopausal women (aged 50-79 years) receiving hormone replacement therapy in a nested case-control study. Of the 23 biomarkers, the following 13 were associated with CHD events: interleukin 6, matrix metalloproteinase 9, HDL-C, LDL-C, total cholesterol, triglycerides, D-dimer, factor VIII, von Willebrand factor, leukocyte count, homocysteine, fasting insulin, and 1 genetic polymorphism (the glycoprotein IIIa leu33pro mutation). Additive risk prediction of the biomarkers over the conventional risk factors was not performed. Ripatti et al. (36) calculated a genetic risk score based on 13 SNPs and observed an association with a first coronary heart disease event, but the genetic risk score did not improve risk prediction compared with risk prediction based on traditional risk factors and

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Biomarkers in premature coronary disease Mansur AP et al. 3. Peden JF, Farrall M. Thirty-five common variants for coronary artery disease: the fruits of much collaborative labour. Hum Mol Genet. 2011;20(R2):R198-R205, http://dx.doi.org/10.1093/hmg/ddr384. 4. Ross R. Atherosclerosis an inflammatory disease. N Engl J Med. 1999;340(2):115-26. 5. Weber C, Noels H. Atherosclerosis: current pathogenesis and therapeutic options. Nat Med. 2011;17(11):1410-22, http://dx.doi.org/10.1038/nm. 2538. 6. Naghavi M, Libby P, Falk E, Casscells SW, Litovsky S, Rumberger J, et al. From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part I. Circulation. 2003;108(14):1664-72, http://dx.doi.org/10.1161/01.CIR.0000087480.94275.97. 7. Naghavi M, Libby P, Falk E, Casscells SW, Litovsky S, Rumberger J, et al. From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part II. Circulation. 2003;108(15):1772-8, http://dx.doi.org/10.1161/01.CIR.0000087481.55887.C9. 8. Sposito AC, Alvarenga BF, Alexandre AS, Arau´jo AL, Santos SN, Andrade JM, et al. Most of the patients presenting myocardial infarction would not be eligible for intensive lipid-lowering based on clinical algorithms or plasma C reactive protein. Atherosclerosis. 2011; 214(1):148-50, http://dx.doi.org/10.1016/j.atherosclerosis.2010.10.034. 9. Lloyd-Jones DM, Wilson PW, Larson MG, Beiser A, Leip EP, D’Agostino RB, et al. Framingham risk score and prediction of lifetime risk for coronary heart disease. Am J Cardiol. 2004;94(1):20-4, http://dx.doi.org/ 10.1016/j.amjcard.2004.03.023. 10. Kistorp C, Raymond I, Pedersen F, Gustafsson F, Faber J, Hildebrandt P, et al. N-terminal pro-brain natriuretic peptide, C-reactive protein, and urinary albumin levels as predictors of mortality and cardiovascular events in older adults. JAMA. 2005;293(13):1609-16, http://dx.doi.org/ 10.1001/jama.293.13.1609. 11. Zethelius B, Berglund L, Sundstro¨m J, Ingelsson E, Basu S, Larsson A, et al. Use of multiple biomarkers to improve the prediction of death from cardiovascular causes. N Engl J Med. 2008;358(20):2107-16. 12. Brown TM, Bittner V. Biomarkers of atherosclerosis: clinical applications. Curr Cardiol Rep. 2008;10(6):497-504, http://dx.doi.org/10.1007/s11886008-0078-1. 13. Roberts R, Stewart AF. The genetics of coronary artery disease. Curr Opin Cardiol. 2012;27(3):221-7, http://dx.doi.org/10.1097/HCO.0b013e 3283515b4b. 14. Pober JS, Lapierre LA, Stolpen AH, Brock TA, Springer TA, Fiers W, et al. Activation of cultured human endothelial cells by recombinant lymphotoxin: comparison with tumor necrosis factor and interleukin 1 species. J Immunol. 1987;138(10):3319-24. 15. Cavender DE, Edelbaum D, Ziff M. Endothelial cell activation induced by tumor necrosis factor and lymphotoxin. Am J Pathol. 1989;134(3):55160. 16. Kratz A, Campos-Neto A, Hanson MS, Ruddle NH. Chronic inflammation caused by lymphotoxin is lymphoid neogenesis. J Exp Med 1996;183(4):1461-72, http://dx.doi.org/10.1084/jem.183.4.1461. 17. Ozaki K, Inoue K, Sato H, Iida A, Ohnishi Y, Sekine A, et al. Functional variation in LGALS2 confers risk of myocardial infarction and regulates lymphotoxin-alpha secretion in vitro. Nature. 2004;429(6987):72-5, http://dx.doi.org/10.1038/nature02502. 18. Ozaki K, Ohnishi Y, Iida A, Sekine A, Yamada R, Tsunoda T, et al. Functional SNPs in the lymphotoxin-alpha gene that are associated with susceptibility to myocardial infarction. Nat Genet. 2002;32(4):650-4, http://dx.doi.org/10.1038/ng1047. 19. Ozaki K, Tanaka T. Genome-wide association study to identify SNPs conferring risk of myocardial infarction and their functional analyses. Cell Mol Life Sci. 2005;62(16):1804-13, http://dx.doi.org/10.1007/ s00018-005-5098-z. 20. Li W, Xu J, Wang X, Chen J, Zhang C, Sun K, et al. Lack of association between lymphotoxin-alpha, galectin-2 polymorphisms and coronary artery disease: a meta-analysis. Atherosclerosis. 2010;208(2):433-6, http://dx.doi.org/10.1016/j.atherosclerosis.2009.08.014. 21. Ryan AW, O’Brien E, Shields D, McManus R. Lack of association between NFKBIL1/LTA polymorphisms and hypertension, myocardial infarct, unstable angina and stable angina in a large Irish population sample. Atherosclerosis. 2008;197(1):465-466, http://dx.doi.org/10. 1016/j.atherosclerosis.2007.03.032. 22. Ingelsson E, Schaefer EJ, Contois JH, McNamara JR, Sullivan L, Keyes MJ, et al. Clinical utility of different lipid measures for prediction of coronary heart disease in men and women. JAMA. 2007;298(7):776-85, http://dx.doi.org/10.1001/jama.298.7.776. 23. Ray KK, Cannon CP, Cairns R, Morrow DA, Ridker PM, Braunwald E. Prognostic utility of apoB/AI, total cholesterol/HDL, non-HDL cholesterol, or hs-CRP as predictors of clinical risk in patients receiving statin therapy after acute coronary syndromes: results from PROVE IT-TIMI 22. Arterioscler Thromb Vasc Biol. 2009;29(3):424-430, http://dx.doi. org/10.1161/ATVBAHA.108.181735. 24. Sierra-Johnson J, Fisher RM, Romero-Corral A, Somers VK, LopezJimenez F, Ohrvik J, et al. Concentration of apolipoprotein B is comparable with the apolipoprotein B/apolipoprotein A-I ratio and better than routine clinical lipid measurements in predicting coronary

to the LTA genomic region, which lies within the HLA region and has an erratic pattern of LD structure, with both short LD islands and long-range haplotypes (47). In our study, Lp(a) and LTA mutations alone were associated with lower risk prediction for CAD compared with conventional risk factors, but the inclusion of each mutation with conventional risk factors resulted in a progressive and statistically significant increase in risk prediction for CAD (Figure 2). The impact of the increased risk prediction based on AUC/C-statistics from 0.779 to 0.802 in clinical practice is uncertain and needs to be confirmed in future studies.

Study limitations Our study has several limitations. This was a case-control study, and in such studies, casual associations are frequent when the sample size is relatively small for the association analysis of complex diseases with genetic mutations of multifactorial traits. Additionally, this case-control study was age-matched but not gender-matched. Therefore, the clinical and laboratory features observed more often in women may have influenced the final results. The analysis of one mutation can lead to results not identified in prospective studies with a larger number of individuals. The analysis of a significantly higher number of mutations, use of better laboratory techniques, and application of more sophisticated statistical models improved the genetic risk prediction for CAD. This study examined a significant number of atherogenic and thrombogenic markers in patients with premature CAD, but the markers represent a small portion of those involved in the atherosclerosis process. The complex interaction between genetic factors and the environment, including the impact of current medications on the treatment of CAD, may have further influenced the data interpretation. Traditional risk factors played a strong role in risk prediction for the onset of premature coronary atherosclerosis in our population. The inclusion of Lp(a) and LTA mutations in the set of conventional risk factors showed an additive but small increase in the risk prediction for premature CAD.

& ACKNOWLEDGMENTS Financial support was provided by ‘‘Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP)’’ number 01/06632-1.

& AUTHOR CONTRIBUTIONS Mansur AP contributed to the study conception and design, data analysis and interpretation, and writing of the manuscript. Takada JY performed the collection, analysis, and interpretation of data. Strunz CM performed the lab analysis and interpretation of data. Avakian SD contributed to the study design, as well as collection, analysis, and interpretation of data. Ce´sar LA performed the analysis and interpretation of data. Ramires JA contributed to the study conception and design, as well as revision and final approval of the manuscript to be published.

& REFERENCES 1. Wang Q. Molecular genetics of coronary artery disease. Curr Opin Cardiol. 2005;20(3):182-8, http://dx.doi.org/10.1097/01.hco.0000160373. 77190.f1. 2. Greenland P, Alpert JS, Beller GA, Benjamin EJ, Budoff MJ, Fayad ZA, et al. 2010 ACCF/AHA guideline for assessment of cardiovascular risk in asymptomatic adults: executive summary: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Circulation. 2010;122(25):2748-64.

1507


Biomarkers in premature coronary disease Mansur AP et al.

25. 26. 27. 28.

29.

30. 31.

32. 33.

34.

35.

36.

CLINICS 2013;68(12):1502-1508

heart disease mortality: findings from a multi-ethnic US population. Eur Heart J. 2009;3096:710-7. Wang TJ, Gona P, Larson MG, Tofler GH, Levy D, Newton-Cheh C, et al. Multiple biomarkers for the prediction of first major cardiovascular events and death. N Engl J Med. 2006;355(25):2631-9. Zethelius B, Berglund L, Sundstro¨m J, Ingelsson E, Basu S, Larsson A, et al. Use of multiple biomarkers to improve the prediction of death from cardiovascular causes. N Engl J Med. 2008;358(20):2107-16. Shlipak MG, Ix JH, Bibbins-Domingo K, Lin F, Whooley MA. Biomarkers to predict recurrent cardiovascular disease: the Heart and Soul Study. Am J Med. 2008;121(1):50-7. Kim HC, Greenland P, Rossouw JE, Manson JE, Cochrane BB, Lasser NL, et al. Multimarker prediction of coronary heart disease risk: the Women’s Health Initiative. J Am Coll Cardiol. 2010;55(19):2080-91, http://dx.doi. org/10.1016/j.jacc.2009.12.047. Blankenberg S, Zeller T, Saarela O, Havulinna AS, Kee F, Tunstall-Pedoe H, et al. Contribution of 30 biomarkers to 10-year cardiovascular risk estimation in 2 population cohorts: the MONICA, risk, genetics, archiving, and monograph (MORGAM) biomarker project. Circulation. 2010;121(22):2388-97, http://dx.doi.org/10.1161/CIRCULATIONAHA. 109.901413. Erqou S, Kaptoge S, Perry PL, Di Angelantonio E, Thompson A, White IR, et al. Lipoprotein(a) concentration and the risk of coronary heart disease, stroke, and nonvascular mortality. JAMA. 2009;302(4):412-23. Bennet A, Di Angelantonio E, Erqou S, Eiriksdottir G, Sigurdsson G, Woodward M, et al. Lipoprotein(a) levels and risk of future coronary heart disease: large-scale prospective data. Arch Intern Med. 2008;168(6):598-608, http://dx.doi.org/10.1001/archinte.168.6.598. Di Angelantonio E, Sarwar N, Perry P, Kaptoge S, Ray KK, Thompson A, et al. Major lipids, apolipoproteins, and risk of vascular disease. JAMA. 2009;302(18):1993-2000. Virani SS, Brautbar A, Davis BC, Nambi V, Hoogeveen RC, Sharrett AR, et al. Associations between lipoprotein(a) levels and cardiovascular outcomes in black and white subjects: the Atherosclerosis Risk in Communities (ARIC) Study. Circulation. 2012;125(2):241-9, http://dx. doi.org/10.1161/CIRCULATIONAHA.111.045120. Sawabe M, Tanaka N, Mieno MN, Ishikawa S, Kayaba K, Nakahara K, et al. Low lipoprotein(a) concentration is associated with cancer and allcause deaths: a population-based cohort study (the JMS cohort study). PLoS One. 2012;7(4):e31954, http://dx.doi.org/10.1371/journal.pone. 0031954. Rossouw JE, Cushman M, Greenland P, Lloyd-Jones DM, Bray P, Kooperberg C, et al. Inflammatory, lipid, thrombotic, and genetic markers of coronary heart disease risk in the women’s health initiative trials of hormone therapy. Arch Intern Med. 2008;168(20):2245-53, http://dx.doi.org/10.1001/archinte.168.20.2245. Ripatti S, Tikkanen E, Orho-Melander M, Havulinna AS, Silander K, Sharma A, et al. A multilocus genetic risk score for coronary heart disease: case-control and prospective cohort analyses. Lancet.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

1508

2010;376(9750):1393-400, http://dx.doi.org/10.1016/S0140-6736(10)6126 7-6. Paynter NP, Chasman DI, Pare´ G, Buring JE, Cook NR, Miletich JP, et al. Association between a literature-based genetic risk score and cardiovascular events in women. JAMA. 2010;303(7):631-7, http://dx.doi.org/10. 1001/jama.2010.119. Thanassoulis G, Peloso GM, Pencina MJ, Hoffmann U, Fox CS, Cupples LA, et al. A genetic risk score is associated with incident cardiovascular disease and coronary artery calcium: the Framingham Heart Study. Circ Cardiovasc Genet. 2012;5(1):113-21, http://dx.doi.org/10.1161/ CIRCGENETICS.111.961342. Samani NJ, Erdmann J, Hall AS, Hengstenberg C, Mangino M, Mayer B, et al. WTCCC and the Cardiogenics Consortium. Genomewide association analysis of coronary artery disease. N Engl J Med. 2007;357(5):44353. Sandhu MS, Waterworth DM, Debenham SL, Wheeler E, Papadakis K, Zhao JH, et al. LDL-cholesterol concentrations: a genome-wide association study. Lancet. 2008;371(9611):483-91, http://dx.doi.org/10.1016/ S0140-6736(08)60208-1. Wang AZ, Li L, Zhang B, Shen GQ, Wang QK. Association of SNP rs17465637 on chromosome 1q41 and rs599839 on 1p13.3 with myocardial infarction in an American caucasian population. Ann Hum Genet. 2011;75(4):475-82, http://dx.doi.org/10.1111/j.1469-1809.2011.00646.x. Angelakopoulou A, Shah T, Sofat R, Shah S, Berry DJ, Cooper J, et al. Comparative analysis of genome-wide association studies signals for lipids, diabetes, and coronary heart disease: Cardiovascular Biomarker Genetics Collaboration. Eur Heart J. 2012;33(3):393-407, http://dx.doi. org/10.1093/eurheartj/ehr225. McPherson R, Pertsemlidis A, Kavaslar N, Stewart A, Roberts R, Cox DR, et al. A common allele on chromosome 9 associated with coronary heart disease. Science. 2007;316(5830):1488-91, http://dx.doi.org/10.1126/ science.1142447. PROCARDIS Consortium. A trio family study showing association of the lymphotoxin-alpha N26 (804A) allele with coronary artery disease. Eur J Hum Genet. 2004;12(9):770-4. Laxton R, Pearce E, Kyriakou T, Ye S. Association of the lymphotoxinalpha gene Thr26Asn polymorphism with severity of coronary atherosclerosis. Genes Immunol. 2005;6(6):539-41, http://dx.doi.org/10. 1038/sj.gene.6364236. Clarke R, Xu P, Bennett D, Lewington S, Zondervan K, Parish S, et al. International Study of Infarct Survival (ISIS) Collaborators. Lymphotoxin-alpha gene and risk of myocardial infarction in 6,928 cases and 2,712 controls in the ISIS case-control study. PLoS Genet. 2006;2(7):e107, http://dx.doi.org/10.1371/journal.pgen.0020107. Palikhe A, Sinisalo J, Seppnen M, Valtonen V, Nieminen MS, Lokki ML. Human MHC region harbors both susceptibility and protective haplotypes for coronary artery disease. Tissue Antigens. 2007;69(1):4755, http://dx.doi.org/10.1111/j.1399-0039.2006.00735.x.


CLINICAL SCIENCE

Reproducibility of ambulatory blood pressure changes from the initial values on two different days Garrett I. Ash, Timothy J. Walker, Kayla M. Olson, Jeffrey H. Stratton, Ana L. Go´mez, William J. Kraemer, Jeff S. Volek, Linda S. Pescatello University of Connecticut, Department of Kinesiology & Human Performance Laboratory, Storrs/Connecticut, United States.

OBJECTIVE: We tested the reproducibility of changes in the ambulatory blood pressure (BP) from the initial values, an indicator of BP reactivity and cardiovascular health outcomes, in young, healthy adults. METHOD: The subjects wore an ambulatory BP monitor attached by the same investigator at the same time of day until the next morning on two different days (day 1 and day 2) separated by a week. We compared the ambulatory BP change from the initial values at hourly intervals over 24 waking and sleeping hours on days 1 and 2 using linear regression and repeated measures analysis of covariance. RESULTS: The subjects comprised 88 men and 57 women (mean age¡SE 22.4¡0.3 years) with normal BP (118.3¡0.9/69.7¡0.6 mmHg). For the total sample, the correlation between the ambulatory BP change on day 1 vs. day 2 over 24, waking, and sleeping hours ranged from 0.37–0.61; among women, the correlation was 0.38–0.71, and among men, it was 0.24–0.52. Among women, the ambulatory systolic/diastolic BP change was greater by 3.1¡1.0/2.4¡0.8 mmHg over 24 hours and by 3.0¡1.1/2.4¡0.8 mmHg over waking hours on day 1 than on day 2. The diastolic ambulatory BP change during sleeping hours was greater by 2.2¡0.9 mmHg on day 1 than on day 2, but the systolic ambulatory BP change during sleeping hours on days 1 and 2 did not differ. Among men, the ambulatory BP change on days 1 and 2 did not differ. CONCLUSION: Our primary findings were that the ambulatory BP change from the initial values was moderately reproducible; however, it was more reproducible in men than in women. These results suggest that women, but not men, may experience an alerting reaction to initially wearing the ambulatory BP monitor. KEYWORDS: Hypertension; Observer Variation; Gender Identity; Clinical Trials as Topic. Ash GI, Walker TJ, Olson KM, Stratton JH, Go´mez AL, Kraemer WJ, et al. Reproducibility of ambulatory blood pressure changes from the initial values on two different days. Clinics. 2013;68(12):1509-1515. Received for publication on February 24, 2013; First review completed on June 3, 2013; Accepted for publication on June 20, 2013 E-mail: garrett.ash@uconn.edu Tel.: 1 860 486 2812

coefficient of variation, which is the standard deviation of the difference expressed as a percentage of the mean value of the BP measurements made on two different days. The reported reproducibility between ambulatory BP measurements made on different days differs among studies. For example, the mean standard deviation of the difference in ambulatory BP readings made on two separate days over 24 hours for the same subject has been found to range from 7– 13 mmHg for systolic and 3–8 mmHg for diastolic ambulatory BP (4–13). Differences in subject characteristics, such as BP status and age, may explain these apparent inconsistencies and the poor reproducibility of ambulatory BP. Another feature of these studies that could contribute to poor ambulatory BP reproducibility is the long interval of time, typically 3 months or longer, that is often employed between BP measurements (6,8,10,13). In studies that use a long interval between ambulatory BP measurements, reproducibility could be modulated by extraneous factors such as season (14), changes in body weight (1,13), and alterations in adherence to lifestyle behavior patterns and pharmacological therapies used to treat hypertension (1). There is also evidence that the reproducibility of BP

& INTRODUCTION Ambulatory blood pressure (BP) monitoring is the standard clinical method for assessing BP status. Ambulatory BP predicts cardiovascular outcomes and target organ damage, assesses circadian BP patterns, and monitors the response to antihypertensive medication (1,2). However, evidence suggests the prognostic value of ambulatory BP monitoring may be limited to the degree of reproducibility of the BP measurements made on different days (3). Ambulatory BP reproducibility is defined as the standard deviation of the difference between two BP measurements made on two different days for the same individual. Ambulatory BP reproducibility can also be defined by 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(12)06

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performed using a mercury sphygmomanometer (W.A. Baum Co. Inc., New York, NY) with a t-tubule (2). When three ambulatory BP measurements within 5 mmHg of the auscultatory measurements had been recorded, the ambulatory BP measurements were averaged and reported as the initial BP value for that day. Upon leaving the laboratory, the subjects were instructed to proceed with normal activities, not to shower or exercise until the next morning, and to keep their arms still and extended when ambulatory BP measurements were being taken. The subjects were asked to carry a standard journal, recording activities performed during each measurement, any unusual physical or emotional events, and sleep and wake times. They were also asked to limit driving while wearing the ambulatory BP monitor; if driving was necessary, they were to rest the cuffed arm against the body at heart level while a reading was being taken. The ambulatory BP monitor was programmed to record the ambulatory BP every 20 minutes during waking hours and every 30 minutes during sleeping hours to minimize sleep disturbance. The monitor obtained a second reading if there was a difference between consecutive readings of systolic ambulatory BP.50 mmHg, diastolic.40 mmHg, or pulse pressure.50 mmHg. The ambulatory BP recordings were acceptable if the monitor obtained at least 80% of the potential BP readings. Investigators omitted readings if the values met the manufacturer’s exclusion criteria of systolic ambulatory BP.220 or ,80 mmHg or diastolic.130 or ,40 mmHg. The ambulatory BP readings taken during each hourly interval were averaged to determine the average ambulatory BP for each hour. In the rare instances in which an hourly average interval of ambulatory BP readings was missing, the investigators averaged the BP values from the hours before and after the missed hour to represent the missing data. The initial ambulatory BP was then subtracted from the average ambulatory BP at each hourly time interval to calculate the ambulatory BP change. The 15 hours when the subjects were awake and ambulating represented the waking hours, the 9 hours when the subjects were sleeping represented the sleeping hours, and the 15 waking plus the 9 sleeping hours represented 24 hours. The average and ambulatory BP changes from the initial value were calculated for days 1 and 2 over 24 hours, the waking hours, and the sleeping hours.

measurements may be compromised by an alerting reaction to initially wearing the monitor (4,9,11–13,15,16). An alerting reaction is defined as a higher ambulatory BP reading on the first measurement than on the second caused by anxiety and sleep disturbances due to initially wearing the monitor; these disturbances typically diminish upon subsequent ambulatory BP assessments. We undertook the present study to quantify reproducibility in the change in ambulatory BP from initial values, an indicator of BP reactivity and predictor of cardiovascular health outcomes (17,18), and the average ambulatory BP on 2 separate days 1 week apart in a sample of young, healthy adults with normal BP. We hypothesized that ambulatory BP would be higher on the first day than on the second.

& METHODS Subjects The participants were recruited from a larger study entitled ‘‘Investigation of whey protein supplementation for physiologic enhancement to resistance training and dietary regimes in young adults’’ (REPS) (National Dairy Council 070996). REPS participants were healthy adults 18– 35 years of age who had not participated in any resistance training programs during the past year and had resting BP ,150/95 mmHg. Prior to participating in REPS, the subjects signed an informed consent form approved by the Institutional Review Board of the University of Connecticut. Upon enrollment, waist circumference was measured at the narrowest part of the torso using a standard flexible tape with a spring-loaded handle (Gulick 4192G, G&S Fibreflex, San Diego, CA). Height was measured with a stadiometer (Seca, Hamburg, Germany), and weight with a calibrated digital scale (OHAUS, Florham Park, NJ). Additionally, the body mass index (BMI) was calculated. Prior to the resistance training and nutrition supplement intervention, the participants were asked to complete two separate ambulatory BP monitoring studies on two different days separated by one week. The ambulatory BP data served as the basis for this sub-study.

Procedures All ambulatory BP monitoring visits occurred in the morning between 7:00 and 11:00 am (mean¡standard error 9:09 am¡0:06). The standard procedures used in our previous ambulatory BP studies (2,19–21) were followed and were performed by the same trained investigator at the same time of day for each subject. The subjects were instructed to refrain from formal exercise and caffeine, respectively, 24 hours prior to and the morning of the attachment. Laboratory visits were postponed if the subject was ill, unusually busy or anxious or planned to spend unusual amounts of time in a motorized vehicle the day of the scheduled attachment. Upon entering the laboratory, the subjects were seated for at least 15 minutes. The investigator took a minimum of three BP readings by auscultation in each arm separated by one minute, alternating between the arms, until three auscultatory readings in each arm agreed within 5 mmHg. The investigator then fitted the subject with an appropriately sized BP cuff and attached the Accutracker II automatic non-invasive ambulatory BP monitor (SunTech Medical Instruments Inc., Raleigh, NC). Upon attachment of the monitor, a calibration check was

Statistical analysis Descriptive statistics were generated for all dependent and independent variables. Analysis of variance was used to test for differences in the subject descriptive characteristics between the sexes. We used the following statistical methods to assess the reproducibility of the average and ambulatory BP change from the initial value on days 1 vs. 2 over 24 hours, the waking hours and the sleeping hours: 1) Pearson correlation coefficients; 2) repeated measures analysis of covariance by sex with BMI and age as covariates; and 3) standard deviation of the difference and coefficient of variation. Significance was set at p,0.05, and all data were reported as the mean¡standard error. All analyses were performed using the Statistical Package for Social Sciences Base 14.0 for Windows (SPSS Inc., Chicago, IL).

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Blood Pressure Reproducibility Ash GI et al.

Table 1 - Physical characteristics (mean¡standard error) of the study sample by sex.

Age (yr) BMI (kg*m22) Waist Circumference (cm) Resting Systolic Blood Pressure (mmHg) Resting Diastolic Blood Pressure (mmHg)

Total (n = 145)

Women (n = 57)

Men (n = 88)

22.4¡0.3 25.2¡0.4 78.0¡0.9 118.3¡0.9 69.7¡0.6

22.5¡0.5 25.1¡0.6 72.8¡1.4 112.3¡1.3 68.9¡1.1

22.3¡0.4 25.2¡0.4 81.4¡1.1 122.3¡0.9* 70.3¡0.7

BMI-body mass index; *p,0.001 women vs. men. The resting systolic/diastolic blood pressure was calculated as the average of the initial systolic/diastolic blood pressure on days 1 and 2.

and by sex. The initial BP was not different on days 1 and 2 in the total sample or by sex (p$0.05). Among the total sample, the ambulatory BP change from the initial value was greater on day 1 than on 2 over 24 hours for the systolic and diastolic BP (p = 0.007 and 0.027, respectively). In addition, the systolic (p = 0.019) but not the diastolic (p$0.05) ambulatory BP change during the waking hours was greater on day 1 than on day 2. The ambulatory BP change from the initial value over the sleeping hours did not differ on days 1 and 2 (p$0.05). The average ambulatory BP over 24 hours was greater on day 1 than on day 2 for both the systolic and diastolic BP (p = 0.007 and p = 0.008, respectively). In addition, the average systolic ambulatory BP over the waking (p = 0.014) and sleeping (p = 0.047) hours was greater on day 1 than on day 2, but the average diastolic ambulatory BP over the waking hours did not differ on the 2 days (p$0.05). Among women, the ambulatory BP change for the systolic and diastolic BP was greater on day 1 than on day 2 over 24 hours (p = 0.004 and p = 0.003, respectively) and over the waking hours (p = 0.007 and p = 0.004, respectively) (Figure 1). In addition, the women’s diastolic (p = 0.011) but not systolic (p$0.05) ambulatory BP change over the sleeping hours was greater on day 1 than on day 2. Their average ambulatory systolic and diastolic BP was greater on day 1 than on day 2 over 24 hours (p = 0.005 and p = 0.024, respectively) and during the waking hours (p = 0.014 and p = 0.039, respectively), and their average systolic (p = 0.014) but not diastolic (p$0.05) ambulatory BP was greater on day 1 than on 2 during the sleeping hours. In contrast, among

& RESULTS Subjects The characteristics of the participants (57 women, 88 men) are shown in Table 1. Age, BMI, waist circumference, and resting ambulatory diastolic BP were not different between sexes (p$0.05). However, the resting ambulatory systolic BP was greater in the men than in the women (p,0.001). The two different ambulatory BP measurements were separated by an average of 7.9¡0.6 days.

Measures of ambulatory BP reproducibility Pearson correlation coefficients. Table 2 displays the Pearson correlation coefficients of the ambulatory BP change from the initial value and the average ambulatory BP on day 1 vs. day 2 over 24 hours, the waking hours, and the sleeping hours. The correlation between the average ambulatory BP change on day 1 vs. day 2 over 24 hours, the waking hours, and the sleeping hours ranged from 0.37–0.61 (p,0.001) in the total sample, 0.38–0.71 (p,0.05) in women, and 0.24–0.52 (p,0.01) in men. The correlation between the average ambulatory BP on days 1 and 2 over 24 hours, the waking hours, and the sleeping hours ranged from 0.62–0.82 (p,0.001) in the total sample, 0.74–0.87 (p,0.001) in women, and 0.50–0.73 in men (p,0.001). Repeated measures analysis of covariance. Table 3 displays the initial BP, the ambulatory BP change from the initial value, the average ambulatory BP, and the mean differences in these values on day 1 vs. day 2 over 24 hours, the waking hours, and the sleeping hours in the total sample

Table 2 - Pearson correlation coefficients between the initial blood pressure, ambulatory blood pressure change from the initial value, and average ambulatory blood pressure on day 1 vs. day 2 over 24 hours, the waking hours, and the sleeping hours. Total (n = 145)

Women (n = 57)

Men (n = 88)

0.80 0.70

0.78 0.82

0.73 0.61

0.40 0.42 0.37 0.39 0.61 0.39

0.41* 0.52 0.38{ 0.47 0.71 0.63

0.40 0.38 0.38 0.36 0.52 0.24{

0.82 0.79 0.81 0.76 0.71 0.62

0.85 0.87 0.84 0.85 0.74 0.78

0.72 0.73 0.70 0.68 0.60 0.50

Initial SBP DBP Ambulatory BP Change from Initial Values 24 hour SBP 24 hour DBP Awake SBP Awake DBP Sleep SBP Sleep DBP Average Ambulatory BP 24 hour SBP 24 hour DBP Awake SBP Awake DBP Sleep SBP Sleep DBP

All correlations p,0.001 unless indicated: *p = 0.001, {p = 0.003, {p = 0.024. SBP-systolic blood pressure; DBP-diastolic blood pressure.

1511


Day 2

Difference*

1512 116.8¡1.1 68.5¡0.9 122.3¡1.2 73.7¡1.0 106.1¡1.2 58.6¡0.9

118.7¡1.2 69.6¡0.8 124.2¡1.3 74.9¡1.0 108.5¡1.3 59.6¡0.8

2.6¡0.2 3.2¡0.2 2.8¡0.2 3.6¡0.3 3.8¡0.3 5.1¡0.3

3.3¡0.3 2.2¡0.2 3.6¡0.3 2.7¡0.2 4.5¡0.3 3.2¡0.2

111.7¡1.3 112.9¡1.3 68.2¡1.1 69.5¡1.1

Day 2

7.0¡1.0 3.9¡1.0 1.4¡0.7 21.0¡0.8 12.5¡0.9 9.5¡1.0 6.7¡0.8 4.2¡0.8 23.2¡1.3 24.7¡1.4 28.6¡1.0 210.8¡1.0

3.2¡0.2 4.8¡0.3

Day 1

4.5¡0.3 3.6¡0.3 4.9¡0.3 3.8¡0.3 4.5¡0.4 4.6¡0.3

3.8¡0.3 3.4¡0.2

Standard Deviation of the Coefficient Difference of Variation

21.9¡0.7{ 21.0¡0.5{ 21.8¡0.7{ 21.1¡0.5{ 22.4¡0.9{ 21.0¡0.6

23.1¡1.0{ 22.4¡0.8{ 23.0¡1.1{ 22.5¡0.8{ 21.5¡1.1 22.2¡0.9{

1.2¡0.9 1.3¡0.7

Difference*

Women (n = 57)

3.0¡0.4 2.1¡0.2 3.3¡0.4 2.5¡0.3 3.8¡0.5 2.6¡0.3

4.3¡0.5 3.4¡0.4 4.6¡0.5 3.7¡0.5 3.9¡0.6 3.7¡0.5

3.7¡0.4 2.9¡0.4

2.5¡0.3 3.1¡0.3 2.7¡0.3 3.4¡0.4 3.5¡0.4 4.5¡0.5

3.3¡0.3 4.2¡0.5

Standard Deviation of the Coefficient Difference of Variation Day 2

128.2¡1.0 127.2¡0.8 70.4¡0.7 69.6¡0.6 134.2¡1.0 133.2¡0.9 75.9¡0.8 74.9¡0.7 117.4¡1.2 116.8¡1.0 60.4¡0.7 60.3¡0.7

5.6¡0.8 5.0¡0.8 20.3¡0.7 20.4¡0.6 11.5¡0.9 11.1¡0.8 5.1¡0.8 4.8¡0.6 25.2¡1.0 25.9¡1.1 210.1¡0.9 29.7¡0.8

122.7¡0.9 122.1¡0.9 70.7¡0.8 70.1¡0.7

Day 1

21.0¡0.8 20.8¡0.5 21.0¡0.8 21.0¡0.6 20.6¡1.0 20.1¡0.7

20.6¡0.9 20.1¡0.8 20.5¡1.0 20.4¡0.8 20.7¡1.0 0.4¡1.0

20.6¡0.8 20.6¡0.7

3.5¡0.3 2.3¡0.2 3.9¡0.3 2.9¡0.2 4.9¡0.4 3.5¡0.3

4.5¡0.4 3.8¡0.3 5.1¡0.4 3.9¡0.4 5.0¡0.5 5.3¡0.4

3.9¡0.3 3.7¡0.3

2.7¡0.2 3.2¡0.3 2.8¡0.3 3.7¡0.3 4.1¡0.4 5.8¡0.4

3.2¡0.3 5.4¡0.4

Standard Deviation of the Coefficient Difference* Difference of Variation

Men (n = 88)

SBP-systolic blood pressure; DBP-diastolic blood pressure. *Difference indicates significance for day 1 vs. day 2 based on the repeated measures analysis of covariance. {p,0.05, {p,0.01. We do not report the coefficient of variation for the ambulatory BP change because the ambulatory BP change adjusts for the initial BP value, as does the coefficient of variation.

Initial SBP 117.2¡0.8 117.5¡0.8 0.3¡0.6 DBP 69.7¡0.7 69.9¡0.6 0.2¡0.5 Ambulatory BP Change From Initial Values 24 hour SBP 6.3¡0.6 4.5¡0.6 21.9¡0.7{ 20.7¡0.5 21.2¡0.6{ 24 hour DBP 0.5¡0.5 Awake SBP 12.0¡0.7 10.3¡0.7 21.7¡0.7{ Awake DBP 5.7¡0.6 4.5¡0.5 21.2¡0.6 Sleep SBP 24.2¡0.8 25.3¡0.9 21.1¡0.8 Sleep DBP 29.5¡0.6 210.2¡0.6 20.7¡0.7 Average Ambulatory BP 24 hour SBP 123.5¡0.8 122.0¡0.7 21.5¡0.5{ 24 hour DBP 70.0¡0.5 69.1¡0.5 20.9¡0.3{ Awake SBP 129.2¡0.8 127.8¡0.7 21.4¡0.6{ Awake DBP 75.5¡0.6 74.4¡0.6 21.1¡0.4 Sleep SBP 112.9¡0.9 111.5¡0.8 21.5¡0.7{ Sleep DBP 60.1¡0.5 59.6¡0.5 20.5¡0.5

Day 1

Total (n = 145)

Table 3 - Comparison of ambulatory blood pressure changes from the initial values and average ambulatory blood pressure (mmHg) on day 1 vs. day 2 over 24 hours, the waking hours, and the sleeping hours (mean¡standard error).

Blood Pressure Reproducibility Ash GI et al. CLINICS 2013;68(12):1509-1515


CLINICS 2013;68(12):1509-1515

Blood Pressure Reproducibility Ash GI et al.

Figure 1 - Average waking ambulatory blood pressure change from the initial value at hourly intervals over 15 hours on 2 different days separated by 1 week among women.

men, the ambulatory BP change from the initial value and the average ambulatory BP did not differ on days 1 and 2 over 24 hours, the waking hours, and the sleeping hours (p$0.05).

Our study is unique because we assessed not only the reproducibility of average ambulatory BP but also ambulatory BP change from initial value as indicators of reproducibility. Based upon strength of the correlations reported in Table 2 and the smaller standard deviation of the difference reported in Table 3, average ambulatory BP appears to be more reproducible than ambulatory BP change. Stergiou and Parati (18) recently stated that the ambulatory BP change from the initial value, which they termed ‘BP reactivity’, was a better indicator of the physiologic response to wearing an ambulatory BP monitor under conditions of daily living than average ambulatory BP. Furthermore, evidence suggests that BP reactivity predicts cardiovascular health outcomes independent of initial BP value (17). For example, the Coronary Artery Risk Development in Young Adults (CARDIA) study measured the BP change from the initial value among young adult women and men in response to playing a video game (22). The CARDIA investigators found that for each 10 mmHg systolic BP increase from the initial value, there was a 24% increased likelihood of developing subsequent coronary artery calcification within 13 years. Thus, determining the reproducibility of not only the measurement of average ambulatory BP but also the ambulatory BP change from the initial value appears to be important. Our study confirms the findings reported in the existing literature that the average ambulatory BP is strongly reproducible among study populations with normal BP regardless of whether the ambulatory BP measurements are separated by several weeks (11) or months (8). We found that the standard deviation of the difference in the average ambulatory BP ranged from 2–4 mmHg and that its coefficient of variation ranged from 3–5% among the subjects in our study who had normal BP (Table 3). In contrast, studies that included subjects with hypertension found higher standard deviation of the difference values ranging from 4–13 mmHg, independent of whether ambulatory BP measurements were separated by several weeks (4,5,7,9,12) or by several months (6,10,13). The greater BP variability among individuals with a higher BP is consistent with recent findings that visit-to-visit (23,24) and

Standard deviation of the difference and coefficient of variation. Table 3 also reports the standard deviation of

the difference and the coefficient of variation for the initial BP, the ambulatory BP change from the initial value, and the average ambulatory BP on days 1 vs. 2 over 24 hours, the waking hours and the sleeping hours in the total sample and by sex. The standard deviation of the difference in the ambulatory BP change from the initial value on day 1 vs. day 2 over 24 hours, the waking hours, and the sleeping hours ranged from 3.6–4.9 mmHg in the total sample, 3.4– 4.6 mmHg in women, and 3.8–5.3 mmHg in men. The standard deviation of the difference in the average ambulatory BP on day 1 vs. day 2 over 24 hours, the waking hours, and the sleeping hours ranged from 2.2– 4.5 mmHg in the total sample, 2.1–3.8 mmHg in women, and 2.3–4.9 mmHg in men. The coefficient of variation between the average ambulatory BP on day 1 vs. day 2 over 24 hours, the waking hours, and the sleeping hours ranged from 2.6–4.5% in the total sample, 2.5–4.5% in women, and 2.7–5.8% in men. We do not report the coefficient of variation for the ambulatory BP change because the ambulatory BP change adjusts for the initial BP values, as does the coefficient of variation.

& DISCUSSION Our primary findings were that the average ambulatory BP was strongly reproducible and that the ambulatory BP change from the initial value was moderately reproducible when measured in young, healthy subjects with normal BP on 2 different days separated by 1 week. An unexpected finding was that the average ambulatory BP was 1–2 mmHg higher and the ambulatory BP change from the initial value was 2–3 mmHg higher on day 1 vs. day 2 among the women but not among the men. This finding suggests that women, but not men, may have experienced an alerting reaction to initially wearing the ambulatory BP monitor (15,16).

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within-visit (25) BP variability for resting BP correlates positively with BP status. The higher ambulatory BP readings observed on day 1 vs. day 2 among women but not among men suggest that the women, but not the men, experienced an alerting reaction to initially wearing the monitor (15,16). Shin et al. (25) found that women exhibited greater within-visit auscultatory BP variability than men, including greater differences between the initial and subsequent BP measurements; this effect was considered to result from an alerting reaction to the initial auscultatory BP measurement. Calvo et al. (15) found that when women and men with hypertension wore an ambulatory BP monitor continuously for 48 hours, the average ambulatory BP over 24 hours and the waking hours was lower on the second day than on the first day. Consistent with these findings, Palatini et al. (13) reported that when women and men with hypertension completed two ambulatory BP monitoring studies separated by three months, the average ambulatory BP declined by 1 mmHg. Similarly, Musso et al. (16) found that when women and men with a normal BP completed four ambulatory BP monitoring studies, each separated by one week, the average ambulatory BP declined approximately 1 mmHg with each successive weekly measurement, reaching statistical significance by the fourth week. Trazzi et al. (4) and Coats et al. (12) also documented trends in ambulatory BP reductions between successive readings, although the reductions did not reach statistical significance. Our study is the first to show that an alerting reaction appears to be gender-dependent and that such a reaction can significantly affect the reproducibility of ambulatory BP. These findings are relevant to both clinical practice and research. For our results indicate that a familiarization ambulatory BP session should occur prior to any clinical decision making regarding the interpretation of the BP response to an experimental perturbation, and perhaps the effectiveness of antihypertensive therapy. A difference in the BP response of 2–3 mmHg, such as we found between the first and second ambulatory BP monitoring studies, could be mistakenly attributed to experimental intervention or antihypertensive treatment when, in fact, it might be due to an alerting reaction to wearing the ambulatory BP monitor for the first time (26). A question raised by our findings of a sex-dependent response is why an alerting reaction was experienced by the women but not by the men. Steptoe et al. (27) found that men exhibited a greater BP response to mental stress interventions administered in the laboratory than did women. Furthermore, laboratory experiments indicate that sympathetic nerve activity is positively associated with BP in men; however, if an association is found among women, it is in the opposite direction to that of men (28,29). In addition, it would seem the men in our study would be more susceptible to experiencing greater BP variability between ambulatory BP measurements made on different days due to having higher resting BP than the women (23– 25). Collectively, these data suggest that the men in our study would have been more likely to experience an alerting reaction to initially wearing the ambulatory BP monitor, whereas we observed the opposite. Previous studies addressing ambulatory BP reproducibility on 2 different days have not found sex differences, possibly due to the use of small sample sizes (4,6–12) and/ or the use of intervals of 3 months or longer between the BP

CLINICS 2013;68(12):1509-1515

measurements (6,8,10,13). However, Muntner et al. (23) found that women exhibited higher day-to-day variability in auscultatory systolic BP than men, findings that are consistent with ours. Similarly, Shin et al. (25) found that women exhibited a higher within-visit variability in auscultatory systolic and diastolic BP measurements than men. State anxiety (30), psychological distress (31), and social alienation (31) influence BP more strongly in women than in men and may drive the sex differences in BP variability. Sex differences in state anxiety also partially explain the higher ‘white-coat hypertension’ among women than men (30,32,33). Although we did not measure sympathetic nerve activity, the differences we observed in ambulatory BP on day 1 vs. day 2 were not accompanied by significant differences in the heart rate (p$0.05, data not shown), suggesting that sex-dependent differences in sympathetic nerve activity do not account for our findings. Hypothalamic-pituitary-adrenal cortical stress reactivity directly correlates with BP stress reactivity (34) and is higher among women than men (35). Therefore, sex differences in cortical system stress reactivity are a plausible physiological mechanism that may underlie the sex differences in the day-to-day BP variability that we and others have observed (23,25,30,32,33). Future mechanistic studies are required to evaluate this possibility and to evaluate other possible mechanisms. A major limitation of our study is that, on average, our subjects had normal BP. We do not know whether our findings regarding the lower reproducibility of ambulatory BP in women than in men are generalizable to individuals with hypertension. Several investigators found a higher ambulatory BP on day 1 vs. day 2 among subjects with hypertension but no sex differences in the reproducibility, possibly due to the use of small sample sizes (4,9,12) and/or long (3-month) intervals between BP measurements (13). Based on previous reports that ambulatory BP reproducibility is lower (4–7,9,10,12,13) and overall BP variability is higher (23–25) in subjects with hypertension than in subjects with normal BP (8,11), we would expect the ambulatory BP to be even less reproducible among women with hypertension than we found to be the case for young women with normal BP. However, further work is needed to determine whether this supposition is correct. In summary, the average ambulatory BP was strongly reproducible, and the ambulatory BP change from the initial value was moderately reproducible in a large sample of young adults with normal BP. A new and unexpected finding was that women, but not men, experienced an alerting reaction of 1–3 mmHg during the initial period of wearing the ambulatory BP monitor. Our observations support the contention that visit-to-visit variability in BP is affected more in women than in men by integrated, complex physiological responses to psychosocial factors (18). The effect we observed could be mitigated by the inclusion of a familiarization ambulatory BP measurement performed prior to the measurements aimed at evaluating the effects of experimental interventions or treatments.

& ACKNOWLEDGMENTS Funding for this research was provided by National Dairy Council Grant 070996. Statistical consultation was provided by Ming-Hui Chen, Ph.D. of the Department of Statistics, University of Connecticut, Storrs, CT. Statistical consultation was funded by the University of Connecticut Center of Health, Intervention, and Prevention.

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Blood Pressure Reproducibility Ash GI et al. 17. Chida Y, Steptoe A. Greater cardiovascular responses to laboratory mental stress are associated with poor subsequent cardiovascular risk status: a meta-analysis of prospective evidence. Hypertension. 2010; 55(4):1026-32, http://dx.doi.org/10.1161/HYPERTENSIONAHA.109.14 6621. 18. Stergiou GS, Parati G. How to best assess blood pressure? The ongoing debate on the clinical value of blood pressure average and variability. Hypertension. 2011;57(6):1041-2. 19. Pescatello LS, Guidry MA, Blanchard BE, Kerr A, Taylor AL, Johnson AN, et al. Exercise intensity alters postexercise hypotension. J Hypertens. 2004;22(10):1881-8, http://dx.doi.org/10.1097/00004872-200410000-000 09. 20. Eicher JD, Maresh CM, Tsongalis GJ, Thompson PD, Pescatello LS. The additive blood pressure lowering effects of exercise intensity on postexercise hypotension. Am Heart J. 2010;160(3):513-20, http://dx.doi.org/ 10.1016/j.ahj.2010.06.005. 21. Pescatello LS, Fargo AE, Leach CN, Jr, Scherzer HH. Short-term effect of dynamic exercise on arterial blood pressure. Circulation. 1991;83(5):1557-61, http://dx.doi.org/10.1161/01.CIR.83.5.1557. 22. Matthews KA, Zhu S, Tucker DC, Whooley MA. Blood pressure reactivity to psychological stress and coronary calcification in the Coronary Artery Risk Development in Young Adults Study. Hypertension. 2006;47(3):391-5, http://dx.doi.org/10.1161/01.HYP. 0000200713.44895.38. 23. Muntner P, Shimbo D, Tonelli M, Reynolds K, Arnett DK, Oparil S. The relationship between visit-to-visit variability in systolic blood pressure and all-cause mortality in the general population: findings from NHANES III, 1988 to 1994. Hypertension. 2011;57(2):160-6, http://dx. doi.org/10.1161/HYPERTENSIONAHA.110.162255. 24. Mancia G, Facchetti R, Parati G, Zanchetti A. Visit-to-visit blood pressure variability, carotid atherosclerosis, and cardiovascular events in the European Lacidipine Study on Atherosclerosis. Circulation. 2012;126(5):569-78, http:// dx.doi.org/10.1161/CIRCULATIONAHA.112.107565. 25. Shin JH, Shin J, Kim BK, Lim YH, Park HC, Choi SI, et al. Within-visit blood pressure variability: relevant factors in the general population. J Hum Hypertens. 2013;27(5):328-34, http://dx.doi.org/10.1038/jhh. 2012.39. 26. ALLHAT Officers and Coordinators for the ALLHAT Collaborative Research Group. The Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial. Major outcomes in high-risk hypertensive patients randomized to angiotensin-converting enzyme inhibitor or calcium channel blocker vs diuretic: The Antihypertensive and LipidLowering Treatment to Prevent Heart Attack Trial (ALLHAT). JAMA. 2002;288(23):2981-97. 27. Steptoe A, Fieldman G, Evans O, Perry L. Cardiovascular risk and responsivity to mental stress: the influence of age, gender and risk factors. J Cardiovasc Risk. 1996;3(1):83-93, http://dx.doi.org/10.1097/ 00043798-199602000-00012. 28. Hart EC, Charkoudian N, Wallin BG, Curry TB, Eisenach JH, Joyner MJ. Sex differences in sympathetic neural-hemodynamic balance: implications for human blood pressure regulation. Hypertension. 2009;53(3):571-6, http://dx.doi.org/10.1161/HYPERTENSIONAHA.108.126391. 29. Casey DP, Curry TB, Joyner MJ, Charkoudian N, Hart EC. Relationship between muscle sympathetic nerve activity and aortic wave reflection characteristics in young men and women. Hypertension. 2011;57(3):4217, http://dx.doi.org/10.1161/HYPERTENSIONAHA.110.164517. 30. Streitel KL, Graham JE, Pickering TG, Gerin W. Explaining gender differences in the white coat effect. Blood Press Monit. 2011;16(1):1-6, http://dx.doi.org/10.1097/MBP.0b013e32833f56c2. 31. Levenstein S, Smith MW, Kaplan GA. Psychosocial predictors of hypertension in men and women. Arch Intern Med. 2001 28;161(10):1341-6, http://dx.doi.org/10.1001/archinte.161.10.1341. 32. Manios ED, Koroboki EA, Tsivgoulis GK, Spengos KM, Spiliopoulou IK, Brodie FG, et al. Factors influencing white-coat effect. Am J Hypertens. 2008;21(2):153-8. 33. Den Hond E, Celis H, Vandenhoven G, O’Brien E, Staessen JA, THOP investigators. Determinants of white-coat syndrome assessed by ambulatory blood pressure or self-measured home blood pressure. Blood Press Monit. 2003;8(1):37-40, http://dx.doi.org/10.1097/00126097200302000-00008. 34. Hamer M, Endrighi R, Venuraju SM, Lahiri A, Steptoe A. Cortisol responses to mental stress and the progression of coronary artery calcification in healthy men and women. PLoS One. 2012;7(2):e31356, http://dx.doi.org/10.1371/journal.pone.0031356. 35. Handa RJ, Burgess LH, Kerr JE, O’Keefe JA. Gonadal steroid hormone receptors and sex differences in the hypothalamo-pituitary-adrenal axis. Horm Behav. 1994;28(4):464-76, http://dx.doi.org/10.1006/hbeh.1994. 1044.

& AUTHOR CONTRIBUTIONS Gomez AL, Kraemer WJ, Volek JS, and Pescatello LS participated in the study design. Ash GI, Walker TJ, Olson KM, Gomez AL, Kraemer WJ, Volek JS, and Pescatello LS conducted the study. Ash GI, Stratton JH, and Pescatello LS performed the statistical analysis. Ash GI, Walker TJ, Olson KM, and Pescatello LS drafted and reviewed the manuscript. All authors reviewed and approved the manuscript.

& REFERENCES 1. Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL Jr, et al. Seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Hypertension. 2003;42(6):1206-52, http://dx.doi.org/10.1161/01.HYP. 0000107251.49515.c2. 2. Pickering TG, Hall JE, Appel LJ, Falkner BE, Graves JW, Hill MN, et al. Recommendations for blood pressure measurement in humans: an AHA scientific statement from the Council on High Blood Pressure Research Professional and Public Education Subcommittee. J Clin Hypertens (Greenwich). 2005;7(2):102-9, http://dx.doi.org/10.1111/j.1524-6175.2005. 04377.x. 3. Palatini P, Mormino P, Santonastaso M, Mos L, Pessina AC. Ambulatory blood pressure predicts end-organ damage only in subjects with reproducible recordings. HARVEST Study Investigators. Hypertension and Ambulatory Recording Venetia Study. J Hypertens. 1999;17(4):46573. 4. Trazzi S, Mutti E, Frattola A, Imholz B, Parati G, Mancia G. Reproducibility of non-invasive and intra-arterial blood pressure monitoring: implications for studies on antihypertensive treatment. J Hypertens. 1991;9(2):115-9, http://dx.doi.org/10.1097/00004872199102000-00003. 5. Stergiou GS, Baibas NM, Gantzarou AP, Skeva II, Kalkana CB, Roussias LG, et al. Reproducibility of home, ambulatory, and clinic blood pressure: implications for the design of trials for the assessment of antihypertensive drug efficacy. Am J Hypertens. 2002;15(2 Pt 1):101-4. 6. Brueren MM, van Limpt P, Schouten HJ, de Leeuw PW, van Ree JW. Is a series of blood pressure measurements by the general practitioner or the patient a reliable alternative to ambulatory blood pressure measurement? A study in general practice with reference to short-term and longterm between-visit variability. Am J Hypertens. 1997;10(8):879-85. 7. Uen S, Fimmers R, Brieger M, Nickenig G, Mengden T. Reproducibility of wrist home blood pressure measurement with position sensor and automatic data storage. BMC Cardiovasc Disord. 2009;9:20, http://dx. doi.org/10.1186/1471-2261-9-20. 8. Wendelin-Saarenhovi M, Isoaho R, Hartiala J, Helenius H, Kivela SL, Hietanen E. Long-term reproducibility of ambulatory blood pressure in unselected elderly subjects. Clin Physiol. 2001;21(3):316-22, http://dx. doi.org/10.1046/j.1365-2281.2001.00332.x. 9. Eguchi K, Hoshide S, Hoshide Y, Ishikawa S, Shimada K, Kario K. Reproducibility of ambulatory blood pressure in treated and untreated hypertensive patients. J Hypertens. 2010;28(5):918-24, http://dx.doi.org/ 10.1097/HJH.0b013e3283378477. 10. Campbell P, Ghuman N, Wakefield D, Wolfson L, White WB. Long-term reproducibility of ambulatory blood pressure is superior to office blood pressure in the very elderly. J Hum Hypertens. 2010;24(11):749-54, http://dx.doi.org/10.1038/jhh.2010.8. 11. Weston PJ, Robinson JE, Watt PA, Thurston H. Reproducibility of the circadian blood pressure fall at night in healthy young volunteers. J Hum Hypertens. 1996;10(3):163-6. 12. Coats AJ. Reproducibility or variability of casual and ambulatory blood pressure data: implications for clinical trials. J Hypertens Suppl. 1990;8(6):S17-20. 13. Palatini P, Mormino P, Canali C, Santonastaso M, De Venuto G, Zanata G, et al. Factors affecting ambulatory blood pressure reproducibility. Results of the HARVEST Trial. Hypertension and Ambulatory Recording Venetia Study. Hypertension. 1994;23(2):211-6. 14. Sega R, Cesana G, Bombelli M, Grassi G, Stella ML, Zanchetti A, et al. Seasonal variations in home and ambulatory blood pressure in the PAMELA population. Pressione Arteriose Monitorate E Loro Associazioni. J Hypertens. 1998;16(11):1585-92. 15. Calvo C, Hermida RC, Ayala DE, Lopez JE, Fernandez JR, Dominguez MJ, et al. The ‘ABPM effect’ gradually decreases but does not disappear in successive sessions of ambulatory monitoring. J Hypertens. 2003;21(12):226573, http://dx.doi.org/10.1097/00004872-200312000-00014. 16. Musso NR, Vergassola C, Barone C, Lotti G. Ambulatory blood pressure monitoring: how reproducible is it? Am J Hypertens. 1997;10(8):936-9.

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CLINICAL SCIENCE

Predictors of in-hospital mortality in patients with ST-segment elevation myocardial infarction undergoing pharmacoinvasive treatment Felipe Jose´ de Andrade Falca˜o,I Cla´udia Maria Rodrigues Alves,I Adriano Henrique Pereira Barbosa,I Adriano Caixeta,I Jose´ Marconi Almeida Sousa,I Jose´ Augusto Marcondes Souza,I Amaury Amaral,II Luiz Carlos Wilke,II Fa´tima Cristina A. Perez,III Iran Gonc¸alves Ju´nior,I Edson Stefanini,I Antoˆnio Carlos CarvalhoI I

Universidade Federal de Sa˜o Paulo, Department of Cardiology, Sa˜o Paulo/SP, Brazil. II Secretaria de Sau´de do Municı´pio de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil. Hospital Municipal Tatuape´, Sa˜o Paulo/SP, Brazil.

III

OBJECTIVES: To identify predictors of in-hospital mortality in patients with acute myocardial infarction undergoing pharmacoinvasive treatment. METHODS: This was an observational, prospective study that included 398 patients admitted to a tertiary center for percutaneous coronary intervention within 3 to 24 hours after thrombolysis with tenecteplase. ClinicalTrials.gov: NCT01791764 RESULTS: The overall in-hospital mortality rate was 5.8%. Compared with patients who survived, patients who died were more likely to be older, have higher rates of diabetes and chronic renal failure, have a lower left ventricular ejection fraction, and demonstrate more evidence of heart failure (Killip class III or IV). Patients who died had significantly lower rates of successful thrombolysis (39% vs. 68%; p = 0.005) and final myocardial blush grade 3 (13.0% vs. 61.9%; p,0.0001). Based on the multivariate analysis, the Global Registry of Acute Coronary Events score (odds ratio 1.05, 95% confidence interval (CI) 1.02-1.09; p = 0.001), left ventricular ejection fraction (odds ratio 0.9, 95% CI 0.89-0.97; p = 0.001), and final myocardial blush grade of 0-2 (odds ratio 8.85, 95% CI 1.34-58.57; p = 0.02) were independent predictors of mortality. CONCLUSIONS: In this prospective study that evaluated patients with ST-segment elevation myocardial infarction treated by a pharmacoinvasive strategy, the in-hospital mortality rate was 5.8%. The Global Registry of Acute Coronary Events score, left ventricular ejection fraction, and myocardial blush were independent predictors of mortality in this high-risk group of acute coronary syndrome patients. KEYWORDS: Myocardial Infarction; Mortality; Pharmacoinvasive Therapy. Falca˜o FJ, Alves CM, Barbosa AH, Caixeta A, Sousa JM, Souza JA, et al. Predictors of in-hospital mortality in patients with ST-segment elevation myocardial infarction undergoing pharmacoinvasive treatment. Clinics. 2013;68(12):1516-1520. Received for publication on April 18, 2013; First review completed on May 22, 2013; Accepted for publication on June 20, 2013 E-mail: felipejaf@gmail.com Tel.: 55 11 5576-4014

tertiary care centers with an around-the-clock (’24/7’) service. Large urban centers and rural areas, where tertiary care hospitals are restricted to specific regions, are less likely to have the capacity to deliver this treatment to the population as a whole (4). Thus, a pharmacoinvasive treatment strategy has emerged as an alternative for STEMI patients admitted to primary care centers. A pharmacoinvasive strategy consists of the use of intravenous thrombolytic therapy in a primary care center followed by immediate transfer to a tertiary hospital, where early, systematic coronary angiography and percutaneous coronary angioplasty should be performed within 3 to 24 hours, even in cases of successful reperfusion (5). In Brazil, despite recent social and medical therapeutic advances, the number of acute myocardial infarctions in the population still reaches 300,000 to 400,000 per year, with STEMI representing 30% to 40% of all cases. Furthermore, mortality rates are still high outside major referral centers,

& INTRODUCTION Primary percutaneous coronary intervention (pPCI) is the standard of care reperfusion therapy for patients with evolving ST-segment elevation myocardial infarction (STEMI) (1-3). Although individual centers have performed pPCI for many years, a significant number of patients with STEMI either do not receive this optimal treatment or do not receive it in a timely manner due to the small number of

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(12)07

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In-hospital mortality in patients with STEMI Falca˜o FJ et al.

(TNK; pain-to-needle time) and between the administration of TNK and coronary angiography (needle-to-balloon time) are expressed in hours. This study was conducted in compliance with the ethical principles of the Helsinki Declaration (2008), as well as local applicable laws and regulations. It was registered in ClinicalTrials.gov with the identifier number NCT01791764.

with an estimated 1 death for every 5 to 7 cases (6). Therefore, we recently developed a public STEMI network based on a pharmacoinvasive treatment strategy in the metropolitan area of Sa˜o Paulo as a possible alternative to pPCI. In the present study, we sought to evaluate the rate and predictors of in-hospital mortality in a large population of STEMI patients who underwent pharmacoinvasive treatment.

Statistical analysis Continuous variables are expressed as the mean and standard deviation, and categorical variables are expressed as absolute numbers and percentages. Fisher’s exact test, Pearson’s chi-square test, or Student’s t-test were used when appropriate. A multivariate logistic regression model was used to identify potential independent predictors of mortality. Odds ratios (ORs) and their respective confidence intervals (95% CIs) were used to quantify the effects. The variables included in the model were age, gender, hemoglobin level, diabetes mellitus, hypertension, current smoking status, chronic renal failure, GRACE score, Killip class, baseline and final TIMI flow, and final myocardial blush grade. SPSSH software version 20 (IBM, Armonk, NY, USA) was used for all analyses, and a final p value less than 0.05 was considered significant.

& METHODS Patients and study protocol Between November 2010 and October 2012, 567 patients with STEMI were transferred to a tertiary hospital (Hospital Sa˜o Paulo, Escola Paulista de Medicina at Federal University of Sa˜o Paulo) as part of a municipal primary care program for STEMI. The structured health care network for patients with STEMI has been previously described in detail elsewhere (7). In brief, patients are first admitted to a primary emergency room facility or rescued by the Emergency Mobile Healthcare Service (SAMU) at home and then referred to our tertiary PCI center. Patients were either treated with thrombolysis or underwent pPCI as appropriate and according to current guidelines (1-3). Those patients receiving thrombolysis were referred for an early invasive coronary angiography procedure regardless of successful reperfusion 3 to 24 hours after thrombolysis (i.e., elective or rescue PCI) according to clinical and electrocardiographic criteria. Coronary angiographies were performed by femoral access, and only bare metal stents were used. After excluding patients who did not receive thrombolysis, those who underwent pPCI, and those with no acute myocardial infarction, 398 patients remained for the present analysis.

& RESULTS The study flowchart is shown in Figure 1. After excluding pPCI patients, patients without CAD, patients triaged to CABG, and patients treated with thrombolytics other than TNK, a total of 398 patients were selected for the current analysis. The mean pain-to-needle and needle-to-balloon times were 4.5¡3.8 and 16.4¡20.6 hours, respectively. Overall, the in-hospital mortality rate was 5.8% (compared with 6.5% of the entire population of 567 patients), and the mean GRACE score was 148.2¡36.9. A comparison of demographic and clinical characteristics in non-survivors and survivors is provided in Table 1. Compared with patients who survived, non-survivors were more likely to be older and to have higher rates of diabetes and chronic renal failure, higher GRACE scores, lower LVEFs, and more evidence of heart failure (Killip class III or IV). Additionally, patients who died had a significantly lower incidence of baseline TIMI grade 2 or 3 flow (39% vs. 68%; p = 0.005) and a lower final myocardial blush grade (Table 1). Two patients (0.5%) had a hemorrhagic stroke, 16 (4%) required blood transfusion, and vascular complications related to the access site were observed in 26 (6.5%) patients. A more detailed analysis of the vascular access and bleeding complications in patients undergoing pharmacoinvasive strategy in our institution has been previously reported (13).

Study objective and definitions The primary objective of this study was to evaluate the rate and predictors of in-hospital mortality in a population of STEMI patients who underwent pharmacoinvasive treatment. Patients were consecutively included and divided into two groups according to mortality (patients who died during the index hospitalization from all causes and patients who survived) and compared according to their clinical, angiographic, and procedural variables as appropriate. Demographic and clinical data were entered into a database prospectively, and all quantitative coronary angiography data were analyzed by one experienced interventional cardiologist. Thrombolysis in myocardial infarction (TIMI) flow and blush grade were assessed as previously reported (8,9). Coronary artery disease (CAD) was defined as single- or multi-vessel disease according to the number of epicardial arteries with at least 1 lesion measuring $70% of the diameter of stenosis. Chronic renal failure was defined as creatinine clearance ,60 mL/min, estimated by the Cockcroft-Gault formula (10). Anemia was defined as plasma hemoglobin measuring less than 13 mg/dL for males or 12 mg/dL for females (11). The Global Registry of Acute Coronary Events (GRACE) score (12) was calculated at admission, and the left ventricular ejection fraction (LVEF) was determined by echocardiography during hospitalization. Successful thrombolysis was defined as a culprit artery with TIMI flow grade 2-3 at the time of coronary angiography. Time intervals between chest pain and the administration of Tenecteplase

Predictors of in-hospital mortality Based on the multivariate regression analysis, only GRACE score, LVEF, and final blush grade 0-2 were independent predictors of mortality (Table 2).

& DISCUSSION Providing optimal care for STEMI patients is challenging in any country, regardless of its wealth and developmental level. This is especially true in large urban centers or rural areas, where a lack of access to tertiary healthcare may make the performance of timely pPCI difficult. The so-called

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CLINICS 2013;68(12):1516-1520

Figure 1 - Flowchart of patients included in the study.

obtained in the TRANSFER AMI study (14), and our own results, one-third of the STREAM patients required rescue catheterization. In the present analysis, LVEF and the GRACE score emerged as significant predictors of mortality. Successful thrombolysis was achieved in over 65% of patients, similar to the rate reported in the STREAM trial, with a mortality rate of only 3.4% in this subgroup (significantly lower than the rate of 10.4% in patients with a baseline TIMI flow of 01). Therefore, this study showing a low mortality rate in patients who underwent successful thrombolysis is encouraging and stresses the potential importance of a pharmacoinvasive treatment strategy, especially in public hospitals and developing countries. Regardless of achieving a similar optimal final TIMI flow after PCI (as a surrogate endpoint for PCI angiographic success) in both groups, grade 3 myocardial blush was observed significantly less often in non-survivors; thus, myocardial blush grade was also an important independent predictorof mortality. Restoring coronary microcirculation through the use of several available strategies and in light of ongoing studies (e.g., intracoronary abciximab and thrombectomy) has been a challenge for interventional cardiologists over the last several decades. Studies evaluating patients who underwent thrombolytic and mechanical reperfusion during STEMI showed a direct relationship between the degree of myocardial perfusion, as detected by the blush angiographic method, and mortality in short and long clinical follow-up periods (22,23). Restoring microcirculation extends beyond the restoration of epicardial flow and depends on the optimization of adjunctive therapy, especially antiplatelet and antithrombotic therapy. Compared with the STREAM trial population, we achieved similar rates of final TIMI flow 2-3 even in a setting of a longer pain-to-needle time. Therefore, achieving

pharmacoinvasive treatment strategy, consisting of thrombolysis with TNK and contemporary antithrombotic therapy administered before transport to a PCI-capable hospital, has emerged as an alternative to pPCI. The recommendation of prompt transfer to a referral center with a catheterization laboratory is deemed fundamental to the strategy’s success, allowing for (i) immediate reperfusion in cases when thrombolysis fails; (ii) treatment for early reocclusion, which can affect as many as 30% of cases (14); and, ultimately, (iii) early PCI (,24 hours) for patients with successful thrombolytic reperfusion but with severe residual stenosis. Compared with pPCI, this strategy has shown similar clinical outcomes (15-17). Accordingly, this strategy is strongly recommended by the European Society of Cardiology and ACC/AHA guidelines (class I and IIa, respectively) (1,3). The present study represents one of the largest real-world studies relevant to this subject thus far and shows an inhospital all-cause mortality rate of 5.8%. Other national, multicenter studies have reported mortality rates of 3.3% in patients who underwent elective PCI after thrombolysis, 7.6% in patients who underwent rescue PCI, and 5.6% in pPCI patients (18,19). In previous randomized multicenter studies comparing a pharmacoinvasive strategy with ischemia-driven PCI, in-hospital mortality rates ranged from 3.0% to 4.5%. These studies, however, also demonstrated shorter pain-to-needle and needle-to-balloon times (2 and 4 hours, respectively), which may explain, at least in part, their lower mortality rates (14,20). A remarkably low mortality rate was also observed in a recently published STREAM trial (21), which compared pPCI with a pharmacoinvasive approach. This study randomized STEMI patients who had a clinical presentation for less than 3 hours and who were at least 60 minutes away from pPCI centers and found an overall mortality rate of 4.7% for both groups. Similar to previous results, such as those

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CLINICS 2013;68(12):1516-1520

In-hospital mortality in patients with STEMI Falca˜o FJ et al.

Table 1 - Baseline, clinical, and procedural characteristics of non-survivors vs. survivors. Variables Age (years) Male CrCl (,60 ml/min) Diabetes mellitus Hypertension Dyslipidemia Currently smoking Anemia Anterior AMI Inferior AMI + RV Killip class III or IV GRACE risk score LVEF (%) Pain-to-needle time (h) Needle-to-balloon time (h) Successful thrombolysis Multi-vessel CAD Final TIMI 2, 3 Final Blush 0-2

Non-survivors (23)

Survivors (375)

p-value

63.78¡11.2 13 (56.5%) 10 (43.5%) 14 (60.9%) 16 (69.5%) 14 (60.9%) 14 (60.9%) 6 (26.1%) 6 (26.1%) 5 (21.7%) 10 (43.5%) 206.2¡39.2 33.9¡20.2 4.8¡3.1 8.0¡6.3 9 (39.1%) 20 (87%) 20 (87%) 20 (87%)

57.63¡11.5 260 (69.3%) 63 (16.8%) 105 (28%) 234 (58.7%) 220 (58.6%) 248 (66.1%) 61 (16.3%) 97 (25.9%) 36 (9.6%) 29 (7.7%) 144.6¡33.7 49.9¡12.5 4.4¡3.9 17.0¡21.1 255 (68%) 240 (64%) 343 (91.4%) 143 (38.1%)

0.011 0.199 0.001 0.001 0.330 0.846 0.797 0.222 0.981 0.075 ,0.0001 ,0.0001 ,0.0001 0.69 0.053 0.005 0.024 0.442 ,0.0001

CrCl: creatinine clearance; AMI: acute myocardial infarction; RV: right ventricular involvement.

a baseline TIMI flow of 3 along with a myocardial blush grade of 3 should be considered to indicate optimal reperfusion. This finding is in accordance with previous studies demonstrating a normal myocardial blush grade as a predictor of survival and left ventricular recovery in patients who undergo pPCI and rescue angioplasty (24-28). Hemorrhagic complications are still a concern associated with this strategy. However, no significant difference was observed in randomized trials between this treatment strategy and other treatments (14,20,21). In the STREAM trial, after the amendment of halving the TNK dosage in patients older than 75 years, there was no significant difference in hemorrhagic events between the groups. Hemorrhagic stroke and blood transfusion occurred in 0.5% and 2.9% of patients, respectively; in our study, these rates were 0.5% and 4.0%, respectively. Although far from ideal because pPCI is considered the standard of care for STEMI patients, a pharmacoinvasive treatment strategy could be an alternative for achieving short pain-to-balloon times and, coupled with a structured healthcare network, could represent a highly important improvement in public health management of STEMI in Brazil. The present study is a step forward in adjusting the healthcare system in accordance with global standards to optimally assist the largest possible number of patients. This study has several limitations. It was an observational and single-center study. A femoral artery approach was used in all cases, and bleeding criteria may underestimate the actual impact of this important complication on mortality. In addition, the use of radial access in pPCI has been associated with lower mortality rates (29,30). The multivariate analysis performed in this study cannot exclude residual confounding due to unmeasured or

unknown variables. Finally, the generalizability of this study is limited because it can only be applied to STEMI patients who undergo a pharmacoinvasive treatment strategy according to the methods described in our protocol. In this real-world, prospective study that included STEMI patients who underwent a pharmacoinvasive treatment strategy, in-hospital mortality rates were low at 5.8%. The GRACE score, LVEF, and myocardial blush grade were powerful predictors of mortality after administration of this pharmacoinvasive treatment strategy in the contemporary reperfusion era.

& AUTHOR CONTRIBUTIONS Falca˜o FJ wrote the manuscript. Alves CM was responsible for data analysis and interpretation. Barbosa AH was responsible for angiographic analysis. Caixeta A performed critical revision of the manuscript for important intellectual content. Sousa JM performed the statistical analysis. Souza JA, Gonc¸alves Ju´nior I, and Stefanini E were responsible for data analysis. Amaral A, Wilke LC, and Perez FC were responsible for data acquisition. Carvalho AC assisted with the study design and reviewed the final version of the manuscript.

& REFERENCES 1. Steg PG, James SK, Atar D, Badano LP, Blomstrom-Lundqvist C, Borger MA, et al. ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation. Eur Heart J. 2012;33(20):2569-619. 2. Piegas LS, Feitosa G, Mattos LA, Nicolau JC, Rossi Neto JM, Timerman A, et al. Sociedade Brasileira de Cardiologia. Diretriz da Sociedade Brasileira de Cardiologia sobre Tratamento do Infarto agudo do Mioca´rdio com Supradesnı´vel do Segmento ST. Arq Bras Cardiol. 2009;93(6 supl.2):e179-e264. 3. O’Gara PT, Kushner FG, Ascheim DD, Casey DE, Jr., Chung MK, de Lemos JA, et al. ACCF/AHA Guideline for the Management of STElevation Myocardial Infarction: Executive Summary: A Report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2013;61(4):485-510, http://dx.doi.org/10.1016/j.jacc.2012.11.018. 4. Boden WE, Eagle K, Granger CB. Reperfusion Strategies in Acute STSegment Elevation Myocardial Infarction. A Comprehensive Review of Contemporary Management Options. J Am Coll Cardiol. 2007;50(10):91729, http://dx.doi.org/10.1016/j.jacc.2007.04.084. 5. Levine GN, Bates ER, Blankenship JC, Bailey SR, Bittl JA, Cercek B, et al. 2011 ACCF/AHA/SCAI Guideline for Percutaneous Coronary Intervention. A Report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice

Table 2 - Independent predictors of mortality according to multivariate logistic regression analysis. Variables GRACE score LVEF Final Blush 0-2

OR

p-value

95% CI

1.057 0.930 8.857

0.001 0.001 0.02

1.023-1.091 0.890-0.972 1.339-58.570

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In-hospital mortality in patients with STEMI Falca˜o FJ et al.

6.

7.

8. 9.

10. 11.

12.

13.

14. 15.

16.

17.

18.

CLINICS 2013;68(12):1516-1520

Guidelines and the Society for Cardiovascular Angiography and Interventions. Circulation. 2011;e574-e651. Linha do Cuidado do Infarto Agudo do Mioca´rdio na Rede de Atenc¸a˜o a`s Urgeˆncias [Path of Care of Acute Myocardial Infarction in Emergency Care Networks, guidelines developed by The Brazilian Health Secretary]. Available at: http://portal.saude.gov.br/portal/arquivos/ pdf/protocolo_sindrome_coronaria.pdf. (Accessed on December 20, 2012). Caluza ACV, Barbosa AH, Gonc¸alves I, Oliveira CAL, Matos LN, Zeefried C, et al. Rede de Infarto com Supradesnivelamento de ST: Sistematizac¸a˜o em 205 Casos Diminui Eventos Clı´nicos na Rede Pu´blica [ST-Elevation Myocardial Infarction Network: Systematization in 205 Cases Reduces Clinical Events in the Public Health Care System]. Arq Bras Cardiol. 2012;99(5):1040-8, http://dx.doi.org/10.1590/S0066-782X2 012005000100. TIMI Study Group. The Thrombolysis in Myocardial Infarction (TIMI) trial. Phase I findings. N Engl J Med. 1985;312(14):932-6. van ’t Hof AW, Liem A, Suryapranata H, Hoorntje JC, de Boer MJ, Zijlstra F. Angiographic assessment of myocardial reperfusion in patients treated with primary angioplasty for acute myocardial infarction: myocardial blush grade. Zwolle Myocardial Infarction Study Group. Circulation. 1998;97(23):2302-6, http://dx.doi.org/10.1161/01. CIR.97.23.2302. Cockcroft DW, Gault MH. Prediction of creatinine clearance from serum creatinine. Nephron. 1976;16(1):31-41, http://dx.doi.org/10.1159/0001 80580. WHO. Hemoglobin concentrations for the diagnosis of anemia and assessment of severity. Vitamin and Mineral Nutrition Information System. Geneva, World Health Organization, 2011 (WHO/NMH/NHD/ MNM/11.1). Available at: http://www.who.int/vmnis/indicators/ haemoglobin.pdf. (Accessed on December 20, 2012). Granger CB, Goldberg RJ, Dabbous O, Pieper KS, Eagle KA, Cannon CP, et al. Predictors of hospital mortality in the global registry of acute coronary events. Arch Intern Med. 2003;163(19):2345-53, http://dx.doi. org/10.1001/archinte.163.19.2345. Gomes Ju´nior MPM, Falca˜o FJA, Alves CMR, Sousa JMA, Herrmann JL, Moreno ACC, et al. Complicac¸o˜es Vasculares em Pacientes Submetidos a Intervenc¸a˜o Corona´ria Percutaˆnea Precoce por Via Femoral apo´s Fibrino´lise com Tenecteplase: Registro de 199 Pacientes. Rev Bras Cardiol Invasiva. 2012;20(3):274-81, http://dx.doi.org/10.1590/S217983972012000300010. Cantor WJ, Fitchett D, Borgundvaag B, Ducas J, Heffernan M, Cohen EA, et al. Routine early angioplasty after fibrinolysis for acute myocardial infarction. N Engl J Med. 2009;360(26):2705-18. Bodı´ V, Rumiz E, Merlos P, Nunez J, Lo´pez-Lereu MP, Monmeneu JV, et al. One-week and 6-month cardiovascular magnetic resonance outcome of the pharmacoinvasive strategy and primary angioplasty for the reperfusion of ST-segment elevation myocardial infarction. Revista Espan˜ola de Cardiologı´a. 2011;4(2):111-20, http://dx.doi.org/10.1016/j. recesp.2010.10.008. Danchin N, Coste P, Ferrieres J, Steg PG, Cottin Y, Blanchard D, et al. Comparison of thrombolysis followed by broad use of percutaneous coronary intervention with primary percutaneous coronary intervention for ST-segment-elevation acute myocardial infarction: data from the french registry on acute ST-elevation myocardial infarction (FAST-MI). Circulation. 2008;118(3):268-76, http://dx.doi.org/10.1161/CIRCULATI ONAHA.107.762765. Armstrong P. WEST Steering Committee. A comparison of pharmacologic therapy with/without timely coronary intervention vs primary percutaneous intervention early after ST-elevation myocardial infarction: the WEST (Which Early ST-elevation myocardial infarction Therapy) Study. Eur Heart J. 2006;27(13):1530-8, http://dx.doi.org/10.1093/ eurheartj/ehl088. Lima EC, Nascimento GA, Pena MI, Vasconcelos VSA, Crepaldi RJQ, Rabelo W, et al. Intervenc¸a˜o Corona´ria Percutaˆnea Eletiva apo´s

19.

20.

21. 22.

23.

24.

25.

26.

27.

28.

29.

30.

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Fibrino´lise: Dados do REMAT (Registro Madre Teresa) [Elective Percutaneous Coronary Intervention after Fibrinolysis: REMAT Data (Madre Teresa Registry)]. Rev Bras Cardiol Invasiva. 2011;19(4):373-8, http://dx.doi.org/10.1590/S2179-83972011000400007. Mattos LA, Sousa AGMR, Pinto IMF, Silva ER, Carneiro JK, Sousa JE, et al. Uma comparac¸a˜o entre a intervenc¸a˜o coronariana percutaˆnea de resgate e prima´ria realizadas no infarto agudo do mioca´rdio: relato multiceˆntrico de 9.371 pacientes. Arq Bras Cardiol. 2004;82(5):434-9, http://dx.doi.org/10.1590/S0066-782X2004000500006. Di Mario C, Dudek D, Piscione F, Mielecki W, Savonitto S, Murena E, et al. Immediate angioplasty versus standard therapy with rescue angioplasty after thrombolysis in the Combined Abciximab REteplase Stent Study in Acute Myocardial Infarction (CARESS-in-AMI): an open, prospective, randomized, multicentre trial. Lancet. 2008;371(9612):55968, http://dx.doi.org/10.1016/S0140-6736(08)60268-8. Armstrong PW, Gershlick AH, Goldstein P, Wilcox R, Danays T, Lambert Y, et al. Fibrinolysis or primary PCI in ST-Segment elevation myocardial infarction. N Engl J Med. 2013;368(15):1379-87. Ito H, Maruyama A, Iwakura K, Takiuchi S, Masuyama T, Hori M, et al. Clinical Implications of the No ‘Reflow’ Phenomenon: A Predictor of Complications and Left Ventricular Remodeling in Reperfused Anterior Wall Myocardial Infarction. Circulation. 1996;93(2):223-8, http://dx.doi. org/10.1161/01.CIR.93.2.223. Henriques JP, Zijlstra L, van ?t Hof AW, de Boer MJ, Dambrink JH, Gosselink M, et al. Angiographic assessment of reperfusion in acute myocardial infarction by myocardial blush grade. Circulation. 2003;107(16):2115-9, http://dx.doi.org/10.1161/01.CIR.0000065221.0643 0.ED. Gibson CM, Murphy SA, Morrow DA, Aroesty JM, Gibbons RJ, Gourlay SG, et al. Angiographic perfusion score: an angiographic variable that integrates both epicardial and tissue level perfusion before and after facilitated percutaneous coronary intervention in acute myocardial infarction. Am Heart J. 2004;148(2):336-40, http://dx.doi.org/10.1016/j. ahj.2003.12.044. Henriques JP, Zijlstra F, van ’t Hof AW, de Boer MJ, Dambrink JH, Gosselink M, et al. Angiographic assessment of reperfusion in acute myocardial infarction by myocardial blush grade. Circulation. 2003;107(16):2115-9, http://dx.doi.org/10.1161/01.CIR.0000065221.064 30.ED. Costantini CO, Stone GW, Mehran R, Aymong E, Grines CL, Cox DA, et al. Frequency, correlates, and clinical implications of myocardial perfusion after primary angioplasty and stenting, with and without glycoprotein IIb/IIIa inhibition, in acute myocardial infarction. J Am Coll Cardiol. 2004;44(2):305-12, http://dx.doi.org/10.1016/j.jacc.2004.03.058. Di Nucci TP, Pimentel Filho WA, Correia MB, Abdalla Filho R, Bocchi EA, Custodio WB, et al. Correlac¸a˜o entre o Grau de Perfusa˜o Mioca´rdica e a Evoluc¸a˜o Clı´nica Tardia de Pacientes Submetidos a Trombo´lise e Implante de Stent. Rev Bras Cardiol Invas. 2006;14(1):56-62. Bellandi F, Leoncini M, Maioli M, Toso A, Gallopin M, Piero Dabizzi R. Markers of myocardial reperfusion as predictors of left ventricular function recovery in acute myocardial infarction treated with primary angioplasty. Clinical Cardiology. 2004;27(12):683-8, http://dx.doi.org/ 10.1002/clc.4960271205. Jolly SS, Yusuf S, Cairns J, Niemela K, Xavier D, Widimsky P, et al. Radial versus femoral access for coronary angiography and intervention in patients with acute coronary syndromes (RIVAL): a randomized, parallel group, multicentre trial. Lancet. 2011;377(9775):1409-20, http:// dx.doi.org/10.1016/S0140-6736(11)60404-2. Romagnoli E, Biondi-Zoccai G, Sciahbasi A, Politi L, Rigattieri S, Pendenza G, et al. Radial Versus Femoral Randomized Investigation in ST-Segment Elevation Acute Coronary Syndrome: The RIFLE-STEACS (Radial Versus Femoral Randomized Investigation in ST-Elevation Acute Coronary Syndrome) Study. J Am Coll Cardiol. 2012;60(24):2481-97, http://dx.doi.org/10.1016/j.jacc.2012.06.017.


CLINICAL SCIENCE

Posterior-only spinal fusion without rib head resection for treating type I neurofibromatosis with intracanal rib head dislocation Dong Sun,# Fei Dai,# Yao Yao Liu, Jian-Zhong Xu* Third Military Medical University, Orthopedic Department of Southwest Hospital, Chongqing, China.

OBJECTIVES: Patients with Type I neurofibromatosis scoliosis with intra-canal rib head protrusion are extremely rare. Current knowledge regarding the diagnosis and treatment for this situation are insufficient. The purpose of this study is to share our experience in the diagnosis and surgical treatments for such unique deformities. METHODS: Six patients with Type I neurofibromatosis scoliosis with rib head dislocation into the spinal canal were diagnosed at our institution. Posterior instrumentation and spinal fusion without intra-canal rib head resection via a posterior-only approach was performed for deformity correction and rib head extraction. The efficacy and outcomes of the surgery were evaluated by measurements before, immediately and 24 months after the surgery using the following parameters: coronal spinal Cobb angle, apex rotation and kyphosis of the spine and the intracanal rib head position. Post-operative complications, surgery time and blood loss were also evaluated. RESULTS: Patients were followed up for at least 24 months post-operatively. The three dimensional spinal deformity was significantly improved and the intra-canal rib head was significantly extracted from the canal immediately after the surgery. At follow-up 24 months after surgery, solid fusions were achieved along the fusion segments, and the deformity corrections and rib head positions were well maintained. There were no surgeryrelated complications any time after the surgery. CONCLUSIONS: Systematic examinations are needed to identify patients with Type I neurofibromatosis scoliosis with rib head dislocation into the canal who can be treated by posterior-only spinal fusion without rib head resection. KEYWORDS: Neurofibromatosis; Rib Head Protrusion; Posterior Spinal Fusion. Sun D, Dai F, Liu YY, Xu JZ. Posterior-only spinal fusion without rib head resection for treating type I neurofibromatosis with intra-canal rib head dislocation. Clinics. 2013;68(12):1521-1527. Received for publication on May 6, 2013; First review completed on May 20, 2013; Accepted for publication on June 20, 2013 E-mail: xjzslw@163.com Tel.: 011 86 23 65340297 *corresponding author # co-first authors

Neurofibromatosis type 1 (NF1), also known as von Recklinghausen disease, involves the spine in 26% to 50% of patients (1,2). The spinal deformity can be divided into two categories: dystrophic and non-dystrophic. Non-dystrophic forms mimic idiopathic scoliosis and are treated as such. Dystrophic deformities, on the other hand, are characterized by relentless curve progression with short segmental and sharply angulated curves, which are often accompanied by severe vertebral body wedging and neural foraminal enlargement (3–6). These dystrophic curves are usually corrected via

combined anterior and posterior fusion, which is also known as circumferential fusion (7–9). In some dystrophic cases, the deformities are so severe that the rib heads on the convex side of the curve penetrate into the spinal canal through the enlarged neural foramen. If unrecognized, the dislocated rib heads could lead to catastrophic injuries by impinging on the spinal cord after corrective procedures (6,10–14). Unfortunately, current knowledge about the diagnosis and surgical management of NF-1 scoliosis with intra-canal rib penetration is insufficient due to the rarity of the condition (6,10–15). Thus far, only 19 cases have been reported in the English literature (Table 1). The purpose of our study is to share our systematic experience with the diagnosis and surgical treatment of this rare and highly risky situation.

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.

& METHODS

& INTRODUCTION

Patient data

No potential conflict of interest was reported.

Following the approval of the Institutional Review Board (IRB) of Southwest Hospital, the records of six patients

DOI: 10.6061/clinics/2013(12)08

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Neurofibromatosis and rib head dislocation Sun D et al.

CLINICS 2013;68(12):1521-1527

Table 1 - Previous Reports of Spinal Canal Rib Penetration in Patients With Neurofibromatosis Type-1. Author

Patients (No.)

Age (y)

Abdulian et al. (10) Capella et al. (15)

1 1

14 15

Dacher et al. (6)

1

10

Deuchi et al. (11)

1

12

Flood et al. (5) Gkioka et al. (4)

1 1

15 13

Kamath et al. (12) Koshhal et al. (32)

1 1

13 16

Major et al. (33)

3

13

Mukhtar et al. (3)

1

5 11 10

Yalcin et al. (14)

3

Ton et al. (24)

4

14 12 6 14 11 11 9

Signs & symptoms

Radiology Exam

None CT No pre-operative CT, MRI symptoms; postoperative weakness and paraparesis Bilateral ankle clonus CT-M and daytime micturition Weakness of the lower CT-M extremities, difficulty walking with eventual paraparesis, hyperesthesia below waist, ankle clonus and knee/ ankle hyperreflexia Knee & ankle clonus CT-M CT,MRI Painful rib hump, gait difficulty, bilateral Bobinski, ankle clonus, right foot drop, decreased sensation/ hyperreflexia in lower limbs, and daytime micturition None CT No pre-operative symptoms; CT postoperative T5 paraparesis Transient loss of sensation CT-M below the waist and inability to move lower extremities after fall on rib hump None CT None CT-M Right leg weakness and CT-M radiculopathy down to toes when he rolled to his right side None CT None CT, MRI None Radiographs &CT Mild lower limb hyperreflexia CT, MRI and ankle clonus None CT,MRI None CT,MRI Right foot weakness, hyperreflexia CT,MRI and clonus

Treatment

Follow-up time

R,ASF,HT,PSF,SSI R,ASF,PSF,SSI

2 years 2 years

R,ASF,PSF

1 year

HT,VCR,R

2 years

R,ASF,PSF R,PSF,SSI

Not mentioned 6 months

ASF,PSF,SSI,R ASF,PSF,R

Not mentioned 5 months

ASF,PSF,SSI,R

Not mentioned

ASF,PSF,SSI,R PSF,R R,PSF

Not mentioned Not mentioned 2 years

R,PSF,SSI R,PSF,SSI PSF,SSI PSF,SSI,R

Not Not Not Not

mentioned mentioned mentioned mentioned

R,PSF,SSI R,ASF,PSF R,ASF,PSF

Not mentioned Not mentioned Not mentioned

ASF: anterior spinal fusion; CT-M: CT myelography; HT: halo traction; PSF: posterior spinal fusion; R: rib head resection; SSI: segmental spinal instrumentation; VCR: vertebral column resection.

(2 males, 4 females; 10–16 years) diagnosed with Neurofibromatosis- type I (NF-1) with rib head dislocation into the spinal canal at our institution between 2008 to 2011 were reviewed. The patient data are shown in Table 1.

represented by its absolute value and direction; the distance was recorded as positive if the intra-canal rib head tip passed the bisecting line of the body; otherwise, it was negative (Figure 1). Finally the correction rate of the coronal Cobb angle, kyphosis and the apex rotation and distance of the intra-canal rib head being retracted out of the canal were calculated. Magnetic resonance imaging (MRI) of the whole spine was performed on all patients to reveal any intra-spinal lesions and assess the relationship between the rib head and the dural sac.

Measurements The following parameters were measured pre- and postoperatively for correction assessment: the coronal and sagittal Cobb angles on plain films, the apex rotation and distance between the intra-canal rib head tip and the line bisecting the vertebral body by computed tomography (CT) scan. The coronal and sagittal deformities were measured by the Cobb method (16,17). The coronal Cobb angle was defined by the angles between the two end vertebrae (the last vertebra that tilted into the curve). Sagittal kyphosis was defined by the angles between the endplates of T2 and T12. The apex rotation was measured using the Aaro-Dahlborn method on a true axial slice (18), in which the axial slices were adjusted to parallel the orientation of the superior endplate. The distance between the intra-canal rib head tip and the line bisecting the vertebral body was measured on a true axial CT slice. It was

Reliability tests The measurements were performed by two orthopedic residents (D.S. &Y.Y.L.) on our PACS system and were confirmed by a senior spine specialist (F.D.). The parameters for each patient were measured five times by each observer, and reliability was determined by intra-observer and interobserver interclass coefficients.

Surgical Procedures The procedure was performed with multimodality spinal cord monitoring. The patient was in the prone position, and

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Figure 1 - True axial CT images of pre- (A) and 24 months post-operation (B) showed the method of recording intra-canal rib head position: Line ‘‘bo’’ is the bisecting line of the vertebral body; line ‘‘a’’ is the perpendicular line of ‘‘bo’’; line ‘‘L"(red line)is the distance between the tip of intra-canal rib head and the bisecting line of vertebral body. The comparison of A and B demonstrated definite intra-canal rib head dislocation before the surgery and significant extraction after the surgery, which was maintained for 24 months after the surgery.

a midline incision was made. After satisfactory exposure, pedicle screws were carefully placed bilaterally at all levels within the two neutral vertebrae except for the apex and its cranial- and caudal-adjacent levels. Afterwards, multi-level Smith-Peterson osteotomy was performed and followed by simultaneous rod rotation on the convex side and translation maneuver on the concave side. Spinal cord status was closely monitored by both transcranial electric motorevoked potential (MEP) and somatosensory-evoked potential (SEP). Alterations in which the MEP wave amplitude decreased more than 75% and SEP amplitude decreased more than 50% compared to baseline were diagnosed as positive changes. Any correction maneuvers were stopped or even reversed if any indication of neurological impairment was noted. In addition, intra-operative ‘‘wake-up’’ tests were performed at the end of the reduction procedure to confirm. The spinal fusions were accomplished in an extended fashion, which means that the one or two levels above and below the neutral vertebrae at each end were instrumented and grafted with autograft and allograft bones. A cell saver was used in all surgeries. After being discharged, the patients were required to wear a thoraciclumbar vest for at least four months to protect the fusion site.

with the ethical standards of the IRB of Southwest Hospital and with the Helsinki Declaration.

& RESULTS Pre-operative examination All six patients displayed multiple cafe au lait spots on the skin and were neurologically asymptomatic. Intra-canal rib head dislocation was detected by pre-operative CT and MRI examinations (Figure 2). There were no signs of cord compression or intra- or para-spinal neurofibromatosis on the pre-operative MRI.

Surgical outcomes All six NF-1 patients with rib head dislocation underwent posterior-only approach correction and instrumentation without rib head resection. The average blood loss was 1020¡80 ml, and the average surgery time was 238¡ 63 minutes. Three of the patients required an intra-operative transfusion, which averaged 667¡56 ml. No neurological impairment, intra-canal hematoma, dural tear or other form of complication was reported after surgery. The spinal deformity parameters, radiology and patients’ postures at pre-operation, immediately post-operation and 24 months post-operation are shown in Table 2, Figures 3 and 4. To evaluate the efficacy of the surgical correction, we compared the deformity parameters pre-operation and immediately after the operation (Table 2 and Figure 3A, B, D, E). The coronal Cobb angle, kyphosis and axial rotation were significantly reduced after the surgery in all patients. The average correction rate of the coronal Cobb angle was 47.4%, and that of kyphosis was 58.6%. The correction rate of apex rotation was 44.4%. These improvements corresponded to a change in the patients’ posture before and after the surgery (Figure 4A, B, D, E). In addition, the intra-spinal

Statistical analysis The measurements were analyzed with SPSS 13.0 (SPSS Inc, Chicago, IL). Paired t-tests were used to determine the difference of parameters. Significance was defined as a p,0.05 and reported as such.

Ethics This study was approved by the Institutional Review Board (IRB) of Southwest Hospital, Third Military Medical University. All procedures in the study were in accordance

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Figure 2 - Pre-operation radiology examination detected intra-canal rib head dislocation. A) Preoperative CT sagittal image showed the intra-canal portion of dislocated rib head (white arrow); B) 3D reconstruction image of the same patient showed rib head(R) dislocation into the canal; C) MRI demonstrated the intra-canal rib head(R) did not compress on the cord(C), and the boundary(white arrow) between the rib head and the cord was clear.

20 to 40 degrees and normal sagittal curvatures (kyphosis less than 50 degrees), which is known as an idiopathic scoliosis-like curve pattern; Type II curves are short, angulated dystrophic curves with angles of kyphosis more than 50 degrees. The latter type is accompanied by ribpenciling, vertebral scalloping, severe apical wedging and rotation, enlargement of the foramina, and para-vertebral tumors (1,2,8,19–21). Occasionally, the rib heads dislocate into the spinal canal and lead to subsequent neurological impairment (10,12,14,15). Due to the rarity of this particular condition, evidence-based diagnosis and surgical treatments on this deformity have been insufficient, which presents risks to patients with this condition (20,24). So far, only 12 studies with a total of 19 involved patients have been previously reported (Table 1). These included 11 males and 8 females with a mean age at presentation of 14.1 years (range: 5 to 16 years). Rib head dislocation occurred on the convex side of the curve, usually around its apex. The mean curvature was 68 degrees (range: 29 to 92 degrees). Eight patients showed preoperative neurologic signs or symptoms, whereas 11 did not. All patients underwent rib head resection, and 11 had combined or staged anterior and posterior spinal fusion and segmental spinal instrumentation. Eight patients underwent stabilization with only a posterior spinal fusion and segmental spinal instrumentation. Among all of the reported cases, only a few were followed up over a long term to evaluate the outcomes. The diagnosis of NF-1 dystrophic scoliosis can be made based on typical clinical features, such as multiple cafe au lait spots on the skin and a characteristic curve pattern on radiology (10,14,15,24). It is fairly important that clinicians

rib head tips were retracted away from canals by 5.18¡2.6 cm. These results suggested that our procedures significantly corrected the spinal deformity and the intracanal rib head dislocation. Because dystrophic NF-1 patients are more likely to develop pseudoarthrosis after posterior-only spinal fusion, all patients were followed up for at least 24 months. All of the deformity parameters at 24 months post-operation were compared with those immediately after surgery (Table 2 and Figure 3B, C, E, F), and there was no significant difference between the two groups. There was also no significant difference in the relative position of the rib heads being extracted from the canal. The maintenance of deformity corrections was also supported by the patients’ posture 24 months after surgery (Figure 4C, F). Solid fusion was confirmed on radiology at 24 months after surgery, and implant failure and screw pull-out (Figure 3 C, F) were not observed. These results indicated that solid spinal fusions were achieved and all of the corrections were wellmaintained 24 months after the surgery.

Reliability The repeated measurements demonstrated good interoperator reliability and intra-observer reliability (Table 3).

& DISCUSSION Multiple manifestations are associated with NF-1, and 26% to 50% of individuals with this disorder show spinal abnormalities. Two general curve patterns have been widely recognized: Type I curves are characterized by scoliosis of

Table 2 - Comparison of pre-and post-operative radiology measurements.

Pre-operation Post-operation 24 Months post-operation *

Cobb angle( ˚ )

Kyphosis( ˚ )

Apex Rotation( ˚ )

Distance of rib extraction(cm)

76.6¡20.2 40.6¡21.5* 42.1¡20.5**

58.8¡26.2 24.8¡4.3* 26.4¡6.8**

10.2¡2.5 5.1¡2.6* 5.6¡2.6**

-5.18¡2.6 5.23¡2.1**

p-value,0.05 when comparing the pre- and post-operative measurements. p-value.0.05 when comparing the 24 month post-operative measurements with those of immediately after operation.

**

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Figure 4 - Pre-operative, immediately post-operative and 24 month post-operative photos of patient postures. A and D) The preoperative anterior and posterior(AP) views and lateral view showed multiple cafe au lait spots on the patient’s skin and significant hyperkyphosis posture; B and E) The immediate postoperative AP and lateral view showed significant improvement of postures; C and F) 24 month post-operative AP and lateral view showed well-maintained body posture in both views; the incision healed well.

rib penetration can be missed pre-operatively even with CT scanning (10). The CT gantry was adjusted individually to parallel the superior endplate of the scoliotic vertebral body, as the intra-canal rib head can be easily under-recognized in routine CT examinations (24). In addition, whole spine MRI was also performed to demonstrate intra-canal and paraspinal soft tissue details. Rib head dislocations were detected on the axial CT slice and 3D reconstruction. All of the dislocated ribs were on the convex side of the curve and were from the levels at the peri-apical regions. Therefore, we suggest that slices from these regions should be carefully inspected. Furthermore, MRI scanning demonstrated the relationship between the cord and intra-canal rib heads, which was also important to clarify the intra-canal deformity and assist with surgical decision-making. It is generally accepted that if any neurological symptoms are present due to spinal cord compression from intra-canal rib head dislocation, it is necessary to resect the compressing part. In contrast, if the patient shows no neurologic symptoms, whether to resect the dislocated rib head in the canal remains controversial. As a result, current surgical approaches to this situation vary greatly among surgeons. Crawford (21,22) and Abdulian et al. (10) believed that the rib head would impale the spinal cord in the correction process; therefore, intra-spinal rib heads needed to be resected before the correction procedure. It was also reported that patients with dislocated rib heads were predisposed to spinal cord injury after a traumatic event (23). The only case without resection of the rib head was reported by Yalcin et al. (14), who claimed that the intracanal rib head had moved away from the spinal cord while

Figure 3 - Pre-operative, immediate post-operative and 24 month post-operative radiographs. The coronal Cobb angles (A-C) were defined by the angles between the two end vertebra(yellow lines), and sagittal kyphosis (D-F) was defined by the angles between T2 and T12(yellow lines). A and D)The preoperative coronal and sagittal images showed sharp and short segmental coronal deformity and significant sagittal hyperkyphosis in the thoracic region; B and E) The immediate post-operative coronal and sagittal images showed significant correction of the deformity in both planes; C and F) The 24 month post-operative coronal and sagittal images showed the well-maintained correction of deformity in both planes, solid fusion within the fused regions was achieved, and all the implants remained in position.

are able to identify this disease early. In contrast, the neurological symptoms of NF-1 with intra-spinal rib head dislocation are varied, ranging from mild sensory and motor deficits to paraplegia and paraparesis, with approximately 60% of documented cases being asymptomatic (12). Therefore, we believe intensive radiology examinations are most reliable for finding intra-canal dislocations. CT myelography may have added benefit in detecting intracanal dislocations but should be weighed against the risks by the clinician, as these risks include adverse reactions to the contrast, iatrogenic headache, nerve injury, or bleeding due to the spinal needle. In our study, we included several steps to aid with the diagnosis and surgical decisionmaking. First, thin slice (1 mm) CT and 3D reconstruction of the entire spine was performed before and after surgery to detect the dislocation pre-operatively and confirm rib head retraction post-operatively, as it has been reported that the

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Table 3 - Reliability tests of measured parameters. Coronal Cobb angle

ICC1 ICC2 1 2

Sagittal Cobb angle

Apex rotation

Distance of rib extraction (cm)

Pre-OP

Post-OP

Pre-OP

Post-OP

Pre-OP

Post-OP

0.91 0.82

0.93 0.85

0.91 0.83

0.88 0.83

0.90 0.81

0.94 0.82

0.91 0.83

Average intra-observer interclass coefficient of two observers Inter-observer interclass coefficient of two observers.

the apex was translated to the concavity. However, the reliability of this claim was questioned because it was based on a single case without any long-term follow up. In our study, we had six consecutive patients with at least 24 months of follow-up. We feel our study guides identification and care of patients with such a deformity. To quantify the extraction effect on the intra-canal rib head, we used the line bisecting the vertebral body on a true axial CT cut as the reference line and measured the distance between this line and the intra-canal rib head. As demonstrated in our postoperative results, the rib head had been extracted out of the canal significantly and remained extracted 24 months after surgery. The benefit of reduction without rib head resection was that we could avoid the risk of impaling the spinal cord or tearing the dural sac while achieving good reduction. Another innovation in the surgical management of our study was that we used a posterior-only approach for spinal fusion and segmental instrumentation. A high incidence of pseudarthrodesis has been reported in dystrophic NF-1 scoliosis, and circumferential spinal fusion via a combined anterior and posterior approach was recommended as the standard treatment (1). At least three groups have reported alarmingly high failure rates (63%–72%) with posterior-only fusion of dystrophic NF-1 scoliosis (8). However, those surgeries frequently involved forms of internal fixation now considered biomechanically inferior to pedicle screw-rod systems (25). An anterior and posterior combined approach necessitates more extensive surgery and therefore carries a higher risk of complications, including decreased respiratory function and excessive bleeding from the plexiform venous channels that are sometimes associated with neurofibromatosis. Several authors (8,18,26,27) have proposed and performed posterior vertebral body resection (pVCR) and circumferential spinal fusion in treating dystrophic scolio-hyperkyphosis. However, this procedure utilizes a challenging technique and should only be performed by a highly experienced surgical team (28). Therefore, the posterior-only approach with extended fusion levels in our study has the advantage of being less invasive and less technique demanding. In our study blood loss and surgery time was limited to approximately 1020 ml and 238 min with a posterior-only approach, which was relatively less invasive than the combined approach (9,2931). The corrections on three planes were also significant, as demonstrated by the results, and were well maintained at the two-year post-operative follow-up. These outcomes validated the efficacy and reliability of the posterior-only approach in treating NF-1 dystrophic scoliosis.

extensive studies should be conducted to better understand the natural history, diagnosis and management of this deformity. In addition, the parameters describing intracanal rib head dislocation still need further verification and improvement. Our study did, however, include more cases of this unique situation than have been previously reported in the English literature; we feel this study was strong enough to provide some primary guidelines in caring for NF-1 dystrophic scoliosis patients without any neurological symptoms. NF-1 dystrophic curves with rib head dislocation into the canal can be treated by extended segmental instrumentation and fusion without rib head resection via a posterior-only approach. Appropriate surgical candidates should be carefully selected prior to any procedure; inclusive preoperative evaluation and sufficient intra-operative exposure and monitoring are necessary to improve surgical outcomes.

& AUTHOR CONTRIBUTIONS Sun D and Dai F contributed equally to the study and are considered cofirst authors. Sun D drafted the manuscript. Dai F and Xu JZ performed surgery and commented on the manuscript. Sun D and Liu YY collected and analyzed the data.

& REFERENCES 1. Tsirikos AI, Saifuddin A, Noordeen MH. Spinal deformity in neurofibromatosis type-1: diagnosis and treatment. Eur Spine J. 2005;14(5):42739, http://dx.doi.org/10.1007/s00586-004-0829-7. 2. Akbarnia BA, Gabriel KR, Beckman E, Chalk D. Prevalence of scoliosis in neurofibromatosis. Spine. 1992;17(8 Suppl):S244-8, http://dx.doi.org/10. 1097/00007632-199208001-00005. 3. Mukhtar IA, Letts M, Kontio K. Spinal cord impingement by a displaced rib in scoliosis due to neurofibromatosis. Can J Surg. 2005;48(5):414-5. 4. Gkiokas A, Hadzimichalis S, Vasiliadis E, Katsalouli M, Kannas G. Painful rib hump: a new clinical sign for detecting intraspinal rib displacement in scoliosis due to neurofibromatosis. Scoliosis. 2006;1:10, http://dx.doi.org/10.1186/1748-7161-1-10. 5. Flood BM, Butt WP, Dickson RA. Rib penetration of the intervertebral foraminae in neurofibromatosis. Spine. 1986;11(2):172-4, http://dx.doi. org/10.1097/00007632-198603000-00016. 6. Dacher JN, Zakine S, Monroc M, Eurin D, Lechevallier J, Le Dosseur P. Rib displacement threatening the spinal cord in a scoliotic child with neurofibromatosis. Pediatric radiology. 1995;25(1):58-9, http://dx.doi. org/10.1007/BF02020851. 7. Winter RB, Lonstein JE, Anderson M. Neurofibromatosis hyperkyphosis: a review of 33 patients with kyphosis of 80 degrees or greater. Journal of spinal disorders. 1988;1(1):39-49. 8. Kim HW, Weinstein SL. Spine update. The management of scoliosis in neurofibromatosis. Spine. 1997;22(23):2770-6. 9. Singh K, Samartzis D, An HS. Neurofibromatosis type I with severe dystrophic kyphoscoliosis and its operative management via a simultaneous anterior-posterior approach: a case report and review of the literature. Spine J. 2005;(4):461-6, http://dx.doi.org/10.1016/j.spinee. 2004.09.015. 10. Abdulian MH, Liu RW, Son-Hing JP, Thompson GH, Armstrong DG. Double rib penetration of the spinal canal in a patient with neurofibromatosis. Journal of pediatric orthopedics. 2011;31(1):6-10, http://dx. doi.org/10.1097/BPO.0b013e3182032029. 11. Deguchi M, Kawakami N, Saito H, Arao K, Mimatsu K, Iwata H. Paraparesis after rib penetration of the spinal canal in neurofibromatous scoliosis. Journal of spinal disorders. 1995;8(5):363-7.

Limitations and future directions Our study is not without limitations. Due to the rarity of NF-1 dystrophic scoliosis with rib head dislocation, more

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12. Kamath SV, Kleinman PK, Ragland RL, Tenreiro-Picon OR, Knorr JR, Davidson RI, et al. Intraspinal dislocation of the rib in neurofibromatosis: a case report. Pediatric radiology. 1995;25(7):538-9, http://dx.doi.org/10. 1007/BF02015788. 13. Legrand B, Filipe G, Blamoutier A, Khouri N, Mary P. Intraspinal rib penetration in four patients in neurofibromatosis vertebral deformities. Revue de chirurgie orthopedique et reparatrice de l’appareil moteur. 2003;89(1):57-61. 14. Yalcin N, Bar-on E, Yazici M. Impingement of spinal cord by dislocated rib in dystrophic scoliosis secondary to neurofibromatosis type 1: radiological signs and management strategies. Spine. 2008;33(23):E8816, http://dx.doi.org/10.1097/BRS.0b013e318184efad. 15. Cappella M, Bettini N, Dema E, Girardo M, Cervellati S. Late postoperative paraparesis after rib penetration of the spinal canal in a patient with neurofibromatous scoliosis. J Orthop Traumatol. 2008;9(3):163-6, http://dx.doi.org/10.1007/s10195-008-0010-x. 16. Mac-Thiong JM, Pinel-Giroux FM, de Guise JA, Labelle H. Comparison between constrained and non-constrained Cobb techniques for the assessment of thoracic kyphosis and lumbar lordosis. Eur Spine J. 2007;16(9):1325-31, http://dx.doi.org/10.1007/s00586-007-0314-1. 17. Sangole AP, Aubin CE, Labelle H, Stokes IA, Lenke LG, Jackson R, et al. Three-dimensional classification of thoracic scoliotic curves. Spine. 2009;34(1):91-9, http://dx.doi.org/10.1097/BRS.0b013e3181877bbb. 18. Lam GC, Hill DL, Le LH, Raso JV, Lou EH. Vertebral rotation measurement: a summary and comparison of common radiographic and CT methods. Scoliosis. 2008;3:16, http://dx.doi.org/10.1186/17487161-3-16. 19. Shen JX, Qiu GX, Wang YP, Zhao Y, Ye QB, Wu ZK. Surgical treatment of scoliosis caused by neurofibromatosis type 1. Chin Med Sci J. 2005;20(2):88-92. 20. Crawford AH, Herrera-Soto J. Scoliosis associated with neurofibromatosis. Orthop Clin North Am. 2007;38(4):553-62, vii, http://dx.doi.org/ 10.1016/j.ocl.2007.03.008. 21. Crawford AH, Parikh S, Schorry EK, Von Stein D. The immature spine in type-1 neurofibromatosis. J Bone Joint Surg Am. 2007;89 Suppl 1:123-42, http://dx.doi.org/10.2106/JBJS.F.00836. 22. Crawford AH, Schorry EK. Neurofibromatosis in children: the role of the orthopaedist. J Am Acad Orthop Surg. 1999;7(4):217-30. 23. Asazuma T, Hashimoto T, Masuoka K, Fujikawa K, Yamagishi M. Acute thoracic myelopathy after a traumatic episode in a patient with neurofibromatosis associated with sharply angular scoliosis: a case

24. 25.

26.

27.

28.

29.

30.

31. 32. 33.

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report. J Orthop Sci. 2003;8(5):721-4, http://dx.doi.org/10.1007/s00776003-0690-2. Ton J, Stein-Wexler R, Yen P, Gupta M. Rib head protrusion into the central canal in type 1 neurofibromatosis. Pediatr Radiol. 2010;40(12):1902-9, http://dx.doi.org/10.1007/s00247-010-1789-1. Kim YJ, Lenke LG, Cho SK, Bridwell KH, Sides B, Blanke K. Comparative analysis of pedicle screw versus hook instrumentation in posterior spinal fusion of adolescent idiopathic scoliosis. Spine. 2004;29(18):2040-8, http://dx.doi.org/10.1097/01.brs.0000138268.12324.1a. Lenke LG, O’Leary PT, Bridwell KH, Sides BA, Koester LA, Blanke KM. Posterior vertebral column resection for severe pediatric deformity: minimum two-year follow-up of thirty-five consecutive patients. Spine. 2009;34(20):2213-21, http://dx.doi.org/10.1097/BRS.0b013e3181b53cba. Li M, Fang X, Li Y, Ni J, Gu S, Zhu X. Successful use of posterior instrumented spinal fusion alone for scoliosis in 19 patients with neurofibromatosis type-1 followed up for at least 25 months. Arch Orthop Trauma Surg. 2009;129(7):915-21, http://dx.doi.org/10.1007/ s00402-008-0696-5. Stoker GE, Lenke LG, Dorward IG. Posterior vertebral column resection for the treatment of dystrophic kyphosis associated with type-1 neurofibromatosis: a case report and review of the literature. Spine. 2012.37(26):E1659-64, http://dx.doi.org/10.1097/BRS.0b013e318 2770aa2. Dobbs MB, Lenke LG, Kim YJ, Luhmann SJ, Bridwell KH. Anterior/ posterior spinal instrumentation versus posterior instrumentation alone for the treatment of adolescent idiopathic scoliotic curves more than 90 degrees. Spine. 2006;31(20):2386-91, http://dx.doi.org/10.1097/01.brs. 0000238965.81013.c5. Kim YJ, Lenke LG, Bridwell KH, Cheh G, Sides B, Whorton J. Prospective pulmonary function comparison of anterior spinal fusion in adolescent idiopathic scoliosis: thoracotomy versus thoracoabdominal approach. Spine. 2008;33(10):1055-60, http://dx.doi.org/10.1097/BRS.0b013e31816 fc3a5. Shamji MF, Isaacs RE. Anterior-only approaches to scoliosis. Neurosurgery. 2008;63(3 Suppl):139-148. Khoshhal KI, Ellis RD. Paraparesis after posterior spinal fusion in neurofibromatosis secondary to rib displacement: case report and literature review. J Pediatr Orthop. 2000;20(6):799-801. Major MR, Huizenga BA. Spinal cord compression by displaced ribs in neurofibromatosis. A report of three cases. J Bone Joint Surg Am. 1988;70(7):1100-2.


BASIC RESEARCH

The putative role of ovary removal and progesterone when considering the effect of formaldehyde exposure on lung inflammation induced by ovalbumin Adriana Lino-dos-Santos-Franco,I,II Renata Midori Amemiya,I Ana Paula Ligeiro de Oliveira,III Amı´lcar Sabino Damazo,V Ana Cristina Breithaupt-Faloppa,IV Luana Beatriz Vitoretti,I Beatriz Golega˜ Acceturi,I Wothan Tavares-de-LimaI I Universidade de Sa˜o Paulo, Institute of Biomedical Sciences, Department of Pharmacology, Sa˜o Paulo/SP, Brazil. II Universidade de Sa˜o Paulo, Faculty of Pharmaceutical Sciences, Department of Clinical and Toxicological Analyses, Sa˜o Paulo/SP, Brazil. III Nove de Julho University, Department of Biophotonics, Sa˜o Paulo/SP, Brazil. IV Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil. V Universidade de Cuiaba´ (UNIC), Faculty of Medical Sciences, Department of Basic Science in Health, Cuiaba/MT, Brazil.

OBJECTIVE: Formaldehyde exposure during the menstrual cycle is known to affect the course of allergic lung inflammation. Because our previous data demonstrated that formaldehyde combined with an ovariectomy reduced allergic lung inflammation, we investigated the putative role of ovary removal and progesterone treatment when considering the effect of formaldehyde on allergic lung inflammation. METHOD: Ovariectomized rats and their matched controls were exposed to formaldehyde (1%, 3 days, 90 min/ day) or vehicle, and immediately after exposure, the rats were sensitized to ovalbumin by a subcutaneous route. After 1 week, the rats received a booster by the same route, and after an additional week, the rats were challenged with ovalbumin (1%) by an aerosol route. The leukocyte numbers, interleukin-10 (IL-10) release, myeloperoxidase activity, vascular permeability, ex vivo tracheal reactivity to methacholine and mast cell degranulation were determined 24 h later. RESULTS: Our results showed that previous exposure to formaldehyde in allergic rats decreased lung cell recruitment, tracheal reactivity, myeloperoxidase activity, vascular permeability and mast cell degranulation while increasing IL-10 levels. Ovariectomy only caused an additional reduction in tracheal reactivity without changing the other parameters studied. Progesterone treatment reversed the effects of formaldehyde exposure on ex vivo tracheal reactivity, cell influx into the lungs and mast cell degranulation. CONCLUSION: In conclusion, our study revealed that formaldehyde and ovariectomy downregulated allergic lung inflammation by IL-10 release and mast cell degranulation. Progesterone treatment increased eosinophil recruitment and mast cell degranulation, which in turn may be responsible for tracheal hyperreactivity and allergic lung inflammation. KEYWORDS: Formaldehyde Exposure; Progesterone; Lung inflammation; Tracheal reactivity; Mast cells; Interleukin-10. Lino-dos-Santos-Franco A, Amemiya RM, Oliveira AP, Damazo AS, Breithaupt-Faloppa AC, Vitoretti LB, et al. The putative role of ovary removal and progesterone when considering the effect of formaldehyde exposure on lung inflammation induced by ovalbumin. Clinics. 2013;68(12):1528-1536. Received for publication on May 10, 2013; First review completed on May 30, 2013; Accepted for publication on June 5, 2013 E-mail: adrilino@usp.br Tel.: 55 11 3091-2197

laboratories (1). FA is also emitted in tobacco smoke, burning fuel, urea-FA foam insulation, cosmetics, solvents and domestic disinfectants (2). FA exposure causes irritation of the eyes and mucous membranes and induces airway inflammation. Many people are exposed to FA, and its role as a risk factor in the development of asthma is still controversial. In previous studies, we reported that both male and female rats developed lung inflammation when exposed to FA inhalation (3,4). Interestingly, ovary removal caused a decrease in the inflammatory response induced by FA exposure (4). Clinical and experimental data have both demonstrated a putative role of female sex hormones (FSHs) in lung

& INTRODUCTION Formaldehyde (FA) is a pollutant that is widely employed in many industries and in anatomy, pathology and histology

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(12)09

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Formaldehyde exposure and ovary removal Lino-dos-Santos-Franco A et al.

inflammation (4–9). Progesterone has been suggested to mediate eosinophilia, airway hyperreactivity and interleukin (IL)-5 production in murine models of allergic lung inflammation (10). Moreover, progesterone skews the differentiation of naı¨ve T helper lymphocytes toward the Th2 lineage in vitro (11). Studies have shown that estradiol and progesterone both exert pro- and anti-inflammatory effects on lung inflammation, depending on the nature of the inflammatory agent (allergic or non-allergic) (4,7,12,13). In this regard, our group has demonstrated that ovariectomy (OVx) reduces lung inflammation when an inflammatory agent is related to ovalbumin (OVA), the allergic stimulus. Moreover, this effect is reversed by estradiol but not by progesterone (5,7). Conversely, pre-treatment with estradiol or progesterone reestablished the inflammatory response when the stimulus was related to FA exposure (a non-allergic stimulus) (4). In other studies, we also demonstrated that pre-exposure to FA in OVA-sensitized and OVA-challenged male rats blunted the allergic lung response (14). Therefore, both FA exposure and ovary removal suppressed the development of an allergic lung response induced by OVA. Considering these earlier results, we investigated the role of ovary removal when considering the effect of FA on lung inflammation induced by OVA. Moreover, because we observed in earlier studies that progesterone had proinflammatory effects in rats submitted to FA inhalation and that its effects were worse than those observed in rats treated with estradiol, we decided to investigate the involvement of progesterone in leukocyte migration into the lung, ex vivo tracheal reactivity to methacholine (MCh), IL-10 release in the lung tissue and mast cell degranulation.

This concentration and duration of FA exposure were found to cause a neutrophilic lung inflammation (14).

Experimental design The rats in the study were randomly assigned to 5 groups: 1) FA/OVA Sham OVx, 2) FA/OVA OVx, 3) OVA/OVA Sham OVx, 4) OVA/OVA OVx and 5) FA/OVA OVx + P. Immediately after the last session of FA or vehicle inhalation (day 10), the OVx or Sham-operated rats were sensitized with 10 mg of OVA and 10 mg of aluminum hydroxide by a subcutaneous route. Seven days after the first OVA sensitization (day 17), the rats subcutaneously received a second sensitization (booster) with OVA. At day 24, a challenge was performed by OVA (1%) inhalation for 15 min. In parallel, the OVx rats were treated with progesterone before each FA inhalation and were sensitized with OVA (day 10), boosted 1 week later (day 17) and challenged with OVA 7 days later (day 24), as described above. Rats from all of the study groups were euthanized by exsanguination of the abdominal aorta under deep anesthesia (choral hydrate, .400 mg/kg ip) 1 day after the OVA challenge. The rats were submitted to FA inhalation 7 days after ovary removal because we had previously demonstrated that the levels of female sex hormones in the serum and the weight of the uterus were both significantly reduced at this time (Figure 1) (4,5,7).

Evaluation of leukocyte recruitment into the lungs Bronchoalveolar lavage (BAL) was performed according to De Lima et al. (15). Polyethylene tubing (1 mm inner diameter) was inserted into the trachea, and the alveolar space was washed by flushing with PBS (20 ml total volume). The recovered BAL fluid was centrifuged (170 x g for 10 min at 20 ˚C), and the resulting cell pellet was resuspended in 1 ml of PBS. Cell suspensions (90 ml) were stained with 10 ml of crystal violet (0.2%), and the total cell number was determined in Neubauer chambers. Neutrophils, eosinophils and mononuclear cells were quantified by morphological criteria after cytospin processing and staining with May-Gru¨nwald-Giemsa.

& MATERIALS AND METHODS Animals Female Wistar rats (160–180 g) were obtained from the Institute of Biomedical Sciences, University of Sa˜o Paulo. The rats were housed in groups (5 rats per cage) in a lightand temperature-controlled room (12/12-h light-dark cycle, 21¡2 ˚C) with free access to food and water. The experiments were approved by the Institutional Animal Care Committee.

Analysis of IgE antibodies IgE antibodies were detected by the Passive Cutaneous Anaphylaxis (PCA) reaction (Mota and Wong, 1969). The skin of non-manipulated rats (recipients) was sensitized with an intradermal injection (100 ml/site) of serially diluted (1:2 up to 1:256) sera from FA/OVA and OVA/OVA rats. After 24 h, the recipient rats received (by an intravenous route) a solution containing 500 mg of OVA plus 2.5 mg of Evans blue (EB) dye, dissolved in NaCl (0.9%). After 30 min, the rats were killed, and the skin was removed. The diameter of the dye stain was measured on the inner surface of the skin. The PCA titers were represented by the highest dilution of the serum that resulted in a dye stain of .5 mm in diameter.

Ovariectomy (OVx) Female rats were anesthetized with intraperitoneal ketamine-xylazine (Konig, Sa˜o Paulo, Brazil) (100 and 20 mg/kg, respectively), and their ovaries were removed. Following the surgery, the rats received a single dose of PentantibioticH (Fort Dodge, IA) (570 mg/kg, intramuscular). Vaginal smears and uterine weight measurements were used to assess the effectiveness of the OVx. Rats that underwent a similar operation, but without ovary removal, were used as the sham-operated controls (Sham-OVx group). A non-manipulated group of rats (Naı¨ve group) was used to obtain the basal parameters.

Exposure to formaldehyde (FA) Lung myeloperoxidase (MPO) activity

Rats (5/chamber) were exposed to daily 90-min sessions of FA or vehicle (water + methanol) inhalation for 3 consecutive days (14). For this procedure, a standard glass chamber (20 l) coupled to an ultrasonic nebulizer device (IcelH, Brazil) was used to generate a constant airstream in an aqueous solution of formalin diluted to 1% FA by weight.

The lungs were removed after perfusion through the pulmonary artery with phosphate-buffered saline (PBS). To normalize the MPO activity among the different groups, the lung tissues were homogenized with 3 ml/g PBS containing 0.5% hexadecyltrimethylammonium bromide and 5 mM

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Figure 1 - Protocol of study. The rats were ovariectomized or not and after 7 days submitted or not to FA inhalation. Subsequently the animals were sensitized and challenged with OVA.

EDTA and were centrifuged at 37,000 x g for 15 min. Samples of the tissue homogenates (10 ml) were incubated for 15 min with H2O2 and o-dianisidine (Sigma, St Louis, MO). The reaction was stopped by the addition of 1% NaN3. The absorbance was determined at 460 nm using a microplate reader (Bio-Tek Instruments, Winooski, VT).

CO2). After the equilibrium period (40 min), the tracheal tension was adjusted to 1 g and the tissue viabilities were assessed by replacing the KH solution with KCl buffer. Subsequently, cumulative dose-response curves to methacholine (MCh) were constructed according to Van Rossum (1963).

Lung microvascular leakage

Determination of IL-10 in lung explants According to the methods of Proust et al. (17), the IL-10 levels were determined in samples of the supernatant from lung explants in culture. To remove the intravascular blood, the lungs were flushed through the right heart with 5 ml of PBS. Next, the remaining parenchyma was chopped randomly into 4 small pieces per well distributed in 24well plates and cultured in 1 ml of Dulbecco-modified Eagle’s medium (DMEM) for 4 h at 37 ˚C with 5% CO2 and 0.5% penicillin-streptomycin (10,000 UI-10 mg/ml). The results were expressed as pg of interleukin produced per mg of lung tissue dry weight. The IL-10 levels were obtained using standard curves via ELISA kits purchased from R&D Systems (Minneapolis, MN).

Lung vascular permeability was assessed using the EB dye extravasation procedure. Briefly, immediately after the OVA challenge, the EB dye was injected (20 mg/kg, iv), and the rats were killed 15 min later. The lungs were perfused through the pulmonary artery with PBS, pH 7.0 containing 5 IU/ml heparin. At this time, a fragment of the lung parenchyma was removed, weighed and incubated overnight in formamide (4 ml/g wet weight) at room temperature. The concentration of EB dye extracted by the formamide was determined by spectrophotometry at 620 nm (Bio-Tek instruments) using a standard curve of EB in formamide (0.3–100 mg/ml). The extravasate dye was expressed as mg/g of dry tissue weight.

Evaluation of mast cell degranulation

Determination of ex vivo tracheal reactivity to methacholine (MCh)

To investigate the role of progesterone and ovary removal on mast cell degranulation, lung fragments were removed from rats and fixed in paraformaldehyde (4%) containing 0.1 M So¨rensen phosphate buffer (SPB), pH 7.4, at 4 ˚C for 2 h. Next, the fragments were washed with SPB, dehydrated through a graded ethanol series and finally embedded in ParaplastH (Sigma, USA). Sections (3-mm thick) were stained with 1% toluidine blue in a 1% borax solution. The lung mast cells were quantified using 10 serial histological

Tracheal rings were mounted for the measurement of isometric force using 2 steel hooks in a 15-ml organ bath (16). The force contraction was registered using a force displacement transducer and a chart recorder (PowerlabH, Labchart, AD Instruments). The tracheal rings were suspended in an organ bath filled with Krebs-Henseleit (KH) buffer at 37 ˚C and continuously aerated (95% O2 and 5%

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sections for every animal (n = 3) (30-mm space between 2 consecutive sections). The analyses were performed by 2 blinded observers using a Zeiss Axioskop II mot plus a high-power objective (x40) and the AxiovisionH software (Carl Zeiss, Germany).

Pharmacological studies Four hours before each FA inhalation session, the groups of OVx rats were subjected to a subcutaneous injection of water-soluble progesterone (200 mg/kg). The controls consisted of OVx rats injected with corresponding volumes of the progesterone vehicle (distilled water). The levels of progesterone in the serum were significantly increased compared with the levels in non-treated OVx rats and did not differ from those in the naı¨ve rats (5).

Statistical analysis The data are expressed as the mean ¡ SEM, and comparisons among the experimental groups were analyzed by one-way ANOVA followed by the Student-NewmanKeuls test for multiple comparisons using GraphPad software V.2.01, GraphPad InstatTM (1990–1993). Statistically significant differences were considered for p-values less than 0.05.

& RESULTS The role of OVx and progesterone when considering the effect of formaldehyde inhalation on leukocyte migration into the lung Figure 2 (panel A) shows that previous FA inhalation in allergic rats (FA/OVA) with an intact ovary (Sham-OVx) caused a significant decrease in the number of leukocytes recovered in the BAL fluid compared with the allergic group (OVA/OVA). Similarly, an OVx in allergic rats (OVA/OVA) caused a significant decrease in the number of leukocytes detected in the BAL fluid compared with the allergic rats with an intact ovary (Sham-OVx OVA/OVA group). However, an OVx in the FA/OVA group did not cause an additional reduction in the number of leukocytes in the BAL relative to the number detected in the FA/OVA group with an intact ovary (Sham-OVx FA/OVA). We also observed that all of the groups in this study produced an increased number of leukocytes in the BAL fluid compared with the naı¨ve group. As shown in panel B, relative to the naı¨ve rats, a significant increase in the number of mononuclear cells, neutrophils and eosinophils is observed in the Sham-OVx OVA/OVA rats. In contrast, an OVx significantly decreased the number of mononuclear cells, neutrophils and eosinophils in allergic rats (OVx OVA/OVA) compared with the animals with an intact ovary. Additionally, previous FA inhalation in allergic rats with an intact ovary (FA/OVA group) reduced the number of mononuclear cells, neutrophils and eosinophils in the BAL compared with the OVA/ OVA group with an intact ovary. An OVx in animals from the FA/OVA group did not cause an additional reduction in the number of mononuclear cells, neutrophils and eosinophils compared with the FA/OVA group with an intact ovary. Interestingly, the OVA/OVA group without an ovary and the FA/OVA group with or without an ovary all presented a leukocyte profile in the BAL that was similar to the one observed in naı¨ve rats.

Figure 2 - The effect of ovariectomy (OVx) and progesterone treatment (P) when considering the results of formaldehyde inhalation on the number of cells recruited in the bronchoalveolar lavage (BAL). Seven days after OVx or Sham-operation (Sham-OVx), the rats were subjected or not to FA inhalation. Subsequently, the rats were sensitized and challenged with OVA. In a parallel study, 7 days after the OVx, the rats were treated with progesterone. Lung inflammation was assessed by quantification of the total number of cells (A) and the total number of differential cells (B and C) present in the BAL 24 h after the OVA challenge. Basal parameters were obtained from non-manipulated rats (naı¨ve). The data are the mean ¡ SEM of 6 animals per group. *p,0.05 relative to the naı¨ve group; hp,0.05 relative to the OVA/OVA Sham-OVx group; dp,0.05 relative to the FA/OVA OVx group. (ANOVA followed by the Student-Newman-Keuls test).

In Panel C, we observe that the treatment of OVx allergic rats with progesterone (P) before FA inhalation increased the number of neutrophils and eosinophils recruited into the

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lung compared with the untreated rats. However, this treatment decreased the number of mononuclear cells relative to that in untreated FA/OVA OVx rats.

The influence of OVx on the effect of formaldehyde on MPO activity and lung microvascular leakage in allergic rats Figure 3 (Panel A) shows that both OVx and previous FA exposure reduced MPO activity in the OVA/OVA group compared with the Sham-OVx group. In contrast, ovary removal did not affect MPO activity in the FA/OVA group. We also observed that all of the groups in this study showed increased MPO activity compared with the naı¨ve group. As shown in panel B, OVx did not interfere with EB extravasation in the OVA/OVA rats, whereas previous FA inhalation reduced EB extravasation compared with the OVA/OVA rats with an intact ovary. In addition, we observed that ovary removal in the FA/OVA group did not alter EB extravasation compared with the animals with an intact ovary.

Figure 4 - The effect of ovariectomy (OVx) and FA exposure on the synthesis of anti-OVA IgE. Seven days after the OVx or ShamOVx, the rats were submitted or not to FA inhalation and then sensitized and challenged with OVA. The IgE titers were determined by passive cutaneous anaphylaxis. The data are the mean ¡ SEM of 5 animals. (ANOVA).

The effect of OVx and FA inhalation on the synthesis of IgE antibodies Figure 4 shows that the titers of anaphylactic antibodies were not modified by OVx or FA inhalation.

The effect of OVx and progesterone on the IL-10 levels in lung explants Figure 5 (Panel A) shows increased IL-10 levels in the supernatant of lung tissue from the OVA/OVA OVx, FA/ OVA Sham-OVx and OVx rats compared with the naı¨ve group. We also observed that the OVx increased the levels of IL-10 in the OVA/OVA group compared with the ShamOVx group. In addition, we did not observe a difference in the IL-10 levels between the FA/OVA Sham-OVx and OVx groups. As shown in panel B, progesterone treatment of the FA/ OVA OVx group did not alter the levels of IL-10 in the lung tissue supernatant compared with the Sham-OVx and OVx untreated rats. Panel C shows that both FA inhalation and OVx per se presented IL-10 levels similar to those obtained in the naı¨ve group.

The role of OVx and progesterone treatment (P) on the effect of formaldehyde exposure on tracheal reactivity in allergic rats As shown in Figure 6 (Panels A and B), previous FA inhalation in animals of the OVA/OVA group with or without an ovary prevented tracheal hyperreactivity to MCh. Similarly, ovary removal in animals from the OVA/OVA and FA/OVA groups reduced tracheal reactivity to MCh (Panels C and D). Panel E shows that treatment with progesterone in FA/ OVA OVx rats caused a tracheal hyperreactivity to MCh compared with the untreated group. We also observed that progesterone per se did not alter tracheal reactivity to MCh.

Figure 3 - The effect of ovariectomy (OVx) when considering the results of FA exposure on myeloperoxidase activity (MPO) and vascular permeability in the lung tissue. Seven days after the OVx or Sham-OVx, the rats were submitted or not to FA inhalation and then sensitized and challenged with OVA. Either 24 h (A) or immediately after the OVA challenge, the MPO activity and vascular permeability were measured. The data are the mean ¡ SEM of 5 animals. *p,0.05 relative to the naı¨ve group; hp,0.05 relative to the OVA/OVA Sham-OVx group; vp,0.05 relative to the OVA/OVA OVx group. (ANOVA followed by the StudentNewman-Keuls test).

The effects of OVx and progesterone on mast cell degranulation in lung tissue As show in Table 1, the histological analysis indicated that in the OVA/OVA Sham-OVx group, the mast cells had

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percentage of mast cell degranulation compared with the OVA/OVA Sham-OVx group. Finally, we observed that treatment with progesterone increased mast cell degranulation compared with the FA/ OVA Sham-OVx and OVx groups.

& DISCUSSION In this study, we investigated the putative role of ovary removal (OVx) and progesterone when considering the effect of FA exposure on lung inflammation induced by OVA. The justification for this study arose from the observation that OVx and FA inhalation reduced leukocyte migration into the lung following an allergic challenge (5,7,14). Here, we confirmed that OVx reduces allergic lung inflammation and added information about the influence of OVx on the effect of FA exposure during an allergic response in the lung. Our data showed that previous FA exposure in allergic female rats reduced cellular recruitment to the lung, MPO activity and lung microvascular permeability and that ovary removal did not modify these parameters. However, we recently observed that OVx prevented lung inflammation induced by FA inhalation in non-allergic rats and that this association increased tracheal reactivity and mast cell degranulation (4). However, in the present work, OVx did not modify the inflammatory parameters in allergic rats previously exposed to FA, and the female sex hormones (FSHs) appeared to play an important role in the ability of FA to induce a lung inflammatory response (4). Because OVx and FA inhalation reduced leukocyte migration into the lung and MPO activity in allergic rats, we hypothesized that FA and the lack of FSHs may act on the same target. Considering that FA inhalation and OVx were performed before the OVA sensitization, we decided to investigate whether FA or OVx could reduce the synthesis of anti-OVA IgE because the OVA response is dependent on IgE synthesis. Our results showed that FA and OVx did not modify IgE synthesis. Therefore, we can infer that the effects of FA and OVx do not correlate with an interference in the induction of an allergic response because IgE synthesis was not modified. In the evaluation of other common pathways involved in the response to both FA and OVx, mast cells have emerged as an important factor because these cells are involved in the actions of FA and OVA in lung tissue (3–5,14). Our results showed that, in fact, FA or OVx reduced mast cell degranulation in allergic rats. Therefore, we can infer that both FA inhalation and the lack of FSHs modulate mast cell degranulation and that the reduced allergic lung inflammation observed in these groups can be attributed to a lower percentage of mast cell degranulation. In addition, we observed that no differences were found between the FA/ OVA OVx and FA/OVA Sham-OVx groups because in the first group, mast cell degranulation was elicited by OVx, but in the second group, mast cell degranulation was triggered by FA exposure. IL-10 has been well established to be an important antiinflammatory interleukin. Increased levels of IL-10 may suppress eosinophil activity (18). Moreover, IL-10 can also modulate allergic lung inflammation through the activation of adhesion molecule expression (19). In the present work, we observed elevated levels of IL-10 in the FA/OVA ShamOVx and OVx groups, a phenomenon that coexisted with a

Figure 5 - The effect of FA inhalation and OVx on IL-10 release in the lung explants. Seven days after the OVx or Sham-OVx, the rats were submitted or not to FA inhalation and then sensitized and challenged with OVA. In a parallel study, the rats 7 days after the OVx were treated with progesterone (P). IL-10 quantification was performed on lung tissue. The data are the mean ¡ SEM of 5 animals. *p,0.05 relative to the naı¨ve group; h p,0.05 relative to the OVA/OVA Sham-OVx group (ANOVA followed by the Student-Newman-Keuls test).

a high percentage of degranulation compared with the naı¨ve group. In contrast, ovary removal in the OVA/OVA rats and FA/OVA rats resulted in a reduction in mast cell degranulation compared with their counterpart animals with an intact ovary. Similarly, previous FA exposure in animals of the OVA/OVA Sham-OVx group reduced the

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Figure 6 - The effects of OVx and FA exposure on tracheal reactivity in allergic rats. Seven days after the OVx or Sham-OVx, the rats were submitted or not to FA inhalation and then sensitized and challenged with OVA. In parallel, the rats 7 days after the OVx were treated with progesterone 4 h before each inhalation of FA. The dose response curve was constructed by the administration of a cholinergic agonist (methacholine). The data are expressed as the mean ¡ SEM of 6 animals. *p,0.05 relative to the naı¨ve group; hp, 0.05 relative to the OVA/OVA Sham-OVx group; dp,0.05 relative to the OVA/OVA OVx group; %p,0.05 relative to the FA/OVA ShamOVx group; vp,0.05 relative to the FA/OVA OVx group. (ANOVA followed by the Student-Newman-Keuls test).

decrease in the number of cells recruited in the BAL, reduced MPO activity and EB extravasation. Similarly, the OVA/OVA OVx group presented increased levels of IL-10 in the lung tissue supernatant. Therefore, we suggest that FA and OVx downregulated the allergic lung inflammation, at least in part, by an IL-10 release mechanism that was mediated in the lung tissue. These results were confirmed in naı¨ve rats that were exposed to FA or submitted to OVx and did not demonstrate increased IL-10 levels in lung explants. Therefore, our data suggest that the effects of FA and OVx during an allergic response are blunted by IL-10 release.

Although we did not quantify the expression of adhesion molecules in the present study, elevated levels of IL-10 can modulate the expression of adhesion molecules, thereby contributing to the suppressed allergic lung response. In another study, we demonstrated that FA inhalation in female rats reduced the expression of ICAM-1 and Mac-1 in granulocytes (4). In the present study, we decided to investigate the impacts of progesterone because it causes increased IL-4 levels and airway hyperresponsiveness (10,20,21). In addition, progesterone exacerbates the airway inflammation

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tracheal hyperresponsiveness (4), whereas in the present study, OVx caused tracheal hyporresponsiveness in allergic rats submitted to FA inhalation. This discrepancy can be explained by a difference between the protocols in each study. In previous studies, the analyses were performed 24 h after the last exposure to FA, but in the present study, the analysis was performed 24 h after the OVA challenge (14 days after FA inhalation). It is important to mention that the relationship among FA, OVx and OVA is the main reason for the results observed in this study. In conclusion, our study revealed that FA and OVx downregulated the allergic lung inflammation through IL10 release and mast cell degranulation mediated in the lung tissue. We observed that reduced allergic lung inflammation induced by FA was not affected by a lack of FSHs but that tracheal hyperresponsiveness was influenced by a lack of FSHs. We also demonstrated that progesterone increased mast cell degranulation, eosinophil recruitment and tracheal responsiveness. Overall, our data have made it possible to understand the role of pollutants on asthma deterioration in women undergoing progesterone therapy.

Table 1 - Mast cell degranulation in samples from the lungs of allergic rats, either ovariectomized (OVx) or sham-operated (Sham-OVx), that were subjected or not to formaldehyde (FA) inhalation (1%, 90 min daily, 3 days). A naı¨ve group was used as the control. In a parallel set, the rats were treated with progesterone before the FA inhalation. The data are the mean ¡ SEM of 5 rats per group. Mast cell number/mm2 Groups Naive OVA/OVA Sham-OVx OVA/OVA OVx FA/OVA Sham-OVx FA/OVA OVx FA/OVA OVx + P

Intact

Degranulated

%

2.15¡0.03 0.36¡0.06* 1.40¡0.07*# 1.62¡0.04*# 1.60¡0.07*h 1.10¡0.03*hc

0.04¡0.04 1.72¡0.07* 0.79¡0.06*# 0.12¡0.03*# 0.30¡0.07*h 1.10¡.0.20*hc

0.0 82.5* 36.1*# 26.5*# 17.2*h 50.0*hc

*

p,0.05 vs. Naı¨ve group; #p,0.05 vs. OVA/OVA Sham-OVx group; h p,0.05 vs. FA/OVA Sham-OVx group; c p,0.05 vs. FA/OVA OVx group.

and remodeling induced by environmental tobacco smoke (22). In previous studies, we showed that the administration of progesterone did not affect the tracheal hyperresponsiveness of OVx rats after exposure to FA inhalation. In contrast, progesterone was effective in increasing leukocyte recruitment into the lung of OVx rats exposed to FA inhalation (4). In the present study, using an allergic model of lung inflammation, we found a similar impact of progesterone on the effect of FA on cellular recruitment, but we detected an opposite effect on tracheal responsiveness. Treatment with progesterone before each FA inhalation session did not modify the number of total leukocytes recovered in the BAL. However, an increased number of eosinophils and neutrophils as well as an elevated percentage of degranulated mast cells were found in animals treated with progesterone. Interestingly, the levels of IL-10 were not modified by progesterone treatment. Our data agree with the results of Hellings et al. (10), which demonstrated that progesterone exogenously induces eosinophilic airway inflammation and increases bronchial reactivity by enhancing systemic IL-5 production. Eosinophils have been known to mediate allergic airway hyperreactivity (23,24). It is well established that the mediators released by eosinophils contribute to airway hyperresponsiveness, and progesterone has been demonstrated to play a role in the degranulation process of eosinophils (22,25). Moreover, the induction of IL-5 by progesterone appears to be a pivotal event by which progesterone mediates the recruitment of eosinophils into the airways (10). Taking these data into account, we might infer that progesterone increased mast cell degranulation and eosinophil recruitment into the lung and that these cells, once activated, could mediate tracheal hyperresponsiveness. Moreover, our data demonstrated that tracheal hyperresponsiveness in the OVA/OVA Sham-OVx group was partially reverted when the rats were submitted to FA inhalation and that OVx caused an additional reduction in the tracheal responsiveness of FA/OVA rats. Data from previous studies showed opposite effects of OVx compared with the results of the present study. OVx in non-allergic rats exposed to FA inhalation determined

& ACKNOWLEDGMENTS This study was sponsored by the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP no. 09/51886-3) and the Conselho Nacional de Pesquisa (CNPq). Adriana Lino dos Santos Franco is a scholarship holder from the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP no. 2011/51711-9), and Renata Midori Amemiya is a scholarship holder from the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP no. 2008/58108-3). Wothan Tavares de Lima is a research fellow from CNPq.

& AUTHOR CONTRIBUTIONS Lino-dos-Santos-Franco A performed the airway reactivity study and wrote the manuscript. Amemiya RM and Vitoretti L performed the OVx and cellular analyses. Acceturi BG performed the OVx. Oliveira AP quantified the IL-10 cytokine results. Breithaupt-Faloppa AC evaluated the MPO activity and helped write the manuscript. Damazo AS performed the mast cell analysis. Lima WT revised the manuscript.

& REFERENCES 1. Flo´-Neyret C, Lorenzi-Filho G, Macchione M, Garcia MLB, Saldiva PHN. Effects of formaldehyde on the frog’s mucociliary epithelium as a surrogate to evaluate air pollution effects on the respiratory epithelium. Braz J Med Biol Res. 2001;34(5):639-43, http://dx.doi.org/10.1590/ S0100-879X2001000500012. 2. Carlson RM, Smith MC, Nedorost ST. Diagnosis and treatment of dermatitis due to formaldehyde resins in clothing. Dermatitis. 2004;15(4):169-75, http://dx.doi.org/10.2310/6620.2004.04021. 3. Lino dos Santos Franco A, Damazo AS, Beraldo de Souza HR, Domingos HV, Oliveira-Filho RM, Oliani SM, et al. Pulmonary neutrophil recruitment and bronchial reactivity in formaldehyde-exposed rats are modulated by mast cells and differentially by neuropeptides and nitric oxide. Toxicol Appl Pharmacol. 2006;214(1):35-42, http://dx.doi.org/10. 1016/j.taap.2005.11.014. 4. Lino-dos-Santos-Franco A, Amemiya RM, Ligeiro de Oliveira AP, Breithaupt-Faloppa AC, Damazo AS, Oliveira-Filho RM, et al. Differential effects of female sex hormones on cellular recruitment and tracheal reactivity after formaldehyde exposure. Toxicol Lett. 2011;205(3):327-35, http://dx.doi.org/10.1016/j.toxlet.2011.06.023. 5. De Oliveira AP, Domingos HV, Cavriani G, Damazo AS, Dos Santos Franco AL, Oliani SM, et al. Cellular recruitment and cytokine generation in a rat model of allergic lung inflammation are differentially modulated by progesterone and estradiol. Am J Physiol Cell Physiol. 2007; 293(3):120-8. 6. Stanford KI, Mickleborough TD, Ray S, Lindley MR, Koceja DM, Stager JM. Influence of menstrual cycle phase on pulmonary function in asthmatic athletes. Eur J Appl Physiol. 2006;96(6):703-10. 7. Ligeiro de Oliveira AP, Oliveira-Filho RM, Da Silva ZL, Borelli P, Tavares De Lima W. Regulation of allergic lung inflammation in rats: interaction

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8.

9.

10.

11.

12.

13.

14.

15.

16.

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17. Proust B, Nahori MA, Ruffie C, Lefort J, Vargaftig BB. Persistence of bronchopulmonary hyper-reactivity and eosinophilic lung inflammation after anti-IL-5 or -IL-13 treatment in allergic BALB/c and IL-4Ralpha knockout mice. Clin Exp Allergy. 2003;33(1):119-31, http://dx.doi.org/ 10.1046/j.1365-2222.2003.01560.x. 18. Fu CL, Ye YL, Lee Y, Chiang BL. Effects of overexpression of IL-10, IL-12, TGF-beta and IL-4 on allergen induced change in bronchial responsiveness. Respir Res. 2006;7(1):72, http://dx.doi.org/10.1186/1465-9921-772. 19. Kelly M, Hwang JM, Kubes P. Modulating leukocyte recruitment in inflammation. J Allergy Clin Immunol. 2007;120(1):3-10, http://dx.doi. org/10.1016/j.jaci.2007.05.017. 20. Mitchell VL, Gershwin LJ. Progesterone and environmental tobacco smoke act synergistically to exacerbate the development of allergic asthma in a mouse model. Clin Exp Allergy. 2007;37(2):276-86, http:// dx.doi.org/10.1111/j.1365-2222.2007.02658.x. 21. Tan K, McFarlane L, Lipworth B. Modulation of airwayr eactivity and peak flow variability in asthmatics receiving the oral contraceptive pill. Am J Respir Crit Care Med. 1997;155(4):1273-7, http://dx.doi.org/10. 1164/ajrccm.155.4.9105066. 22. Mitchell VL, Van Winkle LS, Gershwin LJ. Environmental tobacco smoke and progesterone alter lung inflammation and mucous metaplasia in a mousee model of allergic airway disease. Clin Rev. Allergy Immunol. 2012;43(1-2):57-68, http://dx.doi.org/10.1007/s12016-011-8280-0. 23. Hogan SP, Mould A, Kikutani H, Ramsay AJ, Foster PS. Aeroallergeninduced eosinophilic inflammation, lung damage, and airways hyperreactivity in mice can occur independently of IL-4 and allergen-specific immunoglobulins. J Clin Invest. 1997;99(6):1329-39, http://dx.doi.org/ 10.1172/JCI119292. 24. Balzar S, Strand M, Rhodes D, Wenzel SE. IgE expression pattern in lung: relation to systemic IgE and asthma phenotypes. J Allergy Clin Immunol. 2007;119(4):855-62, http://dx.doi.org/10.1016/j.jaci.2006.12.642. 25. Hamano N, Terada N, Maesako K, Numata T, Konno A. Effect of sex hormones on eosinophilic inflammation in nasal mucosa. Allergy Asthma. 1998;19(5):263-9, http://dx.doi.org/10.2500/108854198778557773.

between estradiol and corticosterone. Neuroimmunomodulation. 2004;11(1):20-7, http://dx.doi.org/10.1159/000072965. Haggerty CL, Ness RB, Kelsey S, Waterer GW. The impact of estrogen and progesterone on asthma. Ann Allergy Asthma Immunol. 2003;90(3):284-91, http://dx.doi.org/10.1016/S1081-1206(10)61794-2. Skobellof EM, Spivey WH, Silverman R, Eskin BA, Harchelroad F, Alessi TV. The effect of the menstrual cycle on asthma presentations in the emergency department. Arch Intern Med. 1996;156(16):1837-40. Hellings PW, Vandekerckhove P, Claeys R, Billen J, Kasran A, Ceuppens JL. Progesterone increases airway eosinophilia and hyper-responsiveness in a murine model of allergic asthma. Clin Exp Allergy. 2003;33(10):1457-63, http://dx.doi.org/10.1046/j.1365-2222.2003.01743.x. Miyaura H and Iwata M. Direct and indirect inhibition of Th1 development by progesterone and glucocorticoids. J Immunol. 2002; 168(3):1087-94. De Oliveira AP, Peron JP, Damazo AS, Franco AL, Domingos HV, Oliani SM, et al. Female sex hormones mediate the allergic lung reaction by regulating the release of inflammatory mediators and the expression of lung E-selectin in rats. Respir Res. 2010;24:11-115. Speyer CL, Rancilio NJ, McClintock SD, Crawford JD, Gao H, Sarma JV, et al. Regulatory effects of estrogen on acute lung inflammation in mice. Am J Physiol Cell Physiol. 2005;288(4):881-90. Lino-dos-Santos-Franco A, Domingos HV, Damazo AS, BreithauptFaloppa A, Ligeiro de Oliveira AP, Costa SKP, et al. Reduced allergic lung inflammation in rats following formaldehyde exposure: long-term effects on multiple effector systems. Toxicology. 2009;256(3):157-63, http://dx.doi.org/10.1016/j.tox.2008.11.011. De Lima WT, Sirois P, Jancar S. Immune-complex alveolitis in the rat: evidence for platelet activating factor and leukotrienes as mediators of the vascular lesions. Eur J Pharmacol. 1992;213(1):63-70. de Lima WT, da Silva ZL. Contractile responses of proximal and distal trachea segments isolated from rats subjected to immunological stimulation: role of connective tissue mast cells. Gen Pharmacol. 1998;30(5):689-95, http://dx.doi.org/10.1016/S0306-3623(97)00377-7.

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Gender differences in microcirculation: Observation using the hamster cheek pouch Diogo Guarnieri Panazzolo,I,II Lucia Henriques Alves da Silva,I,II Fa´tima Zely Garcia de Almeida Cyrino,I Fernando Lencastre Sicuro,I Luiz Guilherme Kraemer-Aguiar,I,III Eliete BouskelaI I

State University of Rio de Janeiro, Biomedical Center, Clinical and Experimental Research Laboratory on Vascular Biology (BioVasc), Rio de Janeiro/RJ, Brazil. II State University of Rio de Janeiro, Graduate Program on Clinical and Experimental Physiopathology (FisClinex), Rio de Janeiro/RJ, Brazil. III State University of Rio de Janeiro, Medical Sciences Faculty, Department of Internal Medicine, Endocrinology, Rio de Janeiro/RJ, Brazil.

OBJECTIVES: Estrogen has been shown to play an important protective role in non-reproductive systems, such as the cardiovascular system. Our aim was to observe gender differences in vivo with regard to the increase in macromolecular permeability and leukocyte-endothelium interaction induced by ischemia/reperfusion as well as in microvascular reactivity to vasoactive substances using the hamster cheek pouch preparation. METHODS: Thirty-six male and 36 female hamsters, 21 weeks old, were selected for this study, and their cheek pouches were prepared for intravital microscopy. An increase in the macromolecular permeability of postcapillary venules was quantified as a leakage of intravenously injected fluorescein-labeled dextran, and the leukocyte-endothelium interaction was measured as the number of fluorescent rolling leukocytes or leukocytes adherent to the venular wall, labeled with rhodamin G, during reperfusion after 30 min of local ischemia. For microvascular reactivity, the mean internal diameter of arterioles was evaluated after the topical application of different concentrations of two vasoconstrictors, phenylephrine (a1-agonist) and endothelin-1, and two vasodilators, acetylcholine (endothelial-dependent) and sodium nitroprusside (endothelial-independent). RESULTS: The increase in macromolecular permeability induced by ischemia/reperfusion was significantly lower in females compared with males [19 (17–22) leaks/cm2 vs. 124 (123–128) leaks/cm2, respectively, p,0.001), but the number of rolling or adherent leukocytes was not different between the groups. Phenylephrine-induced arteriolar constriction was significantly lower in females compared with males [77 (73–102)% vs. 64 (55–69)%, p,0.04], but there were no detectable differences in endothelin-1-dependent vasoreactivity. Additionally, arteriolar vasodilatation elicited by acetylcholine or sodium nitroprusside did not differ between the groups. CONCLUSION: The female gender could have a direct protective role in microvascular reactivity and the increase in macromolecular permeability induced by ischemia/reperfusion. KEYWORDS: Gender Differences; Microcirculation; Hamster Cheek Pouch; Microvascular Reactivity; Macromolecular Permeability. Panazzolo DG, Silva LH, Cyrino FZ, Sicuro FL, Kraemer-Aguiar LG, Bouskela E. Gender differences in microcirculation: Observation using the hamster cheek pouch. Clinics. 2013;68(12):1537-1542. Received for publication on April 11, 2013; First review completed on May 2, 2013; Accepted for publication on June 15, 2013 E-mail: guarnipana@yahoo.com.br Tel.: 55 21 2334-0703

age (1,2). The reasons for these differences are subject to debate, but it is conceivable that the absolute levels of estradiol and the different degrees of estrogen receptor regulation are the two major determinant factors. However, genetic differences may also exert effects independently of gonadal function (3). Estrogen has been shown to play an important protective role in non-reproductive systems such as the cardiovascular system. Estrogen causes vasodilation through non-genomic actions, resulting in rapid increases in nitric oxide (NO) production as well as through genomic actions by inducing transcription of NO synthase. These effects on NO bioavailability provide estrogen with the ability to improve arterial wall responsiveness following vascular injury and to inhibit the development of atherosclerosis by promoting re-endothelization, inhibition of smooth muscle cell proliferation, and

& INTRODUCTION The incidence of cardiovascular disease differs significantly between men and women during their fertile period. Epidemiological studies have revealed that atherosclerosis, hypertension, and peripheral vascular and coronary artery diseases occur with greater prevalence in men and postmenopausal women compared with women at a fertile

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(12)10

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matrix deposition (4). Estrogen may also decrease systemic vascular resistance, improve coronary and peripheral endothelial function, and prevent coronary artery spasm in women with and without coronary atherosclerosis (5). The endothelium effects on the modulation of vasorelaxation are thought to also occur through an endothelium-derived hyperpolarizing factor by inducing vasodilator prostanoids and by inhibiting endothelin-1 production (6,7). Additionally, endothelium action could modulate myogenic vascular responses, resulting in the reduction of microvessel basal tone (8). The majority of these effects have been attributed to estrogen acting on two ligand-activated transcription factors, ERa and ERb, and one G-protein coupled receptor (GPR30 or GPER) expressed on vascular endothelial and smooth muscle cells (4). The purpose of this study was to further explore the previous findings detailing the cardiovascular protective effects of endogenous estrogens and to focus on gender specificities in the in vivo microcirculation.

Cheek pouch preparation Cheek pouch preparations were dissected according to the protocol of Duling (11), as modified by Bouskela and Grampp (12), and mounted in an experimental chamber, where they were continuously superfused at a rate of 4 ml/min by a HEPES-supported HCO23 buffered saline solution (composition in mM: NaCl 110.0, KCl 4.7, CaCl2 2.0, MgSO4 1.2, NaHCO3 18.0, HEPES 15.39, and HEPES Na+-salt 14.61) bubbled with 5% CO2/95% N2 to keep the pH of the superfusate at 7.40. Throughout the experiments, the same gas mixture was also gently blown over the experimental chamber to keep the pO2 level at 12–15 mmHg, monitored by an O2 electrode in the superfusion solution, as previously described (12). The temperature of the solution was kept at 36.0¡0.5 ˚C with a circulating bath (model 8005, Polyscience, Niles, Illinois, USA) and (Leitz optical magnification 4006, Wetzlar, Hessen, Germany) coupled to a closed-circuit TV system and allowed to rest for 30 min. The cheek pouch preparation is stable with respect to microvessel diameter, spontaneous arteriolar vasomotion, arteriolar blood flow, and functional capillary density for at least 4 h of microscopic observation (12).

& METHODS Experiments were performed on 36 male and 36 female hamsters (Mesocricetus auratus), which were divided into six animals/group. Group I was used to evaluate macromolecular permeability, group II was used to evaluate leukocyteendothelium interactions, and the subsequent groups were used to assess microvascular reactivity as follows: group III, phenylephrine (a1-agonist with vasoconstrictor properties); group IV, endothelin-1 (potent endothelial-derived vasoconstrictor); group V, acetylcholine (endothelial-dependent vasodilator); and group VI, sodium nitroprusside (endothelial-independent vasodilator). All animals were at a fertile age and were matched for age, without any attempt to classify females according to their menstrual cycle. Experiments were performed using animals at 21 weeks of age, according to the Guide for Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication No. 85-23, revised in 1996), and the protocol was approved by the Ethical Committee of the State University of Rio de Janeiro. All animals received the same appropriate isocaloric laboratory diet (Nuvital from Nuvilab, Curitiba, PR, Brazil). On the day of the experiment, anesthesia was induced with 0.1-0.2 ml of sodium pentobarbital (Pentobarbital sodique, Sanofi, Paris, France, 60 mg/ml), which was administered intraperitoneally and maintained with a-chloralose [1,2-O(2,2,2-trichlorethyliden) a-D-glucofuranose, Merck, Darmstadt, Germany] at a dose of 150 mg/kg body weight/h, administered through a femoral vein catheter. The femoral artery was also cannulated to enable pressure measurements. Throughout and following surgery, the temperature of the animals was kept at 37.5 ˚C with a heating pad controlled by a rectal thermistor (LTB 750 Thermostat System, Uppsala process data AB, Sweden). A tracheal tube was inserted to facilitate spontaneous breathing. Conceptually, the hamster cheek pouch has both skeletal muscle and cutaneous microcirculatory beds, and in this study, experiments were performed with the cutaneous tissue to facilitate the comparison with data available in the literature (9,10). This part of the pouch is highly vascularized, and all classes of microcirculatory vessels can usually be visualized in the microscopic field with good clarity, thereby allowing for the comparison of effects in different microvascular segments.

Ischemia/reperfusion of the cheek pouch Local ischemia of the cheek pouch was obtained by a cuff made of a thin latex tubing that was mounted around the neck of the everted pouch where it exits the mouth of the hamster; this procedure was previously validated in our laboratory (9,10). The placement of the cuff can be made without any visible interference with local blood flow. The intratubular pressure can be rapidly increased by air compression using a syringe and also rapidly decreased at evacuation. An intratubular pressure of 200-220 mmHg in the cuff resulted in a complete arrest of microvascular blood flow, which eventually returned to a level similar to that observed before occlusion. In these experiments, the duration of the ischemia was 30 min.

Increase in macromolecular permeability after ischemia/reperfusion To quantify the increase in macromolecular permeability, 30 min after completion of the preparative procedure, fluorescein isothiocyanate (FITC)-dextran, with a substitution of two FITC molecules per 1,000 glucose molecules in the polysaccharide chain, was administered at a dose of 25 mg/ 100 g as an intravenous injection of a 5% solution in 0.9% saline. The sites of leakage were defined as fluorescent spots with a diameter of approximately 40 mm found near postcapillary venules. Extravasated material is continuously washed away by the superfusion solution, and consequently, the fluorescent spots gradually decrease (13). Before the ischemic period, hamsters in which the prepared area showed spontaneous nonfading leaks or more than 10 fading leaks were discarded. The number of leaks was counted 10 min after the ischemic period because it was shown that the maximum number of leaks is observed at this time (9,14).

Leukocytes rolling or sticking after ischemia/ reperfusion Three venular segments with a diameter of approximately 40 mm each were selected for observation. Leukocytes were labeled in vivo with rhodamine G (10 mg/100 g body mass) immediately before observation, and images were recorded

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95.2¡1.2 vs. 95.0¡0.8 mmHg (NS). All six study groups were compared according to gender, and the only group that presented any difference in baseline parameters was group V (acetylcholine), in which females had a higher body mass than males (187.7¡10.4 vs. 145.2¡7.2 g; p,0.01). The increase in macromolecular permeability induced by ischemia/reperfusion was significantly lower in females than in males [19 (17–22) vs. 124 (123–128) leaks/cm2; p,0.001; Figure 1], while no gender difference was noted in the number of rolling (p = 0.386) or adherent (p = 0.732) leukocytes (Figure 2). Before the addition of any drug, the baseline arteriolar diameters were similar between the groups. Males and females showed, respectively, the following initial arteriolar diameters: phenylephrine group, 34.6 (27.0–55.1) vs. 30.2 (25.8–36.7) mm (NS); endothelin-1 group, 79.9 (52.7–104.7) vs. 79.80 (59.0–103.5) mm (NS); acetylcholine group, 14.4 (8.7– 19.0) vs. 13.5 (11.8–20.1) mm (NS); and sodium nitroprusside group, 14.5 (11.4–22.3) vs. 13.8 (9.7–21.6) mm (NS). Of note, we observed that arteriolar phenylephrineinduced vasoconstriction was significantly lower in females than in males at 10-5 M [77 (73–102) vs. 64 (55–69)%, p#0.04], while there were no significant differences at other concentrations [1029 M (p = 0.39), 1027M (p = 0.31); Figure 3]. In contrast, arteriolar vasoconstriction due to endothelin-1 revealed no gender differences at all concentrations tested [1029 M (p = 0.24), 1027 M (p = 0.94), 1025 M (p = 0.70); Figure 3]. Arteriolar vasodilation elicited by acetylcholine was not significantly different between males and females at all concentrations tested [1028 M (p = 0.82), 1026 M (p = 0.82), 1024 M (p = 0.70); Figure 4], similar to the results associated with sodium nitroprusside [1028 M (p = 0.82), 1026 M (p = 0.70), 1024 M (p = 0.82); Figure 4].

on VHS tapes for later analysis. Rolling was defined as the occurrence of contact between the leukocyte and the venular wall, i.e., when the speed of the leukocyte in the bloodstream was lower than that observed for red blood cells. A leukocyte was considered as adherent when it did not show any visible movement for at least 30 s (15).

Measurements of arteriolar reactivity For the evaluation of microvascular reactivity, a preparation was considered suitable for experimentation if, under control conditions, there was an indication of good vascular tone (which, in 15 separate control tests, implied that the arteriolar diameter could be increased by 56¡3% through topical application of papaverine, 10 mg/ml), a brisk blood flow in all parts of the vascular bed including the larger veins (where individual erythrocytes should not be discernible in the blood stream images), and no tendency for leukocytes to adhere to the venular wall. Phenylephrine, endothelin-1, acetylcholine, and sodium nitroprusside (Sigma Chemicals, St. Louis, MO, USA) were freshly prepared for each experiment and applied topically, added to the superfusion solution, to the cheek pouch with a syringe infusion pump 22 (model 25-2222, Harvard Apparatus, MA, USA) to avoid systemic effects, such as changes in blood pressure, which could result in biased results. For consecutive measurements, three arterioles were selected in each preparation, taking into account the potential for returning them to exactly the same site (based on, e.g., the presence of fat cells and bifurcations). Experiments were performed by taking 3-min videotape recordings of selected microvessels under initial control conditions (before the addition of any drug) and 10–15 min after each experimental intervention. The cumulative concentrations of phenylephrine and endothelin-1 used were 1029, 1027, and 1025 M, in order to create a dose-response curve, while the concentrations of acetylcholine and sodium nitroprusside used were 1028, 1026, and 1024 M. Diameter measurements were performed using an Image Shearing Device (model 907, Bela Vista, CA, USA).

& DISCUSSION Ischemia of the cheek pouch for 30 min followed by reperfusion is able to induce leukocyte adhesion and increase plasma extravasations in post-capillary venules (16). In this study, we observed a significant decrease in microvascular permeability to macromolecules in females compared with males without a concomitant reduction in the number of rolling or adherent leukocytes. Endothelial cells (ECs) form a barrier between blood and tissue environments and control the movement of blood and immune cells, plasma fluid, small molecules, and proteins between the vascular compartment and the extracellular space. In continuous, non-fenestrated endothelium, the

Statistical analysis StatSoft Statistica 8.0 software was used for statistical analysis, and variables were tested regarding the possible problems associated with data distribution (i.e., normality, kurtosis, skewness, and homoscedasticity). Results are shown as medians and quartile ranges, with the exception of weight and blood pressure, which are shown as the means and standard deviations. Increases in macromolecular permeability, number of adherent or rolling leukocytes, and arteriolar diameters were analyzed by gender using the pairwise non-parametric Mann-Whitney U test, and non-parametric analysis of variance using the KruskalWallis and Dunn’s tests for post hoc comparisons was performed for comparisons of different concentrations and times. The significance level (a) used in all tests was 0.05. Arteriolar diameter values were transformed to percentages relative to the initial diameter measurements taken as baseline values, i.e., before the addition of any drug to each arteriole associated with each preparation.

& RESULTS Seventy-two animals (21 weeks old), 36 males and 36 females, with a mean body mass and blood pressure, respectively, of 124.7¡24.2 vs. 141.1¡39.5 g (NS) and

Figure 1 - An increase in macromolecular permeability induced by ischemia/reperfusion.

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Figure 2 - The number of rolling/adherent leukocytes induced by ischemia/reperfusion.

routes of passage are (a) transcellular through the EC membranes and cytoplasm, (b) transcellular via vesicles that traverse the endothelium, and (c) paracellular at junctions between ECs (17). The paracellular pathway is the most understood route, where junctional complexes are composed of tight (TJ), adherens (AJ), and gap junctions that create a physiological intercellular barrier. It has become increasingly clear that the changes/shifts in the function and regulation of these respective TJ and AJ proteins contribute to paracellular regulation and are important for vascular integrity and function (18,19). Additionally, the role of estrogens in these structures is now under investigation. Mitochondrial oxidative stress caused by reactive oxygen species (ROS) generation after ischemia/reperfusion injury has been shown to regulate the mitochondrial release of cytochrome C into the cytosol, leading to the activation of caspase-3, which cleaves the endothelial cell AJ protein bcatenin (20). The cleavage of b-catenin is thought to be a key regulator of cell窶田ell adhesion and therefore the loss of this important AJ protein results in barrier dysfunction and hyperpermeability. 17b-estradiol has been shown to inhibit important components of the intrinsic apoptotic signaling pathway, causing increased mitochondrial (ROS) formation, decreased mitochondrial transmembrane potential, mitochondrial release of cytochrome C, and activation of caspase-3 (21). In humans, another mechanism contributing to the antioxidant effect of estrogens was observed in young women recruited for in vitro fertilization, where increased estrogen levels led to enhanced manganese superoxide dismutase (SOD) and extracellular (EC)-SOD expression in

circulating monocytes (22). It should also be noted that a previous study demonstrated that the administration of SOD isoforms, specifically EC-SOD and copper-zinc SOD, was able to reduce the post-ischemic permeability increase in the hamster cheek pouch, demonstrating that reperfusion injury could be related to the formation of ROS (23). Although we have not been able to prove the mechanisms involved in estrogen protection, our results suggest that the decreased microvascular permeability observed in females could be dependent on an up-regulation of transmembrane proteins, leading to reinforcement of the structural integrity of TJs and AJs, as well as inhibition of all important components of the intrinsic apoptotic signaling pathway. However, this hypothesis remains to be validated. The control of reactivity and tonus strongly depends on endothelial and smooth muscle cells and their interactions. These cells express adrenergic receptors (a, e, b, and their subtypes) in variable amounts, depending on the vascular bed. Sympathetic neurons release catecholamines, and their effects depend on the type of receptor activated; for example, stimulation of a1 receptors (e.g., by phenylephrine) promotes vasoconstriction (24). Animal and human models have shown that there are gender differences in adrenergic vasomotor responses, although the results are conflicting. Some studies did not show significant differences in vasoreactivity after adrenergic stimuli between genders, while other studies demonstrated that the female vasculature is less responsive to norepinephrineand phenylephrine-induced vasoconstriction (25-27). It is possible that these conflicting results could be explained by different concentrations of circulating sex hormones.

Figure 3 - Arteriolar vasoconstriction induced by phenylephrine and endothelin-1 according to dose.

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Figure 4 - Arteriolar vasodilation induced by acetylcholine and sodium nitroprusside according to dose.

Sex hormone receptors are present in many vascular beds and act through genomic and non-genomic mechanisms. Among these receptors, it appears that the estrogen receptor has the most prominent action due to its potent endothelialdependent vasodilatation effect and vasoconstriction resistance (28). A comparison of castrated and non-castrated male rats demonstrated that there was no difference in vascular contraction following castration; however, when comparing intact and ovarectomized female rats, the latter demonstrated increased vasoconstriction in response to stimuli (8). The present study showed that, in both genders, there was significant, dose-dependent vasoconstriction induced by phenylephrine but that this effect was less pronounced in females, thereby supporting previous findings (25,26). Contrary to previous studies (29-31), we did not detect any gender differences with regard to secondary vascular responses to acetylcholine, sodium nitroprusside, and endothelin-1. These findings could be due to the heterogeneity observed in the microcirculation in vivo, where several physiological mechanisms are involved simultaneously and may reveal different results. Additionally, there are controversial studies concerning the action of endothelin-1, with some researchers observing reduced vasoconstriction in males (30) and other researchers observing it in females (32). In our study, the vasoconstriction elicited by endothelin-1 was not significantly different between males and females. With respect to microvascular reactivity after acetylcholine administration, the literature has more consistent positive findings in females (29), which we were unable to reproduce in our study. It is possible that the higher body mass of females compared with males may have biased our results in acetylcholine group. Finally, it is well-known that the action of an endothelium-independent stimulus is less evident when an atherosclerotic process is already present. It is possible that if the animals had been older or had a pro-atherosclerotic disease, such as diabetes mellitus, we may have found gender differences at microcirculation after sodium nitroprusside administration. Other limitations of the study should also be highlighted. The unawareness of the menstrual cycle period of females may have influenced the microvascular reactivity (33), although some researchers still consider this a controversial issue (34). Moreover, the reduced number of animals in each group may have limited our ability to observe differences. Gender differences in the microvascular bed were observed in this study, and according to our findings, we conclude that the female gender seemed to exhibit

microvascular protection, exhibited as lower microvascular permeability to macromolecules induced by ischemia/ reperfusion and reduced microvascular constriction elicited by a phenylephrine stimulus.

& ACKNOWLEDGMENTS This study was supported by grants from the National Research Council of Brazil (CNPq) and the Agency for Research Support of Rio de Janeiro State (FAPERJ). The authors do not have any conflicts of interest to declare.

& AUTHOR CONTRIBUTIONS Panazzolo DG analyzed the data and wrote the manuscript. Silva LH wrote the manuscript. Cyrino FZ generated data. Sicuri FL analyzed data. Kraemer-Aguiar LG analyzed the data and wrote the manuscript. Bouskela E analyzed the data, wrote the manuscript and designed the research approach.

& REFERENCES 1. Lerner DJ, Kannel WB. Patterns of coronary heart disease morbidity and mortality in the sexes: a 26-year follow-up of the Framingham population. Am Heart J. 1986;111(2):383-90, http://dx.doi.org/10.1016/ 0002-8703(86)90155-9. 2. Kuvin JT, Patel AR, Sliney KA, Pandian NG, Rand WM, Udelson JE, et al. Peripheral vascular endothelial function testing as a noninvasive indicator of coronary artery disease. J Am Coll Cardiol. 2001; 38(7):1843-9, http://dx.doi.org/10.1016/S0735-1097(01)01657-6. 3. Wang J, Bingaman S, Huxley VH. Intrinsic sex-specific differences in microvascular endothelial cell phosphodiesterases. Am J Physiol Heart Circ Physiol. 2010;298(4):H1146-54, http://dx.doi.org/10.1152/ajpheart. 00252.2009. 4. Mendelsohn ME, Karas RH. Molecular and cellular basis of cardiovascular gender differences. Science. 2005;308(5728):1583-7, http://dx.doi. org/10.1126/science.1112062. 5. Leonardo F, Medeirus C, Rosano GM, Pereira WI, Sheiban I, Gebara O, et al. Effect of acute administration of estradiol 17 beta on aortic blood flow in menopausal women. Am J Cardiol. 1997;80(6):791-3, http://dx. doi.org/10.1016/S0002-9149(97)00520-1. 6. Villar IC, Hobbs AJ, Ahluwalia A. Sex differences in vascular function: implication of endothelium-derived hyperpolarizing factor. J Endocrinol. 2008;197(3):447-62, http://dx.doi.org/10.1677/JOE-08-0070. 7. Pearson LJ, Yandle TG, Nicholls MG, Evans JJ. Regulation of endothelin1 release from human endothelial cells by sex steroids and angiotensin-II. Peptides. 2008;29(6):1057-61, http://dx.doi.org/10.1016/j.peptides.2008. 02.003. 8. Orshal JM, Khalil RA. Gender, sex hormones, and vascular tone. Am J Physiol Regul Integr Comp Physiol. 2004;286(2):R233-49. 9. Simoes C, Svensjo E, Bouskela E. Effects of L-NA and sodium nitroprusside on ischemia/reperfusion-induced leukocyte adhesion and macromolecular leakage in hamster cheek pouch venules. Microvasc Res. 2001;62(2):128-35, http://dx.doi.org/10.1006/mvre. 2001.2324. 10. Bouskela E, Cyrino FZ, Lerond L. Leukocyte adhesion after oxidant challenge in the hamster cheek pouch microcirculation. J Vasc Res. 1999;36Suppl 1:11-4, http://dx.doi.org/10.1159/000054069.

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11. Duling BR. The preparation and use of the hamster cheek pouch for studies of the microcirculation. Microvasc Res. 1973;5(3):423-9, http:// dx.doi.org/10.1016/0026-2862(73)90059-9. 12. Bouskela E, Grampp W. Spontaneous vasomotion in hamster cheek pouch arterioles in varying experimental conditions. Am J Physiol. 1992;262(2 Pt 2):H478-85. 13. Svensjo E. Bradykinin and prostaglandin E1, E2 and F2alpha-induced macromolecular leakage in the hamster cheek pouch. Prostaglandins Med. 1978;1(5):397-410, http://dx.doi.org/10.1016/0161-4630(78)90126X. 14. Bouskela E, Cyrino FZ, Conde CM, Garcia AA. Microvascular permeability with sulfonylureas in normal and diabetic hamsters. Metabolism. 1997;46(12 Suppl 1):26-30, http://dx.doi.org/10.1016/S0026-0495(97) 90313-9. 15. Zeintl H, Sack FU, Intaglietta M, Messmer K. Computer assisted leukocyte adhesion measurement in intravital microscopy. Int J Microcirc Clin Exp. 1989;8(3):293-302. 16. Persson NH, Erlansson M, Svensjo E, Takolander R, Bergqvist D. The hamster cheek pouch--an experimental model to study postischemic macromolecular permeability. Int J Microcirc Clin Exp. 1985;4(3):257-63. 17. Bazzoni G, Dejana E. Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis. Physiol Rev. 2004; 84(3):869-901, http://dx.doi.org/10.1152/physrev.00035.2003. 18. Harhaj NS, Antonetti DA. Regulation of tight junctions and loss of barrier function in pathophysiology. Int J Biochem Cell Biol. 2004; 36(7):1206-37. 19. Beyer EC, Gemel J, Seul KH, Larson DM, Banach K, Brink PR. Modulation of intercellular communication by differential regulation and heteromeric mixing of co-expressed connexins. Braz J Med Biol Res. 2000;33(4):391-7. 20. Sugawara T, Noshita N, Lewen A, Gasche Y, Ferrand-Drake M, Fujimura M, et al. Overexpression of copper/zinc superoxide dismutase in transgenic rats protects vulnerable neurons against ischemic damage by blocking the mitochondrial pathway of caspase activation. J Neurosci. 2002;22(1):209-17. 21. Lu A, Frink M, Choudhry MA, Schwacha MG, Hubbard WJ, Rue LW, III, et al. Mitochondria play an important role in 17beta-estradiol attenuation of H(2)O(2)-induced rat endothelial cell apoptosis. Am J Physiol Endocrinol Metab. 2007;292(2):E585-93. 22. Strehlow K, Rotter S, Wassmann S, Adam O, Grohe C, Laufs K, et al. Modulation of antioxidant enzyme expression and function by estrogen.

23.

24. 25. 26. 27.

28.

29.

30.

31.

32.

33.

34.

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Circ Res. 2003;93(2):170-7, http://dx.doi.org/10.1161/01.RES.0000082 334.17947.11. Erlansson M, Bergqvist D, Marklund SL, Persson NH, Svensjo E. Superoxide dismutase as an inhibitor of postischemic microvascular permeability increase in the hamster. Free Radic Biol Med. 1990;9(1):5965, http://dx.doi.org/10.1016/0891-5849(90)90050-S. Guimaraes S, Moura D. Vascular adrenoceptors: an update. Pharmacol Rev. 2001;53(2):319-56. Altura BM. Sex as a factor influencing the responsiveness of arterioles to catecholamines. Eur J Pharmacol. 1972;20(3):261-5. Kalsner S. Steroid potentiation of responses to sympathomimetic amines in aortic strips. Br J Pharmacol. 1969;36(3):582-93. Li Z, Krause DN, Doolen S, Duckles SP. Ovariectomy eliminates sex differences in rat tail artery response to adrenergic nerve stimulation. Am J Physiol. 1997;272(4 Pt 2):H1819-25. Binko J, Murphy TV, Majewski H. 17Beta-oestradiol enhances nitric oxide synthase activity in endothelium-denuded rat aorta. Clin Exp Pharmacol Physiol. 1998;25(2):120-7, http://dx.doi.org/10.1111/j.14401681.1998.tb02188.x. Sader MA, Celermajer DS. Endothelial function, vascular reactivity and gender differences in the cardiovascular system. Cardiovasc Res. 2002;53(3):597-604, http://dx.doi.org/10.1016/S0008-6363(01)00473-4. Barber DA, Sieck GC, Fitzpatrick LA, Miller VM. Endothelin receptors are modulated in association with endogenous fluctuations in estrogen. Am J Physiol. 1996;271(5 Pt 2):H1999-2006. Hayashi T, Fukuto JM, Ignarro LJ, Chaudhuri G. Basal release of nitric oxide from aortic rings is greater in female rabbits than in male rabbits: implications for atherosclerosis. Proc Natl Acad Sci U S A. 1992;89(23):11259-63, http://dx.doi.org/10.1073/pnas.89.23.11259. MacIntyre JN, Slusar JE, Zhu J, Dong AX, Howlett SE, Kelly ME. Ageassociated alterations in retinal arteriole reactivity to endothelin-1 differ between the sexes. Mech Ageing Dev. 2012;133(9-10):611-9, http://dx. doi.org/10.1016/j.mad.2012.08.001. Bungum L, Kvernebo K, Oian P, Maltau JM. Laser doppler-recorded reactive hyperaemia in the forearm skin during the menstrual cycle. Br J Obstet Gynaecol. 1996;103(1):70-5. Ketel IJ, Stehouwer CD, Serne EH, Poel DM, Groot L, Kager C, et al. Microvascular function has no menstrual-cycle-dependent variation in healthy ovulatory women. Microcirculation. 2009;16(8):714-24, http:// dx.doi.org/10.3109/10739680903199186.


BASIC RESEARCH

Biomechanical comparison of the four-strand cruciate and Strickland techniques in animal tendons Raquel Bernardelli Iamaguchi, William Villani, Marcelo Rosa Rezende, Teng Hsiang Wei, Alvaro B. Cho, Gustavo Bispo dos Santos, Rames Mattar Jr. Faculdade de Medicina da Universidade de Sa˜o Paulo (USP), Institute of Orthopedics and Traumatology, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: The objective of this study was to compare two four-strand techniques: the traditional Strickland and cruciate techniques. METHODS: Thirty-eight Achilles tendons were removed from 19 rabbits and were assigned to two groups based on suture technique (Group 1, Strickland suture; Group 2, cruciate repair). The sutured tendons were subjected to constant progressive distraction using a universal testing machine (KratosH). Based on data from the instrument, which were synchronized with the visualized gap at the suture site and at the time of suture rupture, the following data were obtained: maximum load to rupture, maximum deformation or gap, time elapsed until failure, and stiffness. RESULTS: In the statistical analysis, the data were parametric and unpaired, and by Kolmogorov-Smirnov test, the sample distribution was normal. By Student’s t-test, there was no significant difference in any of the data: the cruciate repair sutures had slightly better mean stiffness, and the Strickland sutures had longer time-elapsed suture ruptures and higher average maximum deformation. CONCLUSIONS: The cruciate and Strickland techniques for flexor tendon sutures have similar mechanical characteristics in vitro. KEYWORDS: Tendon Repair; Animal Experimentation; Flexor Tendon. Iamaguchi RB, Villani W, Rezende MR, Wei TH, Cho AB, dos Santos GB, et al. Biomechanical comparison of the four-strand cruciate and Strickland techniques in animal tendons. Clinics. 2013;68(12):1543-1547. Received for publication on February 12, 2013; First review completed on March 17, 2013; Accepted for publication on June 20, 2013 E-mail: biraquel@bol.com.br Tel.: 55 11 3021-4155

Moreover, increasing the number of suture strands prolongs the time of repair and increases the difficulty; consequently, many surgeons prefer four-strand sutures (5,6). Studies of four-strand sutures have reported good strength, but unequal loads can occur when two knots are used because the knot itself is a weak point of the suture (7,8). In recent articles on suture techniques, the cruciate technique has provided good tensile strength and has required greater force for failure and for the formation of gaps, without increasing the operative times (6,7,9–13). The cruciate repair suture was first described by McLarney et al. (10) and was considered the ideal technique by James W. Strickland, possessing the mechanical strength of a fourstrand suture and technical simplicity of a two-strand suture (5,12). Although the cruciate technique provides better mechanical results in vitro, the Strickland technique remains one of the most widely used methods (14). With regard to completing tendon repairs, the circumferential epitendinous suture increases the strength of the tendon suture by 10% to 50% and reduces the gap between the stumps of the tendons (1). The objective of the present study was to compare different four-strand techniques, specifically the cruciate and Strickland sutures, both of which are reinforced by a

& INTRODUCTION Flexor tendon lesions have always been a challenge for hand surgeons. However, due to advances in materials and suture techniques, the functional results of flexor tendon tenorraphies have improved (1,2). New suture techniques are designed to provide sufficient strength during early rehabilitation without increasing the incidence of premature suture rupture or the work required for flexion (3). The strength of a flexor tendon repair is proportional to the number of suture strands that cross the repair site (1,4); however, this biomechanical advantage occurs at the expense of increased suture volume and decreased vascularity of the tendon, resulting in worse clinical outcomes, decreasing the incidence of adherence and increasing the requirement for secondary tenolysis (5).

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(12)11

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continuous epitendinous suture in terms of the maximum load, maximum deformation, time elapsed until rupture and the stiffness of the sutures.

& MATERIALS AND METHODS Nineteen male and female New Zealand albino rabbits, between 3,500 g and 3,900 g, were acquired from the vivarium of the Faculty of Medicine, University of Sa˜o Paulo, and were maintained in a laboratory for musculoskeletal research. The University of Sa˜o Paulo Ethics Committee for Animal Resources approved this animal study. The animals were euthanized with sodium thiopental at 75 mg/kg intraperitoneally, as per instructions from the Brazilian College of Animal Experimentation (COBEA, 2007). Both Achilles tendons from each rabbit were harvested, and the skin was sutured. The tendons were prepared immediately for testing. The animals were disposed of at the Center of Biological Material, University of Sa˜o Paulo. The tendons were divided into two groups, each consisting of 19 experiments. Each tendon was randomly repaired with one of the techniques: Group 1 received a Strickland suture (Figure 1); and Group 2 received a cruciate repair suture (Figure 2). Both groups were reinforced with a circumferential, epitendinous, simple running suture. Each tendon was sectioned into two parts with a number 15 scalpel using a straight transverse cut and was sutured according to the randomization of surgical techniques with a 4–0 Nylon suture and with the core suture placed 7 mm from the cut edge of the tendon. The circumferential, epitendinous, simple running suture was held with 6–0 Nylon and a core suture purchase of 2 mm. The average cross-sectional volume of the tendons in Group 1 was 15.87 mm2 versus 15.65 mm2 in Group 2. The groups were homogeneous with regard to volume. The repaired tendons were tested for failure by constant progressive distraction using a KratosH universal testing machine, equipped with a load cell of 100 kgf and adjusted to a range of 10 kgf (accuracy of 10 gf). The tendon was fixed in the testing machine using two rectangular grasps with a trapezoidal profile; the distal end of the tendon was attached to a fixed section of the machine, and the proximal end was connected to the load cell in the movable part of the machine. The measurement system consisted of one mechanical linear actuator, and the load transducer connected to the proximal end of the Achilles tendon was connected to a computer, using the ADS2000 LynxH data acquisition system. The force and displacement data measured by the system were registered. To measure the gap between the cut edges of the tendon during mechanical testing, the tests were synchronized with a Sony DCR-HC26 digital camera. A two-point template

Figure 2 - Cruciate repair suture.

with known distance was placed beside the tendon as a reference for the gap. Maximum deformation was calculated by setting the gap between the cut edges of the tendons at the time of suture rupture, measured in millimeters. The gap at the repair site was measured using a program that automatically identifies, calculates and records the gap in millimeters, based on the distance between points on the template as a reference. To ensure synchronization between the data from the machine-based tests and measurements from the computer, a light-emitting diode (LED) was placed in the visual field of a digital camera that lit up at the same instant that the computer started to acquire data from the testing machine. This synchronization of equipment allowed us to calculate in seconds the maximum time elapsed until the moment of suture rupture. Based on data from the testing machine, a computer program calculated the maximum load at the time of suture rupture for each test, and the stiffness of the suture was obtained by dividing the maximum load by the maximum gap in Newtons per millimeter.

Ethics The University of Sa˜o Paulo Ethics Committee for Animal Resources approved this animal study.

Statistical analysis In our statistical analysis, the data were parametric and unpaired. The sample distribution was normal, as assessed by the Kolmogorov-Smirnov test, and the variance was homogeneous by Levene’s test. Student’s t-test was employed for quantitative variables. Descriptive and inferential analyses were performed with SPSS software, version 17.0 for Windows.

& RESULTS Group 1 underwent tenorrhaphy by the Strickland method, as follows: the average maximum deformation or gapping of the cut edges of the tendons at the time of suture rupture was 12.68 mm (median 13.05 mm, SD 2.86 mm). Group 2, which underwent cruciate tenorrhaphy, had an average value of 11.74 mm (median 11.51 mm, SD 3.16 mm). In Group 1, the average time that elapsed until the moment of suture rupture was 44.9 seconds (median 46.0 seconds, SD 9.8 seconds), compared with 40.2 seconds in Group 2 (median 38.9 seconds, SD 10.4 seconds). In Group 1, the average maximum force at the time of suture rupture was 34.83 N (median 35.15 N, SD 10.27 N) vs. 35.13 N in Group 2 (median 35.44 N, SD 12.85 N). The mean stiffness in Group 1 was 4.31 N/mm (median 3.92 N/mm, SD 1.35 N/mm), compared with 5.16 N/mm in Group 2 (median 4.87 N/mm, SD 1.45 N/mm).

Figure 1 - Strickland method.

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was performed in both groups, but its presence in mechanical tests hindered the visualization and evaluation of gap formation at the repair site, thereby generating a homogeneous suture and increasing early suture resistance (1). Savage (16) suggested that the ideal suture should withstand a force at the repair site that is five times greater than the force necessary to actively move the tendon without resistance. Initial studies of the cruciate technique (17) demonstrated that it is capable of supporting strength beyond the physiological requirement for active movement. In our study, both sutures attained a strength that exceeded 30 N, sufficient to allow for active rehabilitation protocols, per Viinikiainen et al. (18,19). Because the tests were performed in rabbit tendons in vitro, it was not possible to compare the values of human flexor tendons in the suture tests; these values should approximate one another, although rabbit tendons have less mechanical resistance and smaller diameters. Another limitation of this experimental study, similar to all in vitro studies, was the inability to study the effects of postoperative edema, tendon resistance and gap formation during active movement (17). In this study, we also observed that the cruciate repair suture technique was easier to perform and had a lower volume at the repair site, with one suture knot, and a more homogeneous suture, which was consistent with the literature (6,7,10). During the tests, the cruciate repair suture formed a more homogeneous graph of deformation versus resistance, whereas the Strickland suture had one of its knots rupture and rapidly lose resistance, which might be

In our statistical analysis, by Student’s t-test (2 6 2 table), none of the parameters were statistically significant. The median maximum force that was required for suture rupture and the stiffness of the sutures were greater with cruciate repair (p = 0.94). The Strickland technique resulted in higher median maximum deformation with a wider final gap (p = 0.36) and a longer time elapsing until the moment of suture rupture (p = 0.15). Boxplots for these values were generated for the samples (Figures 3 and 4).

& DISCUSSION Various tendon sutures have been compared with regard to their techniques, materials and use of epitendinous sutures. The ideal suture, according to Strickland (1), must: 1) be easy to perform; 2) be reliable; 3) result in homogeneous coaptation of the cut edges of the tendon; 4) create a lower gap in the suture zone; 5) provide less interference with tendon vascularity; and 6) provide sufficient strength to facilitate early rehabilitation. The ideal suture can be achieved through techniques with a higher number of strands that cross the repair site, which, however, can also lead to increased technical difficulty and more time to perform (13). Moreover, tendons can be injured, with impairments to vascularization, using techniques that use six or more strands (1). The most widely used techniques are the four- and six-strand methods, which are considered superior to two-strand techniques (6,15). The epitendinous suture increases the resistance of the tendon by 10% to 50% and reduces the gap at the repair site of the tendon. In the present study, epitendinous suturing

Figure 3 - Maximum force to suture rupture.

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Figure 4 - Stiffness of the groups.

attributed to the difficulty in creating equal tension between the Kessler suture and the U suture of the Strickland technique. Four-strand cruciate suture techniques are easier to perform, provide less interference with tendon gliding and are sufficiently strong for an early active motion protocol (15,17); in our study, however, there were no significant differences between the Strickland and cruciate repair techniques regarding the maximum load required to rupture the suture, the maximum deformation at failure or the stiffness, which can be explained by the number of tests that were performed with each technique. The cruciate repair suture also had a lower tendency toward gap formation, which will be evaluated in future studies. Croog et al. (6) studied various configurations of the cruciate repair suture and noted that the cross lock increased the overall resistance, as well as the resistance to gap formation (20). Based on our observation that the simple cruciate repair suture had similar resistance compared with the Strickland technique, we recommend using the cross lock cruciate repair suture, which improves suture strength without significantly increasing the technical difficulty. Hand surgeons should aim to simplify tendon sutures and to maintain the suture strength without increasing the technical difficulty (21). Thus, we advocate the cruciate repair suture technique, which yielded results comparable to the Strickland method in our study, in addition to its reported advantages. The technical benefits of the cruciate repair suture under clinical conditions could generate a lower coefficient of friction, thereby reducing failures, which we did not address in our experiments (5,6,7,9,10).

The cruciate repair suture is similar to the Strickland method with regard to the maximum load and the stiffness to suture rupture. Further studies should be conducted to investigate the clinical results of these techniques.

& ACKNOWLEDGMENTS The authors thank Cesar A. M. Pereira for helping to perform the mechanical tests in the Laboratory of Musculoskeletal Research (University of SaËœo Paulo).

& AUTHOR CONTRIBUTIONS Iamaguchi RB contributed to the study design, data collection, assessment of the results, statistical analyses and manuscript preparation. Villani W and Santos GB contributed to the data collection. Rezende MR, Wei TH and Cho AB were responsible for the critical revision. Mattar R supervised the study.

& REFERENCES 1. Strickland JW. Flexor Tendon Injuries: I. Foundations of Treatment. J Am Acad Orthop Surg. 1995;3(1):44-54. 2. Kim HM, Nelson G, Thomopoulos S, Silva MJ, Gelberman RH. Technical and biological modifications for enhanced flexor tendon repair. J Hand Surg Am. 2010;35(6):1031-7, http://dx.doi.org/10.1016/j.jhsa.2009.12. 044. 3. Hwang MD, Pettrone S, Trumble TE. Work of flexion related to different suture materials after flexor digitorum profundus and flexor digitorum superficialis Tendon Repair in Zone II: A biomechanical study. J Hand Surg Am. 2009;34(4):700-4, http://dx.doi.org/10.1016/j.jhsa.2008.12.003. 4. Nelson GN, Potter R, Ntouvali E, Silva MJ, Boyer MI, Gelberman RH, et al. Intrasynovial flexor tendon repair: A biomechanical study of variations in suture application in human cadavera. J Orthop Res. 2012;30(10):1652-9, http://dx.doi.org/10.1002/jor.22108. 5. Thurman RT, Trumble TE, Hanel DP, Tencer AF, Kiser PK. Two-, four-, and six-strand zone II flexor tendon repairs: an in situ biomechanical

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6.

7.

8.

9.

10. 11.

12.

Cruciate vs. Strickland techniques Iamaguchi RB et al. 13. Dona E, Gianoutsos MP, Walsh WR. Optimizing biomechanical performance of the 4-strand cruciate flexor tendon repair. J Hand Surg Am. 2004;29(4):571-80, http://dx.doi.org/10.1016/j.jhsa.2004.04.007. 14. Strickland JW. Flexor tendon repair – Indiana method. The Indiana Hand Center Newsletter. 1993;1:1-19. 15. Wolfe SW, Willis AA, Campbell D, Clabeaux J, Wright TM. Biomechanic comparison of the tenofix tendon repair device with the cruciate and modified Kessler techniques. J Hand Surg Am. 2007;32(3):356-66, http:// dx.doi.org/10.1016/j.jhsa.2006.10.004. 16. Savage R. In vitro studies of a new method of flexor tendon repair. J Hand Surg Br. 1985;10(2):135-41, http://dx.doi.org/10.1016/0266-7681(85)90001-4. 17. Angeles JG, Heminger H, Mass DP. Comparative biomechanical performances of 4-strand core suture repairs for zone II tendon lacerations. J Hand Surg Am. 2002;27(3):508-17, http://dx.doi.org/10. 1053/jhsu.2002.32619. 18. Viinikiainen A, Goransson H, Ryha¨nen J. Primary Flexor Tendon Repair Techniques. Scand J Surg. 2008;97(4):333-40. 19. Viinikiainen A, Goransson H, Houvinen K, Kelloma¨ki M, To¨rma¨ia¨ P, Rokkanen P. The strength of the 6-strand modified kessler repair performed with triple-stranded or triple-stranded bound suture in a porcine extensor tendon model: an ex vivo study. J Hand Surg Am. 2007;32(4):510-7, http://dx.doi.org/10.1016/j.jhsa.2007.01.010. 20. Barrie KA, Tomak SL, Cholewicki J, Wolfe SW. The role of multiple strands and locking sutures on gap formation of flexor tendon repairs during cyclical loading. J Hand Surg Am. 2000;25(4):714-20, http://dx. doi.org/10.1053/jhsu.2000.9414. 21. Cao Y, Tang JB. Biomechanical evaluation of a four-strand modification of the Tang method of tendon repair. J Hand Surg Br. 2005;30(4):374-8, http://dx.doi.org/10.1016/j.jhsb.2005.04.003.

comparison using a cadaver model. J Hand Surg Am. 1998;23(2):261-5, http://dx.doi.org/10.1016/S0363-5023(98)80124-X. Croog A, Goldstein R, Nasser P, Lee SK. Comparative biomechanic performances of locked cruciate four-strand flexor tendon repairs in an ex vivo porcine model. J Hand Surg Am. 2007;32(2):225-32, http://dx. doi.org/10.1016/j.jhsa.2006.11.009. Rees L, Matthews A, Masouros SD, Bull AM, Haywood R. Comparison of 1- and 2- knot, 4- strand, double-modified kessler tendon repairs in a porcine model. J Hand Surg Am. 2009;34(4):705-9, http://dx.doi.org/10. 1016/j.jhsa.2008.12.014. Kim HM, Nelson G, Thornopouulos S, Silva MJ, Das R, Gelberman RH. Technical and biological modifications for enhanced flexor tendon repair. J Hand Surg Am. 2010;35(6):1031-7. quiz 1038, http://dx.doi. org/10.1016/j.jhsa.2009.12.044. Tang JB, Gu YT, Rice K, Chen F, Pan CZ. Evaluation of four methods of flexor tendon repair for postoperative active mobilization. Plast Reconstr Surg. 2001;107(3):742-9, http://dx.doi.org/10.1097/00006534-20010300000014. McLarney E, Hoffman H, Wolfe SW. Biomechanical analysis of the cruciate four-strand flexor tendon repair. J Hand Surg Am. 1999;24(2):295-301. Vigler M, Palti R, Goldstein R, Patel VP, Nasser P, Lee SK. Biomechanical study of cross-locked cruciate versus Strickland flexor tendon repair. J Hand Surg Am. 2008;33(10):1826-33, http://dx.doi.org/10.1016/j.jhsa. 2008.07.009. Waitayawinyu T, Martineau PA, Luria S, Hanel DP, Trumble TE. Comparative biomechanic study of flexor tendon repair using Fiberwire. J Hand Surg Am. 2008;33(5):701-8, http://dx.doi.org/10.1016/j.jhsa. 2008.01.010.

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BASIC RESEARCH

Local and remote ischemic preconditioning protect against intestinal ischemic/reperfusion injury after supraceliac aortic clamping Nilon Erling Junior,I Edna Frasson de Souza Montero,II Paulina Sannomiya,III Luiz Francisco Poli-deFigueiredo (in memoriam)II I

Universidade Federal de Cieˆncias da Sau´de de Porto Alegre, Department of Surgery, Vascular Surgery, Porto Alegre/RS, Brazil. II Faculdade de Medicina da Universidade de Sa˜o Paulo, Department of Surgery, LIM 62, Sa˜o Paulo/SP, Brazil. III Faculdade de Medicina da Universidade de Sa˜o Paulo, Institute of Heart (InCor), LIM 11, Sa˜o Paulo/SP, Brazil.

OBJECTIVES: This study tests the hypothesis that local or remote ischemic preconditioning may protect the intestinal mucosa against ischemia and reperfusion injuries resulting from temporary supraceliac aortic clamping. METHODS: Twenty-eight Wistar rats were divided into four groups: the sham surgery group, the supraceliac aortic occlusion group, the local ischemic preconditioning prior to supraceliac aortic occlusion group, and the remote ischemic preconditioning prior to supraceliac aortic occlusion group. Tissue samples from the small bowel were used for quantitative morphometric analysis of mucosal injury, and blood samples were collected for laboratory analyses. RESULTS: Supraceliac aortic occlusion decreased intestinal mucosal length by reducing villous height and elevated serum lactic dehydrogenase and lactate levels. Both local and remote ischemic preconditioning mitigated these histopathological and laboratory changes. CONCLUSIONS: Both local and remote ischemic preconditioning protect intestinal mucosa against ischemia and reperfusion injury following supraceliac aortic clamping. KEYWORDS: Aorta; Reperfusion; Ischemic Preconditioning; Intestinal Mucosa; Rats. Erling Jr N, Montero EF, Sannomiya P, Poli-de-Figueiredo LF. Local and remote ischemic preconditioning protect against intestinal ischemic/ reperfusion injury after supraceliac aortic clamping. Clinics. 2013;68(12):1548-1554. Received for publication on March 31, 2013; First review completed on April 26, 2013; Accepted for publication on June 20, 2013 E-mail: nilonjr@gmail.com Tel.: 55 51 3303-9000

Strategies to reduce I/R injury are being extensively investigated, particularly ischemic preconditioning (IPC) (7). This strategy of submitting tissues to controlled periods of ischemia and reperfusion prior to the prolonged I/R injury is initially proven to be beneficial when applied to the same tissue (local) (8) and also when applied to a different tissue (remote) (9). Numerous investigators have described the protective effect of IPC on I/R injury in specific organs such as the heart (10) and liver (11). Currently, IPC is considered a ubiquitous phenomenon (12) that involves a complex mechanism of cell signaling (13), with clinical applicability that reaches beyond myocardial protection or organ transplantation (14). Intestinal I/R injury and the use of IPC was initially evaluated by occluding the superior mesentery artery (15,16). The models used thus far typically employ this concept with different combinations of ischemic and reperfusion periods (17). Among studies, the preconditioning stimulus varies from 1 to 4 cycles of ischemia for 1 to 20 minutes and reperfusion for 5 to 10 minutes (18,19). Supraceliac aortic occlusion has been evaluated with regard to the systemic inflammatory response resulting

& INTRODUCTION Surgical correction of disease of the abdominal aorta involving its visceral branches produces intense and abrupt hemodynamic changes induced by aortic clamping and unclamping (1,2). However, the main consequences of temporary visceral aorta occlusion are related to the ischemia and reperfusion (I/R) injury of the splanchnic organs (3). Of the splanchnic organs, the intestine is the most sensitive to I/R injury and plays a pivotal role in the induction of systemic inflammation response syndrome (SIRS) and multiple organ dysfunction (MOD) (4,5), a major cause of morbidity in patients undergoing major aortic repair (6).

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(12)12

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from I/R injury. The inhibition of tumor necrosis factor-a (TNF-a) and interleukin (IL) 1b attenuates pulmonary neutrophil infiltration (20). Additionally, IL-10 administration or its endogenous production seems to be protective (21,22). Intestinal injury in the clinical context of total splanchnic and lower torso I/R has not been sufficiently described. Erling et al. identified that either local or remote IPC modulates the inflammatory response, reducing endothelial dysfunction in the mesenteric circulation after supraceliac aortic clamping (23). However, the possible protective effect of the IPC achieved in the intestine alone (24) has not been studied in the setting of supraceliac occlusion. Using a supraceliac aortic clamping model, this study evaluated the morphologic alterations in the intestinal mucosa, variations in laboratory findings caused by multivisceral I/R injury, and the modulations in outcomes resulting from local or remote IPC.

supraceliac aortic occlusion, followed by 120 minutes of reperfusion. The LIPC (local ischemic preconditioning) group was submitted to 2 cycles of supraceliac occlusion (5 minutes of ischemia and 5 minutes of reperfusion), followed by 20 minutes of supraceliac aortic occlusion and 120 minutes of reperfusion. The RIPC (remote ischemic preconditioning) group was submitted to 2 cycles of infrarenal occlusion (10 minutes of ischemia and 10 minutes of reperfusion), followed by 20 minutes of supraceliac aortic occlusion and 120 minutes of reperfusion (Figure 1A). Hemodynamic stability at the time of supraceliac clamp opening (occluded for 20 minutes) was accomplished by an infusion of 1.5 mL of 0.9% saline. Subsequent doses of 0.5 mL of 0.9% saline were administered after 30, 60, 90, and 120 minutes of reperfusion. This volume expansion was provided at the same time point for all four groups. At the end of the experiment, the animals were exsanguinated by aortic puncture.

& MATERIAL AND METHODS

Blood and tissue sampling Peripheral blood collected from the tail at the beginning and at the end of the experiment was used for hematocrit and leukogram determinations. Arterial blood samples for lactate and blood gas analysis were collected through the carotid catheter at surgical preparation and at the end of experiment. A blood sample for measuring lactic dehydrogenase (LDH) was collected directly by aortic puncture. The bowel was stripped from its mesentery, and three samples of tissue were harvested from the proximal jejunum 3 cm distal to its origin, the middle portion of the small intestine, and the distal ileum 3 cm proximal to the cecum. Tissue was fixed in buffered 10% formalin for 24 hours and then embedded in paraffin wax. Sections of 5 mm were cut and stained with hematoxylin and eosin (HE). An independent pathologist blinded to the experimental group of the samples performed the histological analysis. Images were captured using a high-resolution Samsung camera coupled to a light Nikon E200 microscope and subsequently analyzed using AxioVision-Rel software (Zeiss). Total mucosal thickness, villous height, and crypt depth were evaluated. Each variable was measured three times for all three portions of the intestine, so the final value of a given variable for one specimen is the mean of these nine measurements.

Animal model and surgical preparation The experimental protocol was approved by the Ethical Committee of Federal University of Sao Paulo (CEP 1016/ 06) and was performed according to the National Institutes of Health guidelines on the use of experimental animals. Twenty-eight male Wistar rats weighting 190-250 g were kept in a non-stimulating environment for a week prior to the experiment. The subjects were fasted overnight prior to the procedure, with free access to water. Anesthesia was induced with intraperitoneal sodium pentobarbital (50 mg/ kg). A tracheostomy was performed through a right anterior cervical incision to allow for airway control and spontaneous breathing. The common carotid artery and external jugular vein were dissected and cannulated with polyethylene catheters. Venous access was used to inject solutions, while arterial access was used to monitor mean arterial pressure (MAP) (MP 100, Biopac System Inc., Goleta, CA, USA). A midline abdominal incision was performed, and the aorta was dissected and controlled proximally at the supraceliac portion between the diaphragmatic crura and distally at the aortic bifurcation. The strings utilized for aortic control were used to create a 4 cm long Rumel tourniquet. The abdominal wall was exposured and controlled with the exteriorization of the tourniquets at the top and bottom of the incision to allow for aortic occlusion throughout the experiment. In this model, the supraceliac aortic occlusion produced ischemia in all splanchnic organs and the striate muscle of the lower torso, while the aortic bifurcation occlusion produced ischemia in the striate muscle of the lower torso and the caudal portion of the large bowel (25). Aortic occlusion and flow restoration, which were necessary in some experimental groups, were confirmed by an abrupt rise and fall in the MAP. Heparin (100 IU/kg) was administered intravenously, and the animals were kept warm during the experiment.

Statistical analysis All data are expressed as the mean ยก standard error (SE). A paired t-test was used for repetitive measurements in the same group. Multiple comparisons between groups were performed using one-way ANOVA and post-hoc analysis with the Tukey test. The results were considered significant for p-values less than 0.05.

& RESULTS At baseline, there were no differences between groups regarding weight, MAP, or any other laboratory value (Table 1). The supraceliac aortic occlusion resulted in a significant MAP increase in all groups, while the aortic release significantly decreased MAP to values below the baseline or values at the corresponding time in the control group (Figure 1). Infrarenal aortic occlusion in the RIPC group did not increase MAP, and aortic release caused a transient but non-significant decrease in MAP (Figure 1).

Experimental design and study groups After identical initial surgical preparation and stabilization, the animals were allocated into four experimental groups according to the duration of ischemia and reperfusion. The control group was kept at rest during the entire experiment, without aortic occlusion. The IR (ischemiareperfusion) group was submitted to 20 minutes of

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Figure 1 - Experimental protocol (A) and main arterial pressure variations during the experiment in the control, ischemia and reperfusion (IR), local ischemic preconditioning (LIPC), and remote ischemic preconditioning (RIPC) groups (B). Data are expressed as the mean ยก SE for 7 animals per group. (*) p,0.001: IR, LIPC, and RIPC vs. control, ({) p,0.001: LIPC vs. control.

The results of laboratory testing, summarized in Table 1, showed that there was an increase in hematocrit values at the end of the experiment in groups submitted to aortic occlusion, even in preconditioned animals (p#0.036), compared with baseline values. Compared with the control group, this increase was higher in the IR (p = 0.015) group, but not in the LIPC (p = 0.208) or the RIPC (p = 0.443) groups. The total leukocyte counts at the end of the experiment were increased from baseline values in all groups (p#0.004). Compared with the control group, leukocytosis was more pronounced in the IR group (p = 0.005) and less intense in the LIPC (p = 0.041) and RIPC (p = 0.149) groups. The percentage of polymorphonuclear (PMN) leukocytes increased at the end of the reperfusion compared with baseline values (p#0.002) and increased similarly among all groups (p = 0.647). The results also showed decreases in the percentage of monocytes (p#0.032) and lymphocytes (p#0.002) in all groups at the end of the experiment. The blood gas analysis showed that the pH was constant in the control group throughout the experiment but decreased in other groups (p,0.02). Arterial base excess

(BE) decreased in all groups (p#0.003). Compared with the control group, BE at the end of experiment was lower in the IR (p,0.001), LIPC (p = 0.020), and RIPC (p = 0.024) groups. The arterial partial pressure of CO2 and the HCO3 concentration also decreased during the experiment in all groups (p,0.04). Compared with the control group, both pCO2 and the HCO3 concentration at the end of experiment were lower in the IR, LIPC, and RIPC groups (p#0.003). Compared with the baseline, arterial lactate concentration increased in all groups (p,0.02) at the end of experiment. The increases in the IR, LIPC, and RIPC groups were higher than in the control group (p#0.001), but the values were lower in the LIPC and RIPC groups than in the IR group (p,0.01). Compared with the control group, the LDH concentrations were higher in all three groups submitted to aortic occlusion. Compared with the IR group, the LDH concentrations were lower in the RIPC group (p = 0.007) but were not significantly different in the LIPC group (p = 0.129). The histological analyses of the intestine showed a decrease in villous height in all groups submitted to aortic occlusion when compared with the control group (IR:

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52.28%*¡1.04% 26,442*¡3,500.4 68.14%*¡3.07% 27.42%*¡2.36% 4.42%*¡0.78% 7.318*¡0.024 -11.47*{¡1.14 22.62*{¡1.61 11.8*{¡0.80 3.24*{1¡0.31 48.71%¡0.96% 12,250¡826.5 22.42%¡1.75% 69%¡2.91% 7%¡0.69% 7.415¡0.021 -2.21¡0.80 33.62¡1.57 21.07¡0.59 1.3¡0.13

p,0.001, LIPC: p = 0.013, and RIPC: p = 0.005) (Figure 2A). Crypt depth (Figure 2B) and total mucosal thickness (Figure 2C) were only reduced in the IR group (p,0.001). The villous height-crypt depth ratio was diminished in the IR, RIPC (p#0.001), and LIPC (p,0.035) groups (Figure 2D). Compared with the IR group, the reductions in total mucosal thickness, villous height, crypt depth, and villous height-crypt depth ratio were lower in the LIPC and RIPC groups (p,0.001). Representative photomicrographs are presented in Figure 3.

& DISCUSSION This study indicates that local and remote IPC decreases the intestinal I/R injury resulting from supraceliac aortic clamping. The results of the LIPC group confirmed our hypothesis that the protection achieved in superior mesenteric artery (SMA) occlusion models of I/R and IPC could also apply to the total splanchnic I/R injury of aortic occlusion. The results of the RIPC group show that the method is also protective in this setting, extending the clinical applicability of IPC and strengthening the interest in this technique in aortic surgery. This experiment mimicked the scenario of complex visceral aortic procedures. The redistribution of blood flow in the territories under ischemia (and also during reperfusion) in very different tissues with varying vascular bed resistance and tolerance to the I/R injury makes the local and systemic consequences of supraceliac aortic occlusion different from isolated SMA occlusion. This difference was demonstrated by the near 100% mortality rate in our pilot experiments after 1 h of reperfusion when supraceliac aortic clamping in excess of 20 minutes was tested, in contrast with the 90 minutes of SMA occlusion employed in some models (26,27). The duration and number of IPC cycles were chosen based on the most commonly published models (17,28), in accordance with the findings of our pilot experiments. The intestine is very susceptible to I/R injury, and severe changes occur in the intestinal epithelium after this insult. We objectively measured and quantitatively assessed various components of the intestinal mucosa. This morphometric evaluation showed a reduction in mucosal thickness in the I/ R groups. In accordance with other reports, this reduction was mainly attributable to the loss in villous height, with a relative sparing of the crypt depth (29,30). Our experiment also demonstrated that both forms of IPC consistently decrease the magnitude of the mucosal damage. Laboratory parameters are useful clinical markers of advanced mesenteric ischemia. Lactate and LDH serum levels were also measured to evaluate the I/R injury and the effect of local IPC (31,32). We also observed these changes, with our results indicating markedly high levels of lactate and LDH in the IR group. The preconditioned groups had a reduced increase in the level of these variables, which is in agreement with the minor intestinal mucosal damage that occurred in these groups. Arterial blood gas derangement and leukocytosis, which reflect an increased PMN count, demonstrated the systemic consequences of the I/R injury. Our results indicate that local and remote IPC can modulate the SIRS caused by supraceliac aortic occlusion. This phenomenon could be due to either the systemic anti-inflammatory effect of IPC or a reduced inflammatory response resulting from the halted visceral I/R injury after the IPC. Both mechanisms are likely

57.14%*{¡1.20% 33,478*¡4,296.6 71.57%*¡4.43% 24.42%*¡2.48% 3.85%*¡0.55% 7.280*{¡0.035 -15.81*{¡1.64 19.98*{¡1.27 9.3*{¡0.95 4.85*{¡0.46

49.71%¡1.06% 13,435¡786.7 26.28%¡3.15% 67.71%¡3.41% 6%¡0.69% 7.406¡0.014 -3.2¡0.49 32.57¡1.84 20.1¡0.71 1.37¡0.09 6512¡894{1

53.28%*¡1.06% 29,278*¡2,597.7 75.42%*¡2.77% 20.85%*¡2.48% 3.42%*¡0.71% 7.330*¡0.019 -11.6*{¡1.19 21.61*{¡0.89 11.77*{¡0.59 2.97*{1¡0.25 4812¡1213{1

t180 t180

Ischemic preconditioning in aortic surgery Erling Jr N et al.

t0

49.425¡1.04% 12,392¡1,117.4 20.42%¡1.42% 71.71%¡1.16% 7.42%¡0.64% 7.425¡0.019 -2.67¡0.83 31.45¡0.94 20.25¡0.55 1.57¡0.13 9460¡977{ 49.28%¡0.94% 16,664*¡1,315.9 68.57%*¡6.65% 28.28%*¡6.28% 2.71%*¡0.47% 7.375¡0.017 -6.25*¡0.46 30.44*¡1.70 17.27*¡0.44 1.65*¡0.10 49.42%¡1.84% 12,685.7¡963.5 26.14%¡2.28% 66.57%¡2.25% 7%¡0.72% 7.387¡0.015 -2.21¡0.77 35.97¡1.18 21.58¡0.78 1.18¡0.08 1158¡204 Hematocrit Total leucocyte (cells/mm3) Polymorphonuclear leucocyte Lymphocytes Monocytes pH EB (mEq/L) pCO2 (mmHg) HCO3 (mEq/L) Lactate (mmol/L) LDH (U/L)

t180

t0

IR Control

Table 1 - Blood gas and hematologic profile, lactate, and lactate dehydrogenase levels.

t0

LIPC

t180

RIPC t0

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Ischemic preconditioning in aortic surgery Erling Jr N et al.

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Figure 2 - Histomorphometric analysis of the small bowel demonstrating villous height (A), crypt depth (B), total mucosal thickness (C), and villous height-crypt depth ratio (D) in the control, ischemia and reperfusion (IR), local ischemic preconditioning (LIPC), and remote ischemic preconditioning (RIPC) groups. Data are expressed as the mean ¡ SE for 7 animals per group. (*) p,0.001: IR vs. control, (**) p,0.05: LIPC and RIPC vs. control, ({) p,0.001: LIPC and RIPC vs. IR.

subjects (36). The RIPC method also has a myocardial protective effect when used in percutaneous coronary intervention either for elective procedures (37) or for the management of acute ST-elevation myocardial infarctions (38). In the field of aortic surgery, the first clinical investigation of RIPC was in open aneurysm repair. The authors applied two 10-minute cycles of ischemia by sequentially clamping the right and left common iliac arteries. Remote IPC reduces the incidence of myocardial injury, myocardial infarction, and renal impairment (14). Further studies showed that urinary biomarkers of renal injury are reduced with the RIPC method applied, which utilizes an inflatable tourniquet placed around the thigh, even during less invasive endovascular aneurysm repairs (39).

causes and could take place simultaneously, although the definite protection mechanism of IPC is complex and still subject to debate (17,33), particularly with regard to a remote stimulus (34). The first published clinical application of remote IPC in humans was in children undergoing cardiac surgery (35). Protection was achieved using four 5-minute cycles of lower limb ischemia using a blood pressure cuff. Using the same principle of cuff inflation, but with three 5-minute cycles of upper arm ischemia, remote IPC was tested in adult patients undergoing coronary artery bypass surgery. The primary outcome employed to assess myocardial injury was the ‘‘total area under the curve’’ troponin-T concentration during the 72-h postoperative period, and this value was significantly reduced by 43% in preconditioned

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Ischemic preconditioning in aortic surgery Erling Jr N et al.

Figure 3 - Representative photomicrographs of the small bowel (HE, x100) from the control (A), IR (B), LIPC (C), and RIPC (D) groups. Normal small intestine architecture is shown in the control group (A). The IR group (B) demonstrates a marked loss of villus height, while the LIPC (C) and RIPC (D) groups have less intense findings.

Our experimental protocol has some limitations. We used healthy rats, without the blood loss and fluid shifts that typically characterize aortic surgery in adults. Typically, cardiovascular, pulmonary, and renal dysfunction is present in these patients and contributes substantially to the high rate of observed complications. However, this well-standardized protocol allowed us to control the duration of ischemia and equally apply the method to all experimental groups. Another advantage of this animal model was the direct histopathological evaluation of mucosal injury, instead of using the substitutive outcomes that are usually used in human subjects. In conclusion, this study demonstrates that IPC significantly reduces intestinal I/R injury in a clinically relevant model of supraceliac aortic clamping. The preconditioning stimulus is protective when applied locally or at remote sites. The low cost and simplicity of some forms of RIPC along with the favorable results of this practice highlight the need for further clinical and experimental studies to further elucidate the proper role of this promising strategy in aortic surgery.

approved the final version of the manuscript. Erling Jr N performed the data collection and statistical analysis. Poli-de-Figueiredo LF obtained funding. Erling Jr N, Montero EF, and Poli-de-Figueiredo L assumed overall responsibility.

& REFERENCES 1. Poli de Figueiredo LF, Mathru M, Tao W, Solanki D, Uchida T, Kramer GC. Hemodynamic effects of isovolemic hemodilution during descending thoracic aortic cross clamping and lower torso reperfusion. Surgery. 1997;122(1):32-8, http://dx.doi.org/10.1016/S0039-6060(97)90261-0. 2. Eide TO, Aasland J, Romundstad P, Stenseth R, Saether OD, Aadahl P, et al. Changes in hemodynamics and acid-base balance during crossclamping of the descending thoracic aorta. A study in patients operated on for thoracic and thoracoabdominal aortic aneurysm. Eur Surg Res. 2005;37(6):330-4, http://dx.doi.org/10.1159/000090332. 3. Cornet AD, Kingma SDK, Trof RJ, Wisselink W, Groeneveld ABJ. Hepatosplanchnic ischemia/reperfusion is a major determinant of lung vascular injury after aortic surgery. J Surg Res. 2009;157(1):48-54, http:// dx.doi.org/10.1016/j.jss.2008.09.021. 4. de Arruda MJC, Poggetti RS, Fontes B, Younes RN, Souza AL Jr, Birolini D. Intestinal ischemia/reperfusion induces bronchial hyperreactivity and increases serum TNF-alpha in rats. Clinics. 2006;61(1):21-8, http:// dx.doi.org/10.1590/S1807-59322006000100005. 5. Zanoni FL, Benabou S, Greco KV, Moreno ACR, Cruz JWMC, Filgueira FP, et al. Mesenteric microcirculatory dysfunctions and translocation of indigenous bacteria in a rat model of strangulated small bowel obstruction. Clinics. 2009;64(9):911-9. 6. Back MR, Bandyk M, Bradner M, Cuthbertson D, Johnson BL, Shames ML, et al. Critical analysis of outcome determinants affecting repair of intact aneurysms involving the visceral aorta. Ann Vasc Surg. 2005;19(5):648-56, http://dx.doi.org/10.1007/s10016-005-6843-3. 7. Pasupathy S, Homer-Vanniasinkam S. Surgical implications of ischemic preconditioning. Arch Surg. 2005;140(4):405-9; discussion 410, http://dx. doi.org/10.1001/archsurg.140.4.405. 8. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986;74(5):1124-36, http://dx.doi.org/10.1161/01.CIR.74.5.1124. 9. Przyklenk K, Bauer B, Ovize M, Kloner RA, Whittaker P. Regional ischemic ‘‘preconditioning’’ protects remote virgin myocardium from

& ACKNOWLEDGMENTS This work was supported by a grant from the Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo (FAPESP) 04/15964-6.

& AUTHOR CONTRIBUTIONS Erling Jr N, Montero EF, Sannomiya P, and Poli-de-Figueiredo LF conceived and designed the study. Erling Jr N and Montero EF analyzed and interpreted the data, performed critical review of the manuscript, and

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10. 11. 12.

13. 14.

15.

16.

17.

18. 19.

20.

21.

22.

23.

24. 25.

CLINICS 2013;68(12):1548-1554

subsequent sustained coronary occlusion. Circulation. 1993;87(3):893-9, http://dx.doi.org/10.1161/01.CIR.87.3.893. Bolli R. Preconditioning: a paradigm shift in the biology of myocardial ischemia. Am J Physiol Heart Circ Physiol. 2007;292(1):H19-27. Jaeschke H. Molecular mechanisms of hepatic ischemia-reperfusion injury and preconditioning. Am. J. Physiol. Gastrointest. Liver Physiol. 2003;284(1):G15-26. Pasupathy S, Homer-Vanniasinkam S. Ischaemic preconditioning protects against ischaemia/reperfusion injury: emerging concepts. Eur J Vasc Endovasc Surg. 2005;29(2):106-15, http://dx.doi.org/10.1016/j.ejvs.2004. 11.005. Eltzschig HK, Eckle T. Ischemia and reperfusion--from mechanism to translation. Nat Med. 2011;17(11):1391-401, http://dx.doi.org/10.1038/ nm.2507. Ali ZA, Callaghan CJ, Lim E, Ali AA, Nouraei SAR, Akthar AM, et al. Remote ischemic preconditioning reduces myocardial and renal injury after elective abdominal aortic aneurysm repair: a randomized controlled trial. Circulation. 2007;116(11 Suppl):I98-105. Hotter G, Closa D, Prados M, Ferna´ndez-Cruz L, Prats N, Gelpı´ E, et al. Intestinal preconditioning is mediated by a transient increase in nitric oxide. Biochem Biophys Res Commun. 1996;222(1):27-32, http://dx.doi. org/10.1006/bbrc.1996.0692. Neves J de S, Abraha˜o M de S, Salzedas Netto AA, Montero EF de S, Gonzalez AM. Effects of ischemic preconditioning associated to different preservation solutions in protecting the intestinal graft. Acta Cir Bras. 2011;26(5):396-403, http://dx.doi.org/10.1590/S0102-86502011000500013. Mallick IH, Yang W, Winslet MC, Seifalian AM. Ischemia-reperfusion injury of the intestine and protective strategies against injury. Dig Dis Sci. 2004;49(9):1359-77, http://dx.doi.org/10.1023/B:DDAS.0000042232. 98927.91. Tamion F, Richard V, Lacoume Y, Thuillez C. Intestinal preconditioning prevents systemic inflammatory response in hemorrhagic shock. Role of HO-1. Am J Physiol Gastrointest Liver Physiol. 2002;283(2):G408-414. Wu B, Ootani A, Iwakiri R, Fujise T, Tsunada S, Toda S, et al. Ischemic preconditioning attenuates ischemia-reperfusion-induced mucosal apoptosis by inhibiting the mitochondria-dependent pathway in rat small intestine. Am J Physiol Gastrointest Liver Physiol. 2004;286(4):G580-7, http://dx.doi.org/10.1152/ajpgi.00335.2003. Welborn MB 3rd, Douglas WG, Abouhamze Z, Auffenburg T, Abouhamze AS, Baumhofer J, et al. Visceral ischemia-reperfusion injury promotes tumor necrosis factor (TNF) and interleukin-1 (IL-1) dependent organ injury in the mouse. Shock. 1996;6(3):171-6. Hess PJ, Seeger JM, Huber TS, Welborn MB, Martin TD, Harward TR, et al. Exogenously administered interleukin-10 decreases pulmonary neutrophil infiltration in a tumor necrosis factor-dependent murine model of acute visceral ischemia. J Vasc Surg. 1997;26(1):113-8, http:// dx.doi.org/10.1016/S0741-5214(97)70154-X. Welborn MB 3rd, Moldawer LL, Seeger JM, Minter RM, Huber TS. Role of endogenous interleukin-10 in local and distant organ injury after visceral ischemia-reperfusion. Shock. 2003;20(1):35-40, http://dx.doi. org/10.1097/01.SHK.0000071062.67193.b6. Erling N Jr, Nakagawa NK, Costa Cruz JWM, Zanoni FL, Baptista-Silva JCC, Sannomiya P, et al. Microcirculatory effects of local and remote ischemic preconditioning in supraceliac aortic clamping. J Vasc Surg. 2010;52(5):1321-9, http://dx.doi.org/10.1016/j.jvs.2010.05.120. Moore-Olufemi SD, Kozar RA, Moore FA, Sato N, Hassoun HT, Cox CS Jr, et al. Ischemic preconditioning protects against gut dysfunction and mucosal injury after ischemia/reperfusion injury. Shock. 2005;23(3):258-63. DeSesso JM, Jacobson CF. Anatomical and physiological parameters affecting gastrointestinal absorption in humans and rats. Food Chem Toxicol. 2001;39(3):209-28, http://dx.doi.org/10.1016/S0278-6915(00) 00136-8.

26. Sola A, De Oca J, Gonza´lez R, Prats N, Rosello´-Catafau J, Gelpı´ E, et al. Protective effect of ischemic preconditioning on cold preservation and reperfusion injury associated with rat intestinal transplantation. Ann Surg. 2001;234(1):98-106, http://dx.doi.org/10.1097/00000658-2001070 00-00015. 27. Vlasov TD, Smirnov DA, Nutfullina GM. Preconditioning of the small intestine to ischemia in rats. Neurosci. Behav. Physiol. 2002;32(4):449-53, http://dx.doi.org/10.1023/A:1015896614819. 28. Tapuria N, Kumar Y, Habib MM, Abu Amara M, Seifalian AM, Davidson BR. Remote ischemic preconditioning: a novel protective method from ischemia reperfusion injury--a review. J Surg Res. 2008;150(2):304-30, http://dx.doi.org/10.1016/j.jss.2007.12.747. 29. Higa OH, Parra ER, Ab’Saber AM, Farhat C, Higa R, Capelozzi VL. Protective effects of ascorbic acid pretreatment in a rat model of intestinal ischemia-reperfusion injury: a histomorphometric study. Clinics. 2007;62(3):315-20, http://dx.doi.org/10.1590/S1807-5932200700 0300017. 30. Harkin DW, D’Sa AA, Yassin MM, Hoper M, Halliday MI. Gut mucosal injury is attenuated by recombinant bactericidal/permeability-increasing protein in hind limb ischemia-reperfusion injury. Ann Vasc Surg. 2001;15(3):326-31, http://dx.doi.org/10.1007/s100160010087. 31. Abraha˜o MS, Montero EFS, Junqueira VBC, Giavarotti L, Juliano Y, Fagundes DJ. Biochemical and morphological evaluation of ischemiareperfusion injury in rat small bowel modulated by ischemic preconditioning. Transplant Proc. 2004;36(4):860-2, http://dx.doi.org/10.1016/j. transproceed.2004.03.046. 32. Mallick IH, Yang W, Winslet MC, Seifalian AM. Ischaemic preconditioning improves microvascular perfusion and oxygenation following reperfusion injury of the intestine. Br J Surg. 2005;92(9):1169-76. 33. Downey JM, Davis AM, Cohen MV. Signaling pathways in ischemic preconditioning. Heart Fail Rev. 2007;12(3-4):181-8, http://dx.doi.org/ 10.1007/s10741-007-9025-2. 34. Kanoria S, Jalan R, Seifalian AM, Williams R, Davidson BR. Protocols and mechanisms for remote ischemic preconditioning: a novel method for reducing ischemia reperfusion injury. Transplantation. 2007;84(4): 445-58, http://dx.doi.org/10.1097/01.tp.0000228235.55419.e8. 35. Cheung MMH, Kharbanda RK, Konstantinov IE, Shimizu M, Frndova H, Li J, et al. Randomized controlled trial of the effects of remote ischemic preconditioning on children undergoing cardiac surgery: first clinical application in humans. J Am Coll Cardiol. 2006;47(11):2277-82, http:// dx.doi.org/10.1016/j.jacc.2006.01.066. 36. Hausenloy DJ, Mwamure PK, Venugopal V, Harris J, Barnard M, Grundy E, et al. Effect of remote ischaemic preconditioning on myocardial injury in patients undergoing coronary artery bypass graft surgery: a randomised controlled trial. Lancet. 2007;370(9587):575-9, http://dx.doi.org/10.1016/S0140-6736(07)61296-3. 37. Hoole SP, Heck PM, Sharples L, Khan SN, Duehmke R, Densem CG, et al. Cardiac Remote Ischemic Preconditioning in Coronary Stenting (CRISP Stent) Study: a prospective, randomized control trial. Circulation. 2009;119(6):820-7, http://dx.doi.org/10.1161/CIRCULATIONAHA.108. 809723. 38. Bøtker HE, Kharbanda R, Schmidt MR, Bøttcher M, Kaltoft AK, Terkelsen CJ, et al. Remote ischaemic conditioning before hospital admission, as a complement to angioplasty, and effect on myocardial salvage in patients with acute myocardial infarction: a randomised trial. Lancet. 2010;375(9716):727-34, http://dx.doi.org/10.1016/S0140-6736(09) 62001-8. 39. Walsh SR, Boyle JR, Tang TY, Sadat U, Cooper DG, Lapsley M, et al. Remote ischemic preconditioning for renal and cardiac protection during endovascular aneurysm repair: a randomized controlled trial. J Endovasc Ther. 2009;16(6):680-9, http://dx.doi.org/10.1583/09-2817.1.

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RAPID COMMUNICATION

Association between phase angle, anthropometric measurements, and lipid profile in HCV-infected patients Mariana de Souza Dorna,I Nara Aline Costa,I Erick Prado de Oliveira,II Ligia Yukie Sassaki,I Fernando Gomes Romeiro,I Sergio Alberto Rupp de Paiva,I Marcos Ferreira Minicucci,I Giovanni Faria SilvaI I

Universidade Estadual Paulista Ju´lio de Mesquita Filho (UNESP), Department of Internal Medicine, Botucatu/SP, Brazil. II School of Medicine, Federal University of Uberlandia, Uberlandia/MG, Brazil.

OBJECTIVE: The objective of this study was to investigate the associations between phase angle, anthropometric measurements, and lipid profile in patients chronically infected with the hepatitis C virus. METHODS: A total of 160 consecutive patients chronically infected with the hepatitis C virus and who received treatment at the hepatitis C outpatient unit of our hospital from April 2010 to May 2011 were prospectively evaluated. Bioelectrical impedance analysis, anthropometric measurements, and serum lipid profile analysis were performed. RESULTS: Twenty-five patients were excluded. A total of 135 patients with a mean age of 49.8¡11.4 years were studied. Among these patients, 60% were male. The phase angle and BMI means were 6.5¡0.8˚ and 26.5¡4.8 kg/ m2, respectively. Regarding anthropometric variables, mid-arm circumference, mid-arm muscle circumference, and arm muscle area had a positive correlation with phase angle. In contrast, when analyzing the lipid profile, only HDL was inversely correlated with phase angle. However, in multiple regression models adjusted for age and gender, only mid-arm circumference (p = 0.005), mid-arm muscle circumference (p = 0.003), and arm muscle circumference (p = 0.001) were associated with phase angle in hepatitis C virus-infected patients. CONCLUSIONS: In conclusion, phase angle is positively correlated with anthropometric measures in our study. However, there is no association between phase angle and lipid profile in these patients. Our results suggest that phase angle is related to lean body mass in patients chronically infected with hepatitis C virus. KEYWORDS: Phase Angle; Hepatitis C; Cholesterol; Anthropometry; Bioelectrical Impedance Analysis. Dorna MS, Costa NA, Oliveira EP, Sassaki LY, Romeiro FG, Paiva SA, et al. Association between phase angle, anthropometric measurements, and lipid profile in HCV-infected patients. Clinics. 2013;68(12):1555-1558. Received for publication on June 11, 2013; First review completed on July 7, 2013; Accepted for publication on July 18, 2013 E-mail: mari_dorna@yahoo.com.br Tel.: 014 3822-2969

known, there is not yet a gold standard tool for assessing body composition in these subjects (5). For more than 20 years, bioelectrical impedance analysis (BIA) has been used to assess body composition in several clinical situations. BIA measures the resistance to the movement of an electrical current through the body, allowing for the determination of the fat-free mass and total body water. The body offers two types of resistance to an electrical current, namely, capacitive Xc (reactance) and resistive (simply called resistance) resistance. One measurement of the relationship between capacitance and resistance is the phase angle (PhA), and its association with clinical outcomes has already been shown in different pathological situations (6–12). There are few studies that have evaluated the association between PhA, anthropometric measures, and outcomes in patients with chronic liver disease. Selberg et al. showed that PhA was poorly correlated with disease severity (6). However, in their study, PhA was correlated with muscle mass and strength (6). In addition, Kahraman et al. evaluated 37 consecutive patients with HCV infection and

& INTRODUCTION It is estimated that over 170 million people worldwide have hepatitis C in its chronic form (1). Currently, aside from the established consequence of liver injury, chronic hepatitis C virus (HCV) infection is associated with some metabolic disorders. Several studies have highlighted the association between HCV infection and lipid metabolism; however, the relationship between lipid profile and body composition in HCV carriers is less clear (2–4). Although the importance of nutritional assessment in this population is

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(12)13

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Phase angle and body composition Dorna MS et al.

showed that PhA reduction was associated with increased side effects following pegylated interferon-a and ribavirin treatment (12). Although BIA is non-invasive, easy-to-use, portable, and inexpensive, this device is not available at all centers. Thus, in this scenario, anthropometric measures are one method for evaluating body composition (5). Despite the association between PhA and anthropometric data, chronically infected HCV patients have not yet been evaluated. Therefore, the present study aimed to investigate the associations between PhA, anthropometric measures, and lipid profile in patients chronically infected with HCV.

CLINICS 2013;68(12):1555-1558

between continuous variables. Multiple linear regressions were used for PhA prediction and adjusted for gender and age. Data analysis was performed using Sigma Stat 3.5 for Windows (Systat Software, Inc., San Jose, CA, USA). P values lower than 0.05 were considered statistically significant.

& RESULTS Of the 160 consecutive patients initially evaluated, 25 were excluded (10 had incomplete data, 4 had not undergone liver biopsy, 8 had ascites, 1 was physically disabled, 1 had chronic kidney disease and 1 had heart failure). Thus, a total of 135 patients with a mean age of 49.8¡11.4 y were assessed. Demographic, anthropometric, and laboratory data are listed in Table 1. Of the included patients, 60% were male. The PhA and BMI means were 6.5¡0.8 ˚ and 26.5¡4.8 kg/m2, respectively. With regard to anthropometric variables, MAC, MAMC, and AMA presented a positive correlation with PhA. In contrast, when analyzing the lipid profile, only HDL was inversely correlated with PhA. However, in multiple regression models adjusted for age and gender, only MAC (p = 0.005), MAMC (p = 0.003), and AMA (p = 0.001) were associated with PhA in HCV-infected patients (Table 2).

& MATERIALS AND METHODS Subjects The present study was approved by the ethics committee of our institution, and written informed consent was obtained from all subjects. All patients chronically infected with HCV who underwent treatment at the hepatitis C outpatient unit of our hospital from April 2010 to May 2011 were prospectively evaluated. The inclusion criteria were naı¨ve patients or lack of treatment with pegylated interferon and ribavirin for at least 1 year, completion of liver biopsy, and clinical diagnosis of liver cirrhosis. The exclusion criteria were liver cirrhosis with ascites, hepatitis B virus infection, HIV infection, chronic kidney disease, heart failure, and pregnancy. Bioelectrical impedance analysis and anthropometric measurements were performed during the first hospital visit. The lipid profile was considered in cases where it had been measured up to three months prior to the evaluation.

& DISCUSSION The present study aimed to investigate the associations between PhA, anthropometric measures, and lipid profile in patients chronically infected with HCV. In our study, the mid-arm circumference, mid-arm muscle circumference, and arm muscle area were associated with PhA. There was no association between PhA and lipid profile in these patients. PhA has been suggested as an indicator of cellular health, where higher values reflect higher cellularity, better cell membrane integrity, and better cell function. In healthy adults, age, gender, and BMI are the major determinants of PhA (7). Because PhA is a marker of the amount and quality

Assessment of Body Composition Body height and weight were measured and used to calculate the body mass index (BMI) (13). Mid-arm circumference (MAC) was measured using a measuring tape, as previously described (14). Triceps skinfold (TSF) was measured according to the standardization of Harrison et al. (15). Mid-arm muscle circumference (MAMC) and arm muscle area (AMA) were obtained from the following respective formulas: MAMC = MAC-(p x TSF) and MAMC = MAC-px(TSF/10) (16). All measurements were performed by the same researcher.

Table 1 - Demographic, anthropometric, and laboratory data from 135 patients with HCV.

Bioelectrical Impedance Analysis Variables

BIA was conducted using a tetrapolar single-frequency apparatus (Biodynamic-450, Biodynamics Corporation, USA) that was applied to the skin using adhesive electrodes with the subject lying supine. PhA derived from the BIA was determined as previously described (17), and its values were calculated as follows: PhA = (arc tangent reactance/ resistance * (180 ˚/p)).

Male (n/%) Age (y) PhA ( ˚) Resistance (Ohms) Reactance (Ohms) HDL (g/dL) TG (g/dL) TC (g/dL) LDL (g/dL) BMI (kg/m2) MAC (cm) TSF (mm) MAMC (cm) AMA (mm2)

Lipid Profile Data Total cholesterol (TC), HDL-cholesterol (HDL), and triglycerides (TG) were measured using a dry-chemistry method. LDL-cholesterol (LDL) was calculated by the Friedwald formula.

Results 81 (60%) 49.8¡11.4 6.5¡0.8 538.4¡99.7 60.8¡11.2 50.6¡17.2 105 (79–142) 160.5¡34.7 85.2¡29.8 26.5¡4.8 33.3¡4.0 21 (13–30) 25.9 (23.7–28.7) 45.5 (38–56.2)

PhA: phase angle; total cholesterol: TC; HDL-cholesterol: HDL; triglycerides: TG; LDL-cholesterol: LDL; BMI: body mass index; MAC: midarm circumference; TSF: triceps skinfold; MAMC: mid-arm muscle circumference; AMA: arm muscle area. Data are expressed as the mean ¡ SD, median (lower to upper quartile), or percentage.

Statistical Analysis Data are expressed as the mean ¡ SD or the median (including the lower and upper quartiles). The Spearman correlation was employed to evaluate the association

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Phase angle and body composition Dorna MS et al.

the PhA and lipid profile in these patients has not yet been evaluated. In our study, PhA was not correlated with lipid profile when data were adjusted for age and gender. In agreement with our results, Santarpia et al. demonstrated the absence of a correlation between PhA and cholesterol in patients with advanced cancer (8). An important implication of our results is that when BIA is not available, anthropometric measures, which can be used to estimate lean body mass, could be an alternative method of evaluating body composition in patients chronically infected with HCV. Finally, we should consider the major limitations of the present study, namely, its small sample size and the fact that all of the patients had undergone treatment at a single medical center. In addition, for the BIA, we used a singlefrequency apparatus that may have led to biased results in patients with extracellular fluid overload. In conclusion, PhA was positively correlated with midarm circumference, mid-arm muscle circumference, and arm muscle area in our study. However, there was no association between PhA and lipid profile in these patients. Our results suggest that PhA is related to lean body mass in patients chronically infected with HCV.

Table 2 - Multiple linear regression models for phase angle prediction, adjusted for gender and age. Variables TC (mg/dL) HDL (mg/dL) LDL (mg/dL) TG (mg/dL) BMI (kg/m2) MAC (cm) TSF (mm) MAMC (cm2) AMA (mm2)

Coefficient

Standard Error

p-value

20.002 20.006 20.0002 20.001 0.0208 0.0400 0.0042 0.0564 0.0145

0.002 0.004 0.002 0.001 0.0118 0.0140 0.0062 0.0184 0.0042

0.189 0.075 0.899 0.212 0.079 0.005 0.500 0.003 0.001

Total cholesterol: TC; HDL-cholesterol: HDL; triglycerides: TG; LDLcholesterol: LDL; BMI: body mass index; MAC: mid-arm circumference; TSF: triceps skinfold; MAMC: mid-arm muscle circumference; AMA: arm muscle area.

of soft tissue mass and hydration, it has been considered a useful indicator of nutritional status (7). Several studies found a strong correlation between nutritional status and PhA in patients with gastrointestinal and renal diseases, older adult patients, and patients with anorexia nervosa (18–21). Maggiore et al. demonstrated in hemodialysis patients who albumin, age, subjective global assessment, and protein catabolic rate were associated with PhA (22). However, other studies failed to show these associations (7,23). Despite these controversies concerning the results, a low PhA is associated with malnutrition, and mortality in some disease states. In patients with liver diseases, the association between PhA and nutritional status is not well established. In a study of cirrhotic patients with multiple etiologies, Selberg et al. showed that PhA discriminated poorly between cirrhosis patients of different Child-Pugh classes, although it was positively correlated with muscle area mass and muscle strength (6). It is interesting to note that, in our study, there was no correlation between PhA and BMI, although PhA was positively correlated with MAMC and AMA. These results suggest that PhA is more closely correlated with lean body mass than with fat body mass in patients chronically infected with HCV. In addition to alterations in body composition, HCV infection and its treatment interfere with the lipid profile. Several studies showed that HCV infection may be associated with lower serum cholesterol and triglyceride levels (24–25). Some studies also demonstrated that the plasma lipid profile may be a predictor of therapeutic response in patients with HCV (26–27). Cholesterol and lipoproteins are required not only for the entry of HCV into hepatocytes but also for its viral assembly and replication (27). The LDL receptor was reported to facilitate HCV endocytosis into the liver; thus, high LDL may compete with HCV and limit the spread of the virus in hepatocytes (28). In addition, cholesterol is synthesized in the liver through the mevalonate pathway, which is also important for viral replication (2). As stated previously, higher PhA values were associated with better nutritional status. In addition, higher levels of serum lipids were correlated with better outcomes in patients with HCV infection. Thus, our hypothesis was that PhA may also be associated with lipid profile in these patients. Despite all these studies, the relationship between

& ACKNOWLEDGMENTS This work was supported by CAPES (‘‘Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior’’).

& AUTHOR CONTRIBUTIONS Dorna MS collected the data and contributed to the conception and design of the study. Costa NA and Oliveira EP collected the data. Sassaki LY and Romeiro FG contributed to the design of the study and translation of the manuscript into English. Paiva SA performed the statistical analysis. Minicucci MF and Silva GF contributed to the conception and design of the study.

& REFERENCES 1. Kawaguchi Y, Mizuta T, Oza N, Takahashi H, Ario K, Yoshimura T, et al. Eradication of hepatitic C virus by interferon improves whole-body insulin resistance and hyperinsulinaemia in patients with chronic hepatitis C. Liver Inter. 2009:29(6):871-7, http://dx.doi.org/10.1111/j. 1478-3231.2009.01993.x. 2. Butt AA, Umbleja T, Andersen JW, Sherman KE, Chung TR, ACTG A5178 Study Team. Impact of piginterferon alpha and ribavirin treatment on lipid profiles and insulin resistance in hepatitis C virus/ HIV-coinfected persons: The AIDS clinical trials group A5178 study. CID. 2012;55(5):631-8, http://dx.doi.org/10.1093/cid/cis463. 3. Kuo YH, Chuang TW, Hung CH, Cheng CH, Wang JH, Hu TH, et al. Reversal hypolipidemia in chronic hepatitis C patients after successful antiviral therapy. J Formos Med Assoc. 2012;110(6):363-71. 4. Corey KE, Kane E, Munroe C, Barlow LL, Zheng H, Chung RT. Hepatitis C virus infection and its clearance alter circulating lipids: implications for long term follow-up. Hepatology. 2009;50(4):1030-7, http://dx.doi. org/10.1002/hep.23219. 5. Campillo B. 2010. Assessment of nutritional status and diagnosis of malnutrition in patients with liver disease. In: Nutrition, Diet Therapy and The Liver, First Edition, ed. Preedy, VR; Lakshman R, Srirajaskanthan R, Watson RR. 33-46. CRC Press: Taylor&Francis Group. USA. 6. Selberg O, Selberg D. Norms and correlates of bioimpedance phase angle in healthy human subjects, hospitalized patients, and patients with liver cirrhosis. Eur J Appl Physiol. 2002;86(6):509-16. 7. Norman K, Stoba¨us N, Pirlich M, Bosy-Whestphal A. Bioelectrical phase angle and impedance vector analysis- clinical relevance and applicability of impedance parameters. Clin Nutr. 2012;31(6):854-61, http://dx.doi. org/10.1016/j.clnu.2012.05.008. 8. Santarpia L, Marra M, Montagnese C, Alfonsi L, Pasanisi F, Contaldo F. Prognostic significance of bioelectrical phase angle in advanced caˆncer: preliminar observations. Nutrition. 2009;25(9):930-1, http://dx.doi.org/ 10.1016/j.nut.2009.01.015.

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Phase angle and body composition Dorna MS et al.

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20. Norman K, Smoliner C, Kilbert A, Valentini L, Lochs H, Pirlich M. Disease-related malnutrition but not underweight by BMI is reflected by disturbed electric tissue properties in the bioelectrical impedance vector analysis. Br J Nutr. 2008;100(3):590-5. 21. Scalfi L, Marra M, Caldara A, Silvestri E, Contaldo F. Changes in bioimpedance analysis after stable refeeding of undernourished anorexic patients. Int J Obes Relat Metab Disord. 1999;23(2):133-7, http://dx.doi. org/10.1038/sj.ijo.0800780. 22. Maggiore Q, Nigrelli S, Ciccarelli C, Grimaldi C, Rossi GA, Michelassi C. Nutritional and prognostic correlates of bioimpedance indexes in hemodialysis patients. Kidney Int. 1996;50(6):2103-8, http://dx.doi. org/10.1038/ki.1996.535. 23. Gupta D, Lis CG, Dahlk SL, King J, Vashi PG, Grutsch J, et al. The relationship between bioelectrical impedance phase angle and subjective global assessment in advanced colorectal cancer. Nutr J. 2008;30(7):19, http://dx.doi.org/10.1186/1475-2891-7-19. 24. Perlemuter G, Sabile A, Letteron P, Voga G, Topillo A, Chre´tien Y, et al. Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: a model of viral-related steatosis. FASEB J. 2002;16(2):185-94, http://dx.doi.org/10. 1096/fj.01-0396com. 25. Hsu CS, Liu CJ, Liu CH, Chen CL, Lai MY, Chen PJ, et al. Metabolic profiles in patients with chronic hepatitis C: a case-control study. Hepatol Int. 2008;2(2):250-7, http://dx.doi.org/10.1007/s12072-008-90643. 26. Gopal K, Johnson TC, Gopal S, Wasfish A, Bang CT, Suwandhi P, et al. Correlation between beta-lipoprotein levels and outcome of hepatitis C treatment. Hepatology. 2006;44(2):335-40, http://dx.doi.org/10.1002/ hep.21261. 27. Kuo YH, Chuang TW, Hung CH, Chen CH, Wang JH, Hu TH, et al. Reversal of hypolipidemia in chronic hepatitis C patients after successful antiviral therapy. J Formos Med Assoc. 2011;110(6):363-71, http://dx. doi.org/10.1016/S0929-6646(11)60054-5. 28. Andre´ P, Komurian-Pradel F, Deforges S, Perret M, Berland JL, Sodoyer M, et al. Characterization of low- and very-low-density hepatitis C virus RNA-containing particles. J Virol. 2002;76(14):6919-28, http://dx.doi. org/10.1128/JVI.76.14.6919-6928.2002.

9. Barbosa-Silva MC, Barros AJ. Bioelectrical impedance analysis in clinical practice: a new perspective on its use beyond body composition equations. Curr Opin Clin Nutr Metab Care. 2005;8(3):311-7, http://dx. doi.org/10.1097/01.mco.0000165011.69943.39. 10. Kyle UG, Genton L, Pichard C. Low phase angle determined by bioelectrical impedance analysis is associated with malnutrition and nutritional risk at hospital admission. Clin Nutr. 2013;32(2):194-9. 11. Plauth M, Cabre´ E, Riggio O, Assis-Camilo M, Pirlich M, Kondrup J. Espen Guidelines on Enteral Nutrition: Liver Disease. Clin Nutr. 2006;25(2):285-94, http://dx.doi.org/10.1016/j.clnu.2006.01.018. 12. Kahraman A, Hilsenbeck J, Nyga M, Ertle J, Wree A, Plauth M, et al. Bioelectrical impedance analysis in clinical practice:implications for hepatitis C therapy BIA and hepatitis C. Virol J. 2010;16(7):191. 13. World Health Organization. Obesity:preventing and managing the global epidemic. Geneva: World Health Organization; 1997. 14. Callaway CW, Chumlea WC, Bouchard C, Himes JH, Lohman TG, Martin AD, et al. Circumferences. In: Lohman TG, Roche AF, Martorell R, editors. Anthropometric standardizing reference manual. Champaign, Il: Human Kinetics Books;1991.p.39-54. 15. Harrison GG, Buskirk EK, Carter JEL,Ohmston JFE, Lohman TG, Pollock ML, et al. Skinfold thicknesses and measurements technique. In: Lohman TG, Roche AF, Martorell R, editors. Anthropometric standardizing reference manual. Champaign, Il: Human Kinetics Books;1991. 55-80. 16. Frisancho AR. Anthropometric standards for the assessment of growth and nutritional status. Michigan: The University of Michigan Press; 1990.p.48-53. 17. Kyle UG, Soundar EP, Genton L, Pichard C. Can phase angle determined by bioelectrical impedance analysis assess nutritional risk? A comparison between healthy and hospitalized subjects. Clin Nutr. 2012;31(6):875-81. 18. Oliveira CM, Kubrusly M, Mota RS, Silva CA, Choukroun G, Oliveira VN. The phase angle and mass body cell as markers of nutritional status in hemodialysis patients. J Ren Nutr. 2010;20(5):314-20. 19. Norman K, Smoliner C, Valentini L, Lochs H, Pirlich M. Is bioelectrical impedance vector analysis of value in the elderly with malnutrition and impaired functionality? Nutrition. 2007;23(7-8):564-9, http://dx.doi.org/ 10.1016/j.nut.2007.05.007.

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ERRATA

& CLINICS 2013;68(9):1231-8 Xiao Li Jing is the corresponding author.

& CLINICS 2013;68(8):1128-33 Page 1132 Replace ACKNOWLEDGMENTS This study was supported by the Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Brazil, and by the U.S. Centers for Disease Control and Prevention, Atlanta, GA, USA. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention or the Agency for Toxic Substances and Disease Registry. For ACKNOWLEDGMENTS This study was supported by the Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Brazil, by FAPESP (grant # 2013/08308-4), and by the U.S. Centers for Disease Control and Prevention, Atlanta, GA, USA. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention or the Agency for Toxic Substances and Disease Registry.

& CLINICS 2012;67(6):629-37 Page 629 (Abstract) Replace RESULTS: The degradation of the white scaffold was significantly lower compared with the dark scaffold but was within the acceptable time range for bone-healing processes. The deoxyribonucleic acid and collagen contents increased up to day 28 with no significant difference between the two scaffolds, but the glycosaminoglycan content was slightly higher in the white scaffold throughout 14 days of incubation. Scanning electron microscopy at days 1 and 14 revealed cellular growth and attachment. For RESULTS: The degradation of the white scaffold was significantly lower compared with the dark scaffold but was within the acceptable time range for bone-healing processes. The deoxyribonucleic acid and collagen contents increased up to day 28 with no significant difference between the two scaffolds, but the glycosaminoglycan content was slightly higher in the white scaffold throughout 14 days of incubation. Scanning electron microscopy at day 1 revealed cellular growth and attachment.

Page 631 (Scanning Electron Microscopy (SEM)) Replace Samples of unseeded and seeded (day 1 and 14) white and dark PCLTF scaffolds were processed for SEM. The samples were fixed in 4% glutaraldehyde followed by 1% osmium tetroxide and dehydrated with an ethanol series to 100% before being gold coated. The images of BMSC attachments and cell interactions between each group were compared. For Samples of unseeded and seeded (day 1) white and dark PCLTF scaffolds were processed for SEM. The samples were fixed in 4% glutaraldehyde followed by 1% osmium tetroxide and dehydrated with an ethanol series to 100% before being gold coated. The images of BMSC attachments and cell interactions between each group were compared.

Page 634-5 (Scanning Electron Microscopy (SEM)) Replace The interaction of BMSCs with both PCLTF scaffolds at days 1 and 14 was qualitatively characterized using SEM. The interaction test shows no advantage between white and dark PCLTF scaffolds at these time points. The unseeded scaffolds (both white and dark) have interconnected micropores, as shown in Figure 5A and 5B. The seeded BMSCs attached firmly to the scaffold surface by day 1 after incubation (Figures 5C and D). By day 14 after incubation, a well-formed cellular layer with active extracellular matrix secretion was visible (Figures 5E and F). For The interaction of BMSCs with both PCLTF scaffolds at day 1 after seeding was qualitatively characterized using SEM. The interaction test shows no advantage between white and dark PCLTF scaffolds at these time points. The unseeded scaffolds (both white and dark) have interconnected micropores, as shown in Figure 5A and 5B. The seeded BMSCs attached firmly to the scaffold surface by day 1 after incubation (Figures 5C and D).

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(12)14

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Page 635 Replace Figure 5 For

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