AE&M 61-4 by Sociedade Brasileira de Endocrinologia e Metabologia

ISSN 2359-3997

OFFICIAL JOURNAL OF THE BRAZILIAN SOCIETY OF ENDOCRINOLOGY AND METABOLISM Vol. 61 – No. 04 – August 2017

Archives of Endocrinology

and Metabolism

OFFICIAL JOURNAL OF THE BRAZILIAN SOCIETY OF ENDOCRINOLOGY AND METABOLISM

Archives of

Endocrinology

and Metabolism OFFICIAL JOURNAL OF THE BRAZILIAN SOCIETY OF ENDOCRINOLOGY AND METABOLISM

OFFICIAL JOURNAL OF THE BRAZILIAN SOCIETY OF ENDOCRINOLOGY AND METABOLISM Vol. 61 – No. 04 – August 2017

editorials 313 Facts about the history of the AE&M Antonio R. Chacra

Archives of Endocrinology 316 The obesity puzzle: focus on bone turnover after bariatric surgery Maria Edna de Melo

OFFICIAL JOURNAL 319 Prevalence of musculoskeletal symptoms in obese patients candidates for bariatric surgery and its impact on health related OF THE BRAZILIAN quality of life Gabriela Calenzani, Fabiana Ferreira dos Santos, Veronica Lourenço Wittmer, Grace Kelly Filgueiras Freitas, SOCIETY OFFlávia Marini Paro 326 Association of 5-HT2C (rs3813929) and UCP3 (rs1800849) gene polymorphisms with type 2 diabetes in obese women ENDOCRINOLOGY candidates for bariatric surgery Noa Pereira Prada Schnor, Rozangela Verlengia, Patrícia Fátima Sousa Novais, Alex Harley Crisp, Celso Vieira de Souza Leite, AND METABOLISM Irineu Rasera-Júnior, Maria Rita Marques de Oliveira

original articles

and Metabolism 332 Bone turnover after bariatric surgery

Thalita Lima Melo, Leila Froeder, Leandro da Cunha Baia, Ita Pfeferman Heilberg

337 Association between C1q gene polymorphisms and autoimmune thyroid diseases Qiuming Yao, Jie Li, Xiaofei An, Wenjuan Jiang, Qiu Qin, Ronghua Song, Ni Yan, Danfeng Li, Yanfei Jiang, Wen Wang, Liangfeng Shi, Jin-an Zhang

343 Correlation between parameters of self-monitoring of blood glucose and the perception of health-related quality of life in patients with type 1 diabetes mellitus

Archives of

Juliana Santos Paula, Letícia Dinis Braga, Rodrigo Oliveira Moreira, Rosane Kupfer

348 Substernal goiter and laryngopharyngeal reflux

Mariana Gonçalves Rodrigues, Vergilius José Furtado de Araujo Filho, Leandro Luongo de Matos, Flávio Carneiro Hojaij, Cesar Augusto Simões, Vergilius José Furtado de Araujo Neto, Daniel Marin Ramos, Renata Lorencetti Mahmoud, Letícia de Moraes Mosca, Gustavo Borges Manta, Erivelto Martinho Volpi, Lenine Garcia Brandão, Claudio Roberto Cernea

Endocrinology

354 The role of moderate-to-vigorous physical activity in mediating the relationship between central adiposity and immunometabolic profile in postmenopausal women Tiego A. Diniz,, Fabricio E. Rossi, Loreana S. Silveira, Lucas Melo Neves, Ana Claudia de Souza Fortaleza, Diego G. D. Christofaro, Fabio S. Lira, Ismael F. Freitas-Junior

361 Prevalence of pancreatic autoantibodies in non-diabetic patients with autoimmune thyroid disease and its relation to insulin secretion and glucose tolerance

and Metabolism

Carolina Sallorenzo, Regina Silva, Teresa Kasamatsu, Sérgio Dib

367 Overweight in youth and sleep quality: is there a link?

Susana Rebelo Pacheco, Ana Margalha Miranda, Raquel Coelho, Ana Cristina Monteiro, Graciete Bragança, Helena Cristina Loureiro

374 Related quality of life questionnaire specific to dysthyroid ophthalmopathy evaluated in a population of patients with Graves’ disease

OFFICIAL JOURNAL OF THE BRAZILIAN SOCIETY OF ENDOCRINOLOGY AND METABOLISM

Laura Carolina Delfino, Anabela Zunino, Verónica Sapia, María del Carmen Silva Croome, Verónica Ilera, Alicia Teresa Gauna

382 New insights into the effects of irisin levels in HIV-infected subjects: correlation with adiposity, fat-free mass, and strength parameters Joice Cristina dos Santos Trombeta,, Jonato Prestes, Dahan da Cunha Nascimento, Ramires Alsamir Tibana, Guilherme Borges Pereira, Thiago da Rosa Lima, Géssica Alves Fraga, Roberto Carlos Vieira-Junior, Fabrício Azevedo Voltarelli

reviews 391 The use of aromatase inhibitors in boys with short stature: what to know before prescribing? Alessandra Linardi, Durval Damiani, Carlos A. Longui

398 Medullary thyroid carcinoma – Adverse events during systemic treatment: risk-benefit ratio Léa Maria Zanini Maciel, Patrícia Künzle Ribeiro Magalhães

case report 403 Multifocal oral melanoacanthoma associated with Addison’s disease and hyperthyroidism: a case report Thinali Sousa Dantas, Isabelly Vidal do Nascimento, Maria Elisa Quezado Lima Verde, Ana Paula Negreiros Nunes Alves, Fabrício Bitu Sousa, Mário Rogério Lima Mota

AE&M awards 2016

AND METABOLISM OFFICIAL JOURNAL OF THE BRAZILIAN SOCIETY OF ENDOCRINOLOGY AND METABOLISM

OFFICIAL JOURNAL OF THE BRAZILIAN SOCIETY OF ENDOCRINOLOGY AND METABOLISM

Financial and editorial assistant: Roselaine Monteiro roselaine@endocrino.org.br Rua Botucatu, 572 – conjunto 83 – 04023-062 – São Paulo, SP Telefax: (11) 5575-0311 / 5082-4788

Archives of Endocrinology Online submission / Electronic publishing www.aem-sbem.com • www.scielo.br/abem

and Metabolism

OFFICIAL JOURNAL OF THE BRAZILIAN SOCIETY OF ENDOCRINOLOGY AND METABOLISM

Archives of

Rua Anseriz, 27, Campo Belo 04618-050 – São Paulo, SP. Fone: 11 3093-3300 www.segmentofarma.com.br • segmentofarma@segmentofarma.com.br

Publication code: 22204.8.17

Endocrinology

Informations: Author: José Roberto Aguilar Technique: Acrylic on canvas Measures: 2,5 × 2,5 m Year: 1987 OFFICIAL JOURNAL OF THE BRAZILIAN Collection: Private Collection Foto: Ana PB SOCIETY OF ENDOCRINOLOGY AND METABOLISM

and Metabolism

All adds have to comply with RDC no 96/08 Commercial advising: Estela Kater estela.kater@gmail·com

Circulation of this issue: 3,500 copies Subscription: R$450.00/year – Single issue: R$55.00 Indexed in Biological Abstracts, Index Medicus, Latindex, Lilacs, MedLine, SciELO, Scopus, ISI-Web of Science BRAZILIAN ARCHIVES OF ENDOCRINOLOGY AND METABOLISM Brazilian Society of Endocrinology and Metabolism – São Paulo, SP: Brazilian Society of Endocrinology and Metabolism, volume 5, 1955Six issues/year Continued from: Brazilian Archives of Endocrinology (v. 1-4), 1951-1955 ISSN 2359-3997 (printed issues) ISSN 2359-4292 (online issues) 1. Endocrinology – journals 2. Metabolism – journals I. Brazilian Society of Endocrinology and Metabolism II. Brazilian Medical Association CDU 612.43 Endocrinology CDU 612.015.3 Metabolism

Supported by:

OFFICIAL JOURNAL OF THE BRAZILIAN SOCIETY OF ENDOCRINOLOGY AND METABOLISM

Archives of endocrinology and metabolism Official journal of SBEM – Brazilian Society of Endocrinology and Metabolism (Department of the Brazilian Medical Association), SBD – Brazilian Diabetes Society, ABESO – Brazilian Association for the Study of Obesity and Metabolic Syndrome

2015-2018 EDITOR-IN-CHIEF Marcello D. Bronstein (SP)

CO-EDITORS

REPRESENTATIVES OF COLLABORATING SOCIETIES SBD

Larissa Gomes (SP)

ABESO

Léa Maria Zanini Maciel (SP)

OFFICIAL JOURNALLeandro Kasuki (SP) OF THE BRAZILIANMadson Queiroz Almeida (SP) Brazilian Editorial Commission Manoel Ricardo Alves Martins (CE) Alexander A. L. Jorge (SP) OF SOCIETY Marcio Mancini (SP) Alexandre Hohl (SC) ENDOCRINOLOGY Margaret Cristina S. Boguszewski (PR) Ana Amélia Hoff (SP) AND METABOLISM Maria Candida B. V. Fragoso (SP) Ana Claudia Latronico (SP) Maria Edna de Melo (SP)

and Metabolism

Ana Luiza Silva Maia (RS)

Maria Izabel Chiamolera (SP)

André Fernandes Reis (SP)

Maria Marta Sarquis (MG)

Andrea Glezer (SP)

Mario Saad (SP)

Tânia S. Bachega (SP)

Antônio Marcondes Lerário (SP)

Mário Vaisman (RJ)

INTERNATIONAL ASSOCIATE EDITOR

Antônio Roberto Chacra (SP)

Marise Lazaretti Castro (SP)

Ayrton Custódio Moreira (SP)

Milena Caldato (PA)

Shlomo Melmed (Los Angeles, EUA)

Berenice B. Mendonça (SP)

Raquel Soares Jallad (SP)

Bruno Halpern (SP)

Rodrigo Moreira (RJ)

Carlos Alberto Longui (SP)

Ruth Clapauch (RJ)

Archives of

FOUNDER

ASSOCIATE EDITORS

Waldemar Berardinelli (RJ)

PRESIDENTS OF THE SBEM DEPARTMENTS

EDITORS-IN-CHIEF, EDITORIAL OFFICE*

ADRENAL AND HYPERTENSION

Endocrinology César Luiz Boguszewski (PR)

Sandra R. G. Ferreira (SP)

Clarisse Ponte (CE)

Simão A. Lottemberg (SP)

Delmar Muniz Lourenço Jr. (SP)

Sonir Roberto Antonini (SP)

Luiz Alberto Andreotti Turatti (SP)

Denise Momesso (RJ)

Suemi Marui (SP)

DYSLIPIDEMIA AND ATHEROSCLEROSIS

Eder Carlos R. Quintão (SP)

Madson Queiroz de Almeida (SP)

1951-1955 Waldemar Berardinelli (RJ) Thales Martins (RJ)

DIABETES MELLITUS

1957-1972 Clementino Fraga Filho (RJ)

Cynthia Melissa Valério (RJ)

Edna Nakandakare (SP)

1964-1966* Luiz Carlos Lobo (RJ)

BASIC ENDOCRINOLOGY

Edna T. Kimura (SP)

Maria Izabel Chiamolera (SP)

1966-1968* Pedro Collett-Solberg (RJ) 1969-1972* João Gabriel H. Cordeiro (RJ)

and Metabolism

Weiss (RJ)

PEDIATRIC ENDOCRINOLOGY

Julienne Ângela Ramires de Carvalho (PR)

1983-1990 Antônio Roberto Chacra (SP)

BONE AND MINERAL METABOLISM

1995-2006 Claudio Elias Kater (SP) 2007-2010 Edna T. Kimura (SP) 2011-2014 Sergio Atala Dib (SP)

Elaine Maria Frade Costa (SP)

OFFICIAL JOURNAL OF THE FelipeBRAZILIAN Gaia (SP) SOCIETY OF ENDOCRINOLOGY AND METABOLISM Rita de Cássia Viana Vasconcellos Flavio Hojaij (SP) FEMININE ENDOCRINOLOGY AND ANDROLOGY

1978-1982 Armando de Aguiar Pupo (SP)

1991-1994 Rui M. de Barros Maciel (SP)

Laércio Joel Franco (SP)

Luiz Alberto Andreotti Turatti (SP)

Archives of Endocrinology Bruno Ferraz de Souza (SP) Erika Parente (SP) Francisco Bandeira (PE) Fernanda Vaisman (RJ) Fernando M. A. Giuffrida (BA) João Roberto Maciel Martins (SP) Melanie Rodacki (RJ) Monica R. Gadelha (RJ) Nina Rosa C. Musolino (SP) Poli Mara Spritzer (RS) Ricardo Meirelles (RJ) Rogerio Friedman (RS) Rui M. B. Maciel (SP)

Julio Z. Abucham (SP)

Carolina Aguiar Moreira (PR) NEUROENDOCRINOLOGY

Marcello Delano Bronstein (SP) OBESITY

Maria Edna de Melo (SP) THYROID

Célia Regina Nogueira (SP)

Victória Borba (PR)

International Editorial Commission Andrea Giustina (Itália)

Gil Guerra-Júnior (SP)

Antonio C. Bianco (EUA)

Giovanna Balarini Lima (RJ)

Décio Eizirik (Bélgica)

Gisah M. do Amaral (SP)

Franco Mantero (Itália)

Hans Graf (SP)

Fernando Cassorla (Chile)

José Augusto Sgarbi (SP)

Gilberto Paz-Filho (Austrália)

José Gilberto H. Vieira (SP)

John P. Bilezikian (EUA)

SBEM – Brazilian Society of Endocrinology and Metabolism SBEM BRAZILIAN BOARD OF DIRECTORS 2017-2018 President Vice-President Executive Secretary Adjunct Executive Secretary Treasurer-General Adjunct Treasurer

Fábio Rogério Trujilho Alexandre Hohl Paulo Augusto Carvalho de Miranda Neuton Dornelas Gomes Rodrigo de Oliveira Moreira Marcio Corrêa Mancini

Rua Humaitá, 85, cj. 501 22261-000 – Rio de Janeiro, RJ Fone/Fax: (21) 2579-0312/2266-0170 www.endocrino.org.br sbem@endocrino.org.br

Scientific Departments - 2017/2018 Brazilian Society of Endocrinology and Metabolism ADRENAL AND HYPERTENSION

DIABETES MELLITUS

President Madson Queiroz de Almeida madsonalmeida@usp.br

President Luiz Alberto Andreotti Turatti turatti@uol.com.br Directors Amely Pereira Silva Balthazar Gustavo José Caldas Pinto Costa Sergio Alberto Cunha Vêncio Walter José Minicucci Thaísa Dourado Guedes Treasurer João Eduardo Nunes Salles Alternates Marcos Cauduro Troian Victor Gervásio e Silva

Vice-President Directors

Marivânia da Costa Santos Alexis Dourado Guedes Flávia Amanda Costa Barbosa Milena Coelho Fernandes Caldato Sonir Roberto Rauber Antonini Tânia Aparecida Sanchez Bachega

DYSLIPIDEMIA AND ATHEROSCLEROSIS

BASIC ENDOCRINOLOGY

President Cynthia Melissa Valério cy_valerio@yahoo.com.br

President Maria Izabel Chiamolera mchiamolera@unifesp.br Vice-President Bruno Ferraz de Souza Directors Catarina Segreti Porto Dóris Rosenthal Maria Tereza Nunes Marisa Maria Dreyer Breitenbach Tania Maria Ruffoni Ortiga Alternates Vânia Maria Corrêa da Costa Ubiratan Fabres Machado

Vice-President Directors

Renan Magalhães Montenegro Júnior Fernando de Mello Almada Giuffrida Marcello Casaccia Bertoluci

Scientific Departments - 2017/2018 WOMEN ENDOCRINOLOGY AND ANDROLOGY President Rita de Cássia Viana Vasconcellos Weiss rcvweiss@gmail.com Vice-President Directors Alternates

Dolores Perovano Pardini Amanda Valéria Luna de Athayde Mônica de Oliveira Poli Mara Spritzer Ricardo Martins da Rocha Meirelles Ruth Clapauch Izydorczyk Antônio Mendes Fontanelli Larissa Garcia Gomes

PEDIATRIC ENDOCRINOLOGY President Julienne Angela Ramires de Carvalho julienne@endocrinoped.com.br Vice-President Directors Alternate

Carlos Alberto Longui Aline da Mota Rocha Angela Maria Spinola e Castro Cláudia Braga Monteiro Paulo César Alves da Silva Suzana Nesi França Marilia Martins Guimarães

BONE AND MINERAL METABOLISM

NEUROENDOCRINOLOGY

President Vice-President Directors Alternate

President Marcello D. Bronstein mdbronstein@uol.com.br Vice-President César Luiz Boguszewski Directors Heraldo Mendes Garmes Luciana Ansanelli Naves Lucio Vilar Rabelo Filho Luiz Antonio de Araujo Mônica Roberto Gadelha Alternates Andrea Glezer Manoel Ricardo Alves Martins

Carolina Aguiar Moreira carolina.aguiar.moreira@gmail.com Miguel Madeira Barbara Campolina Carvalho Silva Francisco Alfredo Bandeira e Farias Marise Lazaretti Castro Sergio Setsuo Maeda Victória Zeghbi Cochenski Borba Tatiana Munhoz da Rocha Lemos Costa

OBESITY

THYROID

President Maria Edna de Melo medna@usp.br Vice-President Rosana Bento Radominski Director/Secretary Walmir Ferreira Coutinho Director/Treasurer Erika Paniago Guedes Directors Cintia Cercato Leila Maria Batista Araujo Director/Treasurer Fabio Ferreira de Moura Alternate Jacqueline Rizzolli

President Célia Regina Nogueira nogueira@fmb.unesp.br Vice-President José Augusto Sgarbi Secretary Janete Maria Cerutti Directors Ana Luiza Silva Maia Laura Sterian Ward Patricia de Fátima dos Santos Teixeira Gisah Amaral de Carvalho Mario Vaisman Alternate Danilo Glauco Pereira Villagelin Neto

Permanent Commissions - 2017/2018 Brazilian Society of Endocrinology and Metabolism STRATEGIC PLANNING FOLLOW-UP

HISTORY OF ENDOCRINOLOGY

President Alexandre Hohl alexandrehohl@uol.com.br Members Nina Rosa de Castro Musolino, Airton Golbert, Ricardo Martins da Rocha Meirelles, Ruy Lyra da Silva Filho

President Henrique de Lacerda Suplicy hsuplicy@gmail.com Members Adriana Costa e Forti, Thomaz Rodrigues Porto da Cruz

ENDOCRINOLOGY CAMPAIGNS

President

César Luiz Boguszewski

President Erika Bezerra Parente ebparente@gmail.com Members Érika Paniago Guedes, Teresa Arruti Rey

Members

cesarluiz@hc.ufpr.br Ruy Lyra da Silva Filho, Valéria Cunha C. Guimarães, Ana Cláudia Latrônico

SCIENTIFIC COMISSION President Alexandre Hohl alexandrehohl@uol.com.br Indicated by the directories Joao Eduardo Nunes Salles, Alexis Dourado Guedes, Erika Bezerra Parente, Ana Mayra Andrade de Oliveira, Margaret Cristina da Silva Boguszewski, Guilherme Alcides Flores Rollin, Milena Coelho Fernandes Caldato, Mônica de Oliveira, Nina Rosa de Castro Musolino

SOCIAL COMMUNICATION President Ricardo Martins da Rocha Meirelles r.meirelles@terra.com.br Nominated by the president Alexandre Hohl ABEM Editor Marcello D. Bronstein Members Nina Rosa de Castro Musolino

INTERNATIONAL

NORMS, QUALIFICATION AND CERTIFICATION President Vivian Carole Moema Ellinger vivianellinger@gmail.com Members Ronaldo Rocha Sinay Neves, Marisa Helena César Coral, Maria Emilia Pereira de Almeida, Milena Coelho Fernandes Caldato

JOINT COMMISSION – CAAEP President Julienne Angela Ramires de Carvalho julienne@endocrinoped.com.br Members Marilia Martins Guimarães, Suzana Nesi França

RESEARCH President Members

Freddy Eliaschewitz freddy.g@uol.com.br Antônio Roberto Chacra, Luiz Augusto Tavares Russo

CONTINUOUS MEDICAL EDUCATION

GUIDELINES PROJECT

President Alexandre Hohl alexandrehohl@uol.com.br Members Lireda Meneses Silva, Walter José Minicucci, Cleber Favaro

Coordinator Alexis Dourado Guedes dr.alexis@uol.com.br Adrenal and hypertension Madson Queiroz de Almeida Dyslipidemia and atherosclerosis Cynthia Melissa Valério Diabetes Mellitus Luiz Alberto Andreotti Turatti Basic endocrinology Maria Izabel Chiamolera Feminine and Andrology Rita de Cássia Viana Vasconcellos Weiss Pediatric Endocrinology Julienne Angela Ramires de Carvalho Bone and mineral metabolism Carolina Aguiar Moreira Neuroendocrinology Marcello D. Bronstein Obesity Maria Edna de Melo Thyroid Célia Regina Nogueira

STATUTES, RULES AND REGULATIONS President Nina Rosa de Castro Musolino ninamusolino@gmail.com Members Airton Golbert, Henrique de Lacerda Suplicy, Luiz Henrique Maciel Griz Representative of the Evandro de Souza Portes brazilian directory

PROFESSIONAL ETHICS AND DEFENCE President Itairan da Silva Terres itairan.terres@gmail.com Vice-Inspector Maite Trojaner Salona Chimeno 1ST member Diana Viegas Martins 2ND member João Modesto Filho 3RD member Evandro de Souza Portes 4TH member Marcelo Henrique da Silva Canto Costa 5TH member Luiz Henrique Santos Canani

ENDOCRINE DYSREGULATORS President Elaine Frade Costa elainefradecosta@gmail.com Vice-President Margaret Cristina da Silva Boguszewski Members Tania Aparecida Sanchez Bachega, Ricardo Martins da Rocha Meirelles, Marilia Martins Guimarães, Eveline Gadelha Pereira Fontenele, Maria Izabel Chiamolera

TEMPORARY – SPORT AND EXERCISE ENDOCRINOLOGY - CTEEE President: Yuri Galeno Pinheiro Chaves de Freitas yurigaleno@gmail.com Members: Fábio Ferreira de Moura, Clayton Luiz Dornelles Macedo, Roberto Luís Zagury, Ricardo de Andrade Oliveira, Fulvio Clemo Thomazelli, Felipe Henning Duarte Gaia

TITLE OF SPECIALIST IN ENDOCRINOLOGY AND METABOLISM President: Josivan Gomes de Lima josivanlima@gmail.com Vice-President: Márcio Corrêa Mancini Members: Marise Lazaretti Castro, Mauro Antônio Czepielewski, Milena Coelho Fernandes Caldato, Renan Magalhães Montenegro Júnior, Rogério Friedman

VALORIZATION OF NEW LEADERSHIPS President Joaquim Custodio da Silva Junior jocsjunior@uol.com.br Members Joaquim Custodio da Silva Junior, Eduardo Quadros Araújo, Marcelo Fernando Ronsoni, Manoel Ricardo Alves Martins, Marcio Weissheimer Lauria

Brazilian Societies and Associations for Endocrinology and Metabolism

SBD – BRAZILIAN DIABETES SOCIETY SBD BRAZILIAN BOARD OF DIRECTORS (2016/2017)

President

Luiz Alberto Andreotti Turattii

Vice-Presidents

Reine Marie Chaves Fonseca Solange Travassos de Figueiredo Alves Sergio Alberto Cunha Vêncio Levimar Rocha Araujo Mauro Scharf Pinto

1ST Secretary

Domingos Augusto Malerbi

2ND Secretary

Gustavo J. P. Caldas Costa

1ST Treasurer

Antonio Carlos Lerário

2ND Treasurer

Roberto Abrao Raduan

Supervisory Board

João Eduardo Nunes Salles João Paulo Iazigi Luis Antonio de Araujo

Rua Afonso Brás, 579, cj. 72/74 04511-011– São Paulo, SP Fone/Fax: (11) 3842-4931 secretaria@diabetes.org.br www.diabetes.org.br Administrative Manager: Anna Maria Ferreira

ABESO – BRAZILIAN ASSOCIATION FOR THE STUDY OF OBESITY AND METABOLIC SYNDROME ABESO BRAZILIAN BOARD OF DIRECTORS (2017-2018)

President

Maria Edna de Melo

Vice-President

Alexander Koglin Benchimol

1ST Secretary General

Bruno Halpern

2ND Secretary General

Fábio Ferreira de Moura

Treasurer

Erika Paniago Guedes

Rua Mato Grosso, 306, cj. 1711 01239-040 – São Paulo, SP Fone: (11) 3079-2298/Fax: (11) 3079-1732 Secretary: Renata Felix info@abeso.org.br www.abeso.org.br

editorial

Facts about the history of the AE&M Antonio R. Chacra1

Arch Endocrinol Metab. 2017;61/4

Diretor do Centro de Pesquisa Clínica em Diabetes da Escola Paulista de Medicina da Universidade Federal de São Paulo (EPM/Unifesp). Diretor do Centro de Diabetes do Hospital Sírio-Libanês, São Paulo, SP, Brasil 1

Correspondence to: Antonio R. Chacra clinicachacra@uol.com.br Received on Aug/28/2017 Accepted on Aug/28/2017 DOI: 10.1590/2359-3997000000297

he Archives of Endocrinology and Metabolism – AE&M (formely ABE&M – Arquivos Brasileiros de Endocrinologia e Metabologia) is the official scientific journal of the Brazilian Society of Endocrinology and Metabolism (Sociedade Brasileira de Endocrinologia e Metabologia – SBEM) which is affiliated with the Brazilian Medical Association (Associação Médica Brasileira). Besides SBEM, the alma mater society, the journal also provides a connection for publications with the following medical organizations and associations of interest in the fields of Endocrinology and Metabolism: Brazilian Diabetes Society (Sociedade Brasileira de Diabetes – SBD) and the Brazilian Association for the Study of Obesity (Associação Brasileira para o Estudo da Obesidade – ABESO). This journal is published on a regular basis, with six printed issues per year in addition to online version at AE&M homepage and SciELO. Special issues on seleted topics as well the proceedings of the Brazilian Endocrinology Congresses are also published. It is a peer reviewed journal with a very scientifically respected Editorial Board. AE&M (ISSN 2359-3997 – printed, ISSN 2359-4292 – on line) is indexed on PubMed®/MEDLINE® U.S. National Library of Medicine, ISI-Web of Science, SCOPUS, BIREME-LILACS, Excerpta Medica, Chemical Abstracts, Biological Abstracts, Academic Google. This year we celebrate the 66th anniversary of the foundation of the AE&M. It was with a great joy and surprise that I received a phone call from Marcello Bronstein, the current Editor-in-Chief of the AE&M, asking to write an editorial commenting on some historical facts about the AE&M. I immediately agreed because I do believe that the knowledge of how the Archives have reached this high level of a great scientific journal have developped over the years is important for the continuing progress of the journal. We do not know exactly who conceived the idea of launching a Brazilian journal in the field of endocrine glands. Around the nineteen fifties, there was a pletora of basic scientists in the axis Rio de Janeiro-São Paulo working in the area of internal secretion. Eventually the ABE&M was founded in 1951 by this basic scientist group together with another group of distinguished clinical medical doctors. These scientists doctors are considered the pioneers of Endocrinology in Brazil. They were either envolved in basic science or in clinical endocrinology. In the first group, we should mention Thales Martins and Ribeiro do Valle. Endocrinology at that time was commencing to be characterized as a clinical specialty. Among these medical doctors interested in endocrine disease we should cite Emilio Mattar, João Gabriel H. Cordeiro, Luciano Décourt, Arnaldo Sandoval, Luiz Carlos Lobo and many others, specially from Rio de Janeiro. Regarding the foundation of the ABE&M, all of them were represented

313

History of the AE&M

by Waldemar Berardinelli, a famous endocrinologist from Rio, who is considered the founder and was the first Editor-in-Chief of the journal. Interestigly, the foundation year (1951) of the Archives was also the year that the Journal of Clinical Endocrinology and Metabolism (JCEM) was founded by the Endocrine Society in the United States. Therefore the ABE&M together with the JCEM is one of the oldest medical journal in the field of Endocrinology. We do not know exactly what came first, the society (SBEM) or the journal (ABE&M). But certainly, that time was the heyday of the growth of Endocrinology and the turning point for the recognition of Endocrinology as a medical specialty. Founder, Previous Editors and presente Editor Waldemar Berardinelli (Founder Editor) 19511955 Thales Martins, 1951-1955 Clementino Fraga Filho, 1957-1963 Luiz Carlos Lobo, 1964-1966 Pedro Colletti-Solberg, 1966-1968 JoĂŁo Gabriel H. Cordeiro, 1969-1972 Armando de Aguiar Pupo, 1978-1982 Antonio Roberto Chacra, 1983-1990 Rui Monteiro de Barros Maciel, 1991-1994 Claudio E. Kater, 1995-2006 Edna T. Kimura, 2007-2010 Sergio Atala Dib, 2011-2014 Marcello D. Bronstein, 2015-present We can divide the history of the AE&M in diferente stages accordingly to the tenures of the Editors-in-Chief. The first years are charecterized for publishing mainly the results of original articles related to experimental research. As the clinical specialty of Endocrinology emerged, many clinical articles in the field of physiopathology, diagnosis and treatment of endocrine and metabolic diseases started to increase. For reasons not very well clarified, there was a gap of publications from 1973 to 1977. In 1978, Armando de Aguiar Pupo was indicated as Editor-in-Chief. I remember the great effforts of Armando Pupo to revive the journal, proactively asking us to send manuscripts to be submitted and then published at the Archives. Armando Pupo can be considered the co-founder of this new phase of the ABE&M. We have to be very grateful to Armando Pupo for his dedication in revival the Archives 314

I followed Pupo as the Editor-in-Chief (19831990). During those years Brazil was in economic turmoil and recession, with a high rate of inflation. My main objective at that time was raising funds in order to save the Archives and keep the publication of the journal. I wrote a project and I remember sending the Archives project to a great number of organizations, including pharmaceutical companies. Finances were very bad at that time and we received a lot of refusals. Eventually we got a support from the Brazilian Medical Association and the CNPq (Conselho Nacional de Pesquisa). I am very grateful to CNPq, specially in the person of Reginaldo Holanda e Albuquerque, a great endocrinogist from Brasilia, who helped us to keep the journal alive. During that time, I had also to deal with the legal formalities of the journal and the connection of the Archives to our Brazilian Society of Endocrinology and Metabolism (SBEM). Another task was to make the Archives known to new subscribers and contributors and stimulate receiving manuscripts for publication. Rui Monteiro de Barros Maciel followed me being indicated in 1990 as the new Editor-in-Chief. I remember how glad he was by this nomination. Full of enthusiasm, Rui Maciel had, as one of his main objectives, to use the Archives as a tool to ameliorate the professional capacity of the endocrinologists in Brazil. He stimulated the publications of clinical reviews, cases reports, clinical articles as well clinical guidelines. He was also responsible for introducing editorials commenting the data of original articles. We have also to mention that it was during his tenure that special issues in selected topics started to be published, as well the proceedings of the Brazilian Congresses of Endocrinology and others scientific events organized by SBEM. At that time the Archives also started to receive a great number of manuscripts related to the Post Graduate Courses that have been implanted in Brazil. He acted in a very proactive manner asking colleagues to submitt papers to the Archives. In a personal interview, Rui Maciel pointed out how proud he is for had served as Editor-in-Chief of the Archives, and that he also considers this position as one of the crucial point on his career. After Rui M. B. Maciel the next Editor-in-Chief was Claudio E. Kater. One of the higlights of this period was the starting process of indexation of the journal. This was a very hard work that without Kater would not be possible. Arch Endocrinol Metab. 2017;61/4

History of the AE&M

of plagiarism, including in journals of high impact factor. In order to protect ABE&M from plagiarism, an anti-plagiarism tool was recently acquired, and all manuscripts will be analyzed by it before being published”. The more recent and innovative phase on the history of the Archives started by the time Marcello D. Bronstein was apointed as Editor-in-Chief at the beggining of 2015. Bronstein totally transformed the journal into an international one. The first step was changing the name to “Archives of Endocrinology and Metabolism” (AE&M). As he wrote in his first editorial, “the aim was to make Brazil even more visible in the international scientific community. In fact, I expect that, little by little, colleagues in Brazil and abroad give prestige to our journal by submitting high level articles. This will only be possible if AE&M, by raising its impact fator”. The result of that change has been positive and a great number of articles from several countries around the world have been published. Brazilian authors, as well, are now submitting more and more articles to AE&M. Finally, I would like to congratulate Marcello D. Bronstein for his great achievement as the present Editor-in-Chief of the AE&M and to thank him for inviting and giving me the opportunity of writing this editorial. Disclosure: no potential conflict of interest relevant to this article was reported.

The period of Edna T. Kimura (2007-2010), who followed Kater, was one of the most proficuous in the history of the Archives. In 2009, Edna Kimura expanded the fascicles number/year from six to nine, a great achiement. The following remarkable facts were also achieved during her period and deserve to be mentioned: eletronic edition on the site, www.abem-sbem.org.br was made publically available extending the visibility of ABE&M, that was electronically visible also in the www.scielo.br. Eletronic Submission in 2008-January. First JCR-Web of Science Impact Factor in 2009. Financial support of CNPq (2007-2010). Edna also gave continuity to the publication of the tematic esdtions. Sergio Atala Dib was the next Editor-in-Chief. Regarding the rules for publication, Sergio Atala Dib during his tenure introduced an important issue against plagiarism. That was a great achievement among others that Dib has contributed for the Archives. After completing his four year period as Editor-in-Chief, he wrote a editorial where he enphasised the importance of this measure: “In order to regulate the publishing procedures in ABE&M after publication, the publishing authorization and the copyright cession term, a tool for the transference of the copyright to ABE&M, was created with the aid of the Juridical Department of the Brazilian Society of Endocrinology and Metabolism. In this era when publications became profuse and sophisticated, there is an increase in the prevalence

Arch Endocrinol Metab. 2017;61/4

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editorial

The obesity puzzle: focus on bone turnover after bariatric surgery Maria Edna de Melo1

Grupo de Obesidade e Síndrome Metabólica, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP), São Paulo, SP, Brasil 1

Correspondence to: Maria Edna de Melo Rua Dr. Enéas de Carvalho Aguiar, 255, 5° andar, Bloco 4B, sala 11A 05403-010 – São Paulo, SP, Brasil medna@usp.br Received on Aug/25/2017 Accepted on Aug/25/2017

DOI: 10.1590/2359-3997000000298

316

his issue of the Archives of Endocrinology and Metabolism encompasses three studies related to obesity. The influence of genetics on the development of metabolic obesity complications, quality of life of obese patients and bone metabolism after bariatric surgery are discussed in order to increase the knowledge of these aspects in this complex disease. The article “Prevalence of musculoskeletal symptoms in obese patients candidates for bariatric surgery and its impact on health related quality of life” by Calenzani and cols. emphasizes the relationship between obesity and musculoskeletal symptoms and their influence on physical and mental aspects linked to the quality of life. In addition to the classical metabolic disorders related to obesity that abbreviate patients’ lives, the physical limitation caused by arthritis and joint deformities causes impairment in mobility, increasing sedentary lifestyle, with consequent reduction in energy expenditure (1). Obese patients reported a very high frequency of pain in ankles/feet, knees and lumbar region. These symptoms impair deambulation and, thus obesity withdraws the right to come and go of these patients, determining furthermore impairment in their quality of life (1). Differently from Brilmann and cols. (2), who found a negative correlation between body mass index (BMI) and quality of life scores assessed through SF-36, Calenzani and cols. did not observed this association, that could be consequence of the relatively small and homogenously class 3 obesity sample of patients, as emphasized by the authors (1). The relationship between two main metabolic disorders, diabetes mellitus type 2 (DM2) and systemic arterial hypertension (SAH), with 12 polymorphisms in the genes encoding ghrelin (GHRL, rs26802), uncoupling protein 2 (UCP2, rs659366), uncoupling protein 3 (UCP3, rs1800849), FTO (rs9939609), leptin (LEP, rs7799039), leptin receptor (LEPR, rs1137101), and serotonin receptor (5-HT2C, rs3813929) was investigated by Schnor and cols. and is reported in the paper “Association of 5-HT2C (rs3813929) and UCP3 (rs1800849) gene polymorphisms with type 2 diabetes in obese women candidates for bariatric surgery”. The study, which evaluated 351 obese women candidates for bariatric surgery, reports a 5-fold increased risk of having DM2 in T allele carriers of the rs3813929 and a 3-fold increased risk also for DM2 in CC genotype carriers of the rs1800849 (3). Analyzes of polymorphic variants can frequently lead to conflicting results, either because of the sample size or mainly because of the genetic background. The genetic influence on complex diseases is more associated with the interaction of several polymorphisms than with isolated polymorphisms. Thus, while one single published article reported a similar finding concerning the polymorphism in the UCP3 gene, studies about the polymorphism in the 5-HT2C gene present controversial results (3). Arch Endocrinol Metab. 2017;61/4

The obesity puzzle

Arch Endocrinol Metab. 2017;61/4

loss, implying high NTX (N-telopeptide of type 1 collagen) and low FGF23 (fibroblast growth factor 23) levels, could attenuate nutritional deficiencies, but the controversial leptin effect on BMD and bone microarchitecture calls for the development of further studies (4). Not only weight loss is related to increased risk of bone fractures, but also some drugs used in the treatment of DM2 are associated with bone fracture, such as thiazolidinediones (OR 1.94 [95% CI 1.60, 2.35]) and SGLT2 inhibitors (1.32 [95% CI 1.00, 1.74]), particularly canagliflozin (9). While thiazolidinediones possibly inhibit osteoblasts differentiation and stimulate osteoclasts, increasing reabsorption (10), SGLT2 inhibitors may raise serum phosphate levels, implying in an elevation of FGF23 and, consequently, inhibition of the formation of calcitriol (9). In addition, in diabetic patients the occurrence of hypoglycemia and complications related to DM2, like microangiopathy, increase the risk of falls and consequently bone fractures (9). Faced with the intricate complexity of obesity, each new published study represents a small piece in this vast puzzle. Disclosure: no potential conflict of interest relevant to this article was reported.

REFERENCES 1. Calenzani G, Santos FF, Wittmer VL, Freitas GKF, Paro FM. Prevalence of musculoskeletal symptoms in obese patients candidates for bariatric surgery and its impact on health related quality of life. Arch Endocrinol Metab. 2017;61(3):319-25. 2. Brilmann M, Oliveira MS, Thiers VO. Avaliação da qualidade de vida relacionada à saúde na obesidade. Cad Saude Colet. 2007;15(1):39-54. 3. Schnor NPP, Verlengia R, Novais PFS, Crisp AH, Leite CVS, RaseraJúnior I, et al. Association of 5-HT2C (rs3813929) and UCP3 (rs1800849) gene polymorphisms with type 2 diabetes in obese women candidates for bariatric surgery. Arch Endocrinol Metab. 2017;61(3):326-31. 4. Melo TL, Froeder L, Baia LC, Heilberg IP. Bone turnover after bariatric surgery. Arch Endocrinol Metab. 2017;61(3):332-6. 5. Janicka A, Wren TA, Sanchez MM, Dorey F, Kim PS, Mittelman SD, et al. Fat mass is not beneficial to bone in adolescents and young adults. J Clin Endocrinol Metab. 2007;92(1):143-7. 6. Burghardt A, Issever A, Schwartz A, Davis K, Masharani U, Majumdar S, et al. High-resolution peripheral quantitative computed tomographic imaging of cortical and trabecular bone microarchitecture in patients with type 2 diabetes mellitus. J Clin Endocrinol Metab. 2010;95(11):5045-55. 7. Rubino F, Nathan DM, Eckel RH, Schauer PR, Alberti KG, Zimmet PZ, et al.; Delegates of the 2nd Diabetes Surgery Summit. Metabolic Surgery in the Treatment Algorithm for Type 2 Diabetes: A Joint Statement by International Diabetes Organizations. Diabetes Care. 2016;39(6):861-77.

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In the triad of articles on obesity is also included the study “Bone turnover after bariatric surgery” by Melo and cols., in which patients who had undergone bariatric surgery have an increase in bone turnover, characterized by elevated bone alkaline phosphatase and deoxypyridinoline (4). Interestingly, a high bone mineral density (BMD) was classically propagated as perhaps one of the few benefits of being overweight; however, more recently the mass of adipose tissue was negatively implicated in the bone microarchitecture (5). The relationship between obesity and bone density and/or trabecular bone quality is influenced by body weight load, as well as cytokines and hormones such as insulin and leptin that are synthesized proportionally to the mass of white adipose tissue, with reducing levels after weight loss (4). In addition, patients with DM2 present a significant increase in cortical bone porosity in the radius, as well as a larger volume of the pores in the tibia, even with elevated BMD (6). Thus, the development of research in this area is of paramount importance, especially recently when a reduction in the BMI cutoff point is suggested for indication of bariatric surgery (7). Maghrabi and cols. reported a reduction of more than 9% in femoral bone mineral density two years after bariatric surgery, regardless of the surgical procedure (Roux-en-Y gastric bypass or vertical gastrectomy). However, what is remarkable is that 6/37 (16.2%) patients in surgery group experienced spontaneous fractures, which occurred in only 1/17 (5.9%) in the control group (8). Weight loss, even with lifestyle changes, is associated with a reduction in BMD, as observed in patients participating in the LookAhead Trial (9). As a matter of fact, bariatric surgery determines a state of controlled malnutrition and thus, in addition to weight reduction, patients commonly present with vitamin D deficiency and difficulty in calcium absorption (4), which occurs preferentially in the duodenum, excluded from food transit in the gastric bypass operation. An important point highlighted by Melo and cols. is the poor compliance to oral supplementation of vitamins and minerals, which strengthens the maintenance of nutritional deficiencies, such as hypovitaminosis D (4). Nevertheless, the most relevant finding in this study is the persistence of increased bone turnover even 7 years postoperatively despite the plateau in weight loss that tends to occur twelve months after the procedure. The influence of reduced levels of leptin after weight

The obesity puzzle

9. Schwartz AV. Diabetes, bone and glucose-lowering agents: clinical outcomes. Diabetologia. 2017;60(7):1170-79. 10. Mori H, Okada Y, Tanaka Y. The Effects of Pioglitazone on Bone Formation and Resorption Markers in Type 2 Diabetes Mellitus. Intern Med. 2017;56(11):1301-6.

8. Maghrabi AH, Wolski K, Abood B, Licata A, Pothier C, Bhatt DL, et al. Two-year outcomes on bone density and fracture incidence in patients with T2DM randomized to bariatric surgery versus intensive medical therapy. Obesity (Silver Spring). 2015;23(12):2344-8.

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Arch Endocrinol Metab. 2017;61/4

original article

Prevalence of musculoskeletal symptoms in obese patients candidates for bariatric surgery and its impact on health related quality of life Gabriela Calenzani1, Fabiana Ferreira dos Santos1, Veronica Lourenço Wittmer1, Grace Kelly Filgueiras Freitas1, Flávia Marini Paro1

ABSTRACT Objective: This study was designed to identify the major musculoskeletal symptoms of individuals with obesity, to assess their health-related quality of life, and to evaluate the correlation between the musculoskeletal symptoms and the individuals’ health-related quality of life. Materials and methods: Cross-sectional study. Instruments used: “Nordic Musculoskeletal Questionnaire” and “The Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36)”. Results: In total, 41 subjects were evaluated, of which 90.15% were female. The mean age of the subjects was 40.78 ± 9.85 years and their mean body-mass index was 46.87 ± 8.08. All subjects reported musculoskeletal pain in at least one anatomical region and 80.49% had pain in three or more regions. The activity limitations due to pain were reported by 75.61% of them. The most affected regions by pain were the ankles and/or feet, lower back, knees and wrists/hands/fingers. The most associated regions with activity limitations due to pain were the ankles and/or feet, knees and lower back. The presence of pain showed a negative correlation with the domains physical functioning (PF), role-physical (RP) and body pain (BP). The activity limitations showed a negative correlation with the domains PF, BP, social functioning (SF) and role-emotional (RE). Conclusion: Our data showed a high prevalence of musculoskeletal pain and limitation in activities due to pain in obese subjects. The musculoskeletal symptoms had negative correlations with physical and mental components of the health-related quality of life, highlighting the importance of ensuring that patients with obesity have access to interdisciplinary care, for the prevention and rehabilitation of musculoskeletal disorders. Arch Endocrinol Metab. 2017;61(3):319-25.

Departamento de Educação Integrada em Saúde, Universidade Federal do Espírito Santo (UFES), Vitória, ES, Brasil 1

Correspondence to: Verônica Lourenço Wittmer Universidade Federal do Espírito Santo, Centro de Ciências da Saúde, Departamento de Educação Integrada em Saúde Av. Marechal Campos, 1468 29047-105 – Vitória, ES, Brasil ve_lourenco@yahoo.com.br Received on Feb/11/2016 Accepted on Nov/11/2016 DOI: 10.1590/2359-3997000000237

INTRODUCTION

he incidence of obesity has increased in recent decades, and obesity has become a major global health challenge (1). In Brazil, approximately 50% of men and women over 20 years are overweight, and the prevalence of obesity is 12.5% for men and 16.9% for women (2). Increased body-mass index (BMI) is an important risk factor for several causes of mortality, including cardiovascular disease, many types of cancer, hepatic and kidney diseases, and others (3). A higher prevalence of musculoskeletal symptoms affecting mainly the lower limbs has been reported in obese individuals (4,5). Some of these symptoms manifest as joint discomfort caused mainly by excess weight, which can overload the musculoskeletal structures responsible for stabilizing and moving the Arch Endocrinol Metab. 2017;61/4

body (6). Obesity has been associated with significant impairment in mobility (7) and several musculoskeletal disorders, which can lead to disability and poor quality of life (5,6) and result in high direct and indirect healthcare economic costs (8,9). Some studies have reported that obesity has a negative impact on the patients’ health-related quality of life (10,11) but only a few studies have assessed the impact of musculoskeletal symptoms on the quality of life of obese individuals (12). The purpose of this study was to identify the major musculoskeletal symptoms of individuals with obesity grades II, III and IV, to assess their health-related quality of life, and to evaluate the correlation between the musculoskeletal symptoms and the patients’ quality of life. This information will be useful for the 319

Keywords Obesity; quality of life; musculoskeletal pain; morbid obesity; physical therapy; epidemiology

Pain and quality of life in obese patients

development of preventive and therapeutic measures to improve the musculoskeletal symptoms and quality of life of obese patients.

MATERIALS AND METHODS This was a cross-sectional study and was approved by the Ethics Committee of the Universidade Federal do Espírito Santo (approval number 293/10). The data were collected from April to December 2011. The study population consisted of all patients aged over 18 years who underwent preoperative consultations for bariatric surgery in a Brazilian University Hospital during the study period (n = 41). During the physical therapy consultation of the patients were collected general data, such as age, sex, height, weight, marital status, education, occupation and lifestyle habits. Two questionnaires were administered by the researchers, who were properly trained so that the assessments were standardized. The first questionnaire was the validated Brazilian version of the Nordic Musculoskeletal Questionnaire (NMQ) (13,14), which consists of multiple-choice or binary responses to questions regarding the occurrence of symptoms in anatomical regions. This questionnaire covers the 12 months and the 7 days preceding the assessment, as well as the withdrawal from routine activities due to musculoskeletal symptoms in the 12 months preceding the assessment. The QNSO has been used in other studies of obese individuals (15,16). Immediately after, we used “The Medical Outcomes Study 36-Item Short-Form Health Survey” (SF-36), which was validated and translated into Portuguese (17,18), for the evaluation of the patients’ health-related quality of life. This a multidimensional questionnaire consisting of 36 items, which are grouped into eight scales or domains, four of which include components related to physical health, namely, physical functioning (PF), role-physical (RP), bodily pain (BP), and general health (GH), and four of which include components related to mental health, namely, vitality (VT), social functioning (SF), role-emotional (RE) and mental health (MH). The answers to the questions in each domain are assigned numerical score, which is coded and classified on a scale from 0 to 100. The higher the score, the better the health-related quality of life in that domain (18). Data were analyzed using the Statistical Package for the Social Sciences (SPSS) 18.0, IBM. To measure the 320

degree of association between two metric variables, we used the correlation coefficient. The Shapiro-Wilk test was used to determine whether the data distribution was normal. For variables where the normal hypothesis was not rejected, the t-test was used to analyze the means (parametric analysis). For the domains where the assumption of normality was rejected, we applied the nonparametric Mann-Whitney test. To assess correlations, the Spearman correlation coefficient was used. The data were expressed with standard deviations (SD) and differences were considered significant if p < 0.05.

RESULTS We evaluated 41 obese subjects (grades II to IV), of whom 37 (90.2%) were female and four (9.85%) were male, candidates to bariatric surgery. The average age of the patients was 40.78 ± 9.85 years and ranged from 25 to 65 years. The patients’ mean BMI was 46.87 ± 8.08 kg/m2 and ranged from 35.73 to 65.02 kg/m2. All subjects were followed up by the health interdisciplinary team of the Bariatric Surgery Program in the hospital. The most frequent comorbidities were systemic arterial hypertension (SAH), which was observed in 31 subjects (70.7%), and diabetes mellitus (DM), which was observed in 16 subjects (39%). All individuals with DM also had hypertension. In terms of physical activity, 68.3% reported that they did not participate in any physical activity. Of the 28.3% who performed some activity, 26.8% participated in walking, with frequency ranging from two to five times a week, and 2.4% participate in aquatic exercises, with frequency ranging two to five times a week.

Prevalence of musculoskeletal symptoms In the 12 months preceding the questionnaire, 100% of subjects reported pain in at least one anatomical region and 80.49% had pain in 3 or more anatomical regions. In the previous 7 days, 92.68% reported pain in at least one anatomical region and 60.98% in 3 or more regions. The limitation of activities due to pain in the previous 12 months was reported by 75.61% of patients. Table 1 shows the detailed distribution of patients by number of anatomic regions affected. The anatomical regions most affected by pain in the previous 12 months were the ankles/feet (87.80%), lumbar region (68.29%) and knees (68.29%). In the preceding 7 days, 70.73% had pain in the ankles/ Arch Endocrinol Metab. 2017;61/4

Pain and quality of life in obese patients

feet, 53.66% in the lumbar region, 46.34% in knees and 46.34% in the knees, wrists/hands/fingers. The regions that were most frequently associated with activity limitations due to pain were the ankles/feet (46.34%), knees (43.90%) and lower back (41.46%). The prevalence of pain by anatomic region is presented in detail in Table 2. There was no statistically significant correlation between BMI and musculoskeletal symptoms. There was also no significant correlation between age and musculoskeletal symptoms.

Health-related quality of life

Table 1. Distribution of subjects by number of anatomical regions affected by musculoskeletal symptoms according to the NMQ

Table 3. Scores for the SF-36 domains

None

Pain (last 7 days)

Limitation of activities (last 12 months)

Domains

Mean

Physical functioning (PF)

46.83

28.01

38.25

CI 55.40

Role-physical (RP)

42.68

41.92

29.85

55.52

Bodily pain (BP)

46.93

19.59

40.93

52.92

7.32

24.39

General health (GH)

61.95

22.83

54.96

68.94

44.15

25.07

36.47

51.82

7.32

12.20

14.63

Vitality (VT)

12.20

19.51

9.76

Social functioning (SF)

65.85

32.24

55.99

75.72

12.20

14.63

19.51

Role-emotional (RE)

56.10

39.75

43.93

68.26

17.07

9.76

12.20

Mental health (MH)

55.80

27.76

47.31

64.30

12.20

9.76

4.88

12.20

9.76

4.88

9.76

7.32

14.63

7.32

2.44

SD: standard deviation; CI: confidence interval.

Table 4. Correlations of the SF-36 domains with BMI and age Age Correlation coefficient

p-value

Correlation coefficient

p-value

Physical functioning (PF)

-0.4181

0.007

-0.188

0.239

Role-physical (RP)

-0.218

0.171

-0.092

0.568

Bodily pain (BP)

-0.229

0.15

-0.001

0.995

General health (GH)

-0.123

0.445

-0.002

0.991

Vitality (VT)

0.011

0.946

-0.117

0.467

0.024

0.881

NMQ: Nordic Musculoskeletal Questionnaire.

Table 2. Prevalence of musculoskeletal symptoms by anatomic region according to the NMQ Anatomical regions

Pain (last 12 months)

Pain (last 7 days)

Limitation of activities (last 12 months)

BMI

Domains (SF-36)

Neck

39.02

24.39

9.76

Social functioning (SF)

-0.036

0.824

Shoulders

46.34

36.59

24.39

Role-emotional (RE)

-0.225

0.158

0.086

0.594

Elbows

14.63

9.76

7.32

Mental health (MH)

0.057

0.725

-0.064

0.693

Forearm

17.07

14.63

17.07

Wrists/hands/ fingers

60.98

46.34

26.83

Dorsal region

53.66

43.90

31.71

Lower back

68.29

53.66

41.46

Hips and/or thighs

26.83

21.95

19.51

Knees

63.41

46.34

43.90

Ankles/feet

87.80

70.73

46.34

NMQ: Nordic Musculoskeletal Questionnaire. Arch Endocrinol Metab. 2017;61/4

Statistically significant.

Association between musculoskeletal symptoms and quality of life The presence of pain in the previous 12 months showed a statistically significant correlation with the domains PF and BP. These correlations were negative, indicating that the greater the pain, the worse the quality of life 321

Affected anatomical regions (n)

Pain (last 12 months)

Table 3 contains the descriptive statistics for the SF-36. The domain SF showed the highest score (61.95) and RP showed the lowest (42.68). Table 4 shows the correlation results between SF-36 results, BMI and age. PF was the only domain that showed a statistically significant correlation with age (p = 0.007). Specifically, PF correlated negatively with age, meaning that the older the patient, the poorer the quality of life in this domain. None of the domains showed a statistically significant correlation with BMI.

Pain and quality of life in obese patients

in these domains. The presence of pain in the previous 7 days showed a significant negative correlation with the domains PF, RP and BP (Table 5). Table 5 shows that activity limitations showed a statistically significant correlation with poorer quality of life in the domains RP, BP, SF and RE.

DISCUSSION The results showed that all individuals with obesity of grades II to IV and who were candidates for bariatric surgery had musculoskeletal symptoms, and all subjects reported pain in at least one anatomical region in the 12 months preceding the assessment. The prevalence of pain in the 7 days preceding the assessment was also high (92.68%). A similar prevalence was found in another study, in which 100% of the obese individuals evaluated during preoperative bariatric surgery had musculoskeletal symptoms in at least one region of the body (16). The current study demonstrated that the anatomical areas most often affected by pain in the 12 months and 7 days preceding the assessment were the ankles and/ or feet, lower back, knees and wrists/hands/fingers. A similar prevalence of musculoskeletal symptoms in the ankles/feet and knee was shown in a study conducted in obese patients before and after bariatric surgery. Six months after surgery, the subjects experienced a marked reduction in both frequency and intensity of musculoskeletal pain, which strengthens the association between the symptoms and obesity. However, the study used the modified NMQ, considering only the questions about the presence of pain in the lower limbs in the previous 7 days (19). In addition, their results

differed from our with regard to the high prevalence of hip pain (54.5%) compared with that observed in our population (21.95%). This is in accordance with a study that did not find any association between BMI and the occurrence of hip osteoarthritis (20). Larsson’s results also showed that obese women had more musculoskeletal pain in the lower backs, knees and feet (15). The prevalence of musculoskeletal symptoms in the knees of obese people has been well-established in the literature (5,21). It has been demonstrated that weight gain is associated with adverse effects on the knees, such as pain, stiffness and functional changes (22). Additionally, it has reported that BMI > 30 kg/m² is an important risk factor for the incidence of knee osteoarthritis (20). In our study, back pain was the second most prevalent complaint, with a reported incidence of 68.29% in the past 12 months and 53.66% in the previous 7 days. A meta-analysis published in 2010 showed that obesity increases the risk of lower back pain and is also strongly associated with increased need for health care for the treatment of acute and chronic lower back pain (23). The association between obesity and lower back pain has also been shown in other studies (5,21). Although only a few publications have related obesity to the prevalence of pain in wrist/hand/fingers, a cohort study conducted with 1,675 individuals showed that obesity was a significant independent predictor for the incidence of hand osteoarthritis (22). While the presence of osteoarthritis was not assessed in our study, this finding could be related to the high prevalence of pain in this region found in our results. In

Table 5. Correlations between the SF-36 domains and the presence of musculoskeletal symptoms evaluated by the NMQ Pain (last 12 months) Domains

Correlation coefficient

p-value

Correlation coefficient

p-value

-0.3231

0.039

-0.4951

-0.21

0.187

-0.3321

Bodily pain (BP)

-0.312

0.047

General health (GH)

-0.065

Vitality (VT)

-0.139

Social functioning (SF) Role-emotional (RE)

Physical functioning (PF) Role-physical (RP)

Pain (last 7 days)

Mental health (MH)

Limitations in activities (last 12 months) Correlation coefficient

p-value

0.001

-0.28

0.077

0.034

-0.4181

0.007

-0.4751

0.002

-0.455

0.003

0.685

-0.124

0.441

-0.047

0.771

0.386

-0.177

0.269

-0.162

0.312

-0.166

0.299

-0.201

0.208

-0.319

0.042

0.012

0.94

-0.023

0.886

-0.3641

0.019

-0.064

0.69

-0.164

0.307

-0.12

0.453

NMQ: Nordic Musculoskeletal Questionnaire; Statistically significant. 1

322

Arch Endocrinol Metab. 2017;61/4

addition, obesity is considered an important risk factor for carpal tunnel syndrome (24). A high prevalence of activity limitation due to pain was observed in the current study, and the anatomical regions most frequently associated with limitations were the ankles/feet (46.34%), knees (43.90%) and lower back (41.46%). These findings corroborate Larsson’s conclusion that high BMI value, age and lower body pain could predict functional limitations in obese women (25). Furthermore, it has been demonstrated that increased BMI is strongly associated with foot pain and disability, and this may impair both biomechanical and metabolic mechanisms (26). With regard to quality of life, a study that also used the SF-36 showed a relationship between quality of life and obesity and identified an association between excess weight and low values in the domains PF, RP and BP (27). We must emphasize that these three domains and domain VT in the current study showed the lowest scores in the SF-36, which means that the lowest values were observed in the domains related to physical health components of health-related quality of life. Despite the fact that low values were also observed for mental health components, they were higher than the values for the physical health components. Cameron and cols. showed that obesity was related with impairment in both physical and mental components of health-related quality of life in Australian adults followed over 5 years (28). One factor that could explain the higher values for mental health components found in the current study is the fact that all patients in this study had been receiving psychological care during the months preceding the surgery because this service is part of the bariatric surgery program at our institution. In addition to this, the patients were evaluated a few weeks prior to bariatric surgery after months or years of waiting and preparation, because the study was conducted in a public hospital where there is a queue for bariatric surgery. As such, the expectation of the positive outcome of surgery in the next weeks possibly influenced the mental state of the patients. However, these patients did not receive any health professional care related to the prevention or treatment of musculoskeletal symptoms in the period before surgery. A study conducted with 5,633 people in Sweden showed that the impact of obesity differs by age with relation to physical and mental health domains (25). In our results, there was a statistically significant negative correlation only between age and PF, which was Arch Endocrinol Metab. 2017;61/4

possibly due the small sample size. This suggests that the older obese people had a worse quality of life in this domain than younger obese people. We found no statistically significant correlation between the SF-36 and BMI in our study. However, in the similar study mentioned earlier (27), there were statistically significant negative correlations between BMI and each one of the domains PF, RP and BP. An important difference between this study and ours is that they compared groups with different features. Specifically, group I comprised overweight patients with obesity grades I and II, and group II comprised patients with grade III obesity in the earlier study. In contrast, our sample was composed only of severely obese (grade II), morbidly obese (grade III) or super obese (grade IV) patients, all of whom had indications for bariatric surgery. This means that even the obese grade II individuals presented comorbidities or risk factors that indicated them for surgery. It is also possible that in our study, the correlation between BMI and quality of life did not show significance because our population is less heterogeneous. Another study showed that the degree of obesity had no impact on knee osteoarthritis symptoms of pain, stiffness and functional difficulty; and, in two functional capacity tests, both obese women and women with morbid obesity (29). Sach and cols. reported that the higher the BMI, the worse the HRQL of individuals compared to those with normal BMI (30). Cameron and cols. demonstrated that a bidirectional relationship was observed between the decline of quality of life and increase in weight. Accordingly, reduced quality of life increases the likelihood of increasing weight, and increased weight leads to reduced quality of life (28). Some studies in the literature have reported that musculoskeletal pain is a predictor of poor healthrelated quality of life in obese subjects (5,12,31), but only a few of these studies have correlated the presence of pain with different health-related quality of life domains in obese individuals, and they haven’t correlated the limitation of activities due to pain with the different domains of health-related quality of life. The present study showed a significant negative correlation between the presence of pain and the reduction in quality of life related to health in the domains PF, RP, BP. In other words, the musculoskeletal pain in these obese patients had a negative impact on their quality of life in these domains, suggesting that in our study, the physical health component of the individuals’ health-related quality of life was more 323

Pain and quality of life in obese patients

affected by pain than the mental health component. In addition to this, the results also indicated that the limitation of activities by pain had a statistically significant negative correlation with the domains RP, BP, SF and RE, meaning that limitation of activities due to pain negatively affected both the physical and the mental components of health-related quality of life. Such knowledge can provide evidence to support the adoption of primary, secondary, tertiary health care prevention, and interdisciplinary rehabilitation, to ensure more effective interventions for these patients. The major limitations of this study were the small sample size and the fact that the study was conducted in a single institution. Due to both of these features, generalized conclusions cannot be drawn from this study. Another limitation is that the causes of the musculoskeletal symptoms were not investigated by clinical and radiological evaluation but only by questionnaire. Finally, we did not evaluate other comorbidities that could interfere with the patients’ health-related quality of life. In conclusion, individuals with obesity grades II, III and IV who were candidates for bariatric surgery showed high prevalence of musculoskeletal symptoms and of activity limitation by pain. They also had low scores in all domains of health-related quality of life. Furthermore, the presence of musculoskeletal symptoms had a significant negative correlation with the domains PF, RP, BP, SF and RE. This suggests that the physical and mental components of health-related quality of life were affected by musculoskeletal symptoms. Disclosure: no potential conflict of interest relevant to this article was reported.

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22. Tanamas SK, Wluka AE, Davies-Tuck M, Wang Y, Strauss BJ, Proietto J, et al. Association of weight gain with incident knee pain, stiffness, and functional difficulties: a longitudinal study. Arthritis Care Res (Hoboken). 2013;65:34-43.

4. Krul M, Van der Wouden JC, Schellevis FG, Van Suijlekom-Smit LWA, Koes BW. Musculoskeletal problems in overweight and obese children. Ann Fam Med. 2009;7:352-6.

23. Shiri R, Karppinen J, Leino-arjas P, Solovieva S, Viikari-juntura E. The association between obesity and low back pain: a metaanalysis. Am J Epidemiol. 2010;171:135-54.

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28. Cameron AJ, Magliano DJ, Dunstan DW, Zimmet PZ, Hesketh K, Peeters A, et al. A bi-directional relationship between obesity and health-related quality of life: evidence from the longitudinal AusDiab study. Int J Obes (Lond). 2012;36:295-303.

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29. Vasconcelos KSS, Dias JMD, Dias RC. Impact of the degree of obesity on symptoms and functional capacity of women with knee osteoarthritis. Fisioter Pesqui. 2008;15(2):125-30.

26. Tanamas SK, Wluka AE, Berry P, Menz HB, Strauss BJ, DaviesTuck M, et al. Relationship between obesity and foot pain and its association with fat mass, fat distribution, and muscle mass. Arthritis Care Res (Hoboken). 2012;64:262-8.

30. Sach TH, Barton GR, Doherty M, Muir KR, Jenkinson C, Avery AJ. The relationship between body mass index and healthrelated quality of life: comparing the EQ-5D, EuroQol VAS and SF-6D. Int J Obes (Lond). 2007;31:189-96.

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31. Janke EA, Collins A, Kozak AT. Overview of the relationship between pain and obesity: what do we know? Where do we go next? J Rehabil Res Dev. 2007;44:245-62.

24. Becker J, Nora DB, Gomes I, Stringari FF, Seitensus R, Panosso JS, et al. An evaluation of gender, obesity, age and diabetes mellitus as risk factors for carpal tunnel syndrome. Clin Neurophysiol. 2002;113:1429-34.

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original article

Association of 5-HT2C (rs3813929) and UCP3 (rs1800849) gene polymorphisms with type 2 diabetes in obese women candidates for bariatric surgery Universidade Estadual Paulista Júlio de Mesquita Filho (Unesp), Programa de Pós-Graduação em Alimentos e Nutrição, Araraquara, SP, Brasil 2 Universidade Metodista de Piracicaba (Unimep), Programa de Pós-Graduação em Ciências do Movimento Humano, Piracicaba, SP, Brasil 3 Unesp, Faculdade de Medicina, Botucatu, SP, Brasil 4 Clínica Bariátrica, Piracicaba, SP, Brasil 1

Correspondence to: Maria Rita Marques de Oliveira Universidade Estadual Paulista (Unesp), Instituto de Biociências. Distrito de Rubião Junior, s/n, caixa postal 510 18618-000 – Botucatu, SP, Brasil mrmolive@ibb.unesp.br Received on April/17/2016 Accepted on Nov/25/2016 DOI: 10.1590/2359-3997000000260

Noa Pereira Prada Schnor1, Rozangela Verlengia2, Patrícia Fátima Sousa Novais1, Alex Harley Crisp1, Celso Vieira de Souza Leite3, Irineu Rasera-Júnior4, Maria Rita Marques de Oliveira1

ABSTRACT Objective: Obesity can cause systemic arterial hypertension (SAH) and type 2 diabetes mellitus (DM2) factor that is also influenced by genetic variability. The present study aims to investigate the association between gene polymorphisms related with obesity on the prevalence of SAH and DM2 in the preoperative period and 1 year after Roux-en-Y gastric bypass surgery. Subjects and methods: In total, 351 obese women in a Brazilian cohort completed the study. The clinical diagnosis of SAH and DM2 was monitored from medical records. Twelve gene polymorphisms (rs26802; rs572169; rs7799039; rs1137101; rs3813929; rs659366; rs660339; rs1800849; rs7498665; rs35874116; rs9701796; and rs9939609) were determined using real-time polymerase chain reaction and TaqMan assay. Results: In the preoperative period, prevalence of SAH and DM2 was 57% and 22%, respectively. One year postoperatively, 86.8% subjects had remission of DM2 and 99.5% had control of SAH. Subjects with T allele from the serotonin receptor gene (5-HT2C, rs3813929) had five times greater chance of DM2, and the CC genotype from uncoupling protein 3 gene (UCP3, rs1800849) had three times greater chance in the preoperative period. Conclusion: These findings indicate that polymorphisms rs3813929 and rs1800849 from 5-HT2C and UCP3 genes were related to DM2 prevalence among the Brazilian obese women candidates for bariatric surgery. Arch Endocrinol Metab. 2017;61(3):326-31. Keywords Obesity; type 2 diabetes; hypertension; gene polymorphism

INTRODUCTION

besity prevalence is increasing at a worrying rate; with significant influence on morbidity and mortality rate worldwide (1). Excessive food intake and a sedentary lifestyle contribute to the accumulation of body fat and increase the risk of chronic diseases such as systemic arterial hypertension (SAH) and type 2 diabetes mellitus (DM2) (2). Moreover, genetic factors are recognized as an important influence in increasing susceptibility to obesity and its associated comorbidities (3). A genetic factor of considerable interest is the variation of a gene (DNA sequence bases) between subjects, described as gene polymorphism (4). In this context, some studies have related polymorphism of specifics genes that control appetite, energy metabolism, susceptibility to obesity (5), and chronic diseases such as DM2 and SAH (6). 326

It is recognized that obesity and its associated comorbidities have multifactorial causes. Therefore, it is important to analyze several genes and the interaction between them to better understand the obesity genesis and the contribution of genetic variability. Review studies indicate a large number of gene candidates that may be related to obesity and chronic diseases (7-9). Regarding the treatment of obesity, bariatric surgery is the most long-term effective method of inducing body weight loss and beneficial effects on metabolic disorders (10). However, there is a significant variability in body weight loss and control of comorbidities between subjects undergoing bariatric surgery (11). This suggests that some of these variable responses to bariatric surgery can be explained by genetic factors. In this sense, investigating the relationship between uncoupling protein 3 (UCP3) gene polymorphism Arch Endocrinol Metab. 2017;61/4

Gene polymorphism and bariatric surgery

SUBJECTS AND METHODS This is a prospective study with adult women undergoing RYGB surgery, performed by the same medical staff from June 2010 to May 2013. The subjects were evaluated in the preoperative period and 1 year postoperatively. Self-reported information regarding age, skin color, education, previous pregnancies, family history of obesity, and obesity at early age was recorded. Body weight, BMI, and the presence of SAH and DM2 were provided by medical records. The eligible criteria for participation were: (a) being female; (b) aged between 20 and 50 years; Arch Endocrinol Metab. 2017;61/4

(c) and registered on the waiting line for bariatric surgery. The exclusion criteria were as follows: (a) alcoholism; (b) genetic syndromes associated with obesity; (c) Cushing’s syndrome; (d) hypothyroidism; (e) renal or liver failure; (f) neoplasia; (g) infection with human immunodeficiency virus (HIV); (h) use of corticosteroids; (i) and postmenopausal women using estrogen replacement. In total, 441 women in a Brazilian cohort were eligible for study inclusion. However, subjects who did not attend 1 year postoperatively were not included in data analysis. This study included 351 subjects who completed the study. The number of subjects was determined using the data of the genotype with lower prevalence and a minimum 300 subjects was shown to be necessary. All subjects signed a free-andinformed consent form after being informed about the procedures involved in the research. This study was approved by the local Research Ethics Committee (protocol number: 3303/2009). Body weight was measured using a digital balance (Fizola, SP, Brazil) with a capacity of 200 kg, precise to within 100 g. Height measurements were obtained using a stadiometer (Seca, SP, Brazil) precise to within 0.1 cm. The procedures followed the standard recommendations (14). Excess weight (EW, in kg); weight loss (WL, in kg), and percentage of excess weight loss (%EWL), were calculated using the followings formulas (15): EW = body weight preoperatively – ideal body weight; WL = body weight preoperatively – current body weight; %EWL = percentage of body weight lost in relation to excess weight.

The SAH remission was considered when the subject stopped with anti-hypertensive medications and presented: systolic blood pressure < 140 mmHg and diastolic blood pressure < 90 mmHg (16). The DM2 remission was considered when the subject stopped with anti-diabetic medications and presented: fasting glucose concentration < 126 mg/dL; postprandial glucose < 200 mg/dL after 75g oral glucose load, and hemogloblin A1c level < 6.5% (17). Genomic DNA was isolated from whole blood samples (EDTA-treated), using Illusta blood genomicPrep Kit (GE Healthcare®, New York, USA). The analysis of gene polymorphisms GHRL (rs26802); GHRS (rs572169); LEP (rs7799039); LEPR (rs1137101); 5HT2C (rs3813929); UCP2 327

(rs1800849) and results after biliopancreatic diversion surgery (BDS), Luis and cols. (12) observed no significant association with the control of SAH and DM2 1 year postoperatively. However, another study from Luis and cols. (13) indicated that fat mass and obesityassociated (FTO, rs9939609) gene polymorphism were associated with greater body weight loss 3 months after BDS, but no difference was evident after 9 and 12 months postoperatively. In addition, Luis and cols. (13) observed an association between FTO gene polymorphism (rs9939609) with a decrease in the body mass index (BMI), and improvements of glycated hemoglobin (HbA1c) levels 6 months after mini gastric bypass laparoscopic surgery. Therefore, other studies are warranted to investigate a larger number of gene polymorphisms to expand the evidence regarding the effect of genetic variability as a determinant of SAH and DM2 on obesity and bariatric surgery response. The main benefit of surgical treatment of obesity is the control of comorbidities. Therefore, the current study selected 12 gene polymorphisms related to obesity in order to assess an association with SAH and DM2 prevalence, and the possible influence on bariatric surgery results. There is some evidence that suggests effects on the prevalence of DM2 with polymorphisms of ghrelin (GHRL, rs26802), uncoupling protein 2 (UCP2, rs659366), uncoupling protein 3 (UCP3, rs1800849), FTO (rs9939609), leptin (LEP, rs7799039), leptin receptor (LEPR, rs1137101), and serotonin receptor (5-HT2C, rs3813929) genes. Thus, the current study aimed to investigate the association of 12 gene polymorphisms on the prevalence of SAH and DM2 in the preoperative period and 1 year after Roux-en-Y gastric bypass (RYGB) surgery in obese women.

Gene polymorphism and bariatric surgery

(rs659366); UCP2 (rs660339); UCP3 (rs1800849); SH2B1 (rs7498665); TAS1R2 (rs35874116); TAS1R2 (rs9701796); and FTO (rs9939609), was determined by real-time polymerase chain reaction (RT-PCR) and TaqMan assay (Applied Biosystems®, Branchburg, New Jersey, USA). The RT-PCR was processed in ABI 7500 fast equipment (Applied Biosystems®, Branchburg, NJ, USA) according to the manufacturer’s instructions. A random selection of 10% of the samples was again genotyped to evaluate the reproducibility of genotyping. The agreement of genotype frequencies with Hardy–Weinberg equilibrium expectations was tested by chi-square test. Multiple logistic regressions were performed to evaluate the effects of gene polymorphism (with adjustments for age, skin color, preoperative BMI, previous pregnancy, and age at onset of obesity) taking SAH and DM2 as dependent variables. When the genotype of lower frequency was < 10%, the genotype was combined with a heterozygous genotype. p < 0.05 was considered to be statistically significant.

RESULTS The genotype distributions of gene polymorphisms (rs26802; rs572169; rs7799039; rs1137101; rs3813929; rs659366; rs660339; rs1800849; rs7498665; rs35874116; rs9701796; rs9939609) was within the expectations of the Hardy-Weinberg equilibrium (p > 0.05). Table 1 shows characterization of the study subject. One year postoperatively, of 76 subjects with DM2, only 10 maintained the diagnosis of DM2, representing a control of 86.8%. Regarding SAH, 99.5% of subjects had control of this disease. The median of %EWL was 69.2%; of which 4.3% of the subjects had less than 50% of %EWL and 26.7% had DM2. One subject did not have control of SAH postoperatively and presented 58.7% of %EWL. Because of a high proportion of control of these comorbidities postoperatively, logistic regression analysis was possible only with preoperative data. Among 12 gene polymorphisms analyzed, 5HT2C (rs3813929) and UCP3 (rs1800849) had influence on DM2 prevalence. In the 5HT2C gene, subject carriers of the T allele had a five times greater chance of DM2 compared to CC genotype. In UCP3 gene, CC genotype had three times greater chance of DM2 compared with T allele carriers (Table 2). However, there was no prevalence of 5-HT2C (rs3813929) and UCP3 (rs1800849) gene polymorphisms among 328

diabetic subjects who obtained or did not obtain the control of DM2 postoperatively (Table 3).

DISCUSSION This study investigated the association of 12 gene polymorphisms related to obesity on the prevalence of SAH and DM2 during the preoperative period and 1 year after RYGB surgery in obese women. Our main finding indicated that 5-HT2C (rs3813929) and UCP3 (rs1800849) gene polymorphisms were associated with the prevalence of DM2 in obese women candidates for bariatric surgery.

Table 1. Characteristics of subject study Variables

Median (min–max)

Age (years)

34 (20–50)

Age at onset of obesity (years)

18 (0–40)

Preoperative BMI (kg/m2)

46 (33–73)

BMI postoperatively (kg/m2)

30 (20–55)

%EWL 1 year postoperatively

69 (37–119)

DM2 preoperative – n (%) Yes

76 (22)

275 (88)

DM2 1 year postoperative – n (%) Yes

10 (2.8)

341 (97.2)

SAH preoperative – n (%) Yes

201 (57)

150 (43)

SAH 1 year postoperative – n (%) Yes

01 (0.3)

350 (99.7)

Family history of obesity – n (%) Yes

279 (79.5)

72 (20.5)

Skin Color – n (%) Black

52 (14.8)

White

226 (64.4)

Parda

73 (20.8)

Education – n (%) Middle and High School (incomplete)

123 (35)

High School (complete) and superior

228 (65)

Previous pregnancies – n (%) Yes

263 (74.9)

88 (25.1)

n = 351. BMI: body mass index; %EWL: percentage of excess weight loss.

Arch Endocrinol Metab. 2017;61/4

Gene polymorphism and bariatric surgery

Table 2. Values of odds ratio (OR) and 95% confidence interval (95% CI) for diabetes mellitus type 2 (DM2) and systemic arterial hypertension (SAH) before and after bariatric surgery, according to each gene polymorphism DM2 Preoperative Gene Polymorphism

Genotype

n (%)

35 (10)

1.00

CA + AA

316 (90)

1.045

211 (60)

1.00

GA + AA

140 (40)

0.722

53 (15)

1.00

AG + GG

298 (85)

0.652

73 (21)

1.00

AG + AA

278 (79)

0.989

TT/CT

87 (25)

1.00

264 (75)

0.251

GHRL rs26802 GHSR rs572169 LEP rs7799039 LEPR rs1137101 5–HT2C rs3813929 UCP2 rs659366

49 (14)

1.00

CT + CC

302 (86)

1.330

49 (14)

1.00

AG + GG

302 (86)

0.568

54 (15)

1.00

CT + TT

297 (85)

0.305

223 (64)

1.00

AG + AA

294 (84)

0.815

CC + CT

184 (52)

1.00

167 (48)

1.706

GG + CG

117 (33)

1.00

234 (67)

0.761

71 (20)

1.00

AT + TT

280 (80)

0.963

UCP2 rs660339 UCP3 rs1800849 SH2B1 rs7498665 TAS1R2 rs35874116 TAS1R2 rs9701796

FTO rs9939609

(95% CI)

SAH Preoperative

p-value 0.921

(0.436–2.507)

0.547

(0.250–2.085)

0.318

(0.282–1.508)

0.974

(0.514–1.902)

0.001

(0.109–0.578)

0.505

(0.574–3.082)

0.238

(0.222–1.452)

0.019

(0.113–0.821)

0.594

(0.385–1.726)

0.790

(0.626–1.850)

0.353

(0.428–1.354)

0.911

(0.492–1.882)

(95% CI)

1.00 1.244

(0.593–2.609)

1.00 1.441

(0.566–3.667)

1.00 0.712

(0.388–1.307)

1.00 1.019

(0.596–1.742)

1.00 1.164

(0.702–1.928)

1.00 1.056

(0.511–2.181)

1.00 1.167

(0.567–2.405)

1.00 0.624

(0.341–1.144)

1.00 0.980

(0.544–1.765)

1.00 1.019

(0.658–1.579)

1.00 1.252

(0.788–1.989)

1.00 0.595

(0.348–1.017)

p-value 0.563 0.443 0.274 0.945 0.556 0.884 0.675 0.128 0.946 0.933 0.341 0.058

n = 351. Variables with adjustments for age, skin color, preoperative BMI, previous pregnancy and age at onset of obesity.

Table 3. Prevalence of gene polymorphism for 5–HT2C (rs3813929) and UCP3 (rs1800849) between diabetic subjects who obtained or did not obtain the control of DM2 after bariatric surgery Response Yes

CT + CC

CT + CT

CT + TT

CC + CC

CC + CT

CC + TT

1/65

8/58

4/62

7/59

22/44

24/42

0/10

1/9

3/7

4/6

p-value*

0.868

1.000

0.985

1.000

0.999

This knowledge might be important in identifying, according to genetic variability, subjects who are at higher risk of DM2, because it is not obesity per se that is the lethal factor, but it’s associated chronic diseases. Thus, the analysis of 5-HT2C (rs3813929) and UCP3 (rs1800849) gene polymorphism may theoretically define individualized and preventive measures for DM2 in obese women on a waiting list for bariatric surgery. Arch Endocrinol Metab. 2017;61/4

Unlike the results of other studies (18-23), the current study did not find an association of GHRL (rs26802), UCP2 (rs659366), FTO (rs9939609), LEP (rs7799039), and LEPR (rs1137101) gene polymorphisms with DM2. These controversies, far from discouraging the study of the effects of gene polymorphisms, instigate a deepening of this knowledge, which takes into account the characteristics 329

* Fisher’s exact test.

Gene polymorphism and bariatric surgery

of the populations and the interactions between the different gene polymorphisms. Regarding 5-HT2C gene polymorphism results, serotonin plays an important role in the nervous system, such as sleep regulation, body temperature, appetite and mood, among others (24). Thus, low levels of serotonin or problems in the signaling with the receptor have been linked to an increased desire to eat sweets and carbohydrates (25), which may explain in part the association of 5-HT2C gene polymorphism (rs3813929) with the prevalence of DM2 observed in our study. Similarly, other studies reported that subjects carrying the T allele from 5-HT2C gene polymorphism (rs3813929) were associated with greater chances of DM2. Kring and cols. (26), investigating Caucasian young men, observed an association of T allele from 5-HT2C gene polymorphism with glucose and acute insulin response. On the other hand, Iordanidou and cols. (27) observed that T allele frequency was lower in diabetic subjects compared to nondiabetic subjects. In the present study, the UCP3 gene was also associated with prevalence of DM2, specifically the polymorphism rs1800849. The UCP3 protein is involved in promoting fatty acid oxidation in skeletal muscle and it indirectly influences glucose metabolism (28). In addition, UCP3 regulates the production of reactive oxygen species in the mitochondria (28), a factor that has established an association of this protein with DM2, because oxidative stress pathways play a key role in the development of this chronic disease (29). In this study, women carrying the CC genotype on UCP3 gene polymorphism (rs1800849) showed three times more chance of DM2 compared to allele T carriers. Corroborating our data, Meirhaeghe and cols. (30) showed that UCP3 gene polymorphism (rs1800849) was related to DM2 on a French cohort, and T allele carriers presented lower risk. Regarding data 1 year postoperatively, the 12 gene polymorphisms investigated in our study showed no effect on the control/remission of SAH and DM2. Among the diabetic women in this study, 13% did not obtain the control of this chronic disease; thus, it is plausible to hypothesize that there are a possible genetic factors involved in the lack of this response postoperatively. On the other hand, considering that 1 year postoperatively is a relatively short time period, it is important to do a long-term investigation (> 2 years) to determine these responses. 330

Although the molecular and genetic comprehension of DM2 has advanced rapidly, much of the knowledge remains unknown. Adding to this, the number of bariatric surgeries has risen as an effective treatment option for obesity and its associated comorbidities. Thus, more studies are necessary to investigate the effects of surgical and non-surgical intervention for weight loss and remission of DM2, while larger numbers of subjects, a wider range of gene polymorphism, and analyzing a later postoperative time, could better elucidate these questions. Genetic association studies are relevant to determine the risk of chronic diseases on a specific population and to improve the medical and nutritional treatment. These results enable an individualization of the treatment according to genetic variability. The current study investigates 12 obesity-related gene polymorphisms in obese women candidates for bariatric surgery. In summary, subjects with T allele from 5-HT2C gene polymorphism (rs3813929) had five times greater chance of DM2, and the CC genotype from UCP3 gene polymorphism (rs1800849) had three times greater chance of DM2, in the preoperative period. Our results indicate that 5-HT2C and UCP3 gene polymorphism was related to prevalence of DM2 among Brazilian obese women candidates for bariatric surgery. Disclosure: no potential conflict of interest relevant to this article was reported.

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Gene polymorphism and bariatric surgery

8. Xu X, Zeng H, Xiao D, Zhou H, Liu Z. Genome wide association study of obesity. Zhong Nan Da Xue BaoYi Xue Ban. 2013;38(1):95100. 9. Tan LJ, Zhu H, He H, Wu KH, Li J, Chen XD, et al. Replication of 6 obesity genes in a meta-analysis of genome-wide association studies diverse ancestries. Plos One. 2014;9(5):e96149. 10. Neff KJ, le Roux CW. Bariatric surgery: the indications in metabolic disease. Dig Surg. 2014;31(1):6-12. 11. Benoit SC, Hunter TD, Francis DM, De La Cruz-Munoz N. Use of bariatric outcomes longitudinal database (BOLD) to study variability in patient success after bariatric surgery. Obes Surg. 2014;24(6):936-43. 12. de Luis DA, Pacheco D, Aller R, González Sagrado M, Izaola O, Terroba MC, et al. Influence of -55CT polymorphism of UCP3 gene on surgical results of biliopancreatic diversion. Obes Surg. 2010;20(7):895-9.

2 diabetes and body weight: The Finnish Diabetes Prevention Study. Int J Obes (Lond). 2005;29(10):1245-51. 20. Gueorguiev M, Lecoeur C, Meyre D, Benzinou M, Mein CA, Hinney A, et al. Association studies on ghrelin and ghrelin receptor gene polymorphisms with obesity. Obesity (Silver Spring) 2009;17(4):745-54. 21. Salopuro T, Pulkkinen L, Lindström J, Kolehmainen M, Tolppanen AM, Eriksson JG, et al. Variation in the UCP2 and UCP3 genes associates with abdominal obesity and serum lipids: the Finnish Diabetes Prevention Study. BMC Med Genet. 2009;10:94. 22. Roszkowska-Gancarz M, Kurylowicz A, Polosak J, Mossakowska M, Franek E, Puzianowska-Kuznicka M. Functional polymorphisms of leptin and leptin receptor genes are associated with longevity and with the risk of myocardial infarction and type 2 diabetes mellitus. Endokrynol Pol. 2014;65(1):11-6.

13. de Luis DA, Aller R, Conde R, Izaola O, Pacheco D, Sagrado MG, et al. Effects of RS9939609 gene variant in FTO gene on weight loss and cardiovascular risk factors after biliopancreatic diversion surgery. J Gastrointest Surg. 2012;16(6):1194-8.

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17. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care. 2013;36:S67-74.

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29. Liu J, Li J, Wang CM. The role of uncoupling proteins in diabetes mellitus. J Diabetes Res. 2013;2013:585897. 30. Meirhaeghe A, Amouyel P, Helbecque N, Cottel D, Otabe S, Froguel P, et al. An uncoupling protein 3 gene polymorphism associated with lower risk of developing type II diabetes and with athetogenic lipid profile in a French cohort. Diabetologia. 2000;43(11):1424-8.

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original article

Bone turnover after bariatric surgery Thalita Lima Melo1, Leila Froeder1, Leandro da Cunha Baia1, Ita Pfeferman Heilberg1

ABSTRACT Disciplina de Nefrologia, Universidade Federal de São Paulo (Unifesp), São Paulo, SP, Brasil 1

Correspondence to: Ita Pfeferman Heilberg Disciplina de Nefrologia, Universidade Federal de São Paulo Rua Botucatu, 740 04023-900 – São Paulo, SP, Brasil ita.heilberg@gmail.com Received on Dec/5/2016 Accepted on Mar/10/2017 DOI: 10.1590/2359-3997000000279

Objective: The aim of the present study was to evaluate parameters of bone and mineral metabolism after bariatric surgery. Subjects and methods: This sectional study included data from medical records from 61 bariatric surgery (BS) patients (minimum period of 6 months after the procedure) and from 30 class II and III obese patients as a control group (Cont), consisting of daily dietary intake of macronutrients, calcium and sodium, serum 25(OH)D and parathyroid hormone (PTH) and other biochemical serum and urinary parameters. Bone alkaline phosphatase (BAP), leptin, fibroblast growth factor-23 (FGF-23) and deoxypyridinoline (DPYD) were determined from available banked serum and urinary samples. Results: Mean body mass index (BMI), median energy, carbohydrate, protein and sodium chloride consumption were significantly lower in the BS versus Cont, but calcium and lipids were not. No significant differences were found in ionized calcium, 25(OH)D, PTH and fibroblast growth factor 23 (FGF-23) between groups. Mean serum BAP was significantly higher for BS versus Cont and had a positive correlation with time after the surgical procedure. Mean serum leptin was significantly lower and median urinary DPYD higher in BS versus Cont. Conclusion: The present study showed an increase in bone markers of both bone formation and resorption among bariatric patients up to more than 7 years after the surgical procedure, suggesting that an increased bone turnover persists even at a very long-term follow-up in such patients. Arch Endocrinol Metab. 2017;61(3):332-6. Keywords Gastric bypass; bone turnover; obesity; bone metabolism

INTRODUCTION

he prevalence of obesity has increased during the past decades representing a major public health problem. A high body mass index (BMI) has been directly associated with higher bone mineral density (BMD) given that mechanical loading under physiological conditions possesses a central role on bone mass maintenance. Therefore, it has been well accepted until recently, that obesity was associated with a reduced risk of osteoporosis and that fat mass had a similar beneficial effect on increasing bone mass (1). However, contrasting studies now show that excessive fat mass may not protect against osteoporosis (2) and that lean mass may be a more important determinant of BMD (3). Moreover, adipose tissue expresses and secretes several biologically active hormones and cytokines such as estrogen, leptin, among others, which may affect energy homeostasis and bone metabolism (4). Bariatric surgery (BS) is an effective treatment for clinically severe obesity and Roux-en-Y gastric bypass, the most common technique, induces an important and sustained weight loss improving comorbidities. However, the procedure entails long-term complications such as nutritional deficiencies, nephrolithiasis (5), 332

as well as other disturbances in bone and mineral metabolism, including low bone mass and increased risk of fractures after a substantial weight loss (6-9). Bone loss may be attributed to a reduction of lean body mass during the weight reduction process, previous obesity-induced hypovitaminosis D, which persists even after surgery, and malabsorption of calcium and vitamin D following the procedure (10,11). De Prisco and Levine (6) reported osteomalacia and osteoporosis among bariatric patients up to 12 years after procedure, suggesting that this population is at risk for metabolic bone disease at a long-term basis. Previous studies that evaluated skeletal changes after bariatric surgery through dual-energy x-ray absorptiometry (DXA), quantitative computed tomography (QCT) and high-resolution peripheral computed tomography (HR-pQCT) demonstrated a decreased BMD and also increased bone turnover markers (7,9,12-15). Therefore, bone loss is a multifactorial complication in bariatric patients although the underlying mechanisms are not fully elucidated. The aim of the present study was to evaluate parameters of bone and mineral metabolism after bariatric surgery. Arch Endocrinol Metab. 2017;61/4

Bone turnover after bariatric surgery

A sectional study was carried out by the review of 80 medical records from class II and III obese patients who had undergone bariatric surgery (BS) through Roux-en-Y (RYGB) or biliopancreatic diversion with duodenal switch (BD-DS) between 2007 and 2010 at the Universidade Federal de São Paulo. Eligible patients for inclusion were those with a minimum period of 6 months post-surgery, who had dietary information obtained through 72 hour food recalls, as well as results from serum levels of 25(OH)D and parathyroid hormone (PTH), and other biochemical serum and urinary (retrieved from 24 hour urine specimens) parameters under conditions of discontinuation of multi-vitamins and/or calcium supplementation 72 hours before collection. Serum biochemistry comprised values of creatinine, albumin and ionized calcium and urinary parameters included calcium, sodium and creatinine. This retrospective analysis was followed by a further selection of patients whose banked serum and urine samples were available for the determination of additional mineral metabolism parameters, such as fibroblast grow factor-23 (FGF-23), bone alkaline phosphatase (BAP), deoxypyridinoline (DPYD) as well as leptin. Exclusion criteria were age < 18 years old, abnormal renal function (estimated glomerular filtration rate (eGFR) by CKD-EPI creatinine equation < 60 mL/ min/1.73 m2) and history of medical disorders known to affect bone metabolism (e.g., hyperthyroidism, hyperparathyroidism, renal disease, Paget’s disease, etc.). The study was approved by the local Ethics Committee of the Institution and a written consent was obtained from all participants. The control group (Cont) consisted of data from age-matched obese patients with a body mass index [BMI] 35.0–39.9 (obesity class II) and ≥ 40 kg/m2 (obesity class III) waiting for bariatric surgery, whose similar parameters could be used for comparisons. Serum BAP (Quidel, San Diego, CA), FGF-23 (Kainos Laboratories, Tokyo, Japan), fasting serum leptin (Merck Millipore, Darmstadt, Alemanha) and urinary DPYD (MicroVue DPD EIA, Quidel Corporation, San Diego, USA) were measured by ELISA.

Statistical analyses It was estimated that this sample size would provide 95% of power, at a significance level of 0.01, to detect changes in primary endpoints by including at least Arch Endocrinol Metab. 2017;61/4

15 patients in each group. The software G. Power 3.1.2 was used for the sample calculation (Franz Faul, University of Kiel, Germany). Normally distributed variables are presented as mean ± standard deviation and skewed variables as median [interquartile interval]. Differences between groups were assessed using T tests or Mann Whitney when appropriated. Categorical comparisons were assessed using x2-square test. Spearman’s correlation as used to examine the relationship between change in serum markers of bone turnover and time after procedure. All statistical analyses were performed using SPSS (Statistical Package Social Sciences) software version 18 for Windows (Illinois, MA, USA). A p value ≤ 0.05 was considered statistically significant.

RESULTS After exclusion of missing data on nutritional assessment, biochemical and hormonal parameters, 61 BS (n = 58 RYGB and n = 3 BD-DS) and 30 Cont were available for enrollment in the study. The median [IQR] post-surgical time was 4 [1-7] years and the mean decrease in BMI after surgery was 36% (data not shown in table). As shown in Table 1, no significant differences were observed regarding gender distribution and mean age between BS and Cont. The mean values of BMI, daily dietary intake of energy, carbohydrate, protein and sodium chloride (estimated from 24-hour urinary sodium, for better accuracy) were significantly lower in the BS compared to Cont. Daily consumption of calcium and lipids did not differ between groups. The mean serum BAP was significantly higher for BS versus Cont patients and had a positive correlation with time after procedure (r = 0,278, p = 0,04). Serum leptin was lower in BS group compared to Cont and no significant differences were found in eGFR, ionized calcium, albumin, 25(OH)D, PTH and FGF-23 between groups. Urinary DPYD was higher, calcium and sodium were lower in BS versus Cont.

DISCUSSION The main finding of the present study was an increased bone turnover, disclosed among bariatric patients in the late postoperative period when compared to morbidly obese individuals. Several investigators have reported bone mineral metabolism disturbances following bariatric surgery, including either reduced BMD (8,11-13) or alterations 333

SUBJECTS AND METHODS

Bone turnover after bariatric surgery

in bone biochemical markers of bone turnover (14,15). Although hypovitaminosis D, secondary hyperparathyroidism and mechanical unloading of the skeleton due to weight loss have been implicated, the underlying mechanisms responsible for such changes are not fully elucidated. In the present study, median serum 25(OH)D in BS group was low and not different from Cont before surgery, which agrees well with other reports (9,12). Whereas hypovitaminosis D in class II and III obese patients more often occurs because of the sequestration of vitamin D in adipose tissue (16), the reasons in BS patients include not only pre-operative low levels of 25(OH)D (15), but also low compliance with oral supplements (17). In the current series, despite of the low calcium consumption besides hypovitaminosis D, BS group did not present a higher median serum PTH. Nevertheless, the presence of secondary hyperparathyroidism among bariatric patients is still a matter of debate since some investigators reported normal levels of PTH (8,13,15), while others did not (14,18,19).

The significantly higher serum BAP levels in BS than Cont patients observed in the present study suggests the presence of increased bone formation, in agreement with the data reported by Bruno and cols. (15) and with other investigators, who employed different biochemical markers of bone formation (8,12). Moreover, urinary DPYD was also higher in BS patients than in Cont, indicating an increased bone resorption, as detected by other investigators (20,21), even when using different bone resorption markers as well (8,20,22,23). Therefore, an increased bone turnover has been clearly evidenced by the present findings, which corroborates with numerous reports (8,12,13,15,24), although not with all of them (9,21). We observed a low calcium intake by BS patients (similarly to Cont), as observed by Menegati and cols. (19), what may be a caution against dumping (25). Inadequate calcium intake may contribute to impair bone homeostasis, due to increases in serum PTH and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) (26), and a high sodium consumption may also compromises bone health (27,28). Furthermore, the presence of reduced

Table 1. Nutritional data, serum and urinary parameters Parameter Gender (F/M) Age (years) eGFR

BS [n = 61]

23/7

51/9

0.52

48.0 ± 8.8

47.1 ± 9.9

0.69

102 [85-111]

104 [97-109]

0.32

BMI [kg/m²]

46.5 ± 7.3

31.5 ± 6.0

< 0.001

Energy [kcal]

1739 [1182-2743]

1272 [931-1714]

0.01

199 [149-390]

142 [106-192]

< 0.001

Dietary protein [g]

75 [62-100]

51 [39-70]

< 0.001

Dietary lipids [g]

45 [27-57]

53 [32-76]

0.27

502 [395-1278]

517 [324-740]

0.32

Dietary carbohydrate [g]

Dietary calcium [mg] Dietary NaCl [g]

11 [7-15]

8 [6-11]

0.01

Serum ionized Calcium [mmol/L]

1.3 ± 0.0

0.15

4.2 ± 0.2

0.80

21.0 [17-24]

19.1 [14-26]

0.51

Serum Albumin [g/dL] Serum 25(OH)D [ng/mL]

Cont [n = 30]

Serum PTH [pg/mL]

61.0 ± 22.7

61.0 ± 23.8

0.98

Serum FGF-23 [kRU/mL]

41.1 [27-53]

44.4 [16-58]

0.53

Serum BAP [U/L]

17.9 ± 5.6

24.2 ± 9.6

0.003

Serum Leptin [ng/mL]

44.7 ± 11.6

32.2 ± 14.6

< 0.001

5.1 [3-6]

5.6 [3-9]

0.041

Urinary DPYD [nmol/mmol creat] Urinary calcium [mg/24h]

161 [23-488]

89 [21-270]

0.001

Urinary sodium [mg/24h]

184 [122-252]

141 [105-181]

0.01

1474 ± 511

1112 ± 302

0.000

Urinary creatinine [mg/24h]

Data are presented as mean ± SD or median [interquartile interval]. Cont: control group, BS: bariatric surgery, eGFR: estimated glomerular filtration rate, BMI: body mass index, PTH: parathyroid hormone, FGF-23: fibroblast growth factor-23, BAP: bone alkaline phosphatase, DPYD: deoxypyridinoline.

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Arch Endocrinol Metab. 2017;61/4

remodeling remain not fully elucidated (34). In an experimental study, Tsuji and cols. (35) showed that leptin increased FGF-23 expression in leptin-deficient ob/ob mice, acting directly on bone cells, and suppressed renal 1,25(OH)2D3 synthesis. Conversely, one may speculate that the reduction of leptin following bariatric surgery could be associated with an increased 1,25(OH)2D3, hence contributing to stimulate bone resorption, especially under conditions of low calcium consumption (26). However, in the current series, median values of serum FGF-23 did not differ between groups, even in the presence of lower serum leptin in BS versus Cont. Moreover, levels of 1,25(OH)2D3 were not determined. Other limitations of the present study included the lack of BMD and body composition data from both groups and the absence of enough test results of mineral metabolism parameters among bariatric patients before the surgery, to allow us to make comparisons before and after the procedure. In summary, BS patients exhibited significantly higher serum BAP, urinary DYPD and lower serum leptin levels than Cont, without statistical significance in circulating levels of 25(OH)D and PTH, indicating that other mechanisms, not solely associated to the calciotropic hormones, might be operating to explain the increased bone turnover after bariatric surgery. Such findings are in agreement with other investigators (14,18) and according to Bruno and cols. (15), even the supplementation of vitamin D was not capable to prevent the increase in bone turnover seen after bariatric surgery. In conclusion, the present study showed an increase in bone markers of both bone formation and resorption among bariatric patients up to more than 7 years after the surgical procedure, suggesting that an increased bone turnover persists even at a very longterm follow-up in such patients. Additional studies are still warranted to better elucidate the underlying mechanisms for these findings, aiming to mitigate derangements in bone metabolism and helping to delineate earlier interventions. Acknowledgments: present data were retrieved from a previous study supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp) (Grant 2008/02279-4) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Grant 475681/2007-0). We thank Silvia Regina Moreira for technical assistance. Disclosure: no potential conflict of interest relevant to this article was reported. 335

intestinal calcium absorption even under conditions of both calcium and vitamin D supplementation in bariatric patients (11) may render bone condition even worse. The observed increased bone turnover in BS group could be accounted for by a negative calcium balance. However, given that urinary calcium and sodium were significantly lower in BS versus Cont, the contribution of negative calcium balance did not seem to be important. The present sample consisted of bariatric patients with a median follow-up of 4 years after the procedure with a handful of them attaining more than 7 years of followup. Of note, we found a positive correlation of serum BAP with time after surgery. A substantial increase in bone formation and resorption markers had occurred up to 24 months after gastric bypass despite weight stability, as described by many authors (12,13,29), reinforcing that bone remodeling remains elevated despite weight loss plateaus after the first year and are not exclusively due to mechanical unloading of the skeleton. It is well recognized that the adipose tissue synthesizes and releases a series of hormones and adipokines involved in bone metabolism through direct effects on bone formation and resorption (30). When compared to Cont, bariatric patients from the current series presented a lower mean value of serum leptin, one of the most important cytokines derived from fat tissue, which acts on hypothalamic neurons by stimulating sympathetic postganglionic neurons via axons that enter the bones to inhibit bone formation (31,32). Our findings are in agreement with other reports after bariatric surgery (13,15). Bruno and cols. (15) also showed that a reduction in leptin represented an independent predictor of increase in N-teloptide of type 1 collagen (NTX), a bone resorption marker, among bariatric patients. A more recent study of bone structural changes evaluated through high-resolution peripheral quantitative computed tomography by Frederiksen and cols. (13) revealed an inverse association between leptin and trabecular number. Although there is still some controversy regarding the correlation of leptin with BMD (30), Thomas and cols. (33) noticed a direct association between leptin and BMD only among women, in the general population, suggesting that leptin could mediate at least part of the protective effect of fat mass on the skeleton. However, the reciprocal action between bone and adipose tissue is complex and the underlying mechanisms by which leptin regulates bone

Bone turnover after bariatric surgery

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Arch Endocrinol Metab. 2017;61/4

original article

Association between C1q gene polymorphisms and autoimmune thyroid diseases Qiuming Yao1, Jie Li2, Xiaofei An1, Wenjuan Jiang1, Qiu Qin1, Ronghua Song1, Ni Yan1, Danfeng Li1, Yanfei Jiang1, Wen Wang1, Liangfeng Shi1, Jin-an Zhang1

ABSTRACT Objective: In the present study, we aimed to assess the associations of C1q gene polymorphisms with autoimmune thyroid diseases (AITD) susceptibility. Subjects and methods: A set of 1,003 AITD patients (661 with Graves’ disease and 342 with Hashimoto’s thyroiditis) and 880 ethnicallyand geographically-matched controls from Chinese Han population were included. Five common single nucleotide polymorphisms (SNPs) (rs294185, rs292001, rs682658, rs665691 and rs294179) in C1q gene locus were genotyped. Frequencies of genotypes and alleles were compared between patients and controls, and haplotype analysis was also performed. Results: There was no statistically significant difference between AITD patients and controls in the frequencies of alleles of rs294185 (P = 0.41), rs292001 (P = 0.71), rs682658 (P = 0.68), rs665691 (P = 0.68) and rs294179 (P = 0.69). There was also no statistically significant difference between AITD patients and controls in the frequencies of genotypes of rs294185 (P = 0.72), rs292001 (P = 0.89), rs682658 (P = 0.83), rs665691 (P = 0.90) and rs294179 (P = 0.43). Stratified analyses showed that none of those five SNPs in C1q gene were associated with Graves’ disease or Hashimoto’s thyroiditis (all P values > 0.05). Haplotype analysis revealed that there were no obvious genetic associations of C1q gene polymorphisms with AITD susceptibility. Conclusions: We, for the first time, identified the associations between C1q gene SNPs and AITD, and our findings suggested that five common SNPs in C1q gene were not associated with AITD susceptibility in Chinese Han population. Arch Endocrinol Metab. 2017;61(3):337-42.

Department of Endocrinology, Jinshan Hospital of Fudan University, Shanghai, China 2 Department of Nephrology, Xi’an Central Hospital, Xi’an, China 1

Correspondence to: Jin-an Zhang Department of Endocrinology, Jinshan Hospital of Fudan University, 1508 Longhang Road 201508 – Shanghai, People’s Republic of China zhangjinan@hotmail.com Received on May/26/2016 Accepted on Oct/18/2016 DOI: 10.1590/2359-3997000000256

INTRODUCTION

utoimmune thyroid diseases (AITD) are common autoimmune disorders in endocrinologic system, affecting about 5% of overall population (1). AITD is characterized by immune imbalance and autoantibodies towards thyroid. As the most common type of AITD, Hashimoto’s thyroiditis (HT) mainly causes hypothyroidism, and it is characterized by lymphocytic infiltration and presence of thyroid peroxidase antibodies (TPOAb) or thyroglobulin antibody (TgAb). Graves’ disease (GD) is another main type of AITD, which is characterized by hyperthyroidism due to overproduction of thyroid hormones induced by specific auto-antibodies against thyrotropin receptor (TSHR). It has been known that many factors are involved in the initiation and development of AITD, such as genetic factors, environmental factors and nutritional elements, Arch Endocrinol Metab. 2017;61/4

such as iodine intake and infection (2,3). However, the pathogenesis of AITD remains unclear. In the past decade, several genetic polymorphisms have been found to be associated with AITD susceptibility, such as genetic polymorphisms in the genes encoding TSHR, human leukocyte antigen (HLA) and cytotoxic T lymphocyteassociated antigen-4 (CTLA4) (4-7). Other genetic polymorphisms associated with AITD susceptibility have also been reported, such as polymorphisms in the CD40, IL-17, FCRL3 and protein tyrosine phosphatase-22 (PTPN22) genes (8-11). However, the above-mentioned genetic polymorphisms can only explain part of gene susceptibility to AITD, and other genetic polymorphisms are believed to have important roles in AITD, which need to be explored in future studies. Complement is a vital part of innate immune system in human body, which can be activated by three different pathways (12). The classical pathway 337

Keywords Autoimmune thyroid diseases; C1q; single nucleotide polymorphism

Association of C1q gene with AITD

of complement activation is characterized by the binding of C1q to immune complexes (13). C1q is a recognition component in the classical pathway, and it can help solubilize immune complexes and aid in the clearance of apoptotic debris (14). The gene coding region for C1q is localized on chromosome 1p34–36 and consists of three genes, C1qA, C1qB and C1qC (15). Several single nucleotide polymorphisms (SNPs) have been found in the C1q gene, such as rs292001, rs682658, rs665691 and rs294179. SNPs in the C1q gene have been reported to be associated with several common autoimmune diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis. However, the roles of C1q gene SNPs in AITD susceptibility remain poorly explored. In the present study, the associations of five common SNPs in C1q gene with AITD susceptibility were examined in order to identify additional risk variants for AITD susceptibility. We, for the first time, clarified the roles of C1q gene SNPs in AITD susceptibility in Chinese Han population.

SUBJECTS AND METHODS

Subjects The present study was a case-control study, and all subjects were from Chinese Han population. A set of 1,003 AITD patients (661 with GD and 342 with HT) and 880 ethnically- and geographicallymatched controls were included. All controls were healthy and unrelated to AITD patients. The AITD patients were recruited in the outpatient department of Jinshan Hospital of Fudan University (Shanghai, China). AITD patients were diagnosed according to clinical guidelines, which were described in details in our previously published studies (16). The diagnosis of GD was based on the presence of clinical and laboratory biochemical hyperthyroidism with diffuse goiter, decreased TSH value as well as increased levels of free thyroid hormones and anti-thyroid stimulating hormone receptor antibody (TRAb) (+). HT was diagnosed by the presence of an enlarged thyroid and TPOAb (+) or TgAb (+). The healthy subjects without thyroid goiters, autoimmune diseases and family history were randomly recruited from health examination center of Jinshan Hospital of Fudan University (Shanghai, China) and used as controls. All the controls presented no TRAb(+), TPOAb(+) or TgAb(+). The research was approved by the Ethics 338

Committee of Jinshan Hospital of Fudan University (Shanghai, China), and informed consents were obtained from all included participants.

Genotyping About 2 mL peripheral venous blood was collected from patients and controls. Genomic DNA was extracted from the collected blood samples, and the concentration and purity of DNA samples were determined by Nano Drop 2000 Spectro-photometer (Thermo, USA). From Hapmap CHB database, five SNPs of C1q gene (rs292001/rs682658/rs294185/ rs665691/rs294179) were selected according to the predisigned eligibility criteria as follows: (1) the frequency of minor allele was greater than 0.10; and (2) P value for Hardy-Weinberg equilibrium (HWE) was greater than 0.001. The genotyping of five SNPs of C1q was conducted by using ligase detection reaction (LDR) platform (16). The target DNA sequences of those five SNPs of C1q gene were amplified using multiplex polymerase chain reaction (PCR) method. The sequences of primers for those five SNPs were as follows: (1) rs294185: Forward: 5’-ACCCCAGCTTTGACATTTGC-3’; Reverse: 5’-GGTGTGGTCTCAGTTTTAGG-3’; (2) rs292001: Forward: 5’-TCCTAGTCCAAAGCAGACCA-3’; Reverse: 5’-GTTCAGGTACCACATGTAGG-3’; (3) rs665691: Forward: 5’-AAGCATTCTCAGGGTCCAAG-3’; Reverse: 5’-CCTTAACTGATGGGATGCTC-3’; (4) rs294179: Forward: 5’-GCACATCTTGCCTTTGTCTG-3’; Reverse: 5’-CCTGTGCTGAACTTCAGGAG-3’; (5) rs682658: Forward: 5’-ACTTGGCCCTAGGAGTCCCT-3’; Reverse: 5’-CAGCCCCATAATGCAGTATC-3’.

Statistical analysis Statistical analyses were carried out by using SPSS (version 17.0). Chi-square test was used to detect the difference in the frequencies of genotypes and alleles between patients and controls. The association between SNPs and AITD susceptibility was firstly assessed, and then stratified analyses were performed based on the types of AITD. Haplotype analysis was also conducted using Haploview 4.0 platform. P value less than 0.05 was considered statistically significant. Arch Endocrinol Metab. 2017;61/4

Association of C1q gene with AITD

Demographic and clinical characteristics of subjects Table 1 shows the demographic and clinical characteristics of all the participants in this study. In the GD group, 464 (70.2%) patients were females, 197 (29.8%) patients were males, and the mean age was 36.9 years (Table 1). The HT group consisted of 274 (80.1%) female patients and 68 male patients, and their mean age was 34.8 years (Table 1). In the control group, 587 (66.7%) individuals were females, 293 (33.3%) individuals were males, and the mean age was 38.8 years (Table 1). No significant difference concerning age and gender was observed among those three groups, and all P values were greater than 0.05 (Table 1).

Allele and genotyping results Genotype distributions for the loci of rs294185, rs292001, rs665691, rs294179 and rs682658 were all confirmed to HWE in both patients and controls (P > 0.05). Table 2 shows the frequencies of alleles and genotypes for those five SNPs in patients and controls. No statistically significant difference was observed between AITD patients and controls in the frequencies of alleles of rs294185 (P = 0.41), rs292001 (P = 0.71), rs682658 (P = 0.68), rs665691 (P = 0.68) and rs294179 Table 1. Demographic and clinical characteristics of all the participants in this study Characteristics

GD (N = 661)

HT (n = 342)

Controls (N = 880)

197 (29.8)

68 (19.9)

293 (33.3)

Sex (%) Male

464 (70.2)

274 (80.1)

587 (66.7)

Age (Mean ± SD, year)

Female

36.9 ± 14.5

34.8 ± 13.8

38.8 ± 9.0

Onset age (Mean ± SD, year)

33.9 ± 14.5

32.4 ± 13.4

NA*

Normal

116 (17.6)

48 (14.0)

880 (100.0)

First degree

109 (16.5)

56 (16.4)

0 (0.0)

Second degree

347 (52.5)

213 (62.3)

0 (0.0)

Third degree

89 (13.4)

25 (7.3)

0 (0.0)

Thyroid size (%)

Family history (%) Yes

134 (20.27)

70 (20.47)

0 (0.0)

527 (79.73)

272 (79.53)

880 (100.0)

Yes

115 (17.40)

6 (1.75)

0 (0.0)

546 (82.60)

336 (98.25)

880 (100.0)

Ophthalmopathy (%)

* NA: not available. Arch Endocrinol Metab. 2017;61/4

(P = 0.69) (Table 2). There was also no statistically significant difference between AITD patients and controls in the frequencies of genotypes of rs294185 (P = 0.72), rs292001 (P = 0.89), rs682658 (P = 0.83), rs665691 (P = 0.90) and rs294179 (P = 0.43) (Table 2). Stratified analyses showed that none of those five SNPs in C1q gene were associated with GD or HT (all P values > 0.05) (Table 2).

Haplotype analysis Haplotype analysis suggested strong linkage disequilibrium (LD) in those five SNPs of C1q gene (Table 3). LD mainly existed between rs665691 and rs292001, rs292001 and rs682658, rs665691 and rs682658, rs294185 and rs294179. Table 4 shows the frequency of each haplotype. However, there were no obvious associations of the haplotypes of block 1 and block 2 with GD or HT (Table 4). Figure 1 shows two detected LD blocks according to D’ value, which were rs665691-rs292001-rs682658 and rs294185rs294179, respectively.

DISCUSSION Polymorphisms in the complement C1q gene have been reported to be associated with several types of autoimmune diseases. However, their roles in AITD still remain unclear. In the present study, we aimed to assess the associations of C1q gene polymorphisms with AITD susceptibility. To the best of our knowledge, we, for the first time, clarified the roles of C1q gene SNPs in AITD susceptibility. We recruited a set of 1,003 AITD patients (661 with GD and 342 with HT) and 880 ethnically- and geographically-matched controls from Chinese Han population, and examined the associations of five commonly detected SNPs in C1q gene with AITD susceptibility. In the present study, we showed that AITD patients and healthy controls had statistically similar frequencies of genotypes and alleles of rs294185, rs292001, rs682658, rs665691 and rs294179 (Table 2). Haplotype analysis, which can provide powerful and conducive analyses in identifying genes associated with complex diseases (17), also did not find obvious associations of C1q gene with AITD susceptibility. Therefore, our findings suggested that those five common SNPs in C1q gene (rs294185, rs292001, rs682658, rs665691 and rs294179) were not associated with AITD susceptibility in Chinese Han population. 339

RESULTS

Association of C1q gene with AITD

Table 2. Genotype distributions and allele frequencies of C1q SNPs in AITD patients and controls SNPs

rs294185

rs292001

rs665691

rs294179

rs682658

Allele or genotypes

Controls (%)

AITD (%)

304 (34.5)

361 (36.0)

411 (46.7)

466 (46.5)

GD (%)

P values

235 (35.5) 0.72

HT (%) 126 (36.8)

311 (47.0)

0.78

155 (45.3)

165 (18.7)

176 (17.5)

115 (17.4)

61 (17.8)

1019 (57.9)

1188 (59.2)

781 (59.1)

407 (59.5)

741 (42.1)

818 (40.8)

369 (41.9)

410 (40.9)

398 (45.2)

463 (46.2)

113 (12.8)

130 (12.9)

1136 (64.5)

1283 (64.0)

624 (35.5)

723 (36.0)

368 (41.8)

409 (40.8)

399 (45.3)

463 (46.2)

113 (12.8)

131 (13.0)

1135 (64.5)

1281 (63.9)

625 (35.5)

725 (36.1)

0.41

0.51

541 (40.9) 269 (40.7)

0.89

0.88

307 (46.4) 845 (63.9)

0.72

477 (36.1)

0.90

0.74

477 (36.1)

110 (12.5)

134 (13.4)

405 (46.0)

432 (43.1)

87 (13.2)

365 (41.5)

437 (43.5)

290 (43.8)

625 (35.5)

700 (34.9)

458 (34.6)

1135 (64.5)

1306 (65.1)

116 (13.2)

132 (13.2)

393 (44.7)

461 (45.9)

371 (42.2)

410 (40.9)

269 (40.7)

625 (35.5)

725 (36.1)

478 (36.2)

1135 (64.5)

1281 (63.9)

0.43

0.69

0.81

246 (36.0) 156 (45.6)

0.94

436 (63.7)

0.73

248 (36.3) 47 (13.7)

284 (43.0)

0.49

148 (43.3)

0.66

147 (43.0) 0.62

864 (65.4) 86 (13.0)

0.83

438 (64.0)

46 (13.5)

845 (63.9)

0.97

156 (45.6)

140 (40.9)

307 (46. 4) 85 (12.9)

0.69

0.470

45 (13.2)

269 (40.7) 0.90

277 (40.5)

0.75

141 (41.2)

85 (12.9) 0.71

P values

242 (35.4)

0.95

442 (64.6) 46 (13.5)

306 (46.3)

0.81

155 (45.3)

0.96

141 (41.2) 0.71

844 (63.8)

247 (36.1)

0.78

437 (63.9)

SNP: single nucleotide polymorphism; AITD: autoimmune thyroid diseases; GD: Graves’ disease; HT: Hashimoto’s thyroiditis.

Table 3. Linkage disequilibrium in those five SNPs of C1q gene L1

D’

Control

AITD

Control

AITD

rs665691

rs292001

1.0

0.998

0.991

rs292001

rs682658

0.998

0.993

0.973

0.983

rs665691

rs682658

0.988

0.991

0.975

0.983

rs294185

rs294179

0.997

0.992

0.753

0.767

Haplotypes

Control (N, %)

AITD (N, %)

P values

GD (N, %)

P values

HT (N, %)

P values

CAT

1129 (64.2)

1273 (63.7)

0.74

844 (63.8)

0.83

429 (63.5)

0.73

GGG

619 (35.2)

719 (36.0)

0.62

477 (36.1)

0.62

242 (35.8)

0.78

1018 (57.8)

1185 (59.1)

0.44

779 (58.9)

0.54

405 (59.4)

0.49

624 (35.5)

697 (34.7)

0.65

456 (34.5)

0.58

241 (35.3)

0.94

117 (6.6)

121 (6.0)

0.44

85 (6.4)

0.81

36 (5.3)

0.21

340

Arch Endocrinol Metab. 2017;61/4

rs294179

Block 2 (3 kb) 2

3 63

99 99

63 63

5 99

64 63

Figure 1. Linkage disequilibrium (LD) block defined by the Haploview 4.2. There were five tag SNPs on the LD map of C1q gene. Patterns of LD between the C1q gene SNPs were shown. The strength of LD measured by Dâ&#x20AC;&#x2122; multiplying 100 was displayed in the red diamonds. A larger number in the cell indicates a higher degree of LD.

The complement system is a vital part of immune system in human body, and it is involved in both innate and adaptive immune systems. As an important part of C1, C1q plays important roles in the clearance of apoptotic cells and immune complexes (18). C1q deficiency is the first identified single-gene defect, which causes lupus-like disease (19). Patients with C1q deficiency can develop lupus with high penetrance (18). It has been reported that more than 90% of individuals with complete congenital deficiency of C1q can develop early-onset photosensitive SLE (14). The presence of anti-C1q has been also strongly correlated with hypocomplementemia, disease activity and renal involvement in SLE patients (20). Several SNPs have been also found in the C1q gene, such as rs292001, rs682658, rs665691 and rs294179. It has been reported that the A allele and AA genotype of C1q rs292001 can be considered a risk factor for juvenile SLE and lupus nephritis in a cohort of Egyptian children (21). Goulielmos and cols. reported that C1q rs292001 is associated with type 2 diabetes mellitus (22). Other genetic polymorphisms of C1q have been also associated with susceptibility to autoimmune diseases, such as rheumatoid arthritis (23). Since C1q deficiency can result Arch Endocrinol Metab. 2017;61/4

in increased susceptibility to lupus-like autoimmune disease, the genetic polymorphisms of C1q may also have important roles in SLE (24). In addition, genetic deficiencies of C1q in mice can also lead to autoimmunity (25,26). The above-mentioned findings suggest that C1q is intensively involved in autoimmunity, and genetic polymorphisms in C1q are possibly associated with some types of autoimmune diseases. It has been known that many factors are involved in the initiation and development of AITD, such as genetic factors, environmental factors and nutritional factors. However, the exact pathogenesis of AITD still remains poorly explored. Previous studies have suggested that some genetic factors are intensively involved in the initiation of immune responses against the thyroid gland during the development of AITD, such as HLA and CTLA4 (2). AITD is characterized by autoimmunity, and C1q has been suggested to have some roles in AITD. Potlukova and cols. reported that auto-antibodies against C1q are more prevalent in AITD patients compared with controls (27), while Brohee and cols. reported that circulating immune complexes containing C1q are also more prevalent in AITD patients than controls (28). All of the above evidence suggests that C1q gene is probably an important element associated with AITD. However, our data failed to identify obvious associations of those five different SNPs with AITD susceptibility. Therefore, it is necessary to explore the roles of C1q gene in AITD in future studies. There were several limitations in our study. First, the findings from our study were not sufficient to explore the full roles of C1q gene in AITD, since we only explored five common SNPs in the C1q gene. Future studies can further investigate the associations of other SNPs in the C1q gene with AITD susceptibility. In addition, our study was carried out in only Chinese Han population, which could not be generalized to other ethnical populations. The roles of C1q gene polymorphisms in AITD susceptibility in Caucasian or African populations need to be studied in future studies. Second, the sample size in our study might not be enough to detect a modest association of C1q gene with AITD susceptibility. More studies with larger sample size are still required to further identify those SNPs carrying a smaller risk effect. Finally, we did not analyze the gene-environment interaction in our study due to the limitation of study design. Prospective studies in the future may explore the possible gene341

Block 1 (8 kb)

rs294185

rs682658

rs292001

rs665691

Association of C1q gene with AITD

environment interaction in the associations of C1q gene polymorphisms with AITD susceptibility. In conclusion, our findings suggested that five common SNPs in C1q gene (rs294185, rs292001, rs682658, rs665691 and rs294179) were all not associated with AITD susceptibility in Chinese Han population. Future studies are required to investigate the associations of those C1q gene SNPs with AITD susceptibility in Caucasian or African populations. In addition, it is also necessary to explore the associations of other SNPs in C1q gene with AITD susceptibility in future studies. Acknowledgements: authors are grateful to all the participants in this research. The study was financially supported by grants from the National Natural Science Foundation of China (No. 81471004) and the Key Disciplines Development of Shanghai Jinshan District (No. JSZK2015A02). Disclosure: no potential conflict of interest relevant to this article was reported.

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6. Ban Y, Davies TF, Greenberg DA, Concepcion ES, Tomer Y. The influence of human leucocyte antigen (HLA) genes on autoimmune thyroid disease (AITD): results of studies in HLA-DR3 positive AITD families. Clin Endocrinol (Oxf). 2002;57:81-8.

11. Gu LQ, Zhu W, Zhao SX, Zhao L, Zhang MJ, Cui B, et al. Clinical associations of the genetic variants of CTLA-4, Tg, TSHR, PTPN22, PTPN12 and FCRL3 in patients with Graves’ disease. Clin Endocrinol (Oxf). 2010;72:248-55. 12. Fujita T, Matsushita M, Endo Y. The lectin-complement pathway-its role in innate immunity and evolution. Immunol Rev. 2004;198:185-202. 13. Duncan AR, Winter G. The binding site for C1q on IgG. Nature. 1988;332:738-40. 14. Sontheimer RD, Racila E, Racila DM. C1q, its functions within the innate and adaptive immune responses and its role in lupus autoimmunity. J Invest Dermatol. 2005;125:14-23. 15. Sellar GC, Blake DJ, Reid KB. Characterization and organization of the genes encoding the A-, B-, and C-chains of human complement subcomponent C1q, the complete derived amino acid sequence of human C1q. Biochem J. 1991;274:481-90. 16. Yan N, Meng S, Zhou J, Xu J, Muhali FS, Jiang W, et al. Association between STAT4 gene polymorphisms and autoimmune thyroid diseases in a Chinese population. Int J Mol Sci. 2014;15:12280-93. 17. Shibata S, Saeki H, Tsunemi Y, Kato T, Nakamura K, Kakinuma T, et al. IL-17F single nucleotide polymorphism is not associated with psoriasis vulgaris or atopic dermatitis in the Japanese population. J Dermatol Sci. 2009;53:163-5. 18. Botto M, Walport MJ. C1q, autoimmunity and apoptosis. Immunobiology. 2002;205:395-406. 19. Botto M, Kirschfink M, Macor P, Pickering MC, Wurzner R, Tedesco F. Complement in human diseases: lessons from complement deficiencies. Mol Immunol. 2009;46:2774-83. 20. Flierman R, Daha MR. Pathogenic role of anti-C1q autoantibodies in the development of lupus nephritis-a hypothesis. Mol Immunol. 2007;44:133-8. 21. Mosaad YM, Hammad A, Fawzy Z, El-Refaaey A, Tawhid Z, Hammad EM, et al. C1q rs292001 polymorphism and C1q antibodies in juvenile lupus and their relation to lupus nephritis. Clin Exp Immunol. 2015;182:23-34. 22. Goulielmos GN, Samonis G, Apergi M, Christofaki M, Valachis A, Zervou MI, et al. C1q but not mannose-binding lectin (Mbl2) gene polymorphisms are associated with type 2 diabetes in the genetically homogeneous population of the island of Crete in Greece. Hum Immunol. 2013;74:878-81. 23. Trouw LA, Daha N, Kurreeman FA, Böhringer S, Goulielmos GN, Westra HJ, et al. Genetic variants in the region of the C1q genes are associated with rheumatoid arthritis. Clin Exp Immunol. 2013;173(1):76-83. 24. Walport MJ. Complement and systemic lupus erythematosus. Arthritis Res. 2002;4:S279-93.

7. Kavvoura FK, Akamizu T, Awata T, Ban Y, Chistiakov DA, Frydecka I, et al. Cytotoxic T-lymphocyte associated antigen 4 gene polymorphisms and autoimmune thyroid disease: a meta-analysis. J Clin Endocrinol Metab. 2007;92:3162-70.

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8. Heward JM, Brand OJ, Barrett JC, Carr-Smith JD, Franklyn JA, Gough SC. Association of PTPN22 haplotypes with Graves’ disease. J Clin Endocrinol Metab. 2007;92:685-90.

26. Warren J, Mastroeni P, Dougan G, Noursadeghi M, Cohen J, Walport MJ, et al. Increased susceptibility of C1q-deficient mice to Salmonella enterica serovarTyphimurium infection. Infect Immun. 2002;70:551-7.

9. Ban Y, Tozaki T, Taniyama M, Tomita M, Ban Y. Association of a C/T single-nucleotide polymorphism in the 5’ untranslated region of the CD40 gene with Graves’ disease in Japanese. Thyroid. 2006;16:443-6.

27. Potlukova E, Jiskra J, Limanova Z, Kralikova P, Smutek D, Mareckova H, et al. Autoantibodies against complement C1q correlate with the thyroid function in patients with autoimmune thyroid disease. Clin Exp Immunol. 2008;153:96-101.

10. Yan N, Yu YL, Yang J, Qin Q, Zhu YF, Wang X, et al. Association of interleukin-17A and -17F gene single-nucleotide polymorphisms with autoimmune thyroid diseases. Autoimmunity. 2012;45:533-9.

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original article

Correlation between parameters of self-monitoring of blood glucose and the perception of healthrelated quality of life in patients with type 1 diabetes mellitus Juliana Santos Paula1, Letícia Dinis Braga1, Rodrigo Oliveira Moreira1, Rosane Kupfer1

ABSTRACT Objective: The aim of this study was to evaluate how different parameters of short-term glycemic control would correlate with the perception of health-related quality of life (HRQoL) in patients with type 1 diabetes mellitus (T1D). Subjects and methods: A total of 50 T1D patients aged 18 to 50 years were evaluated with the questionnaires Problem Areas in Diabetes (PAID) scale and Diabetes Quality of Life (DQOL) measure after 30 days of self-monitoring of blood glucose (SMBG). Glycemic control was evaluated using glycated hemoglobin (HbA1c), mean glucose levels (MGL) in the prior month’s data from SMBG (Accu-Check 360o), number of hypoglycemic episodes (< 70 mg/dL and < 50 mg/dL), and glycemic variability (GV). Results: PAID correlated positively with MGL (r = 0.52; p < 0.001) and HbA1c (r = 0.36; p < 0.0097), but not with GV (r = 0.17; p = 0.23) or number of hypoglycemic episodes (r = 0.15; p = 0.17 for glucose < 70 mg/dL and r = 0.02; p = 0.85 for glucose < 50 mg/dL). After multiple linear regression, only MGL remained independently related to PAID scores. DQOL scores had a positive correlation with MGL (r = 0.45; p = 0.001), but not with HbA1c (r = 0.23; p = 0.09), GV (r = 0.20; p = 0.16), or number of hypoglycemic episodes (r = 0.06 p = 0.68). Conclusion: In T1D patients, MGL, but not HbA1c or number hypoglycemic episodes, was the glycemic control parameter that best correlated with short-term perception of HRQoL. Arch Endocrinol Metab. 2017;61(3):343-7.

Serviço de Diabetes, Instituto Estadual de Diabetes e Endocrinologia Luiz Capriglione (IEDE), Rio de Janeiro, RJ, Brasil 1

Correspondence to: Rodrigo Oliveira Moreira Instituto Estadual de Diabetes e Endocrinologia Rua Moncorvo Filho, 90 20211-340 – Rio de Janeiro, RJ, Brasil rom_br@yahoo.com Received on Jan/11//2016 Accepted on Aug/10/2016 DOI: 10.1590/2359-3997000000222

INTRODUCTION

ype 1 diabetes mellitus (T1D) is a chronic disease with high rates of morbidity and mortality. It comprises around 5 to 10% of all cases of diabetes mellitus (DM) (1), and according to estimates from the International Diabetes Federation, 86,000 new patients are diagnosed with T1D every year (2). From the moment of diagnosis, managing DM places a huge burden on the patient, with serious limitations on and changes in lifestyle (3). Treatment consists of multiple injections of insulin, self-monitoring of glucose levels, balanced diet, and regular physical activity. Moreover, patients with DM live in fear of future complications, risk of hypoglycemia, and premature death. Health-related quality of life (HRQoL) is an important health indicator. It is a subjective and multidimensional concept that encompasses a set of interrelated factors, including physical, functional, Arch Endocrinol Metab. 2017;61/4

psychological, and religious aspects (4). Evaluation of HRQoL in patients with chronic diseases and its important relationship with therapeutic management are becoming more valued and recognized as a significant area of scientific knowledge (5). The importance of HRQoL in patients with chronic diseases like DM is such that it has not only been considered a significant predictor of health outcomes, it is now considered a significant health outcome itself (6,7). It is very interesting to speculate how different aspects related to T1D and its treatment would influence the perception of HRQoL in these patients. On one side, recurrent episodes of hypoglycemia and the fear of these episodes have already been demonstrated to impair HRQoL (8). On the other side, hyperglycemia and the fear or microvascular complications have also been related to a worse HRQoL (9,10). However, it is not clear which of these aspects of T1D treatment would more significantly impact the HRQoL of these patients. 343

Keywords Type 1 diabetes mellitus; hypoglycemia; self-monitoring of blood glucose; quality of life

Self-monitoring of blood glucose and quality of life

Our study’s objective was to evaluate how different parameters of short-term glycemic control would correlate with the perception of HRQoL in patients with T1D. The main hypothesis of our study was that the perception of a good glycemic control through self-monitoring of blood glucose (SMBG) might be more important than HbA1c measurement and/ or hypoglycemic episodes in a sample of patients with low socioeconomic status. In this population, the perception of good daily glucose control may be more easily interpreted than biochemical parameters of glycemic control (i.e., HbA1c).

SUBJECTS AND METHODS Study population This was a prospective study involving 50 consecutive outpatient subjects with T1D, aged between 15 and 50 years, performed at the Instituto Estadual de Diabetes e Endocrinologia (IEDE), Brazil, between May and October 2013. Patients were included if they had more than 1 year of diagnosis and were under treatment with a basal-bolus insulin regimen (multiple insulin injections). Patients with type 2 DM (T2D), with serious or limiting complications from DM, end-stage kidney disease, chronic hepatic insufficiency, depression or anxiety, and pregnant women were excluded. Patients who did not have sufficient understanding to fill out the questionnaires or who refused to sign the Informed Consent Form (ICF) were also excluded. The Ethics Committee approved the study’s protocol. All patients read, understood, and signed the ICF form before undergoing clinical evaluation.

Evaluation of glycemic control and quality of life After the initial evaluation, the patients were instructed to perform for the following 30 days at least five measurements of glucose levels a day, before the main meals and when symptoms of hypoglycemia were present. Accu-Check Active was used for the measurements, and data were analyzed using the AccuCheck 360o software. The following parameters were evaluated: mean glucose levels (MGL) and standard deviation (SD), and glycemic variation based on the coefficient of variation (CV) and the number of episodes of hypoglycemia < 70 mg/dL and < 50 mg/dL. Glycemic variability (GV) was also calculated with the formula for the CV = (100 X SD)/mean glucose. 344

After 30 days, glycemic control was evaluated with glycated hemoglobin (HbA1c), which was measured using the high-performance liquid chromatography method. In addition, the participants filled out both questionnaires described below. The Problem Areas in Diabetes (PAID) scale (11) is a specific measurement of psychosocial adjustment to DM. It consists of 20 items covering common problematic situations and negative emotional aspects commonly experienced by these patients. The authors of this questionnaire designed it to evaluate the subject’s emotional understanding of DM. Scores range from 0 to 100. The Diabetes Quality of Life (DQOL) measure (12) consists of 44 items and contains subscales covering five different areas: satisfaction with the treatment, impact, worries about future diabetes complications, worries about the social and vocational aspects of the disease, and general well-being. The total result of the responses for each item of the subscales was calculated. In both questionnaires, a higher result indicates a lower HRQoL.

Statistical analysis The statistical analysis was performed with GraphPad InStat 3.00 (GraphPad Software, San Diego, CA, USA). The Kolmogorov-Smirnov test was used to determine parametric and nonparametric variables. According to the test, age, MGL, and HbA1c levels were considered nonparametric. Spearman test was used for correlation analysis of nonparametric variables and Pearson test for parametric variables. Multiple linear regression was used to identify independent variables of glycemic control that could be related to HRQoL. The level of statistical significance was 5%.

RESULTS Of the 50 patients evaluated, 25 were male. The mean age of the sample was 36.8 ± 11.3 years. No significant differences were observed between genders, and the mean monthly family income (in number of minimum wages) was 2.4 ± 2.3. The patients performed a mean of 131.4 ± 28.4 blood glucose tests during the 30-day period. The HbA1c levels of the sample ranged from 5.8 to 12.9%, with a median of 7.5% (mean = 7.6 ± 1.4%), while MGL ranged from 108.1 to 274.4 mg/dL, with a median of 145.8 mg/dL. The mean CV of the sample was 49.7 ± 8.0. Of the 50 patients, 49 had at least one Arch Endocrinol Metab. 2017;61/4

Self-monitoring of blood glucose and quality of life

80 70 60 PAID

50 40 30 20 10 0

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Figure 1. Correlation between Problem Areas in Diabetes (PAID) scores and mean glucose levels (mg/dL). Increased PAID scores indicate a worse perception of quality of life (r = 0.52; p < 0.0001). 210 180

DQOL

150 120 90 60 30 0

130

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Figure 2. Correlation between the values for the Diabetes Quality of Life (DQOL) measure and mean glucose levels (mg/dL). Increased DQOL scores indicate a worse perception of quality of life (r = 0.45; p = 0.001). Arch Endocrinol Metab. 2017;61/4

DISCUSSION The diagnosis of DM is associated with several modifications in the patient’s daily life. The fear of diabetes complications, as well as the fear of hypoglycemia, may be related to a significant worsening of the HRQoL. We aimed at investigating which parameter of glycemic control was associated with a worse perception of HRQoL. Our main finding was that MGL, but not HbA1c levels or number of hypoglycemic episodes, was the only indicator independently associated with a worse perception of HRQoL. Different studies have already described an association between higher levels of HbA1c and a substantially lower HRQoL (13-17), with higher scores on the PAID (18). Our study did not find an independent correlation between HRQoL and HbA1c levels. One hypothesis to explain this difference could be that, in populations of low socioeconomic status, it may not be so simple for patients to understand the concept of HbA1c. Other studies have explored the relationship between HbA1c and specific mediators of HRQoL in diabetes, and as with our findings, they found such a relationship to be inconsistent or nonexistent (18,19). In our study, higher scores on the DQOL and PAID correlated with high blood glucose levels. Another Brazilian study yielded similar results, showing evidence of a direct relationship between higher glycemic levels in patients who were more dissatisfied with T1D (20). One possible explanation would be that the patients felt uncomfortable when visualizing high glucose levels since hyperglycemia is well known to be associated with complications of the disease. It seems that MGL are more easily interpreted than HbA1c levels as an indicator of glycemic control. We estimated that the main isolated predictor of a reduction in HRQoL in these patients was the appearance of complications arising from long-term hyperglycemia (21,22). We should stress that, by using the term “predictor” to describe any aspect related to HRQoL, we are considering it as an outcome. However, the causal relationship between these variables remains unclear. HRQoL can affect a patient’s perception of the disease, its management, behavior related to self-care, metabolic control and the incidence of complications; all these variables, in turn, can affect the HRQoL (23,24). One can also speculate that hyperglycemia is directly associated with severe symptoms. Although this seems a plausible hypothesis, the vast majority of the patients in this study (42 345

hypoglycemic episode (glucose levels < 70 mg/dL). The mean number of hypoglycemic episodes was 16.5 ± 10.3, ranging from 1 to 38 episodes. Correlation analysis was used to correlate glycemic control parameters and PAID scores. PAID scores correlated positively with MGL (r = 0.52; p < 0.001; Figure 1) and HbA1c levels (r = 0.36; p < 0.0097), but not with GV (r = 0.17; p = 0.23) or the number of hypoglycemic episodes (r = 0.15; p = 0.17 for glucose < 70 mg/dL and r = 0.02; p = 0.85 for glucose < 50 mg/dL). After multiple linear regression (using MGL and HbA1c as independent variables), only MGL remained independently related to PAID (t ratio = 2.769; p = 0.008). Similarly, we also evaluated the correlation between the parameters for glycemic control and the DQOL. We found a statistically significant positive correlation between DQOL scores and MGL (r = 0.45; p = 0.001; Figure 2), but not with HbA1c levels (r = 0.23; p = 0.09), CV (r = 0.20; p = 0.16) or number of episodes of hypoglycemia < 70 mg/dL (r = 0.06 p = 0.68) and < 50 mg/dL (r = 0.08; p = 0.57).

Self-monitoring of blood glucose and quality of life

individuals) had MGL below 200 mg/dL, which are usually asymptomatic. Finally, another hypothesis, albeit unlikely, may also help to explain our results. Patients with a worse HRQoL may be less adherent to treatment, which would lead to a worse glycemic control. Prospective and interventional studies are necessary to clarify the causality of this relationship. Two important studies have already been published investigating the HRQoL in a large sample of Brazilian patients with T1D. The first study evaluated a sample of patients younger than those in our study and demonstrated that a better glycemic control could positively impact the health status of individuals with T1D. Interestingly, glycemic control was evaluated using HbA1c levels. Another important finding in this study was that economic status was inversely related to health status (7). A secondary analysis using the same population demonstrated differences in health status in different Brazilian regions; these differences, however, were not explained by HbA1c levels. The authors suggested that additional factors not evaluated in their study could determine the HRQoL of patients with T1D (25). It is worth noticing that SMBG was not evaluated in these studies. Hypoglycemic episodes are another widely studied variable. The fear of these episodes leads to a lack of adherence to the proposed treatment, in an effort to avoid a recurrence, with a consequent compromise in HRQoL (15,26). An Australian study is in line with the results of our research, finding no evidence of a relationship between hypoglycemic episodes and HRQoL (27). It seems reasonable to speculate that, at least in some populations, the occurrence of hypoglycemia may indicate a better perception of glycemic control. Our study has some limitations. First, only a small number of individuals were evaluated. Furthermore, these individuals were evaluated after only 1 month of SMBG. It seems reasonable to speculate that different results would have been achieved with larger populations and longer follow-up. These two important limitations (i.e., sample size and study length) may also have impacted the relationship between hypoglycemia and HRQoL. Previous studies have already demonstrated that hypoglycemia may have a negative impact in the HRQoL (8). It should be pointed out that we did not differentiate symptomatic from asymptomatic episodes, which could be an important factor linking glycemic control and HRQoL. 346

In conclusion, the relationship between HRQoL and parameters of glucose monitoring in patients with T1D is complex. Our study demonstrates a positive correlation between hyperglycemia and a poorer perception of HRQoL in a sample of Brazilian patients with T1D. Interestingly, it seems that MGL had the most significant correlation with the perception of HRQoL, suggesting that daily glucose testing, and not HbA1c measurement, might be used to investigate the impact of different treatment and interventions in the patient’s perception of the disease. Our results suggest that, when treating a patient with T1D, physicians should optimize glycemic control not only to prevent diabetic complications, but also to improve the patient’s HRQoL. Moreover, more attention should be given to HRQoL in order to optimize glycemic control. Finally, it also indicates that SMBG may be an important tool in patients’ education and may be a simple way to demonstrate the efficacy of the treatment to the patients. Acknowledgments: we acknowledge all physicians from the Diabetes Department of the Instituto Estadual de Diabetes e Endocrinologia, who helped us identifying eligible patients for the study. Funding: no funding was received. Disclosure: Rodrigo O. Moreira has received speaker honorarium from Novartis Pharmaceutics, Novo Nordisk, Merck Serono, Sanofi-Aventis, Ache, and Eli Lilly. Rosane Kupfer has received speaker honorarium from Novartis, Novo Nordisk, Janssen-Cilag, and Sanofi-Aventis. Juliana S. Paula is an employee of Novo Nordisk, Brazil. Leticia D. Braga declares that she has no conflict of interest.

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randomized, prospective, crossover, open clinical trial. Health Qual Life Outcomes. 2015;13:77.

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347

original article

Substernal goiter and laryngopharyngeal reflux Mariana Gonçalves Rodrigues1, Vergilius José Furtado de Araujo Filho2, Leandro Luongo de Matos2, Flávio Carneiro Hojaij3, Cesar Augusto Simões2, Vergilius José Furtado de Araujo Neto2, Daniel Marin Ramos2, Renata Lorencetti Mahmoud2, Letícia de Moraes Mosca2, Gustavo Borges Manta2, Erivelto Martinho Volpi2, Lenine Garcia Brandão2, Claudio Roberto Cernea2

ABSTRACT Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, SP, Brasil 2 Disciplina de Cirurgia de Cabeça e Pescoço do Hospital das Clínicas da FMUSP, São Paulo, SP, Brasil 3 Departamento de Anatomia da FMUSP, São Paulo, SP, Brasil 1

Correspondence to: Mariana Gonçalves Rodrigues Rua Pedro Doll, 310, ap. 62 02404-000 – São Paulo, SP, Brasil mariana.g.rodrigues100@gmail.com Received on Feb/11/2016 Accepted on Dec/23/2016 DOI: 10.1590/2359-3997000000266

Objective: This study aims to compare the prevalence of laryngopharyngeal reflux signs between two groups of patients undergoing thyroidectomy for voluminous goiter: substernal goiters and voluminous cervical goiter without thoracic extension. Subjects and methods: A retrospective case-control study was performed with data retrieved of the charts of the patients submitted to thyroidectomies occurred at a tertiary care center (Head and Neck Surgery Department, University of São Paulo Medical School) between 2010 and 2014. The selected thyroidectomies were allocated in two groups for study: patients with substernal goiters and patients with voluminous cervical goiter without thoracic extension. Cervical goiters were selected by ultrasonography mensuration. Clinical criterion was used to define substernal goiter. Results: The average thyroid volume in patients with substernal goiter was significantly greater than the average volume in patients with only cervical goiter (p < 0.001). The prevalence of signs of reflux laryngitis at laryngoscopy was significantly greater in substernal goiter patients (p = 0.036). Moreover, substernal goiter was considered as the unique independent variable for high reflux laryngitis signs at laryngoscopy (OR = 2.75; CI95%: 1.05-7.20; p = 0.039) when compared to only cervical goiter patients. Conclusion: This study shows a significant association between substernal goiters and signs of laryngopharyngeal reflux at preoperative laryngoscopy. Therefore, when compared with voluminous cervical goiters, the substernal goiters increase the chance of reflux laryngitis signs in patients. Arch Endocrinol Metab. 2017;61(3):348-53. Keywords Substernal goiter; intrathoracic goiter; laryngopharyngeal reflux; gastroesophageal reflux; laryngitis

INTRODUCTION

ubsternal goiter (SG) is the one whose extension exceeds the sternal notch towards the mediastinum. The diagnosis of substernal goiter depends on both clinical and radiological criteria (1,2). The published incidence of substernal goiter varies from 5% to 20%, probably due to a lack of a definitive definition (2). The symptoms and signs associated with a goiter depend on the size and location of the goiter (1), so direct compression of the goiter (mainly if substernal) against the trachea and the esophagus may be responsible for progressive dysphagia for solids, positional dyspnea and dysphonia. Surgery is the treatment of choice for SG (1-3) and requires proper planning. Complications related to surgery for substernal goiter are essentially similar to those of cervical goiter, such as bleeding, recurrent laryngeal nerve injury, hoarseness, and temporary or permanent hypoparathyroidism (1). 348

Systematically preoperative vocal folds examination is recommended (1,2,4-6), because the quality and the functionality of the voice can suffer damage by both the compression of recurrent laryngeal nerve by the SG and the surgery itself (1). In additional, Holler and Anderson (7) recommend that all patients presenting with compressive thyroid symptoms should be evaluated for laryngopharyngeal reflux prior to performing a thyroidectomy, due to the association between these manifestations. Laryngopharyngeal reflux (LPR) is an upperesophageal manifestation of the gastroesophageal reflux disease (GERD), and is also a syndrome associated with symptoms including laryngitis, hoarse voice, chronic cough, and other complaints (8). LPR is believed to be caused by retrograde flow of gastric contents (particularly acid and pepsin) that affect pharyngeal and laryngeal mucosa by direct contact or as a physicoArch Endocrinol Metab. 2017;61/4

Substernal goiter, laryngopharyngeal reflux

SUBJECTS AND METHODS A retrospective case-control study approved by the institutional IRB (number 712/2011) was performed with data retrieved of the charts of the patients submitted to consecutive thyroidectomies occurred at a tertiary care center (Head and Neck Surgery Department, University of São Paulo Medical School) between 2010 and 2014. The selected patients were allocated in two groups for study: GROUP 1 consists of patients with SG, and GROUP 2 consists of patients with CG. Cervical goiters were selected by ultrasonography mensuration. At our Institution, clinical criterion is resorted for substernal goiter diagnosis (caudal extension of thyroid gland below the clavicles or sternal notch on physical examination), while radiological imaging tests determine mediastinal extension of substernal goiter. Goiters with volume at least 40 cc (around 2.5-fold the normal volume of the thyroid gland) were considered voluminous by our service. Patients in antireflux therapy were not selected for this study. Arch Endocrinol Metab. 2017;61/4

Fifty-two patients were selected for Group 1 (SG) and 61 for Group 2 (CG). The following parameters were studied from collected data: demographic data, thyroid function, preoperative compressive symptoms, thyroid gland volume measured by preoperative ultrasonography, vocal fold mobility and suggestive signs of reflux laryngitis in preoperative laryngoscopy (arytenoid hyperemia, laryngeal mucosa edema and vocal folds’ edema). All laryngoscopies were performed by the same medical staff, using flexible laryngoscope Welch Allyn® 3.4 mm with white light. The focus of vocal folds examination was to evaluate their functionality and identify possible injuries. Suggestive signs of reflux laryngitis were considered positive when were identified arytenoid hyperemia, laryngeal mucosa edema and vocal folds edema (posterior laryngitis).

Statistical analysis Distributions were defined as parametric by KolmogorovSmirnov test. The values obtained from the study of each parametric quantitative variable was organized and expressed as mean and standard deviation. Absolute and relative frequencies were used for qualitative variables. Frequency comparisons of a phenomenon between groups of qualitative variables were performed with application of chi-square test or Fisher’s exact test. Student’s t test was used for comparisons between the averages of two qualitative variables. The variables that present p < 0.2 in the univariate analyses were eligible for the multivariate analysis, in which the odds ratio (OR) and confidence interval 95% (CI95%) were calculated using logistic regression. In all analyzes the statistical program SPSS® version 17.0 (SPSS Inc.®, Illinois, USA) was used and in all comparisons was adopted statistical significance level of less than 5% (p ≤ 0.05).

RESULTS Female patients predominated in both groups. There were 45 women in Group 1 and 55 in Group 2. Mean age was also similar between groups, and the sixth decade predominated in both of them. The groups were considered homogeneous and suitable to comparison (Table 1). On preoperative period, 44 patients in Group 1 reported respiratory and/or digestive compressive symptoms, while 47 patients in Group 2 complained of these symptoms on the same period. The average thyroid volume of Group 1 was significantly higher 349

chemical stimulus for laryngeal spasm reflex mediated by the upper laryngeal, recurrent laryngeal and vagus nerves (9-12). The current gold-standard diagnostic test for LPR is dual-probe 24-hour pH monitoring or suggestive laryngoscopy associated with symptoms (9), whereas for GERD it is upper gastrointestinal endoscopy (UGIE) (8). Most patients with LPR do not exhibit GERD typical symptoms (regurgitation and heartburn), because laryngeal epithelium is more sensitive to gastric reflux than esophagus epithelium (13). As laryngeal mucosa is more sensitive, the frequency of reflux episodes and the reflowed volume of gastric content required to produce symptoms are much lower in the LPR when compared to GERD (9,13). Prevalence of LPR symptoms in patients with laryngeal diseases is considered as high as in general population (14,15). Literature concerning goiter and laryngopharyngeal reflux is rare and presents poor or none substernal goiter series (7,16). Most of the published studies fails to achieve statistical significance to assert that the volume of thyroid interferes with the incidence of LPR. This study aims to compare the prevalence of laryngopharyngeal reflux signs between two groups of patients undergoing thyroidectomy for voluminous goiter: substernal goiters (SG) and voluminous cervical goiter (CG) without thoracic extension.

Substernal goiter, laryngopharyngeal reflux

than the other group’s average (respectively, 163.0 vs. 93.4 cc; p < 0.001 – Student’s t test). Suggestive reflux laryngitis signs diagnosed by preoperative laryngoscopy were also significantly higher in Group 1 (41.5% vs. 20.5%, respectively; p = 0.036). Thyroid function, measured by preoperative TSH value, was similar between the two groups. The results concerning autoantibody research, preoperative cytology, use of medication, as well as details about ultrasonography and laryngoscopy could also be found in Table 1.

In order to evaluate a possible confounding bias (the group with substernal goiter has higher reflux laryngitis signs but patients also had greater volume goiter), the goiter’s volume was stratified by the mean of the totality of patients (124.0 cc) and a multivariate analysis was performed (Table 2). Substernal goiter was considered as the unique independent variable for high reflux laryngitis signs at laryngoscopy (OR = 2.75; CI95%: 1.05-7.20; p = 0.039) when compared to only cervical goiter patients.

Table 1. Descriptive data of preoperative thyroidectomy patients with substernal or cervical goiter Variable

Substernal goiter

Cervical goiter

P value

Age**

57.7 ± 11.2 years

51.6 ± 12.6 years

0.100*

Female

87.8%

90.2%

0.687*

Positive anti-thyroid peroxidase

6.1%

17.0%

0.117†

Positive anti-thyroglobulin

12.2%

18.8%

0.376*

Positive TRAB

4.1%

12.2%

0.239†

Preoperative cytology Bethesda I Bethesda II Bethesda III Bethesda IV Bethesda V

2.6% 81.6% 10.6% 5.2% -

1.8% 87.4% 7.2% 1.8% 1.8%

0.853*

Medication Levothyroxine Antithyroid drugs

5.9% 15.4%

15.4% 15.4%

0.403*

Nodules (ultrasonography) Unilateral Bilateral

22.9% 77.1%

14.6% 85.4%

0.296*

163.0 ± 103.6

93,4 ± 61.1

< 0.001#

Compressive symptoms

85.7%

78.3%

0.322*

Laryngopharyngeal reflux signs in pre-operative laryngoscopy

41.5%

20.5%

0.036*

Abnormal vocal fold mobility in pre-operative laryngoscopy

5.0%

11.4%

0.437†

1.31 ± 1.20

114 ± 1.08

0.444#

Goiter volume, cc (ultrasonography)**

Pre-operative TSH (μU/mL)** * Chi-square test; ** Mean ± standard deviation; † Fisher’s exact test; # Student’s t test.

Table 2. Multivariate analysis for predictive factors for reflux laryngitis signs at laryngoscopy in patients with voluminous goiter

Variable

Odds ratio

Confidence interval 95%

P value*

Goiter volume ≤ 124.0 cc > 124.0 cc

1.00 (reference) 2.17

0.81 – 5.84

0.123

Patient’s groups Cervical goiter Substernal goiter

1.00 (reference) 2.75

1.05 – 7.20

0.039

* Logistic regression.

350

Arch Endocrinol Metab. 2017;61/4

DISCUSSION The relationship between thyroid gland diseases and LPR is a recent subject in Head and Neck Surgery literature. This study shows a significant association between substernal goiters and LPR signs in preoperative laryngoscopy. Accordingly, when compared with large cervical goiters, substernal goiters provide a greater chance of signs of reflux laryngitis. To the best of our knowledge, this is the first study to present this relationship with a statistical significance level. Although some authors (7,17) do report reflux laryngitis or LPR series when studying thyroid goiter, they are considered as concomitant diseases, in disagreement with the present findings. For instance, Fiorentino and cols. (17) attribute persistence of dysphagia and dysphonia after thyroidectomy to reflux disease, and concluded that both LPR and goiter compression to anatomical structures could cause such symptoms. Nam and cols. (6) asserted that 35.8% of their preoperative thyroidectomy patients had some laryngeal disease diagnosed by laryngoscopy before surgery, of which 76% had LPR. Such results contribute to LPR relevance in patients undergoing thyroidectomy. In agreement with Nam and cols., LPR occurrence is relevant in both Groups studied; however patients with substernal goiter presented significantly more to have signs of that condition. Hamdan and cols. (16) found a positive relationship between thyroid goiter and LPR, and also that patients with nodular goiters were more likely to present LPR than patients with diffuse goiter, corroborating the findings of our study. LPR symptoms may be present in 33.9% of patients undergoing thyroidectomy (7), consistent with general population prevalence (8). Nevertheless, among more symptomatic patients undergoing thyroidectomy, 68.4% of those who complain of dysphonia and 56% of those who complain with dysphagia have LPR signs and symptoms (7). This increase in percentage of symptomatic individuals was attributed to reflux by Holler and Anderson, which is an acceptable statement. Whereas LPR diagnosis may be based on suggestive laryngoscopy associated with symptoms (9), Holler and Anderson paper and the present one reinforce the association between LPR and thyroid disease, since the first paper studied symptomatic patients, while the present one valued laryngoscopic signs of LPR. Given Arch Endocrinol Metab. 2017;61/4

the fact that the aim of Holler and Anderson’s study, obviously, was not the same as ours, it is possible to point out some differences between them. The first study has no SG sample and has lower thyroid mean volume (38.7 cc vs 163.0 cc for Group 1 and 93.4 cc for Group 2). This series thereby refutes the assumption that goiter and LPR are always concomitants by coincidence, owing to the fact that intrathoracic extension of thyroid gland or its higher volume are significantly more associated with LPR than voluminous goiters restrict to the neck. Usually the LPR is a chronic condition resulting from extra-esophageal repeated exposure to gastric reflux (10). Since the key symptoms of LPR are nonspecific, many patients undergo laryngoscopy (18). Once cancer is ruled out, diagnosis is given when there are erythema, edema, ventricular obliteration, postcricoid hyperplasia and pseudosulcus (18). Laryngoscopy and monitoring pH are the methods used for the diagnosis of LPR (13), though their real specificity and sensitivity values have been interrogated (18). Traditionally, successful empirical treatment with proton pump inhibitors is regarded as a diagnostic method (10); however this method has its limitations and is also questioned by the literature (18,19). In this study, the criteria used to diagnose “reflux laryngitis disease” were the following laryngoscopic findings: arytenoid hyperemia and laryngeal mucosa and the vocal folds edema (posterior laryngitis). Although such signs are very suggestive of reflux laryngitis, other hypotheses must be considered once laryngitis is identified, such as: post-nasal drip, sinusitis, allergies, pulmonary diseases, autoimmune diseases, granulomatous diseases and Reinke’s edema (10,11,18). Once the differential diagnostics have been mentioned, it is important to reinforce that both samples of patients were homogeneous in the present study, so even if some cases assumed as “reflux laryngitis” by examiner belong to other etiologies, their distribution among groups would be hypothetically homogeneous, therefore the findings statistical validity is preserved. Taking into account that laryngopharyngeal mucosa is more sensitive than the esophageal; fewer exposures to contents of gastric reflux would be enough to cause upper-esophageal symptoms of reflux (9,13). Erickson and Sivasankar (20) stated that less than three reflux episodes per week could damage vocal folds mucosa, while zero to 50 reflux episodes per day are considered normal for distal esophagus. For that reason, it is 351

Substernal goiter, laryngopharyngeal reflux

reasonable to assume that whether esophagus peristalsis is impaired, an early manifestation of reflux could occur in upper-esophageal region, such as LPR. The authors of this study believe that higher incidence of signs of reflux laryngitis in substernal goiters group may have an anatomical explanation. It is known that esophagus is innervated by both sympathetic and parasympathetic nerves. The parasympathetics control the peristalsis through the vagus nerve. The nerve fibers to striated muscles depart from the vagus in the upper part of the neck as the recurrent laryngeal nerve. In an extreme situation, a bilateral vagotomy above the origin of the pharyngoesophageal branches could cause abolishment of cervical esophagus peristalsis (21). The vagus nerve also produces excitatory and inhibitory stimuli in the lower esophageal sphincter (21). Therefore, esophageal peristalsis could be impaired if, somehow, the vagus nerve function was compromised. Theoretically, the vagus function could be altered if thyroid gland compresses the nerve against an anatomical structure (the first rib for example), while the inferior pole of the goiter expands and migrates toward thorax (Figure 1). Another plausible hypothesis is that the direct compression of a voluminous goiter against digestive tract would cause an esophageal narrowing and so more food residue would accumulate in the upper part of the esophagus. When esophageal wall compliance is decreased, resistance to the movement of the bolus is increased, and a liquid bolus may flow back up through the ineffective contraction wave (21), accordingly

Figure 1. Preoperative CT of a patient with SG, showing a possible compression of the vagus nerve against the first rib. 352

a substernal goiter could alter esophagus function. Literature (22) also discloses that small physical stimuli in the hypopharynx can cause transient relaxation of the lower esophageal sphincter and thus enable the occurrence of reflux. Hypothetically, the largest food residue in upper-esophagus, secondary to goiter’s compression, could stimulate the hypopharynx and cause episodes of gastroesophageal reflux. As the average SG volume is higher than voluminous CG, it is expected that SG is more likely to cause esophageal stricture. Evidently, these hypotheses have not a practical experimental support yet. The authors report those inferences from their surgical experience and theoretical study of cervical and thoracic anatomy. Moreover, the present series has substernal goiters up to 266.6 cc, which can cause direct compressive symptoms in different cervical structures such as the trachea (23,24), and the superior vena cava (25). Several studies (7,17) recommend treatment of reflux before thyroid surgical approach, because whether the symptoms of cough, hoarseness and dysphagia were due to GERD, an indication of thyroidectomy can be reviewed depending on the original disease of the patient. As far as we know, this study is the first in the literature to show a direct relationship between the extent of intrathoracic goiter and higher incidence of LPR. It is important to state some limitations regarding the present study. For instance, ultrasonography was used to evaluate goiter volume in both groups, but it is known that the intrathoracic portion could be underestimated by this method. Although laryngoscopies were not performed by the same person, they were performed by the same medical staff, whose professionals have equal training and the same diagnosis criteria for standardized possible findings, which include LPR signs. Also, in the present study, postoperative laryngoscopies data was not collected, therefore the authors could not analyze a possible change (or even an improvement) of LPR signs after thyroidectomy in patients who had substernal goiter. Neither dual-probe 24-hour pH monitoring nor patients’ symptoms were used for gastroesophageal reflux diagnosis, however, the present study found out indirect signs of LPR, and the occurrence of those signs was compared between the two groups. Also, the authors propose physiopathological explanations for greater occurrence of LPR signs in substernal goiter group. As well as dualprobe 24-hour pH monitoring, suggestive laryngoscopy Arch Endocrinol Metab. 2017;61/4

Substernal goiter, laryngopharyngeal reflux

associated with symptoms is known as another way to diagnose LPR, but the patients studied were not asked about suggestive symptoms. Nevertheless, the authors believe that despite those limitations, their anatomical hypothesis for greater incidence of LPR signs in people with substernal goiter may show a clinical relevance for future studies. Finally, there is also the possibility of LPR may be associated not only to intrathoracic extent of goiter, but also with the thyroid volume. However, to test this hypothesis, it would be required a study design that includes different thyroids sizes, and not only cases of voluminous goiters as analyzed in this study. In conclusion, this study demonstrated that patients with substernal goiters have significantly higher incidence of laryngoscopic signs of laryngopharyngeal reflux compared with voluminous cervical goiter without thoracic extension and these patients must be investigated for this disease and specific treatment should be considered. This fact also creates opportunities for development of studies on the pathophysiology of this condition in patients with voluminous goiters with intrathoracic extension.

6. Nam IC, Bae JS, Shim MR, Hwang YS, Kim MS, Sun DI. The importance of preoperative laryngeal examination before thyroidectomy and the usefulness of a voice questionnaire in screening. World J Surg. 2012;36(2):303-9.

Acknowledgments: the authors thank Maria Carolina Gonçalves Rodrigues for language editing.

15. Koufman JA, Amin MR, Panetti M. Prevalence of reflux in 113 consecutive patients with laryngeal and voice disorders. Otolaryngol Head Neck Surg. 2000;123(4):385-8.

Fundings: this work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp), research grant funding to Mariana Gonçalves Rodrigues.

16. Hamdan AL, Jabbour J, Al Zaghal Z, Azar ST. Goiter and laryngopharyngeal reflux. ISRN Endocrinol. 2012;2012:208958.

Disclosure: no potential conflict of interest relevant to this article was reported.

REFERENCES 1. Chen AY, Bernet VJ, Carty SE, Davies TF, Ganly I, Inabnet WB 3rd, et al.; Surgical Affairs Committee of the American Thyroid Association. American Thyroid Association statement on optimal surgical management of goiter. Thyroid. 2014;24(2):181-9. 2. Shaha AR. Substernal goiter: what is in a definition? Surgery. 2010;147(2):239-40. 3. Raffaelli M, De Crea C, Ronti S, Bellantone R, Lombardi CP. Substernal goiters: incidence, surgical approach, and complications in a tertiary care referral center. Head Neck. 2011;33(10):1420-5. 4. Farrag TY, Samlan RA, Lin FR, Tufano RP. The utility of evaluating true vocal fold motion before thyroid surgery. Laryngoscope. 2006;116(2):235-8.

8. Groome M, Cotton JP, Borland M, McLeod S, Johnston DA, Dillon JF. Prevalence of laryngopharyngeal reflux in a population with gastroesophageal reflux. Laryngoscope. 2007;117(8):1424-8. 9. Hawkshaw MJ, Pebdani P, Sataloff RT. Reflux laryngitis: an update, 2009-2012. J Voice. 2013;27(4):486-94. 10. Kwon YS, Oelschlager BK, Merati AL. Evaluation and treatment of laryngopharyngeal reflux symptoms. Thorac Surg Clin. 2011;21(4):477-87. 11. Hom C, Vaezi MF. Extraesophageal manifestations of gastroesophageal reflux disease. Gastroenterol Clin North Am. 2013;42(1):71-91. 12. Sataloff RT, Hawkshaw MJ, Gupta R. Laryngopharyngeal reflux and voice disorders: an overview on disease mechanisms, treatments, and research advances. Discov Med. 2010;10(52): 213-24. 13. Coca-Pelaz A, Rodrigo JP, Paccagnella D, Takes RP, Rinaldo A, Silver CE, et al. Reflux and aerodigestive tract diseases. Eur Arch Otorhinolaryngol. 2013;270(2):417-23. 14. Kamani T, Penney S, Mitra I, Pothula V. The prevalence of laryngopharyngeal reflux in the English population. Eur Arch Otorhinolaryngol. 2012;269(10):2219-25.

17. Fiorentino E, Cipolla C, Graceffa G, Cusimano A, Cupido F, Lo Re G, et al. Local neck symptoms before and after thyroidectomy: a possible correlation with reflux laryngopharyngitis. Eur Arch Otorhinolaryngol. 2011;268(5):715-20. 18. Barry DW, Vaezi MF. Laryngopharyngeal reflux: more questions than answers. Cleve Clin J Med. 2010;77(5):327-34. 19. Qadeer MA, Phillips CO, Lopez AR, Steward DL, Noordzij JP, Wo JM, et al. Proton pump inhibitor therapy for suspected GERD-related chronic laryngitis: a meta-analysis of randomized controlled trials. Am J Gastroenterol. 2006;101(11):2646-54. 20. Erickson E, Sivasankar M. Simulated reflux decreases vocal fold epithelial barrier resistance. Laryngoscope. 2010;120(8):1569-75. 21. Mashimo H, Goyal RK. Physiology of esophageal motility. GI Motility online. 2006. 22. Mittal RK, Goyal RK. Sphincter mechanisms at the lower end of the esophagus. GI Motility online. 2006. 23. Stang MT, Armstrong MJ, Ogilvie JB, Yip L, McCoy KL, Faber CN, et al. Positional dyspnea and tracheal compression as indications for goiter resection. Arch Surg. 2012;147(7):621-6. 24. Piao M,YuanY, WangY, Feng C. Successful management of trachea stenosis with massive substernal goiter via thacheobronchial stent. J Cardiothorac Surg. 2013;8:212. 25. Schafranski D, Cavalheiro PR, Stival R. Marañón’s Sign. Internal Medicine. 2011;50(15):1619-9. Copyright© AE&M all rights reserved.

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original article

The role of moderate-to-vigorous physical activity in mediating the relationship between central adiposity and immunometabolic profile in postmenopausal women Universidade de São Paulo (USP), São Paulo, SP, Brasil 2 Grupo de Imunometabolismo e Exercício, Departamento de Educação Física, Universidade Estadual Paulista (Unesp), Presidente Prudente, SP, Brasil 3 Centro de Estudos e Laboratório de Avaliação e Prescrição de Atividades Motoras (Celapam), Departamento de Educação Física, Unesp, Presidente Prudente, SP, Brasil 4 Grupo de Estudos em Atividade Física e Saúde, Departamento de Educação Física, Unesp, Presidente Prudente, SP, Brasil 1

Correspondence to: Tiego A. Diniz Departamento de Educação Física, Universidade Estadual Paulista Rua Roberto Simonsen, 305 19060-900 – Presidente Prudente, SP, Brasil tiegodiniz@gmail.com Received on Sep/13/2016 Accepted on Oct/18/2016 DOI: 10.1590/2359-3997000000259

Tiego A. Diniz1,2, Fabricio E. Rossi2, Loreana S. Silveira2, Lucas Melo Neves1, Ana Claudia de Souza Fortaleza3, Diego G. D. Christofaro4, Fabio S. Lira2, Ismael F. Freitas-Junior3

ABSTRACT Objectives: To analyze the role of moderate-to-vigorous physical activity (MVPA) in mediating the relationship between central adiposity and immune and metabolic profile in postmenopausal women. Materials and methods: Cross-sectional study comprising 49 postmenopausal women (aged 59.26 ± 8.32 years) without regular physical exercise practice. Body composition was measured by dualenergy X-ray absorptiometry. Fasting blood samples were collected for assessment of nonesterified fatty acids, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), adiponectin, insulin and estimation of insulin resistance (HOMA-IR). Physical activity level was assessed with an accelerometer (Actigraph GTX3x) and reported as a percentage of time spent in sedentary behavior and MVPA. All analyses were performed using the software SPSS 17.0, with a significance level set at 5%. Results: Sedentary women had a positive relationship between trunk fat and IL-6 (rho = 0.471; p = 0.020), and trunk fat and HOMA-IR (rho = 0.418; p = 0.042). Adiponectin and fat mass (%) were only positively correlated in physically active women (rho = 0.441; p = 0.027). Physically active women with normal trunk fat values presented a 14.7% lower chance of having increased HOMA-IR levels (β [95%CI] = 0.147 [0.027; 0.811]). Conclusions: The practice of sufficient levels of MVPA was a protective factor against immunometabolic disorders in postmenopausal women. Arch Endocrinol Metab. 2017;61(3):354-60. Keywords Postmenopause; insulin resistance; cytokines; adiponectin; adipose tissue; obesity

INTRODUCTION

enopause contributes to changes in body composition, particularly increased central adiposity (1). It is also associated with decreased physical activity levels (2), which can favor the development of obesity (3). In contrast, engaging in recommended (≥ 150 minutes per week) moderate-to-vigorous physical activity (MVPA) (4) has been associated with improved body composition profile, including decreased body fat and increased lean mass (5,6). Obesity is currently considered a public health problem. It is responsible for the development of chronic low-grade inflammation (7), a condition that is characterized by increased plasma endotoxin (i.e., lipopolysaccharide), saturated fatty acids, and proinflammatory factors, which are involved in the 354

development of morbidities such as type 2 diabetes mellitus, hypertension, dyslipidemia and metabolic syndrome, and decreased anti-inflammatory mediators (8,9). Taken together, these comorbidities may increase the likelihood of cardiovascular diseases, one of the leading causes of death worldwide (10). Insufficient levels of physical activity also increase plasma concentrations of inflammatory mediators such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α, and decrease adiponectin levels and insulin sensitivity (11,12). We have previously shown that postmenopausal women with two or more risk factors, including low MVPA levels, are more likely to have metabolic abnormalities such as insulin resistance (13). As aforementioned, central adiposity is one of the main factors responsible for increased proinflammatory mediators, displaying a linear relationship with Arch Endocrinol Metab. 2017;61/4

MVPA in postmenopausal women

MATERIALS AND METHODS Sample This was a cross-sectional study conducted between 2013–2014 in a city with ~220,000 inhabitants and a human development index of 0.846, located in the west part of the state of São Paulo, southeastern Brazil (14). To be included in the present study, the participants had to meet the following criteria: i) be postmenopausal (absence of a menstrual cycle for at least 1 year and follicle-stimulating hormone [FSH] > 30 IU/L) (15); ii) be older than 50 years on the date of the assessment; iii) have not engaged in regular physical exercise (e.g., walking/running, strength training, etc.) for at least 6 months prior to the study; iv) not be receiving hormone replacement treatment; v) not be using drugs such as beta-blockers, statins, etc.; vi) have signed the written informed consent form for study participation. We performed a power analysis for the design of this study, based on the observation from a previous study that verified a correlation between time of physical activity practice after spinal cord injury and HOMA-IR (r = -0.591; p = 0.033) (16). Using a power of 0.80% and a type I error of 0.05, according to Miot (17), the required sample size was estimated at 21 subjects; however, we opted for over-recruitment. A total of 77 women were evaluated, but only 60 met the study inclusion criteria. After assessing the physical activity levels of the participants using accelerometry, we found that 11 of them had not used the device for the prescribed minimum number of days (4 days during the week and 1 day during the weekend). Thus, the final sample selected for the analysis comprised 49 women. All procedures used in this study met the criteria of the 196/96 Resolution on Ethics in Research of the Brazilian National Health Council (Brasília, DF). All participants included in the study signed an informed consent form approved by the Research Ethics Committee at the university linked to the project (Protocol: 64/2011). Arch Endocrinol Metab. 2017;61/4

Data collection

Anthropometry and body composition During anthropometric measurement, all participants wore light clothing and remained barefoot. The height of the subjects was measured using a fixed stadiometer (Sanny, São Bernardo do Campo, São Paulo, Brazil) with an accuracy of 0.1 cm. Body weight was measured using a digital scale (Filizola PL 50, Filizola Ltda., Brazil) with an accuracy of 0.1 kg. Body fat was analyzed with dual-energy X-ray absorptiometry (DXA; Lunar DPX-NT, version 4.7, General Electric Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). Each examination lasted for approximately 15 minutes. During the examination, the participants were positioned in the supine position on the scanner. The values were expressed as a percentage of body fat (%BF), and trunk and abdominal fat, all in kilograms.

Non-exercise physical activity The habitual levels of physical activity of the participants were assessed using a triaxial accelerometer sensor (Actigraph model GT3X, Actigraph LLC, Pensacola, Florida, United States), which recorded the movements in the three orthogonal planes: vertical, horizontal anteroposterior, and horizontal mediolateral. To carry out the measurements, the accelerometers were attached to an elastic tape and placed on the subjects’ waist, above the hip, at the height of the iliac crest on the right side of the body. The participants were required to use the accelerometer for 7 days, and received instructions for using it during all waking hours, except during bathing or aquatic activities such as swimming (5). A specific software (ActiLife5, data analysis software by Actigraph) was used to process the obtained data, and only results obtained during full monitoring days were analyzed. A non-wear time was defined as at least 60 consecutive minutes with zero counts, with an allowance of up to 2 minutes of counts between 0 and 100 (18). A valid day was defined as ≥ 10 hours of monitor wear time, and only participants with ≥ 5 valid days (including at least 1 day during the weekend) were included in the present analyses (19). Raw measurements from the accelerometer were determined as counts, which was an arbitrary measurement: the greater the number of counts, the higher the level of physical activity. Counts from each sample were added over a specific period of 60 seconds, 355

these mediators. However, little is known about the protective role of MVPA in regards to the association of central adiposity, proinflammatory mediators, and metabolic profile in postmenopausal women. Thus, the purpose of the present study was to analyze the role of MVPA in mediating the association between central adiposity and the immune and metabolic profile of postmenopausal women.

T2DM-associated genetic variants and GDM

called an epoch. The period of 60 seconds was chosen for this study population due to the type of physical activity, which is characterized by a low intensity and long duration pattern (20). In an attempt to obtain a biological value and facilitate the interpretation of data (counts per minute [cpm]) provided by the accelerometer, cpms were translated into physical activity minutes. To classify the intensity of the physical activity, we used the recommendations for accelerometers proposed by Freedson and cols. (21). We defined the physical activities as moderate for values between 1952–5724 cpm (from 3.00 to 5.99 METs), as vigorous for values between 5725–9498 cpm (6.00 to 8.99 METs), and as very vigorous for values above 9499 cpm (≥ 9 METs). We also used Freedson cut points to categorize an epoch as sedentary (< 100 cpm). Thereby, the practice of habitual physical activity was expressed in percentage of time in sedentary behavior and MVPA. In addition, women who engaged in less than 150 minutes of MVPA were considered to be sedentary, according to recommendations by the American College of Sports Medicine (ACSM) (4).

Blood collection and inflammatory and metabolic profile After a 12-hour fast, blood samples were collected by nurses in sterile tubes containing heparin, an anticoagulant for blood samples. Triacylglycerol (mg/dL) and FSH levels were processed in a private laboratory in the city where the study was conducted. Nonesterified fatty acids were assessed by a colorimetric method with a commercial kit (ZenBio Inc., Research Triangle Park, North Carolina, United States). Serum insulin, IL-6, TNF-α, and adiponectin were quantified using enzymelinked immunosorbent assay (ELISA) with a commercial kit (RayBio® Human ELISA Kit, Norcross, Georgia, United States), as per the manufacturer’s manual. In addition, the HOMA-IR and HOMA-β were calculated using a previously reported method (22).

Statistical analysis The Kolmogorov-Smirnov test was used to test the normality of the data. Trunk fat groups were compared by independent Student’s t-test and corrected with Levene’s test for equality of variance. The proportion of women meeting the ACSM physical activity recommendation (MVPA ≥ 150 min/week) in each trunk fat group was 356

evaluated with McNemar’s test. Spearman’s correlation (rho) was used to analyze the relationship between levels of physical activity and markers of systemic inflammation. In addition, odds ratios were obtained using binary logistic regression to analyze the magnitude of possible associations between MVPA practice, inflammation markers, and trunk fat. All analyses were performed using the statistical software SPSS (version 17.0). The level of significance was set at 5%.

RESULTS Mean FSH levels were 59.33 ± 23.89 mUI/mL. Table 1 shows the baseline characteristics of the sample dichotomized by central adiposity. As expected, women with increased trunk fat had increased amount of total body fat and higher insulin resistance when compared with those with normal trunk fat. Interestingly, there was no difference between time in sedentary behavior in the trunk fat groups; however, participants in the normal trunk fat group accumulated approximately 20 minutes more than the other group, although the difference did not reach statistical significance. In addition, of a total of 49 postmenopausal women, 25 (51%) were physically active and 24 (49%) were sedentary according to the ACSM. Table 2 shows the relationship between inflammation markers and central adiposity in 49 postmenopausal women. There were positive and significant correlations between trunk fat and both IL-6 and HOMA-IR. We further investigated the relationship between metabolic and inflammatory profiles and central adiposity dichotomized by MVPA level, according to ACSM recommendations (Figure 1). Interestingly, a positive association between proinflammatory cytokines and trunk fat was only found in sedentary women. Moreover, physically active women presented a positive association between percentage of body fat and adiponectin (p = 0.027; data not shown). We also found a positive and moderate correlation between IL-6 and HOMA-IR (rho = 0.550; p < 0.001), and a weak correlation with HOMA-β (rho = 0.296; p = 0.039). When we performed these analyses dichotomizing the participants by MVPA level, we found higher and positive correlation values between IL-6 and HOMA-IR in sedentary women (rho = 0.739; p < 0.001), in addition to moderate and positive but not significant correlation values among active women (rho Arch Endocrinol Metab. 2017;61/4

MVPA in postmenopausal women

Sedentary

1.0 0.5 0.0

Table 1. Baseline characteristics of the sample dichotomized by central adiposity

Sedentary

p-value

Age (years)

59.80 (6.07)

59.07 (5.24)

0.652

FSH (mUI/mL)

61.48 (19.16)

57.38 (27.82)

0.594

Weight (kg)

60.31 (5.72)

79.66 (8.54)

< 0.001

Height (cm)

154.75 (5.15)

157.70 (6.02)

0.073

BMI (kg.m )

25.20 (2.29)

32.07 (3.40)

< 0.001

Body fat (%)

40.16 (4.69)

48.32 (4.31)

< 0.001

Trunk fat (kg)

11.88 (2.97)

20.68 (2.88)

< 0.001

Abdominal fat (kg)

2.05 (0.86)

3.50 (0.61)

< 0.001

NEFA (µM)

192.45 (48.58)

199.64 (59.47)

0.646

TNF-α (pg/mL)

288.51 (108.86)

234.87 (141.31)

0.143

IL-6 (pg/mL)

105.50 (11.70)

108.12 (8.65)

0.375

Fasting plasma insulin (μIU/ml)

11.81 (9.85)

16.51 (13.92)

0.181

HOMA-IR

2.47 (1.94)

4.57 (3.93)

0.022

30 Active

Adiponectin (μg/mL)

0.24 (0.17)

0.30 (0.35)

0.492

Sedentary behavior (hours/week)

118.45 (12.92)

117.78 (15.92)

0.871

MVPA (min/week)

190.62 (133.39)

171.97 (114.28)

0.601

100

MVPA ≥ 150 min/ week (%)

54.2%

48.0%

0.500†

IL-6 (pg/mL)

140 120 100 80

Sedentary

Trunk fat (kg) rho

p-value

Adiponectin (μg/mL)

0.044

0.766

IL-6 (μg/mL)

0.300

0.033

HOMA-IR

0.295

0.040

NEFA (µM)

0.078

0.595

TNF-α (pg/mL)

-0.114

0.437

NEFA: nonesterified fatty acids; TNF-α: tumor necrosis factor-α; IL-6: interleukin-6; HOMA-IR: homeostatic model assessment-insulin resistance; HOMA-β: homeostatic model assessmentβ-cell function.

Active

TNF-α (pg/mL)

10 20 Trunk fat (kg)

Sedentary

Active

TNF-α (pg/mL)

500 400 300 200

10 20 Trunk fat (kg) Sedentary

Active

300 250 200 150 100

10 20 Trunk fat (kg)

Figure 1. Correlations between markers of inflammation and components of body composition dichotomized by the level of physical activity. Sedentary group (n = 24) and physically active group (n = 25). (A) Trunk fat vs. adiponectin: sedentary (rho = -0.044; p = 0.837) and active (rho = 0.176; p = 0.400). (B) Trunk fat vs. IL-6: sedentary (rho = 0.471; p = 0.020) and active (rho = 0.156; p = 0.456). (C) Trunk fat vs. HOMA-IR: sedentary (rho = 0.418; p = 0.042) and active (rho = 0.198; p = 0.342). (D) Trunk fat vs. TNF-α: sedentary (rho = -0.164; p = 0.443) and active (rho = -0.099; p = 0.637). (E) Trunk fat vs. NEFA: sedentary (rho = 0.221; p = 0.300) and active (rho = -0.114; p = 0.588). 357

Table 2. Correlations between markers of inflammation and central adiposity (n = 49)

NEFA (µM)

FSH: follicle stimulating hormone; BMI: body mass index; NEFA: nonesterified fatty acids; TNF-α: tumor necrosis factor-α alpha; IL-6: interleukin-6; HOMA-IR: homeostatic model assessment-insulin resistance; HOMA-β: homeostatic model assessment-β-cell function; MVPA: moderate-to-vigorous physical activity. † McNemar’s test.

20 10 Trunk fat (kg)

Arch Endocrinol Metab. 2017;61/4

10 20 Trunk fat (kg)

160

Increased trunk fat (kg) (Upper median)

-2

Normal trunk fat (kg) (Lower median)

Variable

Active

1.5 Adiponectin (µg/mL)

= 0.339; p = 0.098). Additionally, when we correlated IL-6 and HOMA-β according to MVPA level, we found that sedentary women presented moderate and positive correlation values (rho = 0.494; p = 0.014), whereas active women presented low correlation values (rho = 0.082; p = 0.697) (Figure 2).

T2DM-associated genetic variants and GDM

All

HOMA-IR

PA + NTF

15 10

PI or HTF

5 0 80

100

120

140

160

PI + HTF

Odds Ratio (95%CI)

B PA + NTF

0 80

100

120

140

160

IL-6 (pg/mL) C

1000

0 2.

5 1.

0 1.

0 0.

HOMA-β

PI + HTF

2000 1500

Odds Ratio (95% CI)

500 0 80

100

120 140 IL-6 (pg/mL)

Sedentary

D HOMA-β

PI or HTF

All 2500

160 Active

2500 2000 1500 1000 500 0

Figure 3. Binary logistic regression between inflammation markers, engaging in moderate-to-vigorous physical activity and trunk fat. PA: physically active women; NTF: normal trunk fat; PI: physically inactive women; HTF: higher trunk fat; 95%CI = 95% Confidence Interval.

DISCUSSION 80

100

120 140 IL-6 (pg/mL)

160

Figure 2. Correlations between Interleukin-6 and insulin resistance index. Sedentary group (n = 24) and physically active group (n = 25). (A) Relationship between IL-6 and HOMA-IR (rho = 0.550; p-value < 0.001). (B) Relationship between IL-6 and HOMA-IR dichotomized according to the physical activity level (sedentary: rho = 0.739; p-value < 0.001 and active: rho = 0,339; p-value = 0.098). (C) Relationship between IL-6 and HOMA-β (rho = 0.296; p-value = 0.039). (D) Relationship between IL-6 and HOMA-β dichotomized according to the physical activity level (sedentary: rho = 0.494; p-value = 0.014 and active: rho = 0.082; p-value = 0.697).

Interleukin-6

HOMA-IR

Figure 3 shows a binary logistic regression analysis between inflammation markers, MVPA level, and trunk fat. Participants who were physically active and had normal trunk fat values presented a 14.7% lower chance of having higher HOMA-IR levels (β [95%CI] = 0.147 [0.027; 0.811]). Having no risk factors (increased trunk fat or low physical activity) did not protect against higher IL-6 levels, although this relationship presented a trend towards significance (β [95%CI] = 0.198 [0.033; 1.181]). 358

1. 5

Active

1. 0

Sedentary

0. 5

0. 0

IL-6 (pg/mL)

The novelty of this study is the investigation of the protective factor of sufficient MVPA levels and normal trunk fat amount in the immunometabolic profile of postmenopausal women. We demonstrated that 1) the HOMA-IR and IL-6 were only associated with trunk fat in sedentary women, and 2) the protective effect of MVPA levels and a normal trunk fat amount was observed in association with the HOMA-IR but not with IL-6 concentrations. Sedentary behavior is recognized as one of the most powerful risk factors for metabolic diseases (23,24). It is also known that the lack of physical activity is independently associated with a worse immunometabolic profile (5,25,26), and when this behavior is clustered with obesity, the risk of death increases 7.5 times (23). On the other hand, independently of sex and age, being moderately active results in 26% and 27% lower chance of death in overweight and obese subjects, respectively (23). In the present study, sedentary women showed a significantly positive association between trunk fat, HOMAIR, and IL-6, which may indicate the occurrence of immunometabolic disorders. These findings are in Arch Endocrinol Metab. 2017;61/4

MVPA in postmenopausal women

Arch Endocrinol Metab. 2017;61/4

study. The sample size indicates a need for caution in extrapolating the results, and the cross-sectional design precludes longitudinal considerations and causal inferences. On the other hand, the positive aspects of this study should be highlighted. Perhaps the most notable was the objective measurement of physical activity using triaxial accelerometer, providing a reliable measurement of habitual physical activity and avoiding eventual mistakes presented in subjective self-reported physical activity questionnaires. Furthermore, the estimation of body composition by DXA is a strength of the study, since this is a reliable and highly precise method to assess the body composition of the studied population (35). In summary, our results suggest that sufficient MVPA levels were a protective factor against immunometabolic disorders in postmenopausal women. Therefore, steps must be taken to stimulate the practice of regular MVPA, especially in this population. Acknowledgments: this work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes). Disclosure: no potential conflict of interest relevant to this article was reported.

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2. Lovejoy J, Champagne C, De Jonge L, Xie H, Smith S. Increased visceral fat and decreased energy expenditure during the menopausal transition. Int J Obes. 2008;32:949-58. 3. Fleg JL, Morrell CH, Bos AG, Brant LJ, Talbot LA, Wright JG, et al. Accelerated longitudinal decline of aerobic capacity in healthy older adults. Circulation. 2005;112:674-82. 4. Haskell WL, Lee IM, Pate RR, Powell KE, Blair SN, Franklin BA, et al. Physical activity and public health: updated recommendation for adults from the American College of Sports Medicine and the American Heart Association. Circulation. 2007;116:1081-93. 5. Buonani C, Rosa CS, Diniz TA, Christofaro DG, Monteiro HL, Rossi FE, et al. Physical activity and body composition in menopausal women. Rev Bras Ginecol Obstet. 2013;35:153-8. 6. Diniz TA, Christofaro DG, Santos VR, Viezel J, Buonani C, Rossi FE, et al. Practice of moderate physical activity can attenuate the loss of lean body mass in menopausal women. Motricidade. 2015;11:151-9. 7.

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agreement with those by Lavoie and cols. (27), who demonstrated that physical activity plays an important role in reducing chronic low-grade inflammation and risk of metabolic and cardiovascular diseases. Furthermore, women with more than one risk factor, including low physical activity levels, are more likely to present metabolic disorders (13). In contrast, physically active women presented no association between trunk fat and immunometabolic profile, possibly indicating that these women were protected against immunometabolic disorders. Physically active women also presented a positive association between percentage of body fat and adiponectin, an anti-inflammatory cytokine secreted mainly by adipocytes (28). Although increased body fat attenuates the expression of adiponectin (9), some types of acute physical activity can increase adiponectin levels independently of weight (29-32), possibly indicating that the anti-inflammatory effect of physical activity overcomes the detrimental effects of excessive fat mass. Analyzing odds ratios, we found that women who were physically active and had normal trunk fat values presented a protective profile against high HOMAIR but not IL-6. We also observed that having only one factor, sufficient physical activity levels (MVPA ≥ 150 min/wk) or normal trunk fat, was not enough to carry over the protective effect on immunometabolic disorders. Our results are in line with those by Knudsen and cols. (33), who found that only 3 days of reduction in physical activity levels led to increased insulin concentration and decreased insulin sensitivity. Interestingly, this pattern occurred regardless of the increase in body fat (visceral and abdominal), indicating the powerful harm of sedentary behavior. In contrast, the authors observed no changes in IL-6 levels in these patients. As already demonstrated, a drastic reduction in physical activity can attenuate the peripheral insulin sensitivity by decreasing the phosphorylation and content of Akt, an important downstream protein in insulin signaling and glucose uptake (34). However, the same authors have also found no relationship between a reduction in physical activity and IL-6 (34). The absence of an association between IL-6 and physical activity in these studies might have been due to the fact that a drastic reduction in physical activity was not enough to increase fat mass, which is one of the factors responsible for the IL-6 increase (24). Despite the significance of our findings, it is important to mention some limitations of our

T2DM-associated genetic variants and GDM

10. Rosamond W, Flegal K, Furie K, Go A, Greenlund K, Haase N, et al. Heart disease and stroke statistics--2008 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation. 2008;117:e25-146. 11. Bruunsgaard H. Physical activity and modulation of systemic lowlevel inflammation. J Leukoc Biol. 2005; 78: 819-35. 12. Petersen AM, Pedersen BK. The anti-inflammatory effect of exercise. J Appl Physiol. 2005;98:1154-62. 13. Diniz TA, Rossi FE, Silveira LS, Christofaro DG, Lira FS, Freitas Jr I. Relationship between moderate-to-vigorous physical activity, abdominal fat and immunometabolic markers in postmenopausal women. Eur J Obstet Gynecol Reprod Biol. 2015;194:178-82. 14. IBGE. Demographic census and population counts: Resident population by sex, status and age groups. 2010. Available from: <http://www.ibge.gov.br/home/estatistica/populacao/censo2010/ default.shtm>. Accessed on: Nov. 15, 2014. 15. WHO. Research on the Menopause in the 1990’s. Proceedings of a meeting. Geneva, Switzerland, 14-17 June 1994. Maturitas. 1996;23:109-259. 16. Koury J, Passos M, Figueiredo F, Chain A, Franco J. Time of physical exercise practice after injury in cervical spinal cordinjured men is related to the increase in insulin sensitivity. Spinal Cord. 2013;51:116-9. 17. Miot HA. Sample size in clinical and experimental studies. J Vasc Bras. 2011;10:275-8. 18. Colley R, Connor Gorber S, Tremblay MS. Quality control and data reduction procedures for accelerometry-derived measures of physical activity. Health Rep. 2010;21:63-9. 19. SaundersTJ,Tremblay MS, Mathieu ME, Henderson M, O’Loughlin J, Tremblay A, et al. Associations of sedentary behavior, sedentary bouts and breaks in sedentary time with cardiometabolic risk in children with a family history of obesity. PloS One. 2013; 8:e79143. 20. Trost SG, McIver KL, Pate RR. Conducting accelerometer-based activity assessments in field-based research. Med Sci Sports Exerc. 2005;37:S531-43.

23. Ekelund U, Ward HA, Norat T, Luan J, May AM, Weiderpass E, et al. Physical activity and all-cause mortality across levels of overall and abdominal adiposity in European men and women: the European Prospective Investigation into Cancer and Nutrition Study (EPIC). Am J Clin Nutr. 2015;101:613-21. 24. Pedersen BK. The diseasome of physical inactivity--and the role of myokines in muscle--fat cross talk. J Physiol. 2009;587:5559-68. 25. Allison MA, Jensky NE, Marshall SJ, Bertoni AG, Cushman M. Sedentary behavior and adiposity-associated inflammation: the Multi-Ethnic Study of Atherosclerosis. Am J Prev Med. 2012;42:8-13. 26. Healy GN, Matthews CE, Dunstan DW, Winkler EA, Owen N. Sedentary time and cardio-metabolic biomarkers in US adults: NHANES 2003-06. Eur Heart J. 2011;32:590-7. 27. Lavoie ME, Rabasa-Lhoret R, Doucet E, Mignault D, Messier L, Bastard JP, et al. Association between physical activity energy expenditure and inflammatory markers in sedentary overweight and obese women. Int J Obes. 2010;34:1387-95. 28. Wang ZV, Scherer PE. Adiponectin, cardiovascular function, and hypertension. Hypertension. 2008;51:8-14. 29. Jurimae J, Purge P, Jurimae T. Adiponectin is altered after maximal exercise in highly trained male rowers. Eur J Appl Physiol. 2005;93:502-5. 30. Markofski MM, Carrillo AE, Timmerman KL, Jennings K, Coen PM, Pence BD, et al. Exercise training modifies ghrelin and adiponectin concentrations and is related to inflammation in older adults. J Gerontol A Biol Sci Med Sci. 2014;69:675-81. 31. Simpson KA, Singh MA. Effects of exercise on adiponectin: a systematic review. Obesity. 2008;16:241-56. 32. Varady KA, Bhutani S, Church EC, Phillips SA. Adipokine responses to acute resistance exercise in trained and untrained men. Med Sci Sports Exerc. 2010;42:456-62. 33. Knudsen SH, Hansen LS, Pedersen M, Dejgaard T, Hansen J, Hall GV, et al. Changes in insulin sensitivity precede changes in body composition during 14 days of step reduction combined with overfeeding in healthy young men. J Appl Physiol. 2012;113:7-15. 34. Krogh-Madsen R, Thyfault JP, Broholm C, Mortensen OH, Olsen RH, Mounier R, et al. A 2-wk reduction of ambulatory activity attenuates peripheral insulin sensitivity. J Appl Physiol. 2010;108:1034-40.

22. Lejskova M, Alusik S, Suchanek M, Zecova S, Pitha J. Menopause: clustering of metabolic syndrome components and population changes in insulin resistance. Climacteric. 2011;14:83-91.

35. Woodrow G. Body composition analysis techniques in the aged adult: indications and limitations. Curr Opin Clin Nutr Metab Care. 2009;12:8-14.

21. Freedson PS, Melanson E, Sirard J. Calibration of the Computer Science and Applications, Inc. accelerometer. Med Sci Sports Exerc. 1998;30:777-81.

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Arch Endocrinol Metab. 2017;61/4

original article

Prevalence of pancreatic autoantibodies in non-diabetic patients with autoimmune thyroid disease and its relation to insulin secretion and glucose tolerance Carolina Sallorenzo1, Regina Silva1, Teresa Kasamatsu1, Sérgio Dib1

ABSTRACT Objective: We evaluated the prevalence of glutamic acid decarboxylase (GADA) and tyrosine phosphatase-protein antibodies (IA2A), their titers and their relation to first phase insulin response (FPIR) and glucose tolerance in autoimmune thyroid diseases (ATDs) patients. Subjects and methods: Graves’ disease (GD; n = 181) and Hashimoto’s thyroiditis (HT; n = 143) patients in addition to healthy controls (n = 93) were studied. Secondly, FPIR and oral glucose tolerance tests (OGTT) were performed in 11 anti-pancreatic islet–cell (+) and in 20 anti-pancreatic–cell (-) patients. Results: There was a non significant trend for higher prevalence of GADA positivity in GD vs HT (7.2% vs 2% p = 0.06), but the GADA titers were higher in HT. We also did not find a significant difference in IA2 prevalence (0.7% vs 0.0%) between these two groups or compared to the control group. In the subsequent analysis, low FPIR was found in 10% of these patients but without statistical difference for OGTT between pancreatic antibody–positive and –negative patients. Conclusion: A trend for greater prevalence of GADA was observed for GD patients than for HT or control. However, the titers of these autoantibodies were higher in HT patients, but there was no significant relation to insulin secretion and glucose tolerance at that moment and stage of autoimmune diseases. Arch Endocrinol Metab. 2017;61(3):361-6.

Divisão de Endocrinologia, Departamento de Medicina, Universidade Federal de São Paulo (Unifesp), São Paulo, SP, Brasil 1

Correspondence to: Sérgio Dib Rua Pedro de Toledo, 781 04039032 – São Paulo, SP, Brasil sergio.dib@unifesp.br Received on Dec/31/2015 Accepted on Feb/1/2017 DOI: 10.1590/2359-3997000000280

INTRODUCTION

utoimmune thyroid diseases (ATDs), represented by Graves’ disease (GD) and Hashimoto’s thyroiditis (HT) are very common, affecting up to 9% of population (1). It has been accepted that endocrine autoimmune diseases coexist in the same person and even in the same families. This association can be explained, in part, by a shared genetic background and is mainly linked to the HLA system and the CTLA-4, CD25, PTPN22 and FOXP3 genes (2). The increased risk of an additional autoimmune disease in a subject with a primary diagnosis of ATD can be quantified (3). Risk assessment of developing type 1A diabetes (T1AD) in adults can begin with the evaluation of genetics and autoantibodies to glutamic acid decarboxylase (GADA) and the protein tyrosine phosphatase antigen IA-2 (IA-2A), among other pancreatic autoantibody sera markers for T1AD (4,5). The risk of clinical disease is related to the titers (6,7) Arch Endocrinol Metab. 2017;61/4

and the number of additional autoantibodies (8). However, the decreasing rate of beta-cell function is not homogeneous, and the subjects at risk for T1AD can express these autoantibodies for years prior to disease diagnosis (8). The metabolic abnormalities of the pre-diabetic period are not well understood. The presence of multiple pancreatic autoantibodies and impaired first phase insulin response (FPIR) are known to be strong risk factors to T1AD clinical development (9). Initial abnormalities in oral glucose tolerance tests (OGTTs) are frequently silent and can precede the diagnosis of T1AD (10). Both OGTT and intravenous glucose tolerance test (IVGTT) may also be modulated by insulin resistance (11). The autoimmune process against islet beta-cells in adults is generally slow and can take years before clinical T1AD (12-15) appears. Also, there are reports on GADA patterns in autoimmune polyendocrine syndrome that can be different from those in sporadic T1AD patients. GADA has been detected in 6-13% of 361

Keywords Autoimmune thyroid disease; pancreatic autoantibodies; glucose tolerance; beta-cell function

Pancreatic and thyroid autoimmunity

GD and in 3-8% of HT patients (16-18). In a previous study, we evaluated the frequency of GADA in GD, HT and T1AD Brazilian patients (19) and found that despite the genetic diversity of our population, the frequency of GADA in Brazilians with these diseases is similar to their pairs from other countries. Few studies have addressed the relation between islet-cell autoimmunity and glucose homeostasis in nondiabetic ATD Brazilian patients. Here, we evaluated the prevalence of GADA and IA2A and their relation to first-phase insulin secretion and glucose tolerance in non-diabetic individuals with ATDs.

SUBJECTS AND METHODS Patients were recruited between March 2001 and July 2003. The study cohort (n = 324) was randomized from a southeast Brazilian population attending the outpatient endocrine clinic of a university hospital for follow-up of their ATDs. All were non-diabetic patients. In addition, as a control group, 93 blood donors, healthy subjects without T1AD, ATD or neither autoimmune disease’s first-degree relatives were studied. All subjects gave informed written consent to participate. The study was approved by the Ethics Committee on Human Research of the University Hospital of São Paulo Federal University. GD was defined by the presence of biochemical hyperthyroidism with the following: I) diffuse uptake on a radionuclide scan/diffuse goiter on ultrasound scan; II) Graves’ ophthalmopathy; III) positive autoantibodies to the thyroid-stimulating receptor or IV) diffuse goiter on physical examination HT diagnosed by documented clinical and biochemical hypothyroidism requiring L-thyroxine replacement therapy and the presence of antithyroperoxidase (TPOAb) and antithyroglobulin antibodies (TgAb) and/or histologically by fine-needle biopsy of the thyroid. All patients were treated for thyroid disease and taking the following medicine: levothyroxine sodium, methimazole and radioiodine (I131) therapy. Eleven euthyroid positive and twenty negative GADA and/or IA2A (a subgroup from the main ATD patient group) with a similar age, gender and BMI were submitted to an OGTT. All but one GADA/IA2Ab+ patient (due to technical problems) were submitted to an IVGTT (20) to evaluate the FPIR. Baseline blood samples were obtained for measurements of glucose, insulin, thyroid hormones and autoantibodies. 362

Glucose was measured using the glucose hexokinase II enzymatic method. Serum insulin was measured using a time-resolved fluoroimmunoassay (AutoDelfia Insulin-Perkin Elmer Life Sciences, Finland) with a reference range: 14.04– 158.4 pmol/L; the intra- and inter-assay coefficients of variation (CV) varied from 1.7% (serum insulin: 180.6 pmol/L) to 2.4% (serum insulin: 33.0 pmol/L) and 3.5% to 2.3%, respectively. TPOAb and TgAb were measured in our laboratory using highly purified recombinant human TPO antigen (Fitzgerald Industries International, Inc, Concord, USA) and a human-purified Tg antigen employing flow cytometry (Luminex) (21). The cut-off limit was 14 and 26 UI/ml for TPOAb and TgAb, respectively. TPOAb intra-assay CVs were 14.3% and 12.6% to mean values of 2.3 ± 0.33 and 621 ± 78 UI/ml, respectively, and inter-assay CVs were 33% and 20% to mean values of 1.8 ± 0.6 and 688 ± 139 UI/ml, respectively. Tg Ab intra-assay CVs were 9.2% and 9.6% to mean values of 2.4 ± 0.22 and 811 ± 78 UI/ml, respectively, and interassays of 36% and 19.6% to mean values of 2.3 ± 0.86 and 905 ± 177 UI/ml, respectively. TSH receptor antibodies (TRAb) were determined using a radioreceptor assay with reagents provided by BRAHMS AG (Henninsdorf, Germany). The normal value was < 10%, and the intra and inter-assay CVs were < 10% and 13.4%, respectively. Serum TSH was measured using an immunofluorimetric assay developed in our laboratory (reference ranges: 0.3–5.0 mUI/L; intra- and interassay CVs of 9.5% and 11.6%, respectively). GADA 65 and IA2A were measured from frozen serum samples using a commercial radioimmunoassay kit RSR (UK, England). The results were expressed in U/ml. The cut-off limit for GADA was 1.72 U/ml and was 0.97U/ml for IA2A, which were estimated to be in the 99th percentile of the results obtained when we analyzed the sera from 194 healthy controls in our laboratory (22). The GADA intra and inter-assay CV were 3.58% and 4.9%, respectively. The IA2A intra- and inter-assay CVs were 4.3% and 3.4%, respectively. The OGTT was performed according to the American Diabetes Association’s standards (23). IVGTT was performed in patients as previously described, after an overnight fast of 10 hours, between 7:00–9:00 am, and blood samples were drawn for measurement of glucose and insulin 5 minutes before and at time 0 and 1, 3, 5, 10 minutes after an IV infusion of 25% glucose Arch Endocrinol Metab. 2017;61/4

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(0.5 g/kg of body weight up to 35 g) (20). FPIR was determined as the sum of 1 plus 3-minute secretion during IVGTT. Insulin resistance was evaluated using a homeostasis model assessment (HOMA) R index calculated from fasting plasma blood glucose and serum insulin concentration according to Matthews and cols. (24).

Statistical analysis Statistical analysis was performed using SPSS version 10.0 for Windows, SPSS Inc. A sample size was calculated to detect type 2 errors, taking into account the low prevalence of GADA and IA2A positivity in subjects with ATD. Data are expressed as the mean (SD) or as median and range. Data that were normally distributed were log transformed for analysis. Statistical differences in antibodies indices between groups were tested using ANOVA and Bonferroni. Homogeneity of variances was tested using Levene’s test. Differences in the frequencies of antibodies between HT, GD and controls were tested by chi-Square and Fisher’s test when appropriate. A p value < 0.05 was considered statistically significant.

GADA and IA2A were detected at 4.9% (16/324) and 0.7% (2/259), respectively, in the ATD patients. We did not find any patient with both antibodies. There was no difference between the clinical characteristics of GADA and/or IA2A positive and negative patients [(age 35 (22–68) vs 43 (16–77) years old, BMI (24.1 ± 3.3 and 27.3 ± 5.4 kg/m2), such as disease duration 3.3 (0.3–20) and 4.0 (0.1–39) years], respectively. None of the 93 control group members was positive for GADA or IA2A. Data on GD and HT individualized are shown in Table 1. Apart from the high variability of GADA titers in both ATD, the values in HT were significantly higher than in GD (p < 0.001) and controls (p < 0.001), but there was no difference between GD and controls (Figures 1 and 2). In patients with positive pancreatic islet cell antibodies, we also analyzed GADA titers according to the length of time since the clinical diagnosis of ATD. We shared them in 4 groups: ≤ 1 yr., > 1 ≤

4 **

RESULTS The clinical features of GD (n = 181), HT (n = 143) and healthy control subjects (n = 93) are represented in Table 1.

Log10 GADA U/ml

-2 47; 75 122; 126 142

-4

Table 1. Clinical and laboratory characteristics in ATD patients and control individuals ATD HT**

N (F/M)

181 (151/30)

143 (132/11)

93 (53/40)

Age (y.o)

40 (16-71)

48 (16-77)a

28 (18-58)

Time from ATD diagnosis (years)

4 (0.2-35)

4 (0.1-39)

BMI (kg/m2)

26 ± 4

28 ± 6.2

25 ± 3.6

GADA (U/ml)

0.05 (0.0-324.89)

0.14 (0.0-57.85)b

0.0 (0.0-1.61)

IA2A (U/ml)

0.02 (0.0-1.46)

0.02 (0.02-1.53)

0.01 (0.01-0.77)

GADA+ (%)

13/181 (7.2)

3/143 (2.0)

0/93 (0.0)

IA2A+ (%)

1/146 (0.7)

1/113 (0)

0/30 (0)

TPOAb (U/ml)

72 (0.1-80)

222 (0.0-3116)

0.8 (0.1-133)

N Group

143 HT

180 GD

93 Control

Figure 1. GADA level distribution in the groups. Values transformed to log10. ** p < 0.001 (HT vs GD and controls) obtained by ANOVA. 0.5 0

(HT vs GD and C; p < 0.01); b(GD vs HT; p < 0.01).

Arch Endocrinol Metab. 2017;61/4

-1.0 -1.5 -2.0 -2.5 -3.0

N Group

113 HT

146 GD

30 Control

Figure 2. IA2A level distribution in the groups. Values transformed to log10. 363

Data are expressed as median (range) or mean ± SD.

Log10 IA2A U/ml

-0.5

* GD: Graves’ disease. ** HT: Hashimoto’s thyroiditis. a

-6

Control

GD*

Pancreatic and thyroid autoimmunity

5 yr., > 5 ≤ 10 yr. and > 10 yrs. The GADA titers were 70.7 ± 38.3, 61.2 ± 107.7, 16.5 ± 15.7, 4.7 ± 0.9 U/ml, respectively, for these groups. The median figures of their ages were in the same order: 22 (22– 54), 29 (23–56), 43 (36–68), 44 (40–48) years old. Patients with less than five years of ATD seemed to have higher titers of GADA and be younger than the others. Eleven positive and 20 negative pancreatic islet cell auto-antibodies euthyroid ATD patients with similar ages, genders and BMIs accepted being tested for FPIR, insulin resistance (HOMA-R) and OGTT (Table 2). Table 2. Clinical and laboratorial features of positive (Ab+) and negative (Ab-) pancreatic islet-cell autoantibodies ATD patients analyzed in relation to βcell function and insulin resistance Ab+

Ab-

Gender (F/M)

8/3

17/3

BMI (kg/m2)

24.3 ± 3.2

24.9 ± 3.2

GADA (U/ml)

14.2 (0.00-324.9)

0.06 (0.00-0.56)

< 0.001

IA2Ab (U/ml)

0.2 (0.01-1.53)

0.0 (0.01-0.37)

< 0.001

FPIR (pmol/L)

786.2 ± 534.0

984.0 ± 717.6

FPG (mmol/L)

4.78 ± 0.43

4.72 ± 0.47

2hPG (mmol/L)

6.07 ± 1.52

5.27 ± 1.31

HOMA-R

1.09 ± 0.6

1.33 ± 0.9

Data were expressed as median values at time of the last evaluation.

Overall, there was not a significant difference between positive and negative GADA/IA2A euthyroid ATD patients in relation to first-phase insulin secretion, insulin resistance or oral glucose tolerance. However, when they were individually analyzed, we saw that 27.2% (3/11) of the GADA/IA2A+ against 5% (1/20) GADA/IA2A- showed low FPIR. Of these GADA/IA2A+/low FPIR, one had GD, with very high GADA titers (324.8 U/ml) and impaired fasting glucose (FPG = 6.83 mmol/L or 123 mg/dl), another had HT with low GADA titers (2.1 U/ml) and was overweight (BMI: 27 kg/m2) and the third subject had HT as well. Both with hypothyroidism had normal glucose tolerance. However, the GADA/IA2A-/low FPIR patient had GD and normal glucose tolerance.

DISCUSSION In this study, we showed that the prevalence of GAD/IA2 autoantibodies in Brazilian, non-diabetic, euthyroid ATD patients is higher than in the healthy population studied. Despite the similar frequency of GAD and IA2 autoantibodies in patients with HT or 364

GD, GADA titers in HT patients were higher than those observed in GD patients. Nevertheless, GAD/ IA2 autoantibodies positivity per se does not seem to be associated with any disturbance in insulin secretion or glucose tolerance in euthyroid ATD patients at the time of this study. GADA and IA2A frequencies were 4.9% and 0.7%, respectively, in the ATD patients. These frequencies were higher than those found previously in our healthy population (0.5% and 0.0%) (22) and demonstrated in similar patients in other studies (22-24). However, when the frequency of GADA in GD was compared to HT, the former (7.2%) tended to be slightly higher than the latter (2%). These data confirm our previous study when we found a higher prevalence of GADA in GD in a small group of patients with the same age and anthropometric clinical characteristics (19). In Swedish patients, GADA was detected in 13% of newly diagnosed GD patients without clinical diabetes mellitus and in 3.4% of HT patients (18,25). In Japanese patients, GADA was found in 6% of GD and 7.9% of HT patients (17) although the time since thyroid disease diagnosis was not reported, and the patients were in different degrees of thyroid function. In our patients, the median time of thyroid disease clinical diagnosis was 4 years, with some of them with more than 30 years. We observed that when ATD were pancreatic isletcell Ab-positive patients, they seemed to have thyroid disease for a shorter time and were younger, although the sample was too small to test this hypothesis statistically. We found higher GADA titers in HT than GD patients despite the same age and thyroid-disease duration in both groups of subjects. The presence of higher GADA at the time of diagnosis in organspecific autoimmune diseases other than T1AD can be explained by the higher influence of an antigenic environment. Furthermore, it has been reported that the GAD65 protein is found in the brain and even in lower concentrations in the thyroid, pituitary, kidney, liver, adrenal, ovary and testes in addition to pancreatic islets, raising the possibility of triggering antibody production based on the polyclonal B-lymphocyte production in ATD patients (17). In fact, subjects with ATD also show a high prevalence of autoantibodies against non-thyroid specific antigens (26). The relevance of GADA to T1AD development must be considered one step of the whole process, such as genes Arch Endocrinol Metab. 2017;61/4

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Arch Endocrinol Metab. 2017;61/4

In summary, the prevalence of GADA/IA2A was higher in non-diabetic ATD patients than in healthy controls. GADA prevalence was similar in HT and GD, but higher titers of these autoantibodies were found in the first. The degree of insulin resistance and glucose tolerance was similar between positive and negative GADA/IA2A euthyroid non-diabetic ATD patients. Moreover, we found only a tendency for low FPIR in GADA/IA2A-positive individuals. This may correspond to subclinical insulitis. Acknowledgements: we would like to thank the participant Walkiria Miranda for assistance in the laboratory. Funding: this study was feasible due to the support of CNPq, Brazil (process number 133045/2001-3). Disclosure: no potential conflict of interest relevant to this article was reported.

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conferring susceptibility to or protecting from T1AD, the presence of others’ pancreatic antibodies, their titer levels, their persistence and the autoantibody epitope specificity. The autoimmune disease is the final phase of a process starting with auto recognition, passing through immunity with the appearance of autoantibodies and finally leading to cell destruction and autoimmune disease (27). A study in a group of Japanese patients who had been diagnosed initially with non-insulin dependent diabetes mellitus (NIDDM) showed that those with higher GADA titers progressed rapidly to an insulindependent state (28-30). However, in a study of stratification of diabetes risk in T1AD’s first-degree relatives, it was found that high titers of IA2A and IAA, though not GADA, were associated with an increased risk for clinical diabetes (29). A study demonstrated that only the simultaneous presence of GADA and “classical” islet cell antibody (ICA) was associated with a loss of beta-cell function during follow-up and an increased risk of progression toward insulin dependency and low BMI (30). We found that ATD patients with positive pancreatic islet cell Ab presented a downward tendency in the FPIR results besides normal FPG and glucose tolerance. It has been shown that positive GADA HT patients had similar FPG and insulin concentrations than negative GADA subjects; however, when submitted to arginine injection, they showed a decline in the insulin secretory capacity and the glucagon response to arginine (25). Insulin resistance in patients with islet autoimmunity could have a genetic-constitutional basis and might lead to the earlier appearance of hyperglycemia when betacell function is compromised by autoimmune disease (31). In a recent study, this research group showed that T1AD relatives that were positive for islet antibodies and progressed most rapidly to diabetes had shown a subtle disturbance of insulin-glucose homeostasis years before the onset of symptoms, distinguished by greater insulin resistance for their level of insulin secretion. The degree of insulin sensitivity estimated by HR was similar between positive and negative GADA/IA2A groups and as expected, showed a positive relation with BMI (rS = 0.52; p = 0.0034). This study was limited by a small sample of GADA/ IA2A-positive AITD patients in relation to FPIR and glucose tolerance and by the short follow-up of these patients.

Pancreatic and thyroid autoimmunity

12. Barova H, Perusicova J, Hill M, Sterzl I, Vondra K, Masek Z. AntiGAD-positive patients with Type 1 diabetes mellitus have higher prevalence of autoimmune thyroiditis than anti-GAD-negative patients with Type 1 and Type 2 diabetes mellitus. Physiol Res. 2004;53:279-86. 13. Baekkeskov S, Aanstoot HJ, Christgau S, Reetz A, Solimena M, Cascalho M, et al. Identification of the 64K autoantigen in insulin dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase. Nature. 1990;347:151-6. 14. Zimmet PZ, Tuomi T, Mackay IR, Rowley MJ, Knowles W, Cohen M, et al. Latent autoimmune diabetes mellitus in adults (LADA): the role of antibodies to glutamic acid decarboxylase in diagnosis and prediction of insulin dependency. Diabet Med. 1994;1:299303.

pacientes portadores de câncer diferenciado de tiroide. Unpublished data. 22. Cesarini PR, Mendonça E, Fernandes V, Silva RC, Morimitsu LK, Garcia FE, et al. Prevalence of immunological markers (antiGAD and anti-IA2) in first degree relatives of patients with type 1 diabetes in Great São Paulo city. Rev Assoc Med Bras. 2003;49: 395-40. 23. ADA. Summary of revision for the 2004 clinical practice recommendations. Diabetes Care. 2004;27:S3. 24. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia. 1985;28:412-9.

15. Takeda H, Kawasaki E, Shimizu I, Ionoue E, Fujiyama M, Murao S, et al.; Ehime Study. Clinical, autoimmune, and genetic characteristics of adult-onset diabetic patients with GAD autoantibodies in Japan (Ehime Study). Diabetes Care. 2002;25:995-1001.

25. Lethagen AL, Ericsson UB, Hallengren B, Groop L, Tuomi T. Glutamic acid decarboxylase antibody positivity is associated with an impaired insulin response to glucose and arginine in nondiabetic patients with autoimmune thyroiditis. J Clin Endocrinol Metab. 2002;87:1177-83.

16. Tuomi T, Bjorses P, Falorni A, Partanen J, Perheentupa J, Lernmark A, et al. Antibodies to glutamic acid decarboxylase and insulin dependent diabetes in patients with autoimmune polyendocrine syndrome type I. J Clin Endocrinol Metab. 1996;81:1488-94.

26. Ishii M, Hasegawa G, Fukui M, Obayashi H, Ohta M, Ogata M, et al. Clinical and genetic characteristics of diabetic patients with high-titer (>10,000 U/ml) of antibodies to glutamic acid decarboxylase. Immunology Letters. 2005;99:180-5.

17. Kawasaki E, Abiru N, Yano M, Uotani S, Matsumoto K, Matsuo H, et al. Autoantibodies to glutamic acid decarboxylase in patients with autoimmune thyroid disease: relation to competitive insulin autoantibodies. J Autoimmunity. 1995;8:633-43.

27. Talal N. Autoimmunity and the immunological network. Arthritis Rheum. 1978;21:853-61.

18. Hallengren B, Falorni A, Landin-Olsson M, Lernmark Ä, Papadopoulos KI, Sundkvist G. Islet cell and glutamic acid decarboxylase antibodies in hyperthyroid patients: at diagnosis and following treatment. J Intern Med. 1996;239:63-8. 19. Silva RC, Sallorenzo C, Kater CE, Dib SA, Falorni A. Autoantibodies against glutamic acid decarboxylase and 21-hydroxylase in Brazilian patients with type 1 diabetes or autoimmune thyroid diseases. Diab Nutr Metab. 2003;16:160-8. 20. Bingley PJ, Colman P, Eisenbarth GS, Jackson RA, McCulloch DK, Riley WJ, et al. Standardization of IVGTT to predict IDDM. Diabetes Care. 1992;15:1313-6.

29. Achenbach P, Warncke K, Reiter J, Naserke HE, Williams AJK, Bingley PJ, et al. Stratification of type 1 diabetes risk on the basis of islet autoantibody characteristics. Diabetes. 2004;53:384-92. 30. Lohmann T, Kellner K, Verlohren HJ, Schroder S, Rotger J, Dahn K, et al. Titre and combination of ICA and autoantibodies to glutamic acid decarboxylase discriminate two clinically distinct types of latent autoimmune diabetes in adults (LADA). Diabetologia. 2001;44:1005-10. 31. Fourlanos S, Narendran P, Byrnes GB, Colman G, Harrison LC. Insulin resistance is a risk factor for progression to Type 1 diabetes. Diabetologia. 2004;47:1661-7.

21. Maciel RMB. Desenvolvimento de um método radioimunológico para a dosagem de Tg sérica, sua aplicação no seguimento de

28. Abiru N, Takino H, Yano M, Kawasaki E, Yamasaki H, Yamaguchi Y, et al. Clinical evaluation of non-insulin-dependent diabetes mellitus patients with autoantibodies to glutamic acid decarboxylase. J Autoimmunity. 1996;9:683-8.

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original article

Overweight in youth and sleep quality: is there a link? Susana Rebelo Pacheco1, Ana Margalha Miranda1, Raquel Coelho1, Ana Cristina Monteiro1, Graciete Bragança1, Helena Cristina Loureiro1

ABSTRACT Objective: Overweight seems to be related to a higher prevalence of sleep disturbances. Decreased sleep duration and altered sleep quality are risk factors for obesity. Our aim was to compare the sleep pattern of overweight children with that of a matched control group and assess the relationship between sleep quality and obesity. Materials and methods: Retrospective cohort study comparing 41 overweight children with a normal-weight control group, both submitted to polysomnography. The samples were matched for age, sex, and apnea-hypopnea index. Body mass index (BMI) z-scores were calculated using World Health Organization (WHO) growth charts. Insulin resistance in the study group was determined using the homeostatic model assessment for insulin resistance (HOMA-IR). Sleep patterns were compared. The statistical analysis was performed using SPSS® version 21. Results: The mean age (± standard deviation) of the population was 10 ± 3.4 years (min. 5 years; max. 17 years). Fifty-six percent of the participants in both groups were girls. N3% was lower in the study group (18.95 ± 6.18%) compared with the control group (21.61 ± 7.39%; t (40) = 2.156, p = 0.037). We found a correlation in the study group between HOMA-IR and N3% (Rs = -0.434, p = 0.008). Conclusion: The present study suggests a link between overweight/obesity and altered sleep quality due to compromised non-rapid eye movement sleep, an indirect marker of sleep quality. There was also a link between slow-wave sleep duration and insulin resistance. We must find a strategy to provide adequate slow-wave sleep duration to reduce the obesity epidemic at young ages. Further research is needed. Arch Endocrinol Metab. 2017;61(3):367-73.

INTRODUCTION

he prevalence of childhood overweight and obesity has significantly increased in the past decades, representing a global epidemic and a major public health concern (1). Childhood obesity contributes to significant physical, psychological, and economic burden. It often continues into adulthood and confers a major risk for insulin resistance, impaired glucose tolerance, hypertension, dyslipidemia, and cardiovascular disease (2). Attempted interventions to improve diet and physical activity patterns among adolescents have had limited success at controlling pediatric obesity (3). Identification of early childhood modifiable risk factors should be a priority (2,3). While the prevalence of obesity has increased, lifestyle changes have led to a decrease in night sleep hours among children and adolescents due to later bedtimes with unchanged rise times. Worldwide Arch Endocrinol Metab. 2017;61/4

Correspondence to: Susana Rebelo Pacheco Hospital Prof. Doutor Fernando Fonseca, E.P.E. IC19, Venteira, Amadora – Portugal suspacheco@gmail.com Received on Jan/19/2016 Accepted on Nov/25/2016 DOI: 10.1590/2359-3997000000265

studies estimate that 20%-30% of children and 6%-37% of adolescents report problems related to prolonged sleep latency, difficulty initiating and maintaining sleep, frequent nocturnal awakenings, and poor sleep quality accompanied by significant daytime impairments (4,5). Short sleep has been identified as a risk factor for obesity in children and adults, and several studies have reported a strong relationship between sleep duration and higher BMI (2). However, it has been difficult to draw immediate conclusions on the consistency of the association, direction of causality, and likely mechanisms involved (6). The underlying pathogenic pathways are complex and unresolved, but it is known that sleep, specially slow-wave sleep (SWS), has a major role in body restorative processes and energy metabolism. Poor sleep might impact glucose regulation, inflammatory markers, and appetite regulating hormones, promoting 367

Keywords Children; obesity; sleep quality

Pediatric Department, Hospital Prof. Doutor Fernando Fonseca, Amadora, Portugal 1

Overweight in youth and sleep quality

food intake (7-9). Although a strong relationship between sleep deprivation, increased food intake, and obesity has been documented, only a small number of studies have yet examined how eating behavior may be related to sleep quality and quantity (9,10). It has been demonstrated that reduced subjective sleep quality scores are associated with behaviors like binge eating and emotional eating (11,12). Other pathogenic mechanisms seem to play a role in the relationship between sleep deprivation and obesity. The autonomic nervous system is an important contributor in coordinating energy homeostasis and plays a role in the pathophysiology of obesity. Sympathovagal imbalance, a marker of autonomic dysfunction, is present in obese children with sympathetic hyperactivity and decreased parasympathetic functioning when compared with normal-weight children (12,13). Autonomic dysfunction has been linked to altered sleep duration, sleep fragmentation, sleep disorders like insomnia, and subjective/objective reports of poor sleep quality (14,15). Cross-sectional studies reveal that shorter sleep duration, poor sleep quality, sleep disturbances, and a delayed sleep phase pattern are associated with larger body composition, greater central adiposity, and increased obesity rates among adults and youths (4,14,16). In addition to these potential consequences, short sleep may contribute to increased risk of cognitive and behavioral problems (17). The aim of the present study was to compare the sleep pattern of overweight children with a matched control group and assess the relationship between sleep quality and overweight/obesity.

MATERIALS AND METHODS Study type Retrospective cohort study comparing 41 overweight and obese children (study group) to 41 non-overweight children (control group).

Study population The study population included children and adolescents submitted to a sleep study. Children were referred to a sleep specialist to exclude sleep disturbances in the context of snoring. Overweight and obese children were selected to make up the study group. The control group was randomly selected from non-overweight 368

patients. Both groups were matched for age, sex, and apnea-hypopnea index (AHI).

Measures

Anthropometrics Children were weighed (to 0.1 kg) with a calibrated scale, and their heights were measured (to 0.1 cm) with a stadiometer at the first sleep appointment by the same observer using the same equipment. The International System of Units was used. Weight was measured in kilograms (kg) and height in centimeters (cm). BMI values were calculated dividing weight (kg) by squared height (m2). Age-sex-adjusted BMI standard deviation scores (z-scores) were computed and distributed using World Health Organization (WHO) growth charts and the official WHO z-score calculator. Overweight was defined as a BMI z-score > 1 and obesity as a BMI z-score > 2. Normal weight was defined as a BMI z-score â&#x2030;¤ 1. These definitions were based on WHO recommendations (18).

Sleep data by polysomnography All children underwent nocturnal type 1 polysomnography (PSG) at our sleep laboratory during an acute illness free period. All PSG were performed using electroencephalography (frontal, central, and occipital channels), chin electromyography, two leg channels, electrooculogram, piezo-electric chest and abdominal belts, simultaneous pressure transducer and thermistor for nasal flow, transcutaneous (Tc) carbon dioxide and oxygen saturation monitoring, snore microphone, electrocardiography, and simultaneous video monitoring. All PSG results were scored by the same professional, adequately qualified in PSG analysis (with a Master degree in sleep sciences). Detailed sleep parameters were measured, including total sleep time (TST); wake after sleep onset (WASO); non-rapid eye movement sleep (NREM) divided into three stages: stages 1 (N1) and 2 (N2), light sleep stages, and stage 3 (N3), SWS; rapid eye movement sleep (REM) latency; sleep efficiency; REM; SWS percentage; snoring percentage; number of arousals per hour; AHI and oxygen saturation nadir. Periodic leg movements (PLM) and PLM arousal index, and parasomnias were also investigated. Sleep architecture was scored using the 2012 American Academy of Sleep Medicine guidelines (AASM 2012) (19). The following definitions were used: 1. Obstructive sleep apnea was Arch Endocrinol Metab. 2017;61/4

Overweight in youth and sleep quality

defined as the cessation of airflow for two respiratory cycles; 2. Hypopnea was defined as a decreased in amplitude of the airflow signal by 50% lasting two respiratory cycles and accompanied by an oxygen desaturation > 3%; 3. An AHI > 1/hr was considered as a diagnosis of obstructive sleep apnea syndrome (OSAS); 4. Habitual snoring was defined as snoring not accompanied by oxygen desaturation, arousals or changes in air flow; 5. Periodic limb movements of sleep were defined as repetitive jerking of the legs during sleep of more than five movements per hour, 0.5–5 sec in duration, in clusters of four or more separated by 5–90 sec intervals; 6. Parasomnias were defined by the occurrence of confused arousal, sleepwalking episodes, sleep terrors, and nightmare disorders according to the International Classification of Sleep Disorders (ICSD), 2nd edition; 7. TST corresponded to the sum of total sleep during stages N1, N2, N3, and REM; 8. WASO encompassed the time spent awake after sleep had been initiated and before final awakening; 9. Sleep efficiency was calculated using the ratio of total minutes of sleep (N1 + N2 + N3 + REM) and time spent in bed; 10. Sleep latency was defined by the time, in minutes, elapsed from lights out to sleep onset; 11. REM latency represented the time from the first epoch of sleep to the first REM sleep.

The normal ranges of sleep variables are presented in Table 1. Weekday sleep duration was calculated using parents or self-reported usual weekday bedtime and wake time.

Evaluation of insulin resistance using the HOMA-IR In the study group, night fasting plasma glucose and insulin levels were measured in our hospital laboratory. Parental consent was obtained for blood sampling at the Endocrinology appointment. No laboratory results from the control group were available, as these are not regular laboratory studies required at a sleep appointment. We evaluated insulin resistance using the homeostasis model assessment for insulin resistance (HOMA-IR). HOMA-IR was calculated with the equation: HOMAIR = fasting insulin (µU/mL) x fasting glucose (mg/dL) / 405. The cutoff level to determine insulin resistance was 3.16 (20).

Statistical analyses Descriptive statistics were computed for all variables. Sleep patterns were compared using Student’s t test for paired samples and Wilcoxon test. We used Spearman’s correlation to correlate the sleep pattern and the

Duration (reported, h) TST (min)

Control group

Study group

Mean

Comparison p*

9.43

1.08

9.26

1.45

0.574

Normal values

> 330

416.41

55.92

426.95

61.62

0.353

WASO (min)

61.40

48.64

67.07

47.25

0.354

Latency (min)

< 30

25.42

26.14

21.07

31.40

0.091

Efficiency (%)

> 85 TST

82.95

10.66

82.89

11.84

0.892

AHI (nr/h)

3.00

2.30

2.85

2.01

0.199

Arousals (nr/h TST)

< 14

12.27

5.17

12.85

5.82

0.444

N1 (%)

0-5

2.98

2.69

3.38

2.33

0.310

N2 (%)

< 55

61.77

8.50

63.95

6.22

0.144

N3 (%)

15-20

21.61

7.39

18.95

6.18

0.037

REM (%)

20-25

13.62

5.47

13.25

4.32

0.726

REM sleep latency (min)

< 120

157.95

74.01

161.57

59.03

0.688

Mean SpO2 (%)

> 95

95.54

2.19

95.59

1.36

Min. SpO2 (%)

> 94

93.12

3.14

92.41

3.41

TST: total sleep time; WASO: wake after sleep onset; AHI: apnea-hypopnea index; N1: stage 1 of non-rapid eye movement sleep; N2: stage 2 of non-rapid eye movement sleep; N3: stage 3 of nonrapid eye movement sleep; REM: rapid eye movement sleep; SpO2: peripheral capillary oxygen saturation; SD: standard deviation. * Statistical comparison of the sleep pattern in the control and study groups. Arch Endocrinol Metab. 2017;61/4

369

Table 1. Normal values of sleep pattern on polysomnography (adapted from the American Academy of Sleep Medicine guidelines) and comparison of sleep patterns between the control and study groups

Overweight in youth and sleep quality

HOMA-IR index. All data were analyzed with IBM SPSS Statistics 21® software (SPSS, Inc., Chicago, IL). We defined a p value of < 0.05 to indicate statistical significance.

RESULTS Descriptive statistics

Descriptive data are presented as a percentage for discrete variables and as mean (± standard deviation [SD]) for continuous variables. Each group (study and control) comprised 41 subjects matched for gender, age, and AHI, who were evaluated with PSG. The age of the participants ranged from 5 to 17 years (mean 10.95 ± 3.4 years), and girls represented 56% of the sample. The mean BMI z-score of our control and study groups were -0.25 ± 0.80 and 2.74 ± 0.56, respectively. Our study group was mostly composed of obese children (90.2% with BMI z-scores > 2; 9.6% with BMI z-scores > 1 and < 2). Means and SDs for measures of sleep are presented in Table 1. The mean sleep duration was similar in the control and study groups (9.43 h vs. 9.26 h). Mean latency was 25.42 and 21.07 minutes in each group, respectively. Sleep latency lasted more than 30 minutes in 17% of the study group (7 children) and in 24.4% of the control group (10 children). Sleep efficiency was < 85% of the TST in 18 study group children (43.9%) and 20 control group children (48.8%). SWS proportion was under 15% in 12 (29.27%) study cases (control group had 10 children – 24.3%) and was higher than 20% in 18 (43.9%) study cases (control group had 24 cases – 58.5%). In 97.6% cases in the study group, the REM proportion was < 20% (90.2% in the control group). In 15 study group children (36.6%) and 14 control group ones (41.1%), counted arousals were > 14 per hour during the PSG. Sleep efficiency was reduced in both groups (study group 82.89 ± 11.84%; control group 82.95 ± 10.66%). The remainder sleep constants were normal in both groups. In the study group, the mean HOMA-IR was 2.38 ± 1.54, and seven cases fulfilled the criteria for insulin resistance.

Comparative statistics The SWS duration (N3) was lower in our study group (study group: 18.95 ± 6.18%; control group: 21.61 ± 7.39%). A statistically significant difference was found 370

comparing SWS in both groups (p = 0.037). No statistical differences were found comparing other sleep constants (p > 0.05). The comparison in sleep patterns between the control and study groups is presented in Table 1. We found a moderately negative correlation (Rs = -0.434) between HOMA-IR and %N3 (p = 0.008). Only a low positive correlation was found between HOMA-IR and %REM (Rs = 0.348, p = 0.037) and number of arousals (Rs = 0.366, p = 0.028). No other statistically different correlations were found comparing HOMA-IR and sleep variables. The correlation between sleep pattern and HOMA-IR in the study group is presented in Table 2. Table 2. Correlation between sleep pattern and HOMA-IR in the study group Study group Correlation

TST (min)

0.014

0.933

WASO (min)

-0.126

0.463

Latency (min)

-0.027

0.875

Efficiency (%)

0.102

0.553

Arousals (nr/h)

0.366

0.028

N1 (%)

0.106

0.540

N2 (%)

0.000

0.999

N3 (%)

-0.434

0.008

REM (%)

0.348

0.037

REM sleep latency (min)

0.115

0.505

Mean SpO2

-0.201

0.239

Min. SpO2

-0.1

0.562

TST: total sleep time; WASO: wake after sleep onset; AHI: apnea-hypopnea index; N1: stage 1 of non-rapid eye movement sleep; N2: stage 2 of non-rapid eye movement sleep; N3: stage 3 of non-rapid eye movement sleep; REM: rapid eye movement sleep; SpO2: peripheral capillary oxygen saturation; SD: standard deviation.

DISCUSSION The increasing prevalence of overweight and obesity in children and youth is a major public health concern, as it is linked to multiple chronic diseases and premature mortality. Interventions to improve lifestyle have been applied and efforts have been made to understand the different pathogeneses of overweight and obesity (13,21). Growing scientific evidence identifies disturbed sleep as a risk factor for obesity in children. In fact, duration of sleep and sleep quality seem to be related to obesity (22-25). Poor sleep has also been associated with increased waist circumference, an index of central adiposity (7,15). Arch Endocrinol Metab. 2017;61/4

Most studies used data collected from self-reporting questionnaires or sleep diaries and described sleep duration without an objective sleep quality analysis. In our study, the mean sleep duration was similar in both groups, with no significant difference found between them (9.43 h vs. 9.26 h, p = 0.574), although other published studies have found that obese children usually sleep less than normal-weight ones (2,4). Sleep duration was the only self-given subjective data. As known from the literature, sleep reports are found to over-report sleep duration. Parents and children were directly asked about the time spent in bed during weekdays. The self-reported time could be a reason for not finding a significant short sleep duration difference between the overweight/obese children and the normal-weight children. In our study, TST was short in both groups, but no significant difference was found between the groups. This may be due to the PSG being performed in a hospital room where the first night effect must be taken into account. Also, the PSG was performed during a weekday. Spruyt and cols. found no significant difference in daily TST but described a shorter TST duration on weekends in obese children (p = 0.03) (24). When analyzing the PSG data from both groups, we noticed that sleep efficiency was reduced in both (study group 82.89 ± 11.84%; control group 82.95 ± 10.66%). This may be related to the first night effect and a shorter sleep duration. We found no significant difference in the number of arousals, a fact that could also explain a reduced efficiency. All other sleep constants were normal in both groups, with the exception of N3%. The study group had a decreased percentage of SWS, and a statistically significant difference was found comparing the SWS results in both groups (18.95 ± 6.18% vs. 21.61 ± 7.39%; p = 0.037). SWS is thought to play an important role in cerebral restoration and recovery, and is involved in maintenance and consolidation of sleep (26). Being the most important sleep phase in regulating metabolism, selective deprivation of SWS is related to insulin metabolism deregulation, increased cortisol secretion, and decreased growth hormone concentration. Consequently, reduced SWS and sleep fragmentation result in disturbed sleep quality and may be related to weight gain and z-score elevation due to an important change in body metabolism (22,26). The literature provides increasing support for the pathophysiology of sympathovagal imbalance Arch Endocrinol Metab. 2017;61/4

having a contributing role in the association between sleep pattern and obesity. SWS has predominantly parasympathetic and reduced sympathetic activity. Sleep disturbances are associated with higher nocturnal arousals, which enhance sympathetic activity leading to increases in urinary catecholamine levels, heart rate, blood pressure, and sympathovagal imbalance among adults and youth (6,13). Although this was not observed in our study, maybe due to the small study sample, it is known that obese children have a higher sympathovagal imbalance (15). Shortened sleep is reported to support obesogenic behaviors with lower levels of physical activity, higher food intake, and selection for junk food and a carbohydrate-rich diet (9,14,27). This seems to be caused by stimulation of neurons of the ventral tegmental area related to the reward sensation after food intake (28). On the other hand, sympathetic stimulation that occurs with sleep deprivation might contribute to metabolic deregulation (29). It is well known in the literature that neurobehavioral changes associated with puberty have a significant influence on sleep organization. The main changes include a delayed sleep phase, which involves a tendency for later bedtimes and rise times; shorter sleep, which is associated with increased levels of daytime sleepiness; and irregular sleep patterns, which involve sleeping very little on weekdays and sleeping longer during weekends to partially compensate for this sleep loss (5,17). However, due to the modest size of the sample and no access to the participant’s Tanner stages, these factors were not taken into account during the statistical analyses in the present study. The mean HOMA-IR in the study group was normal (2.38 ± 1.54), but seven cases fulfilled the criteria for insulin resistance (HOMA-IR > 3.16). Besides, we found a moderately negative correlation (Rs = -0.434) between HOMA-IR and %N3 (p = 0.008). These data seem to reinforce the already described relation between reduced SWS and dysfunctional metabolism. We found no relation between HOMA-IR and TST and efficiency, as mentioned in previous articles (30). Puberty is associated with transient insulin resistance, which normally resolves by the end of this period. Girls are usually more insulin resistant at all Tanner stages than boys (30). This was not taken into account in the present study due to the limitations cited above (small sample size and absent data on Tanner stages). 371

Overweight in youth and sleep quality

We were unable to find other matching published data, mostly because the majority of the studies used exclusively self-reported questionnaires to evaluate sleep quality and no qualitative PSG data. Our study has a number of strengths. First, we used an objective methodology to assess sleep quality using overnight PSG in all patients and no self-reported questionnaire on the perception of sleep quality. This is an important fact, as the majority of published studies do not use these objective data. Second, the study and control group were matched for age, sex, and AHI, limiting confounders. Third, no self-reported height or weight data were used, and BMI was calculated using objective values. The study has some limitations. The sample size (41 children in each group) may be a major limitation. Although we used objective PSG data, the total weekly sleep duration was self-reported, as already described above. Although both groups were matched, other confounders could exist: ethnic, sexual maturity, chronic illness, socioeconomic status, the frequency of snacking and other health-related behaviors or nutritional habits. Another limitation may be that all the studies were performed at the hospital during a single night period; therefore, the first night effect must be taken into account. The PSG data were validated for the age of the population, making the comparison possible. Still, many questions need answers to determine causality and exact changes in sleep pattern. In conclusion, the present study found a preliminary support of altered sleep quality in overweight and obese children compared with normal-weight ones, with an impact on insulin sensitivity and glucose metabolism. This matches previously published information. A link seems to exist between overweight/obesity and altered SWS duration, an indirect marker of sleep quality, related to its metabolic and hormonal homeostatic function. Strategies providing behavioral sleep interventions and adequate SWS duration are a fundamental objective to be pursued in order to reduce the obesity epidemic at young ages. Adequate sleep duration and good sleep quality seem to be low-cost approaches to ensure homeostasis and help control increased weight among our youth. There is still need for further research evaluating the sleep pattern of overweight children and youth, analyzing possible confounding factors in larger populations.

Disclosure: no potential conflict of interest relevant to this article was reported.

Acknowledgments: there were no contributions to the article from individuals other than the authors.

18. World Health Organization (2007). Growth reference data for 5-19 years. Available from: <http://www.who.int/entity/growthref/en/>.

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original article

Related quality of life questionnaire specific to dysthyroid ophthalmopathy evaluated in a population of patients with Graves’ disease Laura Carolina Delfino1, Anabela Zunino1, Verónica Sapia2, María del Carmen Silva Croome1, Verónica Ilera1, Alicia Teresa Gauna1

ABSTRACT División Endocrinología, Hospital de Agudos J. M. Ramos Mejía, Ciudad Autónoma de Buenos Aires, Argentina 2 Servicio de Oftalmología, Hospital de Agudos J. M. Ramos Mejía, Ciudad Autónoma de Buenos Aires, Argentina 1

Correspondence to:

Laura Carolina Delfino Ramón Falcón, 1575, 4° B (1406) Ciudad Autónoma de Buenos Aires Argentina lauracdelfino@gmail.com Received on Jan/19/2016 Accepted on Oct/18/2016 DOI: 10.1590/2359-3997000000252

Objective: The aim of this study was to measure quality of life (QOL) impairment in individuals currently suffering from Graves’ ophthalmopathy (GO) and to determine the correlation of GO-specific QOL scores with disease severity and activity. Subjects and methods: Seventy three GO-specific QOL surveys were prospectively analysed and compared with GO status. The GO-specific QOL survey was translated into Spanish and applied to Argentine patients with Graves’ disease (GD). Results were compared with presence or absence of GO, Clinical Activity Score (CAS), severity score, age, gender and thyroid function. Results: Fifty-six patients answered the survey and underwent complete ophthalmic evaluation, 15 did not have GO and were considered to be a control group. Appearance QOL score for patients with GO (53 ± 31.4) was lower than the control group (88.3 ± 17) (p < 0,000), no difference was observed in functional QOL score. There was a negative correlation between GO severity and both functional (r = -0.575; p < 0.000) and appearance QOL (r = -0.577; p < 0.000). Functional QOL differed between patients with active GO vs control group (p = 0.043). Patients with active and inactive GO had lower appearance QOL scores than control group (p < 0.000, p < 0.001 respectively). Conclusions: GO has significant impact on the life of these Argentine patients. QOL was worse in GO patients than in control group, functional QOL was mostly affected by the activity and appearance QOL was mainly altered by the effects of the disease. Patients with more severe GO had lower scores on both QOL scales. Arch Endocrinol Metab. 2017;61(3):374-81. Keywords Graves ophthalmopathy; Graves’ disease; quality of life; orbitopathy; thyroid autoimmunity

INTRODUCTION

raves’ ophthalmopathy (GO) is a chronic, debilitating infiltrative eye disease that is clinically present in about 50% of patients with Graves’ disease. It is characterized by disfiguring proptosis, pain, redness and swelling of the eyelids, grittiness of the eyes and diplopia. Approximately 3% to 5% of patients suffer severe forms of the disease that could lead to permanent blindness (1,2). In addition, GO may be a severely disabling condition because of its effect on appearance and visual comfort. Although the patient is usually the best person to monitor and judge outcomes in medical care, data concerning patients’ experiences about the disease and response to treatment are not routinely collected (3). Scoring standardized responses to standardized questions is an efficient way to measure health status and related QOL (4). 374

Therefore, Terwee and cols. (5) developed a GO QOL instrument for Dutch patients written in their native language which proved to be reliable among this population. The QOL questionnaire was used to evaluate the change in QOL scores associated with clinical improvement after different treatment modalities (6). Park and cols. (7) used the modified and translated GO-specific QOL survey in Australian patients and their results showed a significant correlation between impaired QOL score and disease severity. Using the Australian GO-GOL version, Wickwar and cols. (8) reported higher levels of potential cases of clinical anxiety and depression in GO than in other chronic diseases or facial disfigurements. GO-QOL visual function scores were explained by age, asymmetrical GO and depressed mood; and appearance scores were explained by gender, appearance-related Arch Endocrinol Metab. 2017;61/4

Quality of life in Graves’ ophthalmopathy

SUBJECTS AND METHODS Design We developed a cross-sectional study at our Endocrinology Division between June 2012 and December 2013. We translated the English version QOL survey provided by the EUGOGO (http://www.eugogo.eu/_downloads/ clinical_evaluation/GO_QOL_EN.pdf) to Spanish. This questionnaire was distributed among Graves’ disease patients. A complete medical history and laboratory tests were performed. Patients were then referred for ophthalmological evaluation. The questionnaire was distributed after the medical evaluation, and returned completed by the patients before leaving the hospital.

Subjects The questionnaire was distributed to 95 Graves’ disease patients: 1) all recently diagnosed Graves’ disease patients (n = 36); 2) all patients prior to radioiodine treatment (n = 49) and 3) all patients with indication of systemic glucocorticoid treatment (n = 10 with severe and active GO and dysthyroid optic neuropathy-DON-). GD diagnosis was performed on the basis of clinical features of thyrotoxicosis, elevated thyroid hormones, suppressed thyrotropin (TSH), positive serum TSH receptor antibodies (TRAb) and diffuse goiter. All patients provided informed written consent, and the institutional ethics committee approval was obtained before starting the study. Glucocorticoid treatment was prescribed for six patients with severe active GO (methylprednisolone 500 mg once weekly for 6 weeks, Arch Endocrinol Metab. 2017;61/4

250 mg once weekly for 6 weeks, total treatment period: 12 weeks) and four patients with sight-threatening GO (methylprednisolone 1g daily for 3 days). Ophthalmologic examination was performed, in all cases, by the same professional. The evaluation included history of present illness, visual acuity, colour vision, pupillary examination, biomicroscopy, funduscopy and full orbital palpebral examination. An automated perimetry and an orbital CT scan were were performed in each case. GO was classified according to the Clinical Activity Score (CAS) and the severity score proposed by the EUGOGO modified consensus (Table 1) (12). Table 1. EUGOGO modified Severity Score Severity Score Sight-threatening GO: patients with dysthyroid optic neuropathy (DON) or corneal ulceration. This category requires immediate intervention Severe GO: patients without risk for vision, but the eye involvement has such an impact on their quality of life that justifies the risk of immunosuppressive therapy (if active) or surgery (if inactive) GO moderate to severe: patients have one or more of the following: palpebral > 2 mm retraction, moderate or severe soft tissue involvement, exophthalmos > 3 mm above normal for race and gender, inconstant, or constant diplopia GO mild: patients whose features of GO have only a minor impact on daily life insufficient to justify immunosuppressive or surgical treatment. They usually have only one or more of the following: minor lid retraction (< 2 mm), mild soft tissue involvement, exophthalmos < 3 mm above normal for race and gender, transient or no diplopia, and corneal exposure responsive to lubricants

Methods Answers on each subscale (functional and appearance) were transformed to scores ranging from 0 (worst) to 100 (best). We registered total QOL scores (average of functional and appearance scale), and also functional QOL scores and appearance QOL scores separately. Serum total T3, total T4, free T4 and TSH were measured by chemiluminescence immunoassay (Immulite 1000, Siemens, USA). Serum TSH receptor antibodies (TRAb) concentrations were determined by RIA (RSR, UK). A value of TRAb ≥ 15% was considered positive. Normal reference ranges were free T4, 0.79 to 1.4 ng/dL; total T4, 5.0 to 11.5 ug/dL; total T3, 85 to 175 ng/dL; and TSH, 0.5 to 4.0 mUI/mL. Statistical analyses were performed using the SPSS software package (version 17.0; SPSS Inc., Chicago, IL, USA). As a measure of reliability, internal consistency, based on item correlations within a subscale, was assessed by calculating Cronbach’s alphas. Continuous variables are reported as mean ± standard deviation (SD) or median (inter-quartile range). Student t test or 375

cognitions and depressed mood. Choi and cols. (9) used the questionnaire among a Korean population and likewise found that the survey was associated with GO activity. Son and cols. (10), using TED-QOL, showed that age, soft-tissue inflammation and motility disorder had a positive correlation with overall and functionrelated QOL; while gender, soft-tissue inflammation and proptosis had a correlation with appearance-related QOL. Finamor and cols. (11) found seriously impaired scores on a 10-item health-related QOL questionnaire in Brazilian patients in the chronic and inactive stages of GO. The aim of the study was to measure QOL impairment in individuals with GO (during the illness) and to determine the correlation of GO-specific QOL scores with disease severity and activity.

Quality of life in Graves’ ophthalmopathy

one way ANOVA used for comparison of continuous variables. For correlations Pearson coefficient was used. Chi-square test was applied for comparing categorical variables. An alpha error of 0.05 was considered.

RESULTS Of the 95 patients who received the questionnaire, 73 completed it adequately (77% response rate). Twentytwo surveys were excluded from analysis: 2 were not correctly identified, 1 survey contained more than 40% missing answers, 1 survey had more than 1 answer in one or more questions and 18 were not returned back to the treating physician (Figure 1). Of those who did not return the survey, 64.3% had no evidence of GO, significantly different to those who answered the questionnaire (26.8%; p = 0.01). Cronbach’s alphas were 0.79 for visual functioning and 0.90 for appearance.

both functional and appearance scales (r = 0,567, p < 0,000). Functional and appearance scores were not significantly different between genders or with smoking or thyroid function (Table 4). There was not significant correlation between age and functional score (r = 0.19, p = 0.09.) or with appearance score (r = 0, p = 0.93). The frequency of responses to each question is shown in Table 5. The activities perceived as being most affected were: watching TV (50.8%), reading (46.5%) and doing something they wanted to do (40.9%). The majority of the participants considered that orbital disease altered their appearance (74.7%) or impaired their self-confidence (54.9%). Seventy percent of the patients felt that their visual function was affected in some way, and 79.5% considered the changes in their appearance to interfere with psychosocial function. Table 2. Demographic characteristic of patients that completed de QOL questionnaire Variable Value (n = 73)

95 Questionnaires

Age (years)

42.67 ± 15.2

Sex

22 Excluded surveys 73 Analyzed surveys

17 Patients no ophthalmic evaluation

Female

80.8%

Male

19.2%

Smokers

40.7%

Thyroid function 56 Patients complete evaluation

15 Control Group

41 Clinical GO

Figure 1. Population.

Hiperthyroidism

63.8%

Hiperthyroidism SC

21.8%

Euthyroidism

11.6%

Hipothyroidism

1.4%

Hipothyroidism SC

1.4%

Treatment Methimazole

Demographic characteristics of the 73 patients included are shown in Table 2. Fifty-six patients underwent complete ophthalmic evaluation, 15 of them did not have Graves’ orbitophathy and were considered to be a control group. The remaining 41 were considered to have clinical GO. The classification according to activity and severity of orbital disease is shown in Table 3. Mean CAS was 2.3 ± 1.8. There were no significant differences in disease severity and activity in relation to gender, thyroid functional status or age. The total mean QOL score over the 73 questionnaires was 67.8 ± 27.3, the means for functional and appearance scores were 67.5 ± 30.1 and 67.4 ± 31.9, respectively. There was a positive correlation between 376

60.5%

Block/replacement

4.7%

No treatment

32.5%

Levothyroxine

4.9%

Previously treated with radioiodine

9.8%

Table 3. Characteristic of orbital disease in 41 patients with clinical GO Activity

Severity

Titled

Total

Inactive

Active

Mild

Moderate

Severe

DON

Total

Arch Endocrinol Metab. 2017;61/4

Quality of life in Graves’ ophthalmopathy

Table 4. Functional and appearance scores according to demographic characteristics and ophthalmic evaluation Functional QOL

0.47

Sex

0.72

Female

65.6 ± 29.4

66.7 ± 32.6

Male

72.1 ± 35.2

70 ± 29.8

Smokers

Appearance QOL

0.58

0.32

Yes

66.7 ± 33.7

58.8 ± 33.9

62.1 ± 30.3

67.7 ± 31.3

Thyroid function

0.19

0.08

Hiperthyroidism

68.9 ± 28.8

72.8 ± 31.3

Hiperthyroidism SC

73.6 ± 27.7

68.7 ± 31.3

Euthyroidism

51.3 ± 38.1

46.2 ± 32.2

Hipothyroidism*

18.7

Hipothyroidism SC*

68.7

56.2

Attend ophthalmic assessment

0.21

0.09

Yes

64.4 ± 32.1

62.5 ± 32.3

74.9 ± 23.1

77.2 ± 26.9

Orbitopathy Control group Severity

75 ± 28.9

88.3 ± 17 0.00

76.9 ± 23.7

0.00

66.3 ± 30.3

Mild Moderate

70.9 ± 16.5

40 ± 33.2

Severe

40.9 ± 29.9

38.7 ± 29.4

6.8 ± 8.9

37.5 ± 8.8

DON Activity Active

46.7 ± 34.3

Inactive

69.2 ± 28.8

0.02

0.00

47.3 ± 26.5 56.7 ± 34.3

Functional and appearance scores are expressed as mean ± SD. * n = 1.

Table 5. Frequency of responses to each question of QOL survey Severely limited

Little limited

Not limited

Missing response

Affected

12,7

14,1

45,1

28,2

26,8

Q2. Driving

8,5

14,1

42,3

35,2

22,6

Q3. Moving around the house

16,9

15,5

64,8

2,8

32,4

Q4. Walking outdoors

14,1

19,7

4,2

33,8

Q5. Reading

19,7

26,8

47,9

5,6

46,5

Q6. Watching TV

21,2

29,6

46,5

2,8

50,8

Q7. Hobbies or pastimes

16,9

15,5

52,1

15,5

32,4

Visual functioning (%)

Q1. Riding a bike

Q8. Hindered from doing something they wanted to do

15,5

25,4

56,3

2,8

40,9

Very much

A little

Missing response

Affected

Q9. Changed appearance

43,7

22,5

2,8

74,7

Q10. Stared in the streets

22,5

15,5

Q11. People react unpleasantly

8,5

16,9

73,2

1,4

25,4

Q12. Influence on self confidence

22,5

32,4

42,3

2,8

54,9

Q13. Socially isolated

15,5

11,3

70,4

2,8

26,8

Q14. Influence on making friends

14,1

71,8

28,2

Q15. Appear less often on photos than before

28,2

11,3

60,6

39,5

Q16. Mask changes in your appearance

25,4

49,3

50,8

Appearance (%)

Limitation in carrying out the following activity

Frequency of responses to each question about visual function and psychosocial consequences as a result of changed appearance expressed as percentage in 73 patients.

Arch Endocrinol Metab. 2017;61/4

377

Quality of life in Graves’ ophthalmopathy

When evaluating only the group of patients with GO (n = 41), 75.6% had altered visual function and 95.1% perceived a disturbance in their appearance. The appearance QOL score of the patients with GO (53 ± 31.4) was significantly lower than the control group (88.3 ± 17) (p < 0.000), but no difference was observed in functional QOL score (60.5 ± 32.6; 75 ± 28.9, respectively) (p = 0.161). The results of the questionnaire were not different between the patients who did not attend ophthalmic assessment (Functional QOL 74.9 ± 23.1; Appearance QOL 77.2 ± 26.9) and those who attended the ophthalmic appointment (Functional QOL 64.4 ± 32.1; Appearance QOL 62.5 ± 32.3) (p = 0.21 and p = 0.09, respectively).

Results of the survey and GO activity There was significant difference in both functional (p = 0.026) and appearance (p < 0.000) QOL scores, among controls, inactive GO and active GO groups. Post hoc analysis for functional QOL showed statistically significant differences between patients with active GO vs the control group (p = 0.043). For appearance QOL, patients with active as well as inactive GO had lower scores than the control group (p < 0.000 and p < 0.001, respectively). No differences were observed between patients with inactive GO vs. those with active GO in this subscale (Figure 2).

In summary, functional QOL was mostly affected by the activity of the disease and appearance QOL was mainly altered just by having/bearing the orbital disease. There was no significant correlation between CAS and the QOL subscales, although there was a tendency for a negative correlation between CAS and functional QOL (p = 0.056) (Figure 3A).

Results of the survey and GO severity There was significant difference in both, functional (p < 0.000) and appearance (p < 0.000) QOL scores, between controls and the different degrees of GO. Post hoc analysis showed that patients with DON had statistically lower functional QOL than patients with mild GO (p < 0.000), moderate GO (p = 0.008) and the control group (p = 0.001). Likewise, patients with severe GO had statistically lower functional scores than those with mild GO (p = 0.016) and controls (p = 0.039). In the appearance subscale, the difference was set between the control group and each one of other groups of patients, those with moderate GO (p = 0.014), severe GO (p = 0.001) and DON (p = 0.031) (Figure 4). There was a negative correlation between the severity of GO and both functional and appearance QOL (p < 0.000) (Figure 3B).

110

110 *

60 50 40

100

Appearance QOL (%)

Functional QOL (%)

100

60 50 40

10 0

0 Control group

Inactive GO Activity

Active GO

Control group

Inactive GO

Active GO

Activity

Figure 2. Differences between control group, inactive and active GO in functional and appearance QOL. * p < 0.05; ** p < 0.001. 378

Arch Endocrinol Metab. 2017;61/4

Quality of life in Gravesâ&#x20AC;&#x2122; ophthalmopathy

Activity

100 90 80 70 60 50 40 30 20 10 0

Severity

Functional QOL

Appearance QOL

Functional QOL

Appearance QOL

Linear (Functional QOL)

Linear (Appearance QOL)

Linear (Functional QOL)

Linear (Appearance QOL)

QOL score

100 90 80 70 60 50 40 30 20 10 0 Control

Mild OG

CAS

Moderate OG

Severe OG

DON

Severity

Figure 3. (A) Pearson Correlation between CAS and Functional QOL (r = -0.301; p = 0.056) and appearance QOL (r = -0.216; p = 0.176). (B) Pearson Correlation between Severity grades and Functional QOL (r = -0.575; p < 0.000) and appearance QOL (r = -0.577; p < 0.000).

110

100

80 Appearance QOL

Functional QOL

100

110

70 60 50

10 0

0 Control group

Mild

Moderate Severity

Severe

DON

Control group

Mild

Moderate Severity

Severe

DON

Patients with indication of immunosuppressive therapy (those with severe and active GO and those with DON), had statistically lower values in both functional (p < 0.000) and appearance (p = 0.009) QOL scores compared with patients that did not require steroid treatment.

DISCUSSION The analysis of this survey in our population showed that more than two thirds of patients with GD (with and without GO) had functional and appearance affectation, regardless of age, sex and thyroid function. The alteration in appearance was present in virtually all Arch Endocrinol Metab. 2017;61/4

patients with GO, with the appearance score significantly lower than controls. Although functional impairment was mentioned by 75.6% of patients with GO, the score was not different from patients without GO. This might be related to the considerable number of mild cases in this study. The functional scale was developed to evaluate the consequences of double vision and decreased visual acuity (5), which are only affected in more severe cases. Therefore, in our patients with DON and severe GO, the functional score was significantly lower than this score in patients with mild/moderate GO or without GO. The present study is, to the best of our knowledge, the first that applied the GO-QOL questionnaire to a control group with GD without clinical GO. 379

Figure 4. Differences between control group and severity grades in functional and appearance QOL. * p < 0.05; # p < 0.00.

Quality of life in Graves’ ophthalmopathy

In this group of patients, 60% felt some impact on daily function and 46.6% felt psychosocial impairment. These results may be explained by the discomfort generated by other symptoms in patients with GD (13) or as a consequence of impaired general health perceptions and effects on quality of life. The results of this survey in this group of patients from Argentina showed that GO has a significant impact on their lives, the majority of respondents reported limitation in daily activities such as reading and watching television, as well as impaired selfconfidence and changes in appearance; similar to those reported by other authors in other countries (7-9). However, we had 35.2% missing answers for driving and 28.2% for cycling. These activities were reported as very frequently affected in other studies (7,14). We consider that this difference may be a consequence of the economic and social characteristics of our hospital populations with limited financial resources. Our patients often live far from their workplace and health care centres and in most cases they work for wages. We believe it would be useful to change these questions for others regarding the use of public transport and the loss of earning capacity, as it is known that GO is an important cause of occupational impairment (15). Active GO patients were more physically impaired than those with inactive or no GO. Unexpectedly, we did not find statistical differences in the psychosocial area between patients with active vs inactive GO. The difference with other studies in a German (14) and a Korean (9) population could possibly be explained by the marked socio-cultural and economic differences of the populations as well as the small number of patients enrolled in this study. We found no differences in functional and appearance scores regarding gender and age. Our results differ from those reported by Wickwar and cols. (8) since they found lower scores in functional QOL in elderly patients and lower scores in appearance QOL in women. The patients were recruited among those assessed to undergo orbital decompressive surgery, thus they included mostly cases with moderate to severe GO. In our sample, there was a high proportion of patients with mild disease, and this fact may partly explain the different results between both studies. In this study, functional QOL was mostly affected by the activity of the disease and appearance QOL was mainly altered simply by the effects of orbital disease. 380

This study was started a year before the Spanish version of the questionnaire was available on the EUGOGO page, so we translated the questionnaire into Spanish. We assumed that the validity and internal consistency of our translation were well preserved as the Cronbach alphas of the Spanish language survey used in this study were comparable with those of the original Dutch GO-QOL survey (5) and those reported by other authors in English and Korean (7-9). The version currently available on the EUGOGO page is not the one we use, although it has no substantial differences with it. Our study has some limitations: first, this was a cross-sectional study with a relatively small number of patients, which makes it hard to clarify a causal relationship; second, among patients who did not return the questionnaire, there was a significant number of patients without GO, which would, at some point, change the results of the control group; third, our results were derived from a relatively specific group of patients seen at a single academic institution, and the condition of patients in our clinic could be different from that of patients in the community setting. In conclusion, GO has significant impact on the quality of life of these Argentine patients. QOL was worse in GO patients than in the control group, functional QOL was mostly affected by activity and appearance QOL was mainly altered simply by the effects of the disease. Patients with more severe GO had lower scores on both QOL scales. To our knowledge, this is the first time this quality of life questionnaire for GO patients has been given to a Latin-American population. It is also the first study that compares Graves’ disease patients with and without clinical GO. This information will be useful for designing formal psychological support and treatment to help those with GO overcome their difficulties. Funding: this research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. Disclosure: no potential conflict of interest relevant to this article was reported.

REFERENCES 1. Burch HB, Wartofsky L. Graves’ ophthalmopathy: current concepts regarding pathogenesis and management. Endocr Rev. 1993;14:747-93. 2. Weetman AP. Thyroid-associated eye disease: pathophysiology. Lancet. 1991;338:25-8. Arch Endocrinol Metab. 2017;61/4

Quality of life in Graves’ ophthalmopathy

3. Kahaly GJ, Petrak F, Hardt J, Pitz S, Egle UT. Psychosocial morbidity of Graves’ orbitopathy. Clin Endocrinol (Oxf). 1991;63:395-402. 4. Wilson IB, Cleary PD. Linking clinical variables with health-related quality of life. A conceptual model of patient outcomes. JAMA. 1995;273:59-65.

10. Son BJ, Lee SY, Yoon JS. Evaluation of thyroid eye disease: quality-of-life questionnaire (TED-QOL) in Korean patients. Can J Ophthalmol. 2014;49(2):167-73.

5. Terwee CB, Gerding MN, Dekker FW, Prummel MF, Wiersinga WM. Development of a disease specific quality of life questionnaire for patients with Graves’ ophthalmopathy: the GO-QOL. Brit J Ophthalmol. 1998;82:773-9.

11. Finamor FE, Martins JRM, Nakanami D, Paiva ER, Manso PG, Furlanetto RP. Pentoxifylline (PTX) – An alternative treatment in Graves’ ophthalmopathy (Inactive phase): assessment by a disease specific quality of life questionnaire and by exophthalmometry in a prospective randomized trial. Eur J Ophthalmol. 2004;14(4):277-83.

6. Terwee CB, Dekker FW, Mourits MP, Gerding MN, Baldeschi L, Kalmann R, et al. Interpretation and validity of changes in scores on the Graves’ ophthalmopathy quality of life questionnaire (GO-QOL) after different treatments. Clin Endocrinol (Oxf). 2001;54:391-8.

12. Bartalena L, Baldeschi L, Dickinson A, Eckstein A, Kendall-Taylor P, Marcocci C, et al. Consensus statement of the European Group on Graves’ orbitopathy (EUGOGO) on management of GO. Eur J Endocrinol. 2008;158:273-85.

7. Park JJ, Sullivan TJ, Mortimer RH, Wagenaar M, Perry-Keene D. Assessing quality of life in Australian patients with Graves’ ophthalmopathy. Brit J Ophthalmol. 2004;88:75-8.

13. Elberling T V, Rasmussen a K, Feldt-Rasmussen U, Hørding M, Perrild H, Waldemar G. Impaired health-related quality of life in Graves’ disease. A prospective study. Eur J Endocrinol. 2004;151:549-55.

8. Wickwar S, McBain HB, Ezra DG, Hirani SP, Rose GE, Newman SP. Which factors are associated with quality of life in patients with Graves’ orbitopathy presenting for orbital decompression surgery? Eye. 2015;29(7):951-7.

15. Ponto KA, Pitz S, Pfeiffer N, Hommel G, Weber MM, Kahaly GJ. Quality of life and occupational disability in endocrine orbitopathy. Deutsches Ärzteblatt International. 2009;106:283-9.

9. Choi YJ, Lim HT, Lee SJ, Lee SY, Yoon JS. Assessing Graves’ ophthalmopathy-specific quality of life in Korean patients. Eye (Lond). 2012;26:544-51.

14. Ponto KA, Hommel G, Pitz S, Elflein H, Pfeiffer N, Kahaly GJ. Quality of life in a German graves orbitopathy population. Am J Ophthalmol. 2011;152:483-90.

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original article

New insights into the effects of irisin levels in HIV-infected subjects: correlation with adiposity, fat-free mass, and strength parameters Joice Cristina dos Santos Trombeta1,2, Jonato Prestes3, Dahan da Cunha Nascimento3, Ramires Alsamir Tibana3, Guilherme Borges Pereira3, Thiago da Rosa Lima2, Géssica Alves Fraga2, Roberto Carlos Vieira-Junior4, Fabrício Azevedo Voltarelli2

Universidade Estadual de Campinas (Unicamp), Campinas, SP, Brasil 2 Universidade Federal de Mato Grosso (UFMT), Cuiaba, MT, Brasil 3 Programa de Pós-Graduação em Educação Física, Universidade Católica de Brasília (UCB), Brasília, DF. Brasil 4 Universidade do Estado de Mato Grosso (Unemat), Caceres, MT, Brasil 1

Correspondence to: Guilherme Borges Pereira Programa de Pós-Graduação em Educação Física, Universidade Católica de Brasília Q.S. 07, Lote 01, EPTC, Bloco G 71966-700 – Taguatinga, Brasília, DF, Brasil gbp.ucb@gmail.com Received on Mar/10/2016 Accepted on Feb/1/2017 DOI: 10.1590/2359-3997000000270

ABSTRACT Objective: Patients infected with the human immunodeficiency virus (HIV) have an increased risk of metabolic disorders and alterations on irisin levels. Therefore, the purpose of the current investigation was to quantify the circulating irisin concentration in HIV-infected subjects under highly active antiretroviral therapy and to determine possible correlations between irisin levels with fat mass, fat-free mass, body mass index (BMI), and muscle strength. Subjects and methods: Cross-sectional study of 10 men (36.7 ± 11.3 years) and 10 women (42.5 ± 10.3 years) infected with HIV, recruited from the Specialized Service Center in the State Center of Reference for High and Medium Complexity. Blood samples were collected to determine plasma irisin levels, glucose, HDL, total cholesterol, triglycerides, and LDL. Body composition (fat mass, fat-free mass) and anthropometrics (body mass index; BMI) were measured by bioelectrical impedance. Muscle strength was assessed using a mechanic hand dynamometer and one maximum repetition tests. Results: Irisin levels correlated positively with fat mass (r = 0.67; p = 0.001) and BMI (r = 0.48; p = 0.036). In contrast, there was an inverse correlation between irisin levels and fat-free mass (r = -0.41; p = 0.008) and five strength parameters: right hand grip (r = -0.46; p = 0.044); left hand grip (r = -0.50; p = 0.027), relative hand grip (r = -0.79; p = 0.001), bench press (r = -0.58; p = 0.009), leg press (r = -0.40; p = 0.085), and biceps curl (r = -0.059; p = 0.009). Conclusion: Irisin levels correlated positively with body fat and negatively with fat-free mass and strength parameters in HIV-infected patients. Female patients infected with HIV receiving highly active antiretroviral therapy have higher levels of irisin compared with men in a similar circumstance. Arch Endocrinol Metab. 2017;61(3):382-90. Keywords Body composition; HIV infection; irisin levels; muscle strength

INTRODUCTION

he skeletal muscle is recognized as an endocrine organ expressing and releasing myokines with autocrine and paracrine effects (1). The peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1-α) is an important molecule expressed in the muscle that plays many biological functions related to energy metabolism (2). Muscle production of PGC1-α is stimulated by exercise and potentially induces the expression and secretion of several muscle gene products such as fibronectin type III domain containing 5 (FNDC5). The FNDC5 gene is processed proteolytically to form irisin, which in turn induces profound changes in subcutaneous adipose tissue by stimulating brown adipose tissue and uncoupling protein 1 (UCP1) expression. Irisin may be a new target 382

for the treatment of metabolic disorders based on its potential to improve the metabolic profile and increase whole body energy expenditure (2). The skeletal muscle is the main source of circulating irisin in animal models and humans (3,4). Rats infected with the human immunodeficiency virus (HIV) lose weight (predominantly due to muscle wasting, with minor changes in fat proportion) (5), which could in part decrease irisin secretion. In a subcohort of morbidly obese subjects with metabolic disorders, circulating irisin levels were significantly associated with FNDC5 gene expression in subcutaneous and visceral adipose tissues instead of muscle mass (6,7). Abnormalities in fat distribution have been described as the most prevalent adverse effect of highly active antiretroviral therapy (HAART) in individuals Arch Endocrinol Metab. 2017;61/4

HIV infection, irisin levels, and body composition

SUBJECTS AND METHODS Participants Twenty volunteers with HIV undergoing HAART were recruited from a Specialized Service Center in the State Center of Reference for High and Medium Complexity Arch Endocrinol Metab. 2017;61/4

(Cuiaba-MT, Brazil). Ten participants were men (36.7 ± 11.3 years), and ten were women (42.5 ± 10.3 years). The participants were older than 18 years, were infected with HIV, and were receiving HAART for ≥ 12 weeks, with a cumulative HAART duration of 6.40 ± 5.93 years. Eligible participants were adults classified as “sedentary” by the International Physical Activity Questionnaire (14,15) and without contraindications for exercise identified during a cardiovascular exercise stress test. All participants were under antiretroviral therapy. The HAART contained a combination of nucleosideanalogue reverse transcriptase inhibitors (NRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), and integrase inhibitors (IIs). Sixteen participants were receiving two types of NRTIs and one type of NNRTI; two participants were receiving two PIs and three NRTIs; one participant was receiving two PIs and two NRTIs; and one participant was receiving two PIs, two NRTIs, and one II. None of the participants showed a positive diagnosis for lipodystrophy syndrome. Potential participants were excluded if presenting an acute or chronic inflammatory condition (besides HIV), pregnant/lactating, having prior myocardial infarction and vascular diseases, receiving systemic chemotherapy or steroids, having uncontrolled diabetes mellitus, using anabolic agents and growth hormone, or showing a CD4+ T cell count below 200 cells/mm3. Table 1 shows the characteristics of the overall cohort. The study design and employed procedures are in accordance with ethical standards and the Declaration of Helsinki. All subjects were fully informed about the risks and benefits associated with the participation in the study and gave their written informed consent for participation. The study was approved by the Research Ethics Committee at Julio Muller University Hospital (N: 673/09).

Clinical assessments The present study was designed to quantify the circulating irisin concentration and determine possible correlations of irisin levels with fat mass, fat-free mass, BMI, and muscle strength in subjects with HIV. All testing and procedures were conducted between 2:00 – 5:30 p.m. Initially, self-reported demographics, and medical and HIV treatment history were obtained and confirmed by medical records. Targeted physical exams were obtained, including anthropometric measurements, maximal strength tests (one-repetition 383

with HIV (8). A working hypothesis for the responsible mechanisms implicates mitochondrial bioenergetics disturbances characterized by mitochondrial toxicity. Mitochondrial toxicity leads to the activation of lipolysis, apoptosis, and abnormal release of proinflammatory cytokines (TNF-alpha, IL-6). This cascade of events may be responsible for the subsequent alteration in the overall metabolism, characterized by insulin resistance, dyslipidemia, and vascular damage, creating an imbalance of the anabolic and catabolic systems and resulting in loss of muscle mass and function (9,10). Further evidence suggests that irisin levels are associated with body mass index (BMI). Stengel and cols. verified that under pathological conditions, obese individuals with BMI ≥ 40 kg/m2 and mean age of 48 years exhibited higher circulating irisin levels when compared with individuals characterized as having a normal weight and being anorexic (11). Furthermore, plasma irisin levels correlated positively with body mass, fat mass, BMI, fat-free mass, waist-to-hip ratio, and insulin levels. The authors speculated that the higher irisin levels found in obese individuals might also represent a physiological attempt to restore glucose tolerance. In addition, irisin correlated positively with fasting insulin, homeostasis model assessment of insulin resistance (HOMA-IR), age, cholesterol ratio, and BMI in patients with gestational diabetes (12). Furthermore, irisin levels are elevated in individuals with HIV compared with those without HIV and are unaffected by lifestyle modifications, including exercise (13). Therefore, the available data regarding irisin and body composition in humans are inconclusive, especially in HIV-infected subjects. Thus, the aim of the present study was to quantify the circulating irisin concentration in individuals with HIV undergoing HAART and identify potential correlations between irisin levels and fat mass, fat-free mass, BMI, and muscle strength. The initial hypothesis was that irisin levels would correlate positively with fat mass and negatively with fat-free mass and strength parameters in this population.

HIV infection, irisin levels, and body composition

maximum, 1RM), and a hand grip strength test using a dynamometer. Prior to the physical evaluation, the participants reported to the laboratory between 7:00 – 10:00 a.m., following an overnight fast for blood sampling. Blood was collected from an antecubital vein for subsequent analysis of biochemical variables. HIV-1 RNA level and CD4+ lymphocyte count were obtained as part of a routine clinical care. All participants were encouraged to avoid smoking, alcohol, and caffeine consumption, as well as unusual physical activity before each test and blood sample collection.

Determination of circulating irisin and other biochemical measurements The participants reported to the laboratory between 08:00–10:00 a.m. after an overnight fast (12 hours), and blood samples (5 mL) were drawn from an antecubital vein into Vacutainer tubes (Becton Dickinson, Brazil). Briefly, samples were centrifuged at room temperature at 2,500 rpm for 15 min, and serum irisin concentrations were determined in duplicate using a commercial enzyme-linked immunosorbent assay (ELISA) kit (MyBioSource Inc., San Diego, CA, USA). Recently, Anastasilakis and cols. found that circulating irisin has a day-night variation with the lowest levels at 6:00 a.m. and the highest ones at 9:00 p.m. However, the blood collection was performed consistently at the same time of the day for individual participants (16). The minimum detectable level was 27.85 ng/mL. The intraassay and interassay coefficients of variation were 2.9–9.5% and 5.9–7.0%, respectively, and the sensitivity was 0.0093 pg/mL. Fasting plasma glucose, highdensity lipoprotein (HDL), total cholesterol, and triglycerides were measured with colorimetric methods using a kit by Linco Research, Inc. (St. Charles, MO, USA) and by an automated analyzer (Human GmbH, Germany), while low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) were estimated by the Friedewald equation (17).

Anthropometric and body composition analysis Height was determined with a stadiometer, and body mass was recorded with a calibrated digital scale (Welmy® W300, São Paulo, Brazil). Body composition was measured using bioelectrical impedance analysis (Maltron® BF 906, São Paulo, Brazil) and absolute fat (kg), fat (%), absolute lean mass (kg), relative (%) lean mass, and BMI were obtained. The participants were 384

advised to avoid alcohol, food or beverages containing stimulants, to ingest a minimum of 2 liters of water, and urinate up to 30 min before the test. On the assessment day, the participants’ earrings, chains and/ or rings and shoes were removed. In order to perform the test, the participants were positioned in dorsal decubitus and electrodes were attached (left side) to their hands and feet.

One-repetition maximum (1RM) muscle strength test The participants performed two sessions of familiarization on bench press, 45° leg press, and biceps curl to establish the correct biomechanics of each movement. After the familiarization period, the participants performed a 1RM test and retest on two nonconsecutive days (minimum of 72 hours between tests). After 5 min of a light treadmill running, the participants performed eight repetitions of a specific warm-up with 50% of the estimated 1RM (according to the previous loads used by the participants in their familiarization sessions), followed by three repetitions with 70% of the estimated 1RM. Consecutive trials were performed for one repetition with progressively heavier loads. The 1RM was determined from three attempts, using 3- to 5-minute rest intervals between each attempt. The intraclass correlation coefficient was r = 0.96 for the tested exercises, thus confirming the test-retest reliability.

Hand grip strength measurement Isometric hand grip strength was determined by a mechanical hand dynamometer (Takei Digital Grip Strength Dynamometer, Model T.K.K., Takei, Scientific Instruments Co., Ltd., Niigata City, Japan). The participants stood up with both arms extended and their forearms in neutral rotation. The dynamometer grip was individually adjusted for each participant so that the interior stem of the dynamometer was positioned over the second phalanges of the index, middle, and annular fingers. The recovery time between measurements was 1 minute. The test was performed over three attempts in both hands. The best score of all three attempts was used as the value for each hand.

Statistical analysis Data are expressed as mean ± standard deviation (18). The Shapiro-Wilk test was applied to verify the normality of the distribution of the variables. Spearman’s test was Arch Endocrinol Metab. 2017;61/4

HIV infection, irisin levels, and body composition

used to verify the correlation of irisin levels with body composition parameters, BMI, strength variables, and HAART. An r value ≥ 0.5 determined a large correlation effect (19). To further investigate the potential predictors involved in irisin level changes, a multiple linear regression model was developed including the factors fat mass and fat-free mass adjusted for sex. The Mann-Whitney test was used for comparisons between the sexes. A post hoc power analysis for multiple linear regression (R² increase) was calculated. Considering our estimated R² of 0.57 for the entire regression model and the three predictors used, we estimated that a total sample size of 16 subjects was required to achieve a power level of 0.97 (Supplement 1). The a priori level of significance was p ≤ 0.05. The analyses were performed with the software SPSS, version 20.0 (SPSS Inc., Chicago, IL, USA), and G*Power 3.1.6 was used to calculate the statistical power (20,21). The authors declare that the study design and procedures employed are in accordance with ethical standards and the Declaration of Helsinki. Each subject was fully informed about the risks associated A 60

with participation in the study and gave their written informed consent for participation. The study was approved by the Research Ethics Committee at Julio Muller University Hospital (protocol number: 673/09).

RESULTS Figure 1 presents the correlation of irisin levels with BMI, fat mass, and fat-free mass. Irisin levels correlated positively with two adiposity parameters: fat (r = 0.67; p = 0.001) and BMI (r = 0.48; p = 0.036). In contrast, there was an inverse correlation between irisin levels and fat-free mass (r = -0.41; p = 0.008) and each of the five strength parameters: relative hand grip (r = -0.79; p = 0.001), right hand grip (r = -0.46; p = 0.044), left hand grip (r = -0.50; p = 0.027), bench press 1RM (r = -0.58; p = 0.009), and biceps curl 1RM (r = -0.59; p = 0.009) (Figure 2). Although irisin levels showed an inverse correlation with leg press 1RM (r = -0.40; p = 0.085), this assessment was not statistically significant (Figure 2). Differences between men and women were observed (Table 1). For the anthropometric measures, B 80

r = 0.67; P = 0.001

r = 0.48; P = 0.036

Fat mass (kg)

BMI (kg/m2)

60 40

0 100

200

300

100

Irisin (ng/mL)

Fat-free mass (kg)

200

300

Irisin (ng/mL)

r = -0.41; P = 0.008

20 0

100

200

300

Irisin (ng/mL)

Figure 1. Correlation between irisin levels, parameters of obesity, and fat-free mass. (A) Correlation between body mass index (BMI) and irisin levels; (B) Correlation between fat mass and irisin levels; (C) Correlation between fat-free mass and irisin levels. Arch Endocrinol Metab. 2017;61/4

385

HIV infection, irisin levels, and body composition

men exhibited higher fat-free mass when compared with women (p = 0.001). For the strength parameters, men exhibited a higher left and right hand grip strength (p = 0.002 and p = 0.004, respectively), bench press 1RM (p = 0.001), leg press 1RM (p = 0.001), and biceps curl 1RM (p = 0.001) when compared with women. For the biochemical markers, women presented higher HDL (p = 0.013) and irisin plasma levels as compared with men (p = 0.022). There were A 1.0

no differences in HIV infection duration between groups (p = 0.124). A numerical but not significant difference between men and women was observed regarding the duration of exposure to HAART (p = 0.062) (Table 1). In addition, the regression model predicting changes in irisin levels varied by 49% (p = 0.002) in regards to fat content and 25% to fat-free mass (p = 0.038), whereas sex was not statistically associated (p = 0.539) (Table 2). D

r = -0.79; P = 0.001

Bench press (kg)

Relative hand grip

0.8 0.6 0.4

r = -0.58; P = 0.009

0.2

0.0 0

200

100

300

100

Irisin (ng/mL)

E 400

r = -0.46; P = 0.044

Leg press (kg)

Right hand grip (kg)

300

200

300

200

300

r = -0.40; P = 0.085

300

200

100

0 0

200

100

300

100

Irisin (ng/mL)

200 Irisin (ng/mL)

Irisin (ng/mL)

r = -0.50; P = 0.027

r = -0.59; P = 0.009

Biceps curl (kg)

Left hand grip (kg)

40 40

30 20

10 0

0 0

200

100 Irisin (ng/mL)

300

100 Irisin (ng/mL)

Figure 2. Correlation between irisin levels and strength variables. (A) Correlation between irisin levels and relative hand grip strength; (B) Correlation between irisin levels and right hand grip strength (kg); (C) Correlation between irisin levels and left hand grip strength (kg); (D) Correlation between irisin levels and bench press 1RM (kg); (E) Correlation between irisin levels and leg press 1RM (kg); (F) Correlation between irisin levels and biceps curl 1RM (kg). 386

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HIV infection, irisin levels, and body composition

Table 1. Baseline characteristics of the participants presented as mean ± standard deviation (SD) Mean ± SD in all subjects

Mean ± SD in men (n = 10)

Mean ± SD in women (n = 10)

70.64 ± 17.88

75.47 ± 12.74

65.91 ± 21.51

1.64 ± 0.10

1.70 ± 0.08

1.57 ± 0.08

BMI, kg/m²

26.53 ± 8.01

26.03 ± 4.83

27.03 ± 10.57

Fat mass, %

30.57 ± 11.29

29.95 ± 8.52

34.19 ± 12.94

Fat mass, kg

23.00 ± 13.88

21.06 ± 10.22

24.95 ± 17.15

Fat-free mass, %

69.47 ± 11.36

73.03 ± 8.57

65.92 ± 13.08

Fat-free mass, kg

48.01 ± 9.11

54.74 ± 6.74*

41.29 ± 5.40

Relative hand grip

0.54 ± 0.14

0.59 ± 0.15

0.48 ± 0.12

Right hand grip, kg

39.05 ± 11.88

46.30 ± 11.97*

31.80 ± 6.17

Left hand grip, kg

35.40 ± 10.53

42.10 ± 9.86*

28.70 ± 6.09

Anthropometric measures Body weight, kg Height, cm

Strength parameters

Bench press 1RM, kg

31.92 ±15.54

43.88 ± 14.03*

21.15 ± 6.00

Leg press 1RM, kg

201.95 ± 79.66

261.11 ± 61.73*

148.70 ± 51.53

Biceps curl 1RM, kg

24.39 ± 9.04

31.05 ± 7.82*

18.40 ± 4.99

115.62 ± 72.41

85.23 ± 77.45*

142.96 ± 58.33

Biochemical measures Irisin, ng/mL Glucose, mg/dL Cholesterol, mg/dL

91.61 ± 9.45

90.77 ± 11.33

92.44 ± 7.73

179.61 ± 46.30

178.88 ± 53.21

180.33 ± 41.52

HDL, mg/dL

43.77 ± 9.58

38.77 ± 8.96*

48.77 ± 7.64

LDL, mg/dL

103.00 ± 41.73

105.12 ± 48.35

101.11 ± 37.79

VLDL, mg/dL

33.00 ± 19.55

34.25 ± 18.66

31.88 ± 21.88

190.00 ± 115.69

210.44 ± 130.32

160.55 ± 102.56

Infection duration, years

7.85 ± 7.10

6.90 ± 8.06

8.80 ± 6.28

HAART

6.05 ± 5.39

4.60 ± 5.03

7.50 ± 5.60

Triglycerides, mg/dL Medication

BMI: body mass index; HDL: high-density lipoprotein; LDL: low-density lipoprotein; VLDL: very-low-density lipoprotein; HAART: highly active antiretroviral therapy. * Significantly different from women (p < 0.05).

Table 2. Independent effect of body composition on irisin lev Fat mass, kg Fat-free mass, kg

B (95% confidence interval)

P value

0.75 (1.70 – 6.03)

0.002*

-0.66 (-10.51 – 0.34)

0.038*

Adjusted for sex (0 = men; 1 = women). * Statistically significant (p < 0.05).

Arch Endocrinol Metab. 2017;61/4

The correlation between irisin levels and HAART was not statistically significant (p = 0.38) (Table 3).

DISCUSSION The aim of this study was to quantify the circulating irisin concentration in patients with HIV undergoing HAART and identify potential correlations between irisin levels and body composition and muscle strength. 387

The results revealed weak positive correlations of HAART with BMI, percentage fat mass, fat mass, and percentage fat-free mass, and a negative and significant correlation with fat-free mass. In addition, there was a weak negative correlation of HAART with relative hand grip (p = 0.86), right hand grip (p = 0.22), and left hand grip (p = 0.16). The correlations between HAART and bench press (p = 0.02), leg press (p = 0.00), and biceps curl (p = 0.04) were negative and significant.

HIV infection, irisin levels, and body composition

Table 3. Correlation between highly active antiretroviral therapy (HAART), obesity parameters, and strength variables R

BMI, kg/mÂ˛

-0.74

0.75

Fat mass, %

0.03

0.87

Fat mass, kg

0.21

0.92

Fat-free mass, %

-0.03

0.85

Fat-free mass, kg

-0.49

0.02*

Relative hand grip

0.04

0.86

Right hand grip, kg

-0.28

0.22

Left hand grip, kg

-0.31

0.16

Bench press 1RM, kg

-0.52

0.02*

Leg press 1RM, kg

-0.65

0.00*

Biceps curl 1RM, kg

-0.46

0.04*

0.21

0.38

Anthropometric measures

Strength parameters

Biochemical measures Irisin, ng/mL * Statistically significant (p < 0.05).

BMI: body mass index; HAART: highly active antiretroviral therapy.

The initial hypothesis was confirmed, and the results revealed a positive correlation of irisin levels with body fat and negative correlation with fat-free mass and strength parameters in HIV-infected patients. It is important to highlight the pathophysiological significance of the correlation between irisin levels, adiposity, fat-free mass, and strength variables. Our results contrast with the idea that the skeletal muscle is the main source of irisin secretion. Although the expression of the FNDC5/irisin gene in the muscle was not determined, irisin correlated positively with fat mass and negatively with fat-free mass. Intriguingly, we observed that 16 weeks of resistance training increased muscle strength and fat-free mass, and decreased fat mass, but did not change the circulating irisin levels in obese older women (22). The source of circulating irisin in humans seems to be multicompartmental. Although the skeletal muscle is the tissue with the highest expression of FNDC5/ irisin (2), other tissues, including the adipose tissue, are responsible for secreting irisin (6,7,23). Additionally, the expression of irisin is upregulated in individuals characterized as obese when compared with others who have a normal weight and are anorexic. Furthermore, irisin correlates positively with fat mass, BMI (11), fasting insulin, and HOMA-IR in women with gestational diabetes (12). Levels of irisin also correlate 388

with reactive oxygen species, which play an important role in the development of insulin resistance and metabolic syndrome and are required for basal insulin signaling (24). Measurement of irisin from glycolytic versus oxidative muscle in response to acute exercise would also be an interesting assessment to compare different sources of irisin production (24). Our findings raise two important hypotheses. Individuals with HIV present upregulated irisin receptors and/or irisin resistance at the receptor level, which may compromise the positive effects of irisin on glucose homeostasis and insulin sensitivity. Although insulin resistance was not analyzed in our study, we speculate that upregulation of the irisin receptor may positively influence the metabolic homeostasis in HIVinfected individuals. This is an interesting topic to be confirmed in future studies. FNDC5/irisin expression has been found to be higher in obese patients with pre-diabetes and reduced by 40% in those with type 2 diabetes (23). In addition, circulating irisin levels have been shown to decrease following bariatric surgery in obese adults (25). Considering the possible benefits of BMI and body mass reduction, and the restoration of metabolic homeostasis, one would expect irisin levels to improve after surgery, but this was not the case (25). Moreover, circulating irisin levels appeared to be higher in women than men (25). This finding is in accordance with our cross-sectional study, in which HIV-infected women presented significantly higher baseline irisin levels as compared with men. Pharmacological treatment may also influence irisin and HDL cholesterol levels. Recent data has revealed that lipid-lowering drugs affect circulating irisin levels in humans and PIs increase HDL cholesterol levels in women infected with HIV (26). In this sense, women presented longer infection and HAART durations when compared with men, although this finding was not significant, while a poor correlation between HAART and irisin levels was observed. Recently, Srinivasa and cols. investigated the effects of lifestyle modifications on circulating irisin and fibroblast growth factor 21 (FGF21) and their relationship with brown adipose tissue gene expression in dorsocervical subcutaneous fat. The authors demonstrated in individuals infected with HIV, a population prone to metabolic complications, that baseline FGF21 levels are increased when compared with a healthy cohort, and that FGF21 levels decrease relatively with lifestyle modifications. Arch Endocrinol Metab. 2017;61/4

HIV infection, irisin levels, and body composition

Arch Endocrinol Metab. 2017;61/4

to include a control group, but due to the patients’ lack of adherence and study dropout, it was impossible to include these data. The dropouts were mainly due to lack of benefits from participation in the study, as reported by the patients. Finally, we only assessed the plasma concentration of circulating irisin, while the expression of FNDC5 in the skeletal muscle and adipose tissue was not determined. In conclusion, irisin levels correlated positively with body fat and negatively with fat-free mass and strength parameters in HIV-infected subjects, which might suggest that the disturbances in adipose tissue and fatfree mass characteristic of the HIV disease could be responsible for the alterations in irisin levels. Future studies should determine the possible clinical relevance of these results in HIV patients. Acknowledgments: we thank the Brazilian foundation Fundação de Amparo à Pesquisa do Estado de Mato Grosso – FAPEMAT (Process number: 331264/2012) for the financial support. The authors alone are responsible for the content and writing of the paper. Disclosure: no potential conflict of interest relevant to this article was reported.

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The effects of increased FGF21 may be to promote increased expression of brown adipose tissue genes in a unique ectopic adipose depot. In contrast, irisin levels are elevated in individuals infected with HIV but are not influenced by lifestyle modifications or associated with brown adipose tissue gene expression (13). In previous studies that included pathological conditions, irisin correlated positively with BMI and fat mass and negatively with fat-free mass (25,27). In sedentary, overweight, and obese individuals, plasma irisin levels were positively associated with maximal contraction force and rate of force development after 12 weeks of resistance and endurance training (23). A decrease in irisin secretion contributes to muscle insulin resistance in high-fat diet mice (28). Thus, it is possible that the muscle/adipose irisin secretion ratio may vary according to the physiological condition of HIV-infected individuals. For example, the skeletal muscle and the adipose tissue are affected during HAART, which could modulate irisin levels. This has been confirmed by the negative correlation between HAART, fat-free mass, and strength parameters. These findings reinforce the need for further studies to elucidate whether irisin is involved in the HIV pathology and to verify the existence of irisin resistance and its correlation with metabolic disturbances present in HIV-infected subjects. Of note, the real effect of irisin in humans remains unclear, as it has been shown that FNDC5 is present in rodents and most primates, while humans present a mutation in the conserved start codon ATG to ATA. Thus, the results of irisin studies in humans should be interpreted with caution, and findings in mice should not be directly translated into humans (29). Moreover, serum irisin was measured by ELISA, which is based on polyclonal antibodies not previously tested for crossreaction with serum proteins (30). However, it has been proposed that the variations in irisin concentrations found with different ELISA kits are consistent across independent studies with different cohorts of patients (31). The ELISA kit used in the present study has not been negatively tested in other studies and has acceptable levels of detection and reliability. The present study has some limitations that should be considered, such as the small sample size and lack of a control group (individuals not infected with HIV or at an early stage of the disease), and a causal-effect relationship should not be interpreted from the results. Although our sample size was reduced, the sample power calculation revealed a moderate to strong effect size for all variables (data not shown). We initially tried

HIV infection, irisin levels, and body composition

11. Stengel A, Hofmann T, Goebel-Stengel M, Elbelt U, Kobelt P, Klapp BF. Circulating levels of irisin in patients with anorexia nervosa and different stages of obesity--correlation with body mass index. Peptides. 2013;39:125-30. 12. Ebert T, Stepan H, Schrey S, Kralisch S, Hindricks J, Hopf L, et al. Serum levels of irisin in gestational diabetes mellitus during pregnancy and after delivery. Cytokine. 2014;65(2):153-8. 13. Srinivasa S, Wong K, Fitch KV, Wei J, Petrow E, Cypess AM, et al. Effects of lifestyle modification and metformin on irisin and FGF21 among HIV-infected subjects with the metabolic syndrome. Clin Endocrinol (Oxf). 2015;82(5):678-85. 14. Bassett DR Jr. International physical activity questionnaire: 12-country reliability and validity. Med Sci Sports Exerc. 2003;35(8):1396. 15. Hallal PC, Gomez LF, Parra DC, Lobelo F, Mosquera J, Florindo AA, et al. Lessons learned after 10 years of IPAQ use in Brazil and Colombia. J Phys Act Health. 2010;7 Suppl 2:S259-64. 16. Anastasilakis AD, Polyzos SA, Saridakis ZG, Kynigopoulos G, Skouvaklidou EC, Molyvas D, et al. Circulating irisin in healthy, young individuals: day-night rhythm, effects of food intake and exercise, and associations with gender, physical activity, diet, and body composition. J Clin Endocrinol Metab. 2014;99(9):3247-55. 17. Martin SS, Blaha MJ, Elshazly MB, Brinton EA, Toth PP, McEvoy JW, et al. Friedewald-estimated versus directly measured lowdensity lipoprotein cholesterol and treatment implications. J Am Coll Cardiol. 2013;62(8):732-9. 18. Chen JQ, HuangYY, Gusdon AM, Qu S. Irisin: a new molecular marker and target in metabolic disorder. Lipids Health Dis. 2015;14:2.

22. Tibana RA, da Cunha Nascimento D, Frade de Souza NM, de Souza VC, de Sousa Neto IV, Voltarelli FA, et al. Irisin Levels Are not Associated to Resistance Training-Induced Alterations in Body Mass Composition in Older Untrained Women with and without Obesity. J Nutr Health Aging. 2017;21(3):241-6. 23. Kurdiova T, Balaz M, Vician M, Maderova D, Vlcek M, Valkovic L, et al. Effects of obesity, diabetes and exercise on Fndc5 gene expression and irisin release in human skeletal muscle and adipose tissue: in vivo and in vitro studies. J Physiol. 2014;592(Pt 5):1091-107. 24. Huh JY, Mantzoros CS. Irisin physiology, oxidative stress, and thyroid dysfunction: What next? Metabolism. 2015;64(7):765-7. 25. Huh JY, Panagiotou G, Mougios V, Brinkoetter M, Vamvini MT, Schneider BE, et al. FNDC5 and irisin in humans: I. Predictors of circulating concentrations in serum and plasma and II. mRNA expression and circulating concentrations in response to weight loss and exercise. Metabolism. 2012;61(12):1725-38. 26. Gouni-Berthold I, Berthold HK, Huh JY, Berman R, Spenrath N, Krone W, et al. Effects of lipid-lowering drugs on irisin in human subjects in vivo and in human skeletal muscle cells ex vivo. PLoS One. 2013;8(9):e72858. 27. Pardo M, Crujeiras AB, Amil M, Aguera Z, Jimenez-Murcia S, Banos R, et al. Association of irisin with fat mass, resting energy expenditure, and daily activity in conditions of extreme body mass index. Int J Endocrinol. 2014;2014:857270. 28. Yang Z, Chen X, Chen Y, Zhao Q. Decreased irisin secretion contributes to muscle insulin resistance in high-fat diet mice. Int J Clin Exp Pathol. 2015;8(6):6490-7. 29. Raschke S, Elsen M, Gassenhuber H, Sommerfeld M, Schwahn U, Brockmann B, et al. Evidence against a beneficial effect of irisin in humans. PLoS One. 2013;8(9):e73680.

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21. Beck TW. The importance of a priori sample size estimation in strength and conditioning research. J Strength Cond Res. 2013;27(8):2323-37.

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Arch Endocrinol Metab. 2017;61/4

review

The use of aromatase inhibitors in boys with short stature: what to know before prescribing? Departamento de Fisiologia, Unidade de Farmacologia, Faculdade de Ciências Médicas da Santa Casa de São Paulo (FCMSCSP), São Paulo, SP, Brasil 2 Departamento de Pediatria, Unidade de Endocrinologia Pediátrica, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP), São Paulo, SP, Brasil 3 Departamento de Fisiologia, Disciplina de Medicina Molecular, Unidade de Endocrinologia Pediátrica, FCMSCSP, São Paulo, SP, Brasil 1

Alessandra Linardi1, Durval Damiani2, Carlos A. Longui3

ABSTRACT Aromatase is a cytochrome P450 enzyme (CYP19A1 isoform) able to catalyze the conversion of androgens to estrogens. The aromatase gene mutations highlighted the action of estrogen as one of the main regulators of bone maturation and closure of bone plate. The use of aromatase inhibitors (AI) in boys with short stature has showed its capability to improve the predicted final height. Anastrozole (ANZ) and letrozole (LTZ) are nonsteroidal inhibitors able to bind reversibly to the heme group of cytochrome P450. In this review, we describe the pharmacokinetic profile of both drugs, discussing possible drug interactions between ANZ and LTZ with other drugs. AIs are triazolic compounds that can induce or suppress cytochrome P450 enzymes, interfering with metabolism of other compounds. Hydroxilation, N-dealkylation and glucoronidation are involved in the metabolism of AIs. Drug interactions can occur with azole antifungals, such as ketoconazole, by inhibiting CYP3A4 and by reducing the clearance of AIs. Antiepileptic drugs (lamotrigine, phenobarbital, and phenytoin) also inhibit aromatase. Concomitant use of phenobarbital or valproate has a synergistic effect on aromatase inhibition. Therefore, it is important to understand the pharmacokinetics of AIs, recognizing and avoiding possible drug interactions and offering a safer prescription profile of this class of aromatase inhibitors. Arch Endocrinol Metab. 2017;61(3):391-7. Keywords Anastrozole; letrozole; short stature; drug interaction; cytochrome P450

Authorship: all authors have participated equally in the article conception, written aspects and final approval of the version sent to be published. Correspondence to: Carlos A. Longui Departamento de Fisiologia, Disciplina de Medicina Molecular, Unidade de Endocrinologia Pediátrica, Faculdade de Ciências Médicas da Santa Casa de São Paulo Rua Dr. Cesário Mota Jr., 112 01221-020 – São Paulo, SP, Brasil carloslongui@msn.com Received on Fev/13/2017 Accepted on Apr/6/2017

INTRODUCTION

romatase is a cytochrome P450 enzyme, identified as the CYP19A1 isoform, which catalyzes the conversion of androstenedione and testosterone to estrone and estradiol, respectively. The enzyme is a complex formed by two proteins: CYP19 and nicotinamide-adenine dinucleotide phosphate reductase (NADPH) (1). In males, androgens are produced both by the adrenal glands and the testes, whereas estrogen is mostly synthesized locally in peripheral tissues from the local aromatization of circulating androgens (2,3). Adipose tissue is the primary site of aromatization, but aromatase can be found at other sites such as the brain (hypothalamus, limbic system, and cerebral cortex), breast, placenta, liver, muscle, bone (osteoblast and chondrocyte), testes (Leydig cell and germ cell), vasculature (smooth muscle cell), and skin (fibroblast Arch Endocrinol Metab. 2017;61/4

and hair follicle) (4,5). Peripherally synthesized estrogen seems to have predominantly local effects (2,3,6). Men with congenital aromatase deficiency have tall stature associated with delayed fusion of the epiphyseal growth plate, osteoporosis, overweight, glucose intolerance, hyperlipidemia, and reduction of fertility. In girls, virilization of the external genitalia can result in atypical genitalia. Hypogonadism can also occur, with inappropriate mammary gland development and primary amenorrhea (7-9). On the other hand, aromatase excess syndrome is caused by sub chromosomal recombination of CYP19A1 gene (duplication, deletion, and inversion), with concomitant recruitment of new promoters. The clinical presentation includes gynecomastia and bone age advancement with potential reduction of final height (10). 391

DOI: 10.1590/2359-3997000000284

Use of aromatase inhibitors

Estrogen, as well as its receptors, plays a key role in bone maturation (11). The use of aromatase inhibitors, with the goal of reducing the estrogen-dependent skeletal maturation rate has been proposed in boys receiving treatment for short stature (12,13). The effects of these drugs upon growth appear to be mediated by the reduction of estrogen concentration at the epiphyseal chondrocyte level, associated with decreased circulating estrogen concentration and consequent reduction in the secretion of both growth hormone (GH) and insulinlike o growth factor 1 (IGF-1) (14,15). Estrogen has apparently small relevance in the regulation of growth in the prepubertal period, but even at low concentration increases growth during puberty, as well as at high concentration determines maturation and closure of the epiphyses at the final stage of puberty (16). Aromatase inhibitors can be divided into type I and type II. Type I inhibitors are steroidal and derivatives of androstenedione, the natural substrate of the aromatase enzyme. They bind irreversibly to the active site, thereby permanently inactivating the enzyme. The inhibitors of type II are nonsteroidal and bind to the heme group of cytochrome P450. Despite the strong binding, these are reversible inhibitors. The third-generation nonsteroidal aromatase inhibitors, anastrozole and letrozole, are the most effective blocking compounds (> 95%) by suppressing the formation of the estrogens estrone (E1) and estradiol (E2) (17). Additionally, type II inhibitors have a longer half-life, which allows for a single daily administration. Due to the higher potency of letrozole, a greater increase in the concentrations of gonadotropins and testosterone has been reported in boys treated with letrozole when compared to the group treated with anastrozole (18). Considering the increased prescription of both aromatase inhibitors in adolescents with short stature, the aim of this review is to discuss the possible drug interactions between anastrozole and letrozole and other drugs. In addition, a brief description of the pharmacokinetic profile of both inhibitors was included in this review in order to understand the emergence of possible drug interactions related to absorption, metabolism, half-life and excretion of these drugs.

PHARMACOKINETICS Anastrozole and letrozole are administered once daily orally at doses of 1 mg and 2.5 mg, respectively. 392

Maximum estradiol suppression is reached around 2 to 4 days for them both. Oral absorption of both inhibitors is not significantly affected by food (18). Anastrozole reaches the equilibrium plasma concentration, also known as steady state, after 7 days, while it takes letrozole around 45-60 days. This fact is due to the probable nonlinear kinetics of letrozole. The half-life of anastrozole after a single 1-mg dose is 48 hours, while that of letrozole is 4 days or more after a 2.5-mg oral dose (19). At therapeutic doses, around 40% of the anastrozole is bound to plasma proteins (20), while this protein binding rate is around 60% for letrozole (21). Anastrozole and letrozole are triazolic derivatives and therefore have a nitrogen-containing heterocyclic ring. Many azole compounds can be inhibitors or inducers of cytochrome P450 and, consequently, interfere with the metabolism of other substances depending on the monoamino oxigenase system. Lipophilic compounds containing heterocyclic nitrogen, such as phenylpyridines and phenylimidazoles, fit this profile (22,23). Anastrozole, via cytochrome P450 (CYP), is metabolized into hydroxyanastrozole or can also undergo N-dealkylation. The isoforms involved were identified mainly as CYP3A4 and, less importantly, CYP3A5 and CYP2C8. Subsequently, hydroxyanastrozole is extensively conjugated by glucuronidation (Figure 1). The main enzyme involved in this step is uridine glucuronyl transferase 1A4 (UGT1A4). UGT1A3 and UGT2B7 can also act in this phase, as a secondary pathway (24,25). Anastrozole can also undergo direct in vitro conjugation (24) without prior involvement of cytochrome P450 enzymes. In addition, anastrozole is also able to inhibit, in vitro, CYP1A2, 2C9, and 3A4 in human microsomes. However, this inhibition occurs to a much lesser extent than that described for aromatase, and the concentrations required for this purpose are not reached in the systemic circulation with the use of therapeutic doses (23). Letrozole is also cleared via hepatic metabolism. In vitro studies have demonstrated that CYP3A4 and CYP2A6 are involved in the metabolism of letrozole, converting it to carbinol (25,26). Carbinol is then conjugated by glucuronyl transferase, more precisely by the UGT2B7 isoform (Figure 2) (27). The drug has a high affinity for CYP2A6 and a low affinity for CYP3A4. Although letrozole is metabolized by CYP2A6, letrozole itself can exert significant inhibition over this Arch Endocrinol Metab. 2017;61/4

Triazole N-dealkylation Anastrozole UGT1A4 UGT2B7

CYP3A4/5 CYP2C8

Glucuronide-N-anastrozole

Hydroxyanastrozole UGT1A4 UGT2B7 Glucuronide-OH-anastrozole

Figure 1. Anastrozole metabolic pathways. Anastrozole can be coupled directly to glucuronide-N-anastrozole or previously converted to hydroxyanastrozole via cytochrome P450 (CYPs) and subsequent conjugation to glucuronide-OH-anastrozole. In addition, anastrozole can undergo dealkylation by CYP3A4, thereby releasing the triazole ring. The main isoforms involved in anastrozole metabolic pathways are in bold. CYP3A4, CYP3A5, and CYP2C8: cytochrome P450 isoforms. UGT1A4 and UGT2B7: uridine glucuronyl transferase (UGT) isoforms.

Triazole N-dealkylation Letrozole

CYP2A6 CYP3A4

Carbinol UGT2B7

Glucuronide-OH-carbinol

Figure 2. Letrozole metabolic pathways. Letrozole is converted previously to carbinol via cytochrome P450 (CYPs) and then, subsequently, the carbinol is conjugated to a glucuronide. Letrozole can undergo dealkylation, thus releasing the triazole ring. The main isoforms involved in the metabolic pathways are in bold. CYP2A6 and CYP3A4: cytochrome P450 isoforms. UGT2B7: uridine glucuronyl transferase (UGT) isoform.

isoform (28). When this occurs, the metabolic pathway is diverted to CYP3A4 (29). This phenomenon seems to explain why letrozole shows nonlinear kinetics. In women with breast cancer following repeated doses of letrozole, there was observed a 28% increase in the area under the curve and a 42% increase in the drugâ&#x20AC;&#x2122;s halflife when comparing such pharmacokinetic parameters to those obtained from a single administration. Furthermore, the maximum letrozole plasma concentration was 107 nmol/L after a single dose and 467 nmol/L after repeated doses (28).

DRUG INTERACTIONS Azole antifungals, commonly used in clinical practice, can interfere with the activity of aromatase (30). The maximum plasma concentrations of oral and topical azole formulations and the IC50 of aromatase were Arch Endocrinol Metab. 2017;61/4

determined after using testosterone as a substrate. Comparison with letrozole inhibitory potency was then performed. Among the azoles studied, miconazole (oral) and bifonazole (topical) were those that approached letrozole in its ability to inhibit aromatase. The other azoles studied were: clotrimazole (topical), itraconazole (oral), ketoconazole (oral) and miconazole (vaginal). They also exerted an inhibitory effect upon aromatase, but on a much smaller scale. Importantly, this ratio takes the maximum plasma concentrations achieved by antifungals into consideration, which means that an increase in drug absorption can induce greater inhibition of aromatase. Furthermore, there is the possibility of inhibition of CYP3A4, especially by ketoconazole, which reduces the clearance of letrozole and anastrozole when used concurrently with ketoconazole (31). Additionally, a study based on enzyme assays showed that some antiepileptic drugs can inhibit aromatase, including lamotrigine, oxycarbamazepine, tiagabine, phenobarbital, phenytoin, and ethosuximide (32). Therefore, it is important to consider that, in clinical practice, treatment with antiepileptics occurs chronically, and that, in some cases, there may be an association between the drugs. Among these, the concomitant use of phenobarbital or valproate has a synergistic effect on the inhibition of aromatase. Regarding to the use of Cannabis, when studying mouse testis microsomes, it was reported that the extract of Cannabis sativa is capable of inhibiting the activity of CYP17A1 (P450c17), the enzyme responsible for the 17Îą-hydroxylation of progesterone and the subsequent formation of androstenedione, one of the substrates of aromatase (33). The authors also observed a similar result with the main constituents of Cannabis sativa, such as tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN). Alcohol consumption could induce an increase in aromatase expression in the adipose tissue of rats (34). Previously, the authors had also described an increased aromatase activity in the hippocampus of rats, induced by both 13% alcohol and red wine (35). In cultured MCF-7 breast cancer cells, there was observed an increase in the expression of aromatase following incubation with ethanol (36). On the other hand, a study conducted with women around 35 years of age showed that red wine consumption during one month induced an increase in plasma testosterone and a decrease in estrogens. The authors suggest that red wine could be a natural inhibitor of aromatase, due 393

Use of aromatase inhibitors

to the presence of chemical compounds such as the flavones and isoflavones. In this case, it is the chemicals present in red wine that predominate, rather than alcohol itself (37). Considered together, these data seem to demonstrate that drugs such as azoles and antiepileptics may potentiate the action of aromatase inhibitors, further inhibiting the enzyme activity. In addition, Cannabis sativa and its correlates can reduce the formation of the substrate androstenedione and also potentiate the action of aromatase inhibitors. On the other hand, alcohol consumption can increase aromatase activity or expression. Relative to the pharmacokinetic profile of anastrozole and letrozole, genetic polymorphisms may induce differences in the metabolic profile of aromatase inhibitors, primarily identified for letrozole. The CYP2A6 and CYP3A isoforms may have their activity increased or decreased because of polymorphisms. With respect to CYP2A6, intermediate and slow metabolizers exhibit higher letrozole plasma concentrations (38). One of the polymorphisms involving CYP2A6 is related to the genetic variation across different ethnic populations. The percentage of slow metabolizers in the Asian population is larger (10-20%) when compared to Caucasians (1.2%) (39). In addition, the CYP2A6 activity in women is greater than in men. Polymorphisms involving CYP3A4 also have relevant clinical significance. The most common one induces an increase in enzyme activity and has a higher prevalence among black (66.7%) as compared to white subjects (4.2%) (40). However, the clinical impact of genetic variants involving CYP3A4 on plasma concentrations of letrozole or anastrozole needs additional studies. Glucuronyltransferases may also have their activity altered due to polymorphisms. Significant individual variations in the conjugation profile, involving glucuronyltransferase isoforms such as UGT1A4 and UGT2B7 are respectively involved in the metabolism of anastrozole and letrozole (41-43). Glucuronidation profile by UGT1A4 was correlated to therapeutic efficacy of anastrozole in women with breast cancer (44). Nevertheless, the clinical significance of individual or racial variability to the conjugation profile of aromatase inhibitors has not been entirely clarified, and neither has the influence on the therapeutic effect and excretion of these drugs. These aspects have not been adequately investigated in the treatment of children with a short stature. 394

By using human microsomes incubated in the presence of anastrozole and specific substrates for CYP1A2 (phenacetin), CYP2A6 (coumarin), CYP2C9 (tolbutamide), CYP2D6 (dextromethorphan), and CYP3A4 (nifedipine), only CYP1A2, CYP2C9 and CYP3A were shown to be inhibited by anastrozole. Plasma concentrations achieved by the chronic administration of therapeutic doses of anastrozole are 30-fold lower than those required for in vitro inhibition (23). Additionally, the administration of anastrozole in healthy adult volunteers did not interfere with the pharmacokinetics or pharmacodynamics of warfarin, an anticoagulant metabolized by both CYP3A4 (R-warfarin) and CYP2C9 (S-warfarin) (45). The American database Micromedex reported only one drug interaction of moderate severity. It is important to emphasize that Micromedex classifies drug interactions as: major, when they represent a danger to life and require immediate medical intervention; moderate, when they may result in an exacerbation of the health condition and the need for a change in drug therapy; minor, which suggest a change in the clinical picture, but does not require a change in treatment; and the contraindicated, when it implies that the concomitant use of the drugs is not recommended. The moderate drug interaction with anastrozole refers to the concomitant use with tamoxifen (nonsteroidal estrogenic antagonist). In women with breast cancer, tamoxifen reduced the anastrozole plasma concentration by approximately 27% (46). With regard to letrozole, Micromedex data show five drug interactions of major severity and three moderate interactions. Relative to the major severity interactions, we have the antineoplastic agent tegafur, in which inhibition of CYP2A6 by letrozole may result in lesser conversion of tegafur to 5-fluouracil and lower drug efficacy. In addition, the inhibition of CYP2C19 by letrozole may induce an increase in cilostazol plasma concentration, a platelet antiaggregant and vasodilator. The other three major severity interactions refer to the inhibition of CYP3A by clarithromycin (macrolide antimicrobial agent), ceritinib and idelalisib (antineoplastic agents), which results in an increase in letrozole plasma concentration. The three moderate severity interactions include: association with tamoxifen, which induces a reduction in plasma letrozole concentration; concomitant use with propranolol, during which the inhibition of Arch Endocrinol Metab. 2017;61/4

Use of aromatase inhibitors

Arch Endocrinol Metab. 2017;61/4

grapefruit, may all inhibit CYP3A4 and reduce the metabolism of anastrozole by this isoform. On the other hand, antiepileptic drugs (carbamazepine, phenobarbital, and phenytoin), dexamethasone, and rifampicin, in addition to St. Johnâ&#x20AC;&#x2122;s wort can all induce CYP3A4 and increase the metabolism of anastrozole (49-51). When considering CYP2A6, drugs such as valproic acid, selegiline and isoniazid may inhibit the activity of this isoform. On the other hand, carbamazepine, phenobarbital, phenytoin, and clonazepam may induce CYP2A6 (50,52). In addition, minor alkaloids present in tobacco can also inhibit CYP2A6 (53,54). Therefore, inducers and inhibitors of CYP2A6 could influence the pharmacokinetic profile of letrozole. Therefore, when using aromatase inhibitors we should consider the concomitance of clinically relevant interfering aspects, such as drug interaction, food and beverage ingestion, use of illicit drugs and plant extracts. Individual metabolic variability dependent on Table 1. Main metabolism enzymes and potential drug interactions associated with inducers and inhibitors of CYP Drugs

Metabolism

CYP

Phase I

Phase II

Inducers

Inhibitors

Anastrozole

CYP3A4

UGT1A4

CYP3A4: Carbamazepine, phenobarbital, phenytoin, dexamethasone, rifampicin, St. Johnâ&#x20AC;&#x2122;s wort

CYP3A4: Fluoxetine, sertraline, paroxetine, venlafaxine, ketoconazole, itraconazole, fluconazole, voriconazole, clarithromycin, erythromycin, chloramphenicol, isoniazid, diltiazem, verapamil, amiodarone, indinavir, saquinavir, cimetidine, nicardipine, grapefruit

Letrozole

CYP2A6 CYP3A4

UGT2B7

UGT2B7, phenytoin, and clonazepam

CYP2A6: Valproic acid, selegiline, isoniazid, letrozole, and tobacco alkaloids

Phase I: metabolism involving cytochrome P450 enzymes (CYP). Phase II: metabolism involving conjugation enzymes. CYP2A6 and CYP3A4: cytochrome P450 isoforms. UGT1A4 and UGT2B7: uridine glucuronyl transferase (UGT) isoforms. The metabolism of letrozole by CYP2A6 is saturable, given that the drug can inhibit this isoform.

395

CYP2C19 by letrozole may increase the concentration of propranolol; the association with ketoconazole, a potent inhibitor of CYP3A4, which can result in an increase in the concentration of letrozole. A study of postmenopausal women with breast cancer showed that tamoxifen reduces letrozole plasma concentration by 38% (47). In human microsomes, letrozole significantly inhibits CYP2A6 and modestly CYP2C19. Carbinol, a metabolite of letrozole, has a less significant inhibitory effect on CYP2B6 and CYP2C19. Other isoforms studied were not altered by incubation with letrozole or carbinol (48). Another relevant aspect is to consider letrozole plasma concentrations achieved during the chronic administration of the drug. The in vitro letrozole concentration that inhibits CYP2C19 is 40-fold greater than that observed in the in vivo steady state. For CYP2A6, such increase is fivefold. Letrozole may also inhibit its own metabolism due to its greater affinity for CYP2A6. This phenomenon is responsible for higher plasma concentrations of the drug and greater individual variability. In relation to the carbinol metabolite, although a modest inhibitory effect on CYP2B6 and CYP2C19 has been observed in vitro, carbinol is rapidly conjugated in vivo by glucuronidation and subsequently excreted by the kidney (28). Therefore, in respect of letrozole, the following considerations are important, namely: I) the nonlinear kinetics of the drug can cause significant variations in its plasma concentration and, thus, drug interactions involving CYP2A6 cannot be discarded; II) with respect to CYP2C19, the drug does not appear to provoke a significant change in the activity of this isoform of cytochrome P450. Considering the cytochrome P450 isoforms involved in the metabolism of anastrozole and letrozole, it is important to note that polymorphisms leading to changes in the activity of CYP2A6 and CYP3A4, as well as drugs, food and herbal essentials can also act as enzyme inducers or inhibitors (Table 1). Therefore, with regard to CYP3A4, antidepressants (fluoxetine, sertraline, paroxetine, venlafaxine), antifungals (ketoconazole, itraconazole, fluconazole, voriconazole), antimicrobials (clarithromycin, erythromycin, chloramphenicol, and isoniazid), antiarrhythmics (diltiazem, verapamil, and amiodarone), protease inhibitors (indinavir, ritonavir, and saquinavir), cimetidine and nicardipine, besides

Use of aromatase inhibitors

genetic background is also a clinically relevant aspect to be considered, with potential impact on the final drug effects. With this information, it is possible to select the AI with the profile that fits better to individual conditions.

17. Boeddinghaus IM, Dowsett M. Comparative clinical pharmacology and pharmacokinetic interactions of aromatase inhibitors. J Steroid Biochem Mol Biol. 2001;79(1-5):85-91.

Fundings: no financial support was received for this publication.

18. Mauras N. Strategies for maximizing growth in puberty in children with short stature. Pediatr Clin North Am. 2011;58(5):1167-79.

Disclosure: no potential conflict of interest relevant to this article was reported.

19. Buzdar AU, Robertson JF, Eiermann W, Nabholtz JM. An overview of the pharmacology and pharmacokinetics of the newer generation aromatase inhibitors anastrozole, letrozole, and exemestane. Cancer. 2002;95(9):2006-16.

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review

Medullary thyroid carcinoma – Adverse events during systemic treatment: risk-benefit ratio Léa Maria Zanini Maciel1, Patrícia Künzle Ribeiro Magalhães1

ABSTRACT Divisão de Endocrinologia, Departamento de Medicina Interna, Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FMRP-USP), Ribeirão Preto, SP, Brasil 1

Correspondence to: Léa Maria Zanini Maciel Av. Bandeirantes, 3900 14049-900 – Ribeirão Preto, SP, Brasil lmzmacie@fmrp.usp.br Received on July/2/2016 Accepted on Dec/26/2016 DOI: 10.1590/2359-3997000000267

Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor originating from parafollicular C cells of the thyroid and associated with mutations in the proto-oncogene REarranged during Transfection (RET).The prognosis of MTC depends on clinical stage, with a 95.6% 10-year survival rate among patients with localized disease and 40% among patients with advanced disease. Standard chemotherapy and radiotherapy have no significant impact on the overall survival of these patients and two tyrosine kinase receptor inhibitors (TKIs), vandetanib and cabozantinib, have been recently approved for the systemic treatment of locally advanced or metastatic MTC. However, since patients with MTC and residual or recurrent disease may have an indolent course with no need for systemic treatment, and since these drugs are highly toxic, it is extremely important to select the patients who will receive these drugs in a correct manner. It is also essential to carefully monitor patients using TKI regarding possible adverse effects, which should be properly managed when occurring. Arch Endocrinol Metab. 2017;61(3):398-402. Keywords Medullary thyroid carcinoma; systemic treatment; tyrosine kinase inhibitors; adverse effects

INTRODUCTION

edullary thyroid carcinoma (MTC) represents 1-2% of all thyroid carcinomas in the United States, a lower frequency than previously reported (35%), primarily due to the higher relative incidence of papillary thyroid cancer (PTC) over the last 3 decades (1). MTC is a rare neuroendocrine tumor originating from parafollicular C cells of the thyroid and associated with mutations in the proto-oncogene REarranged during Transfection (RET) (1,2). MTC is associated with activators of the RET protooncogene, which codes for a membrane receptor of the tyrosine-kinase type of 150-170 kD highly expressed in normal and tumor cells derived from the neural crest, including parafollicular thyroid cells. The RET receptor, after interacting with one of its ligands and undergoing dimerization, activates the RAS-RAF-MAP kinase and PI3K-AKT pathways, being responsible for the transduction of extracellular signals of cell proliferation, growth, differentiation and survival, and cell apoptosis. Genomic mutations activating the RET proto-oncogene are present in most patients with hereditary MTC, with the existence of a genotypephenotype correlation. In addition to the RET, the superexpression of receptors 2 and 3 of the vascular 398

endothelial growth factor (VEGF), which leads to tumor angiogenesis and nutrition, and of the epidermal growth factor receptor (EGFR), involved in tumor proliferation, also contributes to the pathogenesis of MTC (3). MTC occurs in a sporadic or non-hereditary manner in 75-80% of the patients. In the remaining cases (2025%), it is an autosomal dominant hereditary disease with a high degree of penetrance and variability of expression, which may be part of two distinct clinical syndromes depending on the organs involved, i.e., type 2A multiple endocrine neoplasia (MEN2A) or type 2B multiple endocrine neoplasia (MEN2B). MEN2A, which accounts for 95% of MEN2 cases, can be divided into four variants: classical MEN2A (represented by the uniform presence of MTC and the less frequent occurrence of pheochromocytoma or hyperparathyroidism, or both), MEN2A with cutaneous lichen amyloidosis, MEN2A with Hirschsprung’s disease, and familial MTC (families or individuals with RET germline mutations who have MTC but not pheochromocytoma or hyperparathyroidism). Patients with MEN2B develop MTC and pheochromocytoma and exhibit a recognizable phenotype, characterized by a typical facies, ophthalmologic abnormalities (inability to make tears in infancy, thickened and everted eyelids, Arch Endocrinol Metab. 2017;61/4

Treatment of advanced medullary thyroid carcinoma

Arch Endocrinol Metab. 2017;61/4

a palliative treatment for painful bone metastases or for the prevention of other events, such as compression of the spinal cord or fracture. Other measures can also be taken for progressive and symptomatic bone metastatic disease such as radiotherapy and/or antiresorptive medications (intravenous bisphosphonates or RANK ligand inhibitors). Embolization or cryoablation of metastatic liver or bone disease may be beneficial in some cases, relieving pain and treating refractory diarrhea (11). Only a selected group of patients with metastatic MTC should be considered for systemic therapy. These are patients with significant disease progression over a period of fewer than 12-14 months, with symptomatic tumor growth or involvement of vital organs precluding the possibility of localized therapy.

Systemic treatment Knowledge about the pathways involved in MTC tumorigenesis has led to the testing of various tyrosine kinase receptor inhibitors (TKI) for the treatment of this tumor. Vandetanib was the first of them to be approved for the treatment of progressive, locally advanced, and unresectable or metastatic MTC in April 2011 in the United States, in February 2012 in Europe, and in November 2012 in Brazil. Cabozantinib was the other drug approved in the United States in November 2012 and in Europe in March 2014 for the treatment of progressive, locally advanced, and unresectable or metastatic MTC. This medication has not yet been approved by Anvisa and is not available in Brazil for the treatment of MTC.

Vandetanib Vandetanib is a potent TKI of the RET receptor and of receptors 2 and 3 of VEGF (VEGF-2 and VEGF-3) and also has inhibitory activity on EGFR (12,13). Its approval was based on a phase III, randomized, placebo-controlled, double-blind study (ZETA) which demonstrated a longer progression-free survival (PFS) estimated at 30 months in patients with MTC treated with vandetanib compared with the placebo group (PFS = 19 months; p < 0.001) (14). The overall response to vandetanib was 44%. A demonstration of progression before patient admission to the study was not a criterion for entry, a fact that may have contributed to the PFS detected in the placebo group. Additionally, crossover of the placebo group to the group treated with vandetanib was permitted upon demonstration of 399

mild ptosis, and prominent corneal nerves), skeletal malformations (marfanoid body habitus, narrow long facies, pes cavus, pectus excavatum, high-arched palate, scoliosis, and slipped capital femoral epiphyses), and a generalized ganglioneuromatosis throughout the aerodigestive tract (1). MTC has a variable survival depending on the clinical stage. Ten-year survival is 95.6% among patients with localized disease, 75.5% among patients with extension of the disease to nearby tissues or presence of metastatic ganglia, and 40% among patients with distant metastases (4). However, distant metastases are detected in 7-17% of the patients at diagnosis (5). Nevertheless, a substantial number of patients with distant metastases may have an indolent disease course or a slow progression for several years. The main pathway of MTC dissemination is the lymphatic one, with a high frequency of macroscopic or microscopic metastases to regional lymph nodes. The incidence of metastases correlates with the size of the primary tumor, with metastases being present in more than 75% of palpable tumors (6). Other common sites of metastasis are the mediastinum, hilar ganglia, lungs, liver, and bones and, less frequently, skin and brain (7). These tumors secrete calcitonin (Ct) and carcinoembryonic antigen (CEA), which are sensitive biomarkers of the disease. The postoperative Ct and CEA values and their doubling times (DT) are predictors of aggressive tumor behavior. Patients with a Ct-DT of more than 1 year have a 95% 10-year survival rate and a 73% 5-year recurrence-free survival rate. Conversely, patients with Ct-DT of 1 year or less have a 10-year survival rate of 18% and a survival rate without disease recurrence of 20% (8). The only treatment potentially inducing a cure of MTC is surgery, with the most important initial treatment being the full removal of the tumor and suspected ganglia in order to prevent relapses. In about 50% of cases, the disease is unresectable at the time of diagnosis (9). When cervical metastases are detected on the occasion of the initial surgery, the cure rate is low and residual disease will be revealed radiologically or biochemically in 90% of the patients (10). Patients with persistent cervical disease can be placed under observation or submitted to a new surgery if progression is detected. Many patients with distant metastases have indolent disease and do not require systemic therapy for many years. Localized therapy with external irradiation can be considered as

Treatment of advanced medullary thyroid carcinoma

tumor progression, an event that may also have been a confounding factor in the PFS analysis. Although TKIs are better tolerated than cytotoxic chemotherapy, many patients experience side effects. In the ZETA study, treatment with vandetanib led to a low discontinuation rate due to toxicity (12%). The main toxic effects of any grade are listed in Table 1. The adverse effects most commonly detected are diarrhea, skin rash, acne, fatigue, nausea, arterial hypertension, headache, vomiting, prolonged QT in the electrocardiogram (ECG), and photosensitivity. The most common laboratory abnormalities are hypocalcemia, increased levels of hepatic enzymes, hypoglycemia, hypothyroidism, and increased creatinine levels. Rare but serious adverse events may occur, including StevensJohnson syndrome, interstitial pulmonary disease, ischemic stroke, congestive heart failure, and posterior leukoencephalopathy syndrome (15,16). Only 2.5% of the patients treated with vandetanib have been reported to suffer a fatal adverse effect. A reduction of the dose from 300 to 200 mg/day is recommended in the presence of more advanced adverse effects (grade 3 or 4) of any nature. A new reduction of the dose to 100 mg/day should be prescribed if an additional grade 3 or 4 adverse effect should occur (17,18). Before starting treatment, possibly present electrolyte disorders such as hypocalcemia, hypokalemia, Table 1. Toxicity of any degree related to the use of vandetanib

Toxicity Diarrhea, 57% Rash, 53% Acneiform dermatitis/acne, 35% Nausea, 33% Hypertension, 33% Headache, 26% Fatigue, 24% Reduced appetite, 21% Abdominal pain, 21% Dry skin, 15% Vomiting, 15% QTc prolongation*, 14% Photosensitivity reaction, 13% Pruritus, 11% Dyspepsia, 11% Proteinuria, 10% Depression, 10%

Laboratory abnormalities Reduction of calcium, 57% Increase of aspartate transaminase, 51% Reduction of glycemia, 24% Increase of alkaline phosphatase, 53% Increase of creatinine, 16% Increase of bilirubins, 13% Reduction of magnesium, 7% Reduction of potassium, 6% Increase of potassium, 6% Reduction of sodium, 6% Neutropenia, 10% Thrombocytopenia, 9%

QTc: corrected QT interval. * Prolonged QTc > 450 ms occurred in 65% and > 500 ms in 7% using Fridericiaâ&#x20AC;&#x2122;s correction. Adapted from Cabanillas and cols. (17).

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and hypomagnesemia should be corrected, and a baseline ECG should be obtained. These tests should be repeated 2 to 4 weeks and 8 to 12 weeks after the beginning of treatment and then every 3 months for proper monitoring. Throughout treatment, the patient should be instructed not to take any medication without the approval of the physician since some medications can prolong the QT interval. Patients with a history of long QT syndrome or uncorrectable electrolyte disorder should not be treated with this medication. Recently, Fox and cols. (19) conducted a phase I/II trial for adolescents (13-18 years) and children (5-12 years) with metastatic or locally advanced MTC. Sixteen patients were treated with vandetanib 100 mg/ m2/day. The authors concluded that this dosage is well tolerated and highly active for adolescents and children with locally advanced or metastatic MTC and MEN2B.

Cabozantinib Cabozantinib is a multikinase inhibitor of VEGFR2, RET, and hepatocyte growth factor receptor (c-MET), which was approved for the treatment of MTC after the demonstration of PFS advantages in the group treated with the TKI compared with the placebo group (11.2 months versus 4 months, respectively, p < 0.0001) in a phase III, randomized, double-blind study conducted on 330 patients with MTC and radiologically confirmed disease progression (20). The recommended dose is 140 mg/day. However, because of the high toxicity of the drug, lower initial doses have been suggested (100 or 120 mg/day) (21), with later increases to the full dose if the patients tolerate it or if the desired response is not achieved. The drug should be ingested on an empty stomach, and perforations, fistulas, and hemorrhages may occur. The drug should be used with care or avoided in patients with a history of cervical irradiation or disorders of the gastrointestinal mucosa. The side effects more commonly detected with the use of this drug are listed in Table 2.

The choice of a TKI The choice of which of the approved TKIs should be used for the beginning of treatment is unclear. The first study discussing this aspect was published by Cabanillas and cols. in 2014 (17). The idea is that this choice should be centered on the patient, taking into consideration his clinical history, the changes detected upon physical examination, and the results of baseline laboratory tests. Since vandetanib is known Arch Endocrinol Metab. 2017;61/4

Treatment of advanced medullary thyroid carcinoma

Table 2. Toxicity of any degree related to the use of cabozantinib Toxicity Diarrhea, 63% Stomatitis, 51% Hand-foot syndrome, 50% Weight loss, 48% Reduced appetite, 46% Nausea, 43% Fatigue, 41% Dysgeusia, 34% Changes in hair color, 34% Hypertension, 33% Constipation, 27% Abdominal pain, 27% Vomiting, 24% Dysphonia, 20% Headache, 18% Dysphagia, 13% Dyspepsia, 11%

Laboratory abnormalities Increase of alanine transaminase, 86% Increase of aspartate transaminase, 86% Increase of alkaline phosphatase, 53% Reduction of calcium, 52% Reduction of phosphorus, 28% Increase of bilirubins, 25% Reduction of magnesium, 19% Reduction of potassium, 18% Reduction of sodium, 10% Neutropenia, 35% Thrombocytopenia, 35% Increase of TSH, 57%

Adapted from Cabanillas and cols. (17).

Cabozantinib can cause perforation, fistulas, and hemorrhages. When medical history is taken, the patient should be questioned about the presence of hemoptysis and hemorrhages, diverticulitis, chronic gastrointestinal disease (Crohn’s disease and ulcerative colitis), active peptic ulcer, or radiation treatment of the neck or mediastinum, since these patients will have a higher risk of gastrointestinal perforation, hemorrhage, and tracheoesophageal fistulas. Invasion of the trachea, bronchi, and esophagus increases the risk of formation of a fistula. Thus, in these situations, vandetanib will be better indicated. Also, the use of vandetanib is preferable for patients whose profession requires the frequent use of the hands (e.g., musicians) since the hand-foot syndrome is a frequent side effect of cabozantinib. In addition, vandetanib is associated with weight gain and cabozantinib with weight loss (22). A review of the drugs used by the patient is important. The concomitant use of CYP3A4 inhibitors Arch Endocrinol Metab. 2017;61/4

may increase the toxicity of cabozantinib by increasing its plasma concentrations. In contrast, the use of CYP3A4 inducers can reduce the plasma concentrations of vandetanib and, consequently, the efficacy of the drug (23,24).

CONCLUSIONS TKIs represent a highly valuable therapy for patients with metastatic and/or locally advanced MTC. However, it is extremely important to establish an appropriate stratification of the clinical risk of the patient to whom these drugs will be administered, since a significant number of patients with MTC and residual or recurrent disease have an indolent course characterized by stable or slowly increasing Ct and/or CEA levels. The prescription of TKI is not indicated for these patients in view of the toxicity of these drugs, the lack of their curative power, and the need for their chronic use when they are prescribed. In addition, careful monitoring of the patients using these drugs and the management of side effects that might arise with treatment are important for the safe maintenance of the patient and for the determination of the time when the drug loses its efficacy. Disclosure: Léa Maria Zanini Maciel is a principal investigator of AstraZeneca studies (ZETA, KALYPSO, VERIFY and ASTRA). Patrícia Künzle Ribeiro Magalhães is a subinvestigator of AstraZeneca studies (ZETA, KALYPSO, VERIFY and ASTRA).

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to possibly lead to prolongation of the QT interval, “torsade de pointes”, and sudden death, the drug should not be administered to patients with long QT syndrome, arrhythmias, decompensated heart failure, or uncorrectable electrolyte disorders. Also, the drug should not be administered to patients with a corrected QT (QTc) of more than 450 milliseconds and should be prescribed with caution to patients taking an antiarrhythmic drug.

Treatment of advanced medullary thyroid carcinoma

times as prognostic factors in medullary thyroid carcinoma: a structured meta-analysis. Clin Endocrinol (Oxf). 2010;72:534-42. 9. Degrauwe N, Sosa JA, Roman S, Deshpande HA. Vandetanib for the treatment of metastatic medullary thyroid cancer. Clin Med Insights Oncol. 2012;6:243-52.

17. Cabanillas ME, Hu MI, Jimenez C. Medullary thyroid cancer in the era of tyrosine kinase inhibitors: To treat or not to treat – and with which drug – those are the questions. J Clin Endocrinol Metab. 2014;99:4390-6.

10. Machens A, Schneyer U, Holzhausen HJ, Dralle H. Prospects of remission in medullary thyroid carcinoma according to basal calcitonin level. J Clin Endocrinol Metab. 2005;90:2029-34.

18. Wells SA, Gosnell JE, Gagel RF, Moley J, Pfister D, Sosa JA, et al. Vandetanib for the treatment of patients with locally advanced or metastatic hereditary medullary thyroid cancer. J Clin Oncol. 2010;28:767-72.

11. Fromigué J, De Baere T, Baudin E, Dromain C, Leboulleux S, Schlumberger M. Review: chemoembolization for liver metastases from medullary thyroid carcinoma. J Clin Endocrinol Metab. 2006;91:2496-9.

19. Fox E, Widemann BC, Chuk MK, Marcus L, Aikin A, Whitcomb PO, et al. Vandetanib in children and adolescents with multiple endocrine neoplasia type 2B associated medullary thyroid carcinoma. Clin Cancer Res. 2013;19:4239-48.

12. Carlomagno F, Vitagliano D, Guida T, Ciardiello F, Tortora G, Vecchio G, et al. ZD6474, an orally available inhibitor of KDR tyrosine kinase activity. Cancer Res. 2003;170:861-9.

20. Elisei R, Schlumberger MJ, Müller SP, Schöffski P, Brose MS, Shah MH, et al. Cabozantinib in progressive medullary thyroid cancer. J Clin Oncol. 2013;31:3639-46.

13. Wedge SR, Ogilvie DJ, Dukes M, Kendrew J, Chester R, Jackson JA, et al. ZD6474 inhibits vascular endothelial growth factor signaling, angiogenesis, and tumor growth following oral administration. Cancer Res. 2002;62:4645-55.

21. Weitzman SP, Cabanillas ME. The treatment landscape in thyroid cancer: A focus on cabozantinib. Cancer Manag Res. 2015;7:265-78.

14. Wells SA, Robinson BG, Gagel RF, Dralle H, Fagin JA, Santoro M, et al. Vandetanib in patients with locally advanced or metastatic medullary thyroid cancer: A randomized, double-blind phase III trial. J Clin Oncol. 2012;30:134-41. 15. Cabanillas ME, Hu MI, Jimenez C, Grubbs EG, Cote GJ. Treating medullary thyroid cancer in the age of targeted therapy. Int J Endocr Oncol. 2014;1:203-16.

23. Nguyen L, Holland J, Miles D, Engel C, Benrimoh N, O’Reilly T, et al. Pharmacokinetic (PK) drug interaction studies of cabozantinib: Effect of CYP3A inducer rifampin and inhibitor ketoconazole on cabozantinib plasma PK and effect of cabozantinib on CYP2C8 probe substrate rosiglitazone plasma PK. J Clin Pharmacol. 2015;55:1012-23. 24. Martin P, Oliver S, Robertson J, Kennedy S, Read J. Pharmacokinetic drug interactions with vandetanib during coadministration with rifampicin or itraconazole. Drugs R D. 2011;11:37-51.

16. Scheffel RS, Dora M, Siqueira R, Burttet LM, Cerski MR, Scheffel RS, et al. Toxic cardiomyopathy leading to fatal acute cardiac failure related to vandetanib: a case report with histopathological analysis. Eur J Endocrinol. 2013;51-4.

22. Massicotte MH, Borget I, Broutin S, Baracos VE, Leboulleux S, Baudin E, et al. Body composition variation and impact of low skeletal muscle mass in patients with advanced medullary thyroid carcinoma treated with vandetanib: results from a placebocontrolled study. J Clin Endocrinol Metab. 2013;98:2401-8.

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case report

Multifocal oral melanoacanthoma associated with Addison’s disease and hyperthyroidism: a case report Thinali Sousa Dantas1, Isabelly Vidal do Nascimento1, Maria Elisa Quezado Lima Verde2, Ana Paula Negreiros Nunes Alves1, Fabrício Bitu Sousa1, Mário Rogério Lima Mota1

SUMMARY Oral melanoacanthoma is a mucocutaneous, pigmented, rare, benign, and probably reactive lesion. This paper reports for the first time in the literature a case of multifocal oral melanoacanthoma in a patient diagnosed with Addison’s disease and concomitant Graves’ disease with hyperthyroidism.The patient presented with oral pigmented lesions, which were hypothesized to be mucosal pigmentation associated with Addison’s disease. Due to their unusual clinical pattern, these oral lesions were biopsied and diagnosed as oral melanoacanthoma on histopathology and immunohistochemistry for HMB-45. At the moment of this report, the patient was being treated for her systemic conditions, but the lesions had not regressed. Reactive hyperpigmentation of the skin and mucous membranes may be found in Addison’s disease and hyperthyroidism. This case reinforces the hypothesis of a reactive nature for oral melanoacanthoma and highlights the need for investigation of endocrine disorders in patients with multifocal oral melanoacanthoma. Arch Endocrinol Metab. 2017;61(3):403-7.

Divisão de Patologia Oral, Universidade Federal do Ceará (UFC), Fortaleza, CE, Brasil 2 Universidade Federal do Ceará (UFC), Fortaleza, CE, Brasil 1

Correspondence to: Thinali Sousa Dantas Laboratório de Patologia Oral, Universidade Federal do Ceará, Campus Porangabussu, Rua Monsenhor Furtado, s/n 60430-160 – Fortaleza, CE, Brasil thinali@hotmail.com Received on Sep/29/2016 Accepted on Feb/2/2017

INTRODUCTION

utaneous melanoacanthoma was first described as a benign tumor with characteristics of a pigmented variant of seborrheic keratosis, presenting as a proliferation of keratinocytes and dendritic melanocytes in the lower portion of the epithelium (1). However, melanoacanthoma affecting the oral cavity seems to have a more reactive than truly neoplastic characteristic, including a flat or slightly elevated surface, fast growth, preference for involving mucosa exposed to trauma, partial or complete regression after removal of the irritant, as well as association with inflammatory processes. Based on that, the term oral melanoacanthoma has been used to characterize these particular lesions (2-4). The oral manifestation may present as a single or, less frequently, multiple lesions affecting almost exclusively dark-skinned patients with a predilection for poor young females (5-8). Oral melanoacanthoma lesions may vary in color from dark brown to black and are often asymptomatic, although in some cases pain, itching, and burning may Arch Endocrinol Metab. 2017;61/4

occur (5,9). The buccal mucosa is the most affected site, but the lesions may also be found on the palate, gingiva, and lip mucosa (5,7,10). Histologically, oral melanoacanthoma is characte rized by a proliferation of benign dendritic melanocytes across the epithelium, spongiosis, acanthosis, chronic inflammatory infiltrates, and eosinophils in the underlying connective tissue (9,11). The diagnosis of oral melanoacanthoma is based on the histological assessment of the lesion. The Fontana-Masson stain and an immunohistochemical profile for melanocytes, such as the S-100 protein, Melan-A, and HMB-45, can be used to verify the presence of melanin (7,8,10). Several melanocytic lesions can sometimes resemble oral melanoacanthoma, including melanotic macule, melanocytic nevus, atypical melanocytic proliferation/ hyperplasia, and melanoma; (3) therefore, biopsy is indicated to establish the diagnosis (6,10,11). Until the biopsy report becomes available, no further treatment is necessary since the lesion may undergo spontaneous regression, although rarely it may relapse and additional lesions may appear (8,10). 403

DOI: 10.1590/2359-3997000000273

Melanoacanthoma and endocrine disorders

Multiple pigmented lesions of the oral cavity may present clinical features of several systemic disorders such as Addison’s disease, Peutz-Jeghers syndrome, and, to a lesser extent, hyperthyroidism (8,10,12,13). Addison’s disease is an endocrine disorder characterized by a deficient production of adrenal cortex hormones leading to increased secretion of adrenocorticotropic hormone (ACTH) (8,10,14). The most common symptoms of Addison’s disease are fatigue, loss of appetite and weight, weakness, and hypotension. In addition to these symptoms, patients with Addison’s disease also have generalized hyperpigmentation of the skin and oral mucosa (8,10,14). The oral lesions usually precede the cutaneous manifestations, which can be an important diagnostic feature in the early stages of the disease. These lesions affect mainly the lips, buccal mucosa, gingiva, palate, and tongue (8,10,12,13). Treatment of these lesions is not required since they disappear during treatment of the disease itself (8,10). Hyperthyroidism is also an endocrine disorder, in which excessive thyroid hormone is synthesized and released. This disorder may be associated with several etiologies, such as cancer and autoimmune diseases (Graves’ disease) (15). Cutaneous manifestations of hyperthyroidism may appear in the form of hyperhidrosis, myxedema, eczematous dermatitis, alopecia, hyperpigmentation, and telangiectasia. These pigmented lesions affect approximately 2% of the patients with hyperthyroidism. They are poorly described in the literature and rarely involve mucous membranes (13,16). This article aims to describe for the first time in the literature a case of multifocal oral melanoacanthoma in a patient with concomitant Addison’s and Graves’ disease.

CASE REPORT A 50-year-old dark-skinned woman presented to the Stomatology Service of the Federal University of Ceará, Brazil, complaining of two dark patches on her upper lip mucosa. During anamnesis, the patient reported that these patches had grown in recent months, accompanied by a slight itching in the area. She also reported having both Addison’s and Graves’ disease. Information retrieved from her medical history showed that the diagnosis of Graves’ 404

disease with hyperthyroidism was established in 2014 based on clinical characteristics presented on thyroid scintigraphy and blood tests (thyroid-stimulating hormone [TSH] 0.01 mU/L) and anti-TSH receptor antibody [TRAB] 2.68 IU/L). A current serum free thyroxine (FT4) measurement was 2.04 ng/dL. The adrenal insufficiency was diagnosed in the same period that the pigmented lesions had appeared, with a serum cortisol level of 2.3 µg/dL and ACTH level of 84.5 ng/L. At the time of this report, the patient was being treated and monitored for both these diseases. On clinical examination, she presented brown and black patches and plaques sometimes permeated by whitish areas. These lesions were asymptomatic, had varying sizes with irregular and imprecise limits, and were distributed in the upper lip mucosa, gingiva, tongue, and buccal mucosa (Figure 1). The patient used a dental prosthesis, but no trauma from the prosthesis was observed in association with the pigmentation. During the physical examination, these lesions were not observed on the skin. Due to the multifocal involvement and the presence of associated systemic diseases, the hypothesis of a mucosal pigmentation associated with Addison’s disease was proposed. However, due to lack of skin pigmentation, distinct clinical appearance, and history of increased lesion size in recent months, a biopsy of the lesions in the upper lip mucosa was performed. The histopathological report described a stratified squamous epithelium with hyperparakeratinized areas showing acanthosis, spongiosis, exocytosis, vacuolar degeneration, substantial deposition of melanin in all epithelial layers, and a suggestive melanocytic hyperplasia, with occasional dendritic melanocytes in all epithelial layers. The adjacent connective tissue presented subepithelial melanophages and mononuclear cell infiltration (Figure 2 A and B), consistent with oral melanoacanthoma. Immunohistochemistry for HMB-45 showed dendritic melanocytes extending across the epithelium, confirming the diagnosis of oral melanoacanthoma (Figure 2 C and D). At the time of this report, the patient was undergoing treatment with follow-up assessments every 3 months and using Tapazol 5 mg for hyperthyroidism. No medication for Addison’s disease had been initiated, and the cortisol levels were within the normal range (TSH 1.33 mU/L, FT4 0.87 ng/dL, cortisol 13.59 µg/dL). No changes in the clinical appearance of the pigmented lesions were observed during follow-up. Arch Endocrinol Metab. 2017;61/4

Melanoacanthoma and endocrine disorders

Figure 1. Images showing the presence of brown and black patches and plaques distributed in the (A) upper lip mucosa, (B) gingiva, (C) buccal mucosa, and (D) tongue.

Figure 2. Oral melanoacanthoma. (A) Stratified squamous epithelium with hyperparakeratinized areas showing acanthosis, substantial deposition of melanin in basal and suprabasal layers (yellow arrows); (B) dendritic melanocytes (white arrow) in all epithelial layers (hematoxylin-eosin [H&E] stain, original magnification 200X and 400X, respectively); (C) and (D) positive HMB-45 expression in dendritic melanocytes extending across the epithelium (immunohistochemical reaction, original magnification 100X and 400X, respectively). Arch Endocrinol Metab. 2017;61/4

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Melanoacanthoma and endocrine disorders

DISCUSSION Addison’s disease comprises all etiologic factors destroying the adrenal cortex. (17) Patients with this disease may have other autoimmune disorders, classified as autoimmune polyglandular syndromes (17). In addition to Addison’s disease, the patient in the present report also had Graves’ disease, characterized by the presence of autoantibodies binding to the TSH receptor, stimulating the growth of the thyroid gland and secretion of thyroid hormones. Graves’ disease is one of the main causes of hyperthyroidism (18). Reactive mucosal hyperpigmentation may be a common finding in Addison’s disease, since cortisol deficiency leads to increased production of ACTH, a pituitary hormone with a chemical structure similar to that of melanocyte-stimulating hormone (MSH), stimulating the pigmentation of the skin and mucosa (14,19). Hyperthyroidism may also be associated with hyperpigmentation, but pigmented lesions are rarely described in the literature and generally do not involve the mucosa, with only about 2% of the patients having associated skin pigmentation (13,16). Histopathological examination of the pigmented lesions associated with Addison’s disease shows acanthosis and heavy deposition of melanin in the basal and subepithelial layers (20). Pigmented lesions associated with hyperthyroidism share a similar histopathology to those of Addison’s disease, but with a clear deposition of hemosiderin in the connective tissue (13,20). There have been no reports in the literature of multifocal oral melanoacanthoma in a patient with concomitant Addison’s and Graves’ disease with hyperthyroidism. The first description of oral melanoacanthoma was as a single lesion, reported by Schneider in 1981, but reports of multiple lesions are available in the literature (9,21). These lesions are slightly elevated, brownish/ blackish, usually affecting female and melanodermic patients, and requiring no treatment, as in the patient described in this report. The differential diagnosis of these lesions include nevi and melanomas, and a biopsy is required to confirm their diagnoses (6). Unlike skin melanoacanthoma, oral melanoacanthoma appears to have a reactive nature, (2-4) and the case described here reinforce this theory. Increased ACTH levels can induce melanocyte proliferation and melanin secretion (19). In our patient’s case, her ACTH level was 84.5 ng/L. In addition, a possible association between thyroid hormones and their stimulating 406

action on melanocytes has also been described (22,23). These hormonal effects may enhance the proliferation and activation of melanocytes causing melanocytic hyperplasia and deposition of melanin, as seen in the histopathology of oral melanoacanthoma. Besides, several experimental and in vitro studies demonstrate the role of thyroid hormones on epithelial proliferation (24,25), which could be related to the acanthosis observed in oral melanoacanthoma. This case suggests that hormonal changes may facilitate or amplify the emergence of oral melanoacanthoma in patients predisposed to this type of lesion. In 2007, Carlos-Bregni and cols. (6) suggested a link between oral melanotic macule (reactive lesion with prominent basal melanosis) and oral melanoacanthoma. These authors reported that the activation of melanocytes by unknown mechanisms might be the link between these two lesions. Hormonal mechanisms may mediate this process. HMB-45 is an antibody against the GP100 protein, which is present in melanosomes and, more commonly, in proliferating cells. This antibody is an important marker used in the diagnosis of melanomas with cytoplasmic positivity. It has a diagnostic role in helping differentiate melanomas and non-malignant lesions (11,26). It also reacts promptly with melanocytes of oral intramucosal nevi, including blue nevi (11). In our analysis, we found immunoreactivity for HMB-45 in melanocytes without atypia suggestive of malignancy, dendritic and non-dendritic, and in all epithelial layers. Treatment in cases of oral melanoacanthoma is not recommended. However, these lesions must be followed up and may sometimes spontaneously disappear or decrease in size (3,9,27). The lesions in the present case remained unchanged during follow-up; however, changes in the clinical pattern of the lesions and their prognosis were still unavailable due to the short follow-up time and absence of similar cases in the literature. Finally, this report highlights the importance of investigating endocrine disorders (Addison’s and/or Graves’ disease) in patients diagnosed with multifocal oral melanoacanthoma. Disclosure: no potential conflict of interest relevant to this article was reported.

REFERENCES 1. Mishima Y, Pinkus H. Benign mixed tumor of melanocytes and malpighian cells. Melanoacanthoma: Its relationship to Arch Endocrinol Metab. 2017;61/4

Melanoacanthoma and endocrine disorders

Bloch’s benign non-nevoid melanoepithelioma. Arch Dermatol. 1960;81:539-50.

14. Sarkar SB, Sarkar S, Ghosh S, Bandyopadhyay S. Addison’s disease. Contemp Clin Dent. 2012;3(4):484-6.

2. Wright JM, Binnie WH, Byrd DL, Dunsworth AR. Intraoral melanoacanthoma. J Periodontol. 1983;54(2):107-11.

15. De Leo S, Lee SY, Braverman LE. Hyperthyroidism. Lancet. 2016;388(10047):906-18.

3. Buchner A, Merrell PW, Hansen LS, Leider AS. Melanocytic hyperplasia of the oral mucosa. Oral Surg Oral Med Oral Pathol. 1991;71(1):58-62.

16. Locke W. Unusual manifestations of Graves’s disease. Med Clin North Am. 1967;51(4):915-24.

4. Tomich CE, Zunt SL. Melanoacanthosis (melanoacanthoma) of the oral mucosa. J Dermatol Surg Oncol. 1990;16(3):231-6.

17. Fichna M, Fichna P, Gryczyńska M, Walkowiak J, Zurawek M, Sowiński J. Screening for associated autoimmune disorders in Polish patients with Addison’s disease. Endocrine. 2010;37(2):349-60.

5. Rohilla K, Ramesh V, Balamurali PD, Singh N. Oral melanoacanthoma of a rare intraoral site: case report and review of literature. Int J Clin Pediatr Dent. 2013;6(1):40-3.

18. Michels AW, Eisenbarth GS. Immunologic endocrine disorders. J Allergy Clin Immunol. 2010;125(2 Suppl 2):S226-37.

6. Carlos-Bregni R, Contreras E, Netto AC, Mosqueda-Taylor A, Vargas PA, Jorge J, et al. Oral melanoacanthoma and oral melanotic macule: a report of 8 cases, review of the literature, and immunohistochemical analysis. Med Oral Patol Oral Cir Bucal. 2007;12(5):E374-9. 7. Lakshminarayanan V, Ranganathan K. Oral melanoacanthoma: a case report and review of the literature. J Med Case Rep. 2009;3:11. 8. Meleti M, Vescovi P, Mooi WJ, van der Waal I. Pigmented lesions of the oral mucosa and perioral tissues: a flow-chart for the diagnosis and some recommendations for the management. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2008;105(5):606-16. 9. das Chagas e Silva de Carvalho LF, Farina VH, Cabral LAG, Brandão AAH, Coletta R Della, Almeida JD. Immunohistochemical features of multifocal melanoacanthoma in the hard palate: a case report. BMC Res Notes. 2013;6(1):30. 10. Gondak RO, da Silva-Jorge R, Jorge J, Lopes MA, Vargas PA. Oral pigmented lesions: Clinicopathologic features and review of the literature. Med Oral Patol Oral Cir Bucal. 2012;17(6):e919-24. 11. Fornatora ML, Reich RF, Haber S, Solomon F, Freedman PD. Oral melanoacanthoma: a report of 10 cases, review of the literature, and immunohistochemical analysis for HMB-45 reactivity. Am J Dermatopathol. 2003;25(1):12-5.

19. Burk CJ, Ciocca G, Heath CR, Duarte A, Dohil M, Connelly EA. Addison’s disease, diffuse skin, and mucosal hyperpigmenation with subtle “flu-like” symptoms – A report of two cases. Pediatr Dermatol. 2008;25(2):215-8. 20. Chuong R, Goldberg MH. Case 47, part II: Oral hyperpigmentation associated with Addison’s disease. J Oral Maxillofac Surg. 1983;41(10):680-2. 21. Marocchio LS, Júnior DSP, de Sousa SCOM, Fabre RF, Raitz R. Multifocal diffuse oral melanoacanthoma: a case report. J Oral Sci. 2009;51(3):463-6. 22. Slominski A, Wortsman J, Kohn L, Ain KB, Venkataraman GM, Pisarchik A, et al. Expression of hypothalamic-pituitary-thyroid axis related genes in the human skin. J Invest Dermatol. 2002;119(6):1449-55. 23. Redondo P, Guzmán M, Marquina M, Pretel M, Aguado L, Lloret P, et al. [Repigmentation of gray hair after thyroid hormone treatment]. Actas Dermosifiliogr. 2007;98(9):603-10. 24. 24. Safer JD, Crawford TM, Holick MF. A role for thyroid hormone in wound healing through keratin gene expression. Endocrinology. 2004;145(5):2357-61. 25. He Y, Meng Z, Jia Q, Hu F, He X, Tan J, et al. Sleep quality of patients with differentiated thyroid cancer. PLoS One. 2015;10(6):1-9. 26. Elenitsas D, Jaworsky C, Junior BJ. Lever’s histopathology of the skin, eighth edition. Hum Pathol. 1998;29(2):201.

13. Banba K, Tanaka N, Fujioka A, Tajima S. Hyperpigmentation caused by hyperthyroidism: Differences from the pigmentation of Addison’s disease. Clin Exp Dermatol. 1999;24(3):196-8.

27. Buchner A, Merrell PW, Carpenter WM. Relative frequency of solitary melanocytic lesions of the oral mucosa. J Oral Pathol Med. 2004;33(9):550-7.

12. Benmously R, Hammami H, Badri T, Triki S, Mokhtar I, Fenniche S. Une pigmentation cutanée. La Rev Médecine Interne. 2012;33(1):50-1.

Arch Endocrinol Metab. 2017;61/4

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OFFICIAL JOURNAL OF THE BRAZILIAN SOCIETY OF ENDOCRINOLOGY AND METABOLISM

AE&M awards 2016

Archives of Endocrinology

OFFICIAL Since 2001, the Archives of Endocrinology and Metabolism (AE&M) and the Brazilian SocietyJOURNAL of Endocrinology and Metabology award the “AE&M award Professor Thales Martins” and “AE&M award Professor OF THE BRAZILIANWaldemar Berardinelli” to the young researcher with the best original reports published in the AE&M in the areas of basic, SOCIETY OF translational science and clinical practice. ENDOCRINOLOGY The commission was made up by members of the Editorial Board: Marcello D. Bronstein, Bruno Ferraz de Souza, Erika Parente, Francisco Bandeira, Fernanda Vaisman, Fernando M. A. Giuffrida, João Roberto Maciel AND METABOLISM Martins, Melanie Rodacki, Monica R. Gadelha, Nina Rosa C. Musolino, Poli Mara Spritzer, Ricardo Meirelles, Rogerio Friedman, Rui M. B. Maciel, Tânia S. Bachega, who analyzed the reports published in the AE&M, volume 60, 2016. The award ceremony is going to take place during the 33nd Brazilian Congress of Endocrinology and Metabology (CBAEM) on August 16th, 2017, in Fortaleza, CE.

and Metabolism

Archives of

AE&M AWARD PROF. THALES MARTINS

Endocrinology

“Thyroxine increases Serca2 and Ryr2 gene expression in heart failure rats with euthyroid sick syndrome” Authored by Fábio V. G. Campanha, Denise Perone, Dijon H. S. de Campos, Renata de A. M. Luvizotto, Maria T. De Síbio, Miriane de Oliveira, Regiane M. C. Olimpio, Fernanda C. F. Moretto, Carlos R. Padovani, Gláucia M. F. S. Mazeto, Antonio C. Cicogna, Célia R. Nogueira. Arch Endocrinol Metab. 2016;60(6):582-6

and Metabolism OFFICIAL JOURNAL OF THE BRAZILIAN SOCIETY OF ENDOCRINOLOGY AND METABOLISM

AE&M AWARD PROF. WALDEMAR BERARDINELLI

“Evolution to permanent or transient conditions in children with positive neonatal TSH screening tests in Sergipe, Brazil” Authored by Diana M. Matos, Roberto J. R. Ramalho, Bruno M. Carvalho, Maria Augusta C. T. Almeida, Luciana F. D. Passos, Talmay T. S. Vasconcelos, Enaldo V. Melo, Carla R. P. Oliveira, Elenilde G. Santos, Karla F. Resende, Manuel H. Aguiar-Oliveira. Arch Endocrinol Metab. 2016;60(5):450-6

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We emphasize the importance of following these instructions carefully. Failure to do so will delay the processing of your manuscript. Manuscripts should be submitted solely to the AE&M and should not have been published, or be under consideration for publication in any substantial form, in another periodical-either professional or lay. Manuscripts should be submitted in English. Proofreading by a scientific editing service is strongly recommended; the following companies are suggested: Voxmed Medical Communications, American Journal Experts and PaperCheck. Manuscripts that successfully complete the peer-review process and are recommended for publication will only be accepted and published upon receipt of a certificate proving professional academic English proofreading. In extraordinary circumstances, the certificate can be waived by editorial decision. All submissions are initially evaluated in depth by the scientific editors. Papers that do not conform with the general criteria for publication will be returned to the authors without detailed review, typically within three to five days. Otherwise, manuscripts will be sent to reviewers (most commonly two).

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XigDuo XRTM (dapagliflozina + cloridrato de metformina) comprimidos revestidos de liberação prolongada. Indicações: XIGDUO XR é indicado como adjuvante à dieta e exercícios para melhorar o controle glicêmico em adultos com diabetes mellitus tipo 2 quando o tratamento com ambos, dapagliflozina e metformina, é apropriado. XIGDUO XR não é indicado para uso em pacientes com diabetes tipo 1. XIGDUO XR não deve ser usado para o tratamento da cetoacidose diabética. Contraindicações: doença ou disfunção renal moderada a grave (p.ex., níveis de creatinina sérica ≥1,5 mg/dL [homens], ≥1,4 mg/dL [mulheres] ou TFGe <60 mL/min/1,73 m2 ou ClCr <60 mL/min pelo Cockcroft-Gault), inclusive secundária a condições como choque, IAM e septicemia; acidose metabólica aguda ou crônica, incluindo cetoacidose diabética, com ou sem coma, que deve ser tratada com insulina; história de reação de hipersensibilidade grave à substância ativa ou a qualquer um dos excipientes; disfunção hepática. Cuidados e Advertências: acidose láctica (metformina plasmática > 5 µg/mL - maior risco em idosos, disfunção renal, doença hepática, insuficiência cardíaca congestiva, hipoxemia, desidratação, sepse, ingestão excessiva de álcool e uso de contraste intravascular), disfunção renal, disfunção hepática, ingestão excessiva de álcool, cetoacidose (maior risco em disfunções pancreáticas como DM1, pancreatite, cirurgia pancreática, redução da dose de insulina, redução da ingestão calórica, infecções, cirurgias, doenças concomitante e abuso de álcool), níveis de vitamina B12 (risco de redução em pacientes susceptíveis), procedimentos cirúrgicos, alterações no estado clínico, medicações concomitantes que afetem a função renal ou a hemodinâmica ou a eliminação da metformina, administração de meio de contraste intravascular iodado (aumento do risco de insuficiência renal aguda), estados de hipóxia (choque, ICC, IAM, insuficiência renal pré-renal), mau controle glicêmico secundário a febre, trauma, infecção ou cirurgias, pacientes sob risco de depleção de volume intravascular (idosos, uso de diuréticos), uso concomitante com medicamentos que causam hipoglicemia (insulina e sulfonilureias), sepse urinária e pielonefrite, uso em idosos, gravidez, lactação, uso pediátrico, câncer de bexiga ativo. Categoria de Risco na Gravidez: C. Interações Medicamentosas: com dapagliflozina (sem alterações clínicas relevantes, sem necessidade de ajuste de dose): bumetanida, sinvastatina, rifampicina, ácido mefenâmico; com metformina: medicamentos catiônicos (cimetidina), glibenclamida, furosemida, nifedipino; outros medicamentos hiperglicemiantes (tiazidas e outros diuréticos, corticosteroides, fenotiazinas, produtos da tireoide, estrógenos, contraceptivos orais, fenitoína, ácido nicotínico, simpatomiméticos, medicamentos bloqueadores do canal de cálcio e isoniazida). Interferência com teste do 1,5-anidroglucitol (1,5.AG). Reações Adversas: infecção genital, infecção do trato urinário, poliúria, dor nas costas, dor de cabeça, hipoglicemia, desidratação, hipovolemia ou hipotensão, diarreia, náuseas, vômitos, erupção cutânea, redução dos níveis séricos de vitamina B12, aumento do hematócrito. Posologia: deve ser individualizada com base no regime atual do paciente, desde que não exceda a dose máxima recomendada de 10 mg de dapagliflozina e de 2000 mg de cloridrato de metformina de liberação prolongada. XIGDUO XR deve, de modo geral, ser administrado uma vez ao dia com a refeição da noite. Apresentações: XigDuo XR comprimidos revestidos de liberação prolongada de: 5 mg/1000 mg em embalagens com 14 e 60 comprimidos; 10 mg/500 mg em embalagens com 14 comprimidos e 10 mg/1000 mg em embalagens com 14 e 30 comprimidos. USO ADULTO. USO ORAL. VENDA SOB PRESCRIÇÃO MÉDICA. SE PERSISTEREM OS SINTOMAS, O MÉDICO DEVERÁ SER CONSULTADO. Para maiores informações, consulte a bula completa do produto. www.astrazeneca.com.br. Reg. MS – 1.0180.0407 (XIG006_min).

FORXIGA® – (dapagliflozina) comprimidos revestidos. Indicações: FORXIGA é indicado como adjuvante a dieta e exercícios para melhora do controle glicêmico em pacientes com diabetes mellitus tipo 2 em monoterapia ou em combinação com metformina; tiazolidinediona; sulfonilureia; inibidor da DPP4 (com ou sem metformina); ou insulina (isolada ou com até duas medicações antidiabéticas orais), quando a terapia existente juntamente com dieta e exercícios não proporciona controle glicêmico adequado. Indicado em combinação inicial com metformina quando ambas as terapias são apropriadas. FORXIGA não é indicado para uso por pacientes com diabetes tipo 1 e não deve ser utilizado para o tratamento de cetoacidose diabética Contraindicações: hipersensibilidade a dapagliflozina ou aos outros componentes da fórmula. Advertências e Precauções: Foram reportados alguns relatos pós-comercialização de cetoacidose em pacientes diabéticos tipo 1 e tipo 2 em uso de FORXIGA. Embora uma relação causal ainda não tenha sido estabelecida, recomenda-se que pacientes que apresentem sinais de cetoacidose incluindo náusea, vômitos, dor abdominal, prostração ou dispneia sejam avaliados quanto a presença de cetoacidose, mesmo que sua glicemia esteja menor que 250 mg/dL. FORXIGA® deve ser usado com cautela ou ser temporariamente suspenso em pacientes sob risco de depleção de volume, pacientes com hipertensão ou outra doença cardiovascular, infecções do trato urinário, incluindo urosepse e pielonefrite, uso concomitante com medicamentos que podem causar hipoglicemia, gravidez, lactação uso pediátrico, uso geriátrico. Categoria de Risco na Gravidez: C. Reações Adversas: infecção genital, infecção do trato urinário, dor nas costas, poliúria e erupção cutânea. Interações Medicamentosas: (sem alterações clínicas relevantes, sem necessidade de ajuste de dose) metformina, pioglitazona, sitagliptina, glimepirida, voglibose, hidroclorotiazida, bumetanida, valsartana, sinvastatina, rifampicina, ácido mefenâmico. Outras interações: os efeitos da dieta, tabagismo, produtos à base de plantas e uso de álcool sobre a farmacocinética da dapagliflozina não foram especificamente estudados. Interferência com o teste 1,5-anidroglucitol (1,5-AG). Posologia: a dose recomendada de FORXIGA, em monoterapia ou terapia combinada, é 10 mg, uma vez ao dia, a qualquer hora do dia, independentemente das refeições. Para pacientes em risco de depleção de volume devido a condições coexistentes, uma dose inicial de 5 mg de FORXIGA pode ser apropriada. Não são necessários ajustes de dose de FORXIGA com base na função renal ou hepática. Apresentações: embalagens com 30 comprimidos revestidos de 5 mg e embalagens com 14 ou 30 comprimidos revestidos de 10 mg. USO ORAL. USO ADULTO. VENDA SOB PRESCRIÇÃO MÉDICA. SE PERSISTIREM OS SINTOMAS, O MÉDICO DEVERÁ SER CONSULTADO. Para maiores informações, consulte a bula completa do produto. Reg. MS - 1.0180.0404 (FRX013_min).

CONTRAINDICAÇÕES: FORXIGA® é contraindicado a pacientes com conhecida hipersensibilidade à dapagliflozina ou aos outros componentes da fórmula. INTERAÇÕES MEDICAMENTOSAS: em estudos realizados em indivíduos sadios, a farmacocinética CONTRAINDICAÇÕES: doença renal ou disfunção renal da dapagliflozina não foi alterada pela metformina, moderada a grave. INTERAÇÃO MEDICAMENTOSA: pioglitazona, sitagliptina, glimepirida, voglibose, hidroclorotiazida, bumetanida, valsartana ou sinvastatina. cimetidina.

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