www.arquivosonline.com.br Sociedade Brasileira de Cardiologia • ISSN-0066-782X • Volume 104, Nº 2, February 2015

Figure 1 – Ilustration of cell interactions during atheroma formation. Page 170

Editorial Fatty Acid and Cholesterol Concentrations in Usually Consumed The representativeness of the Arquivos Brasileiros de Cardiologia for Fish in Brazil Brazilian Cardiology Science Assessment of Myocardial Infarction by Cardiac Magnetic Resonance Special Article Imaging and Long-Term Mortality Multivariate Analysis for Animal Selection in Experimental Research Review Article Original Articles Microparticles as Potential Biomarkers of Cardiovascular Disease Reproducibility of left ventricular mass by echocardiogram in the Letter to the Editor ELSA-Brasil Acute Effects of Continuous Positive Airway Pressure on Pulse Pressure in CHF Relationship between Neutrophil-To-Lymphocyte Ratio and Electrocardiographic Ischemia Grade in STEMI

Sudden Cardiac Death in Brazil: A Community-Based Autopsy Series Eletronic Pages (2006-2010) Clinicoradiological Session Impact of Light Salt Substitution for Regular Salt on Blood Pressure of Case 2/2015 A 33-Year-Old Woman with Double Right Ventricular Hypertensive Patients Chamber and Ventricular Septal Defect Effects of Ischemic Postconditioning on the Hemodynamic Parameters Case Report and Heart Nitric Oxide Levels of Hypothyroid Rats Giant Left Atrial Thrombus with Double Coronary Vascularization Neural Mechanisms and Delayed Gastric Emptying of Liquid Induced Image Through Acute Myocardial Infarction in Rats Mitral Valve Aneurysm Secondary to Probable Infective Endocarditis A JOURNAL OF SOCIEDADE BRASILEIRA DE CARDIOLOGIA - Published since 1948

Contents

Editorial

The representativeness of the Arquivos Brasileiros de Cardiologia for Brazilian Cardiology Science Paulo Roberto Barbosa Evora e Luiz Felipe P. Moreira ...... page 94

Special Article

Multivariate Analysis for Animal Selection in Experimental Research Renan Mercuri Pinto, Dijon Henrique Salomé de Campos, Loreta Casquel Tomasi, Antonio Carlos Cicogna, Katashi Okoshi, Carlos Roberto Padovani ...... page 97

Original Articles Echocardiography - Adults Reproducibility of left ventricular mass by echocardiogram in the ELSA-Brasil Alexandre Pereira Tognon, Murilo Foppa, Vivian Cristine Luft, Lloyd Ellwood Chambless, Paulo Lotufo, Lilia Maria Mameri El Aouar, Luciana Pereira Fernandes, Bruce Bartholow Duncan ...... page 104

Electrocardiography Relationship between Neutrophil-To-Lymphocyte Ratio and Electrocardiographic Ischemia Grade in STEMI Emre Yalcinkaya, Uygar Cagdas Yuksel, Murat Celik, Hasan Kutsi Kabul, Cem Barcin, Yalcin Gokoglan, Erkan Yildirim, Atila Iyisoy ...... page 112

Epidemiology Sudden Cardiac Death in Brazil: A Community-Based Autopsy Series (2006-2010) Maria Fernanda Braggion-Santos, Gustavo Jardim Volpe, Antonio Pazin-Filho, Benedito Carlos Maciel, José Antonio Marin-Neto, André Schmidt ...... page 120

Hypertension Impact of Light Salt Substitution for Regular Salt on Blood Pressure of Hypertensive Patients Carolina Lôbo de Almeida Barros, Ana Luiza Lima Sousa, Brunella Mendonça Chinem, Rafaela Bernardes Rodrigues, Thiago Souza Veiga Jardim, Sérgio Baiocchi Carneiro, Weimar Kunz Sebba Barroso de Souza, Paulo César Brandão Veiga Jardim ...... page 128

Arquivos Brasileiros de Cardiologia - Volume 104, Nº 2, February 2015 Ischemia/Myocardial Infarction Effects of Ischemic Postconditioning on the Hemodynamic Parameters and Heart Nitric Oxide Levels of Hypothyroid Rats Sajad Jeddi, Jalal Zaman, Asghar Ghasemi ...... page 136

Neural Mechanisms and Delayed Gastric Emptying of Liquid Induced Through Acute Myocardial Infarction in Rats Wilson Ranu Ramirez Nunez, Michiko Regina Ozaki, Adriana Mendes Vinagre, Edgard Ferro Collares, Eros Antonio de Almeida ...... page 144

Metabolism/Nutrition Fatty Acid and Cholesterol Concentrations in Usually Consumed Fish in Brazil Carlos Scherr, Ana Carolina Moron Gagliardi, Marcio Hiroshi Miname, Raul Dias Santos ...... page 152

Cardiovascular Magnetic Resonance Imaging Assessment of Myocardial Infarction by Cardiac Magnetic Resonance Imaging and Long-Term Mortality João Luiz Fernandes Petriz, Bruno Ferraz de Oliveira Gomes, Braulio Santos Rua, Clério Francisco Azevedo, Marcelo Souza Hadlich, Henrique Thadeu Periard Mussi, Gunnar de Cunto Taets, Emília Matos do Nascimento, Basílio de Bragança Pereira, Nelson Albuquerque de Souza e Silva ...... page 159

Review Article

Microparticles as Potential Biomarkers of Cardiovascular Disease Carolina Nunes França, Maria Cristina de Oliveira Izar, Jônatas Bussador do Amaral, Daniela Melo Tegani, Francisco Antonio Helfenstein Fonseca ...... page 169

Letter to the Editor

Acute Effects of Continuous Positive Airway Pressure on Pulse Pressure in CHF Jacobo Bacariza Blanco e Antonio M. Esquinas ...... page 175

Arquivos Brasileiros de Cardiologia - Volume 104, Nº 2, February 2015 Arquivos Brasileiros de Cardiologia - Eletronic Pages

Clinicoradiological Session

Case 2/2015 A 33-Year-Old Woman with Double Right Ventricular Chamber and Ventricular Septal Defect Edmar Atik ...... page e12

Case Report

Giant Left Atrial Thrombus with Double Coronary Vascularization Giuliano Serafino Ciambelli, Mariana Lins Baptista, Vitor Emer Egypto Rosa, Antonio Sérgio de Santis Andrade Lopes, Tarso Augusto Duenhas Accorsi, Flávio Tarasoutchi ...... page e15

Image

Mitral Valve Aneurysm Secondary to Probable Infective Endocarditis Anne Paula Delgado e Pedro Mendes Gama ...... page e18

* Indicate manuscripts only in the electronic version. To view them, visit: http://www.arquivosonline.com.br/2015/english/10402/edicaoatual.asp

Arquivos Brasileiros de Cardiologia - Volume 104, Nº 2, February 2015 www.arquivosonline.com.br A JOURNAL OF SOCIEDADE BRASILEIRA DE CARDIOLOGIA - Published since 1948

Scientific Director Interventionist Cardiology Epidemiology/Statistics Maria da Consolação Vieira Moreira Pedro A. Lemos Lucia Campos Pellanda

Chief Editor Pediatric/Congenital Cardiology Antonio Augusto Lopes Arterial Hypertension Luiz Felipe P. Moreira Paulo Cesar B. V. Jardim Arrhythmias/Pacemaker Associated Editors Mauricio Scanavacca Ergometrics, Exercise and Clinical Cardiology Cardiac Rehabilitation Non-Invasive Diagnostic Methods Ricardo Stein José Augusto Barreto-Filho Carlos E. Rochitte

Surgical Cardiology Basic or Experimental Research First Editor (1948-1953) Paulo Roberto B. Evora Leonardo A. M. Zornoff † Jairo Ramos Editorial Board

Brazil Enio Buffolo (SP) Otoni Moreira Gomes (MG) Aguinaldo Figueiredo de Freitas Junior (GO) Eulógio E. Martinez Filho (SP) Paulo Andrade Lotufo (SP) Alfredo Jose Mansur (SP) Evandro Tinoco Mesquita (RJ) Paulo Cesar B. V. Jardim (GO) Aloir Queiroz de Araújo Sobrinho (ES) Expedito E. Ribeiro da Silva (SP) Paulo J. F. Tucci (SP) Amanda G. M. R. Sousa (SP) Fabio Vilas-Boas (BA) Paulo R. A. Caramori (RS) Ana Clara Tude Rodrigues (SP) Fernando Bacal (SP) Paulo Roberto B. Évora (SP) Andre Labrunie (PR) Flavio D. Fuchs (RS) Paulo Roberto S. Brofman (PR) Andrei Sposito (SP) Francisco Antonio Helfenstein Fonseca (SP) Pedro A. Lemos (SP) Angelo A. V. de Paola (SP) Gilson Soares Feitosa (BA) Protasio Lemos da Luz (SP) Antonio Augusto Barbosa Lopes (SP) Glaucia Maria M. de Oliveira (RJ) Reinaldo B. Bestetti (SP) Antonio Carlos C. Carvalho (SP) Hans Fernando R. Dohmann (RJ) Renato A. K. Kalil (RS) Antonio Carlos Palandri Chagas (SP) Humberto Villacorta Junior (RJ) Ricardo Stein (RS) Antonio Carlos Pereira Barretto (SP) Ínes Lessa (BA) Salvador Rassi (GO) Antonio Claudio L. Nóbrega (RJ) Iran Castro (RS) Sandra da Silva Mattos (PE) Antonio de Padua Mansur (SP) Jarbas Jakson Dinkhuysen (SP) Sandra Fuchs (RS) Ari Timerman (SP) Joao Pimenta (SP) Sergio Timerman (SP) Armênio Costa Guimaraes (BA) Jorge Ilha Guimaraes (RS) Silvio Henrique Barberato (PR) Ayrton Pires Brandao (RJ) Jose Antonio Franchini Ramires (SP) Tales de Carvalho (SC) Beatriz Matsubara (SP) Jose Augusto Soares Barreto Filho (SE) Vera D. Aiello (SP) Brivaldo Markman Filho (PE) Jose Carlos Nicolau (SP) Walter José Gomes (SP) Bruno Caramelli (SP) Jose Lazaro de Andrade (SP) Weimar K. S. B. de Souza (GO) Carisi A. Polanczyk (RS) Jose Pericles Esteves (BA) William Azem Chalela (SP) Carlos Eduardo Rochitte (SP) Leonardo A. M. Zornoff (SP) Wilson Mathias Junior (SP) Carlos Eduardo Suaide Silva (SP) Leopoldo Soares Piegas (SP) Carlos Vicente Serrano Junior (SP) Lucia Campos Pellanda (RS) Exterior Celso Amodeo (SP) Luis Eduardo Rohde (RS) Adelino F. Leite-Moreira (Portugal) Charles Mady (SP) Luís Cláudio Lemos Correia (BA) Alan Maisel (USA) Claudio Gil Soares de Araujo (RJ) Luiz A. Machado Cesar (SP) Aldo P. Maggioni (Italy) Cláudio Tinoco Mesquita (RJ) Luiz Alberto Piva e Mattos (SP) Cândida Fonseca (Portugal) Cleonice Carvalho C. Mota (MG) Marcia Melo Barbosa (MG) Fausto Pinto (Portugal) Clerio Francisco de Azevedo Filho (RJ) Maria da Consolação Moreira (MG) Hugo Grancelli (Argentina) Dalton Bertolim Précoma (PR) Mario S. S. de Azeredo Coutinho (SC) James de Lemos (USA) Dário C. Sobral Filho (PE) Mauricio I. Scanavacca (SP) João A. Lima (USA) Decio Mion Junior (SP) Max Grinberg (SP) John G. F. Cleland (England) Denilson Campos de Albuquerque (RJ) Michel Batlouni (SP) Maria Pilar Tornos (Spain) Djair Brindeiro Filho (PE) Murilo Foppa (RS) Pedro Brugada (Belgium) Domingo M. Braile (SP) Nadine O. Clausell (RS) Peter A. McCullough (USA) Edmar Atik (SP) Orlando Campos Filho (SP) Peter Libby (USA) Emilio Hideyuki Moriguchi (RS) Otavio Rizzi Coelho (SP) Piero Anversa (Italy) Sociedade Brasileira de Cardiologia

President Special Advisor to the Presidency SBC/MG - Odilon Gariglio Alvarenga de Freitas Angelo Amato V. de Paola Fábio Sândoli de Brito

Vice-President Adjunct Coordination SBC/MS - Mércule Pedro Paulista Cavalcante Sergio Tavares Montenegro SBC Newsletter Editor President-Elect Nabil Ghorayeb e Fernando Antonio Lucchese SBC/MT - Julio César De Oliveira Marcus Vinícius Bolívar Malachias Continuing Education Coordination Financial Director Estêvão Lanna Figueiredo SBC/NNE - Jose Itamar Abreu Costa Jacob Atié Norms and Guidelines Coordination Luiz Carlos Bodanese Scientific Director SBC/PA - Luiz Alberto Rolla Maneschy Maria da Consolação Vieira Moreira Governmental Integration Coordination Edna Maria Marques de Oliveira Administrative Director SBC/PB - Helman Campos Martins Emilio Cesar Zilli Regional Integration Coordination José Luis Aziz Assistance Quality Director SBC/PE - Catarina Vasconcelos Cavalcanti Pedro Ferreira de Albuquerque Presidents of State and Regional Brazilian Societies of Cardiology Communication Director SBC/PI - João Francisco de Sousa Maurício Batista Nunes SBC/AL - Carlos Alberto Ramos Macias

Information Technology Director SBC/PR - Osni Moreira Filho José Carlos Moura Jorge SBC/AM - Simão Gonçalves Maduro

Government Liaison Director SBC/RJ - Olga Ferreira de Souza Luiz César Nazário Scala SBC/BA - Mario de Seixas Rocha

Director of State and Regional Affairs SBC/RN - Rui Alberto de Faria Filho Abrahão Afiune Neto SBC/CE - Ana Lucia de Sá Leitão Ramos

Cardiovascular Health Promotion Director SBC/RS - Carisi Anne Polanczyk SBC/CO - Frederico Somaio Neto - SBC/Funcor Carlos Costa Magalhães SBC/DF - Wagner Pires de Oliveira Junior SBC/SC - Marcos Venício Garcia Joaquim Department Director Especializados - Jorge Eduardo Assef SBC/ES - Marcio Augusto Silva SBC/SE - Fabio Serra Silveira Research Director Fernanda Marciano Consolim Colombo SBC/GO - Thiago de Souza Veiga Jardim SBC/SP - Francisco Antonio Helfenstein Fonseca Chief Editor of the Brazilian Archives of Cardiology Luiz Felipe P. Moreira SBC/MA - Nilton Santana de Oliveira SBC/TO - Hueverson Junqueira Neves Presidents of the Specialized Departaments and Study Groups

SBC/DA - José Rocha Faria Neto SBCCV - Marcelo Matos Cascado GECC - Mauricio Wanjgarten

SBC/DECAGE - Josmar de Castro Alves SBHCI - Helio Roque Figueira GEPREC - Glaucia Maria Moraes de Oliveira

SBC/DCC - José Carlos Nicolau SBC/DEIC - Dirceu Rodrigues Almeida Grupo de Estudos de Cardiologia Hospitalar - Evandro Tinoco Mesquita SBC/DCM - Maria Alayde Mendonça da Silva GERTC - Clerio Francisco de Azevedo Filho Grupo de Estudos de Cardio-Oncologia - SBC/DCC/CP - Isabel Cristina Britto Guimarães Roberto Kalil Filho GAPO - Danielle Menosi Gualandro SBC/DIC - Arnaldo Rabischoffsky GEEC - Cláudio José Fuganti GEECG - Joel Alves Pinho Filho SBC/DERC - Nabil Ghorayeb GECIP - Gisela Martina Bohns Meyer GEECABE - Mario Sergio S. de Azeredo SBC/DFCVR - Ricardo Adala Benfati Coutinho GECESP - Ricardo Stein

SBC/DHA - Luiz Aparecido Bortolotto GECETI - Gilson Soares Feitosa Filho GECN - Ronaldo de Souza Leão Lima

SOBRAC - Luiz Pereira de Magalhães GEMCA - Alvaro Avezum Junior GERCPM - Artur Haddad Herdy Arquivos Brasileiros de Cardiologia

Volume 104, Nº 2, February 2015 Indexing: ISI (Thomson Scientific), Cumulated Index Medicus (NLM), SCOPUS, MEDLINE, EMBASE, LILACS, SciELO, PubMed

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The representativeness of the Arquivos Brasileiros de Cardiologia for Brazilian Cardiology Science Paulo Roberto Barbosa Evora1 e Luiz Felipe P. Moreira2 Department of Surgery and Anatomy of Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (USP)1, Ribeirão Preto; Instituto do Coração, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo (USP)2, São Paulo, SP – Brazil

The front page of the Arquivos Brasileiros de Cardiologia field of Cardiology. The Arquivos Brasileiros de Cardiologia (Brazilian Archives of Cardiology) discloses an objective view currently occupies position 95 among 125 journals listed by of the current representativeness of the journal1: ISI with a positive impact factor in the field of cardiovascular “With over 60 years of existence, the Arquivos diseases in 2014. Among these publications, only five have Brasileiros de Cardiologia (Arq Bras Cardiol) is the an impact factor greater than ten (4%); 17 (13.6%) between official scientific publication of Brazilian Society of 5.04 and 7.44; 47 (37.6%) between 2.044 and 4.918; Cardiology (BSC), serving as the main channel for 35 (28%) between 1.965 and 1.018 and; 21 (16.8%) with the dissemination of Brazilian scientific research on an impact factor of 0.011 and 0.912. cardiovascular sciences. Published in two languages Individual analysis of the Arquivos by Web of Science and indexed in major international databases (ISI Web currently discloses the following numbers: 3,383 indexed of Science; Cumulated Index Medicus — MEDLINE; publications, with 2,509 original and review articles. EMBASE; SCOPUS; SCIELO and LILACS), Arquivos Since 2003, these articles have received 7,365 citations Brasileiros de Cardiologia has an average impact factor of (Figure 1), corresponding to a mean rate of 2.64 citations 1.1, according to Thompson Reuters. This rating puts our per article and an H index of 25. The number of journal at a level that is similar to most journals indexed self ‑citations was 1,482, accounting for only 26.2% of in ISI Web of Science in the field of Cardiology.” the total. Among the ten most cited papers, the majority Considering the historical and academic significance of corresponds to guidelines and epidemiological data5-10. It is the Arquivos Brasileiros de Cardiologia, since its indexing worth mentioning the presence of a single article on basic in MEDLINE that started in 1950, some authors have sciences11. The interesting thing is that these articles are assumed the hypothesis that the analysis of publications not totally dependent on national citations. in the last 60 years could reflect the changing trends of The relevance of the Arquivos Brasileiros de Cardiologia heart disease in Brazil. The existence of an epidemiological for Brazilian cardiology science can also be assessed through link between cardiac diseases that are prevalent in Brazil its position as the main vehicle for the dissemination of and the publications of the Arquivos has been suggested. national research at international level. Of 1,581 original This evidence, associated with planning, publications articles published by Brazilian authors and institutions and guideline updates, leaves no doubt about its crucial between 2010 and 2012 in journals indexed in the Web 2-4. importance for Brazilian Cardiology of Science database, it is noteworthy the fact that 27% Regarding the evaluation of its impact, the announcement were published in the Arquivos, whereas 43% of these of the Arquivos’ first Impact Factor by the Journal of Citation articles were published by journals with a higher impact Report of Thompson Reuters occurred in 2010. Since then, factor, located at the upper strata of the CAPES Qualis the values of this bibliometric index, documented by the classification (Figure 2). Web of Science database of the Institute for Scientific Aiming to improve the quality of publications, the Information (ISI), have confirmed the degree of scientific Editorial Board of the journal has adopted a certain degree relevance and the scope of studies carried out in Brazil, of academic prioritization. Currently, 60% of the articles as well as those performed in international centers that published in the Arquivos originate from academic studies have been published in our journal. The ratings place at post-graduate level. Although greater receptivity has the Arquivos at the same level of approximately 30% of been observed from Post-Graduate Programs in Cardiology, international journals indexed in the ISI database in the unfortunately this decision has not reflected the positive variation of the Impact Factor in recent years, leading to the suggestion that the Brazilian academic community has Keywords not prioritized the Arquivos Brasileiros de Cardiologia as an Periodicals as Topic; Journal Impact Factor; Portals for option to have their best works published. However, we Scientific Journals; Bibliometrics. believe that this trend can be reversed, being one of the motivations of this editorial, seeking to encourage post- Mailing Address: Paulo Roberto Barbosa Evora • graduate programs to give higher priority to the Archives. Rua Rui Barbosa 367, 15, Centro. Postal Code 14015-120, Ribeirão Preto, SP – Brazil Finally, there is a clear need for greater participation in the E-mail: [email protected]; [email protected] area of Basic Science and applied research in Cardiology, as well as the publication of a greater number of controlled DOI: 10.5935/abc.20150015 clinical trials and meta-analyses in our journal.

94 Evora and Moreira The Arquivos and Brazilian Cardiology

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Items published per year Citations in each year

350 1400

300 1200

250 1000

200 800

150 600

100 400

50 200

0 0 2011 2011 2002 2003 2004 2005 2006 2007 2008 2009 2010 2012 2013 2014 2002 2003 2004 2005 2006 2007 2008 2009 2010 2012 2013 2014 2015

Figure 1 – Number of original and review articles published and number of citations obtained by the same articles in the Web of Science database during the analyzed period.

Period 2010-12 (1581 articles) Qualis-CAPES classification

12% A1 19% 18% A2 12% B1 12% 27% ABC

B2

B3

Figure 2 – Percentage of original and review articles published by Brazilian authors and institutions, according to the Qualis-CAPES classification. ABC: Brazilian Archives of Cardiology

Arq Bras Cardiol. 2015; 104(2):94-96 95 Evora and Moreira The Arquivos and Brazilian Cardiology

Editorial

References

1. Arquivos Brasileiros de Cardiologia. [Acesso em 2015 jan 10]. Disponível 7. Guedes DP, Guedes JE, Barbosa DS, de Oliveira JA, Stanganelli LC. em: http://www.arquivosonline.com.br/2014/ Cardiovascular risk factors in adolescents: biological and behavioral indicators. Arq Bras Cardiol. 2006;86(6):439-50. 2. Amorim DS. Publications of the Arquivos Brasileiros de Cardiologia in the period 1968-1977. Arq Bras Cardiol. 1980;34(1):1-7. 8. Gus I, Harzheim E, Zaslavsky C, Medina C, Gus M. Prevalence, awareness, and control of systemic arterial hypertension in the state of Rio Grande do 3. Evora PR, Nather JC, Rodrigues AJ. Prevalence of heart disease demonstrated Sul. Arq Bras Cardiol. 2004;83(5):429-33; 424-8. in 60 years of the Arquivos Brasileiros de Cardiologia. Arq Bras Cardiol. 2014; 102(1):3-9. 9. Pitanga FJ, Lessa I. Anthropometric indexes of obesity as an instrument of screening for high coronary risk in adults in the city of Salvador--Bahia. Arq 4. Mansur AJ, Abud AS, Albuquerque CP. Publication trends in quarterly, Bras Cardiol. 2005;85(1):26-31. bimonthly and monthly cycles of publication during the five decades of Brazilian Archives of Cardiology. Arq Bras Cardiol. 2000;75(1):1-7. 10. Ribeiro RQ, Lotufo PA, Lamounier JA, Oliveira RG, Soares JF, Botter DA. Additional cardiovascular risk factors associated with excess weight in 5. Jardim PC, Gondim M do R, Monego ET, Moreira HG, Vitorino PV, Souza children and adolescents: the Belo Horizonte heart study. Arq Bras Cardiol. WK, et al. High blood pressure and some risk factors in a Brazilian capital. 2006 ;86(6):408-18. Arq Bras Cardiol. 2007;88(4):452-7. 11. Higuchi ML, Gutierrez PS, Bezerra HG, Palomino SA, Aiello VD, Silvestre 6. Giuliano I de C, Coutinho MS, Freitas SF, Pires MM, Zunino JN, Ribeiro RQ. JM, et al. Comparison between adventitial and intimal inflammation of Serum lipids in school kids and adolescents from Florianópolis, SC, Brazil-- ruptured and nonruptured atherosclerotic plaques in human coronary Healthy Floripa 2040 study. Arq Bras Cardiol. 2005;85(2):85-91. arteries. Arq Bras Cardiol. 2002;79(1):20-4.

96 Arq Bras Cardiol. 2015; 104(2):94-96 Special Article

Multivariate Analysis for Animal Selection in Experimental Research Renan Mercuri Pinto1, Dijon Henrique Salomé de Campos2, Loreta Casquel Tomasi2, Antonio Carlos Cicogna2, Katashi Okoshi2, Carlos Roberto Padovani1 Departamento de Bioestatística - Instituto de Ciências Biológicas da Universidade Estadual Paulista (Unesp)1; Departamento de Clínica Médica - Faculdade de Medicina de Botucatu - Universidade Estadual Paulista (Unesp)2, Botucatu, São Paulo - Brazil

Abstract Background: Several researchers seek methods for the selection of homogeneous groups of animals in experimental studies, a fact justified because homogeneity is an indispensable prerequisite for casualization of treatments. The lack of robust methods that comply with statistical and biological principles is the reason why researchers use empirical or subjective methods, influencing their results. Objective: To develop a multivariate statistical model for the selection of a homogeneous group of animals for experimental research and to elaborate a computational package to use it. Methods: The set of echocardiographic data of 115 male Wistar rats with supravalvular aortic stenosis (AoS) was used as an example of model development. Initially, the data were standardized, and became dimensionless. Then, the variance matrix of the set was submitted to principal components analysis (PCA), aiming at reducing the parametric space and at retaining the relevant variability. That technique established a new Cartesian system into which the animals were allocated, and finally the confidence region (ellipsoid) was built for the profile of the animals’ homogeneous responses. The animals located inside the ellipsoid were considered as belonging to the homogeneous batch; those outside the ellipsoid were considered spurious. Results: The PCA established eight descriptive axes that represented the accumulated variance of the data set in 88.71%. The allocation of the animals in the new system and the construction of the confidence region revealed six spurious animals as compared to the homogeneous batch of 109 animals. Conclusion: The biometric criterion presented proved to be effective, because it considers the animal as a whole, analyzing jointly all parameters measured, in addition to having a small discard rate. (Arq Bras Cardiol. 2015; 104(2):97-103) Keywords: Multivariate Analysis; Animals; Epidemiology, Experimental; Aortic Valve Stenosis.

Introduction From the biological viewpoint, the most interesting process Due to lack of statistical knowledge, several researchers of homogenization should jointly consider all parameters choose to use empirical or subjective decision-making assessed in the experimental unit, because most of those are methods, ignoring the casualization process, a basic principle correlated, and the best way to understand the behavior of for reliability of findings, which influences the results. the animal consists in a set of numerical data representing all its biological characteristics. Thus, statistical methods that A very common example of that influence occurs in consider the animal as a whole rather than in a fragmented the process of sample homogenization, which is important way are required, because the organism reacts as a whole to for randomization in research involving animals as an any intervention or treatment. experimental unit. Regarding that process, several researchers usually adopt a fragmented or intentional approach, using It is worth noting that, when there is dependence between only a convenient parameter to classify the group as variables, multivariate analysis of data should be used, homogeneous, which results in a biased and inappropriate because, as such, the significance index of inferential statistics is not inflated, a problem that occurs when several univariate homogenization, in addition to favoring the possibility of 1 discarding animals due to a simple spurious value rather than analyses are performed simultaneously . to biological dissimilarity. This study’s objective was to develop a multivariate statistical model for the selection of a homogeneous group of Mailing Address: Renan Mercuri Pinto • animals for experimental research that follows biological and Rua Thomaz Ceneviva, 117, Vila Anita. Postal Code 13484-295, Limeira, statistical principles, in addition to elaborating a computational SP - Brazil package to apply that model. E-mail: [email protected]; [email protected] Manuscript received June 26, 2014; revised manuscript September 27, To exemplify the development and application of the 2014; accepted September 30, 2014. criterion, the set of echocardiographic data of animals undergoing surgery for induction of supravalvular aortic stenosis DOI: 10.5935/abc.20140219 (AoS), provided by the Research Group on Experimental

97 Pinto et al. Selection of Animals in Experimental Research

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Cardiology of the Botucatu Medical School (FMB) of the the Ethics Committee for Animal Experimentation of the FMB State University of São Paulo (Unesp), was used, aiming at - Unesp (protocol number 850/2010). Of the set comprising overcoming the difficulties in selecting a homogeneous group 115 experimental units, 31 parameters regarding body of animals by using the simple casualization process, for later mass and structural and functional variables of transthoracic submission to treatments of interest. echocardiography were used (Table 1). The literature has plenty of experimental studies on Methods the development of cardiac remodeling due to pressure overload2-6. The AoS model has been widely used to promote the gradual development of left ventricular (LV) hypertrophy in Animals and experimental protocol rats5. In that model, pressure overload installs and progressively The set of echocardiographic data was obtained from male increases as the animals grow, partially similar to AoS in Wistar rats undergoing surgery for induction of supravalvular men. Some advantages of that experimental procedure are AoS and provided by the Research Group on Experimental the absence of myocardial anatomical lesions and its low Cardiology of the FMB - Unesp. This study was approved by operational cost4.

Table 1 – Identification of the parameters measured

Index Identification Description

X1 BM (g) body mass

X2 HR (bpm) heart rate

X3 LVDD (mm) left ventricular diastolic diameter

X4 LVSD (mm) left ventricular systolic diameter

X5 PWDT (mm) left ventricular posterior wall diastolic thickness

X6 PWST (mm) left ventricular posterior wall systolic thickness

X7 IVSDT (mm) interventricular septum diastolic thickness

X8 IVSST (mm) interventricular septum systolic thickness

X9 AOD (mm) aorta diameter

X10 LAD (mm) left atrial diameter

X11 LAD/AOD left atrial diameter to aorta diameter ratio

X12 LVDD/BM (mm/kg) left ventricular diastolic diameter to body mass ratio

X13 LAD/BM (mm/kg) left atrial diameter to body mass ratio

X14 CO (mL/min) cardiac output

X15 CI (mL/min/kg) cardiac index

X16 EFS endocardial fractional shortening

X17 MFS midwall fractional shortening

X18 LVM (g) left ventricular mass

X19 LVMI (g/kg) left ventricular mass index

X20 E wave (cm/s) early diastolic velocity of mitral flow

X21 A wave (cm/s) late diastolic velocity of mitral flow

X22 E/A E wave to A wave ratio

X23 LVRT left ventricular relative wall thickness

X24 LVPWSV (mm/s) left ventricular posterior wall shortening velocity

X25 IVRT (ms) left ventricular isovolumetric relaxation time

X26 R-R (s) interval between two consecutive cardiac cycles

X27 IVRTn left ventricular isovolumetric relaxation time normalized to heart rate

X28 Tei-a (ms) isovolumetric contraction time + ejection time + IVRT

X29 Tei-b (ms) ejection time

X30 MPI myocardial performance index

X31 LVEF left ventricular ejection fraction

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Induction of supravalvular aortic stenosis (50 mg/kg/ip) and xylidine chloride (10 mg/kg/ip), and put in Aortic stenosis was induced according to a previously the left lateral decubitus position. A Vivid S6 echocardiography described method4-7. The three- to four-week-old animals, device (General Electric Medical Systems, Tirat Carmel, Israel) weighing 70-90 g, underwent anesthesia with the association of equipped with a 12-MHz electronic transducer was used. ketamine chloride (60 mg/kg) and xylidine chloride (10 mg/kg), To measure the cardiac structures, M-mode images were used by intraperitoneal (ip) route. Then median thoracotomy was and the ultrasound beam was guided by the two-dimensional performed, the ascending aorta dissected and a silver clip (inner image with the transducer in the parasternal short axis view. diameter of 0.6 mm) placed approximately 3 mm from its The monodimensional image of the left ventricle was obtained root (Figure 1). The thoracic wall was closed, and the sternum, by positioning the M-mode cursor right below the mitral valve 8 muscle layers and skin were sutured with 5.0-mononylon plane between the papillary muscles . The images of the aorta thread. During surgery, the animals were manually ventilated and left atrium were also obtained in the parasternal short axis with positive pressure, 100% oxygen. After the end of surgery, view with the M-mode cursor positioned at the aortic valve they received, subcutaneously, 1 mL of warm saline solution, level. Later, the cardiac structures were measured manually and were placed on a warm surface to recover from anesthesia. with the aid of a pachymeter in at least five consecutive cardiac cycles. The LV diastolic diameter (LVDD), the LV posterior wall diastolic thickness (PWDT) and the interventricular Echocardiographic assessment septum diastolic thickness (IVSDT) were measured at the time Echocardiographic assessment was performed six weeks corresponding to the maximum LV diameter. The LV systolic after AoS induction. To undergo that test, the rats were diameter (LVSD) and the LV posterior wall systolic thickness anesthetized with the association of ketamine chloride (PWST) and the interventricular septum systolic thickness

Figure 1 - Placement of the silver clip in the aortic valve.

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(IVSST) were measured at the time corresponding to the and covariances of standardized data (variance matrix) was minimum LV diameter. The LV systolic function was assessed by considered, which is equivalent to the correlation matrix (R) of calculating the midwall fractional shortening (MFS), {[(LVDD + the initial set9. ½ PWDT + ½ IVSDT) – (LVSD + ½ PWST + ½ IVSST)]/(LVDD The multivariate technique used for data analysis consists in + ½ PWDT + IVSDT)}, and the LV posterior wall shortening the principal components analysis (PCA), whose basic principle velocity (LVPWSV), maximum tangent of the posterior wall relies on the reduction of the parametric space with no loss of systolic movement. Studying the LV diastolic function, the the data set variance structure (in the present study, biological peak velocities of mitral flow corresponding to the initial filling characteristics of the animal) and no loss of the general biological phase (E wave) and to the late filling phase, consequent to atrial information of the animal. That statistical technique, proposed contraction (A wave), were measured, and the E wave/A wave by Karl Pearson in 1901, consists in transforming an initial set ratio was calculated. The flows related to the diastolic function of interrelated variables into another set of non-interrelated were obtained by positioning the transducer on the region variables, which is an orthogonal linear combination of the corresponding to the tip of the heart in the four-chamber image; initial set10. the flows were measured on the echocardiographic monitor. The principal components (PC) are presented in a decreasing order of importance for the data set variance structure, that Statistical model is, the first explains the maximum possible variance, and the The statistical model developed to establish the procedure second explains the maximum variance still retained in the of exploratory multivariate analysis of data and assess the set after discounting the effect of the first, and so on, up to the homogeneity of the batch involves simultaneously all variables last component. The greater the retention of total variance in a measured and considers the entire data variation structure, that smaller number of linear combinations, the better the practical is, the variation inside the variables (intravariability) and the application of the procedure to experimental data, although that variation between variables (intervariability). It is worth noting condition is not a deterrent factor for the use of the criterion to that the global variation structure can be well represented identify spurious values11. by descriptive measures of data variability, more specifically, An interesting mathematical property of the PC consists in variances and covariances. all of them being non-interrelated and, thus, regarding data In addition, the model is elaborated to use all animals included normality, independent of each other. This ensures a system in the research, in the cardiac remodeling group, which survived of orthogonal axes for the graphic representation of animals the surgical procedure previously described. Thus, there was no (experimental units), which can be complemented with statistical specific criterion to determine the number of animals that should inference to identify those that can be ruled out due to the high participate in the study. probability of the occurrence of spurious values as compared to Initially, because the parameters were presented in different the population of origin. measure units, the data were standardized and became There are several criteria to determine the number of dimensionless, that is, each measure was presented as a descriptive axes (PC) considered relevant to the orthogonal value that represents how far away it is from the mean of the system. The present study adopted the Kaiser criterion, also respective parameter. Then, to build the statistical model, known as the latent root criterion, in which a component named multihomogen criterion, the matrix structure of variances accounts for a meaningful amount of variance when it has an

35.000 5 19 43 30.000 53 25.000 64 20.000 110 15.000 10.000 5.000 Distance from the centroid 0.000 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 111 101 106 Animals

Figure 2 - Mahalanobis distance between the animals and the centroid of the group.

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eigenvalue greater than 1.0. Therefore such a component is As such, spurious animals were those registered under the worthy of being retained12. numbers 5, 19, 43, 53, 64 and 110, being the homogeneous For the methodological practice of homogenization in batch formed by the 109 animals inside the confidence region, experimental or observational research, the desirable situation which can be submitted to the experimental models via the for biological information, mainly for the graphical visualization simple casualization process. Of the spurious animals, the one of the procedure, consists in having a maximum number of PC registered as 19 was the most dissimilar to the total group, while (also known as descriptive axes) of three. However, for more that registered as 110 was the least dissimilar. than three dimensions (a common situation when assessing a considerable number of characteristics in the experimental Discussion unit), the technique remains the same, except for graphical visualization, which becomes unfeasible. The conclusion of the assessment of the amount of PC required to retain the relevant variability of this study was that the data set After assigning the animals to the Cartesian system, regarding the animals induced to AoS is a complex structure, from determined by the descriptive axes selected, the confidence the statistical viewpoint. Although its parametric space has been (ellipsoid) region for the mean profile of the animals’ reduced substantially, eight descriptive axes were necessary to form homogeneous responses is built. The 100(1 − α)% the new Cartesian system, making the graphical visualization of 2 confidence region is constructed by using Hotelling T the method impossible. statistics transformed into Fisher-Snedecor’s F distribution13. It is worth considering that the statistical technique used to Despite that limitation, the graph in Figure 2 can visually establish the confidence region involves the construction of translate each animal’s behavior regarding the group. The ordinate inclusion intervals, considering a jointly confidence level for axis represents the Mahalanobis distance of each animal from all variables, that enables verifying if the animals are inside or the set centroid. Thus, knowing the distance from the centroid outside the ellipsoid10. to the outline of the ellipsoid (represented by the red horizontal line), the animals with discrepant behavior are evidenced. They If the response vector of the animal studied is inside the should be more deeply assessed to verify their non-homogeneous confidence ellipsoid built (that is, if the animal’s generalized characteristics regarding the group. Mahalanobis distance from the centroid of an ellipsoid is shorter than the distance from the outline), the animal is Regarding the development of the statistical model to classified as belonging to the homogeneous batch; on the detect the non-homogeneous animals, there is no limitation contrary (greater distance), it is identified as spurious, and, to the application of the procedure in the search for spurious thus, does not participate in the simple casualization process animals. From the practical perspective of using the results of of treatments14. the model proposed to new studies, it is worth noting that the process is limited to the study of animals submitted to AoS By using the statistical procedures approached in this under experimental conditions similar to the one described. section, the generalized algorithm was developed, as well as As such, further studies/tests would be necessary to corroborate the computational package, making the use of the criterion the reliability of the model for other samples. proposed possible. It was named multihomogen package, elaborated for the Rstudio statistical program (made available In the process of selecting homogeneous groups in experimental free of charge on-line: http://www.R-project.org), and can be research, when using procedures with no concern about the directly and costlessly obtained by emailing the authors15. general structure of variability, the number of experimental units (animals) discarded is relevant when compared to the amount of spurious animals identified by the criterion proposed in this study. Results This can be understood by the fact that the method proposed To use the software developed and characterize the characterizes the discard by considering the animal, while those homogeneity of an experimental group of animals, the with no concern about the general structure of variability consider multihomogen criterion was applied to the set of data relating the discard fragmented according to the variable. to the parameters assessed in Wistar rats undergoing AoS in The contribution of the method is explained by the improvement the study previously mentioned. From the quadratic matrix of in the quality of homogenization, by ensuring greater reliability to variance (order 31), involving the correlations, eight descriptive the animals’ biological characteristics in the inclusion of similar axes were established, corresponding to eight PC selected based animals, thus motivating a smaller discard rate, maximizing the on the Kaiser criterion, representing the accumulated variance homogeneous batch, for posterior submission to treatments by of the set of data in 88.71% (Table 2). use of the simple casualization process of the animals. Based on the new data structure, in the system generated by the eight descriptive axes, the Mahalanobis distances of Conclusions each animal were determined in relation to the centroid of the set of all 115 animals. The multivariate statistical procedure The criterion presented, named multihomogen criterion, established an animal’s maximum distance from the centroid for proved to be effective in selecting homogeneous groups of animals it to be located inside the ellipsoid, built to a 95% confidence for experimental research, because it considers the biological level, as being 17.419. Thus, animals whose distance values situation of the animal as a whole, analyzing jointly all parameters were greater than that, shown in Figure 2, were considered to measured, in addition to having a small discard rate. be outside the confidence ellipsoid, being indicated as special As such, this biometrical tool serves to researchers of the (spurious) as compared to those inside that ellipsoid. biological and health areas. Regarding specifically cardiac

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Table 2 – Eigenvalues corresponding to the components selected based on the Kaiser criterion

Eigenvalue Explained variance (%) Accumulated variance (%)

9.400 30.32 30.32

5.121 16.52 46.84

3.995 12.89 59.73

2.541 8.20 67.93

2.094 6.75 74.68

1.839 5.93 80.61

1.456 4.70 85.31

1.055 3.40 88.71

remodeling, in which animals are submitted to AoS, the results Potential Conflict of Interest of this study confirm the expectations presented initially in the No potential conflict of interest relevant to this article discussions involving the experiments previously conducted by the was reported. Research Group on Experimental Cardiology of the FMB - Unesp.

Author contributions Sources of Funding This study was funded by Fundação de Amparo à Conception and design of the research:Pinto RM, Padovani Pesquisa do Estado de São Paulo (FAPESP). CR. Acquisition of data: Campos DHS, Tomasi LC, Cicogna AC, Okoshi K. Analysis and interpretation of the data: Pinto This study was partially funded by Coordenação de RM, Campos DHS, Tomasi LC, Cicogna AC, Okoshi K, Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Padovani CR. Statistical analysis: Pinto RM, Padovani CR. Obtaining financing:Pinto RM, Padovani CR. Writing of the Study Association manuscript: Pinto RM, Campos DHS, Tomasi LC, Padovani CR. Critical revision of the manuscript for intellectual content: This article is part of the dissertation of master submitted Pinto RM, Cicogna AC, Okoshi K, Padovani CR. Orientation by Renan Mercuri Pinto, from Universidade Estadual / Supervision: Padovani CR. Paulista “Júlio de Mesquita Filho”.

References

1. Messeti AV, Padovani CR. Estudo da divergência genética em girassol por 5. Bregagnollo EA, Zornoff LA, Okoshi K, Sugizaki M, Mestrinel MA, Padovani CR, meio de técnicas multivariadas. Energ Agric Botucatu. 2009;24(2):14-28. et al. Myocardial contractile dysfunction contributes to the development of heart failure in rats with aortic stenosis. Int J Cardiol. 2006;117(1):109-14. 2. Rodrigues MA, Bregagnollo EA, Montenegro MR, Tucci PJ. Coronary vascular and myocardial lesions due to experimental constriction of the 6. Mendes OC, Sugizaki MM, Campos DS, Damatto RL, Leopoldo AS, Lima- abdominal aorta. Int J Cardiol. 1992;35(2):253-7. Leopoldo AP, et al. Exercise tolerance in rats with aortic stenosis and ventricular diastolic and/or systolic dysfunction. Arq Bras Cardiol. 2013;100(1):44-51. 3. Okoshi K, Ribeiro HB, Okoshi MP, Matsubara BB, Gonçalves G, Barros R, et al. Improved systolic ventricular function with normal 7. Mendes Ode C, Campos DH, Damatto RL, Sugizaki MM, Padovani CR, Okoshi myocardial mechanism compensated cardiac hypertrophy. Jpn Heart J. K, et al. Remodelamento cardíaco: análise seriada e índices de detecção precoce 2004;45(4):647-53. de disfunção ventricular. Arq Bras Cardiol. 2010;94(1):62-70.

4. Bregagnollo EA, Mestrinel MA, Okoshi K, Carvalho FC, Bregagnollo IF, 8. Litwin SE, Katz SE, Weinberg EO, Lorell HB, Aurigemma GP, Douglas PS. Serial Padovani CR, et al. Relative role of left ventricular geometric remodeling echocardiographic-Doppler assessment of left ventricular geometry and function and of morphological and functional myocardial remodeling in the in rats with pressure overload hypertrophy chronic angiotensin-converting transition from compensated hypertrophy to heart failure in rats with enzyme inhibition attenuates the transition to heart failure. Circulation. supravalvar aortic stenosis. Arq Bras Cardiol. 2007;88(2):225-33. 1995;91(10):2642-54.

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9. Mingoti SA. Análise de dados através de métodos de estatística multivariada: 13. Johnson RA, Wichern DW. Applied multivariate statistical analysis. 6th ed. New uma abordagem aplicada. Belo Horizonte: Editora UFMG; 2007. Jersey: Prentice Hall; 2007.

10. Morrison DF. Multivariate statistical methods. 3rd ed. New York: McGraw Hill, 14. Mahalanobis PC. Historic note on the D2 statistic. Sankhya. 1948;9:237-40. Inc; 2004. 15. The R Core Team. R: a language and environment for statistical computing: 11. Silva NR, Padovani CR. Utilização de componentes principais em reference index. Viena: Foundation for Statistical Computing; 2013. experimentação agronômica. Energ Agric Botucatu. 2006;21(4):98-113.

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103 Arq Bras Cardiol. 2015; 104(2):97-103 Original Article

Reproducibility of Left Ventricular Mass by Echocardiogram in the ELSA-Brasil Alexandre Pereira Tognon1,2, Murilo Foppa1, Vivian Cristine Luft1,7, Lloyd Ellwood Chambless3, Paulo Lotufo4, Lilia Maria Mameri El Aouar5, Luciana Pereira Fernandes6, Bruce Bartholow Duncan1 Programa de Pós-Graduação em Epidemiologia, Faculdade de Medicina, Universidade Federal do Rio Grande do Sul1, Porto Alegre, Rio Grande do Sul; Grupo de Pesquisa Cardiovascular do Hospital São Vicente de Paulo2, Passo Fundo, Rio Grande do Sul; The University of North Carolina. Chapel Hill3, NC, Estados Unidos; Faculdade de Medicina da Universidade de São Paulo4, São Paulo; Universidade Federal do Espírito Santo5, Vitória, Espírito Santo; Universidade Federal da Bahia6, Salvador, Bahia; Centro de Estudos em Alimentação e Nutrição, HCPA/ UFRGS7, Porto Alegre, Rio Grande do Sul, RS - Brazil

Abstract Background: Echocardiography, though non-invasive and having relatively low-cost, presents issues of variability which can limit its use in epidemiological studies. Objectives: To evaluate left ventricular mass reproducibility when assessed at acquisition (online) compared to when assessed at a reading center after electronic transmission (offline) and also when assessed by different readers at the reading center. Method: Echocardiographers from the 6 ELSA-Brasil study investigation centers measured the left ventricular mass online during the acquisition from 124 studies before transmitting to the reading center, where studies were read according to the study protocol. Half of these studies were blindly read by a second reader in the reading center. Results: From the 124 echocardiograms, 5 (4%) were considered not measurable. Among the remaining 119, 72 (61%) were women, mean age was 50.2 ± 7.0 years and 2 had structural myocardial abnormalities. Images were considered to be optimal/ good by the reading center for 110 (92.4%) cases. No significant difference existed between online and offline measurements (1,29 g, CI 95% −3.60-6.19), and the intraclass correlation coefficient between them was 0.79 (CI 95% 0.71-0.85). For images read by two readers, the intraclass correlation coefficient was 0.86 (CI 95% 0.78-0.91). Conclusion: There were no significant drifts between online and offline left ventricular mass measurements, and reproducibility was similar to that described in previous studies. Central quantitative assessment of echocardiographic studies in reading centers, as performed in the ELSA-Brasil study, is feasible and useful in clinical and epidemiological studies performed in our setting. (Arq Bras Cardiol. 2015; 104(2):104-111) Keywords: Hypertrophy Left Ventricular; Echocardiography; Dimensional Measurement Accuracy; Multicenter Studies; Reproducibiity of Results.

Introduction obtaining good-quality images for all individuals and the 5-7 Echocardiography is the most used noninvasive imaging variability among repeated measurements . method in clinical cardiology for functional and structural The assessment of the variability of the left ventricular mass (LVM) evaluation, and it is also employed in clinical and is important to ensure the quality control of echocardiographic epidemiological research1-4. Left ventricular hypertrophy is measurements. In addition, it can be performed in the clinical a condition that can be measured by echocardiography and setting and integrates several measurements acquired during is used in clinical practice as a predictor of cardiovascular examination. Notably, its calculation method may potentially cause events2. Although echocardiography is the cheapest and errors in the LVM estimate with a magnitude of 10E3. most accessible cardiovascular imaging method, its use in To ensure more precise and accurate measurements, the epidemiological studies is limited owing to the difficulty in American Society of Echocardiography defends a centralized reading of echocardiography exams in multicenter trials8. This is recommended because it has been shown that echocardiography exams performed locally have a lower Mailing Address: Alexandre Pereira Tognon • Rua Teixeira Soares 808, Hospital São Vicente de Paulo – Hemodinâmica. prognostic impact than those performed in a central Centro. Postal Code 99010-080. Passo Fundo, RS – Brazil. laboratory9. These recommendations were implemented in the E-mail: [email protected] Brazilian Longitudinal Study of Adult Health (ELSA-Brasil)10, Manuscript received March 05, 2014, revised July 26, 2014, accepted August 25, 2014. which generated an imaging protocol and digital videos corresponding to three cardiac cycles that were stored and DOI: 10.5935/abc.20140183 transmitted to the reading center.

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Digital technology has revolutionized the acquisition, LVM was calculated by making linear measurements of transmission, and storage of exams and aids in the processing the final diastolic diameter and the LV parietal thickness on and use of images. However, even if these technological the two-dimensional mode and M-mode. For this purpose, advances are incorporated into clinical practice, their impact in the formula and the definition criteria of the leading measurement reading variability in the research environment edge technique proposed by the American Society of remains unknown. Therefore, the objective of this study Echocardiography were used, as follows12: was to assess the reproducibility of echocardiographic LVM = 0.8 × [1.04 (LVIDD + PWTD + IVSTD)³ − measurements that allow the estimation of LVM by comparing (LVIDD)³] + 0.6 g, values obtained locally (online, in the acquisition centers) where LVM: left ventricular mass; LVIDD: left ventricular with those obtained in the reading centers (offline) and with internal diameter in diastole; PWTD: posterior wall thickness in duplicate measurements performed by different readers at diastole; and IVSTD: interventricular septum thickness in diastole. the reading center. Furthermore, the study aimed to assess the reproducibility of the measurements of the left atrium For other measurements and Doppler studies, we used the 10 and aorta and those obtained by transmitral flow Doppler parameters defined in the ELSA-Brasil protocol . and mitral annular tissue Doppler imaging. Statistical Analysis Methods Statistical analysis was performed with SPSS 17.0 software for Windows. The categorical variables were expressed as the ELSA-Brasil is a prospective multicenter trial involving absolute and relative frequency and the numeric variables as 15,105 voluntary participants aged between 35 and means ± standard deviations. 74 years and consisting of the staff of public universities and The differences between echocardiographic measurements research institutes. The goal of ELSA-Brasil was to investigate 13 the epidemiological, clinical, and molecular aspects of were represented using Bland–Altman plots and tested by nontransmissible chronic diseases, particularly cardiovascular variance analysis. Measurement variability was described diseases and diabetes10. Data were collected between using the intraclass correlation coefficient (ICC), also known August 2008 and December 2010, and approximately as the reliability coefficient. ICC was calculated from the estimated variance components in models that considered 10,000 participants were subjected to an echocardiographic the following to determine the reproducibility among the exam as the standard procedure. The ELSA-Brasil protocol readers of the reading center: 1) the effect of the reading was approved by the Research Ethics Committees of all center versus the acquisition center as the fixed variable and institutions involved, and all the participants signed an the effect of individuals on which these measurements were informed consent form. performed as the random variable and 2) the effect of the All the tests were performed at the investigation centers first read at the reading center in relation to the second read using the same model equipment (Toshiba Aplio XG) and by at the reading center as the fixed variable and the effect of well-trained echocardiographists who were later certified individuals on which these measurements were performed by the reading center involved in the study. The ELSA‑Brasil as the random variable. protocol followed the methods recommended by the The mean differences and ICCs were calculated using 95% European and North American Echocardiography Societies8,11. confidence intervals (CIs). We selected the sequences of three consecutive heartbeats in each echocardiographic window that best represented existing A sample size of 100 exams was estimated to obtain 90% findings and recorded them in a standard digital format for potency and identify a mean difference of 10 g in LVM among medical imaging (DICOM). acquisition measurements and reading center measurements, with a significance level of 0.055. For convenience purposes For analysis of reproducibility, in addition to image and to compensate for losses, 124 individuals were selected for acquisition according to the established protocol, the the reproducibility study, representing at least 20 individuals of echocardiographists of the investigation center performed each of the six investigation centers of the ELSA-Brasil study. echocardiographic measurements during image acquisition, Between July and November 2009, each center consecutively according to current practice (online measurements). included its participants until the local target was reached, Subsequently, the data files were sent to the reading following the visit schedule of ELSA. center, together with a completed form with an evaluation of image quality and online measurements. At the reading center, the images were analyzed in a ComPACS 10.5 Results workstation (Medimatic SrL, Italy). The offline measurements Among the selected participants, five exams were considered were performed at the reading center by another skilled to be nonmeasurable (4%). Of the 119 participants, 72 (61%) echocardiographist (reader) blinded to previous readings. were female, with a mean age of 50.2 ± 7.0 years (minimum The offline readings involved a second qualitative assessment and maximum of 35 and 68 years, respectively), mean height of as well as all echocardiographic measurements using 1.64 ± 0.09 m (minimum and maximum of 1.44 and 1.83 m, procedures similar to those adopted in the investigation respectively), and mean body mass index of 26.6 ± 4.0 kg/m² center. A few exams (n = 68) were read again by a second (minimum and maximum of 19.2 and 36.9 kg/m², respectively). reader at the reading center blinded to the initial online Of the participating individuals, 54 (45.3%) were of Caucasian assessment and to the initial offline assessment. ethnicity, 32 (26.9%) were of Middle Eastern/South Asian

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ethnicity, 31 (26.1%) were of African ethnicity, and two (1.7%) No clinically significant differences were found among the were of Eastern/Southeast Asian ethnicity. Only two individuals means of measurements obtained at the investigation center were diagnosed with cardiomyopathy. (online) and those obtained at the reading center (offline) for The quality of parasternal longitudinal images of 115 (96.6%) most measurements (Table 1). tests and 106 (92.4%) tests was considered to be excellent/ Figure 1 shows the differences between values obtained good by the acquisition center and reading center, respectively. at the acquisition centers and those obtained at the reading Agreement was found for 106 (89.1%) quality evaluations. center on the basis of the mean LVM readings, estimated

Table 1 – Echocardiographic measurements obtained at the acquisition and reading centers and respective reproducibility measurements

Values Reproducibility measurement Measurement n Acquisition center Reading center Mean difference (95% CI) ICC (95% CI)

Two-dimensional mode Aortic root diameter (cm) 117 3.00 ± 0.45 2.98 ± 0.38 0.02 (−0.03-0.08) 0.75 (0.66-0.82) LA diameter (cm) 118 3.40 ± 0.54 3.50 ± 0.46 −0.10 (−0.18-0.02) 0.63 (0.51-0.73) Septal thickness(cm) 119 0.88 ± 0.17 0.87 ± 0.17 0.00 (−0.03-0.03) 0.56 (0.42-0.67) LV posterior wall thickness (cm) 119 0.84 ± 0.16 0.86 ± 0.15 −0.02 (−0.05-0.01) 0.56 (0.43-0.68) Diastolic LV diameter (cm) 119 4.58 ± 0.50 4.52 ± 0.47 0.06 (0.00-0.11) 0.79 (0.71-0.85) Systolic LV diameter (cm) 118 2.92 ± 0.46 2.82 ± 0.43 0.10 (0.03-0.17) 0.67 (0.55-0.76) LV mass 119 131.29 ± 42.98 129.99 ± 40.79 1.29 (−3.60-6.19) 0.79 (0.72-0.86) LA area (cm²) 101 16.33 ± 3.85 16.63 ± 3.68 −0.30 (−0.97-0.37) 0.59 (0.45-0.71) LV ejection fraction 118 65.08 ± 10.87 67.31 ± 8.95 −2.22 (−4.29-0.16) 0.35 (0.19-0.50)

M-mode Aortic root diameter (cm) 100 2.99 ± 0.40 2.98 ± 0.42 0.01 (−0.03-0.05) 0.89 (0.84-0.92) LA diameter (cm) 100 3.55 ± 0.46 3.68 ± 0.46 −0.13 (−0.18-0.08) 0.86 (0.80-0.90) Septal thickness(cm) 86 0.88 ± 0.18 0.87 ± 0.19 0.01 (−0.02-0.04) 0.67 (0.53-0.77) LV posterior wall thickness (cm) 86 0.84 ± 0.16 0.85 ± 0.17 −0.01 (−0.04-0.02) 0.60 (0.44-0.72) Diastolic LV diameter (cm) 86 4.72 ± 0.57 4.71 ± 0.56 0.01 (−0.04-0.06) 0.92 (0.88-0.95) Systolic LV diameter (cm) 86 2.85 ± 0.54 2.82 ± 0.58 0.03 (−0.02-0.08) 0.92 (0.88-0.95) LV mass 86 139.52 ± 55.18 138.87 ± 53.05 0.66 (−4.89-6.20) 0.89 (0.83-0.92) LV ejection fraction 86 69.98 ± 7.62 70.33 ± 9.18 −0.35 (−1.59-0.89) 0.77 (0.66-0.84)

Mitral Doppler E wave 119 0.72 ± 0.17 0.72 ± 0.15 0.01 (−0.01-0.02) 0.88 (0.83-0.92) Deceleration time 117 217.50 ± 45.55 226.35 ± 42.31 −8.86 (−16.22-1.49) 0.58 (0.45-0.69) A wave 119 0.59 ± 0.16 0.60 ± 0.15 −0.01 (−0.02-0.01) 0.90 (0.86-0.93) E/A ratio 119 1.30 ± 0.45 1.26 ± 0.41 0.04 (0.01-0.07) 0.93 (0.90-0.95)

Medial mitral annular TDI s’ velocity 111 7.56 ± 1.34 7.49 ± 1.14 0.07 (−0.13-0.26) 0.65 (0.52-0.74) e’ velocity 112 9.86 ± 2.74 9.92 ± 2.61 −0.06 (−0.34-0.23) 0.84 (0.78-0.89) a’ velocity 113 9.71 ± 2.36 9.63 ± 1.74 0.07 (−0.28-0.43) 0.57 (0.43-0.68)

Lateral mitral annular TDI s’ velocity 111 8.38 ± 2.06 8.35 ± 1.82 0.03 (−0.31-0.37) 0.57 (0.43-0.68) e’ velocity 113 11.98 ± 2.63 11.97 ± 2.55 0.01 (−0.23-0.26) 0.87 (0.82-0.91) a’ velocity 113 9.14 ± 2.57 9.23 ± 2.51 −0.09 (−0.25-0.06) 0.95 (0.92-0.96) ICC: intraclass correlation coefficient; difference: value obtained at the reading center − value obtained at the acquisition center; LV: left ventricle; LA: left atrium; TDI: tissue Doppler imaging. The values obtained at the acquisition and reading centers are expressed as the mean ± standard deviation.

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1.5 0.3

0.2 1.0 0.1

0.5 0.0

-0.1 0.0

-0.2

Difference in posterior wall (cm) Difference in LV diameter (cm) Difference in LV -0.5 -0.3

-1.0 -0.4

3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 Mean LV diameter (cm) Mean posterior wall (cm)

0.5 150 0.4

0.3 100 0.2

0.1 50 0.0

-0.1 0 -0.2

Difference in LV mass (g) Difference in LV

Difference in IV septum (cm) -0.3 -50 -0.4 -0.5 -100

0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 050 100 150 200 250 300 350 400 Mean IV septum (g) Mean LV mass (g)

Figure 1 – Differences between the measurements from which LVM was estimated, performed from images obtained on the two-dimensional mode in the acquisition and reading centers (y axis) and the mean of both measurements (x axis). A: Left ventricular diameter; B: left ventricular posterior wall thickness; C: septal thickness; D: left ventricular mass. The solid line indicates the mean difference between both measurements, and the dashed line indicates the mean ± two standard deviations. using two-dimensional measurements (Bland–Altman plots). For the remaining assessments, the distribution of the There was no notable tendency among online and offline differences was of the same magnitude, as well as the measurements. In 95% samples, the differences were less distribution of the ICC values (Table 1). than 0.7 cm, 0.3 cm, and 0.4 cm for the LV diastolic diameter, Table 2 shows the measurements obtained by different septum thickness, and posterior wall thickness, respectively. readers at the reading center. Although significant differences LVM was calculated from these three variables and showed a were observed between measurements, their magnitude was discrepancy of 54 g or lower in 95% of samples. There were relatively low from a clinical point of view. no association trends between magnitude and amplitude Figure 2 shows the measurement differences obtained in both differences in these variables. readings, according to the mean LV values. The LVM difference The exclusion of the images that the reading center did not surpass 58 g in 95% samples. There were no association considered to be of regular quality (n = 9) did not change trends between magnitude and amplitude differences in these the mean differences of the following parameters measured variables. After the exclusion of regular-quality exams (n = 8), online and offline: LV diameter (0.07 cm, 95% CI: 0.01 to the results were quite similar in terms of the mean differences 0.13), posterior LV wall thickness (−0.02 cm, 95% CI: −0.05 to between both offline readings of LV diameter (0.04 cm, 0.01), interventricular septum thickness (0 cm, 95% CI: −0.03 95% CI: −0.02 to 0.10), posterior LV wall thickness (−0.08 cm, to 0.04), and LVM estimate (2.27 g, 95% CI: −2.77 to 7.31). 95% CI: −0.11 to −0.04), interventricular septum thickness Moreover, the results were quite similar after the exclusion in (−0.07 cm, 95% CI: −0.10 to −0.04), and LVM estimate relation to ICC (0.80 for LV diameter, 0.48 for posterior LV wall (−13.2 g, 95% CI: −18.5 to −7.96) and the respective ICC values thickness, 0.52 for interventricular septum thickness, and 0.80 (0.89 for LV diameter, 0.57 for posterior LV wall thickness, 0.79 for LVM estimate). for interventricular septum thickness, and 0.90 for LVM estimate).

Arq Bras Cardiol. 2015; 104(2):104-111 107 Tognon et al. Reproducibility of echocardiography in ELSA-Brasil

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Table 2 – Echocardiographic measurements obtained at the reading center, repeated in different moments by different readers

Values Reproducibility measurement

Measurement First reading at the Second reading at the n Mean difference (95% CI) ICC (95% CI) reading center reading center

Measurement Aortic root diameter (cm) 63 3.02 ± 0.37 3.02 ± 0.37 0.00 (−0.07-0.07) 0.74 (0.61-0.84) LA diameter (cm) 66 3.58 ± 0.43 3.63 ± 0.43 −0.05 (-0.11-0.01) 0.84 (0.75-0.90) Septal thickness(cm) 66 0.87 ± 0.19 0.94 ± 0.18 -0.07 (−0.09-0.04) 0.84 (0.75-0.90) LV posterior wall thickness (cm) 66 0.87 ± 0.17 0.93 ± 0.15 −0.06 (−0.10-0.02) 0.53 (0.33-0.68) Diastolic LV diameter (cm) 66 4.60 ± 0.47 4.55 ± 0.50 0.05 (−0.01-0.11) 0.88 (0.82-0.93) Systolic LV diameter (cm) 66 2.85 ± 0.50 2.90 ± 0.52 −0.05 (−0.10-0.00) 0.92 (0.86-0.95) LV mass 66 134.80 ± 46.22 145.30 ± 43.66 −10.50 (−16.40-4.60) 0.86 (0.78-0.91) LA area (cm²) 59 16.80 ± 4.03 18.07 ± 3.19 −1.27 (−1.94-0.60) 0.75 (0.61-0.84) LV ejection fraction 66 68.04 ± 8.97 65.79 ± 9.44 2.25 (0.76-3.75) 0.78 (0.67-0.86)

M-mode Aortic root diameter (cm) 57 3.00 ± 0.40 2.98 ± 0.40 0.02 (−0.03-0.07) 0.88 (0.80-0.93) LA diameter (cm) 57 3.73 ± 0.47 3.77 ± 0.47 −0.04 (−0.10-0.01) 0.89 (0.83-0.94) Septal thickness(cm) 35 0.86 ± 0.14 1.04 ± 0.18 −0.18 (−0.24-0.13) 0.54 (0.26-0.74) LV posterior wall thickness (cm) 35 0.83 ± 0.16 1.01 ± 0.19 −0.18 (−0.22-0.13) 0.74 (0.55-0.70) Diastolic LV diameter (cm) 35 4.90 ± 0.60 4.79 ± 0.63 0.11 (0.03-0.18) 0.93 (0.87-0.97) Systolic LV diameter (cm) 35 2.99 ± 0.77 3.12 ± 0.65 −0.13 (−0.22-0.04) 0.88 (0.88-0.97) LV mass 35 143.42 ± 41.58 178.04 ± 47.58 −34.61 (-42.89-26.34) 0.86 (0.73-0.92) LV ejection fraction 35 68.81 ± 11.93 63.92 ± 9.26 4.89 (2.20-7.58) 0.73 (0.53-0.86)

Mitral Doppler E wave 66 0.72 ± 0.15 0.72 ± 0.16 0.00 (−0.02-0.02) 0.91 (0.86-0.94) Deceleration time 66 222.59 ± 44.18 214.03 ± 35.73 8.56 (0.42-16.69) 0.66 (0.50-0.78) A wave 66 0.58 ± 0.15 0.58 ± 0.15 0.01 (−0.01-0.02) 0.92 (0.87-0.95) E/A ration 66 1.31 ± 0.41 1.32 ± 0.42 −0.02 (−0.03-0.00) 0.99 (0.98-0.99)

Medial mitral annular TDI s’ velocity 64 7.69 ± 1.13 7.74 ± 1.18 −0.06 (−0.31-0.19) 0.62 (0.45-0.75) e’ velocity 64 10.09 ± 2.49 9.70 ± 2.54 0.39 (0.03-0.76) 0.83 (0.74-0.89) a’ velocity 64 9.49 ± 1.61 9.33 ± 1.55 0.16 (0.00-0.31) 0.92 (0.88-0.95)

Lateral mitral annular TDI s’ velocity 64 8.50 ± 1.74 9.11 ± 1.83 −0.61 (−1.01-0.20) 0.59 (0.40-0.73) e’ velocity 64 12.41 ± 2.36 12.36 ± 2.36 0.05 (−0.16-0.27) 0.93 (0.89-0.96) a’ velocity 63 9.42 ± 2.37 9.22 ± 2.18 0.20 (0.00-0.40) 0.94 (0.90-0.96) ICC: intraclass correlation coefficient; difference: value obtained at the reading center − value obtained at the acquisition center; LV: left ventricle; LA: left atrium; TDI: tissue Doppler imaging. The values obtained at the acquisition and reading centers are expressed as the mean ± standard deviation.

In addition to LV measurements using the two-dimensional measurements but also for measurements performed by mode, we investigated the reproducibility of other distinct readers of the reading center (Tables 1 and 2). measurements performed as part of the echocardiography protocol of ELSA-Brasil. We observed CCI values in the same range for measurements obtained using the M-Mode and Discussion transmitral flow Doppler and mitral annular tissue Doppler In this study, the differences observed between online imaging. This was true not only for offline and online and offline measurements were practically null. The intraclass

108 Arq Bras Cardiol. 2015; 104(2):104-111 Tognon et al. Reproducibility of echocardiography in ELSA-Brasil

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0.6 0.5 0.4 0.5 0.3 0.2 0.1 0.0 0.0 -0.1 -0.2 -0.3

Difference in posterior wall (cm)

Difference in LV diameter (cm) Difference in LV -0.4

-0.5 -0.5 -0.6

3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 Mean LV diameter (cm) Mean posterior wall (cm)

0.3 100

0.2

50 0.1

0.0 0 -0.1

-0.2 mass (g) Difference in LV Difference in IV septum (cm) -50

-0.3

-0.4 -100

0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 50 100 150 200 250 300 350 400 Mean IV septum (cm) Mean LV mass (g)

Figure 2 – Differences between the measurements from which LVM was estimated, performed by different readers at the reading center (y axis) from images obtained on the two-dimensional mode, and the mean of both measurements (x axis). A: Left ventricular diameter; B: left ventricular posterior wall thickness; C: septal thickness; D: left ventricular mass. The solid line indicates the mean difference between both measurements, and the dashed line indicates the mean ± two standard deviations. correlation coefficient, used for reproducibility assessment, was With regard to other assessed echocardiographic considered to be satisfactory for the LVM estimates obtained in the measurements, we also observed small differences among acquisition and reading centers (ICC = 0.79). For most tests, the the readers, and the reproducibility estimates were better online and offline LVM estimates did not surpass 50 g. In a similar when both the assessments were performed at the reading comparative study involving 274 hypertensive individuals14, ICC center. In general, these values were similar and had the same was not reported for the comparison of measurements performed result patterns for the LVM findings. A similar performance in the acquisition centers and the reading center; however, the was observed in other studies assessing the reproducibility of Pearson correlation was 0.76 and the mean difference was echocardiographic measurements5,6,15. 8 ± 20 g (p < 0.001), and this discrepancy was similar to that Although the reading center was apparently more obtained in the present study for most individuals. demanding in the assessment of image quality than the With regard to reproducibility of measurements performed investigation center (92% versus 97% for images considered at the reading center by different readers, despite the existence to have excellent/good quality), the proportion of exams of differences between the mean LVM estimates (−10.50 g, considered to be nonmeasureable was much lower than that CI 95%: −16.40 to −4.60), the magnitude of this difference observed in other population studies. In the Framingham Heart was relatively small from the clinical point of view, and ICC Study, only 28% of the echocardiography exams performed of both estimates was quite satisfactory (ICC = 0.86). In a during the first 5 months were considered to be acceptable, previous report,14 the agreement between the readers of reaching an acceptability of 74% to 81% in 2 years16. In the the reading center was only calculated for 10 high-quality Atherosclerosis Risk in Communities (ARIC) study, despite the echocardiography exams after repeated measurements, and fact that the echocardiogram was acquired in one study center, it was considered to be very satisfactory (ICC = 0.96 to 0.99, only 70% of the exams were considered to be appropriate for depending on the reader). estimating LVM1,7. This phenomenon may explain the variability

Arq Bras Cardiol. 2015; 104(2):104-111 109 Tognon et al. Reproducibility of echocardiography in ELSA-Brasil

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in our study, and we can assume that the echocardiographist Several variability sources persisted in the present study, of the investigation center caused more interference in the even in the exams performed with current technology and measurements owing to the larger sample size when performing guided by a research protocol. This may indicate the need the exams. In contrast, it is possible that the image quality for an increased sample size to identify LVM differences assessment was too permissive. Considering the increased among population and/or therapeutic interventions. number of images whose measurement was difficult in The strategy of performing echocardiography measurements comparison with that in other studies, the technological benefits in reading centers (offline), as used in ELSA-Brasil, is feasible may have not been sufficient to compensate for the inadequate and can be important in clinical and epidemiological studies. image quality. This is also true for institutes where exams are analyzed by the echocardiographist during image acquisition (online). Study Limitations Among the limitations of the study, the first noteworthy Funding aspect is that the reproducibility was not evaluated in The ELSA-Brasil study was funded by the Health Ministry all investigation centers; therefore, it is not possible to (Department of Science and Technology), the Ministry of infer whether the measurements repeated at the reading Science and Technology (FINEP– Funding Authority for Studies center are more reproducible than those repeated at each and Projects, and the National Council for Scientific Research), acquisition center. Potential difficulties in the standardization process numbers: 01 06 0010.00 RS, 01 06 0212.00BA, of acquisition readings in different centers are one of the 01 06 0300.00 ES, 01 06 0278.00 MG, 01 06 0115.00SP, main reasons to perform all measurements in a single and 01 06 0071.00 RJ. center. The reproducibility of offline measurements was only determined in 50% of the samples. This percentage is significant in comparison with that observed previously14, and Acknowledgments it seems to be appropriate because there are fewer sources We are grateful to the participants of the ELSA-Brasil study of variability in these comparisons. and the researchers who made their achievement possible. Another limitation of the study is that data collected for the evaluation of reproducibility were from the initial phases, Author contributions during the learning curve, as observed in the Framingham Heart Study16. This may have caused systematic temporal Conception and design of the research: Tognon AP, Foppa drifts, consequently influencing the results, corroborating the M, Duncan BB; Acquisition of data: Foppa M, El Aoua LMM, need for permanent quality control protocols in ELSA-Brasil Fernandes LP; Analysis and interpretation of the data: Tognon and subsequent studies, and it may help implement additional AP, Luft VC, Chambless L; Statistical analysis: Tognon AP, reading control measures that are effective for most exams that Chambless L; Obtaining financing: Lotufo P, Duncan BB; Writing were included and read in the present study. of the manuscript: Tognon AP, Foppa M; Critical revision of the manuscript for intellectual content: Luft VC, Chambless L, ICC of the LVM measurement obtained on the M-mode Lotufo P, El Aoua LMM, Fernandes LP, Duncan BB. (86 exams, 69.4%) was higher than that obtained on the two‑dimensional mode (119 exams, 96.0%). These findings suggest that exams using poor-quality images are less Potential Conflict of Interest reproducible; however, when these were excluded, there was No potential conflict of interest relevant to this article was no significant improvement in reproducibility. We focused reported. our conclusions on the two-dimensional measurements because they best reflect current practices. Moreover, higher reproducibility does not necessarily imply increased accuracy Sources of Funding because of the systematic errors inherent to the M-mode17. This study was funded by Ministério da Saúde (Departamento de Ciência e Tecnologia), Ministério da Ciência e Tecnologia (Financiadora de Estudos e Projetos e Conclusion Conselho Nacional de Pesquisa). There were no relevant systematic differences in the offline and online LVM echocardiographic measurements. The reproducibility measurements were similar to those Study Association found in previous studies, despite the fact that these studies This article is part of the thesis of master submitted by avoided the exclusion of a high percentage of images. Alexandre Pereira Tognon, from Alexandre Pereira Tognon.

110 Arq Bras Cardiol. 2015; 104(2):104-111 Tognon et al. Reproducibility of echocardiography in ELSA-Brasil

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1. Burchfiel CM, Skelton TN, Andrew ME, Garrison RJ, Arnett DK, Jones DW, of Echocardiography Standards for echocardiography core laboratories: et al. Metabolic syndrome and echocardiographic left ventricular mass in endorsed by the American College of Cardiology Foundation. J Am Soc blacks: the Atherosclerosis Risk in Communities (ARIC) Study. Circulation. Echocardiogr. 2009;22(7):755-65. 2005;112(6):819-27. 9. Hole T, Otterstad JE, St John Sutton M, Froland G, Holme I, Skjaerpe T. 2. Levy D, Garrison RJ, Savage DD, Kannel WB, Castelli WP. Prognostic Differences between echocardiographic measurements of left ventricular implications of echocardiographically determined left ventricular mass in dimensions and function by local investigators and a core laboratory in a the Framingham Heart Study. N Engl J Med. 1990;322(22):1561-6. 2-year follow-up study of patients with an acute myocardial infarction. Eur J Echocardiogr. 2002;3(4):263-70. 3. Gardin JM, Wagenknecht LE, Anton-Culver H, Flack J, Gidding S, Kurosaki T, et al. Relationship of cardiovascular risk factors to echocardiographic left 10. Aquino EM, Barreto SM, Bensenor IM, Carvalho MS, Chor D, Duncan BB, ventricular mass in healthy young black and white adult men and women. et al. Brazilian Longitudinal Study of Adult Health (ELSA-Brasil): objectives The CARDIA study. Coronary Artery Risk Development in Young Adults. and design. Am J Epidemiol. 2012; 175(4):315-24. Circulation. 1995;92(3):380-7. 11. Lang RM, Bierig M, Devereux RB, Flachskampf FA, Foster E, Pellikka PA 4. Gardin JM, McClelland R, Kitzman D, Lima JA, Bommer W, Klopfenstein HS, et al. et al. Recommendations for chamber quantification. Eur J Echocardiogr. M-mode echocardiographic predictors of six- to seven-year incidence of coronary 2006;7(2):79-108. heart disease, stroke, congestive heart failure, and mortality in an elderly cohort (the Cardiovascular Health Study). Am J Cardiol. 2001;87(9):1051-7. 12. Gottdiener JS, Bednarz J, Devereux R, Gardin J, Klein A, Manning WJ, et al. American Society of Echocardiography recommendations for use of echocardiography in 5. De Simone G, Muiesan ML, Ganau A, Longhini C, Verdecchia P, Palmieri V, clinical trials. J Am Soc Echocardiogr. 2004;17(10):1086-119. et al. Reliability and limitations of echocardiographic measurement of left ventricular mass for risk stratification and follow-up in single patients: the 13. Bland JM, Altman DG. Statistical methods for assessing agreement between RES trial. Working Group on Heart and Hypertension of the Italian Society two methods of clinical measurement. Lancet. 1986;1(8476):307-10. of Hypertension. Reliability of M-mode Echocardiographic Studies. J Hypertens. 1999;17(12Pt2):1955-63. 14. Gosse P, Guez D, Gueret P, Dubourg O, Beauchet A, de Cordoue A et al. Centralized echocardiogram quality control in a multicenter study of regression 6. Gottdiener JS, Livengood SV, Meyer PS, Chase GA. Should echocardiography of left ventricular hypertrophy in hypertension. J Hypertens. 1998;16(4):531-5. be performed to assess effects of antihypertensive therapy? Test-retest reliability of echocardiography for measurement of left ventricular mass and 15. Ogah OS, Adebanjo AT, Otukoya AS, Jagusa TJ. Echocardiography in Nigeria: use, function. J Am Coll Cardiol. 1995;25(2):424-30. problems, reproducibility and potentials. Cardiovasc Ultrasound. 2006;4:13.

7. Skelton TN, Andrew ME, Arnett DK, Burchfiel CM, Garrison RJ, Samdarshi 16. Savage DD, Garrison RJ, Kannel WB, Anderson SJ, Feinleib M, Castelli TE, et al. Echocardiographic left ventricular mass in African-Americans: WP. Considerations in the use of echocardiography in epidemiology. The the Jackson cohort of the Atherosclerosis Risk in Communities Study. Framingham Study. Hypertension. 1987; 9(2 Pt 2):II40-4. Echocardiography. 2003;20(2):111-20. 17. Foppa M, Duncan BB, Rohde LE. Echocardiography-based left ventricular 8. Douglas PS, DeCara JM, Devereux RB, Duckworth S, Gardin JM, Jaber mass estimation. How should we define hypertrophy? Cardiovasc WA, et al. Echocardiographic imaging in clinical trials: American Society Ultrasound. 2005;3:17.

Arq Bras Cardiol. 2015; 104(2):104-111 111 Original Article

Relationship between Neutrophil-To-Lymphocyte Ratio and Electrocardiographic Ischemia Grade in STEMI Emre Yalcinkaya1, Uygar Cagdas Yuksel2, Murat Celik2, Hasan Kutsi Kabul2, Cem Barcin2, Yalcin Gokoglan2, Erkan Yildirim2, Atila Iyisoy2 Aksaz Military Hospital - Department of Cardiology1; Gulhane Military Medical Academy - School of Medicine - Department of Cardiology2

Abstract Background: Neutrophil-to-lymphocyte ratio (NLR) has been found to be a good predictor of future adverse cardiovascular outcomes in patients with ST-segment elevation myocardial infarction (STEMI). Changes in the QRS terminal portion have also been associated with adverse outcomes following STEMI. Objectives: To investigate the relationship between ECG ischemia grade and NLR in patients presenting with STEMI, in order to determine additional conventional risk factors for early risk stratification. Methods: Patients with STEMI were investigated. The grade of ischemia was analyzed from the ECG performed on admission. White blood cells and subtypes were measured as part of the automated complete blood count (CBC) analysis. Patients were classified into two groups according to the ischemia grade presented on the admission ECG, as grade 2 ischemia (G2I) and grade 3 ischemia (G3I). Results: Patients with G3I had significantly lower mean left ventricular ejection fraction than those in G2I (44.58 ± 7.23 vs. 48.44 ± 7.61, p = 0.001). As expected, in-hospital mortality rate increased proportionally with the increase in ischemia grade (p = 0.036). There were significant differences in percentage of lymphocytes (p = 0.010) and percentage of neutrophils (p = 0.004), and therefore, NLR was significantly different between G2I and G3I patients (p < 0.001). Multivariate logistic regression analysis revealed that only NLR was the independent variable with a significant effect on ECG ischemia grade (odds ratio = 1.254, 95% confidence interval 1.120–1.403, p < 0.001). Conclusions: We found an association between G3I and elevated NLR in patients with STEMI. We believe that such an association might provide an additional prognostic value for risk stratification in patients with STEMI when combined with standardized risk scores. (Arq Bras Cardiol. 2015; 104(2):112-119) Keywords: ST elevation myocardial infarction; neutrophil-to-lymphocyte ratio; electrocardiographically grade 3 ischemia.

Introduction The neutrophil-to-lymphocyte ratio (NLR), a combination of two independent markers of inflammation, is regarded as a Early risk stratification is recommended in daily clinical simple, non-specific marker of inflammation1. In this context, practice to predict the infarct size, success of epicardial the NLR has been evaluated in various studies of coronary recanalization and risk of adverse outcomes in patients artery disease (CAD), especially STEMI, and emerged as a new, presenting with ST-segment elevation myocardial infarction inexpensive risk assessment tool for patients with STEMI prior (STEMI). In addition to the current risk scores, interest has to revascularization2-5. Additionally, a number of parameters on recently been directed to the development of a set of bedside the presenting electrocardiogram (ECG) have previously been tools obtained from admission data to promptly predict the associated with adverse outcomes following STEMI6. One of prognosis of arterial recanalization. Electrocardiographic and these parameters is the grade of ischemia, which can be readily biochemical parameters are the mostly studied admission determined by the presence of distortion of the terminal portion parameters to define the prognosis of patients with STEMI. of the QRS complex on admission ECG without requiring any 7 Complete blood count (CBC) analysis is routinely performed measurement . Increased ischemia grade has been found in association with failure of myocardial reperfusion independent on admission and carries important prognostic information. of other key predictors of outcomes in STEMI patients8. Although both NLR and ischemia grade have been shown to be useful in predicting adverse outcomes in patients with STEMI, Mailing Address: Emre Yalcinkaya • their mechanisms are unclear and therefore, NLR and ischemia Aksaz Asker Hastanesi Kardiyoloji Bolumu Marmaris - Mugla. 48750 – Turkey grade are not integrated in any risk scoring system. Email: [email protected] Manuscript received May 30, 2014; revised August 24, 2014; accepted We hypothesized that the prognostic value of leukocyte August 25, 2014. subtypes may at least be partially linked to a possible association with ECG ischemia grades in STEMI. In our study, DOI: 10.5935/abc.20140179 we sought to identify available clinical and ECG characteristics

112 Yalcinkaya et al. NLR and Ischemia Grade in STEMI

Original Article on admission that might predict myocardial reperfusion and blinded to the patients’ angiographic and clinical data analyzed investigate whether NLR is related to ischemia grade on the the ECGs. After excluding patients with G1I, the remaining were admission ECG in patients with STEMI. classified into G2I and G3I according to their ischemia grade.

Methods Biochemical analysis Blood samples were drawn from the antecubital vein Patients within 1 hour of admission. White blood cells (WBC) and subtypes were measured as part of the automated CBC analysis We investigated 253 patients who underwent primary before pPCI and prior to starting any medication to prevent percutaneous coronary intervention (pPCI) between February interference with the results. The NLR was automatically 2011 and June 2013 after diagnosis of STEMI. The diagnosis computed as the neutrophils to lymphocytes ratio, both of myocardial infarction (MI) was determined by the obtained from the same blood sample. Other biochemical occurrence of classic symptoms of coronary ischemia within measurements including cardiac biomarkers, renal function, 12 hours, elevation in cardiac biomarkers and detection of electrolytes and lipid panel were measured using standard ST-segment elevation in two contiguous leads, as well defined laboratory methods. by the guidelines of the American College of Cardiology and the European Society of Cardiology9,10. Clinical and demographic characteristics were obtained from patients’ Angiographic analysis medical records retrieved from the computerized hospital After an administration of bolus injection of intravenous database. The following variables were retrospectively heparin (70 U/kg), a coronary angiography was performed using analyzed: age, cardiovascular risk factors (hypertension, the Judkins technique as immediately as possible. The decision on smoking, diabetes mellitus and hypercholesterolemia), the type of coronary revascularization (pPCI or urgent coronary cardiovascular history (prior coronary revascularization, artery bypass grafting) was established according to current congestive heart failure and MI), and relevant family history guidelines9,10. The pPCI only targeted the infarct-related artery of CAD, as well as systolic and diastolic blood pressures (IRA). Direct stenting was performed whenever possible, while and heart rate on admission. Exclusion criteria included the remaining cases were managed with balloon predilatation. clinical evidence of active infection, systemic inflammatory A pPCI was considered successful when associated with disease, hematological disease, end-stage liver and kidney angiographic success and relief of presenting symptoms, without diseases, systemic autoimmune disease, known malignancy, adverse procedural results (coronary dissection, no-reflow, isolated posterior MI, presence of left bundle branch block, coronary emboli, residual thrombus or > 50% residual stenosis) paced / ventricular rhythm or grade 1 ischemia (G1I) on the or major adverse outcome (stroke, emergent coronary artery presenting ECG, lack of laboratory data and unavailable ECG bypass graft or death). Stented patients received adjunctive dual records. A total of 50 patients who met the exclusion criteria antiplatelet therapy with clopidogrel and aspirin. The selection were not considered for the study. The final cohort analyzed criterion for drug-eluting or bare‑metal stent and the use of consisted of 203 patients. The local ethics committee of our platelet glycoprotein IIb/IIIa receptor antagonists were left to the institute approved the study protocol. operator’s discretion.

Electrocardiographic analysis Statistical analysis On admission, 12 lead ECGs were immediately obtained The data were tested for normal distributions using the at 25 mm/sec paper speed, and 10 mV gain. STEMI was Kolmogorov–Smirnov test. Continuous variables were presented determined by the occurrence of ST segment elevation, of as mean ± standard deviation (SD) and categorical variables 0.2 mV measured at the J point in leads V1-V3 or 0.1 mV on at as percentages. Independent samples t test and chi-square least two contiguous leads of the remaining leads. The first ECG test were used to compare quantitative and categorical data, obtained after pPCI was analyzed for ST segment resolution (STR). respectively, between groups. Univariate correlation with NLR As recommended by guidelines9,10, we evaluated the percentage was performed with Spearman’s and Pearson’s correlation difference between the sum of the ST-segment elevation on coefficients. Following univariate correlations, a multivariate the ECGs performed on admission and after the procedure. linear regression model with a backward selection process was We defined as a complete STR the finding of a resolution applied to identify independent predictors of NLR. The effects ≥ 70%, as partial STR a resolution between 30% and 69%, and of various variables on ECG ischemia grade were evaluated as having no STR a resolution < 30%. The grade of ischemia with backward stepwise multivariate logistic regression analysis. was analyzed on the admission ECG. A G1I was defined as Differences were considered statistically significant when the the presence of symmetrical, tall and peaked T waves, grade 2 p value was < 0.05. The Statistical Package for Social Sciences ischemia (G2I) as ST elevation without distortion of the terminal (SPSS, Chicago, Illinois, USA) version 20 was used for all portion of the QRS complex (J-point elevation > 1.0 mm but calculations and statistical analyses. < 50% of the R-wave amplitude) and grade 3 ischemia (G3I) as ST elevation with distortion of the terminal portion of the QRS complex (ST–J-point amplitude > 50% of the R wave in leads Results with qR configuration, or absence of the S wave in leads with A total of 203 patients (163 men, mean age = 59.78 ± Rs configuration), as previously described7,11-13. Two investigators 13.49 years) with STEMI were included in this study. Patients

Arq Bras Cardiol. 2015; 104(2):112-119 113 Yalcinkaya et al. NLR and Ischemia Grade in STEMI

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were classified into two groups according to the ischemia in the univariate regression model for prediction of NLR grade on admission ECG. Group 1 consisted of 126 patients and other variables (NLR, age and gender) considered as with G2I (103 men, mean age = 59.15 ± 13.36 years) and predictors of ECG ischemia grade were evaluated with group 2 consisted of 77 patients with G3I (60 men, mean age univariate logistic regression analysis. Variables that remained = 60.83 ± 13.71 years). Baseline clinical and demographic in the univariate regression model (p < 0.05) were included characteristics were similar in both groups (Table 1). in the backward stepwise multivariate regression analysis to A transthoracic echocardiography, performed on admission determine independent predictors of ECG ischemia grade. on all patients, showed that patients with G3I had significantly Multivariate logistic regression analysis showed that only NLR lower mean left ventricular ejection fraction than those with G2I (odds ratio = 1.254, 95% confidence interval 1.120–1.403, (44.58 ± 7.23 vs. 48.44 ± 7.61, p = 0.001). All patients had p < 0.001) emerged as an independent variable after subtotal or total occlusion of the IRA before pPCI procedure. demonstrating a significant effect on the ECG ischemia The LAD was defined as the IRA in 39.7% of patients with grade (Table 4). G2I and 59.7% of patients with G3I (p = 0.031), therefore, the frequency of anterior MI was significantly higher than that of non-anterior MI in patients with G3I compared with G2I Discussion patients (46 patients vs. 50 patients, p = 0.002). pPCI with In this study, we showed that NLR was independently stent implantation was performed in 147 (72.1%) patients associated with ECG ischemia grade in patients with STEMI. participating in the study. With the growing understanding of the pathophysiological There were no statistically significant differences between role of WBC counts in the STEMI process, many studies groups in terms of time from symptoms to admission, that have focused on this topic have shown that the door‑to‑needle time, PCI with stent implantation, type increase in WBC count is associated with worse clinical of stent used and medication administered (including outcomes and all-cause mortality in patients with glycoprotein IIb/IIIa inhibitors and dual antiplatelet therapy). STEMI14,15. Nonetheless, WBC subtypes have been found For STR, there was a statistically significant difference to be superior to WBC in modulating the inflammatory between the groups. Partial and no STR were more frequent response in the setting of STEMI. Neutrophils are the first in G3I patients, whereas the rate of complete STR was higher leukocytes to be found in the damaged myocardial area16. in G2I patients (p = 0.008). As expected, in-hospital mortality Activated neutrophils exacerbate the inflammatory response rate increased proportionally to the increase in grade of through secretion of a variety of inflammatory mediators ischemia (p = 0.036). including myeloperoxidase, elastase, oxygen free radicals 17-19 There were significant differences in percentages of and arachidonic acid derivates . These neutrophil- lymphocytes (p = 0.010) and neutrophils (p = 0.004), mediated inflammatory processes that occur during STEMI consequently, NLR was significantly different in G2I and cause additional tissue damage, plaque disruption, activation G3I patients (p < 0.001). In contrast, WBC did not differ of coagulation pathways and thrombosis, microvascular between groups. After categorizing the patients according to plugging, myocyte necrosis and enlargement of the infarct 20,21 their STR findings, we found that patients with no STR had size . In contrast to neutrophils, lymphocytes infiltrating higher NLR values compared with those with complete and the ischemic myocardium represent the regulatory arm 22 partial STR. Mean NLR value was 3.55 ± 2.48 for patients of the inflammatory and cytotoxic response and play a with complete STR, 6.26 ± 3.62 for those with partial significant role in the healing process of the heart during 23 STR and 8.44 ± 5.67 for those with no STR (p < 0.001). the course of STEMI . However, in patients presenting with NLR was also higher in patients in whom in-hospital MI, low lymphocyte count – particularly CD4+ count – is a mortality occurred than the remaining patients (6.42 ± 6.11 common finding24. Consequently, the inflammatory process vs. 4.03 ± 2.84, p = 0.007). Other hematological and occurring during the course of STEMI is mediated by the biochemical parameters were similar in both groups (Table 2). complex interaction between innate neutrophil mediated reactive immune responses and subsequent lymphocyte In univariate correlation analysis, STR, ischemia grade, mediated adaptive immune responses. Also, many previous time from symptoms to admission, in-hospital mortality, studies have demonstrated that NLR has superior predictive hospitalization duration, admission systolic blood pressure, and serum glucose and urea correlated significantly with value compared with counts of leukocytes and their NLR (p < 0.05 for all). Variables that correlated significantly subtypes in predicting worse clinical outcomes in patients 2-4,25 with NLR and other variables (hyperlipidemia, left ventricular with STEMI . As a result of these studies, the addition of ejection fraction, anterior MI and infarct related artery) that NLR to conventional risk factors for early risk stratification exhibited significant differences between the G2I and G3I of STEMI patients has been suggested. groups were included in the univariate regression analysis. Various parameters on the admission ECG, such as sum To determine the independent variables likely to predict NLR, of ST elevation, number of Q waves and bundle branch including variables that remained in the univariate regression block, have been correlated with short- and long-term model (p < 0.05), a backward multivariate linear regression adverse outcomes in patients with STEMI26,27. In addition, analysis was performed. We found that ECG ischemia grade recent studies have focused on one such presenting (β = 1.017, p = 0.001), STR (β = 2.527, p < 0.001) and ECG parameter, the determination of distortion of the in-hospital mortality (β = -2.445, p = 0.025) were significant terminal portion of the QRS complex in leads in which independent predictors of NLR (Table 3). Variables included ST segment elevations are seen. The local Purkinje fibers

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Table 1 – Basal demographic and clinical characteristics

Grade 2 Ischemia (n = 126) Grade 3 Ischemia (n = 77) p Age, (years) 59.15 ± 13.36 60.83 ± 13.71 0.391 Male, n (%) 103 (81.7) 60 (77.9) 0.312 Hypertension, n (%) 56 (44.4) 38 (49.4) 0.296 Hyperlipidemia, n (%) 39 (31.2) 15 (19.5) 0.047 Diabetes mellitus, n (%) 30 (24) 21 (27.3) 0.360 Smoking, n (%) 37 (29.4) 22 (28.6) 0.517 Previous coronary artery disease, n (%) 32 (25.4) 15 (19.5) 0.213 Admission systolic blood pressure, (mmHg) 129.79 ± 23.63 133.89 ± 22.83 0.231 Admission diastolic blood pressure, (mmHg) 76.24 ± 13.80 78.86 ± 16.47 0.229 Admission heart rate, (bpm) 76.69 ± 11.79 78.92 ± 11.51 0.188 Time from symptoms to admission (hour) 4.36 ± 3.18 5.15 ± 3.88 0.129 Hospitalization (days) 5.26 ± 1.23 5.44 ± 1.38 0.359 Left ventricular ejection fraction (%) 48.44 ± 7.61 44.58 ± 7.23 0.001 Anterior myocardial infarction, n (%) 50 (39.7) 46 (61.0) 0.002

Infarct-related artery Left anterior descending artery 50 (39.7) 46 (59.7) Circumflex artery 26 (20.6) 14 (18.2) 0.031 Right coronary artery 46 (36.5) 15 (19.5) LMCA/SVG/intermediate/diagonal/ obtuse 4 (3.2) 2 (2.6) PCI with stent implantation, n (%) 87 (69.0) 60 (77.9) 0.112

ST-segment resolution, n (%) Complete 109 (86.5) 54 (70.1) Partial 14 (11.1) 15 (19.5) 0.008 No 3 (2.4) 8 (10.4)

In-hospital mortality, n (%) 5 (4.0) 9 (11.7) 0.036 LMCA: left main coronary artery, SVG: saphenous vein graft, PCI: percutaneous coronary intervention.

are less sensitive to ischemia and conduct impulses more microcirculatory and increased tissue damages occur in slowly than the contracting myocytes28. Increases in the patients with anterior STEMI because of the usually larger R-wave amplitude and decreases in the S-wave amplitude myocardial territory involved in the infarction of the left and, therefore, changes in the terminal portion of the anterior descending coronary artery. In patients with STEMI, QRS develop due to prolonged delay in the electrical the grade of ischemia has emerged as an independent, conduction of the Purkinje system induced by ischemia29,30. strong predictor of adverse procedural outcome and With respect to these changes, the grade of ischemia can mortality to a much greater extent than other initial ECG be readily determined by changes in the terminal portion parameters32,33. Many studies have demonstrated that of the QRS on the surface ECG of STEMI patients and patients with G3I on admission ECG have larger infarcts, does not require any calculations. Three different ischemic poor tissue myocardial perfusion grades irrespective of ECG patterns can be seen in the leads corresponding the reperfusion modality, failure of STR, higher rates of to the ischemic zone7 (G1I, G2I and G3I). Among these reinfarction, lower left ventricular ejection fraction and ischemia grades, G3I has been correlated with more severe therefore, increased mortality and longer hospital stay as ischemia, more tissue damage, higher peak CKMB levels30, compared with G2I patients30,34. less collateral circulation31 and pre-infarction angina which STR is considered a simple and perhaps more accurate might precondition the heart and prevent G3I changes by marker of microvascular reflow and adequate myocardial either metabolic protective mechanisms or residual blood reperfusion after pPCI in STEMI35. Recently, numerous supply12. This study also found that the frequency of G3I studies have pointed to STR as a stronger prognostic was higher in patients presenting with anterior STEMI marker in identifying tissue reperfusion than epicardial than in those with non-anterior STEMI. We propose that TIMI flow grade in the IRA35,36. We therefore used STR

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Table 2 – Hematological and biochemical parameters

Grade 2 Ischemia (n = 126) Grade 3 Ischemia (n = 77) p Serum glucose (mg/dL) 141.36 ± 56.51 155.85 ± 60.00 0.085 Urea, (mg/dL) 37.78 ± 15.92 41.88 ± 19.18 0.102 Creatinine, (mg/dL) 1.08 ± 0.33 1.19 ± 0.81 0.194 Serum uric acid, (mg/dL) 6.08 ± 1.64 5.98 ± 2.09 0.759 High-density lipoprotein cholesterol, (mg/dL) 41.08 ± 8.41 39.68 ± 8.07 0.260 Low-density lipoprotein cholesterol, (mg/dL) 118.44 ± 35.55 112.49 ± 40.70 0.296 Triglycerides, (mg/dL) 140.48 ± 77.64 151.47 ± 141.43 0.494 Total serum cholesterol, (mg/dL) 186.99 ± 39.37 177.10 ± 45.20 0.117 White blood cells, (103 µL) 11. 40 ± 33.34 12.30 ± 40.52 0.088 Neutrophils, (103 µL) 7.45 ± 3.15 8.95 ± 4.03 0.004 Lymphocytes, (103 µL) 2.86 ± 1.55 2.28 ± 1.47 0.010 Neutrophil / lymphocyte ratio 3.40 ± 2.38 5.51 ± 3.90 < 0.001 Hemoglobin, (g/dL) 14.12 ± 1.93 14.05 ± 2.16 0.800 Hematocrit, (%) 41.51 ± 5.19 41.19 ± 5.61 0.682 Platelet count, (103 µL) 252.11 ± 72.25 254.54 ± 73.70 0.818 Mean platelet volume, (fL) 8.24 ± 1.01 8.27 ± 0.95 0.833 Red cell distribution width (%) 12.78 ± 1.27 13.09 ± 1.82 0.170

Table 3 – Univariate and multivariate regression models based on independent variables likely to predict the neutrophil-to-lymphocyte (NLR) ratio

Univariate Analysis Multivariate Analysis* Independent variables Beta p Beta p STR 0.432 < 0.001 2.527 < 0.001 Electrocardiographic ischemia grade 0.321 < 0.001 1.497 0.001 Time from symptoms to admission (min) 0.162 0.028 0.052 0.383 In-hospital mortality 0.189 0.007 -2.445 0.025 Hospitalization duration (days) 0.165 0.019 0.114 0.558 Admission SBP -0.151 0.033 -0.016 0.081 Serum glucose 0.159 0.023 0.003 0.555 Serum urea 0.187 0.008 0.023 0.080 Hyperlipidemia -0.081 0.251 -0.009 0.985 LVEF -0.123 0.087 -0.029 0.319 Anterior MI 0.012 0.870 0.566 0.230 Infarct-related artery -0.018 0.803 -0.377 0.428 * = p value at the last step with the independent variables that remained in model. NLR: neutrophil-to-lymphocyte ratio; STR: ST-segment resolution; SBP: systolic blood pressure; LVEF: left ventricular ejection fraction; MI: myocardial infarction.

to evaluate tissue reperfusion due to its ease of use and Although quick restoration of epicardial coronary artery is known superiority. Similar to previous studies, our present achieved with pPCI in patients with STEMI, the prognosis is study revealed that patients with G3I on admission ECG mainly dependent upon restoration of an adequate perfusion had partial or no STR after pPCI. Additionally, we found of areas with microvascular integrity. Separately, both elevated that patients with no STR had higher NLR values compared NLR and G3I on admission are predictors of poor tissue- with patients with complete and partial STR. level reperfusion and poor prognostic markers in patients

116 Arq Bras Cardiol. 2015; 104(2):112-119 Yalcinkaya et al. NLR and Ischemia Grade in STEMI

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Table 4 – Effects of different variables on ECG ischemia grade in multivariate logistic regression analyses

Covariates Adjusted OR 95% CI p NLR 1.254 1.120-1.403 < 0.001 Anterior MI 2.016 0.979-4.151 0.050 Admission SBP 1.015 1.000-1.030 0.075 Hyperlipidemia 2.107 0.927-4.789 0.051 LVEF 0.952 0.906-1.000 0.185 OR: odds ratio; 95% CI: 95% confidence interval; NLR: neutrophil-to-lymphocyte ratio; MI: myocardial infarction; SBP: systolic blood pressure; LVEF: left ventricular ejection fraction.

with STEMI, probably due to severe ischemic damage to the unpredictability may be accurate for counts of WBCs and microvasculature. This hypothesis has been explained in part their subtypes, but not for the NLR, which is the reason we by the poorer collateral supply and the larger clot burden seen evaluated NLR only once on admission. in these patients. Activation of inflammation in the infarcted region may contribute to plugging in the microvasculature and developing no-reflow phenomenon via platelet-neutrophil Conclusion interaction in patients with STEMI37. We found that there is an association between G3I Lucchesi et al38 showed that activated leukocytes might and elevated NLR, both easily ascertainable through modulate the electrical activity of the myocardium by low‑cost methods. We speculate that this association is releasing oxygen free radicals. In the Efficacy of Vasopressin likely caused by the same mechanism (adequate perfusion Antagonism in Heart Failure Outcome Study with Tolvaptan of areas with microvascular integrity). The finding of this (EVEREST) study, lymphopenia was associated with a association may help elucidate the adverse cardiovascular widened QRS39. Also, a longer occlusion period causes outcomes seen in patients with STEMI. We believe that the additional microvascular damage and, therefore, worse incorporation of NLR and ECG ischemia grade as covariables in final flow rates. However, to our knowledge, no prior well‑validated standardized risk scores will provide additional study has reported the association between NLR and ECG prognostic value for risk stratification of patients with STEMI. grades of ischemia, and our present study is the first in this However, these findings must be confirmed in additional regard. We demonstrated that there is a significant positive studies with larger numbers of patients. correlation between NLR and ECG ischemia grades, and we therefore speculate that the worse effect of G3I on clinical outcome might be at least partially explained by Author contributions the prognostic value of NLR. Conception and design of the research, Analysis and interpretation of the data and Critical revision of the manuscript Limitations for intellectual content: Yalcinkaya E, Yuksel UC, Celik M, Kabul HK, Barcin C, Gokoglan Y, Yildirim E, Iyisoy A; Acquisition of The present study has a number of limitations. The major data: Yalcinkaya E, Celik M, Gokoglan Y, Yildirim E; Statistical limitations are the study’s retrospective design, its location analysis: Yalcinkaya E, Yuksel UC, Celik M, Barcin C; Writing on a single tertiary center and the relatively small number of of the manuscript: Yalcinkaya E, Barcin C. patients, which may affect study generalizability. However, our study may serve as an inspiration for additional prospective studies with larger sample sizes. Next, we did not assess TIMI Potential Conflict of Interest flow grade routinely. Since many studies have shown that STR No potential conflict of interest relevant to this article is a more powerful prognostic predictor than epicardial TIMI was reported. flow grade, and with a better correlation with late mortality, we chose to use STR and believe our findings are clinically important. Finally, the choice of the ideal blood collection Sources of Funding time is challenging since neutrophils have a short life in the There were no external funding sources for this study. circulation (around 7 hours), and because the exact time to peak inflammatory response after STEMI and cut-off points of NLR remains unknown. Although many studies recommend Study Association serial sampling for better prognostication, we believe this This study is not associated with any thesis or dissertation work.

Arq Bras Cardiol. 2015; 104(2):112-119 117 Yalcinkaya et al. NLR and Ischemia Grade in STEMI

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Arq Bras Cardiol. 2015; 104(2):112-119 119 Original Article

Sudden Cardiac Death in Brazil: A Community-Based Autopsy Series (2006-2010) Maria Fernanda Braggion-Santos1, Gustavo Jardim Volpe1, Antonio Pazin-Filho2, Benedito Carlos Maciel1, José Antonio Marin-Neto1, André Schmidt1 Divisão de Cardiologia do Departamento de Clínica Médica - Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo1, Ribeirão Preto, SP; Divisão de Emergências Clínicas do Departamento de Clínica Médica - Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo2, Ribeirão Preto, SP - Brazil

Abstract Background: Sudden cardiac death (SCD) is a sudden unexpected event, from a cardiac cause, that occurs in less than one hour after the symptoms onset, in a person without any previous condition that would seem fatal or who was seen without any symptoms 24 hours before found dead. Although it is a relatively frequent event, there are only few reliable data in underdeveloped countries. Objectives: We aimed to describe the features of SCD in Ribeirão Preto, Brazil (600,000 residents) according to Coroners’ Office autopsy reports. Methods: We retrospectively reviewed 4501 autopsy reports between 2006 and 2010, to identify cases of SCD. Specific cause of death as well as demographic information, date, location and time of the event, comorbidities and whether cardiopulmonary resuscitation (CPR) was attempted were collected. Results: We identified 899 cases of SCD (20%); the rate was 30/100000 residents per year. The vast majority of cases of SCD involved a coronary artery disease (CAD) (64%) and occurred in men (67%), between the 6th and the 7th decades of life. Most events occurred during the morning in the home setting (53.3%) and CPR was attempted in almost half of victims (49.7%). The most prevalent comorbidity was systemic hypertension (57.3%). Chagas’ disease was present in 49 cases (5.5%). Conclusion: The majority of victims of SCD were men, in their sixties and seventies and the main cause of death was CAD. Chagas’ disease, an important public health problem in Latin America, was found in about 5.5% of the cases. (Arq Bras Cardiol. 2015; 104(2):120-127) Keywords: Death, Sudden, Cardiac / epidemiology; Brazil; Autopsy.

Introduction are used4, studies based on retrospective analysis of death 5 Sudden cardiac death (SCD) is an unexpected event certificates or even absence of a structured system to report from a cardiac cause1. According to the World Health cases of SCD in some regions. Such difficulties are more evident 6 Organization (WHO), SCD is a natural event that occurs in developing countries . within less than one hour of symptom onset in individuals The most affected individuals are men between the sixth without any potentially fatal precondition. However, 40% and seventh decades of life7. The epidemiology of SCD is of cases are not witnessed and, in these situations, the closely correlated with coronary artery disease (CAD) and up victims must have been seen asymptomatic in the last to 80% of the victims have CAD4. Risk factors such as systemic 24 hours before the event2. arterial hypertension, diabetes and smoking increase the risk An incidence of SCD between 180.00-400.00 cases / year3 is of SCD8, as well as advanced left ventricular dysfunction estimated in the United States. However, an accurate estimate (ejection fraction < 30%)4. In young individuals, the most is not possible for several reasons: different MSCD definitions common diagnoses are hypertrophic cardiomyopathy, coronary artery anomalies, arrhythmogenic dysplasia of 9 Mailing Address: Maria Fernanda Braggion-Santos • the right ventricle and channelopathies . In Latin America, Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Chagas disease is a significant cause of SCD10. Universidade de São Paulo – Divisão de Cardiologia Avenida Bandeirantes 3900, Campus Universitário, Monte Alegre. Circadian and seasonal SCD variations have been Postal Code 14048-900 Ribeirão Preto, SP - Brazil described. A marked variation in the occurrence of SCD with E-mail: [email protected]; [email protected] peaks in the morning can be observed on Mondays, in the Manuscript received March 28, 2014; revised manuscript June 15, 2014; 11 accepted June 27, 2014. winter months . Stressful situations such as terrorist attacks or events such as the FIFA World Cup have been associated DOI: 10.5935/abc.20140178 to increased SCD rates12,13.

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The high incidence of this event, combined with low Results survival rates, makes SCD a relevant health issue7. In Latin America, few studies have been performed to date, with small Sudden cardiac death cases populations and specific heart diseases14,15. Between 2006 and 2010, 4,501 autopsies were Our objective was to describe the characteristics of performed in Ribeirão Preto. Initially, 2,053 cases SCD in the city of Ribeirão Preto, state of Sao Paulo, Brazil were selected as possible SCD; however, after careful (approximately 600,000 inhabitants) in five years, according analysis, 718 cases were excluded for not meeting all the to the autopsy reports of the Death Verification Service of the criteria for SCD, as well as 256 cases due to incomplete Countryside (SVOi). information. In 180 cases, a noncardiac cause of sudden death was identified: 99 cases of acute aortic syndrome, Methods 58 cases of pulmonary embolism, 21 cases of hemorrhagic CVA and two cases of asthma. Therefore, 899 cases Ribeirão Preto is a medium-sized city located in the state (20% of all autopsies) were defined as SCD (Figure 1). of São Paulo, Brazil. It has a public body linked to the School SCD rates ranged from 28/100,000 inhabitants in 2009 of Medicine of Ribeirão Preto, Universidade de São Paulo, to 32/100,000 inhabitants in 2007 and 2008 (Figure 2). the Death Verification Service of the Countryside (SVOi). There was no difference in SCD rates between the years responsible for the autopsies of victims of non-violent deaths (p = 0.88). referred from any health service of the city, in accordance with applicable federal laws, as requested by the health professional who assisted the victim. Demographic characteristics of the population In our study, we assessed the autopsy request forms and The demographic characteristics of the study population complete reports of autopsies performed by SVOi in victims are summarized in Table 1. Men were more affected than from the city of Ribeirão Preto between 2006 and 2010. women (67% x 33%). Most of the victims were Caucasians All cases that met SCD criteria were included, according to (75% x 25%), between the sixth and seventh decade of life the most widely used definition of the WHO: unexpected (mean age 62.7 ± 13.2 years). death within one hour of symptom onset, or in cases of unwitnessed death, when the victim was seen in good Cause of death 2 health in the 24 hours prior to the event . The most prevalent cause of death was acute coronary Data related to the event were collected, such as the syndrome, accounting for approximately two-thirds (64%) cause of death reported by the pathologist who performed of all cases of SCD. The second cause was cardiomyopathy the investigation, demographic characteristics and (32%), including both etiologies, ischemic and nonischemic. comorbidities of the victims, date, time and place of the A diagnosis of myocardial bridge in eight cases, a case of event and cardiopulmonary resuscitation (CPR) maneuvers myocarditis confirmed by histopathological analysis and, performed. The study was approved by the Research Ethics in another case, severe hypoplasia of the left anterior Committee of our institution. descending coronary artery in a 35-year-old woman were also observed. In 24 victims (3%) the cause of death could Exclusion criteria not diagnosed, with these cases being considered sudden death of unknown cause (Table 2). Initially, deaths of newborns and children were excluded. Then, all individuals with causes of death not compatible with sudden death were excluded: infectious diseases, advanced Risk factors for sudden cardiac death malignancies, abdominal diseases, such as bowel obstruction More than one-fifth of the patients (21.1%) victims of SCD or perforated gastric ulcer, cachexia and prolonged bed rest. had some type of previously known cardiac disease. Several Subsequently, cases of sudden death from noncardiac causes risk factors for SCD were found in these patients, with the were also excluded: pulmonary embolism, acute aortic most prevalent being systemic arterial hypertension (57.3%). syndromes, asthma or hemorrhagic cerebrovascular accident Furthermore, we observed 56 patients (6.2%) with a history (CVA). Finally, cases with no clinical history consistent with of alcohol abuse (Table 3). SCD, according to the WHO criteria employed in our study, or with incomplete information were also excluded. Chagas Disease Forty-nine patients (5.5%) had a diagnosis of Chagas Statistical Analysis disease, including those with previously known chagasic The data were expressed as absolute values and cardiomyopathy and those with positive serology at the percentages. ANOVA parametric test and Bonferroni time of the autopsy. It is noteworthy the fact that the post‑test were used to analyze continuous variables, proportion of patients with Chagas disease decreased from while the chi-square test was used to evaluate categorical 2006 to 2010 (12 patients: 7.1% in 2006, 13 patients: variables. All statistical analyzes were performed using 7.0% in 2007, 11 patients: 5.8% in 2008; five patients: commercially available statistical software (InStat, version 3.0% in 2009, eight patients: 4.3% in 2010), but the 3.0, GraphPad Software Inc, USA). A p value < 0.05 was difference over the years did not reach statistical considered statistically significant. significance (p = 0.14).

Arq Bras Cardiol. 2015; 104(2):120-127 121 Braggion-Santos et al. Sudden cardiac death in Brazil

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4501 Autopsies

2448 Deaths from other causes*

2053 Possible sudden cardiac deaths

1154 Excluded 718 Lack of criteria for SCD 256 Incomplete information 180 Non-cardiac causes of sudden**

899 Sudden cardiac deaths

Figure 1 – Study flowchart. * Deaths from other causes: infectious diseases, advanced malignancies, abdominal diseases, cachexia, prolonged bed rest. ** Non-cardiac causes of sudden death: acute aortic syndromes, pulmonary embolism, hemorrhagic stroke, asthma.

Temporal variation of sudden cardiac death Cardiopulmonary resuscitation The analysis of cases of SCD showed a circadian variation, In total, 447 patients of 899 received CPR maneuvers with fewer cases during the night (p < 0.05 in comparison (49.7%). In 2006, CPR was performed in 43.4% of patients, to other periods of the day) followed by a significant increase and this proportion increased over the years, reaching 54.4% in the early hours of the morning, which was the period with of the victims submitted to CPR in 2010. An increasing the highest number of SCD events (p < 0.05 in comparison tendency in CPR maneuvers is observed (Figure 4), but with to other periods of the day). It was not possible to determine marginal statistical significance (p = 0.05). the hour of the event in 10% of cases, (Figure 3A). Further analyses showed no predominance of any day Additional considerations of the week (p = 0.79, Figure 3B) or any month of the Although SCD is included in the International Classification year (p = 0.06); however, the events tended to occur more of Diseases 10 (ICD-10), code I46-117, only 3.6% of the forms frequently in the months of May and June (Figure 3C). completed by physicians who treated the victims and 2.2% of the autopsy reports issued by the pathologists specified this diagnosis as "cause of death". Finally, in 26 cases (2.9%) Place of death microscopic examination of the heart was performed based More than half of the deaths occurred at home on the diagnostic hypothesis of myocarditis; however, the (53.3%). Of the remaining, the deaths occurred in diagnosis was confirmed in only one case. emergency rooms (37.8%), where most patients arrived in cardiorespiratory arrest, called "death on arrival"16. It is worth noting that 8.2% of all events occurred in public Discussion places and six men died (0.7%) during physical activity To the best of our knowledge, this is the first study that practice (mean age 35 years). characterizes different aspects related to SCD in Brazil.

122 Arq Bras Cardiol. 2015; 104(2):120-127 Braggion-Santos et al. Sudden cardiac death in Brazil

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40 p = 0.88

36

32

28

per 100,000 inhabitants 24

20 2006 2007 2008 2009 2010 Year

Figure 2 – SCD rates (per 100,000 inhabitants) in Ribeirão Preto from 2006 to 2010. The mean rate of SCD was 30/100,000 inhabitants per year, ranging from 28/100,000 inhabitants in 2009 to 32/100,000 inhabitants in 2007 and 2008.

Because there are scarce data on this topic, both in Brazil and of our country; however, as there is no reliable data available, in other Latin American countries, it is very important to know both related to our population and to other developing countries, the rates of SCD in our community, the event circumstances one cannot establish adequate comparisons. and the characteristics of the victims, so that prevention In our study, most of the victims were males, Caucasians, aged strategies are developed and tested, of which some are specific 60 to 80 years. According to recent data, SCD occurs most often to our population. between the sixth and seventh decade of life7, in accordance with The rate of SCD in Ribeirão Preto was approximately our findings. Regarding the distribution of SCD by gender, there 30/100,000 inhabitants. In other communities around the world, was a change in the pattern over the years, with an increase in the incidence of SCD ranged from 37/100,000 inhabitants in the proportion of events in women3. Previous studies showed a Okinawa, Japan18 and 90-100 / 100,000 in Maastricht, in the women / men ratio of 25:754; however, in more recent studies, Netherlands19, with most of the studies reporting similar SCD the women/men ratio increased to 40:607, probably due to the incidence, little over 50/100,000 residents: in Oregon, United increase in CAD prevalence and mortality among women over States (53/100,000 residents)20 and a community located in the years24. We found 67% of events in men, similar to what western Ireland (51.2 / 100,000)21. is described in the literature. Finally, most SCD victims were In comparison to data from other communities, the mean Caucasians (75%). However, it is important to note that we have rate of SCD in our population was lower. There are several a heterogeneous racial distribution in different regions of Brazil possible reasons for this difference. First, we analyzed only data and thus, this finding should not be extrapolated or considered from the SVOi, and thus, our data may be underestimated representative of the entire country. because not all deaths that occurred in Ribeirão Preto during Acute coronary syndrome was responsible for most cases of the analyzed period were referred for autopsy. Moreover, SCD (64%), and cardiomyopathy was the second most common SCD survivors were not considered. Another reason for this cause in our series (32%), including myocardial diseases of difference was the exclusion of cases with inconclusive data. ischemic and nonischemic etiology. Studies have shown that up However, it is known that the epidemiology of SCD is strongly to 80% of cases of SCD are associated with CAD. Approximately associated to the incidence of CAD and, in Brazil, CAD mortality 10-15% of cases occur in patients with myocardial diseases is estimated at 48 / 100,000 inhabitants22, whereas in the United such as hypertrophic cardiomyopathy, idiopathic dilated States, it is approximately three times higher (135/100,000)23. cardiomyopathy, right ventricular arrhythmogenic dysplasia or These factors may be related to the lower economic development infiltrative myocardial diseases3,4.

Arq Bras Cardiol. 2015; 104(2):120-127 123 Braggion-Santos et al. Sudden cardiac death in Brazil

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Table 1 – Demographic characteristics of victims of SCD in Ribeirão Table 2 – Distribution of SCD causes among SCD victims in Ribeirão Preto between 2006-2010 Preto: 2006-2010

Characteristics N.º % Cause of death N.º % Gender Acute coronary syndrome 576 64.1 Male 599 67 Cardiomyopathies* 289 32.1 Female 300 33 Myocardial bridge 8 0.9

Age, years Myocarditis 1 0.1 10-19 3 0.3 Coronary artery anomaly 1 0.1 20-29 7 0.7 Undetermined 24 2.7 30-39 33 3.5 * Ischemic and nonischemic cardiomyopathies; SCD: sudden cardiac death 40-49 114 12.5 50-59 190 21 Table 3 – Risk factors for SCD among victims of SCD in Ribeirão 60-69 241 27 Preto: 2006-2010 70-79 231 26 80 80 9 Risk factors for SCD % N.º

Ethnicity SAH 57.3 515 Caucasian 675 75 Structural heart disease 21.1 190 Black 100 11 Diabetes 15.3 138 Mixed-race 119 13 Smoking 8.8 79 Asians 5 1 Obesity 8.6 77 SCD: sudden cardiac death. Dyslipidemia 1.2 11 Alcohol abuse 6.2 56 Chagas disease 5.5 49 In our study it was not possible to separate SCD victims SAH: systemic arterial hypertension; SCD: sudden cardiac death due to ischemic or nonischemic myocardial diseases, due to the difficulty in differentiating some cases according to the information provided in the analyzed reports. As an example relatives. Furthermore, we observed that systemic arterial of this limitation, some SCD victims had CAD and Chagas hypertension was the most prevalent risk factor among SCD disease, and both diseases can induce lethal arrhythmias, victims (57.3%) and hypertensive patients, especially those making it impossible to define what the pathology responsible with left ventricular hypertrophy, had a higher risk of SCD for the event was. This may explain the lower rates of CAD than the overall population27. as cause of SCD and the higher number of events per myocardial diseases. Other risk factors such as diabetes, smoking, obesity and dyslipidemia, were also found, but in smaller proportions, Regarding Chagas disease, 49 SCD victims (5.5%) had suggesting that these data are underestimated, for some this diagnosis in their autopsy reports. Although the WHO reasons described as follows: first, retrospective data were certified in 2006 that the vector-borne transmission of Chagas collected and there were no specific questionnaires in disease was eliminated in our country, Brazil is considered the analyzed forms. The victims’ medical records were as having high prevalence of human infection (1% of our not evaluated. Finally, some diseases, such as diabetes or 25 population, 1.9 million infected individuals in 2005) , with dyslipidemia, may be underdiagnosed in our population, chagasic cardiomyopathy still having a very important role in as diagnosis depends on laboratory tests that are often not cardiovascular mortality in our country, especially in cases of routinely performed. Alcohol abuse was detected in 6.2% 10 sudden death . of the cases, similar to the study carried out in a community The cause of death could not be identified in 24 cases located in western Ireland21. (2.2%). The victims had a structurally normal heart and some It is known that the temporal distribution of SCD has of these cases could be related to genetic diseases such an established pattern, with most events occurring in the as channelopathies, undiagnosed accessory pathways or morning, on Mondays during winter months11. In our series, 26 coronary vasospasm . there were a higher number of events in the morning, with The presence of structural heart disease, such as previous no difference regarding the days of the week or months of myocardial infarction or left ventricular dysfunction is an the year, with the latter being explained by the absence of important risk factor for SCD3. In our study, more than 20% well-defined seasons in our region. The higher number of of the victims had some type of heart disease reported by events in the morning was previously attributed to increased

124 Arq Bras Cardiol. 2015; 104(2):120-127 Braggion-Santos et al. Sudden cardiac death in Brazil

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A 35 30 p < 0.05 25 20 15 % of patients 10 5 0 06-12h 12-18h 18-24h 24-06h Unknown Period of day B 20 p = 0.79 15

10

% of patients 5

0 Sun Mon Tue Wed Thu Fri Sat Day of the week C 14 12 p = 0.06 10 8 6

% of patients 4 2 0 Jan Feb Mar Apr May Jun Jul Aug Sept Oct Nov Dec Months of the year

Figure 3 – Histogram of the temporal variation of SCD. A) Histogram of the circadian variation of SCD. The morning period is the period with the most SCD events (p < 0.05 in comparison to other periods). B) Histogram of the weekly variation of SCD. There was no difference between days of the week (p = 0.79). C) Histogram of monthly variation of SCD. SCD events tended to occur more frequently in the months of May and June, but without statistical significance (p = 0.06). sympathetic discharge that occurs at waking up, causing lacking regarding events such as complete profile of risk the higher number of CVAs, acute coronary syndromes factors for SCD victims, better description of the clinical and sudden deaths28. manifestations presented by victims before death in witnessed Regarding the place of death, more than half of the events cases and CPR procedures performed or autopsy protocols. occurred at home (53.3%), which is known to be associated with Although the institution of reference for performing lower success rates after attempted CPR29. It can be observed autopsies of our region is the SVOi, not all cases of SCD are that 49.7% of victims received CPR maneuvers, a result similar referred to this service, as, according to Brazilian laws, if the to that found in Maastricht, Netherlands, where 51% of SCD death resulted from non-traumatic causes and if there is a victims received CPR maneuvers19. Despite not reaching physician aware of the case that is willing to sign the death statistical significance, the number of SCD victims that received certificate, referral of the victim to the autopsy examination CPR increased over the years, which may reflect improvements is not mandatory. Finally, there are not specific forms for the in the health system, with a larger number of ambulances and investigation of SCD in SVOi. emergency rooms in public and private hospitals. Although the study has several limitations, we believe our findings provide very important information about the Study limitations characteristics of SCD in Brazil, also showing flaws in our As mentioned before, SCD rates may be underestimated. medical documentation, which should be readily improved, Because this is a retrospective study, some specific data are e.g., by establishing specific autopsy protocols30.

Arq Bras Cardiol. 2015; 104(2):120-127 125 Braggion-Santos et al. Sudden cardiac death in Brazil

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100 p = 0.05 80

60

40

% of patients 20

0 2006 2007 2008 2009 2010 Year

Figure 4 – Proportion of SCD victims submitted to CPR maneuvers in Ribeirão Preto (2006-2010). Altogether, 447 of the 899 victims received CPR (49.7%). There was a clear tendency to an increase in the number of patients that received CPR between 2006 and 2010 (p = 0.05).

Conclusion Schmidt A; Statistical analysis: Braggion-Santos MF, Schmidt Sudden cardiac death accounted for about 20% of all A; Writing of the manuscript: Braggion-Santos MF, Volpe GJ; Critical revision of the manuscript for intellectual content: non‑traumatic deaths in this large Brazilian community, Pazin-Filho A, Maciel BC, Marin-Neto JA, Schmidt A. based on autopsy reports. Most SCD cases occurred due to ACS in men between the sixth and seventh decade of life. Most events occurred in the morning, at home and Potential Conflict of Interest CPR maneuvers were performed on half of the victims. No potential conflict of interest relevant to this article This study is particularly significant because it is the first to was reported. comprehensively evaluate the scenario of SCD in Brazil and to contribute to the development of preventive strategies in our social context. Sources of Funding There were no external funding sources for this study. Author contributions Conception and design of the research: Braggion-Santos Study Association MF, Volpe GJ, Pazin-Filho A, Maciel BC, Marin-Neto JA, This article is part of the thesis of master submitted by Maria Schmidt A; Acquisition of data: Braggion-Santos MF; Analysis Fernanda Braggion Santos, from Faculdade de Medicina de and interpretation of the data: Braggion-Santos MF, Volpe GJ, Ribeirão Preto da Universidade de São Paulo.

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Arq Bras Cardiol. 2015; 104(2):120-127 127 Original Article

Impact of Light Salt Substitution for Regular Salt on Blood Pressure of Hypertensive Patients Carolina Lôbo de Almeida Barros, Ana Luiza Lima Sousa, Brunella Mendonça Chinem, Rafaela Bernardes Rodrigues, Thiago Souza Veiga Jardim, Sérgio Baiocchi Carneiro, Weimar Kunz Sebba Barroso de Souza, Paulo César Brandão Veiga Jardim Liga de Hipertensão Arterial da Faculdade de Medicina da Universidade Federal de Goiás, Goiânia, GO - Brazil

Abstract Background: Studies have shown sodium restriction to have a beneficial effect on blood pressure (BP) of hypertensive patients. Objective: To evaluate the impact of light salt substitution for regular salt on BP of hypertensive patients. Methods: Uncontrolled hypertensive patients of both sexes, 20 to 65 years-old, on stable doses of antihypertensive drugs were randomized into Intervention Group (IG – receiving light salt) and Control Group (CG – receiving regular salt). Systolic BP (SBP) and diastolic BP (DBP) were analyzed by using casual BP measurements and Home Blood Pressure Monitoring (HBPM), and sodium and potassium excretion was assessed on 24-hour urine samples. The patients received 3 g of salt for daily consumption for 4 weeks. Results: The study evaluated 35 patients (65.7% women), 19 allocated to the IG and 16 to the CG. The mean age was 55.5 ± 7.4 years. Most participants had completed the Brazilian middle school (up to the 8th grade; n = 28; 80.0%), had a family income of up to US$ 600 (n = 17; 48.6%) and practiced regular physical activity (n = 19; 54.3%). Two patients (5.7%) were smokers and 40.0% consumed alcohol regularly (n = 14). The IG showed a significant reduction in both SBP and DBP on the casual measurements and HBPM (p < 0.05) and in sodium excretion (p = 0.016). The CG showed a significant reduction only in casual SBP (p = 0.032). Conclusions: The light salt substitution for regular salt significantly reduced BP of hypertensive patients. (Arq Bras Cardiol. 2015; 104(2):128-135) Keywords: Hypertension; Sodium; Blood Pressure; Diet.

Introduction implies reduced heart attack risk6. Chang et al7 have Excessive salt intake has been identified as an important detected that mortality due to CVD decreased significantly risk factor for cardiovascular disease (CVD). Sodium restriction in individuals who consumed salt with lower sodium and has a favorable influence on blood pressure (BP) control, higher potassium levels. thus being a potentially powerful tool for systemic arterial Research studies assessing the effect of the reduction in hypertension (SAH) prevention and control1. sodium consumption on BP have been treated with great Several studies have claimed that a reduction in sodium- importance by the scientific community. For this reason, it is rich foods consumption causes a significant decrease in BP of important to perform new experimental studies in order to find hypertensive patients2-4. Intersalt was one of the first studies complementary and more current results on the association to evaluate sodium intake from 24-hour urine samples, between those variables8. demonstrating a positive association between high sodium Therefore, evaluating the impact of a low-sodium salt 5 consumption and BP increase . consumption on BP may contribute to better control it. The present High sodium excretion is related to a higher risk of death study aimed at evaluating whether light salt substitution for regular due to CVD. On the other hand, higher potassium excretion salt could reduce the BP of hypertensive patients.

Methods Mailing Address: Carolina Lôbo de Almeida Barros • This was a single-blind randomized controlled trial. Rua T37 n. 3832 St. Bueno. Postal Code 74.230-022, Goiânia, GO - Brazil. E-mail: [email protected] Sample characterization Manuscript received May 14, 2014; revised mansucript June 11, 2014; accepted July 22, 2014 Sample size was calculated for comparison of means, with a statistical power of 90%, two-tailed hypothesis test, standard DOI: 10.5935/abc.20140174 deviation of 12.7 mmHg and an expected difference of 13.1

128 Barros et al. Salt intake and blood pressure

Original Article mmHg in systolic blood pressue (SBP)3, with a significance visits, had their casual BP measured, and underwent HBPM level of 5%. The necessary sample size was of 20 individuals and 24-hour urine sample collection, following the same in each group. procedures used before the intervention. Hypertensive individuals of both sexes, aged between Casual BP was measured by the same researcher, at least 20 and 65 years, were recruited and regularly followed three times and at 1-minute intervals, until the differences up at a multiprofessional service for hypertension care. between the measurements were lower than 4 mmHg9. Patients were dwellers of the metropolitan region of For the purpose of analysis, the mean of the last two values Goiânia, Brazil, on stable doses of antihypertensive drugs was considered. for at least 30 days, with uncontrolled hypertension HBPM was accomplished according to the III Brazilian 9 (BP ≥ 140 x 90 mmHg) in their last visit. Guidelines for HBPM10. Twenty-four measurements were Patients with acute or subacute (up to 3 months performed, being three in the morning and three in the before the beginning) and unstable chronic diseases were afternoon for four days. The examinations with a recovery excluded, as were those having their meals prepared with percentage higher than 70% were considered adequate. a salt different from that provided in this study more than All the SBP and DBP measurements were obtained by once a week. using a semi-automatic digital device (OMRON 705 CPINT, Illinois, USA), and patients were duly guided on how to use Sample selection and randomization the device for HBPM. Initially, patients were identified from medical charts, The process of 24-hour urine sample collection according to the inclusion and exclusion criteria. Then, they initiated with the second urine elimination of the first day were invited to visit a nutrition office to receive the necessary and finished at the first elimination of the following day, information about this research and to provide their written approximately at the same time. Urine was analyzed at informed consent, when agreeing to participate in this study. the General Hospital’s laboratory of the Federal University The project was approved by the Research Ethics Committee of Goiás using the ion-selective membrane technique11 from the General Hospital of the Federal University of to quantify sodium and potassium levels in human urine. Goiás (protocol number 193/2011) and was conducted in accordance with the Declaration of Helsinki. Intervention The studied population was assigned to two homogeneous All patients were instructed to consume only the groups [Intervention Group (IG) and Control Group (CG)], provided salt throughout this study. In addition, they according to the order of their visit with the nutritionist: the were instructed to reduce sodium-rich food consumption first patient was included in IG and the second in CG, and during the study period, being particularly warned about so forth. The IG participants received light salt, whereas CG industrialized foods12,13. participants received placebo (regular salt), for four weeks (28 days). All participants from both groups remained on The patients received the salt after the first BP evaluation regular follow-up. and urine collection. The daily recommendation adopted was 3 g of salt per person, proposed by the VI Brazilian Guidelines on Hypertension9. To calculate the amount of Study design salt consumed per patient, the number of people usually The variables collected in the inclusion visit were sex, sharing the same meals was considered. Ten percent of the age, schooling level (complete middle school; complete amount of salt per patient was added, anticipating a higher high school; and complete college), family income (up to consumption due to family routine changes on weekends US$ 600; from US$ 600 to US$ 1200; and US$ 1200 or (greater number of people). more), regular physical activity practice (at least 30 minutes, Every patient received 28 small plastic bags containing three times a week), smoking habit and alcoholism the daily amount of salt, with a tag showing the initials of (alcoholic beverage intake frequency equal to or higher the patient’s name, date of use and the amount of salt. than once a month. The salt provided showed no identification of its The variables collected prior to and following the composition. Therefore, participants were not aware of intervention included weight, height, body mass index the type of salt they were receiving. The researcher was (BMI), 24-hour urine sodium and potassium (mEq/day), the only person who knew that information, characterizing casual BP and home blood pressure monitoring (HBPM). the study as a simple-blind investigation. Patients were recruited from May to October 2012. Two The light salt composition (per gram) was as follows: visits were scheduled during this study, the first corresponding 130 mg of sodium, 346 mg of potassium and 44 mcg of to the participants’ selection and randomization and the iodine. The regular salt contained (per gram) 390 mg of second, 28 days after the first, during the post-intervention sodium and 25 mcg of iodine. return. In the initial week of study, patients had their casual BP measured, and underwent HBPM for four days and 24- hour urine sample collection. Then, they started to consume Statistical analysis the prescribed salt and carried it on for 28 days. During the The data were analyzed using the SPSS software, version last week of the study, patients returned to their second 20.0 (SPSS Inc, Chicago, USA). Qualitative variables were

Arq Bras Cardiol. 2015; 104(2):128-135 129 Barros et al. Salt intake and blood pressure

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presented as mean and standard deviation, according to Adverse effects their absolute and relative frequencies. Shapiro-Wilks W This clinical trial detected an adverse effect among test was used to verify data normality. In the presence of IG patients. Due to its peculiar taste, light salt had low normal distribution, independent data, as well as paired acceptance by 89.5% of IG individuals, who claimed there data, were compared by using Student t test; Wilcoxon’s were taste changes in the prepared foods. Nevertheless, test was used when paired data were skewed. patients agreed to use that salt throughout the study period. The 5% significance level was adopted for all tests. The above mentioned adverse effect was considered mild and study interruption was not necessary. Results Of the 1800 medical registrations at our service, 265 Discussion corresponded to patients with uncontrolled BP, of whom, This study sought to reduce the dietary sodium intake of only 56 were eligible. This could be explained by the fact CG patients to less than the 5 g of salt per day (2 g of sodium) that our service provides care to an elderly population with proposed by the World Health Organization14, considering a high incidence of chronic diseases, which were exclusion that the 3 g provided corresponded to the consumption of criteria of this study. 1.2 g of sodium from regular salt. Of the 56 eligible patients, 38 agreed to participate On the other hand, light salt (with approximately 67% and were selected for the study from May to October 2012 less sodium than the regular one) promoted a mean intake (19 in each group). The calculated sample (40 individuals) of 390 mg of sodium per day. This sodium restriction was was not reached, because most of the patients reported capable of promoting the BP and sodium excretion control personal problems or difficulties as a justification for not observed in the IG, suggesting that, at least in the short-term, participating. In addition, after beginning the research and the use of light salt has proven to be an efficient strategy due to patients’ personal reasons (illness in family or lack for SAH treatment. of time), there was a follow-up loss of three CG individuals Despite demonstrating a significant BP reduction after prior to the intervention, which resulted in a final sample decreasing salt intake, the mean sodium excretion values in of 35 patients (19 in IG and 16 in CG) (Figure 1). the present study were higher than 125 mEq (IG) and 180 mEq (CG), indicating that the provided sodium amount (1.2 g At the beginning of the study, the groups were from regular salt and 390 mg from light salt) may not have homogeneous in regard to socio-demographic variables. corresponded to that consumed by the patients. This shows The mean age was 55.5 ± 7.4 years, and 65.7% of the difficulty of changing a lifestyle, because salt consumption the sample corresponded to women. The majority had is a strong habit around the world. Ortega et al15 have found completed the Brazilian middle school, that is, up to the a dietary salt intake of 9 g/day on Spanish adults between 18 8th grade (n = 28; 80.0%), had a family income of up to and 60 years, with 168 mmol/d sodium excretion on 24-hour US$ 600 (n = 17; 48.6%) and practiced regular physical urine. It reflects ingestion above the recommended 5 g per activity (n = 19; 54.3%). Two patients (5.7%) were smokers day, just as in the present study. and 40.0% consumed alcohol regularly (n = 14). Unfortunately, the design of this study could not follow Regarding the clinical variables, both groups were also patients on their routine to check if there was a complete homogeneous at the beginning of the study (Table 1). adhesion regarding their sodium intake, although they The recovery percentage of BP measurements was 100%. had been strongly instructed about how to use the given salt and how to avoid all sodium-rich food. It seems they After intervention may have eaten less sodium than usual, but not exactly as recommended, justifying sodium excretion above Seven days from the end of the research, one CG expected. Besides, 24-hour excretion values showed only patient asked for more salt. At the end of the study, casual the previous-day sodium and potassium intake, which BP measurements (SBP and DBP) and 24-hour sodium could have been atypical as compared to that of other days, excretion significantly differed between IC and CG. even with all the recommendations. The same argument However, no significant differences in BMI values, HBPM may apply to the unchanged potassium excretion of IG, and potassium urine excretion were observed between the in addition to the fact that the light salt potassium content groups (Table 2). is low to cause a significant difference in urine excretion, The intragroup analysis demonstrated that the mean BP which could vary more with a higher than usual potassium- measurements of IG patients were significantly reduced rich food ingestion. after using light salt. Likewise, sodium excretion was Resistance to lifestyle changes to control SAH is significantly decreased in that group. No change was common among hypertensive patients16. In addition, observed in 24-hour urine potassium excretion (Table 3). another limiting aspect that impairs adhesion to SAH Concerning the CG individuals, the only significant control was detected among IG patients, because 89.5% difference after using regular salt was a SBP reduction, reported identifying a bitter taste in their foods. Although because the other variables (casual DBP, SBP and DBP by the same patients reported that the 28-day period of diet HBPM and urine sodium and potassium values) remained was acceptable, they were reluctant to comply with the similar (Table 4). suggestion of maintaining light salt after the conclusion

130 Arq Bras Cardiol. 2015; 104(2):128-135 Barros et al. Salt intake and blood pressure

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Assessed for eligibility (n=56)

Excluded (n = 8) - Declined to participe (n = 14) Enrollment - Other reasons (n = 4)

Randomized (n = 38)

Allocation Intervention Group – IG (n = 19) Control Group – CG (n = 19)

Lost to follow (n = 3) Lost to follow-up (n = 0) Follow-up - Personal reasons

Analysis Analised (n = 16) Analised (n = 19)

Figure 1 - Flow diagram of patients’ randomization.

Table 1 - Mean values of the evaluated variables before intervention, according to groups. Goiânia city, Goiás state, Brazil, 2012 (n = 35)

CG (n= 16) IG (n= 19) p*

Mean ± SD Mean ± SD

BMI 31.00 ± 5.97 29.38 ± 5,55 0.411

Casual BP: SBP 143.44 ± 13.99 142.95 ± 14.86 0.921 DBP 91.19 ± 9.10 89.79 ± 9.10 0.654

HBPM: SBP 131.63 ± 14.36 134.47 ± 17.00 0.600 DBP 79.38 ± 11.65 77.95 ± 10.23 0.702 Sodium 213.56 ± 89.99 205.87 ± 131.50 0.844 Potassium 54.41 ± 17.01 74.53 ± 76.14 0.309 CG: control group; IG: intervention group; BMI: body mass index (kg/m²); BP: blood pressure; DBP: diastolic blood pressure (mmHg); HBPM: home blood pressure monitoring; Potassium: 24-hour urine potassium (mEq/day); SBP: systolic blood pressure (mmHg); SD: standard deviation; Sodium: 24-hour urine sodium (mEq/day). *Student t test for independent samples.

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Table 2 - Mean values of the evaluated variables after intervention, according to groups. Goiânia city, Goiás state, Brazil, 2012 (n = 35)

CG IG IG x CG p*

Mean ± SD Mean ± SD Difference

BMI 31.19 ± 6.14 29.44 ± 5.50 (1.75) 0.379 Casual BP: SBP 137.19 ± 20.22 127.11 ± 15.64 (12.47) 0.034 DBP 82.75 ± 12.12 75.95 ± 9.47 (7.58) 0,046

HBPM: SBP 131.06 ± 15.53 127.47 ± 15.33 (4.78) 0.365 DBP 80.13 ± 11.75 73.42 ± 10.78 (7.19) 0.074 Sodium 182.59 ± 76.74 127.11 ± 57.39 (55.59) 0.023 Potassium 55.33 ± 18.43 48.05 ± 19.29 (7.03) 0.296 CG: control group; IG: intervention group; BMI: body mass index (kg/m²); BP: blood pressure; DBP: diastolic blood pressure (mmHg); HBPM: home blood pressure monitoring; Potassium: 24-hour urine potassium (mEq/day); SBP: systolic blood pressure (mmHg); SD: standard deviation; Sodium: 24-hour urine sodium (mEq/day). *Student t test for independent samples.

Table 3 - Mean values of casual BP and HBPM in the IG (light salt) before and after intervention. Goiânia city, Goiás state, Brazil, 2012 (n = 19)

IG (before) IG (after) Before x After p Mean ± SD Mean ± SD Difference Casual BP: SBP 142.95 ± 14.86 127.11 ± 15.64 (-15.84) 0.002* DBP 89.79 ± 9.10 75.95 ± 9.47 (-13.84) 0.000** HBPM: SBP 134.47 ± 17.00 127.47 ± 15.33 (-7.00) 0.012** DBP 77.95 ± 10.23 73.42 ± 10.78 (-4.53) 0.003** Sodium 205.87 ± 131.50 127.11 ± 57.39 (-78.76) 0.016 ** Potassium 74.53 ± 76.14 48.05 ± 19.29 (-9.40) 0.157** IG: intervention group; BMI: body mass index (kg/m²); BP: blood pressure; DBP: diastolic blood pressure (mmHg); HBPM: home blood pressure monitoring; Potassium: 24-hour urine potassium (mEq/day); SBP: systolic blood pressure (mmHg); SD: standard deviation; Sodium: 24-hour urine sodium (mEq/day). * Wilcoxon’s test. **Student t test for matched samples.

Table 4 - Mean values of casual BP and HBPM in the CG (regular salt) before and after intervention. Goiânia city, Goiás state, Brazil, 2012 (n = 16)

CG (before) CG (after) Before x After p Mean ± SD Mean ± SD Difference Casual BP: SBP 143.44 ± 13.99 137.19 ± 20.22 (-6.25) 0.032** DBP 91.19 ± 9.10 82.75 ± 12.12 (-8.44) 0.055* HBPM: SBP 131.63 ± 14.36 131.06 ± 15.53 (-0.57) 0.858** DBP 79.38 ± 11.65 80.13 ± 11.75 (+0.75) 0.587** Sodium 213.56 ± 89.99 182.59 ± 76.74 (-30.97) 0.175** Potassium 54.41 ± 17.01 55.33 ± 18.43 (+0.92) 0.796** CG: control group; BMI: body mass index (kg/m²); BP: blood pressure; DBP: diastolic blood pressure (mmHg); HBPM: home blood pressure monitoring; Potassium: 24-hour urine potassium (mEq/day); SBP: systolic blood pressure (mmHg); SD: standard deviation; Sodium: 24-hour urine sodium (mEq/day). * Wilcoxon’s test. **Student t test for matched samples.

132 Arq Bras Cardiol. 2015; 104(2):128-135 Barros et al. Salt intake and blood pressure

Original Article of the study, even after acknowledging the better control Another meta-analysis allowed the conclusion that, when of their BP. compared to the current recommendations, a reduction in The possible explanation for the taste change of light salt salt consumption to 3 g per day may have much better results is the potassium added to its composition. In spite of this and, thus, that should be used as the daily intake goal around 4 negative effect, the scientific community often discusses the world . Such conclusion reinforces this study’s findings not only the beneficial effect on BP of reducing sodium to obtain more favorable results on BP control. intake, but also the positive impact that potassium intake The sodium excretion values found in the present study may have on SAH control17,18. suggest that the patients may have consumed amounts Similarly to the present study, Lotaif et al17 used a salt higher than 3 g of the provided salt, as already mentioned. with lower sodium content and added potassium in order Nevertheless, BP control at the end of the intervention to verify its effect on BP. Patients were separated into a demonstrated that the intake prior to the study was probably control group (regular salt) and an intervention group (light excessive as compared to that during the study. Therefore, salt), and had their BP assessed by using Ambulatory Blood the reduction in salt consumption had beneficial effects on Pressure Monitoring (ABPM), as well as their blood and urine SAH, as seen in this study’s results. sodium and potassium levels assessed. The intervention These data reinforce the relevance of the present study, group showed BP reduction. On the other hand, sodium once dietary sodium restriction has been constantly referred levels showed no change in any of the groups, whereas the to as a factor contributing to SAH control, and, consequently, intervention group showed a significant increase only in to reduce mortality due to CVD2-4. plasma potassium levels. The authors attributed BP control Therefore, changes in eating habits, such as decreasing to potassium supplementation and not to the reduction in the consumption of addicted salt and processed food, which sodium intake. represents more than 75% of sodium intake of the North The literature, however, still lacks conclusive studies American’s diet22, proposed in this study, are a valuable tool on the need for medical supplementation of potassium to for SAH control and reduction in the risk of mortality due to control BP, being a balanced diet, rich in fruits and vegetables, CVD, which is the major cause of death in the world. 14 the best way to obtain that mineral . The significant reduction in casual SBP in the CG, with In accordance with the results of the study by Lotaif et al17, no changes in HBPM values, may have occurred due to the potassium excretion values obtained at the beginning and attenuation of the white-coat effect in the study. end of this study were similar. It indicates that, in the present One limitation of this study was the change in food taste study, salt intake from light salt may not have influenced the attributed to light salt by a large number of IG patients. total balance ingested by patients, since the daily potassium This fact did not interfere with the use of light salt during need for adult individuals, which contributes to BP control, this study, but it may be a limiting factor regarding long‑term 19 is 4.7 g . adhesion. Finally, salt sensitivity, which varies among Therefore, the potassium content consumed by the IG individuals, may have influenced the patients’ BP levels. may not have accounted for the BP reduction of those patients, because the additional amount ingested from light salt was only 1038 mg. This fact reinforces the hypothesis Conclusion that the BP control of this study was due to sodium In conclusion, potassium-enriched light salt substitution reduction, much more effective with the use of light salt for regular salt was efficient in reducing hypertensive patients’ and, additionally, confirmed with the significant reduction BP in this study. Thus, the long-term implementation of in sodium excretion of IG patients. that change could be interesting to reduce population Such reduction in sodium intake is an important factor hypertension and even mortality due to CVD. Health care considering the reduction in CVD mortality, as shown in the professionals could use these results to explain to their study with individuals from five kitchens of a retirement home patients how sodium intake can raise BP and encourage them in northern Taiwan, who were separated into two groups. The to reduce it by using light salt. Furthermore, hospitals could first group was given a potassium-enriched lower sodium salt, control BP of hypertensive patients by using that type of salt and the other group, only regular salt. A significant reduction in their diet during hospitalization and light salt’s industry in CVD mortality was observed in the experimental group7. could create strategies so that its flavor is not a limiting factor for its use, since the benefits of its consumption were evident. A systematic review has shown randomized trials of dietary counseling methods on sodium intake reduction to treat hypertension and identified a significant reduction in BP Author contributions 20 and sodium excretion when sodium intake was restricted . Conception and design of the research: Barros CLA, Sousa Sodium intake reduction has also been proven to be ALL, Jardim TSV, Jardim PCBV; Acquisition of data: Barros efficient in the SAH control of 169 individuals, separated CLA, Chinem BM, Rodrigues RB; Analysis and interpretation into two groups, one with low sodium intake and the other of the data: Barros CLA, Sousa ALL, Chinem BM, Rodrigues with placebo, submitted to a double-blind crossover study21. RB, Jardim TSV, Souza WKSB, Jardim PCBV; Statistical analysis: In the present study, the decrease in BP reached 15.84 x Barros CLA, Sousa ALL, Jardim PCBV; Obtaining Financing: 13.84 mmHg (IG) and 6.25 x 8.44 mmHg (CG), although Barros CLA, Jardim PCBV; Writing of the manuscript: Barros the reduction was significant only in IG. CLA, Sousa ALL, Jardim PCBV; Critical revision of the

Arq Bras Cardiol. 2015; 104(2):128-135 133 Barros et al. Salt intake and blood pressure

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manuscript for intellectual content: Barros CLA, Sousa ALL, Sources of Funding Chinem BM, Rodrigues RB, Jardim TSV, Carneiro SB, Souza This study was partially funded by CAPES. WKSB, Jardim PCBV.

Study Association Potential Conflict of Interest This article is part of the dissertation of Carolina Lôbo de No potential conflict of interest relevant to this article was Almeida Barros by Programa de Pós-Graduação em Nutrição reported. e Saúde da Universidade Federal de Goiás.

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3. Pimenta E, Gaddam KK, Oparil S, Aban I, Husain S, Dell’Italia LJ, et al. 14. World Health Organization. (WH). Diet, nutrition and the prevention of chronic Effects of dietary sodium reduction on blood pressure in subjects with diseases. Geneva; 2003. (WHO Technical Report Series, 916) resistant hypertension: results from a randomized trial. Hypertension. 2009;54(3):475-81. 15. Ortega RM, López-Sobaler AM, Ballesteros JM, Pérez-Farinós N, Rodríguez- Rodréguez E, Aparicio A, et al. Estimation of salt intake by 24 h urinary 4. Sarno F, Jaime PC, Ferreira SR, Monteiro CA. [Sodium intake and sodium excretion in a representative sample of Spanish adults. Br J Nutr. metabolic syndrome: a systematic review]. Arq Bras Endocrinol Metabol. 2011;105(5):787-94. 2009;53(5):608-16. 16. Dosse C, Cesarino CB, Martin JF, Castedo MC. Factors associated to patients’ 5. Elliot P, Stamler J, Nichols R, Dyer AR, Stamler R, Kestellot H. Intersalt noncompliance with hypertension treatment. Rev Lat Am Enfermagem. revisited: further analyses of 24 hour sodium excretion and blood 2009;17(2):201-6. pressure within and across populations. Intersalt Cooperative Research Group. BMJ. 1996;312(7041):1249-53. Erratum in: BMJ. 17. Lotaif LA, Kohlmann Junior O, Zanella MT, Kohlmann NE, Ribeiro AB. Efeito 1997;315(7106):458. da suplementação de potássio através do sal de cozinha na hipertensão arterial primária leve a moderada. J Bras Nefrol. 1995;17(4):214-8. 6. O’Donnell MJ, Yusuf S, Mente A, Gao P, Mann JF, Teo K, et al. Urinary sodium and potassium excretion and risk of cardiovascular events. JAMA. 18. Van Horn L, Mccoin M, Kris-Etherton PM, Burke F, Carson JA, Champagne CM, et 2011;306(20):2229-38. al. The evidence for dietary prevention and treatment of cardiovascular disease. J Am Diet Assoc. 2008;108(2):287-331. 7. Chang HY, Hu YW, Yue CS, Wen YW, Yeh WT, Hsu LS, et al. Effect of potassium-enriched salt on cardiovascular mortality and medical 19. Institute of Medicine. Dietary Reference Intakes for Water, Potassium, Sodium, expenses of elderly men. Am J Clin Nutr. 2006;83(6):1289-96. Chloride, and Sulfate. Food and Nutrition Board. Washington, DC: National Academy Press; 2004. 8. Whelton PK. Urinary sodium and cardiovascular disease risk. JAMA. 2011;306(20):2262-64. 20. Ruzicka M, Hiremath S, Steiner S, Helis E, Szczotka A, Baker P, et al. What is the feasibility of implementing effective sodium reduction strategies to 9. Sociedade Brasileira de Cardiologia, Sociedade Brasileira de treat hypertension in primary care settings? A systematic review. Hypertens. Hipertensão, Sociedade Brasileira de Nefrologia. VI Diretrizes brasileiras 2014;32(7):1388-94. de hipertensão. Arq Bras Cardiol. 2010;95(1 supl. 1):1-51. Erratum in: Arq Bras Cardiol. 2010;95(4):553. 21. He FJ, Marciniak M, Visagie E, Markandu ND, Anand V, Dalton RN, et al. Effect of modest salt reduction on blood pressure, urinary albumin, and pulse 10. Sociedade Brasileira de Cardiologia, Sociedade Brasileira de wave velocity in white, black, and asian mild hypertensives. Hypertension. Hipertensão, Sociedade Brasileira de Nefrologia. V Diretrizes Brasileiras 2009;54(3):482-8. de Monitorização Ambulatorial da Pressão Arterial (MAPA) e III Diretrizes Brasileiras de Monitorização Residencial de Pressão Arterial (MRPA). Arq 22. Antman EM, Appel LJ, Balentine D, Johnson RK, Steffen LM, Miller EA, et Bras Cardiol. 2011;97 (3 supl.3):1-24. al. Stakeholder Discussion to reduce population-wide sodium intake and decrease sodium in the food supply: a Conference Report From the American 11. Oesch U, Ammann D, Simon W. Ion-selective membrane electrodes for Heart Association Sodium Conference 2013 Planning Group. Circulation. clinical use. Clin Chem. 1986;32(8):1448-59. 2014;129(25):e660-79.

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Arq Bras Cardiol. 2015; 104(2):128-135 135 Original Article

Effects of Ischemic Postconditioning on the Hemodynamic Parameters and Heart Nitric Oxide Levels of Hypothyroid Rats Sajad Jeddi1,2, Jalal Zaman1,2, Asghar Ghasemi1,2 Endocrine Physiology Research Center - Research Institute for Endocrine Sciences - Shahid Beheshti University of Medical Sciences1, Tehran, Iran; Endocrine Research Center - Research Institute for Endocrine Sciences - Shahid Beheshti University of Medical Sciences2, Tehran - Iran

Abstract Background: Ischemic postconditioning (IPost) is a method of protecting the heart against ischemia-reperfusion (IR) injury. However, the effectiveness of IPost in cases of ischemic heart disease accompanied by co-morbidities such as hypothyroidism remains unclear. Objective: The aim of this study was to determine the effect of IPost on myocardial IR injury in hypothyroid male rats. Methods: Propylthiouracil in drinking water (500 mg/L) was administered to male rats for 21 days to induce hypothyroidism. The hearts from control and hypothyroid rats were perfused in a Langendorff apparatus and exposed to 30 min of global ischemia, followed by 120 min of reperfusion. IPost was induced immediately following ischemia. Results: Hypothyroidism and IPost significantly improved the left ventricular developed pressure (LVDP) and peak rates of positive and negative changes in left ventricular pressure (±dp/dt) during reperfusion in control rats (p < 0.05). However, IPost had no add-on effect on the recovery of LVDP and ±dp/dt in hypothyroid rats. Furthermore, hypothyroidism significantly

decreased the basal NO metabolite (NOx) levels of the serum (72.5 ± 4.2 vs. 102.8 ± 3.7 µmol/L; p < 0.05) and heart (7.9 ± 1.6

vs. 18.8 ± 3.2 µmol/L; p < 0.05). Heart NOx concentration in the hypothyroid groups did not change after IR and IPost, whereas these were significantly (p < 0.05) higher and lower after IR and IPost, respectively, in the control groups. Conclusions: Hypothyroidism protects the heart from IR injury, which may be due to a decrease in basal nitric oxide (NO) levels in the serum and heart and a decrease in NO after IR. IPost did not decrease the NO level and did not provide further cardioprotection in the hypothyroid group. (Arq Bras Cardiol. 2015; 104(2):136-143) Keywords: Ischemic Postconditioning; Reperfusion; Hypothyroidism; Nitric Oxide; Rats.

Introduction upon initiation of reperfusion after prolonged coronary artery 3,4 Acute myocardial infarction (AMI) is the principle cause of occlusion . Although IPost has various clinical applications, human mortality worldwide1,2, and its prevalence is increasing most investigations currently conducted on IPost involve because of aging and co-morbid diseases such as obesity, healthy myocardium. Coronary artery disease frequently 5 diabetes, and thyroid disorders1-3. AMI is often induced co-exists with other morbidities ; therefore, further research by the partial occlusion of coronary arteries at the site of a on the pathological condition before the use of the IPost in 5 ruptured atherosclerotic plaque1,4. Although reperfusion can clinical conditions is necessary . rescue the ischemic myocardium from unavoidable death, it Hypothyroidism is a major thyroid gland disease that also can also induce side effects, known as ischemia-reperfusion affects the heart and has been implicated in an increase (IR) injuries4. The myocardial response to ischemia can in morbidity6. Low thyroid hormone levels could affect be modulated by different interventions such as ischemic the response of the heart to IR injuries6. It is therefore postconditioning (IPost)3,5. IPost is an effective mechanism of essential to determine the clinical utility of cardioprotective protecting the myocardium from IR injuries and is induced interventions such as IPost in hypothyroid states to design by cycles of brief IR periods that are immediately performed future management strategies. Nitric oxide (NO) is mainly synthesized by NO synthase enzymes in the heart and plays an important role in cardiac functions. Ischemia of the heart leads to an increase in NO Mailing Address: Asghar Ghasemi • production that might contribute to IR injury. However, Endocrine Physiology Research Center,Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Nº 24, Parvaneh no studies have examined the changes in NO content in Street, Velenjak. Tehran – Iran the hearts of hypothyroid rats. Therefore, the aim of this Email: [email protected]; [email protected] study was to determine the response of the heart to IPost Manuscript received May 18, 2014; revised manuscript July 21, 2014; accepted August 08, 2014. in a propylthiouracil-induced hypothyroidism rat model.

In addition, changes in NO metabolites (NOx) following IR DOI: 10.5935/abc.20140181 injury and IPost were also assessed.

136 Jeddi et al. Ischemic postconditioning in hypothyroid rats

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Methods parameters, including left ventricular developed pressure (LVDP) as an index of systolic function, the peak rates of Animals positive changes in the left ventricular pressure (+dP/dt) as an index of contraction, negative changes in the left Forty-eight 2-month-old male Wistar rats were obtained ventricular pressure (−dp/dt) as an index of relaxation, and from the laboratory animal house of the Research Institute for left ventricular end diastolic pressure (LVEDP) as an index Endocrine Sciences, Shahid Beheshti University of Medical of contracture. Initially, the average LVEDP was adjusted Sciences. We set a 2-sided α of 0.05 and a power of 90%, to 5–10 mmHg in all hearts by filling the latex balloon with and the sample size of each group was calculated to be 8 water; LVEDP, LVDP, and ± dp/dt were digitalized by a data 222 222 7 (Z1/ab21 + Z)× (S12 + S) (1.96 + 1.29)´ (13.52 + 9.07 ) using the formula : n = = acquisition system (PowerLab,= AD 7.75 Instruments,~ 8 Australia). 2 2 d Post-ischemic(89 70)hemodynamic parameters were assessed by 222 222 (Z1/ab21 + Z)× (S12 + S) (1.96 + 1.29)´ (13.52 + 9.07 ) 2 the recovery of LVEDP, LVDP, and ± dp/dt and expressed n = = = 7.75~ 8; where µ1, S1 2 2 d (89 70) in relation to their baseline values. 2 and µ2, S2 are the means and variances of the two groups, 2 2 respectively, and d = (µ1 – µ2) . Rats were housed in an Measurement of NOx animal room with a temperature of 22 ± 3°C and a relative Serum and heart NOx were measured using the Griess humidity of 50 ± 6% and given free access to standard rat method. Briefly, after 2 h of reperfusion, myocardial chow (Pars Co., Tehran) and tap water during the study. samples from the left ventricle (LV) of the hearts were The animals were adapted to an inverse 12:12 h light/dark rinsed, homogenized in PBS (1:5, w/v), and centrifuged at cycle. All experimental procedures employed, as well as 15,000 g for 20 min. The supernatant was deproteinized rat care and handling, were in accordance with guidelines by adding zinc sulfate (15 mg/mL). The serum samples provided by the local ethics committee of the Research were also deproteinized by zinc sulfate (15 mg/mL) and Institute for Endocrine Sciences, Shahid Beheshti University centrifuged at 10,000 g for 10 min. A 100-μL aliquot of the of Medical Sciences. Hypothyroidism was induced by adding supernatant or serum sample was transferred to a microplate propylthiouracil (PTU) (500 mg/L) to the drinking water for well and 100 μL of vanadium (III) chloride (8 mg/mL) was 21 days5,6. To determine the efficacy of the PTU treatment, added to each well to reduce nitrate to nitrite. Afterwards, changes in serum total T4, T3, and TSH levels and citrate 50 μL of sulfanilamide (2%) and 50 μL of N-1-(naphthyl) synthase (CS) activity in the soleus muscle (Srere et al8 ethylenediamine (0.1%) were added to the samples and method8) were assessed in two study groups (control and incubated for 30 min at 37°C; absorbance was read at hypothyroid). After the establishment of the hypothyroid a wavelength of 540 nm using an ELISA reader (BioTek, model, animals in the control and hypothyroid groups Powerwave XS2,). NOx concentrations were determined were further divided into three subgroups [i.e., control, from the linear standard curve established using 0–100 μM control‑IR (C-IR), and control‑IPost (C-IPost), and hypothyroid, sodium nitrate. Serum and tissue NOx levels were expressed hypothyroid-IR (H‑IR), and hypothyroid-IPost (H-IPost)] as µmol/L. Inter-assay coefficient of variation was 4.1%. for in vitro experiments. Serum total T4, total T3, and TSH levels were measured at end of the treatment period using enzyme-linked immunosorbent assay (ELISA) kits. Inter-assay Statistical analysis coefficients of variation were 3.2% for total T4, total T3, and All values were expressed as the mean ± SEM. Statistical TSH levels. analysis was performed using the SPSS software (SPSS, Chicago, IL, USA; version 20); repeated measurement Experimental protocol ANOVA was used to compare hemodynamic parameters at All rats were anesthetized by intraperitoneal injection of various time points. One-way ANOVA with Tukey’s post-hoc ketamine/xylazine (50 mg/kg and 10 mg/kg, respectively). test was used to compare heart NOx levels among different The hearts of the control and hypothyroid rats were groups. The student’s sample t-test was used to compare immediately isolated and placed in an ice-cold perfusion serum NOx levels, T3, T4, TSH, and CS activity between buffer; after aorta cannulation, the hearts were perfused control and hypothyroid groups. Two-sided p-values < 0.05 in the Langendorff apparatus using Krebs–Henseleit buffer were considered statistically significant.

[containing 118 mM NaCl, 25 mM NaHCO3, 4.7 mM KCl, 1.2 mM MgCl , 2.5 mM CaCl , 1.2 mM KH PO , and 2 2 2 4 Results 11 mM glucose at a constant pressure (75 mmHg) and a pH level of 7.4], and the Krebs solution was oxygenated Serum thyroid hormone levels and soleus muscle CS activity significantly decreased, whereas serum TSH significantly with 95% O2 and 5% CO2. The hearts were stabilized for 20 min to obtain the baseline data. In the C-IR and increased in hypothyroid rats. In addition, weight changes H-IR groups, after 20 min of stabilization, the hearts were significantly lower in hypothyroid rats (Table 1). were subjected to 30 min of global ischemia, followed Basal hemodynamic parameters were significantly lower by 120 min of reperfusion. IPost was induced by 6 cycles in the hypothyroid group than in the controls (Table 2). of 10-s reperfusion-10-s ischemia immediately following When ischemia was induced by pausing coronary perfusion, the 30-min global ischemia; a latex balloon was inserted the LVDP, ± dp/dt, and heart rate rapidly decreased and into the left ventricle to measure various hemodynamic ceased in the isolated hearts.

Arq Bras Cardiol. 2015; 104(2):136-143 137 Jeddi et al. Ischemic postconditioning in hypothyroid rats

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Table 1 – Characteristics of control and hypothyroid rats

Controls (n = 8) Hypothyroid rats (n = 8) Weight change (g) 20.3 ± 3.2 8.1 ± 3.5* T3 (nmol/L) 0.76 ± 0.06 0.20 ± 0.04* T4 (nmol/L) 49.43 ± 2.34 17.65 ± 3.42* TSH (ng/mL) 6.8 ± 0.6 29.62 ± 3.7* Citrate synthase activity (µmol/mL/min) 1.2 ± 0.3 0.45 ± 0.01* Data are expressed as mean ± SEM.; * p < 0.05.

Table 2 – Baseline cardiac function

Controls (n = 8) Hypothyroid rats (n = 8) LVEDP (mmHg) 8.5 ± 2.2 8.8 ± 2.8 LVDP (mmHg) 96.8 ± 7.6 74.0 ± 6.3* +dp/dt (mmHg/s) 3135 ± 211 2352 ± 434* –dp/dt (mmHg/s) 2215 ± 185 1684 ± 124* Heart rate (pulse/min) 283.3 ± 10.4 170.2 ± 11.3* Data are expressed as the mean ± SEM. LVEDP: left ventricular end diastolic pressure; LVDP: left ventricular developed pressure: and the peak rates of positive and negative changes in left ventricular pressure (±dp/dt); *p < 0.05.

The hearts from the H-IR group showed significant Baseline LVEDP, LVDP, and ± dp/dt were lower in recovery in post-ischemic LVDP and ± dp/dt after 30 min the hypothyroid groups, compared to the controls, of ischemia and 120 min of reperfusion compared to those and these results were similar to those of previous from the C-IR group. IPost significantly improved the LVDP studies5,6,9,10. Several changes, including upregulation of and ± dp/dt during reperfusion in the C-IPost group. In phospholamban and V3 isomyosin and downregulation contrast, IPost failed to increase recovery of LVDP and ± of SR Ca2+-ATPase and ryanodine receptor occur in the dp/dt in the H-IPost group (Figure 1). During the 30-min heart of hypothyroid rats; therefore, cardiac dysfunction ischemia, the hypothyroid group displayed a significant is a frequent consequence of hypothyroidism5,6,9,10. decrease in LVEDP, compared to the controls. IPost In response to IR, the hypothyroid group showed an significantly prevented the reperfusion-induced increase increased recovery of LVEDP, LVDP and ± dp/dt, which in LVEDP in the C-IPost group (Figure 2). indicates that hypothyroidism can protect the heart from IR 6,11 Serum and heart NOx levels were significantly lower in injury. These findings are supported by previous studies . the hypothyroid group, compared to the control. IR and The protective mechanisms against IR in the hearts of IPost had no effect on heart NOx levels in the hypothyroid the hypothyroid group have not been entirely elucidated group, whereas in the control group, IR induced a marked and might be due to changes in metabolism6,11,12. Previous increase in heart NOx levels and IPost significantly studies have shown that in hypothyroid rats, ATP, oxygen, decreased the IR-induced increase in heart NOx levels and glycogen levels slowly decrease during ischemia and are (Figure 3). higher at the start of reperfusion6,11-13. On the other hand, the hearts of hyperthyroid rats, despite having a high metabolism, were protected from IR injury; therefore, tolerance of the Discussion hypothyroid group to IR injury cannot be solely explained by Our findings indicate that hypothyroidism decreases the low metabolism during ischemia14. injuries induced by IR in the rat heart, which may be due Different molecular pathways, including total c-jun to the reduction in NO. IPost provides protection against IR NH2-terminal kinases, mitogen-activated protein kinases, injury in control rats, whereas no add-on effect was observed and NO play an essential role in the response of the in hypothyroid rats. heart to IR injury6,15,16. Our results showed that the The decrease in weight change and CS activity in the baseline serum and heart levels of NOx were lower in soleus muscle and the decrease in the level of circulating the hypothyroid groups; NOx levels increased after IR thyroid hormone with elevated TSH levels all indicate that in control group, whereas no change was observed in hypothyroidism has been successfully induced. hypothyroid group. A decrease in heart NOx levels in fetal

138 Arq Bras Cardiol. 2015; 104(2):136-143 Jeddi et al. Ischemic postconditioning in hypothyroid rats

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Control Hypotyroid

100 100 Ischemia Reperfusion Ischemia Reperfusion 80 80

60 60

40 40 H-IR C-IR H-Ipost

LVDP (% of baseline) 20 C-Ipost LVDP (% of baseline) 20

0 0 0306090 120 150 0306090 120 150 A1 Time (min) A2 Time (min)

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60 60 H-IR 40 40 H-Ipost C-IR

20 +dp/dt (% of baseline) 20

+dp/dp (% of baseline) C-Ipost 0 0 0306090 120 150 0306090 120 150 B1 Time (min) B2 Time (min)

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40 40 C-IR H-IR

-dp/dp (% of baseline) H-Ipost -dp/dp (% of baseline) 20 C-Ipost 20

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60 60 H-IR 40 40 C-IR H-Ipost

HR (% of baseline)

HR (% of baseline) 20 C-Ipost 20

0 0 0306090 120 150 0306090 120 150 D1 Time (min) D2 Time (min)

Figure 1 – Recovery of cardiac function after IR injury; A. Left ventricular developed pressure (LVDP); B. Peak rates of positive changes in left ventricular pressure (+dp/dt); C. Peak rates of negative changes in left ventricular pressure (−dp/dt); D. Heart rate; Control-IR, C-IR; Control-IPost, C-IPost; Hypothyroid-IR, H-IR; Hypothyroid-IPost, H-IPost; values are expressed as the mean ± SEM; (n = 8 rats); *p < 0.05 as compared to the C-IR group.

Arq Bras Cardiol. 2015; 104(2):136-143 139 Jeddi et al. Ischemic postconditioning in hypothyroid rats

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60 Ischemia Reperfusion

40

LVEDP (mmHg) 20

C-IR H-Ipost

C-Ipost H-IR 0 03060 90 120 150 Time (min)

Figure 2 – Change in LVEDP (ischemic contracture) during the experiment; left ventricular end diastolic pressure (LVEDP); values are expressed as mean ± SEM;(n = 8 rats); *p < 0.05 as compared to the C-IR group. Control-IR, C-IR; Control-IPost, C-IPost; Hypothyroid-IR, H-IR; Hypothyroid-IPost, H-IPost;

hypothyroid rats has been previously reported17, suggesting have lost its efficacy in a hypothyroid group. Our findings were that low levels of NOx during the experimental period similar to the results of previous studies on diabetic rats, which might be a significant element of hypothyroid-induced show that preconditioning and IPost lose their protective effects cardioprotection. against IR injuries in the hearts of diabetic rats4,29. The function of NO in myocardial IR injury has not The mechanism behind the effect of IPost in the been clearly demonstrated and is a very complex issue. unhealthy myocardium has not been established. Several Some studies have described the protective role of NO, studies have indicated that IPost and preconditioning whereas others have reported a detrimental role18-20. protect the hearts of healthy rats from IR injury by Recent experimental studies have indicated that the NO decreasing its NO content during ischemia18,24,30,31. Similarly, level of heart tissues is within a low range at baseline and in the present study, the application of six cycles of IPost increases during ischemia because it triggers the enzymatic significantly decreased the heart NOx levels after a 30-min (through NO synthase 3) and non-enzymatic (tissue acidosis) ischemia in the C-Ipost group. However, this reduction was production of NO. Although a small increase in NO not observed in the H-IPost group, and the application of content may be cardioprotective, a large increase appears Ipost did not result in an additional decrease in the NOx to be detrimental19,21-25; the detrimental effect of NO in level in the hearts of hypothyroid group. These results the heart in response to IR is mediated by peroxynitrite18. demonstrate the inefficiency of IPost in providing additional At high levels, NO reacts with superoxide and produces protective effects against IR injury in the hypothyroid peroxynitrite, which is a highly toxic agent that could induce group. Both IPost and hypothyroidism reduced the levels apoptosis in heart cells. Thus, it could be hypothesized of NOx in response to IR and protected the heart from IR that hypothyroidism protects the heart from ischemia by injury, indicating that NOx is a critical component of the decreasing NO production and subsequently reducing the protection response. 18,20,24-28 levels of nitro-oxidative stress . With regard to the limitations of this study, we did not In the present study, IPost protected the heart against IR measure NO synthase activity, which might play a role in injury in the C-IPost group through the recovery of LVEDP, the cardioprotective effects of IPost on hypothyroid rats18,32. LVDP and ± dp/dt, whereas no significant effects on the hearts In addition, our results were limited to male rats, whereas in the H-IPost group were observed, indicating that IPost might hypothyroidism is more prevalent among females33.

140 Arq Bras Cardiol. 2015; 104(2):136-143 Jeddi et al. Ischemic postconditioning in hypothyroid rats

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40

30

m

x 20

Heart NO (10 mol/L)

0

C-IR H-IR Control C-Ipost H-Ipost Hypotyroid A1

150

100

mmol/L

50

Serum NOx ( )

0

A2 d Control Hypotyroi

Figure 3 – Change in NOx levels in the control and hypothyroidism groups in the heart (above) and serum (below). Control-IR, C-IR; Control-IPost, C-IPost; Hypothyroid-IR, H-IR; Hypothyroid-IPost, H-IPost; values are expressed as the mean ± SEM; (n = 8 rats); *p < 0.05 as compared to the control group. # p < 0.05 as compared to the C-IR group.

Conclusion Obtaining financing: Ghasemi A; Critical revision of the manuscript for intellectual content: Ghasemi A. Hypothyroidism increased the recovery of LVEDP, LVDP, and ± dp/dt following IR in the rat heart, which might be due to a decrease in the basal levels of NOx and NOx Potential Conflict of Interest after the IR period. Although IPost imparted protective No potential conflict of interest relevant to this article was effects, these were not related to further decreases in reported. NOx levels. The abolished protective effect of IPost in the hypothyroid group could be attributable to the impairment Sources of Funding of the NO pathway. This study was funded by Endocrine Research Center - Research Institute for Endocrine Sciences - Shahid Beheshti Author contributions University of Medical Sciences. Conception and design of the research: Jeddi S, Ghasemi A; Acquisition of data: Jeddi S, Zaman J; Analysis and Study Association interpretation of the data: Jeddi S; Statistical analysis and This article is part of the thesis of Doctoral submitted by Sajad Writing of the manuscript: Jeddi S, Zaman J, Ghasemi A; Jeddi, from Shahid Beheshti University of Medical Sciences.

Arq Bras Cardiol. 2015; 104(2):136-143 141 Jeddi et al. Ischemic postconditioning in hypothyroid rats

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Arq Bras Cardiol. 2015; 104(2):136-143 143 Original Article

Neural Mechanisms and Delayed Gastric Emptying of Liquid Induced Through Acute Myocardial Infarction in Rats Wilson Ranu Ramirez Nunez, Michiko Regina Ozaki, Adriana Mendes Vinagre, Edgard Ferro Collares, Eros Antonio de Almeida Universidade Estadual de Campinas, Campinas, SP - Brazil

Abstract Background: In pathological situations, such as acute myocardial infarction, disorders of motility of the proximal gut can trigger symptoms like nausea and vomiting. Acute myocardial infarction delays gastric emptying (GE) of liquid in rats.

Objective: Investigate the involvement of the vagus nerve, α 1-adrenoceptors, central nervous system GABAB receptors and also participation of paraventricular nucleus (PVN) of the hypothalamus in GE and gastric compliance (GC) in infarcted rats. Methods: Wistar rats, N = 8-15 in each group, were divided as INF group and sham (SH) group and subdivided. The infarction was performed through ligation of the left anterior descending coronary artery. GC was estimated with pressure-volume curves. Vagotomy was performed by sectioning the dorsal and ventral branches. To verify the action

of GABAB receptors, baclofen was injected via icv (intracerebroventricular). Intravenous prazosin was used to produce chemical sympathectomy. The lesion in the PVN of the hypothalamus was performed using a 1mA/10s electrical current and GE was determined by measuring the percentage of gastric retention (% GR) of a saline meal. Results: No significant differences were observed regarding GC between groups; vagotomy significantly reduced % GR

in INF group; icv treatment with baclofen significantly reduced %GR. GABAB receptors were not conclusively involved in delaying GE; intravenous treatment with prazosin significantly reduced GR% in INF group. PVN lesion abolished the effect of myocardial infarction on GE. Conclusion: Gastric emptying of liquids induced through acute myocardial infarction in rats showed the involvement of the vagus nerve, alpha1- adrenergic receptors and PVN. (Arq Bras Cardiol. 2015; 104(2):144-151) Keywords: Rats; Gastric Emptying; Myocardial Infarction; Midline Thalamic Nuclei Receptors, GABA; Gastrointestinal Motility.

Introduction hypothalamus, which under certain conditions can modify gastric motility and GE2-4. In rats, GABA receptors are The gastric emptying (GE) process results from mechanism B located in the presynaptic afferent endings of the vagus that actions that inhibit or stimulate the motor activity of the project into the solitary tract nucleus5. Presynaptic GABA stomach, pylorus and duodenum. The central nervous B receptors are involved in regulation of neurotransmitter system connects itself with the enteric nervous system release, as the effect of (an agonist for these receptors) is through the vagus nerve and sympathetic nervous system, to reduce the release of stimulatory and inhibitory synaptic participating in GE control1.The vagus nerve controls food transmitters6,7. In pathological situations, such as acute movement throughout the digestive tract. If this mechanism myocardial infarction, disorders of motility of the proximal is impaired, the stomach muscles and intestines do not gut can trigger symptoms like nausea and vomiting8. function normally and food transportation slows down or stops completely. The dorsal vagal complex consists of the Experimental studies on the association of myocardial 9,10 solitary tract nucleus with neurons that receive afferent infarction and gastric emptying are very rare, and one 10 information, area postrema and the dorsal nucleus of the study considered that delayed gastric emptying may be vagus, where stimulatory and inhibitory motoneurons are due to stress caused by ischemia. However, the underlying mechanisms of delayed gastric emptying were not located, with the axons being efferent pathways of the addressed. Therefore, the present study aimed to determine vagus nerve2. The dorsal vagal complex is more influenced the involvement of neural mechanisms related to delayed by higher structures, such as the paraventricular nucleus of gastric emptying.

Methods Mailing Address: Eros Antonio de Almeida • Professor Jorge Nogueira Ferraz, Jardim Chapadão. Postal Code 13070-120, Male Wistar rats (n = 8-15) were used to perform this Campinas, SP – Brazil experiment, weighing 220 to 300 g, supplied by the Central Email: [email protected]; [email protected] Animal Facility of Universidade Estadual de Campinas. Manuscript received June 04, 2014; revised manuscript August 28, 2014; accepted September 02, 2014. The study protocol was approved by SBCAL (Brazilian Society of Laboratory Animal Sciences) (www.ib.unicamp.br/ DOI: 10.5935/abc.20140190 ceea/principios) (Protocol Nº. 1021-2).

144 Nunez et al. Gastric emptying and myocardial infarction

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Rats had an adjustment period of four weeks to laboratory animal corresponded to the mean of three measurements conditions with controlled temperature (22 - 26°C) and at each point of IGP. To estimate the GC, curves of volume/ artificial light cycle of 12 hours and were given water pressure were constructed and calculated through the ad libitum. In surgical procedures such as vagotomy or following formula: implantation of cannula into the lateral brain ventricle or Compliance (mL/mmHg) = V – V / P – P , electrolytic lesion of the paraventricular nucleus, the rats 1 0 1 0 Where V = initial volume and V = final volume; were previously sedated with intra-peritoneal (ip) injection 0 1 of thiopental 75mg/Kg. After the procedures or the study, P0 = initial IGP and P1 = final IGP. the animals were kept in individual cages, receiving water and food ad libitum. In order to study GC, the animals were Gastric emptying anesthetized with ip administration of ketamine (85mg/kg) A saline solution containing phenol red (6mg/dl) with a + xylazine (10mg/kg). volume of 1.5 mL/100g bodyweight was used as a test meal The drugs prazosin (PRA) and baclofen (BAC) (both from in the GE study. Gastric emptying was assessed indirectly in Sigma, USA) were diluted at the time of the study, using sterile awaken rats, to determine the percentage of gastric retention saline as vehicle (V). (% GR) of test meal, after ten minutes of the orogastric administration, using a standardized technique13. Myocardial infarction The rats were placed vertically and the test meal infused Myocardial infarction was induced by ligation of the by gavage to reach the stomach. After the administration, left anterior descending coronary artery, according to the the animals remained in the cage for 8 min and 30 sec. technique recommended by Johns & Olson11. The rats were After being anesthetized with ether, the orogastric tube anesthetized with ether and thoracotomy was performed. was introduced, keeping the animals anesthetized by ether Through gentle pressure applied to the right hemithorax, inhalation. The abdomen was opened longitudinally and the heart was exposed and a ligature was performed around the pylorus clamped, exactly 10 minutes after orogastric the proximal left coronary artery in its proximal segment, infusion. All steps were timed. The gastric residue was between the pulmonary artery cone and the left atrial apex. aspirated and then five washes were performed with Only the animals with major infarction, i.e., involving 40% or 2 mL of distilled water at each time, taking care to always more of the entire area of the left ventricle, were considered aspirate using the same syringe. Complete emptying was for the study. Twenty-four hours after the surgical procedure, confirmed by direct visualization of the viscera. The probe studies were performed by measuring gastric compliance was then removed and negative pressure was applied to the (GC), vagotomy, intracerebroventricular injection with euthanized animal. The gastric residue obtained plus the washings were transferred to a 25 mL graduated cylinder, GABAB, intravenous treatment with prazosin and electrolytic paraventricular nucleus lesions. and the aspiration tools (probe and syringe) were washed three times with one mL of water each time, and the volume In order to measure GC, rats were divided into three groups was added to the beaker. twenty-four hours before: rats were submitted to myocardial infarction (INF), and also to simulated infarction (SH) and naive To determine the percentage of gastric retention (GR%) (NA). Only the INF and SH groups were used to determine GE. 2.0 mL were taken from the total volume recovered, and Rats in all groups were fasted and GE or GC were evaluated transferred in duplicate to 10 mL volumetric flasks, to which twenty-four hours after these groups were formed, between 5.0 mL of a trisodium phosphate solution were added at a concentration of 27.5 g/L. The same procedure was 2:00 pm and 5:00 pm, and access to water was cancelled an performed with one mL of the test meal. The final volume hour before the test. was completed to 10 mL with distilled water. The readings were made in a spectrophotometer (Spectrophotometer B Gastric Complacence 382, Micronal) at a wavelength of 560 nM. GR was calculated The technique described by Bustorff-Silva et al12 was used using the following formula: to measure GC. In brief, anesthetized rats were submitted to GR = vrg × arg ÷ vrp × arp and the result was multiplied the following procedures: tracheotomy, abdominal incision, by 100. pylorus ligation, fixation of distal esophagus with an orogastric Where: vrg = volume of gastric residue; arg = gastric polyethylene tube filled with saline solution and connected residue absorbance; vrp = volume of the test meal; arp = through a three-way stopcock to an infusion pump (model absorbance of the test meal. LF 2001 Lifemed, Brazil) and a pressure monitor (Biomotor 7.0, BESE, Belo Horizonte - Brazil). Thirty minutes after these procedures, saline solution at 37 °C was infused into the Vagotomy stomach of each animal, at a rate of 1.5 mL/100g weight/ Two weeks before the vagotomy study, rats underwent min intermittently, every 20 seconds (s) at 1-minute intervals. subdiaphragmatic vagotomy (VGX), in which the dorsal and Every 20s (1/3 the volume) the infusion was stopped and the ventral branches of vagus nerve were sectioned, while other system was balanced for 50s, while recording intragastric animals were submitted to sham procedure (VGS), constituting pressure (IGP). Intragastric pressure corresponding to 1/3, the controls. Twenty-four hours before the procedure of GE, 2/3 and the total volume were recorded. The procedure was animals VGX and VGS, were submitted to surgical infarction, repeated twice with 30-min intervals. The results of each while others were submitted to the same simulated surgery.

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Intracerebroventricular baclofen injection Statistical Analysis

To study the involvement of GABAB receptors in the central The SAS (Statistical Analysis System) for Windows software, nervous system eight days before the GE study, each animal, version 9.2 (SAS Institute Inc., 2002-2008, Cary, NC, USA) underwent implantation of a cannula (21G) in the right lateral was used in the statistical analysis. 14 ventricle, using techniques and coordinates related to bregma , The results of GC and GR are presented as mean ± SEM. 15 as previously described . Twenty-four hours prior to the GE Statistical analysis was performed using ANOVA, followed study, rats were submitted to surgical infarction (INF), while by Tukey test when necessary. It was established the value of others were submitted to the same simulated surgery (SH). α = 0.05 for both tests. Ten minutes before GE assessment, the two groups were treated with an intracerebroventricular (icv) injection of 10μL of saline solution (V) or an equal volume of solution containing 1µg of Results 16 BAC. The dose of BAC used was based on the literature . There were no significant differences in GC in the comparison GE was evaluated ten minutes after the end of the injection. between groups (mean ± SEM of group INF = 0.16 ± 0.03 mL / mmHg, N = 9, group SH = 0.17 ± 0.03 mL/mmHg N = 9, Involvement of alpha1-adrenergic receptors group NA = 0.16 ± 0.01 mL / mmHg, N = 8). The percentage To study the involvement of alpha-1adrenergic receptors, of infarcted area of left ventricle, mean ± SEM, in rats of INF both INF and SH animals , were treated intravenously (iv) group was 47.8 ± 2.7%. through a tail vein, with saline as vehicle (V) or prazosin The results of the study prior to the vagotomy are shown at a dose of 1mg/kg, 24 hours after the surgery, and dose in Figure 1. There were significant differences between GR% given was based on the literature17. GE was evaluated fifteen of the animals in the VGS+SH vs. VGS+INF groups (mean minutes after the injection. ± SEM = 36.6 ± 2.0%, N = 10; and 48.0 ± 2.3%, N = 15, respectively), which indicated that infarction determined Paraventricular nucleus of the hypothalamus lesion delayed GE in animals with sham vagotomy when compared To evaluate the involvement of the paraventricular to their controls. Moreover, the VGX+INF subgroup presented nucleus, ten days prior to the GE study , the animals were significantly lower GR% (28.7 ± 2.8%, N = 11) than the VGS submitted to a restricted PVN lesion (group PVNX) at two + INF group and it did not differ from the VGX + SH group points, bilaterally, with passage of a 1 mA/10 s electrical (25.9 ± 1.5%, N = 10). However, the previous vagotomy current injury, using nickel and chromium electrodes with also significantly reduced GR% in SH group (VGX + SH) 0.25 mm in diameter. The coordinates in relation to the when compared to the VGS group (VGS + SH). Therefore, bregma, were the following: anterior-posterior (AP) -1.2 and the reduction in GR% caused by vagotomy in INF group was -1.5 mm, lateral ± 0.5 mm, vertical 7.8 mm and 8.0 points 40% higher. In this study, the infarcted area of left ventricle, corresponding to AP, as shown in another study15. In rats mean ± SEM, in VGS + INF animals was 51.7 ± 2.3% and with sham lesion (PVNS), the same coordinates were used, in VGX + INF animals was 50.2 ± 2%. except the vertical one, in which the depth was 7.5 mm, Intracerebroventricular treatment with BAC (Figure 2) without the passage of an electrical current. significantly reduced GR% in the control group (SH+BAC Twenty-four hours before the GE procedure, PVNX = 17,8 ± 2,6%, N = 11) when compared to treatment and PVNS animals were divided into INF and SH groups. with V (SH + V = 32,0 ± 2,9%, N = 9), as well as in the After the experiments, all rats were euthanized. In the INF +BAC group when compared to INF+V (26.0 ± 3.3%, INF group, the hearts were removed, sectioned in the N = 11 and 40.2 ± 2 1%, N = 10, respectively). Although sagittal plane, using the left auricle, the interventricular myocardial infarction increased the GR% in the group of rats sulcus and left ventricular outflow tract as reference points. treated with vehicle (INF + V), this result did not significantly Then, the two halves were fixed in 10% formalin and differ from its control group (SH + V). Additionally, it was embedded in paraffin. The histological sections stained found that the mean reduction in GR% determined by BAC with hematoxylin-eosin were used to determine % of was 25% higher in the SH groups than that observed in the infarction area in relation to the entire area of the left INF groups. The infarcted area of the left ventricle, mean ± ventricle, using a standard technique18. SEM, in INF + V animals was 51.2 ± 2.7% and 52,2 ± 3 % In the PVNX group, the brains were removed, fixed, in INF + BAC animals. embedded in paraffin and histological sections were stained with Figure 3 shows results from the iv treatment with PRA, which toluidine blue. To confirm the site of injury, the sections, under significantly reduced GR% in infarcted group (INF + PRA = microscopic view, were compared to the Paxinos & Watson19 atlas 22.0 ± 1.5%, 10) when compared to the vehicle-treated group in another study performed in the same laboratory15. (INF + V = 42.1 ± 2.4%, 10). The same phenomenon occurred In animals with implantation of a cannula into the lateral among animals from the SH group (SH + PRA = 22.3 ± 1.8%, ventricle, the assessment was made with an icv injection of N = 12 vs SH + V = 30.5 ± 1.3%, N = 11); however, GR% 10μL of an Evans Blue solution at 1% at the end of the GE reduction was 77% higher in the INF group. The infarcted area study. In this group, after euthanization, the brains were of the left ventricle, in mean ± SEM, in INF + V animals was removed and fixed in 10% formalin, and coronal sections 55.1 ± 1.2% and 55.2 ± 1.2% in INF + PRA animals. were obtained and confirmed when the dye injected icv was Figure 4 shows the results of the lesion seen in the found in the fourth ventricle. paraventricular nucleus in a prior study. Rats with electrolytic

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75 * * *

50

25

Gastric retention (%)

0 VgS VgX VgS VgX Surgery SH INF Groups

Figure 1 – Results of gastric retention (%) in groups of rats with myocardial infarction (INF) and vagotomy (Vg) * p < 0.05.

75 * * 50

25

Gastric retention (%)

0 SH INF SH INF Group icv V BAC

Figure 2 – Results of gastric retention (%) in groups of rats with myocardial infarction and inserted metal probe, with baclofen (BAC) or saline (V) injected into the lateral ventricle of the brain * p <0.05.

PVN lesion and infarction showed a significantly lower Discussion GR% (PVNX + INF = 25.0 ± 3.0%, N = 8) compared to Decrease in gastric tone determines slower GE of liquid20. animals with sham lesion (PVNS) and infarction (PVNS+INF Therefore, under the experimental conditions of this study, = 41,2 ±1,7%,N=8). The same lesion did not reduce GR% acute myocardial infarction did not induce changes in in control animals (PVNX + SH = 28.5 ± 2.9%, N = 8 vs. gastric tone in rats. However, as GE is a coordinated action PVNS + SH = 32.1 ± 1.9%, N = 10). The infarcted area of of the stomach, pylorus and duodenum21, it is possible the left ventricle, in mean ± SEM, in PVNS + INF animals that the determinant factor of delayed GE in myocardial was 52.2 ± 0.4% and 55.4 ± 0.8% in PVNX + INF animals. infarction did not act directly on the stomach. Camurça et As for the histological assessment of the brains after the PVN al9 detected that the transit of liquid in the small intestine lesion, only lesions involving 100% of the paraventricular is slower in acute myocardial infarction, in addition to region were considered for this study. delayed GE. It was unclear whether the two disorders are

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75 * * * 50

25

Gastric retention (%)

0 SH INF SH INF Group V PRA iv

Figure 3 – Results of gastric retention (%) in groups of rats with myocardial infarction that received prazosin (PRA) or Saline (V) injection * p < 0.05.

75 * * 50

25

Gastric retention (%)

0 PVNS PVNX PVNS PVNX Surgery SH INF groups

Figure 4 – Results of gastric retention (%) in groups of rats with myocardial infarction and lesions of the paraventricular nucleus * p < 0.05.

dependent on the same phenomenon or if the effect on GE verified the involvement of GABAB receptors in delayed GE is a consequence of what occurs after the stomach. As no through INF. Baclofen in the central nervous system increases GE changes were observed in GC, an alternative speculation of liquids through its capacity to block the dorsal vagal complex, to explain delayed GE of liquid in myocardial infarction mechanical inhibitory stimuli that act on proximal stomach, would be an increase the resistance, of unknown nature, conveyed through the afferent vagus nerve fibers22-25. As a to the meal flow into the small intestine. result, there is an increased tone in this segment of functional Intracerebroventricular treatment with Baclofen significantly stomach, leading to faster GE of a saline meal. What was reduced GR% in control group (SH) when compared to controls expected, considering that the infarction did not modify gastric treated with saline and, similarly, in the infarcted group (INF) compliance and may have resulted in an increased tone with (Figure 1). In comparative terms, it was surprising that the largest the same intensity in controls and infarcted groups, overcoming reduction occurred in the control groups. As a result, it was not the condition that determined the delayed GE in infarction.

148 Arq Bras Cardiol. 2015; 104(2):144-151 Nunez et al. Gastric emptying and myocardial infarction

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The previous sub-diaphragmatic section of the ventral Practically, PVN is involved in food intake, response to stress and dorsal branches of the vagus nerve significantly reduced and it also modulates metabolic rate and thermoregulation, GR in infarcted animals and also in SH animals (Figure 3). and participates in regulation of cardiovascular function The result observed between sham animals was expected, as and the autonomic nervous system35. This hypothalamic this type of vagotomy can increase gastric emptying of liquid structure participates in cardiovascular autonomic increasing the tone of the proximal stomach26. However, GR% regulation36,37. Heart failure has been associated with reduction through vagotomy in infarcted animals was higher changes in specific brain areas, as well as the activation of (approximately 40%), when compared to the mean reductions neurons in PVN, which are related to abnormalities in the of GR% between the two groups. production of vasopressin, blood volume regulation and 38 Vagotomy modifies the motor activity of the stomach by sympathetic stimulation (excitation) . blocking the arrival of inhibitory afferent stimuli to the solitary In this study, infarcted animals with electrolytic lesion of tract and abolishes the efferent stimuli, which originate in the PVN showed significantly lower GR when compared the dorsal vagal nucleus1,27,28. Afferent fibers of the vagus to infarcted animals with sham lesion. This type of lesion nerve carry sensory information from other regions of the does not reduce GR% in control animals (Figure 4). gastrointestinal tract and the efferent pathways innervate from These results indicate that the PVN lesion abolished the effect the gastric fundus to the descending colon29-30. of recent myocardial infarction on GE in rats. This finding is an evidence of the important participation of PVN in GE delay Thus, regarding the procedure employed in this study, it is in myocardial infarction. It is possible that the partial results unknown whether we are blocking afferent or efferent stimuli obtained with vagotomy and alpha1-adrenergic blockade and in which part of the gastrointestinal tract. However, results occurred due to the fact that each one of these procedures suggest the participation, at least partially, of the vagus nerve affected only one part of the set of changes under PVN effect, in delayed GE induced by myocardial infarction. determining delayed GE in myocardial infarction. The role of alpha1-adrenoceptors as stimulatory receptors particularly involves smooth muscle contraction, especially the contraction of vascular smooth muscle fibers, determining Conclusion local vasoconstriction and acting on blood pressure control31. The results of this study suggest the involvement of the Prazosin is a peripheral antagonist that binds to these receptors vagus nerve, the alpha1-adrenergic receptors and the PVN in in vessels32,33 and has no significant influence on gastric tone the delayed GE induced by recent myocardial infarction in rats. and phasic contractions in the stomach34. Intravenous treatment with Prazosin significantly reduced Author contributions the effect of myocardial infarction on GE (Figure 3). This fact is Conception and design of the research: Nunes WRR, related to the one proposed by Camurça et al9, that delayed GE Collares EF, Almeida EA; Acquisition of data: Nunes WRR, in myocardial infarction would result of increased sympathetic Ozaki MR, Vinagre AM, Collares EF; Analysis and interpretation activity as it was observed in the present study, with a possible of the data: Nunes WRR, Ozaki MR, Collares EF, Almeida EA; involvement of the vascular system. Statistical analysis: Nunes WRR, Vinagre AM; Writing of the In addition, it was also found that there was a significant manuscript: Nunes WRR; Critical revision of the manuscript GR reduction in SH group, although the reason for this for intellectual content: Ozaki MR, Collares EF, Almeida EA. effect remains unknown. In a previous study, carried out in the same laboratory, on the effect of myocardial infarction on GE10, it was found that sham animals showed a non- Potential Conflict of Interest significant GR increase, when compared with the naive group. Fundação Coordenação de Aperfeiçoamento de This fact was attributed to the combined effect of anesthesia Pessoal de Nível Superior (Capes); Conselho Nacional de + surgery; these procedures were performed twenty-four Desenvolvimento Científico e Tecnológico (CNPq). hours before the sham group was created. This combined effect, if confirmed in this study, might be less intense at Sources of Funding vascular level in the SH group than in the INF group and This study was funded by CAPES. it can also explain the results of treatment with prazosin. Nevertheless, GR reduction induced through Prazosin was 77% higher in infarcted animals, suggesting involvement of Study Association alpha1-adrenoceptors in this condition. This article is part of the thesis of Doctoral submitted by The paraventricular nucleus (PVN) is a major integrative Wilson Ranu Ramirez Nunes, from Universidade Estadual region of the hypothalamus that maintains homeostasis. de Campinas.

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Arq Bras Cardiol. 2015; 104(2):144-151 151 Original Article

Fatty Acid and Cholesterol Concentrations in Usually Consumed Fish in Brazil Carlos Scherr1, Ana Carolina Moron Gagliardi2, Marcio Hiroshi Miname2, Raul Dias Santos2 Instituto do Coração e do Diabetes1, Rio de Janeiro, RJ; Unidade Clínica de Lípides - Instituto do Coração (InCor) - Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo2, São Paulo, SP - Brazil

Abstract Background: Several studies have demonstrated clinical benefits of fish consumption for the cardiovascular system. These effects are attributed to the increased amounts of polyunsaturated fatty acids in these foods. However, the concentrations of fatty acids may vary according to region. Objective: The goal of this study was to determine the amount of,cholesterol and fatty acids in 10 Brazilian fishes and in a non-native farmed salmon usually consumed in Brazil. Methods: The concentrations of cholesterol and fatty acids, especially omega-3, were determined in grilled fishes. Each fish sample was divided in 3 sub-samples (chops) and each one was extracted from the fish to minimize possible differences in muscle and fat contents. Results: The largest cholesterol amount was found in white grouper (107.6 mg/100 g of fish) and the smallest in badejo (70 mg/100 g). Omega-3 amount varied from 0.01 g/100 g in badejo to 0.900 g/100 g in weakfish. Saturated fat varied from 0.687 g/100 g in seabass to 4.530 g/100 g in filhote. The salmon had the greatest concentration of polyunsaturated fats (3.29 g/100 g) and the highest content of monounsaturated was found in pescadinha (5.98 g/100 g). Whiting and boyfriend had the best omega-6/omega 3 ratios respectively 2.22 and 1.19, however these species showed very little amounts of omega-3. Conclusion: All studied Brazilian fishes and imported salmon have low amounts of saturated fat and most of them also have low amounts of omega-3. (Arq Bras Cardiol. 2015; 104(2):152-158) Keywords: Fatty Acids; Cholesterol; Fishes; Coronary Artery Disease / prevention & control; Dyslipidemias.

Introduction cardiovascular morbidity. In addition, numerous studies Several studies have reported that cardiovascular have showed that fish consumption has positive effects on diseases are associated with lifestyles, particularly food the lipoprotein metabolism, clotting and platelet function, 6 habits1. In 2002, Hu and Willet2 published a broader endothelial function, and arterial stiffness . review on those studies and concluded that the following Finally, it is important to point out that all fishes do three dietary strategies were effective in preventing not contain similar omega-3 concentration. For example, coronary artery disease: 1) replacing saturated fats or fishes from icy and deep waters exhibit better protective trans fats with poly/monounsaturated fat; 2) increasing effects because of the phytoplankton diversity they feed on. the omega-3 fatty acid consumption; 3) and consuming Therefore, protective effects should not be generalized for more fruits, vegetables, nuts, whole grains, and avoiding all species or on the way they are prepared or cooked7,8. refined carbohydrates. The present study aimed to determine the composition There are extensive evidence associating the increase of fatty acids and cholesterol in the most consumed fishes in omega-3 consumption with lower risks of cardiovascular in Brazil. Fishes from all over the country as well as the diseases3-5. From the epidemiological point of view, increased non-native farmed salmon, which is mostly imported from fish consumption is associated with lower mortality and Chile, were analyzed.

Mailing Address: Ana Carolina Moron Gagliardi Miguel • Material and Methods Rua Bernardo Ferraz de Almeida, 187 apt.º 11, Jardim Faculdade. Fishes were obtained from local markets in various regions Postal Code 18030-290, Sorocaba, São Paulo - Brazil. of the country. Fish processing was performed at the Meat E-mail: [email protected] Manuscript received December 27, 2013, revised August 21, 2014, Technology Center (Centro de Tecnologia de Carnes) of the accepted August 25, 2014. Institute of Food Technology (Instituto de Tecnologia de Alimentos). A standard protocol was used for this type of DOI: 10.5935/abc.20140176 study in which fishes were cleaned and processed to provide

152 Scherr et al. Fatty acids and cholesterol levels in fishes of Brazil

Original Article approximately 1 kg/preparation. Then, the preparations After the grilling process, the composition of fats was were homogenized in a cutter and vacuum packed for a analyzed by gas chromatography, consisting of the separation single serving. and subsequent quantification of fatty acids. Specific methods 9-11 12-14 Three fishes of the salmon and namorado species, were used to evaluate total lipids , cholesterol , and fatty respectively, were used, and a sample from each fish was acids13. As a standard, cholesterol was quantified in mg/100 g cut, totaling to three samples per species. Each sample was of fish and fatty acids in g/100 g of fish. Then, the ratio of the divided in three subsamples (chops), and a section from fatty acids omega-6/omega-3 was calculated. each was cut from the beginning, middle, and end of the The results were evaluated on the basis of the fish. Parts such as the head and tail that are usually not recommendation by the National Agency of Sanitary consumed by people were discarded and the remaining parts Surveillance (Anvisa, Agência Nacional de Vigilância Sanitária), of the sample were cut from the one-third initial, middle, which suggest a 2,000 kcal diet a day, including a lipid intake and end of the fish. This procedure intended to minimize of up to 55 g/day; however, it should include < 22 g/day of problems regarding possible differences between fats and saturated fatty acids and up to 300 mg/day of cholesterol15. muscles, which could influence the final analysis results. For the other species, 1 kg of sample, which consisted of Statistical analysis several fishes belonging to the same species for each type of preparation, was used. Data are showed as mean ± standard deviation. The normality of the data was tested by the Kolmogorov– Smirnov test. ANOVA test and post-hoc Bonferroni correction Preparation of samples were used to compare the quantification of total lipids, fatty Tissue compositions for cholesterol and fatty acids acids, and cholesterol among the 10 different types of grilled from 10 fishes were analyzed; of these, nine are found in fishes for the seven variable studied. Results were considered Brazil and one is mostly imported from Chile (non-native significant at 5% significance level. farmed salmon). Of the fishes found in Brazil, three inhabit freshwater (filhote, trout, and sea bass), three inhabit salty waters of the Brazilian coast (namorado, weakfish, and Results sardines), and three inhabit the offshore area (whiting, Table 1 shows the total lipid composition in fishes assessed. robalo, and white grouper). These fishes were chosen Table 2 shows the differences that were statistically significant because of their high consumption and distribution offshore among the analyzed fishes. Significant differences in the (three), in the coast (three) and in freshwater (Belém, concentrations of cholesterol and fatty acids in different Manaus, and São Paulo). species were observed. The average content of cholesterol All fishes were grilled (standard preparation), and ranged from 70 mg/100 g in whiting to 107.6 mg/100 g in 1 kg samples were separated without adding any other white grouper (P < 0.05). Therefore, to overcome the daily ingredient. The grill was heated and fishes were placed recommended amounts of cholesterol, at least 428 g/day or on it for approximately 20 min until one side was golden 279 g/day of whiting or white grouper, respectively, should brown. They were turned only once with a total time of be consumed16. However, there was no significant difference approximately 40 min on the grill. between the species in the highest cholesterol content (white

Table 1 - Total lipids found in the fishes assessed

Cholesterol Saturated fats Polyunsaturated fats Monounsaturated fats (mg/100 g) (g/100 g) (g/100 g) (g/100 g) p < 0,01* p < 0,01* p < 0,01* p < 0,01* Whiting 70,03 ± 1,68 0,69 ± 0,03 0,03 ± 0,00 0,37 ± 0,01 White grouper 107,61 ± 2,91 1,56 ± 0,02 0,44 ± 0,12 2,50 ± 0,05 Filhote 94,31 ± 0,88 4,53 ± 0,07 1,84 ± 0,03 3,73 ± 0,07 Namorado 73,49 ± 0,80 0,70 ± 0,02 0,08 ± 0,01 0,63 ± 0,01 Weakfish 84,90 ± 2,34 2,13 ± 0,07 1,26 ± 0,09 5,98 ± 0,25 Seabass 88,12 ± 3,84 1,76 ± 0,02 0,18 ± 0,03 1,21 ± 0,04 Robalo 73,76 ± 2,50 0,68 ± 0,02 0,07 ± 0,10 0,34 ± 0,005 Sardine 86,05 ± 1,55 1,85 ± 0,05 0,02 ± 0,00 0,60 ± 0,03 Trout 86,82 ± 3,50 2,57 ± 0,04 1,60 ± 0,06 4,03 ± 0,07 Salmon 93,33 ± 18,42 2,57 ± 0,66 3,11 ± 0,72 2,41 ± 0,71 * ANOVA: difference among the various fishes assessed.

Arq Bras Cardiol. 2015; 104(2):152-158 153 Scherr et al. Fatty acids and cholesterol levels in fishes of Brazil

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Table 2 – Breakdown of the differences found in the post-hoc Bonferroni correction among fishes assessed

Comparison among fishes/type Cholesterol Saturated fats Polyunsaturated fats Monounsaturated fats of fat assessed White grouper, filhote, White grouper, filhote, weakfish, seabass, namorado, weakfish, Whiting White grouper, filhote Weakfish, trout, salmon trout, salmon seabass, sardine, trout, salmon Whiting, filhote, namorado, Whiting, namorado, Whiting, filhote, namorado, robalo, trout, White grouper Weakfish, trout, salmon weakfish, seabass, robalo, robalo, sardine, trout salmon sardine, trout Whiting, white grouper, Whiting, white grouper, Whiting, namorado, Whiting, white grouper, namorado, weakfish, namorado, weakfish, Filhote namorado, seabass, robalo, robalo seabass, robalo, sardine, trout, salmon seabass, robalo, sardine, sardine, salmon salmon White grouper, filhote, White grouper, filhote, weakfish, seabass, Filhote, weakfish, trout, White grouper, filhote, Namorado trout sardine, trout, salmon salmon weakfish, trout, salmon Whiting, white grouper, Namorado, seabass, robalo, filhote, namorado, seabass, Weakfish White grouper Whiting, filhote, namorado, robalo sardine, salmon robalo, sardine, trout, salmon Whiting, white grouper, Filhote, weakfish, trout, Seabass White grouper Filhote, namorado, robalo, trout, salmon filhote, weakfish, robalo, salmon trout, salmon White grouper, filhote, White grouper, filhote, White grouper, filhote, weakfish, seabass, Filhote, weakfish, trout, Robalo weakfish, seabass, trout, salmon sardine, trout, salmon salmon salmon Filhote, weakfish, trout, White grouper, filhote, Sardine White grouper Filhote, namorado, robalo, trout, salmon salmon weakfish, trout, salmon Whiting, white grouper, Whiting, filhote, namorado, Whiting, white grouper, filhote, namorado, Trout White grouper namorado, seabass, robalo, weakfish, seabass, robalo, seabass, robalo, sardine, salmon sardine, salmon sardine, salmon Lines show statistical differences (p value < 0.05).

grouper versus filhote, p = 0.776; white grouper vs. salmon, (0.79 g/100 g), and filhote (0.38 g/100 g) had the highest omega-3 p = 0.511). concentration. Salmon is best known as a source of omega-3. Saturated fat analysis revealed that the content of saturated According to other studies, the ideal ratio between fats were not similar among fishes, ranging from 0.68 g/100 g omega-6 and omega-3 is 1/1 or 2/117. Thus, whiting (2.22) in sea bass to 4.53 g/100 g in filhote. Therefore, to exceed and namorado (1.19) had the best ratios; there is no statistic the recommended saturated fat levels, it would be necessary difference between them (p = 0.228). The worst ratios were to consume 3,200 g/day of sea bass or 486 g/day of filhote16. found in trout (9.03) and sea bass (5.25), with a statistic Unsaturated fatty acid analysis showed that salmon, difference of p < 0.05. However, despite their good ratios, filhote, and trout (p < 0.01 vs. other fish species) are the whiting and namorado had very low omega-3 concentrations. species with the highest concentration of polyunsaturated fats, whereas weakfish, trout, and filhote have the most significant Discussion concentration of monounsaturated fats (p < 0.01 vs. other In this study, we quantified fats found in the most consumed fish species). fishes in Brazil. Analyses were done once fishes were grilled, Table 3 shows the concentrations of omega-6 and omega-3 and our data showed important variations among species. and their ratio. Variables were statistically different among Our results show that all fishes had low saturated fat levels; fishes (p < 0.01). Table 4 shows the differences that were however, they also had small omega-3 concentrations. statistically significant among the fishes analyzed. Differences in cholesterol concentrations among fishes were Most fishes had low omega-3 concentrations. For example, observed as well. the omega-3 concentration in whiting was 0.009 g/100 g, Cholesterol intake, particularly from saturated fats, requiring an individual to consume at least 20 kg/day of is associated with increasing LDL-cholesterol (LDL-C) whiting to achieve the recommended amount of omega-3 concentrations in the blood16, which is the opposite when (2 g/day)16. On the other hand, weakfish (0.9 g/100 g), salmon poly/monounsaturated fats are consumed. Both cholesterol

154 Arq Bras Cardiol. 2015; 104(2):152-158 Scherr et al. Fatty acids and cholesterol levels in fishes of Brazil

Original Article and dietary fats exert effects upon cholesterolemia by cholesterol concentrations and greater polyunsaturated fat modulating the expression of LDL receptors in the liver16. concentrations. Furthermore, there are possible functional Dietary cholesterol intake can change blood LDL-C effects of omega-3 sourced from deep sea fishes, such as 6,16 concentrations. It is not recommended to consume antiplatelet and antiarrhythmic actions . > 200 mg/day of cholesterol in a healthy diet16. Therefore, The Chicago Western Electric study, which assessed white grouper should be consumed as part of a primary 1,822 men between 40–55 years old, for 30 years, showed cardiovascular prevention and should not exceed 186 g/day that fish consumption was inversely associated with mortality in patients who are undergoing secondary cardiovascular from coronary disease18. Similar data were found in the prevention. However, it is important to emphasize that cohort of Zutphen in The Netherlands, where 852 middle- cholesterolemia is determined mostly because of saturated aged men were studied for 20 years. The consumption of at fatty acid consumption instead of dietary cholesterol16. least 30 g/day of freshwater fish, regardless the species, was Therefore, some questions remain regarding whether fishes associated with 50% reduction in mortality from coronary consumption with the highest content of cholesterol has heart disease19. deleterious effects on the cardiovascular health. Positive results were also discussed in a major review Brazilian guidelines for fat consumption and cardiovascular by Mozaffarian (2011), regarding sudden cardiac death health16 states that more mono/poly-unsaturated fats (20% and higher fish consumption. This review was focused on and 10%, respectively) should be consumed, with reduced polyunsaturated fat intake, particularly omega-320. amounts of saturated fat (up to 10% of total caloric intake)16. The Nurse’s Health Study, performed in the United Based on this guideline, filhote is the species that better States with 85,000 women, showed that the consumption matches these recommendations. of two to four servings of fish per week reduced one-third Regarding omega-3, weakfish presented the highest risks of heart diseases. There were benefits in even those tissue concentrations, making it a good source of omega-3, who consumed fish only one to three times a month. As a particularly if at least 222 g/day of this fish is consumed. result of this research, the American Heart Association Second comes the salmon, and a consumption of 253 g/day began recommending the consumption of two servings of is recommended to achieve the recommendations of 2 g/day fish per week21. of omega-3 to prevent cardiovascular diseases. On the Even in lower-risk groups, such as young women, studies other hand, whiting had such low omega-3 concentrations have found beneficial effect on reducing cardiovascular risks that at least 20 kg of this fish would have to be consumed in those who consume more fish compared with those who to achieve levels that are usually associated with the eat little or do not consume fish at all22. reduction of cardiovascular risks. This amount is unfeasible Evidence of fish consumption benefits also exist in for human consumption on a daily basis; the same applies secondary cardiovascular prevention, as shown in the Diet to sea bass andnamorado. and Reinfarction Trial. They studied 2,000 men in secondary Epidemiological and intervention studies associate fish prevention for myocardial infarction for 2 years. Randomized consumption with better lipid profiles and reduced risk into four groups, those who consumed more fish had 29% of cardiovascular disease18-23. This could be explained lower mortality compared with the controls and had fewer by the fact that fish have the lowest saturated fat and fatal infarcts23.

Table 3 – Concentration of omega-3 and omega-6 fatty acids and their ratio in fishes assessed

Omega-6 Omega-3 Ratio omega 6/3 (g/100 g) (g/100 g) p < 0,01* p < 0,01* p < 0,01* Whiting 0,02 ± 0,00 0,009 ± 0,00 2,22 ± 0,00 White grouper 0,16 ± 0,02 0,27 ± 0,10 0,64 ± 0,20 Filhote 1,46 ± 0,01 0,38 ± 0,01 3,85 ± 0,13 Namorado 0,04 ± 0,005 0,04 ± 0,01 1,19 ± 0,17 Weakfish 0,36 ± 0,02 0,90 ± 0,11 0,40 ± 0,07 Seabass 0,15 ± 0,02 0,03 ± 0,01 5,25 ± 1,14 Robalo 0,016 ± 0,005 0,01 ± 0,00 0,18 ± 0,06 Sardine 0,02 ± 0,00 0,09 ± 0,00 0,22 ± 0,00 Trout 1,44 ± 0,05 0,16 ± 0,01 9,03 ± 0,43 Salmon 0,29 ± 0,07 0,79 ± 0,19 0,36 ± 0,016 *ANOVA: difference among the various fishes assessed.

Arq Bras Cardiol. 2015; 104(2):152-158 155 Scherr et al. Fatty acids and cholesterol levels in fishes of Brazil

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Table 4 – Breakdown of the differences found in the post hoc analysis with Bonferroni correction among fishes assessed

Omega-6 Omega-3 Ratio omega 6/3 p < 0,01* p < 0,01* p < 0,01* White grouper, filhote, weakfish, seabass, White grouper, filhote, weakfish, seabass, robalo, Whiting White grouper, filhote, weakfish, salmon trout, salmon sardine, trout, salmon Whiting, filhote, namorado, weakfish, White grouper Whiting, weakfish, seabass, salmon Whiting, filhote, seabass, trout robalo, sardine, trout, salmon Whiting, white grouper, namorado, Whiting, namorado, weakfish, seabass, Whiting, white grouper, namorado, weakfish, Filhote weakfish, seabass, robalo, sardine, trout robalo, sardine, salmon seabass, robalo, sardine, trout, salmon White grouper, filhote, weakfish, seabass, Namorado Filhote, weakfish, salmon Filhote, seabass, trout trout, salmon Whiting, white grouper, filhote, namorado, Whiting, white grouper, filhote, namorado, Weakfish Whiting, filhote, seabass, trout seabass, robalo, sardine, trout seabass, robalo, sardine, trout Whiting, filhote, namorado, weakfish, Whiting, white grouper, filhote, namorado, Seabass White grouper, filhote, weakfish, salmon robalo, sardine, trout, salmon weakfish, robalo, sardine, trout, salmon White grouper, filhote, weakfish, seabass, Robalo Filhote, weakfish, salmon Whiting, filhote, seabass, trout trout, salmon White grouper, filhote, weakfish, seabass, Sardine Filhote, weakfish, salmon Whiting, filhote, seabass, trout trout, salmon Whiting, white grouper, namorado, White grouper, filhote, namorado, weakfish, robalo, Trout weakfish, seabass, robalo, sardine, Weakfish, salmon sardine, trout, salmon salmon Whiting, white grouper, filhote, namorado, Whiting, white grouper, filhote, namorado, Salmon Whiting, filhote, seabass, trout seabass, robalo, sardine, trout, salmon weakfish, seabass, robalo, sardine, trout Lines show statistical differences (p value < 0.05).

Fish intake has been recommended as part of a Mediterranean- Despite our findings, we believe that substitution of foods style diet, and its benefits in cardiovascular prevention was shown such as meats and dairy products, which have the highest in the Italian cohort GISSI-Prevenzione. This study comprised saturated fat content, with fish is recommended as part of a 172 centers that followed up with 11,323 men and women for diet to prevent cardiovascular diseases. approximately 6 and a half years; all patients presented a history of myocardial infarction. They were encouraged to increase fish, fruits and raw or cooked vegetables, and olive oil consumptions. Limitations Individuals who followed the recommendations had lower risk In this study, we assessed only some types of fish and only of cardiovascular events24. This study also provided patients with one preparation method. It is possible that the preparation supplemental eicosapentaenoic acid and docosahexaenoic acid method may influence the chemical composition of at an average dosage of 850 mg/day. Omega-3 consumption cholesterol and fatty acids of fishes. In addition, there may from deep sea fishes was associated with a significant reduction be variations in the composition of fats in fishes of the same in deaths from coronary heart diseases (30% reduction) and species or in those captured from different locations along the sudden cardiac death (45% reduction). Brazilian coast due to the variation in the phytoplankton28,29. A review spanning seven cohorts and one control case study also showed that the amount of omega-3 intake was correlated Conclusion with greater benefits for those who consumed > 250 mg/day of Fishes analyzed in our study presented low saturated fat fish compared with those who consumed less25,26. content but some may have high cholesterol concentrations, Therefore, not only is fish consumption beneficial in itself as is the case of white grouper. Omega-3 concentrations were but their omega-3 concentration can have positive effect. low in most fishes analyzed. This, in turn, is related to the species and its origin. In our study, all species presented low omega-3 concentrations, which may be related to the type of food consumed by them27 and incorporated Author contributions into their fatty tissue. Conception and design of the research, Acquisition of data Altogether, our study showed low omega-3 concentrations in and Obtaining financing: Scherr C; Analysis and interpretation most Brazilian fishes assessed the differences in fats among the of the data, Statistical analysis and Critical revision of the species. Therefore, questions remain whether all fishes bring the manuscript for intellectual content: Scherr C, Gagliardi ACM, same beneficial effects or only specific species, such as the ones Miname MH, Santos RD; Writing of the manuscript: Scherr that live in cold water27. C, Gagliardi ACM, Santos RD.

156 Arq Bras Cardiol. 2015; 104(2):152-158 Scherr et al. Fatty acids and cholesterol levels in fishes of Brazil

Original Article

Potential Conflict of Interest Study Association No potential conflict of interest relevant to this article was This study is not associated with any thesis or dissertation reported. work.

Sources of Funding This study was funded by Instituto Nacional de Metrologia e Estatística.

References

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158 Arq Bras Cardiol. 2015; 104(2):152-158 Original Article

Assessment of Myocardial Infarction by Cardiac Magnetic Resonance Imaging and Long-Term Mortality João Luiz Fernandes Petriz1,2,3, Bruno Ferraz de Oliveira Gomes2, Braulio Santos Rua2, Clério Francisco Azevedo3, Marcelo Souza Hadlich1,3, Henrique Thadeu Periard Mussi1,2, Gunnar de Cunto Taets3, Emília Matos do Nascimento1, Basílio de Bragança Pereira1, Nelson Albuquerque de Souza e Silva1 Universidade Federal do Rio de Janeiro (UFRJ) / Instituto do Coração Edson Saad - Programa de Pós Graduação em Medicina (Cardiologia) 1; Hospital Barra D’Or2; Instituto D’Or de Pesquisa e Ensino3, Rio de Janeiro, RJ − Brazil

Abstract Background: Cardiac magnetic resonance imaging provides detailed anatomical information on infarction. However, few studies have investigated the association of these data with mortality after acute myocardial infarction. Objective: To study the association between data regarding infarct size and anatomy, as obtained from cardiac magnetic resonance imaging after acute myocardial infarction, and long-term mortality. Methods: A total of 1959 reports of “infarct size” were identified in 7119 cardiac magnetic resonance imaging studies, of which 420 had clinical and laboratory confirmation of previous myocardial infarction. The variables studied were the classic risk factors – left ventricular ejection fraction, categorized ventricular function, and location of acute myocardial infarction. Infarct size and acute myocardial infarction extent and transmurality were analyzed alone and together, using the variable named “MET-AMI”. The statistical analysis was carried out using the elastic net regularization, with the Cox model and survival trees. Results: The mean age was 62.3 ± 12 years, and 77.3% were males. During the mean follow-up of 6.4 ± 2.9 years, there were 76 deaths (18.1%). Serum creatinine, diabetes mellitus and previous myocardial infarction were independently associated with mortality. Age was the main explanatory factor. The cardiac magnetic resonance imaging variables independently associated with mortality were transmurality of acute myocardial infarction (p = 0.047), ventricular dysfunction (p = 0.0005) and infarcted size (p = 0.0005); the latter was the main explanatory variable for ischemic heart disease death. The MET-AMI variable was the most strongly associated with risk of ischemic heart disease death (HR: 16.04; 95%CI: 2.64-97.5; p = 0.003). Conclusion: The anatomical data of infarction, obtained from cardiac magnetic resonance imaging after acute myocardial infarction, were independently associated with long-term mortality, especially for ischemic heart disease death. (Arq Bras Cardiol. 2015; 104(2):159-168) Keywords: Myocardial Infarction/physiology; Magnetic Resonance Imaging; Diagnostic Imaging; Mortality; Risk Factor.

Introduction assessment. However, some knowledge gaps regarding the The size and morphology of myocardial fibrosis or association of AMI anatomical data provided by CMRI with long-term mortality still exist, as well as regarding its ability to necrosis area are potentially associated with the occurrence add relevant prognostic information. The number of studies of ventricular dysfunction and ventricular arrhythmias, which is limited, and most of them had short follow-up periods and are themselves associated with mortality in patients after acute involved North American and European populations4-12. myocardial infarction (AMI)1,2. The introduction of cardiac magnetic resonance imaging (CMRI) in the assessment of AMI3 was a major advance. It permits thorough documentation Objective of myocardial infarct size and anatomy, improving both To verify the association of data regarding infarct size and the diagnostic ability and the perspective of post-AMI risk anatomy with overall mortality, circulatory system disease mortality, and ischemic heart disease (IHD) mortality in patients undergoing CMRI after AMI.

Mailing Address: João Luiz Fernandes Petriz • Rua Pio Borges de Castro, 429, Barra da Tijuca. Postal Code 22793-081, Rio de Janeiro, RJ − Brazil Methods E-mail: [email protected]; [email protected] Manuscript received February 02, 2014; revised manuscript received July 08, 2014; accepted August 11, 2014. Study design and population Between June 2001 and December 2010, 7119 CMRI DOI: 10.5935/abc.20140177 records and 1959 reports with the term “infarct size” (IS)

159 Petriz et al. Magnetic resonance imaging and mortality after AMI

Original Article were found in a tertiary health care network of imaging grade-3 MET-AMI for two abnormal variables; grade-4 laboratories and hospitals. There were 489 patients MET‑AMI for three abnormal variables. undergoing CMRI after hospitalization, with documented The presence of microvascular obstruction (no reflow) was elevation of troponin levels, of whom 420 were selected for documented whenever described in the test report. diagnostic confirmation of AMI from their medical records; patients with non-ischemic myocardial injury were excluded. Statistical analysis This study was approved by the Research Ethics Committee of University Hospital Clementino Fraga Filho (HUCFF), Data analysis was carried out to evaluate the three Federal University of Rio de Janeiro (UFRJ). endpoints: overall mortality, circulatory system disease death, and IHD death, as codified in the Declaration of Death (DD) by the 10th Review of the International Classification Variables analyzed of Diseases (ICD-10). Mann-Whitney, chi-square or Fisher’s The demographic and clinical variables analyzed were: exact test were used for the univariate analysis; the log age; gender; previous AMI; diabetes mellitus; systemic rank test was used for the analysis of Kaplan-Meier curves. hypertension; smoking habit; dyslipidemia; angiographic Multivariate analysis was carried out sequentially by means of classification of obstructive coronary artery disease; an initial selection of variables using elastic net regularization treatment with CABG; serum creatinine levels; and peak (EM), which were analyzed by the Cox model. The survival serum troponin I level. Mortality and the basic cause of death tree was used for the identification of the main explanatory were obtained from probabilistic relationship of identification variables for each endpoint. The significance level was set data of the study population, with 1,485,735 records of the at 5%. The Pearson method was used for the analysis of Mortality Information System (Sistema de Informação de correlations between numeric variables. Mortalidade -SIM) between 2001 and 2012.

Cardiac magnetic resonance imaging findings Results CMRI findings were obtained from the medical records The mean follow-up period from the day CMRI was of each patient. The resonance techniques routinely used performed until the occurrence of death or the end of the for the assessment of patients with AMI were cine-magnetic observation period was 6.4 ± 2.9 years. The median time resonance, for left ventricular (LV) functional assessment, between admission for AMI and performance of CMRI was and late enhancement technique13, for the assessment of 13 days, and 278 patients (66.1%) underwent the imaging myocardial necrosis or fibrosis. test within the first 30 days after AMI. There were 76 deaths (18.09%), of which 34 (44.7%) were for circulatory system LV function was obtained from LV ejection fraction (EF) diseases and 22 (29%), for IHD. (Simpson’s method) and from the LV function category by subjective visual analysis, and was classified as normal or mild, moderate or severe dysfunction. Demographic and clinical data AMI location was based on the 17-segment model used Demographic and clinical characteristics of the study for myocardial segmentation nomenclature14. The presence population, by subgroup of survivors and of all-cause deaths, of transmural AMI was defined by the involvement of > 50% are shown in Table 1. of the segment15,16. The AMI type, regarding transmurality, The subgroup of deaths compared to that of survivors was thus classified into three categories: transmural (TM), showed: higher mean age; higher prevalences of diabetes non‑transmural (NTM), and mixed (when both AMI types mellitus, of previous AMI and two-vessel coronary artery were present). The percentage of infarcted LV mass was disease; higher median peak troponin levels; and higher calculated using the semi-quantitative visual score method, percentage of cases with serum creatinine levels > 2 mg/dL. as described elsewhere17. The AMI transmurality index (TI) A greater number of myocardial revascularization procedures was obtained by calculating the ratio between the number was observed in the subgroup of survivors (73.3% vs. 47.4%). of segments with transmural infarction (SegTM AMI) and the The angiographic profile of single-vessel disease was more total number of segments with AMI (NSegAMI). frequent among survivors, and only 19.8% did not undergo The MET-AMI variable was elaborated to express AMI cardiac catheterization. size and complexity, combining three categorized primary CMRI data in the study population, in the subgroup of variables: AMI transmurality, IS, and NSegAMI. survivors and in the all-cause death group are shown in Table 2. The definitions of abnormality (negative vs positive) for Comparing the subgroup of deaths with that of survivors, the variables comprising MET-AMI were: (1) transmurality, if a higher prevalence of mixed AMI was observed in the mixed AMI present; (2) categorization of components of the group of deaths. Number of segments with TM AMI, total MET-AMI composite variable was obtained from the most number of segments with AMI, and IS were significantly accurate cut-off value found in the ROC (Receiver Operating higher in the subgroup of deaths. Characteristic) curve, i.e., IS ≥ 14% and NSegAMI ≥ 4. The Pearson test showed positive linear correlations The MET-AMI variable was graded according to the between IS and SegTM AMI (r = 0.83) and between IS and number of abnormal components: grade-1 MET-AMI for no NSegAMI (r = 0.78). LVEF showed a negative linear association abnormality; grade-2 MET-AMI for one abnormal variable; with IS (r = - 0.57).

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Table 1 – Demographic and clinical characteristics of the study population and of the subgroups of survivors and all-cause deaths

Variables Total population (n = 420) Survivors (n = 344) Deaths (n = 76) p value Age, mean ± SD (years) 62.3 ± 12.0 60.4 ± 12.4 72.6 ± 12.1 < 0.001 Male gender 325 (77.3) 270 (78.4) 55 (72.3) 0.248 SH 312 (74.2) 254 (73.8) 58 (76.3) 0.655 Diabetes, n (%) 127 (30.2) 91 (26.4) 36 (47.3) < 0.001 Cigarette smoking, n (%) 136 (32.3) 119 (34.6) 17 (22.3) 0.039 Dyslipidemia, n (%) 212 (50.4) 179 (52.0) 33 (43.4) 0.174 Previous AMI, n (%) 207 (49.2) 153 (44.4) 54 (71.0) < 0.001

Angiographic profile of CAD, n (%) 0.566 Single-vessel 176 (41.9) 147 (42.7) 29 (38.2) Two-vessel 72 (17.1) 56 (16.3) 16 (21.0) Three-vessel or multivessel 89 (21.2) 73 (21.2) 16 (21.0) CAT not performed 83 (19.8) 68 (19.7) 15 (19.8)

Treatment during hospitaltization, n (%) < 0.001 Medical only 132 (31.4) 92 (26.8) 40 (52.6) Revascularization by PCI 226 (53.8) 197 (57.3) 29 (38.2) Surgical revascularization 62 (14.7) 55 (15.9) 7 (9.2)

Laboratory data Peak troponin I level, median (ng/mL) 12.9 12.2 19.1 0.614 Serum creatinine, mean ± SD (mg/dL) 1.06 ± 0.45 1.0 ± 0.4 1.1 ± 0.5 0.085 Serum creatinine (> 2 mg/dL), n (%) 11 (2.61) 7 (2.03) 4 (5.26) 0.037 SD: standard deviation; SH: systemic hypertension; AMI: acute myocardial infarction; CAD: coronary artery disease; CAT: catheterization; PCI: percutaneous coronary intervention.

Associations of variables with the endpoints studied type of treatment, serum creatinine level, IS (%), AMI of anterior location, moderate or severe LV dysfunction, AMI transmurality, previous AMI, AMI-CMDRI time (days), male Endpoint all-cause mortality gender, SegNTM AMI and no reflow. The multivariate Cox model was used for the all-cause death The Cox model identified, among the demographic or endpoint (time elapsed between performance of CMRI and clinical variables, age (p < 0.0001) and serum creatinine occurrence of death), with the variables selected by EN (Table 3). level (p = 0.001) as independent variables. Among CMRI Among the CMRI data, the model identified the following variables, the independent variables identified were ventricular independent variables: moderate or severe ventricular dysfunction (hazard ratio − HR: 3.34; 95% confidence dysfunction, IS and NTM AMI. The model also identified the interval − 95% CI: 1.35-8.24; p = 0.008), IS (HR: 1.10; following clinical or demographic variables as independent 95%CI: 1.05‑1.15; p = 0.0001) and previous AMI (HR: 2.87; variables: age, diabetes mellitus and serum creatinine level. 95%CI: 1.04‑7.89; p = 0.041). The presence of mixed AMI was An inverse association was also found between treatment also a factor identified with significance < 10% (p = 0.055). with CABG and mortality. The MET-AMI variable was tested again using the Cox The MET-AMI variable was tested using the multivariate multivariate model. Table 4 shows the independent variables Cox model again. However, this was proven to be a significant identified in the new model. Moderate/severe LV dysfunction, variable for overall mortality (p = 0.25). age, serum creatinine level, and grades 3 and 4 MET-AMI were The survival tree identified age at CMRI, with a cut-off independent variables related to the endpoint circulatory point at 69.7 years, as the most relevant explanatory variable system disease death. for overall mortality. Survival tree for the endpoint circulatory system disease Endpoint mortality for circulatory system diseases mortality The variables selected by EN for the endpoint mortality The survival tree (Figure 1) identified ventricular function, for circulatory system diseases were: age, diabetes mellitus, categorized as moderate or severe dysfunction vs mild

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Table 2 – Values of variables obtained from cardiac magnetic resonance imaging (CMRI) in the study population, and in the subgroups of survival and all-cause deaths

CRMI variables Total population (n = 420) Survivors (n = 344) Deaths (n = 76) p value Anterior AMI, n (%) 218 (51.9) 175 (50.8) 43 (56.5) 0.37 LV IS, median (%) 11.0 10.5 15.0 0.006 LV IS, mean ± SD (%) 14.3 ± 11.0 13.5 ± 10.6 17.7 ± 12.1 0.003

AMI type 0.07 Transmural, n (%) 99 (23.6) 84 (24.4) 15 (19.7) Non-transmural, n (%) 165 (39.2) 141 (40.9) 24 (31.5) Mixed transmurality, n (%) 156 (37.2) 119 (34.6) 37 (48.6) NSegTM AMI, mean ± SD 2.3 ± 2.6 2.1 ± 2.5 3.0 ± 2.9 0.013 NSegNTM AMI, mean ± SD 1.8 ± 1.6 1.7 ± 1.6 1.9 ± 1.6 0.411 NSegAMI, mean ± SD 4.1 ± 2.7 3.9 ± 2.5 4.9 ± 3.0 0.003 Transmurality index 0.44 ± 0.41 0.43 ± 0.41 0.49 ± 0.3 0.29 Presence of no reflow, n (%) 28 (6.7) 22 (6.4) 6 (8.6) 0.82

LV systolic function, n (%) < 0.0001 Normal 221 (52.6) 199 (57.8) 22 (28.9) Mild dysfunction 75 (17.8) 64 (18.6) 11 (14.4) Moderate dysfunction 47 (11.2) 38 (11.0) 9 (11.8) Severe dysfunction 77 (18.3) 43 (12.5) 34 (44.7) LV ejection fraction, mean ± SD (%) 51.0 ± 17.0 53.7 ± 15.6 39.8 ± 18.3 < 0.0001 AMI: acute myocardial infarction; IS: infarct size; LV: left ventricle; SD: standard deviation; NSegTM AMI: number of segments with transmural myocardial infarction; NSegNTM AMI: number of segments with non-transmural acute myocardial infarction; NSegAMI: total number of segments with acute myocardial infarction.

Table 3 – Cox model for the all-cause mortality endpoint

Variables Coefficient SE p value HR 95%CI

Clinical and demographic data Age (years) 0.082 0.012 < 0.0001 1.08 1.06-1.11 Diabetes mellitus 0.516 0.246 0.036 1.67 1.03-2.71

Treatment Medical only Reference PCI -0.368 0.291 0.21 0.69 0.39-1.22 CABG -0.883 0.429 0.039 0.41 0.18-0.96 Serum creatinine (mg/dL) 0.510 0.223 0.022 1.67 1.08-2.58 Previous AMI 0.486 0.296 0.10 1.63 0.91-2.90

CMRI data Infarct size (%) 0.048 0.014 0.0008 1.05 1.02-1.08 Anterior AMI 0.486 0.277 0.079 1.63 0.94-2.80 Moderate / severe LV dysfunction 1.054 0.304 0.0005 2.87 1.58-5.20

Type of AMI Transmural Reference Non-transmural 0.867 0.436 0.047 2.38 1.01-5.60 Mixed 0.495 0.318 0.11 1.64 0.88-3.06 SE: stand error of the coefficient; HR: hazard ratio; 95%CI: 95% confidence interval; PCI: percutaneous coronary intervention; CABG: coronary artery by-pass grafting; AMI: acute myocardial infarction; CMRI: cardiac magnetic resonance imaging; LV: left ventricle.

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Table 4 – Cox model for the endpoint circulatory disease mortality, with the MET-AMI variable included

Variables Coefficient SE p value HR 95%CI Age (years) 0.077 0.019 < 0.0001 1.08 1.04-1.12 Diabetes mellitus 0.441 0.390 0.26 1.55 0.72-3.34

Treatment Medical Reference 0.13 PCI -0.830 0.479 0.083 0.44 0.17-1.12 CABG -0.885 0.599 0.13 0.41 0.13-1.33 Serum creatinine level (mg/dL) 0.899 0.303 0.003 2.46 1.36-4.44 Anterior AMI 0.347 0.423 0.41 1.41 0.62-3.24 Moderate/severe LV dysfunction 1.152 0.420 0.006 3.16 1.39-7.20 Previous AMI 0.949 0.516 0.066 2.58 0.94-7.11 AMI-CMRI time (days) -0.0016 0.001 0.10 1.00 1.00-1.00 Male gender -0.533 0.448 0.23 0.59 0.24-1.41 No reflow 0.513 0.701 0.46 1.67 0.42-6.59

Variable MET-AMI 1 Reference 2 0.028 0.733 0.97 1.03 0.24-4.33 3 1.399 0.612 0.022 4.05 1.22-13.44 4 2.152 0.618 0.0005 8.60 2.56-28.90 SE: standard error of the coefficient; HR: hazard ratio; 95%CI: 95% confidence interval; PCI: percutaneous coronary intervention; CABG: coronary artery by-pass grafting; LV: left ventricle; AMI: acute myocardial infarction; CMRI: cardiac magnetic resonance imaging.

dysfunction or normal, as the most relevant explanatory The Cox model applied to these variables showed that, variable for the classification of survivors and of circulatory among CMRI variables, IS was the one that demonstrated system disease deaths. The age variable was also relevant independent value (HR: 1.09; 95%CI: 1.03-1.15; in the model. p = 0.0015). Among the clinical and demographic variables, independent value was identified for age (HR: 1.08; 95%CI: Joint assessment of MET-AMI and ventricular function 1.03-1.13; p = 0.001), serum creatinine level (HR: 2.62; variables 95%CI: 1.35-5.03; p = 0.004) and previous AMI (HR: 2.87; Figure 2 shows the Kaplan-Meier survival curves for the 95%CI: 1.04‑7.89; p = 0.041). endpoint circulatory system disease mortality, classified in Table 5 shows the results of the Cox regression model four groups according to relevant changes of the MET-AMI applied to the MET-AMI variable. The MET-AMI variable composite variable (defined by cohort ≥ 3) and of left showed an increasing risk ratio, according to its grade, ventricular function (defined by the presence of moderate or showing independent value for grades 3 or 4, and was the severe ventricular dysfunction). variable with the highest risk ratio in the model. A significant reduction in survival was observed in the group with relevant changes in both variables in comparison Survival tree to the subgroup with no changes. The subgroups with only The survival tree (Figure 3) identified IS as a relevant one abnormal variable had intermediate survival, however explanatory variable, with a cut-off point at 21.0% for the significantly shorter than that of the subgroup with no endpoint IHD mortality. abnormalities.

Endpoint ischemic heart disease mortality Discussion The variables selected by EN for the endpoint IHD Most of the studies assessing data obtained from CMRI after mortality were age, diabetes mellitus, serum creatinine level, AMI adopted composite endpoints involving the occurrence of IS (%), moderate or severe LV dysfunction, AMI transmurality, decompensated heart failure, reinfarction, ventricular arrhythmias, previous AMI, and SegNTM AMI. and death. Their mean follow-up period was 2 years7,18.

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1.00

0.95

0.90 0.89 0.85 0.84 0.80

0.75 0.73 0.70

0.65 group A group B

Accumulated probability of survival 0.60 group C group D 0.57 0.55 p < 0.0001 (Log Rank) 0.50

0 360 720 10801440 1800 2160 2520 2880 3240 3600 3960 4320 Follow-up period (days)

Figure 2 – Kaplan-Meier survival curves for the endpoint circulatory system disease mortality, according to the MET-AMI and left ventricular function variables. Group A: dysfunction absent and grades 1 or 2 MET-AMI; Group B: dysfunction absent and grades 3 or 4 MET-AMI; Group C: moderate or severe dysfunction and grades 1 or 2 MET-AMI; Group D: moderate or severe dysfunction and grades 3 or 4 MET-AMI.

Kwong et al19 reported a relevant study on the value of with mortality; also, cigarette smoking was more prevalent in late enhancement documented by CMRI in a population the subgroup of survivors. These findings may be potentially with suspected coronary artery disease, and verified that attributed to the high prevalence of more severely ill patients the presence of subclinical AMI, as detected in CMRI, was with previous coronary artery disease (49.2% with previous the factor more significantly associated with the risk of AMI in this study), who had probably quit smoking before events and mortality. Differently, the focus of the present the index AMI episode. study was to evaluate the role of CMRI after clinical Four clinical variables seemed always significant to predict documentation of AMI. post-AMI death, whether in ours or in other studies24,25, Other studies have evaluated patients with ischemic heart namely: age, renal dysfunction (as expressed by serum disease in more advanced stages and with longer follow-up creatinine levels), history of previous AMI, left ventricular periods4,8, however shorter than that of the present study. dysfunction, and diabetes mellitus. Another differential aspect of this study was the assessment The association between the type of AMI and mortality of the basic death cause using DD data. Cheong et al4 similarly was verified for NTM or mixed AMI, reinforcing the potential obtained mortality data using the social security computed anatomical risk for arrhythmic or ischemic phenomena system, however without the definition of death cause. reported for NTM infarction11 or heterogeneous scar26,27. The study population showed demographic and clinical IS remained a variable of AMI magnitude, with an characteristics consistent with those found in national studies independent value in the present study, in corroboration and registries20-22, as well as in international data23. The mean with most of the studies5,6,8,10,11,18. The independent value age of 62 years is similar to that found for patients with AMI for ventricular dysfunction was more frequently found in of the ACCEPT registry21. studies that also adopted the endpoint all-cause mortality4,8; Bello et al8 investigated the importance of late however, it was also significant to predict short-term enhancement provided by CMRI in 100 patients with composite cardiovascular events after AMI7,12. This was also coronary artery disease in a stable stage and, in corroboration verified in the present study, in which age and ventricular with our findings, observed an independent association of function were identified as explanatory variables for the the ventricular function, IS, and diabetes mellitus variables endpoint all-cause mortality.

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1 Lv function p < 0.001

{3, 4} {1, 2}

2 Age at test p = 0.007

£ 69.4 > 69.4

Node 3 (n = 87) Node 4 (n = 37) Node 5 (n = 296) 1 1 1

0.8 0.8 0.8

0.6 0.6 0.6

0.4 0.4 0.4

0.2 0.2 0.2

0 0 0 020406080100 120 020406080100 120 020406080 120100

Figure 1 – Survival tree for the endpoint circulatory system disease deaths. (1) Normal LV function; (2) Mild LV dysfunction; (3) Moderate LV dysfunction; (4) Severe LV dysfunction. LV: left ventricle.

Table 5 – Cox model for the endpoint ischemic heart disease mortality, with inclusion of the MET-AMI variable

Variables Coefficient SE p value HR 95%CI Age (years) 0.064 0.021 0.003 1.07 1.02-1.11 Diabetes mellitus 0.690 0.463 0.14 1.99 0.80-4.95 Serum creatinine level (mg/dL) 0.797 0.342 0.019 2.22 1.13-4.34 Moderate/severe LV dysfunction 0.623 0.485 0.20 1.86 0.72-4.82 Previous AMI 1.310 0.574 0.022 3.71 1.20-11.4

MET-AMI variable Grade 1 Reference Grade 2 1.222 0.973 0.21 3.39 0.50-22.8 Grade 3 2.056 0.906 0.023 7.82 1.32-46.2 Grade 4 2.775 0.921 0.003 16.04 2.64-97.5 SE: standard error of the coefficient; HR: hazard ratio; 95%CI: 95% confidence interval;AMI: acute myocardial infarction; LV: left ventricle.

The DETERMINE multicenter randomized clinical trial28 In the present study, we observed a 14% cut-off value for IS evaluated the prophylactic use of implantable cardiac for the endpoint overall mortality, by the ROC curve; however, defibrillator in patients after AMI guided by the IS value found for the endpoint IHD mortality, the survival tree identified IS on CMRI, adopting a cut-off value of 10% for CMRI in the late as a relevant variable for a higher cut-off value (21.0%). phase or 15% for CMRI in the acute phase, based on previous Isquierdo et al12 studied 440 patients in the acute phase findings29 of the arrhythmogenic risk of an AMI scar. of AMI in a 2-year follow-up. The occurrence of composite

165 Arq Bras Cardiol. 2015; 104(2):159-168 Petriz et al. Magnetic resonance imaging and mortality after AMI

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1 IS p < 0.001

£ 21 > 21

Node 2 (n = 325) Node 3 (n = 95) 1 1

0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2

0 0 020406080100 120 020406080 120100

Figure 3 – Survival tree for the endpoint circulatory system disease mortality. IS: infarct size.

endpoints (deaths or severe ventricular arrhythmias) was Dra. Mariana M. Canário da Silva, Dra. Daniele Guedes, independently associated with IS; however they occurred Dr. André Casarsa Marques, Dr. Rafael Aron Abtibol and predominantly in the presence of ventricular dysfunction. Dr. Ricardo G. Gusmão Oliveira (Hospital Barra D’Or-RJ); Differently, in the present study, in the subgroup without Dr. Denilson Campos de Albuquerque, Dra. Fernanda significant ventricular dysfunction, the presence of ≥ grade-3 Tovar Moll and Dr. Jorge Moll Neto (Instituto D’Or de MET-AMI was associated with shorter survival. The longer Ensino e Pesquisa-RJ). follow-up period and the high risk ratio observed for this variable may justify this finding. Conclusions limitations The general findings of this study reinforce the ability of clinical and left ventricular function data to predict Retrospective study that assessed cases from the partial case series of patients hospitalized for AMI, with a subgroup the risk after acute myocardial infarction. However, the selected as having a better prognosis, for which CMRI was anatomical data provided by cardiac magnetic resonance indicated. A population at different stages after AMI was imaging regarding acute myocardial infarction showed an studied, since CMRI was adopted as the reference. independent association with long-term mortality, especially for ischemic heart disease death, and thus can contribute for risk identification in selected cases. Acknowledgments The authors express your sincere appreciation to following contributors: Ângela Maria Cascão (Secretaria Author contributions Estadual de Saúde - RJ); Dr. Jorge Moll Filho (Rede D’Or Conception and design of the research: Petriz JLF, Souza e São Luiz); Dr. Fábio Vilas-Boas Pinto (Hospital Espanhol Silva NA; Acquisition of data: Petriz JLF, Gomes BFO, Rua BS, da Bahia); Dr. Carlos Eduardo Rochitte (InCor-USP); Dr. Azevedo CF, Hadlich MS, Mussi HTP, Taets GC; Analysis and Bernardo Rangel Tura (INC - RJ); Dra. Gláucia M. Moraes interpretation of the data: Petriz JLF, Souza e Silva NA; Statistical de Oliveira e Estat. Rosângela Martins (UFRJ); Eng. Luis analysis: Petriz JLF, Azevedo CF, Nascimento EM, Pereira BB; Antônio Mendonça (IME); Profa. Maria Lúcia Brandão Obtaining Financing: Petriz JLF; Writing of the manuscript: (Faculdade de Educação - UFF); Dra. Martha Savedra, Dr. Petriz JLF, Souza e Silva NA; Critical revision of the manuscript Plínio Resende do Carmo Jr, Dra. Andréa C. Parise Fontes, for intellectual content: Petriz JLF, Souza e Silva NA.

Arq Bras Cardiol. 2015; 104(2):159-168 166 Petriz et al. Magnetic resonance imaging and mortality after AMI

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Potential Conflict of Interest Study Association No potential conflict of interest relevant to this article was This article is part of the doctoral thesis of the authors João reported. Luiz Fernandes Petriz (doctoral student), Basílio de Bragança Pereira and Nelson Albuquerque de Souza e Silva (counselors) at the Programa de Pós-Graduação em Medicina Cardiologia Sources of Funding da Faculdade de Medicina/ Instituto do Coração Edson Saad This study was partially funded by CAPES. - Universidade Federal do Rio de Janeiro (UFRJ).

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Arq Bras Cardiol. 2015; 104(2):159-168 168 Review Article

Microparticles as Potential Biomarkers of Cardiovascular Disease Carolina Nunes França1,2, Maria Cristina de Oliveira Izar1, Jônatas Bussador do Amaral1, Daniela Melo Tegani1, Francisco Antonio Helfenstein Fonseca1 Universidade Federal de São Paulo – UNIFESP1, Universidade de Santo Amaro – UNISA2, São Paulo, SP – Brazil

Abstract from cell destruction or only markers of apoptosis. In 1996, however, Raposo et al.5 suggested that MP played an Primary prevention of cardiovascular disease is a important role in adaptative immune response. Since then, choice of great relevance because of its impact on health. several studies have shown the importance of MP as vectors Some biomarkers, such as microparticles derived from of intracellular exchange of biological information, by use different cell populations, have been considered useful in of identification, characterization and quantification of the assessment of cardiovascular disease. Microparticles MP in several situations, such as obesity, diabetes mellitus, are released by the membrane structures of different cell infarction, depression, cancer, HIV and renal failure. types upon activation or apoptosis, and are present in the plasma of healthy individuals (in levels considered As the atherosclerotic process develops, monocytes physiological) and in patients with different pathologies. accumulate lipoproteins and change into cholesterol-rich Many studies have suggested an association between macrophages, which undergo apoptosis, releasing a high microparticles and different pathological conditions, amount of lipids to the extracellular medium, causing a mainly the relationship with the development of vicious cycle of inflammation, oxidative stress, apoptosis cardiovascular diseases. Moreover, the effects of different of endothelial cells or endothelial erosion (Figure 1). lipid-lowering therapies have been described in regard This culminates in atherothrombotic outcomes, such as to measurement of microparticles. The studies are still myocardial infarction or ischemic cerebral vascular accident, controversial regarding the levels of microparticles which result from the contact of inner plaque substances with that can be considered pathological. In addition, the blood, producing immediate coagulation and consequent 6 methodologies used still vary, suggesting the need for total and sudden obstruction of the vessel . standardization of the different protocols applied, aiming Healthy individuals and those with different diseases have at using microparticles as biomarkers in clinical practice. MP in their plasma7. Inflammatory stimuli to the release of MP include Gram-negative bacterial lipopolysaccharides (LPS) and cytokines, such as tumor necrosis factor alpha (TNF-α), Introduction interleukine 6 (IL-6) and interleukine 1 (IL-1β). After activation, the cells change their asymmetrical conformation, exposing Microparticles (MP) are defined as a population of phosphatidylserine, an aminophospholipid responsible for 8 vesicles derived from different cell types (Table 1) after high procoagulant capacity . activation or apoptosis, measuring from 50 nm to 1000 nm, and containing cell material, such as proteins, mRNA and Microparticles derived from different cell lipoproteins, which are fundamental to the identification of those vesicles by use of different techniques, such as populations flow cytometry1. All blood cells produce MP, the greatest amount being released by platelets, platelet MP (PMP), Platelet microparticles corresponding to 70%-90% of the total amount of MP in 2-4 the plasma of healthy individuals . The PMP are the most abundant in human plasma4,9. Until the 1990s, no biological importance had been Several studies have shown the relationship between those given to MP, which were considered inert particles resulting PMP and blood coagulation10, inflammatory processes11, thrombosis and tumor progression12, as well as the interaction between leukocytes and endothelial cells13. Keywords However, some studies have suggested that PMP have an Cardiovascular Diseases; Biomarkers, Pharmacological important role in tissue regeneration, because they are strongly 14 / analysis; Cell-Derived Microparticles; Atherosclerosis / associated with angiogenesis . Some authors have shown that, prevention & control. in vitro, PMP promote budding of aortic rings by activating the signaling pathways of PI3-kinase and Extracellular Signal- Mailing Address: Carolina Nunes França • Regulated Kinase (ERK)13, in addition to promoting post- Rua Pedro de Toledo, 276, Vila Clementino, Postal Code 04039030, 15 São Paulo, SP - Brazil ischemic revascularization . E-mail: [email protected]; [email protected] Manuscript received September 10, 2014; revised manuscript September 19, 2014; accepted October 21, 2014 Endothelial microparticles Endothelial MP (EMP) represent a smaller population of DOI: 10.5935/abc.20140210 MP in plasma, but have been associated with cardiovascular

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Table 1 – Antigens on the surface of microparticles derived from platelets, endothelium and monocytes

Microparticles Surface antigens References PMP CD31, CD41, CD42, CD61, CD62P, CD63 3, 8, 10, 39, 58, 62 CD31, CD51, CD54, CD62E,CD105, CD106, CD144, CD146, E-selectina, 8, 9, 23, 34, 36, 39, 42, 43, EMP VE-caderina 58-61

MMP CD14, CD54 28, 29, 31

PMP: platelet microparticles; EMP: endothelial microparticles; MMP: monocyte microparticles62

Figure 1 – Ilustration of cell interactions during atheroma formation.

disease, mainly endothelial dysfunction8. Similarly to PMP, manner to the concentration of EMP. Those same some studies have suggested a relationship between authors had previously shown that 105 EMP/mL impaired EMP and angiogenesis16, tumor growth17 and increased endothelium-dependent relaxation, which was not seen oxidative stress18. with 104 EMP/mL27. Some studies have shown the importance of EMP in the proliferation and differentiation of endothelial progenitor Monocyte microparticles 19 cells, which are essential for vascular regeneration , Similarly to PMP, the MP originated from monocytes, indicating a possible protective function related to vascular monocyte MP (MPM), can contain procoagulant 20 regeneration, repair and protection . substances and be related to endothelial dysfunction28 and Mezentnev et al.21 have assessed in vitro different sepsis29. The study by Wang et al.30 has shown that MPM patterns of angiogenesis (cell division rate, capillary can activate endothelial cells, because MPM contain IL-1β, formation and apoptosis of endothelial cells), comparing which enhances the inflammatory process. physiological levels of EMP present in healthy individuals Hoyer et al.31 have assessed the role of MPM in (between 103 and 104 EMP/mL)22,23 and pathological vascular inflammation and reported that the treatment concentrations (present in individuals with cardiovascular of ApoE -/- mice with MPM promoted the formation disease, 105 EMP/mL)24-26. Those authors have reported of atherosclerotic plaque in the mice and increased that pathological levels of EMP affected all parameters the accumulation of macrophages in the vascular wall. associated with angiogenesis in a directly proportional Those authors have suggested an important interaction

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between MPM and inflammatory cells in the atherosclerotic electrocardiography). The main finding was that the disease of ApoE -/- mice. MP subpopulations assessed (PMP and EMP) showed higher levels within the coronary arteries as compared Microparticles and coronary disease to those in aortic blood. In addition, a greater release of both MP subpopulations was observed in the impaired Several studies have suggested a direct relationship coronary artery than in ascending aorta, indicating local between the increase in MP and development of coronary MP production. Those authors have suggested that their disease. Augustine et al.32, assessing patients undergoing findings can support the hypothesis that MP act as active dobutamine stress echocardiography, have reported an elements in the embolization and pathophysiology of elevation in MP derived from different cell types (platelets, microvascular obstruction. erythrocytes and endothelial cells) immediately after the test 38 followed by a rapid MP clearance from the circulation during Kaabi et al. have assessed the relationship between the next hour in response to cardiac stress. Those authors have the levels of MP and treatment of stable coronary artery suggested that the release of MP is a protective mechanism to disease patients with external counterpulsation (ECP). clear cell stress in those patients. That therapy has been considered effective and safe for patients with refractory angina pectoris. Those authors Sarlon-Bartoli et al.33 have measured the plasma levels of have found an increase in PMP after ECP therapy, and no leukocyte-derived MP (LMP) in 42 individuals with carotid difference in EMP and MPM levels. artery stenosis greater than 70%. They have shown that 11 patients with unstable plaque had increased levels of the Willians et al. have assessed platelet activation and CD11bCD66b+ and CD15+ LMP, suggesting that even less depression levels in coronary artery disease patients, frequently found subpopulations of MP in plasma, as compared because depression, even mild, is an independent to PMP, can provide important information regarding clinical predictor of increased mortality after myocardial infarction. studies on atherosclerotic plaque vulnerability in patients with Those authors have reported that patients with moderate high-grade carotid stenosis. depression and high levels of TNF-α, IL-6 and PCR also released more PMP, indicating that a pro-inflammatory Morel et al.34 have assessed the levels of LMP and component could change platelet function in those patients. EMP within occluded coronary arteries of ST-segment 39 elevation myocardial infarction patients treated with Bernal-Mizrachi et al. have shown that, depending on primary angioplasty and have compared them with the the cell stimulus (activation or apoptosis), different surface levels of MP in peripheral blood. Those authors have proteins are expressed. Those authors have conducted a reported an increase in MP within arteries, indicating the study analyzing two subpopulations of EMP (CD31+/CD42- importance of those vesicles in the development of coronary and CD51+) in coronary artery disease patients and have atherothrombosis. reported that CD31+/CD42- EMP were more frequently expressed in acute events (myocardial infarction and Faille et al.35 have measured CD11b+ MP (monocyte unstable angina), and CD51+ EMP were released in similar marker) in patients with acute coronary syndrome with no amounts both in acute and chronic events (stable angina). ST-segment elevation on the electrocardiogram, aiming at assessing whether the quantification of those MP could contribute to the identification of patients at higher risk Microparticles and diabetes mellitus for a recurring cardiovascular event within one month Some studies have shown higher concentrations of after coronary stent implantation. A smaller amount of PMP related to diabetes mellitus. Ogata et al.40 have CD11b+ MP was found in individuals with recurring assessed the levels of PMP in 92 patients with diabetic cardiovascular event as compared to that in patients with retinopathy. Those authors have reported increased release no complications, suggesting greater capture of those MP of PMP in those patients as compared to that in healthy in sites of atherosclerotic lesions. individuals, and the increase was higher the more severe Jeanneteau et al.36 have assessed in rats and humans the retinopathy. In atherothrombosis, MP are related to the role of MP in the mechanism of remote ischemic the release of cytokines by leukocytes and endothelial conditioning (RIC), which has been described as an cells, monocyte recruitment to the atherosclerotic infarction-related cardioprotective strategy. No differences plaque, smooth muscle cell proliferation, angiogenesis were found in the total number of MP in the group of and increased oxidative stress. In addition, MP can be animals undergoing RIC as compared to the control group. signalers of cell homeostasis, promoting balance between 12 After phenotypic characterization of MP, elevations in the cell stimulus, proliferation and apoptosis . endothelial and Annexin V+ (apoptotic) subpopulations Lumsden et al.41 have assessed patients with type 2 were observed in the RIC group. Similarly, elevations in diabetes mellitus after acute coronary syndrome (six months EMP and Annexin V+ MP were found in the group of prior to event), who had reduced levels of EMP and no individuals submitted to RIC. PMP changes. Those authors have suggested that those Porto et al.37 have assessed the concentrations of MP unexpected findings, which disagree with most studies in in ST-segment elevation myocardial infarction patients the literature, can result from concomitant medications 42-44 undergoing primary percutaneous coronary intervention, used by patients . and the relationship of those vesicles with microvascular Other studies have reported that the increase in the obstruction (defined by multiple angiography and expression of adhesion molecules is associated with the

171 Arq Bras Cardiol. 2015; 104(2):169-174 França et al. Microparticles and Cardiovascular Disease

Review Article activation of monocytes, which can bind to endothelial cells Pinheiro et al.59 have assessed the effect of the antiplatelet in vessel walls, leading to diabetic retinopathy progression. drug clopidogrel in association or not with rosuvastatin Those data suggest that measuring the levels of MPM can (40 mg) on the levels of EMP and PMP in patients with be a useful biomarker of diabetic retinopathy progression45. stable coronary disease on statins for at least three months. Those authors have identified an increase in the levels of Microparticles in endothelial dysfunction and PMP after suspension of rosuvastatina and maintenance dyslipidemia of only clopidogrel for four weeks and a tendency towards greater release of EMP in those patients. They have suggested Leroyer et al.46 have shown that MP originated from that an increase in the apoptosis of platelets occurred, macrophages (CD40+) can promote angiogenesis within and that rosuvastatin might have a protective effect on the plaque, suggesting that these MP can determine plaque the endothelium when associated with clopidogrel. In a vulnerability. However, studies assessing the relationship similar study, França et al.60 have assessed the influence of between MP and angiogenesis have proved contraversial and atorvastatin (80 mg) in association or not with clopidogrel inconclusive, because some MP have been reported to be in patients with stable coronary disease. Those authors have able to stimulate angiogenesis (PMP, for example)13,47, while suggested higher vascular stability promoted by atorvastatin other studies have reported that MP can both stimulate and after identifying an inverse relationship between the plasma inhibit angiogenesis, depending on cell origin48-50. concentration of atorvastatin and the levels of PMP. Some studies have shown that individuals with metabolic Another study61 has assessed the effect of the treatment syndrome have increased levels of MP as compared to with vitamin C for five days on the levels of MP in patients those of healthy individuals, and that MP are related to with diabetes, dyslipidemia, or at least two risk factors for endothelial dysfunction, due to decreased eNOS expression post‑infarction cardiovascular disease. A reduction in the amount and increased release of reactive oxygen species51. of EMP and PMP was observed, which has been associated with The first study to show the direct effect of MP on vascular the reduction in oxidative stress caused by vitamin C. function has been developed by Boulanger et al.52 Those authors Thus, the studies have shown that MP can be useful markers have assessed whether the MP present in peripheral blood of not only to assess cardiovascular disease, but also cancer10,17, patients with non-ischemic syndrome and after acute myocardial sepsis22 and other illnesses. However, the MP levels considered infarction would influence the endothelium-dependent physiological and pathological are still controversial. response in aortic rings of rats. The MP of post-infarction Although flow cytometry is considered a reference for the individuals have been reported to reduce acetylcholine‑induced identification and phenotypic characterization of MP, the vascular relaxation (by influencing the nitric oxide pathway), studies have used several methods (time of centrifugation and suggesting that MP could contribute to the endothelial incubation with antibodies, different markers), which makes dysfunction observed after the acute event. the comparison between publications in the literature difficult. Diehl et al.53 have analyzed different subpopulations In conclusion, the search for new biomarkers that might of MP in individuals with pulmonary hypertension, and be related to cardiovascular disease has increased the interest have reported increased levels of LMP, EMP and PMP, in MP derived from different cells, especially platelets. indicating higher inflammatory and procoagulant activity, However, studies are still controversial, and further research which can be related to thromboembolic complications on standardization of more sensitive techniques to obtain, and endothelial dysfunction in those patients. characterize and quantify those vesicles is required, so that The improvement in endothelial function promoted by the findings can be applied to clinical practice. calcium channel blockers has been well described in the literature. Nomura et al.54 have shown a reduction in EMP in patients with type 2 diabetes mellitus after treatment with Author contributions the calcium channel blocker, nifedipine. Similar result has Acquisition of data: França CN. Writing of the manuscript: been reported by the same group55 with patients with type França CN, Amaral JB, Tegani DM. Critical revision of the 2 diabetes mellitus and hypertension after treatment with manuscript for intellectual content: Izar MCO, Fonseca FAH. benidipine, belonging to the same drug class. The effect of Figure Confection:Amaral JB. the renin-angiotensin system blocker valsartan on the levels of MPM in individuals with type 2 diabetes mellitus has also been assessed by the same group56, who has reported that the drug Potential Conflict of Interest inhibited the release of MPM. Those results have suggested No potential conflict of interest relevant to this article was that the renin-angiotensin system blocker can contribute to reported. the treatment of atherosclerosis. Sources of Funding Microparticles and lipid-lowering therapies There were no external funding sources for this study. New strategies that can either inhibit MP functions or provide greater clearance have been searched. Several studies have assessd the effect of different lipid-lowering strategies on Study Association the amount of MP released by different cell types57,58. This study is not associated with any thesis or dissertation work.

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38. Kaabi AA, Traupe T, Stutz M, Buchs N, Heller M. Cause or effect of 52. Boulanger CM, Scoazec A, Ebrahimian T, Henry P, Mathieu E, Tedgui A, atherogenesis: compositional alterations of microparticles from CAD et al. Circulating microparticles from patients with myocardial infarction patients undergoing external counterpulsation therapy. Plos One. cause endothelial dysfunction. Circulation. 2001;104(22):2649-52. 2012;7(10):e46822. 53. Diehl P, Aleker M, Helbing T, Sossong V, Germann M, Sorichter S, 39. Bernal-Mizrachi L, Jy W, Jimenez JJ, Pastor J, Mauro LM, Horstman LL, et et al. Increased platelet, leukocyte and endothelial microparticles al. High levels of circulating endothelial microparticles in patients with predict enhanced coagulation and vascular inflammation in pulmonary acute coronary syndromes. Am Heart J. 2003;145(6):962-70. hypertension. J Thromb Thrombolysis. 2011;31(2):173-9.

40. Ogata N, Imaizumi M, Nomura S, Shozu A, Arichi M, Matsuoka M, et 54. Nomura S, Inami N, Kimura Y, Omoto S, Shouzu A, Nishikawa M, et al. al. Increased levels of platelet-derived microparticles in patients with Effect of nifedipine on adiponectin in hypertensive patients with type 2 diabetic retinopathy. Diab Res Clin Pract. 2005;68(3):193-201. diabetes mellitus. J Hum Hypertens. 2007;21(1):38-44.

41. Lumsden NG, Andrews KL, Bobadilla M, Moore XL, Sampson AK, Shaw 55. Nomura S, Shouzu A, Omoto S, Nishikawa M, Iwasaka T. Benidipine JA, et al. Endothelial dysfunction in patients with type 2 diabetes post improves oxidized LDLdependent monocyte and endothelial dysfunction acute coronary syndrome. Diab Vasc Dis Res. 2013;10(4):368-74. in hypertensive patients with type 2 diabetes mellitus. J Hum Hypertens. 42. Tramontano AF, Lyubarova R, Tsiakos J, Palaia T, Deleon JR, Ragolia L. 2005;19(7):551-7. Circulating endothelial microparticles in diabetes mellitus. Mediators 56. Nomura S, Shouzu A, Omoto S, Nishikawa M, Fukuhara S, Iwasaka Inflamm. 2010;2010:250476. T. Effect of valsartan on monocyte/endothelial cell activation markers 43. Koga H, Sugiyama S, Kugiyama K, Watanabe K, Fukushima H, Tanaka T, and adiponectin in hypertensive patients with type 2 diabetes mellitus. et al. Elevated levels of VE-cadherin-positive endothelial microparticles Thromb Res. 2006;117(4):385-92. in patients with type 2 diabetes mellitus and coronary artery disease. J 57. Camargo LM, França CN, Izar MC, Bianco HT, Lins LS, Barbosa SP, et al. Am Coll Cardiol. 2005;45(10):1622-30. Effects of simvastatin/ezetimibe on microparticles, endothelial progenitor 44. Feng B, Chen Y, Luo Y, Chen M, Li X, Ni Y. Circulating level of cells and platelet aggregation in subjects with coronary heart disease microparticles and their correlation with arterial elasticity and under antiplatelet therapy. Braz J Med Biol Res. 2014;47(5):432-7. endothelium-dependent dilation in patients with type 2 diabetes 58. Lins LC, França CN, Fonseca FA, Barbosa SP, Matos LN, Aguirre AC, et al. mellitus. Atherosclerosis. 2010;208(1):264-9. Effects of ezetimibe on endothelial progenitor cells and microparticles 45. Ogata N, Nomura S, Shouzu A, Imaizumi M, Arichi M, Matsumura M. in high-risk patients. Cell Biochem Biophys. 2014;70(1):687-96. Elevation of monocyte-derived microparticles in patients with diabetic 59. Pinheiro LF, França CN, Izar MC, Barbosa SP, Bianco HT, Kasmas SH, et retinopathy. Diabetes Res Clin Pract. 2006;73(3):241-8. al. Pharmacokinetic interactions between clopidogrel and rosuvastatin: 46. Leroyer AS, Rautou PE, Silvestre JS, Castier Y, Leseche G, Devue C, et effects on vascular protection in subjects with coronary heart disease. al. CD40 ligand- microparticles from human atherosclerotic plaques Int J Cardiol. 2012;158(1):125-9. stimulate endothelial proliferation and angiogenesis a potential 60. França CN, Pinheiro LF, Izar MC, Brunialti MK, Salomão R, Bianco HT, mechanism for intraplaque neovascularization. J Am Coll Cardiol. et al. Endothelial progenitor cell mobilization and platelet microparticle 2008;52(16):1302-11. release are influenced by clopidogrel plasma levels in stable coronary 47. Brill A, Elinav H, Varon D. Differential role of platelet granular mediators artery disease. Circ J. 2012;76(3):729-36. in angiogenesis. Cardiovasc Res. 2004;63(2):226-35. 61. Morel O, Jesel L, Hugel B, Douchet MP, Zupan M, Chauvin M, et 48. Mostefai HA, Agouni A, Carusio N, Mastronardi ML, Heymes C, al. Protective effects of vitamin C on endothelium damage and Henrion D, et al. Phosphatidylinositol 3-kinase and xanthine oxidase platelet activation during myocardial infarction in patients with regulate nitric oxide and reactive oxygen species productions by sustained generation of circulating microparticles. J Thromb Haemost. apoptotic lymphocyte microparticles in endothelial cells. J Immunol. 2003;1(1):171-7. 2008;180(7):5028-35. 62. Montoro-Garcia S, Shantsila E, Hernández-Romero D, Jover E, Valdés 49. Agouni A, Mostefai HA, Porro C, Carusio N, Favre J, Richard V, et al. Sonic M, Marín F, et al. Small-size platelet microparticles trigger platelet and hedgehog carried by microparticles corrects endothelial injury through monocyte functionality and modulate thrombogenesis via P-selectin. Br nitric oxide release. FASEB J. 2007;21(11):2735-41. J Haematol. 2014;166(4):571-80.

Arq Bras Cardiol. 2015; 104(2):169-174 174 Letter to the Editor

Acute Effects of Continuous Positive Airway Pressure on Pulse Pressure in CHF Jacobo Bacariza Blanco e Antonio M. Esquinas Unidade de Terapia Intensiva, Hospital de São Bernardo EPE1, Setúbal - Portugal; Unidade de Terapia Intensiva, Hospital Morales Meseguer2, Murcia - Spain

Today’s reality on non-invasive ventilation (NIV) use the stroke volume (SV) and improves left systolic output. has nothing but four key A level indications supported by The afterload reduction is secondary to the systemic evidence-based medicine. These, which would be chronic vasodilatation effect as a response to intrathoracic pressure obstructive pulmonary disease (COPD) exacerbation, elevation. As a final result, HR, MAP, SAP and PP decrease, cardiogenic pulmonary edema, pulmonary infiltrates in protecting myocardial oxygenation and reducing the immunocompromised patients, and the weaning of already myocardial infarction risk. In the right heart, translung extubated COPD patients, are the so called “the fabulous pressure reduces preload secondary to the vena cava 1 four” . But is this the maximum therapeutic potential of squeeze and elevates afterload3 by the increase in pulmonary NIV? Probably not. If so, which would be the next one vascular resistances. As a result a “dDown”4 effect and on this selected “fabulous four” group? Maybe it is stable right SV reduction occur, reducing vascular congestion and chronic heart failure (CHF). If so, we would be facing a lung edema, and once again improving oxygenation and new frontier, yet unexplored, of those chronically stable not ventilation. Regarding respiratory effects, there will be direct respiratory but cardiac patients, opening new applications, oxygenation by O administration and also the alveolar none existent up to today. 2 recruitment effect. As a final result, PaO and mixed venous 2 2 Quintão et al. move on into the next step to conquer oxygen (SVO ) raise, and RR and HR decrease. this new frontier, the NIV application on stable CHF. 2 2 They do so, analyzing the NIV (Continuous Positive Airway In a study by Quintão et al. , hemodynamic Pressure - CPAP) effects on pulse pressure (PP), as a risk monitoring was not continuous, but manually measured factor with independent negative predictive value for (sphygmomanometer); thus, a continuous monitoring adverse cardiovascular events, followed by left ventricular might offer more accurate and precise data. Actually dysfunction, especially type II, caused by acute myocardial additional monitoring with echocardiography will allow ischemia. They prove not only to affect PP reduction, but to expand data, calculate ejection fraction and SV, also heart rate (HR), mean arterial pressure (MAP), systolic which will allow to establish the relationship between PP blood pressure (SBP) and respiratory rate (RR). reduction and ventricular output improvement. The trial The results will be explained through the relationship lasted 30 minutes, enough to confirm the hypothesis, but between positive pressure ventilation effects3 on the a longer time will allow maximum effect assessment to cardiorespiratory system. In the left heart, pulmonary vein possibly define the best CPAP potential on this matter.

compression followed to translung pressure increases, Finally, although a CPAP pressure of 6 cm H20 is in fact improving venous return and so the preload. In addition, this the usual level used in those studies, a bigger pressure of

translung pressure increase contributes to squeeze the heart 8 cm H20 will probably have a bigger effect, as we usually chambers and discharge them, this “dUp”4 effect increases see in everyday work.

Keywords Heart Failure; Continuous Positive Airway Pressure; Pulse. Mailing Address: Jacobo Bacariza Blanco • Rua Miradouro do Sado N9 7 Dto, Nossa Senhora da Anunciada, Setúbal - Portugal Email: [email protected] Manuscript received October 10, 2014; revised manuscript December 02, 2014; accepted December 02, 2014 DOI: 10.5935/abc.20140217

175 Blanco et al. Chronic Heart Failure

Letter to the Editor

References

1. Nava S. Behind a mask: tricks, pitfalls, and prejudices for noninvasive Hemodynamic monitoring using echocardiography in the critically III. ventilation. Respir Care. 2013;58(8):1367-76. Philadelphia: Springer; 2011.

2. Quintão M, Chermont S, Marchese L, Brandão L, Bernardez SP, Mesquita ET, 4. Polanco PM, Pinsky MR. Cardiovascular issues in respiratory care: clinical et al. Acute effects of continuous positive airway pressure on pulse pressure applications of heart lung interactions. In: Yearbook respiratory care clinics in chronic heart failure. Arq Bras Cardiol. 2014;102(2):181-6. and applied technologies. Murcia: Esquinas Antonio; 2008. p. 396-401.

3. Vieillard-Baron A. Heart lung interactions in mechanical ventilation. In: Backer D, Cholly BP, Slama M, Vieillard-Baron A, Vignon P (eds).

Reply Letter to Editor I am very thankful to Dr. Blanco for his comments regarding similar form to ours5,6. Our group has studied hemodynamic our study. Non-invasive ventilation (NIV) has been our focus parameters beat to beat and will publish the results soon. of study, especially with chronic heart failure (CHF), thus, an In our CPAP experience in CHF, we also observed that the opportunity to discuss it is always welcome. In our experience, main hemodynamic changes occur between 10 and 20 minutes, we have observed benefits with the use of NIV with lower CPAP and, after that, there is very few or no significant difference levels to exercise tolerance in CHF patients1. Other authors2,3 from baseline. Furthermore, 30-minute protocols for CPAP have published studies before showing hemodynamic effects have proven to be enough to provide satisfactory results, even with lower CPAP levels as adequate and safer. This gave us in patients with CHF exacerbation7. Patients with CHF undergo an incentive to use that form, since our patients were stable many phases of both functional and respiratory worsening in and, therefore, no upper levels were necessary, which could the course of their illness. Non‑invasive ventilation with CPAP be uncomfortable, and, consequently, promote increase of may be a method available to improve quality of life. Our group some parameters. Thus, we observed in a scale of 3-6 cm has also used CPAP as a non‑pharmacological resource for the

H2O the greatest CPAP pressure, which showed a decrease in relief of dyspnea to reduce any minimum hemodynamic load hemodynamics parameters with the least discomfort sensation, caused by the mechanism of that syndrome. In HF outpatient as in other studies4. The hemodynamics parameters were context, we used that device as a complementary treatment for measured in periods, but not continuously. In fact, in previous HF. Our future results will demonstrate the magnitude of the use studies they were measured continuously in many forms, by of this non-pharmacological resource in different hemodynamic use of catheterization or echocardiography, and others, in a variables and the benefits to the quality of life of patients with HF.

References

1. Chermont S, Quintão MM, Mesquita ET, Rocha NN, Nóbrega AC. on cardiac autonomic modulation in chronic heart failure and chronic Noninvasive ventilation with continuous positive airway pressure acutely obstructive pulmonary disease. Arch Med Sci. 2010;6(5):719-27. improves 6-minute walk distance in chronic heart failure. J Cardiopulm Rehabil Prev. 2009;29(1):44-8. 5. Steiner S, Schannwell CM, Strauer BE. Left ventricular response to continuous positive airway pressure: role of left ventricular geometry. 2. Bradley TD, Holloway RM, McLaughlin PR, Ross BL, Walters J, Liu PP. Cardiac Respiration. 2008;76(4):393-7. output response to continuous positive airway pressure in congestive heart failure. Am Rev Respir Dis. 1992;145(2 Pt 1):377-82. 6. Azevedo JC, Carvalho ER, Feijó LA, Oliveira FP, Menezes SL, Murad H. Effects of the continuous positive airway pressure on the airways of patients with 3. Yoshida M, Kadokami T, Momii H, Hayashi A, Urashi T, Narita S, et al. chronic heart failure. Arq Bras Cardiol. 2010 Jul;95(1):115-21. Enhancement of cardiac performance by bilevel positive airway pressure ventilation in heart failure. J Card Fail. 2012;18(12):912-8. 7. Bellone A, Barbieri A, Ricci C, Iori E, Donateo M, Massobrio M, et al. Acute effects of non-invasive ventilatory support on functional mitral regurgitation 4. Reis MS, Sampaio LM, Lacerda D, De Oliveira LV, Pereira GB, Pantoni CB, in patients with exacerbation of congestive heart failure. Intensive Care Med. et al. Acute effects of different levels of continuous positive airway pressure 2002;28(9):1348-50.

Arq Bras Cardiol. 2015; 104(2):175-176 176 Clinicoradiological Session

Case 2/2015 A 33-Year-Old Woman with Double Right Ventricular Chamber and Ventricular Septal Defect Edmar Atik Instituto do Coração, Faculdade de Medicina, Universidade de São Paulo – USP, São Paulo, SP - Brazil

Clinical data: non-progressive shortness of breath on was a 3-mm foramen ovale with bidirectional predominantly moderate exertion for 1 year. A heart murmur had been heard left‑to‑right shunt. RV systolic pressure = 110 mmHg. at one month of age, and the patient had been followed up Cardiac catheterization (Figure 2) Pressure values were: RV irregularly ever since. She did not report cyanosis and had inflow tract = 98/13; RV outflow tract = 25/7; PT = 20/7-12; recently been prescribed with hydrochlorothiazide. Laboratory LV = 124/11; Ao = 126/64; PC = 11 mmHg. tests showed H = 4,700,000/mm3; Hct = 41%; Hg = 13.4 g/dL. Clinical diagnosis: Severe RV inflow tract stenosis with Physical examination: normal breathing, acyanotic, perimembranous ventricular septal defect of little impact, normal pulses. Weight: 62 kg; height: 163 cm; blood pressure: with no hypoxemia and/or heart failure, in natural course. 110/60 mmHg; heart rate: 78 bpm; oxygen saturation: 97%; Clinical reasoning: The clinical elements of long-standing aorta not palpable on the suprasternal notch. right obstructive congenital heart defects without hypoxemia The apical impulse was not palpable in the precordium, present as shortness of breath and are not accompanied by and there were no systolic impulses on the left sternal hematocrit elevation. For this reason, with preserved cardiac border. Normal heart sounds; grade 3/4 course systolic output, their outcome is favorable thanks to compensatory murmur on the mid-left sternal border accompanied by myocardial hypertrophy. The normal heart sounds suggest thrill. The liver was not palpable. normal arterial position, and the preserved pulmonary flow results from an adequate compensatory mechanism. Laboratory tests The severe systolic murmur in the mid-left sternal border Electrocardiogram showed normal sinus rhythm and signs suggests the presence of an obstructive lesion in the RV inflow of right ventricular (RV) overload. Low-voltage QRS complex tract and differentiates from the murmur of the ventricular with qR morphology in V1, RS in V6 with final conduction septal defect for being coarser and stronger. Ventricular disturbance through the right bundle branch. QRSA: -70o, septal defects of little impact do not result in any functional disturbance, and their auscultatory manifestation mingles TA: + 30o. with that of the obstructive defect. RV systolic overload on Chest radiograph showed normal cardiac silhouette the electrocardiogram suggests the diagnosis of an obstructive (cardiothoracic ratio of 0.46). The pulmonary vascular lesion on the right. Chest radiograph showing normal network was normal and the arch of the pulmonary artery cardiac silhouette is consistent with good RV function, and was concave (Figure 1). the normal pulmonary vascular network results from the Echocardiogram (Figure 2) showed right ventricular maintained antegrade flow. inflow tract stenosis causing an intraventricular gradient of Differential diagnosis: Heart diseases with RV outflow 80 mmHg, with a 13-mm diameter ventricular septal defect tract obstruction may have a similar presentation, except for partially occluded by the tricuspid valve, thus resulting in an the systolic murmur, which is more intense in the upper left effective 4.7‑mm orifice. There was RV hypertrophy with mildly sternal border, irradiating to the neck vessels, albeit mildly. enlarged right cardiac chambers. Gradient between ventricles Management: Because of the long-standing systolic was 82 mmHg. There was a small aneurysm formation in the impact with severe hypertrophy and nearly systemic RV ventricular septum. Measurements were as follows: left ventricle pressures, indication of operation becomes mandatory, aiming (LV) = 45 mm; left atrium (LA) = 32; Ao = 26; septum = at relieving the chamber obstruction, which would imply a posterior wall = 7 mm. LV ejection fraction = 66%. There greater possibility of development of myocardial fibrosis, heart failure, arrhythmias and earlier death. Commentaries: Obstructive congenital heart defects Keywords usually manifest early in life by means of a heart murmur. Double chambered right ventricle, right ventricle inflow Cardiac compensation with maintained antegrade flow is tract stenosis, ventricular septal defect, hipertrophy of achieved by myocardial hypertrophy. The consequences of right ventricle. this phenomenon are directly proportional to the degree Mailling Address: Edmar Atik • of obstruction. In the present case, this obstruction, which Rua Dona Adma Jafet, 74, conj. 73, Bela Vista. Postal Code 01308-050. was more intense in adulthood and whose manifestation of São Paulo, SP – Brazil symptoms had been more recent, progressed throughout E-mail: [email protected]; [email protected] Manuscript received July 23, 2014; revised manuscript July 31, 2014; time, which permitted a good outcome during this period. accepted July 31, 2014. Hence, the patient had a favorable course up to the fourth decade of life. Most of these patients are treated in childhood DOI: 10.5935/abc.20140218 and very uncommonly in adulthood. These defects result e12 Edmar Atik Right Ventricular Inflow Tract Stenosis

Clinicoradiological Session

On expiration and hypertrophic shape

Figure 1 – Chest radiograph showing normal cardiac silhouette, with a round shape resulting from myocardial hypertrophy due to an obstructive lesion on the right side.

Figure 2 – (A) 4-chamber apical view echocardiogram shows ventricular septal defect with septal aneurysm (arrow); (B and C) short-axis cross-sectional view showing right ventricular inflow tract stenosis (arrows, in colors). (D) Angiography showing septal aneurysm (arrow) and (E) right ventricular inflow tract stenosis (arrow) with marked hypertrophy.

Arq Bras Cardiol. 2015; 104(2):e12-e14 e13 Edmar Atik Right Ventricular Inflow Tract Stenosis

Clinicoradiological Session

from an impaired growth of the trabecular myocardium with ventricular septal defects (80% of cases)1,2. Even when during the early fetal formation, and the non-uniformity their impact is minor, these obstructive anomalies should be in its position, closer to the tricuspid or pulmonary valve, treated earlier to prevent an unfavorable outcome in relation which causes the ventricle to divide into two parts – a to the development of myocardial fibrosis, arrhythmias and proximal and a distal part. They are frequently associated heart failure.

References

1. López-Fernández S, Molina-Lerma MJ, García-Orta R, Medina-Benitez A. 2. Lee WJ, Song BG, Kang GH, Park YH, Chun WJ, Oh JH. A case of Double-chambered right ventricle in adults: an ‘uncommon’ entity, new asymptomatic isolated double-chambered right ventricle in an adult man. ways of imaging. Eur Heart J. 2013;34(11):801. J Clin Ultrasound. 2013;41(9):579-81.

e14 Arq Bras Cardiol. 2015; 104(2):e12-e14 Case Report

Giant Left Atrial Thrombus with Double Coronary Vascularization Giuliano Serafino Ciambelli, Mariana Lins Baptista, Vitor Emer Egypto Rosa, Antonio Sérgio de Santis Andrade Lopes, Tarso Augusto Duenhas Accorsi, Flávio Tarasoutchi Instituto do Coração (INCOR) - Hospital das Clínicas from the Medical School at USP, São Paulo, SP - Brazil

Introduction to second sound, rumbling 2+/6+ diastolic murmur Rheumatic valvular disease, especially mitral stenosis (MS) in the mitral area and pulmonary auscultation with and atrial fibrillation (AF), are the main factors related to the fine crepitation in both bases. The electrocardiogram formation of left atrial (LA) thrombi1-6. Its incidence can range presented AF rhythm. The thoracic x-ray showed bilateral from 16 to 64%, and the most affected site is the left atrial pulmonary congestion and increased cardiothoracic index. appendage1,7. The transthoracic echocardiogram showed mitral valve with commissural fusion, thickened cusps and reduced Systemic embolism is responsible for 10 to 45% of valve opening, mean LA-LV diastolic gradient of 4 mmHg the complications, being the most frequent clinical and maximum of 16 mmHg, with valve area of 1.2 cm2, 1 presentation . Despite being rare, the mechanical mitral which is compatible with moderate rheumatic compromise. valve obstruction caused by thrombus may be considered A 51 mm LA and the presence of hyperechoic image as potentially severe, especially among those patients with from the left atrial roof to its lateral wall were described, previous MS. The clinical manifestations of this condition measuring 65 x 54 mm (Figure 1A and B). There was also are variable, presenting from worsened functional class severe pulmonary arterial hypertension (SPAP 66 mmHg) (NYHA) to cardiogenic shock2. and important increase in the left chambers, eccentric The left atrial thrombus related to MS, even if hypertrophy and left ventricular systolic dysfunction (35% rarely, may present as a large and organized mass with ejection fraction), with diffuse hypokinesis. undistinguishable characteristics from vascularized The patient was submitted to cardiac catheterization, tumors, especially the atrial myxoma. Clinical and which excluded obstructive coronary lesions and showed echocardiographic aspects may not be sufficiently specific the presence of extensive vascularization of the atrial to distinguish one from the other safely, and additional mass, with irrigation originated from the left (Figure 1C) examinations are often required7. and right (Figure 1D) coronaries. Cardiac nuclear magnetic This is the case of a patient with moderate rheumatic resonance showed important LA increase, with thickened MS and no anticoagulation for AF, with a large thrombus walls and image compatible with large thrombus adhered organized in LA mimicking an atrial tumor, with difficult clinical to its walls (roof, floor and lateral wall), besides positive differentiation by complementary examinations. parietal diffuse enhancement, compatible with hypertrophy and atrial fibrosis, probably associated with MS. However, Case Report in the interface between the thrombus and the lateral LA wall, there was a positive perfusion, so it was not possible It is the case of a 57-year-old female patient, with to prevent other expansive processes in the atrial wall. dyspnea that progressed to orthopnea, paroxysmal The late left ventricular enhancement was negative. nocturnal dyspnea and lower extremity edema for two months. She had been diagnosed with rheumatic By considering the presence of an image in the LA in a valvulopathy for seven years, permanent AF with no patient with moderate MS and no anticoagulation for AF, anticoagulation; she was a smoker. In the physical there was the hypothesis of a giant thrombus. Due to the examination, she presented with good general status, progression of the symptoms, size of thrombus and risk of 44 bpm heart rate, 150 x 80 mmHg blood pressure, embolism or mechanical obstruction of the mitral valve, the cardiac auscultation with hyperphonetic sound, second surgical treatment was chosen. normophonetic sound, presence of opening snap close The patient was submitted to surgery, therefore, the mass that presented red coloration, with soft and friable consistency was removed, which confirmed the diagnosis Keywords of left atrial giant thrombus and impairment of the entire Mitral Valve Stenosis / surgery; Thrombosis / surgery; LA posterior wall, weighing 80 g (Figure 2A and B). Myxoma / surgery; Heart Atria; Coronary Circulation. Anterior and posterior commissurotomy was conducted in the mitral valve. The postoperative transthoracic Mailing Address: Giuliano Serafino Ciambelli • Av. Dr. Enéas de Carvalho Aguiar, 44 echocardiogram showed mitral valve with mean LA-LV InCor HCFMUSP - Andar AB - Unidade Clínica de Valvopatias diastolic gradient of 4 mmHg and maximum diastolic Postal Code 05403-900, São Paulo, SP – Brazil gradient of 12 mmHg, and also a 1.6 cm2 valvular area. E-mail: [email protected]; [email protected] Manuscript received November 30, 2013; revised manuscript April 08, 2014; There was still systolic dysfunction (EF 42%) resulting accepted May 05, 2014. from diffuse hypokinesis. The patient evolved well in the postoperative period and received asymptomatic hospital DOI: 10.5935/abc.20140128 discharge, on warfarin anticoagulant. e15 Ciambelli et al. Giant thrombus with double coronary vascularization

Case Report

Figure 1 – A) Echocardiographic image of the apical four-chamber view demonstrating extensive mass in the LA measuring 65 x 54 mm; B) echocardiographic image of the apical four-chamber view demonstrating extensive mass in the LA; C) Cineangiocoronariography with right anterior oblique incidence showing right coronary circulation to the atrial mass (arrow).

Figure 2 – A) Surgical view of the mass after opening the left atrium (arrow); B) Thrombus extracted from the let atrium weighing 80 grams.

Discussion In this case, atrial mass was related to MS with AF MS and its marked atrial increase predisposes to AF with no anticoagulation and to increased LA, being in 40 to 75% of the symptomatic patients. Its occurrence diagnosed as thrombus by the echocardiogram. However, increases with age and with the level of valvular the vascularized mass in the preoperative catheterization obstruction3,4,8. In this condition, blood stasis on the left created doubts as to the nature of the mass. The discovery atrial appendage and LA favors the formation of multiple of a large mass in the LA in the echocardiogram forces thrombi in those areas, therefore, it is very important to the doctors to distinguish the cardiac myxoma and the identify them due to the risk of systemic embolism and, thrombus, which are the most common aspects in cases of less frequently, mechanical valve obstruction2,8. round masses in this chamber8.

Arq Bras Cardiol. 2015; 104(2):e15-e17 e16 Ciambelli et al. Giant thrombus with double coronary vascularization

Case Report

The myxoma is located in the LA in 90% of the cases, been described in association with atrial thrombus among and there are also numberless reports of abnormal coronary patients with MS3,4,9. Most of the time, the irrigation of these vascularization in these tumors, thus being the main organized thrombi originates from the circumflex artery, differential diagnosis4. The precise diagnosis is important so, the double irrigation by the left and right coronaries9 is due to the different therapeutic proposals. The myxoma extremely rare, as observed in this case. requires surgical resection, while the thrombus can be solved MS and AF are the main factors related to the formation of with anticoagulation3. Nuclear magnetic resonance plays an thrombi in the LA, and sometimes they cannot be distinguished important role in this differentiation; however, the organized from vascularized tumors. Even though the patient had thrombus may acquire the same characteristics of the image sufficient thrombogenic substrate, subsidiary tests brought of a myxoma4. Therefore, the presence of vascularization is up this diagnostic doubt, especially due to the preoperative not pathognomonic for atrial myxoma and cannot be used angiographic finding. Surgery was the best strategy both in the alone to tell a thrombus from a myxoma. diagnostic and in the therapeutic approach. This case leads to Giant thrombi that develop inside the atrial chamber the diagnosis of giant left atrial thrombus with double coronary are usually immovable, well organized and fibrotic, with vascularization, thus configuring the second case in the world a close relation to the wall. They have an unfavorable and the first one in Brazil. response to thrombolytic therapy, which is not safe due to the high risks of systemic embolism9. The rare cases Author contributions described in literature suggest that surgical removal should Conception and design of the research, Acquisition of be the treatment of choice2,10. data, Analysis and interpretation of the data, Writing of The mechanical obstruction of the mitral valve is rare, the manuscript and Critical revision of the manuscript for however, potentially severe, especially for people with intellectual content: Ciambelli GS, Baptista ML, Rosa VEE, previous MS. In this case, the patient had presented Lopes ASSA, Accorsi RAD, Tarasoutchi F. symptomatic moderate MS for two months (NYHA III) and pulmonary arterial hypertension (SPAP 66 mmHg), which were sufficient to indicate surgical treatment. Besides this Potential Conflict of Interest indication, there was the presence of a large mass in the LA, No potential conflict of interest relevant to this article which could not only have contributed with the symptoms was reported. presented by the patient, but also added the risk of systemic embolism. With regard to left ventricular dysfunction, the other Sources of Funding etiologies were ruled out, therefore, such a dysfunction may There were no external funding sources for this study. be justified by preload reduction and by changes in ventricular geometry caused by the calcification and immobility of the mitral valve ring11,12. Study Association In literature, angiographic findings of neovascularization or This study is not associated with any thesis or coronary artery fistula formation for the LA have occasionally dissertation work.

References

1. Judas T, Almeida AR, Celeiro MR, Cotrim C, Miranda R, Almeida S, et al. 7. Rost C, Daniel WG, Schmid M. Giant left atrial thrombus in moderate mitral Trombo auricular esquerdo gigante, um achado inesperado. Rev Port Cardiol. stenosis. Eur J Echocardiogr. 2009;10(2):358-9. 2011;30(6):621-6. 8. Choi BH, Ko SM, Hwang HK, Song MG, Shin JK, Kang WS, et al. Detection of left 2. Kakkavas AT, Fosteris MK, Stougiannos PN, Paschalis AK, Damelou AN, atrial thrombus in patients with mitral stenosis and atrial fibrillation: retrospective Trikas AG. A giant, free-floating mass in the left atrium in a patient with atrial comparison of two-phase computed tomography, transoesophageal fibrillation. Hellenic J Cardiol. 2011;52(5):462-5. echocardiography and surgical findings. Eur Radiol. 2013;23(11):2944-53.

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Mitral Valve Aneurysm Secondary to Probable Infective Endocarditis Anne Paula Delgado and Pedro Mendes Gama Serviço de Cardiologia - Centro Hospitalar Tondela – Tondela, Viseu - Portugal

We report the case of a 54-year-old male with a history of leaflet of the mitral valve. Transesophageal echocardiography chest trauma in 2011, complicated by bacteremia caused by (TEE) confirmed the presence of the aneurysm, which caused methicillin-sensitive Staphylococcus aureus (MSSA). In 2012, severe valvular insufficiency. the patient was diagnosed with spondylodiscitis, and in 2013, Although infective endocarditis was never confirmed, after he was referred to cardiology for dyspnea. Transthoracic reviewing his medical records, the authors concluded that this echocardiography (TTE) showed an aneurysm of the posterior condition was probably associated with mitral valve aneurysm.

Keywords Thoracic Injuries; Heart Valve Diseases; Mitral Valve/ physiopathology; Endocarditis. Mailing Address: Anne Paula Delgado Bohlen • Edifício EuroViso, LT E. nº 403, 4º Post., Santa Eugénia. Postal Code 3500‑034, Viseu – Portugal E-mail: [email protected] Manuscript received September 07, 2014; revised manuscript September 17, 2014; accepted September 17, 2014. DOI: 10.5935/abc.20150014

Figure 1 – Transthoracic echocardiography (TTE): apical 4-chamber view – image of the calcified nodule on the posterior leaflet (P1–P2 transition), under which lies an aneurysm of the mitral valve in diastole (A) and systole (B) – white arrow. C. Transesophageal echocardiography (TEE): mid-esophageal 5-chamber view (0°) – image of saccular aneurysm on the posterior leaflet of the mitral valve (green arrow). D. TEE: mid-esophageal 2-chamber view: severe mitral regurgitation identified by color Doppler – blue arrow.

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