Clinics E-Book September 2011 by eBook Clinics

CLINICS Editor Mauricio Rocha-e-Silva Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Area Editors Anuar Ibrahim Mitre Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

Heitor Franco de Andrade Jr. 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

Berenice Bilharinho Mendonça Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

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

Ruy Jorge Cruz Junior University of Pittsburgh Pittsburgh, PA, USA

Bruno Zilberstein 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

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

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

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

Silvia Vanessa Lourenço Faculdade de Odontologia da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

Maria Cecı´lia Solimene Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Sophie Franc¸oise Mauricette Derchain Faculdade de Cieˆncias Me´dicas, Universidade Estadual de Campinas Campinas, SP, Brazil

Newton Kara-Junior Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Suely Kazue Nagahashi Marie 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

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

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

Vale´ria Aoki Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Edmund Chada Baracat Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Eliete Bouskela Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil Eloisa Silva Dutra de Oliveira Bonfa´ Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Geraldo Busatto Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Gustavo Franco Carvalhal Faculdade de Medicina da Pontifıćia Universidade Cato´lica do Rio Grande do Sul Porto Alegre, Rio Grande do Sul, Brazil

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

Editorial Board Abhijit Chandra King George’s Medical College Lucknow, India Adamastor Humberto Pereira Universidade Federal do Rio Grande do Sul Porto Alegre, RS, Brazil Adauto Castelo Universidade Federal de Saõ Paulo Saõ Paulo, SP, Brazil Ademar Lopes Fundaçaõ Antoˆnio Prudente, Hospital do Caˆncer Saõ Paulo, SP, Brazil

Daniel Romero Munõz Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Edmund Neugebauer Witten/Herdecke University Witten, North Rhine - Westphalia, Germany Egberto Gaspar de Moura Jr. Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil Ernest Eugene Moore University of Colorado Denver Denver, CO, USA

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

Euclides Ayres Castilho 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

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

Andrea Schmitt University of Goettingen Goettingen, Germany

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

Arnaldo Valdir Zumiotti Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Artur Brum-Fernandes Universite´ de Sherbrooke Que´bec, Canada´ Ashok Agarwal The Cleveland Clinic Foundation Cleveland, Ohio, USA Carmita Helena Najjar Abdo Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Cesar Gomes Victora Faculdade de Medicina da Universidade Federal de Pelotas Pelotas, RS, Brasil

Hiroyuki Hirasawa Chiba University School of Medicine Chiba, Japan Irismar Reis de Oliveira Faculdade de Medicina da Universidade Federal da Bahia Salvador, BA, Brasil Irshad Chaudry University of Alabama Birmingham, AL, USA Ivan Cecconello Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ke-Seng Zhao Southern Medical University Guangzhou, China

Laura Cunha Rodrigues London School of Hygiene and Tropical Medicine - University of London London, UK Marcelo Zugaib Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Marco Martins Amatuzzi Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Maria Aparecida Shikanai Yasuda Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Mauro Perretti William Harvey Research Institute London, UK Michael Gregory Sarr Mayo Clinic Rochester, MN, USA Milton de Arruda Martins Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

Olavo Pires de Camargo Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Pedro Puech-Leaõ Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Peter Libby Brigham and Women’s Hospital Boston, Boston, MA, USA Philip Cohen University of Houston Health Center Houston, Texas, USA Rafael Andrade-Alegre Santo Toma´s Hospital Republic of Panama´, Panama´ Ricardo Antonio Refinetti Faculdade de Medicina da Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil Roberto Chiesa San Raffaele Hospital Milan, Italy Ronald A. Asherson Netcare Rosebank Hospital Rosebank, Johannesburg, South A´frica

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

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

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

Tarcisio Eloy Pessoa de Barros Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

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

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

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

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

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

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

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

Editorial Assistant Nair Gomes Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

CEP 05403-010 Sa˜o Paulo/SP Tel.: +55-11-3069-6235 Submission: http://mc.manuscriptcentral.com/clinics

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

PUBLICATION INFORMATION AND EDITORIAL POLICIES CLINICS publishes peer-reviewed articles of interest to clinicians and researchers in the medical sciences. CLINICS is registered with PubMed Central and SciELO. This complies with the policies of funding agencies, such as, the Wellcome Trust, the Research Councils UK - RCUK, the National Institutes of Health (NIH), and the DFG, German Research Foundation, which request or require deposition of the published articles that they fund into such publicly available databases. CLINICS supports the position of the International Committee of Medical Journal Editors (http://www.icmje.org/) on trial registration. All trials initiated after January 1, 2010 must be registered prospectively (before patient recruitment begins) in a publicly accessible registry. Trials initiated before January 1, 2007 must be registered before submission to our journals. See the ICMJE faq on trial registration for further details. Visit http://www.who.int/ ictrp/network/list_registers/en/index.html for the WHO’s list of approved registries. CLINICS suggests: http://www.clinicaltrials. gov/, a user friendly site.

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Books: Turner WW, Valentine RJ, Wright CB. Acute vascular insufficiency due to drug injection. In: Rutherford RB, editor. Vascular Surgery. 4th ed. Philadelphia: Saunders; 1995. p. 680.

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Journal articles: Wolosker N, Nakano L, D’Hippolito G, Rosoky RA, Borri ML, Wolosker AM. Gadolinium magnetic angioresonance in the study of aortoiliac disease. Angiology. 2003;54:163–8. Lee JT, Ling D, Heiken JP, Glazer HS, Sicard GA, Totty WG, et al. Magnetic resonance imaging of abdominal aortic aneurysms. Am J Roentgenol. 1984;143:1197–202. Citation of unpublished material or personal communication. should be noted parenthetically in the text and not added to the reference list. TABLES: Should be incorporated into the Main Document, after the end of the reference list. Tables should be constructed using the Table feature in your word processor or using a spreadsheet program such as Excel. Tables should be numbered in order of appearance in the text with Arabic numerals. Each table should have a title and, if necessary, an explanatory legend. All tables must be referred to and succinctly described in the text. Under no circumstances should a table repeat data that are presented in an illustration. Statistical measures of variation (i.e., standard deviation, standard error) should be identified, and decimal places in tabular data should be restricted to those with mathematical and statistical significance. FIGURES: Photographs, illustrations, charts, drawings, line graphs, etc are all defined as Figures. We do not publish Pictures Graphics, Photos, etc. Number your figures consecutively in Arabic numerals in order of appearance. Upload each Figure individually into the system. Do not incorporate Figures into the Main Document. Legend(s) should be descriptive and allow examination of the figure without reference to text. Legends should be incorporated into the Main Document, after the Tables (if any) or after the references. Photographic illustrations should should be of professional quality and uploaded as *.jpeg files, and. They must be clear even after reduction in size for publication. Typewritten or hand lettering is unacceptable, as are figures generated by dot matrix printers. Generally, figures will be reduced to fit one column of text. Actual magnification of all photomicrographs should be provided, preferably by placing a length scale on the print. Line graphs and charts should never be sent as jpeg illustrations. It is usually best to prepare these as ExcelH files. When ready copy the line graph or chart to a word.doc sheet. For each submitted line graph or chart, upload the corresponding ExcelH data matrix as a supplementary file. Identify it as ‘‘Fig_N_excel’’ and upload as supplementary file. TITLE PAGE: Please submit a separate file containing: (A) the full title; (B) a running title of not more than 45 characters; (C) Scientific address where project was executed; (D) authors in the order in which they are to appear if the article if accepted opposite the name of each author specify his her scientific address; (C) his/her contribution for the project and production of the article. Authors should have participated sufficiently in the work to take public responsibility for appropriate portions of the content; (E) Acknowledgments: Contributors who do not meet the criteria for authorship should be listed in the acknowledgments section. All

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ISSN-1807-5932

CLINICS CONTENTS Clinics 2011 66(9):1513–1663

CLINICAL SCIENCES

Adiposity and postural balance control: Correlations between bioelectrical impedance and stabilometric signals in elderly Brazilian women ´ rika de Carvalho Rodrigues, Juliana Fla´via de Oliveira, Arthur de Sa´ Ferreira, Mı´riam Raquel Meira Mainenti, E Cristina Ma´rcia Dias, Andre´ Luı´s dos Santos Silva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1513

The prevalence of aminoglycoside-modifying enzyme genes (aac (69)-I, aac (69)-II, ant (20)-I, aph (39)-VI) in Pseudomonas aeruginosa Farzam Vaziri, Shahin Najar Peerayeh, Qorban Behzadian Nejad, Abbas Farhadian . . . . . . . . . . . . . . . . . . . . . . . 1519

Impaired hemodynamic response to mental stress in subjects with prehypertension is improved after a single bout of maximal dynamic exercise Renata Frauches Medeiros, Bruno Moreira Silva, Fabricia Junqueira Neves, Natalia Galito Rocha, Allan Robson Kluser Sales, Antonio Claudio Nobrega . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1523

Prevalence of metabolic syndrome and related factors in Taiwanese high-tech industry workers Tzung-Yi Tsai, Jung-Feng Cheng, Yu-Min Lai . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1531

Organic grape juice intake improves functional capillary density and postocclusive reactive hyperemia in triathletes Mariana Correa Gonc¸alves, Flavia Fioruci Bezerra, Elis Cristina de Araujo Eleutherio, Eliete Bouskela, Josely Koury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1537

Decreased high-density lipoprotein cholesterol levels in polyarticular juvenile idiopathic arthritis Roberta Gonc¸alves Marangoni, Andre L. Hayata, Eduardo F. Borba, Pedro M. Azevedo, Eloisa Bonfa´, Claudia Goldenstein-Schainberg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1549

Acute exercise improves cognition in the depressed elderly: the effect of dual-tasks Paulo Eduardo Vasques, Helena Moraes, Heitor Silveira, Andrea Camaz Deslandes, Jerson Laks . . . . . . . . . . . . . . 1553

Antibody response following Hepatitis B vaccination in peritoneal dialysis patients: does normalized urea clearance matter? Erkan Dervisoglu, Melih Simsek, Ahmet Yilmaz. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1559

A comparative study of pelvic floor muscle training in women with multiple sclerosis: its impact on lower urinary tract symptoms and quality of life Ade´lia Correia Lu´cio, Maria Carolina Perissinoto, Ricardo Aydar Natalin, Alessandro Prudente, Benito Pereira Damasceno, Carlos Arturo Levi D’ancona . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1563

End of life in the neonatal intensive care unit Helena Moura, Vera Costa, Manuela Rodrigues, Filipe Almeida, Teresa Maia, Hercı´lia Guimara˜es . . . . . . . . . . . . 1569

Is allergic rhinitis a trivial disease? Dirceu Sole´, Ineˆs Cristina Camelo-Nunes, Gustavo F. Wandalsen, Nelson A. Rosa´rio, Emanuel C. Sarinho, Brazilian ISAAC Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1573

Endoscopic ultrasound-guided biopsies for mediastinal lesions and lymph node diagnosis and staging Jose´ Celso Ardengh, Ricardo H. Bammann, Matheus de Giovani, Filadelfio Venco, Artur A. Parada . . . . . . . . . . . 1579

Why are there defaulters in eye health projects? Regina Noma, Regina de S. Carvalho, Newton Kara-Jose´ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1585

Analysis of angiogenic factors and cyclooxygenase-2 expression in cartilaginous tumors – clinical and histological correlation Francisco Fontes Cintra, Mauricio Etchebehere, Jose´ Carlos Barbi Gonc¸alves, Alejandro Enzo Cassone, Eliane Maria Ingrid Amstalden . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1591

Prenatal tobacco exposure is related to neurobehavioral modifications in infants of adolescent mothers Marina C. M. Barros, Sandro S. Mitsuhiro, Elisa Chalem, Ronaldo R. Laranjeira, Ruth Guinsburg . . . . . . . . . . . . 1597

BASIC RESEARCHES

The effects of antidepressants and pilocarpine on rat parotid glands: an immunohistochemical study ˆ ngela Naval Machado, Tatiana Maria Folador Mattioli, Silvana da Silva, Ana Maria Trindade Gre´gio, Maria A Antoˆnio Adilson Soares de Lima, Luciana Reis Azevedo-Alanis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1605

High-energy extracorporeal shockwave therapy in a patellar tendon animal model: a vascularizationfocused study Fernando Travaglini Penteado, Fla´vio Faloppa, Guilherme Giusti, Vinıćius Ynoe Moraes, Joaõ Carlos Belloti, Joaõ Baptista Gomes dos Santos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1611

Exercise training attenuates acute hyperalgesia in streptozotocin-induced diabetic female rats Denise M. Rossi, Vitor E. Valenti, Marcelo T. Navega . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1615

The effects of pneumoperitoneum and controlled ventilation on peritoneal lymphatic bacterial clearance: experimental results in rats Armando Angelo Casaroli, Lycia M. J. Mimica, Belchor Fontes, Samir Rasslan . . . . . . . . . . . . . . . . . . . . . . . . . . 1621

REVIEW

Brazilian guidelines for the diagnosis and treatment of hereditary angioedema Pedro Giavina-Bianchi, Alfeu T. Franc¸a, Anete S. Grumach, Abı´lio A. Motta, Fa´tima R. Fernandes, Regis A. Campos, Solange O. Valle, Nelson A. Rosa´rio, Dirceu Sole´ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1627

RAPID COMMUNICATIONS

Hyperglycemia and postoperative outcomes in pediatric neurosurgery Eduardo Mekitarian Filho, Werther Brunow de Carvalho, Se´rgio Cavalheiro, Nelson Kazunobu Horigoshi, Norberto Antonio Freddi, Gil Kruppa Vieira . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1637

Association of MMP-8 polymorphisms with tendinopathy of the primary posterior tibial tendon: a pilot study Alexandre Leme Godoy-Santos, Rafael Trevisan, Tu´lio Diniz Fernandes, Maria Cristina L. G. dos Santos . . . . . . . 1641

CASE REPORTS

Paroxysmal atrial fibrillation and intermittent left bundle branch block: an unusual electrocardiographic presentation of mad honey poisoning Ayhan Saritas, Hayati Kandis, Davut Baltaci, Ismail Erdem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1651

Choreoathetosis after subarachnoid hemorrhage related to an aneurysm of the posterior fossa Ju´lio Leonardo Barbosa Pereira, Lucas Alverne Freitas de Albuquerque, Mauro Cruz Machado Borgo, Gerival Vieira Junior, Paulo Pereira Christo, Gerva´sio Teles C. de Carvalho . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1655

Extracorporeal membrane oxygenation as a bridge to pulmonary transplantation in Brazil: Are we ready to embark upon this new age? Marcelo Park, Eduardo Leite Vieira Costa, Luciano Cesar Pontes Azevedo, Jose´ Eduardo Afonso Junior, Marcos Naoyuki Samano, Carlos Roberto Ribeiro Carvalho, Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1659

ERRATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1663

CLINICS 2011;66(9):1513-1518

DOI:10.1590/S1807-59322011000900001

CLINICAL SCIENCE

Adiposity and postural balance control: Correlations between bioelectrical impedance and stabilometric signals in elderly Brazilian women Mı´riam Raquel Meira Mainenti, E´rika de Carvalho Rodrigues, Juliana Fla´via de Oliveira, Arthur de Sa´ Ferreira, Cristina Ma´rcia Dias, Andre´ Luı´s dos Santos Silva Augusto Motta University Center (UNISUAM) - Postgraduate Program of Rehabilitation Sciences, Rio de Janeiro, Brazil.

OBJECTIVE: The purpose of this study was to investigate the correlation between body adiposity and postural control in elderly women. INTRODUCTION: Aging and obesity account for a significant portion of healthcare spending. Life expectancy is increasing worldwide, and Rio de Janeiro has the largest proportion of elderly residents of all Brazilian states. METHODS: A total of 45 women underwent bioelectrical impedance analysis, waist circumference measurements, weight and height measurements, and stabilometric tests in eight different stance conditions (opened and closed bases with both eyes opened and closed and right and left tandem and unilateral stances with eyes opened). During unilateral stances, the number of hand or foot contacts was counted. RESULTS: Weight, body mass index, waist circumference, fat percentage, and fat mass showed statistically significant (p,0.05) and positive correlations with the number of contacts made during unilateral stances. The subjects with greater fat mass showed significantly higher anterior-posterior standard deviation and range when their eyes were closed. The sway area was also greater for this group in opened base when their eyes were closed. DISCUSSION: The results relating body adiposity and postural control can be explained by the difficulty of maintaining a greater quantity of body fat mass within the limits of the individual support base, especially while assuming a unilateral stance. CONCLUSION: The subjects with a greater fat mass exhibited poor balance control, indicating that body adiposity level was associated with postural control in the elderly women examined in the present study. KEYWORDS: Body composition; Body fat distribution; Biomechanics; Musculoskeletal equilibrium; Aging. Mainenti MRM, Rodrigues EC, Oliveira JF, Ferreira AS, Dias CM, Silva ALS. Adiposity and postural balance control: Correlations between bioelectrical impedance and stabilometric signals in elderly Brazilian women. Clinics. 2011;66(9):1513-1518. Received for publication on January 25, 2011; First review for publication on March 28, 2011; Accepted for publication on May 12, 2011 E-mail: miriam.mainenti@hotmail.com Tel.: 55 21 3882-9841

established that aging is associated with modifications in body composition, including decreased lean mass4,5 and increased truncal fat.6 This ‘‘sarcopenic obesity’’ influences normal walking speed and mobility disability,7 modifies balance control,8 and decreases stability limits in those with advanced obesity.9 The body mass index (BMI) is a simple proportion of weight-for-height that is commonly used to classify individuals as overweight or obese. Despite the fact that it is a widely accepted measurement,10 BMI is unable to separately quantify body fat composition. The use of body fat measures instead of weight measures is preferable for determining an individual’s possible health risks for cardiovascular diseases even when weight is corrected for height. The bioelectrical impedance method is a fast, noninvasive, easy-to-implement, and low-cost technique that is used to estimate body fat.11 This method requires little collaboration from the patient and reduces inter- and

INTRODUCTION The number of individuals over age 60 is increasing worldwide. In Rio de Janeiro, Brazil, 12.8% of the population is considered to be elderly, and nationwide statistics show that the number of elderly people living alone increased by 16% between 1991 and 2000.1 The number of obese individuals in Brazil is also increasing; 38.8 million Brazilian adults are either overweight or obese, corresponding to 40% of the adult population.2 In the United States, 66.3% of all adults over 20 years old and 71% of those over 60 years old are overweight or obese.3 It has been well

Copyright ß 2011 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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intra-observer errors;11 in addition, the equipment is lightweight, portable, and produces fast results.12 In this method, an imperceptible electric current is applied via surface electrodes and passes through the body; the conductance is then measured. Impedance measurements allow fat-free mass and fat mass to be estimated using validated equations.11,13 Another measurement that is positively correlated with intra-abdominal fat mass is waist circumference, which constitutes a simple method of fat mass measurement that is frequently used by health professionals. Waist circumference is strongly associated with truncal fat, regardless of the site of measurement chosen,14 and increases with age.15 Quiet standing postural control is possible, due to the integration of different sensory systems that deliver information to the central nervous system about how body parts are placed relative to the environment. Stabilometry, an objective method used to evaluate body stability, measures successive two-dimensional coordinates based on the center-of-foot pressure on a force platform and calculates a variety of variables.16 One of the most dangerous aspects of changes in body stability for the elderly is an increased risk of falls. This concern explains the focus on postural balance and aging in the literature over the last ten years;17-21 many researchers have attempted to understand which characteristics significantly disturb balance control and, consequently, tend to increase the incidence of falls. The evaluation of the relationship between balance and adiposity in elderly subjects is important for providing information to health professionals about the characteristics that influence balance control. Aging and obesity are conditions that account for a significant portion of healthcare spending.22 Therefore, this research may promote progress in the treatment and prevention of falls, indirectly enhancing the quality of life of elderly individuals. The aim of the present study was to investigate the association between body adiposity as analyzed by body composition and distribution measurements and postural control in elderly women living in Rio de Janeiro, Brazil. It was hypothesized that subjects with a higher level of body adiposity would have a greater center-of-foot pressure displacement during quiet stance.

limbs, as assessed by physical examination (inspection and palpation); and 6) when the recommendations described below were not followed.

Recommendations and Preliminary Evaluations Body composition and stabilometric assessments were carried out at the Laboratory of Human Movement Analysis (Postgraduate Program of Rehabilitation Sciences, UNISUAM). When the evaluation was scheduled, the following recommendations were discussed with each subject: 1) no alcohol consumption or exercise within 24 hours prior to taking the test; 2) no caffeine or food consumption for four hours prior to the test; 3) the consumption of two-four glasses of water within the two hours prior to the test; 4) bathroom use within the 30 minutes prior to the assessment. The subjects’ weight, rounded to the nearest 0.1 kg, was measured using an analog balance scale (R110, Welmy, Santa Ba´rbara d’Oeste, Sa˜o Paulo, Brazil), and the subjects’ height, rounded to the nearest 0.005 m, was also assessed using this device, which includes a stadiometer. Using the weight and height measurements, the subject’s BMI was calculated using the standard method (BMI = Weight/ Height2).10 Lower limb dominancy was also assessed by asking subjects which leg they preferred to use for kicking a ball.

Bioelectrical Impedance Analysis Body composition analysis was performed using a bioelectrical impedance analyzer (BIA 310e, Biodynamics, Seattle, Washington, USA). The test current was set at 800 mA and 50 kHz, which were well below the Association for Advancement of Medical Instrumentation’s standard for "Safe Current Limits". The tetrapolar resistance and reactance measurements were collected in a standardized manner; the subjects were asked to rest for five minutes prior to the exam on an examination table. They stood barefoot without any metal objects close to them, and the feet and hands were at least 30 cm and 15 cm apart, respectively. Two electrodes were applied to the dorsal surface of the right hand, and two electrodes were placed on the dorsal surface of the right foot. Resistance and reactance provided by the analyzer were used to estimate the fat-free mass (kg). Additional body composition variables were analyzed, including fat mass (kg) and fat percentage (%). The equation selected to predict the fat-free mass23 was previously validated in elderly samples,11,24 including a Brazilian sample of subjects aged 60-81 years:11

MATERIALS AND METHODS Study Protocol and Subjects This cross-sectional study evaluated 45 women enrolled in the program ‘‘Open University for the Elderly’’ (Universidade Aberta a` Terceira Idade, UNATI) at the Augusto Motta University Center (Centro Universita´rio Augusto Motta, UNISUAM) in Bonsucesso, a northern region of Rio de Janeiro, Brazil. Inclusion criteria were as follows: women over 60 years old who were participating in the UNATI program. For exclusion purposes, all of the elderly women underwent a screening evaluation before the main evaluation, which was conducted by the examiner. Exclusion criteria were as follows: 1) the presence of any implantable electronic or metallic device, such as a pacemaker; 2) musculoskeletal impairments, such as hip or knee pain during 60 seconds of orthostatic posture; 3) neurological diseases, such as stroke, Parkinson disease, Alzheimer disease and amyotrophic lateral sclerosis; 4) acute dizziness; 5) abnormal accumulation of fluid (edema), mainly in the

FFM ~ { 4:104 z 0:518 H2 =R z 0:231 ðBWÞ z 0:130 ðXcÞ z 4:229 ðGÞ, where FFM = fat-free mass (kg), H = stature (cm), R = resistance (V), BW = body weight (kg), Xc = reactance (V) and G = gender (0 for females and 1 for males). Waist circumference was also measured at the narrowest point between the lower costal border and the iliac crest.25 A flexible steel tape (Terrazul, Cambuci, Sa˜o Paulo, Brazil) was used to verify this girth to the nearest 0.1 cm.

Stabilometry Postural sway was quantified using a force platform (AccuSway Plus, AMTI, Watertown, Massachusetts, USA),

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support during right or left unilateral stances was higher in the Low Fat Mass Group (Table 1). The High Fat Mass Group showed significantly higher values for anterior-posterior standard deviation and range during OBEC and CBEC. The effective area was also greater for this group during OBEC (Table 2). A statistically significant difference in path length and average velocity was not observed between groups in any trial. Right and left tandem variables were analyzed only for those participants who used no foot or hand support to avoid falls. For this reason, the sample size for this investigation was smaller (19 vs. 15 and 19 vs. 16, respectively) (Table 2). When the whole sample was considered, fat percentage and fat mass showed significant and positive correlations with the number of contacts during right and left unilateral stances even when controlled for age and height. Weight, BMI, and waist circumference were also statistically correlated with contacts used during left unilateral stance and non-dominant unilateral stance (Table 3). Only eight individuals performed the right unilateral stance without foot or hand contacts, and eleven attained the left unilateral stance without foot or hand contacts. Because the sample size was small (eight and eleven), the stabilometric data were not analyzed for these trials.

and center-of-foot pressure signals were recorded using Balance Clinic Software (AMTI, USA). All participants performed the following eight trials: opened base, eyes open (OBEO); opened base, eyes closed (OBEC); closed base, eyes open (CBEO); closed base, eyes closed (CBEC); tandem right, eyes open; tandem left, eyes open; right unilateral stance, eyes open; left unilateral stance, eyes open. They were asked to maintain a standing posture while remaining as still as possible and to look straight ahead at a specific point on the wall at their eye level when their eyes were opened. Each trial lasted 60 seconds, and a randomized block design (four possible sequences of the described trials) was used to minimize fatigue and learning effects. The calculated stabilometric variables included the lateral standard deviation, anterior-posterior standard deviation, lateral range, anterior-posterior range, effective area, path length, and average velocity. During tandem and unilateral stances, the number of foot or hand contacts needed by the subject to avoid a fall was also recorded.

Statistical Analysis Variable distributions were analyzed using KolmogorovSmirnov tests; if a meaningful number of variables did not have a normal distribution, nonparametric tests were selected. Variable values are presented as the median (first - third quartile). The sample was divided into two groups using the fat mass median. The Low Fat Mass Group was comprised of those elderly women with a fat mass #29.08 kg. The High Fat Mass Group was comprised of subjects with a fat mass .29.08 kg. Two different analyses were performed; the Low Fat Mass Group was compared with the High Fat Mass Group using the Mann-Whitney Test for numerical variables and the chi-square test for categorical variables. The correlation between body composition variables and the number of foot and hand contacts required to avoid falls was evaluated using the Spearman Correlation Test.26 Statistical Package for Social Sciences (SPSS) 13.0 software for Windows was used for the statistical analysis, and significance was assigned when the p-value was less than 0.05.

DISCUSSION This study showed that an association exists between body adiposity and postural balance control. Greve and coworkers8 analyzed the association between BMI and a general instability index in another type of sample (young male subjects), and their results showed a positive correlation that corroborates the data presented here. Barbosa and co-workers27 evaluated BMI in relation to selected physical performance tasks, and their results showed a significant association between balance and BMI for elderly women (60-79 years old). Although the studies cited above indicate that a relationship exists between fatness and balance, BMI has a poor diagnostic performance for identifying excess body fat, particularly in the elderly.28 The distinction between fat and lean mass has considerable importance because these factors are directly associated with the independence of elderly individuals.11 A study carried out in Canada showed that by 60 to 69 years of age, the percentage of subjects at high risk for health problems (classified using waist circumference values) was more than twice as high as that of individuals between 20 and 39 years (65% for females).15 The results reported here revealed a significant association between waist circumference and the number of foot or hand contacts needed by the subject to avoid a fall during left unilateral stance. Whether the association between waist circumference and instability is a result of an increased upper circumference area based on the inverted pendulum model29 or the increased total mass that must be controlled by the neuromuscular system is still unclear and should be investigated with prospective studies. Bioelectrical impedance analysis, a method previously used in elderly subjects,5,11,23 showed a high median fat percentage (44.28%), classifying the group as obese ($35% for women, independent of age),30 as observed in other studies evaluating elderly individuals.5,11 The fat-free mass results were similar to those of another study carried out in

Ethics According to the recommendations of the Helsinki Declaration, the protocol was approved by the UNISUAM Ethics Committee (protocol number 003/10), and written informed consent was obtained from all participants before their participation in this study.

RESULTS General and anthropometric characteristics and body composition variables derived from bioelectrical impedance measurements of the studied sample are presented in Table 1. As expected, the High and Low Fat Mass Groups were statistically different in terms of their weight, BMI, waist circumference, fat percentage, fat mass, and fat-free mass. Subject age, a possible confounding variable, was not statistically different between these groups. There was a considerable number of women with right leg dominance (88.89%) in the sample, and, when separated into Low and High Fat Mass Groups, this pattern was preserved (86.96% and 90.91%, respectively). Although not statistically significantly different, the number of subjects not using any

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Table 1 - Anthropometric characteristics of the studied sample. Variable Weight (kg) 2

BMI (kg/m ) Age (years) Waist (cm) Fat % Fat mass (kg) FFM (kg) Right leg dominance Subjects not using support during RUS Subjects not using support during LUS

Sample (n = 45)

Low Fat Mass Group (n = 23)

High Fat Mass Group (n = 22)

67.00 (59.10-77.10) 28.08 (25.30-32.60) 66 (62-72) 89.00 (82.30-93.50) 44.28 (41.64-48.44) 29.08 (25.26-36.57) 37.16 (34.35-39.63) 40 (88.89%) 8 (17.78%) 11 (24.44%)

59.20 (57.50-64.00) 25.39 (24.56-26.84) 69 (60-73) 82.60 (80.08-87.50) 42.06 (40.19-43.99) 25.66 (23.39-27.88) 34.92 (34.12-36.71) 20 (86.96%) 6 (26.09%) 7 (30.43%)

77.10 (70.08-88.78)* 32.60 (31.05-35.38)* 66 (62-71) 92.00 (89.20-106.25)* 48.44 (45.18-50.35)* 36.57 (32.49-43.36)* 39.61 (37.51-45.88)* 20 (90.91%) 2 (9.09%) 4 (18.18%)

Values are expressed as the median (1st – 3rd quartile) for numerical variables and as the absolute number (percentage) for categorical variables. BMI = Body Mass Index; RUS = right unilateral stance; LUS = left unilateral stance; Fat % = Fat percentage; FFM = Fat-free mass. * p,0.05 (Low Fat Mass Group vs. High Fat Mass Group; Mann Whitney Test for numerical variables and chi-square test for categorical variables).

southern Brazil11 but were slightly lower when compared to data collected in a US study,5 probably due to the use of different equations11,31 to calculate this variable.

Weight, body mass index, waist circumference, fat percentage, and fat mass were positively and significantly correlated with the number of contacts needed to avoid falling during unilateral stance, but only the fat percentage and the fat mass were associated with the number of contacts during right and left unilateral stance. The greater responsiveness observed during left stance may be related to the greater number of individuals with right leg dominance (Table 1). Supporting the body for a one-minute period should be more difficult with the non-dominant leg than with the dominant leg. This correlation becomes stronger when the results for the non-dominant unilateral stance are analyzed (Table 3). When controlling body sway becomes more difficult, it becomes easier to detect differences due to other causes, such as adiposity. When the entire sample was divided into two groups based on median fat mass, greater instability was observed in the High Fat Mass Group for some stabilometric variables when the subjects’ eyes were closed, which is a situation in which visual feedback is eliminated and better integration of somatosensory and vestibular inputs are required to ensure adequate postural control. In addition to the results

Table 2 - Stabilometric variables for High and Low Fat Mass Groups.

Variable X SD (mm)

Trials

OBEO OBEC CBEO CBEC Tandem Right Tandem Left Y SD OBEO (mm) OBEC* CBEO CBEC* Tandem Right Tandem Left X Range OBEO (mm) OBEC CBEO CBEC Tandem Right Tandem Left Y Range OBEO (mm) OBEC* CBEO CBEC* Tandem Right Tandem Left Effective Area OBEO (mm2) OBEC* CBEO CBEC Tandem Right Tandem Left

Low Fat Mass Group (n = 23) 0.21 0.19 0.52 0.63 0.62 0.60 0.37 0.40 0.54 0.52 0.74 0.52 1.07 1.16 2.98 3.85 3.92 3.80 1.98 2.23 3.08 3.11 4.00 3.54 0.57 0.64 1.81 1.97 3.52 2.10

(0.15-0.24) (0.16-0.25) (0.38-0.56) (0.51-0.76) (0.51-0.74) (0.52-0.76) (0.30-0.39) (0.31-0.48) (0.40-0.62) (0.45-0.58) (0.51-0.97) (0.44-0.88) (0.94-1.34) (1.02-1.34) (2.29-3.36) (3.06-5.01) (3.29-4.56) (3.21-4.59) (1.67-2.47) (1.92-2.71) (2.19-3.43) (2.70-3.70) (2.98-6.90) (3.08-5.48) (0.37-0.65) (0.41-0.98) (1.09-2.10) (1.60-3.25) (1.74-4.93) (1.60-3.94)

High Fat Mass Group (n = 22) 0.20 0.25 0.45 0.61 0.65 0.70 0.40 0.52 0.51 0.66 0.65 0.67 1.07 1.38 2.82 3.50 3.71 4.05 2.26 3.09 2.81 3.71 3.87 4.40 0.59 1.04 1.48 2.52 2.81 3.16

(0.15-0.24) (0.17-0.39) (0.36-0.54) (0.47-0.82) (0.53-0.81) (0.58-0.78) (0.34-0.46) (0.45-0.65) (0.43-0.58) (0.54-0.72) (0.39-0.77) (0.52-1.02) (0.92-1.44) (1.14-2.17) (2.41-3.69) (2.74-4.72) (3.26-4.70) (3.52-4.50) (1.85-2.82) (2.82-3.97) (2.12-3.36) (3.31-4.46) (2.59-5.00) (3.21-5.42) (0.42-0.77) (0.78-1.87) (1.05-2.18) (1.54-3.59) (1.38-4.04) (1.78-5.21)

Table 3 - Correlations observed during unilateral stance: Number of foot or hand contacts vs. body composition and general variables. Variable Weight (kg) Height (m) BMI (kg/cm2) Age (years) Waist (cm) Fat % Fat mass (kg) FFM (kg)

Data are presented as the median (1st - 3rd quartile). *p#0.01 (Mann Whitney Test). SD = Standard Deviation; OBEO = Opened Base, Eyes Open; OBEC = Opened Base, Eyes Closed; CBEO = Closed Base, Eyes Open; CBEC = Closed Base, Eyes Closed.

RUS

LUS

NDUS

0.17 -0.10 0.30 0.26 0.22 0.46* 0.31* -0.03

0.30* -0.02 0.36* 0.30 0.42* 0.43* 0.38* 0.16

0.33* 0.02 0.37* 0.31* 0.41* 0.44* 0.40* 0.19

The presented values are the Spearman Correlation Coefficients. * p,0.05; § 0.05,p,0.10. BMI = Body Mass Index; Fat % = Fat percentage; FFM = Fat-free mass; RUS = right unilateral stance; LUS = left unilateral stance; NDUS = Non-dominant unilateral stance.

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dietitians. Regular evaluation of adiposity levels in elderly individuals should be adopted using such simple but reliable techniques as bioelectrical impedance and waist circumference measurements. Furthermore, balance and mobility tests must also be performed using stabilometry or other methods used in clinical practice, such as the Berg balance scale and timed up-and-go test. It is important to emphasize that the results of the present study are limited to the selected population, which consists of a sample that was readily recruited from female participants in a university program for the elderly. Although the use of bioelectrical impedance to measure body composition is a limitation of this study, given that there are more accurate methods for assessing body fat levels, bioelectrical impedance is widely used in clinics and health promotion centers.

for effective area, the higher values for anterior-posterior standard deviation and range demonstrate that the women with higher fat mass scores oscillate their center-of-foot pressure more than the women with lower fat mass scores as predicted by our hypothesis. The observed differences were mainly in anterior-posterior direction, which was expected, due to the loss of ankle flexibility in elderly individuals. Furthermore, the lack of ankle flexibility influences anterior-posterior stability more than does lateral stability.32 Postural control differences in tandem positions were not observed. Considering that only 34 and 35 individuals (for the right and left conditions, respectively) successfully completed 60 seconds in this stance, the exclusion of subjects with poor performance could have influenced the final results. These trials were excluded when the hand support or foot adjustment added noise to the signal for the center-of-foot displacement. However, women with poor performance in tandem stances were not considered for analysis if these signals had been excluded, and only those with less body sway remained in the analysis. This decision could have influenced the power of the study to distinguish an association between adiposity and body sway. A study carried out in Sa˜o Paulo, Brazil, determined that postural control in quiet standing may not influence the quality or pattern of movement during functional activity;33 thus, tandem positions were expected to show greater differences than quiet stance trials. The correlations of fat mass and waist circumference with unilateral stance behavior can be explained by the smaller range of the stability limit previously documented in obese individuals.9 These correlations and the differences in certain stabilometric variables between the High and Low Fat Mass Groups attest to the relationship between adiposity and instability in the present elderly sample. Thus, it can be speculated that a higher quantity of fat mass may influence the localization and displacement of the center-of-foot pressure, altering stabilometric variables and causing the subject to make a greater number of hand and foot contacts to avoid falls during unilateral stance. After the sixth decade, it is well established that there is a reduction in total body mass, which is primarily influenced by the decrease in bone and muscle mass.34 Furthermore, aging is associated with fat redistribution; visceral fat increases, whereas subcutaneous fat in other regions of the body (abdomen, thigh, calves) decreases.22 These changes may disturb postural control. Health professionals should be concerned with overweight and obese elderly individuals. Without specific intervention, aging will increase body fat, decreasing the level of total physical activity even further and increasing the accumulation of adipose tissue. In accordance with the reported results, this higher amount of fat mass can increase instability and disturb postural control in the elderly, which could lead to a greater number of falls. A focus on the prevention and treatment of obesity in elderly subjects delays the risk of metabolic complications, reduces the risk of falls and, consequently, enhances the quality of life of the elderly population. Because obesity occurs when energy intake consistently exceeds energy expenditure, interventions must emphasize nutrition reeducation and promote an adequate level of physical activity and a healthy lifestyle, as recommended by a multi-professional team, including physicians, physical therapists, psychologists, physical educators and

CONCLUSIONS This study shows that there is an association between body adiposity and postural balance control in elderly women. The women with greater fat masses presented higher values for stabilometric variables and required a greater number of contacts to avoid falling, reflecting less efficient postural control.

REFERENCES 1. IBGE – Brazilian Institute of Geography and Statistics. Perfil dos Idosos Responsa´veis pelos Domicı´lios no Brasil. Estudos & Pesquisas – Informac¸a˜o Demogra´fica e Socioeconoˆmica. 2002;9. 2. Sanches GDS, Gazoni FM, Konishi RK, Guimara˜es HP, Vendrame LS, Lopes RD. Intensive care of postoperative patients in bariatric surgery. Rev bras ter intensiva. 2007;19:205-9, doi: 10.1590/S0103-507X2007000200011. 3. Ogden CL, Carroll MD, Curtin LR, McDowell MA, Tabak CJ, Flegal KM. Prevalence of Overweight and Obesity in the United States, 1999-2004. JAMA. 2006;295:1549-55, doi: 10.1001/jama.295.13.1549. 4. West NA, Haan MN. Body Adiposity in Late Life and Risk of Dementia or Cognitive Impairment in a Longitudinal Community-Based Study. J Gerontol A Biol Sci Med Sci. 2009;64:103-9, doi: 10.1093/gerona/gln006. 5. Chumlea WC, Guo SS, Kuczmarski RJ, Flegal KM, Johnson CL, Heymsfield SB, et al. Body composition estimates from NHANES III bioelectrical impedance data. Int J Obes (Lond). 2002;26:1596–609, doi: 10.1038/sj.ijo.0802167. 6. Wu CH, Heshka S, Wang J, Pierson Jr RN, Heymsfield SB, Laferre`re B, et al. Truncal fat in relation to total body fat: influences of age, sex, ethnicity and fatness. Int J Obes (Lond). 2007;31:1384-91, doi: 10.1038/sj. ijo.0803624. 7. Stenholm S, Alley D, Bandinelli S, Griswold ME, Koskinen S, Rantanen T, et al. The effect of obesity combined with low muscle strength on decline in mobility in older persons: results from the InCHIANTI Study. Int J Obes (Lond). 2009;33:635–44, doi: 10.1038/ijo.2009.62. 8. Greve J, Alonso A, Bordini ACPG, Camanho Gl. Correlation Between Body Mass Index and Postural Balance. Clinics. 2007;62:717-20, doi: 10. 1590/S1807-59322007000600010. 9. Blaszczyk JW, Cieslinska-Swider J, Plewa M, Zahorska-Markiewicz B, Markiewicz. A Effects of excessive body weight on postural control. J Biomech. 2009;42:1295-300, doi: 10.1016/j.jbiomech.2009.03.006. 10. WHO - World Health Organization. Obesity – Preventing and Managing the Global Epidemic. 1998;1-158. 11. Reach CR, Cordeiro BA, Petroski EL, Vasconcelos FAG. Validation of Bioelectrical Impedance for the Prediction of Fat-free Mass in Brazilian Elderly Subjects. Arq Bras Endocrinol Metab. 2008;52:1163-71, doi: 10. 1590/S0004-27302008000700013. 12. Kyle UG, Bosaeus I, De Lorenzo AD, Deurenberg P, Elia M, Go´mez JM, et al. Bioelectrical impedance analysis - part I: review of principles and methods. Clin Nutr. 2004;23:1226-43, doi: 10.1016/j.clnu.2004.06.004. 13. Deurenberg P, Deurenberg-Yap M. Validation of skinfold thickness and hand-held impedance measurements for estimation of body fat percentage among Singaporean Chinese, Malay and Indian subjects. Asia Pacific J Clin Nutr. 2002;11:1-7, doi: 10.1046/j.1440-6047.2002.00258. x. 14. Wang JA, Thornton JC, Bari S, Williamson B, Gallagher D, Heymsfield SB, et al. Comparisions of Waist circumferences measured at 4 sites. Am J Clin Nutr. 2003;77:379-84.

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25. ISAK (International Society for the Advancement of Kinanthropometry). International Standards for Anthropometric Assessment. Australia: ISAK, 2001. 26. Medronho RA, Bloch KV, Luiz RR, Werneck GL. Epidemiologia. 2 ed. Saõ Paulo: Atheneu, 2008. 27. Barbosa AR, Souza JMP, Lebraõ ML, Marucci MFN. Nutritional Status and Physical Performance of Elderly in the City of Saõ Paulo. Rev Assoc Med Bras. 2007;53:75-9, doi: 10.1590/S0104-42302007000100024. 28. Romero-Corral A, Somers VK, Sierra-Johnson J, Thomas RJ, Bailey KR, Collazo-Clavell ML, et al. Accuracy of Body Mass Index to Diagnose Obesity In the US Adult Population. Int J Obes (Lond). 2008;32:959-66, doi: 10.1038/ijo.2008.11. 29. Winter DA, Patla AE, Prince F, Ishac M, Gielo-Perczak K. Stiffness Control of Balance in Quiet Standing. J Neurophysiol. 1998;80:1211-21. 30. Deurenberg P, Andreoli A, Borg P, Kukkonen-Harjula K, de Lorenzo A, van Marken WD, et al. The validity of predicted body fat percentage from body mass index and from impedance in samples of five European populations. Eur J Clin Nutr. 2001;55:973–9, doi: 10.1038/sj.ejcn. 1601254. 31. Sun SS, Chumlea WC, Heymsfield SB, Lukaski HC, Schoeller D, Friedl K, et al. Development of bioelectrical impedance analysis prediction equations for body composition with use of a multicomponent model for use in epidemiologic surveys. Am J Clin Nutr. 2003;77:331-40. 32. Shumway-Cook A, Woollacott M. Motor control: Translating Research into Clinical Practice. USA: Lippincott Williams & Wilkins, 2007. 33. Moya GBL, Siqueira CM, Caffaro RR, Fu C, Tanaka C. Can quiet standing posture predict compensatory postural adjustment? Clinics. 2009;64:791-6, doi: 10.1590/S1807-59322009000800014. 34. McArdle WD, Katch FI, Katch VL. Exercise Physiology – Energy, Nutrition and Human Performance. 6th ed. Philadelphia: Lippincot Williams & Wilkins. 2007;868-966.

15. Shields M, Tremblay MS, Laviolette M, Craig CL, Janssen I, Gorber SC. Fitness of Canadian adults: Results from the 2007-2009 Canadian Health Measures Survey. Health Rep. 2010;21:21-35. 16. Mainenti MRM, Oliveira LF, Lima MAMT, Nadal J. Stabilometric signal analysis in tests with sound stimuli. Exp Brain Res. 2007;181:229-36, doi: 10.1007/s00221-007-0921-4. 17. Cavalheiro GL, Almeida MFS, Pereira AA, Andrade AO. Study of agerelated changes in postural control during quiet standing through Linear Discriminant Analysis. Biomed Eng Online. 2009;8:35, doi: 10.1186/1475925X-8-35. 18. Viljanen A, Kaprio J, Pyykko¨ L, Sorri M, Pajala S, Kauppinen M, et al. Hearing as a Predictor of Falls and Postural Balance in Older Female Twins. J Gerontol A Biol Sci Med Sci. 2009;64A:312-7, doi: 10.1093/ gerona/gln015. 19. Schrager MA, Kelly VE, Price R, Ferrucci L, Shumway-Cook A. The Effects of Age on Medio-lateral Stability during Normal and Narrow Base Walking. Gait Posture. 2008;28:466–71, doi: 10.1016/j.gaitpost.2008.02.009. 20. Abrahamova´ D, Hlavacˇka E. Age-Related Changes of Human Balance during Quiet Stance. Physiol Res. 2008;57:957-64. 21. Ruwer SL, Rossi AG, Simon LF. Balance in the elderly. Rev Bras Otorrinolaringol. 2005;71:298-303, doi: 10.1590/S0034-72992005000300006. 22. Zamboni M, Mazzali G, Zoico E, Harris TB, Meigs JB, Di Francesco V, et al. Health consequences of obesity in the elderly: a review of four unresolved questions. Int J Obesity (Lond). 2005;29:1011–29, doi: 10. 1038/sj.ijo.0803005. 23. Kyle UG, Genton L, Karsegard L, Slosman DO, Pichard C. Single prediction equation for bioelectrical impedance analysis in adults aged 20–94 years. Nutrition. 2001;17:248-53, doi: 10.1016/S0899-9007(00)00553-0. 24. Genton LC, Karsegard VL, Kyle UG, Hans DB, Michel JP, Pichard C. Comparison of four bioelectrical impedance analysis formulas in healthy elderly adults. Gerontology. 2001;47:315–23, doi: 10.1159/000052821.

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DOI:10.1590/S1807-59322011000900002

CLINICAL SCIENCE

INTRODUCTION: Pseudomonas aeruginosa (P. aeruginosa) is one of the primary opportunistic pathogens responsible for nosocomial infections. Aminoglycosides are an important component of antipseudomonal chemotherapy. The inactivation of drugs by modifying enzymes is the most common mechanism of aminoglycoside resistance. OBJECTIVES: The inactivation of aminoglycosides by modifying enzymes is the primary resistance mechanism employed by P. aeruginosa. The aim of the present study was to investigate the occurrence of aminoglycoside resistance and the prevalence of four important modifying enzyme genes (aac (69)-I, aac (69)-II, ant (20)-I, aph (39)-VI) in P. aeruginosa in Iran. METHODS: A total of 250 clinical isolates of P. aeruginosa were collected from several hospitals in seven cities in Iran. Antimicrobial susceptibility tests (using the disk diffusion method and E-tests) were performed for all 250 isolates. In addition, all isolates were screened for the presence of modifying enzyme genes by polymerase chain reaction. RESULTS: The resistance rates, as determined by the disk diffusion method, were as follows: gentamicin 43%, tobramycin 38%, and amikacin 24%. Of the genes examined, aac (69)-II (36%) was the most frequently identified gene in phenotypic resistant isolates, followed by ant (20)-I, aph (39)-VI, and aac (69)-I. CONCLUSIONS: Aminoglycoside resistance in P. aeruginosa remains a significant problem in Iran. Therefore, there is considerable local surveillance of aminoglycoside resistance. KEYWORDS: Pseudomonas aeruginosa; Antibiotic; Resistance; aac (69)-II; ant (20)-I. Vaziri F, Peerayeh SN, Nejad QB, Farhadian A. The prevalence of aminoglycoside-modifying enzyme genes (aac (69)-I, aac (69)-II, ant (20)-I, aph (39)-VI) in Pseudomonas aeruginosa. Clinics. 2011;66(9):1519-1522. Received for publication on February 1, 2011; First review completed on March 3, 2011; Accepted for publication on May 16, 2011 E-mail: najarp_s@modares.ac.ir / vaziri@modares.ac.ir Tel.: 00 98 021 82883870

methylases.9 Among these mechanisms, the inactivation of drugs by plasmid- or chromosome-encoded modifying enzymes is the most common. These modifying enzymes include aminoglycoside phosphoryl transferase (aph), aminoglycoside acetyltransferase (aac), and aminoglycoside nucleotidyl transferase (ant).10-12 Four of these enzymes, encoded by aac (69)-I, aac (69)-II, ant (20)-I, and aph (39)-VI, are of particular significance because they are among the most common modifying enzymes present in P. aeruginosa, and their substrates are the most important antipseudomonal aminoglycosides. aac (69)-I confers resistance to tobramycin and amikacin, aac (69)-II and ant (20)-I inactivate tobramycin and gentamicin, and amikacin is the substrate of aph (39)-VI.13,14 The aim of the present nationwide study was to investigate the occurrence of aminoglycoside resistance and the prevalence of the resistance-modifying enzyme genes, aac (69)-I, aac (69)-II, ant (20)-I and aph (39)-VI, in P. aeruginosa isolated from several hospitals in seven Iranian cities.

INTRODUCTION Pseudomonas aeruginosa (P. aeruginosa) is one of the primary opportunistic pathogens responsible for nosocomial infections. The most important problem in the eradication of P. aeruginosa is the frequently observed multi-drug resistance of the species.1 In addition, P. aeruginosa can also acquire resistance to various antimicrobial agents, such as aminoglycosides, b-lactams2 and fluoroquinolones.3 Aminoglycosides are an important component of antipseudomonal chemotherapy,4 and they exhibit synergy with b-lactams.5 Resistance to aminoglycosides occurs via enzymatic modification, impermeability, the activity of efflux pumps (MexXY-OprM),6 the PhoP-PhoQ system,7 ndvBdependent biofilm formation,8 and the activity of 16s rRNA Copyright Ă&#x; 2011 CLINICS â&#x20AC;&#x201C; This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 1 - The antimicrobial resistance rate of 250 P. aeruginosa isolates as determined by the disk diffusion method AMI: amikacin, GEN: gentamicin, TOB: tobramycin, IMI: imipenem, PIP: piperacillin, TIC: ticarcillin, CAZ: ceftazidime, CIP: ciprofloxacin.

tobramycin (10 mg)] and for five other antibiotics [imipenem (10 mg), piperacillin (100 mg), ticarcillin (75 mg), ceftazidime (30 mg), and ciprofloxacin (5 mg)]. All drugs were obtained from Mast laboratories (Merseyside, United Kingdom). For all 250 isolates, the minimum inhibitory concentrations (MIC) of amikacin, gentamicin, and tobramycin were determined using the E-test (Biodisk, Dalvagen, Sweden) according to CLSI guidelines. P. aeruginosa (ATCC 27853) served as a control for the disk diffusion and E-tests.

METHODS AND MATERIALS Collection of bacterial isolates A total of 250 non-duplicate, clinical isolates of P. aeruginosa were collected from a nationwide distribution of several hospitals in seven cities in Iran (Tehran, Shiraz, Zahedan, Tabriz, Sannandaj, Sari, and Ahvaz) between May 2007 and January 2008. Strain data and the demographic and clinical data for each patient were regularly forwarded to our laboratory. The study population was 62% male and 38% female. The specimens were isolated from urine (38%), wounds (18%), the trachea (18%), blood (10%), sputum (9%), and other sources (7%). Isolate confirmations were conducted using conventional biochemical tests, and then the isolates were stored at –76 ˚C in glycerol skim milk broth.

Polymerase chain reaction amplification Polymerase chain reaction (PCR) was used to screen all 250 isolates for the presence of the modifying enzyme genes, aac (69)-I, aac (69)-II, ant (20)-I and aph (39)-VI. The total template DNA for the PCR amplification was extracted from the supernatant of a mixture of P. aeruginosa cells produced by the boiling method. PCR amplification was performed using 2.5 mL of the template DNA, 1 mL of each primer,16 19.5 mL master mix, and 1 mL of Taq DNA polymerase (CinnaGen) in a total volume of 25 mL. A thermocycler (Mastercycler gradient; Eppendorf, Hamburg, Germany) was programmed

Antimicrobial susceptibility testing Antimicrobial susceptibility tests were performed using the disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI) guidelines15 for three aminoglycosides [gentamicin (10 mg), amikacin (30 mg), and

Table 1 - Aminoglycoside susceptibility profiles according to the disk diffusion method and MIC results (E-test) for 250 P. aeruginosa isolates (according to CLSI guidelines).15 E-test2

Disk Diffusion1

No. (%) of isolates

Gentamicin Tobramycin

Resistant

Intermediate

Susceptible

108(43.2) 95(38)

8(3.2) 7(2.8)

134(53.6) 148(59.2)

Resistant

Intermediate

Susceptible

MIC(mg/ml)

–1024 128 64–128 16–64 10 8

41 45

49 37

MIC(mg/ml)

.256 Amikacin

59(23.6)

7(2.8)

184(73.6)

2–4

8 5

98 103

44 52

MIC(mg/ml)

–256 128 64–128 17

MIC(mg/ml)

4–16

134

1. Zone diameter (mm) for gentamicin and tobramycin, R:,12, I:13-14, S:.15; for amikacin, R:,14, I:15-16, S:.17. 2. MIC breakpoint(mg/ml) for gentamicin and tobramycin, R:.16, I:8; S:,4; for amikacin, R:.64, I:32, S:,16.

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MIC 50% (ug/mL)

MIC 90% (ug/mL).

4 4

64 64

MIC 50% (ug/mL)

MIC 90% (ug/mL).

128

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Aminoglycoside resistance in Pseudomonas aeruginosa Vaziri F et al.

the disk diffusion method, 135 isolates were resistant to aminoglycosides. The resistance rates according to the E-test (base on MICs) were as follows: gentamicin 40%, tobramycin 36%, and amikacin 21%. The aminoglycoside susceptibility profiles according to the disk diffusion and E-test results for the 250 isolates are listed in Table 1. PCR analysis revealed the absence of resistance genes in susceptible isolates. The prevalence of aminoglycoside resistance genes in the 135 resistant isolates (as determined by the disk diffusion method) was as follows: aac (69)-II was detected in 36% of the resistant isolates, ant (20)-I was detected in 28%, aph (39)-VI in 11%, and aac (69)-I was found in 7% of the resistant isolates (Figure 2). Interestingly, individual aminoglycoside-resistant isolates carried multiple (two to four) modifying enzyme genes. Six isolates harbored ant(20)-I and aac(69)-II; three harbored aph(39)-VI and ant(20)-I; two harbored ant(20)-I and aac(69)-I; and three isolates harbored ant(20)-I, aac(69)-II and aph(39)-VI. Only one isolate harbored all four genes. Several previous studies have examined the occurrence of aminoglycoside resistance mechanisms in P. aeruginosa isolated from different countries. The overall incidence of aminoglycoside resistance found in our study (according to the disk diffusion test and the E-test) was much higher than the incidence that has been reported previously in different countries worldwide.14,16,17 However, Estahbanati and coworkers reported that 53.3% of clinical isolates from Iranian burn patients were resistant to amikacin, and 90.7% were resistant to gentamicin, a result that reveals a high level of aminoglycoside resistance in their study.18 The aminoglycoside resistance rate was almost as high in our isolates, and most of the resistant isolates harbored modifying enzyme genes. In addition, none of the susceptible isolates harbored these resistance genes. These results highlight the importance of aminoglycoside-modification-related mechanisms in aminoglycoside resistance in P. aeruginosa. Two genes, aac (69)-II and ant (20)-I, were the most frequent resistance genes observed in these isolates. These results are similar to what has been observed in different studies in other countries.19-21 All isolates harboring the aac (69)-II gene were resistant to gentamicin and tobramycin (100% concordance), which indicates that aac (69)-II is a significant determinant of gentamicin and tobramycin resistance in P. aeruginosa. It is important to mention that we encountered an unexpected phenotype in some isolates (Table 2). For example, when an isolate harbored only the aph (39)-VI gene, which has amikacin as a substrate, resistance to gentamicin, tobramycin, and amikacin was observed. We presume that the reason for

Figure 2 - Lane 1, 1000 bp DNA size marker; Lane 2, aac(69)-II (125 bp); Lane 5, aph(39)-VI (800 bp); Lane 7, ant(20)-I (524 bp); Lanes 8 and 9, aac(69)-I (392 bp).

with the appropriate conditions. Then, 5 ml of each PCR product was analyzed by electrophoresis on a 1% (w/v) TAE agarose gel (Fermentas UAB, Vilnius, Lithuania) containing 0.1 ml/ml ethidium bromide. The amplicons were then visualized on a UV transilluminator and photographed (BioDoc-Analyse; Biometra, Goettingen, Germany).

Statistical analysis All statistical analyses were performed using Statistical Package for Social Sciences (SPSS) software (version 11.5) for Windows (x2-test and Fisherâ&#x20AC;&#x2122;s exact test). P-values of ,0.05 were considered significant.

RESULTS AND DISCUSSION The resistance rates as determined using the disk diffusion method are presented in Figure 1. According to

Table 2 - Prevalence of aminoglycoside modifying enzymes genes and the correlation between these genes and phenotypic patterns in aminoglycoside resistant P. aeruginosa isolates. Gene aac(69)-I aac(69)-II ant(20)-I aph(39)-VI aac(69)-II+ ant(20)-I ant(20)-I+ aph(39)-VI aac(69)-I+ ant(20)-I ant(20)-I + aac(69)-II +aph(39)-VI aac(69)-II ant(20)-I+ + aph(39)-VI + aac(69)-I

no. of isolates (%)1

Expected resistance2

Observed resistance phenotypes (no. of isolates)3

9(7%) 49(36%) 38(28%) 15(11%) 6(4%) 3(2%) 2(1%) 3(2%) 1(,1%)

TOB,AMI GEN,TOB GEN,TOB AMI GEN,TOB AMI,GEN,TOB AMI,GEN,TOB AMI,GEN,TOB AMI,GEN,TOB

Unexpected resistance to GEN(2) Unexpected resistance to AMI(16) As expected(38) Unexpected resistance to GEN and TOB(2) As expected(6) As expected(3) As expected(2) As expected(3) As expected(1)

1. According to the disk diffusion method, a total of 135 resistant isolates were identified. 2. AMI: amikacin, GEN: gentamicin, TOB: tobramycin. 3. Described in greater detail in the text.

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this phenomenon might be the action of other resistance mechanisms, such as impermeability, efflux pumps, or other types of modifying enzymes. On the other hand, we detected 15 isolates which co-harbored two, three, or four aminoglycoside-modifying enzyme genes simultaneously, which is in contrast to several studies conducted in the USA and Europe, which reported that the majority of isolates exhibit only a single aminoglycoside modifying gene.22 Miller et al. brought together the results of several studies of aminoglycoside resistance in P. aeruginosa carried out worldwide.22 Their results indicated that in Europe, aac(69)II was the most prevalent resistance gene (32.5%), followed by ant(20)-I (16.9%). These results are in concordance with our study. In contrast to our results, a Korean nationwide study of 250 isolates of P. aeruginosa reported that aph(39)-VI, ant(20)-I, and aac(69)-I were all prevalent, but none harbored aac(69)-II.16 The difference in the distribution of modifying enzymes may derive from differences in aminoglycoside prescription patterns, the selection of bacterial population or geographical differences in the occurrence of aminoglycoside resistance genes.

7. 8.

10. 11. 12. 13.

CONCLUSIONS In conclusion, although aminoglycosides remain useful antipseudomonal agents, resistance to these drugs continues to be a major issue, especially in Iran. Because these aminoglycoside resistance genes are usually located on mobile genetic elements (i.e., plasmid, transposon, or integrons),23,24 there is a growing concern that they could easily spread and be disseminated among other bacteria. Integrons that carry gene cassettes encoding both aacs and carbapenemases will only exacerbate this problem.25 The design of novel aminoglycosides with stronger affinity for their targets and resistance to these modifying enzymes is inevitable,26 and the new generation of anti-Pseudomonas therapy is forthcoming.27 Aminoglycoside resistance among clinical isolates of P. aeruginosa promises to become a major clinical concern in the future, and continuous local surveillance of aminoglycoside resistance is crucial.

14. 15. 16.

17.

18. 19.

ACKNOWLEDGEMENTS

20.

This study was funded by a M.Sc. grant from the Tarbiat Modares University, Tehran, Iran.

21. 22.

REFERENCES 1. Lambert PA. Mechanisms of antibiotic resistance in Pseudomonas aeruginosa. J R Soc Med. 2002;95(suppl 41):22–6. 2. Park YJ. Prevalence of Ambler class A and D b-lactamases among clinical isolates of Pseudomonas aeruginosa in Korea. J Antimicrob Chemother. 2005;56:122-7, doi: 10.1093/jac/dki160. 3. Jalal S, Ciofu O, Høiby N, Gotoh N, Wretlind B. Molecular mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrob Agents Chemother. 2000;44:710–2. 4. Giamarellou H. Therapeutic guidelines for Pseudomonas aeruginosa infections. Int J antimicrob Agen. 2000;16:103-6, doi: 10.1016/S09248579(00)00212-0. 5. Dubois V, Arpin C, Dupart V, Scavelli A, Coulange L, Andre C, et al. blactam and aminoglycoside resistance rates and mechanisms among Pseudomonas aeruginosa in French general practice (community and

23. 24. 25. 26. 27.

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private healthcare centres). J Antimicrob Chemother. 2008;62:316-23, doi: 10.1093/jac/dkn174. Islam S, Oh H, Jalal S, Karpati F, Ciofu O, Høiby N, et al. Chromosomal mechanisms of aminoglycoside resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Clin Microbiol Infect. 2009;15:60–6, doi: 10.1111/j.1469-0691.2008.02097.x. Macfarlane E, Kwasnicka A, Hancock R. Role of Pseudomonas aeruginosa PhoP-PhoQ in resistance to antimicrobial cationic peptides and aminoglycosides. Microbiol. 2000;146:2543–54. Sadovskaya I, Vinogradov E, Li J, Hachani A, Kowalska K, Filloux A. High-level antibiotic resistance in Pseudomonas aeruginosa biofilm: the ndvB gene is involved in the production of highly glycerol-phosphorylated b-(1R3)-glucans, which bind aminoglycosides. Glycobiol. 2010;20:895–904, doi: 10.1093/glycob/cwq047. Doi Y, Ghilardi AC, Adams J, de Oliveira GD, Paterson DL. High prevalence of metallo- b-lactamase and 16S rRNA methylase coproduction among imipenem- resistant Pseudomonas aeruginosa isolates in Brazil. Antimicrob Agents Chemother. 2007;51:3388–90, doi: 10.1128/ AAC.00443-07. Shaw KJ, Rather PN, Hare RS, Miller GH. Molecular genetics aminoglycoside resistance genes and familial relationship of the aminoglycoside modifying enzymes. Microbiol Rev. 1993;57:138–63. Smith CA, Baker EN. Aminoglycoside antibiotic resistance by enzymatic deactivation. Curr Drug Targets Infect Disord. 2002;2:143–60, doi: 10. 2174/1568005023342533. Yamane K, Wachino J, Doi Y, Kurokawa H, Arakawa Y. Global spread of multiple aminoglycoside resistance genes. Emerg Infect Dis. 2005;11: 9513. Miller GH, Sabatelli FJ, Naples L, Hare RS, Shaw KJ. The most frequently occurring aminoglycoside resistance mechanisms— combined results of surveys in eight regions of the world. J Chemother. 1995;7(Suppl. 2):17– 30. Poole K. Aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2005;49:479–87, doi: 10.1128/AAC.49.2. 479-487.2005. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; seventeenth informational supplement. CLSI document M100-S17. CLSI, 2007 Wayne, PA. Kim JY, Park YJ, Kwon HJ, Han K, Kang MW, Woo GJ. Occurrence and mechanisms of amikacin resistance and its association with b-lactamases in Pseudomonas aeruginosa: a Korean nationwide study. J Antimicrob Chemother. 2008;62:479-83, doi: 10.1093/jac/dkn244. Cavallo J, Hocquet D, Plesiat P, Fabre R, Roussel-Delvallez M. Susceptibility of Pseudomonas aeruginosa to antimicrobials: a 2004 French multicentre hospital study. J Antimicrob Chemother. 2007;59: 1021–4, doi: 10.1093/jac/dkm076. Estahbanati H, Kashani P, Ghanaatpisheh F. Frequency of Pseudomonas aeruginosa serotypes in burn wound infections and their resistance to antibiotics. Burns. 2002;28:340–8, doi: 10.1016/S0305-4179(02)00024-4. Busch-Sorensen C, Sonmezoglu M, Frimodt-Moller N, Hojbjerg T, Miller GH, Espersen F. Aminoglycoside resistance mechanisms in Enterobacteriaceae and Pseudomonas spp. from two Danish hospitals: correlation with type of aminoglycoside used. APMIS. 1996;104:763–8. Over U, Gur D, Unal S, Miller GH. The changing nature of aminoglycoside resistance mechanisms and prevalence of newly recognized resistance mechanisms in Turkey. Clin Microbiol Infect. 2001;7:470-8. Phillips I, King A, Shannon K. Prevalence and mechanisms of aminoglycoside resistance. A ten-year study. Am J Med. 1986;80:48–55. Miller GH, Sabatelli FJ, Hare RS, Glupczynski Y, Mackey P, Shlaes D, et al. The most frequent aminoglycoside resistance mechanisms changes with time and geographic area: a reflection of aminoglycoside usage patterns. Clin Infect Dis. 1997;24:S46-62, doi: 10.1093/clinids/24. Supplement_1.S46. Park YJ. Aminoglycoside Resistance in Gram-negative Bacilli. Korean J Clin Microbiol. 2009;12:57-61. Ramirez MS, Tolmasky ME. Aminoglycoside modifying enzymes. Drug Resist. 2010;13:151–71, doi: 10.1016/j.drup.2010.08.003. Strateva T, Yordanov D. Pseudomonas aeruginosa – a phenomenon of bacterial resistance. J Medic Microbiol. 2009;58:1133–48, doi: 10.1099/ jmm.0.009142-0. Haddad J, Kotra LP, Llano-Sotelo B, Kim C, Azucena EF, Liu M, et al. Design of novel antibiotics that bind to the ribosomal acyltransfer site. J Am Chem Soc. 2002;124:3229-37, doi: 10.1021/ja011695m. Page M, Heim J. Prospects for the next anti-Pseudomonas drug. Curr Opin Pharmacol. 2009;9:558–65, doi: 10.1016/j.coph.2009.08.006.

CLINICS 2011;66(9):1523-1529

DOI:10.1590/S1807-59322011000900003

CLINICAL SCIENCE

Impaired hemodynamic response to mental stress in subjects with prehypertension is improved after a single bout of maximal dynamic exercise Renata Frauches Medeiros, Bruno Moreira Silva, Fabricia Junqueira Neves, Natalia Galito Rocha, Allan Robson Kluser Sales, Antonio Claudio Nobrega Department of Physiology and Pharmacology & Postgraduate Program in Cardiovascular Sciences, Fluminense Federal University, Niteroi/RJ, Brazil.

INTRODUCTION: High blood pressure during mental stress in subjects with prehypertension is associated with blunted vasodilation in skeletal muscles, which might be improved by an acute bout of exercise. OBJECTIVE: To investigate the hemodynamic responses to mental stress before and after a bout of exercise in subjects with prehypertension. METHOD: Eighteen subjects with prehypertension and 16 with normotension underwent a mental stress test before and after a maximal cardiopulmonary exercise test on a treadmill. Blood pressure was measured by auscultation, and forearm blood flow was measured by venous occlusion plethysmography; from these measurements, the vascular conductance was calculated. RESULTS: Subjects with prehypertension had a higher mean blood pressure during mental stress (prehypertension 112¡2 vs. normotension 101¡3 mm Hg, p,0.05), and their vascular conductance did not increase (baseline 0.025¡0.004 vs. mental stress 0.022¡0.003 a.u., p.0.05). After the exercise bout, the mean blood pressure during mental stress was lower in subjects with prehypertension (before exercise 112¡2 vs. after exercise 107¡2 mm Hg, p,0.05), and vascular conductance increased (baseline 0.011¡0.001 vs. mental stress 0.024¡0.004 a.u., p,0.05). CONCLUSION: Subjects with prehypertension had elevated blood pressure and a blunted vasodilator response during mental stress, but their blood pressure was attenuated and their vasodilator response was normalized after a single bout of maximal dynamic exercise. KEYWORDS: Psychological stress; Blood pressure; Blood flow; Physical exercise; Hypertension. Frauches RM, Silva BM, Neves FJ, Rocha NG, Sales ARK, Nobrega AC. Impaired hemodynamic response to mental stress in subjects with prehypertension is improved after a single bout of maximal dynamic exercise. Clinics. 2011;66(9):1523-1529. Received for publication on February 8, 2011; First review completed on March 16, 2011; Accepted for publication on May 16, 2011 E-mail: anobrega@id.uff.br Tel.: 55 21 2629-2404

Mental or psychological stress occurs on a daily basis, and the ability of organisms to respond to it is natural and necessary. The cardiovascular system participates extensively in the response to stressful situations, and some of its adjustments involve increases in systemic blood pressure2,3 and vasodilation in skeletal muscles.4,5 However, it has been shown that subjects with PHT have an augmented blood pressure response to mental stress,6-8 which has been associated with an increased risk for the development of HT.2,3 Santangelo et al.6 suggested that one mechanism that might lead to this enhanced pressor response is blunted muscle vasodilation during mental stress. More recently, Schwartz et al.7 found that blunted vasodilation in skeletal muscles in subjects with PHT was not associated with muscle sympathetic nerve activity. Therefore, it is important to assess the impact of factors that might attenuate or reverse the enhanced pressor response and blunted muscle vasodilation during mental stress in subjects with PHT.

INTRODUCTION Arterial hypertension (HT) affects approximately one billion people worldwide, and as the population ages, this number tends to increase.1 The classification of prehypertension (PHT) was introduced in the Seventh Report of the Joint National Committee on the Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. This classification focuses attention on a segment of the population at elevated risk of developing cardiovascular disease and for whom therapies that prevent or delay the onset of HT would be valuable.1

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Prehypertension and stress reactivity Medeiros RF et al.

CLINICS 2011;66(9):1523-1529

was calculated to provide an index of insulin resistance.17 Cholesterol and its subfractions, triglycerides and C-reactive protein were determined by the dry chemistry method.

Dynamic exercise involving large muscle groups requires complex integrative cardiovascular responses,9 and some effects of exercise on the cardiovascular system can still be observed hours after the exercise ceases.10-12 It has been shown that an acute bout of dynamic exercise decreases blood pressure reactivity to psychosocial stressors in subjects with borderline HT.8,13 However, the mechanism that mediates this response is not known. Specifically, it is not known whether the blunted muscle vasodilation during mental stress in subjects with PHT is improved after a bout of dynamic exercise. Thus, the purpose of this study was to evaluate blood pressure and vascular responses to mental stress experienced before and after a bout of dynamic exercise in subjects with PHT.

Anthropometric analysis Anthropometric assessments included measurements of body weight (adult mechanical balance 110 CH, Welmy, Santa Barbora d’ Oeste, SP, Brazil) and height followed by a calculation of the body mass index. The waist circumference was obtained with a tape measure at the point midway between the last costal margin and the iliac crest with the subject standing and breathing normally.18

Experimental protocol MATERIALS AND METHODS

After the initial clinical and laboratory screening, there was an experimental visit that occurred in the morning, one hour after a standardized light breakfast with low-fat foods selected by a dietitian. Women were evaluated from the first until the 12th day after the onset of menstruation. Subjects did not drink alcohol or caffeinated beverages and did not perform intense physical activity for at least 24 hours before the experimental visit. For the experiment, the subjects were placed in supine position in a quiet air conditioned room (¡24 ˚C). After instrumentation, they rested quietly for ten minutes. The experimental protocol consisted of blood pressure and forearm vascular reactivity assessments at baseline and during mental stress, and these measurements were performed before and 60 minutes after a maximal cardiopulmonary exercise test.

Sample Subjects were recruited through advertisements in the university and in local newspapers. The sample was composed of 34 subjects, of whom 18 had PHT and 16 had normotension (NT). Eligibility for the study was verified through a clinical history assessment (performed using a questionnaire consisting of questions on cardiovascular history and risk factors for cardiovascular disease),14 a physical examination, two blood pressure measurements per day performed on two different days, biochemical blood analyses, a resting electrocardiogram, and maximal cardiopulmonary exercise testing. All of the subjects fulfilled the following criteria: age between 18 and 49 years, women had regular menstrual cycles, no chronic diseases, no recent infections, body mass index between 18.5 and 29.9 kg/m2, glycemia lower than 5.6 mmol/l,15 total cholesterol lower than 6.21 mmol/l,16 low-density lipoprotein lower than 4.14 mmol/l,16 triglycerides lower than 2.26 mmol/l,16 non-smoker, not using medications other than oral contraceptives, normal resting and exercise electrocardiogram, and sedentary (not engaged in exercise activities lasting 30 minutes or more, three times per week during the last three months). The study procedures were approved by the Ethics Committee of the Antonio Pedro University Hospital, and written informed consent was obtained from all participants prior to the experimental procedures.

Hemodynamic measurements Forearm blood flow (FBF) was measured by venous occlusion plethysmography. The subjects were placed in the supine position with the right arm supported comfortably above the level of the heart. Two cuffs were used: one (8-cm width) was placed around the right wrist, and the other (10cm width) was placed around the right upper arm. The last cuff was attached to a rapid cuff inflator (EC6; Hokanson, Bellevue, Washington, USA). A mercury-in-silastic strain gauge was placed at the widest girth of the right forearm. The diameter of the strain gauge was 2 cm less than the widest girth of the forearm. The FBF was calculated using a semi-automatic method that has high intra- and interevaluator reproducibility (intraclass correlation coefficients between 0.98 and 0.99).19 To measure the BF, the wrist cuff was inflated to 220 mmHg one minute before the onset of the FBF measurement to isolate the vascular circulation of the hand, and it was kept inflated throughout the blood flow measurement.20 At the beginning of all measurements, the evaluator generated a standard calibration pulse of 1 millivolt. The FBF was measured by rapidly inflating the cuff placed around the right upper arm to 50 mmHg (in less than 0.5 s), maintaining this pressure for 10 s and then rapidly deflating it to 0 mmHg for 10 s, thus completing a 20-s cycle. Six cycles of 20 s were performed to determine the baseline FBF, totaling 120 s of baseline measurement. The blood pressure was obtained once during the two minutes of baseline FBF recording. The measurement of FBF during the mental stress test was similar to that during the baseline measurement, but it was performed in nine 20-s cycles (10-s inflation to 50 mmHg followed by 10-s deflation to 0 mmHg), totaling

Blood pressure assessment Blood pressure was measured in the right arm in the supine position after ten minutes of quiet rest. Measurements were performed with a calibrated mercury sphygmomanometer and a cuff size that was appropriate for the arm circumference of each subject. According to The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure,1 NT was defined as systolic blood pressure (SBP) ,120 mm Hg and diastolic blood pressure (DBP) ,80 mm Hg, and PHT was defined as SBP between 120 and 139 and/ or DBP between 80 and 89 mmHg. The classification was based on the average of four SBP and DBP measurements, where two measurements were taken during each of two visits to the laboratory. The mean blood pressure (MBP) was calculated with the equation [SBP + (2?DBP)]/3.

Biochemical blood analyses Plasma glucose was measured with an enzymatic in-vitro test, and serum insulin was determined using an electrochemiluminescence assay. The homeostasis model assessment

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180 s of recording of the mental stress FBF. The mean value of each minute of recording was obtained for three cycles, totaling three FBF measurements. Blood pressure was measured three times (once per minute) during the mental stress test using the auscultatory method. The FBF was divided by the mean blood pressure to calculate the forearm vascular conductance (FVC). In addition, the heart rate (HR) was recorded continuously during the mental stress test (Polar Vantage NV, Electro Oy, Kempele, Finland).

Table 1 - Anthropometric, biochemical, and hemodynamic characteristics of subjects with prehypertension or normotension. Variable N (male%) Age (years) Weight (Kg) BMI (Kg/m2) Waist circumference (cm) Fasting glucose (mmol/l) Fasting insulin (pmol/l) HOMA-IR* Cholesterol (mmol/l) HDL (mmol/l) LDL (mmol/l) Triglyceride (mmol/l) CRP (mg/L)* SBP (mm Hg) DBP (mm Hg) MBP (mm Hg) VO2peak (ml/kg min)

Mental stress test One of the evaluators explained the traditional colorword conflict task (Stroop Color test) to the subjects.21 A continuous auditory conflict with different names of colors was used to increase the difficulty of the mental stress. Each subject was asked to assess the difficulty of the task according to previously established levels of difficulty: 0 = not stressful; 1 = slightly stressful; 2 = stressful; 3 = very stressful; and 4 = extremely stressful.22

Exercise bout

Prehypertension

Normotension

p-value

18 (33.3%) 34¡2 69.5¡2.7 25.4¡0.5 80¡2 4.8¡0.1 41.3¡6.8 0.9¡1.9 4.6¡0.2 1.5¡0.1 2.6¡0.2 1.1¡0.1 0.4¡0.4 126¡2 78¡2 95¡2 32¡2

16 (12.5%) 33¡3 68.8¡2.4 24.9¡0.6 81¡2 4.6¡0.1 42.8¡6.7 1.2¡0.8 4.7¡0.2 1.5¡0.1 2.8¡0.1 0.8¡0.1 0.5¡0.5 111¡2 70¡2 84¡2 31¡2

0.15 0.80 0.44 0.93 0.27 0.55 0.73 0.65 0.66 0.20 0.28 0.73 0.65 ,0.01 0.02 ,0.01 0.67

Data are the mean ¡ SEM or (*) median ¡ interquartile range. BMI indicates body mass index; HOMA, homeostasis model assessment; HDL, high-density lipoprotein; LDL, low-density lipoprotein; CRP, C-reactive protein; SBP, systolic blood pressure; DPB, diastolic blood pressure; MBP, mean blood pressure; VO2peak, peak oxygen uptake.

The exercise bout consisted of a standard cardiopulmonary exercise test performed on a treadmill (Master Super ATL, Inbramed, Porto Alegre, RS, Brazil). The protocol was established based on the predicted maximum aerobic capacity (maximal oxygen uptake predicted, based on age, weight, and sex) of each individual.23 The test consisted of a warm-up for three minutes at 3 km/h and 0% grade, followed by a ramp protocol with a linear increase in speed and grade every minute and recovery for five minutes at 4 km/h and 0% grade. The ramp protocol was individualized according to the predicted maximal exercise capacity to reach volitional fatigue at approximately ten minutes. The subjects were verbally encouraged to exercise until exhaustion. All subjects met at least two of the following criteria to confirm that maximal effort was attained: 1) respiratory exchange ratio .1.1; 2) HR within ¡10 beats/minute of the age-predicted maximum [210 – (age?0.65)]; 3) a perceived effort score of 10 on the Borg 0-10 scale.24 Ventilation, oxygen uptake, and carbon dioxide output were measured with each breath (CPX Ultima Gas Exchange System; Medgraphics, St Paul, Minnesota, USA). Breath-by-breath ventilation and expired gases were averaged over 20 s to identify the peak oxygen consumption (VO2peak), which was considered to be the highest oxygen uptake value during the exercise bout.

would be expected. However, there were no differences between the groups regarding anthropometric, biochemical, or exercise capacity variables. Figure 1 shows the absolute values of the hemodynamic variables at baseline and during mental stress, which were obtained before and after a single bout of maximal dynamic exercise. Before the exercise bout, the group with PHT had higher FBF and FVC values at baseline than the group with NT. SBP, DBP, MBP, and HR increased during mental stress in both groups, whereas FBF and FVC increased only in the group with NT. In addition, the group with PHT had higher SBP, DBP, and MBP during mental stress compared with the group with NT. After the exercise bout, baseline SBP, FBF, and FVC decreased only in the group with PHT; DBP and MBP increased only in the group with NT; and HR increased in both groups compared with the values obtained before the exercise bout. Furthermore, all variables increased during mental stress in both groups. However, in the PHT group, SBP, DBP, and MBP were lower and HR was higher during the mental stress after the exercise bout than before. In the NT group, SBP was higher but HR, FBF, and FVC were lower during the mental stress after the exercise bout than before. Finally, the SBP during mental stress was higher in the group with PHT than in the group with NT. There was no difference in mental stress scores before and after exercise within the groups (PHT: before exercise 2¡0 vs. after exercise 2¡2, p = 0.58; NT: before exercise 2¡2 vs. after exercise 2¡2, p = 0.81). Figure 2 shows the percent change of hemodynamic variables in response to mental stress before and after a single bout of maximal dynamic exercise. Before the exercise bout, FBF and FVC percent change values were lower in the PHT group than in the NT group. After the exercise bout, FBF and FVC percent change values in the PHT group increased relative to the values obtained before the exercise bout, and thus, there was no longer any difference between the groups. It is worth noting that there was 80% power in the FVC percent change analysis, which was the main

Statistical analysis The Shapiro–Wilk’s test was used to verify the variables’ distribution. Group characteristics were compared using Student’s t-test, the Mann–Whitney’s test or the Chi-square test when appropriate. FBF and FVC data were logtransformed to normalize their distribution. A two-way analysis of variance (ANOVA) adjusted for gender followed by the Newman-Keuls’ post hoc test was used to compare hemodynamic data before and after exercise between the NT and PHT groups. Statistical significance was established at p,0.05 in two-tailed comparisons. All analyses were performed using the software STATISTICA (version 8; StatSoft Inc., Tulsa, Oklahoma, USA).

RESULTS As shown in Table 1, the resting SBP, DBP, and MBP were higher in subjects with PHT than in subjects with NT, as

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Figure 1 - Absolute hemodynamic responses to mental stress, adjusted for gender, before and after an exercise bout in subjects with prehypertension or normotension. Values are the mean ยก SEM. *, p,0.05 vs. within-group baseline; {, p,0.05 vs. same condition between groups; {, p,0.05 vs. before-exercise within group.

Santangelo et al.6 and Schwartz et al.7 found that part of the enhanced pressor response to mental stress in subjects with PHT was associated with blunted forearm vasodilation. In addition, Scharwrtz et al.7 showed that blunted vasodilation in response to mental stress in subjects with PHT was not mediated by sympathetic nerve activity in the muscle. In the present study we also observed an attenuated forearm vasodilator response to mental stress, which corroborates previous findings. Subjects with PHT had a higher blood flow and vascular conductance at rest, which reduced their vasodilator reserve (i.e., the change from baseline to maximal vasodilation), thus impairing their vasodilator response to mental stress. A reduction in the vasodilator reserve was shown previously in the forearm7 and coronary25 circulation of subjects with PHT, and this reduction seems to be partially mediated by passive arterial distention as a consequence of increased perfusion pressure.26 Additionally, this mechanism seems to present at the onset of HT pathophysiology.27 Further evolution of the disease induces arterial remodeling to decrease the tensile stress in the arterial wall,27 which occurs by means of an increase in the intima-media thickness and a decrease in the vessel lumen.26 The increased blood flow in the muscle vascular bed during mental stress is dependent on the integrity of the vascular endothelium, and nitric oxide (NO) production by the endothelium seems to be a cornerstone mechanism. Studies have shown that during mental stress, sympathetic cholinergic fibers,4 beta 2-adrenergic receptor stimulation by adrenaline28,29 and increased shear stress30 stimulate the release of NO, leading to vasodilation. Thus, when

endpoint of the present study. Moreover, the DBP and MBP percent change values in the NT group decreased compared with the values obtained before the exercise bout, which was not observed in the PHT group.

DISCUSSION The present study investigated hemodynamic responses to a mental stress test performed before and after a bout of maximal dynamic exercise in subjects with PHT or NT. The hypothesis of this study was that subjects with PHT and NT have different levels of vascular reactivity to mental stress and that this difference would disappear after a bout of exercise. This approach revealed two major findings. First, subjects with PHT had higher blood pressure during mental stress than subjects with NT, and the exercise bout reduced their blood pressure during the mental stress test. Second, subjects with PHT presented a blunted vasodilator response to mental stress, and the exercise bout normalized this response. Previous studies have also shown elevated blood pressure levels during mental stress in subjects with PHT.6-8 However, the mechanisms that mediate this phenomenon are still unclear. Schwartz et al.7 and the present study found that heart rate increases during mental stress were similar in subjects with PHT versus those with NT. However, no study has yet determined whether the increased stroke volume and cardiac output during mental stress are enhanced in subjects with PHT. Therefore, it is possible that increased cardiac output in response to mental stress might contribute to the augmented blood pressure levels in subjects with PHT.

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Figure 2 - Percent change of hemodynamic responses to mental stress, adjusted for gender, before and after an exercise bout in subjects with prehypertension or normotension.

NG-monomethyl-L-arginine was administered to healthy subjects to inhibit NO production, the increase in forearm vascular conductance during mental stress was blunted by approximately 45%5 to 65%.4 In this context, it was shown that subjects with HT have impaired NO-dependent vasodilation during mental stress. Considering that there is evidence that subjects with PHT also present a certain degree of endothelial dysfunction,31 it is conceivable that impaired endothelial-dependent vasodilation might also be involved in the blunted vasodilation during mental stress in subjects with PHT.

The present study showed that the enhanced pressor response to mental stress in subjects with PHT was attenuated after a bout of exercise, which is consistent with similar findings from a meta-analysis that evaluated 15 randomized controlled studies that investigated the acute effects of aerobic exercise on blood pressure responses to mental stress in subjects with NT and HT.13 Boone et al.8 and the present study found that the HR response to mental stress was not affected by an acute exercise bout in subjects with PHT. No study has yet evaluated stroke volume and cardiac output responses to mental stress in this population

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following an exercise bout. However, West et al.32 observed a significant reduction in the total vascular resistance during mental stress in subjects with borderline HT after an exercise bout. In this context, the present study adds the finding that the forearm vasodilator response during mental stress was enhanced after an acute bout of exercise in subjects with PHT, which suggests that there was enhanced vasodilation in the forearm skeletal muscles. Three primary mechanisms might be involved in the enhanced vasodilation in response to mental stress in skeletal muscles following an acute bout of exercise. First, it was observed that the exercise bout reduced SBP at rest in subjects with PHT, which might have attenuated the passive arterial distension caused by increased perfusion pressure27 and thus might be associated with the reduction in baseline blood flow. However, regardless of the cause, reduced baseline blood flow increases the vasodilator reserve, thereby facilitating vasodilation during mental stress. Second, there is evidence that following a bout of dynamic exercise, endothelial-dependent vasodilation is improved, even in vascular bed segments that were not directly involved in the exercise.12,33 This phenomenon has been attributed to a systemic increase in the expression and phosphorylation of endothelial nitric oxide synthase34,35 as a consequence of the systemic increase in shear stress during exercise, which leads to an increase in the bioavailability of NO.33 In addition, it was shown that beta 2- adrenergic receptor responsiveness is enhanced after an acute bout of exercise,36 which can also improve the vasodilator response to mental stress. Third, Brownley et al.36 showed that a reduced noradrenaline response to a stress task was associated with an attenuated post-exercise stress-related blood pressure response. Therefore, it is possible that a reduction in sympathetic vasoconstrictor activity might also have contributed to this effect. The results of this study have some clinical implications. The novel result of this study is that a single bout of exercise reversed an impaired vasodilator response to mental stress, as indicated by a lower blood pressure increase. Considering that an enhanced blood pressure response to mental stress is associated with a greater risk for the development of cardiovascular disease, our results suggest that by correcting the vasodilator response to mental stress, as the exercise bout did, it is possible to attenuate the blood pressure increase during mental stress in subjects with PHT. Further studies should determine whether chronic exposure to exercise leads to vascular adaptations that correct the vasodilator response, and whether other non-pharmacological vasodilatory interventions such as anti-oxidant and/or L-arginine supplements would yield similar benefits. The results presented here should be interpreted considering certain limitations. Blood flow was measured by venous occlusion plethysmography, which takes into account the blood flow to all tissues in the evaluated segment. The tissue that contributes most to the total blood flow measured by plethysmography is skeletal muscle,37 but skin blood flow can also contribute to the results to a small extent.37 Therefore, the temperature in the experimental room was maintained at a fairly constant level to minimize variations in skin blood flow. This technique has been validated,38 and is widely used to measure blood flow in skeletal muscle. It is possible that repeated exposure to mental stress could lead to habituation. However, Hammer et al. showed that repeated exposure to the same test that

was used in the present study did not change cardiovascular responses to mental stress.39 In addition, in the present study, there was no change in stress perception scores before and after exercise in either group. Hence, a repeated exposure to mental stress might not have influenced the results that were observed in the present study. Another limitation is the low number of men in the sample. This limitation is minimal because there was no significant difference in the number of men between the groups. Finally, we used a maximal dynamic exercise test to investigate the effects of exercise on cardiovascular responses to mental stress. This method has the advantage of providing a standardized exercise stimulus. It was shown previously that a bout of maximal dynamic exercise increases endothelial-mediated vasodilation for at least 60 minutes12 and increases the systemic production of NO,33 which supports our study mechanistically. However, this exercise protocol is not used during training programs. Thus, further studies should investigate whether a regular session of aerobic exercise training yields similar benefits in subjects with PHT.

CONCLUSION The present study showed that subjects with PHT have elevated blood pressure and blunted vasodilation during mental stress compared with subjects with NT, and a single bout of maximal dynamic exercise reduced their blood pressure and normalized their vasodilator response to mental stress.

ACKNOWLEDGMENTS The authors thank Felipe de Sa´ Pereira, Thales Coelho Barbosa, Tatiane Marinz de Souza and Roge´rio Barbosa M. Barros for their assistance during the experiments and Labs D’OR for biochemical blood analyses.

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25. Laine H, Raitakari OT, Niinikoski H, Pitkanen OP, Iida H, Viikari J, et al. Early impairment of coronary flow reserve in young men with borderline hypertension. J Am Coll Cardiol. 1998;32:147-53, doi: 10.1016/S07351097(98)00222-8. 26. Boutouyrie P, Bussy C, Lacolley P, Girerd X, Laloux B, Laurent S. Association between local pulse pressure, mean blood pressure, and large-artery remodeling. Circulation. 1999;100:1387-93. 27. Mayet J, Hughes A. Cardiac and vascular pathophysiology in hypertension. Heart. 2003;89:1104-9, doi: 10.1136/heart.89.9.1104. 28. von zur Muhlen B, Millgard J, Sarabi M, Lind L. Ambulatory blood pressure and endothelium-dependent vasodilation in hypertensive patients. Blood Press. 2000;9:110-5, doi: 10.1080/080370500453438. 29. Halliwill JR, Lawler LA, Eickhoff TJ, Dietz NM, Nauss LA, Joyner MJ. Forearm sympathetic withdrawal and vasodilatation during mental stress in humans. J Physiol. 1997;504:211-20, doi: 10.1111/j.1469-7793. 1997.211bf.x. 30. Reims HM, Sevre K, Hoieggen A, Fossum E, Eide I, Kjeldsen SE. Blood viscosity: effects of mental stress and relations to autonomic nervous system function and insulin sensitivity. Blood Press. 2005;14:159-69, doi: 10.1080/08037050510034176. 31. Cugini P, Baldoni F, De Rosa R, Pandolfi C, Colotto M, Buccarella PA, et al. Higher blood pressure load (baric impact) in normotensives with endothelial dysfunction: a paraphysiological status of ‘‘pre-hypertension’’. Clin Ter. 2002;153:309-15. 32. West SG, Brownley KA, Light KC. Postexercise vasodilatation reduces diastolic blood pressure responses to stress. Ann Behav Med. 1998;20:7783, doi: 10.1007/BF02884452. 33. Allen JD, Cobb FR, Gow AJ. Regional and whole-body markers of nitric oxide production following hyperemic stimuli. Free Radic Biol Med. 2005;38:1164-9, doi: 10.1016/j.freeradbiomed.2004.12.018. 34. Boo YC, Sorescu G, Boyd N, Shiojima I, Walsh K, Du J, et al. Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A. J Biol Chem. 2002;277:3388-96, doi: 10.1074/jbc.M108789200. 35. Zhang QJ, McMillin SL, Tanner JM, Palionyte M, Abel ED, Symons JD. Endothelial nitric oxide synthase phosphorylation in treadmill-running mice: role of vascular signalling kinases. J Physiol. 2009;587:3911-20, doi: 10.1113/jphysiol.2009.172916. 36. Brownley KA, Hinderliter AL, West SG, Girdler SS, Sherwood A, Light KC. Sympathoadrenergic mechanisms in reduced hemodynamic stress responses after exercise. Med Sci Sports Exerc. 2003;35:978-86, doi: 10. 1249/01.MSS.0000069335.12756.1B. 37. Cooper KE, Edholm OG, Mottram RF. The blood flow in skin and muscle of the human forearm. J Physiol. 1955;128:258-67. 38. Matthews JN, Altman DG, Campbell MJ, Royston P. Analysis of serial measurements in medical research. BMJ. 1990;300:230-5, doi: 10.1136/ bmj.300.6719.230. 39. Hamer M, Boutcher YN, Park Y, Boutcher SH. Reproducibility of skeletal muscle vasodilatation responses to Stroop mental challenge over repeated sessions. Biol Psychol. 2006;73:186-9, doi: 10.1016/j.biopsycho. 2006.03.003.

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DOI:10.1590/S1807-59322011000900004

CLINICAL SCIENCE

Prevalence of metabolic syndrome and related factors in Taiwanese high-tech industry workers Tzung-Yi Tsai,I Jung-Feng Cheng,II Yu-Min LaiIII,IV I

Department of Research, Buddhist Dalin Tzu Chi General Hospital, Taiwan. II Department of Nursing, Buddhist Dalin Tzu Chi General Hospital, Taiwan. Department of Family Medicine, Buddhist Dalin Tzu Chi General Hospital, Taiwan. IV School of Medicine, Tzu Chi University, Hualien, Taiwan.

III

OBJECTIVES: In light of the increasing number of high-tech industry workers and the differences in their working conditions compared to those of the general population, the health status of these workers merits serious attention. This study aimed to explore the prevalence of metabolic syndrome and its correlates among Taiwanese high-tech industry workers. METHODS: This cross-sectional study included 4,666 workers who participated in labor health examinations at a hospital in southern Taiwan in 2008. Participants with metabolic syndrome were defined using the criteria proposed by the Taiwan National Department of Health in 2007. Factors associated with metabolic syndrome were determined using multiple logistic regression analysis. RESULTS: The overall prevalence of metabolic syndrome was 8.2%, and the prevalence was higher in men than in women (14.0% vs. 2.3%, p,0.01). Male gender, advanced age, elevated white blood count, and elevated levels of blood biochemistry markers, such as alanine aminotransferase and uric acid, can independently predict metabolic syndrome. CONCLUSIONS: The prevalence of metabolic syndrome among high-tech industry workers is lower than in the general population. Our study’s findings may facilitate early health assessments and the provision of proper workplace health promotion programs to reduce the risks faced by high-risk workers. KEYWORDS: High-tech industry; Metabolic syndrome; Workplace health promotion. Tsai TY, Cheng JF, Lai YM. Prevalence of metabolic syndrome and related factors in Taiwanese high-tech industry workers. Clinics. 2011;66(9):15311535. Received for publication on March 28, 2011; First review completed on May 2, 2011; Accepted for publication on May 16, 2011 E-mail: dm732024@tzuchi.com.tw Tel.: 886 5 2648000 ext. 5968

mellitus, hypertension, and pyelonephritis, had surpassed cancer mortality (27.3%), revealing that metabolic abnormalities have become a major health challenge. Given its threat to the well-being of humans, a syndrome related to insulin resistance abnormalities was first proposed by Reaven in 1988.4 Subsequently, in 1998, the WHO introduced the first internationally recognized term for metabolic abnormalities, metabolic syndrome (MS), which is characterized by a core set of disorders, including glucose intolerance, central obesity, dyslipidemia, and hypertension co-occurring within an individual.5 Although there is still some debate as to the precise definition of MS, its prevalence around the world has been estimated at approximately 7% to 58% according to the Adult Treatment Panel III definition, and these figures were still rising when the criteria were specified by the WHO.6 Recently, investigators have reported on the challenge that MS poses for human health. For example, MS is not only an indication of the likelihood of developing coronary heart disease or type II diabetes but is also associated with a threefold greater risk of cardiovascular death, as compared to those without the disorder.5 Additionally, MS is related to cerebral atrophy, which may trigger depression and cognitive impairment.7,8 Most recently, studies on the

INTRODUCTION As a result of economic growth and associated sociodemographic shifts, changes in lifestyle and diet have led to an elevated risk with respect to chronic diseases, especially cardiovascular disease. According to the World Health Organization (WHO), cardiovascular disease is the leading cause of death worldwide, claiming 17.5 million victims yearly.1 This high mortality rate creates an enormous financial burden. In the European Union, the yearly direct and indirect economic cost of cardiovascular disease is as high as 169 billion EUR.2 In contrast, after reviewing the top ten causes of mortality in Taiwan, we found that chronic diseases had replaced infectious diseases as the main cause of death. For example, according to statistics reports from the Taiwan National Department of Health in 2008,3 the mortality attributed to metabolic abnormalities (29.5%), including cerebrovascular disease, heart disease, diabetes

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the feet touching each other and both arms hanging freely. Hemoglobin concentration, ALT, AST, triglycerides, fasting blood glucose, UA, WBC, and HDL-C were evaluated by automatic bio-assays (Olympus AU-640, Tokyo, Japan). MS was defined as the presence of three or more of the following five criteria proposed by the Taiwan National Health Department:18 (1) fasting blood glucose $100 mg/dl or self-reported diabetes mellitus; (2) blood pressure $130/ 85 mmHg or self-reported hypertension; (3) HDL-C cholesterol ,40 mg/dl in men or ,50 mg/dl in women; (4) triglycerides $150 mg/dl; and (5) waist circumference $90 cm in men or $80 cm in women.

correlation between malignancy and MS have shown that patients with MS have a 1.6-fold higher risk of death from cancer than those without MS.9 Therefore, clarifying the factors related to MS and to identify high-risk cases early may be of utmost importance for public health. Workers are an important part of any institution. Since the promulgation of the Ottawa Charter for Health Promotion by the WHO in 1986, global support for workplace health promotion has encouraged employers to hold health promotion activities to ensure employee health.10 With increasing competition in the global market and the rapid development of technology, all countries (including Taiwan) seek to foster growth in high-tech industries. Facilitated by support from the government, high-tech products comprised 71.2% of all exports from Taiwan in 2006 with a value of US$159.5 billion.11 Taiwan is a pivotal site for high-tech industry development in the world, but increasing demand for high-tech products may increase the already heavy workload for workers in this industry. Therefore, the health status of high-tech industry workers requires immediate attention. Many previous studies have focused on musculoskeletal discomfort,12 workplace stress,13 skin disease,14 and lumbar pain,15 among other issues, but there is a relative paucity of data pertaining to metabolic abnormalities in the workplace. Most importantly, the medical expenses related to MS are considerable and thus cannot be overlooked. Goetzel et al.16 found that obesity poses a burden of US$644 per worker on companies, due to medical expenses and loss of productivity. Moreover, this cost rises to US$3,719 with the occurrence of MS,17 which may have a major impact on the fiscal performance of the company. Therefore, our study aimed to understand the prevalence of MS and related factors in high-tech industry workers, which should facilitate the development of strategies for promoting appropriate workplace health.

Statistical analysis Descriptive and inferential analyses were conducted in accordance with the studyâ&#x20AC;&#x2122;s aims and the nature of the variables. For descriptive analysis, subjects were grouped according to the presence of MS. Differences in various factors between groups are expressed with averages, standard deviations (SDs), and percentages. Besides gender, which is shown as a percentage, other variables with continuous distributions are expressed as averages with SD. For inferential analysis, t-tests and chi-square tests were used to explore the relationship between analytical variables and MS. Multiple logistic regression analysis with the simultaneous entry method was further used to determine risk factors and their adjusted odds ratios (AORs). All analyses were conducted with Statistical Package for Social Sciences (SPSS) version 15.0 software (SPSS, Inc., Chicago, IL), and p,0.05 was considered significant.

RESULTS The 4,666 high-tech industry workers enrolled in this study included 2,344 men and 2,322 women. Among subjects, the overall prevalence of MS was 8.2% (382/ 4,666) with 14.0% (328/2,344) in men and 2.3% (54/2,322) in women, revealing a higher MS prevalence in men (p,0.01). Based on the 2007 population census published by the Department of Health,19 the age-standardized prevalence was 5.9%, which was also higher in men than in women (10.1% vs. 1.7%, p,0.01). Additionally, men also showed higher BMIs, WBCs, liver enzyme levels, creatinine, UA, and hemoglobin, as compared to women (p,0.01). Subjects with MS averaged 33.5 years in age, as opposed to 30.2 years for those without MS. On average, workers with MS had significantly higher BMIs and abnormal blood samples, as compared to those without MS (Table 1). Our analysis also showed that the most common metabolic abnormalities among men and women with MS was higher waist circumference, which was followed by irregular blood pressure and hypertriglyceridemia; these disorders had prevalences of 82.0%, 81.4%, and 73.2% in males and 98.2%, 88.9%, and 64.8% in females, respectively (Table 2). Multivariate analysis revealed that men had an AOR of 3.94 (95% confidence interval (CI) = 2.2926.78) for developing MS, as compared to women. A one-year increase in age was associated with a 6% (95% CI = 1.04-1.09) increase in the risk of MS. An increase in BMI of one raised the AOR for MS by 2% (95% CI = 1.0121.02). Regarding blood biochemistry markers, an increase of one unit of ALT increased the AOR for MS by 30% (95% CI = 1.2621.35). An increase of one unit of UA or WBC was associated with an increase of

METHODS Research design and participants This study was cross-sectional. The subjects were workers from a company in the integrated circuit industry; the company contracted a hospital in Taiwan for health examination services in 2008. A total of 4,666 workers providing informed consent were included in this study, and the study was reviewed and approved by the Institutional Review Board of the target hospital.

Data characteristics and diagnostic criteria Analytical data were obtained from the health examination database. These data included gender, age, height, body weight, blood pressure, waist circumference, and blood biochemistry markers (i.e., white blood cell count (WBC), hemoglobin concentration, fasting blood glucose levels, high-density lipoprotein cholesterol (HDL-C), triglycerides, uric acid (UA), creatinine, aspartate aminotransferase (AST), and alanine aminotransferase (ALT)). The body mass index (BMI) was calculated by dividing weight (in kg) by height (in m2). Systolic and diastolic blood pressures were measured using an automatic sphygmomanometer (UDEX-IIa, UEDA Corp., Tokyo, Japan) and recorded in the seated position after participants had rested for at least 5 min. Waist circumference was measured with a heavyduty inelastic plastic fiber tape measure to the nearest 0.5 cm, while the subject stood balanced on both feet with

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Table 1 - Characteristics of study participants. MS cases (n = 382)

Variables

Non-MS cases (n = 4284)

Mean (SD) Gender Male Female Age BMI Liver Enzymes AST (IU/L) ALT (IU/L) Creatinine (mg/dl) UA (mg/dl) WBC (103 cells/ml) Hemoglobin (g/dl)

n (%)

Mean (SD)

2016 (86.01) 2268 (97.67)

,0.01

33.52 (5.76) 28.50 (3.68)

30.17(5.36) 22.97(3.75)

,0.01 ,0.01

29.84 (20.04) 49.99 (34.89) 1.12 (0.17) 7.10 (1.48) 7.93 (1.95) 15.05 (1.40)

19.39 (8.39) 22.29 (18.05) 0.99 (0.33) 5.63 (1.45) 7.13 (1.87) 14.00 (1.52)

,0.01 ,0.01 ,0.01 ,0.01 ,0.01 ,0.01

between age and predisposition for MS,21-22 further research is needed to elucidate whether the leading metabolic abnormalities vary with age. Men were at a 3.47-fold greater risk of MS than women, which is in line with previous studies.22 Two reasons may account for this phenomenon. First, men are less aware of self-care than women. For instance, not only do fewer women smoke and drink, but they are more prone to seek medical counsel or an examination in the event of physical discomfort. Thus, the risks for chronic diseases, such as hypertension and diabetes, are usually lower in women than in men.25 Second, this study was mostly conducted on younger subjects, and younger women may benefit from higher estrogen levels, which decrease serum low-density lipoprotein-cholesterol (LDL-C) and curb coronary thrombosis and atherosclerosis by regulating vascular smooth muscle and endothelial cells. Our findings also show that age was positively correlated with the risk of MS, which is in line with previous studies.20-21 It can be inferred that, with age, blood vessels gradually lose elasticity and gain resistance, slowing blood flow. Moreover, with poor circulation, fat is prone to accumulate in the abdomen and release free fatty acids into the serum, leading to higher insulin resistance, elevated serum triglyceride levels, increased small dense LDL-C levels, and, consequently, a greater risk of MS.26 This study also showed that a higher BMI was associated with a greater AOR for developing MS, which confirms a previous report.21 BMI can be used to assess body

DISCUSSION The overall MS prevalence was 8.2% (14.0% in men and 2.3% in women). Because the average age of the subjects, who ranged in age from 31 to 40 years, was younger than in most other epidemiological studies, it is difficult to directly compare our results with MS prevalence reported elsewhere. However, as compared to the prevalence in similarly aged men and women (10.1%225.8% and 2.1%219.7%, respectively20-22), the MS prevalence among high-tech industry workers was lower than in the general population. This difference may be attributed to the healthy worker effect: these workers not only must demonstrate a betterthan-average health status to qualify for work but also are screened by health examinations before being allowed to enter the high-tech industry. We found that the most common manifestations of MS in both men and women were high waist circumference followed by high blood pressure and hypertriglyceridemia. However, Pan23 found that blood pressure, triglyceride, and HDL-C disorders were the most common metabolic abnormalities among male and female adults in Taiwan. Another study of 1,562 Taiwanese teenagers indicated that triglycerides, HDL-C, and waist circumference were the most common abnormal indicators in male adolescents, as compared to HDL-C, blood pressure, and waist circumference in female adolescents.24 Findings based on the same ethnic group, therefore, may vary due to age discrepancies with respect to the leading types of metabolic abnormalities (one third of Pan’s subjects were aged 45 years or above). Despite several studies showing a significant correlation

Table 3 - Factors associated with MS according to multiple logistic regression analysis. Variables Gender Male Female Age BMI Liver enzymes AST (IU/L) ALT (IU/L) Creatinine (mg/dl) UA (mg/dl) WBC (103 cells/ml) Hemoglobin (g/dl)

Table 2 - Sex-specific prevalence of individual metabolic abnormalities among MS cases.

High triglycerides High fasting glucose Abdominal obesity Low HDL–C High blood pressure

n (%)

328 (13.99) 54 (2.33)

14% (95% CI = 1.0321.27) and 12% (95% CI = 1.0421.19), respectively, in the risk of being diagnosed with MS (Table 3).

Metabolic abnormality

p-value

Male (n = 328)

Female (n = 54)

95% CI

73.17 49.09 82.01 39.63 81.40

68.37–77.97 43.68–54.50 77.85–86.17 34.34–44.92 77.17–85.61

64.81 55.56 98.15 0 88.89

52.07–77.55 42.31–68.81 94.56–101.74 0 80.51–97.27

p,0.05; p,0.01.

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Crude OR (95% CI)

Adjusted OR (95% CI)

6.83 (5.09–9.12) 1 1.11 (1.08–1.12) 1.04 (1.03–1.04)

3.94 (2.29–6.78)** 1 1.06 (1.04–1.09)** 1.02 (1.01–1.02)*

1.06 1.36 4.62 1.87 1.21 1.71

1.00 (0.96–1.01) 1.30 (1.26–1.35)** 0.73 (0.30–1.76) 1.14 (1.03–1.27)** 1.12 (1.04–1.19)** 1.00 (0.88–1.13)

(1.05–1.07) (1.32–1.39) (2.83–7.58) (1.74–2.01) (1.16–1.27) (1.57–1.86)

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generalizable. More diverse samples collected by a nationwide survey or random sampling should be considered in future research. Second, as a cross-sectional study, we cannot infer causality from our findings. A longitudinal study design is needed to investigate any causal relationships among the factors included in this study. Third, data on potential factors, such as smoking, alcohol, education, exercise habits, family history, and worksite pressure were not collected in this study; lack of information about these factors may have caused some inferential error. Future studies are needed to understand the impact of the abovementioned confounding variables on MS. Moreover, issues related to the medical costs induced by MS also warrant further evaluation. Despite these methodological concerns, to our knowledge, this was the first evidence-based study to estimate the prevalence of MS in Taiwanese high-tech industry workers. It also involved an analysis of corresponding health check-up data, which can be useful in assessing the risk factors for MS because the data were obtained from standardized laboratory tests.

fat content, which serves as an indicator of obesity. In cases of obesity, adipose cells release free fatty acids and tumor necrosis factor-a (TNF-a), which blocks phosphatidylinositide-3-kinase-dependent signal transduction pathways and reduces glucose uptake in the liver and skeletal muscles.6,27 To compensate, pancreatic b cells are induced to secrete excessive insulin, leading to hyperglycemia or type II diabetes. We discovered that ALT can better assess the risk of MS than AST, which is in line with both cross-sectional and cohort studies.28-29 This finding stems from the storage of ALT in hepatic cells; therefore, ALT is a more effective marker for detecting pathogenic hepatic fatty changes caused by obesity, as compared to AST.29 Two reasons may explain the positive relationship between ALT and the risk of MS. First, abnormally elevated ALT is often found in people under tremendous workplace pressure or extreme fatigue.30 High psychological pressure may activate the sympathetic system to raise arterial blood pressure and reduce renal blood flow.31 Consequently, the patient develops vasculitic reactions and elevated serum UA levels, increasing the risk of MS. Second, elevated ALT can also indicate abnormal liver function caused by fatty liver.28 Once a patient has developed fatty liver, fat accumulation has usually already occurred in the muscle and other organs, such as the pancreas. As these tissues release free fatty acids that inhibit the insulin-based regulation of the uptake and utilization of glucose, metabolic abnormalities subsequently emerge.27 This study found a positive correlation between WBC and the risk of MS, which was in line with previous literature reports.32-33 A higher WBC may be related to bacterial infection or vasculitis caused by hyperglycemia and dyslipidemia. In an inflammatory state, vascular endothelial cells secrete proinflammatory cytokines, such as interleukin6 or TNF-a, which can activate and attract massive numbers of white blood cells to the damaged region within the lumen of the vessel.33 Following infiltration into the tunica media, white blood cells absorb oxidized LDL-C and become foam cells, which may greatly increase the risk of thromboembolism. Additionally, Rho kinase secreted by white blood cells can serve as another potential pathogenic mechanism. Liu et al.32 have found that Rho kinase can block insulin signaling by stimulating the phosphorylation of insulin receptor substrate-1. This action prevents the translocation of glucose to the cell membrane by glucose transporter 4, thereby leading to insulin resistance. Our study revealed that workers with MS have a higher risk of elevated UA, which is in accordance with a previous report.34 The mechanism involved may be related to the ability of high levels of UA to inhibit the production of nitric oxide (NO) by endothelial cells. NO can serve as an inhibitor of thrombosis and inflammatory reactions, further preventing the growth and transfer of vascular smooth muscle cells.35 As for hemoglobin levels, we discovered that hemoglobin was of borderline significance. Because the specific biological mechanisms by which hemoglobin levels increase the risk of MS remain unclear, further study is needed to clarify the effect of hemoglobin levels on MS, especially among diverse participants. The results from this study should be interpreted with the following limitations in mind. First, subjects were drawn from a single company in southern Taiwan, and, therefore, the inferences drawn from the results are not completely

CONCLUSION This study explored the prevalence of MS and related factors among high-tech industry workers. We found that factors contributing to a high risk of MS included being male and older, as well as having a higher BMI, increased ALT, elevated UA, and higher WBC. If our study is replicated in other occupational settings, the results may have significant economic and public-health implications for other shift-work occupational groups, such as the military, medical service personnel, and police officers. We believe that our findings support the need to improve workplace health promotion in the high-tech industry. For example, appropriate on-site diet and exercise programs aimed at high-tech industry workers should be implemented. In addition, occupational managers should actively offer in-service education concerning MS prevention to workers and monitor their physical and mental health, especially in high-risk cases. In summary, the identification of at-risk workers and disease management programs addressing MS-related factors are of great importance in employer-based interventions.

ACKNOWLEDGMENT This study was supported by grants from the Buddhist Dalin Tzu Chi General Hospital.

REFERENCES 1. World Health Organization. Cardiovascular Diseases. Available online: http://www.who.int/mediacentre/factsheets/fs317/en/index.html. Accessed 2010 Apr 25. 2. Leal J, Luengo-FernaÂ´ndez R, Gray A, Petersen S, Rayner M. Economic burden of cardiovascular diseases in the enlarged European Union. Eur Heart J. 2006;27:1610-9, doi: 10.1093/eurheartj/ehi733. 3. Department of Health. 2008 Statistics of Cause of Death. Available online: http://www.doh.gov.tw/CHT2006/DM/DM2_2_p02.aspx? class_no = 440&now_fod_list_no = 10642&level_no = 3&doc_no = 73104. Accessed 2010 Jun 20. 4. Reaven GM. Banting lecture 1988.Role of insulin resistance in human disease. Diabetes. 1988;37:1595-607, doi: 10.2337/diabetes.37.12.1595. 5. Gami AS, Witt BJ, Howard DE, Erwin PJ, Gami LA, Somers VK, et al. Metabolic syndrome and risk of incident cardiovascular events and death: a systematic review and meta-analysis of longitudinal studies. J Am Coll Cardiol. 2007;49:403-14, doi: 10.1016/j.jacc.2006.09.032. 6. Cornier MA, Dabelea D, Hernandez TL, Lindstrom RC, Steig AJ, Stob NR, et al. The metabolic syndrome. Endocr Rev. 2008;29:777-822, doi: 10. 1210/er.2008-0024.

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7. Koponen H, Jokelainen J, KeinaÂ¨nen-Kiukaanniemi S, Kumpusalo E, Vanhala M. Metabolic syndrome predisposes to depressive symptoms: a population-based 7-year follow-up study. J Clin Psychiatry. 2008;69:17882, doi: 10.4088/JCP.v69n0202. 8. Yaffe K, Weston AL, Blackwell T, Krueger KA. The metabolic syndrome and development of cognitive impairment among older women. Arch Neurol. 2009;66:324-8, doi: 10.1001/archneurol.2008.566. 9. Jaggers JR, Sui X, Hooker SP, LaMonte MJ, Matthews CE, Hand GA, et al. Metabolic syndrome and risk of cancer mortality in men. Eur J Cancer. 2009;45:1831-8. 10. McMahon A, Kelleher CC, Helly G, Duffy E. Evaluation of a workplace cardiovascular health promotion programme in the Republic of Ireland. Health Promot Int. 2002;17:297-308, doi: 10.1093/heapro/17.4.297. 11. Directorate General of Budget, Accounting and Statistics, ROC. National Statistics. Available online: http://www.stat.gov.tw/public/Data/ 77316232871.pdf. Accessed 2010 Jun 8. 12. Lin RT, Chan CC. Effectiveness of workstation design on reducing musculoskeletal risk factors and symptoms among semiconductor fabrication room workers. Int J Ind Ergon. 2007;37:35-42, doi: 10.1016/j. ergon.2006.09.015. 13. Huang C. Y., Su, S. B., Guo, H. R., & Hsu, S. Y. Using Karasekâ&#x20AC;&#x2122;s model to evaluate work related stress among male rotating shift workers in the high-tech industry in Taiwan. Chin J Occup Med. 2009;16:109-17. 14. Shiao JSC, Sheu HM, Chen CJ, Tsai PJ, Guo YL. Prevalence and risk factors of occupational hand dermatoses in electronics workers. Toxicol Ind Health. 2004;20:1-7, doi: 10.1191/0748233704th193oa. 15. Hsu WH, Wang MJ. Physical discomfort among visual display terminal users in a semiconductor manufacturing company: a study of prevalence and relation to psychosocial and physical/ergonomic factors. AIHA J. 2003;64:276-82, doi: 10.1080/15428110308984818. 16. Goetzel RZ, Gibson TB, Short ME, Chu BC, Waddell J, Bowen J, et al. A multi-worksite analysis of the relationships among body mass index, medical utilization, and worker productivity. J Occup Environ Med. 2010;52:S52-8, doi: 10.1097/JOM.0b013e3181c95b84. 17. Schultz AB, Edington DW. Metabolic syndrome in a workplace: prevalence, co-morbidities, and economic impact. Metab Syndr Relat Disord. 2009;7:459-68, doi: 10.1089/met.2009.0008. 18. National Department of Health. The Definition of the Metabolic Syndrome in Adults. Available online: http://www.bhp.doh.gov.tw/ BHP/do/chinese/Press/View;jsessionid = EB0141BDFD1C5CCCE6369 69C323CEE3C?p_No = 200711816324963WA3X+. Accessed 2009 Aug 20. 19. National Department of Health. Health Statistics Indicators in 2008. Available online: http://www.doh.gov.tw/CHT2006/DM/DM2_2. aspx?now_fod_list_no = 10338&class_no = 440&level_no = 3. Accessed 2008 December 5. 20. Chuang SY, Chen CH, Chou P. Prevalence of metabolic syndrome in a large health check-up populations in Taiwan. J Chin Med Assoc. 2004;67:611-20.

21. Hwang LC, Bai CH, Chen CJ. Prevalence of obesity and metabolic syndrome in Taiwan. J Formos Med Assoc. 2006;105:626-35, doi: 10.1016/ S0929-6646(09)60161-3. 22. Lohsoonthorn V, Lertmaharit S, Williams MA. Prevalence of metabolic syndrome among professional and office workers in Bangkok, Thailand. J Med Assoc Thai. 2007;90:1908-15. 23. Pan WP. Metabolic syndrome-an important but complex disease entity for Asians. Acta Cardiol Sin. 2002;18:24-6. 24. Chin HC, Chu NF, Shen MH, Wu DM. Prevalence of metabolic syndrome among junior high school students in Taipei. Taiwan J Fam Med. 2007;17:27-37. 25. Minh HV, Byass P, Chuc NTK, Wall S. Gender differences in prevalence and socioeconomic determinants of hypertension: findings from the WHO STEPs survey in a rural community of Vietnam. J Hum Hypertens. 2006;20:109-15, doi: 10.1038/sj.jhh.1001942. 26. Ai M, Otokozawa S, Asztalos BF, Ito Y, Nakajima K, White CC, et al. Small dense LDL cholesterol and coronary heart disease: Results from the Framingham Offspring study. Clin Chem. 2010;56:967-76, doi: 10. 1373/clinchem.2009.137489. 27. Arner P. Insulin resistance in type 2 diabetes: role of fatty acids. Diabetes Metab Res Rev. 2002;18:S5-9, doi: 10.1002/dmrr.254. 28. Perera S, Lohsoonthorn V, Jiamjarasrangsi W, Lertmaharit S, Williams MA. Association between elevated liver enzymes and metabolic syndrome among Thai adults. Diabetes Metab Clin Res Rev. 2008;2:171-8, doi: 10.1016/j.dsx.2008.04.012. 29. Goessling W, Massaro JM, Vasan RS, D9Agostino RB Sr, Ellison RC, Fox CS. Aminotransferase levels and 20-year risk of metabolic syndrome, diabetes, and cardiovascular disease. Gastroenterology. 2008;135:193544, doi: 10.1053/j.gastro.2008.09.018. 30. Lin YC, Chen JD, Chen CJ. Abnormal liver function and central obesity associate with work-related fatigue among the Taiwanese workers. World J Gastroenterol. 2008;14:6541-5. 31. Mancia GA, Bousquet PB, Elghozi JL, Esler MD, Grassi GA, Julius SE, et al. The sympathetic nervous system and the metabolic syndrome. J Hypertens. 2007;25:909-20, doi: 10.1097/HJH.0b013e328048d004. 32. Liu PY, Chen JH, Lin LJ, Liao JK. Increased rho kinase activity in a Taiwanese population with metabolic syndrome. J Am Coll Cardiol. 2007;49:1619-24, doi: 10.1016/j.jacc.2006.12.043. 33. Oda E, Kawai R. Comparison between high-sensitivity c-reactive protein (hs-CRP) and white blood cell count (WBC) as an inflammatory component of metabolic syndrome in Japanese. Intern Med. 2010;49:117-24, doi: 10.2169/internalmedicine.49.2670. 34. Sui X, Church TS, Meriwether RA, Lobelo F, Blair SN. Uric acid and the development of metabolic syndrome in women and men. Metabolism. 2008;57:845-52, doi: 10.1016/j.metabol.2008.01.030. 35. Maxwell AJ, Bruinsma KA. Uric acid is closely linked to vascular nitric oxide activity : Evidence for mechanism of association with cardiovascular disease. J Am Coll Cardiol. 2001;38:1850-8, doi: 10.1016/S07351097(01)01643-6.

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DOI:10.1590/S1807-59322011000900005

CLINICAL SCIENCE

Organic grape juice intake improves functional capillary density and postocclusive reactive hyperemia in triathletes Mariana Correa Gonc¸alves,I Flavia Fioruci Bezerra,I Elis Cristina de Araujo Eleutherio,II Eliete Bouskela,III Josely KouryI I Department of Basic and Experimental Nutrition, State University of Rio de Janeiro, Rio de Janeiro/RJ, Brazil. II Department of Biochemistry, Federal University of Rio de Janeiro, Rio de Janeiro/RJ, Brazil. III Laboratory for Clinical and Experimental Research in Vascular Biology, State University of Rio de Janeiro, Rio de Janeiro/RJ, Brazil.

OBJECTIVE: The aim of this study was to evaluate the effect of organic grape juice intake on biochemical variables and microcirculatory parameters in triathlon athletes. INTRODUCTION: The physiological stress that is imposed by a strenuous sport, such as a triathlon, together with an insufficient amount of antioxidants in the diet may cause oxidative imbalance and endothelial dysfunction. METHODS: Ten adult male triathletes participated in this study. A venous blood sample was drawn before (baseline) and after 20 days of organic grape juice intake (300 ml/day). Serum insulin, plasma glucose and uric acid levels, the total content of polyphenols, and the erythrocyte superoxide dismutase activity were determined. The functional microcirculatory parameters (the functional capillary density, red blood cell velocity at baseline and peak levels, and time required to reach the peak red blood cell velocity during postocclusive reactive hyperemia after a one-min arterial occlusion) were evaluated using nailfold videocapillaroscopy. RESULTS: Compared with baseline levels, the peak levels of serum insulin (p = 0.02), plasma uric acid (p = 0.04), the functional capillary density (p = 0.003), and the red blood cell velocity (p,0.001) increased, whereas the plasma glucose level (p,0.001), erythrocyte superoxide dismutase activity (p = 0.04), and time required to reach red blood cell velocity during postocclusive reactive hyperemia (p = 0.04) decreased after organic grape juice intake. CONCLUSION: Our data showed that organic grape juice intake improved glucose homeostasis, antioxidant capacity, and microvascular function, which may be due to its high concentration of polyphenols. These results indicate that organic grape juice has a positive effect in endurance athletes. KEYWORDS: Endurance athletes; Nailfold videocapillaroscopy; Glucose homeostasis; Antioxidant capacity; Microcirculatory function. Gonc¸alves MC, Bezerra FF, Eleutherio ECC, Bouskela E, Koury J. Organic grape juice intake improves functional capillary density and postocclusive reactive hyperemia in triathletes. Clinics. 2011;66(9):1537-1541. Received for publication on March 30, 2011; First review completed on April 28, 2011; Accepted for publication on May 16, 2011 E-mail: jckoury@gmail.com Tel.: 55 21 23340679

oxide bioactivities in the dermal microcirculation.4 These observations have considerable physiological impact and may indicate that endurance training modifies the thermoregulatory control of skin blood flow and reduces the risk of cardiovascular disease in athletes. Circulatory adaptations are important for exercise maintenance in athletes.4 However, several mechanisms can destroy or disrupt these adaptations. For instance, the production of intracellular reactive oxygen species may increase because of the alteration of the metabolic oxidation rate in the organism.1 To avoid oxidative damage, reactive oxygen species are counteracted by a sophisticated antioxidant defense system that includes enzymatic scavengers, such as superoxide dismutases (SOD), catalases, and peroxidases, as well as several other nonenzymatic low

INTRODUCTION The triathlon is a strenuous sport that includes three modalities: swimming, cycling, and running. Physiological stress and muscle damage may occur during training and competitions.1 Studies indicate that continuous exercise and a heavy training load improve enzymatic antioxidant protection2 and promote vasodilatation of the cutaneous microcirculation in humans3,4 possibly because of augmented nitric

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molecular weight molecules, such as glutathione and uric acid, which are synthesized by cells, and ascorbate, carotenoids and polyphenols, which are acquired from the diet.5 Additionally, a paradox seems to arise when the antioxidant capacity is unbalanced, and enhanced oxidative stress may lead to cellular damage and endothelial dysfunction.6 Grape juice is a relevant source of polyphenolic compounds.7 Organic agriculture, which does not use pesticides during cultivation, yields a higher content of secondary metabolites.8 Polyphenol-rich beverages prevent platelet aggregation,9 increase plasma antioxidant capacity,10 improve hyperglycemic control,11 and protect against cardiovascular diseases.12 Taken together, these factors improve microvascular endothelial function.2,3,4 However, no data are available on the effects of organic grape juice (OGJ) on glucose homeostasis, antioxidant capacity and cutaneous microcirculatory function in athletes. The aim of this study was to evaluate the effects of the inclusion of OGJ in the diet of triathletes on glucose homeostasis, antioxidant status, and cutaneous microcirculatory function.

water. Aliquots of plasma, serum, and erythrocytes were stored at –20 ˚C until analysis.

Laboratory assays Serum insulin was measured using a radioimmunoassay with a commercial kit (MP, Biomedicals, USA). Plasma glucose was determined by enzymatic method (Gold Analisa, catalog number 434, RJ, Brazil), plasma uric acid was determined by colorimetric analysis (Gold Analisa, catalog number 451, RJ, Brazil) and the total content of polyphenols was determined by Folin–Ciocalteu method employing galic acid as standard as previously described.13 The superoxide dismutase activity in erythrocytes (E-SOD) was determined with a commercial kit based on the enzymatic colorimetric method (Stressgen, catalog number 900-157, MI, USA). The intra-assay variation coefficient for all measurements was lower than 5%. All of the assays were run in duplicate except for the assay measuring serum insulin levels, which was run in triplicate. The homeostasis model assessment for the insulin resistance (HOMA2-IR) index was obtained using the HOMA calculator v.2.2.2 (2009).14

METHODS

Anthropometric measurements

Subjects Male triathletes of the same team (n = 13) were selected. After passing the exclusion criteria (use of vitamin and/or mineral supplements), ten triathletes participated in the study. These athletes were adults (34¡7 years) and had been members of the team for the previous five years. Information concerning the time (hours) of weekly exercise practice was obtained using individual structured interviews. During the study, the athletes were instructed not to change their dietary pattern except for exclusion of grapes and their derivatives from their daily intake. They were also instructed to maintain their rigorous weekly training program (22¡3 h/w; 30 km of cycling, 7 km of running and 2 km of swimming per day).

The total body mass was measured to the nearest 0.1 kg using a portable scale. The height was determined to the nearest 0.5 cm using a stadiometer. The subcutaneous body fat was assessed using the skin-fold measurement technique with a Lange skinfold caliper. The localization of ten sites (pectoral, midaxillar, subscapular, abdominal, suprailiac and iliospinal muscles, triceps, biceps, quadriceps and calf) and the execution of measurements (in triplicate) were performed by a trained anthropometrist, as previously described.15 The percent body fat was calculated.16 The fat-free mass was estimated by subtracting the fat mass from the total body mass.

Experimental design

The dietary habits of triathletes were assessed using a 24h recall for three days applied before and during the study, including two weekdays and one weekend day. Food pictures and rulers were provided to help estimate the amount of food and beverages consumed. A well-trained dietitian verified and quantified the food records. The dietary nutrient analysis was conducted using the Avanutri software version 3.1.5 (AVANUTRI, RJ, Brazil).

Dietary assessment

The Ethical Committee of the Universidade do Estado do Rio de Janeiro in Brazil (COEP070/2007) approved the study protocol. The study consisted of a 20-day intervention pilot trial in a selected group of ten adult male elite triathletes living and training in the same city and environment (Rio de Janeiro, Brazil) who agreed to follow the study design under the same training conditions. Triathletes consumed 300 ml of OGJ daily for 20 days. OGJ was produced by a Brazilian industry from V. labrusca, which is a variety of Bordeaux and is cultivated in southern Brazil. The total content of polyphenols in OGJ (5.32 mg/ ml) was determined using high-performance liquid chromatography (HPLC).7

Microvascular Function Assessment Nailfold videocapillaroscopy was performed by the same observer and analyzed according to a standardized and wellvalidated methodology using the fourth finger of the left hand in a temperature-controlled environment (24˚C).17 All of the subjects had fasted for 12 h and abstained from exercise for 20 h. Using the Cap Image software,18 the following microvascular parameters were determined: (a) the functional capillary density (FCD), which is the number of capillaries/mm2 with red blood cell flux, was evaluated using a magnification of 250x in a 3-mm area of the distal row of capillaries in three different areas; (b) the diameters of afferent, efferent, and apical capillaries were determined based on morphological parameters; (c) the red blood cell velocity at rest (RBCV) and its peak after an one-min arterial occlusion (RBCVmax) were determined; and (d) the time

Sample collection Fasting (12 h) blood samples were obtained after 20 h of exercise in the periods before (baseline) and after 20 days of OGJ intake. Blood samples (10 ml) were collected by venous puncture into tubes with or without heparin as an anticoagulant. Blood samples with heparin were centrifuged at 1,8006 g for 10 min to separate the plasma and blood cells. After the removal of the buffy coat, erythrocytes were washed three times with ice-cold 0.9% NaCl, and the cells were lysed with an equal volume of ice-cold deionized

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Organic grape juice and microcirculatory function Goncยธalves MC et al.

E-SOD activity decreased (p = 0.03) (Table 1) in relation to the antioxidant status. After OGJ intake, FCD (p,0.001) and RBCVmax (p,0.001) increased, whereas TRBCVmax decreased (p,0.05) compared with respective baseline levels (Table 2).

taken to reach RBCVmax (TRBCVmax) was noted. With the exception of the FCD, the microvascular parameters were measured at a magnification of 680x before and after the postocclusive reactive hyperemia (PORH) response. FCD, RBCV, RBCVmax, and TRBCVmax are considered functional parameters. Before performing the RBCV measurements on an individual capillary loop, a pressure cuff (1-cm wide) was placed around the proximal phalanx and connected to a mercury manometer. The basal RBCV was measured three times, and the intra-assay variation coefficient was 17.1%. Each variable was tested once at PORH. The nailfold videocapillaroscopy exam was repeated on nine subjects on different days, and the inter-assay variation coefficient of the tested functional parameters ranged from 2.0% to 9.0%.

DISCUSSION Physical exercise and polyphenol intake may increase antioxidant capacity2,10 and cardiovascular health,12,22 including the health of the cutaneous vascular bed.4 To our knowledge, the present study is the first one to show that OGJ intake for short period may improve glucose homeostasis, antioxidant capacity and microcirculatory parameters in elite triathletes. Circulatory adaptation in response to intense exercise is an important determinant of an athleteโ s performance. It is well recognized that routine exercise improves microcirculatory parameters in locally active muscle and in the whole organism4,22 by promoting vasodilatation that is observed in the cutaneous microcirculation,23,24 glucose uptake,25 and the endogenous antioxidant status.26 Exogenous antioxidants that are provided by an antioxidant-rich diet can interact with endogenous antioxidants and reduce the risk of cellular injury that occurs following intense exercise. Improving the antioxidant capacity by polyphenol intake may be determined using different biomarkers of oxidative stress.27 E-SOD is a cytosolic antioxidant enzyme that is responsible for superoxide anion dismutation into oxygen and hydrogen peroxide28 and is sensitive to the intake of polyphenols in humans.10,29 Uric acid is the principal component of plasma antioxidant capacity30 as a potent scavenger of peroxyl and hydroxyl radicals, especially during metabolic stress.31 In the present study, the increased plasma uric acid concentration was possibly due to the fructose concentration in OGJ, which accelerates purine metabolism.32 Decreased E-SOD activity may be caused by the reduction of intra- and extracellular oxidative imbalances. The consumption of grapes and its derivatives has been associated with lower levels of plasma glucose.26 In the present study, we observed a reduction in fasting plasma glucose and an increase in serum insulin in athletes after OGJ intake. These results may be mediated by different mechanisms, such as the insulin-mimetic effects of polyphenols, including the increased transport and oxidation of glucose as well as glycogen synthesis,33 the resveratrol-mediated activation of sirtuin 1,34 which improves insulin sensitivity and increases

Statistical analysis The data were represented as the meanยกstandard deviation. After grape juice intake, FCD, RBCV, TRBCVmax, and plasma polyphenols were not normally distributed and were log10-transformed before statistical analysis. After OGJ intake, changes in biochemical variables and microcirculatory parameters from baseline were evaluated using the paired t-test. The acceptable level for statistical significance was established at p,0.05. The statistical analysis was conducted using Sigma Stat software (3.5, Ashburn, USA).

RESULTS The baseline total body mass, percent fat mass, and fat-free mass were 71ยก4.0 kg, 10ยก3.4%, and 63.4ยก3.9 kg, respectively, and they did not change during the study. The mean energy (4121ยก1407 kcal) and macronutrient (62ยก25%, 15ยก5% and 23ยก5% for carbohydrates, proteins, and lipids, respectively) intake were adequate19 and similar throughout the study. OGJ intake led to an increase in total plasma polyphenol concentration after 20 days of OGJ intake (p = 0.048). Although the difference was small, this result has clinical relevance because polyphenols have a low bioavailability, and the higher plasma concentration after OGJ showed adherence to OGJ intake in the athleteโ s diet. During the study, all athletes were normouricemic (,0.42 mmol/l),20 normoglycemic (,5.6 mmol/l)21 and were not insulin-resistant (Table 1). After 20 days of OGJ intake, the fasting plasma glucose concentration decreased (p,0.001), and the fasting serum insulin concentration increased (p = 0.03). However, HOMA did not change. The plasma uric acid concentration increased (p = 0.01) and

Table 2 - Microcirculatory parameters of triathletes before (baseline) and 20 days after OGJ intake (n = 10).

Table 1 - Biochemical parameters for glycemic homeostasis and antioxidant status in triathletes before (baseline) and after 20 days of organic grape juice intake (n = 10). Baseline Glucose (mg/dl) Insulin (mU/ml) HOMA2-IR Uric acid (mmol/l) E-SOD (U/mg protein) Plasma polyphenols (mg of galic acid/ml)

89.4ยก8.9 13.3ยก2.7 1.7ยก 0.1 0.3ยก0.07 27.8ยก6.3 0.2ยก0.03

After grape juice intake 67.1ยก11.2* 16.9ยก3.7* 1.9ยก0.4 0.4ยก0.06* 24.3ยก2.5* 0.3ยก0.04*

Microcirculatory parameters

Baseline

After grape juice intake

Functional capillary density (n/mm2) Afferent diameter (mm) Apical diameter (mm) Efferent diameter (mm) RBCV (mm/s) RBCVmax (mm/s) TRBCV max (mm/s)

10.7ยก2.3 17.6ยก2.9 23.7ยก 4.1 19.8ยก4.7 0.3ยก0.05 0.3ยก0.02 5.9ยก2.8

14.9ยก2.7 * 17.9ยก2.0 23.7ยก1.4 20.9ยก3.3 0.3ยก0.01 0.4ยก0.01* 4.0ยก1.1 *

* p#0.05 by paired t-test. FCD: functional capillary density; RBCV: red blood cell flux velocity at rest; RBCVmax: red blood cell flux velocity after 1-min arterial occlusion; TRBCVmax: time required to reach peak red blood cell velocity.

* p#0.05 by paired t-test. HOMA2-IR: homeostasis model assessment for insulin resistance; E-SOD: superoxide dismutase activity in erythrocytes.

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2. Koury JC, de Olilveria AV, Jr, Portella ES, de Oliveira CF, Lopes GC, Donangelo CM. Zinc and copper biochemical indices of antioxidant status in elite athletes of different modalities. Int J Sport Nutr Exerc Metab. 2004;14:358-72. 3. Wang JS. Effects of exercise training and detraining on cutaneous microvascular function in man: the regulatory role of endotheliumdependent dilation in skin vasculature. Eur J Appl Physiol. 2005;93:42934, doi: 10.1007/s00421-004-1176-4. 4. Boegli Y, Gremion G, Golay S, Kubli S, Liaudet L, Leyvraz PF, et al. Endurance Training Enhances Vasodilation Induced by Nitric Oxide in Human Skin. J Invest Dermatol. 2003;121:1197-204, doi: 10.1046/j.15231747.2003.12518.x. 5. Benzie IF. Evolution of antioxidant defense mechanisms. Eur J Nutr. 2000;9:53-61, doi: 10.1007/s003940070030. 6. Knez WL, Coombes JS, Jenkins DG. Ultra-endurance exercise and oxidative damage: implications for cardiovascular health. Sports Med. 2006;36:429–41, doi: 10.2165/00007256-200636050-00005. 7. Dani C, Oliboni LS, Vanderlinde R, Bonatto D, Salvador M, Henriques JA. Phenolic content and antioxidant activities of white and purple juices manufactured with organically- or conventionally-produced grapes. Food Chem Toxicol. 2007;45:2574-80, doi: 10.1016/j.fct.2007.06.022. 8. IFOAM. International Federation of Organic Agriculture Movements, 2009. ,Available in: http://www.ifoam.org.. Accessed April/26/2009. 9. Vitseva O, Varghese S, Chakrabarti S, Folts JD, Freedman JE. Grape seed and skin extracts inhibit platelet function and release of reactive oxygen intermediates. J Cardiovasc Pharmacol. 2005;46:445-51, doi: 10.1097/01. fjc.0000176727.67066.1c. 10. Prior RL, Gu L, Wu X, Jacob RA, Sotoudeh G, Kader AA, et al. Plasma Antioxidant Capacity Changes Following a Meal as a Measure of the Ability of a Food to Alter In Vivo Antioxidant Status. J Am Coll Nutr. 2007;26:170–81. 11. Banini AE, Boyd LC, Allen JC, Allen HG, Sauls DL. Muscadine grape products intake, diet and blood constituents of non-diabetic and type 2 diabetic subjects. Nutrition. 2006;22:1137–45, doi: 10.1016/j.nut.2006.08. 012. 12. Leifert WR, Abeywardena MY. Cardioprotective actions of grape polyphenols. Nutr Res. 2008;28:729–37, doi: 10.1016/j.nutres.2008.08.007. 13. Singleton VL, Rossi JA. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic. 1965;20:14458. 14. HOMA Calculator. The Oxford Center for Diabetes. Endocrinology & Metabolism, Diabetes Trial Unit. , Available from: http://www.dtu.ox. ac.uk. Accessed April/26/2009. 15. Lohman TG. Anthropometric standardization reference manual. In: Lohman TG, Roche A, Martorell R, editor. Human Kinetics, Champaign, IL. 1998;99-102 16. Jackson AS, Pollock ML. Generalized equations for predict body density of men. Br J Nutr. 1978;40:497–504, doi: 10.1079/BJN19780152. 17. Kraemer-Aguiar LG, Maranha˜ o PA, Sicuro FL, Bouskela E. Microvascular dysfunction: a direct link among BMI, waist circumference and glucose homeostasis in young overweight/obese normoglycemic women? Int J Obes. 2010;34:111-7, doi: 10.1038/ijo.2009.209. 18. Klyscz T, Junger M, Jung F, Zeintl H. CapImage – ein neuartigens computerunterstu¨ tztes Videoanalysesystem fu¨ r die dynamische Kapillarmikroskopie. Biomedizinische Technik. 1997;42:168-75, doi: 10. 1515/bmte.1997.42.6.168. 19. American Dietetic Association Dietitians of Canada. Joint Position Statement. Nutrition and athletic performance. Med Sci Sports Exerc. 2009;41:709-731, doi: 10.1249/MSS.0b013e31890eb86. 20. Bergamini C, Cicoira M, Rossi A, Vassanelli C. Oxidative stress and hyperuricaemia: pathophysiology, clinical relevance, and therapeutic implications in chronic heart failure. Eur J Heart Fail. 2009;11:444-452, doi: 10.1093/eurjhf/hfp042. 21. American Diabetes Association. Diagnosis and Classification of Diabetes Mellitus. Diabetes Care. 2010;33:S62-S69, doi: 10.2337/dc10-S062. 22. Green DJ, Spence A, Halliwill JR, Cable T, Thijssen D. Exercise and vascular adaptation in asymptomatic humans. Exp Physiol. 2011;96:57-70. 23. Franzoni F, Galetta F, Morizzo C, Lubrano V, Palombo C, Santoro G, et al. Effects of age and physical fitness on microcirculatory function. Clin Sci. 2004;106:329–335, doi: 10.1042/CS20030229. 24. Franzoni F, Plantinga Y, Femia FR, Bartolomucci F, Gaudio C, Regoli F, et al. Plasma antioxidant activity and cutaneous microvascular endothelial function in athletes and sedentary controls. Biomed Pharmacother. 2004;58:432-36. 25. Riddell M, Perkins BA. Exercise and glucose metabolism in persons with diabetes mellitus: perspectives on the role for continuous glucose monitoring. J Diabetes Sci Technol. 2009;3:914-923. 26. Zunino SJ, Storms DH, Stephensen CB. Diets rich in polyphenols and vitamin A inhibit the development of type I autoimmune diabetes in nonobese diabetic mice. J Nutr. 2007;137:1216-1221. 27. Rivero-Pe´rez MD, Mun˜iz P, Gonzalez-Sanjose´ ML. Antioxidant profile of red wines evaluated by total antioxidant capacity, scavenger activity, and biomarkers of oxidative stress methodologies. J Agric Food Chem. 2007;55:5476-5483, doi: 10.1021/jf070306q.

the number of mitochondria in hepatocytes, and increased GLUT-4 expression in skeletal muscle.35 Nailfold videocapillaroscopy is a highly reproducible noninvasive method that can be useful to provide quantitative information on skin microcirculation.17 Microcirculatory function is strongly influenced by body mass,17 diabetes, and oxidative status.36 Altered microvascular structures may contribute to insulin resistance by limiting the rate of glucose delivery and insulin-mediated glucose uptake.37 These alterations may also reduce athletic performance. In the present study, microcirculatory parameters (FCD, RBCVmax, and TRBCVmax) were altered after OGJ intake. A significant increase in FCD indicated an improvement of tissue nutrition. The postocclusive reactive hyperemia response (PORH), which is normally used to induce capillary recruitment, is thought to be determined at the level of small arterioles and to be independent of the autonomic nervous system. After the onset of reperfusion, blood flow sharply increases, followed by a gradual return to its baseline level, and is influenced by the accumulation of vasodilator metabolites (including nitric oxide) and the formation of reactive oxygen species, which are normally washed out or destroyed by circulating blood and smooth muscle cell reactivity. During reperfusion, the myogenic response, which is mediated by the rapid stretching of microvascular smooth muscle cells, is responsible, at least partially, for the return of blood flow to its baseline values. The role of the endothelium in modulating the myogenic response remains controversial. In the current study, we detected an increase in RBCVmax and a decrease in TRBCVmax after OGJ intake. These results clearly indicate an amelioration of PORH, which is accompanied by a decrease in its duration. These results may be caused by improvements in glucose homeostasis that in turn improve endothelial function through reduced peroxynitrite formation, restored eNOS expression and, therefore, increased production and availability of NO. In conclusion, our results suggest that the consumption of organic red grape juice, which is a source of polyphenols, may benefit the antioxidant capacity, glucose homeostasis and microcirculatory parameters in endurance athletes. Although limited by the small sample size, our results are consistent with other studies and may suggest that the consumption of grape juice can positively affect an athlete’s health and reduce the risk of cardiovascular diseases. Further studies are required to clarify the role of polyphenols in glucose homeostasis and the antioxidant capacity based on the microvascular parameters in athletes using different modalities. Clinical trials using OGJ may be used to investigate the possible benefits of OGJ on athletic performance and health.

ACKNOWLEDGMENTS The authors would like to thank the participating athletes for their motivation and cooperation. This work was supported by Fundaçaõ de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ). The organic grape juice used in this study was a kind gift from Aliança Winery, Rio Grande do Sul, Brazil. Finally, we would like to thank Dr. Caroline Dani (Centro Universita´rio Metodista IPA, Brazil) for measuring polyphenols in grape juice.

REFERENCES 1. Palazzetti S, Richard MJ, Favier A, Margaritis I. Overloaded training increases exercise-induced oxidative stress and damage. Can J Appl Physiol. 2003;28:588-604, doi: 10.1139/h03-045.

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28. C ¸ imen MY. Free radical metabolism in human erythrocytes. Clin Chim Acta. 2008;390:1-11, doi: 10.1016/j.cca.2007.12.025. 29. Fernańdez-Pachoń MS, Berna´ G, Otaolaurruchi E, Troncoso AM, Martıń F, Garcıá-Parrilla MC. Changes in antioxidant endogenous enzymes (activity and gene expression levels) after repeated red wine intake. J Agric Food Chem. 2009;12:6578-83, doi: 10.1021/jf901863w. 30. Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of ‘‘antioxidant power’’: the FRAP assay. Anal Biochem. 1996;239:70-6, doi: 10.1006/abio.1996.0292. 31. Sevanian A, Davies KJ, Hochstein P. Serum urate as an antioxidant for ascorbic acid. Am J Clin Nutr. 1991;54:1129S-1134S. 32. Ohno M, Ka T, Inokuchi T, Yamamoto A, Takahashi S, Tsutsumi Z, et al. Effects of exercise and grape juice ingestion in combination on plasma concentrations of purine bases and uridine. Clin Chim Acta. 2008;388:167-72, doi: 10.1016/j.cca.2007.10.032. 33. Pinent M, Blay M, Blade MC, Salvado MJ, Arola L, Ardevol A. Grape seed-derived procyanidins have an antihyperglycemic effect

34.

35.

36. 37.

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in streptozotocin-induced diabetic rats and insulinomimetic activity in insulin-sensitive cell lines. Endocrinology. 2004;145:4985-90, doi: 10.1210/ en.2004-0764. Franco JG, de Moura EG, Koury JC, Trotta PA, Cordeiro A, Souza LL et al. Resveratrol reduces lipid peroxidation and increases sirtuin 1 expression in adult animals programmed by neonatal protein restriction. J Endocrinol. 2010;207:319-28, doi: 10.1677/JOE-10-0124. Chi TC, Chen WP, Chi TL, Kuo TF, Lee SS, Cheng JT, et al. Phosphatidylinositol-3-kinase is involved in the antihyperglycemic effect induced by resveratrol in streptozotocin-induced diabetic rats. Life Sci. 2007;80:1713-20, doi: 10.1016/j.lfs.2007.02.002. Abi-Chahin TC, Hausen MA, Mansano-Marques CM, Halfoun VL. Microvascular reactivity in type 1 diabetics. Arq Bras Endocrinol Metabol. 2009;53:741-6. Irving RJ, Walker BR, Noon JP, Watt GC, Webb DJ, Shore AC. Microvascular correlates of blood pressure, plasma glucose, and insulin resistance in health. Cardiovasc Res. 2002;53:271-6, doi: 10.1016/S0008-6363(01)00450-3.

CLINICS 2011;66(9):1543-1548

DOI:10.1590/S1807-59322011000900006

CLINICAL SCIENCE

Simultaneous transfer of cholesterol, triglycerides, and phospholipids to high-density lipoprotein in aging subjects with or without coronary artery disease Carolina H. M. Azevedo,I,III Maurıćio Wajngarten,II Ana C. Lo Prete,I,III Jayme Diament,I Raul C. MaranhaõI,III I Lipid Metabolism Laboratory, Heart Institute (InCor), Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brazil. II Cardiogerontology Section, Heart Institute (InCor), Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brazil. III Faculty of Pharmaceutical Sciences, Universidade de Saõ Paulo, Saõ Paulo/SP, Brazil.

OBJECTIVE: To verify whether the capacity of high-density lipoprotein (HDL) to simultaneously receive nonesterified cholesterol, triglycerides, cholesteryl esters, and phospholipids changes with aging and the presence of coronary artery disease. DESIGN: Cross-sectional study with biochemical analyses. SUBJECTS: Eleven elderly patients with coronary artery disease (74¡5 years) were compared with the following groups of non-coronary artery disease subjects (referred to as ‘‘healthy’’): 25 young (25¡5 years), 25 middle-aged (42¡6 years), and 25 elderly subjects (75¡8 years). METHODS: Plasma samples were incubated with a nanoemulsion labeled with radioactive lipids; the transfer of the lipids from the nanoemulsion to the HDL was measured in chemically precipitated HDL. HDL size and paraoxonase-1 activity were also determined. RESULTS: The transfer of cholesteryl esters and phospholipids to high-density lipoprotein was significantly greater (p,0.001) in healthy elderly subjects than in the middle-aged and younger subjects. Non-esterified cholesterol and triglyceride transfer was not different among these three groups. The HDL size was significantly greater (p,0.001) in healthy elderly subjects than in the middle-aged and younger subjects. The paraoxonase-1 activity was similar among the groups. Compared with healthy elderly subjects, coronary artery disease elderly subjects had significantly less (p,0.05) transfer of non-esterified cholesterol, triglycerides, and cholesteryl esters to the HDL and a significantly smaller (p,0.05) HDL size. CONCLUSION: Because lipid transfer is enhanced in healthy elderly subjects but not in those with coronary artery disease, increasing lipid transfer to HDL may be a protective mechanism against the disease. KEYWORDS: Aging; cholesteryl ester transfer protein (CETP); transfer proteins; lipoproteins; nanoparticles. Azevedo CHM, Wajngarten M, Lo Prete AC, Diament J, Maranha˜o RC. Simultaneous transfer of cholesterol, triglycerides, and phospholipids to highdensity lipoprotein in aging subjects with or without coronary artery disease. Clinics. 2011;66(9):1543-1548. Received for publication on April 4, 2011; First review completed on May 9, 2011; Accepted for publication on May 16, 2011 E-mail: ramarans@usp.br Tel.: 55 11 3069-5574

having a low HDL cholesterol level was the predominant characteristic of subjects older than 60 years with a history of myocardial infarction compared to subjects older than 60 years without previous cardiovascular events.4 The HDL fraction is composed of heterogeneous particles with sizes ranging from 7 to 14 nm.1,5 In addition to its roles in reverse cholesterol transport and cholesterol esterification, HDL has antioxidant activity that is mostly due to its association with paraoxonase 1 (PON 1) and anti-inflammatory, antithrombotic, and vasodilation activities that may account for the antiatherogenic action of the lipoprotein.6,7 Nascent HDL is produced in the liver and intestine as discoid particles composed of phospholipids and nonesterified cholesterol. These particles are progressively

INTRODUCTION Both the high-density lipoprotein (HDL) cholesterol concentration in the plasma and the concentration of the main HDL apolipoprotein, apo A1, negatively correlate with the incidence of coronary artery disease, and both are biomarkers of healthy aging.1-4 Data from the Prospective Cardiovascular Mu¨nster (PROCAM) study have shown that

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transformed into a spherical shape by the acquisition of lipids and the esterification of cholesterol by lecithincholesterol acyltransferase (LCAT), with apo A1 as a cofactor. Because most of the apo A1 that is present in the plasma is contained in the HDL fraction and because LCAT is also associated with HDL, cholesterol esterification occurs mainly in this lipoprotein fraction.7 HDL is thus constantly being remodeled, and lipid transfers are essential for the roles of this lipoprotein in cholesterol esterification and reverse cholesterol transport. Both processes are intertwined and are necessary for cholesterol homeostasis. Lipid transfers between lipoprotein classes are bidirectional and depend on the structures of the donor and acceptor lipoproteins and the activity of the transfer proteins, i.e., cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP). The concentrations of the donor and acceptor lipoprotein classes may also influence the process.8-11 Because the lipid transfer process is a determinant of HDL composition and metabolism, it is also possible that in addition to the HDL function in reverse cholesterol transport, other atheroprotective functions of the lipoprotein may be affected by this process. The current study was designed to verify the effects of aging on the capacity of HDL to simultaneously receive cholesteryl esters (CEs), phospholipids (PLs), triglycerides (TGs), and non-esterified cholesterol (NEC). A standard artificial nanoemulsion was used as the donor of radioactive lipids to HDL in an in vitro assay12 with plasma from subjects within different age ranges: young, middle-aged, and elderly subjects. The HDL size and PON 1 activity were also measured. A group of elderly patients with coronary disease was also studied to investigate whether a different pattern of lipid transfer to HDL was associated with the disease.

Table 1 - Physical characteristics and plasma biochemical parameters of the study subjects. Parameters

Young (n = 25)

Age (years) 25¡5 Gender (M/F) 10/15 26¡4 BMI (kg/m2) Cholesterol Total (mg/dL) 113¡18 HDL (mg/dL) 41¡6 LDL (mg/dL) 96¡23 Triglycerides (mg/dL) 86¡15 Apo A1 (mg/dL) 128¡30 Apo B (mg/dL) 70¡18

Middle-aged (n = 25)

Elderly (n = 25)

CAD Elderly (n = 11)

42¡6 9/16 25¡5

75¡8* 11/14 27¡3

74¡5 5/6 26¡4

173¡43 40¡7 102¡44 100¡20 135¡14 88¡15

190¡40* 50¡12* 104¡19 101¡30 141¡40 80¡15

201¡30 42¡6** 143¡25** 123¡46 100¡60 87¡40

The data are expressed as the means ¡ S.D.; * p,0.001 when compared to the young and middle-aged groups; ** p,0.05 when compared to the non-CAD elderly group. BMI, body mass index; HDL, high-density lipoprotein; LDL, low-density lipoprotein; apo A1, apolipoprotein A1; Apo B, apolipoprotein B.

informed consent was obtained from all of the subjects following a complete description of the protocol.

Plasma lipids and apolipoproteins Plasma total cholesterol (CHOD-PAP; Roche, Basel, Switzerland) and triglyceride (Triglyceride Rapid; Roche) levels were determined by enzymatic methods using a Cobas Bio analyzer (Roche, Basel, Switzerland). HDL cholesterol was measured using the same method used for total cholesterol after lipoprotein precipitation with magnesium phosphotungstate. The low-density lipoprotein (LDL) cholesterol level was estimated with the Friedewald formula.13 Apo A1 and apo B levels were determined using an immunoturbidimetric assay (Roche) on a Cobas MIRA analyzer.

METHODS AND MATERIALS

Nanoemulsion preparation

Study subjects

The lipid donor nanoemulsion was prepared from a lipid mixture composed of 40 mg cholesteryl oleate, 20 mg egg phosphatidylcholine, 1 mg triolein, and 0.5 mg cholesterol, all purchased from Sigma Chemical Company (St. Louis, MO). The lipids were emulsified by prolonged ultrasonic irradiation in aqueous media and a two-step ultracentrifugation of the crude emulsion with density adjustment by addition of KBr to obtain the nanoemulsion, as previously described.14,15 The nanoemulsion fraction was dialyzed against a 0.9% NaCl solution. Trace amounts of [12, 2d(n)-3H]-cholesteryl oleate and glycerol tri[9, 10(n)-3H] oleate or 4-14C-cholesterol and L-3-phosphatidylcholine, 1-stearoyl-2-[1-14C] arachidonyl. (Amersham, Little Chalfont, Buckinghanshier, UK) were added to the initial solution.

The volunteer participants in the study were recruited at the outpatient clinics and at the Division of Geriatric Cardiology of the Heart Institute of the Medical School Hospital of the University of Sa˜o Paulo, where they were referred for routine check-ups and clinical and laboratory tests and were pronounced healthy. Three groups of subjects paired for gender and body mass index (BMI) were studied: young (aged 20-25 years; 10 men and 15 women), middle-aged (aged 39-54 years; 9 men and 16 women), and elderly (aged 65-82 years; 11 men and 14 women) subjects. A fourth group (aged 65-81 years; 5 men and 6 women) was composed of 11 elderly patients with coronary artery disease (CAD). The presence of CAD was confirmed by coronary angiography performed within six months prior to the study. None of the participants were smokers; obese; alcoholics; or had liver, renal, metabolic, inflammatory, or neoplastic disease. Their physical characteristics and plasma biochemical parameters are shown in Table 1. All of the patients in the non-CAD elderly and CAD elderly groups were receiving standard maintenance hypertension treatment with a beta-adrenergic blocker or an angiotensinconverting enzyme (ACE) inhibitor. The elderly CAD subjects were also taking statin medications, which were discontinued for at least 30 days before the study. The Ethics Committee of the Medical School Hospital of the University of Sa˜o Paulo approved the study, and written

Lipid transfer from the nanoemulsion to HDL This assay was previously described by Lo Prete et al.12 In brief, the nanoemulsion labeled with 3H-CE and 14C-PL or with 14C-NEC and 3H-TG was incubated with whole plasma followed by chemical precipitation of the apo B-containing lipoproteins and the nanoemulsion. The supernatant containing the lipids that shifted from the nanoemulsion to the HDL was then measured for radioactivity in a scintillation solution. The results of the radioactive transfer from the nanoemulsion to the HDL were expressed as the percentage

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Aging, heart disease, and lipid transfers to HDL Azevedo CHM et al.

of the total incubated radioactivity found in the HDLcontaining supernatant.12

PON 1 activity was assayed using 12-h fasting blood serum, according to the method described by Mackness et al.,16 by adding serum to Tris-HCl buffer (100 mmol/L, pH 8.0) containing 2 mmol/L CaCl2 and 1.1 mmol/L paraoxon (0,0-diethyl-0-p-nitrophenylphosphate; Sigma). The rate of generation of p-nitrophenol was determined at 37 ˚C with the use of a spectrophotometer at 405 nm.

between HDL cholesterol, cholesterol total concentration and HDL size, as shown in Figure 1. No correlation was found between age and the TG, PL, or NEC transfer. The PL transfer correlated positively with the HDL cholesterol level, whereas the NEC, CE, and TG transfers did not. With respect to other plasma lipids, a positive correlation was found between the TG transfer and the apo B concentration. No correlation was found between the CE, PL, NEC, or TG transfer and the total, LDL or very-low-density lipoprotein (VLDL) cholesterol or TG. The transfer of the four lipids did not correlate with the plasma glucose level or the patient’s BMI.

HDL diameter

DISCUSSION

PON 1 activity

The HDL size was measured by use of a ZetaPALS zeta potential analyzer (Brookhaven Instruments, Holtsville, NY), as previously described.17

In this study, the group of non-CAD elderly subjects showed greater transfers of CE and PL and larger HDL particle sizes than did the middle-aged and young subjects. In the CAD elderly subjects, less CE, TG, and NEC was transferred, and the HDL particle size was smaller than in the non-CAD elderly subjects. These novel observations were obtained by means of the in vitro method recently described by Lo Prete et al.,12 in which the simultaneous transfer of all four main lipids is tested, specifically focusing on HDL and measuring the particle size of the entire HDL fraction using laser light scattering.17 In reverse cholesterol transport, cholesterol from the peripheral tissues is transported by HDL and taken up by the SR-B1 receptors in the liver, together with the HDL particles.18 Alternatively, HDL cholesterol is transferred to other lipoprotein classes, such as VLDL and VLDL catabolic products, such as LDL, that are taken up together by the liver, which excretes the lipoprotein cholesterol content into the bile. During this process, the transfer proteins play a major role. CETP and PLTP are the major proteins involved in these lipid shifts. CETP specifically mediates the transfer of cholesteryl esters and triglycerides and is associated with HDL in the plasma.19 In contrast, PLTP facilitates the transfer of surface phospholipids from other lipoproteins to HDL and influences HDL remodeling by promoting the formation of larger HDL particles, such as HDL2b and HDL2a, at the expense of the small subfraction HDL3 (see ref. 20 for a review). Lipid transfers between lipoprotein classes are bidirectional but frequently result in lipid enrichment or the depletion of lipid species of a given lipoprotein class.7,21 Cholesterol is stored in cells in its esterified form. After the hydrolysis of CE by cytoplasmic esterases, NEC is transferred from cells to HDL by the ABCA1 system, which comprises membrane transporters that mediate the efflux of NEC and PL to HDL.22 The cholesterol from peripheral tissues transported in HDL can be transferred to the other lipoproteins by CETP or be taken up by hepatic SR-B1 receptors.22 Both the ABCA1 system and the SR-B1 receptors, which were not evaluated here, are thus important contributors to the continuously changing HDL composition and may have influenced the results of the lipid transfer assay in this study. Both the concentration of HDL in the plasma and the qualitative and functional aspects of this lipoprotein are important for its anti-atherogenic actions.23,24 Patients whose HDL contains the apo A1 Milano mutation exemplify a population that is protected from cardiovascular events despite low levels of circulating HDL. Similarly, individuals with high levels of HDL that is prone to modification may be less protected than individuals with average levels of HDL

Statistical analyses A power calculation indicated that, with n.23 in each group, we would have 80% power (at a significance level of 0.05) to detect a difference in lipid transfer values between the study groups. The differences between the results were evaluated using analysis of variance (ANOVA) and Student’s t-test. Significant correlations were identified using ANOVA and Pearson’s test. In all of the analyses, a difference of p,0.05 was considered statistically significant. The data are expressed as the means ¡ standard deviations. GraphPad Prism version 3.0 was used to assist with the analyses.

RESULTS Table 1 shows the plasma lipid and apolipoprotein concentrations of the study subjects. The HDL cholesterol levels were greater in the non-CAD elderly than in the middle-aged and young subjects (p,0.001) but did not differ between the young and middle-aged groups. The LDL cholesterol, triglycerides, apo A1, and apo B levels were not different among these three groups. In the CAD elderly group, the HDL cholesterol level was higher and the LDL cholesterol level was lower than in the non-CAD elderly group (p,0.001). Table 2 shows that the non-CAD elderly group had larger HDL particles when compared with the middle-aged and young subjects. The CAD elderly group had smaller HDL particles than the non-CAD elderly group. The PON 1 activity was similar among the four groups. Table 2 also shows the transfer of the four lipids from the artificial nanoemulsion to the HDL fraction. The transfer of CE and PL was greater in the non-CAD elderly subjects than in the middle-aged and young subjects (p = 0.0368), whereas the latter two groups did not differ in the transfer of CE or PL. The NEC and TG transfer rates were equal in these three study groups. Compared with the non-CAD elderly group, the CAD elderly had decreased transfer of NEC, TG, and CE to HDL (p,0.001), but the PL transfer was equal. The CE, TG, and NEC transfers were positively correlated with each other. In contrast, the PL transfer correlated positively with the CE transfer but showed no correlation with the TG or NEC transfer. With respect to the correlation analysis of the lipid transfer values versus the physical characteristics and laboratory data of the subjects, positive correlations were found between age and the CE transfer and

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that is more resistant to modification.9 The capacity to receive lipids is a fundamental feature of HDL because the metabolism and function of this lipoprotein in the reverse cholesterol transport fundamentally depend on lipid exchanges.25 In the in vitro assay with whole plasma used in this study, many factors may have influenced the transfer of lipids from the standard donor nanoemulsion to HDL, such as the concentration, composition, and structure of both the HDL and the other acceptor lipoprotein classes, which contain apo B. The latter lipoproteins compete with HDL to receive lipids from the nanoemulsion and may thus influence the HDL lipid acceptance results. CETP and PLTP are paramount in this process because they facilitate the transfer of the lipids. The concentration of apo B, which is present in LDL and VLDL and LDL cholesterol, was not different among the study groups, but the HDL cholesterol level, which was higher in the non-CAD elderly, could have influenced the transfer results, which would favor an increase in lipid acceptance by HDL. There are no consistent reports in the literature on the effect of aging on the action of transfer proteins. An increase in core lipids, such as CE, may increase the size of the particles,24 as was found in this study in the elderly group. The increase in the PL transfer found in the

Table 2 - The transfer of lipids from the artificial nanoemulsion to HDL, HDL size and PON1 activity in the study subjects. PARAMETERS Lipid transfers (%) Non-esterified cholesterol Cholesteryl ester Triglycerides Phospholipids HDL diameter (nm) PON 1 (nmol min-1 mL-1)

Young (n = 25)

Middle-aged (n = 25)

Elderly (n = 25)

CAD Elderly (n = 11)

9.6¡1.7

10.1¡1.2

10.0¡2.4

8.2¡1.3**

3.7¡1.0 6.5¡ 1.3 18.7¡4.6 8.9¡ 0.3 97¡ 44

4.1¡0.7 6.9¡1.3 18.3¡4.0 8.9¡ 0.3 85¡ 40

5.3¡1.8* 7.3¡2.4 20.6¡5.3* 9.7¡ 1.6* 85¡ 44

3.1¡2.3** 5.1¡1.6** 19.9¡2.3 8.7¡0.7** 76¡42

The data are expressed as the means ¡ S.D.; * p,0.001 when compared to the young and middle-aged groups; ** p,0.05 when compared to the non-CAD elderly group. HDL, highdensity lipoprotein; PON 1, paraoxonase 1. The transfer of lipids from the donor nanoemulsion to HDL is expressed as the % of the total radioactivity from the whole plasma that was recovered in the HDL fraction after chemical precipitation of the apo B-containing lipoproteins and the nanoemulsion.

Figure 1 - Correlations between age and HDL cholesterol (A), total cholesterol (B), HDL size (C), and the transfer of cholesteryl esters (D).

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Aging, heart disease, and lipid transfers to HDL Azevedo CHM et al. 2. Heijmans BT, Beekman M, Houwing-Duistermaat JJ, Cobain MR, Powell J, Blauw GJ, et al. Lipoprotein particle profiles mark familial and sporadic human longevity. PLoS Med. 2006;3:495-9, doi: 10.1371/journal. pmed.0030495. 3. Terry DF, Evans JC, Pencina MJ, Murabito JM, Vasan RS, Wolf PA, et al. Characteristics of Framingham offspring participants with long-lived parents. Arch Intern Med.2007;12:438-41, doi: 10.1001/archinte.167.5. 438. 4. Assmann G, Schulte H, von Eckardstein A, Huang Y. High-density lipoprotein cholesterol as a predictor of coronary heart disease risk. The PROCAM experience and pathophysiological implications for reverse cholesterol transport. Atherosclerosis. 1996;124:S11-S20, doi: 10.1016/ 0021-9150(96)05852-2. 5. Kwiterovich Jr PO. The metabolic pathways of high-density lipoprotein, low-density lipoprotein, and triglycerides: a current review. Am J Cardiol. 2000;86:452-7. 6. Falk E. Pathogenesis of Atherosclerosis. J Am Coll Cardiol. 2006;47(8C Suppl):C7-12. 7. Wang CS, Briggs MR. HDL: the metabolism, function, and therapeutic importance. Chem Rev. 2004;104:119-23. 8. Ferretti G, Bacchetti T, Ne`gre-Salvayre A, Salvayre R, Dousset N, Curatola G. Structural modifications of HDL and functional consequences. Atherosclerosis. 2006;184:1-7, doi: 10.1016/j.atherosclerosis. 2005.08.008. 9. Norata GD, Pirillo A, Catapano AL. Modified HDL: biological and physiopathological consequences. Nutr Metab Cardiovasc Dis. 2006;16: 371-7, doi: 10.1016/j.numecd.2006.01.012. 10. Stein O, Stein Y. Lipid transfer proteins (LTP) and atherosclerosis. Atherosclerosis. 2005;178:217-30, doi: 10.1016/j.atherosclerosis.2004.10. 008. 11. Rashid S, Watanabe T, Sakaue T. Mechanisms of HDL lowering in insulin resistant, hypertriglyceridemic states: the combined effect of HDL triglyceride enrichment and elevated hepatic lipase activity. Clin Biochem. 2003;36:421-6, doi: 10.1016/S0009-9120(03)00078-X. 12. Lo Prete AC, Dina CH, Azevedo CH, Puk CG, Lopes NH, Hueb WA, et al. In vitro simultaneous transfer of lipids to HDL in coronary artery disease and in statin treatment. Lipids. 2009;44:917-24, doi: 10.1007/ s11745-009-3342-2. 13. Friedewald WT, Levy RI, Fredrickison DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without the use of the preparative ultracentrifugue. Clin Chem. 1972;18:499-502. 14. Maranhaõ RC, Cesar TB, Pedroso-Mariani SR. Metabolic behavior in rats of a nonproteinmicroemulsion resembling LDL. Lipids. 1993;28:691-6, doi: 10.1007/BF02535988. 15. Dantas SA, Ficker ES, Vinagre CG, Ianni BM, Maranhaõ RC, Mady C. Metabolism of a lipid nanoemulsion resembling low-density lipoprotein in patients with grade iii obesity. Clinics. 2010;65:23-7, doi: 10.1590/ S1807-59322010000100005. 16. Mackness MI, Harty D, Bhatnagar D, et al. Serum paraoxonase activity in familial hypercholesterolaemia and insulin-dependent diabetes mellitus. Atherosclerosis. 1991;86:193-9, doi: 10.1016/0021-9150(91)90215-O. 17. Lima E, Maranhaõ RC. Rapid, simple laser-light scattering method for HDL particle size in whole plasma. Clin Chem. 2004;50:1086-91, doi: 10. 1373/clinchem.2004.032383. 18. vonEckardstein AV, Nofer JR, Assmann G. High density lipoproteins and arteriosclerosis: Role of cholesterol efflux and reverse cholesterol transport. Arterioscler Thromb Vasc Biol. 2001;21:13-7, doi: 10.1161/01. ATV.21.1.13. 19. Chapman MJ, Le Goff W, Guerin M, Kontush A. Cholesteryl ester transfer protein: at the heart of the action of lipid-modulating therapy with statins, fibrates, niacin, and cholesteryl ester transfer protein inhibitors. Eur Heart J. 2010;31:149-64, doi: 10.1093/eur heartj/ehp399. 20. Tzotzas T, Desrumaux C, Lagrost L. Plasma phospholipid transfer protein (PLTP): review of an emerging cardiometabolic risk factor. Obesity reviews. 2009;10:403-11, doi: 10.1111/j.1467-789X.2009. 00586.x. 21. Lewis GF, Rader DJ. New insights into the regulation of HDL metabolism and reverse cholesterol transport. Circ Res. 2006;96:122-9. 22. Rader DJ, Alexander ET, Weibel GL, Billheimer J, Rothblat GH. The role of reverse cholesterol transport in animals and humans and relationship to atherosclerosis. J Lipid Res. 2009;50:S189-94, doi: 10.1194/jlr.R800088JLR200. 23. Cutri BA, Hime NJ, Nicholls SJ. High-density lipoproteins: an emerging target in the prevention of cardiovascular disease. Cell Res. 2006;16:799804, doi: 10.1038/sj.cr.7310097. 24. Barter PJ, Rye KA. Relationship between the concentration and antiatherogenic activity of high-density lipoproteins. Curr Opin Lipid. 2006;17:399-404, doi: 10.1097/01.mol.0000236365.40969.af. 25. Cheung MC, Wolfbauer G, Brown BG, Albers JJ. Relationship between plasma phospholipid transfer protein activity and HDL subclasses among patients with low HDL and cardiovascular disease. Atherosclerosis. 1999;142:201-5, doi: 10.1016/S0021-9150(98)00190-7.

non-CAD elderly group should favor the expansion of the surface monolayer because the HDL particles swell with the greater influx of core CE. Therefore, the existence of larger HDL particles in the non-CAD elderly subjects can be attributed to the increased PLTP activity observed with the increased transfer of PL to HDL in this group. It is noteworthy that a positive correlation was also observed between age and CE transfer, HDL size and HDL cholesterol. The increase in HDL cholesterol in the absence of apo A1 is in accordance with our hypothesis that the cholesterol content in the HDL particles, and not the particle number, was greater in the elderly. The smaller amount of transfer of CE, TG, and NEC to HDL in the group of elderly patients with CAD than in the non-CAD subjects suggests that an increase in the transfer of those lipids may protect against CAD. Diminution of HDL size in elderly patients with CAD may be secondary to the decreased lipid transfers. A diminished ability of HDL to receive NEC may reflect disturbances in the function of the lipoprotein; because most of the cholesterol esterification process occurs in the HDL fraction, diminution of the NEC influx to the lipoprotein would correspond to decreased cholesterol esterification. As expected, the HDL cholesterol level was lower in the CAD elderly group, and this result may be one of the causative factors for the decreased lipid transfers to the lipoprotein. Because of the dynamic nature of HDL metabolism, it is difficult to hypothesize whether the diminished influx of lipids was one of the causes of CAD in the CAD elderly group or only a marker that reflected the exposure of the lipoprotein to the metabolic disturbances occurring in the disease.26 In any event, the small number of subjects in the CAD group, as indicated by the power analysis, may have been a limitation when comparing that group to the non-CAD group. In this study, PON 1 activity was not different among the subject groups; however, the activity of this enzyme can widely vary (up to 40-fold) in a given population because of environmental factors and polymorphisms.27

CONCLUSION In conclusion, our results show that in elderly subjects who did not have CAD, the transfer of CE, the major component of the HDL core, and PL, the main HDL surface layer component, to HDL was higher than in younger subjects. In elderly subjects with CAD, the transfer of CE, TG, and NEC to HDL was decreased compared to the nonCAD elderly subjects. Those differences were also correlated with HDL particle size and suggest that aging is accompanied by changes in lipid transfer and HDL size that, in addition to HDL cholesterol levels, could also be determinants of the absence or presence of CAD in the elderly.

FUNDING This study was supported by the Fundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo (FAPESP), Saõ Paulo, SP, Brazil. Dr. Maranhaõ has a research award from the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq, Brası´lia, Brazil), and Dr. Azevedo had a scholarship from CNPq.

REFERENCES 1. Natarajan P, Ray KK, Cannon CP. High-density lipoprotein and coronary heart disease: current and future therapies. J Am Coll Cardiol. 2010;55:1283-99.

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27. Costa LG, Vitalone A, Cole TB, Furlong CE. Modulation of paraoxonase (PON1) activity. Biochem Pharmacol. 2005;69:541-50, doi: 10.1016/j.bcp. 2004.08.027.

26. Berrougui H, Isabelle M, Cloutier M, Grenier G, Khalil A. Age-related impairment of HDL-mediated cholesterol efflux. J Lipid Res. 2007;48:32836, doi: 10.1194/jlr.M600167-JLR200.

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DOI:10.1590/S1807-59322011000900007

CLINICAL SCIENCE

Decreased high-density lipoprotein cholesterol levels in polyarticular juvenile idiopathic arthritis Roberta Gonçalves Marangoni, Andre L. Hayata, Eduardo F. Borba, Pedro M. Azevedo, Eloisa Bonfa´, Claudia Goldenstein-Schainberg Rheumatology Division, Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brazil.

OBJECTIVES: To investigate the prevalence of dyslipoproteinemia in a homogeneous cohort of polyarticular juvenile idiopathic arthritis patients. METHODS: Based on the National Cholesterol Education Program, fasting lipoprotein levels and risk levels for coronary artery disease were determined in 28 patients with polyarticular juvenile idiopathic arthritis. The exclusion criteria included diabetes, thyroid dysfunction, smoking, proteinuria, lipid-lowering drugs, and hormone/diuretic therapy. Disease activity, disease duration, and therapy with corticosteroids and/or chloroquine were defined at the time of lipid measurements. RESULTS: Dyslipoproteinemia was identified in 20 of the 28 (71%) patients with polyarticular juvenile idiopathic arthritis. The primary lipoprotein risk factor was decreased high-density lipoprotein cholesterol (57%), followed by elevated levels of low-density lipoprotein cholesterol (18%), triglycerides (14%), and total cholesterol (7%). The male patients had decreased high-density lipoprotein cholesterol levels than the female patients (p,0.05). The incidence of decreased high-density lipoprotein cholesterol levels did not seem to be affected by disease activity or therapy because the incidence was similar in patients with active or inactive disease, with or without corticosteroid use and with or without chloroquine use. In addition, the frequency of decreased high-density lipoprotein cholesterol levels was similar in patients with short (#5 years) vs. long (.5 years) disease duration. CONCLUSIONS: Dyslipoproteinemia is highly prevalent in patients with polyarticular juvenile idiopathic arthritis and is primarily related to decreased high-density lipoprotein cholesterol levels; therefore, early intervention is essential. KEYWORDS: Lipids; Atherosclerosis; Dyslipidemia; Rheumatic Disease; Juvenile. Marangoni RG, Hayata AL, Borba EF, Azevedo PM, Bonfa´ E, Goldenstein-Schainberg C. Decreased high-density lipoprotein cholesterol levels in polyarticular juvenile idiopathic arthritis. Clinics. 2011;66(9):1549-1552. Received for publication on February 24, 2011; First review completed on March 29, 2011; Accepted for publication on May 17, 2011 E-mail: cgs@usp.br Tel.: 55 11 30617492

RA has its own pattern of dyslipoproteinemia (DL), and— though they are controversial—most studies have demonstrated decreased HDL levels in these patients.7,8 In juvenile idiopathic arthritis (JIA), the data regarding DL prevalence are scarce and do not conclusively define the role of JIA in this metabolic disturbance.9-14 One study found altered lipid levels in both patients with active and inactive JIA,9 whereas another study found that decreased HDL and elevated triglyceride (TG) levels were strongly correlated with the inflammatory state of the disease.12 These contradictory findings may be explained by the broad spectrum of JIA subtypes because specific clinical and laboratory parameters can influence the lipid profile.15 Furthermore, an altered lipid profile can occur in chronic inflammatory states that favor atherosclerosis.16 Therefore, we evaluated the prevalence of DL in a homogeneous group of polyarticular JIA patients to define CAD lipoprotein risk in this patient population.

INTRODUCTION Overwhelming evidence indicates that abnormal lipoprotein levels play an important role in atherosclerotic processes that can be related to autoimmune disease. Indeed, the risk to develop atherosclerosis increases progressively with increasing low-density lipoprotein cholesterol (LDL) levels and declines with increasing levels of high-density lipoprotein cholesterol (HDL).1 In adult patients with rheumatoid arthritis (RA), coronary artery disease (CAD) is the leading cause of shortened life expectancy relative to the general population,2-4 and nearly half of these deaths can be attributed to CAD that is linked to inflammation and elevated Creactive protein (CRP) levels.5,6 Another important factor that certainly contributes to the development of atherosclerosis in rheumatological diseases is abnormal lipid levels. Of note,

METHODS Study population: One hundred and eighty JIA patients who were regularly followed at the Rheumatology Division

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of Hospital das Clı´nicas da Universidade de Sa˜o Paulo were initially evaluated for this study. All of the patients fulfilled the International League of Associations for Rheumatology (ILAR) criteria for JIA.17 Forty JIA patients were eligible for this study after excluding patients with diabetes mellitus, hypertension, renal disease, infection, thyroid or hepatic dysfunction, pregnancy, smoking, or the intake of drugs that could interfere with lipid metabolism (such as hormones, lipid-lowering agents, diuretics, and prednisone .10 mg/day). We then excluded JIA patients with systemic or oligoarticular JIA, enthesopathy-related arthritis (ERA), psoriatic arthritis, and inflammatory bowel disease to define a homogeneous group of 28 patients with polyarticular onset JIA. Corticosteroid and diphosphate chloroquine intake was calculated for each patient by analyzing the mean dosage over the previous three months and the stable dosage at least one month prior to inclusion in this study. The local ethics committee approved this study, and written, informed consent was obtained from each patient. JIA disease activity: Clinical JIA disease activity was defined as the presence of clinically active synovitis in at least one joint together with an elevated erythrocyte sedimentation rate (ESR) (with normal defined as ,12 mm/h) and/or elevated CRP levels (with normal defined as ,5 mg/dl].

RESULTS We found a clear female predominance (75%) among the 28 polyarticular onset JIA patients. The mean age of all 28 patients was 19.9¡5.8 years, with 11 (39%) patients younger than 20 years and 17 (61%) patients older than 20 years. The mean weight and body mass index were 54¡10.2 kg and 21¡3.9 kg/m2, respectively. The mean disease duration was 9.4¡5.4 years. Upon joining the study, 17 of the patients (60.7%) had active JIA with a mean of four active joints (range 1-10 joints) and mean ESR and CRP levels of 26.4¡27.0 mm/h (range 1-110 mm/h) and 15.5¡23.2 mg/dl (range 0.26-76.9 mg/dl), respectively. At the time of their evaluation, 16 (57.1%) patients were taking diphosphate chloroquine at a mean dose of 227.2¡56 mg/ day, range 50-250 mg/day (4 mg/kg/day), and nine (32.1%) patients were taking a low dose of prednisone with a mean dose of 5.8¡2.5 mg/day, range 2.5-10 mg/day (0.1 mg/kg/ day). Additionally, 12 (42.8%) of the patients were on immunosuppressive therapy as follows: methotrexate at 18.7¡7.1 mg/day, range 10-30 mg/day (0.4 mg/kg/day), sulfasalazine at 2.2¡0.6mg/day, range 1.5-3.0 mg/day (0.04 mg/kg/day) and azathioprine at 75¡35.3 mg/day, range 50-100 mg/day (1.2 mg/kg/day). None of the patients were receiving biological therapy. All of the patients had a serum creatinine level below 1.3 mg/dl and a proteinuria level of less than 0.3 g (0.05¡0.01 g/24 h). As shown in Table 1, the lipoprotein levels revealed a mean TC level of 150.3¡26.0 mg/dl, LDL level of 83.6¡23.1 mg/dl, HDL level of 50.6¡11.6 mg/dl, and TG level of 81.6¡49.4 mg/dl. According to the normal lipoprotein levels adjusted for the age range,23,24 57% of the subjects (16/28) had decreased HDL levels. In contrast, abnormal TC, LDL, and TG levels were observed in only 7 (2/28), 18 (5/28), and 14% (4/28) of the patients, respectively. Remarkably, the male patients had lower HDL levels than the female patients (42.6¡12.6 mg/dl vs. 53.3¡10.1 mg/dl, respectively; p,0.05) (Table 1). Decreased HDL levels were not associated with disease activity (p = 0.07), corticosteroid (p = 1.0), or chloroquine therapy (p = 0.4). The frequency of patients with decreased HDL was similar in patients with a short (#5 years) or long (.5 years) disease duration (50% vs. 50%, respectively; p = 1.0). A trend towards increased LDL levels was observed for the patients who were taking corticosteroid (OR: 9.0, 95% CI: 0.8 to 103.7, p = 0.05) but not for the patients were taking chloroquine or who had active JIA (p.0.05). Moreover, neither disease activity nor therapy influenced either the TC or TG levels (p.0.05). An evaluation of the stratified lipoprotein risk factors for CAD revealed that 71% (20/28) of the patients had risk factors, with sixteen patients having one factor and four patients having two or more. Among the sixteen patients with a single lipoprotein risk factor, 81% (13/16) were attributed to decreased HDL levels.

Biochemical analysis Plasma lipid levels: Each subject followed their own regular diet, and blood samples for lipoprotein evaluation were obtained after a 12-h overnight fast. Plasma total cholesterol (TC) and TG levels were measured enzymatically (Boehringer Mannheim, Buenos Aires, Argentina and Merck, Darmstadt, Germany, respectively) using an RA 1000 analyzer (Technicon Instruments Corp., Tarrytown, NY, USA).18,19 HDL levels were obtained after precipitating very low-density lipoprotein cholesterol (VLDL) and LDL with phosphotungstic acid and magnesium chloride.20 VLDL and LDL levels were estimated for samples with TG levels that were lower than 400 mg/dl.21 VLDL levels were calculated by multiplying the TG level by 0.45, and LDL levels were estimated using the following equation: LDL = TC – (HDL+TG)/5.21 Lipoprotein risk levels for CAD: Lipoprotein risk levels for the JIA patients were determined based on the updated recommendations from the Brazilian consent guidelines for the detection and treatment of DL, which were established by the Brazilian Society of Cardiology and the Brazilian Society of Pediatrics.22 These guidelines are in accordance with the National Cholesterol Education Program (NCEP) for subjects who are older than 20 years of age23 and for children and adolescents (NCEP-Peds) ranging from 2 to 19 years of age.24 Statistical analysis: Values are expressed as the mean¡ standard deviation (SD). Statistical comparisons were performed using the Mann-Whitney test, Kruskal-Wallis test, Student’s t-test or x2 test (Pearson Chi-square or Fisher’s exact test), as appropriate. The relative risks for DL according to chloroquine and steroid intake and disease activity and duration were calculated by estimating the odds ratio (OR) and 95% confidence interval (95% CI) using unconditional binary logistic regression. Differences with a p-value ,0.05 were considered to be significant. All of the analyses were performed using the Statistical Package of Social Sciences, version 15.0 for Windows (SPSS Inc., Chicago, IL, USA).

DISCUSSION In this study, we found decreased HDL levels in polyarticular onset JIA patients, and this finding was not related to disease activity, disease duration, or therapy. Furthermore, we demonstrate here for the first time that male patients with polyarticular onset JIA have significantly lower HDL levels than female patients.

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Decreased HDL levels in JIA Marangoni RG et al.

Table 1 - Characteristics and lipid profiles of 28 patients with polyarticular JIA. Variable Age, years Age at diagnosis, years Disease duration, years Total cholesterol, mg/dl Triglycerides, mg/dl HDL, mg/dl LDL, mg/dl CV risk factor, number

JIA patients (n = 28) 19.86¡5.79 (5-29) 10.00¡5.03 (1-16) 9.36¡5.41 (1-25) 150.29¡26.04 (104-206) 81.61¡49.36 (33-255) 50.61¡11.55 (30-74) 83.61¡23.06 (41-138) 0.96¡0.92 (0-4)

Female gender (n = 21)

Male gender (n = 7)

p-value{

20.00¡5.64

19.43¡6.68

0.826

9.62¡4.96

11.14¡5.43

0.498

9.71¡5.12

8.29¡6.52

0.555

150.71¡26.44

149.00¡26.78

0.883

72.62¡35.00

108.57¡75.82

0.265

53.29¡10.11

42.57¡12.62

0.031

83.29¡22.49

84.57¡26.58

0.901

0.90¡0.89

1.14¡1.07

0.640

Except where indicated otherwise, data are expressed as mean¡standard deviation; the values in parentheses indicate the range. LDL – decreased-density lipoprotein cholesterol; HDL – high-density lipoprotein cholesterol; CV – cardiovascular. {p-values represent the correlation between female and male patients. The p-value in bold is statistically significant.

remission within ten years of the initial diagnosis of polyarticular JIA,31 most children will enter adulthood with ongoing active arthritis. In fact, more than 30% of our JIA patients still had active JIA five years after onset; thus, chronic inflammation seems to be a relevant issue of concern. Although the relationship between physical activity and HDL was not addressed in this study, we can speculate that persistent active disease associated with physical incapacity due to functional impairment may play an important role in the dyslipidemic status that we observed in our JIA patients because a sedentary lifestyle is a contributing factor for decreased HDL. Moreover, decreased physical activity was recently associated with proinflammatory HDL in patients with SLE, which suggests that exercise can modulate the development of cardiovascular disease in this group of patients, possibly by decreasing inflammatory mediators.32 Alternatively, the role of drug intake may be a component of the abnormal lipid levels that we observed. Our findings cannot be explained by chloroquine treatment, which has a lipid-lowering effect that promotes an increase in HDL levels.33 Moreover, decreased HDL levels cannot be related to steroid use, which enhances the levels of all lipid fractions.34 Furthermore, although diet intake was not specifically investigated, the majority of the children that we evaluated had a normal BMI, and we excluded children with diabetes or other factors that could have interfered with our evaluation of lipoprotein levels and CAD risk stratification. In conclusion, we found a elevated prevalence of altered lipoprotein profiles in polyarticular onset JIA that was primarily related to decreased HDL levels and male gender, thereby emphasizing the need for early intervention in order to minimize this perturbation. Additional studies are needed to determine the role of diet, statins, and exercise in preventing cardiac events in JIA patients, with the aim of effectively assessing whether physical fitness and drug intake will have beneficial effects on future cardiovascular disease in these patients.

In contrast to previous studies, the lipid profile that we evaluated was focused on a homogeneous population of JIA patients with polyarticular onset. JIA encompasses a broad spectrum of diseases, and a systemic onset has a pattern of inflammatory markers—with oligoarticular and polyarticular JIA subtypes having distinct markers—that can influence abnormal lipid levels.15 In this respect, there are differing reports of abnormal lipoprotein levels in JIA patients and a lack of a definitive prevalence of DL for this disease.9-14 Ilowite and colleagues9 were the first to describe an altered lipoprotein profile in both active and inactive JIA patients that was characterized by decreased HDL levels and increased TG and VLDL levels. Interestingly, these abnormalities were primarily observed in those patients with an active, systemic subtype of the disease.9 In addition, an elevated TG level was the only finding that was observed in 99 Polish children with JIA (compared to agematched healthy controls) when several subtypes of the disease were evaluated.10 In contrast, no significant differences in TG, HDL, TC, or apolipoprotein B levels were detected between 37 Turkish JIA patients and 18 controls, although an inverse correlation between apolipoprotein A1 and inflammatory markers (ESR and CRP) was observed.11 However, a study of 26 Greek children with JIA found decreased HDL and elevated TG levels that were strongly correlated with the inflammatory state of the disease.12 Overall, ethnic differences, lifestyle, and environmental factors as well as genetic background may help explain these varied observations that are related to DL in patients with juvenile arthritis.25,26 Our results are in agreement with the majority of previous studies that have shown decreased HDL levels in patients with JIA. Decreased HDL levels have been described in patients with systemic lupus erythematosus (SLE); indeed, our group has previously reported decreased HDL levels in 88% of patients with juvenile SLE and a trend for an association of this reduced level with the active disease.30 In contrast to some studies that have demonstrated a correlation with active disease,9,11,12 we found no association between decreased HDL cholesterol levels and disease activity; in this regard, abnormal lipid levels in active JIA may be related to the inflammatory state of the disease.9,11,12 Considering the 15% probability of achieving disease

ACKNOWLEDGMENTS This work was supported by the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPQ grants #303165/2008-1 to EFB and

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18. Siedel J, Ha¨gele EO, Ziegenhorn J, Wahlefeld AW. Reagent for the enzymatic determination of serum total cholesterol with improved lipolytic efficiency. Clinical Chemistry. 1983;29:1075–80. 19. Fossati P, Prencipe L. Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clinical Chemistry. 1982;28:2077–80. 20. Warnick GR, Cheung MC, Albers JJ. Comparison of current methods for high-density lipoprotein cholesterol quantitation. Clinical Chemistry. 1979;25:596–604. 21. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clinical Chemistry. 1972;18:499–502. 22. Sposito AC, Caramelli B, Fonseca FA, Bertolami MC, Afiune Neto A, Souza AD, et al. IV Brazilian Guideline for Dyslipidemia and Atherosclerosis prevention: Department of Atherosclerosis of Brazilian Society of Cardiology. Arq Bras Cardiol. 2007;88(Suppl. I):2-19, doi: 10. 1590/S0066-782X2007000700002. 23. National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adults Treatment Panel III). 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;106:3143-421. 24. American Academy of PEDIATRICS. National Cholesterol Education Program (NCEP): report of the expert panel on blood cholesterol levels in children and adolescents. Pediatrics. 1992;89:525-84. 25. Parnell LD, Lee YC, Lai CQ. Adaptive genetic variation and heart disease risk. Curr Opin Lipidol. 2010;21:116-22, doi: 10.1097/MOL.0b013e328 3378e42. 26. Chandalia M, Mohan V, Adams-Huet B, Deepa R, Abate N. Ethnic difference in sex gap in high-density lipoprotein cholesterol between Asian Indians and Whites. J Investig Med. 2008;56:574–80. 27. Hahn BH, Grossman J, Ansell BJ, Skaggs BJ, McMahon M. Altered lipoprotein metabolism in chronic inflammatory states: proinflammatory high-density lipoprotein and accelerated atherosclerosis in systemic lupus erythematosus and rheumatoid arthritis. Arthritis Res Ther. 2008;10:213, doi: 10.1186/ar2471. 28. Svenungsson E, Gunnarsson I, Fei GZ, Lundberg IE, Klareskog L, Frostega˚rd J. Elevated triglycerides and low levels of high-density lipoprotein as markers of disease activity in association with upregulation of tumor necrosis factor alpha/tumor necrosis factor receptor system in systemic lupus erythematosus. Arthritis Rheum. 2003;48:253340, doi: 10.1002/art.11264. 29. Borba EF, Bonfa´ E. Dyslipoproteinemias in systemic lupus erythematosus: influence of disease, activity, and anticardiolipin antibodies. Lupus. 1997;6:533-9, doi: 10.1177/096120339700600610. 30. Hayata AL, Borba EF, Bonfa´ E, Kochen JA, Goldenstein-Schainberg C. The frequency of high/moderate lipoprotein risk factor for coronary artery disease is significant in juvenile-onset systemic lupus erythematosus. Lupus. 2005;14:613-7, doi: 10.1191/0961203305lu2177oa. 31. Oen K, Malleson PN, Cabral DA, Rosenberg AM, Petty RE, Cheang M. Disease course and outcome of juvenile rheumatoid arthritis in a multicenter cohort. J Rheumatol. 2002;29:1989-99. 32. Volkmann ER, Grossman JM, Sahakian LJ, Skaggs BJ, FitzGerald J, Ragavendra N, et al. Low physical activity is associated with proinflammatory high-density lipoprotein and increased subclinical atherosclerosis in women with systemic lupus erythematosus. Arthritis Care Res (Hoboken). 2010;62:258-65, doi: 10.1002/acr.20076. 33. Borba EF, Bonfa´ E. Long-term beneficial effect of chloroquine diphosphate on lipoprotein profile in lupus patients with and without steroid therapy. J Rheumatol. 2001;28:780-5. 34. Ettinger WH, Klinefelter HF, Kwiterovich PO. Effects of short term, low dose corticosteroids on plasma lipoprotein lipids. Atherosclerosis. 1987;63:167-72, doi: 10.1016/0021-9150(87)90117-1.

#305468/2006-5 to EB) and a Federico Foundation Grant (to EFB and EB).

REFERENCES 1. Natarajan S, Glick H, Criqui M, Horowitz D, Lipsitz SR, Kinosian B. Cholesterol measures to identify and treat individuals at risk for coronary heart disease. Am J Prev Med. 2003;25:5-7, doi: 10.1016/ S0749-3797(03)00092-8. 2. Wolfe F, Mitchell DM, Sibley JT, Fries JF, Bloch DA, Williams CA, et al. The mortality of rheumatoid arthritis. Arthritis Rheum. 1994;37:481-94, doi: 10.1002/art.1780370408. 3. Maradit-Kremers H, Nicola PJ, Crowson CS, Ballman KV, Gabriel SE. Cardiovascular death in rheumatoid arthritis. Arthritis Rheum. 2005; 52:722-32, doi: 10.1002/art.20878. 4. Kremers HM, Crowson CS, Therneau TM, Roger VL, Gabriel SE. High ten-year risk of cardiovascular disease in newly diagnosed rheumatoid arthritis patients: a population-based cohort study. Arthritis Rheum. 2008;58:2268-74, doi: 10.1002/art.23650. 5. Gasparyan AY, Stavropoulos-Kalinoglou A, Mikhailidis DP, Toms TE, Douglas KM, Kitas GD. The Rationale for Comparative Studies of Accelerated atherosclerosis in Rheumatic Diseases. Curr Vasc Pharmacol. 2010;8:437-49, doi: 10.2174/157016110791330852. 6. Thomas E, Symmons DP, Brewester DH, Black RJ, Macfarlane GJ. National study of cause-specific mortality in rheumatoid arthritis, juvenile chronic arthritis and other rheumatic conditions: a 20-year follow-up study. J Rheumatol. 2003;30:958-65. 7. Del Rincon I, Escalante A. Atherosclerotic cardiovascular disease in rheumatoid arthritis. Curr Rhematol Rep. 2003;5:278-86, doi: 10.1007/ s11926-003-0006-8. 8. Van Doornum S, McColl G, Wicks IP. Accelerated atherosclerosis. Arthritis Rheum. 46:862-873, doi: 10.1002/art.10089 9. Ilowite NT, Samuel P, Beseler L, Jacobson MS. Dyslipoproteinemia in juvenile rheumatoid arthritis. J Pediatrics. 1989;114:823-6, doi: 10.1016/ S0022-3476(89)80148-9. 10. Musiej-Nowakowska E, Zawadzka F, Weso3owska H, Miko3ajew M. Serum lipid concentration in juvenile rheumatoid arthritis. Acta Univ Carol. 1991;37:46-9. 11. Bakkaloglu A, Kirel B, Ozen S, Saatc¸i U, Topaloglu, Besbas N. Plasma lipids and lipoproteins in juvenile chronic arthritis. Clin Rheumatol. 1996;15:341-5, doi: 10.1007/BF02230355. 12. Tselepis AD, Elisaf M, Besis S, Karabina SA, Chapman MJ, Siamopoulou A. Association of the inflammatory state in active juvenile rheumatoid arthritis with hypo-high-density lipoproteinemia and deuced lipoprotein associated platelet-activating factor acetylhydrolase activity. Arthritis Rheum. 1999;42:373-83, doi: 10.1002/1529-0131(199902)42:2,373::AIDANR21.3.0.CO;2-3. 13. Pietrewicz E, Urban M. [Early atherosclerosis changes in children with juvenile idiopathic arthritis] Pol Merkur Lekarski. 2007;22:211-4 [Article in Polish]. 14. Gonc¸alves M, D’Almeida V, Guerra-Shinohara EM, Galdieri LC, Len CA, Hila´rio MO. Homocysteine and lipid profile in children with Juvenile Idiopathic Arthritis. Pediatr Rheumatol Online J. 2007;5:2, doi: 10.1186/ 1546-0096-5-2. 15. Thomas E, Barret JH, Donn RP, Thomson W, Southwood TR. Subtyping of juvenile idiopathic arthritis using latent class analysis. British Paediatric Rheumatology Group. Arthritis Rheum. 2000;43:1496-503. 16. Libby P, Ridker PM, Maseri A. Inflammation and Atherosclerosis. Circulation. 2002;105:1135-43, doi: 10.1161/hc0902.104353. 17. Petty RE, Southwood TR, Manners P, Baum J, Glass DN, Goldenberg J, et al. International League of Associations for Rheumatology classification of juvenile idiopathic arthritis: second revision, Edmonton, 2001. J Rheumatol. 2004;31:390–2.

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DOI:10.1590/S1807-59322011000900008

CLINICAL SCIENCE

Acute exercise improves cognition in the depressed elderly: the effect of dual-tasks Paulo Eduardo Vasques,I,II Helena Moraes,I,II Heitor Silveira,I,II Andrea Camaz Deslandes,I,II,III,IV Jerson LaksI,V I

Center for Alzheimer’s Disease and Related Disorders, Institute of Psychiatry, Federal University of Rio de Janeiro/RJ, Brazil. II Neuroscience of Exercise Laboratory, Gama Filho University, Rio de Janeiro/RJ, Brazil. III Biometrics Laboratory, Federal University of Rio de Janeiro/RJ, Brazil. IV National School of Public Health, Rio de Janeiro/RJ, Brazil. V Brazilian National Research Council - Researcher II.

OBJECTIVE: The goal of this study was to assess the acute effect of physical exercise on the cognitive function of depressed elderly patients in a dual-task experiment. INTRODUCTION: Physical exercise has a positive effect on the brain and may even act as a treatment for major depressive disorder. However, the effects of acute cardiovascular exercise on cognitive function during and after one session of aerobic training in elderly depressive patients are not known. METHODS: Ten elderly subjects diagnosed with major depressive disorder performed neuropsychological tests during and after a moderate physical exercise session (65-75%HRmax). A Digit Span Test (Forward and Backward) and a Stroop Color-Word Test were used to assess cognitive function. The elderly participants walked on an electric treadmill for 30 minutes and underwent the same cognitive testing before, during, immediately after, and 15 minutes after the exercise session. In the control session, the same cognitive testing was conducted, but without exercise training. RESULTS: The results of the Digit Span Test did not change between the control and the exercise sessions. The results of the Stroop Color-Word Test improved after physical exercise, indicating a positive effect of exercise on cognition. CONCLUSIONS: These data suggest that the cognitive functions of depressed elderly persons, especially attention and inhibitory control, are not impaired during and after an acute session of physical exercise. In contrast, the effect of dual-tasks showed beneficial results for these subjects, mainly after exercise. The dual-task may be a safe and useful tool for assessing cognitive function. KEYWORDS: Aerobic exercise; Major depression; Attention; Memory; Older. Vasques PE, Moraes H, Silveira H, Deslandes AC, Laks J. Acute exercise improves cognition in the depressed elderly: the effect of dual-tasks. Clinics. 2011;66(9):1553-1557. Received for publication on April 3, 2011; First review completed on April 25, 2011; Accepted for publication on May 19, 2011 E-mail: pevasques@yahoo.com.br Tel.: 55 21 22540615

intimately associated with the severity of disease and the impairment of daily activities during and after a depressive crisis, including during remission. In addition to the various treatments proposed for depression, physical exercise may have beneficial effects as an add-on therapy.3,4 However, activities that demand divided attention in daily life (e.g., walking) present an increased risk of falls and impaired cognition. Combined concurrent tasks (dual-task interventions) have been studied in subjects with neuropsychiatric disorders and in normal subjects, although few data exist on depression in the elderly. Studies have produced divergent results regarding other variables such as age and the presence of motor or neuropsychiatric disorders.5-8 Some hypotheses and theories attempt to explain cognitive and motor performance for the dual-task paradigm. Dietrich proposed the transient hypofrontality hypothesis,9 which suggests that the cognitive functions associated with the frontal areas are impaired during physical exercise because the brain prioritizes motor control and the

INTRODUCTION Depression is one of the most common disorders associated with aging. Depression has a high prevalence of symptoms related to social, physical, and neurophysiological factors.1 Cognition is a function that is altered in depression and has received increasing interest.2 Neurophysiological changes that occur in depressive states, such as prefrontal cortex and cingulate activation, are partly responsible for reduced attention, memory, and visuospatial capacities. Depression also affects executive processing, causing difficulties in planning strategies and mental flexibility, which, in turn, lead to impaired motivational and decision-making functions.2 Impaired cognition is thus

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The DST assesses short-term memory, working memory, and attention. It consists of two subtests, one of which requires the patient to orally repeat a sequence of digits forward and another one that requires that the patient repeat a sequence backward. The SCW is a test of selective attention26 in which patients are required to name the ink color in which incongruent color names are printed. The Victoria version was used because of its short administration time (the approximate time required is 5 minutes) and its sensitivity to frontal lobe disorders. The Victoria version uses three 21.5614 cm cards presented in the following order: part D, part W, and part C. Each card has six rows of four items. Part D contains colored dots (red, green, blue, and yellow), and the task on this card is to name the colors as quickly as possible (congruence). Part W has words printed in the following colors: red, green, blue, or yellow. The examinee must name the colored ink in which each word is printed as quickly as possible. In part C, the words red, green, blue and yellow are printed in a color not denoted by the word (e.g., the word red is printed in green ink, etc.), and the examinee must name the colored ink in which the word is printed as quickly as possible (incongruence). For each part, time, and number of errors are considered in the score. A ratio index (C/D) of interference was used.27,28

maintenance of vital functions (e.g., blood pressure and temperature control). In contrast, the bottleneck theory assumes that dual tasks are processed by the same neural networks, resulting in impaired execution of the second task.10 Indeed, Bloem et al. noted that young adults adopt a ‘‘posture first’’ strategy, in which they automatically prioritize motor tasks rather than cognitive tasks.11 Conversely, elderly subjects and neurological patients (Alzheimer’s disease, Parkinson’s disease, and stroke patients) focus on the cognitive task when performing dual-tasks, thereby increasing the risk of falls.8 An increasing body of data has shown that moderate physical exercise has preventive and therapeutic effects on several clinical and mental disorders,3,12 especially mild to moderate depression.4,13 Although much is already known about the effects of routine exercise on cognitive function, the acute effect of exercise remains to be studied in depth. Diverging results might be partly explained by factors such as the following: the time of assessment (during or after exercise); the type, intensity, and duration of the exercise; and the type of cognitive task under study.14,15 Studies with young subjects have shown that reaction time, attention, and working memory improve after one session of aerobic exercise.16,17 However, little is known about the effect of acute exercise in the elderly with or without psychiatric disorders. Hoffman et al. observed that routine physical exercise did not improve the cognition of depressed elderly.18 Another study examined the effect of acute exercise on the cognition of adults with depression19 and showed that patients improved their attention and inhibitory control immediately after an exercise session. To the best of our knowledge, however, there are no studies on combined cognitive and motor tasks in depressed elderly individuals. The present study aimed to assess cognitive performance during and after one session of physical exercise among elderly persons with major depression. We hypothesized that the most complex cognitive functions would worsen during and improve after moderate physical exercise.

Experimental Procedure The subjects were tested on two different occasions. In the first session, all cognitive tests were conducted, followed by a 30-minute walk on an electric treadmill (BH FitnessH Explorer Pro Action). After a 5-minute warm-up, subjects trained for 25 minutes at 65%-75% of the maximum heart rate estimated for their age (220-age).29 The cognitive tests were conducted again at the 20th minute of the training, immediately after the completion of the training, and again 15 minutes later. One month after this first trial, the cognitive tests were conducted again using the same schedule without submitting the subjects to any physical exercise. This project was approved by the Ethics Committee of the Institute of Psychiatry of the Federal University of Rio de Janeiro. All subjects signed informed consent forms before any procedure was undertaken.

MATERIALS AND METHODS This study included a series of elderly subjects (n = 10) diagnosed according to the DSM-IV criteria20 with major depression with mild to moderate severity who had already been participating in a exercise program for at least six months. Exclusion criteria consisted of illiteracy, any presentation of degenerative neurological dementia, history of stoke, or any limitation of gait. All patients were weighed and measured for height using a mechanic anthropometric scale (WelmyH, Brazil). Depressive mood was assessed using the Brazilian validated version of the Hamilton Depression Scale (HAMD).21,22 The HAMD evaluates the severity of 17 depressive symptoms with a range of zero to 49. Patients with scores from seven to 13 are considered mildly depressed, from 14 to 18 moderately depressed, from 19 to 22 severely depressed, and more than 23 very severely depressed. Cognition was assessed by a battery of tests including the Mini Mental State Examination (MMSE), the Digit Span Test (DST)23 and the Stroop Color-Word Test (SCW). The MMSE Brazilian validated version assesses global cognitive functions, with scores ranging from zero to 30.24,25

Statistical Analysis Descriptive data are shown as the mean and standard deviation (SD) or median and confidence intervals (CI). Shapiro-Wilk and Levene tests were used to assess the normality and the homogeneity of variances, respectively. Because the data were characteristically non-parametric, two analyses using Friedman’s repeat measures were performed

Table 1 - Sociodemographic characteristics of the sample (n = 10). Mean (standard deviation) Age (years) Education (years) Height (cm) Weight (kg) HAMD MMSE

71.5 (6.0) 7.4 (4.7) 158.7 (8.0) 70.21 (7.8) 14.1 (3.8) 27.1 (2.2)

HAMD: Hamilton Depression Scale; MMSE: Mini Mental State Examination.

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Acute exercise improves cognition Vasques PE et al.

Figure 1 - Comparison of the performance in the Digit Span Test among different time points (means ยก SD).

(p = 0.009) and for the post-15 minutes time point vs. the during-exercise time point (p = 0.013) for the congruent subitem, whereas in the incongruent trial, there was improvement in the results for the immediately after time point vs. the before exercise time point (0.017), for the post15 minutes time point vs. the before exercise time point (p = 0.005) and for the post-15 time point vs. the immediately after time point (p = 0.028) (Figure 2). Furthermore, there was a trend toward improvement for the post-15 time point vs. the during-exercise time point (p = 0.059). Control Session: There were no statistically significant differences in the DST results for the different time points (forward p = 0.069, x2 = 7.091; backward p = 0.857, x2 = 0.767). Although the congruent subitem of the SCW did not present a statistically significant difference (p = 0.062, x2 = 7.320), the incongruent task showed a difference among the time points (p = 0.015, x2 = 10.440), with improvement in the immediately after time point vs. the before exercise time point (p = 0.005) and a trend toward improvement for the during exercise time point vs. the before exercise time point (p = 0.059).

for the exercise and control sessions. This analysis compared the cognitive test results among the time points (before, during, immediately after, and 15 minutes after exercising or at the corresponding times during the control session). Statistical Package for Social Sciences (SPSS) version 15.0 was used, and the level of significance was p,0.05.

RESULTS The patients had a mean age of 71.5 (6.0) years and a mean HAMD score of 14.1 (3.8). The descriptive statistics for the sample are listed in Table 1. Exercise Session: There were no statistically significant differences among the time points with regard to the results of the DST Forward and Backward (p = 0.692, x2 = 1.456; p = 0.569, x2 = 2.016, respectively) (Figure 1). The SCW results were statistically significantly different among the time points for both the congruent and incongruent subtests (p = 0.038, x2 = 8.400; p = 0.011, x2 = 11,160, respectively). There was improvement in the results for the post-15 minutes time point vs. the before exercise time point

Figure 2 - Comparison of performance in the Stroop Test among different time points (means ยก SD). * p,0.05 compared to before exercise, ** p,0.05 compared to before and during exercise, *** p,0.05 compared to before and immediately after exercise.

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DISCUSSION

CONCLUSION

This study aimed to assess changes in cognitive performance in depressed elderly persons during and after a session of physical exercise. To the best of our knowledge, this is the first study to examine this issue using a dual-task paradigm (motor and cognitive stimuli). We found an improvement in attention and inhibitory control but not in working memory after a 30-minute moderately intense walk on a treadmill. These results only partially confirmed our hypothesis because we found changes only after but not during the exercise. Our results corroborate those of other studies of depressive subjects that have found improvements in attention and inhibitory control and no change in working memory.19 The improvements in attention and inhibitory control immediately after and 15 minutes after exercise can be associated with activated brain areas such as the anterior cingulate, which present impaired activation in depressive subjects. Moreover, exercise may promote the activation of the reticular formation that is responsible for the modulation of attention and arousal. Bartholomew et al.30 have shown that acute exercise can improve the mood of depressed individuals in addition to yielding cognitive improvement. Studies with young and elderly healthy subjects have observed that cognitive function is immediately improved after physical exercise.16,19 This effect appears to occur only when aerobic exercise is applied.16 The improvement in cognition and mood after exercise in depressive and healthy subjects has been attributed to the acute release of neurotransmitters.31 However, recent data have confirmed that acute exercise also increases the levels of neurotrophic factors, such as BDNF (Brain-Derived Neurotrophic Factor).32 Higher levels of BDNF immediately after exercise may enhance neurogenesis, neuronal plasticity, learning abilities, memory, and mood.33 Dual tasks that comprise only cognitive tasks are more difficult to perform than dual tasks that include one physical task, especially for depressive subjects.34 Cognitive performance during exercise is still a matter of debate. Some studies have shown an impaired performance in complex tasks that depend on frontal functions,7,35 whereas other studies have shown improvement.5,6 Contrary to our expectations, we did not observe differences in cognitive function during exercise. However, this study was conducted with physically active patients, which might explain why we observed no impairment during exercise. Because the subjects included in this study were trained and adapted to the motor tasks, the processing demand required to perform the task was reduced (Task Automatization Hypothesis), thus minimizing the detrimental effect of the dual task. In the present study, we used a session with no exercise to control for possible responses generated by the learning effect. However, we did not observe significant changes in the test scores during this session except for the SCW incongruence subitem for the immediately after time point vs. the before exercise time point. This response may not be explained by the learning effect because the post-15 and during test results were not different. Hence, we suggest that the responses were influenced by physical exercise. This study has some limitations that should be taken into consideration. As a preliminary report, we assessed a small number of patients. Further studies that focus on healthy controls and that use direct markers of exercise intensity such as oxygen consumption are warranted.

In conclusion, our study provides evidence that the cognitive function of depressed elderly persons is enhanced immediately after and 15 minutes following a session of physical exercise. Moreover, cognitive function is not impaired during the dual-task activity (motor and cognitive tasks). The dual task may be a safe and useful tool to assess cognitive function during and after exercise.

ACKNOWLEDGMENTS This work was supported by the Brazilian National Council of Research and the Foundation for Research Support of Rio de Janeiro. This project was conducted at the Center for Alzheimerâ&#x20AC;&#x2122;s Disease and Related Disorders, Institute of Psychiatry, Federal University of Rio de Janeiro, Brazil.

REFERENCES 1. Laks J, Engelhardt E. Peculiarities of geriatric psychiatry: a focus on aging and depression. CNS Neurosci Ther. 2010;16:374-9, doi: 10.1111/j. 1755-5949.2010.00196.x. 2. Davidson RJ, Lewis DA, Alloy LB, Amaral DG, Bush G, Cohen JD, et al. Neural and behavioral substrates of mood and mood regulation. Biol Psychiatry. 2002;52:478-502, doi: 10.1016/S0006-3223(02)01458-0. 3. Deslandes A, Moraes H, Ferreira C, Veiga H, Silveira H, Mouta R, et al. Exercise and mental health: many reasons to move. Neuropsychobiology. 2009;59:191-8, doi: 10.1159/000223730. 4. Deslandes AC, Moraes H, Alves H, Pompeu FAMS, Silveira H, Mouta R, et al. Effect of aerobic training on EEG alpha asymmetry and depressive symptoms in the elderly: a 1-year follow-up study. Braz J Med Biol Res. 2010;43:585-92, doi: 10.1590/S0100-879X2010007500041. 5. Pesce C, Cereatti L, Casella R, Baldari C, Capranica L. Preservation of visual attention in older expert orienteers at rest and under physical effort. J Sport Exerc Psychol. 2007;29:78-99. 6. Davranche K, Audiffren M. Facilitating effects of exercise on information processing. J Sports Sci. 2004;22:419-28, doi: 10.1080/02640410410001675289. 7. Dietrich A, Sparling PB. Endurance exercise selectively impairs prefrontal-dependent cognition. Brain Cogn. 2004;55:516-24, doi: 10. 1016/j.bandc.2004.03.002. 8. Yogev-Seligmann G, Hausdorff JM, Giladi N. The role of executive function and attention in gait. Mov Disord. 2008;15;23:329-42, doi: 10. 1002/mds.21720. 9. Dietrich A. Functional neuroanatomy of altered states of consciousness: the transient hypofrontality hypothesis. Conscious Cogn. 2003;12:231-56, doi: 10.1016/S1053-8100(02)00046-6. 10. Pashler H. Dual-task interference in simple tasks: data and theory. Psychol Bull. 1994;116:220-44, doi: 10.1037/0033-2909.116.2.220. 11. Bloem BR, Valkenburg VV, Slabbekoorn M, Willemsen MD. The Multiple Tasks Test: development and normal strategies. Gait Posture. 2001;14:191-202, doi: 10.1016/S0966-6362(01)00141-2. 12. Wichi RB, Angelis K, Jones L, Irigoyen MC. A brief review of chronic exercise intervention to prevent autonomic nervous system changes during the aging process. Clinics. 2009;64:253-8, doi: 10.1590/S180759322009000300017. 13. Silveira H, Deslandes AC, Moraes H, Mouta R, Ribeiro P, Piedade R, et al. Effects of exercise on electroencephalographic mean frequency in depressed elderly subjects. Neuropsychobiology. 2010;61:141-7, doi: 10. 1159/000279304. 14. Brisswalter J, Collardeau M, ReneÂ´ A. Effects of acute physical exercise characteristics on cognitive performance. Sports Med. 2002;32:555-66, doi: 10.2165/00007256-200232090-00002. 15. Tomporowski PD. Effects of acute bouts of exercise on cognition. Acta Psychol (Amst). 2003;112:297-324, doi: 10.1016/S0001-6918(02)00134-8. 16. Pontifex MB, Hillman CH, Fernhall B, Thompson KM, Valentini TA. The effect of acute aerobic and resistance exercise on working memory. Med Sci Sports Exerc. 2009;41:927-34, doi: 10.1249/MSS.0b013e3181907d69. 17. Pesce C, Capranica L, Tessitore A, Figura F. Focusing of visual attention under submaximal physical load. Int J Sport Exerc Psychology. 2003;1:275-92. 18. Hoffman BM, Blumenthal JA, Babyak MA, Smith PJ, Rogers SD, Doraiswamy PM, et al. Exercise fails to improve neurocognition in depressed middle-aged and older adults. Med Sci Sports Exerc. 2008;40:1344-52, doi: 10.1249/MSS.0b013e31816b877c. 19. Kubesch S, Bretschneider V, Freudenmann R, Weidenhammer N, Lehmann M, Spitzer M, et al. Aerobic endurance exercise improves executive functions in depressed patients. J Clin Psychiatry. 2003;64:1005-12, doi: 10.4088/JCP.v64n0905.

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Acute exercise improves cognition Vasques PE et al.

20. American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders,ed 4. Washington;American Psychiatric Association, 1994. 21. Hamilton M. Rating scale for depression. J Neurol Neurosurg Psychiatry. 1960;23:56-62, doi: 10.1136/jnnp.23.1.56. 22. Moreno RA, Moreno DH. Escalas de depressaõ de Montgomery & Asberg (MADRS) e de Hamilton (HAM-D). Revista de Psiquiatria Clıńica. 1998;25:1-17. 23. Wechsler D. Wechsler Memorv Scale-Revised. New York: Psychological Corporation. 1987;1-150 24. Folstein MF, Folstein SE, McHugh PR. ‘‘Mini-mental state’’. A practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res. 1975;12:189-98. 25. Brucki SM, Nitrini R, Caramelli P, Bertolucci PH, Okamoto IH. Suggestions for utilization of the mini-mental state examination in Brazil. Arq Neuropsiquiatr. 2003;61:777-81, doi: 10.1590/S0004282X2003000500014. 26. Stroop JR. Studies of interference in serial verbal reactions. J Exp Psychol. 1935;18:643-62, doi: 10.1037/h0054651. 27. Spreen O., Strauss E.A. Compendium of Neuropsychological Tests. 3rd ed. New York; Administration, Norms, and Commentary; 1998. 28. Silberman CD, Laks J, Capitaõ CF, Rodrigues CS, Moreira I, Vasconcellos LFR, et al. Frontal functions in depressed and nondepressed Parkinson’s

29. 30. 31.

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33. 34. 35.

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disease patients: impact of severity stages. Psychiatry Res. 2007;149:28589, doi: 10.1016/j.psychres.2006.04.020. Karvonen MJ, Kentala E, Mustala O. The effects of training on heart rate: A longitudinal study. Ann Med Exp Biol Fenn. 1957;35:307-15. Bartholomew JB, Morrison D, Ciccolo JT. Effects of Acute Exercise on Mood and Well-Being in Patients with Major depressive disorder. Med Sci Sports Exerc. 2005;37:2032-7, doi: 10.1249/01.mss.0000178101.78322.dd. Boecker H, Henriksen G, Sprenger T, Miederer I, Willoch F, Valet M, et al. Positron emission tomography ligand activation studies in the sports sciences: measuring neurochemistry in vivo. Methods. 2008;45:307-18, doi: 10.1016/j.ymeth.2008.07.003. Gustafsson G, Lira CM, Johansson J, Wise´n A, Wohlfart B, Ekman R, et al. The acute response of plasma brain-derived neurotrophic factor as a result of exercise in major depressive disorder. Psychiatry Res. 2009;169:244-8, doi: 10.1016/j.psychres.2008.06.030. Cotman CW, Berchtold NC, Christie LA. Exercise builds brain health: key roles of growth factor cascades and inflammation. Trends Neurosci. 2007;30:464-72, doi: 10.1016/j.tins.2007.06.011. Nebes RD, Butters MA, Houck PR, Zmuda MD, Aizenstein H, Pollock BG, et al. Dual-task performance in depressed geriatric patients. Psychiatry Res. 2001;102:139-51, doi: 10.1016/S0165-1781(01)00244-X. Davranche K, McMorris T. Specific effects of acute moderate exercise on cognitive control. Brain Cogn. 2009;69:565-70, doi: 10.1016/j.bandc.2008. 12.001.

CLINICS 2011;66(9):1559-1562

DOI:10.1590/S1807-59322011000900009

CLINICAL SCIENCE

Antibody response following Hepatitis B vaccination in peritoneal dialysis patients: does normalized urea clearance matter? Erkan Dervisoglu,I Melih Simsek,II Ahmet YilmazI I

Departments of Nephrology, Kocaeli University School of Medicine, Kocaeli, Turkey. II Internal Medicine, Kocaeli University School of Medicine, Kocaeli, Turkey.

OBJECTIVES: Data on the factors that contribute to the antibody response to hepatitis B virus vaccination in peritoneal dialysis patients are scarce. The current study was conducted on a group of peritoneal dialysis patients to learn how the response to hepatitis B virus vaccination varies according to the patient’s clearance of urea normalized to total body water (Kt/V). METHODS: A convenience sample of 33 peritoneal dialysis patients (13 women and 20 men, with a mean age of 49¡12 years) was administered double doses (20 mg IM in each deltoid muscle) of recombinant hepatitis B vaccine at 0, 1, 2, and 6 months. Response to immunization was measured at one to three months after the final dose of vaccine. The subjects were divided into groups according to the level of antibodies to hepatitis B surface antigen (anti-HBs), including non-responders (,10 IU/L), weak responders (10-100 IU/L), and good responders (.100 IU/L). RESULTS: Among non-responders, weak responders, and good responders, significant differences were found in age (54¡12 vs. 56¡9 vs. 45¡12 years, respectively; p = 0.049) and recombinant human erythropoietin use (20 vs. 29 vs. 76%, respectively; p = 0.016). No significant differences in weekly total Kt/V (p = 0.704), weekly peritoneal Kt/V (p = 0.064) and residual glomerular filtration rate (p = 0.355) were found across the three groups. CONCLUSIONS: Delivered clearance measured by weekly peritoneal Kt/V and total clearance measured by weekly total Kt/V did not predict the response to hepatitis B virus vaccination in patients on peritoneal dialysis. KEYWORDS: Continuous ambulatory peritoneal dialysis; Hepatitis B virus; Vaccination; Dialysis adequacy; Kt/V. Dervisoglu E, Simsek M, Yilmaz A Antibody response following Hepatitis B vaccination in peritoneal dialysis patients: does normalized urea clearance matter? Clinics. 2011;66(9):1559-1562. Received for publication on April 4, 2011; First review completed on April 25, 2011; Accepted for publication on May 20, 2011 E-mail: dervisoglue@yahoo.com Tel.: 90 2623037041

Uremic patients elicit weak response to vaccination with recombinant HBV vaccine.5 To improve the immunogenicity of HBV vaccination for dialysis-dependent patients, the vaccine should be administered at a dose of 40 mg, in contrast to the 20 mg dose for healthy adults, through the intramuscular route.6 Moreover, instead of a three-dose schedule (i.e., 0, 1, and 6 months), a four-dose schedule (i.e., 0, 1, 2, and 6 months) is recommended.6 Blood antibody levels of hepatitis B antigen (anti-HBs) $10 IU/L are considered protective in healthy individuals; however, some authors believe that higher levels (.100 IU/L) are desirable in patients on chronic HD.7,8 Factors associated with a favorable response to HBV vaccination in HD patients include young age, good nutritional status, and effective dialysis.1,5,9 Dialysis adequacy is defined as the weekly clearance of urea normalized to total body water (Kt/V). To date, only four studies have described the effects of dialysis adequacy on HBV vaccination response in PD patients. The results of these studies, however, have been inconclusive.10-13 We conducted this study on a group of PD patients undergoing PD to demonstrate how the response to HBV vaccination varies with weekly Kt/V.

INTRODUCTION Control of a hepatitis B virus (HBV) infection has been a continuous challenge in the management of patients with end-stage renal disease (ESRD). Patients undergoing hemodialysis (HD) are particularly at risk for HBV infection.1 In contrast to HD patients, patients on peritoneal dialysis (PD) appear to be at low risk for HBV infection. Nevertheless, all susceptible ESRD patients, including those on PD, are encouraged to be vaccinated because it is likely that they will eventually need HD when PD becomes unfeasible, either temporarily or permanently.2 Furthermore, the peritoneal dialysate of patients positive for the hepatitis B surface antigen (HBsAg) contains sufficient infectious particles to cause hepatitis outbreaks in dialysis units.3,4

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WindowsH (SPSS Inc., Chicago, USA). Data are expressed as the mean ยก standard deviation (SD), unless otherwise stated. The normality of data distributions was determined using the Kolmogorov-Smirnov test. Comparisons between groups were made using Studentโ s t-test and analysis of variance (ANOVA) for normally distributed variables, while the Mann-Whitney U test and Kruskall-Wallis variance analysis were used for parametric variables with non-normal distributions. The chi-square test was used to analyze categorical data. A p-value of less than 0.05 was considered statistically significant.

METHODS AND MATERIALS The Faculty of Medicine Ethics Committee of Kocaeli University School of Medicine approved this prospective observational study protocol, and all of the patients provided written informed consent before entry into the study. Both incident and prevalent PD patients over the age of 18 years with ESRD at our university hospital clinic were screened for HBV markers. Patients with negative HBsAg, negative antibodies to hepatitis B surface antigen (anti-HBs), and negative antibodies to hepatitis B core antigen (anti-HBc) were included in the study. Patients using immunosuppressive agents or patients with a history of malignancy, human immunodeficiency virus (HIV) infection, alcoholic liver disease, HBV vaccination, abnormal liver function results during the six months prior to recruitment, or positivity for HBsAg, anti-HBs, or anti-HBc at any time in the past were excluded. Peritoneal dialysis patients were on a standard continuous ambulatory peritoneal dialysis (CAPD) program, with four or five administrations daily of 2000 mL each. Between January 2009 and May 2010, all participants were administered double doses (20 mg IM in each deltoid muscle) of recombinant hepatitis B vaccine (Euvax B, LG Life Sciences, Jeonbuk-do, Korea) at 0, 1, 2, and 6 months. The antibody response was determined by measuring the HBsAg antibody 1 to 3 months after the last dose of vaccine. Anti-HBs titers were measured using a commercial enzyme immunoassay kit (Cobas, Roche Diagnostics GmBH, Mannheim, Germany). Demographic and clinical data, including age, gender, body mass index (weight/height2), primary renal disease, recombinant human erythropoietin (rHuEpo) use, and time on dialysis, were recorded at study entry. Hemoglobin, urea, creatinine, albumin, and high sensitivity C-reactive protein (hs-CRP) were measured in venous blood samples. Patients who had anti-HBs antibody levels ,10 IU/L after the fourth dose of vaccine were considered nonresponders. Responders were divided into weak responders (an anti-HBs level after the last vaccine dose of 10-100 IU/L) and good responders (an anti-HBs level after the last vaccine dose .100 IU/L).5 All participants underwent assessments of peritoneal membrane function and residual renal function during the vaccination process. Peritoneal membrane function was evaluated by the standard peritoneal equilibrium test (PET) using a 2.5% glucose PD solution.14 Adequacy was calculated as weekly total Kt/V (weekly clearance of urea normalized to total body water). Peritoneal Kt/V was calculated by performing a 24-hour collection of dialysate effluent and measuring its urea content and taking a plasma sample during the PET. The dialysate-to-plasma creatinine (D/P Cr) concentration ratio at four hours of dwell was used to describe the peritoneal small solute transport rate. The protein equivalent of nitrogen appearance (PNA) was calculated using the Randerson15 formula and normalized by the ideal body weight (nPNA). Residual renal function was evaluated by collecting all urine over the same 24-hour period as the dialysate collection. Residual glomerular filtration rate (GFR; mL/minute) was calculated as the mean of the renal urea and creatinine clearances.16

RESULTS Thirty-five patients gave written informed consent to participate in the study. Two patients did not complete the vaccination program; one patient underwent transplantation, and one patient died. Therefore, 33 patients (13 women and 20 men, mean age 49ยก12 years) were included in the final analysis. Only one patient was positive for antibodies to hepatitis C virus (anti-HCV). The demographic, clinic, and laboratory features of the patients are shown in Table 1. After the fourth double dose of the vaccine, a response (antiHBs titer $10 IU/L) was observed in 28 (84.8%) of the patients. No significant differences were observed for age, time on dialysis, body mass index, urea, creatinine, hemoglobin, serum albumin, nPNA, or hs-CRP between responders and non-responders (Table 2). Similarly, weekly total Kt/V (p = 0.573), weekly peritoneal Kt/V (p = 0.385) and residual GFR (p = 0.404) were not significantly different between responders and non-responders. The response rate to immunization was 86% among the 29 CAPD patients with a weekly Kt/V of 1.7 or greater. This rate was not significantly different as compared to the 75% response rate among the four CAPD patients with lower values of weekly Kt/V (p = 0.558). Of the 28 patients responding to vaccination, seven patients (25%) were weak responders, and 21 patients (75%) were good responders. Time on dialysis, body mass index, urea, creatinine, hemoglobin, serum albumin, nPNA, and hs-CRP were not significantly different among non-responders, weak Table 1 - Demographic, clinical, and laboratory characteristics of 33 CAPD patients at study onset. Values are expressed as the mean ยก SD, unless otherwise noted.

Statistical analysis Statistical analyses were performed using Statistical Package for Social Sciences (SPSS) version 13.0 for

Variables

n = 33

Age Gender (F/M) Cause of ESRD (%) Hypertensive nephropathy Diabetic nephropathy Chronic glomerulonephritis Polycystic kidney disease Reflux nephropathy Amyloidosis Unknown Time on dialysis (months) Diabetes, n (%) Body mass index (kg/m2) Urea (mg/dL) Creatinine (mg/dL) Serum albumin (g/dL) Hemoglobin (g/dL) Hs-CRP (mg/dL)

49ยก12 13/20 8 (24%) 5 (15%) 3 (9%) 3 (9%) 2 (6%) 1 (3%) 11 (33%) 28ยก23 7(21) 28.1ยก4.9 105ยก40 6.7ยก3.5 3.8ยก0.5 11.3ยก1.9 1.06ยก1.61

SD, standard deviation; ESRD, end stage renal disease; hs-CRP, high sensitive C-reactive protein.

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Normalized urea clearance and response to hepatitis B vaccine in peritoneal dialysis Dervisoglu E et al.

DISCUSSION

Table 2 - Responses to four-dose HBV vaccination schedule measured by anti-HBs titers and clinical, laboratory, dialysis adequacy, and residual renal function results in 33 peritoneal dialysis patients. Values are expressed as the mean ¡ SD, unless otherwise noted. Variables Subjects (n) Age (years) Time on dialysis (months) Erythropoietin use, n (%) Diabetes, n (%) Body mass index (kg/m2) Urea (mg/dL) Creatinine (mg/dL) Hemoglobin (g/dL) Serum albumin (g/dL) hs-CRP (mg/dL) Weekly total Kt/V Peritoneal Kt/V RRF (mL/min) Patients with RRF, n (%) D/P creatinine nPNA, g/kg/day

Non-responders ,10 IU/L

Responders $10 IU/L

p-value

5 54¡12 18¡17 1 (20) 2 (40) 29.1¡3.2 89¡28 6.3¡5.7 10.6¡1.3 3.5¡0.7 1.8¡1.4 2.9¡1.4 1.5¡0.5 4.7¡2.9 4 (80%) 0.75¡0.18 1.1¡0.5

28 48¡12 29¡23 18 (64) 5 (18) 28.0¡5.2 108¡41 6.8¡3.1 11.4¡1.9 3.8¡0.4 0.9¡1.6 2.6¡1.1 1.7¡0.5 6.5¡4.0 23 (82%) 0.73¡0.35 1.2¡0.4

0.296 0.379 0.065 0.265 0.641 0.348 0.291 0.498 0.138 0.079 0.573 0.385 0.404 0.909 0.300 0.979

In the present study, we found an overall response rate of 84.8%, which is comparable to the rates reported in the literature for peritoneal dialysis patients (53% to 93.3%).2,10,11,17,18 Delivered clearance measured by weekly peritoneal Kt/V, total clearance measured by weekly total Kt/V, and residual GFR did not predict response to HBV vaccination in the study’s patients treated with CAPD. Regarding the effect of weekly total Kt/V on HBV vaccination response, Dacko et al. examined the influence of residual renal function and dialysis adequacy on HBV vaccination response in 32 peritoneal dialysis (PD) patients. Consistent with our findings, they found that the incident residual renal function and the incident weekly Kt/V in well-dialyzed peritoneal dialysis patients did not predict responses to the hepatitis B vaccine.10 Similarly, Chow et al. did not find a relationship between weekly total Kt/V and immune response for 52 patients with ESRD on PD.12 More recently, this same group of researchers evaluated the factors associated with hepatitis B vaccine response in 87 PD patients and found that higher nPNA, but not weekly Kt/V, was significantly associated with better vaccination response.13 In contrast to our findings, however, Svac et al. found a vaccination response rate of 78% for 40 PD patients with a weekly Kt/V greater than 1.7 (n = 28) compared to a response rate of just 8% in patients with a weekly Kt/V below 1.7 (p = 0.0003).11 The limitations of their study, however, included a lack of information on the vaccination schedule and dose; in addition, the overall seroconversion in their patients was only 53%. The proportion of patients receiving inadequate dialysis in their study (30%) was larger than in our study (12%), making comparisons difficult. Based on our data and those of other reliable studies, total clearance in peritoneal dialysis as measured via weekly total Kt/V is not predictive of responses to HBV vaccination in PD patients. In ESRD patients, the inadequate antibody response following HBV vaccination is multifactorial.1 In addition

hs-CRP, high sensitive C-reactive protein; Kt/V, clearance of urea normalized to total body water; RRF, residual renal function (patients with RRF had a daily diuresis over 200 mL); D/P, dialysate/plasma; nPNA, normalized protein equivalent of nitrogen appearance.

responders and good responders (Table 3). A statistically significant difference was observed across non-responders, weak responders and good responders in age (54¡12 vs. 56¡9 vs. 45¡12 years, respectively; p = 0.049) and rHuEpo use (20 vs. 29 vs. 76%, respectively; p = 0.016). There were no statistically significant differences in weekly total Kt/V (p = 0.704), weekly peritoneal Kt/V (p = 0.064) and the residual glomerular filtration rate (p = 0.355) across non-responders, weak responders and good responders.

Table 3 - Responses to four-dose HBV vaccination schedule measured by anti-HBs titers and clinical, laboratory, dialysis adequacy, and residual renal function values in 33 peritoneal dialysis patients. Values are expressed as the mean ¡ SD, unless otherwise noted. Statistically significant values are underlined. Variables Subjects (n) Age (years) Time on dialysis (months) Erythropoietin use, n (%) Diabetes, n (%) Body mass index (kg/m2) Urea (mg/dL) Creatinine (mg/dL) Hemoglobin (g/dL) Serum albumin (g/dL) hs-CRP (mg/dL) Weekly total Kt/V Peritoneal Kt/V RRF (mL/min) Patients with RRF, n (%) D/P creatinine nPNA, g/kg/day

Non-responders ,10 IU/L

Weak responders 10-100 IU/L

Good Responders .100 IU/L

p-value

5 54¡12 18¡17 1 (20) 2 (40) 29.1¡3.2 89¡28 6.3¡5.7 10.6¡1.3 3.5¡0.7 1.8¡1.4 2.9¡1.4 1.5¡0.5 4.7¡2.9 4 (80%) 0.75¡0.18 1.1¡0.5

7 56¡9 32¡22 2 (29) 0 (0) 27.9¡3.8 96¡42 6.9¡4.0 11.6¡1.8 3.9¡0.5 0.8¡0.7 2.8¡1.4 2.0¡0.7 8.3¡3.8 5 (71%) 0.82¡0.61 1.4¡0.6

21 45¡12 29¡24 16 (76) 5 (24) 28.0¡5.6 112¡41 6.8¡2.8 11.4¡2.1 3.8¡0.4 1.0¡1.9 2.5¡0.9 1.6¡0.4 6.0¡4.0 18 (86%) 0.70¡0.28 1.2¡0.4

0.049 0.554 0.016 0.220 0.897 0.437 0.551 0.690 0.337 0.212 0.704 0.064 0.355 0.693 0.317 0.798

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to age, many variables such as albumin levels,9 hepatitis C infection,7 and erythropoietin deficiency1 may influence the response of a patient to hepatitis B vaccine. In addition, cellmediated immunity factors such as impairment of monocyte function, reduced T cell proliferation, and decreased production of interleukin 2 are related to poor antibody response.19,20 In the present study, we found that younger PD patients had significantly better responses to HBV vaccination. Similarly, a meta-analysis of 17 clinical trials showed an increased response to hepatitis B virus vaccination among younger dialysis patients;21 these findings may be due to age-associated changes in immune status. Our study also showed that rHuEpo use in PD patients was associated with better response to hepatitis B virus vaccination, a finding that suggests an immunomodulating effect of rHuEpo.22 As found in other studies,7,10,23 the duration of dialysis therapy as well as the levels of hemoglobin and serum albumin failed to predict responses to the hepatitis B vaccine in PD patients in this study. Fernandez et al.9 and Kara et al.24, however, found that malnutrition in HD patients, which is defined by low serum albumin levels, negatively influenced responses to HBV vaccination. We could not determine this association in our study because we lacked a subgroup of malnourished patients. Only one patient in our study had antibodies to hepatitis C virus, making it difficult to determine the effects of HCV positivity on hepatitis B vaccination responses. The positive effects of dialysis adequacy, which are characterized by single-pool Kt/V, on the response to HBV in HD patients have been well documented.5 However, given the results of both the present study and previous research,10,12,13 this association may not be present in PD patients. Clearance delivered continuously, as in PD, is more efficient than the same amount of clearance delivered intermittently, as in hemodialysis; thus, the Kt/V values are not comparable.25 A limitation of our study is that the small number of patients (n = 33) resulted in a small number of patients in each vaccination response subgroup, which precluded reliable statistical analysis of any differences. In addition, the persistence of protective antibodies 12 months after completion of the fourth dose was not measured.

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3. Goodman W, Gallagher N, Sherrard D. Peritoneal dialysis fluid as a source of hepatitis antigen. Nephron. 1981;29:107-9, doi: 10.1159/ 000182324. 4. Oâ&#x20AC;&#x2122;Connor J. Priorities for immunization against hepatitis. Br Med J. 1982;284:1876, doi: 10.1136/bmj.284.6332.1876. 5. Kovacic V, Sain M, Vukman V. Efficient haemodialysis improves the response to hepatitis B virus vaccination. Intervirology. 2002;45:172-6, doi: 10.1159/000065873. 6. Recommendations for preventing transmission of infections among chronic hemodialysis patients. Centers for Disease Control and Prevention [editorial]. MMWR Recomm Rep. 2001;50:1-43. 7. Navarro JF, Teruel JL, Mateos ML, Marcen R, Ortuno J. Antibody level after hepatitis B vaccination in hemodialysis patients: influence of hepatitis C virus infection. Am J Nephrol. 1996;16:95-7, doi: 10.1159/ 000168977. 8. Kong NC, Beran J, Kee SA, Miguel JL, Sanchez C, Bayas JM, et al. A new adjuvant improves the immune response to hepatitis B vaccine in hemodialysis patients. Kidney Int. 2008;73:856-62, doi: 10.1038/sj.ki. 5002725. 9. Fernandez E, Betriu MA, Gomez R, Montoliu J. Response to the hepatitis B virus vaccine in haemodialysis patients: influence of malnutrition and its importance as a risk factor for morbidity and mortality. Nephrol Dial Transplant. 1996;11:1559-63. 10. Dacko C, Holley JL. The influence of nutritional status, dialysis adequacy, and residual renal function on the reponse to hepatitis B vaccination in peritoneal dialysis patients. Adv Perit Dial. 1996;12:315-7. 11. Svac J, Skladany L, Sekerkova Z, Javorsky P, Leskova L, Mizla P, et al. Peritoneal dialysis is the better therapy choice for successful antihepatitis B vaccination. Adv Perit Dial. 2005;21:151-3. 12. Chow KM, Law MC, Leung CB, Szeto CC, Li PK. Antibody response to hepatitis B vaccine in end-stage renal disease patients. Nephron Clin Pract. 2006;103:89-93, doi: 10.1159/000092016. 13. Chow KM, Lo SH, Szeto CC, Yuen SK, Wong KS, Kwan BC, et al. Extrahigh-dose hepatitis B vaccination does not confer longer serological protection in peritoneal dialysis patients: a randomized controlled trial. Nephrol Dial Transplant. 2010;25:2303-9, doi: 10.1093/ndt/gfq094. 14. Twardowski ZJ. Clinical value of standardized equilibration tests in CAPD patients. Blood Purif. 1989;7:95-108, doi: 10.1159/000169582. 15. Bernardini JY, Kelman B, Piraino B. Urea kinetics and nitrogen appearance rates in diabetic patients on peritoneal dialysis. Perit Dial Int. 1195;15:259-63. 16. van Olden RW, Krediet RT, Struijk DG, Arisz L. Measurement of residual renal function in patients treated with continuous ambulatory peritoneal dialysis. J Am Soc Nephrol. 1996;7:745-50. 17. Khan AN, Bernardini J, Rault RM, Piraino B. Low seroconversion with hepatitis B vaccination in peritoneal dialysis patients. Perit Dial Int. 1996;16:370-3. 18. Mitwalli A. Responsiveness to hepatitis B vaccine in immuncompromised patients by doubling the dose scheduling. Nephron. 1996;73:41720, doi: 10.1159/000189103. 19. Mauri JM, Valles M. Effects of recombinant interleukin-2 and revaccination for hepatitis B in previously vaccinated, non-responder, chronic uraemic patients. Collaborative Group of Girona. Nephrol Dial Transplant. 1997;12:729-32, doi: 10.1093/ndt/12.4.729. 20. el-Reshaid K, al-Mufti S, Johny KV, Sugathan TN. Comparison of two immunization schedules with recombinant hepatitis B vaccine and natural immunity acquired by hepatitis B infection in dialysis patients. Vaccine. 1994;12:223-34, doi: 10.1016/0264-410X(94)90198-8. 21. Fabrizi F, Martin P, Dixit V, Bunnapradist S, Dulai G. Meta-analysis: the effect of age on immunological response to hepatitis B vaccine in endstage renal disease. Aliment Pharmacol Ther. 2004;20:1053-62, doi: 10. 1111/j.1365-2036.2004.02264.x. 22. Katz O, Gil L, Lifshitz L, Prutchi-Sagiv S, Gassmann M, Mittelman M, et al. Erythropoietin enhances immune responses in mice. Eur J Immunol. 2007;37:1584-93, doi: 10.1002/eji.200637025. 23. Afsar B, Elsurer R, Eyileten T, Yilmaz MI, Caglar K. Antibody response following hepatitis B vaccination in dialysis patients: does depression and life quality matter? Vaccine. 2009;27:5865-9, doi: 10.1016/j.vaccine. 2009.07.055. 24. Kara IH, Yilmaz ME, Suner A, Kadiroglu AK, Isikoglu B. The evaluation of immune responses that occur after HBV infection and HBV vaccination in hemodialysis patients. Vaccine. 2004;22:3963-7, doi: 10. 1016/j.vaccine.2004.04.001. 25. Blake PG. Adequacy of peritoneal dialysis and chronic peritoneal dialysis prescription. In: Daugirdas JT, Blake PG, Ing TS, editors. Handbook of Dialysis. 4th ed. Philadelphia;Lippincott Williams & Wilkins; 2007.p.387-409.

CONCLUSIONS We demonstrated that delivered clearance (measured by weekly peritoneal Kt/V), total clearance (measured by weekly total Kt/V), and residual GFR did not predict responses to HBV vaccination in patients on CAPD. The influence of dialysis adequacy on responses to hepatitis B vaccination would be best studied in a larger group of PD patients with wide variations in Kt/V and residual renal function.

REFERENCES 1. Unger JK, Peters H. Hepatitis B in chronic kidney disease: moving toward effective prevention. Kidney Int. 2008;73:799-801, doi: 10.1038/ki. 2008.57. 2. Chau KF, Cheng YL, Tsang DN, Choi KS, Wong KM, Chak WL, et al. Efficacy and side effects of intradermal hepatitis B vaccination in CAPD patients: a comparison with the intramuscular vaccination. Am J Kidney Dis. 2004;43:910-7, doi: 10.1053/j.ajkd.2003.12.052.

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DOI:10.1590/S1807-59322011000900010

CLINICAL SCIENCE

A comparative study of pelvic floor muscle training in women with multiple sclerosis: its impact on lower urinary tract symptoms and quality of life Ade´lia Correia Lućio, Maria Carolina Perissinoto, Ricardo Aydar Natalin, Alessandro Prudente, Benito Pereira Damasceno, Carlos Arturo Levi D’ancona Serviço de Urologia - Hospital das Clıńicas Universidade de Campinas (Unicamp), Cidade Universita´ria Zeferino Vaz, Distrito de Baraõ Geraldo, Campinas/ SP, Brazil.

OBJECTIVE: To compare pelvic floor muscle training and a sham procedure for the treatment of lower urinary tract symptoms and quality of life in women with multiple sclerosis. METHODS: Thirty-five female patients with multiple sclerosis were randomized into two groups: a treatment group (n = 18) and a sham group (n = 17). The evaluation included use of the Overactive Bladder Questionnaire, Medical Outcomes Study Short Form 36, International Consultation on Incontinence Questionnaire Short Form, and Qualiveen questionnaire. The intervention was performed twice per week for 12 weeks in both groups. The treatment group underwent pelvic floor muscle training with assistance from a vaginal perineometer and instructions to practice the exercises daily at home. The sham group received a treatment consisting of introducing a perineometer inside the vagina with no exercises required. Pre- and post-intervention data were recorded. RESULTS: The evaluation results of the two groups were similar at baseline. At the end of the treatment, the treatment group reported fewer storage and voiding symptoms than the sham group. Furthermore, the differences found between the groups were significant improvements in the following scores in the treatment group: Overactive Bladder Questionnaire, International Consultation on Incontinence Questionnaire Short Form, and the General Quality of Life, and Specific Impact of Urinary Problems domains of the Qualiveen questionnaire. CONCLUSIONS: The improvement of lower urinary tract symptoms had a positive effect on the quality of life of women with multiple sclerosis who underwent pelvic floor muscle training, as the disease-specific of quality of life questionnaires demonstrated. This study reinforces the importance of assessing quality of life to judge the effectiveness of a treatment intervention. KEYWORDS: Vaginal perineometer; Sham treatment; Quality of life questionnaires; Pelvic floor exercises; Neurogenic bladder. Lucio AC, Perissinoto MC, Natalin RA, Prudente A, Damasceno BP, D’Ancona CAL. A comparative study of pelvic floor muscle training in women with multiple sclerosis: its impact on lower urinary tract symptoms and quality of life. Clinics. 2011;66(9):1563-1568. Received for publication on February 24, 2011; First review completed on March 20, 2011; Accepted for publication on May 24, 2011 E-mail: adeliac@fcm.unicamp.br Tel.: 55 19 33885074

Lower urinary tract symptoms (LUTS) are highly prevalent and affect approximately 50 to 90% of these patients throughout the course of the disease.2-4 Most patients report a combination of both storage and voiding symptoms5,6 caused by parasympathetic dysfunction due to brain and spinal cord damage.7 There are several treatments for this condition, such as anti-cholinergic drugs, botulinum toxin, electrical stimulation, surgical intervention, and pelvic floor muscle training (PFMT). PFMT was developed by Kegel in 1948 and was primarily used for treatment of stress urinary incontinence (SUI). Few studies have evaluated PFMT for the treatment of patients with MS.3,8 These symptoms are not life-threatening, and, thus, are often neglected by health professionals. However, bladder dysfunction is responsible for a significant negative impact on the quality of life (QoL) of affected patients.1,9

INTRODUCTION Multiple sclerosis (MS) is a chronic neurological disease involving the deterioration of the white matter pathways in the brain and spinal cord. MS is generally described as either relapsing-remitting (the most common presentation form), characterized by episodes of neurological dysfunction followed by remissions, or as primary progressive, where patients present a continuous and progressive decline in their neurological function.1,2

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fatigue, or deterioration in coordination, he or she was reevaluated by the neurologist using the EDSS. If his or her EDSS score increased by more than 0.5 relative to their initial score, he or she was removed from the study. All of the patients were blind to the randomization and were randomly allocated to one of the following two groups according to a computer-generated randomization list: treatment (GI, n = 18) or sham (GII, n = 17). The patients were evaluated before and after the intervention. All of the assessments were performed by a physiotherapist who was blinded to the patient group assignments. The patients were unaware of which group they were participating in until the end of the study. Two different informed consent procedures were prepared explaining each treatment and provided to the patients only after the randomization. Patient QoL was assessed using the Medical Outcomes Study Short Form 36 (SF-36) questionnaire,17 the International Consultation on Incontinence Questionnaire Short Form (ICIQ-SF)10 and the Qualiveen,9 a diseasespecific questionnaire. The SF-36 is a generic questionnaire with 36 questions and eight multi-item scales containing physical function (ten items), social function (two items), mental health (five items), role limitation due to physical problems (four items), role limitation due to emotional problems (three items), vitality (four items), bodily pain (two items), and general health perceptions (five items). An additional one-item measure of any self-evaluated change in health status is also available. Each domain is coded, summed, and transformed into a scale from 0 (worst) to 100 (best). The ICIQ-SF is a brief and subjective questionnaire that is used to assess the level and impact of urinary incontinence in the patients’ lives; it is comprised of three questions that target the daytime frequency, severity of incontinence, and QoL impact of the incontinence along with an eight-item scale assessing the possible causes or situations related to the urinary incontinence. The final ICIQ-SF score is the sum of the total scores, ranging from 0 to 21; the higher the score, the greater the impact on QoL. The Qualiveen is an extensive questionnaire specifically developed for use with patients with urinary dysfunction due to neurological disorders. It is divided into two major sections: the Specific Impact of Urinary Problems on Quality of Life (SIUP) and General Quality of Life (GQoL). The SIUP section is split into four domains (inconvenience, restrictions, fears, and impact on daily life), with a total of 30 questions. Each answer has five quantified items using a five-category ordinal Likert scale, with values ranging from 0 (no impact) to 4 (great negative impact). The average for each domain is calculated and used to determine the final SIUP score, which also ranges from 0 to 4, with 4 being the greatest negative impact. The GQoL section has nine questions, also using a five-category ordinal Likert scale, with values ranging from -2 to +2 (very badly to very well, respectively). The final general QoL value is calculated as the average of the nine questions, also ranging from -2 to +2. All of the questionnaires used in this study were translated into and validated in Portuguese.9,10,13,17 The questionnaires were filled out by the patients at the physiotherapist’s office, but, if necessary, the assistance of the blind (to group affiliation) physiotherapist, who conducted the evaluations, was allowed. Pelvic floor musculature was evaluated according to the PERFECT scheme18 (explained as follows) by digital

Health care professionals recognizing the impact of urinary disorders on QoL in patients with MS is essential for enabling appropriate investigations and judging the effectiveness of the treatment intervention.1,9,10 Thus, the aim of this study was to compare PFMT and a sham procedure for the treatment of LUTS and their effects on QoL. The effects of PFMT on disability were discussed in another study.11

MATERIALS AND METHODS We performed a prospective randomized controlled trial at the Neurourology Clinic of the Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil. The study was approved by the Institutional Ethics Committee (protocol number 242/2006), and all of the subjects provided informed consent. The clinical history and a neurological examination, including a Kurtzke’s Expanded Disability Status Scale (EDSS) evaluation, were assessed for each patient. The EDSS is a neurological scale that grades the level of disability in MS with a score that ranges from 0 (normal neurological findings) to 10 (death due to MS).12 A questionnaire containing items asking about the presence (’’yes’’ or ‘‘no’’) of daytime urinary frequency and urgency, urge incontinence, nocturnal enuresis, nocturia, hesitancy, a slow urine stream, and incomplete emptying was provided to the patients. The Overactive Bladder Questionnaire (OAB-V8)13 is a self-administered questionnaire designed to rate how disturbed patients are regarding four OAB symptoms: urinary frequency, urgency, nocturia, and urge incontinence. The patients respond to each item using a 6-point Likert scale ranging from 0 (not at all) to 5 (a very great deal). The subjects were considered to have a likely diagnosis of LUTS if their total score was more than eight. The OAB-V8 is a questionnaire that is commonly used to assess overactive bladder symptoms, but it is also an important tool for evaluating the patients’ self-perception of the symptoms caused by lower urinary tract dysfunction. The inclusion criteria were as follows: women with a definitive diagnosis of MS14 with a stable disease over the previous four months; a relapsing-remitting form of MS; 18 years of age or older; an EDSS score12 less than or equal to 6.5; the cognitive capacity to complete the assessment and treatment protocol; reporting lower urinary tract symptoms (nine or more positive responses on the OAB-V8 questionnaire). The exclusion criteria were as follows: pregnancy, previous vaginal prolapse surgery, stress urinary incontinence surgery, Caesarean section or vaginal delivery in the six months prior to enrollment, MS relapse during treatment (defined as any change in symptoms according to the EDSS evaluation), pelvic organ prolapse (detected during the vaginal examination) of grade II or more,15 urinary tract infection (confirmed by lab tests), and a postmenopausal status due to a reduction in muscle strength after menopause.16 Volunteers taking anti-cholinergic or other medications for the treatment of LUTS were permitted to participate if they had been taking the medication for at least three months prior to enrollment and if the dosage would not change over the duration of their participation in the study. Similarly, if any participant reported any worsening of double or blurred vision, increased muscle weakness,

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the desired sample size was determined using an SAS program (fpower). The sample size needed was calculated on the basis of the pilot study, with four patients in GI and five patients in GII. The evaluations chosen for this test were the following: pad testing, bladder diary (number of pads) and the ICIQ-SF. By setting the alpha at 5% and the power at 90%, the results of the sample size calculation showed that 10 patients were necessary for each group. To compare the baseline measurements between the two groups, a Mann-Whitney test was employed, and repeatedmeasures ANOVA was used to compare the measurements between groups. To compare proportions, we used the chisquare or Fisher’s exact test. For all of the statistical tests, the significance criterion of p,0.05 was used.

examination, which includes assessments of the following: power (P), scored from 0 (no contraction) to 5 (contraction against strong resistance) according to the modified Oxford grading system; endurance (E), noted in seconds and referring to the duration that a maximal contraction could be held; repetitions (R), recorded from muscle exhaustion and described as the number of times (at maximum 10) that contraction could be repeated without losing both power and endurance; the number of fast contractions (F) with every contraction timed (ECT). The interventions (treatment and sham) were performed by a single physiotherapist for GI and GII over a period of 12 weeks, with the participants in both groups attending twice per week for 30 minutes per session. The GI patients underwent an intervention that consisted of PFMT in the supine position with the assistance of a perineometer (Perina, Quark, Sa˜o Paulo, Brazil). The patients were instructed to practice the exercises learned during the intervention at home three times daily, without the assistance of any device and in various positions (such as sitting and standing). They were also advised to integrate the exercises into their daily activities. The regimen was evaluated weekly according to a vaginal assessment using the PERFECT scheme and by the physiotherapist responsible for the treatment; the data were recorded by the blind physiotherapist before and after the intervention. The training focused on improving pelvic floor muscle awareness and contraction strength, and the exercises were individualized according to the degree of pelvic floor weakness, the loss of proprioception and the patient’s tolerance. The GII patients received a sham procedure that solely consisted of introducing a perineometer into the vagina. The patients were asked to keep the device in place for 30 minutes, with no contraction required. No instructions regarding performing the exercises at home were given. The physiotherapist was present during all of the sham procedures. Statistical analysis was performed using the SAS (Statistical Analysis System) system for Windows. The data from nine patients were used to perform a pilot study, and

RESULTS Between July 2007 and December 2008, a total of forty-two patients consented, out of which thirty-five fulfilled the inclusion criteria. The exclusions were resulted from the following: the impossibility of attending the treatment twice per week (six patients), a relapse of MS (three patients), voluntary dropout (three patients), treatment denial (one patient), and a urinary tract infection diagnosis before (one patient) or during (one patient) the intervention. (Figure 1). The patients who were excluded from the study but wanted to continue the treatment were treated by the physiotherapy staff. The baseline demographic data and the initial assessment are shown in Table 1. No statistical differences were found between groups. The numbers of patients in each group complaining about storage and voiding symptoms before and after the intervention, based on their initial clinical history, are shown in Table 2. After the treatment, no differences in the EDSS assessment were found between the groups. In contrast, there were significant differences in the OABV8 assessment between the groups (p,0.0001) (Figure 2). In the SF-36 assessment, no differences were found between GI and GII.

Figure 1 - The excluded patients throughout the study.

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Table 1 - The mean, standard deviation (SD) and p-value of the baseline, demographic data and the initial assessments in the treatment (GI) and sham (GII) groups, as determined by the Mann-Whitney test. Data Age in years (SD); range: 20-49 Body mass index (kg/m); range: 16.6-33.3 Parity; range: 0-3 EDSS; range: 1.5 - 6.5 Duration of urinary disorders (months); range: 6- 132 Duration of MS since the onset of the disease (years); range: 3 - 20 OAB-V8; range: 10 - 40 ICIQ-SF; range: 0 - 18 Qualiveen – SIUP; range: 0.3 - 3 Qualiveen – GQoL; range: - 1.2 - 1.6 SF-36 – PF; range: 0 - 100 SF-36 – SF; range: 12.5 - 75 SF-36 – MH; range: 32 - 76 SF-36 – RP; range: 0 - 100 SF-36 – RE; range: 0 - 100 SF-36 – VT; range: 25 - 80 SF-36 – BP; range: 0 - 100 SF-36 – GH; range: 35 - 90

GII

p-value

36.0 (7.2) 23.4 (3.1) 1.3 (1.3) 3.4 (1.5) 36.5 (37.4) 9.1 (5.8) 23.8 (8.5) 11.4 (5.5) 1.7 (0.6) 0.2 (0.8) 39.2 (19.7) 46.2 (21.3) 54.8 (11.7) 38.5 (42.8) 61.5 (40.5) 56.9 (10.3) 44.4 (24.0) 61.5 (15.3)

34.7 (8.8) 23.8 (3.6) 1.1 (1.2) 3.3 (1.5) 31.5 (20.8) 6.8 (3.5) 27.1 (10.1) 11.1 (5.4) 1.8 (0.9) 0.3 (1.0) 33.6 (30.8) 41.1 (14.2) 53.4(14.5) 37.5 (40.1) 54.8 (46.4) 51.8 (13.8) 46.0 (26.1) 52.5 (12.4)

0.69 0.97 0.83 0.77 0.93 0.39 0.38 0.89 0.72 0.80 0.30 0.59 1.00 1.00 0.64 0.48 0.63 0.12

SIUP, specific impact of urinary problems on quality of life; GQoL, general quality of life; PF, physical function; SF, social function; MH, mental health; RP, role limitation due to physical problems; RE, role limitation due to emotional problems; VT, vitality; BP, bodily pain; GH, general health perceptions.

The OAB-V8 was very important for analyzing urinary symptoms in these patients with MS. This questionnaire is commonly used to assess overactive bladder symptoms, but it was observed that it is an important tool for evaluating the patients’ self-perception of LUTS. Although movement disorders, depressive moods, and fatigue affect the QoL of people with MS,19,21,22 urinary problems also have a major impact on the health-related QoL of these patients.1 The patients who underwent PFMT presented improvement in their LUTS and QoL compared with the sham group. It would seem reasonable to assume that a decrease in LUTS would increase QoL. However, a randomized controlled trial3 found encouraging results regarding the treatment of LUTS but unclear results regarding any improvement in QoL. QoL should be evaluated using generic and diseasespecific21,10 instruments. The most commonly used instrument to assess general QoL is the SF-36.5,20,22,23 In our study, this questionnaire was not adequately sensitive to detect

In the ICIQ-SF assessment, significant differences (p = 0.0003) were found between the two groups (Figure 3). In the Qualiveen questionnaire, the Specific Impact of Urinary Problems on Quality of Life (SIUP) domain showed significant differences (p = 0.0001) between the groups. In the same manner, the General QoL domain of the same questionnaire showed significant differences (p = 0.0443) between GI and GII (Figure 4). Compliance was based on the patients’ attendance at the clinic sessions, and there was no difference in compliance (p = 0.9622) between the patients in GI (mean+SD = 21.5+1.8) vs. GII (mean+SD = 21.5+1.8) across the 24 sessions.

DISCUSSION In the patients who were analyzed in this study, the impairment measured by EDSS was unchanged. Rehabilitation will not improve the neurological damage, but it will certainly reduce the disability and help patients face the disability with a better outlook.19,20 Both groups had the same visit schedule. Thus, the observed improvement was not due to the relationship between the health care professionals and their patients.

Table 2 - The number of patients complaining about storage and voiding symptoms before and after the intervention in the treatment (GI) and sham (GII) groups. GI SYMPTOMS Frequency Urgency Urge urinary incontinence Nocturnal enuresis Nocturia Hesitancy Slow stream Incomplete emptying

GII

Baseline

Final

Baseline

Final

p-value

13 13 12

4 4 4

14 14 13

14 13 13

,0.0001 0.0013 0.0013

8 12 10 8 8

2 2 3 5 3

9 12 8 6 7

10 11 9 6 7

0.0034 0.0010 0.0313 0.8163 0.2365

Figure 2 - The mean and standard deviation of the OAB-V8 before and after the intervention in treatment (GI) and sham (GII) groups. Mean: 23.84 initial and 5.92 final in GI; 27.14 initial and 28.21 final in GII (p,0.0001).

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Pelvic floor muscle training: its impact on LUTS and QoL Lucio AC et al.

CONCLUSION The improvement of LUTS had a positive effect on QoL in women with MS who underwent PFMT compared with a sham group. This study reinforces the importance of assessing QoL to judge the effectiveness of a treatment intervention. Furthermore, a disease-specific QoL tool should be used to identify the specific problems that contribute to negative impacts on QoL in patients with MS.

REFERENCES 1. Quarto G, Autorino R, Gallo A, De Sio M, D’Armiento M, Perdona S, et al. Quality of life in women with multiple sclerosis and overactive bladder syndrome. Int Urogynecol J. 2007;18:189-94, doi: 10.1007/s00192-006-0131-9. 2. Litwiller SE, Frohman EM, Zimmern PE. Multiple sclerosis and the urologist. J Urol. 1999;161:743-57, doi: 10.1016/S0022-5347(01)61760-9. 3. McClurg D, Ashe RG, Marshall K, Lowe-Strong AS. Comparison of pelvic floor muscle training electromyography biofeedback, and neuromuscular electrical stimulation for bladder dysfunction in people with multiple sclerosis: a randomized pilot study. Neurourol Urodyn. 2006;25:337-48, doi: 10.1002/nau.20209. 4. McClurg D, Ashe RG, Lowe-Strong AS. Neuromuscular electrical stimulation and the treatment of lower urinary tract dysfunction in multiple sclerosis – A double blind, placebo controlled randomised clinical trial. Neurourol Urodyn. 2008;27:231-7, doi: 10.1002/nau.20486. 5. Bonniaud V, Bryant D, Parratte B, Gallien P, Guyatt G. Qualiveen: A urinary disorder-specific instrument for use in clinical trials in multiple Sclerosis. Arch Phys Med Rehabil. 2006;87(12):1661-3, doi: 10.1016/j. apmr.2006.08.345. 6. Haslam C. Managing bladder symptoms in people with multiple sclerosis. Nurs Times. 2005;101:48-52. 7. Fingerman JS, Finkelstein LH. The overactive bladder in multiple sclerosis. JAOA. 2000;100:9-12. 8. De Ridder D, Vermeulen C, Ketelaer P, Van Poppel H, Baert L. Pelvic floor rehabilitation in multiple sclerosis. Acta Neurol Belg. 1999;99:61-4. 9. D’Ancona CA, Tamanini JT, Botega N, Lavoura N, Ferreira R, Leitaõ V, et al. Quality of life of neurogenic patients: translation and validation of the Portuguese version of Qualiveen. Int Urol Nephrol. 2009;41:29-33, doi: 10.1007/s11255-008-9402-3. 10. Tamanini JT, Dambros M, D’Ancona CA, Palma PC, Rodrigues-Netto NJr. Responsiveness to the Portuguese version of the international consultation on incontinence questionnaire – short form (ICIQ-SF) after stress urinary incontinence surgery. Int Braz J Urol. 2005;31:482-9, doi: 10.1590/S1677-55382005000500013. 11. Lućio AC, Campos RM, Perissinotto MC, Miyaoca R, Damasceno BP, D’Ancona CA. Pelvic floor muscle training in the treatment of lower urinary tract dysfunction in women with multiple sclerosis. Neurourol Urodyn. 2010;29:1410-3, doi: 10.1002/nau.20941. 12. Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology. 1983;33:1444-52. 13. Acquadro C, Kopp Z, Coyne KS, Corcos J, Tubaro A, Choo MS, et al. Translating overactive bladder questionnaires in 14 languages. Urology. 2006;67:536-40, doi: 10.1016/j.urology.2005.09.035. 14. Polman CH, Reingold SC, Edan G, Filippi M, Hartung HP, Kappos L, et al. Diagnostic criteria for multiple sclerosis: 2005 revisions to the ‘‘McDonald Criteria’’. Ann Neurol. 2005;58:840-6, doi: 10.1002/ana.20703. 15. Swift SE. The distribution of pelvic organ support in a population of female subjects seen for routine gynaecologic health care. Am J Obstet Gynecol. 2000;183:277-85, doi: 10.1067/mob.2000.107583. 16. Elliott KJ, Sale C, Cable NT. Effects of resistance training and detraining on muscle strength and blood lipid profiles in postmenopausal women. Br J Sports Med. 2002;36:340-4, doi: 10.1136/bjsm.36.5.340. 17. Ciconelli RM, Ferraz MB, Santos W, Meinaõ I, Quaresma MR. Traduçaõ para a lıńgua portuguesa e validaçaõ do questiona´rio gene´rico de avaliaçaõ de qualidade de vida SF-36 (Brasil SF-36). Rev Bras Reumatol. 1999;39:143-50. 18. Laycock J, Jerwood D. Pelvic floor muscle assessment: The PERFECT scheme. Physiotherapy. 2001;87:631-41, doi: 10.1016/S00319406(05)61108-X. 19. Attarian H. Importance of sleep in the quality of life of multiple sclerosis patients: a long under-recognized issue. Sleep Med. 2009;10:7-8, doi: 10. 1016/j.sleep.2008.02.002. 20. Patti F, Ciancio MR, Reggio E, Lopes R, Palermo F, Cacopardo M, et al. The impact of outpatient rehabilitation on quality of life in multiple sclerosis. J Neurol. 2002;249:1027-33, doi: 10.1007/s00415-002-0778-1. 21. Lobentanz IS, Asenbaum S, Vass K, Sauter C, Klosch G, Kollegger H, et al. Factor influencing quality of life in multiple sclerosis patients: disability, depressive mood, fatigue and sleep quality. Acta Neurol Scand. 2004;100:6-13, doi: 10.1111/j.1600-0404.2004.00257.x. 22. Hemmett L, Barnes M, Russell N. What drives quality of life in multiple sclerosis? QJM. 2004;97:671-6, doi: 10.1093/qjmed/hch105.

Figure 3 - The mean and standard deviation of ICIQ - SF before and after the intervention in the treatment (GI) and sham (GII) groups. Mean: 11.38 initial and 5.07 final in GI and 11.07 initial and 12.71 final in GII (p = 0.0003).

any improvement in QoL, which is predictable given that this is a general questionnaire and does not specifically measure the impact of urinary urgency, frequency, nocturia, and incontinence on QoL. Studies have shown that MS patients have a worse QoL compared with non-MS populations,24 which explains the importance of identifying specific problems that have negative impacts on QoL. The Qualiveen questionnaire showed that PFMT improves LUTS and contributes to both an improved General Quality of Life domain and a Specific Impact of Urinary Problems on Quality of Life domain. The findings of this study show that, although it is subjective, the assessment of QoL provides important additional information about the effects of the proposed treatment, the measure of the rehabilitation outcomes and the patients evaluation of their own health. This information will help health professionals to choose the best treatment to obtain the most improvement.

Figure 4 - The means of the final evaluations of the Qualiveen domains (Specific Impact of Urinary Problems on Quality of Life (SIUP) and General Quality of Life (GQoL)) before and after the intervention in the treatment (GI) and sham (GII) groups. Mean: 1.65 initial and 0.78 final in GI and 1.77 initial and 2.05 final in GII in the SIUP domain (p = 0.0001). Mean: 0.23 initial and 0.81 final in GI and 0.27 initial and 0.15 final in GII in GQoL (p = 0.0443).

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24. Nortvedt MW, Riise T, Myhr K-M, Nyland HI. Quality of life in multiple sclerosis: Measuring the disease effects more broadly. Neurology. 1999;53:1098-103.

23. Hopman WM, Coo H, Edgar CM, McBride EV, Day AG, Brunet DG. Factors associated with health-related Quality of Life in Multiple Sclerosis. Can J Neurol Sci. 2007;34:160-6.

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DOI:10.1590/S1807-59322011000900011

CLINICAL SCIENCE

End of life in the neonatal intensive care unit Helena Moura,I Vera Costa,II Manuela Rodrigues,II Filipe Almeida,III Teresa Maia,II Hercı´lia Guimara˜esII,III I

Serviço de Neonatologia do Centro Hospitalar Porto, Porto/Portugal. II Serviço de Neonatologia do Hospital Saõ Joaõ E.P.E, Porto/Portugal. de Medicina do Porto, Porto/Portugal.

III

Faculdade

PURPOSE: Death at the beginning of life is tragic but not uncommon in neonatal intensive care units. In Portugal, few studies have examined the circumstances surrounding the final moments of neonates. We evaluated the care given to neonates and their families in terminal situations and the changes that had occurred one decade later. DESIGN AND METHODS: We analyzed 256 charts in a retrospective chart review of neonatal deaths between two periods (1992-1995 and 2002-2005) in a level III neonatal intensive care unit. RESULTS: Our results show differences in the care of dying infants between the two periods. The analysis of the 2002-2005 cohort four years revealed more withholding and withdrawing of therapeutic activities and more effective pain and distress relief; however, on the final day of life, 95.7% of the infants received invasive ventilatory support, 76.3% received antibiotics, 58.1% received inotropics, and 25.8% received no opioid or sedative administration. The 2002-2005 cohort had more spiritual advisor solicitation, a higher number of relatives with permission to freely visit and more clinical meetings with neonatologists. Interventions by parents, healthcare providers and ethics committees during decision-making were not documented in any of the charts. Only eight written orders regarding therapeutic limitations and the adoption of palliative care were documented; seven (87.5%) were from the 2002-2005 cohort. Parental presence during death was more frequent in the latter four years (2002-2005 cohort), but only 21.5% of the parents wanted to be present at that moment. CONCLUSION: Despite an increase in the withholding and withdrawing of therapeutic activities and improvements in pain management and family support, many neonates still receive curative and aggressive practices at the end of life. KEYWORDS: Neonatal death; NICU; End of life; Ethics in neonatology; Intensive care. Moura H, Costa V, Rodrigues M, Almeida F, Maia T, Guimara˜es H. End of life in the neonatal intensive care unit. Clinics. 2011;66(9):1569-1572. Received for publication on February 25, 2011; First review completed on April 26, 2011; Accepted for publication on May 25, 2011 E-mail: herciliaguimaraes@gmail.com Tel.: +351 919317720

prolong pain and suffering.3-9 However, the treatment decisions for children with life-limiting conditions often involve ethical, moral, legal, and emotional conflicts between the parties involved in decision making. Neonatal physiological resilience complicates predicting survival and outcomes, and no one wants to give up fighting for a child’s opportunity to live. Caregivers and parents tend to ceaselessly extend the process of dying, with exponential costs in the form of pain, suffering, and loneliness.10,11 End-of-life care requires holistic and consistent support of the family. The knowledge and communication skills of the medical caregivers can greatly influence the ability of the parents to effectively cope with their loss around the time of death and after returning to their home.12 Understanding how these infants and families are treated in the final moments of an infant’s short life can provide accurate information that can lead to the development of an action plan for accurate policies, educational practices, and research.

INTRODUCTION Recent advances in prenatal and neonatal care have increased the survival of many infants who, in previous decades, would not have been treated because of a presumed lack of viability. A significant number of extremely preterm infants and newborns with lethal congenital malformations or lethal chromosomal anomalies still face terminal illnesses in the neonatal intensive care unit (NICU).1 More infants die in the first 28 days of life than during any other period of childhood, and neonatal palliative care is rarely offered.2 When science and technology are insufficient for the rehabilitation and cure of certain neonatal diseases, intensive treatments may be either inappropriate or inhumane. Physicians and nurses frequently are willing to withhold additional treatment and withdraw life support when the infant is severely injured or marginally viable, when death is imminent, or when further medical interventions would only

STUDY DESIGN AND METHODS In this study, we evaluated the care given to neonates and their families in terminal situations and compared the

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practical attitudes of the staff during two periods: from 1 January 1992 to 31 December 1995 and, one decade later, from 1 January 2002 to 31 December 2005. The charts of the newborns who died in a level III NICU at Sa˜o Joa˜o Hospital were retrospectively reviewed. This NICU provides care to all premature neonates and newborns affected by respiratory, cardiac, neurological, metabolic, renal, and gastrointestinal, diseases as well as newborns recovering from surgery. It is a reference center for neonatal cardiology and surgery in northern Portugal. Our goal was to analyze the documented end-of-life care of neonates and their families, with a focus on five areas: 1) therapeutic activities, 2) pain and distress relief, 3) family support, 4) decision-making, and 5) parental presence during death. A simple data collection methodology was developed and piloted by the researchers, and its reliability was tested. The categories registered in the chart review included the following: the infant’s birth weight and gestational age, resuscitation in the delivery room, the cause of death, any prenatal diagnosis, the hospitalization period, any medical interventions in the final 24 hours of life, any decisions to withdraw or withhold medical interventions, any documented orders to limit medical interventions or to initiate palliative care, the use of a neonatal pain scale, opioid and sedative administration, referrals to chaplains or other spiritual advisors, psychosocial support, permission for siblings and other relatives to visit the infant, interdisciplinary meetings between parents and neonatologists, advice from an ethics committee or judicial support during decisionmaking, and parental presence during the death of the infant. Each chart was reviewed by two researchers. To identify the mode of death, the researchers used the classifications of Garros, Rosychuk and Cox13 to gather the data and allocate the neonatal deaths into three groups: cardiopulmonary resuscitation failure (CPR failure), withholding and/or withdrawal of therapy (W/W), and do-not-resuscitate (DNR). A total of 313 deaths occurred from 1 January 1992 to 31 December 1995 and from 1 January 2002 to 31 December 2005; of these, 256 (81.8%) charts were examined. Incomplete charts were excluded. The data were analyzed using Statistical Package for Social Sciences (SPSS) (version 14.0). We used descriptive statistics and a Chi square test to identify any differences between the cohorts; p,0.05 was required to achieve statistical significance.

Table 1 - Modes of death in the NICU in the two cohorts. 1992-1995 Modes of death Failed CPR Withholding and/or withdrawal of therapy Do-not-resuscitate Total

2002-2005

n (%)

Total

p-value*

94 (57.7)

41 (44.1)

135 (52.7)

0.036

54 (33.1) 15 (9.2) 163 (100)

48 (51.6) 4 (4.3) 93 (100)

102 (39.8) 19 (7.4) 256 (100)

0.044 0.147

*Chi-squared test.

only one chart, in 1994. In the eight (7.8%) charts in which the mode of death was W/W, the following was written: ‘‘comfort care only’’, ‘‘decided to reduce ventilatory measures’’, and ‘‘decided to withdraw vital support’’. In the 2002-2005 cohort, there were seven orders to limit therapeutic activities. In 92.6% of the charts, the charts from both cohorts, no specific order was found.

Pain and distress relief In the 1992-1995 cohort, the charts of 151 (9.6%) infants mentioned the use of a neonatal pain scale; 123 (75.5%) and 71 (43.6%) infants received opioids and sedatives, respectively. In the 2002-2005 cohort, the charts of 89 (95.7%) infants mentioned the use of a neonatal pain scale; 71 (76.3%) and 54 (58.1%) infants received opioids and sedatives, respectively (Table 3).

Family support In the later cohort, 28 (30.1%) charts mentioned a chaplain visiting the NICU. Permission for siblings and other family members to freely visit the infant was documented in 30 (32.3%) charts. There was no referral to psychosocial family support or support groups (Table 4). The reviewed charts focused on the neonate’s physical condition and therapeutic procedures; the emotional support provided by the caregivers (physicians and especially nurses) to the neonate, parents, and others family members during the grieving process was not recorded.

Decision-making There were no data regarding parental participation during decision-making. However, 56 (60.2%) charts in the 2002-2005 cohort mentioned that the parents had meetings with their neonatologists to discuss the clinical situation of their infants. In the first cohort, 62 (38%) parents communicated with their neonatologists (p,0.001). Regarding the involvement of other entities in the decision-making

RESULTS In both cohorts, approximately 50% of the neonates were born after 37 weeks of gestation and weighed more than 2,500 g at birth. Death occurred within the first six days of life in 59.5% of the 1992-1995 cohort and 62.5% of the 20022005 cohort.

Table 2 - Therapeutic activities in the final 24 hours in the two cohorts. 1992-1995

Therapeutic activities

Therapeutic activities in the final 24 hours

In the final four years (2002-2005), W/W was found in 51.6% of the charts, CPR failure was found in 44.1% and DNR was found in 4.3% (Table 1). In the final 24 hours of life, in both cohorts, 93.8% of the infants died with ventilatory support, 75.8% died with antibiotics, 48.8% died with inotropic support, 1.2% died with peritoneal dialysis, 25.4% died with parenteral nutrition, and 16.4% died with enteral nutrition (Table 2). DNR orders were documented in

Ventilation Antibiotics Inotropics Peritoneal dialysis Parenteral nutrition Enteral nutrition *Chi-squared test.

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n (%) 151 (92.6) 123 (75.5) 71 (43.6) 1 (0.6) 37 (22.7) 26 (16)

2002-2005

n (%)

Total

89 (95.7%) 240 (93.8%) 71 (76.3%) 194 (75.8%) 54 (58.1) 125 (48.8) 2 (2.2) 3 (1.2) 28 (30.1) 65 (25.4) 16 (17.2) 42 (16.4)

p-value* 0.331 0.563 0.056 0.311 0.256 0.549

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process, some of the reviewed charts mentioned interdisciplinary meetings to discuss better intervention plans or the solicitation of an ethics committee. An appeal to the Minor Court was found in one chart in the 1992-1995 cohort.

Table 3 - Pain and distress management. 1992-1995 n (%)

Parental presence during infant death

n (%)

Neonatal pain scale 151 (9.6) Administration of opioids 123 (75.5) Administration of sedatives 71 (43.6)

In 20 (25.1%) of the reviewed charts in the 2002-2005 cohort, one or both parents were with their infant when death occurred, as compared to 3.7% in the first period (p,0.001). Fifty-four (58.1%) parents in the 2002-2005 cohort did not want to be present during the infantâ&#x20AC;&#x2122;s death. In 19 (20.4%) charts, there were no data regarding parental communication concerning the death of their infant.

2002-2005 Total

p-value*

89 (95.7) 240 (93.8) ,0.001 71 (76.3) 194 (75.8) ,0.001 54 (58.1) 125 (48.8) 0.007

*Chi-squared test.

aggressive procedures until death. A new attitude toward the concept of total pain as a life-limiting condition in the NICU is needed. Appropriate environmental, behavioral, and pharmacological approaches to prevent, reduce, or eliminate the pain and discomfort of neonates at the end of life should be provided. Educational programs should be promoted to improve caregiversâ&#x20AC;&#x2122; pain and symptom assessment and management skills. Institutional policies and NICU guidelines for the management of neonatal pain under lifelimiting conditions should also be implemented. Providing compassionate and family-centered end-of-life care to infants and their families in the NICU should be a mandatory component of optimal neonatal palliative care.12 Honest and complete information, adequate communication, careful coordination, emotional expression and support by the staff, preservation of the integrity of the parent-child relationship and support of religious faith are important priorities that have been identified by grieving parents.17 When a child dies, the family needs intense and long-term psychosocial support as well as cultural and spiritual comfort. Nevertheless, no charts documented any emotional and social support given to families or referrals to grief counseling groups.14 A neonatal death in the NICU results in a great deal of spiritual distress and can initiate a serious crisis of meaning and connection, which highlights the need for religious and spiritual support to families and caregivers.18 Our data support the findings of Robinson et al.19, who stated that parents draw on and rely on their spirituality to guide them during end-of-life decisionmaking, to find meaning in their loss, and to sustain them emotionally. One decade later, we observed a major increase in the number of chaplain interventions in response to familiesâ&#x20AC;&#x2122; spiritual needs, which suggests more acceptance and integration of spiritual issues during end-of-life care in the NICU and emphasizes the significance of having access to a clergyperson as an important part of good care. Facilitating sibling visits or asking parents for other relatives to join them in the NICU is an opportunity to evaluate family dynamics, integrate the family into the dying process. In the final four years of the study, we observed a

DISCUSSION In this study, we found that more than half of the newborns died within the first six days of life and that neonatal palliative care was rarely offered in the NICU. However, there appears to be a strong tendency to not hasten the dying process of a child facing imminent death by withholding and withdrawing medical interventions, which causes more harm than benefits to the neonate and family. This tendency is consistent with published European studies, in our opinion.3-9 Additionally, in 92.6% of the charts, no clear written orders to discontinue life support or withhold resuscitation were found, which has also been previously reported.13,14 Nevertheless, even as caregivers have more regularly given life-limiting therapeutic orders in the 2002-2005 cohort, it remains crucial to have explicit records and written institutional policies permitting the forgoing of life-supporting treatment of neonates in terminal situations. In this context, the American Academy of Pediatrics15 has stated that when a life-sustaining medical treatment is to be forgone, the attending physician should write an order in the medical record, and the chart notes must include the following information: the diagnosis, the prognosis, or other decisions, the contents of any meetings with the involved parties, any disagreements or unresolved issues, and the recommendations of the treatment team and any consultants. Despite recent advances in neurobiology indicating that fetuses and newborns are able to experience acute, persistent or chronic pain and express abnormal biochemistry as well as physical and behavioral changes during pain, neonates in the NICU remain vulnerable to many invasive therapeutic procedures without adequate pain relief.16 Although we observed the development of a neonatal pain scale assessment tool and a significant increase in the administration of pain and discomfort medication one decade later, 25.8% of the infants under life-limiting conditions did not receive any sedatives or opioids during their dying processes (Table 3). Today, sedation and analgesia are widely used in neonatal procedures and in situations that require comfort or palliative care.26 Additionally, 95.7% of the infants remained connected to a ventilator until death, 76.3% received antibiotics, and 58.1% received inotropics. These procedures involve repetitive secretion aspiration, tracheal intubation or frequent and potentially painful intravenous punctures. These results show that our NICU urgently needs to develop neonatal palliative care, adopt guidelines and maintain regular staff education to improve the end-of-life care of newborns. Wolfe et al.11 and McCallum et al.10 reported the introduction and persistence of submitting children to

Table 4 - Family support. 1992- 1995

Chaplain/spiritual counselor Psychosocial support Visitation by siblings or other family members *Chi-squared test.

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2002-2005

n (%)

Total

p-value*

16 (9.8) 0 (0.0)

28 (30.1) 0 (0.0)

44 (17.2) 0.0 (0.0)

,0.001

19 (11.7)

30 (32.3)

49 (19.1)

,0.001

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2. Moro T, Kavanaugh K, Okuno-Jones S, Vankleef JA. Neonatal End-ofLife Care: A Review of the Research Literature. J Perinat Neonatal Nurs. 2006;20:262-73. 3. Cuttini M, Nadai M, KaminsKi M, Hansen G, de Leeuw R, Lenoir S, et al. Endof- Life decisions in neonatal intensive care: physicians’self-reported practices in seven European countries. Lancet. 2000;335:2112-8, doi: 10. 1016/S0140-6736(00)02378-3. 4. McHaffie HE, Cuttini M, Brolz-Voit G, Randag L, Mousty R, Duguet AM, et al. Withholding/withdrawing treatment from neonates: legislation and official guidelines across Europe. J Med Ethics. 1999;24:440-6, doi: 10. 1136/jme.25.6.440. 5. Wall SN, Partridge JC. Death in the Intensive Care Nursery: Physician Practice of Withdrawing and Withholding Life Support. Pediatrics. 1997;99:64-70, doi: 10.1542/peds.99.1.64. 6. Roy R, Aladangady N, Costeloe K, Larcher V. Decision making and modes of death in a tertiary neonatal unit. Arch Dis Child Fetal Neonatal Ed. 2004;89:F527-F530. 7. Singh J, Lantos J, Meadow W. End-of-Life After Birth: Death and Dying in a Neonatal Intensive Care Unit. Pediatrics. 2004;114:1620-6, doi: 10. 1542/peds.2004-0447. 8. Grupo de Trabajo de la Sociedad Espan˜ ola de Neonatologıá sobre Limitacio´ n del Esfuerzo Terapeútico y Cuidados Paliativos en recień nacidos. Decisiones de limitacio´ n del esfuerzo terapeûtico en recieńnascidos crı´ticos: studio multiceńtrico. An Esp Pediatr. 2002;57: 547-53. 9. Barton L, Hodgman JE. The contribution of Withholding or withdrawing care to newborn mortality. Pediatrics. 2005;116:1487-91, doi: 10.1542/ peds.2005-0392. 10. McCallum DE, Byrne P, Bruera E. How children die in hospital. J Pain Symptom Manage. 2000;20:417-23, doi: 10.1016/S0885-3924(00)00212-8. 11. Wolfe J, Grier HE, Klar N, Levin SB, Ellenbogen JM, Salem-Schatz S, et al. Symptoms and Suffering at the End of Life in Children with Cancer. N Engl J Med. 2000;342:326-33, doi: 10.1056/NEJM200002033420506. 12. Harris L, Douma C. End-of-life Care in the NICU: A Family-centered Approach. NeoReviews. 2010;11:e194-9, doi: 10.1542/neo.11-4-e194. 13. Garros D, Rosychuk RJ, Cox PN. Circunstances Surrounding End of Life in a Pediatric Intensive Care Unit. Pediatrics. 2003;112: 371-80, doi: 10. 1542/peds.112.5.e371. 14. Abe N, Catlin A, Mihara D. End-of-life in the NICU. A Study of Ventilator Withdraw. Am J Matern Child Nurs. 2001;26:141-6, doi: 10. 1097/00005721-200105000-00010. 15. American Academy of Pediatrics. Committee on Bioethics.Guidelines on Forgoing Life-Sustaining Medical Treatment. Pediatrics. 1994;93:532-7. 16. Simons SHP, Van Dijk M, Anand KS, Roofthooft D, Van Lingen RA, Tibboel D. Do we Still Hurt Newborn Babies?. Arch Pediatr & Adolesc Med. 2003;157:1058-64, doi: 10.1001/archpedi.157.11.1058. 17. Meyer EC, Ritholz MD, Burns JP, Truog RD. Improving the Quality of Endof- Life Care in the Pediatric Intensive Care Unit: Parents Priorities and Recommendations. Pediatrics. 2006;117:649-57, doi: 10.1542/peds.2005-0144. 18. Catlin EA, Guillemin JH, Thiel MM, Hammond S, Wang ML, ODonnell J. Spiritual and Religious Components of Patient Care in the Neonatal Intensive Care Unit: Sacred Themes in a Secular Setting. Journal of Perinatology. 2001;21:426-30. 19. Robinson MR, Thiel MM, Backus MM, Meyer EC. Matters of Spirituality at the end of life in the pediatric intensive care unit. Pediatrics. 2006;118: e719-e29, doi: 10.1542/peds.2005-2298. 20. Rushton C, Catlin A. Pediatric Palliative Care: The Time is Now. Pediatric Nursing. 2002;28:57-60. 21. McHaffie HE, Lyon AJ, Fowlie PW. Lingering death after treatment withdrawal in the neonatal intensive care unit. Arch Dis Child Fetal Neonatal Ed. 2001;85:F8-F12. 22. Contro N, Larson J, Scofield S, Sourkes B, Cohen H. Family Perspectives on the Quality of Pediatric Palliative Care. Arch Pediatr Adolesc Med. 2002;156:14-19. 23. Cook LA, Watchko JF. Decisions making for the critically ill neonate near the end of life. Journal of Perinatology. 1996;16:133-6. 24. Meert KL, Thurston CS, Briller SH. The spiritual needs of parents at the time of their child’s death in the pediatric intensive care unit and during bereavement: a qualitative study. Pediatr Crit care Med. 2005;6:420-7, doi: 10.1097/01.PCC.0000163679.87749.CA. 25. Meert KL, Thurston CS, Sarnaik AP. End-of-life decision-making and satisfaction with care: parental perspectives. Pediatr Crit Care Med. 2000;1:179-185, doi: 10.1097/00130478-200010000-00017. 26. Catlin A, Carter B. Creation of a Neonatal End-of-Life Palliative Care Protocol. Neonatal Network. 2002;21:37-49.

higher frequency of visits by siblings and other family members. However, no charts documented how the siblings and other relatives coped with the death, although sibling support is an important component of compassionate and holistic neonatal palliative care.20 Access to honest and complete information, appropriate communication, and coordination of care are important priorities that have been identified by parents to achieve quality care under end-of-life conditions.17,21,22 One decade later, our data revealed a positive evolution in the communication between parents and their neonatologists. Although this issue is critically significant in defining what is in the child’s best interest, no charts documented parental involvement in decision-making. Additionally, we found no information concerning interdisciplinary meetings, although it has been found that the parents are critically important to the proper management of the illness.20 The opinion of an ethics committee was not sought in any of the charts in our study, consistent with a study by Cook and Watchko.23 Their physical presence during infant death is a spiritual need felt by grieving parents.24 Our data showed that in the final four years (more than in the previous decade), 21.5% of the parents were present at the moment of death, which suggests better support and preparation for infant death. Meert, Thurston and Sarnaik showed that more than 50% of parents studied stayed with their child in the PICU at the time of death. Although none regretted being present, 63% of the parents who were not present later wished that they had been present.25

CONCLUSION Despite an increase in the withholding of therapy and reduction in therapeutic approaches, and improvements in pain management and family support, a large number of neonates in intensive care units still receive curative and aggressive treatments at the end of life. Additionally, psychosocial support for families and bereavement followup care, such as interdisciplinary meetings for discussing the best intervention plan to follow, are woefully lacking. A holistic and specialized team approach to care for newborns with life-limiting diseases is suggested to improve the conditions of the infants and families during the dying process. Caregivers should be trained in the basic principles of pediatric palliative care and should be prepared with the skills, confidence and expertise to provide consistent and high-quality end-of-life care. This research concerns only what was documented in neonatal charts; therefore, we cannot know if additional measures were taken during the actual care of the infants and their families. However, it is crucial that consistent and complete documentation be taken during end-of-life care. Research into the care of infants who do not benefit from intensive and life-extending support is needed to develop action plans that ensure that every child receives the best possible care, regardless of the outcome.

REFERENCES 1. Instituto Nacional de Estatı´stica (Portugal). Mortalidade Neonatal 2009. 2010.

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DOI:10.1590/S1807-59322011000900012

CLINICAL SCIENCE

Is allergic rhinitis a trivial disease? Dirceu Sole´,I Ineˆs Cristina Camelo-Nunes,I Gustavo F. Wandalsen,I Nelson A. Rosa´rio,II Emanuel C. Sarinho,III Brazilian ISAAC Group I

Division of Allergy, Clinical Immunology and Rheumatology, Department of Pediatrics, Federal University of Sa˜o Paulo (UNIFESP), Sa˜o Paulo/SP, Brazil. Department of Pediatrics, Federal University of Parana´, Parana´, Brazil. III Department of Pediatrics, Federal University of Pernambuco, Recife/PE, Brazil.

BACKGROUND: Asthma and rhinitis often coexist, which potentially increases the disease severity and can negatively impact a patients’ quality of life. However, there are few reports based on data obtained from the International Study of Asthma and Allergies in Childhood examining asthma severity in combination with rhinitisrelated symptoms. OBJECTIVE: To demonstrate whether current rhinitis and current rhinoconjunctivitis are associated with the development of asthma or its increasing severity in Brazilian adolescents. METHODS: The prevalence of current asthma was correlated with the prevalence of current rhinitis and current rhinoconjunctivitis in adolescents (13 to 14 year olds) from 16 Brazilian centers (based on Spearman’s rank correlation index). The influence of current rhinitis and current rhinoconjunctivitis on asthma presentation was also evaluated using the chi-squared test and was expressed as odds ratios with 95% confidence intervals (95%CI). RESULTS: A significant positive correlation was observed between the prevalence of current asthma and current rhinitis (rs = 0.82; 95%CI: 0.60–0.93, p,0.0001) and between the prevalence of current asthma and current rhinoconjunctivitis (rs = 0.75; 95%CI: 0.47–0.89, p,0.0001). Current rhinitis was associated with a significantly increased risk of current asthma and of more severe asthma. Similar results were observed for current rhinoconjunctivitis. CONCLUSION: In this epidemiologic study of Brazilian adolescents, the presence of current rhinitis and current rhinoconjunctivitis was associated with a high risk of developing asthma and increased asthma severity. The mutual evaluation of rhinitis and asthma is necessary to establish an adequate treatment plan. KEYWORDS: Epidemiology; Asthma; Rhinitis; Rhinoconjunctivitis; ISAAC. Sole´ D, Camelo-Nunes IC, Wandalsen GF, Rosa´rio NA, Sarinho EC, Brazilian ISAAC’s Group. Is allergic rhinitis a trivial disease? Clinics. 2011;66(9):15731577. Received for publication on February 11, 2011; First review completed on April 19, 2011; Accepted for publication on May 30, 2011 E-mail: dirceusole.dped@epm.br / sole.dirceu@gmail.com Tel.: 55 11 5579 1590

The prevalence of rhinitis and related symptoms found by the International Study of Asthma and Allergies in Childhood (ISAAC) in Phases One (Ph1) and Three (Ph3) was quite high and variable around the world, with a sustained high trend.7,8 Asthma and rhinitis often coexist, which potentially increases the disease severity and negatively impacts the quality of life.9 AR is considered a common condition with a high morbidity, and it is associated with a reduced quality of life due to its coexistence with other diseases, such as chronic sinusitis and asthma.2,3 AR often precedes the onset of clinical asthma and has been identified as a risk factor for the development of asthma in children10-14 and adults.13,15-17 Several studies have reported a prevalence of AR in asthma patients of 80 to 90%.1-3,10-12 The concomitance of AR and asthma has been associated with an increase in the healthcare costs associated with asthma14,18-22 and an impairment in the quality of life.9,19,23 A retrospective study of asthmatic patients, aged 16 to 55 years, showed that patients with asthma alone had significantly fewer asthma-related visits to general practitioners, lower asthma-related drug costs, and

INTRODUCTION In the last decade, allergic rhinitis (AR) has become prominent among allergic diseases due to its prevalence, negative impact on quality of life, and associated comorbidities.1,2 Several epidemiological, etiological, anatomical, and therapeutic similarities between asthma and rhinitis have been reported.1-3 It has been hypothesized that both asthma and AR are manifestations of a single inflammatory process present throughout the airway and that they represent a continuum of disease.3-5 Brown et al. examined bronchial biopsies from adults with AR and observed an intermediate state of airway inflammation between that seen in healthy individuals and subjects with clinical asthma.6

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Information regarding the number of schools and students in each area was obtained from the appropriate Municipal Education Secretary’s official records. The data obtained were transcribed to a database (Epi-Info) supplied by ISAAC’s coordinators. The frequency of affirmative answers to specific questions was analyzed. ADs were identified as having current asthma if they answered ‘‘yes’’ to the question ‘‘Have you had a wheezing episode in the last 12 months?’’; as having current rhinitis if they answered ‘‘yes’’ to the question ‘‘Have you had nasal problems (sneezing; runny or blocked nose) in the last 12 months without a cold?’’; and as having current rhinoconjunctivitis if they answered ‘‘yes’’ to the question ‘‘Have you had nasal problems (sneezing; runny or blocked nose) with itchy and watery eyes in the last 12 months?’’.29 ADs were identified as having severe asthma if they answered ‘‘yes’’ to the question ‘‘Have you had wheezing severe enough to limit speech in the last 12 months?’’ or at least two of the following questions: ‘‘Have you had more than 12 wheezing episodes in the last 12 months?’’, ‘‘Have you had wheezing with exercise?’’, and ‘‘Have you had nocturnal coughing without a cold?’’ (atypical form of asthma). Asthma diagnosed by a physician was considered a medical diagnosis.30 To analyze the correlation between the prevalence of current rhinitis and current rhinoconjunctivitis with current asthma, Spearman’s rank correlation coefficient was used. The influence of current rhinitis and current rhinoconjunctivitis on asthma presentation was analyzed using the chisquare test and is expressed as the odds ratio (OR) with 95% confidence intervals (95%CI). The study was approved by all local ethics committees. In all tests, the level of rejection of the null hypothesis was 5%.

fewer hospitalizations due to asthma than patients with rhinitis associated with asthma.24,25 Similar results have been observed in asthmatic children.26 In a previous study of 6,520 children and adolescents enrolled in ISAAC Ph1, we evaluated the effects of rhinitis alone and rhinitis associated with atopic eczema on asthma severity in patients identified as asthmatics (23.2%). A higher prevalence of asthma and severe asthma was observed in children with rhinitis and/or atopic eczema.27 To date, few studies using the data from the ISAAC have examined asthma severity in combination with rhinitisrelated symptoms. Thus, the aim of this study was to determine whether current rhinitis or current rhinoconjunctivitis are risk factors for the development of asthma or increased asthma severity in Brazilian adolescents.28

METHODS The data presented in this epidemiological study were previously published and came from 16 centers in 14 Brazilian cities.28,29 Adolescents (ADs, 13 to 14 years old) were selected following the ISAAC Ph3 protocol.30 The cities, states, and regions/areas in which the study took place were Manaus (Amazonas, Northern [N]); Caruaru (Pernambuco [PE], Northeastern [NE]); Aracaju (Sergipe, NE); Feira de Santana (Bahia [BA], NE); Salvador (BA, NE); Vito´ria da Conquista (BA, NE); Brası´lia (Distrito Federal, MiddleWestern); Nova Iguaçu (Rio de Janeiro, Southeastern [SE]); Saõ Paulo (West and South, Saõ Paulo [SP], SE); Santo Andre´ (SP, SE); Curitiba (Parana´, Southern [S]); Itajaı´ (Santa Catarina, S); Porto Alegre (Rio Grande do Sul [RS], S) and Santa Maria (RS, S). The data from all centers were approved by the ISAAC International Data Center and were considered ISAAC’s official centers (Table 1). ISAAC’s written questionnaire (WQ), previously translated and validated for the Brazilian culture,31-33 was completed by 46,770 ADs. The participants were selected from adolescents who attended public and private schools located in the participating cities. Only the asthma and rhinitis core questionnaires were considered in this study.

RESULTS The prevalence of current asthma, current rhinitis, and current rhinoconjunctivitis was lower in Nova Iguac¸u and higher in Salvador and Vito´ria da Conquista (Table 1). A significant positive correlation was observed between the

Table 1 - Prevalence of current asthma, current rhinitis, and current rhinoconjunctivitis in Brazilian adolescents from ISAAC Phase Three centers, based on responses to the ISAAC written questionnaire (determined with Spearman’s correlation coefficient). Center Manaus Caruaru Aracaju Feira de Santana Salvador Vito´ria da Conquista Brası´lia Nova Iguaçu Saõ Paulo West Saõ Paulo South Santo Andre´ Curitiba Itajaı´ Porto Alegre Santa Maria – rural Santa Maria – urban

Current asthma (%)

Current rhinitis (%)

Current rhinoconjunctivitis (%)

3,009 3,026 3,041 1,732 3,020 1,679 3,009 3,185 3,181 3,161 3,232 3,628 2,737 3,007 3,057 3,066

18.1 17.9 18.7 21.5 24.6 30.5 19.7 11.8 21.9 18.7 23.2 18.9 12.3 18.2 15.3 16.7

23.0 25.5 25.6 33.0 44.2 39.8 29.3 17.4 30.1 27.4 28.4 39.2 22.1 32.1 20.6 24.3

12.8 15.4 17.4 17.2 24.4 24.4 15.4 8.9 19.8 12.2 13.8 17.2 12.9 15.9 9.6 11.4

Spearman’s correlation coefficient: Current asthma VS. current rhinitis: rs = 0.82 (95%CI: 0.60–0.93), p,0.0001. Current asthma vs. current rhinoconjunctivitis: rs = 0.75 (95%CI: 0.47–0.89), p,0.0001.

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rhinoconjunctivitis in all participating centers, with OR ranging from 2.77 (Vito´ria da Conquista) to 4.38 (Itajaı´) (Table 3). Nocturnal coughing in the last year was significantly associated with current rhinoconjunctivitis in all centers, with OR ranging from 2.79 (Curitiba) to 5.45 (Nova Iguac¸u) (Table 3). Physician-diagnosed asthma was significantly associated with current rhinoconjunctivitis in all centers, with OR ranging from 2.02 (Manaus) to 4.20 (Nova Iguac¸u) (Table 3).

prevalence of current asthma and current rhinitis and between the prevalence of current asthma and current rhinoconjunctivitis (Table 1). Current rhinitis was associated with a significantly increased risk (odds ratio [OR]) of current asthma in all participating centers, with OR ranging from 2.19 (Curitiba) to 4.36 (Nova Iguaçu) (Table 2). Reports of current rhinitis were also associated with a significant risk of having 12 or more episodes of acute asthma in nine of 16 centers, with OR ranging from 0.68 (Salvador) to 8.03 (Saõ Paulo West) (Table 2). Sleep disturbance was significantly associated with current rhinitis in 13 of 16 centers, with OR ranging from 0.92 (Itajaı´) to 4.45 (Nova Iguaçu) (Table 2). Similar results were found for the risk of speech difficulty due to an acute asthma attack, which was observed in nine of 16 centers and ranged from 0.72 (Itajaı´) to 5.18 (Nova Iguaçu) (Table 2). The risk of wheezing with exercise in the last year was significantly associated with current rhinitis in all evaluated centers and ranged from 2.38 (Aracaju) to 3.25 (Salvador) (Table 2). The risk of nocturnal cough in the last year was significantly associated with current rhinitis in all centers and ranged from 2.99 (Curitiba) to 4.50 (Nova Iguaçu) (Table 2). The risk of physician-diagnosed asthma was significantly associated with current rhinitis in all centers and ranged from 1.82 (Manaus) to 3.44 (Vito´ria da Conquista) (Table 2). Current rhinoconjunctivitis was associated with a significantly increased risk of current asthma in all participating centers that ranged from 2.73 (Curitiba) to 6.04 (Nova Iguaçu) (Table 3). It was also associated with a significant risk of having 12 or more episodes of acute asthma in 10 of 16 participating centers that ranged from 1.31 (Porto Alegre) to 8.85 (Saõ Paulo West) (Table 3). Sleep disturbance was significantly associated with current rhinoconjunctivitis in 13 of 16 participating centers, with OR ranging from 1.27 (Itajaı´) to 5.41 (Nova Iguaçu) (Table 3). A similar result was observed in 13 of 16 centers for speech difficulty due to an acute asthma attack, with OR ranging from 1.23 (Itajaı´) to 7.18 (Nova Iguaçu) (Table 3). Wheezing caused by exercise in the last year was also significantly associated with current

DISCUSSION In this multicenter study, we evaluated the majority of the Brazilian ADs (46,770 or 80.4%) enrolled in ISAAC Ph3, representing the different regions of Brazil. The mean index of return of the completed WQ was high (approximately 93%).30 Our study has some limitations arising from its ecological nature. Ecological studies are inherently limited because their analyses are based on a general population rather than individuals. No individual information is available regarding confounding factors that might explain the associations between the studied variable and the outcome. Therefore, such potential confounding factors can be neither examined nor controlled for in the analysis. Nevertheless, such studies can provide useful information about the potential impact of a disease or drug on a population. Another point to consider is the validity of self-reported information (such as the responses to the ISAAC WQ) in studies using questionnaires; the possibility of inaccuracy and bias must be considered.31 However, the ISAAC has strengths that improve its reliability; these strengths include its sample size, comprehensiveness, and high response rates; its inclusion of hitherto unstudied populations; and its use of an identical, standardized, simple and validated questionnaire based on asthma and rhinitis symptoms.31 The prevalence of current asthma is assumed to be high in patients with current AR.1-3 In this study, we observed a mean prevalence of current asthma in AD with current rhinitis of 33.0% and a mean prevalence of current asthma in AD with current rhinoconjunctivitis of 40.9%. Differences in

Table 2 - Odds ratio (OR) and 95% confidence interval (95% CI) for wheezing-related symptoms in Brazilian adolescents with current rhinitis from ISAAC Phase Three centers.

Center Manaus Caruaru Aracaju Feira Santana Salvador Vito´ria Conquista Brası´lia Nova Iguaçu Saõ Paulo West Saõ Paulo South Santo Andre´ Curitiba Itajaı´ Porto Alegre Santa Maria Santa Maria - rural

.12 wheezing Wheezing last episodes last year year OR (95%CI) OR (95%CI) 4.18 (3.43–5.09)* 3.30 (2.72–4.01)* 3.03 (2.50–3.70)* 3.67 (2.90–4.66)* 3.80 (3.18–4.53)* 3.47 (2.80–4.30)* 3.67 (3.05–4.43)* 4.36 (3.44–5.52)* 3.75 (3.14–4.46)* 2.91 (2.42–3.51)* 3.32 (2.80–3.94)* 2.19 (1.85–2.59)* 3.94 (3.11–4.99)* 3.65 (3.02–4.43)* 3.74 (3.03–4.62)* 3.59 (2.94–4.38)*

2.41 (1.02–5.69)* 1.06 (0.46–2.44) 1.61 (0.59–4.39) 6.93 (1.58–30.5)* 0.68 (0.33–1.44) 2.90 (0.82–10.32) 3.96 (1.12–14.1)* 2.35 (0.80–6.90) 8.03 (2.95– 21.8)* 1.69 (0.71–4.02)* 2.62 (1.08–6.40)* 1.68 (0.71–3.99) 1.83 (0.92–3.64) 1.70 (1.04–2.76)* 2.76 (1.30–5.86)* 2.07 (1.16–3.71)*

Sleep disturb. last year OR (95%CI)

Speech problems last year OR (95%CI)

1.58 (1.12–2.21)* 1.44 (1.03–2.02)* 0.99 (0.71–1.37) 1,81 (1.19–2.74)* 1.71 (1.23–2.37)* 1.71 (1.19–2.45)* 1.85 (1.33–2.57)* 4.45 (3.31–5.99)* 3.36 (2.75– 4.12)* 1.56 (1.12–2.18)* 1.61 (1.21–2.16)* 1.93 (1.41–2.66)* 0.92 (0.60–1.42) 1.93 (1.37–2.73)* 1.15 (0.79–1.66) 1.82 (1.28–2.58)*

2.37 (1.57–3.55)* 1.37 (0.89–2.11) 0.83 (0.56–1.23) 1.35 (0.81–2.24) 1.57 (1.03–2.40)* 1.75 (1.11–2.75)* 1.59 (1.01–2.52) 5.18 (3.46–7.75)* 4.27 (3.11–5.84)* 1.70 (1.02–2.85) 1.59 (1.03–2.45)* 2.24 (1.45–3.47)* 0.72 (0.39–1.35) 1.19 (0.77–1.85) 2.20 (1.35–3.59)* 1.82 (1.12–3.00)*

l *p,0.05.

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Asthma ever OR (95%CI)

Wheezing with exercise last year OR (95%CI)

Nocturnal coughing last year OR (95%CI)

1.82 (1.49–2.22)* 2.45 (2.03–2.97)* 2.56 (2.08–3.14)* 2.08 (1.53–2.82)* 2.47 (2.00–3.06)* 3.44 (2.55–4.63)* 2.42 (1.97–2.97)* 2.97 (2.22–3.98)* 2.77 (2.16–3.54)* 2.50 (1.97–3.14)* 2.28 (1.78–2.91)* 2.40 (1.91–3.02)* 3.25 (2.54–4.17)* 2.40 (2.00–2.87)* 2.61 (2.12–3.22)* 2.92 (2.30–3.71)*

2.92 (2.40–3.54)* 3.12 (2.56–3.80)* 2.38 (1.93–2.83)* 2.94 (2.40–3.65)* 3.25 (2.78–3.80)* 3.16 (2.57–3.88)* 2.77 (2.31–3.33)* 3.00 (2.40–3.70)* 3.18 (2.65–3.81)* 2.40 (1.98–2.90)* 2.53 (2.10–3.05)* 2.52 (2.13–2.98)* 3.16 (2.56–3.90)* 2.88 (2.41–3.44)* 2.78 (2.29–3.37)* 2.72 (2.23–3.33)*

3.56 (2.98–4.26)* 3.05 (2.57–3.61)* 3.07 (2.60–3.64)* 3.46 (2.80–4.28)* 3.24 (2.77–3.80)* 3.13 (2.55–3.85)* 4.25 (3.60–5.02)* 4.50 (3.70–5.49)* 3.49 (2.98–4.09)* 3.37 (2.87–3.97)* 3.30 (2.81–3.87)* 2.99 (2.59–3.44)* 3.86 (3.20–4.67)* 3.31 (2.82–3.89)* 3.83 (3.22–4.55)* 3.95 (3.29–4.74)*

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Table 3 - Odds ratio (OR) and 95% confidence interval (95% CI) for wheezing-related symptoms in Brazilian adolescents from ISAAC Phase Three centers with current rhinoconjunctivitis.

.12 wheezing Wheezing last year episodes last year OR (95%CI) OR (95%CI) 4.63 (3.68–5.83)* 3.21 (2.58–4.00)* 2.93 (2.37–3.61)* 3.73 (2.85–4.87)* 3.40 (2.84–4.07) * 3.18 (2.52–4.01) * 4.11 (3.32–5.08)* 6.04 (4.58–7.97)* 3.62 (2.99–4.37)* 4.17 (3.32–5.23)* 3.70 (3.00–4.56)* 2.73 (2.25–3.31)* 4.71 (3.62–6.12)* 4.18 (3.37–5.18)* 4.32 (3.33–5.60)* 5.26 (4.14–6.68)*

2.57 (1.15–5.76)* 1.48 (0.63–3.50) 2.15 (0.80–5.84) 2.19 (0.87–5.55) 1.33 (0.64–2.77) 4.67 (1.48–14.69)* 4.56 (1.56–13.33)* 3.68 (1.16–11.6)* 8.85 (3.59– 21.8)* 1.49 (0.61–3.61)* 3.17 (1.40–7.20)* 2.32 (1.02–5.24)* 2.37 (1.20–4.70)* 1.31 (0.82–2.10) 1.96 (0.93–4.15) 3.34 (1.88–5.94)*

Sleep disturb. last year OR (95%CI)

Speech problems last year OR (95%CI)

1.57 (1.09–2.27)* 1.38 (0.96–1.98) 1.61 (1.13–2.29)* 1.52 (0.99–2.33) 1.87 (1.39–2.52)* 2.88 (2.00–4.16)* 1.84 (1.30–2.60)* 5.41 (3.88–7.54)* 3.80 (3.07–4.68)* 2.09 (1.45–3.01)* 2.04 (1.48–2.82)* 1.74 (1.25–2.42)* 1.27 (0.81–2.01) 1.65 (1.16–2.34)* 1.43 (0.93–2.20)* 2.14 (1.45–3.17)*

3.06 (2.03–4.61)* 2.57 (1.66–3.99)* 1.33 (0.89–2.01) 1.35 (0.81–2.24) 1.65 (1.14–2.38)* 2.10 (1.38–3.22)* 2.75 (1.74–4.33)* 7.18 (4.71–10.90)* 5.47 (4.02–7.44)* 2.31 (1.38–3.89)* 2.21 (1.42–3.42)* 1.72 (1.13–2.61)* 1.23 (0.65–2.32) 1.61 (1.04–2.49)* 2.16 (1.30–3.62)* 3.03 (1.85–4.95)*

Asthma ever OR (95%CI)

Wheezing from exercise last year OR (95%CI)

Nocturnal coughing last year OR (95%CI)

2.02 (1.60–2.57)* 2.44 (1.96–3.03)* 2.50 (2.00–3.12)* 2.18 (1.54–3.08)* 2.21 (1.80–2.75)* 2.83 (2.11–3.80)* 2.60 (2.05–3.29)* 4.20 (3.00–5.80)* 2.79 (2.15–3.60)* 2.94 (2.23–3.87)* 2.88 (2.18–3.80)* 2.72 (2.13–3.48)* 3.91 (2.97–5.14)* 2.71 (2.19–3.35)* 3.18 (2.47–4.10)* 2.94 (2.20–3.97)*

3.38 (2.69–4.25)* 3.24 (2.60–4.04)* 3.24 (2.63–3.98)* 3.13 (2.42–4.04)* 2.93 (2.47–3.48)* 2.77 (2.20–3.48)* 3.30 (2.67–4.09)* 4.00 (3.00–5.25)* 3.27 (2.69–3.97)* 3.23 (2.55–4.08)* 3.21 (2.57–4.00)* 2.95 (2.44–3.57)* 4.38 (3.44–5.57)* 3.80 (3.09–4.70)* 3.72 (2.94–4.72)* 3.19 (2.47–4.11)*

4.08 (3.22–5.17)* 3.20 (2.60–3.92)* 3.98 (3.25–4.86)* 3.96 (3.05–5.13)* 3.12 (2.63–3.71)* 2.92 (2.32–3.67)* 3.77 (3.05–4.65)* 5.45 (4.15–7.17)* 3.73 (3.11–4.47)* 4.83 (3.85–6.05)* 3.93 (3.20–4.84)* 2.79 (2.34–3.33)* 3.83 (3.05–4.82)* 4.19 (3.42–5.15)* 3.93 (3.12–4.95)* 3.58 (2.80–4.60)*

p,0.05.

and 7 of 16 centers, respectively. Current rhinoconjunctivitis was a significant risk factor for speech difficulty and for sleep disturbance in 13 of 16 centers. In 12 of 16 centers, the risk was significantly higher for ADs who had both conditions. The ISAAC protocol indicates that the combination of nasal and ocular symptoms make this question more specific for the diagnosis of AR and lessen the chance of bias.36 Because asthma is a heterogeneous disease with various clinical presentations, we analyzed other indications of severe asthma, such as having had more than 12 wheezing episodes in the last 12 months. A significantly higher risk of having more than 12 wheezing episodes in the last year was associated with current rhinitis in nine of 16 centers and was associated with current rhinoconjunctivitis in ten of 16 centers. Other criteria included wheezing with exercise and coughing during the night without having a cold. Both were significantly associated with rhinitis and rhinoconjunctivitis in all centers participating in this study. Although wheezing with exercise and nocturnal cough without a cold are atypical asthma presentations, both were significantly associated with current rhinitis and current rhinoconjunctivitis. In a program for asthma and AR control, Branda˜o et al. observed that a low education level, chronic rhinitis, and more severe asthma were risk factors for hospitalizations38 and emergency room visits due to increased asthma severity.37 Treatment for rhinitis was associated with a reduction in asthma severity. In conclusion, this epidemiologic study of Brazilian adolescents revealed that both current rhinitis and current rhinoconjunctivitis were associated with a high risk of developing asthma and with more severe asthma. The evaluation of both rhinitis and asthma is necessary for the development of an adequate treatment plan.

the definition of current asthma, current rhinitis or current rhinoconjunctivitis could explain the differences between study findings. In this study, we used definitions used by ISAAC coordinators and in studies worldwide.8,9,30,35 The differences in sensitivity and specificity for diagnosing asthma and rhinitis via a written questionnaire could also have affected the results. Despite these differences, as previously reported, this study found a significant correlation between the prevalence of current asthma and current rhinitis (rs = 0.82; 95%CI: 0.60–0.93) and current asthma and current rhinoconjunctivitis (rs = 0.75; 95%CI: 0.47–0.89), demonstrating that these diseases are correlated in the studied population. The association between an asthma diagnosis and current rhinitis or current rhinoconjunctivitis ranged from 1.82 to 3.44 and from 2.02 to 4.20, respectively, which reinforces the association between asthma and AR. Rhinitis in asthmatic patients is a risk factor for severe asthma and poorly controlled asthma. In a recent prospective study, severely asthmatic patients who were followed for one year had a 12.6 times greater risk of having uncontrolled asthma, a 3.8 times greater risk of having more visits to the emergency room, a 2.9 times lower risk of a 10% improvement in airway obstruction, and a 2.9 times lower risk of a 50% reduction in the emergency room visits if they had moderate/severe allergic rhinitis.25 In a previous study, we identified rhinitis as a risk factor for severe asthma in children and adolescents enrolled in ISAAC Ph1.27 The prevalence of severe asthma was 1.6 times higher in children with asthma and rhinitis living in the southern area of the city of Sa˜o Paulo; this prevalence is lower than the prevalence in the same area in the present study. Regarding asthma severity, the ISAAC protocol defined severe asthmatics as those children who had wheezing severe enough to limit speech in the last 12 months or had awoken in the night due to wheezing in the last 12 months. 25,35 As defined using sleep disturbance, speech difficulty, or both sleep disturbance and speech difficulty, a significantly higher risk of severe asthma was observed in AD with current rhinitis in 13, 9,

APPENDIX The Brazilian ISAAC’s Group comprises: Maria Socorro Cardoso (Federal University of Amazon, Manaus); Almerinda R Silva (Federal

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Is allergic rhinitis a trivial disease? Sole´ D et al. 18. Schramm B, Ehlken B, Smala A, Quednau K, Berger K, Nowak D. Cost of illness of atopic asthma and seasonal allergic rhinitis in Germany: 1-yr retrospective study. Eur Respir J. 2003;21:116-22, doi: 10.1183/09031936. 03.00019502. 19. Halpern MT, Schmier JK, Richner R, Guo C, Togias A. Allergic rhinitis: a potential cause of increased asthma medication use, costs, and morbidity. J Asthma. 2004;41:117-26, doi: 10.1081/JAS-120026069. 20. Magnan A, Meunier JP, Saugnac C, Gasteau J, Neukirch F. Frequency and impact of allergic rhinitis in asthma patients in everyday general medical practice: a French observational cross-sectional study. Allergy. 2008;63:292-8, doi: 10.1111/j.1398-9995.2007.01584.x. 21. Kang HY, Park CS, Bang HR, Sazonov V, Kim CJ. Effect of allergic rhinitis on the use and cost of health services by children with asthma. Yonsei Med J. 2008;49:521-9, doi: 10.3349/ymj.2008.49.4.521. 22. Taegtmeyer AB, Steurer-Stey C, Spertini F, Bircher A, Helbling A, Miedinger D, et al. Allergic rhinitis in patients with asthma: the Swiss LARA (Link Allergic Rhinitis in Asthma) survey. Curr Med Res Opin. 2009;25:1073-80, doi: 10.1185/03007990902820733. 23. Magnan A, Meunier JP, Saugnac C, Gasteau J, Neukirch F. Frequency and impact of allergic rhinitis in asthma patients in everyday general medical practice: a French observational cross-sectional study. Allergy. 2008;63:292-8, doi: 10.1111/j.1398-9995.2007.01584.x. 24. Price D, Zhang Q, Kocevar VS, Yin DD, Thomas M. Effect of a concomitant diagnosis of allergic rhinitis on asthma-related health care use by adults. Clin Exp Allergy. 2005;35:282-7, doi: 10.1111/j.1365-2222.2005.02182.x. 25. Ponte EV, Franco R, Nascimento HF, Souza-Machado A, Cunha S, Barreto ML, et al. Lack of control of severe asthma is associated with coexistence of moderate-to-severe rhinitis. Allergy. 2008;63:564-9, doi: 10. 1111/j.1398-9995.2007.01624.x. 26. Thomas M, Kocevar VS, Zhang Q, Yin DD, Price D. Asthma-related health care resource use among asthmatic children with and without concomitant allergic rhinitis. Pediatrics. 2005;115:129-34. 27. Sole´ D, Camelo-Nunes IC, Wandalsen GF, Melo KC, Naspitz CK. Is rhinitis alone or associated with atopic eczema a risk factor for severe asthma in children? Pediatr Allergy Immunol. 2005;16:121–5, doi: 10. 1111/j.1399-3038.2005.00227.x. 28. Sole´ D, Camelo-Nunes IC, Wandalsen GF, Rosa´ rio Filho NA, Naspitz CK; Brazilian ISAAC’s Group. Prevalence of rhinitis among Brazilian schoolchildren: ISAAC phase 3 results. Rhinology. 2007;45:122-8. 29. Sole´ D, Camelo-Nunes IC, Wandalsen GF, Mallozi MC, Naspitz CK; Brazilian ISAAC’s Group. Is the prevalence of asthma and related symptoms among Brazilian children related to socioeconomic status? J Asthma. 2008;45:19-25, doi: 10.1080/02770900701496056. 30. Ellwood P, Asher MI, Beasley R, Clayton TO, Stewartt AW. ISAAC Steering Committee - The international study of asthma and allergies in childhood (ISAAC): phase three rationale and methods. Int J Tuberc Lung Dis. 2005;9:10-16. 31. Williams H, Stewart A, von Mutius E, Cookson D, Anderson H R, The International Study of Asthma and Allergies in Childhood (ISAAC) Phase One and Three Study Groups. Is eczema really on the increase worldwide? J Allergy Clin Immunol. 2008;121:947-54, doi: 10.1016/j.jaci.2007.11.004. 32. Sole´ D, Vanna AT, Yamada E, Rizzo MC, Naspitz CK. International Study of Asthma and Allergies in Childhood (ISAAC) written questionnaire: validation of the asthma component among Brazilian children. J Invest Allergol Clin Immunol. 1998;8:376-82. 33. Vanna AT, Yamada E, Arruda LK, Naspitz CK, Sole D. International Study of Asthma and Allergies in Childhood: validation of the rhinitis symptom questionnaire and prevalence of rhinitis in schoolchildren in Saõ Paulo, Brazil. Pediatr Allergy Immunol. 2001;12:95-101, doi: 10.1034/ j.1399-3038.2001.012002095.x. 34. Yamada E, Vanna AT, Naspitz CK, Sole´ D. International Study of Asthma and Allergies in Childhood (ISAAC): validation of the written questionnaire (eczema component) and prevalence of atopic eczema among Brazilian children. J Investig Allergol Clin Immunol. 2002;12: 34-41. 35. Asher MI, Montefort S, Bjo¨ rksteń B, Lai CK, Strachan DP, Weiland SK, et al. Worldwide time trends in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and eczema in childhood: ISAAC Phases One and Three repeat multicountry cross-sectional surveys. Lancet. 2006;368:733-43, doi: 10.1016/ S0140-6736(06)69283-0. 36. Asher MI, Keil U, Anderson HR, Beasley R, Crane J, Martinez F, et al. International Study of Asthma and Allergies in Childhood (ISAAC): rationale and methods. Eur Respir J. 1995;8:483-91, doi: 10.1183/09031936.95.08030483. 37. Brandaõ HV, Cruz CS, Pinheiro MC, Costa EA, Guimara˜es A, SouzaMachado A, et al. Risk factors for ER visits due to asthma exacerbations in patients enrolled in a program for the control of asthma and allergic rhinitis in Feira de Santana, Brazil. J Bras Pneumol. 2009;35:1168-73, doi: 10.1590/S1806-37132009001200002. 38. Brandaõ HV, Cruz CMS, Santos IS Jr, Ponte EV, Guimara˜es A, Cruz AA. Hospitalizations for asthma: impact of a program for the control of asthma and allergic rhinitis in Feira de Santana, Brazil. J Bras Pneumol. 2009;35:723-9, doi: 10.1590/S1806-37132009000800002.

University of Pernambuco, Caruaru); Jackeline Motta and Ricardo Gurgel (Federal University of Sergipe, Aracaju); Leda Solano de Freitas (Federal University of Bahia, Salvador); Wellington Borges (Hospital de Base do Distrito Federal, Brası´lia); Fa´bio Kuschnir and Antoˆnio Jose´ Ledo Alves da Cunha (Federal University of Rio de Janeiro, Nova Iguaçu); Antoˆnio C Pastorino and Cristina Miuki A Jacob (State University of Saõ Paulo, Saõ Paulo); Karyn Chacon de Mello (Federal University of Saõ Paulo, Saõ Paulo); Ca´ssia Gonzalez and Neusa F Wandalsen (ABC Foundation School of Medicine, Santo Andre´); Carlos Riedi (Federal University of Parana´, Curitiba); Claúdia Benhardt (Federal University of Santa Catarina, Itajaı´); Gilberto B Fischer (Medical Federal Foundation of Rio Grande do Sul, Porto Alegre); Vitor E. Cassol (Federal University of Rio Grande do Sul, Santa Maria, Brazil).

REFERENCES 1. Bousquet J, Van Cauwenberge P, Khaltaev N, and the Aria Workshop Group for the World Health Organization. Allergic rhinitis and its impact on asthma. J Allergy Clin Immunol. 2001;108:S147–334, doi: 10. 1067/mai.2001.118891. 2. Bousquet J, Khaltaev N, Cruz AA, Denburg J, Fokkens WJ, Togias A, et al. Allergic Rhinitis and its Impact on Asthma (ARIA) 2008 update (in collaboration with the World Health Organization, GA(2)LEN and AllerGen). Allergy. 2008;63 Suppl 86:8-160, doi: 10.1111/j.1398-9995.2007. 01620.x. 3. Cruz AA, Popov T, Pawankar R, Annesi-Maesano I, Fokkens W, Kemp J, et al. Common characteristics of upper and lower airways in rhinitis and asthma: ARIA update, in collaboration with GA(2)LEN. Allergy. 2007;62 Suppl 84:1-41, doi: 10.1111/j.1398-9995.2007.01551.x. 4. Togias A. Rhinitis and asthma: evidence for respiratory system integration. J Allergy Clin Immunol. 2003;111:1171-83, doi: 10.1067/mai.2003. 1592. 5. Braunstahl GJ. United airways concept: what does it teach us about systemic inflammation in airways disease? Proc Am Thorac Soc. 2009;6:652-4, doi: 10.1513/pats.200906-052DP. 6. Brown JL, Behndig AF, Sekerel BE, Pourazar J, Blomberg A, Kellyz FJ, et al. Lower airways inflammation in allergic rhinitics: a comparison with asthmatics and normal controls. Clin Exp Allergy.2007;37:688-95, doi: 10.1111/j.1365-2222.2007.02695.x. 7. Bjo¨rkste´n B, Clayton T, Ellwood P, Stewart A, , Strachan D; ISAAC Phase III Study Group. Worldwide time trends for symptoms of rhinitis and conjunctivitis: Phase III of the International Study of Asthma and Allergies in Childhood. Pediatr Allergy Immunol. 2008;19:110-24, doi: 10. 1111/j.1399-3038.2007.00601.x. 8. Aı¨t-Khaled N, Pearce N, Anderson HR, Ellwood P, Montefort S, Shah J, et al. Global map of the prevalence of symptoms of rhinoconjunctivitis in children: The International Study of Asthma and Allergies in Childhood (ISAAC) Phase Three. Allergy. 2009;64:123-48, doi: 10.1111/j.1398-9995. 2008.01884.x. 9. Hansen JW, Thomsen SF, Nolte H, Backer V. Rhinitis: a complication to asthma. Allergy. 2010;65:883-8, doi: 10.1111/j.1398-9995.2009.02290.x. 10. Leynaert B, Neukirch F, Demoly P, Bousquet J. Epidemiologic evidence for asthma and rhinitis comorbidity. J Allergy Clin Immunol. 2000;106:S201–5, doi: 10.1067/mai.2000.110151. 11. Peroni D, Piacentini G, Alfonsi L, Zerman L, Di Blasi P, Visona’ G, et al. Rhinitis in pre-school children: prevalence, association with allergic diseases and risk factors. Clin Exp Allergy. 2003;33:1349–54, doi: 10. 1046/j.1365-2222.2003.01766.x. 12. Hamouda S, Karila C, Connault T, Scheinmann P, de Blic J. Allergic rhinitis in children with asthma: a questionnaire-based study. Clin Exp Allergy. 2008;38:761-6, doi: 10.1111/j.1365-2222.2008.02953.x. 13. van den Nieuwenhof L, Schermer T, Bosch Y, Bousquet J, Heijdra Y, Bor H, et al. Is physician-diagnosed allergic rhinitis a risk factor for the development of asthma? Allergy. 2010;65:1049-55, doi: 10.1111/j. 1398-9995.2009.02316.x. 14. Pinto Pereira LM, Jackman J, Figaro N, Babootee N, Cudjoe G, Farrell S, et al. Health burden of co-morbid asthma and allergic rhinitis in West Indian children. Allergol Immunopathol (Madr). 2010;38:129-34, doi: 10. 1016/j.aller.2009.09.002. 15. Guerra S, Sherrill DL, Martinez FD, Barbee RA. Rhinitis as an independent risk factor for adult-onset asthma. J Allergy Clin Immunol. 2002;109:419–25, doi: 10.1067/mai.2002.121701. 16. Shaaban R, Zureik M, Soussan D, Neukirch C, Heinrich J, Sunyer J, et al. Rhinitis and onset of asthma: a longitudinal population-based study. Lancet. 2008;372:1049-57, doi: 10.1016/S0140-6736(08)61446-4. 17. Bisaccioni C, Aun MV, Cajuela E, Kalil J, Agondi RC, Giavina-Bianchi P. Comorbidities in severe asthma: frequency of rhinitis, nasal polyposis, gastroesophageal reflux disease, vocal cord dysfunction andbronchiectasis. Clinics. 2009;64:769-73, doi: 10.1590/S1807-59322009000800010.

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DOI:10.1590/S1807-59322011000900013

CLINICAL SCIENCE

OBJECTIVES: To disseminate transesophageal ultrasound-guided fine needle aspiration (EUS-FNA) as an alternative to investigate mediastinal tumoral lesions because it is an underused modality that has been available in Brazil for more than 15 years. METHODS: Descriptive analysis of a single endoscopy service’s experience since 1997 in the accomplishment of EUSFNA for mediastinal staging of previously known malignancies (Group 1) or diagnostic definition of suspect lymph nodes and masses (Group 2). RESULTS: EUS-FNA was performed in 51 patients between 26 and 87 years of age. The diameter of the lesions ranged between 1.1 and 9.8 cm (mean 3.9 cm). Their location corresponded to the following stations: higher paratracheal (4 cases), lower paratracheal (7), aortic window (12), para-aortic (6), subcarinal (9), paraesophageal (8), and hilar (5). In Group 1, 17 patients had previously diagnosed primary lung (9), breast (4), kidney (2), colon (1), and bladder (1) cancer. Fifteen of these punctures were positive for malignity. Two others were later submitted to mediastinoscopy, which identified metastases not detected by EUS-FNA. Group 2 comprised 34 patients. Among these patients, EUS-FNA diagnosed 22 neoplasms, five cases of tuberculosis and two duplication cysts. Cytology was inconclusive or without a specific diagnosis in five other cases. Mediastinoscopy identified two undiagnosed cases of oat-cell carcinoma, one lymphoma and one cryptococcosis, and confirmed one reactive lymphadenitis. There were no complications related to the method. CONCLUSIONS: EUS-FNA obviated the need for surgical procedures in 86.3% of cases. Therefore, oncologists, pulmonologists, and thoracic surgeons should always remember the technique’s potential and availability. KEYWORDS: Endoscopic ultrasound; Mediastinoscopy; Mediastinal Lymphadenopaty; Lung cancer, Staging; Mediastinal Tumor. Ardengh JC, Bammann RH, Giovani M, Venco F, Parada AA. Endoscopic ultrasound-guided biopsies for mediastinal lesions and lymph node diagnosis and staging. Clinics. 2011;66(9):1579-1583. Received for publication on March 28, 2011; First review completed on April 26, 2011; Accepted for publication on May 30, 2011 E-mail: jcelso@uol.com.br Tel.: 55 11 5055-7134

ultrasound and air.1 The first sectorial echobronchoscope was launched on the international market only in the middle of the first decade of 2000. Gastrointestinal endosonography (known as EUS), on the other hand, has been in use as a routine procedure for more than 15 years at large hospitals performing high-complexity procedures, including those in Brazil.2-4 Its diagnostic and therapeutic range has been well established for pancreatic and pelvic diseases; mediastinal lesions can also be approached through the intrathoracic esophagus.5,6 Fineneedle aspiration (FNA) of masses and lymph nodes through the esophageal wall has been performed at specialized centers, with minimal risks of infection or bleeding and without great technical difficulty.5-8 The importance and usefulness of EUS for the mediastinal staging of primary lung cancer has been well known since 1996.6 The main limitation of EUS is its inability to access the anterior mediastinum because of the interference of air present in the trachea.1,7,8

INTRODUCTION The association between endoscopic techniques and ultrasound first developed in the 1980s. The repercussions and clinical impact of this minimally invasive technological advance have been broadly highlighted in the international scientific literature and more recently expanded to pulmonology and thoracic oncology. Endobronchial ultrasound (known as EBUS) has faced greater technical bottlenecks, related to the smaller diameters of the bronchoscope, its working channel, the patients’ airways and, especially, the interface between the

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This study aims to assess the performance of EUS-guided FNA in diagnosing mediastinal tumor lesions (including lymph node enlargements) and to describe some advantages and particularities of the technique.

purposes, 23 (45.1%) happened in the final four years of the research period, and these cases were mostly referred by pulmonologists and thoracic surgeons. Endoscopic alterations (extrinsic compression) were found in 24 (47.1%) patients, three of whom already displayed esophageal stenosis. In Group 1 (with previously known malignant disease, forwarded for mediastinal staging), 17 patients were included, 9 with primary lung tumors, 4 with breast tumors, 2 with kidney tumors, 1 with a colon tumor, and 1 with a bladder tumor. Out of these 17 patients, a previous PET scan had been done in only 4, all of whom were considered ‘‘positive’’ for the suspected mediastinal lesion. The diameter of the punctured lesions ranged from 1.1 to 6.8 cm, with an average of 3.7 cm. Their location (Mountain, 1997)9 corresponded to stations 2R (2 cases), 2L (1), 4R (1), 4L (2), #5 (1), #6 (1), #7 (3), #8 (2), 10R (1), and 10L (3). EUS-FNA demonstrated metastatic involvement in 15 out of 17 (88.2%) patients in Group 1. One case was negative, and another was inconclusive—the respective lymph node stations sampled by EUS were the paraesophageal (#8) and the left hilar (10L). Both cases were later submitted to classical cervical mediastinoscopy, which identified metastases in lower paratracheal lymph nodes (#4) that were previously undetected through EUS. Group 2 (undiagnosed lymph node enlargements or mediastinal masses) comprised 34 patients. The diameter of the punctured lesions varied from 1.6 to 9.8 cm (average 4.0 cm). Their location (Mountain, 1997)9 corresponded to stations 2L (one case), 4R (3), 4L (1), #5 (11), #6 (5), #7 (6), #8 (6), and 10L (1). Among the 34 patients in Group 2, 22 (64.7%) ‘‘new’’ tumors were diagnosed through EUS-FNA, including epidermoid carcinoma (10), adenocarcinoma (5), oat-cell (3), lymphoma (2), sarcoma (1), and neuroendocrine carcinoma (1). Other diagnoses established in this group included tuberculosis (5) and duplication cyst (2). Cytology was not malignant (but without a specific diagnosis) in three cases and inconclusive in two others—these five patients were later submitted to mediastinoscopy, which identified two other cases of oat-cell carcinoma, one non-Hodgkin Bcell lymphoma, and one ganglionic cryptococcosis, in addition to confirming one case of non-specific reactive lymphadenitis. Figure 1 displays a flow chart that summarizes the procedures and diagnoses in this study. Figure 2A illustrates a clinical case from Group 1; Figure 2B illustrates a clinical case from Group 2. There were no complications related to the method.

MATERIAL AND METHODS This observational, retrospective, and cross-sectional experience analysis reports the experience of a single endosonography service linked to a private hospital in Sao Paulo City between February 1997 and January 2011. All clinical data (including copies of radiological and endosonography images) were obtained from the service’s computerized database. The demands for EUS for mediastinal assessment purposes were spontaneous because the patients’ own physicians referred them due to pathological findings on chest-Computerized tomography (CT) and, in some more recent cases, on Positron emission tomography (PET) scans. For the sake of this study, patients were classified into two groups according to the purpose of the examination: Group 1—EUS-FNA performed for mediastinal staging of previously known malignant tumors; and Group 2—EUSFNA performed for diagnostic definition of lymph nodes or suspected mediastinal masses. No technical or logistic differences occurred when the procedure was accomplished in both groups, which always followed the same service routine. All examinations took place in an outpatient setting, under general anesthesia, starting with conventional upper digestive endoscopy. Then echoendoscopy was used to identify the mediastinal lesions previously detected on radiology exams. Under a direct and real-time ultrasound view, one single lesion (the largest in cases of multiple identified lesions) was punctured with a dedicated 22-gauge endoscopic needle. Once guided into the target lesion, the needle was moved back and forth within the mass while applying suction with a 20-ml syringe. At least three needle punctures were made to obtain adequate tissue specimens. Frozen-section examination was not performed during the procedure in any of the cases. The aspirated material was fixed in formaldehyde and analyzed through the cell-block technique. In case of inconclusive cytopathology results, the patient’s physician-in-charge was asked for further information on clinical monitoring, other diagnostic methods, and the respective final diagnosis in each case. Approval for this study was obtained from the local Institutional Review Board in compliance with the National Health Council Resolution 196/96.

RESULTS

DISCUSSION

Out of 1,639 gastrointestinal endosonographies performed during the study period, 51 (3.1%) looked for mediastinal lesions. This series involved 37 (72.5%) men and 14 women between 26 and 87 years old (median 65 years). Out of these 51 patients, 23 (45.1%) manifested thoracic symptoms (dysphagia, dyspnea, thoracic pain), 22 (43.1%) reported nonspecific signs and symptoms (fever and weight loss), and 6 (11.8%) were asymptomatic. The forwarding physicians included 22 (43.1%) oncologists, 18 (35.3%) clinical pulmonologists and thoracic surgeons, and 11 (21.6%) others (general clinicians, digestive surgeons, and cardiologists). It should be highlighted that out of the 51 EUS performed for mediastinal assessment

Despite its technical and commercial availability, EUS is still rather underused in the treatment of thoracic illnesses. Aside from its well-established importance for lung cancer staging,6,10 its indication extends to other clinical situations, such as mediastinal lymph node enlargement of unknown causes or primary tumor masses and cystic lesions (for diagnostic or symptom relief purposes).11 Considering each patient’s final diagnosis as the gold standard, the general sensitivity of EUS-FNA in our study was 88.0%, with 11.7% false negative cases. These rates still apply if the sample is limited to the 17 cases in Group 1. In a recent meta-analysis6 restricted to lung cancer cases, the general EUS-FNA sensitivity was 84% for metastasis

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EUS-FNA for mediastinal diagnosis and staging Ardengh JC et al.

Figure 1 - Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) results and complementary mediastinoscopy performed in 51 patients.

- Mediastinoscopy continues to be an obligatory complementary method whenever the above techniques reveal a negative result.6,14,15 Some authors defend the position that if the main goal is the diagnostic confirmation of suspected metastatic disease detected through CT or PET scan, then endosonography methods (EUS and/or EBUS), if available, are an excellent alternative, with high sensitivity and low morbidity levels. However, if the main goal of invasive staging is to confirm the absence of mediastinal involvement, in most cases, surgical mediastinoscopy seems to be the best option.13

detection (N2 and/or N3), with a global false-negative rate of 19%. The only other Brazilian publication found that addressed this issue12 assessed 25 EUS-FNA performed for the sake of diagnostic clarification of mediastinal masses and lymph nodes. Most (48%) lesions were neoplastic, while 24% were inflammatory or infectious. Normal lymphatic tissue was obtained in three cases (12%) and, in four others (16%), insufficient material was sampled. No data are available on other complementary methods used to define the diagnosis for inconclusive cases. The comparison between different methods (EUS, EBUS, and surgical mediastinoscopy) in mediastinal staging for primary lung cancer has been a recurrent and widely discussed theme. This technical choice depends, among other factors, on the patient’s clinical condition, the degree of suspected mediastinal involvement, the location of the primary tumor, the histological type, diameter and level of the biopsied lymph nodes, the number of samples obtained and, most importantly, the availability of different methods at each institution, as well as the respective results the local team has achieved.6,13 A larger number of recent EUS have been performed at the request of chest physicians—we believe this change resulted from these specialists’ recent contact with the largescale dissemination of EBUS in the international literature, particularly regarding clinical repercussions. Both EUS and EBUS are recommended by the main thoracic oncology guidelines on the invasive mediastinal staging of primary lung cancer.6,14,15

Based on Mountain’s former lymph node map9 (which was the gold standard used during the study period), EUS can assess and obtain samples from the upper and lower paratracheal levels (stations #2 and #4), aortic window (#5), subcarinal level (#7), paraesophageal level (#8), inferior pulmonary ligament (#9), and pulmonary hilum (#10). It should be noted that EUS also permits staging (and biopsying) of primary pulmonary lesions when located near (or eventually invading) the mediastinum.19 It has also been capable of detecting (and biopsying) metastatic disease in subdiaphragmatic lesions, such as those affecting the left adrenal gland, celiac lymph nodes, and liver.6,8 The experience reported here includes a considerable number (six cases) of samples obtained from the para-aortic level (station #6), which deserves more careful and detailed analysis. Strictly speaking, station #6 corresponds to the lymph nodes anterior and lateral to the ascending aorta, between a line tangent to the superior border and another to the inferior border of the aortic arch.9 Hence, although station #6 can indeed be visualized through EUS, it is rather difficult to obtain samples through the esophageal route, as that would imply transection of the pulmonary artery or the aorta itself with the puncture needle. This location may therefore have not been very precise under the EUS view, so that some lymph nodes attributed to the para-aortic position (if not all of them) may include lesions from stations 4L, #5 and even #8. Because esophageal endosonography does not offer easy anatomical reference points, the endoscopist’s experience

Yet other facts and peculiarities should be reminded: - Any invasive sampling method is more specific than CT scan and PET scan alone.16 - The association between EUS and EBUS in the same patient reaches accuracy levels of more than 95%.17,18 These rates are quite encouraging, but combining both sets of equipment, logistics, training, and the availability of human and technical resources can hardly be justified in commercial terms.

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Figure 2 - A A PET scan shows a 2-cm hypermetabolic lymph node (station 10R) in an 81-year-old male patient who had already been submitted to a lower right lobe segmentectomy (adenocarcinoma) 18 months prior. EUS-FNA (notice the needle piercing the lesion) demonstrated metastatic disease. B A 46-year-old male presented chronic cough attributed to gastroesophageal reflux. Subsequent hoarseness led to a CT scan that showed a large mediastinal mass in the subcarinal and paraesophageal stations. EUS-FNA was effectively used to diagnose epidermoid carcinoma.

ability to avoid surgical procedures (mediastinoscopy, videothoracoscopy or even exploratory thoracotomy) in a considerable number of patients—86.3% of cases in this study.

and knowledge of regional topography are fundamental for a successful examination. Identifying and sampling lesions located at the subcarinal level (#7), for example, will hardly represent any difficulty because of its central position, which is always anterior to the middle esophagus. It is known that FNA of station #7 guided by endosonography techniques (both EUS and EBUS) does not obtain better results than a simple, ‘‘blind’’ transtracheal puncture.20 The pulmonary hilar levels (#10), on the other hand, are frequently mixed up with the inferior paratracheal stations (#4), especially on the right. Such inadequate staging can radically change therapeutic decisions—this means a possible N2 false-positive result. In lung cancer patients, if the lymph nodes of stations 10 R/L (classified as N1) are unintentionally interpreted as belonging to stations 4 R/L (classified as N2), a malignant aspirate may exclude the option for a radical surgical resection and the potential for cure.8,13 There are further issues related to the routine adopted at our service, which remains limited to the puncturing of a single suspect lesion (the largest in case of multiple identified lesions). Hence, it is recommended that, in all cases, samples be obtained from at least two lymph node zones as recently mapped by the International Association for the Study of Lung Cancer (IALSC),21 always including the subcarinal zone (station #7), to improve prognostic definitions. Equipment costs (especially disposable needles) and the learning curve for use of the technique are highlighted as the main difficulties that EBUS will still have to face before achieving greater availability and widespread use.1 International and Brazilian experiences with EUS-FNA, on the other hand, have already demonstrated the method’s

CONCLUSION EUS-FNA is an excellent alternative for mediastinal lesion diagnosis and staging. Not only endoscopists but also oncologists, pulmonologists, and thoracic surgeons should consider its reliable potential and current availability.

REFERENCES 1. Bugalho A, Doris MK, Hamacher J, Eberhardt R, Herth FJ. Ecoendoscopia broˆnquica: aspectos pra´ticos e aplicabilidade clı´nica. Rev Port Pneumol. 2008;14:55-88. 2. Ardengh JC, Paulo GA, Ferrari Jr AP. Pancreatic carcinomas smaller than 3.0 cm: endosonography (EUS) in diagnosis, staging and prediction of resectability. HPB (Oxford). 2003;5:226-30. 3. Ardengh JC, Paulo GA, Ferrari Jr AP. EUS-guided FNA in the diagnosis of pancreatic neuroendocrine tumors before surgery. Gastrointestinal Endoscopy. 2004;60:381-4. 4. Silva AF, Moura EGH, Artifon ELA, Sakai P, Maluf-Filho F, Matuguma SE, et al. Effectiveness of the echoendoscopic puncture in the diagnosis of solid pancreatic mass. ABCD Arq Bras Cir Dig. 2009;22:192-6. 5. Maluf-Filho F, Dotti CM, Farias AQ, Kupski C, Chaves DM, Artifon E, et al. I Consenso Brasileiro de Ecoendoscopia. Arq Gastroenterol. 2007;44:353-8, doi: 10.1590/S0004-28032007000400014. 6. Detterbeck FC, Jantz MA, Wallace M, Vansteenkiste J, Silvestri GA. Invasive mediastinal staging of lung cancer. ACCP evidence-based clinical practice guidelines (2nd edition). Chest. 2007;132:202S-220S, doi: 10.1378/chest.07-1362. 7. Larsen SS, Krasnik M, Vilmann P, Jacobsen GK, Pedersen JH, Faurschou P, et al. Endoscopic ultrasound guided biopsy of mediastinal lesions has a major impact on patient management. Thorax. 2002;57:98–103, doi: 10. 1136/thorax.57.2.98. 8. Annema JT, Veersteegh MI, Veselic M, Welker L, Mauad T, Sont JK, et al. Endoscopic ultrasound added to mediastinoscopy for preoperative

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11. 12. 13. 14. 15.

EUS-FNA for mediastinal diagnosis and staging Ardengh JC et al.

staging of patients with lung cancer. JAMA. 2005;294:931–6, doi: 10. 1001/jama.294.8.931. Mountain CF, Dresler CM. Regional lymph node classification for lung cancer staging. Chest. 1997;111:1718–23, doi: 10.1378/chest.111.6.1718. Cerfolio RJ, Bryant AS, Eloubeidi MA. Routine Mediastinoscopy and Esophageal Ultrasound Fine-Needle Aspiration in Patients With Non-small Cell Lung Cancer Who Are Clinically N2 Negative. Chest. 2006;130:1791-5, doi: 10.1378/chest.130.6.1791. Wildi SM, Hoda RS, Fickling W, Schmulewitz N, Varadarajulu S, Roberts SS, et al. Diagnosis of benign cysts of the mediastinum: the role and risks of EUS-FNA. Gastrointest Endosc. 2003;58:362-8. Roldan LF, Artifon ELA, Maluf-Filho F, Chaves DM, Matuguma SE, Souza TF, et al. Ecopunc¸a˜o endosco´pica no diagno´stico das massas mediastinais: ana´lise de 25 casos. GED Gastroenterol Endosc Dig. 2006;25:1-4. Toloza EM, Harpole L, Detterbeck F, McCrory DC. Invasive Staging of Non-small Cell Lung Cancer: A Review of the Current Evidence. Chest. 2003;123:157S-166S, doi: 10.1378/chest.123.1_suppl.157S. National Comprehensive Cancer Network (NCCN). Clinical Practice Guidelines in Oncology. V.2.2010. http://www.nccn.org/professionals/ physician_gls/PDF/nscl.pdf Accessed September 10, 2010. De Leyn P, Lardinois D, Van Schil PE, Rami-Porta R, Passlick B, Zielinski M, et al. ESTS guidelines for preoperative lymph node staging for non-small cell lung cancer. Eur J Cardiothorac Surg. 2007;32:1-8, doi: 10.1016/j.ejcts. 2007.01.075.

16. Yasufuku K, Nakajima T, Motoori K, Sekine Y, Shibuya K, Hiroshima K, et al. Comparison of endobronchial ultrasound, positron emission tomography and CT for lymph node staging of lung cancer. Chest. 2006;130:710-8, doi: 10.1378/chest.130.3.710. 17. Herth FJ, Lunn W, Eberhardt R, Becker HD, Ernst A. Transbronchial versus transesophageal ultrasound-guided aspiration of enlarged mediastinal lymph nodes. Am J Respir Crit Care Med. 2005;171:1164-7, doi: 10.1164/rccm.200411-1560OC. 18. Rintoul RC, Skwarski KM, Murchison JT, Wallace WA, Walker WS, Penman ID. Endobronchial and endoscopic ultrasound-guided real-time fine-needle aspiration for mediastinal staging. Eur Respir J. 2005;25:41621, doi: 10.1183/09031936.05.00095404. 19. Varadarajulu S, Schmulewitz N, Wildi SM, Roberts S, Ravenel J, Reed CE, et al. Accuracy of EUS in staging of T4 lung cancer. Gastrointest Endosc. 2004;59:345–8, doi: 10.1016/S0016-5107(03)02541-0. 20. Herth F, Becker H, Ernst A. Conventional vs Endobronchial UltrasoundGuided Transbronchial Needle Aspiration: a Randomized Trial. Chest. 2004;125:322-5, doi: 10.1378/chest.125.1.322. 21. Rusch VW, Asamura H, Watanabe H, Giroux DJ, Rami-Porta R, Goldstraw P. The IASLC Lung Cancer Staging Project – a proposal for a new International Lymph Node Map in the forthcoming seventh edition of the TNM Classification for Lung Cancer. J Thorac Oncol. 2009;4:568-77, doi: 10.1097/JTO.0b013e3181a0d82e.

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DOI:10.1590/S1807-59322011000900014

CLINICAL SCIENCE

Why are there defaulters in eye health projects? Regina Noma,I Regina de S. Carvalho,II Newton Kara-Jose´I I

Ophthalmology, Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brazil. II Pedagogue, Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brazil.

PURPOSE: To identify barriers to attendance for eye examination of schoolchildren. METHODS: Cross-sectional study. Students in grades 1-4 in elementary school in Guarulhos (Brazil) were screened and referred for ophthalmic examination in 2006. Facilities offered in this project were: examination arranged during weekends, free transportation, spectacle donation and two different opportunities for exam. A questionnaire was applied, by interview, to a sample consisted of students’ parents attended in a community project who missed the first call and attended the recall, to identify the reasons for non-attendance. RESULTS: The sample consisted of 767 parents or guardians, corresponding to an equal number of schoolchildren. Personal characteristics of the students: 49.2% male and 50.8% female, 60.2% of them had never received previous ophthalmologic evaluation. Reported reasons for no-show to the project: parents had not received appropriate orientation (35.6%), loss of working day (20.6%), illness (12.4%), had another appointment (10.0%). The need for eyeglasses was higher in the recall. CONCLUSIONS: A significant number of parents did not take their children for ophthalmological exams, even when a second opportunity was offered in projects with transportation facilities, free exams performed during weekends and spectacle donation. The main causes of absenteeism were lack of awareness and work. For 87.1% of the absenteeism cases, the difficulties could be overcome via improved structuring of the first call. A recall increases attendance coverage of target population by only 15.2% (59.3 to 74.5%). Notably, the eye exam campaign was the first exam for most of the absent students. KEYWORDS: Children; Vision screening; Acess to Care; Refractive error; Ophthalmic evaluation. Noma R, Carvalho RS, Kara-Jose´ N. Why are there defaulters in eye health projects? Clinics. 2011;66(9):1585-1589. Received for publication on April 1, 2011; First review completed on April 27, 2011; Accepted for publication June 1, 2011 E-mail: reginanoma@hotmail.com Tel.: 55 15 3212 5009

useful to the managers and those involved in the public health area, as guidance for future actions. The campaigns are also a means to educate the population to adopt preventive actions and to demonstrate the acceptance rate of the proposed treatment.5 Brazil has a extensive experience in school-based community campaigns, which have been orchestrated since 1970s.6-12 One of the problems related to these projects in several countries worldwide is the high level of absenteeism, which varies from 31.2 to 68.7%.11-17 The main reasons provided by the parents or guardians to not attend the visit are: lack of guidance (day, time and place of the exam) from the school; financial difficulty in taking transportation to the locale of the exam; distance from the locale of the visit; not having someone else to look after other children; weather changes; preference to have a visit scheduled by his/her private medical practitioner; disease; trip; forgetfulness; lack of awareness of the importance of the ophthalmologic exam or denial of the child’s low vision.7,12,13,17-20 To reduce absenteeism, some campaigns in Brazil offer facilities such as: free transportation and exam dates on the weekend, so the parents do not need to lose a work day; a second chance for an exam and the performance of the exam close to the school area where the visual screening was performed.12

INTRODUCTION The lack of optical correction is the main cause of low vision and the second cause of blindness worldwide.1 Although it is easily corrected, the optical correction problem is complex and depends on the following: suspected problem, demand, availability, access to medical assistance, acquisition and use of spectacles, and replacement in case of loss or damage.2,3 In Brazil, 78.8% of the population depends on the public health system.4 Despite the great improvement over recent years, the Public Health System (SUS) still presents a low availability of specialized services, as well as difficulties with access to optical correction and related compliance conditions.4 When the public health system is poor, the communities’ campaigns are a way to investigate a given problem’s frequency, the existing coverage and the importance and enforceability of the solution. This increase in support is

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PURPOSE To identify the reasons for non-attendance at the community projects that offer transportation facilities, meal, free ophthalmological exams performed during the weekends, a second opportunity for examination and spectacle donation.

METHODS The city of Guarulhos belongs to the metropolitan region of Sa˜o Paulo (Brazil), located 17 km from the capital and is the 12th most populated city of the country. In Guarulhos, 97.9% of the children are enrolled in schools.21 A cross-sectional, descriptive study investigated students in grades 1-4 in elementary school in Guarulhos, who were submitted to visual acuity screening by trained teachers in 2006. Children were referred to complete ophthalmologic evaluation if they presented visual acuity equal to or less than 0.7 in at least one eye or with a visual difference between the eyes of two lines or more; presence of strabismus; asthenopia; or use of spectacles.22 The exam was scheduled for the weekends (Saturday and Sunday) in the student’s own municipality. Free transportation and meals were offered. The spectacles were donated and delivered to the schools. For students who were absent at the first call, a second chance for an exam was offered, at the same facilities. A questionnaire, validated by an exploratory study and pre-tested in previous campaigns, was prepared. The following variables were studied:

Personal characteristics of the students: gender, age (in years); Ophthalmological evaluation previously received by the student (yes/no), type of health service used (public, covenant, private); Reasons for absence at the ophthalmological exam (did not receive guidelines/transmittal guide; could not miss the day of work; child’s or family’s disease; other appointment; did not have someone else to look after the other children; recent or scheduled ophthalmological exam); Need of optical correction.

The instrument was applied through an interview with the parents or guardians who took his/her children to the second-chance exam. This study was approved by the Investigational Review Board for Research Project Analysis of the Clinical Directory of Hospital das Clı´nicas and Faculdade de Medicina da Universidade de Sa˜o Paulo/SP – Study Protocol n˚ 0557/07. Informed consent was obtained from each parent or guardian who participated in the study. The parents or guardians were informed that the not answering to the questionnaire would not affect the Campaign service.

RESULTS Fifty-one thousand, five hundred and nine (51,509) students were screened, and 14.651 (28.4%) were referred for an ophthalmological exam. Among these patients, 8.683

Figure 1 - Flow chart of the Eye-to-Eye Campaign attendance – Guarulhos 2006.

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Table 1 - Coverage of ophthalmological service – Eye-to-Eye Campaign - Guarulhos, 2006 and 2007. Call Screened First call Recall TOTAL

Referred

Attended

Absenteeism %

Coverage %

Coverage increasing after recall%

14,651 5,968 14,651

51,509 8,683 2,228 10,911

40.7 62.7 25.5

59.3 15.2 74.5

15.2

number of students who attended, the first call was, approximately, four times more efficient than the recall, although the cost for staging it was similar. The main reason for non-attendance was lack of awareness or failure to receive the notice about the day and place where the exam would be performed (35.6%) – Table 3. Each school was in charge of this communication step. Similar study performed in Sa˜o Paulo six years ago reported 53.7% absenteeism at the first call and 54.3% at the second call. It also identified failure to receive an orientation or transmittal guide as the main reason for the absence.13 This issue could easily be resolved with a referral letter, explaining that the child failed the school vision screening test, how the vision test was done and the importance of the follow-up eye examination. It is also important to confirm receipt of the letter and awareness of the information. However, a study performed in North Carolina, revealed that 35% did not attend exams, even after they had received the referral letter.20 These findings show that interventions to improve follow-up on school vision screening referrals represent an important component of screening programs. It also calls attention to the need to reinforce the involvement of the teachers and directors at the school in the ocular heath program, as well as the need to further develop the campaign’s logistic protocol. In previous campaigns, difficulties related to transportation were also shown as an important limiting factor,13,18 however, the offering of free transportation and the performance of the exam close to the screening place did not influence the level of absenteeism (Table 3). Even with the exam during the weekend, 20.6% of the parents reported that they did not attend the project because they could not miss work (Table 3); however, they were able to overcome the problem and attended the recall. These patients were probably encouraged by the results from students who attended the first examination. The fact that 10.0% of the parents did not attend the exam due to other appointments (Table 3) indicates the

(59.3%) attended the first call. The 5.968 absent students were recalled, and 2.228 (37.3%) attended this secondchance exam (Figure 1). Among the 2.228 students who attended the second-chance exam, 1.461 (65.6%) were released during the screening period. The questionnaire was administered to 767 students who attended the secondchance exam and underwent the complete ophthalmological examination (Figure 1). Among the students examined, 50.8% were female, with ages ranging from seven to ten years old. For 461 students (60.1%), it was the first opportunity for an ophthalmological exam (Table 2). Among the 39.9% who had already submitted to an ophthalmological exam, 48.0% used the public health service (Table 2). The reasons for not attending the exam are shown in Table 3. The likelihood of a spectacle prescription at each appointment is described in Table 4. The recall facilitated a 15.2% (59.3% to 74.5%) increase in campaign coverage. Overall, 10,911 students were examined; 74.5% of those were referred for the exam (Table 1).

DISCUSSION Ophthalmological campaigns for students performed in the last 40 years in Brazil6,12 introduced children and their parents to facilities to increase the attendance. However, the projects still present a high percentage of absenteeism, which results in unnecessary costs and loss of exam opportunities for the children. Even with the access to facilities, attendance during the weekends, free transportation, spectacle donation, and two opportunities for free exams, 25.5% of the parents did not take their children for the examination. Ultimately, 51,509 students were screened; 14,651 (28.4%) were referred for an ophthalmological exam and 59.3% (8,683) attended the first call. Of the 5,968 who missed the first call, only 37.3% (2,228) attended the recall. The recall increased overall attendance from 59.3% to 74.5% (15.2%) – Table 1. Regarding the

Table 3 - Reason for not attending the first appointment.

Table 2 - Previous ophthalmological evaluations of the students.

Reason for non-attendance

Previous ophthalmological exam f Previous ophthalmological exam Yes No Service Public Service Covenant System Private System Do not remember

Did not know Work Disease Other appointment Forgot Missed the time Did not have anyone else to look after the other children Think the child has good vision Medical appointment scheduled Bad weather Lack of money TOTAL

% n = 767

306 461

39.9 60.1 n = 306

147 81 74 4

48.0 26.5 24.2 1.3

1587

273 158 95 77 48 39 36

35.6 20.6 12.4 10.0 6.3 5.1 4.7

18 17 4 2 767

2.3 2.2 0.5 0.3 100.0

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CLINICS 2011;66(9):1585-1589

Considering the extent of existing ocular health coverage, the routine eye exam required at the time the child starts going to school becomes even more important, not only to detect refractive errors, but also due to its educational role within the community. It is necessary to educate the population regarding the importance of ophthalmological exams and the adoption of ocular health-promoting behaviors,35 which would increase the attendance at campaigns and the patient-motivated search for health services. The perception of physical and mental health is one of the most significant human values. One study showed that a significant number of parents (29%) felt there was no need for a professional eye exam. Another 38% expressed lack of confidence in the screening results. These parents stated that they saw no signs of vision problems or that the child denied vision difficulties.30 It is noted that, after six years, the rates and reasons for absenteeism are the same and remain unaddressed. The measures used to increase the attendance did not have detectable influence on the rate of absenteeism. To increase attendance, restructuring of the campaigns is recommended, with emphasis on improved informationsharing, teacher involvement and community education regarding the importance of the exam. Optimal informationsharing could influence even the 25.5% of parents who did not take their children to the second-chance exam. Aside from the technical-scientific studies, a reorientation of the research in the health area, including politicalinstitutional aspects and assessment of the incorporation of new technologies, should be instituted. The promotion of strategic research is important to identify the priority areas that demand resource capitation and immediate application of the results. This study shows that restructuring of the campaigns could improve the efficiency of the campaigns (of attended students) by up to 15.2%.

Table 4 - Percentage of spectacles prescribed at each appointment – Guarulhos 2006. Appointment Spectacles

Yes No Total

First n 2,069 6,614 8,683

% 23.8 76.2 100.0

Recall n 713 1,515 2,228

Total

% n 32.0 2.782 68.0 8,129 100.0 10,911

% 25.5 74.5 100.0

p-value

,0.001

non-prioritization of the exam by the parents, even in cases when a visual problem was suspected by teachers. In a study conducted in Sa˜o Paulo six years ago, 19.4% could not miss work; 8.5% did not have money for transportation and 9.0% missed the exams due to other appointments.13 It was noted that 83.9% of the reasons for missing the first call could have been avoided. Similarly, in this study, the difficulties that justified absence at the first call were overcome for 87.1% of those who attended the recall. Only reasons related to disease (12.4%) and bad weather (0.5%) are insurmountable impediments that could justify absenteeism on the day of the exam. However, the absenteeism at the recall was much higher (62.7% at the recall and 49.7% at the first call) (Table 1). Non-attendance at the first call was also not related to the optical correction needed, as the results showed that those who were absent had a higher frequency of visual problems (Table 4). These findings confirm that there are additional barriers inhibiting follow-up after visual referral, as has been demonstrated by several studies.19,20,30 The use of the health system involves not only availability and access, but also the behavior necessary to look for existing services.2,23 Other studies showed that, after suspicion of possible ocular problems based on visual acuity screening exam, several parents or guardians waited up to four years to book an exam.20,24 Medical service barriers can be related to the user (lack of knowledge, fear, poverty, emotional difficulties, geographical distance, and cultural and behavioral aspects) and to the providers of these services (lack of motivation, training, material resources, and communication).19,20,25-30 The results of this study confirmed results of previous studies19,20,24,30 and support the hypothesis that there are multiple factors affecting follow-up compliance after failed school vision screenings. Parental reasons for not following up on referrals are complex and interventions must address multiple barriers.20,30 Among the students screened due to suspicion of visual problems and who attended the second call, this campaign was the first opportunity for ophthalmological exam among 60.1% of the cases, which indicates insufficient coverage provided by the health system (Table 2). This condition should be considered by the health managers involved with ophthalmological services. There is no consensus about the recommended age for the first ophthalmological exam. In some countries, it is recommended for patients between four and seven years old31 or is mandatory when registering the child in school.32-34 In Brazil, several attempts to institute this policy had little success.12

CONCLUSION A significant number of parents did not take their children for ophthalmological exams, even when facilities (free transportation, free exam performed over the weekend, spectacle donation, and second opportunity for exam) were offered. The main causes of absenteeism were lack of awareness and work. For 87.1% of the absenteeism cases, the difficulties could have been overcome via improved structuring of the first call. A recall increases attendance coverage of the target population by only 15.2% (59.3 to 74.5%). Notably, the eye exam campaign was the first exam for most of the absent students.

ACKNOWLEDGMENTS Contributors who do not meet the criteria for authorship should be listed in the acknowledgments section. All contributors must give written permission to be acknowledged.

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5. Carvalho RS, Kara Jose N, Arieta CEL, Castro RS. Campanhas comunita´rias - conceitos ba´sicos. In: Kara-Jose´ N, Gonçalves ER, Carvalho RS. Olho no Olho. Rio de Janeiro: Cultura Me´dica. 2006;7-16. 6. Kara-Jose´ N, Ferrarini M, Temporini E. Avaliaçaõ do desenvolvimento do plano de oftalmologia sanita´ria escolar em treˆs anos da sua aplicaçaõ no Estado de Saõ Paulo. Arq Bras Oftalmol. 1977;40:9-15. 7. Russ HHA, Temporini ER, Kara-Jose´ N. Impacto da Campanha Olho no Olho em escolas de ensino fundamental: percepçaõ do pessoal de ensino. Arq Bras Oftalmol. 2004;67:359-63, doi: 10.1590/S0004-27492004000200023. 8. Kara-Jose N, Holzchuh N, Temporini ER. Refractive errors in school children in the city of Sao Paulo, Brazil. Bol Oficina Sanit Panam. 1984;96:326-33. 9. Kara-Jose´ N, Pereira VL, Melo HFR, Uruaneja AJ, Brasil-Jr W. Criaçaõ do nućleo de prevençaõ a` cegueira. Arq Bras Oftalmol. 1982;50:145-7. 10. Macchiaverni Filho N, Kara-Jose N, Rueda G, Pereira VL, Costa MN, Rangel FF, et al. Levantamento oftalmolo´gico em escolares da primeira a quarta se´ries do primeiro grau na cidade de Paulıńia, Saõ Paulo. Arq Bras Oftalmol. 1979;42:289-94. 11. Castro RS. Triagem visual e assisteˆncia oftalmolo´gica em pre´ – escolares da cidade de Limeira, SP, 1995. Campinas: Universidade Estadual de Campinas - UNICAMP. 1997. 12. Carvalho RS, Kara Jose N, Gonçalves ER. Avaliaçaõ das Campanhas de Prevençaõ e Reabilitaçaõ Visual Olho no Olho - 1998 a 2001. In: Kara Jose N, Gonçalves ER, Carvalho RS. Olho no Olho ‘‘Campanha Nacional de Prevençaõ e Reabilitaçaõ Visual do Escolar’’. Rio de Janeiro: Cultura Me´dica. 2006;119-29. 13. Alves MR, Temporini ER, Kara Jose N. Atendimento oftalmolo´gico de escolares do sistema pu´blico de ensino no municı´pio de Saõ Paulo aspectos me´dico-sociais. Arq Bras Oftalmol. 2000;63:359-63. 14. Estacia P, Stramari LM, Schuch SB, Negrello D, Donato L. Prevaleˆncia de erros refrativos em escolares da prineira se´rie do ensino fundamental da regiaõ nordeste do Rio Grande do Sul. Rev Bras Oftalmol. 2007;66:297303, doi: 10.1590/S0034-72802007000500002. 15. Kara-Jose N, de Carvalho KM, Caldato R, Pereira VL, de Oliveira AM, da Fonseca Neto JC. Treatment and incidence of amblyopia in the preschool population, Campinas, Sao Paulo, Brazil. Bol Oficina Sanit Panam. 1984;96:31-7. 16. Constanti F, Costa M, Salgado M, Bastos C, Benchimol E. Avaliaçaõ das alteraço˜es oculares encontradas na aplicaçaõ do projeto de oftalmologia saninta´ria escolar. Rev Bras Oftalmol. 1989;48:39-42. 17. Abud AB, Ottaiano JAA. Aspectos soćioeconoˆmicos que influenciaram no comparecimento ao exame oftalmolo´gico de escolares com alteraço˜es visuais. Arq Bras Oftalmol. 2004;67:773-9, doi: 10.1590/S000427492004000500015. 18. Cavalcante S, Kara Jose N, Temporini ER. Percepçaõ de pais de escolares da 1a se´rie do ensino fundamental a respeito da campanha ‘‘Olho no Olho’’ 2000, na cidade de Maceio´ - Alagoas Arq Bras Oftalmol. 2004;67:87-91, doi: 10.1590/S0004-27492004000100015.

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DOI:10.1590/S1807-59322011000900015

CLINICAL SCIENCE

Analysis of angiogenic factors and cyclooxygenase-2 expression in cartilaginous tumors – clinical and histological correlation Francisco Fontes Cintra,I,II Mauricio Etchebehere,III Jose´ Carlos Barbi Gonc¸alves,V Alejandro Enzo Cassone,IV Eliane Maria Ingrid AmstaldenI I

Pathology Department, Universidade Estadual de Campinas, Campinas/SP, Brazil. II Orthopedics Department, Casa de Sau´de Campinas, Campinas/SP, Brazil. Orthopedics Department; UNICAMP, Universidade Estadual de Campinas, Campinas/SP, Brazil. IV Oncological Orthopedics Unit, Department of Orthopedics, Domingos Boldrini Children’s Cancer Center, Campinas/SP, Brazil. V Orthopedics Department, Centro Me´dico de Campinas, Campinas/SP, Brazil. III

OBJECTIVES: To study the role of angiogenesis and cyclooxygenase-2 expression in cartilaginous tumors and correlate these factors with prognosis. INTRODUCTION: For chondrosarcoma, the histological grade is the current standard for predicting tumor outcome. However, a low-grade chondrosarcoma can follow an aggressive course—as monitored by sequential imaging techniques—even when it is histologically indistinguishable from an enchondroma. Therefore, additional tools are needed to help identify the biological potential of these tumors. The degree of angiogenesis that is induced by the tumor could assist in this task. Angiogenesis can be quantified by measuring the expression of vascular endothelial growth factor and CD34, and cyclooxygenase-2 can induce angiogenesis by stimulating the production of proangiogenic factors. METHODS: In total, 21 enchondromas and 58 conventional chondrosarcomas were studied by examining the clinical and histopathological findings in conjunction with the immunostaining markers of angiogenesis and cyclooxygenase-2 expression. RESULTS: The significant variables that were associated with poor outcome were 1) higher-grade chondrosarcomas, 2) tumors that developed in flat bones, and 3) over-expression of CD34 (with a median count that was higher than 5.9 vessels in 5 high power fields). Moreover, CD34 expression (measured using the Chalkley method) revealed significantly higher microvessel density in flat bone chondrosarcomas. DISCUSSION: Previous studies have shown a positive correlation between Chalkley microvessel density and histological grade; however, in our sample, we found that the former is predictive of the outcome. Chondrosarcomas in flat bones have been shown to correlate with a poor prognosis. We also found that CD34 microvessel density values were significantly higher in flat-bone chondrosarcomas. This could explain—at least in part—the more aggressive biological course that is taken by these tumors. CONCLUSIONS: These results provide evidence that CD34 microvessel density in chondrosarcomas can be helpful in predicting patient outcome and may add to our understanding of chondrosarcoma pathogenesis. KEYWORDS: Histological grades; Flat bones; CD34; VEGF; Prognosis. Cintra FF, Etchebehere M, Gonc¸alves JCB, Cassone AE, Amstalden EMI. Analysis of angiogenic factors and cyclooxygenase-2 expression in cartilaginous tumors – clinical and histological correlation. Clinics. 2011;66(9):1591-1596. Received for publication on April 24, 2011; First review completed on May 26, 2011; Accepted for publication on June 1, 2011 E-mail: ingrid@fcm.unicamp.br Tel.: 55 19 32893897

tumors and 10–20% of malignant tumors.1,2 The World Health Organization (WHO) Classification of Bone Tumors (2002), which is based on the histological grade of the tumor, has been accepted as the standard for predicting tumor outcome.3,4 The histological grading system for chondrosarcomas (CSs) categorizes these tumors into three grades that are based on cellularity, nuclear atypia, and pleomorphism.4 Low-grade CSs tend to grow slowly and are associated with a 90% five-year survival rate but can recur and metastasize. In contrast, high-grade tumors have a

INTRODUCTION Among bone tumors, cartilage-producing tumors rank as the third most frequent type, representing 30% of benign

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expression. The aim of this study was to evaluate the value of these findings as a prognostic tool in CSs and as a possible aid in the differential diagnosis between an ECs and a low-grade CSs.

higher incidence of metastasis and a 45% five-year survival rate.3-6 In some borderline tumors (i.e., a grade 1 CS), distinguishing a CS from an enchondroma (EC) may be difficult when using only a routine histopathological examination, as the current diagnostic criteria are not definitive. Furthermore, these tumors have a broad array of clinical, radiographic, and histological presentations that cause difficulties in both diagnosis and treatment.3-7 Thus, it is important to establish additional tools that could help predict a tumor’s biological potential. The degree of angiogenesis that the tumor elicits might assist in making this distinction. Angiogenesis is a fundamental step in both neoplastic transformation and the regulation of tumor growth, as demonstrated by Folkman8 and Dvorak et al.9 The interaction of vessels with cartilaginous tissue is not a physiological step, except during skeletal development. For example, in enchondral ossification (the period in which the growth plate is active), vessels can be seen penetrating the zones of hypertrophied cartilage during matrix ossification.10,11 However, normal adult cartilage is avascular. Thus, the presence of vessels within cartilaginous tissue is associated with pathological conditions, such as osteoarthritis and tumors.10,11 Angiogenesis (or neoangiogenesis) can be quantified by measuring the expression of certain molecules. Vascular endothelial growth factor (VEGF) is one of the most important angiogenic factors that have been described. Among its many functions are the formation, organization, and migration of blood vessels. VEGF also promotes the degradation of soft tissues around the sprouting endothelium and increases the permeability of venules. An increase in VEGF staining has been described in many epithelial, germ cell, lymphoid, melanocytic, and mesenchymal tumors.12-16 CD34 is a surface glycophosphoprotein that is expressed in small-vessel endothelial cells and is associated with angiogenesis.16-20 Cyclooxygenases (COXs) are enzymes that catalyze the synthesis of prostaglandins from arachidonic acid. Cyclooxygenase-2 (COX-2) is associated with inflammatory and mitogenic stimuli, resulting in increased prostaglandin synthesis in both inflamed and neoplastic tissues.21-23 COX2 also plays an important role in carcinogenesis, as it can induce angiogenesis by stimulating the production of proangiogenic factors, such as VEGF;21-23 in addition, COX-2 can inhibit tumor cell apoptosis and immune surveillance, thus increasing the tumor’s invasive and metastatic potential.21-23,25 Indeed, recent studies of various human tumors showed that COX-2 over-expression is associated with poor prognosis.21 In musculoskeletal tumors (e.g., osteosarcomas, rhabdomyosarcomas, and CSs), studies of COX-2 overexpression have yielded conflicting results.21-29 In fact, neovascularization is only one of the many elements involved in neoplastic transformation and the progression of a tumor to a higher histological grade. However, because inhibiting angiogenesis is so important in the preservation of intact cartilage (as seen in the osteoarthritis therapeutic approach),10,11,15,16 it is possible that the lack of this inhibition is a significant component in the pathogenesis of cartilage tumors. The present report is a retrospective study of 21 ECs and 58 conventional CSs, in which we addressed the clinical and follow-up data and histopathological findings with regard to immunostaining for markers of angiogenesis and COX-2

MATERIALS AND METHODS Patients and tumor samples From 1988 through 2007, 141 patients with a primary EC or CS were admitted to the University Hospital – Unicamp (Campinas-Brazil) and Centro Me´dico de Campinas (Brazil). The tumor specimens were routinely fixed in 10% formalin and later decalcified with hydrochloric acid and ethylenediaminetetraacetic acid. The embedded tumor tissue from each patient was obtained from the Department of Pathology. The clinical, radiographic, and follow-up information were obtained from the patients’ medical records. The routine staining of the tumor sections was reviewed to confirm the diagnosis. The histological grade (on a scale of 1 to 3) of each CS was determined based on nuclear size and staining (hyperchromasia) and cellularity according to the WHO Classification of Bone Tumors (2002).1 Pathological material with insufficient tissue samples for new cuts or in poor fixative condition (n = 54) and chondrosarcoma variants (mesenchymal: n = 4; de-differentiated: n = 2; clear cell: n = 2), were excluded from the analysis. Therefore, a total of 79 patients (30 men and 49 women, ranging from 5 to 87 years of age, with a median age of 42 years) entered the study. There was no loss of follow-up data. The patient data are summarized in Table 1. Twenty-one ECs that developed in the long (n = 5) and short bones (n = 16) were studied. Among the CSs were 31 grade 1 CSs that developed in the long (n = 23) and flat bones (n = 8). Twenty-four grade 2 CSs developed in the long (n = 18) and flat bones (n = 6), and 3 grade 3 CSs developed in the long (n = 1) and flat bones (n = 2). With the goal of correlating the clinical and histological results, the patients were divided into the following three groups: group I (n = 21) comprised the patients with an EC; group II (n = 31) comprised the patients with a grade 1 CS; and group III (n = 27) comprised the patients with a grade 2 or 3 CS. Because flat-bone CSs are associated with poor prognosis,1 the benign and malignant tumor anatomical sites were also divided into three groups. Seventeen tumors (16 ECs and 1 high-grade CS) developed in small bones (metacarpal bone, n = 11; hand phalanx, n = 4; metatarsal bone, n = 1; calcaneus, n = 1). Forty-eight tumors (5 ECs and 43 CSs) developed in long bones (femur, n = 18; fibula, n = 3; tibia, n = 10; humerus, n = 13; radius, n = 3; lumbar vertebra, n = 1). Fourteen tumors (all CSs) developed in flat bones (ribs, n = 6; scapula, n = 2; pelvis, n = 6). All of the ECs (n = 21) were treated with curettage. The CSs were removed by either wide resection or amputation, with a wide margin in 12 cases, a marginal resection in 26 cases, and an intralesional resection or curettage in 18 cases (all 18 of which were grade 1 CS cases).30 Instead of surgery, two extremely large pelvic tumors (in the sacrum and iliac bone) were treated using chemotherapy and/or radiotherapy after establishing the diagnosis by biopsy. Four of the patients underwent chemo/radiotherapy for local recurrence and metastasis after undergoing a marginal resection. Chondrosarcoma size and the adequacy of the

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less than 10% of all tumor cells stained; 2+ = 10–50% of all tumor cells stained; and 3+ = more than 50% of all tumor cells stained. For VEGF, cellularity was quantified by counting the relative number of immunostained cells per 5 ‘‘hot-spot’’ high-power fields (HPFs) (at 400x magnification) and was expressed as a percentage of the total number of neoplastic cells according to the method of Ayala et al.12 For the CD34 analysis, the sections were scanned using a low-power view (at 50x magnification). Five areas displaying the highest number of immunostained microvessels (i.e., hot-spots) were identified.17,18 Next, one 400x microscopic field (corresponding to an area of 0.1449 mm2) was chosen within each hot-spot. A 25-point Chalkley eyepiece graticule (Leitz Orthoplan, Leica) was applied to each selected hot-spot field (corresponding to a Chalkley grid area of 0.041 mm2). The graticule was positioned such that the immunostained vessels hit the maximum number of points. The final Chalkley count for an individual tumor was taken as the mean value of the five graticule counts. The generally accepted criteria for determining a vessel’s profile17,18 were followed and included any stained endothelial cells or endothelial cell clusters that were separated from the adjacent microvessels. A visible lumen was not a requirement for a structure to be counted as a microvessel. Necrotic or sclerotic areas within the tumor and non-tumor areas that were adjacent to the tumor were excluded from the vessel counts.31-33

Table 1 - Correlation between variables in groups I, II, and III. Group I (n = 21 ECs) Gender Male Female Mean age at Dx (years) Follow-up (months) Site Small bone Long bone Flat bone COX-2 No expression ,10% 10-50% .50% VEGF #10.5% .10.5% CD34 #5.9 .5.9 Outcome

Group II (n = 31 Group III (n = 27 Low-Grade Moderate/HighCSs) Grade CSs)

7 (33.33%) 14 (66.67%) 29.6 98

13 (41.93%) 18 (58.07%) 43 66

10 (37.03%) 17 (62.97%) 50 71

16 (76.2%) 5 (23.8%) 0

0 23 (74.20%) 8 (25.8%)

1 (3.7%) 20 (74.1%) 6 (22.22)

2 (9.5%) 9 (42.9%) 8 (38.1%) 2 (9.5%)

20 (64.5%) 7 (22.6%) 4 (12.9%) 0

15 (55.56%) 6 (22.22) 3 (11.11%) 3 (11.11%)

11 (52.38%) 10 (47.69%)

17 (54.84%) 14 (45.16%)

14 (51.85%) 13 (48.15%)

21 (100%) 27 (87%) 0 4 (13%) 100% cured 3 (9.6%) poor

20 (74.1%) 7 (25.9%) 11 (40.7%) poor

Dx: diagnosis; ECs: enchondromas; CSs: chondrosarcomas; Mod: moderate.

surgical margins were excluded from analysis, as 18 of the 58 CS patients were treated with curettage. The follow-up interval was recorded from the time of the surgery until January 2009. The minimum follow-up period was 24 months (or shorter in cases of death), and the median follow-up was 77 months (with a range of 4-250 months).

Statistical analysis Chi-squared or Fisher’s tests were used to compare outcome, gender, site, follow-up span, and COX-2 expression. For the VEGF and CD34 results, Mann-Whitney test, Kruskal-Wallis test or analysis of variance (ANOVA) with transformation by ranks was used, followed by Tukey’s test to identify differences when necessary. To test the linear association between two immunomarkers, the Spearman correlation coefficient was determined. A multiple logistic regression analysis (the generalized log model) was used to identify factors that differentiate the histological grade of the tumor. The process of selecting the variables that were used was stepwise. The level of significance for the statistical tests was set at p,0.05. The data analysis was performed using the SAS System for Windows version 9.2 (SAS Institute Inc., Cary, NC, USA).

Immunohistochemical technique The primary antibodies that were used included antiCD34 (Mo a Hu CD34 Class II, Clone QBEnd 10, Dako Cytomation, Carpenteria, CA, USA) at a dilution of 1550, anti-VEGF (A-20 rabbit polyclonal IgG, 200 mg/ml, Santa Cruz Biotech., Inc, Santa Cruz, CA, USA) at a dilution of 15200, and anti-COX-2 (Cyclooxygenase-2 antibody clone4h12, Diagnostic Byosystem, Pleasanton, CA, USA) at a dilution of 1550. Epitope retrieval was achieved by steaming with citrate buffer (at 95 ˚C). The EnVision + Dual Link System HRP polymer (Dako) was used as a reaction amplifier. The antibody complex was visualized with 3,39diaminobenzidine tetrahydrochloride (DAB) staining according to the manufacturer’s instructions. The sections were counterstained with Mayer hematoxylin. The appropriate negative and positive controls were included in each assay.

RESULTS The results are summarized in Tables 1 and 2. The following 14 patients had poor outcomes: three in group II (metastasis: n = 1; local recurrence: n = 2) and 11 in group III (local recurrence: n = 4; death: n = 7). None of the ECs recurred. None of the patients whose CS was treated with curettage presented a tumor recurrence or poor outcome. There were no significant differences among the three groups with regard to the duration of follow-up (p = 0.7431). A poor outcome was significantly associated with the following: 1) moderate/high-grade CS (p,0.0001); 2) tumors that developed in a flat bone (p,0.0001); and 3) overexpression (p,0.02) of CD34 (when the number of immunostained vessels was higher than the median of 5.9 vessels in 5 high-power fields) (Figures 1A and B). No significant correlations were found between outcome and age

Immunohistochemical analysis All of the immunostained sections were evaluated simultaneously (using a double-headed microscope) by two investigators (EMIA and FFC) who were unaware of the clinical status of the patient being studied. With regard to COX-2, a consensus judgment was adopted as the proper tumor immunohistochemical score according to the method of Endo et al.,22 with adaptations characterized by the mean of the proportion of stained tumor cells in 5 ‘‘hot-spot’’ high-power fields (at 400x magnification) as follows: 0 = no stained tumor cells; 1+ =

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Table 2 - Correlation between variables in groups II (grade I CSs) and III (high-grade CSs). Outcome Gender Male Female Age (years) #51 .51 Histological Grade Grade 1 Grade 2 or 3 Site small bone long bone flat bone CD34 MVD #5.9 .5.9 COX-2 no expression ,10% 10-50% .50% VEGF expression #10.5% .10.5%

Good (n = 44) Poor (n = 14)

p-value 0.7293*

18 (40.1%) 26 (39.9%)

5 (35.7%) 9 (64.3%)

28 (63.6%) 16 (36.4%)

7 (50%) 7(50%)

28 (63.6%) 16 (36.4%)

3 (21.4%) 11 (78.6%)

1 (2.3%) 36 (81.8%) 7 (15.9%)

0 7 (50%) 7 (50%)

37 (84%) 7 (16%)

7 (50%) 7 (50%)

28 (63.6%) 10 (22.7%) 5 (11.3%) 1 (2.4%)

7 (50%) 3 (21.4%) 2 (14.3%) 2 (14.3%)

22 (50%) 22 (50%)

9 (64.3%) 5 (35.7%)

0.1966*

,0.0001*

0.0104¥

0.3364*

0.2094¥

*- Fischer test. ¥ - Mann-Whitney and ANOVA test.

(p = 0.1966), gender (p = 0.7293), COX-2 expression (p = 0.3364), or VEGF expression (p = 0.2094). An overexpression of VEGF (when the percentage of immunostained cells was higher than the median of 10.5% in five high-power fields) was found in 48% of low-grade CSs (group II) and in 59% of moderate/high-grade CSs (group III). None of the variables proved useful for differentiating between low-grade CSs and ECS (p = 0.7152). A multivariate analysis of CD34 immunostaining (using Mann-Whitney, ANOVA, and Tukey’s tests) revealed a positive association between higher microvessel density (MVD) and flat bone sites (p,0.01). Indeed, flat bone CSs (Figure 1B) had a mean of 6.1 CD34-positive vessels in the Chalkley count compared with 4.7 and 4.6 CD34-positive vessels in the long and small bones, respectively. Although COX-2 expression was not correlated with patient outcome, its over-expression (Figure 1C) (more than 10% positive COX-2 cells in five high-power fields) was positively correlated (p,0.02) with increased CD34-positive MVD (using an ANOVA test followed by the KruskalWallis test). Among the EC cases, we found that 19/21 (90%) of the tumors stained positively for COX-2 (Fig. 1D). In 10 of the 21 ECs (47%), an over-expression (i.e., .10% of positive cells) was found; VEGF over-expression was found in 13 (61%) of the ECs. Intra-tumoral CD34-positive vessels were found in 100% of the ECs but displayed weak CD34 expression (none had a Chalkley count above 5.5).

Figure 1 - (A) A grade I femur chondrosarcoma (CS) with low CD34 microvascular density (CD34 positive MVD). Bar = 0.02 mm. (B) A grade II iliac bone CS with high CD34 positive MVD. Bar = 0.04 mm. A magnified view is shown in the inset (bar = 0.02 mm). (C) A grade 3 humerus CS. Bar = 0.02 mm. Bizarre cells are shown in the inset (bar = 0.01 mm) with diffuse strong immunoreactivity to COX-2 antibody. (D) A hand phalanx enchondroma with uniform and diffuse immunoreactivity to COX-2 antibody. Bar = 0.02 mm.

prognosis of CS patients have been sought and include the evaluation of DNA synthesis and content; flow cytometry using molecular markers, such as p53 and MIB-1; cytogenetic features; histomorphometry; and radiographic classifications. Nevertheless, none of these methods is considered to be definitive.1,3-7 The present study provides immunohistochemical evidence that MVD—as evaluated by CD34 staining—is significantly associated with poor prognosis. Our results differ from those of Nakagawa et al.,25 who found no correlation between CD34 expression and outcome. The two additional predictive variables that we identified (i.e., histological grade and tumor site in flat bones) are well known in the medical literature.1,4,5 However, in our study, we also found that CD34 positive MVD was significantly higher in flat bone CSs. In animal experiments, flat and long bones had the same density of vessels.34 However, in adults, flat bones have functional bone marrow that contains stem cells and produces many growth factors16 that could elicit tumor angiogenesis. The role of COX-2 in cartilage tumors is controversial. Endo et al. in 2006 and Schrage et al. in 201022,23 have reported that COX-2 over-expression is associated with poor

DISCUSSION Histological grading of CSs is considered to be the most useful predictor of outcome,1,3,4 but this is a subjective procedure, and the current criteria are not definitive.5,6 Therefore, supplemental methods for assessing the

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Angiogenesis and COX - 2 in cartilaginous tumors Cintra FF et al. English grammar. This study was supported by FAPESP (grant number 09/51473-0).

prognosis in CSs. Our results are in line with those of Sutton et al.,21 who studied 32 cartilaginous tumors by Western blot analysis and found no statistical correlation between the expression of COX-2 and the following variables: age, sex, stage, anatomical site, metastasis development, or survival rate. In addition, Nakagawa et al.25 studied 101 CS specimens and found no correlation between COX-2 expression and prognosis. We previously studied 53 CSs (seven of which were unconventional and included four mesenchymal, two de-differentiated and one clear-cell).35 In the previous sample, we found that a COX-2 expression frequency above 50% was associated with poor prognosis; all of the six patients with either mesenchymal or dedifferentiated CSs died within 24 months of their diagnosis. In the present study, we added 12 additional conventional CS patients and excluded the unconventional tumors from the sample and found no significant correlation between COX-2 expression and outcome. Another difference between our findings and those of Endo et al.22 was that they did not find any COX-2 staining in enchondromas, whereas 90% of our enchondroma specimens had positive COX-2 staining. Contradictory results have also been obtained from studying other bone tumors, such as osteosarcomas. Xu et al.26 found that COX-2 over-expression was associated with a better prognosis. In contrast, Dinckens et al.27 found no correlation between COX-2 over-expression and patient outcome. Finally, Masi et al.28 and Liao et al.29 reported that cases of osteosarcoma had worse prognosis and decreased patient survival rates whether COX-2 was overexpressed. We suggest the following two reasons for these discordant results: 1) the COX-2 antibody clone that we used was different from that used by Endo et al.; and/or 2) any problems with the fixation or decalcification of the samples could have biased the results. Nevertheless, we found a positive correlation between CD34 positive MVD and COX-2 expression, which is in line with the well-known relationship between COX-2 and angiogenesis. Therefore, this enzyme might have at least an indirect role in patient prognosis. With respect to VEGF expression, our quantitative results are similar to those of Ayala et al.12 and Kalinski et al.,13,14 which were descriptive studies that found a higher rate of VEGF expression in moderate- and high-grade CSs. In our sample, no significant correlation was observed between VEGF expression and outcome.

REFERENCES 1. Dorfman HD, Czerniak B. Bone cancers. Cancer. 1995;75:203-10, doi: 10. 1002/1097-0142(19950101)75:1+,203::AID-CNCR2820751308.3.0.CO;2-V. 2. Terek RM. Angiogenesis in chondrosarcoma. Curr Opin Orthop. 2002;13:449-53, doi: 10.1097/00001433-200212000-00012. 3. Chow WA. Update on chondrosarcomas. Curr Opin Oncol. 2007;19:371– 6, doi: 10.1097/CCO.0b013e32812143d9. 4. Amstalden EMI. Tumores do sistema osteoarticular. In: Billis A, Vassallo J, editors. Patologia diagno´stica de tumores. 2nd ed. Campinas. Impressa˜o Digital do Brasil Gra´fica e Editora. 2004;345-52. 5. Galant C, Malghem J, Sibille C, Docquier PL, Delloye C. Current limitations to the histopathological diagnosis of some frequently encountered bone tumours. Acta Orthop Belg. 2008;74:1-6. 6. Wang XL, De Beuckeleer LH, De Schepper AM, Van Marck E. Low-grade chondrosarcoma vs enchondroma: challenges in diagnosis and management. Eur Radio. 2001;11:1054-7, doi: 10.1007/s003300000651. 7. Ollivier L, Vanel D, Lecle`re J. Imaging of chondrosarcomas. Cancer Imaging. 2003;4:36–8, doi: 10.1102/1470-7330.2003.0022. 8. Folkman J. Tumor angiogenesis. Adv Cancer Res. 1985;43:175-203, doi: 10.1016/S0065-230X(08)60946-X. 9. Dvorak HF, Brown LF, Detmar M, Dvorak AM. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability and angiogenesis. Am J Pathol. 1995;146:1029-39. 10. McGough R, Aswad B, Terek RM. Pathologic neovascularization in cartilage tumors. Clin Orthop. 2002;397:76-82, doi: 10.1097/00003086200204000-00011. 11. Murata M, Yudoh K, Masuko K. The potential role of vascular endothelial growth factor (VEGF) in cartilage: how the angiogenic factor could be involved in the pathogenesis of osteoarthritis? Osteoarthritis Cartilage. 2008;16:279-86, doi: 10.1016/j.joca.2007.09.003. 12. Ayala G, Liu C, Nicosia R, Horowitz S, Lackman R. Microvasculature and VEGF expression in cartilaginous tumors. Hum Pathol. 2000;31:341– 6, doi: 10.1016/S0046-8177(00)80248-8. 13. Kalinski T, Krueger S, Sel S, Werner K, Ropke M, Roessner A. Differential expression of VEGF-A and angiopoietins in cartilage tumors and regulation by interleukin-1b. Cancer. 2006;106:2028-38, doi: 10.1002/ cncr.21848. 14. Kalinski T, Sel S, Kouznetsova I, Ro¨pke M, Roessner A. Heterogeneity of angiogenesis and blood vessel maturation in cartilage tumors. Pathol Res Pract. 2009;205:339-45, doi: 10.1016/j.prp.2008.12.008. 15. Furumatsu T, Nishida K, Kawai A, Namba M, Inoue H, Ninomiya Y. Human chondrosarcoma secretes vascular endothelial growth factor to induce tumor angiogenesis and stores basic fibroblast growth factor for regulation of its own growth Int J Cancer. 2002;97:313-22. 16. McGough RL, Lin C, Meitner P, Aswad BI, Terek RM. Angiogenic cytokines in cartilage tumors. Clin Orthop Relat Res. 2002;397:62-9, doi: 10.1097/00003086-200204000-00009. 17. Krause DS, Fackler MJ, Civin CI, May WS. CD34: structure, biology, and clinical utility. Blood. 1996;87:1-13. 18. Fina L, Molgaard HV, Robertson D, Bradley NJ, Monaghan P, Delia D, et al. Expression of the CD34 gene in vascular endothelial cells. Blood.1990;15:2417-26. 19. Hansen S, Sørensen FB, Vach W, Grabau DA, Bak M, Rose C. Microvessel density compared with the Chalkley count in a prognostic study of angiogenesis in breast cancer patients. Histopathology. 2004;44(5):428-36, doi: 10.1111/j.1365-2559.2004.01848.x. 20. Vermeulen PB, Gasparini G, Fox SB, Toi M, Martin L, McCulloch P, et al. Quantification of angiogenesis in solid human tumours: an international consensus on the methodology and criteria of evaluation. Eur J Cancer. 1996;32A:2474-84, doi: 10.1016/S0959-8049(96)00379-6. 21. Sutton KM, Wright M, Fondren G, Towle CA, Mankin HJ. Cyclooxygenase-2 expression in chondrosarcoma. Oncology. 2004;66: 275-80, doi: 10.1159/000078327. 22. Endo M, Matsumura T, Yamaguchi T, Yamaguchi U, Morimoto Y, Nakatani F, et al. Cycloxygenase-2 overexpression associated with a poor prognosis in chondrosarcomas. Hum Pathol. 2006;37:471-6, doi: 10.1016/ j.humpath.2005.12.001. 23. Schrage YM, Machado I, Meijer D, Briaire-de Brujin I, van den Akker BE, Taminau AH, et al. COX-2 expression in chondrosarcoma: a role for celecoxib treatment? Eur J Cancer. 2010;46:616-24, doi: 10.1016/j.ejca. 2009.11.002. 24. Liu JF, Fong YC, Chang CS, Huang CY, Chen HT, Yang WH, et al. Cycloxygenase-2 enhances alpha2beta1 integrin expression and cell migration via EP1 dependent signaling pathway in human chondrosarcoma cells. Mol Cancer. 2010;9:43, doi: 10.1186/1476-4598-9-43. 25. Nakagawa SA, Lopes A, Lopes de Carvalho A, Rossi BM, Werneck da Cunha I, Soares FA, et al. Nitric oxide synthase, cycloxigenase-2, nitrotyrosine, and angiogenesis in chondrosarcoma and their relation

CONCLUSIONS Chalkley MVD—as evaluated through CD34 antibody expression—may be a useful tool to help predict patient outcome in chondrosarcoma (CS) cases. These data can be considered for selective therapeutic inhibitory targeting. The higher MVD in flat bone CSs could explain the poorer outcome of these tumors relative to long/short bone CSs. None of the variables that were examined in this study were found to be useful in distinguishing a low-grade chondrosarcoma from an enchondroma.

ACKNOWLEDGEMENTS The authors are grateful to Ana Cla´udia S. Piaza and Luzia A. M. Reis for technical assistance, Cleide Ap. M. Silva and Helymar C. Machado for assistance with the statistical analyses, Adilson A. Piaza for assistance with photographic documentation and Ms. Diane Ellis, for her assistance with

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26.

27.

28.

29. 30.

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to prognosis. Bone Joint Surg Am. 2010;92:1738-46, doi: 10.2106/JBJS.H. 00717. Xu Z, Choudhary S, Voznesensky O, Mehrotra M, Woodard M, Hansen M, et al. Overexpression of COX-2 in human osteosarcoma cells decreases proliferation and increases apoptosis. Cancer Res. 2006;66:6657-64, doi: 10. 1158/0008-5472.CAN-05-3624. Dickens DS, Kozielski R, Leavey PJ, Timmons C, Cripe TP. Cycloxygenase-2 expression does not correlate with outcome in osteosarcoma or rhabdomyosarcoma. J Pediatr Hematol Oncol. 2003;25:282-5, doi: 10.1097/00043426-200304000-00003. Masi L, Recenti R, Silvestri S, Pinzani P, Pepi M, Paglierani M, et al. Expression of cycloxygenase-2 in osteosarcoma of bone. Immunohistochem Mol Morphol. 2007;15:70-6, doi: 10.1097/01.pai.0000213131. 63417.fa. Liao Y, Li F, Hu X. Expression and clinical significance of OPN and COX2 in osteosarcoma. Chin Ger J Clin Oncol. 2007;6:378-82, doi: 10.1007/ s10330-007-0004-9. Etchebehere M, de Camargo OP, Croci AT, Oliveira CR, Baptista AM. Relationship between surgical procedure and outcome for patients with

31. 32.

33.

34.

35.

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grade I chondrosarcomas. Clinics. 2005;60:121-6, doi: 10.1590/S180759322005000200007. Fox SB, Harris AL. Histological quantitation of tumour angiogenesis. APMIS. 2004;112:413-30, doi: 10.1111/j.1600-0463.2004.apm11207-0803.x. Nico B, Benagiano V, Mangieri D, Maruotti N, Vacca A, Ribatti D. Evaluation of microvascular density in tumors: pro and contra. Histol Histopathol. 2008;23:601-7. Sharma S, Sharma MC, Sarkar C. Morphology of angiogenesis in human cancer: a conceptual overview, histoprognostic perspective and significance of neoangiogenesis. Histopathology. 2005;46:481-9, doi: 10. 1111/j.1365-2559.2005.02142.x. Pannarale L, Morini S, Dâ&#x20AC;&#x2122;Ubaldo E, Gaudino E, Marinozzi G. SEM corrosion-casts study of the microcirculation of the flat bones in the rat. Anat Rec. 1997;247:462-71, doi: 10.1002/(SICI)1097-0185(199704)247: 4,462::AID-AR4.3.0.CO;2-T. Cintra FF, Cassone AE, GoncÂ¸alves JCB, Etchebehere M, Amstalden EMI. Prognostic significance of cyclooxygenase-2 (COX-2) expression in cartilaginous tumors. Histopatholgy. 2010;57:20.

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DOI:10.1590/S1807-59322011000900016

CLINICAL SCIENCE

Prenatal tobacco exposure is related to neurobehavioral modifications in infants of adolescent mothers Marina C. M. Barros,I Sandro S. Mitsuhiro,II Elisa Chalem,III Ronaldo R. Laranjeira,II Ruth GuinsburgI I

Division of Neonatal Medicine, Department of Pediatrics, Federal University of Saõ Paulo (UNIFESP), Saõ Paulo/SP, Brazil. II National Institute of Science and Technology for Policies on Alcohol and Drugs - INPAD/UNIFESP/CNPq, Saõ Paulo/SP, Brazil. III Research Unit on Alcohol and Other Drugs of UNIFESP, Federal University of Saõ Paulo (UNIFESP), Saõ Paulo/SP, Brazil.

INTRODUCTION: Prenatal tobacco exposure interferes with neonatal outcomes. OBJECTIVE: To determine the neonatal neurobehavioral effects of in utero tobacco exposure. METHODS: This prospective cross-sectional study included healthy, term, with birth weight appropriate for gestacional age neonates without exposure to alcohol, drugs, or infections, born to adolescent mothers without psychiatric disorders or post-traumatic stress. Infants were classified according to in utero tobacco exposure, as identified by the Composite International Diagnostic Interview administered to mothers. Neurobehavior was assessed by the Neonatal Intensive Care Unit Network Neurobehavioral Scale. Both tools were administered between 24 and 72 hours after birth. Neurobehavioral outcomes were compared between exposed and nonexposed infants by ANOVA. The associations between neurobehavioral scores and number of cigarettes smoked were studied by linear correlation. RESULTS: During the study, 928 newborns of adolescent mothers were born, and 388 were included in the study. Of these, 23 were exposed to tobacco, and 365 neonates were not exposed. There were no differences between the groups in gestational age, birth weight, post-natal age at the exam, or time between last feeding and exam. Exposed neonates showed higher scores on arousal (p = 0.004), excitability (p = 0.003), and stress/abstinence signals (p = 0.019) and a lower score on regulation (p = 0.025). After adjusting for the type of anesthesia, mode of delivery, gender, age at neurologic exam, exam duration and time between last feeding and exam, differences in arousal and excitability remained significant. The mean number of cigarettes consumed daily was positively correlated with lethargy (p = 0.013) and inversely with attention (p = 0.043). CONCLUSIONS: Neonates exposed in utero to tobacco showed worse neurobehavioral performance between 24 and 48 hours of life. KEYWORDS: Tobacco; Prenatal exposure delayed effects; Pregnancy in adolescence; Infant; Newborn behavior. Barros MCM, Mitsuhiro SS, Chalem E, Laranjeira RR, Guinsburg R. Prenatal tobacco exposure is related to neurobehavioral modifications in infants of adolescent mothers. Clinics. 2011;66(9):1597-1603. Received for publication on March 26, 2011; First review completed on May 10, 2011; Accepted for publication on June 3, 2011 E-mail: marinamoraesbarros@uol.com.br Tel.: 55 11 5084-0535

smokers by the time they completed high school.4 In a study published in 2010, 14.2% of 1,367 private-school–attending female adolescents from 28 schools in Sa˜o Paulo, Brazil, reported that they had used tobacco in the past month.5 Tobacco use during pregnancy is a public health problem. Approximately 13% of American women report daily tobacco use during the last three months of pregnancy.6 Bloch et al. examined pregnant women’s use of cigarettes and other tobacco products in nine nations in Latin America, Asia, and Africa. A face-to-face survey was administered to 7,961 pregnant women (more than 700 per site) between Oct 2004 and Sept 2005. The highest levels of current smoking were found in Uruguay (18.3%), Argentina (10.3%), and Brazil (6.1%).7 Furthermore, among substances of abuse, tobacco is the one that women are least prone to

INTRODUCTION In 2007, there were 183,440 births to mothers aged 15–19 years in the United States, a birth rate of 42.5 per 1,000 women in this age group.1 In Brazil in 2007, 21% of births occurred in adolescent mothers (10 to 20 years old).2 Adolescents have a high prevalence of substance abuse.3 Despite a significant decline in current smoking frequency since 1996, in 2008, one-fifth of young Americans were

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discontinue during pregnancy.8 Regarding data on pregnancy during adolescence and smoking during pregnancy, a Canadian study of 1,134 women done by Johnson et al. found that smokers during pregnancy were 2.4 times more likely to be under 25 years of age.9 In addition, pregnancy during adolescence is associated with lower socio-economic level, lack of employment, and the presence of a dysfunctional family.10 Therefore, the relationship among smoking, pregnancy, and adolescence is complex and mediated by several contextual factors that are not completely understood. Maternal smoking during gestation can interfere with fetal growth and development in different ways. Tobacco metabolites are vasoconstrictors, reducing the uterine blood flow and causing fetal intrauterine growth retardation.11 In the central nervous system, nicotine upregulates nicotinic cholinergic receptor binding sites, interfering with synaptic activity,12 leading to fetal neuronal damage and loss. Even low levels of cigarette smoking during gestation can interfere with fetal growth and cerebral development because the fetal nicotine concentration is 15% higher than the maternal concentration.13 Although there are many studies on the effects of maternal smoking during gestation, few of them focus on neonatal neurobehavioral consequences. Using different tools to assess neonatal behavior, these studies show that newborn infants of mothers who use tobacco during pregnancy are excitable and irritable, have more disturbances of tone and abnormal reflexes, need more handling, and are less alert.14-16 Among the different tools used in the literature, the Neonatal Intensive Care Unit Neurobehavior Network Scale (NNNS) was specifically designed to characterize the neonatal effects of prenatal drug exposure. Only one study has been published using the NNNS to assess the neurobehavioral effects of antenatal tobacco exposure in infants during the first days of life. Law et al.17 found that infants exposed to tobacco during gestation were more excitable and hypertonic; they required more handling and showed more stress/abstinence signs than non-exposed neonates in the first 48 hours of life. This study aimed to determine whether prenatal exposure to tobacco interferes with the neurobehavior of newborn infants of adolescent mothers assessed between 24 and 72 hours of life and to determine whether a dose–response relationship exists between the number of cigarettes smoked during gestation and neurobehavioral assessment.

19 years, 11 months and 29 days; and the infant was a fullterm newborn, defined as having a gestational age between 37 weeks and 41 weeks and six days, according to the best obstetric estimate or by the New Ballard method.18 Neonates with conditions or risk factors that could potentially interfere with their neurobehavioral assessment were excluded. The following groups of neonates were excluded: those whose mothers had positive serology for syphilis, toxoplasmosis, cytomegalovirus or human immunodeficiency virus; those whose mothers were diagnosed with maternal depression, anxiety and/or post-traumatic stress disorders during pregnancy; those whose mothers used antidepressant medications during gestation; those whose mothers received opioids, sedatives, and/or anticonvulsants 24 hours prior to delivery or systemic anesthesia during delivery; neonates with antenatal exposure to alcohol, marijuana, cocaine or other illicit drugs; multiple births; neonates with Apgar scores less than 3 in the 1st minute or less than 7 in the 5th minute of life; infants with major congenital malformations or genetic syndromes; those small or large for gestational age; and/or those with any clinical problems, defined by a need for an incubator, oxygen, vascular access, oral tube and/or any medication. Intra-uterine exposure to alcohol, marijuana, cocaine or other illicit drugs was identified by the Composite International Diagnostic Interview (CIDI 2.1).19 Marijuana and cocaine use during gestation were also identified by toxicological analysis of maternal hair and newborn infant meconium samples. A 3-cm segment of hair near the mother’s scalp was analyzed by semi-quantitative enzymatic immunoassay after initial decontamination. All positive results were confirmed by gas chromatography and mass spectrometry. Meconium samples were collected in the first 48 h and analyzed by a homogeneous semiquantitative enzymatic immunoassay. Neonates and their mothers were studied by a team of five neonatologists, three psychologists and two psychiatrists, and the following steps were performed: 1) Maternal interview by the neonatologists to collect data related to the socio-demographic and obstetrical characteristics of the mothers; 2) administration of CIDI 2.1 to the mothers by the psychologists; 3) clinical examination of the neonate to collect data related to the birth and clinical course until inclusion in the study; 4) neurobehavioral assessment of the infants by the neonatologists with NNNS;20 5) collection of maternal hair and neonatal meconium samples for analysis regarding the presence of marijuana and cocaine metabolites. CIDI is a standardized and structured interview that identifies psychiatric disorders according to the International Classification of Diseases (CID-10) and the 4th edition of the Diagnostic and Statistical Manual of Mental Disorders as follows: depression; anxiety; post-traumatic stress disorder (PTSD); mania; bipolar disorder; psychotic, dissociative, somatoform or eating disorders; and tobacco, alcohol, or illicit drug use. Tobacco use during gestation was classified as harmful tobacco use (a pattern of psychoactive drug use that causes damage to health, either mental or physical) or as tobacco dependence syndrome (a cluster of physiological, behavioral and cognitive phenomena that indicate the use of a substance takes on a much higher priority for a given individual than other behaviors that once had greater value).21

MATERIALS AND METHODS This cross-sectional study with prospective data collection was performed in the Maternity Hospital Mario de Moraes Altenfelder Silva, a third-level, city-owned hospital in Saõ Paulo, Brazil, between July 2001 and November 2002. The study was approved by the ethical committees of the hospital and the Federal University of Saõ Paulo and was funded by the State of Saõ Paulo Research Support Foundation (FAPESP – grant number 2000/10.293-5). After birth, one of the investigators explained the study and read the informed consent to each adolescent mother. After resolving possible doubts, the investigator asked her to sign the consent form. Term neonates were included in the study based on the following criteria: a maternal informed consent form had been signed; the mother was an adolescent aged 10 years to

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performed using Statistical Package for Social Sciences (SPSS) version 10.0 (SPSS Inc., Chicago, IL).

NNNS evaluates neurological integrity, behavioral function, and the presence of stress and abstinence signs in newborn infants. The NNNS was administered after 24 hours of life, when the global stress response to the birth process was already attenuated, and before 72 hours of life. The exam was carried out in a specific warm, calm room free of intense light by one of four neonatologists previously trained in its procedures. The principal investigator was certified to perform the NNNS at Rhode Island Hospital, Brown University, RI. The other three were trained by the principal investigator. All investigators were masked to tobacco exposure status when performing NNNS. After the complete NNNS evaluation, the items assessed were grouped into 13 variables as described by Boukydis et al.:22 habituation, attention, arousal, regulation, handling, quality of movement, excitability, lethargy, non-optimal reflexes, asymmetry, hypertonicity, hypotonicity, and stress/abstinence signals. Normative data for healthy term infants in the first three days of life were obtained from Tronick et al. (2004).23

RESULTS From July 2001 to November 2002, 3,685 infants were born in the study hospital, and 928 (25%) of them had teenage mothers. Among these, 131 were preterm, five were postterm, and 404 met one or more exclusion criteria: positive serology for congenital infections (26); maternal use of opioids/sedatives 24 hours prior to delivery (7); maternal use of alcohol (except occasional use) or any marijuana and/ or cocaine use (52); maternal depression, anxiety and/or post-traumatic stress disorder (166); multiple gestations (6); Apgar score less than 3 at 1 minute or less than 7 at 5 minutes (16); major congenital malformations or genetic syndromes (2); small or large for gestational age (203); and clinical problems (65). Therefore, 388 infants were studied: 23 infants with antenatal exposure to tobacco and 365 who had not been exposed. Among smoking pregnant women, five had tobacco dependence syndrome, and 18 were harmful tobacco users. They smoked an average of 14¡9 cigarettes per day during pregnancy, varying from 2 to 40. Characteristics of the mothers who smoked and those who did not smoke during gestation are displayed in Table 1. Mothers who smoked during gestation had had fewer years of schooling (p = 0.001) and more prenatal care visits (p = 0.020). Both groups were similar regarding maternal age, race, marital status, socioeconomic level, per capita income, delivery mode, and anesthesia type. Neonatal characteristics are shown in Table 2. The neonates of the two groups were similar in gestational age, gender, birth weight, length, head circumference, 1stand 5th-minute Apgar scores, post-natal age at the neurologic exam, time between last feeding and the neurologic exam and length of hospital stay. The neurologic exam of infants exposed to tobacco during gestation lasted longer than of those not exposed (25.7¡7.5 vs. 22.4¡5.2 minutes; p = 0.004). The neurobehavioral scores of the two studied groups are shown in Table 3. Infants exposed to tobacco had higher scores on arousal (4.13¡0.78 vs. 3.69¡0.69; p = 0.004), excitability (3.83¡2.19 vs. 2.64¡1.83; p = 0.003), and stress and/or abstinence signals (0.09¡0.06 vs. 0.06¡0.05; p = 0.019) and lower scores on regulation (5.67¡1.01 vs. 6.05¡0.77; p = 0.025). No differences were observed in the other NNNS

STATISTICAL ANALYSES Maternal and neonatal characteristics and NNNS scores were compared between tobacco-exposed and non-exposed neonates using the chi-square or Student’s t test. The associations of independent factors with the mean scores of the NNNS variables were tested by analysis of variance (ANOVA). The independent variables analyzed were tobacco exposure (absent vs. present), anesthesia (absent or local vs. regional), type of delivery (vaginal vs. cesarean section), gender (female vs. male), age of the neonate at neurobehavioral exam (#33 or .33 hours), time between last feeding and neurologic exam (#21 or .21 minutes), and exam duration (#25 or .25 minutes). For the last three variables, cutoffs were chosen based on the median value obtained for the whole studied population. Relationships between maternal cigarette consumption during gestation and neurobehavioral scores were assessed by linear regression analysis. The sample size was determined according to the need to include 15 neonates for each independent variable analyzed in the regression models. Each of the 13 NNNS variables was considered a dependent variable. At least 11 independent categorical variables and five continuous independent variables were analyzed. Therefore, the minimum sample size was calculated as 240. All statistical procedures were

Table 1 - Demographic characteristics of the adolescent mothers. Exposure to Tobacco during gestation

Maternal age 10 to 14 years 15 to 19 years White race Married Years in school Low socioeconomic level Per capita income (US$ per month) Prenatal care Present Number of visits Vaginal delivery Regional anesthesia

Absent n = 365

Present n = 23

p-value

17.0¡1.5 22 (6%) 343 (94%) 179 (49%) 230 (63%) 7.1¡2.2 327/350 (93%) 194¡133

17.3¡1.4 1 (4%) 22 (96%) 7 (30%) 33 (61%) 5.6¡2.2 22/23 (96%) 141¡87

0.284 1.000

352 (96%) 6.7¡2.7 265 (73%) 278 (76%)

21 (91%) 8.23¡4.8 15 (65%) 17 (74%)

0.221 0.020 0.474 0.803

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Table 2 - Neonatal demographic characteristics and neonatal data at the time of NNNS assessment. Exposure to Tobacco during gestation

Gestational age (weeks) Mean¡sd Median (minimum–maximum) Birthweight (g) Mean¡sd Median (minimum–maximum) Length (cm) Head circumference (cm) Male Apgar at 1 minute Mean¡sd Median (minimum–maximum) Apgar at 5 minutes Mean¡sd Median (minimum–maximum) Post-natal age (hours) Mean¡sd Median (minimum–maximum) Exam duration (minutes) Mean¡sd Median (minimum–maximum) Time after feeding (minutes) Mean¡sd Median (minimum–maximum) Hospital stay (days)

Absent (n = 365)

Present (n = 23)

39.4¡1.1 39.4 (37.0–41.9)

39.5¡1.0 39.4 (37.1–41.6)

3237¡292 3225 (2600–4010) 48.9¡1.5 34.4¡1.1 193 (53%)

3261¡308 3245 (2550–3770) 49.4¡1.4 34.4¡1.1 16 (70%)

8.1¡1.3 9 (3–10)

8.1¡1.2 8 (4–9)

9.6¡0.6 10 (8–10)

9.5¡0.6 10 (8–10)

33.3¡6.9 32.4 (24–51.3)

33.9¡6.9 34.5 (24.2–46)

22.4¡5.2 20 (10–43)

25.7¡7.5 25 (10–45)

49.0¡54.7 30 (2–300) 2.5¡0.9

36.5¡46.2 15 (5–150) 2.4¡0.5

p-value 0.588

0.704

0.109 0.948 0.135 0.817

0.506

0.660

0.004

0.287

0.900

effects of prenatal tobacco exposure on the neurobehavior of newborn infants of adolescent mothers. In the present study, we administered the NNNS on the second or third day of life to evaluate the neurobehavior of infants of adolescent mothers who smoked tobacco during gestation. Compared to non-exposed infants, infants exposed to tobacco aroused easily, were more excitable, had poorer self-control, and had more stress and/or abstinence signals. After controlling for possible confounders, arousal and excitability scores remained significantly different from the controls. Not only were these items associated with antenatal tobacco exposure, but attention and lethargy scores were also correlated with the number of cigarettes smoked during pregnancy, with a decrease in neurologic performance as the number of cigarettes smoked during pregnancy increased. Other studies that have used different neurobehavioral exams have shown similar results. Newborn infants

items: habituation, attention, handling, quality of movements, lethargy, non-optimal reflexes, asymmetry, hypertonicity, and hypotonicity. When adjusted for type of anesthesia, delivery mode, gender, age at the neurologic exam, exam duration, and time between the last feeding and the exam, differences in arousal and excitability remained significant (Table 3). Linear regression analysis showed that the number of cigarettes smoked during gestation was positively correlated with the lethargy score (Score = 0,148 6 number of cigarettes + 1.961; r = 0.545; p = 0.013) and inversely correlated with the attention score (Score = 20.069 6 number of cigarettes + 6.574; r = 0.496; p = 0.043) (Figures 1 and 2).

DISCUSSION Because 21% of the births in Brazil occur in adolescents,2 a group with frequent tobacco use, it is important to know the Table 3 - NNNS scores in the studied population.

Exposure to Tobacco during gestation

Habituation Attention Arousal Regulation Handling Quality of movement Excitability Lethargy Non-optimal reflexes Asymmetry Hypertonicity Hypotonicity Stress/abstinence signals *

Absent n = 365

Present n = 23

p-value

p-value*

6.90¡1.44 (n = 222) 5.75¡1.32 (n = 337) 3.69¡0.69 6.05¡0.77 0.35¡0.26 5.25¡0.45 2.64¡1.83 4.60¡2.72 9.27¡1.34 0.75¡1.10 0.18¡0.38 0.11¡0.35 0.06¡0.05

6.72¡1.73 (n = 12) 5.50¡1.35 (n = 20) 4.13¡0.78 5.67¡1.01 0.41¡0.26 5.15¡0.54 3.83¡2.19 4.09¡2.35 9.17¡1.39 0.48¡0.79 0.26¡0.45 0.22¡0.42 0.09¡0.06

0.686 0.417 0.004 0.025 0.290 0.280 0.003 0.376 0.728 0.239 0.321 0.154 0.019

0.784 0.462 0.031 0.085 0.637 0.460 0.037 0.389 0.597 0.132 0.307 0.139 0.055

adjusted for type of anesthesia, delivery mode, gender, age at neurologic exam, exam duration, and time between last feeding and the exam.

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Figure 1 - Correlation between the number of cigarettes consumed during gestation (X) and neonatal attention score (Y).

Figure 2 - Correlation between the number of cigarettes consumed during gestation (X) and neonatal lethargy score (Y).

exposed to tobacco during gestation and evaluated by the Neonatal Behavioral Assessment Scale (NBAS) between days two and six of life had worse performance on attention tasks, were more difficult to comfort and showed weaker autonomic regulation compared with non-exposed infants.24,25 In another study, Fried et al.14 evaluated infants at 30 days of life and noted associations between prenatal tobacco exposure and hypertonicity and excitability using the Prechtl scale. Oyemade et al.26 administered the NBAS to infants exposed in utero to tobacco and observed worse performance on orientation tasks. On the other hand, some studies did not find neurobehavioral alterations in infants exposed to tobacco during pregnancy.27-29 Many of these studies evaluated tobacco effects as covariates or analyzed tobacco exposure together with other drugs, such as cocaine, alcohol, caffeine, and/or marijuana. Using the NNNS to evaluate the neurobehavior of infants exposed to tobacco during gestation, Law et al.17 found that these infants were more excitable and hypertonic, required more handling and showed more stress/abstinence signs in the first 48 hours of life. They also showed relationships between higher maternal salivary cotinine level and stress and/or abstinence signals and excitability scores. The number of cigarettes smoked per day during pregnancy was also correlated with neonatal stress and abstinence signs. Stroud et al.30 evaluated term infants exposed in utero to tobacco, aged 10 to 27 days, with the NNNS. Compared to non-exposed infants, the exposed infants had a greater need for handling, worse self-regulation, and a tendency to show greater excitability and arousal. The authors noted that these results suggest that the behavioral effects of nicotine are not only due to a withdrawal process but a persistent deregulation, indicating early vulnerability to later neurobehavioral deficits. This hypothesis is supported by the fact that the half-life of nicotine in newborn infants is approximately 9â&#x20AC;&#x201C;11 hours, and the patterns of maternal smoking effects during pregnancy on infant neurobehavior at 10 to 27 days were different from those at one to two days of life, when infants showed stress/abstinence signals and increased muscle tension.30 Yolton et al.31 assessed infants exposed to tobacco in utero at five weeks after birth with the NNNS. After controlling for possible confounders, a higher maternal cotinine level was associated with increased

arousal and excitability and decreased self-regulation in white infants. In contrast, among black infants, a higher cotinine level was associated with decreased arousal and excitability and increased self-regulation and hypotonicity. These effects may reflect racial differences in nicotine metabolism. Our study reinforces the previous results that newborn infants antenatally exposed to tobacco arouse easily, are more excitable, and have worse self-regulation and more stress and abstinence signals. After adjustment for covariates, tobacco-exposed infants showed higher arousal and excitability scores, suggesting that antenatal tobacco exposure may have neurotoxic effects. Nicotine acts on neurotransmitter receptors in the fetal brain, interfering with cell proliferation and differentiation and with synaptic activity.12 In one study, newborn infants of adolescent mothers, assessed between two and five days of life by the Neonatal Brazelton Scale, habituated easier but were less alert and had poorer performance on orientation tasks compared with infants of non-adolescent mothers.32 The combination of an excitable and easily aroused infant and an adolescent mother could interfere with motherâ&#x20AC;&#x201C;infant bonding during a critical period of development, leading to long-term behavioral deficits.12,33-35 In the present study, mothers smoked an average of 14.1 cigarettes per day, more than reported by Law et al.17 (12.9 cigarettes before pregnancy recognition, 7.5 in the first trimester, 3.8 in the second, and 2.8 in the third). They found that the motherâ&#x20AC;&#x2122;s salivary cotinine level was associated with a higher frequency of stress/abstinence signs and increased excitability scores. In our study, the number of cigarettes consumed during gestation was related to lower attention and higher lethargy scores. We did not verify the number of cigarettes consumed during the different trimesters of gestation, and we did not measure cotinine levels, but salivary cotinine level only reflects tobacco use within the last two days.36 Because the infants in our study were evaluated between 24 and 72 hours of life, it is unlikely that the tobacco effects on newborn neurobehavior were related to passive inhalation, as smoking is not allowed in the hospital. The large number of exclusion criteria and the adjustment for confounders in the statistical analysis are among the strengths of our study, which allowed us to verify the

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8. Kahn RS, Certain L, Whitaker R. A reexamination of smoking before, during, and after pregnancy. Am J Public Health. 2002;92:1-11. 9. Johnson IL, Ashley MJ, Reynolds D, Goettler F, Lee-Han H, Stratton J, et al. Prevalence of smoking associated with pregnancy in three Southern Ontario Health Units. Can J Public Health. 2004;95:209-13. 10. Gama SGN, Szwarcwald CL, Leal MC. Pregnancy in adolescence, associated factors, and perinatal results among low-income post-partum women. Cad. Sau´ de Pu´ blica 2002;18:153-61, doi: 10.1590/S0102311X2002000100016. 11. Suzuki K, Minei LJ, Johnson EE. Effect of nicotine upon uterine blood flow in the pregnant rhesus monkey. Am J Obstet Gynecol. 1980;136:1009–13. 12. Slotkin TA. Fetal nicotine or cocaine exposure: Which one is worse? J Pharmacol Exp Ther. 1998;285:931–45. 13. Nau H, Hansen R, Steldinger R. Extent of nicotine and cotinine transfer to the human fetus, placenta and amniotic fluid of smoking mothers. Dev Pharmacol Ther. 1985;8:384–95. 14. Fried PA, Watkinson B, Dillon RF, Dulberg CS. Neonatal neurological status in a low-risk population after prenatal exposure to cigarettes, marijuana, and alcohol. J Dev Behav Pediatr. 1987;8:318–26, doi: 10.1097/ 00004703-198712000-00003. 15. Godding V, Bonnier C, Fiasse L, Michel M, Longueville E, Lebecque P, et al. Does in utero exposure to heavy maternal smoking induce nicotine withdrawal symptoms in neonates? Pediatr Res. 2004;55:645–51, doi: 10. 1203/01.PDR.0000112099.88740.4E. 16. Stroud LR, Paster RL, Goodwin MS, Shenassa E, Buka S, Niaura R, et al. Maternal smoking during pregnancy and neonatal behavior: a large scale community study. Pediatrics. 2009;123:e842–e848, doi: 10.1542/peds. 2008-2084. 17. Law KL, Stroud LR, LaGasse LL, Niaura R, Liu J, Lester BM. Smoking during pregnancy and newborn neurobehavior. Pediatrics. 2003;111: 1318–23, doi: 10.1542/peds.111.6.1318. 18. Ballard JL, Khoury JC, Wedig K, Wang L, Eilers-Walsman BL, Lipp R. New Ballard Score, expanded to include extremely premature infants. J Pediatr. 1991;119:417-23, doi: 10.1016/S0022-3476(05)82056-6. 19. World Health Organization. Composite International Diagnostic Interview (CIDI). Geneve: WHO, 1997. 20. Lester BM, Tronick EZ. The neonatal intensive care unit network neurobehavioral scale procedures. Pediatrics. 2004;113:641-67. 21. World Health Organization. The ICD-10 classification of mental and behavioral disorders: clinical descriptions and diagnostic guidelines. Geneva; 1992. 22. Boukydis CFZ, Bigsby R, Lester BM. Clinical use of the neonatal intensive care unit network neurobehavioral scale. Pediatrics. 2004;113:679-89. 23. Tronick EZ, Olson K, Rosenberg R, Bohne L, Lu J, Lester BM. Normative Neurobehavioral Performance of Healthy Infants on the Neonatal Intensive Care Unit Network Neurobehavioral Scale. Pediatrics. 2004;113:676-8. 24. Saxton DW. The behavior of infants whose mothers smoke in pregnancy. Early Hum Dev. 1978;2:363-9, doi: 10.1016/0378-3782(78)90063-4. 25. Picone TA, Allen LH, Schramm MM, Olsen PN. Pregnancy outcome in North American women: I. Effects of diet, cigarette smoking, and psychological stress on maternal weight gain. Am J Clin Nutr. 1982;36:1205-13. 26. Oyemade UJ, Cole OJ, Johnson AA, Knight EM, Westney OE, Laryea H, et al. Prenatal substance abuse and pregnancy outcomes among AfricanAmerican women. J Nutr. 1994;124:994S-9S. 27. Richardson GA, Day NL, Taylor PM. The effect of prenatal alcohol, marijuana, and tobacco exposure on neonatal behavior. Infant Behav Dev. 1989;12:199-209, doi: 10.1016/0163-6383(89)90006-4. 28. Epsy KA, Riese ML, Francis DJ. Neurobehavior in preterm neonates exposed to cocaine, alcohol and tobacco. Infant Behav Dev. 1997;20:297309, doi: 10.1016/S0163-6383(97)90002-3. 29. Jacobson SW, Fein GG, Jacobson JL, Schwartz P, Dowler J. Neonatal correlates of prenatal exposure to smoking, caffeine and alcohol. Infant Behav Dev. 1984;7:253-65, doi: 10.1016/S0163-6383(84)80041-7. 30. Stroud LR, Paster RL, Papandonatos GD, Niaura R, Salisbury AL, Battle C, et al. Maternal smoking during pregnancy and newborn neurobehavior: effects at 10 to 27 days. J Pediatr. 2009;154:10–6, doi: 10.1016/j.jpeds.2008. 07.048. 31. Yolton K, Khoury J, Xu Y, Succop P, Lanphear B, Bernert JT, et al. Lowlevel prenatal exposure to nicotine and infant neurobehavior. Neurotoxicol Teratol. 2009;31:356-63, doi: 10.1016/j.ntt.2009.07.004. 32. Thompson Jr RJ, Cappleman MW, Zeitschel KA. Neonatal behavior of infants of adolescent mothers. Develop Med Child Neurol. 1979;21:47482, doi: 10.1111/j.1469-8749.1979.tb01651.x. 33. Wakschlag LS, Lahey BB, Loeber R, Green SM, Gordon RA, Leventhal BL. Maternal smoking during pregnancy and the risk of conduct disorder in boys. Arch Gen Psychiatry. 1997;54:670-6. 34. Leech SL, Richardson GA, Goldschmidt L, Day NL. Prenatal substance exposure: effects on attention and impulsivity of 6-year-olds. Neurotoxicol Teratol. 1999;21:109-18, doi: 10.1016/S0892-0362(98)00042-7.

specific effects of tobacco use during pregnancy on neonatal neurobehavior. At the same time and for the same reason, tobacco-exposed infants represented only 6% of our unbalanced sample, which may be a limitation of the study. A post-hoc analysis to verify the power to detect differences between exposed and non-exposed infants in the variables that remained significant after adjustment for confounders (arousal and excitability) was performed. We found that the power of the study was greater than 90% for the differences and standard deviations observed, with an alpha error of 5%. Moreover, for all studied variables, 18 exposed and non-exposed infants would be required to detect a difference of 1 point in the score, with a standard deviation of 0.8, a power of 80% and an alpha error of 5%. The cross-sectional design of this study is another limitation. It would be interesting to longitudinally follow the neurobehavioral skills of newborn infants of adolescent mothers exposed and not exposed in utero to tobacco. A single neurobehavioral evaluation should only be used as a screening tool to identify neurobehavioral problems in these groups of newborns.

CONCLUSION Neonates exposed to tobacco during pregnancy showed worse neurobehavioral performance between 24 and 48 hours of life. Our findings raise concerns about the interaction between families and infants exposed in utero to tobacco, regarding their neurodevelopment. These data reinforce the importance of prenatal care and should encourage pregnant women to reduce or quit tobacco consumption during gestation.

ACKNOWLEDGMENTS We are grateful to the staff of the Maternity Hospital Mario de Moraes Altenfelder Silva for their help during the data collection. FUNDING: This research was fully funded as a thematic project by FAPESP (State of Sa˜o Paulo Research Support Foundation), grant number 2000/10293-5.

REFERENCES 1. Martin JA, Hamilton BE, Sutton PD, Ventura SJ, Matheus TJ, Kirmeyer S, et al. Births: Final data for 2007. Natl Vital Stat Rep. 2010;58:1-44. Available from: http://www.cdc.gov/nchs/data/nvsr/nvsr58/nvsr58_24.pdf 2. Brasil - Ministe´rio da Sau´de – DATASUS [homepage on the Internet]. Informac¸o˜es de Sau´de-Estatı´sticas Vitais – Nascidos Vivos – 1994 a 2007 [cited 2010 October 4]. Available from: http://tabnet.datasus.gov.br/ cgi/tabcgi.exe?sinasc/cnv/nvuf.def. 3. Evans DL, Foa EB, Gur RE, Hendin H, O9Brien CP, Seligman ME, et al. Preventing adolescent mental health disorders: what we know and what we don’t know. A research agenda for improving the mental health of our youth. New York: Oxford University Press, 2005. 4. Johnston LD, O9Malley PM, Bachman JG, Schulenberg JE. Monitoring the Future - National Survey Results on Drug Use, 1975-2008.Volume II. College Students and Adults Ages 19–50. National Institute on Drug Abuse. National Institutes of Health. U.S. Department of Health & Human Services 2009. pg.306 5. Sanchez ZM, Opaleye ES, Martins SS, Ahluwalia JS, Noto AR. Adolescent gender differences in the determinants of tobacco smoking: a cross sectional survey among high school students in Sa˜o Paulo. BMC Public Health. 2010;10:748, doi: 10.1186/1471-2458-10-748. 6. Department of Health and Human Services. Centers for Disease Control and Prevention. Tobacco Use and Pregnancy [cited 2010 October 4]. Available from: http://www.cdc.gov/reproductivehealth/tobaccousepregnancy/ index.htm 7. Bloch M, Althabe F, Onyamboko M, Kaseba-Sata C, Castilla EE, Freire S, et al. Tobacco use and secondhand smoke exposure during pregnancy: an investigative survey of women in 9 developing nations. Am J Public Health. 2008;98:1833-40, doi: 10.2105/AJPH.2007.117887.

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Prenatal Tobacco Exposure and Neonatal Behavior Barros MCM et al. 36. Benowitz JP. Metabolism of nicotine to cotinine studied by a dual stable isotope method. Clin Pharmacol Ther. 1994;56:483â&#x20AC;&#x201C;93, doi: 10.1038/clpt. 1994.169.

35. Fergusson DM, Woodward LJ, Horwood LJ. Maternal smoking during pregnancy and psychiatric adjustment in late adolescence. Arch Gen Psychiatry. 1998;55:721-7, doi: 10.1001/archpsyc.55.8.721.

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DOI:10.1590/S1807-59322011000900017

BASIC RESEARCH

The effects of antidepressants and pilocarpine on rat parotid glands: an immunohistochemical study ˆ ngela Naval Tatiana Maria Folador Mattioli,I Silvana da Silva,I Ana Maria Trindade Gre´gio,II Maria A III III II Machado, Antoˆnio Adilson Soares de Lima, Luciana Reis Azevedo-Alanis I

Department of Stomatology, School of Dentistry, Pontifical Catholic University of Parana´, Curitiba, Brazil. II School of Dentistry, Pontifı´cia Universidade Cato´lica do Parana´, Brazil. III Department of Stomatology, School of Dentistry, Universidade Federal do Parana´, Brazil.

OBJECTIVES: To evaluate the effects of antidepressants and pilocarpine on the quantity of myoepithelial cells and on the proliferation index of the epithelial cells of rat parotid glands. INTRODUCTION: Hyposalivation, xerostomia, and alterations in saliva composition are important clinical side effects related to the use of antidepressants. METHODS: Ninety male Wistar rats were allocated to nine groups. The control groups received saline for 30 (group C30) or 60 days (group C60) or pilocarpine for 60 days (group Pilo). The experimental groups were administered fluoxetine (group F30) or venlafaxine for 30 days (group V30); fluoxetine (group FS60) or venlafaxine (group VS60) with saline for 60 days; or fluoxetine (group FP60) or venlafaxine (group VP60) with pilocarpine for 60 days. Parotid gland specimens were processed, and the immunohistochemical expression of calponin and proliferating cell nuclear anti-antigen on the myoepithelial and parenchymal cells, respectively, was evaluated. Analysis of variance (ANOVA), Tukey HSD and Games-Howell tests were applied to detect differences among groups (p,0.05). RESULTS: Compared with the controls, chronic exposure to antidepressants was associated with an increase in the number of positively stained cells for calponin. In addition, venlafaxine administration for 30 days was associated with an increase in the number of positively stained cells for proliferating cell nuclear anti-antigen. Fluoxetine and pilocarpine (group FP60) induced a significant decrease in the number of positively stained cells for calponin compared with all other groups. CONCLUSIONS: The number of positively stained cells for calponin increased after chronic administration of antidepressants. The proliferation index of the epithelial cells of rat parotid glands was not altered by the use of antidepressants for 60 days. KEYWORDS: Antidepressants; Immunohistochemistry; Salivary glands; Rats; Saliva. Mattioli TMF, Silva S, Gre´gio AMT, Machado MAN, Lima ADS, Azevedo-Alanis LR. The effects of antidepressants and pilocarpine on rat parotid glands: an immunohistochemical study. Clinics. 2011;66(9):1605-1610. Received for publication on March 10, 2011; First review completed on April 5, 2011; Accepted for publication on May 23, 2011 E-mail: l.azevedo@pucpr.br Tel.: 55 41 3271-2592

antidepressants differ from the classic tricyclic antidepressants and from the monoamine oxidase inhibitors, which are irreversible, because of their enhanced pharmacological selectivity and diminished side effects.5 In a Cochrane systematic review (1966-2004), the adverse effects of fluoxetine (dry mouth sensation, dizziness, and sudoresis) were compared with the adverse effects of the most recent antidepressants (venlafaxine, reboxetine, phenelzine, and nefazodone), and the adverse effects were shown to be less pronounced in the more recent drugs.6 Xerostomia is defined as a subjective sensation of dry mouth reported by the patient. It can result from a reduction in saliva secretion, but it can also occur in the presence of a normal salivary flow rate.7 Stimulated salivary flow rate (SSFR) values of #0.7 mL/min are considered to indicate hyposalivation.8 Hyposalivation, xerostomia, and alterations in the saliva composition are important side effects related to the use of psychotropic medications, including

INTRODUCTION The current generation of antidepressants includes drugs that only act on one neurotransmitter, such as the serotonin (fluoxetine) or the noradrenaline (reboxetine) selective reuptake inhibitors, and drugs that that act on multiple neurotransmitters (venlafaxine) without targeting other cerebral receptors that are not related to depression, such as histamine and acetylcoline.1-2 Venlafaxine is a serotonin and noradrenaline reuptake inhibitor, and it exerts a weak activity as a dopamine reuptake inhibitor, which is only clinically significant at high doses.3,4 The current

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antidepressants.7,9-14 The mechanism by which these side effects occur has still not been completely clarified; however, histomorphometric and gravimetric studies have contributed to a better understanding of the cytotoxic effects of psychotropic drugs on the salivary glands.12-15 Gre´gio et al.,12 studied the effects of chronic administration of a benzodiazepine (diazepam) and an antidepressant (amitryptiline) on the parotid glands of rats and observed hyposalivation and hypertrophy of the serous cells. These findings suggested a possible inhibition of the activity of the myoepithelial cells (originating from nervous stimulation), a decrease in the number of myoepithelial cells following chronic administration of psychotropic drugs, or an alteration in the number of acinar and ductal cells. The myoepithelial cells, which are located between the basal lamina and the plasmatic membrane of the acinar cells and intercalated ducts, are nonmuscular cells that exert contractile functions and contribute to the emptying of the secretion from the secretory units and the ducts.16,17 Calponin (C) is a protein located in myoepithelial cells that is involved in the regulatory system of smooth muscle contraction.18 Acinar and ductal cells are epithelial cells with proliferative potential for maintenance and regeneration.19-23 The aim of this study was to evaluate the quantity of myoepithelial cells and the proliferation index of acinar and ductal cells of the parotid glands of rats treated with fluoxetine and venlafaxine using immunoreactions with calponin and proliferating cell nuclear anti-antigen (PCNA).

We used tissue microarrays (TMAs) containing ten cylinders of paraffinized salivary glands (3 mm in diameter) organized in lines and columns.24,25 Each TMA exhibited ten specimens of salivary glands from the rats of each group. For immunohistochemistry, anti-calponin, and anti-PCNA (DakoCytomationH; Dako North America, Carpinteria, CA, USA) antibodies were used. The secondary antibody was EnVisionH+Dual Link/Peroxidase (DakoCytomationH), and the antigen-antibody reactions were revealed with a DAB substrate-chromogen system (DakoCytomationH). The slides were counterstained with Harris hematoxylin. We used an anti-calponin (C) monoclonal antibody (Dako Corporation) for myoepithelial cell staining and an antiPCNA antibody (Dako Corporation) for the staining of proliferating acinar and ductal cells. PCNA is a monoclonal antibody that allows the study of cell kinetics. The immunoreactions with C and PCNA used antibody dilutions of 15800 and 15400, respectively. Slides were visualized by only one examiner using an OlympusH BX50 optical microscope (Olympus Corporation, Ishikawa, Japan) with a 40X objective. The immunohistochemical expression of C in the myoepithelial cells and PCNA in the acinar and ductal cells was analyzed by interpreting the entire TMA area. The presence or absence of PCNA and C antibody staining was evaluated. Cells that exhibited any expression for PCNA or for C in the glandular epithelium were considered positive (represented by a brownish staining) regardless of the staining intensity. There were 24 histological fields in each cylinder of paraffinized tissue. Before counting the number of stained cells, we verified the number of fields (out of 24) that could be evaluated. Therefore, the integrity and quality of the tissue and the presence of technical artifacts were taken into consideration. After evaluating the 24 histological fields of all cylinders, the numerical indices for the positive staining of cells for C and for PCNA were obtained. To determine the average value of stained cells for C and PCNA from each cylinder, the average value of stained cells in a cylinder was represented by the sum of the number of stained cells for the antibody in each evaluated field divided by the number of evaluated fields. After obtaining the

MATERIALS AND METHODS This study was approved by the Research Ethics Committee at Universidade Tuiuti do Parana´ (CEP-UTP n. 55/2003). Parotid glands from male Wistar rats (Rattus norvegicus albinus, Rodentia, mammalian) with an approximate weight of 250 g were embedded in paraffin blocks from Laborato´rio de Patologia Experimental, Pontifı´cia Universidade Cato´lica do Parana´. Ninety animals were allocated to nine groups, and each group was composed of ten animals. The animals received different treatments, which are described in Table 1.15

Table 1 - Control and experimental groups according to the drugs used. Group

Drugs

Period of treatment (days)

Dose

Administration

123456-

C30 C60 Pilo F30 V30 FS60

10 10 10 10 10 10

7- VS60

8- FP60

9- VP60

Saline Saline Pilocarpine1 Fluoxetine2 Venlafaxine3 Fluoxetine Saline Venlafaxine Saline Fluoxetine Fluoxetine + Pilocarpine Venlafaxine Venlafaxine + Pilocarpine

1-30 1-60 1-60 1-30 1-30 1-30 31-60 1-30 31-60 1-30 31-60 1-30 31-60

0.1 mL 0.1 mL 0.05 mL 20 mg/kg 40 mg/kg 20 mg/kg 0.1 mL 40 mg/kg 0.1 mL 20 mg/kg 20 mg/kg/0.05 mL 40 mg/kg 40 mg/kg/0.05 mL

Intraperitoneal Intraperitoneal Topical Intramuscular Intramuscular Intramuscular Intraperitoneal Intramuscular Intraperitoneal Intramuscular Intramuscular and Topical Intramuscular Intramuscular and Topical

N = sample size. 1 Gel base prepared with 1% pilocarpine hydrochloride (Gerbras Quı´mica e Farmaceûtica Ltda., Saõ Paulo, Brazil). 2 Injectable fluoxetine (20040625, Galena Quı´mica e Farmaceûtica Ltda., Campinas, Brazil). 3 Injectable venlafaxine (D/VN/002/02, Galena Quı´mica e Farmaceûtica Ltda., Campinas, Brazil).

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average value for each cylinder, the values were added together and divided by ten (equivalent to the total number of TMAs), which resulted in the average value of stained cells for each group. In addition to the C and PCNA variables, this study also used the average values for the SSFR and cellular volume (CV) determined in a study by da Silva et al.15 Because the present study investigated the same sample as the da Silva et al. study, the SSFR and CV findings were compared with the immunohistochemical staining results for C and PCNA.

(p,0.05). Conversely, the highest average values for CV were observed in groups F30 and V30.

Groups treated for 60 days

The data were analyzed using Statistical Package for Social Sciences (SPSS) software version 15.0 for Windows. The normality analysis was performed using the Kolmogorov-Smirnov test, and the Levene test was used to analyze the variance of homogeneity. For the groups with a normal distribution, analysis of variance (ANOVA) at one criterion was performed. When ANOVA at one criterion showed differences among the groups and treatment, the Tukey HSD multiple comparison test was used for the variables that presented variance of homogeneity among the groups. For the variables that did not present variance homogeneity, the Games-Howell test was used. The level of significance for all the statistical tests was set at 5% (p,0.05).

Table 3 shows the average values, standard deviations and p-values for the studied variables in the groups treated for 60 days (C60, Pilo, FS60, VS60, FP60, and VP60). There were significant differences among the groups treated for 60 days for the CV, SSFR, and C variables (p,0.05). There was a significant increase in the number of positively stained cells for C in the group treated with fluoxetine and saline (FS60) compared with the control group (C60) (p = 0.0258). In addition, there was a significant decrease in the number of myoepithelial cells in group FP60 compared with all groups (p,0.05). SSFR was significantly increased in the group treated with venlafaxine and pilocarpine (VP60) compared with the group treated with venlafaxine and saline (VS60) (p = 0.0214). In addition, the highest SSFR average value was found in the Pilo group. Moreover, the lowest average value for CV was found in the Pilo group, whereas the highest CV value was found in the VP60 group (p,0.05). Figures 1-4 illustrate photomicrographs of the parotid glands of the rats in the experimental and control groups.

RESULTS

DISCUSSION

Three out of nine groups (C30, F30, and V30) were treated for 30 days, and six groups (C60, Pilo, FS60, VS60, FP60, and VP60) were treated for 60 days. The studied variables were C and PCNA, and they were compared with the CV and SSFR variables.15

The anticholinergic actions of psychotropic drugs have not been completely clarified. Many studies have suggested that central nervous system (CNS) drugs have pharmacological actions on the salivary glands. The attempts to clarify the xerostomic effects of such drugs have been of great scientific value, both in explaining the physiopharmacological effects and in bringing additional benefits to patients using these drugs. In the present study, the chronic use of fluoxetine and venlafaxine for 30 days was associated with a decrease in SSFR, an increase in CV and an increase in the number of myoepithelial cells in rats. In addition, venlafaxine was associated with an increase in acinar and ductal cell proliferation in the parotid glands. Interestingly, the use of pilocarpine with venlafaxine for 60 days re-established a normal SSFR in rats. The number of myoepithelial cells remained stable in the parotid glands of rats that were treated for 60 days with venlafaxine irrespective of whether it was administered with saline or pilocarpine. Pilocarpine used in association with fluoxetine for 60 days did not induce changes in SSFR, but it was associated with a severe

Statistical analysis

Groups treated for 30 days Table 2 shows the average values, standard deviations, and p-values for the studied variables in the groups treated for 30 days (C30, F30, and V30). There were significant differences among the groups treated for 30 days for the SSFR, CV, PCNA, and C variables (p,0.05). Compared with the control group (C30), there was a significant increase in the number of myoepithelial cells (positively stained cells for C) in the groups treated with antidepressants (V30 and F30) (p,0.05). In addition, there was a significant increase in the number of positively stained cells for PCNA in the group treated with venlafaxine (V30) compared with the C30 and F30 groups (p,0.05). SSFR was significantly decreased in the experimental (V30 and F30) groups compared with the control group

Table 2 - Mean values (X) and standard deviations (SD) of the studied variables in the groups treated for 30 days with saline (C30), fluoxetine (F30), or venlafaxine (V30). Groups

C30

F30

X SSFR (mL/min) CV (mm3)d PCNA C

SD a,b

0.051¡0.026 6,965.683¡3,792.951a,b 55.8¡14.382 a 13.9¡2.644 a,b

SD a

0.014 ¡ 0.006 10,384.311¡4,869.539a 59.4¡16.900 b 20¡4.216 a

SD b

0.026 ¡ 0.022 11,945.927¡7,891.179b 89.5¡16.400 a,b 19.8¡5.007 b

d Values obtained from the study by da Silva et al. (2009)15. SSFR – stimulated salivary flow rate; CV – cellular volume; PCNA – proliferating cell nuclear antigen; C – calponin. a,b Groups followed by the same letter differed statistically from each other. * Statistically significant difference among groups (p,0.05).

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p-value

V30

0.001* 0.000* 0.000* 0.003*

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Table 3 - Mean values (X) and standard deviations (SD) of the studied variables in the groups treated for 60 days with saline (C60), pilocarpine (Pilo), fluoxetine and saline (FS60), venlafaxine and saline (VS60), fluoxetine and pilocarpine (FP60), or venlafaxine and pilocarpine (VP60). Groups

C60 X

SSFR (mL/min) CV (mm3) d PCNA C

PILO SD

0.052¡0.029

FS60 SD

0.067¡0.028

a,b

VS60 SD

0.036¡0.017

FP60 SD

0.020¡0.004

a,c

p- value

VP60 SD

0.034¡0.014

SD c

0.000*

10,194.315¡ 4,456.345b,c,d,e,f 43.6¡18.007 18.2¡6.250 e

0.000*

0.055¡ 0.026

6,505.564¡ 3,343.475d 50¡17.531 13.6¡4.789 c,f

5,825.418¡ 1,968.070ab 46.8¡13.506 22.1¡7.385 a

6,809.347¡ 3,189.246e 36.7¡16.687 24.2¡7.955 b,c

7,525.112¡ 3,196.085ac 52.5¡18.710 19.7¡4.322 d

7,519.797¡ 4,272.808f 52¡17.153 7¡1.563 a,b,d,e,f

0.296ns 0.000*

Values obtained from the study by da Silva et al. (2009)15. SSFR – stimulated salivary flow rate; CV – cellular volume; PCNA – proliferating cell nuclear antigen; C – calponin. a,b,c,d,e,f Groups followed by the same letter differed statistically from each other. * Statistically significant difference among the groups (p,0.05). ns – No statistically significant difference among the groups (p.0.05). d

CV values in the parotid glands of rats that received antidepressants for 30 days (groups F30 and V30) appeared to represent acinar and ductal cell hypertrophy. Serous cell hypertrophy, which is characterized by widened acinar cells and secretory granule accumulation, has previously been reported following the chronic use of psychotropic drugs.12,26 The present study showed the anticholinergic effects of chronic fluoxetine and venlafaxine administration for 30 days . With 60-day treatment, the SSFR reduction was not significantly different than the control group. Studies have shown that the prolonged use of psychotropics causes alterations in receptor sensitivity.24 In addition, therapeutic effects and side effects gradually decrease after continuous or repeated psychotropic administration, which may cause desensitization, resistance, or tolerance. Physiological adaptation may also occur, and many side effects of drugs tend to diminish over time despite continuous use of a drug.27 Therefore, the least pronounced SSFR decrease in rats treated for 60 days may be a consequence of tolerance or adaptation to psychotropic drugs. The absence of a significant difference in the mean SSFR values among the C60, FS60, and VS60 groups suggested that the normal SSFR of the rats was re-established 30 days after suspending the medication (Table 3). These data must be carefully analyzed, however, because a decrease in SSFR

decrease in the number of myoepithelial cells in the parotid glands of rats. Considering these results, three main points must be discussed: the mechanisms of action of the medications on the CNS, physiological adaptation to the use of psychotropic medications, and alterations in receptor sensitivity due to the chronic use of medication. Fluoxetine and venlafaxine have anticholinergic effects of different intensities. Because fluoxetine is a selective serotonin reuptake inhibitor, it may cause greater serotonin availability in the synaptic gap, which alters the binding of acetylcholine to the muscarinic receptors (M3) present in the salivary glands. Thus, fluoxetine may decrease the quantity of salivary secretion. Venlafaxine is an atypical antidepressant and acts as a reuptake inhibitor of serotonin, noradrenaline, and dopamine.11 Venlafaxine does not show affinity for a1 adrenergic receptors, M3 muscarinic receptors, or histamine receptors. Interestingly, the actions of venlafaxine become unpredictable because it can act through multiple targets, which can elicit results ranging from a decrease in salivary secretion to alterations in salivary composition.13,21 Although antidepressant medications do not seem to block saliva production, they interfere with the binding of acetylcholine to M3 receptors. Thus, saliva is produced, but it cannot be excreted.12 Compared with the control groups (C30), the increase in

Figure 1 - Rat parotid gland with normal parenchyma. Immunohistochemical staining of PCNA on epithelial cells (C60, original magnification 400X).

Figure 2 - Immunohistochemical staining of PCNA on epithelial cells (FP60, original magnification 400X).

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Figure 3 - Immunohistochemical staining of calponin on myoepithelial cells (Pilo, original magnification 400X).

Figure 4 - Immunohistochemical staining of calponin on myoepithelial cells (V30, original magnification 400X).

greater than 50% brings relevant prejudicial consequences (C60 – 0.052 mL/min; VS60 – 0.020 mL/min). SSFR reduction in rats undergoing chronic treatment with fluoxetine may still be an indirect consequence of serotonin action at the 5-HT (5-hydroxytryptamine) receptors present in peripheral microcirculation. Indeed, fluoxetine increases serotonin availability, and serotonin may bind to 5-HT receptors, which would alter the blood flow in the salivary glands and, consequently, the quantity and composition of the salivary flow.28 In the present study, pilocarpine exerted a significant secretagogue effect, which showed its cholinergic agonist capacity and corroborated the findings of Davies and Shorthose.29 Pilocarpine was associated with an increase in SSFR and efficiently treated the hyposalivation caused by venlafaxine (groups VS60 and VP60). Conversely, when pilocarpine was used in association with fluoxetine (group FP60), there was no SSFR increase compared with the group that did not receive pilocarpine (group FS60 – Table 3). Pilocarpine is a parasympathomimetic agent that acts as a nonselective muscarinic receptor agonist.30,31 Thus, pilocarpine promotes stimulation of the exocrine glands and salivary secretion, which is efficient for patients without extensive glandular parenchymal destruction.30 In the present study, chronic administration of either fluoxetine or venlafaxine for 30 days was associated with an increase in the number of myoepithelial cells in the parotid glands of rats. These results contradict Gre´gio et al.12 who suggested that the hyposalivation and serous cell hypertrophy observed after chronic treatment with benzodiazepines and antidepressants in rats could be explained by a decrease in the number of myoepithelial cells.12 We hypothesized that a deficit in saliva elicits a compensatory activity that increases the number of myoepithelial cells, which have contractile functions, to assist in releasing the retained saliva. The myoepithelial cells accelerate the initial saliva emptying flow rate and promote the drainage of any adjacent extracellular fluid. The dendrites of the myoepithelial cells fold around the duct system, and their function is to compress the acinar and ductal cells.16,17 The parenchymatous cells (i.e., acinar, myoepithelial, and ductal) show a proliferative potential for maintenance and regeneration of the adult parenchymatous cell population in normal glands. In addition, parenchymatous cells show a

proliferative capacity when submitted to physical injury, such as ductal obstruction.19,20,32,33 This study was performed because there were no immunohistochemical studies of myoepithelial, acinar and ductal cells following the use of psychotropic drugs. There have been immunohistochemical studies on the proliferative capacity of parenchymatous cells in induced glandular atrophy,19,22,23,33 and a comparative analysis between the present study and the studies of induced glandular atrophy may be established. Interestingly, an animal model of induced atrophy of the parotid gland’s main duct did not prevent the gland from producing saliva. This saliva, however, was not excreted due to a physical obstruction in the main excretory duct. In an animal model of chronic antidepressant administration, we suggested that the saliva was continuously produced, but it was not secreted because of neurochemical interference from the psychotropic drugs. In animal models submitted to injuries (physical and neurochemical), there were significant increases in the number of myoepithelial cells.22,23,33 Because calponin is not a cellular proliferation marker, this study determined the numerical index of positively stained cells for calponin, which specifically identifies the myoepithelial cells in the glandular parenchyma. Due to the methodology that was employed, it was not possible to directly infer the cell proliferation, but it was possible to suggest that there was an alteration in the myoepithelial cellular proliferation rate among the studied groups. In the groups of rats treated for 30 days, venlafaxine (V30) seemed to induce an increase in acinar and ductal cell proliferation compared with saline (control group). In an attempt to compensate for the significant SSFR reduction that resulted from the chronic use of venlafaxine (V30), mitoses may have occurred to create new acini and ducts (Table 2). The intense proliferative capacity of acinar and ductal cells has already been reported in studies of glandular atrophy induced by obstruction.19 Moreover, the V30 group showed the highest mean CV value, which suggested the presence of cellular hypertrophy. It is worth emphasizing that there is a critical size for each cell, above which the stimulus for hypertrophy does not cause adaptative reactions, and the cell goes through mitosis to compensate for the excessive cytoplasm.34 We believe that the proliferative capacity of the acinar and ductal cells and the presence of cellular hypertrophy may have contributed

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to the increase in the mean value of positively stained cells for PCNA in the V30 group. For the groups treated for 60 days, there were not any significant differences in PCNA. Generally, chronic use of antidepressants has not been associated with significant aggression to the acinar and ductal cells. We believe that the proliferative potential of acinar and ductal cells assists in maintaining and regenerating the adult parenchymatous cellular population. Although rat models have some limitations, diseases and/or therapeutic simulations in animal models are important tools for examining disease prognoses and investigating the side effects of drugs.

14.

15.

16.

17. 18.

CONCLUSIONS

19.

The number of positively stained cells for C was shown to be increased with the chronic use of venlafaxine or fluoxetine. The proliferation index of the epithelial cells of rat parotid glands, however, was not altered by the use of antidepressants for 60 days.

20.

ACKNOWLEDGEMENTS

21.

The authors would like to thank the employees of the Laborato´rio de Patologia Experimental, Pontifı´cia Universidade Cato´lica do Parana´. This study was supported by CNPq (grant 474790/2004-5).

22.

REFERENCES

23.

1. Sansone RA, Sansone LA. Pain, pain, go away: antidepressants and pain management. Psychiatry (Edgmont). 2008;5:16-9. 2. Cusack B, Nelson A, Richelson E. Binding of antidepressants to human brain receptors: focus on newer generation compounds. Psychopharmacology (Berl). 1994;114:559-65, doi: 10.1007/BF02244985. 3. Dhir A, Kulkarni SK. Antidepressant-like effect of 17 beta-estradiol: involvement of dopaminergic, serotonergic, and (or) sigma-1 receptor systems. Can J Physiol Pharmacol. 2008;86:726-35, doi: 10.1139/Y08-077. 4. Spina E, Santoro V, D9Arrigo C. Clinically relevant pharmacokinetic drug interactions with second-generation antidepressants: an update. Clin Ther. 2008;30:1206-27, doi: 10.1016/S0149-2918(08)80047-1. 5. Stahl SM. Psychopharmacology of antidepressants. London: Martin Dunitz. 1997;114. 6. Cipriani A, Brambilla P, Furukawa TA, Geddes J, Gregis M, Hotopf M, et al. Fluoxetine versus other types of pharmacotherapy for depression. Cochrane Database of Systematic Reviews. In: The Cochrane Library. 2006;3. 7. Guggenheimer J, Moore PA. Xerostomia: etiology, recognition and treatment. J Am Dent Assoc. 2003;134:61-9. 8. Tenovuo J. Salivary parameters of relevance for assessing caries activity in individuals and populations. Community Dent Oral Epidemiol. 1997;25:82-6, doi: 10.1111/j.1600-0528.1997.tb00903.x. 9. Ferguson MM. Pilocarpine and other cholinergic drugs in the management of salivary gland dysfunction. Oral Surg Oral Med Oral Pathol. 1993;75:186-91, doi: 10.1016/0030-4220(93)90092-I. 10. Scully C. Drug effects on salivary glands: dry mouth. Oral Dis. 2003;9:165-76, doi: 10.1034/j.1601-0825.2003.03967.x. 11. Keene JJ Jr, Galasko GT, Land MF. Antidepressant use in psychiatry and medicine: importance for dental practice. J Am Dent Assoc. 2003;134:719. 12. Gre´gio AMT, Durscki JRC, Lima AAS, Machado MAN, Igna´cio SA, Azevedo LR. Association of amitriptyline and diazepam on the histomorphometry of rat parotid glands. Pharmacologyonline. 2006;2:96-108. 13. de Almeida P del V, Gre´gio AM, Brancher JA, Igna´cio SA, Machado MA, de Lima AA, et al. Effects of antidepressants and benzodiazepines on stimulated salivary flow rate and biochemistry composition of the saliva.

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Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2008;106:58-65, doi: 10.1016/j.tripleo.2007.11.008. Zaclikevis MV, D’Agulham AC, Bertassoni LE, Machado MA, de Lima AA, Gre´gio AM, et al. Effects of benzodiazepine and pilocarpine on rat parotid glands: histomorphometric and sialometric study. Med Chem. 2009;5:74-8, doi: 10.2174/157340609787049262. da Silva S, de Azevedo LR, de Lima AA, Ignaćio SA, Machado MA, ZacliKevis MV, et al. Effects of fluoxetine and venlafaxine and pilocarpine on rat parotid glands. Med Chem. 2009;5:483-90, doi: 10. 2174/157340609789117868. Klein RM. Development, structure and function of the salivary glands. In: Avery JK. Oral development and histology. USA: New York. 2001;292-331. Berkovitz BKB, Holland GR, Moxham BJ. Oral anatomy, embryology and histology. Chicago: Mosby. 2002;255-67. Zarbo RJ, Prasad AR, Regezi JA, Gown AM, Savera AT. Salivary gland basal cell and canalicular adenomas: immunohistochemical demonstration of myoepithelial cell participation and morphogenetic considerations. Arch Pathol Lab Med. 2000;124:401-5. Burford-Mason AP, Cummins MM, Brown DH, MacKay AJ, Dardick I. Immunohistochemical analysis of the proliferative capacity of duct and acinar cells during ligation-induced atrophy and subsequent regeneration of rat parotid gland. J Oral Pathol Med. 1993;22:440-6, doi: 10.1111/j. 1600-0714.1993.tb00122.x. Norberg L, Dardick I, Burford-Mason AP. Differentiating myoepithelial and acinar cells in rat neonatal parotid gland and histogenetic concepts for salivary gland tumors. J Oral Pathol Med. 1996;25:474-80, doi: 10. 1111/j.1600-0714.1996.tb00300.x. Denys D, van der Wee N, van Megen HJ, Westenberg HG. A double blind comparison of venlafaxine and paroxetine in obsessive-compulsive disorder. J Clin Psychopharmacol. 2003;23:568-75, doi: 10.1097/01.jcp. 0000095342.32154.54. Takahashi S, Schoch S, Walker NI. Origin of acinar cell regeneration after atrophy of the rat parotid induced by duct obstruction. Int J Exp Pathol. 1998;79:293-301, doi: 10.1046/j.1365-2613.1998.710405.x. Burgess KL, Dardick I, Cummins MM, Burford–Mason AP, Bassett R, Brown DH. Myoepithelial cells actively proliferate during atrophy of rat parotid gland. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1996;82:674-80, doi: 10.1016/S1079-2104(96)80443-4. Rocha RM, Andrade VP, Nunes CB, Rocha GFS, Sanches FSF, Oliveira FS, et al. Construçaõ de arrays de tecido com equipamento alternativo e de baixo custo para estudo imuno-histoquı´mico de tumores mama´rios. J Bras Patol Med Lab. 2006;42:477-82, doi: 10.1590/S1676-2444200 6000600012. Schuler S, Gurmini J, Cecı´lio WA, Viola de Azevedo ML, Olandoski M, de Noronha L. Hepatic and thymic alterations in newborn offspring of malnourished rat dams. J Parenter Enteral Nutr. 2008;32:184-9, doi: 10. 1177/0148607108314387. Martinez-Madrigal F, Micheau C. Histology of the major salivary glands. Am J Surg Pathol. 1989;13:879-99, doi: 10.1097/00000478-19891000000008. Gorenstein C, Scavone C. Avanços em psicofarmacologia - mecanismos de açaõ de psicofa´rmacos hoje. Rev Bras Psiquiatr. 1999;2:64-73, doi: 10. 1590/S1516-44461999000100012. Rang HP, Dale MM, Ritter JM, Flower RJ. Pharmacology. London: Churchill Livingstone. 2007;557-74. Davies AN, Shorthose K. Parasympathomimetic drugs for the treatment of salivary gland dysfunction due to radiotherapy. Cochrane Database Syst Ver. 2007;18:19. Grisius MM. Salivary gland dysfunction: a review of systemic therapies. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2001;92:156-62, doi: 10.1067/moe.2001.116601. Ship JA, Pillemer SR, Baum BJ. Xerostomia and the geriatric patient. J Am Geriatr Soc. 2002;50:535-43, doi: 10.1046/j.1532-5415.2002.50123.x. Denny PC, Ball WD, Redman RS. Salivary glands: a paradigm for diversity of gland development. Crit Rev Biol Med. 1997;8:51-75, doi: 10. 1177/10454411970080010301. Miguel MC, Andrade ES, Taga R, Pinto LP, Souza LB. Hyperplasia of myoepithelial cells expressing calponin during atrophy of the rat parotid gland induced by duct ligation. Histochem J. 2002;34:499-506, doi: 10. 1023/A:1024761923303. Cotran RS, Kumar V, Collins T, Robbins SL. Robbins pathologic basis of disease. 6th ed. Philadelphia: W. B. Saunders Co. 1999;p.336.

CLINICS 2011;66(9):1611-1614

DOI:10.1590/S1807-59322011000900018

BASIC RESEARCH

High-energy extracorporeal shockwave therapy in a patellar tendon animal model: a vascularizationfocused study Fernando Travaglini Penteado, Fla´vio Faloppa, Guilherme Giusti, Vinıćius Ynoe Moraes, Joaõ Carlos Belloti, Joaõ Baptista Gomes dos Santos Federal University of Saõ Paulo - Orthopedics and Traumatology, Saõ Paulo, Saõ Paulo, Brazil.

OBJECTIVE: The aim of this study was to analyze the effect of high-energy extracorporeal shockwave therapy on tendon angiogenesis in the patellar tendons of rabbits. We sought to investigate whether different voltage and number pulses modify the angiogenesis pattern. INTRODUCTION: High-energy extracorporeal shockwave therapy is an option in the treatment of orthopedic diseases such as chronic tendonitis. Despite its potential clinical applicability, there have been few studies on this technique that examine both its clinical effectiveness and its effect on angiogenesis. METHODS: High-energy extracorporeal shockwave therapy was applied at the tibial insertion of the left patellar ligament in 30 rabbits that were separated into six groups that differed in terms of the voltage and number of pulses that were applied by high-energy extracorporeal shockwave therapy. The tibial insertion in the right legs of the animals was used as the control. After six weeks, we performed histological analysis on the region and quantified the number of blood vessels. RESULTS: No significant differences in the number of blood vessels between the left and right patellar tendons were found within groups. Additionally, no significant differences in the number of blood vessels in the left patellar tendons were found between groups. CONCLUSIONS: The application of high-energy extracorporeal shockwave therapy did not cause a change in vascularization in the patellar tendon in rabbits. KEYWORDS: High-energy shock waves; Patellar ligament; Neovascularization; Animal model. Travaglini PF, Faloppa F, Giusti G, Moraes VY, Belloti JC, Santos JBG. High-energy extracorporeal shockwave therapy in a patellar tendon animal model: a vascularization-focused study. Clinics. 2011;66(9):1611-1614. Received for publication on March 1, 2011; First review completed on April 18, 2011; Accepted for publication on May 30, 2011 E-mail: fernandopenteado@ig.com.br Tel.: 55 11 55797049

rate and high success rate, and has a relatively low cost.6 The utilization of HEST for the treatment of musculoskeletal diseases began in 1986 when the first studies were performed regarding its effects on bone. Animal model studies have shown that HEST does not cause harmful changes to normal bone and increases the osteogenic potential through the activation of osteoblasts.4 HEST generates shockwaves that are transmitted through different tissues, and the processes by which this occurs are known.21 Most experimental work has aimed to analyze the effect of HEST on bone.8-10 In contrast, few studies have focused on the effects of HEST on soft tissue.11,12 Thus, little is known about this subject. To better understand HEST as a modality for the treatment of soft-tissue musculoskeletal disorders, basic science research should focus on local changes induced by HEST and seek to determine which are responsible for the improvements demonstrated in clinical studies.13-16 One possibility for such an HEST-induced change is that HEST could stimulate the formation of new blood vessels in the

INTRODUCTION Treatment with high-energy extracorporeal shockwave therapy (HEST) is currently used for the treatment of musculoskeletal diseases.1-5 Among its main indications are the treatment of nonunion, chronic insertional enthesopathies such as plantar fasciitis, lateral and medial epicondylitis, calcaneal tendon tendinitis, and calcareal tendinitis of the shoulder. It is a therapeutic option for cases in which conservative treatment has failed and surgery is potentially indicated.1,4-7 There are some theoretical advantages of HEST in comparison to surgical treatment. Namely, HEST is noninvasive and fast, and it does not require hospitalization, has a low complication

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Table 1 - Distribution of groups in terms of energy level in kilovolts (kV) and the number of pulses applied. Energy Pulses 1000 4000

14 kV

21 kV

28 kV

Group 1 Group 4

Group 2 Group 5

Group 3 Group 6

Table 2 - Blood vessels per field from HEST-treated (left) and untreated (right) patellar tendons, indicating the differences between them. Groups N

tendon and that it is this increased vascularization that leads to clinical improvement.6,15,16 There are few experimental studies showing changes in tendon vascularity after the application of extracorporeal shockwaves (ESW),17,18 but there is evidence that HESTinduced tissue changes are dose dependent.19 To find better evidence of HEST-induced angiogenesis in soft tissues, we designed this study to quantify the effects of HEST on tendons. The aims of this study were to examine the effect of HEST on the vascularity of the patellar tendon at its insertion at the anterior tibial tuberosity and to determine how varying the voltage and number of pulses applied changed this effect.

MATERIALS AND METHODS A total of 30 six-week-old New Zealand white female rabbits were divided randomly into six groups of five animals. The groups received HEST that differed by voltage and the number of pulses (Table 1). The apparatus used for the study was the OssaTronH (Barueri, SP, Brazil), which was developed specifically for the treatment of musculoskeletal disorders. This apparatus uses electrohydraulic principles to generate shockwaves. For the application of HEST, animals were anesthetized by intramuscular injection of 50 mg/kg ketamine hydrochloride and 5 mg/kg diazepam. After the animals had been anesthetized, the left knees were shaved. The application site, the anterior tibial tuberosity, was located with the aid of fluoroscopy and marked with a surgical marker pen. The animals were placed on a table in the supine position, and the device was positioned to direct the center of focus precisely to the marked site. Shockwaves were applied to the animal’s left knee. After the procedure, intramuscular analgesics were provided to alleviate pain. The animals were sacrificed by carbon dioxide inhalation six weeks after HEST treatment, and the left and right patellar ligaments were harvested. The right patellar ligament was used as a control. The patellar ligaments were separated, fixed in 10% formalin, dehydrated in increasing alcohol concentrations, cleared in xylene, and embedded in paraffin in a manner that allowed the resulting blocks to provide an assessment of the entire ligament in the frontal plane. The paraffin blocks were cut into 5 mm sections that were stained with hematoxylin-eosin. With the aid of a digital system (Image Tool 3.0; Department of Dental Diagnostic Science at The University of Texas Health Science Center, San Antonio, Texas), we analyzed the entire length of the ligament insertion at the margin (Figure 1). To cover the entire length of the insertional ligament margin, sections were divided into fields (6–10 fields per section) and amplified 200 times. On the scanned images, all obvious vascular structures were marked, i.e., those with patent lumens, those lined by endothelium, or those containing red blood cells in the lumen. All of these structures were counted, and the end result was expressed

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Vessels/Field R Vessels/Field L 4.00 4.28 7.19 3.71 6.93 5.87 5.00 7.43 4.56 8.22 7.33 7.92 5.43 7.62 5.62 5.86 10.00 7.28 7.21 9.00 * 10.86 4.29 4.10 2.62 7.79 4.57

3.92 3.14 8.81 5.64 7.57 * 6.71 9.50 6.00 7.14 9.50 3.50 * 6.37 5.30 5.67 7.30 3.71 10.08 4.43 5.00 * 8.00 7.17 5.37 * 10.57 11.00 4.94 6.44 4.50 5.37 5.00

R-L difference p-value 0.08 1.14 -1.62 -1.93 0.22 -3.63 -1.00 0.29 -4.94 1.85 2.03 2.25 -1.87 3.91 -4.46 1.43 2.00 0.11 -3.36 0.14 -0.65 -2.34 -1.88 2.42 -0.43

0.465 (1)

0.225 (1)

0.273 (1)

0.751 (1)

0.285 (1)

0.500 (1) 0.438 (2)

(*) Excluded from the statistical analysis because of the lack of a matching sample on the other side. (1) Wilcoxon tests; (2) Kruskall-Wallis test.

in absolute numbers (Figure 2). Two independent observers, blinded to the distribution of the groups, examined the images.

Statistical Analysis The data were analyzed first to evaluate the distribution. The Shapiro-Wilk test showed the data to be normally distributed (p = 0.407). However, due to the small nature of the groups sampled, we chose to perform nonparametric testing. We calculated the mean number of vessels per slide, and this value was used to represent each subject. We determined the ratio between the total number of vessels and the total number of fields for each sample. This ratio (vessels per field) was used for statistical analysis. To standardize the groups, we calculated the difference in the number of vessels between the right and left patellar tendons of each animal. These differences were used to compare the groups. The nonparametric Wilcoxon test was used to compare the means of the number of vessels per field on the left and right legs within each group. The Kruskall-Wallis test was performed to compare groups when comparisons were made for more than two groups. In all statistical tests, an a of 5% (p,0.05) was considered significant. The University’s ethics research committee approved this study, which was conducted at the Department of Surgery of the Hand and Upper Limb, Department of Orthopedics and Traumatology, Escola Paulista de Medicina, Universidade Federal de Sa˜o Paulo.

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Shockwave therapy for patellar tendon angiogenesis Travaglini PF et al.

Figure - 1A (left): Histological section of a patellar tendon stained with hematoxylin-eosin (2006). Checkpoint arrows represent identified blood vessels. Figure - 1B (right): Patellar tendon section stained with hematoxylin-eosin (106).

the location of the ailment. Under these standards, the amount of energy to be applied for the treatment of most enthesopathies is 1000 pulses of 16 kV.6 One experimental study cautioned about the harmful effects of using greater than 1000 pulses of 21 kV because there was evidence that such a dose could result in tendon tissue damage.5 In our study, we used a broad spectrum of pulse-voltage combinations, with values both within the recommended range and above, to test the effects that HEST would have at different doses. The tibial insertion of the patellar ligament was chosen as the application site for HEST because it is a structure that is easy to find and is relatively broad. These characteristics allowed for proper positioning of the device, which allowed us to focus the application of HEST correctly. In addition, because this ligament has a large insertion area and is well defined at one side, the demarcation of its margins was straightforward. While it is not confirmed that the improvement of pain after ESW treatment of chronic enthesopathies is because of the induction of angiogenesis in the tendon, there are several reports that support this idea.17,18 A histochemical study indicated that ESW appeared to induce the early release of vascular growth factors from the tendon. These growth factors induced angiogenesis, which led to increased blood supply to the tendon, which in turn supported tissue regeneration.18 Hsu et al. studied the effects of ESW in a rabbit model of patellar tendinitis induced by the injection of collagenase and found an increase in vascularization at 16 weeks.31 It is noteworthy that while all three of these studies showed an increase in tendon vascularization after ESW treatment, our results did not demonstrate such an increase. It should also be noted that only one of these three studies is comparable to our study in terms of the methodology used. Each type of tissue has a particular impedance value. The impedance of bone is much greater than that of water, and therefore, bone absorbs large amounts of energy from shockwaves. However, the impedance of a healthy tendon is very similar to that of water, and thus, a tendon absorbs less energy than bone. However, a calcified tendon has a higher level of impedance because of the presence of calcium crystals. This could explain why HEST is more effective in cases of chronic enthesopathies because the tendons in these cases tend to be tougher and more calcified than in acute enthesopathies.6 We believe that more reliable results could be obtained if we use an experimental model of induced calcification in the tendon that simulates cases of chronic, calcified enthesopathies. There is definitely a need for more research on this topic.

RESULTS Twenty-five animals were included in the studyâ&#x20AC;&#x2122;s final analysis. Five animals were excluded because of problems related to histological analysis. Intergroup analyses indicated that there was some difference within the control groups (Kruskall-Wallis, X2 = 13.52; p = 0.031). This result led us to perform comparisons of the mean difference between the left and right patellar tendons of each animal (R-L difference). There were no statistically significant differences among groups in terms of the R-L difference (Table 2, Kruskal-Wallis; X2 = 4.71; p = 0.438).

DISCUSSION It is known that the etiology of insertional enthesopathies is likely multifactorial, but the exact causes remain unknown.5,8,19 It is thought that a degenerative process that is accompanied by inflammation is important in pathogenesis.20 Authors have stated that the vascular changes and hyaline degeneration observed in surgical biopsies from insertional enthesopathy patients suggest that degeneration has a greater role than inflammation in disease pathogenesis and is the main etiological factor.20,21 However, there is insufficient evidence to determine the best method for treatment of the disease. As such, there are a considerable number of options for treatment, including nonsteroidal anti-inflammatory and hormonal therapy, detention, infiltration, acupuncture, and surgery.5,21 Currently, we use ESW therapy as an alternative treatment for insertional enthesopathies and have achieved good results with this approach.3,5,22-30 However, the mode of action behind this treatment is still unknown. It has been suggested that the pain in enthesopathies is the result of reduced vascularization in degenerative tendon tissue and that extracorporeal shockwave therapy could increase neovascularization and promote tissue regeneration.21 There is little in the literature regarding experimental research that is focused on understanding the effects of ESW on tendons. One author concluded that the effects of ESW are dependent on the amount of energy applied,5,19 while others have observed an increased vascularization of treated tendons after ESW therapy.1,31 Our work was designed to verify whether ESW causes changes in tendon vascularization, and if so, whether these changes would be dosedependent. The International Society for Musculoskeletal Shockwave Therapy advocates a normalization of the amount of energy to be applied in each case, based on the disease in question and

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CONCLUSION 17.

Within the energy range tested, high-energy shockwave therapy did not cause significant changes in the vascularity of insertional patellar tendons in a rabbit model.

18.

REFERENCES 19.

1. Wang CJ. An overview of shock wave therapy in musculoskeletal disorders. Chang Gung Med J. 2003;26:220-32. 2. Loew M, Jurgowski W, Thomsen M. [Effect of extracorporeal shockwave therapy on tendinosis calcarea of the shoulder. A preliminary report]. Urologe A. 1995;34:49-53. 3. Thiel M, Nieswand M, Dorffel M. The use of shock waves in medicine–a tool of the modern OR: an overview of basic physical principles, history and research. Minim Invasive Ther Allied Technol. 2000;9:247-53. 4. Haupt G. Use of Extracorporeal Shock Waves in the Treatment of Pseudarthrosis, Tendinopathy and Other Orthopedic Diseases. The Journal of urology. 1997;158:4-11, doi: 10.1097/00005392-199707000-00003. 5. Rompe JD, Ku¨llmer K, Vogel J, Eckardt A, Wahlmann U, Eysel P, et al. [Extracorporeal shock-wave therapy. Experimental basis, clinical application]. Orthopade. 1997;26:215-28. 6. Coombs R, Schaden W, Zhou S. Muskuloskeletal Shockwave Therapy. Greenwich Medical Media Ltd. London. 2000. 7. Ramos LAo, Carvalho RrTd, Garms E, Navarro MS, Abdalla RJ, Cohen Ms. Prevalence of pain on palpation of the inferior pole of the patella among patients with complaints of knee pain. Clinics. 2009;64:199-202, doi: 10.1590/S1807-59322009000300009. 8. Delius M, Draenert K, Al Diek Y, Draenert Y. Biological effects of shock waves: in vivo effect of high energy pulses on rabbit bone. Ultrasound Med Biol. 1995;21:1219-25, doi: 10.1016/0301-5629(95)00030-5. 9. Wang CJ, Wang FS, Yang KD. Biological effects of extracorporeal shockwave in bone healing: a study in rabbits. Arch Orthop Trauma Surg. 2008;128:879-84, doi: 10.1007/s00402-008-0663-1. 10. Gollwitzer H, Roessner M, Langer R, Gloeck T, Diehl P, Horn C, et al. Safety and effectiveness of extracorporeal shockwave therapy: results of a rabbit model of chronic osteomyelitis. Ultrasound Med Biol. 2009;35:595-602, doi: 10.1016/j.ultrasmedbio.2008.10.004. 11. Bosch G, Lin YL, van Schie HT, van De Lest CH, Barneveld A, van Weeren PR. Effect of extracorporeal shock wave therapy on the biochemical composition and metabolic activity of tenocytes in normal tendinous structures in ponies. Equine Vet J. 2007;39:226-31, doi: 10. 2746/042516407X180408. 12. Qin L, Wang L, Wong MW, Wen C, Wang G, Zhang G, et al. Osteogenesis induced by extracorporeal shockwave in treatment of delayed osteotendinous junction healing. J Orthop Res. 2010;28:70-6. 13. Steinacker T, Steuer M. [Use of extracorporeal shockwave therapy (ESWT)in sports orthopedics]. Sportverletz Sportschaden. 2001;15:45-9, doi: 10.1055/s-2001-14817. 14. Zwerver J, Dekker F, Pepping GJ. Patient guided Piezo-electric Extracorporeal Shockwave Therapy as treatment for chronic severe patellar tendinopathy: A pilot study. J Back Musculoskelet Rehabil. 2010;23:111-5. 15. Rasmussen S, Christensen M, Mathiesen I, Simonson O. Shockwave therapy for chronic Achilles tendinopathy: a double-blind, randomized clinical trial of efficacy. Acta Orthop. 2008;79:249-56, doi: 10.1080/ 17453670710015058. 16. Vulpiani MC, Trischitta D, Trovato P, Vetrano M, Ferretti A. Extracorporeal shockwave therapy (ESWT) in Achilles tendinopathy.

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26.

27.

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A long-term follow-up observational study. J Sports Med Phys Fitness. 2009;49:171-6. Wang CJ, Huang HY, Pai CH. Shock wave-enhanced neovascularization at the tendon-bone junction: an experiment in dogs. J Foot Ankle Surg. 2002;41:16-22, doi: 10.1016/S1067-2516(02)80005-9. Wang CJ, Wang FS, Yang KD, Weng LH, Hsu CC, Huang CS, et al. Shock wave therapy induces neovascularization at the tendon-bone junction. A study in rabbits. J Orthop Res. 2003;21:984-9, doi: 10.1016/ S0736-0266(03)00104-9. Rompe JD, Kirkpatrick CJ, Kullmer K, Schwitalle M, Krischek O. Doserelated effects of shock waves on rabbit tendo Achillis. A sonographic and histological study. J Bone Joint Surg Br. 1998;80:546-52, doi: 10.1302/ 0301-620X.80B3.8434. Regan W, Wold LE, Coonrad R, Morrey BF. Microscopic histopathology of chronic refractory lateral epicondylitis. The American Journal of Sports Medicine. 1992;20:746-9, doi: 10.1177/036354659202000618. Maier M, Milz S, Wirtz DC, Rompe JD, Schmitz C. [Basic research of applying extracorporeal shockwaves on the musculoskeletal system. An assessment of current status]. Orthopade. 2002;31:667-77, doi: 10.1007/ s00132-002-0328-7. Geddes LA, Tacker WA, Rosborough JP, Moore AG, Cabler PS. Electrical dose for ventricular defibrillation of large and small animals using precordial electrodes. J Clin Invest. 1974;53:310-9, doi: 10.1172/ JCI107552. Loew M, Jurgowski W, Mau HC, Thomsen M. Treatment of calcifying tendinitis of rotator cuff by extracorporeal shock waves: a preliminary report. J Shoulder Elbow Surg. 1995;4:101-6, doi: 10.1016/S10582746(05)80062-X. Haake M, Sattler A, Gross MW, Schmitt J, Hildebrandt R, Muller HH. [Comparison of extracorporeal shockwave therapy (ESWT) with roentgen irradiation in supraspinatus tendon syndrome–a prospective randomized single-blind parallel group comparison]. Z Orthop Ihre Grenzgeb. 2001;139:397-402, doi: 10.1055/s-2001-17981. Maier M, Steinborn M, Schmitz C, et al. Extracorporeal shock-wave therapy for chronic lateral tennis elbow–prediction of outcome by imaging. Arch Orthop Trauma Surg. 2001;121:379-84, doi: 10.1007/ s004020100261. Schmitt J, Tosch A, Hunerkopf M, Haake M. [Extracorporeal shockwave therapy (ESWT) as therapeutic option in supraspinatus tendon syndrome? One year results of a placebo controlled study]. Orthopade. 2002;31:652-7, doi: 10.1007/s00132-002-0325-x. Buchbinder R, Green S, White M, Barnsley L, Smidt N, Assendelft WJ. Shock wave therapy for lateral elbow pain. Cochrane Database Syst Rev. 2002:CD003524. Peers KH, Lysens RJ, Brys P, Bellemans J. Cross-sectional outcome analysis of athletes with chronic patellar tendinopathy treated surgically and by extracorporeal shock wave therapy. Clin J Sport Med. 2003;13:7983, doi: 10.1097/00042752-200303000-00003. Gerdesmeyer L, Wagenpfeil S, Haake M, Maier M, Loew M, Wo¨rtler K, et al. Extracorporeal shock wave therapy for the treatment of chronic calcifying tendinitis of the rotator cuff: a randomized controlled trial. JAMA. 2003;290:2573-80, doi: 10.1001/jama.290.19.2573. Harniman E, Carette S, Kennedy C, Beaton D. Extracorporeal shock wave therapy for calcific and noncalcific tendinitis of the rotator cuff: a systematic review. J Hand Ther. 2004;17:132-51, doi: 10.1197/j.jht.2004.02. 003. Hsu RW, Hsu WH, Tai CL, Lee KF. Effect of shock-wave therapy on patellar tendinopathy in a rabbit model. J Orthop Res. 2004;22:221-7, doi: 10.1016/S0736-0266(03)00138-4.

CLINICS 2011;66(9):1615-1619

DOI:10.1590/S1807-59322011000900019

BASIC RESEARCH

Exercise training attenuates acute hyperalgesia in streptozotocin-induced diabetic female rats Denise M. Rossi,I Vitor E. Valenti,II Marcelo T. NavegaI I Departamento de Educaçaõ Especial, Faculdade de Filosofia e Cieˆncia, UNESP, Marı´lia/SP, Brasil. II Departamento de Patologia, Faculdade de Medicina, Universidade Saõ Paulo, Saõ Paulo/SP, Brasil.

OBJECTIVES: We investigated the effects of chronic (eight weeks) low- to moderate-intensity swimming training on thermal pain sensitivity in streptozotocin-induced diabetic female rats. METHODS: Female Wistar rats (n = 51) were divided into the following groups: trained streptozotocin-induced diabetic rats [hyperglycemic trained (HT)], sedentary streptozotocin-induced diabetic rats [hyperglycemic sedentary (HS)], normoglycemic trained rats (NT) and normoglycemic sedentary rats (NS). Diabetes was induced by a single injection of streptozotocin (50 mg/kg, i.p.). One day after the last exercise protocol (60 min/day, five days/week for eight weeks) in the trained groups or after water stress exposure (ten min/twice a week) in the sedentary groups, the rats were subjected to a hot plate test. RESULTS: After eight weeks of swimming training, streptozotocin-induced diabetic rats presented a significantly lower body mass (trained: 219.5¡29 g, sedentary: 217.8¡23 g) compared with the normoglycemic groups (trained: 271¡24 g, sedentary: 275.7¡32 g). Interestingly, we did not find differences in blood glucose levels (mg/dl) between the trained and sedentary groups of the hyperglycemic or normoglycemic rats (HT: 360.2¡66.6, HS: 391.7¡66.7, NT: 83.8¡14.0, NS: 77.5¡10.1). In the hot plate test, the rats from the HT group presented a significantly lower latency than the other rats (HT: 11.7¡7.38 s, HS: 7.02¡7.38 s, NT: 21.21¡7.64 s, NS: 22.82¡7.82 s). CONCLUSION: Low-to-moderate swimming training for a long duration reduces thermal hyperalgesia during a hot plate test in streptozotocin-induced diabetic female rats. KEYWORDS: Diabetes mellitus; Diabetic neuropathy; Pain sensitivity; Physical exercise; Swimming training; Hot plate test. Rossi DM, Valenti VE, Navega MT. Exercise training attenuates acute hyperalgesia in streptozotocin-induced diabetic female rats. Clinics. 2011;66(9):1615-1619. Received for publication on April 12, 2011; First review completed on June 1, 2011; Accepted for publication on June 1, 2011 E-mail: denisemartineli@hotmail.com Tel.: 55 14 3402-1300

diabetic patients with neuropathy. Diabetic neuropathy may be classified as peripheral, autonomic, proximal, focal, or multifocal.4 Diabetic peripheral neuropathy has been reported to be the most common neuropathy, and diabetic peripheral neuropathy leads to pain and sensitivity loss, which reduces life expectancy and increases health costs.4,6 One of the most elusive symptoms is pain, which is typically characterized by mechanical and thermal hyperalgesia or allodynia.2 Common complaints include a constant burning discomfort, severe hyperesthesia, deep aching pain, stabbing or ‘‘electric shock’’-like sensations and sensations in the lower limbs.5 Early prevention of diabetic neuropathy is necessary to avoid serious complications that can begin in the asymptomatic period of onset. Exercise is useful for lowering plasma glucose levels both during exercise and following exercise. In addition, exercise has been shown to increase insulin sensitivity. Skeletal muscles are the major site for metabolic fuel consumption in the resting state, and increased muscle activity during vigorous aerobic exercise greatly increases fuel requirements.7 Studies in animal models have indicated

INTRODUCTION According to the International Diabetes Federation,1 diabetes mellitus (DM) is a great challenge for public health. Diabetes is a serious problem that is increasing worldwide, and estimates predict that 380 million people will be affected in 2025. Experimental research on diabetic neuropathy is usually carried out using genetic animal models or chemically induced diabetic animal models to provide information on the underlying mechanisms and to evaluate potential therapies, and both models have enhanced our understanding of the disease.2 Approximately 60 to 70% of people with diabetes present some neuropathy3, and studies have estimated that 50 to 70% of all nontraumatic amputations in the USA are

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glucose levels $250 mg/100 mL. Plasma glucose levels in the normoglycemic animals remained normal for the duration of the study.11,14

a general improvement in the ability of diabetic animals to conduct and sustain chronic physical exercise, especially regarding the metabolism of energy substrates and secreting hormones.8 Gender differences have been evaluated in experimentally induced pain paradigms through diverse methods, including pressure, electrical, ischemic, and thermal chemical stimuli. According to a review published by Fillingim et al.,9 females have a lower pain threshold than males, and evidence has shown that sexual hormones are involved in this process. Although the mechanisms involving the role of estrogen in pain perception are not entirely clear, changes in plasma estrogen levels can influence pain sensitivity by either decreasing or increasing excitability of nerve fibers and brain cells.10 Taken together, these data support the difference between males and females regarding pain sensitivity and suggest that females exhibit greater sensitivity to painful stimulation.9 Data regarding thermal sensitivity in diabetic female rats and the effects of exercise, however, are lacking; thus, the present study examined female rats. Although previous studies have already examined the effects of physical exercise on pain sensitivity,8 to the best of our knowledge, there is no evidence regarding the effects of physical exercise on pain sensitivity in diabetic female animals. Moreover, exercise training is a nonpharmacological and noninvasive treatment method. In addition, new data are always well received in both scientific and clinical practice. Therefore, this study was undertaken to evaluate the effects of swimming training on the thermal sensitivity of streptozotocin-induced diabetic female rats using a hot plate test.

Exercise training protocol Exercise training was performed in a swimming pool (112 cm680 cm650 cm) filled with tap water that was warmed to approximately 27 ˚C, and the exercise intensity was progressively increased. In the first week, the rats swam for ten minutes every day. In the second week, the swimming time was increased each day until the animals could swim for 60 min while wearing a caudal dumbbell weighing 5% of their body weight (overload).15 In each exercise session, eight to ten rats were placed together in the swimming pool to perform the exercise. During the exercise period, the normoglycemic groups were exposed to water stress for ten minutes, twice a week. This swimming protocol has previously been characterized as low to moderate intensity (with a long duration) based on improvements in muscle oxidative capacity.15

Hot plate test One day after the end of the exercise training protocol period, a hot plate test was conducted as previously described.16 This test measures the time that elapses before the rat demonstrates hind paw licking/shaking and jumping, which indicate pain in response to the applied heat. The hot plate was maintained at 50¡1 ˚C, and the animals were placed into a Perspex cylinder on the heated stage. Response latency was measured by recording the time between placement in the cylinder and shaking or licking the paws. The cut-off time was set at 35 seconds to minimize skin injury.

METHODS Statistical analysis

Animals

We applied the Shapiro-Wilk test to verify the normality of the distributions. A two-way analysis of variance (ANOVA) was used to verify the differences between the normal distributions, and the Kruskal–Wallis test was used to assess differences between nonparametric distributions. The differences were considered significant when the probability of a Type I error was lower than 5% (p,0.05).

A total of 51 female Wistar rats (160-250 g) were used in the present study. The care and use of the animals followed the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health (National Institutes of Health Publication No. 96-23, revised 1996) and was approved by the Ethics Committee in Research of the Faculdade de Medicina de Marı´lia (protocol number 428/ 09). The rats were randomly distributed into four groups: normoglycemic sedentary (NS, n = 15), normoglycemic trained (NT, n = 16), hyperglycemic sedentary (HS, n = 9) and hyperglycemic trained (HT, n = 11). The rats were housed in plastic cages (3-4 rats/cage) under a 12-h dark– light cycle at 22 ˚C and 60% air humidity with free access to water and rodent pellet chow. At the end of the experiment, the rats were sacrificed by CO2 exposure.

RESULTS STZ-induced diabetic animals presented lower body mass values (HT: 219.8+29 g, HS: 217.8+23 g) compared with the normoglycemic animals (NT: 271+24 g, NS: 275.7+32 g; p = 0.0001). Figure 1 shows that the rats in the STZ-induced diabetic groups (i.e., HS and HT) presented higher blood glucose levels than the normoglycemic rats. Figure 2 shows that the exercise training protocol influenced the response latency in the HT group. Indeed, the response latency in the HT group was higher than the response latency in the HS group.

Streptozotocin-induced diabetes Diabetes was induced in the hyperglycemic groups by a single intraperitoneal (i.p) injection of streptozotocin (STZ, 50 mg/kg). The STZ was dissolved in a citrate buffer solution (0.1 mol/L citric acid, 0.1 mol/L sodium citrate, pH 4.5). Streptozotocin destroys pancreatic beta cells and induces hyperglycemia. In the normoglycemic groups, we applied a single injection of citrate buffer. Two days after STZ treatment, blood glucose levels were measured to confirm a hyperglycemic state. Blood was obtained from a small nick in the tail and measured with a glucometer (Optium Xceed). The hyperglycemic rats presented blood

DISCUSSION The aim of the present study was to evaluate the effects of swimming on thermal sensitivity in STZ-induced diabetic female rats. We observed that the HS rats had the lowest latency time of the four groups of rats. Our data suggested that swimming attenuates the hyperalgesia caused by acute hyperglycemia in STZ-induced diabetic rats.

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Figure 1 - Blood glucose levels hot plate of the trained STZ-induced diabetic (HT, n = 11), sedentary STZ-induced diabetic (HS, n = 9), normoglycemic trained (NT, n = 14) and normoglycemic sedentary (NS, n = 16) groups before the hot plate test. *p,0.0001: different from NS and NT.

peripheral neuropathy development in STZ-induced diabetic male rats. Similar to our findings, they observed that the swimming exercise protocol did not influence blood glucose levels; however, the exercise restored body weight, compound muscle action potential amplitude and potential latency, which are parameters that define motor dysfunction in diabetic animals. In addition, Cunha et al.20 observed that STZ induces mechanical hypernociception, which does not depend on hyperglycemia. Our findings revealed new aspects on how exercise influences diabetic rats. Unlike previous studies, we evaluated female rats and thermal sensitivity rather than motor dysfunction. Furthermore, we found that swimming training improved acute hyperalgesia without influencing blood glucose levels.

Chronic hyperglycemia is an important factor that can trigger many complications. Previous studies have suggested that attenuation of chronic hyperglycemia may slow or even prevent microvascular and macrovascular complications, such as retinopathy, nephropathy and neuropathies.8 Although the pathogenesis of diabetic neuropathy is multifactorial, increased blood glucose levels are likely the major factor. Moreover, acute hyperglycemia is followed by hyperalgesia, whereas chronic hyperglycemia is followed by hypoalgesia due to impairment of the peripheral neurons.17 Previous studies have evaluated the effects of exercise on diabetic nerve regeneration.18 Selagzi and colleagues19 investigated the effects of swimming training on diabetic

Figure 2 - Response latency of the trained STZ-induced diabetic (HT, n = 11), sedentary STZ-induced diabetic (HS, n = 9), normoglycemic trained (NT, n = 14) and normoglycemic sedentary (NS, n = 16) groups. The Kruskal Wallis test was used. *p,0.01: different from NS and NT; #p,0.05: different from HS.

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difference in the normoglycemic groups, in which the effect was observed 24 h after the last day of physical exercise. Except for strict control of the blood glucose level, there are few effective ways to influence or delay the natural progression of diabetic neuropathy because of the limitations of current drug therapies. Despite the challenges ahead, the future promises more effective treatments for diabetes and its complications with an aim toward improving patients’ quality of life. The present study showed that a swimming exercise protocol influences the thermal nociceptive stimulus in STZ-induced diabetic female rats by attenuating the hyperalgesia caused by the hyperglycemic state. We suggest that exercise provides protection against diabetic neuropathy in rats as a result of multiple mechanisms and provides a means of avoiding the potential deterioration of diabetic neuropathy. The majority of the data examining the effects of exercise on STZ-induced diabetic animals come from male animals. The reason for this is that the pathogenetic mechanisms in males are not influenced by fluctuations in hormonal activity.31 Interestingly, no study has shown that exercise training can improve acute hyperalgesia in STZ-induced diabetic female rats, and it is still unclear if molecular mechanisms in different genders can be influenced by hormones to improve diabetic neuropathy in exercising rats. The molecular mechanisms underlying the different findings of the present study in female rats require further investigation. There were several issues in the present study that warranted discussion. First, we did not confirm the involvement of peripheral neurons affected by hyperglycemia. Second, although we observed that exercise attenuated acute hyperalgesia in STZ-induced diabetic female rats, which is a feature of early diabetic neuropathy, we should be careful in extrapolating our data to humans with diabetic neuropathy. Third, we did not measure any parameters to determine whether the rats were effectively trained, such as resting bradycardia or citrate synthase enzyme activity. We controlled training progression by starting with ten minutes of exercise a day for the first week. In the second week, the session times were increased by ten minutes per day until they reached 60 minutes, at which point the rats wore caudal dumbbells weighing 5% of their body weight. Throughout the sessions, the examiner observed the rats to ensure that they were swimming. This is an accepted training protocol in the literature.13,32,34 Finally, because the hot plate test was performed one day after the end of training, the findings could be the result of the acute effects of the last exercise session. Nevertheless, if the animals were tested one week or more after the last exercise session, the effects of two months of exercise training could still be partially restored. In conclusion, low to moderate swimming training for a long duration in STZ-induced diabetic female rats attenuates thermal hyperalgesia in a hot plate test.

Some hypotheses may be raised regarding the observed effects of swimming on thermal sensitivity. Because studies have shown that hypernociception does not depend on a hyperglycemic state,20 we believe that exercise training attenuates hyperalgesia via mechanisms that are not related to a decrease in blood glucose levels. Koltyn et al.21 indicated that the increase in endogenous opioid levels induced by exercise may lead to a feedback inhibition of acute pain in peripheral, spinal and supraspinal neurons. In addition, Stagg and colleagues22 reported that aerobic exercise increased the endogenous opioid concentration in brainstem regions involved in pain regulation and decreased neuropathic pain in a rat neuropathic pain model. It is also plausible that the antioxidant effects of chronic exercise23 attenuated the acute hyperalgesia caused by the hyperglycemic-induced nerve injuries; however, the present study did not measure the antioxidant or prooxidant enzyme activities. The hot plate test was applied 24 h after the last swimming protocol. Therefore, our data could also be the result of an acute effect of the last exercise training because thermal sensation usually increases during exercise.24 Furthermore, we speculated that short-term influences on thermal sensation were due to increases in local skin and brain temperatures. A previous investigation suggested that high brain temperature influences the functions related to the nervous system (alertness, contentment, calmness, and thermal comfort).25 Nonetheless, the literature is unclear on the influence of body temperature on perception.26 Future studies should be performed to investigate the acute effects of exercise during hot plate testing of STZ-induced diabetic animals. The data from our study indicate that swimming exercise (mild to moderate) is able to reduce thermal hyperalgesia in STZ-induced hyperglycemic female rats. These findings corroborate the results of Bento-Silva et al.,27 which demonstrated that low-intensity swimming exercise decreases the nociception induced by thermal and chemical stimulus after exercise; however, their methodology was different with respect to the intensity, the duration of exercise and the duration of painful stimuli. Moreover, Kuphal et al.28 demonstrated that swimming reduces hyperalgesia induced by intraplantar injection of formalininduced partial injury in rodent peripheral nerves. Therefore, the difference between our study and BentoSilva et al. study,27 suggests us to be careful in interpreting our findings. Unlike our results for the normoglycemic groups (i.e., swimming did not affect the latency during the hot plate test), Mazzardo-Martins et al.29 found that high-intensity swimming reduced acute pain stimulated by intraperitoneal injection of acetic acid (chemical noxious stimulus) in normoglycemic rats. It is believed that the method used to analyze pain sensitivity may influence this result because Koltyn et al.21 indicated that the antinociceptive effect of exercise is more consistently found in studies using electrical or pressure stimulation compared with studies that used temperature to produce pain. Interestingly, the effects of exercise on pain sensitivity are not consistent in the literature, and the controversies seem to be related to different methodologies. Indeed, there are differences in the type, intensity and duration of exercise beyond the pain stimulus protocol.30 Furthermore, the antinociceptive effect of exercise is likely short term, assuming the absence of a

ACKNOWLEDGEMENTS This research was supported by grants from Fundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo (FAPESP). *Study performed at Faculdade de Filosofia e Cieˆncia, UNESP, Marı´lia.

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Exercise training attenuates acute hyperalgesia Rossi DM et al. 19. Malysz T, Ilha J, Nascimento PS, Angelis KD, Schaan BD, Achaval M. Beneficial effects of treadmill training in experimental diabetic nerve regeneration. Clinics. 2010;65:1329-37, doi: 10.1590/S1807-59322010001200017. 20. Selagzi H, Buyukakilli B, Cimen B, Yilmaz N, Erdogan S. Protective and therapeutic effects of swimming exercise training on diabetic peripheral neuropathy of streptozotocin-induced diabetic rats. J Endocrinol Invest. 2008;31:971-8. 21. Cunha JM, Funez MI, Cunha FQ, Parada CA, Ferreira SH. Streptozotocin-induced mechanical hypernociception is not dependent on hyperglycemia. Braz J Med Biol Res. 2009;42:197-206, doi: 10.1590/ S0100-879X2009000200008. 22. Koltyn KF. Analgesia following exercise: a review. Sports Med. 2000;29:85-98, doi: 10.2165/00007256-200029020-00002. 23. Stagg NJ, Mata HP, Ibrahim MM, Henriksen EJ, Porreca F, Vanderah TW, et al. Regular exercise reverses sensory hypersensitivity in a rat neuropathic pain model: role of endogenous opioids. Anesthesiology. 2011;114:940-8, doi: 10.1097/ALN.0b013e318210f880. 24. Perse M, Injac R, Strukelj B, Cerar A. Effects of high-fat mixed-lipid diet and exercise on the antioxiidant system in skeletal and cardiac muscles of rats with colon carcinoma. Pharmacol Rep. 2009;61:909-16. 25. Hellstrom B, Berg K, Vogt Lorentzen F. Human peripheral rewarming during exercise in the cold. J Appl Physiol. 1970;29:191–9. 26. Salerian AJ, Saleri NG, Salerian JA. Brain temperature may influence mood: a hypothesis. Med Hypotheses. 2008;70:497–500, doi: 10.1016/j. mehy.2007.06.032. 27. Muller MD, Muller SM, Ryan EJ, Bellar DM, Kim CH, Glickman EL. Pain and thermal sensation in the cold: the effect of interval versus continuous exercise. Eur J Appl Physiol. 2010 Nov 17. [Epub ahead of print]. 28. Bento-Silva MT, Santos MAP, Almeida FEC. Exercise of light intensity decrease pain induced for thermal and chemical stimulus in rats. Braz J Biomotric. 2010;4:14-23. 29. Kuphal KE, Fibuch EE, taylor BK. Extended swimming exercise reduces inflammatory and peripheral neuropathic pain in rodents. J Pain. 2007;8:989-97, doi: 10.1016/j.jpain.2007.08.001. 30. Mazzardo-Martins L, Martins DF, Marcon R, Santos UD, Speckhann B, Gadotti VM, et al. High-intensity extended swimming exercise reduces pain-related behavior in mice: involvement of endogenous opioids and the serotonergic system. J Pain. 2010;11:1384-93, doi: 10.1016/j.jpain.2010. 03.015. 31. Souza JB. Poderia a atividade fı´sica induzir analgesia em pacientes com dor croˆnica? Rev Bras Med Esp. 2009;14:2. 32. Leinwand LA. Sex is a potent modifier of the cardiovascular system. J Clin Invest. 2003;112:302-7. 33. Ogihara CA, Schoorlemmer GH, Levada AC, Pithon-Curi TC, Curi R, Lopes OU, et al. Exercise changes regional vascular control by commissural NTS in spontaneously hypertensive rats. Am J Physiol Regul Integr Comp Physiol. 2010;299:R291-7, doi: 10.1152/ajpregu.00055.2009. 34. Serra AJ, Santos MH, Bocalini DS, Antoˆnio EL, Levy RF, Santos AA, et al. Exercise training inhibits inflammatory cytokines and more than prevents myocardial dysfunction in rats with sustained beta-adrenergic hyperactivity. J Physiol. 2010;588:2431-42, doi: 10.1113/jphysiol.2010.187310. 35. Bocalini DS, Carvalho EV, de Sousa AF, Levy RF, Tucci PJ. Exercise training-induced enhancement in myocardial mechanics is lost after 2 weeks of detraining in rats. Eur J Appl Physiol. 2010;109:909-14, doi: 10. 1007/s00421-010-1406-x.

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DOI:10.1590/S1807-59322011000900020

BASIC RESEARCH

The effects of pneumoperitoneum and controlled ventilation on peritoneal lymphatic bacterial clearance: experimental results in rats Armando Angelo Casaroli,I Lycia M. J. Mimica,II Belchor Fontes,III Samir RasslanIV I

Departamento de Cirurgia da Santa Casa de Miserico´rdia de Saõ Paulo, Saõ Paulo/SP, Brazil. II Disciplina de Microbiologia e Imunologia da Faculdade de Cieˆncias Me´dicas da Santa Casa de Saõ Paulo, Saõ Paulo/SP, Brazil. III Laborato´rio de Investigaçaõ Me´dica (LIM-62), Disciplina de Cirurgia Geral do Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brazil. IV Disciplinas de Cirurgia Geral e do Trauma do Departamento de Cirurgia da Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brazil.

OBJECTIVE: To evaluate the effect of pneumoperitoneum, both alone and in combination with controlled ventilation, on peritoneal lymphatic bacterial clearance using a rat bacterial peritonitis model. METHOD: A total of 69 male Wistar rats were intraperitoneally inoculated with an Escherichia coli solution (109 colony-forming units (cfu)/mL) and divided into three groups of 23 animals each: A (control group), B (pneumoperitoneum under 5 mmHg of constant pressure), and C (endotracheal intubation, controlled ventilation, and pneumoperitoneum as in Group B). The animals were sacrificed after 30 min under these conditions, and blood, mediastinal ganglia, lungs, peritoneum, liver, and spleen cultures were performed. RESULTS: Statistical analyses comparing the number of cfu/sample in each of the cultures showed that no differences existed between the three groups. CONCLUSION: Based on our results, we concluded that pneumoperitoneum, either alone or in association with mechanical ventilation, did not modify the bacterial clearance through the diaphragmatic lymphatic system of the peritoneal cavity. KEYWORDS: Diaphragm; Pneumoperitoneum; Mechanical Ventilation; Peritoneal Infection; Rats. Casaroli AA, Mimica LMJ, Fontes B, Rasslan S. The effects of pneumoperitoneum and controlled ventilation on peritoneal lymphatic bacterial clearance: experimental results in rats. Clinics. 2011;66(9):1621-1625. Received for publication on March 16, 2011; First review completed on April 15, 2011; Accepted for publication on June 2, 2011 E-mail: armandangelo@uol.com.br Tel.: 55 11 30696555

secretions.5 Although this concept has faced opposition,6 it is supported by studies reporting improved survival in animal peritonitis models following procedures designed to prevent bacterial clearance, such as scarification of the diaphragmatic surface and using substances to block the diaphragmatic pores.7 Other studies have observed that carbon particles injected into the mouse peritoneum are found in Kupffer cells 20 min later, possibly after absorption by the diaphragm and migration through blood and lymphatic transport systems.8 Thus, the possible role of the diaphragm in this process has remained unclear. More recent studies have shown that a CO2 insufflation-induced pneumoperitoneum increases bacterial (E. coli) translocation in rats,9 and others have observed that a CO2 pneumoperitoneum is associated with positive-end expiratory pressure and has neither a positive nor a negative impact on the systemic expansion of intra-abdominal E. coli infection.10 Despite frequent and progressive clinical utilization of video-laparoscopy in urgent abdominal surgery, certain questions concerning the possible effects of pneumoperitoneum on patients with abdominal infections remain unanswered. It has been argued that the increase in abdominal pressure induced by the pneumoperitoneum

INTRODUCTION The use of video-laparoscopy has significantly contributed to urgent abdominal surgery, both as a diagnostic method in acute cases and as an alternative, and even preferred, procedure for treating certain diseases.1,2 For many years, the presence of peritonitis was considered to be a significant risk factor for laparoscopy. However, research examining the use of video-laparoscopy in the presence of peritoneal infection has reported favorable clinical results.2,3 The presence of bacteria in the peritoneal cavity and its septic consequences have motivated recent research.4 Historically, the concept that bacterial clearance by the diaphragm is a negative rather than a positive prognostic factor was first suggested by the reduced mortality of peritonitis patients who were maintained in a semi-seated position that decreases diaphragmatic contact with peritoneal

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using a standard small-animal fan. The rats in Group C were maintained under controlled respiration with a minute volume of 400 mL and an average respiratory frequency of 40 incursions per minute. The pneumoperitoneum was created after the bacterial inoculation while the rats remained under anesthesia. Animal electronic insufflators were used to distend the peritoneal cavity at a constant 5-mmHg pressure and a flux of 0.2-0.5 mL/min. All of the groups were observed for 30 min. The pneumoperitoneum was then interrupted in Groups B and C, and the animals were sacrificed using a lethal dose of anesthesia. Blood cultures were grown on a hemolisobac (PROBAC) medium, and organ tissue cultures were grown on cysteine lactose electrolyte-deficient Agar (CLED-Agar).

could promote greater absorption of bacteria and toxins from the peritoneal cavity, which could increase the risk of septic shock if they enter the bloodstream.11 The distension of the peritoneal cavity produced by pneumoperitoneum causes alterations in diaphragmatic movements, intra-abdominal and intra-thoracic pressures, and respiratory dynamics, and these factors are usually considered to be involved in lymphatic drainage of the peritoneum. However, the true effect of pneumoperitoneum on the diaphragmatic lymphatic drainage system remains unclear. Similarly, it is not known whether the combination of controlled ventilation and constant-pressure pneumoperitoneum, which is commonly employed in video-laparoscopic surgeries, increases or decreases the removal of substances from the peritoneal cavity. In many experimental animal studies involving sepsis and pneumoperitoneum, controversy concerning the repercussions of video-surgery in the presence of intra-abdominal infection remains.12-15 Thus, the objective of this study was to use an experimental rat peritoneal contamination model to evaluate the influence of pneumoperitoneum, both alone and in combination with controlled ventilation, on lymphatic bacterial clearance from the peritoneal cavity.

Statistical analysis The presence or absence of bacteria in the blood samples was expressed as the percentage of animals with positive cultures in each group, and the results were analyzed using the likelihood-ratio test. The Kruskal-Wallis test and an analysis of variance (ANOVA) model were used to compare the mean number of cfu in the cultures per gram of tissue collected between the groups. Statistical significance was set at p,0.05.

MATERIALS AND METHODS A total of 69 adult male Wistar rats, weighing 200-300 g, was maintained in the laboratory on ad libitum water and standard diet for less than seven days. They were randomly divided into three groups of 23 rats each. All of the animals were anesthetized using intramuscular injections of ketamine (100 mg/kg) and xylazine (10 mg/kg) in the medial face of the thigh, and a 0.5-cm incision was made beneath the umbilical cicatrix to expose the delicate aponeurotic muscle layer. While light traction was maintained on the abdominal wall, a peritoneal puncture was performed with an 18-G Teflon catheter-embedded needle. After removing the metallic needle, the peritoneal cavity was inoculated with 1 mL of 109 colony forming units (cfu)/mL E. coli (ATCC pattern) solution. The Teflon catheter was maintained in all of the groups, with and without pneumoperitoneum, until the end of the experiment. After anesthesia and asepsis, the Group A rats were maintained under spontaneous respiration for a period of 30 min, the Group B rats were maintained under spontaneous respiration and pneumoperitoneum for a period of 30 min, and the Group C rats were intubated and maintained under controlled respiration and pneumoperitoneum for a period of 30 min. Instead of the usual tracheotomy, the orotracheal intubation in Group C used a technique to produce airways in small animals that was developed specifically for this experiment. To induce less surgical trauma, endotracheal access was obtained by direct laryngoscopy rather than a usual tracheotomy. The laryngoscopy used an optical video-laparoscope with a 5-mm diameter and a 30 Ë&#x161; angulated lens. The video-laparoscope was connected to a micro-camera system, light fountain, and video monitor. The anesthetized animal was maintained in the dorsal decubitus position, and its snout was lightly tractioned and lifted to create a space to introduce the optics. The optical system was used to correctly orient a number six Levine tube with a 5-cm extension in the transglottic position. Following the endotracheal intubation, the animals were maintained on mechanical ventilation

RESULTS The blood culture measurements were expressed as cfu/ mL of blood and analyzed as the percentage (%) of animals with positive cultures in each group. The other culture results were expressed as the number of cfu/g of tissue collected (Table 1). No significant differences between Groups A, B, and C were found for any of the cultures (Table 1 and Figures 1, 2, and 3).

DISCUSSION In the context of video-laparoscopic surgery, the role of CO2 insufflation-induced pneumoperitoneum in combination with mechanical ventilation in the dissemination of peritoneal bacterial infection and sepsis remains controversial.5,16,17 When bacterial contamination is introduced into the peritoneal cavity, three major defense mechanisms are activated to remove the infection: bacterial clearance by the diaphragmatic lymphatic system, phagocytosis by local macrophages, and migration of neutrophils to the abdomen. Lymphatic drainage and macrophage activity are the first lines of defense against bacteria following peritoneal contamination.5,18,19 Due to its considerable level of communication with the rich lymphatic system, the diaphragmatic peritoneum confers the particular function of bacterial clearance from the peritoneum upon the diaphragm. Bacteria are removed from the peritoneal cavity through this lymphatic system and reach the mediastinal lymph nodes through the retrosternal nodes.20,21 A decrease in diaphragmatic mobility causes a decline in bacterial clearance from the peritoneum.22,23 Thus, the dampening effect of mechanical ventilation on diaphragmatic dynamics could be expected to attenuate this bacterial clearance mechanism.19 Another factor that interferes with the absorption of substances by

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Table 1 - Comparison of the blood (qualitative) and other tissue (quantitative) culture results between the three groups. Animal groups All animals were bacterially inoculated. (n = 23)

Diaphragm Mean (SD) Mediastinal lymph nodes Mean (SD) Liver Mean (SD) Spleen Mean (SD) Peritoneum Mean (SD) Lung Mean (SD) Blood n (%) 95% CI

Comparison of the three groups

No pneumoperitoneum

With pneumoperitoneum

Pneumoperitoneum + Mechanical ventilation

p-value

42.06105 (53.36105)

48.46105 (66.56105)

25.26105 (35.86105)

0.4379*

141.3610 (476.4610 )

38.1610 (77.5610 )

20.2610 (51.9610 )

0.8254*

12.06105 (27.96105)

12.96105 (21.16105)

21.26105 (40.06105)

0.5405**

10.8610 (25.3610 )

31.8610 (61.3610 )

17.6610 (35.9610 )

0.2561**

51.86105 (79.76105)

78.76105 (120.76105)

32.86105 (49.96105)

0.1841*

6.6610 (16.0610 )

3.8610 (7.8610 )

5.5610 (16.5610 )

0.8049**

7 (30.4%)

8 (34.8%)

7 (30.4%)

0.9354***

[19.44%; 68.06%]

[28.09%; 78.58%]

[19.44%; 68.06%]

CI - Confidence interval. *p-value for the nonparametric Kruskal-Wallis test. **p-value for the ANOVA model. ***p-value for the likelihood ratio test. There were no significant differences between the groups for any of the variables in the Table.

the combined effect of these two antagonistic factors on diaphragm-mediated bacterial removal remains unclear. In this context, we hypothesized that these two opposing factors compete to define the lymphatic bacterial clearance from the peritoneal cavity. On the one hand, the pneumoperitoneum increases the intra-abdominal pressure, which favors bacterial clearance via the lymphatic system. On the other hand, lengthening of the diaphragmatic surface and controlled respiration decrease the lymphatic pumping action of the diaphragm,22 which results in reduced

the diaphragmatic lymphatic system is abdominal pressure; there is a direct correlation between abdominal pressure and diaphragmatic lymphatic bacterial clearance.24,25 The gaseous pneumoperitoneum used in laparoscopic surgery causes elevated intra-abdominal pressure and leads to increased diaphragmatic bacterial clearance from the peritoneal cavity. It also leads to peritoneal distension, which promotes diaphragmatic lengthening and interferes with diaphragmatic movement. This condition may reduce bacterial clearance from the peritoneal cavity.26 However,

Figure 1 - Box-plots for diaphragmatic and mediastinal lymph node tissue cultures. Groups: A = no pneumoperitoneum, B = pneumoperitoneum, and C = pneumoperitoneum + mechanical ventilation.

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Figure 2 - Box-plots for liver and spleen tissue cultures. Groups: A = no pneumoperitoneum, B = pneumoperitoneum, and C = pneumoperitoneum + mechanical ventilation.

between the groups in the numbers of E. coli cfu in the diaphragmatic or mediastinal lymph nodes, which participate in the most relevant mechanism for bacterial clearance. The similarities between the three groups in this study may have resulted from complex, synergistic effects of the pneumoperitoneum and mechanical ventilation on the diaphragmatic lymphatic system, as discussed above. Furthermore, no significant differences between the three groups were observed in our hemoculture results. Similar hemoculture results have been observed in other studies.28,29 Additionally, no evidence of hemodynamic alterations or increased bacteremia or endotoxemia have been

bacterial clearance. The 30-min period used in this study was based on previous experiments showing that peritoneal lymphatic clearance of particulate matter is quickly initiated, becoming detectable in minutes, and absorbs most of the material in approximately 30 min.27,28 Thus, controlled ventilation combined with pneumoperitoneum was used to simulate the conditions of laparoscopic surgical procedures that employ pneumoperitoneum and mechanical ventilation. We found no significant differences between the three groups based on blood, liver, lung, spleen, and peritoneum cultures. We also did not observe significant differences

Figure 3 - Box-Plots for Peritoneum and Lung tissue cultures. Groups: A = no pneumoperitoneum, B = pneumoperitoneum, and C = pneumoperitoneum + mechanical ventilation.

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reported in several other studies that examined the effect of pneumoperitoneum on the dissemination of peritoneal bacteria in animal models.12,29,30 However, other studies have reported an increased incidence of bacteremia one hour after insufflation of the peritoneal cavity.31,32 Overall, no clear consensus can be obtained from the results of experiments that have investigated this issue.

14.

15. 16.

CONCLUSION

17.

Based on our results, we conclude that pneumoperitoneum, either alone or in combination with mechanical ventilation, does not modify the lymphatic clearance of peritoneal bacteria through diaphragmatic drainage. More studies are required to clarify the conflicting results observed in the literature on this topic.

18. 19. 20.

REFERENCES

21.

1. Salky BA, Edye MB. The role of laparoscopy in the diagnosis and treatment of abdominal pain syndromes. Surg Endosc. 1998;12:911-4, doi: 10.1007/s004649900744. 2. Navez B, Tassetti V, Scohy JJ, Mutter D, Guiot P. Laparoscopic Management of acute peritonitis. Br J Surg. 1998;85:32–6, doi: 10.1046/ j.1365-2168.1998.00531.x. 3. Geis WP, Kim HC. Use of laparoscopy in the diagnosis and treatment of patients with surgical abdominal sepsis. Surg Endosc. 1995;9:178-82, doi: 10.1007/BF00191962. 4. Souza YM, Fontes B, Martins JO, Sannomiya P, Brito GS, Younes RN, Rasslan S. Evaluation of the effects of ozone therapy in the treatment of intra-abdominal infection in rats. Clinics. 2010;65:195-202, doi: 10.1590/ S1807-59322010000200012. 5. Maddaus MA, Ahrenholz D, Simmons R. The biology of peritonitis and implications for treatment. Surg Clin North Am. 1988; 68:431–443. 6. Steinberg B. Infections of the Peritoneum. New York, Paul Hoeber Inc, 1944;455. 7. Silva LN, Cardoso MB, Gondek JFL, Esmanhotto LB, Sebastia˜o APM, Simo˜es JC. Peritonite aguda experimental em ratos modelo de bloqueio transdiafragma´ tico com membrana celulo´ sica. Rev Col Bras Cir. 1998;25:113-7, doi: 10.1590/S0100-69911998000200008. 8. Abu-Hijleh MF, Habbal OA, Moqattash ST. The role of the diaphragm in lymphatic absorption from the peritoneal cavity. J Anat. 1995;186:453–67. 9. Horattas MC, Haller N, Ricchiuti D. Increased transperitoneal bacterial Translocation in laparoscopic surgery. Surg Endosc. 2003;17:1464-7, doi: 10.1007/s00464-001-8289-1. 10. Barbaros U, Ozarmagan S, Erbil Y, Bozbora A, Cakar N, Eraksoy H, Kapran Y, Kiran B. Effects of pneumoperitoneum created through CO2 insufflation and parameters of mechanical ventilation (PEEP application) on systemic dissemination of intraabdominal infections. Surg Endosc. 2004;8:501-7, doi: 10.1007/s00464-003-9107-8 11. Greif WM, Forse RA. Hemodynamic effects of the laparoscopic pneumoperitoneum during sepsis in a porcine endotoxic shock model. Ann Surg. 1998;227:474-80, doi: 10.1097/00000658-199804000-00004. 12. Collet D, Vitale GC, Reynolds M, Klar E, Cheadle WG. Peritoneal host defenses are less impaired by laparoscopy than by open operation. Surg Endosc. 1995;9:1059-64, doi: 10.1007/BF00188987. 13. Bustos B, Go´mez-Ferrer F, Balique JG, Porcheron J, Gobernado M, Canto´n E. Laparoscopy and septic dissemination caused by perioperative

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perforation of the occluded small bowel: an experimental study. Surg Laparosc Endosc. 1997;7:228-31, doi: 10.1097/00019509-199706000-00010. Dugue L, Fritsch S, Felten A, Gossot D, Colomer S, Celerier M, et al. Effects of intraperitoneal insufflation on hematogenous seeding of abdominal infections: Preliminary results of an experimental study in rats. Ann Chir. 1995;49:423–6. Jacobi CA, Ordemann J, Bohm B, Zieren HU, Volk HD, Lorenz W, et al. Does laparoscopy increase bacteremia and endotoxemia in a peritonitis model? Surg Endosc. 1997;11:235-8, doi: 10.1007/s004649900333. Balague´ PC, Trias M. Laparoscopic surgery and surgical infection. J Chemother. 2001;13 Spec. 1:17-22. Targarona EM, Rodrı´guez M, Camacho M, Balague´ C, Gich I, Vila L, et al. Immediate peritoneal response to bacterial contamination during laparoscopic surgery. Surg Endosc. 2006;20:316-21, doi: 10.1007/s00464005-0367-3. Leak LV. Interaction of mesothelium to intraperitoneal stimulation. I. Aggregation of peritoneal cells. Lab Invest. 1983;48:479–91. Skau T, Nystro¨m PO, Ohman L, Stendahl O. Bacterial clearance and granulocyte response in experimental peritonitis. J Surg Res. 1986;40:1320, doi: 10.1016/0022-4804(86)90139-3. Tsilibary EC, Wissig SL. Light and electron microscope observation of the lymphatic drainage units of the peritoneal cavity of rodents. Am J Anat. 1987;180:195-207, doi: 10.1002/aja.1001800209. Abu-Hijleh MF, Scothorne RJ. Regional Lymph Drainage Routes From the Diaphragm in the Rat. Clin Anat. 1994;7:181-8, doi: 10.1002/ca. 980070403. Bettendorf U. Lymph flow mechanism of the subperitoneal diaphragmatic lymphatics. Lymphology. 1978;11:111-6. Mengle HA. Effect of anaesthetics on lymphatic absorption from the peritoneal cavity in peritonitis: an experimental study. Arch Surg. 1937;34:39-52. Forey H. Reactions of, and absorption by lymphatics, with special reference to those of the diaphragm. Br J Exp Pathol. 1927;28:479-90. Zink J, Greenway CV. Control of ascites absorption in anesthetized cats; effects of intraperitoneal pressure, protein and furosemide diuresis. Gastroenterology. 1977;73:1119-24. SareM, YesiladaO, Gu¨relM, BalkayaM, YologluS, FiskinK. Effects of CO2 insufflation on bacterial growth in rats with Escherichia coli–induced experimental peritonitis. Surg Laparosc Endosc. 1997;7:38-41, doi: 10. 1097/00019509-199702000-00010. Courtice FC, Steinbeck AW. The lymphatic drainage of plasma from the peritoneal cavity of the cat. Aust J Exp Biol Med Sci. 1950;28:161–9, doi: 10.1038/icb.1950.15. Hau T, Simmons RL. Mechanisms of the adjuvant effect of hemoglobin in experimental peritonitis. III. The influence of hemoglobin on phagocytosis and intracellular killing by human granulocytes. Surgery. 1980;87:588-92. Gurtner GC, Robertson CS, Chung CS, Ling TK, Ip SM, Li AK. Effect of carbon dioxide pneumoperitoneum on bacteraemia and endotoxaemia in an animal model of peritonitis. Br J Surg. 1995;82:844-8, doi: 10.1002/bjs. 1800820639. Silva FCS. Ana´lise da concentraçaõ sanguıńea de bacte´rias durante insuflaçaõ peritoneal com dio´xido de carbono em ca˜es com peritonite bacteriana. Saõ Paulo, 1997 (Doctarate thesis, University of Saõ Paulo School of Medicine). Ipek TM, Paksoy T, Colak E, Polat E, Uygun N. Effect of carbon dioxide pneumoperitoneum on bacteremia and severity of peritonitis in an experimental model. Surg Endosc. 1998;12:432-5, doi: 10.1007/ s004649900697. Jacobi CA, Ordemann J, Zieren HU, Volk HD, Bauhofer A, Halle E, et al. Increased Systemic Inflammation After Laparotomy vs Laparoscopy in an Animal Model of Peritonitis. Arch Surg. 1998;133:258-62, doi: 10.1001/ archsurg.133.3.258.

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DOI:10.1590/S1807-59322011000900021

REVIEW

Brazilian guidelines for the diagnosis and treatment of hereditary angioedema Pedro Giavina-Bianchi,I,II Alfeu T. Franc¸a,I,III Anete S. Grumach,I,IV Abı´lio A. Motta,I,II Fa´tima R. Fernandes,I,V Regis A. Campos,I,VI Solange O. Valle,I,III Nelson A. Rosa´rio,I,VII Dirceu Sole´I,VIII I

Associaçaõ Brasileira de Alergia e Imunopatologia (ASBAI), Saõ Paulo/SP, Brazil. II Division of Clinical Immunology and Allergy, Department of Internal Medicine, University of Saõ Paulo, Saõ Paulo/SP, Brazil. III Department of Clinical Immunology, Federal University of Rio de Janeiro, RJ, Brazil. IV Primary Immunodeficiency Outpatient Group, Department of Dermatology, University of Saõ Paulo, Saõ Paulo/SP, Brazil. V Department of Allergy and Clinical Immunology, Hospital do Servidor Pu´blico Estadual de Saõ Paulo – FMO, SP, Brazil. VI Department of Clinical Immunology, Medical School, Federal University of Bahia, Salvador/BA, Brazil. VII Department of Pediatrics, Federal University of Parana´, Curitiba/PR, Brazil. VIII Division of Allergy, Clinical Immunology and Rheumatology, Department of Pediatrics, Federal University of Saõ Paulo-Paulista School of Medicine, Saõ Paulo/SP, Brazil.

Hereditary angioedema is an autosomal dominant disease characterized by edema attacks with multiple organ involvement. It is caused by a quantitative or functional deficiency of the C1 inhibitor, which is a member of the serine protease inhibitor family. Hereditary angioedema is unknown to many health professionals and is therefore an underdiagnosed disease. The causes of death from hereditary angioedema include laryngeal edema with asphyxia. The estimated mortality rate in patients in whom the disease goes undetected and who are therefore incorrectly treated is 25-40%. In addition to edema of the glottis, hereditary angioedema often results in edema of the gastrointestinal tract, which can be incapacitating. Patients with hereditary angioedema may undergo unnecessary surgical interventions because the digestive tract can be the primary or only organ system involved, thus mimicking acute surgical abdomen. It is estimated that patients with hereditary angioedema experience some degree of disability 20-100 days per year. The Experts in Clinical Immunology and Allergy of the ‘‘Associaçaõ Brasileira de Alergia e Imunopatologia - ASBAI’’ developed these guidelines for the diagnosis, therapy, and management of hereditary angioedema. KEYWORDS: Hereditary angioedema; C1 inhibitor; Asphyxia; Acute surgical abdomen; Guidelines; Consensus. Giavina-Bianchi P, França AT, Grumach AS, Motta AA, Fernandes FR, Campos RA. Brazilian guidelines for the diagnosis and treatment of hereditary angioedema. Clinics. 2011;66(9):1627-1636. Received for publication on April 27, 2011; First review completed on April 28, 2011; Accepted for publication on May 1, 2011 E-mail: saudesos@saudesos.com.br Tel.: 55 11 30713189

Sir William Osler (1849-1919), a Canadian physician who lived in the United States and England, was renowned for his many contributions to medicine, including his participation in the description of HAE and for stating that ‘‘Medicine is both a Science and an Art’’. To develop evidence-based guidelines is to practice medicine as a science. To follow such guidelines and consensuses while treating patients and their various phenotypes in a personalized manner is to practice medicine as an art. HAE is a disease that is unknown to many health professionals and is therefore underdiagnosed. The prevalence of HAE is approximately 1550,000 (with estimates ranging from 1510,000 to 15150,000); the disease affects various ethnic groups and accounts for 2% of all cases of angioedema.4-7 As is true for other autosomal dominant diseases, the children of a patient with HAE have a 50% chance of carrying the anomalous gene. Although a family history is characteristic of the disease and should alert physicians to a possible diagnosis of HAE, in 20-25% of cases, a family history of disease is absent, and new, spontaneous mutations can be observed.8 Approximately 200 HAE-associated mutations have been identified to date. Although genetic defects are found in patients of both genders with equal

DEFINITION What is hereditary angioedema? Hereditary angioedema (HAE) is an autosomal dominant disease that is characterized by edema attacks with multiple organ involvement. It is caused by quantitative or functional deficiency of the C1 inhibitor (C1-INH), which is a member of the serine protease inhibitor family.

INTRODUCTION Why should we study HAE? HAE was first described as a clinical entity by Quincke in 1882, and its hereditary nature was established by Osler in 1888.1,2 The biochemical change associated with HAE, C1INH deficiency, was not identified until 75 years later, in 1963.3

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reduced C1-INH synthesis (a quantitative defect).28 Low serum levels of C1-INH trigger the attacks. In patients who present with type II HAE (15-20%), adequate quantities of C1-INH are produced, although the functional capacity of the protein is partially diminished.29 Therefore, the enzyme has a qualitative (i.e., functional) defect. Type III HAE is rarer than types I and II and principally affects females. It is characterized by normal C1-INH levels and activity.30 This group includes diseases that vary in their etiopathogenesis; coagulation disorders and links to the endocrine system have been identified in this group.9,31,32 Acquired angioedema (AAE), which has a manifestation similar to that of HAE, is noteworthy among the differential diagnoses of HAE.33 AAE is characteristically associated with lymphoproliferative and autoimmune diseases in which there is consumption of C1-INH, which is caused by the activation of C1-INH or by the production of anti-C1INH autoantibodies.34

frequency, the phenotype is more common in female patients, for whom the disease is more severe.9 The time elapsed between the onset of symptoms and diagnosis, as well as the time between diagnosis and the initiation of treatment, play important roles in HAE-related morbidity and mortality.10-13 Therefore, physicians should be aware of the clinical profile and laboratory tests that confirm a diagnosis of HAE and inform decisions regarding its treatment. The cause of death from HAE is laryngeal edema with asphyxia, and the estimated mortality rate in patients in whom the disease goes undetected and who are therefore incorrectly treated is 25-40%.7,10-15 Patients with HAE are commonly hospitalized and admitted to intensive care units, and the disease accounts for 15,000-30,000 emergency room visits per year in the United States.16 The two most severe clinical manifestations of HAE are edema of the larynx and edema of the bowel wall. Delayed diagnosis increases morbidity, thereby affecting the quality of life of patients and their families.17,18 A study involving two families showed that nine out of ten family members who were hospitalized for symptoms of HAE were discharged with diagnoses other than HAE.19 In addition to life-threatening edema of the glottis, HAE often results in edema of the gastrointestinal tract, which can be incapacitating. Patients with HAE may undergo unnecessary surgical interventions because the digestive tract can be the primary or only organ system involved, thus mimicking acute surgical abdomen.20,21 It is estimated that patients with HAE experience some degree of disability 20100 days per year.17

DIAGNOSIS What is the typical clinical manifestation of HAE? The patientâ&#x20AC;&#x2122;s clinical history is the principal component of an HAE diagnosis. Patients with HAE present with attacks of nonpruritic edema of the skin and submucosa that affects various organs.11-13 The most commonly affected sites are the face, extremities, genitalia, oropharynx, larynx, and digestive system. However, rarer clinical manifestations, including intense headaches caused by brain edema, urinary retention, and acute pancreatitis, can also occur.12,35 The incidence and severity of the clinical manifestations of HAE vary among individuals. It has been reported that 5% of patients with HAE are asymptomatic, and 25% develop sporadic symptoms.6,11,12,36-38 A retrospective study analyzing 131,110 attacks in 221 patients with HAE reported that laryngeal edema occurred in less than 1% of the attacks, although over 50% of the patients had previously experienced that symptom at least once.12 Left untreated, HAE attacks typically last 48-72 h. Although many attacks occur spontaneously, a number of triggering factors have been identified, including minor trauma, stress, infection, menstruation, pregnancy, alcohol consumption, a change in temperature, exercise, the use of angiotensin-converting enzyme (ACE) inhibitors, and the use of estrogen (including contraceptives and hormone replacement therapy).6,11,12,36-39 Serpiginous erythema can occur as a prodromal manifestation that precedes angioedema in some patients; however, the concomitant presence of pruritic urticaria makes the diagnosis of HAE unlikely.6,11,12,36-38 However, some cases of HAE coexisting with urticaria have been reported.40 A family history of clinical manifestations that are similar to those seen in the patient supports a diagnosis of HAE, although this type of family history is absent in approximately 25% of cases.8 In children, the clinical manifestations of HAE generally develop before six years of age. However, the onset of clinical manifestations in infants is rare, and few cases have been described. Attacks of laryngeal edema are particularly rare before three years of age and tend to occur later than other manifestations. During adolescence, there are substantial changes in disease activity, particularly in girls, for

PATHOPHYSIOLOGY What is the cause of HAE? Patients with HAE present with a quantitative or qualitative deficiency of C1-INH, which is a serine protease inhibitor. This enzyme inhibits the C1r and C1s esterases, which bind to and activate C1q. Without such inhibition, the complement system becomes excessively activated.22,23 C1-INH also inhibits the lectin pathway of complement system activation and participates in the regulation of the contact, coagulation, and fibrinolysis systems. Deficiency of C1-INH results in increased bradykinin production. Episodes of angioedema were initially attributed to factors that were formed during complement system activation, including a C2 fragment (C2 kinin) that is associated with vasodilation and increased vascular permeability.24 Recent evidence has shown that bradykinin is one of the principal mediators of HAE.25-27 It has been shown that bradykinin levels are higher in blood drawn from angioedematous sites than in blood drawn from the systemic circulation.25 Knockout mice with concomitant deficiencies of C1-INH and bradykinin receptor B2 (BDKRB2) display decreased vascular permeability, which demonstrates that the bradykinin/BDKRB2 pathway plays an important role in the development of angioedema.26

CLASSIFICATION What are the types of HAE? Currently, HAE is divided into three groups (Table 1). Most patients (80-85%) present with type I HAE, with

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Table 1 - Classification of hereditary angioedema. TYPE

DEFECT

Type I â&#x20AC;&#x201C; Quantitative Type II - Functional; Qualitative Type III

Decreased C1 inhibitor synthesis Decreased C1 inhibitor function Normal C1 inhibitor levels and function A- Estrogen-dependent or estrogen-related B- Mutation of factor XII (Hageman factor) C- Idiopathic

Following (or concomitant with) the determination of serum levels of C4, quantitative and qualitative determinations of C1-INH should be performed. All health professionals and family members who are involved in providing care for patients with HAE must ensure that such tests are available. Although the quantitative determination of C1INH is a relatively easy test to perform, the determination of functional enzymatic activity (qualitative test) should be performed at a referral laboratory.6,36-38 Importantly, the determination of functional activity is only necessary when C1-INH levels are normal (Figure 1). If clinical suspicion remains despite normal C4 levels and normal (quantitative/qualitative) C1-INH levels, these tests should be performed again during an angioedema attack.6,36-38 If the test results are again normal, a diagnosis of type III HAE is suggested.30

whom disease progression is worse because of their menstrual cycles and their use of contraceptives containing estrogen.6,11,12,36-38 Although the manifestations of type III HAE are similar to those of the other HAE types, there are certain features unique to this type. Symptom onset generally occurs later in life, the course of the disease tends to be more benign, and tongue involvement is common. Purpura is occasionally seen at sites that are affected by angioedema. However, the most striking characteristic of type III HAE is a personal and family history of associations between the disease and female gender and between the disease and estrogen administration. In AAE, the onset of symptoms also occurs later, there is no family history of angioedema, and the associations between AAE and lymphoproliferative diseases and between AAE and autoimmune diseases should be investigated.33,34

What are the diagnostic criteria for HAE? Criteria to standardize the diagnosis of HAE have been proposed (Table 3).36 According to these criteria, the presence of HAE is confirmed when patients meet one major clinical criterion and one biochemical criterion. It should be noted that those criteria are not absolute and that the patientâ&#x20AC;&#x2122;s clinical history takes precedence, principally in locations where the laboratory tests are not available and in suspected cases of type III HAE. In selected cases, we can presume a diagnosis of HAE and perform a therapeutic test.

How can laboratory tests confirm the diagnosis of HAE? Individuals who are clinically suspected of having HAE and individuals with a family history of HAE should be investigated (Table 2). The principal screening test is the determination of serum levels of C4.6,36-38 Quantitative or qualitative C1-INH deficiency leads to the permanent activation of the complement system, accompanied by C4 consumption, even when patients are not experiencing an angioedema attack. In 2-5% of cases, C4 levels normalize during the intercrisis period.41 C3 turnover is greater than C4 turnover. In addition, there are other proteins that, together with C1-INH, regulate the consumption of C3, the levels of which are generally normal in HAE patients. Therefore, the determination of C3 levels is unnecessary except in patients who are suspected of having AAE, principally in the presence of autoimmune diseases.6,36-38 In AAE, there is activation and consumption of complement components, and 75% of patients present with reduced serum levels of C1q.42 Therefore, the determination of C1q levels can aid in the differentiation between HAE and AAE.

TREATMENT Guidance To improve the quality of life of patients and their families and to avoid severe complications, the most important initial measure is to provide guidance regarding the course of HAE and the triggering factors for attacks. Patients should be given relevant information (in writing) regarding the disease and the steps that must be taken in the case of an attack (an action plan). There are nonpharmacological approaches that can reduce HAE severity and therefore merit attention.

Table 2 - Laboratory diagnosis of angioedema. Type of Angioedema Type I HAE Type II HAE Type III HAE AAE ACE inhibitor Idiopathic

C1-INH level

C1-INH function

C1q

Low Normal Normal Low Normal Normal

Low Low Normal Low Normal Normal

Normal Normal Normal Normal/Low Normal Normal

Normal Normal Normal Low Normal Normal

C1-INH: C1 inhibitor, HAE: hereditary angioedema, AAE: acquired angioedema, ACE: angiotensin-converting enzyme.

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Table 4 - General recommendations for the medical treatment of attacks of hereditary angioedema. Take a personal and family history of the acute attack profile Use oxygenation and pulse oximetry Hydrate Use antispasmodics and analgesics as needed Avoid the use of angiotensin-converting enzyme inhibitors Increase the dose of chronic medication to control the attack Use acute phase control agents (fresh plasma, C1 inhibitor, bradykinin receptor antagonist, or kallikrein inhibitor) if available

peptidase-4 inhibitors, which can worsen the clinical profile of HAE.47 Figure 1 - Diagnostic algorithm of hereditary angioedema. C1INH: C1 inhibitor, HAE: hereditary angioedema, AAE: acquired angioedema, NL: normal.

Genetic screening of family members All first-degree relatives of patients with HAE should be screened for HAE. In pediatric patients with a family history of HAE, the HAE diagnostic tests should be performed at six months of age and repeated at one year.48 Patients and their families should receive genetic counseling and guidance regarding the inheritance pattern of the disease.49

Because of the significant morbidity and mortality associated with HAE, a strategy involving careful treatment and prevention of attacks is essential for appropriate patient management (Table 4). The experience at large centers has demonstrated that 25-40% of patients can develop asphyxia and die if they are left untreated.7,10-15

Pharmacological treatment The pharmacological treatment of HAE can be divided into three modalities: long-term prophylaxis, short-term prophylaxis, and treatment of attacks. The therapies recommended here were classified according to the strength of the recommendation, which is based on the level of scientific evidence supporting such a recommendation (Table 5).

Prevention of attacks The risk of attacks can be reduced by identifying and eliminating the factors that trigger them. Stress and trauma are clearly triggering factors for edema in HAE, so patients should monitor those factors. High-impact sports and hobbies that pose a risk of trauma should be avoided. To prevent infections, which constitute a trigger of attacks, immunization is indicated. Hepatitis B vaccination is recommended because blood products might be used during the treatment of HAE attacks.43

How should long-term prophylaxis be performed? The objective of long-term prophylaxis in HAE is to reduce the frequency and severity of attacks. These two variables can vary widely. Patients may be asymptomatic or experience attacks twice per week with symptoms that are practically continuous. Therefore, the first question regarding long-term prophylaxis should be whether patients actually require this type of treatment. In general, individuals with frequent symptoms or a history of angioedema attacks involving the upper airways should be treated prophylactically. Patients who are candidates for this type of treatment are those who present with more than one severe attack per month or those who experience more than five attacks per month.6,36-38 Although the number and severity of angioedema attacks play important roles in this evaluation, the impact of angioedema episodes on the patientâ&#x20AC;&#x2122;s quality of life is a decisive aspect. Another important point to be considered is whether patients have access to appropriate medical care in the case of a severe episode of angioedema.50 In Brazil, two treatment modalities are available for longterm prophylaxis: attenuated androgens and antifibrinolytic agents. The most effective and best tolerated therapy for longterm prophylaxis of HAE is attenuated androgens that increase the levels of C1-INH and C4 and reduce the number of attacks of angioedema (grade B recommendation).51 The drugs used include danazol, stanozolol, and oxandrolone, which are less virilizing than methyltestosterone. Oxandrolone is especially recommended for use in children.52

Drugs that can worsen or prolong an HAE attack The drugs that most commonly worsen or prolong the clinical profile of HAE or AAE are ACE inhibitors, angiotensin II receptor antagonists, medications containing estrogen, and certain oral hypoglycemic agents. Because ACE inhibitors increase the half-life of bradykinin, their use should be avoided.44 Angiotensin II receptor antagonists can also worsen the clinical profile of HAE, although they do so less commonly than ACE inhibitors.45 Contraceptives containing progesterone should be preferentially prescribed over those containing estrogen.16,46 Sitagliptin phosphate, which is an oral hypoglycemic agent indicated for the treatment of type 2 diabetes, belongs to a class of oral hypoglycemic agents designated dipeptidyl Table 3 - Diagnostic criteria for hereditary angioedema.36 I - Primary clinical criteria a) Non-inflammatory subcutaneous angioedema lasting longer than 12 h b) Abdominal pain of undefined organic etiology lasting longer than 6 h c) Recurrent laryngeal edema II - Secondary clinical criteria a) Family history of hereditary angioedema III - Biochemical criteria a) Quantitative C1 inhibitor ,50% in two distinct samples b) Functional C1 inhibitor ,50% in two distinct samples c) Mutation in the C1 inhibitor gene

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Table 5 - Evidence levels and degrees of recommendation for therapies.

Levels of evidence Level 1

Meaning One or more randomized controlled clinical trials of sufficient sample size with a narrow confidence interval Meta-analysis of randomized controlled clinical trials Good quality cohort study Low-quality randomized clinical trial (small sample size and .20% lost to follow-up) Case-control studies Meta-analysis of case-control studies Case series Low-quality cohort studies Low-quality case-control studies Based on expert opinions, experimental studies or physiology

3 4

The best strategy should be chosen based on the patient’s clinical status. Therefore, we should consider whether it is more important to control the angioedema attacks as fast as possible or to minimize the potential adverse effects of the drug. In both protocols, the final dose should be the lowest dose that provides adequate prophylaxis, generally ranging from the administration of 50 to 200 mg/day or on alternate days. Although androgens increase the levels of C1-INH and C4, symptomatic benefits are generally achieved at doses lower than those required to significantly alter the levels of complement components. Therefore, it is important that the drug dosage is based on the patient’s clinical symptoms rather than his or her biochemical parameters. It should be emphasized that androgens are ineffective in controlling attacks of HAE because it takes approximately 48 h for the drugs to begin to take effect.55 Most patients tolerate androgens at the aforementioned doses. However, sustained use at higher doses generally results in significant adverse effects. The adverse effects of androgens are related to the dosage, with the major adverse effects being hepatotoxicity and virilization.56 Other adverse reactions include weight gain, headache, menstrual changes, acne, changes in libido, anxiety, mood disorders, hypertension, myopathy, changes in lipid profiles, and hematuria.57 Although there is evidence of changes in patients’ lipid profiles, the association between danazol and atherosclerosis is controversial.57-59 Danazolinduced hematuria results from mild cystitis or bladder telangiectasia.60 These adverse effects tend to disappear after the discontinuation of the drug. In individuals receiving androgens, the hepatic enzyme levels should be evaluated every six months. If liver damage develops, the dose should be reduced or the drug should be discontinued until these tests yield normal results. Because hepatic adenomas and hepatocellular carcinoma have previously been reported as consequences of the use of androgens, liver ultrasounds should also be routinely performed every six months.61,62 The development of liver tumors in patients who are using danazol has been associated with the use of higher doses (400-800 mg), a longer duration of administration, and the lack of monitoring for liver injury.63 Although many patients complain of androgen-induced adverse events, most patients with HAE can benefit, at least moderately, from androgen use, and the risk profile is acceptable. Danazol is contraindicated during pregnancy because there have been documented cases of virilization in female neonates. However, discontinuation of the drug by gestational week eight has been shown to avert that alteration.64-66 Other contraindications for danazol use include breastfeeding and prostate cancer, as well as kidney, liver, and heart failure.

Grade/Strength of recommendation Grade A B

Meaning Corresponding to level of evidence 1 Corresponding to level of evidence 2 or 3 Extrapolated from level 1 studies involving populations other than the current population Corresponding to level of evidence 4 Extrapolated from level 2 or 3 studies involving populations other than the current population Corresponding to level of evidence 5

Adapted from: Levels of evidence and grades of recommendation, Oxford Center for Evidence-Based Medicine, May 2001. http://www.cebm.net/levels_of_evidence.

In Brazil, the most widely used attenuated androgen is danazol, which is also the most widely available (its availability is guaranteed by the High-Cost Drug Program); in addition, well-controlled studies have demonstrated the clinical efficacy of this drug and have shown that it improves biochemical parameters.51,53,54 Treatment with danazol can be started at high doses (600 mg/day), which can later be reduced. Another option is to start at a low dose (50-200 mg) and increase the dose as needed. Therefore, two protocols have been established: 6 a)

Milan Protocol—Initiation at a high dose with a subsequent reduction: -

400-600 mg/day of danazol for one month Wean by 1/3, or 100 mg, every month At 200 mg/day, taper by 50 mg every two months At 100 mg/day, taper by 50 mg every three months Minimum dose of 50 mg/day, five days per week If the symptoms recur, re-induce remission with the initial dose and wean to a higher maintenance dose than the previously administered dose.

Budapest Protocol—Initiation at a low dose with a subsequent increase: -

If there is no response, increase to 400 mg/day for two-four weeks If controlled at 200 mg, reduce to 100 mg/day for one month If controlled at 100 mg, reduce to 50 mg/day or 100 mg on alternate days If there are prodromal symptoms of attack, double the dose for several days.

2.5 mg/kg of danazol (maximum of 200 mg) administered daily for one month If there is no response, increase to 300 mg/day for two-four weeks

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In addition to prophylaxis, patients should remain under observation for 36 h and should have easy access to rescue medication.5 Therapeutic intervention should be performed before the occurrence of events that can provoke HAE attacks. Favorable results have been obtained with the use of attenuated androgens, antifibrinolytic agents, fresh frozen plasma, and C1-INH concentrates (Table 6). Fresh frozen plasma can be administered intravenously at a dose of 2 U the night before surgery or on the day of surgery (grade D recommendation). Epsilon-aminocaproic acid is effective when it is administered several days before the triggering event (grade C recommendation). Androgens, which have a grade C recommendation, are used three-five days before surgery at doses of 10 mg N kg21 N day21, with a maximum dose of 600 mg/day.76 For prophylaxis in adults and children, C1-INH concentrates are safe and effective at doses of 500-1,000 U when administered intravenously (grade C recommendation). Some of these products have also been tested and are currently used in pregnant women. Initially, in the 1970s, C1-INH concentrate was established as the emergency treatment for HAE. With the introduction of viral inactivation processes, C1-INH concentrate also became a first-line drug for the prophylaxis of acute HAE attacks. There are two C1-INH concentrates currently in use in the United States: CinryzeH and BerinertH. Because of regulatory issues, CinryzeH is indicated for short-term prophylaxis of HAE but not for the treatment of attacks, and BerinertH is indicated for the treatment of acute HAE attacks.

Antifibrinolytic agents (epsilon-aminocaproic acid and tranexamic acid) are generally effective (grade B recommendation) in preventing HAE attacks.67-71 These drugs antagonize the fibrinolytic system by blocking plasmin formation and inhibiting the proteolytic activity of plasminogen activators and, consequently, clot dissolution. The mechanism by which antifibrinolytic agents prevent HAE attacks remains unknown. Unlike attenuated androgens, antifibrinolytic agents do not increase the serum levels of C1-INH or C4. The therapeutic dose of epsilon-aminocaproic acid is 1 g, administered orally three to four times per day, and it can be as high as 8 g/day.55 Tranexamic acid should be administered at a dose of 0.5-0.75 mg/kg two to three times per day; the drug is more potent than aminocaproic acid and has a lower incidence of adverse effects.72 The side effects of antifibrinolytic agents include nausea, diarrhea, vertigo, postural hypotension, fatigue, muscle weakness, cramps, and increased muscle enzyme levels.50 Other adverse effects that are associated with the inhibition of plasmin include the increased occurrence of thrombosis and increased tumor growth. Because of the risks of teratogenicity, the use of antifibrinolytic agents during pregnancy is restricted.50 As occurs with attenuated androgens, the time of onset of the therapeutic effects of antifibrinolytic agents is approximately 48 h after administration. Therefore, both classes of drugs are of little use in providing immediate symptom relief. Because anabolic androgens are more effective in controlling HAE, they generally constitute the treatment of choice (grade C recommendation). Antifibrinolytic agents should be reserved for patients who do not tolerate anabolic androgens or for cases in which anabolic androgens are contraindicated. In severe cases for which the maximum dose of androgen is not sufficient to control the attacks, antifibrinolytic agents can be used in combination with androgens. Long-term prophylaxis of HAE is satisfactory for most patients but has the disadvantage of requiring daily medication. In some patients, the use of androgens or antifibrinolytic agents is impractical because of the adverse reactions that they provoke (principally in females) or because of a lack of response. In addition, neither class of drugs is safe for use during pregnancy. Although they are still unavailable in Brazil, C1-INH concentrates, which have a grade B recommendation, have been used elsewhere for the long-term prophylaxis of HAE.73-75 Administered intravenously at regular intervals, approximately three times per week, C1-INH concentrates constitute a viable therapeutic alternative for individuals in whom other therapies cannot be used or are ineffective. Regardless of the drug chosen, the effectiveness of each drug depends on treatment adherence, which must be encouraged and evaluated.

How should HAE attacks be treated? The treatment of acute HAE attacks depends on their severity (Table 7). Severe attacks and attacks involving the respiratory tract require urgent treatment because of the potential for morbidity and mortality. Although episodes of peripheral edema rarely require treatment, the early administration of danazol can shorten the duration of attacks and uncomfortable symptoms (grade D recommendation). Patients who are taking attenuated androgens prophylactically should double the dose for a few days after identifying an attack in any part of the body.76 Therapy with antifibrinolytic agents, such as tranexamic acid administered orally every 3-4 h, has also been recommended for mild crisis periods (grade C recommendation). Abdominal attacks are extremely painful and can be accompanied by vomiting, diarrhea, or both. When patients present with severe abdominal attacks, symptomatic treatment with the administration of fluids, antiemetics, and analgesics is indicated. Antispasmodics and narcotics may be required to treat intense pain.37 Dysphonia and dysphagia are indicative of progression to a severe laryngeal attack. Such attacks develop slowly over the course of approximately 8 h on average, and dysphagia and voice changes generally precede laryngeal obstruction.16 However, there have been reports of rapid-onset laryngeal edema, and physicians should bear this in mind when evaluating such patients. More severe cases require immediate intubation. In such cases, oxygen therapy is indicated, and pulse oximetry should be monitored. During laryngoscopy and intubation, the need for tracheostomy should be evaluated.77 In cases of laryngeal edema, it may be prudent to perform prophylactic

How should short-term prophylaxis be performed? Short-term prophylaxis is indicated for patients undergoing major surgical procedures (e.g., surgery with orotracheal intubation), other surgical procedures (principally craniofacial procedures), invasive diagnostic procedures (e.g., endoscopy), or major dental procedures (all patients should be informed of the higher risk of attacks during major dental procedures).

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Table 6 - Drugs used for the short-term prophylaxis and treatment of acute attacks of hereditary angioedema. Short-term prophylaxis

Trade name

Dose

Adverse events

Tranexamic acid

TransaminH HemoblockH

Diarrhea

High-dose attenuated androgens

Danazol Oxandrolone

1 g administered orally every 4 h (or 0.5 g administered intravenously every 4 h) for 18 h 10 mg N kg21 N day21 with a maximum dose of 600 mg/day for 3-5 days before the procedure 500-1,000 U on the day before the procedure or on the day of the procedure 10 ml/kg

C1-INH concentrate* and recombinant C1-INH*

BerinertH CinryzeH

Fresh plasma

Weight gain, voice changes, increased hair growth, and menstrual irregularity -

Hyperosmolarity

Treatment of acute attacks C1-INH concentrate* and recombinant C1-INH* Fresh plasma Bradykinin receptor Antagonist

Kallikrein inhibitor* *

BerinertH CinryzeH Icatibant (FirazyrH)

Ecallantide (KalbitorH)

1,000 U administered intravenously or 10-20 U/kg 10 ml/kg 30 mg/dose and repeat an initial dose and a second dose if the attack persists 20 U/kg or 30 mg/dose and repeat the second dose if the attack persists

Hyperosmolarity Local hyperemia

Hypersensitivity reactions

Drugs that have not yet been approved in Brazil.

The fractional catabolic rate of C1-INH is 2.5% of the plasma pool per hour. However, in patients with HAE, the half-life of C1-INH is longer than predicted by the fractional catabolic rate (.48 h vs. 28 h). This difference is probably attributable to the observation that exogenous C1-INH reduces the consumption of endogenous C1-INH, preventing C1 autoactivation.50,79,80 RhucinH, which was developed recently, is a recombinant C1-INH derived from the milk of transgenic rabbits.75 Although its half-life is only 3 h, RhucinH has been shown to produce a response similar to that produced by other commercially available C1-INH preparations in the treatment of HAE attacks (grade B recommendation).75,81 Further studies are needed to assess the effects of Rhucin. In addition to C1-INH replacement therapy, blocking the synthesis and effects of bradykinin constitute other pharmacological approaches to HAE attacks. Recent studies have confirmed the efficacy of a kallikrein inhibitor and a BDKRB2 antagonist. The BDKRB2 antagonist, icatibant, is highly specific for BDKRB2, binding to it with the same affinity as bradykinin and inhibiting a variety of BDKRB2-mediated effects.82 The drug is administered subcutaneously, and its plasma halflife is 2-4 h. Icatibant is degraded by peptidases, and the products of icatibant degradation are excreted by the

intubation as an early measure to maintain airway patency and avoid tracheostomy (grade D recommendation).77 In cases of severe acute attacks, the treatment of choice is C1-INH replacement therapy, which can be achieved by the intravenous infusion of 1,000 U of a C1-INH concentrate (grade A recommendation), by the infusion of recombinant C1-INH, or, as reported recently, by a kallikrein inhibitor or a BDKRB2 antagonist (grade B recommendation). In countries where the aforementioned drugs are not available, fresh plasma or solvent/detergent-treated plasma, which contains C1-INH, can be used (grade D recommendation). However, this type of treatment can worsen HAE attacks because fresh plasma also contains the remaining complement, coagulation, and contact system components. In addition, there are concerns regarding the safety of fresh plasma (e.g., transfusion-related acute lung injury, anaphylaxis, and viral transmission), and the need for a relatively large volume of plasma might be problematic in an emergency setting or in patients who cannot tolerate significant volume expansion. Therefore, when C1-INH concentrate, or bradykinin receptor antagonist, or kallikrein inhibitor are available, they are preferred over plasma transfusion.37,78,79 The symptoms generally improve 30-40 min after the infusion of C1-INH concentrate. A second, identical, dose can be administered if necessary.

Table 7 - Parameters for the treatment of acute attacks in patients with hereditary angioedema.6,38 Treatment

Wait and see (spontaneous resolution) Tranexamic acid C1-INH concentrate, bradykinin receptor antagonist, kallikrein inhibitor ICU (intubation/tracheostomy)

Edema of the skin

Abdominal attack

Laryngeal edema

Torso and extremities

Face and neck region

ยก + ยก

2 + +

C1-INH: C1 inhibitor, ICU: intensive care unit. + indicated. ยก consider indication. 2 contraindicated.

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kidneys. Icatibant was initially shown to be effective in treating seasonal allergic rhinitis and asthma. Subsequently, two double-blind, randomized, multicenter trials (the For Angioedema Subcutaneous Treatment trial, parts 1 and 2) found that a single dose of icatibant was effective in 90% of the attacks of HAE (grade B recommendation).83 One perspective that merits investigation is the home treatment of HAE attacks with icatibant. Icatibant has been approved in Brazil, and its use is indicated for the treatment of HAE attacks. Although the drug can be stored at room temperature, in Brazil, it is suggested that the drug be stored at 4 ˚C. Ecallantide is a high-potency recombinant protease inhibitor that binds to and inhibits kallikrein, thus decreasing bradykinin production. Because of its short half-life when it is administered subcutaneously, ecallantide has been evaluated for use in acute HAE attacks only. Multicenter phase III clinical trials have found a significant reduction in the severity of acute attacks after ecallantide administration compared to a placebo (grade B recommendation).84 Side effects were rare and included dyspnea, oropharyngeal edema, and prolonged prothrombin and thrombin times. There have also been isolated reports of anaphylactic reactions following ecallantide administration; therefore, the use of the drug is restricted to hospital environments.85 To date, no studies been conducted to compare the efficacy of C1-INH concentrate, icatibant, and ecallantide in the treatment of HAE attacks.86 Regarding pharmacological treatment, it should be highlighted that, unlike anaphylaxis and edemas associated with the degranulation of mast cells and basophils, C1-INH deficiency-induced angioedema does not respond significantly to the administration of antihistamines, glucocorticoids, or epinephrine.77

analgesia is recommended for surgical delivery; general anesthesia and orotracheal intubation should be avoided (grade C recommendation).

Suspected cases of acute abdomen Some HAE attacks mimic acute surgical abdomen, and negative exploratory laparotomy results are common in such patients.20,21 However, a diagnosis of HAE raises the concern that if there is a true abdominal emergency requiring surgery, the timing of the surgical procedure may be delayed.

CONCLUSION The Experts in Clinical Immunology and Allergy of the ‘‘Associac¸a˜o Brasileira de Alergia e Imunopatologia ASBAI’’ developed these guidelines for the diagnosis, therapy, and management of hereditary angioedema. We hope that these guidelines will aid readers in the diagnosis and treatment of HAE, which is a neglected disease. A Portuguese version of this consensus is published in the ‘‘Revista Brasileira de Alergia e Imunopatologia’’. 88

REFERENCES ¨ ber akutes umschriebenes Hauto¨dem. Monatsh Prakt 1. Quincke HI. U Dermatol. 1882;1:129-31. 2. Osler W. Hereditary angioneurotic edema. Am J Med Sci. 1888;95:362-7, doi: 10.1097/00000441-188804000-00004. 3. Donaldson VH, Evans RR. A biochemical abnormality in hereditary angioneurotic edema: absence of serum inhibitor of C’1-esterase. Am J Med. 1963;35:37-44, doi: 10.1016/0002-9343(63)90162-1. 4. Freiberger T, Kola´rova´ L, Mejstrı´k P, Vyskocilova´ M, Kuklı´nek P, Litzman J. Five novel mutations in the C1 inhibitor gene (C1INH) leading to a premature stop codon in patients with type I hereditary angioedema. Hum Mutat. 2002;19:461, doi: 10.1002/humu.9029. 5. Gompels MM, Lock RJ, Abinun M, Bethune CA, Davies G, Grattan C, et al. C1 inhibitor deficiency: consensus document. Clin Exp Immunol. 2005;139:379-94, doi: 10.1111/j.1365-2249.2005.02726.x. 6. Bowen T, Cicardi M, Bork K, Zuraw B, Frank M, Ritchie B, et al. Hereditary angioedema: a current state-of-the-art review, VII: Canadian Hungarian 2007 International Consensus Algorithm for the Diagnosis, Therapy, and Management of Hereditary Angioedema. Ann Allergy Asthma Immunol. 2008;100(1Suppl 2):S30-40, doi: 10.1016/S10811206(10)60584-4. 7. Agostoni A, Cicardi M. Hereditary and acquired C1-inhibitor deficiency: biological and clinical characteristics in 235 patients. Medicine (Baltimore). 1992;71:206-15. 8. Pappalardo E, Cicardi M, Duponchel C, Carugati A, Choquet S, Agostoni A, et al. Frequent de novo mutations and exon deletions in the C1 inhibitor gene of patients with angioedema. J Allergy Clin Immunol. 2000;106:1147-54, doi: 10.1067/mai.2000.110471. 9. Bouillet L. Hereditary angioedema in women. Allergy Asthma Clin Immunol. 2010;6:17, doi: 10.1186/1710-1492-6-17. 10. Cicardi M, Bergamaschini L, Marasini B, Boccassini G, Tucci A, Agostoni A. Hereditary angioedema: an appraisal of 104 cases. Am J Med Sci. 1982;284:2-9, doi: 10.1097/00000441-198207000-00001. 11. Bork K, Meng G, Staubach P, Hardt J. Hereditary Angioedema: New Findings Concerning Symptoms, Affected Organs, and Course. Am J Med. 2006;119:267-74, doi: 10.1016/j.amjmed.2005.09.064. 12. Tanno LK, Pinto LH, Motta AA, Kalil J, Giavina-Bianchi P. Hereditary Angioedema: Clinical Characteristics and Outcome of 36 Patients. J Allergy Clin Immunol. 2007;119(1S):S275, doi: 10.1016/j.jaci.2006.12. 445. 13. Grumach AS. Angioedema Heredita´rio, EPM, Sa˜o Paulo. 2008. 14. Fay A, Abinun M. Current management of hereditary angio-oedema (C’1 esterase inhibitor deficiency). J Clin Pathol. 2002;55:266-70, doi: 10.1136/ jcp.55.4.266. 15. Bork K, Hardt J, Schicketanz KH, Ressel N. Clinical studies of sudden upper airway obstruction in patients with hereditary angioedema due to C1 esterase inhibitor deficiency. Arch Intern Med. 2003;163:1229-35, doi: 10.1001/archinte.163.10.1229. 16. Moore GP, Hurley WT, Pace SA. Hereditary angioedema. Ann Emerg Med. 1988;17:1082-6, doi: 10.1016/S0196-0644(88)80450-5. 17. Lumry WR, Castaldo AJ, Vernon MK, Blaustein MB, Wilson DA, Horn PT. The humanistic burden of hereditary angioedema: Impact on

SPECIAL SITUATIONS Pediatric patients In pediatric patients, antifibrinolytic agents are the drugs of choice for long-term prophylaxis because of their safety profiles (grade C recommendation). Studies have reported that tranexamic acid is better tolerated than epsilonaminocaproic acid. When these agents are not sufficient, attenuated androgens may be required. The use of minimum maintenance doses for control has been found to have no negative impact on growth, and the only adverse effect that was observed was late menarche with subsequent menstrual irregularity, which was attributed to the use of danazol (200 mg/day).87 During the first two years of prophylaxis, it is recommended that laboratory tests be performed every three-four months and that abdominal ultrasounds be performed every six months. The clinical course of HAE should be monitored, as should any adverse effects of treatment.

Pregnancy and delivery During pregnancy and, if possible, even before conception, the ideal situation is that no prophylactic drug be used. Attenuated androgens are contraindicated, and tranexamic acid can be used if it is administered with caution (grade C recommendation). The treatment of HAE attacks does not change during pregnancy. Attacks during vaginal delivery are rare. However, when they do occur, they are severe. Regional

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18. 19.

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health-related quality of life, productivity, and depression. Allergy Asthma Proc. 2010;31:407-14, doi: 10.2500/aap.2010.31.3394. Bygum A, Andersen KE, Mikkelsen CS. Self-administration of intravenous C1-inhibitor therapy for hereditary angioedema and associated quality of life benefits. Eur J Dermatol. 2009;19:147-51. Winnewisser J, Rossi M, Spa¨th P, Bu¨rgi H. Type 1 hereditary angiooedema. Variability of clinical presentation and course within two large kindreds. J Inter Med. 1997;241:39-46, doi: 10.1046/j.1365-2796.1997. 76893000.x. Bork K, Staubach P, Eckardt AJ, Hardt J. Symptoms, course, and complications of abdominal attacks in hereditary angioedema due to C1 inhibitor deficiency. Am J Gastroenterol. 2006;101:619-27, doi: 10.1111/j. 1572-0241.2006.00492.x. Farkas H, Harmat G, Kaposi PN, Kara´di I, Fekete B, Fu¨st G, et al. Ultrasonography in the diagnosis and monitoring of ascites in acute abdominal attacks of hereditary angioneurotic oedema. Eur J Gastroenterol Hepatol. 2001;13:1225-30, doi: 10.1097/00042737-200110000-00016. Rosen FS, Pensky J, Donaldson V, Charache P. Hereditary angioneurotic edema: two genetic variants. Science. 1965;148:957-8, doi: 10.1126/ science.148.3672.957. Ziccardi RJ. Spontaneous activation of the first component of human complement (C1) by an intramolecular autocatalytic mechanism. J Immunol. 1982;128:2500-4. Fields T, Ghebrehiwet B, Kaplan AP. Kinin formation in hereditary angioedema plasma: evidence against kinin derivation from C2 and in support of ‘‘spontaneous’’ formation of bradykinin. J Allergy Clin Immunol. 1983;72:54-60, doi: 10.1016/0091-6749(83)90052-0. Nussberger J, Cugno M, Amstutz C, Cicardi M, Pellacani A, Agostoni A. Plasma bradykinin in angio-oedema. Lancet. 1998;351:1693-7, doi: 10. 1016/S0140-6736(97)09137-X. Han ED, MacFarlane RC, Mulligan AN, Scafidi J, Davis AE 3rd. Increased vascular permeability in C1 inhibitor-deficient mice mediated by the bradykinin type 2 receptor. J Clin Invest. 2002;109:1057-63. Nussberger J, Cugno M, Cicardi M. Bradykinin-mediated angioedema. N Engl J Med. 2002;347:621-2, doi: 10.1056/NEJM200208223470820. Zuraw BL, Herschbach J. Detection of C1 inhibitor mutations in patients with hereditary angioedema. J Allergy Clin Immunol. 2000;105:541-6, doi: 10.1067/mai.2000.104780. Bowen B, Hawk JJ, Sibunka S, Hovick S, Weiler JM. A review of the reported defects in the human C1 esterase inhibitor gene producing hereditary angioedema including four new mutations. Clin Immunol. 2001;98:157-63, doi: 10.1006/clim.2000.4947. Bork K, Barnstedt SE, Koch P, Traupe H. Hereditary angioedema with normal C1-inhibitor activity in women. Lancet. 2000;356:213-7, doi: 10. 1016/S0140-6736(00)02483-1. Cichon S, Martin L, Hennies HC, Mu¨ller F, Van Driessche K, Karpushova A, et al. Increased activity of coagulation factor XII (Hageman Factor) causes hereditary angioedema type III. Am J Hum Genet. 2006;79:1098104, doi: 10.1086/509899. Serrano C, Guilarte M, Tella R, Dalmau G, Bartra J, Gaig P, et al. Oestrogen-dependent hereditary angio-oedema with normal C1 inhibitor: description of six new cases and review of pathogenic mechanisms and treatment. Allergy. 2008;63:735-41, doi: 10.1111/j.1398-9995.2007. 01579.x. Sheffer AL, Austen KF, Rosen FS, Fearon DT. Acquired deficiency of the inhibitor of the first component of complement: report of five additional cases with commentary on the syndrome. J Allergy Clin Immunol. 1985;75:640-6, doi: 10.1016/0091-6749(85)90087-9. Cugno M, Castelli R, Cicardi M. Angioedema due to acquired C1inhibitor deficiency: a bridging condition between autoimmunity and lymphoproliferation. Autoimmun Rev. 2008;8:156-9, doi: 10.1016/j. autrev.2008.05.003. ´. Marıń Garcıá D, Ceballos Torres A, Ruiz Serrato A, Garcıá Ordoń˜ez MA Acute pancreatitis associated with hereditary angioedema. Gastroenterol Hepatol. 2010;33:633-7, doi: 10.1016/j.gastrohep.2010.07.005. Agostoni A, Aygo¨ren-Pu¨rsu¨n E, Binkley KE, Blanch A, Bork K, Bouillet L, et al. Hereditary and acquired angioedema: problems and progress: proceedings of the third C1 esterase inhibitor deficiency workshop and beyond. J Allergy Clin Immunol. 2004;114:S51-131, doi: 10.1016/j.jaci. 2004.06.047. Bowen T, Cicardi M, Farkas H, Bork K, Kreuz W, Zingale L, et al. Canadian 2003 International Consensus Algorithm For the Diagnosis, Therapy, and Management of Hereditary Angioedema. J Allergy Clin Immunol. 2004;114:629-37, doi: 10.1016/j.jaci.2004.06.043. Bowen T, Cicardi M, Farkas H, Bork K, Longhurst HJ, Zuraw B, et al. 2010 International consensus algorithm for the diagnosis, therapy and management of hereditary angioedema. Allergy Asthma Clin Immunol. 2010;6:24, doi: 10.1186/1710-1492-6-24. França AT, Valle SOR. Urtica´ria e Angioedema. 2a ediçaõ. Editora Revinter, RJ. 2006;p193. Jorge AS, Dortas SD, Valle SO, França AT. Hereditary angioedema and chronic urticaria: is there a possible association? J Investig Allergol Clin Immunol. 2009;19:327-8.

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77. Jensen NF, Weiler JM. C1 esterase inhibitor deficiency, airway compromise, and anesthesia. Anesth Analg. 1998;87:480-8. 78. Sheffer AL. Hereditary angioedema: optimal therapy. J Allergy Clin Immunol. 2007;120:756-7, doi: 10.1016/j.jaci.2007.08.010. 79. Bernstein JA. Hereditary angioedema: a current state-of-the-art review, VIII: current status of emerging therapies. Ann Allergy Asthma Immunol. 2008;100 1Suppl 2:S41-6, doi: 10.1016/S1081-1206(10)60585-6. 80. Farkas H, Jakab L, Temesszentandra´si G, Visy B, Harmat G, Fu¨st G, et al. Hereditary angioedema: a decade of human C1-inhibitor concentrate therapy. J Allergy Clin Immunol. 2007;120:941-7, doi: 10.1016/j.jaci.2007. 06.026. 81. Lock RJ, Gompels MM. C1-inhibitor deficiencies (hereditary angioedema): where are we with therapies? Curr Allergy Asthma Rep. 2007;7:264-9, doi: 10.1007/s11882-007-0039-6. 82. Bork K, Frank J, Grundt B, Schlattmann P, Nussberger J, Kreuz W. Treatment of acute edema attacks in hereditary angioedema with a bradykinin receptor-2 antagonist (Icatibant). J Allergy Clin Immunol. 2007;119:1497-503, doi: 10.1016/j.jaci.2007.02.012. 83. Cicardi M, Banerji A, Bracho F, Malbra´n A, Rosenkranz B, Riedl M, et al. Icatibant, a new bradykinin-receptor antagonist, in hereditary angioedema. N Engl J Med. 2010;363:523-31, doi: 10.1056/NEJMoa0905079. 84. Cicardi M, Levy RJ, McNeil DL, Li HH, Sheffer AL, Campion MMS, et al. Ecallantide for the treatment of acute attacks in hereditary angioedema. N Engl J Med. 2010;363:532-41, doi: 10.1056/NEJMoa0906393. 85. Frank MM. Hereditary angioedema: a current state-of-the-art review, VI: novel therapies for hereditary angioedema. Ann Allergy Asthma Immunol. 2008;100 1Suppl 2:S23-9, doi: 10.1016/S1081-1206(10)60583-2. 86. Giavina-Bianchi P, Motta A, Kalil J. Therapeutic agents for hereditary angioedema. N Engl J Med. 2011;364:84-6, doi: 10.1056/NEJMc1010085. 87. Farkas H, Harmat G, Fu¨st G, Varga L, Visy B. Clinical management of hereditary angio-oedema in children. Pediatr Allergy Immunol. 2002;13:153-61, doi: 10.1034/j.1399-3038.2002.01014.x. 88. Giavina-Bianchi P, Franc¸a AT, Grumach AS, Motta AA, Fernandes FR, Campos RA, et al. Diretrizes do diagno´stico e tratamento do angioedema heredita´rio. Revista Brasileira de Alergia e Imunopatologia. 2010;33:241252.

66. Castro-Magana M, Cheruvanky T, Collipp PJ, Ghavami-Maibodi Z, Angulo M, Stewart C. Transient adrenogenital syndrome due to exposure to danazol in utero. Am J Dis Child. 1981;135:1032-4. 67. Frank MM, Sergent JS, Kane MA, Alling DW. Epsilon aminocaproic acid therapy of hereditary angioneurotic edema: A double-blind study. N Engl J Med. 1972;286:808-12, doi: 10.1056/NEJM197204132861503. 68. Sheffer AL, Austen KF, Rosen FS. Tranexamic acid therapy in hereditary angioneurotic edema. N Engl J Med. 1972;287:452-4, doi: 10.1056/ NEJM197208312870907. 69. Blohme´ G. Treatment of hereditary angioneurotic oedema with tranexamic acid. A random double-blind cross-over study. Acta Med Scand. 1972;192:293-8. 70. Champion RH, Lachmann PJ. Hereditary angio-oedema treated with Eaminocaproic acid. Br J Dermatol. 1969;81:763–5, doi: 10.1111/j.13652133.1969.tb15938.x. 71. Nilsson IM, Andersson L, Bjo¨rkman SE. Epsilon-aminocaproic acid (EACA) as a therapeutic agent based on 5 year’s clinical experience. Acta Med Scand Suppl. 1966;448:1–46. 72. Sheffer AL, Fearon DT, Austen KF, Rosen FS. Tranexamic acid: preoperative prophylatic therapy for patients with hereditary angioneurotic edema. J Allergy Clin Immunol. 1977;60:38-40, doi: 10.1016/ 0091-6749(77)90080-X. 73. Bork K, Hardt J. Hereditary angioedema: long-term treatment with one or more injections of C1 inhibitor concentrate per week. Int Arch Allergy Immunol. 2011;154:81-8, doi: 10.1159/000319213. 74. Tallroth GA. Long-Term Prophylaxis of Hereditary Angioedema with a Pasteurized C1 Inhibitor Concentrate. Int Arch Allergy Immunol. 2010;154:356-359, doi: 10.1159/000321830. 75. Zuraw BL, Busse PJ, White M, Jacobs J, Lumry W, Baker J, et al. Nanofiltered C1 inhibitor concentrate for treatment of hereditary angioedema. N Engl J Med. 2010;363:513-22, doi: 10.1056/ NEJMoa0805538. 76. Banerji A, Sloane DE, Sheffer AL. Hereditary Angioedema: a current state-of-the-art review, V: attenuated androgens for the treatment of hereditary angioedema. Ann Allergy Asthma Immunol. 2008;(100 1Suppl 2):S19-22, doi: 10.1016/S1081-1206(10)60582-0.

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DOI:10.1590/S1807-59322011000900022

RAPID COMMUNICATION

Hyperglycemia and postoperative outcomes in pediatric neurosurgery Eduardo Mekitarian Filho,I Werther Brunow de Carvalho,II Se´rgio Cavalheiro,III Nelson Kazunobu Horigoshi,IV Norberto Antonio Freddi,V Gil Kruppa VieiraVI I

Pediatrics and Sciences Applied to Pediatrics, Universidade Federal de Saõ Paulo (UNIFESP), Pediatric Emergency Department, University Hospital of Universidade de Saõ Paulo (USP) and Admissions Unit of Hospital Israelita Albert Einstein, Pediatric Intensivist, Santa Catarina Hospital, Saõ Paulo/SP, Brazil. II Neonatology and Intensive Care, Department of Pediatrics, School of Medicine, USP. Head of Pediatric ICU, Santa Catarina Hospital, Saõ Paulo/SP, Brazil. III Neurosurgery, UNIFESP, Saõ Paulo/SP, Brazil. IV Attending Physician, Pediatric ICU of Santa Catarina Hospital, Saõ Paulo/SP, Brazil. V Sciences, USP. Pediatrician, Hospital Israelita Albert Einstein. Head of Pediatric ICU, Santa Catarina Hospital, Saõ Paulo/SP, Brazil. VI University Hospital, University of Saõ Paulo/SP, Brazil. Email: emf2002@uol.com.br Tel.: 55 11 3091 9333/9451

glucose values were collected from patients during their stay in the PICU and during their stay in the pediatric ward after transfer from the PICU. Hyperglycemia was defined as a blood glucose level $150 mg/dL. The three outcomes evaluated in the study were the durations of mechanical ventilation, PICU, and hospital stay. Hyperglycemia was studied along with other likely risk factors in these patients, such as fever, laryngitis, infection, hypothermia, packed red blood cells, and hormonal disorders. First, a univariate analysis was performed to identify the main risk factors associated with the three outcomes, and the statistically relevant risk factors were separated and analyzed in a multivariate analysis. The level of significance was set at 5%. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) 18.0 (Chicago, Illinois). The study was approved by the Ethics in Research Committee of the institution where it was conducted.

INTRODUCTION Recent studies in adults have demonstrated the deleterious effects of hyperglycemia in intensive care patients and its substantial impact on crucial outcomes such as mortality. As a result, there is an increasing interest in the impact of this complication on the outcomes of critical illness in children.1 Hyperglycemia is prevalent among critically ill children and may be associated with poor outcomes and higher morbidity during hospitalization.2 Few prospective studies have analyzed the occurrence of hyperglycemia and the use of intensive insulin therapy in critically ill children. A recent single-center trial demonstrated the efficacy and safety of an insulin therapy protocol. The trial results indicated improved survival rates among the patients who received the treatment.3 A clear association between hyperglycemia and poorer outcomes has been demonstrated in some specific clinical situations, such as septic shock, cardiac surgery, and traumatic brain injury.4-7 However, hyperglycemia has yet to be fully studied in the context of pediatric surgery, and no pediatric neurosurgical studies have analyzed the association between hyperglycemia, morbidity, and mortality. This study evaluated the postoperative glucose levels of children who underwent neurosurgeries for different indications and analyzed the association of these levels with lengths of mechanical ventilation, intensive care, and hospital stay.

RESULTS The charts of 198 patients were analyzed during the study. The most frequent surgeries were brain tumor resection (37.4%), craniosynostosis (31.3%), ventriculoperitoneal shunting (16.7%), craniotomy for craniofacial dysostosis (4.5%), spinal arthrodesis (4%), epilepsy surgery (2%), and brain revascularization for Moyamoya disease (1.5%). A total of 139 glucose measurements were recorded for the patients included in the study. Hyperglycemia was diagnosed in 62.6% of the patients. The patient glucose level upon admission to the PICU and the highest glucose level noted in the first 24 hours post-admission were recorded. The mean glucose level was recorded following the 24-hour measurement. The results of the glucose measurements according to surgical diagnosis are listed in Table 1. A univariate analysis identified a positive association between hyperglycemia and a prolonged duration of PICU stay (3.88 days vs. 2.46 days, p = 0.042). However, hyperglycemia was not associated with prolonged hospitalization or the duration of mechanical ventilation required (Table 2). The multivariate analysis did not identify any positive associations between hyperglycemia and any of the three outcomes studied (Table 3).

MATERIAL AND METHODS The study was conducted in the Pediatric Intensive Care Unit (PICU) of Santa Catarina Hospital, Sao Paulo, Brazil, a tertiary multidisciplinary hospital with 16 PICU beds. This retrospective cohort study evaluated all patients admitted to the PICU who underwent neurosurgical procedures from May 2004 to May 2009. Both plasma and capillary blood

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Table 1 - Glucose levels according to surgical diagnosis. Diagnosis Ventriculoperitoneal shunting Craniostenosis Supratentorial brain tumors Infratentorial brain tumor Craniotomy for craniofacial dysostosis Spinal arthrodesis Epilepsy surgery Brain revascularization for arteriopathy Spinal cord tumors

Glucose level at admission * Highest glucose level in 24 hours * 135.4ยก66.5 159.2ยก69.9 160.5ยก83.3 131.2ยก30.8 161.8ยก39.2 149.8ยก61.8 154.3ยก41.1 148ยก28.3 128.3ยก30.1

133.6ยก38.5 150.4ยก64.2 163.3ยก47.9 151ยก44.7 152.7ยก14.2 238ยก172.5 138ยก9.9 N/E N/E

Mean glucose level * 117.9ยก23.6 118ยก39.9 131ยก31.9 126.1ยก36.9 155.1ยก42.3 112.2ยก13.1 110.9ยก11.9 N/E N/E

Mean ยก Standard Deviation (mg/dL). N/E โ Blood glucose measurement not evaluated due to the small number of samples.

highest glucose level within the first 24 hours following surgery was analyzed separately from the glucose level at admission. There was a progressive reduction in glucose levels in all surgical groups during the course of hospitalization. Mild hyperglycemia was identified and its rapid progression to normal levels may be explained by the low preoperative morbidity of the population studied, as most procedures were elective and performed in patients with a low anesthetic risk. According to this hypothesis, the patients were more likely not to present the main consequences of severe hyperglycemia, such as osmotic diuresis, dehydration, and metabolic disorders. When considering the results of the entire study population, hyperglycemia was not severe enough in this scenario to cause adverse effects and outcomes related to this metabolic disorder. It was not possible to evaluate the correlation between hyperglycemia and mortality because only two deaths occurred during the study. Nearly 59% of the patients received one or more doses of systemic corticosteroids during surgery or their ICU stay, while 93% of the patients who underwent brain tumor surgeries received them. There are no current recommendations regarding the routine postoperative use of corticosteroids, which are administered with the aim of reducing the brain edema associated with resection. Nevertheless, corticosteroids are well known to increase glucose levels, which may have contributed to our findings. We did not identify a significant association between hyperglycemia and the three outcomes examined, which may be explained by the small sample size and the relatively few glucose measurements taken in the study population. As hyperglycemia was common in this group, special attention should be paid to it to prevent adverse events, especially because other studies with larger series of patients found a significant association between hyperglycemia and worse outcomes in surgical patients. There were a few limitations associated with this study, particularly due to the retrospective analytical design and consequent lack of a previous protocol of glucose measurement. Moreover, some of the surgery subgroups contained few patients, which made it difficult to establish an association between glucose levels and outcomes. The treatment of hyperglycemia in pediatric intensive care must be further analyzed, as there is no consensus on the benefits of several procedures, such as insulin therapy and strict glycemic control in these situations. In children, organic components such as beta cells, liver, kidneys, and muscles have not been exposed to oxidative, lipotoxic or

DISCUSSION To the best of our knowledge, the present study is the first to analyze the effects and the incidence of hyperglycemia in a pediatric neurosurgery environment. The highest glucose levels were found in patients that underwent surgery for craniosynostosis, supratentorial tumor resection, and craniotomies; these procedures are more likely to involve longer surgical times and higher incidence of complications, such as bleeding and infection. These higher incidences of complications increase the risk of hyperglycemia. Univariate analysis indicated an association between hyperglycemia and prolonged duration of stay in the PICU. However, no association was found between hyperglycemia and the total duration of hospital stay or the length of mechanical ventilation time required. Multivariate analysis indicated no statistically relevant associations between hyperglycemia and any of the outcomes. The occurrence of severe hyperglycemia (above 300 mg/dL) was rare. Hyperglycemia is common in critical illness as a consequence of organic stress and counter-regulatory hormones and is typically interpreted as a secondary event in critical care. Several studies have tried to identify an association between hyperglycemia and a worse prognosis in children, but their findings should be interpreted as markers of severe illness. As the patientโ s condition improves, it is likely that their glucose levels will revert to the normal range. As a secondary event, patients with other complications that may affect glucose levels, either directly or indirectly, such as fever, infection or bleeding, are more likely to develop hyperglycemia and, consequently, worse outcomes such as a prolonged ICU stay. Such factors may explain why hyperglycemia was found to be associated with a prolonged stay in the PICU in the univariate but not in the multivariate analysis. Up to 50% of pediatric patients who undergo neurosurgeries may develop several postoperative complications,8 and hyperglycemia is an important event that may impact on their clinical course. Stress hyperglycemia is associated with insulin resistance and frequently affects adults and children. Some of the factors that may contribute to stress hyperglycemia are high glucose infusion rates, the increase in the production of endogenous glucose due to action of the counter-regulatory hormones (cortisol, glucagon, and catecholamine), and changes in glucose transport.9 Changes in insulin counter-regulatory hormones due to surgical stress have not been clearly defined; therefore, the

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Hyperglycemia and pediatric neurosurgery Filho EM et al.

Table 2 - Results of univariate analysis. Outcome

PICU LOS Hosp. LOS MV time

PICU LOS Hosp. LOS

Risk Factor Fever No Yes No Yes No Yes Use of PRBC No Yes No Yes No Yes Laryngitis No Yes No Yes No Yes Coag. Disorders No Yes No Yes No Yes Inappropriate ADH Syndrome No Yes No Yes No Yes Seizures No Yes No Yes No Yes Hyperglycemia No Yes No yes No yes CSF Leakage No yes No yes No yes ICH No yes No yes No Yes Infection No Yes No Yes

Table 2 Cont.

Median

p-value

2.7 5.08 5.96 9.9 5.83 8.48

1.9 8.11 4.61 10.97 8.49 11.57

138 60 138 60 138 60

0.006

3.13 3.73 7.23 7.08 5.54 7.8

2.94 6.26 6.79 7.95 5.55 12.43

102 96 102 96 102 96

2.9 6.33 6.6 10.3 6.21 9.02

2.29 10.89 5.66 13.06 8.83 12.91

168 30 168 30 168 30

0.027

3.32 5.44 7.08 8.78 5.92 21.72

4.82 5.05 7.4 6.4 6.58 31.15

189 9 189 9 189 9

0.065

3.4 4.67 7.4 6.3 6.61 8.33

4.87 1.15 7.41 0.58 9.64 1.15

195 3 195 3 195 3

0.037

3.03 12.63 6.65 19.25 6.56 8.5

2.62 19.33 5.79 21.07 9.75 3.02

190 8 190 8 190 8

0.003

2.46 3.88 5.15 8.46 5.27 9.74

1.43 3.76 2.02 7.49 1.71 15.6

52 87 52 87 52 87

0.042

3.24 9.17 6.7 21.67 6.32 16.83

4.72 5.6 6.75 11.47 8.49 27.05

192 6 192 6 192 6

0.001

2.97 12.78 6.72 16.33 6.49 9.67

2.54 17.95 5.78 21.14 9.67 6.96

189 9 189 9 189 9

,0.001

3.17 8.56 6.58 19.33

4.7 5.22 6.74 9.46

189 9 189 9

,0.001

Outcome

Risk Factor

Median

p-value

MV time

No Yes Hypothermia No Yes No Yes No Yes

6.18 16.17

8.8 18.15

189 9

0.001

3.5 3 7.37 6.06 7.05 4.47

5.14 2.86 7.8 4.28 10.38 1.88

166 32 166 32 166 32

0.65

0.001

PICU LOS

,0.001

Hosp. LOS MV time

0.468

0.365 0.038

Legend – PICU LOS – Pediatric Intensive Care Unit Length of stay, SD – standard deviation, MV – mechanical ventilation, PRBC – packed red blood cells, CSF – cerebrospinal fluid, ICH – intracranial hypertension, ADH – antidiuretic hormone.

0.623 0.017

hyperglycemic stress for extended periods, and the effects of hyperglycemia on these patients may differ from those seen in adult populations.10-12 Therefore, changes in glucose levels that develop after neurosurgery should be better studied to outline preventive measures, such as determination of glucose infusion rates, prevention of unnecessary use of systemic corticosteroids, and early diagnosis of infection.

0.059 0.036

0.247 0.006

CONCLUSIONS Hyperglycemia is frequent in children following neurosurgery and was not found to be associated with the durations of mechanical ventilation, PICU stay, or hospital stay in this sample of pediatric patients. In pediatric patients, glucose levels should be carefully and accurately controlled after surgery, as this may reduce morbidity and hospitalization time. Further studies are necessary to elucidate the role of hyperglycemia in pediatric neurosurgical patients.

0.214 0.022

,0.001 0.002

Table 3 - Results of multivariate analysis. Outcome

Risk Factor

0.073

Regression

p-value

Analysis (Wald Test)

0.078

PICU LOS

,0.001 0.152

Hospital LOS

0.011 MV time

0.039

,0.001

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Fever Laryngitis Hyperglycemia Coagulation disorders Infection Use of steroids Fever Laryngitis Coagulation disorders Infection Use of steroids Fever Laryngitis Use of PRBC Coagulation disorders Infection Use of steroids

7.72 9.13 3.2 0.23

,0.001 0.003 0.062 0.63

4.1 2.96 7.54 3.39 0.31

0.043 0.085 0.006 0.066 0.576

8.99 0.02 3.88 4.29 5.27 14.11

0.003 0.894 0.049 0.038 0.022 ,0.001

2.36 48.24

0.125 ,0.001

Hyperglycemia and pediatric neurosurgery Filho EM et al.

CLINICS 2011;66(9):1637-1640

REFERENCES 7.

1. Preissig CM, Rigby MR. Pediatric critical illness hyperglycemia: risk factors associated with development and severity of hyperglycemia in critically ill children. J Pediatr. 2009;155:734-9, doi: 10.1016/j.jpeds.2009.05.007. 2. Hirshberg E, Larsen G, Van Duker HV. Alterations in glucose homeostasis in the pediatric intensive care unit: hyperglycemia and glucose variability are associated with increased mortality and morbidity. Pediatr Crit Care Med. 2008;9:361-6, doi: 10.1097/PCC. 0b013e318172d401. 3. Verhoeven JJ, Brand JB, van de Polder MM, Joosten KFM. Management of hyperglycemia in the pediatric intensive care unit; implementation of a glucose control protocol. Pediatr Crit Care Med. 2009;10:648-52, doi: 10. 1097/PCC.0b013e3181ae787b. 4. Branco RG, Garcia PC, Piva JP, Casartelli CH, Seibel V, Tasker RC. Glucose level and risk of mortality in pediatric septic shock. Pediatr Crit Care Med. 2005;6:470-2, doi: 10.1097/01.PCC.0000161284.96739.3A. 5. Yates AR, Dyke PC 2nd, Taeed R, Hoffman TM, Hayes J, Feltes JF, et al. Hyperglycemia is a marker for poor outcome in the postoperative pediatric cardiac patient. Pediatr Crit Care Med. 2006;7:351-5, doi: 10. 1097/01.PCC.0000227755.96700.98. 6. Chiaretti A, Piastra M, Pulitano S, Pietrini D, De Rosa G, Barbaro G, et al. Prognostic factors and outcome of children with severe head injury: an

9. 10. 11. 12.

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8-year experience. Childs Nerv Syst. 2002;18:129-36, doi: 10.1007/s00381002-0558-3. Smith RL, Lin JC, Adelson PD, Kochanek PM, Fink EL, Wisniewski S, et al. Relationship between hyperglycemia and outcome in children with severe traumatic brain injury. Pediatr Crit Care Med. 2012;13 (in press). Ferna´ndez de Sevilla Estrach M, Cambra Lasaosa FJ, Segura Matute S, Guille´n Quesada A, Palomeque Rico A. Postoperatorio de tumores cerebrales en la unidad de cuidados intensivos pedia´tricos. An Pediatr (Barc). 2009;70:282-6, doi: 10.1016/j.anpedi.2008.10.015. Ognibene KL, Vawdrey DK, Biagas KV. The association of age, illness severity, and glycemic status in a pediatric intensive care unit. Pediatr Crit Care Med. 2012;13 (in press). Kyle UG, Bu JAC, Kennedy CE, Jefferson LS. Organ dysfunction is associated with hyperglycemia in critically ill children. Intensive Care Med. 2010;36:312-20, doi: 10.1007/s00134-009-1703-1. Klein GW, Hojsak JM, Rapaport R. Hyperglycemia in the pediatric intensive care unit. Curr Opin Clin Nutr Metab Care. 2007;10:187-92, doi: 10.1097/MCO.0b013e3280147d3e. Rosenbaum M, Nonas C, Horlick M, Fennov I, Vargas I, Schachner H, et al. Beta-cell function and insulin sensitivity in early adolescence: association with body fatness and family history of type 2 diabetes mellitus. J Clin Endocrinol Metab. 2004;98:5469-76, doi: 10.1210/jc.20040971.

CLINICS 2011;66(9):1641-1643

DOI:10.1590/S1807-59322011000900023

RAPID COMMUNICATION

Association of MMP-8 polymorphisms with tendinopathy of the primary posterior tibial tendon: a pilot study Alexandre Leme Godoy-Santos,I Rafael Trevisan,I Tu´lio Diniz Fernandes,I Maria Cristina L. G. dos SantosII I

Instituto de Ortopedia e Traumatologia – Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brazil. Departamento de Biologia Celular - Setor de Cieˆncias Biolo´gicas, Universidade Federal do Parana´, Curitiba/PR, Brazil.

Email: alexandrelemegodoy@gmail.com Tel.: 55 11 30696888

several inflammatory diseases and is related to cardiovascular disease,9 bronchiectasis,13 pulmonary insufficiency,14 periodontitis,15-17 melanoma,18 cancer of the head and neck,19 and diabetic wound healing.20 The MMP-8 gene, located on chromosome 11, contains functional polymorphisms in the promoter region, including the substitution of a cytosine by a thymine at position 799 (rs11225395).21 Alterations in this gene have been associated with chronic dilatation of the bronchi13 and breast cancer.22 The discovery of genetic markers of tendinopathy risk could allow for the identification of susceptible individuals and, thus, early therapeutic interventions. MMPs play key roles in tissue destruction and may have important roles in the pathogenesis of tendinopathy. Therefore, we hypothesized that the -799C/T polymorphism in MMP-8 was associated with tendinopathy of the primary posterior tibial tendon and could be a risk factor for this condition.

INTRODUCTION Tendon disorders are common and are treated on a daily basis in orthopedic foot and ankle practices. Some tendons are particularly vulnerable to primary degenerative changes, such as the patellar, Achilles, rotator cuff, biceps, posterior tibial, and fibular tendons.1 Significant advances in histopathology and research imaging techniques have contributed to an improved understanding of the pathophysiology of tendon degeneration.2 However, both mechanical factors and vascular and neurological disorders have limitations in explaining the etiology of many cases.1,3,4 It is known that intense physical activity may predispose an individual to tendinopathy, but some individuals have a predisposition with no clinically recognized cause. The literature suggests that individual characteristics, including genetic inheritance, may influence the likelihood of developing tendinopathy. Thus, there is a group of individuals with a genetic background that causes increased susceptibility to diseases of the tendon. Matrix metalloproteinases (MMPs) are a pivotal family of zinc-dependent enzymes responsible for the degradation of extracellular matrix components, including basement membrane collagen, interstitial collagen, fibronectin and various proteoglycans, during normal remodeling, repair processes, development and inflammation. MMPs are expressed in response to specific stimuli by resident connective tissue cells and by the major inflammatory cell types that invade tissues during remodeling events, including tendinopathy. MMP-8, or collagenase-2, was initially discovered in neutrophils, in which it was thought to be exclusively produced, but this enzyme was subsequently shown to be expressed by a variety of other cell types, including endothelial cells, smooth muscle cells, macrophages, polymorphonuclear leukocytes, gingival fibroblasts, keratinocytes, chondrocytes, odontoblasts5-10 and oral11 cancer cells. It has been suggested that MMP-8 degrades type I collagen, thereby contributing to tissue degradation and remodeling.12 MMP-8 is an important mediator of tissue destruction in

MATERIALS AND METHODS Subject selection A total of 64 subjects were recruited from the patient pool at the Foot and Ankle Group, Department of Orthopedics, at the Traumatology Hospital of the University of Sa˜o Paulo, Brazil. After a brief explanation of the study, as recommended by the the hospital’s Research Ethics Committee, subjects were identified according to age, sex, diagnosis, pathology, medical history, postoperative complications, medication use, personal history of systemic diseases, and family history of infectious and inflammatory diseases. The subjects were divided into two groups, with 66% female and a mean age of 51 years: Test group - 14 patients undergoing surgical procedures who had pathological diagnoses of degenerative lesions of the posterior tibial tendon. Control group - 50 patients with posterior tibial tendon integrity and with no signs of degeneration based on magnetic resonance images produced to investigate complaints about the ankle and foot. All the subjects were in good general health and did not display any of the following exclusion criteria: rheumatic diseases, immunological diseases, diabetes, hepatitis or prior or current infections in the topography of the foot and ankle.

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CLINICS 2011;66(9):1641-1643

DNA sampling and extraction

Table 1 - The distribution of the MMP-8 alleles and genotypes in the control and test groups.

Buccal epithelial cells were sampled as described by Trevilatto and Line (2000),23 and DNA extraction was performed as by Aidar and Line (2007).24 The DNA concentration was estimated by measuring the absorbance ratio at 260/280 nm.

MMP-8

Control Group n

Allele C T Genotype C/C T/T C/T

Polymerase chain reaction and restriction endonuclease digestion The MMP-8 genotypes were determined using the polymerase chain reaction - restricted fragment length polymorphism (PCR-RFLP) assay. The PCR primers used for amplifying the MMP-8 polymorphism were the forward primer 59-CAGAGACTCAAGTGGGA-39 and the reverse primer 59-TTTCATTTGTGGAGGGGC-39. The PCR reactions were performed in a total volume of 10 ml that contained 400 ng of genomic DNA, 5 ml of SYBR GREEN JUMPSTART TAQ READY MIX (Amersham Pharmacia-Biotech, Uppsala, Sweden) and 200 nmol of each primer. A 6-ml aliquot of each PCR product was then digested with 1 unit of SfcI enzyme at 37 Ë&#x161;C overnight.

n = 100 71.0 29.0 n = 50 33 66.0 12 24.0 05 10.0

71 29

Test Group n

p-value

(chi-squared)

35.7 64.3

p = 0.0006

n = 28 10 18 n = 14 03 07 04

(Fisherâ&#x20AC;&#x2122;s exact test) 21.4 50.0 28.6

p = 0.0036

type V collagen gene (COL5A1) was associated with Achilles tendon pathologies, with the A2 allele appearing to play a protective role. The same group also demonstrated the role of tenascin-C in Achilles tendon pathologies.29 By analyzing a guanine-thymine (GT)n repeat polymorphism in the tenascin-C gene, the authors showed that individuals with 12 or 14 GT repeats had a 6-fold higher risk of developing lesions in the Achilles tendon. A G/T polymorphism in the alpha-1 chain of type I collagen has been associated with anterior cruciate ligament rupture30 but not with Achilles tendon injuries.31 Raleigh and collaborators32 found that variants within the MMP-3 gene were associated with Achilles tendinopathy but not Achilles tendon rupture. The presence of cholesterol deposits in the tendons (tendon xanthomas) of familial hypercholesterolemia patients has been associated with polymorphisms in the reverse cholesterol transport and low-density lipoprotein oxidation pathways.33 In the present pilot study, the -799C/T polymorphism in the promoter region of the MMP-8 gene was associated with tendinopathy of the primary posterior tibial tendon. The C allele was observed in the majority of the control group, whereas the T allele was more frequent in the test group. Patients bearing the T allele were more likely to have lesions in the posterior tibial tendon. It is possible that this allele can provide the molecular basis for a more intense degradation of the extracellular matrix, which could lead to an increased susceptibility to lesions in the posterior tibial tendon. The clinical significance of these results, however, must be interpreted with caution because the data were derived from only 64 patients (including only 14 in the test group), which provided a moderate power to detect a statistical relationship between the polymorphism and the disease. Indeed, genetic polymorphisms probably influence the degeneration of tendons through the cumulative effect of multiple polymorphisms that involve complex interactions between multiple genes.34 Understanding the importance of each polymorphic allele is necessary for assessing the contribution of each polymorphism to the disease phenotype.35 Further investigations should determine the roles of several genetic polymorphisms in maintaining homeostasis of the tendon and in tendon pathologies. Because MMP-8 affects the degradation of a large number of extracellular proteins and influences the degradation and remodeling of injured tissues, the study of this gene is important for a better understanding of the process of tendon degeneration. The determination of the genetic patterns of patients with tendinopathy could enable the identification of individuals at higher risk of developing this disease and those with

Gel electrophoresis The entire digest was electrophoresed on a 10% vertical non-denaturing polyacrylamide gel at 20 mA. The gel was stained with ethidium bromide.

Statistical analysis The significance of the differences in the observed frequencies of polymorphisms between both groups was assessed using the chi-squared test, with a p,0.05 indicating statistical significance.

RESULTS The primers used for PCR efficiently amplified the MMP8 locus of interest, and the SfcI enzyme digestion cleaved the PCR products into two fragments when the polymorphic site contained the C allele (but not T). Electrophoresis produced DNA bands of 32 and 74 bp for C alleles and a band of 106 bp for T alleles, whereas the heterozygote displayed a combination of both alleles (32, 74, and 106 bp). There was a significant difference in the allele frequencies of the control and test groups (p = 0.0006). In the control group, the C allele was observed with a frequency of 71%, whereas in the test group, the T allele was present at a frequency of 64.3%. The C/C genotype was found in 66% of the control group, whereas the T/T genotype was observed in 50% of the test group (p = 0.0036). The frequencies of the different alleles and genotypes of the MMP-8 gene are shown in Table 1.

DISCUSSION Posterior tibial tendon dysfunction has been associated with obesity, hypertension, and diabetes.25 There has also been an association with decreased vascular supply.26,27 However, some patients have a predisposition for this condition without a clinically recognized cause, which suggests that genetic inheritance may play an important role in tendinopathy. Some studies have already shown the influence of genetic polymorphisms on tendinopathy. Mokone and collaborators28 demonstrated that a polymorphism in the alpha 1

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CLINICS 2011;66(9):1641-1643

MMP-8 ploymorphisms and tendinopathy Godoy-Santos AL et al. 15. Chen Hy, Cox Sw, Eley Bm, Ma¨ntyla¨ P, Ro¨nka¨ H, Sorsa T. Matrix metalloproteinase-8 levels and elastase activities in gingival crevicular fluid from chronic adult periodontitis patients. J Clin Periodontol. 2000;27:366-9, doi: 10.1034/j.1600-051x.2000.027005366.x. 16. Tervahartiala T, Pirila¨ E, Ceponis A, Maisi P, Salo T, Tuter G, et al. The in vivo expression of the collagenolytic matrix metalloproteinases (MMP-2, -8, -13, and -14) and matrilysin (MMP-7) in adult and localized juvenile perio do n titis . J Dent R e s . 2 000 ;7 9: 19 69 -7 7, do i: 10 .1 17 7/ 00220345000790120801. 17. Kiili M, Cox Sw, Chen Hy, Wahlgren J, Maisi P, Eley Bm, et al. Collagenase-2 (MMP-8) and collagenase-3 (MMP-13) in adult periodontitis: molecular forms and levels in gingival crevicular fluid and immunolocalisation in gingival tissue. J Clin Periodontol. 2002;29:224-32, doi: 10.1034/j.1600-051x.2002.290308.x. 18. Vihinen P, Koskivuo I, Syrja¨nen K, Tervahartiala T, Sorsa T, Pyrho¨nen S. Serum matrix metalloproteinase-8 is associated with ulceration and vascular invasion of malignant melanoma. Melanoma Res. 2008;18:26873, doi: 10.1097/CMR.0b013e3283090031. 19. Ko¨hrmann A, Kammerer U, Kapp M, Dietl J, Anacker J. Expression of matrix metalloproteinases (MMPs) in primary human breast cancer and breast cancer cell lines: New findings and review of the literature. BMC Cancer. 2009;90:188, doi: 10.1186/1471-2407-9-188. 20. Kumar Ms, Vamsi G, Sripriya R, Sehgal PK. Expression of matrix metalloproteinases (MMP-8 and -9) in chronic periodontitis patients with and without diabetes mellitus. J Periodontol. 2006;77:1803-8, doi: 10. 1902/jop.2006.050293. 21. Wang H, Parry S, Macones G. Functionally significant SNP MMP8 promoter haplotypes and preterm premature rupture of membranes (PPROM). Hum Mol Genet. 2004;13:2659–69, doi: 10.1093/hmg/ddh287. 22. Decock J, Long JR, Laxton RC, Shu XO, Hodgkinson C, Hendrickx W, et al. Association of matrix metalloproteinase-8 gene variation with breast cancer prognosis. Cancer Res. 2007;67:10214-21, doi: 10.1158/00085472.CAN-07-1683. 23. Trevilatto PC, Line SR. Use of buccal epithelial cells for pcr amplification of large DNA fragments. J Forensic Odontostomatol. 2000;18:6–9. 24. Aidar M, Line SR. Asimple and cost-effective protocol nfor DNA isolation from buccal epithelial cells. Braz Dent J. 2007;18:148-52, doi: 10. 1590/S0103-64402007000200012. 25. Holmes GB Jr., Mann RA. Possible epidemiological factors associated with rupture of the posterior tibial tendon. Foot Ankle 1992;13:70-9. 26. Frey C, Shereff M, Greenidge N. Vascularity of the posterior tibial tendon. J Bone Joint Surg Am. 1990;72:884-8. 27. Petersen W, Hohmann G, Stein V, Tillmann B. The blood supply of the posterior tibial tendon. J Bone Joint Surg Br 2002;84:141-4. 28. Mokone GG, Schwellnus MP, Noakes TD, Collins M. The COL5A1 gene and Achilles tendon pathology. Scand J Med Sci Sports. 2006;16:19-26, doi: 10.1111/j.1600-0838.2005.00439.x. 29. Mokone GG, Gajjar M, September AV, Schwellnus MP, Greenberg J, Noakes TD, et al. The guanine-thymine dinucleotide repeat polymorphism within the tenascin-C gene is associated with achilles tendon injuries. Am J Sports Med. 2005;33:1016-21, doi: 10.1177/0363546504271986. 30. Posthumus M, September AV, Keegan M, O’Cuinneagain D, van der Merwe W, Schwellnus MP, et al. Genetic risk factors for anterior cruciate ligament ruptures: COL1A1 gene variant. Br J Sports Med. 2009;43:352-6, doi: 10.1136/bjsm.2008.056150. 31. Posthumus M, September AV, Schwellnus MP, Collins M. Investigation of the Sp1-binding site polymorphism within the COL1A1 gene in participants with Achilles tendon injuries and controls. J Sci Med Sport. 2009;12:184-9, doi: 10.1016/j.jsams.2007.12.006. 32. Raleigh SM, van der Merwe L, Ribbans WJ, Smith RK, Schwellnus MP, Collins M. Variants within the MMP3 gene are associated with Achilles tendinopathy: possible interaction with the COL5A1 gene. Br J Sports Med. 2009;43:514-20, doi: 10.1136/bjsm.2008.053892. 33. Oosterveer DM, Versmissen J, Yazdanpanah M, Defesche JC, Kastelein JJ, Sijbrands EJ. The risk of tendon xanthomas in familial hypercholesterolaemia is influenced by variation in genes of the reverse cholesterol transport pathway and the low-density lipoprotein oxidation pathway. Eur Heart J. 2010;31:1007-12, doi: 10.1093/eurheartj/ehp538. 34. Magra M, Maffulli N. Genetic aspects of tendinopathy. J Sci Med Sport. 2008;11:243-7, doi: 10.1016/j.jsams.2007.04.007. 35. Adamo CT, Mailhot JM, Smith AK, Borke JL. Connexin-43 expression in oral-derived human osteoblasts after transforming growth factor-beta and prostaglandin E2 exposure. J Oral Implantol. 2001;27:25-31, doi: 10. 1563/1548-1336(2001)027,0025:CEIOHO.2.3.CO;2.

impaired regeneration capabilities. Thus, any identified genetic markers may contribute to the appropriate preoperative selection, the preparation of strategies for prevention, and individualized therapy to modulate the effects of the genetic markers and increase the success rate of treatments. Thus, it is important that a larger test group be studied in the future to more conclusively identify the influence of the -799C/T MMP-8 polymorphism on the disease phenotype.

CONCLUSION The utilized primers were effective for the PCR-RFLP analysis of the -799C/T polymorphism in the MMP-8 gene. The preliminary results indicate that this polymorphism may be a risk factor for tendinopathy and could be used as a genetic marker for the primary lesions of the posterior tibial tendon.

REFERENCES 1. Riley G. The pathogenesis of tendinopathy. A molecular perspective. Rheumatology. 2004;43:131-42. 2. James SL, Bates BT, Osternig LR. Injuries to runners. Am J Sports Med. 1978;6:40-50, doi: 10.1177/036354657800600202. 3. Rolf C, Movin T. Etiology, histopathology and outcome of surgery in achillodynia. Foot Ankle Int. 1997;18:565-9. 4. McLauchlan GJ, Handoll HHG. Interventions for treating acute and chronic Achilles tendinitis. Cochrane Database Syst Rev. 2001;2:CD000232. 5. Cole AA, Chubinskaya S, Schumacher B, Huch K, Szabo G, Yao J, et al. Chondrocyte matrix metalloproteinase-8. Human articular chondrocytes express neutrophil collagenase. J Biol Chem. 1996;271:11023-6, doi: 10. 1074/jbc.271.18.11023. 6. Palosaari H, Wahlgren J, Larmas M, Ro¨nka¨ H, Sorsa T, Salo T, et al. The expression of MMP-8 in human odontoblasts and dental pulp cells is down-regulated by TGF-beta1. J Dent Res. 2000;79:77-84, doi: 10.1177/ 00220345000790011401. 7. Prikk K, Maisi P, Pirila¨ E, Sepper R, Salo T, Wahlgren J, et al. In vivo collagenase-2 (MMP-8) expression by human bronchial epithelial cells and monocytes/macrophages in bronchiectasis. J Pathol. 2001;194:232-8, doi: 10.1002/path.849. 8. Wahlgren J, Maisi P, Sorsa T, Sutinen M, Tervahartiala T, Pirila¨ E, et al. Expression and induction of collagenases (MMP-8 and -13) in plasma cells associated with bone-destructive lesions. J Pathol. 2001;194:217-24, doi: 10.1002/path.854. 9. Herman Mp, Sukhova Gk, Libby P, Gerdes N, Tang N, Horton Db, et al. Expression of neutrophil collagenase (matrix metalloproteinase-8) in human atheroma: a novel collagenolytic pathway suggested by transcriptional profiling. Circulation. 2001;104:1899-904, doi: 10.1161/ hc4101.097419. 10. Pirila¨ E, Ramamurthy Ns, Sorsa T, Salo T, Hietanen J, et al. Gelatinase A (MMP-2), collagenase-2 (MMP-8), and laminin-5 gamma2-chain expression in murine inflammatory bowel disease (ulcerative colitis). Dig Dis Sci. 2003;48:93-8, doi: 10.1023/A:1021790532723. 11. Moilanen M, Pirila¨ E, Gre´nman R, Sorsa T, Salo T. Expression and regulation of collagenase-2 (MMP-8) in head and neck squamous cell carcinomas. J Pathol. 2002;97:72-81, doi: 10.1002/path.1078. 12. Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest. 1994;94:2493–503, doi: 10.1172/JCI117619. 13. Lee J, Kim HR, Min JW, Park JS, Jin SM, Han SK, et al. Lack of association between matrix metalloproteinase 8 promoter polymorphism and bronchiectasis in Koreans. J Korean Med Sci. 2007;22:667-71, doi: 10. 3346/jkms.2007.22.4.667. 14. Roderfeld M, Rath T, Schulz R, Seeger W, Tschuschner A, Graf J, et al. Serum matrix metalloproteinases in adult CF patients: Relation to pulmonary exacerbation. J Cyst Fibros. 2009;8:338-47, doi: 10.1016/j.jcf. 2009.06.001.

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DOI:10.1590/S1807-59322011000900024

RAPID COMMUNICATION

Trunk stabilization among women with chronic lower back pain: a randomized, controlled, and blinded pilot study Silvia Ferreira Andrusaitis, Guilherme Carlos Brech, Gabriela Faller Vitale, Ju´lia Maria D9 Andreá Greve Laborato´rio de Estudos do Movimento do Instituto de Ortopedia e Traumatologia do Hospital das Clıńicas - Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brazil. Email: guilhermebrech@yahoo.com.br / guibrech@gmail.com Tel.: 55 11 3069-6000

included in the study, patients needed to be free from vestibular abnormalities and musculoskeletal disorders of the hips and lower limbs. In addition, the study had a control group composed of five women between the ages of 30 and 55 who did not present with lower back pain but who fulfilled the same inclusion criteria. The exclusion criteria were the following: abandonment of the physical therapy, more than three consecutive absences from the treatment sessions, worsening of the symptoms, and a patient’s desire, for any reason, to have her data excluded from the study without this harming the continuity of her treatment. After the study had been explained to the patients and they had signed the consent form, they were assessed in accordance with the evaluation protocol. Randomization was performed by means of a draw using opaque envelopes containing folded papers that allocated patients to one of two treatment groups: group A (strengthening) or B (stabilization). All the volunteers were assessed in relation to balance (questionnaire and balance tests at a force plate) and pain scales at the time that they were selected for the study. Three groups were created, consisting of groups A and B, each with five patients with lower back pain, and a control group with five volunteers. The patients in groups A and B were evaluated with respect to the balance and pain scales before and after the treatment, whereas the patients in the control group were only evaluated before the treatment.

INTRODUCTION Treatment for mechanical lower back pain is a challenge in Western society, in which its occurrence can now be considered to have reached epidemic proportions. The origin of such pain and the factors that cause it to become chronic and recurrent remain poorly understood. Abnormalities in motor control and trunk muscle function have been found in individuals with chronic lumbar pain.1-4 Many studies have demonstrated that the deep muscles of the lumbar column and abdomen, especially the multifidus and transversus abdominis, present late activation, weakness, and diminished resistance during episodes of lower back pain.5 These changes persist even when the painful condition goes into remission, and they contribute to episodes of lower back pain recurrence.1,5,6 However, it is still difficult to determine whether the neuromuscular imbalance occurs because of the pain or whether the imbalance causes the pain.1,6 Although the mechanisms that lead to these abnormalities are incompletely understood, rehabilitation programs aiming to stabilize the lumbar spine and improve its musculature and proprioceptive action have been used with positive effects on pain and functional capacity in individuals with mechanical lower back pain.7 This pilot study compared stabilization exercises with strengthening exercises for the trunk and hips in women with chronic lower back pain in terms of their effects on pain, functional capacity, and postural balance.

Evaluation protocol

MATERIALS AND METHODS

The evaluation protocol was administered one week before the treatment was started and one week after it was terminated by two experienced evaluators who had been trained to handle the assessment instruments and were blinded regarding the treatment groups. All the patients in the treatment groups (both A and B) gave responses to the Oswestry Disability Questionnaire using the version translated and validated for the Portuguese language,8 and they completed a visual analog pain scale (VAS)9 regarding the frequency and intensity of their lower back pain. In addition to the questionnaire and pain scale, all the volunteers (groups A and B and the control group) underwent four balance tests on the Balance MasterH System (Neurocom International, Inc., Clackamas, Oregon, USA).

Fifteen female volunteers were prospectively studied in a randomized, controlled and blinded manner between April 2008 and April 2010 with approval from the ethics committee (no. 1248/07). This study included ten women between the ages of 30 and 55 years who had been referred for physical therapy because of nonspecific, chronic lower back pain. These women had sedentary habits, had no significant radiological abnormalities, and had no neurological impairments. To be

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the patients performed exercises in accordance with their treatment group. In group A, the exercises had the goal of strengthening the abdominal, back, and hip muscles. The patients performed an average of three series of ten repetitions of each exercise. Increases in the number of exercises performed in each session (or load progression) occurred according to individual tolerance. In group B, stabilization exercises were taught, starting with the dorsal decubitus and progressing to the ventral decubitus, in seated, four-support, and standing positions. Increases in the number of exercises performed in each session (or load progression) occurred according to individual tolerance. All the sessions were conducted individually with the same physical therapist, who was blinded to the results of the initial assessment.

The tests undertaken were the following. Modified clinical test of sensory interaction and balance (mCTSIB). Static balance was assessed using the modified

clinical test of sensory interaction and balance, which consists of an assessment of body sway under four sensory conditions while the individual remains on a force platform: eyes open and closed on a stable surface and eyes open and closed on an unstable surface. Each condition was repeated three times for ten seconds, and the average of the attempts was used. This test measures the displacement velocity at the individual’s center of pressure in degrees per second. A force platform with four coupled sensors is used for the test. Decreases in the displacement velocity are considered to be a positive outcome. The variables studied were the mean sway velocities with eyes open and with eyes closed on a stable surface (firm surface) and an unstable surface (foam surface) and the mean sway velocities in the anteroposterior (mean-Y) and side-to-side (mean-X) directions with eyes open and with eyes closed on a stable surface (firm surface) and an unstable surface (foam surface). The following three tests were undertaken to assess the subjects’ functional limitations affecting the activities of daily life. Single-leg stance test. This test was conducted with the subject standing on one leg on a force platform under four conditions: with eyes open or closed and on the left or right leg. Similar to the mCTSIB, each condition was repeated three times for ten seconds, and the mean from the attempts was used. The variables studied were the mean sway velocity with eyes open and closed on the left and right legs. Get-up-and-go test. The get-up-and-go test was conducted on a platform with the individual initially in a seated position on a bench 30 cm in height without a seat back. The patient’s knees were flexed at 90 ˚, and her feet were separated by 10 cm at the heels for base support. Her arms were kept along the sides of her body. The patient was instructed to stand up quickly but safely. There were three repetitions of the movement separated by 30-second intervals. The parameters measured were the mean weight transfer time, the mean rising index and the mean sway velocity while rising. Step-up test. In this test, the patients were instructed to climb a step 10 cm in height while putting only one foot on the step. They were told that the other foot should go directly to the platform without contacting the step. When both feet had reached the platform after crossing the step, the patients were then supposed to remain in as static a position as possible. In this protocol, three attempts were made for each leg, beginning with the left leg. The variables evaluated were the mean weight transfer index (mean liftup index), the mean movement time, and the mean impact index. In all the balance tests conducted in this study, the volunteers were only allowed a maximum of three attempts to perform each test.

Statistical analysis Fifteen women participated in this study: ten with lower back pain and five without any history of pain. A descriptive analysis was performed on the following sample parameters: number of cases (N), median, minimum, and maximum. To meet the aims of the study, the 10 patients were evaluated using the scale values at the time that they were selected, and tests (Kolmogorov-Smirnov) were performed to determine whether the results presented a normal distribution.10 To compare the scales between the groups, the KruskalWallis test was used before the treatment, and the MannWhitney test was used after the treatment.10 To compare the scales in each group between the evaluations before and after the treatment, the Wilcoxon paired signed-rank test was used.10 The data were analyzed using a significance level of 5%.

RESULTS This study was conducted with five patients in each of three groups: group A (strengthening), group B (stabilization), and a control group. Descriptive information for the groups (median, minimum, and maximum values), including age, start of symptoms, pain scale (VAS), and Oswestry questionnaire data before and after treatment, is shown in Table 1. A Pearson correlation analysis between the pain scale and the Oswestry questionnaire responses revealed that higher pain intensity (as assessed using the VAS) correlated with higher Oswestry scores (r = 0.754 and p = 0.007) (Table 2). Group B presented significant reductions in both pain (intensity and frequency) (p,0.043) and disability (measured by the Oswestry questionnaire) (p,0.05) after the treatment. However, group A did not show any significant changes (p.0.05). Comparisons between the two groups at each assessment time showed that after the treatment, the Oswestry values of group B were lower than those of group A (p = 0.016) (Table 3). With regard to the balance measurements, only the mean weight transfer time increased significantly from before to after the treatment in group B (p = 0.043). The other measurements did not change and did not differ between the groups (p.0.05) (Tables 4 to 7).

Treatment protocol For both groups A and B, the treatment consisted of a 40minute physical therapy session three times a week for a total of 20 sessions. All the sessions began with a ten-minute warm-up on an ergometric bicycle. Following the warm-up,

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Table 1 - Descriptions of group A, group B and the control group (median, minimum and maximum) in relation to age, onset of symptoms, pain scale (VAS) and Oswestry questionnaire before and after treatment. Variable

Group

Period A

Age (years) Onset of symptoms (months) VAS intensity (cm) VAS frequency (cm) Oswestry Questionnaire

Before Before After Before After Before After

Median

Min

Max

Median

Min

Max

Median

Min

Max

46.2 78 4.83 3.59 8.74 5.31 19.8 18.2

37 6 4.3 0.15 6.8 0.3 11 5

53 240 5.5 7.5 9.3 9.1 33 37

46.2 7.2 5.08 0.23 6.19 2.09 11.8 3.4

39 3 0.5 0 0.7 0 4 1

51 12 7.7 0.5 9.9 5.09 23 18

46.6

exercises, the patients underwent balance assessments before and after the treatment. They were compared with each other and with a group of women without any history of lumbar pain. The principal result was that the group B (stabilization) patients presented significant reductions after the treatment in both pain (measured using the VAS) and functional capacity (measured using the Oswestry questionnaire). These two evaluations correlated positively with one another. In Table 2, it can be seen that higher pain intensity scores (as assessed using the VAS) correlated with higher Oswestry scores (r = 0.754 and p = 0.007). Similar results were found by Goldby et al.12 in a study in which trunk stabilization exercises were compared with manual therapy and an educational program for patients with chronic lumbar pain. Those authors observed that stabilization exercises were significantly superior to other interventions with regard to pain and function. However, Cairns et al.13 did not find any significant difference between programs using stabilization exercises, general exercises and manual therapy and programs using only general exercises, and manual therapy. Both of their groups presented notable reductions in pain and improvements in functional capacity. They offered two hypotheses to explain these results: the stabilization exercises may not have been as effective as expected, or the general exercises may have actually provided the positive results that were observed. In the present study, for group A (strengthening), there was no significant change from before to after the treatment. Comparisons between the groups at the two assessment times showed that after the treatment, group B9s values were lower than group A9s, although group B9s values had also been lower before the treatment. Furthermore, the patients in group A presented symptoms of longer duration (a mean of 78 months) than group B9s symptoms (a mean of 7.2 months), which may have influenced group A negatively with regard to the response to treatment. In relation to the balance measurements, in the get-upand-go test (Table 6), the mean weight transfer time variable, which measures the time required to transfer the body from a seated position to a standing position, increased significantly from before to after the treatment in group B. The higher the score on this test, the slower this process, thereby signifying a reduction in the ability to move the center of gravity forward and an increased need to prolong the muscular contraction. Changes in the strength or flexibility of the hip or trunk or difficulty in moving the pelvis forward might delay this response. Despite the poor result from this particular test, the difficulty group B

DISCUSSION Physical exercise is one of the most widely used methods for the rehabilitation of individuals with chronic low back pain. The primary goals of treating lumbar pain with physical exercise are to improve muscle strength, to maintain or improve flexibility, to heal tissue lesions and to promote spinal segment stability.7,11,12 There are many exercise programs for lower back pain, and they differ in terms of the duration, frequency, and intensity of the training and especially regarding the type of exercise and the way it is performed. 11,13 The term ‘‘stabilization exercise’’ is a generic term for any type of exercise that challenges the stability of the spine while training muscle activity patterns and postures that ensure sufficient stability without unnecessarily overloading tissue.14,15 Trunk stabilization exercises are based on co-contraction of the abdominal and multifidus muscles, and they are performed in a variety of body positions. It has been well documented that individuals with chronic lower back pain present differences in the activation patterns of these muscles (along with histomorphological abnormalities) compared with the muscles of individuals without a history of lumbar pain. 16 Stabilization exercises thus aim to improve these muscle activation patterns, thereby diminishing both incapacity and lumbar pain through improvements in trunk muscle contraction.12,15 The results of studies relating to improvements in pain and functional capacity in lower back pain are contradictory. In a literature review, Macedo et al.17 found that stabilization exercise programs effectively diminished pain and functional incapacity, but they did not outperform other treatments. Another point of contention is whether stabilization training actually interferes with motor control. In this study, women with chronic lower back pain were divided into two exercise groups (A and B) and were compared in terms of improvements in pain and functional capacity. To ascertain the effects of the postural control Table 2 - Pearson correlations between the pain scale results and the Oswestry questionnaire responses. Correlation VAS frequency Oswestry Questionnaire

r p r p

Control

VAS intensity

VAS frequency

0.535 0.090 0.754 0.007

0.409 0.211

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Table 3 - Pain scale and Oswestry questionnaire data for the treatment groups and the results of the comparative tests. Group Variable

Period

VAS intensity p* VAS frequency p* Oswestry Questionnaire

p-value#

Median

Min

Max

Median

Min

Max

Before After

4.6 4.2

5.5 7.5

5 5

5 0.2

5 5

0.548 0.056

9.15 7.5

9.3 9.1

5 5

8.4 0.3

9.9 5.9

5 5

0.841 0.095

Before After

19 15

33 37

5 5

10 2

0.5 0 0.043 0.7 0 0.043 4 1 0.039

7.7 0.5

Before After

4.3 0.15 0.225 6.8 0.3 0.138 11 5 0.461

23 8

5 5

0.151 0.016

p* # Result of the Mann-Whitney test. * Result of the Wilcoxon paired test. All significant values are in bold.

patients had in moving the pelvis may have reflected the process of learning to stabilize the trunk and consequently the pelvis. Excessive muscle co-contraction increases joint

stability, but it also increases joint overload and reduces the efficiency of balance strategies, with a negative repercussion on hip joint mobility.15,18,19

Table 4 - Descriptions of the clinical tests of sensory interaction and balance measurements in the treatment groups and the results of the comparative tests. Group Variable

Mean-Firm-EO p* Mean-Firm-EC p* Mean-Foam-EO p* Mean-Foam-EC p* Firm-EO-Mean-X p* Firm-EO-Mean-Y p* Firm-EC-Mean-X p* Firm-EC-Mean-Y p* Foam-EO-Mean-X p* Foam-EO-Mean-Y p* Foam-EC-Mean-X p* Foam-EC-Mean-Y

Control

Period

p-value#

Median

Min

Max

Median

Min

Max

Median

Min

Max

Before After

0.2

0.1

0.2

0.4 0.2

0.7 0.5

5 5

0.3 0.2

5 5

0.515 0.421

0.2

0.1

0.3

0.3 0.3

0.8 1.6

5 5

0.3 0.3

0.4 0.4

5 5

0.519 1.000

Before After

0.6

0.5

0.9

0.7 0.6

1 1.2

5 5

0.8 0.6

1.1 1.2

5 5

0.335 0.548

Before After

1.1

0.7

1.5

1.4 1.2

2.1 2.3

5 5

1.4 1.1

1.8 1.7

5 5

0.344 0.421

Before After

22.2

0.8

20.5 20.1

1.2 1.8

5 5

20.7 21

0.4 0.5

5 5

0.249 0.310

Before After

20.2

21.9

0.8

22.1 21.6

1.1 20.2

5 5

21 20.8

0.1 0.5

5 5

0.346 0.548

Before After

20.8

22.1

0.5

20.1 0.1

1.1 1.4

5 5

21.5 20.8

0.5 0

5 5

0.171 0.056

Before After

20.2

22.2

1.6

21.6 21.3

0.7 20.3

5 5

21.2 21

0.5 1.5

5 5

0.465 0.548

Before After

21.5

0.1

21.1 20.7

0.9 0

5 5

20.8 21.4

20.2 20.9

5 5

0.599 0.056

Before After

2.9

1.8

5.1

2.8 2.6

3 3.4

5 5

3.2 2.4

4.6 3.1

5 5

0.295 0.690

Before After

20.5

21.5

20.4

21.1 21.2

0.5 0.2

5 5

20.8 21.2

20.2 0.3

5 5

0.293 0.690

Before After

3.5

2.9 2.5

3.1 4.3

5 5

2.6 2.6

0.2 0.2 0.317 0.2 0.2 0.157 0.5 0.5 0.683 1.1 0.8 0.279 22.1 22.5 0.893 21.7 21.9 0.498 22.1 22.9 0.893 21.4 21.8 0.715 22.1 22.5 0.176 1.5 0.9 0.345 21.1 21.9 0.498 1.6 0 0.588

0.4 0.4

Before After

0 0.1 0.680 0.1 0.2 0.461 0.4 0.3 0.408 1 1 0.705 20.6 20.6 0.269 23 22.4 0.893 20.7 20.5 0.343 22.5 21.9 0.893 22.3 21.2 0.498 23.1 1.3 0.345 22.1 21.3 0.588 22 1.1 0.465

3.9 2.9

5 5

0.175 0.841

p* # Result of the Kruskal-Wallis test (before) or the Mann-Whitney test (after). * Result of the Wilcoxon paired test.

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Table 5 - Step-up balance measurements in the treatment groups and the results of comparative tests. Group Variable

Mean lift-up index - left p* Mean lift-up index - right p* Mean movement time - left p* Mean movement time - right p* Mean impact index - left p* Mean impact index - right

Control

Period

p-value#

Median

Min

Max

Median

Min

Max

Median

Min

Max

Before After

22 19

29 38

4 5

19 21

5 5

1.000 0.690

20 19

26 36

4 5

19 17

23 23

5 5

0.323 0.548

Before After

1.45

1.07

1.82

1.465 1.51

2.4 3.21

4 5

1.63 1.61

2.07 1.78

5 5

0.623 0.841

Before After

1.55

0.96

1.74

1.45 1.5

1.71 2.95

4 5

1.69 1.63

1.8 1.85

5 5

0.624 0.421

Before After

25.5 19

36 36

4 5

20 17

24 25

5 5

0.805 0.548

Before After

21.5 18

32 50

4 5

21 18

16 14 0.892 15 14 0.414 1.5 1.49 0.588 1.66 1.44 0.345 14 9 0.345 17 14 0.461

29 25

Before After

16 8 0.593 17 7 0.180 1.42 1.29 0.109 1.36 1.35 0.593 15 6 0.593 15 9 0.414

28 29

5 5

0.327 1.000

p* # Result of the Kruskal-Wallis test (before) and the Mann-Whitney test (after). * Result of the Wilcoxon paired test.

balance among patients with lower back pain who underwent an exercise program. There are several hypotheses that might help explain this result. In principle, the step-up test and the get-up-and-go test are sensitive to changes in balance and mobility that may have arisen through many conditions, not only lumbar pain but also conditions associated with aging. The postural balance abnormalities in individuals with lower back pain may be subtle, and the equipment used in this study might not have been sensitive enough to pick up differences between the groups. Such hypotheses were also raised by Kuukkanen et al.,22 who came to the conclusion that balance abnormalities could be observed both in patients with significant balance abnormalities and in patients with severe lower back pain. There are some limitations that must be highlighted in this study, including the small number of subjects in the sample and the differences in the duration of symptoms between the groups.

The other measurements from the other tests did not change and did not differ between the groups. Studies have shown that individuals with chronic lumbar pain show postural balance abnormalities relative to individuals without any history of pain, especially under conditions that make greater postural demands. Although Mientjes et al.20 and Della Volpe et al.21 did not find any significant difference in static balance in individuals with lumbar pain, when the patients were subjected to more challenging postures, such as closing the eyes and remaining upright on an unstable surface, the subjects with lower back pain presented greater sway than did the control group. In the present study, such abnormalities were not observed. The three groups (A, B, and control) behaved similarly in relation to the four balance tests. After the treatment, groups A and B presented no changes in their results relative to before the intervention. Similar data were obtained by Kuukkanen et al.,22 who did not observe any differences in

Table 6 - Get-up-and-go test results for the treatment groups and the results of the comparative tests. Group Variable

Mean weight transfer time p* Mean rising index p* Mean sway velocity while rising

Control

Period

p-value#

Median

Min

Max

Median

Min

Max

Median

Min

Max

Before After

0.24

0.17

0.68

0.655 0.55

0.71 0.89

4 5

0.21 0.26

5 5

0.050 0.310

16.5 8

20 29

4 5

20 16

23 21

5 5

0.319 0.548

Before After

4.3

2.65 2.8

2.8 3.1

4 5

4.4 4.6

0.16 0.23 0.043 10 11 0.223 2.6 1.5 0.715

0.59 0.71

Before After

0.58 0.29 1.000 8 7 0.655 1.4 2.3 0.285

4.9 5.4

5 5

0.086 0.690

p* # Result of the Kruskal-Wallis test (before) and the Mann-Whitney test (after). * Result of the Wilcoxon paired test.

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Table 7 - Single-leg stance test results for the treatment groups and the results of the comparative tests. Group Variable

Mean-EO-L p* Mean-EC-L p* Mean-EO-R p* Mean-EC-R

Control

Period

p-value#

Median

Min

Max

Median

Min

Max

Median

Min

Max

Before After

1.1

0.5

1.3

0.8 0.8

1 1.2

5 5

0.9 0.9

5 5

0.599 0.548

5.2

1.4

3.6 4.9

12 12

4 5

8.4 8.6

12 8.7

5 5

0.805 0.548

Before After

0.7

0.5

1.8

0.7 0.7

0.8 0.9

4 5

0.9 0.7

1.4 1.1

5 5

0.788 0.841

Before After

5.7

1.2

1.75 2.1

12 12

4 5

8.7 12

0.7 0.6 0.131 1.6 1.5 0.138 0.7 0.7 0.109 1.3 1.7 0.715

1.2 1

Before After

0.7 0.5 0.461 1.3 2 1.000 0.7 0.7 1.000 1.3 1.3 0.593

12 12

5 5

0.539 0.690

p* # Result of the Kruskal-Wallis test (before) and the Mann-Whitney test (after). * Result of the Wilcoxon paired test.

8. Vigatto R, Alexandre NM, Correa Filho HR. Development of a Brazilian Portuguese version of the Oswestry Disability Index: cross-cultural adaptation, reliability, and validity. Spine (Phila Pa 1976). 2007;32:481-6, doi: 10.1097/01.brs.0000255075.11496.47. 9. Dolan P, Greenfield K, Nelson R, Nelson I. Can exercise therapy improve the outcome of microdiscectomy? Spine (Phila Pa 1976). 2000;25:1523-32, doi: 10.1097/00007632-200006150-00011. 10. Kirkwood B, Sterne J. Essentials of medical statistics. 2nd ed. Oxford: Blackwell Science. 2003. 11. Kofotolis N, Kellis E. Effects of two 4-week proprioceptive neuromuscular facilitation programs on muscle endurance, flexibility, and functional performance in women with chronic low back pain. Phys Ther. 2006;86:1001-10. 12. Goldby LJ, Moore AP, Doust J, Trew ME. A randomized controlled trial investigating the efficiency of musculoskeletal physiotherapy on chronic low back disorder. Spine (Phila Pa 1976). 2006;31:1083-93, doi: 10.1097/ 01.brs.0000216464.37504.64. 13. Cairns MC, Foster NE, Wright C. Randomized controlled trial of specific spinal stabilization exercises and conventional physiotherapy for recurrent low back pain. Spine (Phila Pa 1976). 2006;31:E670-81, doi: 10.1097/01.brs.0000232787.71938.5d. 14. Akuthota V, Nadler SF. Core strengthening. Arch Phys Med Rehabil. 2004;85(Suppl 1):S86-S92, doi: 10.1053/j.apmr.2003.12.005. 15. Kavcic N, Grenier S, McGill SM. Quantifying tissue loads and spine stability while performing commonly prescribed low back stabilization exercises. Spine (Phila Pa 1976). 2004;29:2319-29, doi: 10.1097/01.brs. 0000142222.62203.67. 16. Mayer J, Mooney V, Dagenais S. Evidence-informed management of chronic low back pain with lumbar extensor strengthening exercises. Spine J. 2008;8:96-113, doi: 10.1016/j.spinee.2007.09.008. 17. Macedo LG, Maher CG, Latimer J, McAuley JH. Motor control exercise for persistent, nonspecific low back pain: A systematic review. Phys Ther. 2009;89:9-25, doi: 10.2522/ptj.20080103. 18. Henry SM, Hitt JR, Jones SL, Bunn JY. Decreased limits of stability in response to postural perturbations in subjects with low back pain. Clin Biomech. 2006;21:881-92, doi: 10.1016/j.clinbiomech.2006.04.016. 19. Brumagne S, Janssens L, Knapen S, Clayes K, Suuden-Johanson E. Persons with recurrent low back pain exhibit a rigid postural control strategy. Eur Spine J. 2008;17:1177-84, doi: 10.1007/s00586-008-0709-7. 20. Mientjes MIV, Frank SJ. Balance in chronic low back pain patients compared to healthy people under various conditions in upright standing. Clin Biomech (Bristol, Avon). 1999;14:710-6, doi: 10.1016/ S0268-0033(99)00025-X. 21. Della Volpe R, Popa T, Ginanneschi F, Spidalieri R, Mazzochio R, Rossi A. Changes in coordination of postural control during dynamic stance in chronic low back pain patients. Gait Posture. 2006;24:349-55, doi: 10. 1016/j.gaitpost.2005.10.009. 22. Kuukkanen TM, Malkia EA. An experimental controlled study on postural sway and therapeutic exercise in subjects with low back pain. Clin Rehabil. 2000;14:192-202, doi: 10.1191/026921500667300454.

CONCLUSION The trunk stabilization exercises were more effective at relieving pain and improving functional capacity than the strengthening exercise program in the patient sample we studied. With regard to the balance measurements, only the mean weight transfer time presented a significant increase, which was seen in group B after the intervention. The other balance measurements did not change and did not differ between the three groups evaluated. Stabilization exercises appear to be an important tool for improving lower back pain. This pilot study has helped us to improve the assessment methodology, both in relation to balance and in relation to lower back pain itself. The duration of symptoms needs to be investigated as an important prognostic factor for lumbar pain. Further studies should be conducted taking these factors into account and incorporating adequate sample sizes.

REFERENCES 1. Tsao H, Galea MP, Hodges PW. Reorganization of the motor cortex is associated with postural control deficits in recurrent low back pain. Brain. 2008;131:2161-71, doi: 10.1093/brain/awn154. 2. Suni J, Rinne M, Natri A, Statistisian MP, Parkkari J, Alaranta H. Control of the lumbar neutral zone decreases low back pain and improves selfevaluated work ability – A 12-month randomized controlled study. Spine. 2006;31:E611-E20, doi: 10.1097/01.brs.0000231701.76452.05. 3. Panjabi MM. Clinical spinal instability and low back pain. Journal of Electromyography and Kinesiology. 2003;13:371-9, doi: 10.1016/S10506411(03)00044-0. 4. Ebenbichler GR, Odsson LIE, Kollmitzer J, Erim Z. Sensory motorcontrol of the lower back: implications for rehabilitation. Med Sci Sports Exerc. 2001;33:1889-98, doi: 10.1097/00005768-200111000-00014. 5. Barr KP, Griggs M, Cadby T. Lumbar stabilization – core concepts and current literature, part 1. Am J Phys Med Rehabil. 2005;84:473-80, doi: 10. 1097/01.phm.0000163709.70471.42. 6. McGill SM, Karpowicz A. Exercises for spine stabilization: motion/ motor patterns, stability progressions, and clinical technique. Arch Phys Med Rehabil. 2009;90:118-26, doi: 10.1016/j.apmr.2008.06.026. 7. Barr KP, Griggs M, Cadby T. Lumbar stabilization – a review of core concepts and current literature Part 2. Am J Phys Med Rehabil. 2007;86:72-80, doi: 10.1097/01.phm.0000250566.44629.a0.

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CASE REPORT

Paroxysmal atrial fibrillation and intermittent left bundle branch block: an unusual electrocardiographic presentation of mad honey poisoning Ayhan Saritas,I Hayati Kandis,I Davut Baltaci,II Ismail ErdemIII I Duzce University School of Medicine, Department of Emergency Medicine, Duzce, Turkey. II Duzce University School of Medicine, Department of Family Medicine, Duzce, Turkey. III Duzce University School of Medicine, Department of Cardiology, Duzce, Turkey.

Email: a_saritas_@hotmail.com Tel.: 0 380 5421390/5820

INTRODUCTION

DISCUSSION

Mad honey poisoning occurs through the intake of honey made from the nectar of Rhododendron species containing grayanotoxins, a family of lipid-soluble toxins responsible for the clinical manifestations of mad honey intoxication syndrome. These plants grow in the eastern parts of the Black Sea region of Turkey and in various other parts of the world (e.g., South America, Europe, and Japan).1 Previous reports concerning mad honey poisoning have generally described various degrees of atrioventricular (AV) block, sinus bradycardia, asystole, nodal rhythm with ventricular parasystole, and even acute myocardial infarction.2,3 The diagnosis is often made based on a history of honey intake. We report a unique case of a patient who presented paroxysmal atrial fibrillation and intermittent left bundle branch block related to mad honey poisoning.

Grayanotoxins are responsible for the clinical manifestations of mad honey poisoning. These toxins bind to sodium channels in the cell membrane, increasing the permeability of these channels and are thus responsible for inotropy. The signs and symptoms generally may occur within minutes and to up to two hours or more after ingestion.1,4,5 Wellknown clinical manifestations of mad honey poisoning include hypotension, bradycardia, arrhythmia, loss of consciousness, weakness, nausea, and vomiting. To the best of our knowledge, no case of paroxysmal atrial fibrillation and intermittent left bundle branch block associated with mad honey poisoning has previously been reported.

CONCLUSION We report a case of a 70-year-old man with severe hemodynamic instability following the ingestion of mad honey and his unusual electrocardiographic manifestations: paroxysmal atrial fibrillation and intermittent left bundle branch block.

CASE REPORT A 70-year-old man was admitted to our emergency department with weakness, palpitation, nausea and vomiting that arose from eating a few teaspoons of mad honey. He did not have any cardiovascular or systemic disease and was not taking any medication. On physical examination, he was confused, his blood pressure was 60/20 mmHg, and his heart rate was 105 beats/min. The neurological examination was normal. Upon admission, his electrocardiography revealed paroxysmal atrial fibrillation and intermittent left bundle branch block (Figure 1). There was no structural cardiac abnormality found from transthoracic echocardiography. His hypotension improved rapidly after saline infusion. During observation, his rhythm returned to a normal sinus rhythm six hours after admission (Figure 2), and he was discharged on the second day of his hospital stay.

REFERENCES 1. Gunduz A, Bostan H, Turedi S, Nuhog˘lu I, Patan T. Wild flowers and mad honey. Wilderness Environ Med. 2007;18:69–71, doi: 10.1580/06WEME-LE-042R.1. 2. Aliyev F, Tu¨rkoglu C, Celiker C. Nodal rhythm and ventricular parasystole: an unusual electrocardiographic presentation of mad honey poisoning. Clin Cardiol. 2009;32:52-54, doi: 10.1002/clc.20438. 3. Akinci S, Arslan U, Karakurt K, Cengel A. An unusual presentation of mad honey poisoning: acute myocardial infarction. Int J Cardiol. 2008;129:56-8, doi: 10.1016/j.ijcard.2007.06.129. 4. Gunduz A, Turedi S, Uzun H, Topbas M. Mad honey poisoning. Am J Emerg Med. 2006;24:595–8, doi: 10.1016/j.ajem.2006.01.022. 5. Yilmaz O, Eser M, Sahiner A, Altintop L, Yesildag O. Hypotension, bradycardia and syncope caused by honey poisoning. Resuscitation. 2006;68:405–8, doi: 10.1016/j.resuscitation.2005.07.014.

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Figure 1 - Paroxysmal atrial fibrillation and intermittent left bundle branch block upon admission.

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Figure 2 - Normal sinus rhythm 6 hours after admission.

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DOI:10.1590/S1807-59322011000900026

CASE REPORT

Choreoathetosis after subarachnoid hemorrhage related to an aneurysm of the posterior fossa Ju´lio Leonardo Barbosa Pereira,I Lucas Alverne Freitas de Albuquerque,I Mauro Cruz Machado Borgo,I Gerival Vieira Junior,I Paulo Pereira Christo,II Gerva´sio Teles C. de CarvalhoIII I

Neurosurgery, Santa Casa de Belo Horizonte, Belo Horizonte/MG, Brazil. II Research Program of the Santa Casa, Belo Horizonte/MG, Brazil. Neurosurgery, Santa Casa de Belo Horizonte and Faculdade de Cieˆncias Medicas de Minais Gerais, Belo Horizonte/MG, Brazil.

III

Email: lucasalverne@yahoo.com.br Tel.: 31 3238-8100

prior to the endovascular aneurysm repair, which was completed uneventfully. A control CT scan did not reveal hydrocephalus. At the one-year follow-up visit, the patient did not exhibit any choreoatetoid movements; however, the cerebellar alterations remained, with an important static and dynamic imbalance and significant dysmetria. Magnetic Resonance Imaging (MRI) at the one year follow-up revealed cerebellar atrophy (Figure 1B).

INTRODUCTION Non-traumatic subarachnoid hemorrhage (NSAH) is a neurological emergency with a high rate of death and complications.1,2 A ruptured intracranial aneurysm accounts for approximately 80% of NSAH cases. The classic clinical manifestations of NSAH are headache, nausea and vomiting, focal neurological signs, meningeal irritation, and a reduction in the level of consciousness. In the present work, we describe a case of choreoathetosis that developed in a young patient who presented with a subarachnoid hemorrhage (SAH) related to an aneurysm of the posterior fossa. We also review the corresponding literature.

DISCUSSION Stroke-related movement disorders are uncommon (3.6%) and are very rare in SAH cases . Chorea, tremor, dystonia, Parkinsonism, and myoclonus have all been associated with cerebral infarcts and hemorrhaging.3 Movement disorders, which represent a portion of the clinical spectrum of acute stroke, may be delayed or progressive. The first case of a movement disorder (chorea) after a SAH was reported by Sakai et al.4 and occurred eight days after the SAH onset. The CT scans revealed a SAH with ventricular dilation and periventricular lucency involving the bilateral caudate nuclei. The chorea was attributed to the vasospasm and hydrocephalus.4 In another case, reported by Morigaki et al.,5 the involuntary movements began shortly after the SAH onset. There was no acute hydrocephalus, and the authors attributed the symptoms to a hematoma on the corpus callosum that resulted from a rupture of an aneurysm of the distal accessory anterior cerebral artery. The hyperkinetic involuntary movements were suggested to have occurred due to the interruption of the cortico-striato-pallido-thalamo-cortical feedback loop. Alarcoń et al. analyzed 1,500 consecutive stroke patients over a period of ten years to identify patients with a movement disorder, which was observed in only 56 patients (3.6%). Chorea was the most common movement disorder (35.7%). Thirty-nine (69.6%) patients experienced an ischemic stroke, 14 (25%) experienced a parenchymal hemorrhage, and only three (5.3%) experienced a SAH. All of the patients with a movement disorder that was secondary to a SAH in the Alarcoń et al. series presented with a tremor (Table 1) as their principal involuntary movement.3 The time that elapses between a stroke onset and the development of a movement disorder is variable. Alarcoń et al. observed involuntary movements that began on the first day of the stroke in 12.5% of their patients but reported

CASE REPORT A 17-year-old boy, with no previous co-morbidity or neurological disability, reported the sudden onset of a severe headache associated with an alteration of his level of consciousness and meningeal signs. Upon admission to our department, three days after the acute event, he was confused, dysarthric, exhibited choreoatetoid movements of the distal upper limbs, exhibited postural instability, and exhibited the inability to walk, stand up, or sit up without help. The choreoathetosis began just after the onset of the SAH. The results of the motor, sensitivity , and reflex examinations were normal. The patient’s hyperkinesia ceased with sleep. In addition, there was no family history of movement disorders. A computed tomography (CT) scan revealed a hemorrhage in the fourth ventricle but no evidence of a parenchyma lesion. A cerebral angiography (Figure 1A) revealed a small saccular aneurysm of the basilar artery, which was located close to the emergence of the anterior inferior cerebellar artery (AICA). No evidence of vasospasm was observed. On the fourth day after the ictus, the patient was administered haloperidol, and a progressive reduction in the frequency of choreoatetoid movements was observed. However, the dysarthria and postural instability remained. The patient was administered phenytoin for seven days

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Figure 1 - A) Cerebral angiography at admission revealed a small saccular aneurysm of the basilar artery near the emergence of the anterior inferior cerebellar artery (AICA). B) T2-weighted Magnetic Resonance Imaging (MRI) at the one-year follow-up revealed cerebellar atrophy.

Table 1 - Movement disorders after SAH. Author (Year)

Age

Sex

Time from SAH to MD

Aneurysm localization

MD Recovery

Hypothesis

Sakai et al. (1991)4 Alarcoń et al. (2004)3 Alarcoń et al. (2004)3 Alarcoń et al. (2004)3 Morigaki et al. (2008)5 Pereira et al. (2011)

Chorea

8 days

ICA - AchoA

Total

Vasospasm Hydrocephalus

Tremor

Intraventricular hemorrhage

Tremor

Hydrocephalus

Tremor, dystonia, ataxia and dysmetria Choreoathetosis

Ictus

Distal accessory ACA

Total

Corpus callosum hematoma

Choreoathetosis

Ictus

Basilar artery at AICA emergence

Total

Disturbance of the dentatorubro-thalamo-cortical pathways

F = female; M = male; MD = movement disorder; AICA = anterior inferior cerebellar artery; ICA – AchoA = internal carotid artery - anterior choroidal artery; ACA = anterior cerebral artery. * Only time in days (18.7 days, SD = 12.8 days) reported between the diagnosis of stroke and the onset of abnormal movements in a group of 14 patients with tremor after stroke. Six patients experienced an ischemic stroke, five had parenchymal hemorrhage, and three experienced a subarachnoid hemorrhage. ** not mentioned.

cases in which the abnormal movement occurred much later, including Parkinsonism that began ten months after the stroke.3 While analyzing the literature data on movement disorders after a SAH (Table 1), we observed a total of six patients (including this reported case). Of these six patients, five were female and one was male, with a mean age of 58.6 years (SD 21.6, ranging from 17 to 74 years). Chorea or choreoathetosis was observed in three of these cases; tremor was observed in the other three cases. One patient in the tremor group exhibited associated dystonia. Our case is very atypical because he is the youngest patient ever described and was the only male to present with SAHrelated involuntary movement. In our case, no vasospasm, hydrocephalus, or even direct injury to the basal ganglia was observed that could explain the choreoathetosis. We suggest that the hematoma that was

located in the fourth ventricle may have disturbed the dentato-rubro-thalamo-cortical pathways, leading to transient choreoatetoid movements. Another hypothesis, without evidence of a vasospasm, is that the movement disorder was secondary to an ischemic lesion in the thalamogeniculate artery (a branch of the posterior cerebral artery), which disturbed the posterior ventral thalamic areas related to the basal ganglia circuit.

REFERENCES 1. Brisman JL, Song JK, Newell DW. Cerebral aneurysms. N Engl J Med. 2006;355:928-39, doi: 10.1056/NEJMra052760. 2. Suarez JI, Tarr RW, Selman WR. Aneurysmal subarachnoid hemorrhage. N Engl J Med. 2006;354:387-96, doi: 10.1056/NEJMra052732. 3. Alarco´n F, Zijlmans JCM, Duen˜as G, Cevallos N. Post-stroke movement disorders: report of 56 patients. J Neurol Neurosurg Psychiatry. 2004;75:1568–74, doi: 10.1136/jnnp.2003.011874.

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Choreoathetosis after subarachnoid hemorrhage Pereira JLB et al. 5. Morigaki R, Uno M, Matsubara S, Satoh K, Nagahiro S. Choreoathetosis due to rupture of a distal accessory anterior cerebral artery aneurysm. Cerebrovascular Diseases. 2008;25:285-7, doi: 10.1159/000119640.

4. Sakai K, Kyoshima K, Ohigashi Y, Unoki T, Kobayashi S, Meguro M. Generalized choreic movement associated with subarachnoid hemorrhage. No To Shinkei. 1991;43:875â&#x20AC;&#x201C;80.

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DOI:10.1590/S1807-59322011000900027

CASE REPORT

Extracorporeal membrane oxygenation as a bridge to pulmonary transplantation in Brazil: Are we ready to embark upon this new age? Marcelo Park,I,III Eduardo Leite Vieira Costa,II,III Luciano Cesar Pontes Azevedo,I,III Jose´ Eduardo Afonso Junior,IV Marcos Naoyuki Samano,IV Carlos Roberto Ribeiro Carvalho,II ECMO Group I

Intensive Care Unit – Emergency Department – Hospital das Clıńicas, Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brazil. Respiratory Intensive Care Unit – Hospital das Clıńicas, Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brazil. III Intensive Care Unit – Hospital Sı´rio-Libaneˆs, Saõ Paulo/SP, Brazil. IV Pulmonary Transplantation Group – Heart Institute (InCor) – Hospital das Clıńicas, Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brazil. II

Email: mpark@uol.com.br Tel.: 55 11 3069-6457

the cannulas. Arterial blood gases improved, promoting a comfortable spontaneous breathing pattern during mechanical ventilation. Trivial anticoagulation with heparin was started and titrated to a ratio of activated thromboplastin time of 1.5–2.3. The clinical characteristics during the patient’s respiratory intensive care unit stay are shown in Table 1. The patient was maintained awake with a Richmond Agitation Sedation Scale score ranging from -1 to 0. The patient’s condition gradually improved, and daily weaning from extracorporeal membrane oxygenation was performed by zeroing the sweeper flow of the oxygenation membrane. Our criteria for extracorporeal membrane oxygenation removal are as follows: (1) the patient is awake and comfortable throughout the test and (2) PaO2 $55 mmHg and PaCO2 #60 mmHg (or pH$7.30 in patients with chronic hypercapnia) after one hour of ventilation with PEEP #10 cm H2O, FIO2 #0.6 and a tidal volume #6 mL/ kg (or a driving pressure #12 cm H2O). We abort the test when (1) the peripheral oxygen saturation is less than 85%, (2) the patient presents clinical signs of dyspnea, or (3) the

INTRODUCTION Currently, there are few centers able to perform pulmonary transplantation in Brazil.1 The number of patients in need of this procedure and their disease severity has increased in recent years; however, the reduced number of lung donors has resulted in a high mortality rate for patients on the waiting list.2 Some of these patients with exacerbations of severe chronic pulmonary disease can be temporarily supported by extracorporeal membrane oxygenation systems, prolonging life until a donor lung is available for transplantation.3 Those patients who are mechanically supported should receive the donated lungs with priority and should be shifted forward on the waiting list.4 In Brazil, there are no extracorporeal membrane oxygenation centers and, consequently, a prioritization system for patients awaiting lung transplantation using extracorporeal membrane oxygenation does not exist.5 This report describes our experience with an exacerbated severe hypoxemic patient awaiting lung transplantation and using extracorporeal membrane oxygenation in a tertiary center in Brazil.

CASE REPORT An eighteen-year-old woman was admitted to the respiratory intensive care unit of the Hospital das Clıńicas de Saõ Paulo, Brazil, with the diagnosis of pneumonia. The chest X-ray is shown in Figure 1. She had a previous history of advanced cystic fibrosis, was awaiting lung transplantation and was in the fifth position on the Saõ Paulo State waiting list. During the first day of her respiratory intensive care unit stay, her status deteriorated, she was intubated, and mechanical ventilation was started. In spite of the ventilatory support, she developed severe hypoxemic and hypercapnic respiratory failure (Table 1), with no signs of hemodynamic compromise. Femoro-femoral venousvenous extracorporeal membrane oxygenation support was then initiated after ultrasound-guided placement of

Figure 1 - Chest X-ray before the installation of ECMO.

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Table 1 - Clinical data and arterial blood gases. Data Mechanical ventilation Ventilatory mode Peak pressure (min – max) - cm H2O PEEP (min – max) - cm H2O £ FIO2 (min – max) ¥ Respiratory rate (min – max) - breaths/min ECMO J Blood flow (min – max) - L/min Sweeper flow (min – max) - L/min FIO2 Routine blood gas PaO2 - mm Hg PaCO2 - mm Hg SBE - mEq/L * pH Patient data RASS (min – max) 1 Lung injury score " Total SOFA score 1 Respiratory SOFA Cardiovascular SOFA Hematological SOFA Hepatic SOFA Neurological SOFA Renal SOFA

Pre-ECMO

Day 1

Day 2

Day 3

Day 4

Day 18

VCV I 28 - 28 20 – 20 0.6 – 0.7 22 - 30

PSV I 28 - 33 20 – 25 0.7 – 0.7 25 - 36

PSV I 28 - 33 20 - 25 0.6 – 0.7 18 - 34

PSV I 28 - 28 20 - 20 0.6 – 0.6 22 - 39

PSV I 26 - 28 18 - 20 0.6 – 0.6 24 – 36

PSV I 24 - 24 10 – 10 0.6 – 0.6 22 - 38

6.0 – 6.5 6.0 – 10.0 1.0

6.0 – 6.0 6.0 – 6.0 1.0

5.5 – 6.0 4.0 – 6.0 1.0

5.0 – 5.5 2.5 – 4.0 1.0

5.0 – 5.0 2.5 – 2.5 1.0

42 118 1.8 7.10

50 51 6.1 7.41

62 38 -1.0 7.43

74 53 0.9 7.33

55 51 -0.8 7.32

81 51 2.1 7.36

-1 - 0 4.00 10 4 3 1 1 0 1

0-0 4.00 9 4 3 1 0 0 1

0–0 4.00 6 4 0 1 0 0 1

0–0 3.75 4 4 0 0 0 0 0

-5 - 0 3.00 4 4 0 0 0 0 0

SBE denotes standard base excess. # RASS denotes Richmond agitation sedation score. 1 SOFA denotes sequential organ failure assessment. This is a score to diagnose and quantify organ failure, which ranges from 0 to 24. " The lung injury score is Murray`s score, which quantifies the severity of lung injury based on the respiratory compliance, PEEP, number of quadrants of chest X-ray infiltrated and PaO2/FIO2 ratio. I VCV denotes volume-controlled ventilation. I PSV denotes pressure-support ventilation. £ PEEP denotes positive end-expiratory pressure. ¥ FiO2 denotes inspiratory fraction of oxygen. J ECMO denotes extracorporeal membrane oxygenation.

in the respiratory intensive care unit. Despite her clinical improvement, she remained completely dependent on extracorporeal membrane oxygenation support (i.e., the use of extracorporeal membrane oxygenation as a bridge to lung transplantation). On the 17th day of her stay in the respiratory intensive care unit, the Ministry of Health approved prioritization of the patient on the lung transplantation list on an exceptional basis due to the circumstances. During this period, no transplant had been performed from donors with the same blood type as the patient. Unfortunately, the patient died on the 18th day of her stay in the respiratory intensive care unit. In summary, we believe that our experience with this case should motivate revision of the current legislation regulating lung transplants in Brazil, as well as the procurement, conservation, and reconditioning of organ systems. The scenario in which patients with exacerbations of chronic severe pulmonary diseases will become dependent on the support of extracorporeal devices will become frequent. Should the clinical conditions allow, prioritization on the waiting list for lung transplantation for these patients should be carefully considered.

staff deems such an action to be necessary. The patient remained on extracorporeal membrane oxygenation support for 18 days with no adverse events, but she never tolerated more than five minutes of the weaning test. After 18 days of extracorporeal membrane oxygenation support, the patient died.

DISCUSSION In the case presented, before the initiation of extracorporeal membrane oxygenation support, we promoted an extensive discussion between the respiratory intensive care unit and transplantation teams in order to define the focus of care. The background of the discussion was the absence, in Brazil, of prioritization criteria for patients on the waiting list for lung transplantation who require extracorporeal membrane oxygenation support when weaning from extracorporeal membrane oxygenation is considered difficult or impossible. The final decision was to start extracorporeal membrane oxygenation support and to file a special request to the Ministry of Health, asking to prioritize the patient on the lung transplantation waiting list. Our expectation was that with full intensive care support and antibiotics, the patient would gradually improve toward a clinical condition that was sufficient to allow lung transplantation. In the meantime, our request was analyzed by the Ministry of Health. The extrapulmonary organ dysfunctions of the patient quickly resolved; from a clinical standpoint, she was able to undergo lung transplantation from the third day of her stay

APPENDIX The ECMO group comprises: Luciano Cesar Pontes Azevedo, Marcelo Park, Andre´ Luiz de Oliveira Martins, Eduardo Leite Vieira Costa, Guilherme Paula Pinto Schettino, Marcelo Brito Passos Amato, Carlos Roberto Ribeiro Carvalho, Mauro Tucci, Alexandre Toledo Maciel,

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Extracorporeal membrane oxygenation Park M et al. 3. Haneya A, Philipp A, Mueller T, Lubnow M, Pfeifer M, Zink W, et al. Extracorporeal circulatory systems as a bridge to lung transplantation at remote transplant centers. Ann Thorac Surg. 2011;91:250-5, doi: 10.1016/ j.athoracsur.2010.09.005. 4. Orens JB, Estenne M, Arcasoy S, Conte JV, Corris P, Egan JJ, et al. International guidelines for the selection of lung transplant candidates: 2006 update--a consensus report from the Pulmonary Scientific Council of the International Society for Heart and Lung Transplantation. J Heart Lung Transplant. 2006;25:745-55, doi: 10. 1016/j.healun.2006.03.011. 5. Ministe´rio da Sau´de. PORTARIA No- 2.600. www.abto.org.br/download/ Portaria2600_GM pdf 2009.

Fernanda Maria Queiroz Silva, Leandro Utino Taniguchi, Edzaˆngela Vasconcelos and Adriana Sayuri Hirota.

REFERENCES 1. Associaçaõ brasileira de transplantes de orgaõs. Registro Brasileiro de Transplantes. www.abto.org.br 2010. 2. Pego-Fernandes PM, Mariani AW, de Medeiros IL, Pereira AE, Fernandes FG, do Valle UF, et al. Ex vivo lung evaluation and reconditioning. Rev Bras Cir Cardiovasc. 2010;25:441-6, doi: 10.1590/ S0102-76382010000400006.

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ERRATA CLINICS 2011;66(4):673-679 Page 673 Replace Khuraskan Branch For Khorasgan Branch

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