AE&M 61-5 by Sociedade Brasileira de Endocrinologia e Metabologia

ISSN 2359-3997

OFFICIAL JOURNAL OF THE BRAZILIAN SOCIETY OF ENDOCRINOLOGY AND METABOLISM Vol. 61 – No. 05 – October 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. 05 – October 2017

memorial 409 Prof. Dr. Bernardo Leo Wajchenberg Antonio Carlos Lerario

Archives of Endocrinology editorials

OFFICIAL JOURNAL OF THE BRAZILIAN 414 Biochemical diagnosis of acromegaly without a typical clinical phenotype: what are the concerns? Antonio Ribeiro-Oliveira Jr., Ariel Barkan SOCIETY OF original articles ENDOCRINOLOGY 416 Clinical utility of F-FDG PET/CT in the follow-up of a large cohort of patients with high-risk differentiated thyroid carcinoma Ji H. Yang, Rui M. B. Maciel, Claudia C. D. Nakabashi, Carolina C. P. S. Janovsky, Rosalia AND P. Padovani, Danielle Macellaro, METABOLISM Cléber P. Camacho, Akemi Osawa, Jairo Wagner, Rosa Paula M. Biscolla 411 Is the 18F-FDG PET/CT the definite resource to detect the recurrence on high-risk thyroid cancer patients? Carlos Alberto Buchpiguel

and Metabolism 18

426 Long-term follow-up of patients with elevated IGF-1 and nadir GH > 0.4 µg/L but < 1 µg/L Pedro Weslley Rosario, Maria Regina Calsolari

432 Timing of thyroid ultrasonography in the etiological investigation of congenital hypothyroidism Maria de Fátima Borges, Nathalie de Almeida Sedassari, Anelise de Almeida Sedassari, Luis Ronan Marquez Ferreira de Souza, Beatriz Pires Ferreira, Beatriz Hallal Jorge Lara, Heloísa Marcelina Cunha Palhares

438 IL-6, TNF-α, and IL-10 levels/polymorphisms and their association with type 2 diabetes mellitus and obesity in Brazilian individuals Kathryna Fontana Rodrigues, Nathalia Teixeira Pietrani, Adriana Aparecida Bosco, Fernanda Magalhães Freire Campos, Valéria Cristina Sandrim, Karina Braga Gomes

Archives of

447 Use of prophylactic oral calcium after total thyroidectomy: a prospective study Erwin Langner, Alfio José Tincani, André del Negro

455 Serum nesfatin-1 levels are decreased in pregnant women newly diagnosed with gestational diabetes

Esra Nur Ademoglu, Suheyla Gorar, Muge Keskin, Ayse Carlioglu, Rifki Ucler, Husamettin Erdamar, Cavit Culha, Yalcin Aral

Endocrinology

460 Novel immunoassay for TSH measurement in rats

Thalita G. Alves, Maria Clara de C. Melo, Teresa S. Kasamatsu, Kelen C. Oliveira, Janaina Sena de Souza, Rodrigo Rodrigues da Conceição, Gisele Giannocco, Magnus R. Dias-da-Silva, Maria Izabel Chiamolera, José Gilberto Vieira

464 Serum ghrelin levels in papillary thyroid carcinoma

and Metabolism

Bekir Ucan, Mustafa Sahin, Muhammed Kizilgul, Mustafa Ozbek, Seyda Ozdemir, Mustafa Calıskan, Erman Cakal

470 Screening tests for distal symmetrical polyneuropathy in Latin American patients with type 2 diabetes mellitus Nicolás Gómez-Banoy, Virginia Cuevas, Fernando Soler, Maria Fernanda Pineda, Ismena Mockus

476 Endothelial dysfunction in children with type 1 diabetes mellitus

Antonella Márcia Mercadante de Albuquerque do Nascimento, Inês Jorge Sequeira, Daniel França Vasconcelos, Lenora Gandolfi, Riccardo Pratesi, Yanna Karla de Medeiros Nóbrega

OFFICIAL JOURNAL OF THE BRAZILIAN SOCIETY OF ENDOCRINOLOGY AND METABOLISM 484 Effects of energetic restriction diet on butyrylcholinesterase in obese women from southern Brazil – A longitudinal study Willian dos Santos, Luciane Viater Tureck, Louise Farah Saliba,Caroline Schovanz Schenknecht, Débora Scaraboto, Ricardo Lehtonen R. Souza, Lupe Furtado-Alle

reviews 490 The clinical genetics of phaeochromocytoma and paraganglioma P. T. Kavinga Gunawardane, Ashley Grossman

501 Mechanisms involved in hearing disorders of thyroid ontogeny: a literature review Caio Leônidas Oliveira de Andrade, Gabriela Carvalho Machado, Luciene da Cruz Fernandes, Jamile Morais de Albuquerque, Luciana Lyra Casais-e-Silva, Helton Estrela Ramos, Crésio de Aragão Dantas Alves

case report 506 Use of cinacalcet and sunitinib to treat hypercalcaemia due to a pancreatic neuroendocrine tumor Hernan Valdes-Socin, Matilde Rubio Almanza, Mariana Tomé Fernández-Ladreda, Daniel Van Daele, Marc Polus, Marcela Chavez, Albert Beckers

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

OFFICIAL JOURNAL OF THE BRAZILIAN SOCIETY OF ENDOCRINOLOGY AND METABOLISM

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and Metabolism

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and Metabolism

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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

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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

INTERNATIONAL

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

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

memorial

Prof. Dr. Bernardo Leo Wajchenberg Antonio Carlos Lerario1

Arch Endocrinol Metab. 2017;61/5

Professor livre-docente em Endocrinologia da Faculdade de Medicina da Universidade de São Paulo (FMUSP). Diretor da Sociedade Brasileira de Diabetes (SBD)

Correspondence to: Antonio Carlos Lerario aclerario@terra.com.br Received on Oct/17/2017 Accepted on Oct/17/2017 DOI: 10.1590/2359-3997000000302

he responsability of writing a memorial honoring a great friend and master is always considered an arduous and sad task. But, in this case, I could realize that it was actually an honor to have the opportunity of sharing with the community of endocrinologists, particularly the younger ones, a little bit of the biography from one of the most prestigious and famous clinical Endocrinologist in our country. Born in São Paulo, Bernardo Leo Wajchenberg has shown since childhood to have a “brilliant mind”, starting at the first years of his elementary education, when he was considered the best student of his class. During medical school at São Paulo University (USP), he was distinguished as one of the best students, being awarded with 11 of 13 prizes granted by São Paulo Medical School. And, in 1950, at the conclusion of the medical course, he received the Rockefeller Foundation Prize as the outstanding graduate in this medical school. After completing residency in Internal Medicine at Clinics Hospital, School of Medicine, University of São Paulo (FMUSP), he ventured to USA to become a Research Fellow at Lilly Lab, at the University of Minnesota, followed by a WK Kellogg Fellowship at the University of Michigan. He returned to his alma mater (FMUSP) where he has progressed from Junior staff to Chief of Diabetes and Adrenal Unit, then to Chair of Endocrinology and Metabolism Division, posteriorly to Chief of the Laboratory of Medical Research and, finally, he became Vice-Chairman of the Medicine Department. In 1971 in parallel to his activities at Clinics Hospital, Dr. Wajchenberg organized and became responsible of a Laboratory of Radioimmunoassay in Brazil at the Nuclear and Energy Research Institute, University of São Paulo, a new technique recently developed in United States for quantification of several hormones (not available at this time), with the assistance of Dr. Rosalyn Yallow, who was later awarded with the Nobel Prize in Medicine. Despite the administrative load during his long academic career, Dr. Wajchenberg continued working in medical care with his assistants, directing clinical rounds, teaching residents and medical fellows, mentoring

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memorial

After his retirement of the São Paulo University Medical School and after becoming Professor of Medicine, Dr. Wajchenberg continued working as Coordinator of the Diabetes and Cardiology Service of the Heart Institute, at Clinics Hospital. In 2010 he assumed the role of Principal Investigator of the The BARI 2D Study Group in Brazil, a multicenter trial (sponsored by the National Institute of Health) evaluating the relative safety and efficacy of an insulinproviding vs. insulin-sparing regimen in the treatment of type 2 diabetes complicated by coronary heart disease. At that time he also returned to his passion of teaching new students and fellows, participating in rounds and, mainly, coordinating patient care in the outpatient clinic. Besides academic activities Dr. Wajchenberg also promoted a great contribution for the development of Diabetes and Endocrinology specialties in Brazil and in Latin America, participating actively in the foundation and management of several associations, serving as President of Brazilian Endocrine Society, Brazilian Diabetes Society and Latin American Diabetes Association. He was also part of the Executive Committee of the International Diabetes Federation. As a master, mentor and a physician, Dr. Wajchenberg was described by most of his subordinates as a tireless worker, an enthusiastic leader, a perfectionist and a man of principles and integrity. At the time of his retirement we counted more than 200 endocrinologists from different parts of Brazil that initiated their medical education with him in São Paulo, and are now disseminating the new advances in the physiopathology and treatment of endocrinology in different medical centers of the country.

post graduating doctors and acting incessantly as an active researcher in different areas of endocrinology, such as diabetes, adrenal, obesity and calcium metabolism, thyroid and steroid hormones metabolism. During his career he published more than 250 papers in peer reviewed publications in three different languages. From research perspective, Dr. Wajchenberg has moved seamlessly from bench to bedside, contributing to the improvement of knowledge of the physiopathology and the treatment of metabolic and endocrinological diseases. Some of his accomplishments are the use o,p’-DDD for treating adrenocortical carcinoma, in the metabolic effects of sulphonylureas, unraveling the mechanisms of phenformin and on the physiopathology of visceral fat and metabolic syndrome. The quality of his research can be attested by the impressive number of 11,124 citations observed in peer-reviewed publications of other authors. Two of his review articles published in the Journal Endocrine Reviews in 2000 and 2007, respectively, were cited over 3,200 times in papers of other authors. Other publications signed by Dr. Wajchenberg includes several chapters in medical books and his position as Editor for two editions of the book entitled “Tratado de Endocrinologia Clínica” (Treatise on Clinical Endocrinology; 1992 and 2014). Recognizing the breadth, depth and longevity of his illustrious career (over 50 years), The American Endocrine Society distinguished Dr. Bernardo Leo Wajchenberg with the Distinguished Physician Award. The other prizes that he was granted include: Francisco Arduino Prize (from Brazilian Diabetes Society, in 2003), José Scherman Prize (from State Institute of Diabetes and Endocrinology; IEDE, in 2011).

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editorial

Is the 18F-FDG PET/CT the definite resource to detect the recurrence on high-risk thyroid cancer patients? Carlos Alberto Buchpiguel1

Arch Endocrinol Metab. 2017;61/5

1 Departamento de Radiologia e Oncologia da Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, SP, Brasil

Correspondence to: Carlos Alberto Buchpiguel buch@usp.br Received on Oct/13/2017 Accepted on Oct/13/2017 DOI: 10.1590/2359-3997000000300

hyroid cancer is the most common endocrine neoplasm (1). Besides its good prognosis and indolent clinical course, more aggressive stages pose some challenges and may impair the morbidity/mortality rate in high-risk patients. So in those cases, is mandatory to optimize the diagnostic work-up in order to detect recurrences and metastases as early as possible for an effective therapeutic planning (2). High-risk patients and undifferentiated tumors loose the capacity of trapping iodine-131, making not only the diagnosis but also the treatment of those patients a challenge. Even when conventional imaging is not able to localize the recurrence besides the rise of the serum thyroglobulin (Tg) levels, it is clinically valuable to pursue other alternatives to localize the sites of recurrent disease. Actually, neck ultrasound and chest computed tomography are efficient to detect the most common sites of recurrent thyroid tumor (3). However, it is not rare to see high-risk patients with elevated Tg and no signs of recurrence on conventional imaging, including iodine-131 whole body survey (WBS) (4). PET-CT emerged as a molecular imaging tool, where the disease is detected more due to the molecular profile and/or metabolic cellular signaling than structural or functional abnormalities. The high rate of anaerobic glycolysis is one of the main features of various malignant tumors, and that is the reason for using fluordesoxyglucose labeled with fluoride-18 (FDG), a common positron emitter produced on Cyclotrons (5). Dedifferentiating thyroid tumors overexpress GLUT (glucose transporter proteins located on the cellular membrane) and also hexokinase-II (HK-II) that are the two major conditions for promoting and facilitating glucose uptake in the malignant cells. A reasonable number of publications are seen in the literature showing the value of FDG-PET in the evaluation of patients with thyroid carcinoma. A recent meta-analysis published by Haslerud and cols. (6) showed a pooled sensitivity and specificity of 79.4% for detecting recurrent well-differentiated thyroid carcinoma (WDTC). After the year 2000, the majority of PET scanners were shipped with a CT integrated to the equipment (PET-CT). That technological advance brought an increase of specificity since it was possible to correlate the molecular findings with the exact anatomical location and structural abnormality seen on CT. That’s why the more recent systematic reviews evaluating the use of PET-CT in thyroid cancer showed better accuracy values as compared to “old” meta-analysis (7). A very interesting paper published in this issue of Archives of Endocrinology and Metabolism (AE&M) by Yang and cols. (8) is the first series enrolling a reasonable number of Brazilian patients to be evaluated by PET-CT in detecting recurrent thyroid cancer. It is really an interesting contribution since it divided the patients in three different groups. The third group was the one where we wouldn’t expect great

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performance for PET-CT, as shown by the authors, since they included differentiated tumors with elevated serum Tg and positive WBS (2). The first group was divided in two, 1A (elevated Tg and negative conventional imaging and WBS) and 1B (elevated TG, and WBS not compatible with conventional imaging finding or level of Tg). Here we can make some comments. The greater impact would be detecting foci of recurrence where no other test is able to do. We would expect to see better incremental diagnostic value of PET-CT in those cases where no abnormalities are seen on US, CT or MRI. However, in that group the authors could only include nine patients, a very small number that precludes stronger conclusion regarding the value of the method tested in this article. Also the authors did not comment in that particular group how many patients had the PET-CT scans done under TSH stimulation or not. They stated that in significant percentage of patients it was not applied TSH stimulation for the PET scans. It is true that it is controversial the value of TSH stimulation in increasing the accuracy of PET in thyroid cancer, however, there has been no enough evidence in the literature yet to rule out any value of that stimulation for difficult and small-size disease detection. So we could conclude that in this small group of patients the PET did not add any clinical value and in a worst scenario lead to unnecessary biopsy caused by false-positive findings in cervical lymph nodes in three patients. In the group IB, many patients showed alterations on conventional imaging at the same location seen of FDG-PET. If the location was the same of FDG-PET, and not compatible with the WBS, could we assume that the CT or MRI finding was enough to confirm the recurrence? Moreover, it is very well know that CT has a better detection rate of lung metastasis compared to PET, since size of the nodule is a limitation factor for the resolution of the modern PET scanners (9). Even though the small nodules detected by CT might be unspecific or indeterminate only by anatomical analysis, under the circumstance of rising Tg and highrisk profile of the patient for recurrence, it would be fair to consider those findings at least suspicious for recurrent disease. Moreover, to be detected by PET, those findings in the lungs must be large or the lung nodules numerous enough to be depicted by PET. The authors stated very well the limitations of the study, and then, in this group 1B could the CT/MRI alone be effective enough to change the clinical management in certain number of those thirteen patients, with 412

no incremental information provided by PET? The question regarding that comment concerns the costeffectiveness of doing PET-CT in all patients, including those with abnormalities already seen on conventional structural cross-sectional imaging. The same issues can be discussed for the Group 2. Many patients showed abnormalities on lungs by CT and also by PET. Has PET also provided real incremental value towards the CT findings or just confirmed the CT abnormalities as related to the thyroid cancer recurrence? Another limitation is the small number of patients regarding each group with unfavorable histology, a limitation that is expected considering the low prevalence of those histological types. There is no data in the literature evaluating the GLUT and HK-II expression in the various aggressive histology thyroid tumors. So, some conclusions must be taken with caution regarding the power of sample and considering some methodological aspects inherent to a retrospective study. Nevertheless, this is the first large cohort Brazilian study evaluating the clinical value of PET-CT in high-risk thyroid cancer patients, and hopefully will stimulate other groups to replicate that study to confirm the very interesting findings published in this issue of the AE&M. Disclosure: no potential conflict of interest relevant to this article was reported.

REFERENCES 1. Kitahara CM, Devesa SS, Sosa JA. Increases in Thyroid Cancer Incidence and Mortality-Reply. JAMA. 2017;318(4):390-1. 2. Haugen BR, Alexander EK, Bible KC, Doherty GM, Mandel SJ, Nikiforov YE, et al. 2015 American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer: The American Thyroid Association Guidelines Task Force on Thyroid Nodules and Differentiated Thyroid Cancer. Thyroid. 2016;26(1):1-133. 3. Machado MR, Tavares MR, Buchpiguel CA, Chammas MC. Ultrasonographic Evaluation of Cervical Lymph Nodes in Thyroid Cancer. Otolaryngol Head Neck Surg. 2017;156(2):263-71. 4. Kendi ATK, Mudalegundi S, Switchenko J, Lee D, Halkar R, Chen AY. Assessment of the Role of Different Imaging Modalities with Emphasis on Fdg Pet/Ct in the Management of Well Differentiated Thyroid Cancer (WDTC). J Thyroid Disord Ther. 2016;5.pii:202. 5. Okada J, Oonishi H, Yoshikawa K, Imaseki K, Uno K, Itami J, et al. FDG-PET for the evaluation of tumor viability after anticancer therapy. Ann Nucl Med. 1994;8(2):109-13. 6. Haslerud T, Brauckhoff K, Reisæter L, Küfner Lein R, Heinecke A, Varhaug JE, et al. F18-FDG-PET for recurrent differentiated thyroid cancer: a systematic meta-analysis. Acta Radiol. 2016;57(10): 1193-200. Arch Endocrinol Metab. 2017;61/5

F-FDG PET/CT & high-risk thyroid cancer

7. Leboulleux S, Schroeder PR, Schlumberger M, Ladenson PW. The role of PET in follow-up of patients treated for differentiated epithelial thyroid cancers. Nat Clin Pract Endocrinol Metab. 2007;3(2):112-21.

9. Lohrmann C, Weber WA. What is the clinical value of PET/CT in the diagnosis of pulmonary nodules? Zentralbl Chir. 2014;139(1): 108-13.

8. Yang JH, Maciel RMB, Nakabashi CCD, Janovsky CCPS, Padovani RP, Macellaro D, et al. Clinical utility of 18F-FDG PET/CT in the

follow-up of a large cohort of patients with high-risk differen tiated thyroid carcinoma. Arch Endocrinol Metab. 2017;61(5): 416-25.

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editorial

Biochemical diagnosis of acromegaly without a typical clinical phenotype: what are the concerns? Antonio Ribeiro-Oliveira Jr.1, Ariel Barkan2

Laboratory of Endocrinology, Federal University of Minas Gerais (UFMG), Belo Horizonte, MG, Brazil 2 Department of Medicine, Division of MEND, and Department of Neurosurgery, University of Michigan, Ann Arbor, MI, USA 1

Correspondence to: Antonio Ribeiro-Oliveira Jr. antoniorojr@gmail.com Received on Oct/21/2017 Accepted on Oct/21/2017

DOI: 10.1590/2359-3997000000301

414

he importance of early diagnosis and prompt treatment of acromegaly is beyond question (1). In this issue of Archives of Endocrinology and Metabolism, Rosario and Calsolari (2) report the results of a five year follow-up investigation of patients with a suspicious clinical scenario of acromegaly coupled to mildly to modestly elevated IGF-1 but with questionable glucose-suppressed GH. It included 16 women and one man aged 30 to 55 years who were found to have some clinical findings potentially attributable to acromegaly (glucose intolerance, headaches, arthritic pains, etc.) but no acromegalic phenotype. They had circulating IGF-1 levels 1.08 to 1.53 times the upper limit of the normal range for age in two measurements outside puberty or pregnancy, but their glucose-suppressed GH went down below the currently agreed cut-off point of 1 µg/L but was still higher than the strictest criterion of 0.4 µg/L. All the 17 selected patients had a pituitary MRI performed and non-pituitary acromegaly was excluded through chest and abdominal contrast-enhanced tomography. All of them were not treated for acromegaly (either surgically or medically) but were just followed for up to 5 years and dynamic facial changes was evaluated through longitudinal comparison of photographs. Initially, just one patient had a visible pituitary microadenoma that did not change on follow-up. After five years of follow-up, these patients remained without an acromegalic phenotype and IGF-1 spontaneously returned to normal in 5 out of 17 patients, as confirmed by two measurements. Regarding the other 12 patients with persistently elevated IGF-1, none of them showed an increase of IGF-1 higher than 20%, and 2 of them suppressed GH in oGTT to lower than 0.4 µg/L, while no tumor was further detected. Therefore, after a follow-up of five years, we are still uncertain of the diagnosis in the remaining 10 patients, which means ~60% of the cohort of patients with elevated IGF-1 levels without GH suppression in oGTT to below 0.4 µg/L. Importantly, in this population without a clinical phenotype of acromegaly, the oGTT cut-off of 1 µg/L, as suggested by the current Endocrine Society consensus (3) would have ended up further investigation at the initial presentation, although high IGF-1 levels would still be without an explanation. The five years follow-up was able to confirm that there was no appearance of facial acromegalic changes or further deterioration of biochemical parameters in these patients. Conversely, over one third of the patients were able to normalize either IGF-1 or reach GH suppression levels < 0.4 µg/L during an oGTT after five years of first assessment. Although quite unlikely, these patients should always be evaluated in the context of genetic syndromes associated with acromegaly, when alteration of biochemical parameters may be subtle (4). Another possibility discussed by the authors, is that these patients may have had slightly elevated IGF-1 level expected in 2.5% of normal population due to statistical distribution of “normalcy”. Arch Endocrinol Metab. 2017;61/5

Acromegaly without a typical phenotype

confirmed somatotropinomas are “silent”, i.e. found in patients without any phenotypical manifestations (8,9). In these cases, we are completely in the dark: should we treat the patient or the biochemical/radiological finding? Holdaway and cols. (10) introduced the “high IGF” parameter as a predictor of higher mortality in acromegaly, but we still do not know exactly how high it should be to have clinical impact on morbidities and mortality justifying active normalization of IGF-1 levels, especially in patients whose clinical diagnosis of acromegaly is not firmly defined. Cushing disease is famous for being diagnostically the most difficult variety of pituitary tumors. Acromegaly is rapidly gaining on it. Disclosure: no potential conflict of interest relevant to this article was reported.

REFERENCES 1. Ribeiro-Oliveira A Jr, Barkan A. The changing face of acromegaly-advances in diagnosis and treatment. Nat Rev Endocrinol. 2012;8(10):605-11. 2. Rosario PW, Calsolari MR. Long-term follow-up of patients with elevated IGF-1 and nadir GH > 0.4 µg/L but < 1 µg/L. Arch Endocrinol Metab. 2017;61(5):426-31. 3. Katznelson L, Laws ER Jr, Melmed S, Molitch ME, Murad MH, Utz A, et al.; Endocrine Society. Acromegaly: an endocrine society clinical practice guideline. J Clin Endocrinol Metab. 2014;99(11):3933-51. 4. Gadelha MR, Kasuki L, Korbonits M. The genetic background of acromegaly. Pituitary. 2017;20(1):10-21. 5. Ribeiro-Oliveira A Jr, Faje AT, Barkan AL. Limited utility of oral glucose tolerance test in biochemically active acromegaly. Eur J Endocrinol. 2011;164(1):17-22. 6. Butz LB, Sullivan SE, Chandler WF, Barkan AL. “Micromegaly”: an update on the prevalence of acromegaly with apparently normal GH secretion in the modern era. Pituitary. 2016;19(6):547-51. 7. Lonser RR, Kindzelski BA, Mehta GU, Jane JA Jr, Oldfield EH. Acromegaly without imaging evidence of pituitary adenoma. J Clin Endocrinol Metab. 2010;95(9):4192-6. 8. Sakharova AA, Dimaraki EV, Chandler WF, Barkan AL. Clinically silent somatotropinomas may be biochemically active. J Clin Endocrinol Metab. 2005;90(4):2117-21. 9. Wade AN, Baccon J, Grady MS, Judy KD, O’Rourke DM, Snyder PJ. Clinically silent somatotroph adenomas are common. Eur J Endocrinol. 2011;165(1):39-44. 10. Holdaway IM, Bolland MJ, Gamble GD. A meta-analysis of the effect of lowering serum levels of GH and IGF-I on mortality in acromegaly. Eur J Endocrinol. 2008;159(2):89-95. Copyright© AE&M all rights reserved.

The authors suggest that the oGTT criterion of GH < 1 µg/L may be optimal for excluding acromegaly in patients without convincing clinical findings and with only mildly elevated IGF-1. However, the combination of comorbidities of acromegaly found in these patients still raises suspicion that a mild form of the disease might have been present. First, it relates to purely technical issues. There had recently been a lot of concerns as related to GH and IGF-1 assays (1). For example, we still do not have glucose suppression criteria that are specific for age, gender and body mass index. For the IGF-1, the authors are to be congratulated for using normative parameters derived from a large population (4,350 adults) from the same locality. However, although the authors seem to be aware of the assay overestimation drift with the Siemens IGF-1 assay, the observed normalization of IGF-1 in the subsequent assessment five years later in 30% of patients could be related to the correction of the assay overestimation drift instead of a true normalization. However, the most important is the realization that the classical diagnostic criteria of acromegaly no longer apply. Recent technical refinements have demonstrated that a significant proportion of patients with acromegaly differ dramatically from “classical” clinical, biochemical and radiological presentations. Indeed, patients with a typical clinical phenotype coupled to high IGF-1 levels, but with plasma GH in the “normal” range may have glucose-suppressed GH below 1 µg/L in ~50% of the cases and below 0.4 µg/L in as many as 30% of the cases (5). Although rare, these cases of “micromegaly” are usually, albeit not necessarily, coupled to the presence of microadenomas (5,6). In these cases when pituitary MRI confirms the presence of the tumor, neurosurgical approach is usually followed by amelioration of the signs and symptoms as well as confirmation of the somatotroph tumor by pathological analysis and immunohistochemistry. Occasionally, cases of acromegaly without a visible tumor have been reported (7). In these cases, the best approach is yet to be defined, although neurosurgical successes have been reported (7). Perhaps, the most difficult is the situation where biochemically and immunochemically –

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original article

Clinical utility of 18F-FDG PET/CT in the follow-up of a large cohort of patients with high-risk differentiated thyroid carcinoma Ji H. Yang1, Rui M. B. Maciel1, Claudia C. D. Nakabashi1, Carolina C. P. S. Janovsky1, Rosalia P. Padovani1, Danielle Macellaro1, Cléber P. Camacho1, Akemi Osawa2, Jairo Wagner2, Rosa Paula M. Biscolla1

ABSTRACT Centro de Doenças da Tireoide e Laboratório de Endocrinologia Molecular e Translacional, Divisão de Endocrinologia, Departamento de Medicina, Escola Paulista de Medicina, Universidade Federal de São Paulo (EPM-Unifesp), São Paulo, SP, Brazil 2 Departamento de Imagem, Hospital Israelita Albert Einstein (HIAE), São Paulo, SP, Brazil 1

Correspondence to: Rui M. B. Maciel Laboratório de Endocrinologia Molecular e Translacional, Divisão de Endocrinologia, Departamento de Medicina, Escola Paulista de Medicina, Universidade Federal de São Paulo Rua Pedro de Toledo, 669, 11th Floor 04039-032 – São Paulo, SP, Brazil rui.maciel@unifesp.br

Objective: To evaluate the clinical utility of 18F-FDG PET/CT in patients with high-risk DTC. Subjects and methods: Single-center retrospective study with 74 patients with high-risk differentiated thyroid cancer (DTC), classified in 4 groups. Group 1: patients with positive sTg or TgAb, subdivided in Group 1A: negative RxWBS and no foci of metastases identified at conventional image (n = 9); Group 1B: RxWBS not compatible with suspicious foci at conventional image or not proportional to sTg level (n = 13); Group 2: patients with histological findings of aggressive DTC variants (n = 21) and Group 3: patients with positive RxWBS (n = 31). Results: 18F-FDG PET/CT identified undifferentiated lesions and helped restage the disease in groups 1B and 2. The scan helped guide clinical judgment in 9/13 (69%) patients of group 1B, 10/21 (48%) patients of group 2 and 2/31 (6%) patients of group 3. There was no clinical benefit associated with group 1A. 18F-FDG PET/CT was associated with progressive disease. Conclusion: 18F-FDG PET/CT is a useful tool in the follow-up of patients with high-risk DTC, mainly in the group of RxWBS not compatible with suspicious foci at conventional image or not proportional to sTg level and in those with aggressive DTC variants. Additionally, this study showed that 18F-FDG PET/CT was associated with progression and helped display undifferentiated lesions guiding clinical assessments regarding surgeries or expectant treatments. Arch Endocrinol Metab. 2017;61(5):416-25. Keywords Differentiated thyroid carcinoma, 18F-FDG PET/CT, radioiodine (RAI), whole-body scan (WBS), thyroglobulin (Tg)

Received on Nov/12/2016 Accepted on Mar/18/2017 DOI: 10.1590/2359-3997000000285

INTRODUCTION

he routine follow-up of patients with differentiated thyroid cancer (DTC) after surgery and radioiodine (RAI) remnant ablation comprises the measurement of serum thyroglobulin (sTg), cervical ultrasound (US), whole-body scan with 131I (WBS) or conventional imaging techniques, such as computed tomography (CT) or magnetic resonance imaging (MRI). However, serum Tg levels may not be a reliable tool in some patients, mainly due to the presence of anti-thyroglobulin antibodies (TgAb) (1) or to undifferentiated tumors that do not secrete Tg. Similarly, WBS may fail to localize residual thyroid tissue in less differentiated tumors due to its impaired ability to concentrate RAI (2). 416

In recent years, combined 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) has emerged as a valuable tool in the follow-up of thyroid cancers. By correlating the metabolic information of 18F-FDG PET with the morphologic resolution of CT and due to the enhanced glucose metabolism in thyroid cancers, particularly in less differentiated tumors, this imaging technique has been employed beyond the classical indication of DTC patients with positive sTg and negative WBS. Furthermore, current applications extend to disease extension, including the detection of undifferentiated metastases (3), guidance of therapy assessments and prediction of prognosis (4-6). Some studies have shown that 18F-FDG PET and 18F-FDG PET/CT can induce changes in clinical management plans in 10-78% of Arch Endocrinol Metab. 2017;61/5

FDG PET/CT in differentiated thyroid cancer

patients with DTC (7-12), thereby improving clinical judgment. In the Brazilian population, there has been only one study using 18F-FDG PET/CT in thyroid cancer patients with negative WBS and positive sTg in a small patient sample (13). The potential to induce changes in the clinical management and the lack of other studies in our country motivated this work. Therefore, the aim was to evaluate the clinical utility of the 18F-FDG PET/CT in a large cohort of patients with DTC in various groups.

SUBJECTS AND METHODS A total of 644 patients with DTC were referred, evaluated, treated and followed by a single team of physicians at the associated Thyroid Disease Centers in the Division of Endocrinology, Department of Medicine, Escola Paulista de Medicina, Universidade Federal de São Paulo and the Instituto Israelita de Ensino e Pesquisa Albert Einstein (in São Paulo, Brazil). In this population, 80 patients were submitted to 18F-FDG PET/CT scans from February 2008 to June 2013. Six patients were lost to follow-up; the medical records of the remaining 74 patients (who performed 95 total 18 F-FDG PET/CT scans) were analyzed retrospectively. This study was approved by the Institutional Ethics Committee.

According to the medical indications of 18F-FDG PET/CT, the 74 patients were classified in 4 groups (Figure 1, part I); clinical and epidemiological information is shown in Table 1: • Group 1. Patients with positive sTg or TgAb were subdivided in Group 1A (n = 9): negative post-therapeutic -131 whole body scan (RxWBS) and no foci of metastases identified at conventional image, and Group 1B (n = 13): RxWBS not compatible with suspicious foci at conventional image or not proportional to sTg level. • Group 2. Patients with histological findings of aggressive DTC variants (n = 21): oncocytic (n = 3), poorly differentiated areas (n = 2), tall-cell (n = 4), diffuse sclerosing (n = 4), insular (n = 5) and solid variant (n = 3) with incomplete biochemical or structural disease. • Group 3. Patients with positive RxWBS (n = 31): in this group, 18F-FDG PET/CT was performed to detect additional foci of undifferentiated metastases. In the beginning of the study, 59 of 95 18 F-FDG PET/CT scans were performed after TSH stimulation (Tg/TSH): hypothyroidism, TSH > 30 mcUI/mL or after recombinant human TSH, rhTSH, Genzyme Transgenics Corp., Cambridge, 644 patients with DTC FDG-PET/CT (n = 80)

Insufficient data (n = 6)

FDG-PET/CT (n = 74)

Group 1 Positive sTg or TgAb (n = 22)

Group 2 Aggressive variants (n = 21)

1A. patients with positive sTg or TgAb, negative RxWBS and no foci of metastases identified at CI (n = 9)

1B. patients with positive sTg or TgAb, and RxWBS not compatible with suspicions foci at CI and/or not proportional to sTg level (n = 13)

Clinical relevance n = 0/9

Clinical relevance n = 9/13 (69%)

Clinical relevance n = 10/21 (48%)

Group 3 Positive RxWBS (n = 31)

Clinical relevance n = 2/31 (6%)

* CI: conventional images.

Figure 1. Schematic representation of the groups (I) and the clinical relevance (II). Arch Endocrinol Metab. 2017;61/5

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Massachusetts. Over the course of follow-up, the literature demonstrated that despite studies showing that the number of positive scans and standard uptake value (SUV) increase under rhTSH stimulation, there was no conclusive evidence that those findings improve clinical management (12,14). Consequently, the remaining 36 scans were performed using LT4 (Tg/LT4). 18F-FDG PET/CT imaging and analysis were performed in accordance with the protocol described by Yamaga and cols. (13). For the predictive value analysis, the following criteria defined by Hooft and cols. (15) were used: 1) histology/cytology; 2) US-FNAC for cervical lesions; 3) focal 131I-uptake; 4) pathognomonic bone scan or MRI for bone metastases; 5) CT/MRI for brain metastases; and 6) progression of radiological documented lesions suspect for malignancy. The results were considered positive in the presence of 18F-FDG uptake in suspected lesions (visualized on conventional image) or in those patients with biochemical disease.

Table 1. Clinical and epidemiological data Clinical variables

Results

Sex

Female n = 57/Male n = 17

Total thyroidectomy

Yes n = 74

Lymph node resection

Yes: 57/No: 17

Age at diagnosis (years)

Median 40.9 (12-82)

Age at PET/CT (years)

Median 47.6 (16-85)

Follow up after PET/CT (months)*

Median 32.4 (7-72)

Group 1A (n = 9) sTg (ng/mL) TgAb+ Median of accumulated activity (mCi)

n = 6: 0.2-4.9 (Tg/LT4)/3.9-7.4 (Tg/TSH) n=2 350 (200-750)

Group 1B (n = 13) sTg (ng/mL) TgAb+ Median of accumulated activity (mCi)

n = 7: 0,6-91 (Tg/LT4)/2.4-292 (Tg/TSH) n=5 375 (230-700)

Group 2 (n = 21) sTg (ng/mL) TgAb+ Median of accumulated activity (mCi)

n = 20: 0.1-898 (Tg/LT4)/0.1-1000 (Tg/TSH) n=1 250 (100-1000)

Group 3 (n = 31) sTg (ng/mL) TgAb+ Median of accumulated activity (mCi)

n = 30: 0.1-47 (Tg/LT4)/0.2-168 (Tg/TSH) 1 450 mCi (150-800)

RESULTS

* From the first FDG-PET/CT; sTg: serum thyroglobulin; Tg/LT4: unstimulated Tg; Tg/TSH: stimulated Tg.

418

The results were considered negative if there was no 18 F-FDG uptake. All patients underwent cervical US as the routine serial assessment, and suspicious cervical lesions were submitted to US-guided fine-needle aspiration cytology (US-FNAC) (16). Conventional imaging was performed during the follow-up if necessary (high levels of sTg measurements, WBS uptake, lung, retropharyngeal or bone suspicious metastases). Serum Tg levels were measured by a highly sensitive chemiluminescence assay (Tg Access immunoassay, Beckman Coulter, Brea, CA) with a functional sensitivity of 0.1 ng/mL. TSH levels were measured using a thirdgeneration assay that provided a functional sensitivity of 0.05 mUI/mL (17). According to the combined and serial data of conventional imaging, the patientâ&#x20AC;&#x2122;s clinical status was classified as stable or progressive disease. Progressive disease was defined as an increase in tumor size during the follow-up, and stable disease was defined as stability of the lesions. Then, we analyzed the association between 18F-FDG uptake and progressive disease. We also studied the association between the PET/CT results and sTg levels using the cutoff recommended by ATA, 10 ng/mL. For the continuous variables, the difference between positive and negative 18F-FDG PET/CT groups was assessed using the Mann-Whitney test. The ROC curve was used for the continuous variables to calculate the best cut-off point. The chi-square test was used to determine the differences in the frequency of the categorical variables. A p < 0.05 result was considered significant.

F-FDG PET/CT results and clinical relevance

Group 1A: patients with positive sTg or TgAb, negative RxWBS and no foci of metastases identified at conventional image (n = 9) Ten scans were performed in this group. Although 18 F-FDG PET/CT displayed 6 cervical positive lesions in only 3 patients, none was confirmed as metastasis based on US-FNAC. The sTg levels in this group were 0.2-4.9 ng/mL (Tg/LT4) and 3.9-7.4 ng/mL (Tg/TSH). In conclusion, 18F-FDG PET/CT did not provide additional information in this group of patients. Arch Endocrinol Metab. 2017;61/5

FDG PET/CT in differentiated thyroid cancer

Group 1B: patients with positive sTg or TgAb and RxWBS not compatible with suspicious foci at conventional image or not proportional to sTg level (n = 13)

results (Table 2, patients 1-3); in other three patients, the second 18F-FDG PET/CT was useful to show progression of the metastases (Table 2, patients 4, 6-7). The 18F-FDG PET/CT displayed, in all, cervical, mediastinal or retropharyngeal uptake in 4 patients who had confirmed metastatic lesions based on histological results (Table 2, patients 1-4). One patient (Table 2, patient 4) and the remaining 5 (Table 2, patient 5-9) presented diffuse pulmonary 18F-FDG uptake. In group 1B, we considered that the 18F-FDG PET/CT results helped localize metastases in 9/13 (69%).

Twenty 18F-FDG PET/CT scans were performed in this group of 13 patients, and nine of 13 presented positive 18 F-FDG PET/CT scans (Table 2, patients 1-9). In those patients, the results helped clarify lesions visualized in conventional images that were RAI negative; in three of them, a second 18F-FDG PET/CT demonstrated the efficacy of the surgery indicated after the first scan

Table 2. Clinical and imaging characteristics of group 1B No

RxWBS uptake

PET

Tg/LT4

Tg/TSH

CT/RM findings

PET-CT findings

Management

Clinical Relevance

Negative

Thyroid bed Mediastinum Retropharingeaum

Surgery

Yes

8.7

Thyroid bed Retropharingeaum

Expectant

1.9

Mediastinum

Surgery

Negative

A B

3.8

Cervical

Expectant

Negative

0.5*

Cervical

Surgery

0.1*

Retropharingeaum

Expectant

1.5

Thyroid bed Lung

Thyroid bed

Expectant

173

Thyroid bed Mediastinum Lung

Surgery

0.5*

Paratracheal Lung Esophagus

Expectant

Yes

Cervical

Negative

Yes Yes

0.4*

Lung

Expectant

0.1*

Lung

Progression of the lesions**

Expectant

292

Lung

Expectant

Lung

Progression of the lesions**

Expectant

1000*

Hilar mass lung

Lung atelectasis

Expectant

Yes

Mediastinum Paratracheal Lunge

Expectant

Yes

Negative

5000*

Cervical

0.8*

Cervical

0.6

4.8

Mediastinum Lung

Expectant

Negative

0.8

2.4

Mediastinum

None

Expectant

Negative

3.7

5.1

Lung

None

Expectant

Cervical

Lung

Expectant

Yes

PET A, B: 1 , 2 scans, respectively; RxWBS: Post-Therapeutic -131 Whole Body Scan; Tg/LT4: unstimulated Tg (ng/mL); st

Tg/TSH: stimulated Tg (ng/mL). * TgAb positive; ** In relation to 1st PET/CT.

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FDG PET/CT in differentiated thyroid cancer

Group 2: patients with aggressive variants at the histological findings with incomplete biochemical or structural disease (n = 21) Twenty-seven scans were performed in this group of 21 patients. In 10 of 21 patients, 18F-FDG PET/CT provided relevant information. Oncocytic variant (n = 2): The scan was indicated for an undetectable sTg and positive RxWBS cervical metastasis uptake, and there was 18F-FDG uptake in the cervical subcutaneous tissue. The other patient presented with RAI negative but suspicious pulmonary

lesions on conventional images, and the 18F-FDG PET/CT scan showed lung uptake. Both lesions were confirmed as metastases based on histological analysis (Table 3, patients 1-2), indicating that 18F-FDG PET/CT provided relevant information. Poorly differentiated areas on histology (n = 1): the patient presented a positive RxWBS lumbar vertebrae metastasis with accelerated elevation of sTg, and the positive 18F-FDG uptake suggested possible tumor undifferentiation. This patient also presented with cerebral metastasis and had been treated with cerebral and vertebrae radiotherapy (Table 3, patient 4).

Table 3. Clinical and imaging characteristics of group 2 No

Histology

RxWBS uptake

PET

Tg/LT4

Tg/TSH

CT/RM findings

PET-CT findings

Management

Clinical Relevance

Oncocytic

Cervical

0.6

5.4

Subcutaneous

Surgery

Yes

Oncocytic

Cervical

278

Lung

Surgery

Yes

Oncocytic

Scapula

0.1

0.6

Scapula

RAI

Poorly differentiated

Spine

767

Vertebra L4

RAI, RT

Yes No

Poorly differentiated

Cervical

0.1

Retropharingeaum

None

Expectant

Insular

Ø RAI

511

1000

Lung

None

RAI

Lung

None

RAI

Insular

Ø RAI

3.3

Lung

None

RAI

Tall cell

Cervical Mediastinum Focal lung

134

898

7 8

Cervical Focal lung

Negative

Tall cell Tall cell

Cervical

Lung

Expectant

Lung

Surgery

Brain

379

Kidney, L2

Surgery

3* Mediastinum Lung

Cervical Mediastinum Lung

Surgery TKI

Insular

Lung Lung

Mediastinum Lung

Progression of the lesions**

Expectant

Lung

Surgery

104

635

Lung

Expectant

Lung

Expectant

Yes Yes

Yes

Yes Yes

Solid trabecular

Cervical

Lung

Expectant

Yes

Solid trabecular

Negative

0.2

None

Expectant

Solid trabecular

Mediastinum

0.2

Mediastinum

Cervical

Expectant

Diffuse sclerosing

Lung

0.1

2.7

Thyroid bed

Expectant

Diffuse sclerosing

Negative

1.9

271

Paratracheal

None

PEI

Diffuse sclerosing

Cervical

0.2

0.1

None

Expectant

Diffuse sclerosing

Negative

1.9

4.3

None

Expectant

Lung

Insular

Cervical

428

Insular

Ø RAI

Tall cell

Cervical

2.4

None

Expectant

Cervical

Expectant

Cervical

Expectant

PET A, B, C, D: 1 , 2 , 3 and 4 scans, respectively; PEI: percutaneous injection of ethanol; RAI: radioiodine; RxWBS: Post-Therapeutic -131 Whole Body Scan; Ø RAI: without radioiodine; RT: radiotherapy; Tg/LT4: unstimulated Tg (ng/mL); Tg/TSH: stimulated Tg (ng/mL); TKI: tyrosine-kinase inhibitor. * TgAb positive; ** In relation to 1st PET/CT. st

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FDG PET/CT in differentiated thyroid cancer

Other aggressive variants (n = 7): 18F-FDG PET/CT provided relevant information in 7 patients. Two patients without previous RAI treatment underwent paired 18F-FDG PET/CT and DxWBS scans. A 18F-FDG negative scan combined with positive RAI uptake endorsed the first RAI treatment indication (Table 3, patients 6-7). The other 5 patients had 18 F-FDG uptake in pulmonary nodules, suggesting undifferentiated metastases (Table 3, patients 8-12). Three patients underwent resection of the 18F-FDG positive metastases (Table 3, patients 8-10), but despite treatment, the disease progressed in all patients, and one patient presented with renal metastasis confirmed on histopathology, cerebral metastasis refractory to radiotherapy and death (Table 3, patient 8, Figure 2). In this group, the 18F-FDG PET/CT results contributed to the clinical management in 10/21 patients (48%). A

Group 3: patients with positive RxWBS (n = 31) This group included 31 patients with positive WBS who underwent 39 scans. There was no additional 18 F-FDG uptake suggestive of metastases in respect to RxWBS or conventional images. However, 2 patients with previous RxWBS positive pulmonary disease and increasing sTg/TgAb levels showed pulmonary 18 F-FDG uptake, suggesting tumor undifferentiation. In this group of 31 patients, the 18F-FDG PET/CT results delineated prognosis in 2 patients (6%). The clinical relevance of each group is summarized in Figure 1, part II.

F-FDG PET/CT result as predictor of progressive disease 18

From the 95 18F-FDG PET/CT scans, we found 61 positive, 28 negative and 6 indeterminate scans.

Figure 2. 18F-FDG PET/CT images show increased metabolic activity in 62-y-woman: A. Expansive lesion on frontal lobe of the brain; B. Lytic lesion on L2 vertebral body; C. Right lower renal mass. (Table 3, patient 8). Arch Endocrinol Metab. 2017;61/5

421

FDG PET/CT in differentiated thyroid cancer

All eighteen patients who presented with progressive disease showed 18F-FDG uptake (100%) compared to twenty-nine of fifty-six patients who were stable (52%). The 18F-FDG uptake was associated with progressive disease (p = 0.0004).

F-FDG PET/CT results and sTg measurements

The median serum Tg/LT4 was 9.4 ng/mL (0.1-898) in the positive PET/CT patients, compared to 0.8 ng/mL (0.1-44) in the PET/CT negative group (p = 0.001). In analyzing the Tg/TSH level, the median level in the positive PET/CT patients was 26 ng/mL (0.9-1,000), in contrast to 5.1 ng/mL (0.1-271) in the PET/CT negative group (p = 0.003) (Table 4A). In analyzing each group separately, there was a significant difference between the positive and negative PET/CT results in group 1 (for both Tg/LT4 and Tg/TSH) and group 2 (only for Tg/LT4). If we used the sTg level of 10 ng/mL, there were abnormal PET/CT results in 52% of all patients with Tg/LT4 levels ≤ 10 ng/mL and in 90% of patients with Tg/LT4 levels of > 10 ng/mL

(p < 0.001) (Table 4B). For Tg/TSH, the outcome was 38% in Tg/TSH levels of ≤ 10 ng/mL versus 74% if Tg/ TSH was > 10 ng/mL (p < 0.009). For each group, separately, there was a significant difference in only group 1 (for both Tg/LT4 and Tg/TSH) and group 2 (only for Tg/LT4).

DISCUSSION High metabolic activity revealed by 18F-FDG avidity represents advanced tumor and undifferentiation. In these cases, poorly differentiated follicular cells might lose the ability to concentrate RAI, synthesize sTg, and progressively enhance glucose metabolism due to high cell activity and metabolic demand. In this way, 18F-FDG PET/CT has become a powerful tool to improve staging and tumor aggressiveness and investigate undifferentiated lesions that do not take up radioiodine, denoting important diagnostic and prognostic implications (18-20).

Table 4 – A. The median of thyroglobulin level from 18F-FDG PET/CT positive (FDG +) and negative (FDG -) patients. B. 18F-FDG PET/CT positive result according to the cutoff of sTg = 10 ng/mL. A All patients Group 1 Group 2 Group 3

FDG +

FDG -

Tg/LT4 (ng/mL)

9.4 (0.1-898)

0.8 (0.1–44)

< 0.01

Tg/TSH (ng/mL)

26 (0.9-1000)

5.1 (0.1–271)

< 0.01

Tg/LT4 (ng/mL)

6.9 (0.5-91)

1.4 (0.1-4.9)

< 0.05

Tg/TSH (ng/mL)

72.5 (3.9-292)

5.1 (2.4-7.4)

< 0.05

Tg/LT4 (ng/mL)

59.1 (0.2-898)

0.2 (0.1-44)

< 0.05

Tg/TSH (ng/mL)

95 (0.4-1000)

0.6 (0.1-271)

> 0.05

Tg/LT4 (ng/mL)

2.2 (0.1-47)

1.0 (0.1-7.9)

> 0.05

Tg/TSH (ng/mL)

13.9 (1.1-168)

8.0 (0.7-38)

> 0.05

Tg/LT4: unstimulated Tg; Tg/TSH: stimulated Tg.

B F-FDG PET/CT positive result

Tg ≤ 10 ng/mL All patients

Group 1 Group 2 Group 3

Tg > 10 ng/mL

Tg/LT4

27/52 (52%)

Tg/LT4

21/22 (95%)

< 0.01

Tg/TSH

10/26 (38%)

Tg/TSH

20/27 (74%)

< 0.01

Tg/LT4

6/13 (46%)

Tg/LT4

5/5 (100%)

< 0.05

Tg/TSH

1/8 (13%)

Tg/TSH

5/5 (100%)

< 0.01

Tg/LT4

5/11 (45%)

Tg/LT4

13/14 (93%)

< 0.01

Tg/TSH

4/7 (57%)

Tg/TSH

7/9 (78%)

> 0.05

Tg/LT4

17/29 (59%)

Tg/LT4

3/3 (100%)

> 0.05

Tg/TSH

5/11 (45%)

Tg/TSH

8/13 (62%)

> 0.05

Tg/LT4: unstimulated Tg; Tg/TSH: stimulated Tg.

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The classical indication to perform 18F-FDG PET/CT in thyroid cancer patients is positive sTg measurements with negative WBS uptake (6). In the literature, 18F-FDG PET/CT provides additional information not revealed by traditional images in 2171% of patients, mostly in negative WBS (7,8), and in 13-50% of patients with positive TgAb (21,22). In our study, we analyzed the classical indication 18 of F-FDG PET/CT in 22 patients with positive sTg or TgAb, negative RxWBS and no foci of metastases identified at conventional image (Group 1A, n = 9) and those with positive sTg or TgAb and RxWBS not compatible with suspicious foci at conventional image or not proportional to sTg level (Group 1B, n = 13). In group 1A, 18F-FDG PET/CT did not detect additional metastases. The low sTg levels (0.2-4.9 ng/mL), even under stimulation (3.9-7.4 ng/mL), combined with microscopic metastasis might explain the lack of 18 F-FDG-avid lesions, as 18F-FDG is limited in detecting minimal disease (under 1.0 cm). However, the 18F-FDG PET/CT scan helped unveil undifferentiated cervical, lung and mediastinal metastases in 9 patients (69%) in group 1B. The positive FDG uptake observed in this subgroup was associated with higher levels of sTg (Tg/ LT4: 0.6-91 ng/mL and Tg/TSH: 2.4-292 ng/mL) and higher dimensions of metastasis. In the subgroup 1B, surgery was possible in 4 of 9 patients with positive 18F-FDG PET/CT scan results. As described by Hall and Kloos (3), the ideal of 18F-FDG, to identify resectable lesions to pursue a cure, should be attempted as undifferentiated lesions are less likely to respond to RAI, and additional surgery can lead to a higher rate of full remission during follow-up (23). The other 6 patients presented diffuse pulmonary 18F-FDG uptake, and there was no role for surgery. The other aspect to consider is the behavior of aggressive histological variants. Those variants have unfavorable prognosis as they feature low iodine avidity and aggressive clinical behavior with more local and distance recurrences, less disease-free intervals and shorter survivals, requiring close follow-up and continued surveillance to pursue occult metastases. Publications regarding 18F-FDG PET/CT and aggressive variants describe these subtypes as more 18 F-FDG-avid than RAI tumors. Concerning oncocytic cell tumors, 80% of patients have no iodine-avid tumor (24), and therefore, 18F-FDG PET/CT is a valuable tool for screening occult recurrence, evaluating prognosis, and providing additional images not presented by WBS Arch Endocrinol Metab. 2017;61/5

or conventional image (25,26). In regard to the other aggressive subtypes, few studies consider 18F-FDG PET/CT as a useful guide in the management of insular (27), sclerosing diffuse (28) and tall cell (29) variants. Treglia and cols. (30) concluded that the 18 F-FDG PET scan usefulness is clear for the oncocytic cell, uncertain for poorly differentiated cancers and suggestive in the other aggressive forms. The concept of tumor undifferentiation was also observed in patients in group 2 (n = 21). In our study, the results corroborate the findings in the literature. The higher FDG uptake presented in this group can be attributed to more undifferentiated thyroid tumors with more avid uptake for 18F-FDG and high levels of sTg. Pryma and cols. (25) suggested that 18F-FDG PET/CT could be indicated in oncocytic cell carcinoma in postoperative staging and as follow-up in patients with an increase in sTg or recurrent disease, whereas Nascimento and cols. (29) recommended routine early postoperative 18 F-FDG PET/CT concomitantly with RxWBS in all patients with aggressive histological DTC. 18 F-FDG and RAI may function as complementary tools in DTC (9,31) to investigate additional undifferentiated metastases. However, in our cohort, we did not find additional metastases visualized by WBS. WBS-positive patients have no classical indication for 18F-FDG PET/CT and cost-efficacy must be considered in WBS positive group patients. ATA recommends 18F-FDG PET/CT in highrisk DTC patients with elevated sTg, generally Tg/ TSH > 10 ng/mL (6). If Tg/TSH is ≤ 10 ng/mL, the sensitivity of PET/CT is low, ranging from less than 10% to 30% (6). In our data, 38% of all scans performed with Tg/TSH of ≤ 10 ng/mL and 13% of group 1 were positive, similar to the literature data. In contrast, 74% of PET/CT performed with Tg/TSH of > 10 ng/mL provided positive results (as was the case for 100% of group 1). Regarding unstimulated Tg analysis, Tg/LT4 > 10 ng/mL was associated with higher lesion detection in overall patients and groups 1 and 2 when compared to Tg/LT4 ≤ 10 ng/mL. As a matter of fact, more important than the influence of rhTSH or thyroid hormone withdrawal in 18F-FDG PET/CT is the presence of high levels of Tg (Tg/ TSH or Tg/LT4 > 10 ng/mL). Over the last years, it has been demonstrated that both strategies, with or without TSH stimulation, do not considerably lead to management changes (12,32). 423

FDG PET/CT in differentiated thyroid cancer

Additional factors than sTg that influence 18F-FDG PET/CT sensitivity are tumor de-differentiation and larger tumor burden (6), as we have seen in our results. Additionally, the scanning is limited in detecting minimal disease (generally less than 1 cm) and well-differentiated metastases, resulting in false negative outcomes. It is well known that inflammatory lesions can take up FDG, and there may be false positive results. All these features should be considered with care to avoid misjudgments. The frequency of false positive lesions in the literature varies among studies from 0 to 39% (6), and this high number justifies the PET/CT results through the combined data of clinical, laboratorial, conventional image and cytological/histological information to guide ongoing clinical assessments. The present work has some limitations. First, this was a retrospective study, and direct comparison of the detection rate of metastases between 18F-FDG PET/CT and other diagnostic methods was not the design of this study. Additionally, we had no cytological or pathological confirmation of all lesions with 18F-FDG uptake. Based on the serial evaluation of thyroid cancer patients with laboratorial and image exams to assess tumor growth, invasive procedure to confirm the metastases is seldom necessary. In conclusion, 18F-FDG PET/CT results changed the management in 28% (21/74) of patients, mostly in 1B group, patients with RxWBS not compatible with suspicious foci at conventional image or not proportional to sTg level (69%, 9/13) and in group 2, patients with aggressive histological variant patients (48%, 10/21), confirming the literature indications that 18F-FDG PET/CT is more useful in these two groups of patients. For group 1A patients, with positive sTg or TgAb, negative RxWBS and no foci of metastases identified at the conventional image, 18F-FDG PET/CT was not useful, probably due to low levels of sTg and low tumor burden. Additionally, this study showed that 18F-FDG uptake was associated with progressive disease and helped display undifferentiated lesions guiding clinical assessments with respect to surgeries or expectant treatments.

R.M.B.M.) and grant 25000.168513/2008-11 from the Brazilian Ministry of Health.

Acknowledgments: the authors thank the team of Thyroid Diseases Centers at Universidade Federal de São Paulo and Instituto Israelita de Ensino e Pesquisa Albert Einstein, Elza Setsuko Ikejiri, Maria da Conceição Oliveira Mamone, Felipe Augusto Brasileiro Vanderlei and Jairo Tabacow Hidal.

11. Pomerri F, Cervino AR, Al Bunni F, Evangelista L, Muzzio PC. Therapeutic impact of (18)F-FDG PET/CT in recurrent differentiated thyroid carcinoma. Radiol Med. 2014;119(2):97-102.

Financial disclosure: the research is supported by the São Paulo State Research Foundation (Fapesp) grant 2006/60402-1 (to 424

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

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original article

Long-term follow-up of patients with elevated IGF-1 and nadir GH > 0.4 µg/L but < 1 µg/L Pedro Weslley Rosario1, Maria Regina Calsolari1

ABSTRACT Objective: To report the results of initial investigation and after 5 years of patients with a suspicious clinical scenario for acromegaly, elevated IGF-1, and nadir GH during an oral glucose tolerance test (OGTT) > 0.4 µg/L but < 1 µg/L. Subjects and methods: Seventeen patients who had elevated IGF-1 (outside puberty and pregnancy) in two measurements and GH between 0.4 and 1 µg/L during OGTT were selected. Results: During initial assessment, only one patient had microadenoma on magnetic resonance imaging (MRI) of the pituitary. In this patient, IGF-1 returned to normal spontaneously after 5 years. In the remaining 16 patients, spontaneous normalization of IGF-1 was observed in four and IGF-1 continued to be elevated in 12 after 5 years. None of the latter patients developed a phenotype of acromegaly, changes in physiognomy or increase in IGF-1 and no tumor was detected by imaging methods. Two patients had nadir GH < 0.4 µg/L, while the nadir GH remained between 0.4 and 1 µg/L in 10 patients. Conclusion: In patients (notably young adult or adult women) without a typical phenotype in whom IGF-1 is measured due to a suspicious clinical scenario and is found to be slightly elevated, even if confirmed and in the absence of other causes, a nadir GH cut-off value of 0.4 µg/L instead of 1 µg/L in the OGTT might be inadequate for the diagnosis. Arch Endocrinol Metab.

1 Serviço de Endocrinologia, Santa Casa de Belo Horizonte, MG, Brasil

Correspondence to: Pedro Weslley Rosario Instituto de Ensino e Pesquisa, Santa Casa de Belo Horizonte Rua Domingos Vieira, 590 30150-240 – Belo Horizonte, MG, Brasil pedrowsrosario@gmail.com Received on Sept/08/2016 Accepted on Jun/11/2017 DOI: 10.1590/2359-3997000000295

2017;61(5):426-31 Keywords Acromegaly; diagnosis; elevated IGF-1; GH suppression

INTRODUCTION

ntreated acromegaly is associated with higher morbidity and mortality (1-3). The chance of treatment success, which would result in the improvement or reversal of complications, increases if the disease is diagnosed early (1-3). An early diagnosis of acromegaly is therefore desirable and has been encouraged (1-5). Regarding diagnostic investigation, important points need to be addressed. First, acromegaly is not always accompanied by a typical phenotype as highlighted by some authors: “acromegaly is a clinical syndrome that may not manifest with clear diagnostic features” (1); “some patients with acromegaly have mild or absent clinical features” (2); “we suggest the measurement of IGF-1 in patients without the typical manifestations of acromegaly, but who have several associated conditions” (3), and “the diagnosis does not require the presence of typical phenotypic features” (4). Thus, patients with a suspicious clinical scenario should be investigated even in the absence of a typical phenotype (1-5). Second, while normal IGF1 virtually excludes the diagnosis of acromegaly (6), 426

elevated concentrations of IGF-1 outside puberty and pregnancy strongly support the hypothesis. Third, the diagnosis of acromegaly is confirmed when elevated IGF-1 is associated with lack of GH suppression during an oral glucose tolerance test (OGTT). In fact, other conditions associated with the lack of GH suppression do not increase IGF-1 but rather reduce it (7). Fourth, magnetic resonance imaging (MRI) of the pituitary should be obtained, but the absence of adenoma on MRI does not rule out the diagnosis of acromegaly as stated by some authors: “occasionally patients will not have imaging evidence of a pituitary adenoma” (8); “some patients with acromegaly have small or undetectable tumour” (2), and “the diagnosis is a biochemical one and does not require the presence of a pituitary tumor on MRI” (4). A GH cut-off value of 1 µg/L in the suppression test has traditionally been used for the diagnosis of acromegaly (9). However, patients with acromegaly and GH (basal or nadir) concentrations < 1 µg/L are not uncommon (2,10,11). At present, most authors consider nadir GH levels > 0.3 µg/L or 0.4 µg/L during OGTT sufficient for the diagnosis of acromegaly Arch Endocrinol Metab. 2017;61/5

Acromegaly or laboratory false-positive

SUBJECTS AND METHODS Patients First, 4,350 adults (age between 18 and 70 years, excluding pregnant women and patients with known pituitary disease) underwent acromegaly screening: 2,270 patients with type 2 diabetes mellitus or glucose intolerance (19), 178 patients who reported “enlargement of their extremities” (20), and 1,902 patients with two or more comorbidities related to acromegaly [including arterial hypertension in 1,806 patients (21)]. In patients with elevated IGF-1, a new measurement was obtained and was combined with the measurement of GH during an OGTT. For this study, patients with a suspicious clinical scenario (1-5) according to the definition below (1,3,5), who had a diagnosis of acromegaly (i.e., elevated IGF-1 in two measurements outside puberty and pregnancy associated with lack of GH suppression during OGTT) based on the cut-off value of 0.4 µg/L (1,4,12-18) but not 1 µg/L (3,9), were selected. The study and its respective protocol were approved by the Ethics Committee of our institution.

Definitions A typical acromegalic phenotype was defined i) by an endocrinologist with experience in the disease (P.W.R.), ii) based on ectoscopy, and iii) considering acral enlargement and maxillofacial changes (3). A suspicious clinical scenario was defined in the presence of two or more comorbidities related to acromegaly according to the Canadian Consensus (5), American Association of Clinical Endocrinologists Arch Endocrinol Metab. 2017;61/5

(1), and Endocrine Society (3). The comorbidities considered were (1,3,5): i) nonspecific chronic headache (for example, migraine and hypertensive headache were not considered); ii) generalized and persistent excessive sweating; iii) diffuse arthralgias associated with some radiologic alteration (22) in the absence of known rheumatological disease (reported by the patient, suspected, or confirmed in the medical record); iv) chronic fatigue not explained by any other underlying disease (among the diagnoses reported by the patient or present in the medical record); v) bilateral paresthesias (Carpal tunnel syndrome); vi) recently diagnosed diabetes mellitus; vii) recently diagnosed arterial hypertension requiring antihypertensive medication.

Follow-up During initial assessment, the patients were submitted to MRI of the pituitary using gadolinium as contrast agent. Patients without adenoma on MRI underwent chest and abdominal contrast-enhanced computed tomography (CT). These patients were not treated for acromegaly. The patients were reevaluated clinically and by laboratory testing (serum IGF-1) after 5 years. The presence of a typical phenotype (see above) and changes in physiognomy were evaluated by comparing current photographs and those obtained at the time of initial assessment. Patients with persistently elevated IGF-1 were submitted to a new GH suppression test and 3-tesla MRI of the pituitary (23) using gadolinium as contrast agent. Patients with elevated IGF-1, in the absence of GH suppression and adenoma on MRI, were again submitted to chest and abdominal CT. The samples were collected in the morning after an approximately 10-h fast, with the subject resting for 20 min before and during the OGTT. For the OGTT, GH was measured before and 30, 60, 90 and 120 min after the oral administration of 75 g anhydrous glucose. GH was measured with a chemiluminescence assay (Immulite, Diagnostic Products Corporation, Los Angeles, CA) with an analytical sensitivity ≤ 0.05 µg/L. The standard provided by the kit was calibrated against the World Health Organization (WHO) 2nd International Standard (IS) 98/574. The results are expressed as µg/L. IGF-1 was also measured with a chemiluminescent assay (Immulite 2000, Diagnostic Products Corporation, Los Angeles, CA) (analytical sensitivity of 25 μg/L) using antibodies highly specific 427

in patients with elevated IGF-1 (1,4,12-18). Although increasing sensitivity, it is important to evaluate whether this cut off does not lead to unnecessary investigations, equivocal diagnoses and, consequently, treatments that are not indicated. It should be remembered that, in opposition to reducing the cut off, the Endocrine Society still considers GH < 1 µg/L in the OGTT sufficient for exclusion of acromegaly (3). The objective of this study was to report the results of initial investigation and after 5 years of patients with a suspicious clinical scenario (1-5) and elevated IGF-1, who would have a diagnosis of acromegaly based on the nadir GH cut-off value of 0.4 µg/L (1,4,12-18) but not 1 µg/L (3,9).

Acromegaly or laboratory false-positive

for IGF-1 and previously established reference values stratified by age based on a sample of 1,000 subjects rigorously selected in the same town where the study was conducted (24). “Functional separation” (acidification followed by saturation with IGF-2) was the technique used to exclude interference from IGFbinding proteins (IGFBPs). We highlight that on initial assessment the measurements were made before the period in which overestimated IGF-1 values began to be observed (2527). On last assessment, the measurements were made in 2015 using lots that, according to the manufacturer (25) and in our laboratory (28), are in alignment with the medians of the reference range data published in the Instructions For Use.

RESULTS The study included 16 women and one men aged 30 to 55 years (median 41 years). Initial IGF-1 ranged from 1.08 to 1.53 times the upper limit of the normal range (ULN) for age (24). The frequency of comorbidities is showed in the Table 1. On initial assessment and 5 years later, none of the patients had kidney or liver failure or malnutrition, or was using oral estrogen. Seven patients had diabetes mellitus, but were compensated (29) at the time of IGF-1 measurement and OGTT. Thyroid dysfunction and pregnancy (in premenopausal women) were excluded in all patients. During initial assessment, a lesion suggestive of microadenoma was detected in one patient by MRI of the pituitary (hypointense nodule measuring 4 mm in diameter and showing no contrast enhancement after the administration of gadolinium) (Table 2). Other hormone hypersecretions were excluded in this patient. The woman was not submitted to surgical or Table 1. Frequency of comorbidities

Comorbidity

Number of patients (%)

Recently diagnosed arterial hypertension

15 (88.2%)

Recently diagnosed diabetes mellitus

12 (70.6%)

Nonspecific chronic headache

10 (58.8%)

Bilateral paresthesias (carpal tunnel syndrome)

9 (53%)

Generalized and persistent excessive sweating

6 (35.3%)

Chronic fatigue not explained by any other underlying disease

6 (35.3%)

Diffuse arthralgias with some radiologic alteration in the absence of known rheumatological disease

5 (29.4%)

428

Table 2. Results of the patients with microadenoma on MRI Initial assessment

Last assessment

Sex

Female

Age

50 years

56 years

Clinical scenario

Carpal tunnel syndrome, hypertension, headache, dyslipidemia, glucose intolerance

Hypertension, dyslipidemia, glucose intolerance

Serum IGF-1

1.28 x ULN

0.9 x ULN

Nadir GH

0.65 µg/L

Not performed

MRI

Microadenoma (4 mm)

ULN: upper limit of normal range; MRI: magnetic resonance imaging; DM: diabetes mellitus; GI: glucose intolerance.

medicamentous treatment for acromegaly based on the absence of a typical phenotype, low GH concentrations (nadir < 1 µg/L during OGTT), and good control of comorbidities with conventional treatments. After 5 years, the patient exhibited no changes in physiognomy, remained without a phenotype, and had normal IGF-1 (confirmed in two measurements). MRI was repeated in this case to exclude tumor apoplexy and the lesion was found to be unchanged. MRI did not detect adenoma or pituitary enlargement, and chest and abdominal CT did not reveal a tumor during initial assessment in 16 patients. After 5 years, these patients did not develop changes in physiognomy and remained without a phenotype. IGF-1 spontaneously returned to normal in 4 patients (confirmed in two measurements) and elevated IGF-1 persisted in 12. Regarding the 12 patients with persistently elevated IGF-1, the last IGF-1 ranged from 1.1 to 1.61 times the ULN, already considering the current age of the patient. Comparing the final and initial concentrations, none of the patients exhibited a significant increase in IGF-1, i.e., increment > 20% [limit defined based on the variation found in 100 healthy subjects rigorously selected and in stable conditions, who were submitted to IGF-1 measurement at an interval of 3 months using the same assay as employed in this study (24)]. In a new OGTT, GH suppression was achieved in 2 patients and 10 continued with nadir GH between 0.4 and 1 µg/L. MRI of the pituitary (obtained for all patients) and chest and abdominal CT (obtained for the 10 patients without GH suppression) again revealed no tumor. Thus, 7 patients no longer had a diagnosis of acromegaly (based on spontaneous normalization of IGF-1 in 5 and on GH suppression in 2). None of the 10 patients with persistently elevated IGF-1 and nadir Arch Endocrinol Metab. 2017;61/5

GH > 0.4 µg/L after 5 years developed a phenotype of acromegaly, changes in physiognomy or increase in IGF-1 and no tumor was detected by the imaging methods.

DISCUSSION There is consensus that not only patients with typical phenotypic features should be investigated for acromegaly (1-5). Although not presenting the typical acromegalic phenotype, the patients included in this study had two or more comorbidities commonly found in “active” acromegaly (1,3,5), and additional criteria were required to consider them compatible with this condition (see Subjects and Methods). Moreover, the age range of our patients (30-60 years) coincides with that of a higher incidence of the disease. Consequently, there was a suspicious clinical scenario justifying investigation for acromegaly (1-5). Elevated IGF-1 does not always indicate acromegaly, but its specificity increases when measured outside puberty and pregnancy (situations characterized by physiological elevation of this hormone). Furthermore, the results should be confirmed in a subsequent measurement. One cause of falsely elevated IGF-1 are inadequate limits of normality. When defined using an inadequately selected sample or an insufficient number of subjects, the upper limit may be underestimated and, consequently, an individual with normal IGF-1 would be erroneously classified as having elevated IGF-1. In the present study, the definition of elevated IGF-1 was based on the limits established for a sample of 1,000 subjects from the same town as the patients included in this study. This sample was selected rigorously (exclusion of interfering conditions and medications and extremes of body mass index) and stratified by decade of life (24) according to current recommendations. Hence, in the present study “elevated IGF-1” refers to the measurement obtained outside puberty and pregnancy, confirmed in two measurements, and based on adequate normative information. Although theoretically possible, heterophile antibodies are not cited as possible agents that interfere with serum IGF-1. Moreover, the only case report in the literature mentioning interference of these antibodies with the Immulite assay inexplicably found a reduction in IGF-1 (30). The assay used does not show cross-reactivity to insulin or IGF-II and eventual interference from IGFBPs would cause a reduction in IGF-1 (24). Overweight/obese subjects Arch Endocrinol Metab. 2017;61/5

have higher hepatic sensitivity to GH. However, there is no elevation of serum IGF-1 (31). It has also been suggested that genotype d3 of the GH receptor (d3-GHR) increases sensitivity to this hormone (32). However, to our knowledge, there is no study reporting an association between the presence of d3-GHR and elevated IGF-1 in individuals without acromegaly and not treated with GH. The diagnosis of acromegaly is made when elevated IGF-1 is associated with the “absence of GH suppression” during an OGTT. In fact, other conditions that can cause a lack of GH suppression do not result in IGF-1 elevation, but rather reduce it (7). Nevertheless, these conditions were excluded in our patients. Most authors define a nadir > 0.4 µg/L as “lack of GH suppression” (1,4,12-18) and many others recommend even lower cut offs, 0.3 µg/L (4,12,14,15,17), 0.25 µg/L (13), and 0.2 µg/L (16). Since the patients of the present study had i) “a suspicious clinical scenario” (1-5); ii) “elevated IGF-1” in two measurements and excluding other causes; iii) “lack of GH suppression” during OGTT using a cut off accepted by most authors (1,4,1218), and iv) considering that “the diagnosis is a biochemical one and does not require the presence of a pituitary tumor on MRI” (4) since “some patients with acromegaly have small or undetectable tumour” (2) and “occasionally patients will not have imaging evidence of a pituitary adenoma” (8), they could have been diagnosed with acromegaly during initial assessment and submitted to medicamentous treatment or exploratory transsphenoidal surgery (8). However, follow-up suggests that these patients probably did not have acromegaly, First, considering the interval between the onset of manifestations and diagnosis, with the typical phenotype already present (33,34), the absence of this phenotype and of changes in physiognomy after 5 years makes the disease unlikely. Second, spontaneous normalization or absence of an increase in IGF-1 after this period also weakens the diagnosis. Even in patients with persistently elevated IGF-1, GH concentrations in the OGTT continued to be < 1 µg/L. Third, 3-tesla MRI of the pituitary (23) and chest and abdominal CT were negative in the initial and last assessment (in patients in whom biochemical alterations persisted). There is no question that the recommendation of investigating acromegaly in patients with a suspicious clinical scenario, even in the absence of a typical phenotype (1-5), is interesting for early diagnosis. 429

Acromegaly or laboratory false-positive

However, since comorbidities associated with acromegaly are also common in the adult population (e.g., diabetes mellitus, arterial hypertension, carpal tunnel syndrome, nonspecific headache), it is expected that this investigation does not confirm the disease in most patients (low pre-test probability). By definition, approximately 2% of the normal population has “elevated IGF-1”. Obviously, the concentrations in these individuals do not deviate much from the ULN, but IGF-1 is also not very elevated in patients with acromegaly and nadir GH between 0.4 and 1 µg/L [up to 2/3 have IGF-1 ≤ 2 x ULN (2,10,11)], and the intensity of IGF-1 elevation is therefore little useful for this distinction. Although increasing sensitivity, the reduction in the nadir GH cut off from 1 µg/L to 0.4 µg/L may decrease the specificity of the diagnostic criterion. Indeed, even after the exclusion of interfering conditions and using sensitive assays calibrated against the second IS 98/574 for hGH, normal individuals, notably young adult and adult women, may have nadir GH > 0.4 µg/L in the OGTT (6,35-38). In the present series, all but one patient were women ≤ 55 years. Thus, the high prevalence of comorbidities associated with acromegaly in the general adult population, the fact that even individuals without disease can have slightly elevated IGF-1 and nadir GH > 0.4 µg/L in the OGTT and the rarity of acromegaly may explain why even the combination of findings (suspicious clinical scenario (1-5), elevated IGF-1, nadir GH > 0.4 µg/L) can have a low positive predictive value. In patients without a typical phenotype, notably young adult or adult women, in whom IGF-1 is measured due to a suspicious clinical scenario (1-5) and is found to be slightly elevated, even if confirmed and in the absence of other causes, a GH cut-off value of 0.4 µg/L (1,4,12-18) instead of 1 µg/L (3) in the OGTT might be inadequate for the diagnosis of acromegaly. We do not know whether these individuals correspond to the portion of the “normal” population that exhibits concentrations outside the reference range or have GH hypersecretion, although not tumoral. Additionally, we do not know whether these persistently elevated concentrations of IGF-1 increase the risk of comorbidities despite the absence of acromegaly, remembering that all of these patients had comorbidities. Since GH hypersecretion should be investigated in all patients with pituitary incidentaloma, even in the absence of a phenotype (3,4,39), the conclusion of the present 430

study may also have implications for the diagnosis of clinically silent somatotropinoma, remembering that this diagnosis could change expectant management (in the case of non-functional incidentaloma) to surgery or medicamentous treatment (in the case of somatotropinoma) (39). 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.

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17. Chanson P, Bertherat J, Beckers A, Bihan H, Brue T, Caron P, et al. French consensus on the management of acromegaly. Ann Endocrinol (Paris). 2009;70:92-106.

29. Rosario PW, Calsolari MR. Safety and specificity of the growth hormone suppression test in patients with diabetes. Endocrine 2015;48:329-33.

18. Vieira Neto L, Abucham J, Araujo LA, Boguszewski CL, Bronstein MD, Czepielewski M, et al. Recommendations of Neuroendocrinology Department from Brazilian Society of Endocrinology and Metabolism for diagnosis and treatment of acromegaly in Brazil. Arq Bras Endocrinol Metabol. 2011;55:91-105.

30. Brugts MP, Luermans JG, Lentjes EG, van Trooyen-van Vrouwerff NJ, van der Horst FA, Slee PH, et al. Heterophilic antibodies may be a cause of falsely low total IGF1 levels. Eur J Endocrinol. 2009;161:561-5.

19. Rosario PW. Frequency of acromegaly in adults with diabetes or glucose intolerance and estimated prevalence in the general population. Pituitary. 2011;14:217-21. 20. Rosario PW, Calsolari MR. Screening for acromegaly by application of a simple questionnaire evaluating the enlargement of extremities in adult patients seen at primary health care units. Pituitary 2012;15:179-83. 21. Rosario PW, Calsolari MR. Screening for acromegaly in adult patients not reporting enlargement of the extremities, but with arterial hypertension associated with another comorbidity of the disease. Arq Bras Endocrinol Metab. 2014;58:807-11. 22. Killinger Z, Payer J, Lazúrová I, Imrich R, Homérová Z, Kužma M, et al. Arthropathy in acromegaly. Rheum Dis Clin North Am. 2010;36:713-20. 23. Stobo DB, Lindsay RS, Connell JM, Dunn L, Forbes KP. Initial experience of 3 Tesla versus conventional field strength magnetic resonance imaging of small functioning pituitary tumours. Clin Endocrinol (Oxf). 2011;75:673-7. 24. Rosario PW. Normal values of serum IGF-1 in adults: results from a Brazilian population. Arq Bras Endocrinol Metabol. 2010;54: 477-81. 25. Siemens Healthcare Diagnostics. All IMMULITE Platforms for IGF-I Shift in Patient Medians and Supply Disruption. Urgent Field Safety Notice 4005, November 2012. 26. Algeciras-Schimnich A, Bruns DE, Boyd JC, Bryant SC, La Fortune KA, Grebe SK. Failure of current laboratory protocols to detect lot-to-lot reagent differences: findings and possible solutions. Clin Chem. 2013;59:1187-94. 27. Bancos I, Algeciras-Schimnich A, Grebe SK, Donato LJ, Nippoldt TB, Erickson D. Evaluation of variables influencing the measurement of insulin like growth factor-1 (IGF-1). Endocr Pract. 2014;20:421-6.

32. Jorge AA, Arnhold IJ. Growth hormone receptor exon 3 isoforms and their implication in growth disorders and treatment. Horm Res. 2009;71(Suppl 2):55-63. 33. Nachtigall L, Delgado A, Swearingen B, Lee H, Zerikly R, Klibanski A. Changing patterns in diagnosis and therapy of acromegaly over two decades. J Clin Endocrinol Metab. 2008;93:2035-41. 34. Reid TJ, Post KD, Bruce JN, Nabi Kanibir M, Reyes-Vidal CM, Freda PU. Features at diagnosis of 324 patients with acromegaly did not change from 1981 to 2006: acromegaly remains under-recognized and under-diagnosed. Clin Endocrinol. 2010;72:203-8. 35. Markkanen H, Pekkarinen T, Välimäki MJ, Alfthan H, KauppinenMäkelin R, Sane T, et al. Effect of sex and assay method on serum concentrations of growth hormone in patients with acromegaly and in healthy controls. Clin Chem. 2006;52:468-73. 36. Arafat AM, Möhlig M, Weickert MO, Perschel FH, Purschwitz J, et al. Growth hormone response during OGTT: the impact of assay method on the estimation of reference values in patients with acromegaly and in healthy controls and the role of gender, age, and BMI. J Clin Endocrinol Metab. 2008;93:1254-62. 37. Rosario PW, Furtado MS. Growth hormone after oral glucose overload: revision of reference values in normal subjects. Arq Bras Endocrinol Metabol. 2008;52:1139-44. 38. Colao A, Pivonello R, Auriemma RS, Grasso LF, Galdiero M, Pivonello C, et al. Growth hormone nadir during oral glucose load depends on waist circumference, gender and age: normative data in 231 healthy subjects. Clin Endocrinol (Oxf). 2011;74:234-40. 39. Freda PU, Beckers AM, Katznelson L, Molitch ME, Montori VM, Post KD, et al. Pituitary incidentaloma: an endocrine society clinical practice guideline. J Clin Endocrinol Metab. 2011;96: 894-904.

28. Rosario PW, Gadelha MR. Current reliability of the Immulite® assay for measurement of serum IGF-1 in the Brazilian adult population. Arch Endocrinol Metab. 2015;59:195-6.

31. Schneider HJ, Saller B, Klotsche J, März W, Erwa W, Wittchen HU, et al. Opposite associations of age-dependent insulinlike growth factor-I standard deviation scores with nutritional state in normal weight and obese subjects. Eur J Endocrinol. 2006;154:699-706.

Arch Endocrinol Metab. 2017;61/5

431

original article

Timing of thyroid ultrasonography in the etiological investigation of congenital hypothyroidism Maria de Fátima Borges1, Nathalie de Almeida Sedassari1, Anelise de Almeida Sedassari1, Luis Ronan Marquez Ferreira de Souza2, Beatriz Pires Ferreira1, Beatriz Hallal Jorge Lara1, Heloísa Marcelina Cunha Palhares1

ABSTRACT Disciplina de Endocrinologia, Universidade Federal do Triângulo Mineiro (UFTM), Uberaba, MG, Brasil 2 Disciplina de Radiologia e Diagnóstico por Imagem, UFTM, Uberaba, MG, Brasil 1

Correspondence to: Maria de Fátima Borges Disciplina de Endocrinologia, Hospital de Clínicas Rua Getúlio Guaritá, s/n 38025-440 – Uberaba, MG, Brasil borgmf@uol.com.br Received on Aug/2/2016 Accepted on Oct/18/2016 DOI: 10.1590/2359-3997000000239

Objectives: To describe the findings of thyroid ultrasonography (T-US), its contribution to diagnose congenital hypothyroidism (CH) and the best time to perform it. Subjects and methods: Forty-four patients with CH were invited to undergo T-US and 41 accepted. Age ranged from 2 months to 45 years; 23 patients were females. All were treated with L-thyroxine; 16 had previously undergone scintigraphy and 30 had previous T-US, which were compared to current ones. Results: At the current T-US, the thyroid gland was not visualized in its normal topography in 10 patients (24.5%); 31 T-US showed topic thyroid, 17 with normal or increased volume due to probable dyshormonogenesis, 13 cases of hypoplasia and one case of left-lobe hemiagenesis. One patient had decreased volume due to central hypothyroidism. Scintigraphy scans performed 3-4 years earlier showed 100% agreement with current results. Comparisons with previous T-US showed concordant results regarding thyroid location, but a decrease in current volume was observed in eight due to the use of L-thyroxine, calling the diagnosis of hypoplasia into question. Conclusions: The role of T-US goes beyond complementing scintigraphy results. It allows inferring the etiology of CH, but it must be performed in the first months of life. An accurate diagnosis of CH will be attained with molecular study and the T-US can guide this early assessment, without therapy withdrawal. Arch Endocrinol Metab. 2017;61(5):432-7 Keywords Congenital hypothyroidism; thyroid dysgenesis; thyroid ectopia; dyshormonogenesis; thyroid ultrasound

INTRODUCTION

n the early years of life, thyroid ultrasonography (T-US) can be performed on individuals with suspected congenital hypothyroidism (CH), assisting in the etiological diagnosis. Thyroid dysgenesis includes: athyrosis, as an “empty” thyroid area with or without ectopic tissue (agenesis), and thyroid hypoplasia. When the thyroid, visualized at the ultrasound assessment, has normal or increased volume, one of several forms of dyshormonogenesis is suggested (1,2). The diagnosis of CH through screening programs has greatly contributed to the start of treatment within an adequate time frame and the etiological diagnosis has been sought by these programs aiming at genetic counseling (3-7). Due to the need to initiate treatment within the first month after birth aiming to preserve the child’s neuronal development, the investigation of CH etiology is usually delayed until the age of 3, when treatment withdrawal for one month does not result in much impairment or, in many cases, is not performed due 432

to the lack of resources in the region where the child resides or due to limited mobility to the capital cities, which are the centers of screening programs (6,7). The aim of the present study was to describe the ultrasonographic findings and their contribution to the diagnosis of CH in a series of 41 patients, as well as evaluate the most appropriate time to perform the T-US.

SUBJECTS AND METHODS Forty-four individuals with a previous diagnosis of CH were invited to undergo T-US at the Imaging Service of Universidade Federal do Triângulo Mineiro (UFTM). Of these patients, 23 were from the State’s Newborn Screening Program (SNSP) and were being followed at the Municipal Health Unit of Uberaba, while another 21 patients being followed at the Department of Endocrinology of UFTM were from Uberaba and other municipalities in the Triangulo Mineiro region, state of Minas Gerais, Brazil. A total Arch Endocrinol Metab. 2017;61/5

Ultrasonography in congenital hypothyroidism

of 41 patients, 23 females and 18 males, aged 0.2 to 45 years (median: 6 years) accepted the invitation to undergo T-US. The study was approved by the local University Medical Ethic Committee. All patients were undergoing treatment since the diagnosis, with the exception of one patient who had a positive newborn screening test, but whose TSH levels normalized after medication withdrawal, being considered a case of transient hyperthyrotropinemia (case 21). Of the patients referred by the SNSP (n: 23), 15 had already undergone etiological investigation after treatment withdrawal between 3 and 4 years old, having been submitted to T-US and scintigraphy. Of the patients from UFTM (n: 21), 15 had undergone T-US and of these, only two had undergone scintigraphy. Therefore, of the total of 41 patients, 30 had undergone T-US and 17 scintigraphy, prior to the study (Tables 1 and 2). Of the 30 patients with previous T-US, test results found in the medical records of 23 patients allowed a comparison with the current results, as they contained thyroid descriptions and volumes (Tables 1 and 2). All children participating in the study underwent T-US, which evaluated the following thyroid gland characteristics: position, texture, volume and additional findings. The examinations were performed with patients in the supine position with neck hyperextension (1). The assessment started with the cervical region evaluation from the base of the tongue to the thyroid, characterizing its lobes and the isthmus regarding their depth, length and width, evaluating the neck position, echotexture, calculated dimensions and volumes according to the formula π/6 x depth x length

x width (8). Total thyroid volume was obtained by adding both lobes, whereas the isthmus was disregarded when determining the volume. Color Doppler was also used to assess its behavior. If any thyroid lesion was identified, it was recorded and analyzed separately. The ultrasound assessments were performed in the Radiology and Imaging Diagnostic Department of Hospital de Clinicas, UFTM. The ultrasound equipment used for the assessments was a Philips device, model HD11 (Philips Healthcare, The Netherlands) with a 7.5-MHz frequency linear transducer (3-13 MHz). An Accuvix V10 device (Samsung Medison Heathcare) with a 7.5 MHz frequency linear transducer (6-12 MHz) was also used. Reference values used for individuals aged ≥ 6 years to evaluate the volumes obtained were the ones described by Chanoine and cols. (9), and Zimmermann and cols. (10), i.e., thyroids with a total volume < 1.5 mL were considered compatible with low volume and, therefore, hypoplastic. In individuals aged up to 5 years, the reference values used were those obtained at the service itself from 30 normal children and stratified by age group, as follows: from 2 months to 3 years (n: 15) ranging from 0.52 to 1.3 mL; and from 3.1 to 5 years (n: 15) ranging from 0.98 to 2.7 mL. In adults, the volume considered normal was 10.3 ± 5.1 mL (11).

Statistical analysis Numerical data are shown as median, minimum and maximum values. Student’s t test was employed when comparing volumes obtained at prior x current T-US, with P < 0.05 being considered significant.

Table 1. Data from congenital hypothyroidism patients whose thyroids were not visualized on Ultrasonography (T-US) or with ectopic thyroid confirmed by scintigraphy (131I) Initial T-US Gender

Current T-US

Local

Total volume (mL)

131

Local

Diagnosis

0.81

7.0

SME

Ectopic

4.4

SLE

Ectopic

3.6

Dysgenesis

1.0

15.7

Dysgenesis

1.2

32.0

SLE

Ectopic

21.0

Agenesis

Dysgenesis

20.8

Dysgenesis

14.0

Dysgenesis

0.7

Dysgenesis

2.2

Dysgenesis

TT: time of treatment (years); E: ectopic; SME: submentonian ectopia; NV: not visualized; SLE: sublingual ectopia; T: topic. Arch Endocrinol Metab. 2017;61/5

433

Case N.

Ultrasonography in congenital hypothyroidism

Table 2. Sonographic (T-US) and scintigraphic (131I) data of congenital hypothyroidism patients with topic thyroids and etiological diagnosis Initial T-us

Case N.

Gender

Current T-uS Total volume (mL)

131

Local

Diagnosis

3.0

LLH

8.1

LLH

4.34*

3.0

0.70*

9.0

4.35*

3.0

2.80*

9.0

0.26

4.0

0.20

4.7

0.73

3.5

1.00

6.0

1.79*

3.0

1.20*

8.5

2.50

4.0

0.50

4.0

0.80

4.6

0.89

3.5

1.00

7.9

13.2*

3.0

6.50*

10.8

3.00

3.0

1.41

3.0

1.50

6.9

Not described

3.0

14.20

14.0

2.30*

3.0

0.50*

14.4

1.10

6.0

0.94

7.0

3.00

11.0

1.70

0.1

2.10

5.9

0.50

0.1

0.85

12.5

0.90

1.0

1.00

4.9

7.70

8.0

8.50

13.0

4.50

1.8

11.00*

0.1

3.00*

2.0

5.40*

37.0

2.00*

45.0

1.90

5.0

2.00

20.0

2.35

2.0

2.40

11.4

5.50

6.0

0.20

0.2

Local

Total volume (mL)

1.85

16 17

1.90

1.5

13.00*

0.1

3.00*

0.5

1.90

1.0

0.80

0.9

TT: time of treatment (years); T: topic; LLH: left lobe hemiagenesis; D: dyshormonogenesis; H: hypoplasia; TH: transient hyperthyrotropinemia; CH: central hypothyroidism. * Decrease in volume from previous to current T-US.

RESULTS Of the 44 patients invited to participate in the study, 41 underwent T-US. The thyroid gland was not visualized in its bed in 10 cases (24.5%), which were considered as thyroid dysgenesis (Table 1). Of these, four had undergone scintigraphy, of which three had an ectopic thyroid and one thyroid agenesis. In this group, eight patients had previously undergone T-US. In case 1 the thyroid was located in the submentonian region, which was confirmed by scintigraphy. In two cases (cases 4 434

and 5) the thyroid was topic, but was not seen in the current T-US. In case 4, the thyroid was very small and its volume might have reduced even further with L-thyroxine treatment, but in case 5, the thyroid gland was located in the sub-lingual region at the scintigraphy, showing a disagreement between the initial T-US and the scintigraphy (Table 1). Thirty-one patients had topic thyroids (75.6%), with volumes ranging from 0.2 to 14.2 mL (median: 1.95 mL). One patient had left-lobe hemiagenesis, Arch Endocrinol Metab. 2017;61/5

Ultrasonography in congenital hypothyroidism

confirmed by a previous T-US. Thirteen patients (cases 12, 14, 15, 16, 18, 19, 22, 24, 25, 28, 33, 34 and 37) had homogeneous thyroids with reduced volumes for their ages, which could be considered hypoplastic if they had not undergone a previous T-US for comparison. In this group, 11 had undergone previous T-US and in six, thyroid volume was normal for age. Of these six patients, four showed a reduction in volume over the course of treatment with L-thyroxine (cases 12, 16, 24 and 33) and case 14 was diagnosed with central hypothyroidism. Thus, taking into account these particularities, only seven of the 13 patients could be actually considered as having true hypoplasia and, therefore, thyroid dysgenesis, being one patient with central hypothyroidism and the remaining five with dyshormonogenesis (Table 2). As for the other cases of topic thyroid (n = 17) (Table 2), the patients showed normal or increased thyroid volumes according to the reference standards for age. In this group, 11 had previously undergone T-US, confirming the diagnosis of dyshormonogenesis. It was also observed that four cases had a reduction in thyroid volume with treatment, but still kept within normal limits for age. Thus, of the total of 31 patients, one case of thyroid hemiagenesis, seven cases of true hypoplasia, one case of central hypothyroidism, one of transient hyperthyrotropinemia (21 cases) and 21 cases of dyshormonogenesis were identified (Figure 1). In the group of topic thyroid, scintigraphy with 123 I had been performed on 12 patients, and showed 100% agreement with the results obtained from the current T-US, considering the thyroid location. When analyzing the two groups (topic and ectopic thyroid), scintigraphy showed to be more accurate in locating the remaining ectopic gland. It also allowed identifying the iodine organification defect in three cases through

the perchlorate test performed in 11 patients with topic thyroid. The remaining patients did not undergo scintigraphy due to younger ages or because they did not have access to the exam.

DISCUSSION A child with a confirmed diagnosis of CH needs prompt treatment with L-thyroxine and the etiological research may be delayed until 3 years of age, considering that the first concern is to preserve the child’s developing central nervous system, growth and cognitive capacity (12-16). Some doctors advise parents that, at some point, the drug will be discontinued and the etiology of CH will be investigated (6,7). In some cases, this information generates a degree of anxiety and an indeterminate diagnosis can lead to difficulties in the relationship between doctor and parents, who also inquire about treatment duration. The physician should convey confidence and reassure the parents that there is a best moment for this investigation to occur. In clinical practice, the institution of L-thyroxine treatment in a timely manner was a breakthrough of the screening programs and, until recently, there were no resources for the study of CH etiology, considered a research subject limited to only a few centers of excellence and this is not the case in many Brazilian states (3-7). This series includes some children who were investigated between 3 and 4 years of age, in the city of Uberaba, from SNSP-MG, but there are adults aged 45 years that received the diagnosis of CH at birth, have been using L-thyroxine adequately and the T-US was the only test they had access to. In adulthood, the patients themselves ask their doctors why they have the disease, express their desire to know the nature of the defect and whether they could pass it on to offspring,

N: 44 invited

10 thyroid glands not visualized (dysgenesis – 24.5%)

21 non-ectopic thyroids with normal or increased volume (dyshormonogenesis – 51.0%) 1 case of central hypothyroidism (2.5%)

3 did not accept the invitation

7 hypoplasic topic thyroids 1 left lobe hemiagenesis (dysgenesis 19.5%)

1 case of transient hyperthyrotropinemia (2,5%)

Figure 1. Patient distribution according to thyroid location after ultrasonography and etiological diagnosis. Arch Endocrinol Metab. 2017;61/5

435

41 underwent T-US

Ultrasonography in congenital hypothyroidism

prompting the physician to attain a more accurate diagnosis to support genetic counseling. As it is currently carried out, the etiological research of CH is difficult and inaccurate, as well as expensive and limited to large centers in the capital cities of Brazil. Thyroid scintigraphy, considered the “gold standard” for the gland identification, could be performed in the neonatal period when treatment has not been started yet, but would require prompt availability of a Nuclear Medicine Service, only available in certain cities (2). Most often, it is carried out much later in life, after medication withdrawal for at least a month (6). The T-US complements the scintigraphy; it is very effective for thyroid identification and can be performed in the neonatal period, after treatment has been instituted (2). When the gland is located in its normal topography, the T-US can also disclose its characteristics, and according to the volume found, it may indicate whether it is normal, increased or decreased, or whether there is hemiagenesis of one of the lobes (1,17). In the present study, we observed very good agreement between the two tests regarding thyroid location. In 10 cases the thyroid was not visualized by T-US, probably due to dysgenesis. Among the 31 cases of topic thyroid, 21 had normal or increased volume for age, probably due to dyshormonogenesis, 7 had topic thyroid with reduced volume, configuring thyroid hypoplasia, one case showed central hypothyroidism, one case had transient hyperthyrotropinemia and one case had left lobe hemiagenesis, a rare variant of dysgenesis (18). However, to attain a secure diagnosis, the T-US should be performed as soon as possible, and not much later as it was done, after long-term use of thyroid hormone (2). In 22 cases of topic thyroid gland, thyroid volume obtained through the previous T-US was compared with the current one, demonstrating volume reduction in eight. This probably occurred due to L-thyroxine use since birth, which reduces volume and changes characteristics of the gland (19). These data indicate that the best time to perform the T-US is within the first few months after birth, when the thyroid still retains its original features and when it is topic and has reduced volume, CH may be due to hypoplasia or central hypothyroidism. A patient with central hypothyroidism (case N. 14) had decreased thyroid volume, decreased TSH with low free T4 and a prior investigation had revealed a TSH-receptor defect (20,21). Increased 436

thyroid volume was also observed in some cases, which can be explained by the fact that thyroid volume increases with age, especially after 8 years old (22). A T-US limitation is that when the thyroid is ectopic, its location is not always defined, in which case scintigraphy is superior, being effective in accurately locate the gland along the embryonic migration path (17). In a previous study involving a few cases of this series (6) scintigraphy effectively located two ectopic thyroids in 15 cases and when the perchlorate test was performed, it indicated iodine organification defect in three cases. In other cases of dyshormonogenesis, scintigraphy associated with the perchlorate test, as well as the T-US do not have the power to diagnose the cause of dyshormonogenesis. Further progress toward etiological diagnosis will come with molecular studies of congenital thyroid defects (23-25) and the T-US can direct the initial evaluation, without requiring therapy interruption. The collection of material for DNA extraction can be carried out by a small laboratory and the material may be sent to referral centers closer to the municipality where the child resides. Until recently, thyroid dysgenesis was considered a sporadic event (26). In recent years, there have been reports of families with multiple affected cases and molecular biology studies have demonstrated the involvement of genes (TTF1 and 2, PAX8, TSH-R), which encode highly conserved transcription factors that result in agenesis, ectopia and thyroid hypoplasia, when inactivated (27,28). Furthermore, the description of other affected genes responsible for cases of dyshormonogenesis (25) will make the etiological diagnosis much more practical and objective. Currently, the main proteins responsible for most of the critical biochemical steps in thyroid hormone synthesis have been identified, with increasing knowledge of the phenotype/genotype correlation, allowing a more accurate diagnosis to be attained (25). Even the transient hyperthyrotropinemia diagnosed in one of our cases can interfere with cognitive development and subsequently express as manifest hypothyroidism and has been considered a thyroid hormonogenesis defect (29). We conclude that the role of T-US is more than a complementary test to scintigraphy. Its early performance, after the diagnosis of CH, in the first years of life allows suggesting the diagnosis of hypoplasia or dyshormonogenesis according to the observed volume Arch Endocrinol Metab. 2017;61/5

Ultrasonography in congenital hypothyroidism

Funding statement: this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. Disclosure: no potential conflict of interest relevant to this article was reported.

REFERENCES 1. Garel C, Léger J. Thyroid imaging in children. Endocr Dev Basel. 2007;10:43-61. 2. Léger J, Olivieri A, Donaldson M, Torresani T, Krude H, van Vliet G, et al.; ESPE-PES-SLEP-JSPE-APEG-APPES-ISPAE; Congenital Hypothyroidism Consensus Conference Group. European Society for Paediatric Endocrinology consensus guidelines on screening, diagnosis, and management of congenital hypothyroidism. J Clin Endocrinol Metab. 2014;99:363-84. 3. Maciel LM. Hipotireoidismo congênito. Projeto Diretrizes – Associação Médica Brasileira e Conselho Federal de Medicina. 2011. Available at: <http://www.projetodiretrizes.org.br/ans/diretrizes/ hipotireoidismo_congenito.pdf>. Accessed on: Apr. 23, 2014. 4. Pezzuti IL, Lima PP, Dias VM. Congenital hypothyroidism: the clinical profile of affected newborns identified by the Newborn Screening Program of the State of Minas Gerais, Brazil. J Pediatr (Rio J). 2009;85:72-9. 5. Delange F. Neonatal screening for congenital hypothyroidism: results and perspectives. Horm Res. 1997;48:51-61. 6. Palhares HM, Silva LC, Sato LM, Lara BH, Miranzi SSC, Silva AP, et al. Incidence of congenital hypothyroidism in the city of Uberaba/ Minas Gerais and etiological evaluation of the affected subjects. Arq Bras Endocrinol Metab. 2012;56:305-12. 7. Dias VM, Campos AP, Chagas AJ, Silva RM. Congenital hypothyroidism: etiology. J Pediatr Endocrinol Metab. 2010;23:815-26. 8. Brunn J, Block U, Ruf G, Bos I, Kunze WP, Scriba PC. Volumetrie der Schilddrüsenlappen mittels Riel-time-Sonographie. Dtsch Med Wochenschr. 1981;106:1338-40. 9. Chanoine JP, Toppet V, Lagasse R, Spehl M, Delange F. Determination of thyroid volume by ultrasound from the neonatal period to late adolescence. Eur J Pediatr. 1991;150:395-9. 10. Zimmermann MB, Hess SY, Molinari L, Benoist B, Delange F, Braverman LE, et al. New reference values for thyroid volume by ultrasound in iodine-sufficient school children: a World Health Organization/Nutrition for Health and Development Iodine Deficiency Study Group Report. Am J ClinNutr. 2004;79:231-7. 11. Berghout A, Endert E, Ross A, Hogerzeil HV, Smits NJ, Wiersinga WM. Thyroid function and thyroid size in normal pregnant women living in an iodine replete area. Clin Endocrinol (Oxf). 1994;41:375-9.

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12. Balhara B, Misra M, Levitsky LL. Clinical monitoring guidelines for congenital hypothyroidism: laboratory outcome data in the first year of life. J Pediatr. 2011;158:532-7. 13. Klein AH, Meltzer S, Kenny FM. Improved prognosis in congenital hypothyroidism treated before age three months. J Pediatr. 1972;81:912-5. 14. Silva KA, Harper A, Downs A, Blasco PA, Lafranchi SH. Neurodevelopment outcomes in congenital hypothyroidism: comparison of initial T4 dose and time to reach target T4 and TSH. J Pediatr. 2005;147:775-80. 15. Rose SR, Brown RS, Foley T, Kaplowitz PB, Kaye CI, Sundararajan S, et al. American Academy of Pediatrics; Section on Endocrinology and Committee on Genetics, American Thyroid Association; Public Health Committee, Lawson Wilkins Pediatric Endocrine Society. Update of newborn screening and therapy for congenital hypothyroidism. Pediatrics. 2006;117:2290-303. 16. LaFranchi SH. Approach to the diagnosis and treatment of neonatal hypothyroidism. J Clin Endocrinol Metab. 2011;96:2959-67. 17. De Bruyn R, Ng WK, Taylor J, Campbell F, Mitton SG, DicksMireaux C, et al. Neonatal hypothyroidism: comparison of radioisotope and ultrasound imaging in 54 cases. Acta Paediatr Scand. 1990;79:1194-8. 18. Castanet M, Leenhardt L, Léger J, Simon-Carre A, Lyonnet S, Pelet A, et al. Thyroid hemiagenesis is a rare variant of thyroid dysgenesis with a familial component but without Pax8 mutations in a cohort of 22 cases. Pediatr Res. 2005;57:908-13. 19. Bubuteishvili L, Garel C, Czernichow P, Léger J. Thyroid abnormalities by ultrasonography in neonates with congenital hypothyroidism. J Pediatr. 2003;143:759-64. 20. Biebermann H, Schöneberg T, Krude H, Schultz G, Gudermann T, Gruters A. Mutations of the human thyrotropin receptor gene causing thyroid hypoplasia and persistent congenital hypothyroidism. J Cin Endocrinol Metab. 1997;82:3471-80. 21. Santos A, Lara BH, Palhares HM, Ferreira BP, Domené H, Scaglia PA, et al. C105fs114x thyrotropin beta-subunit gene mutation resulting in congenital central hypothyroidism: a genetic study of a Brazilian Family. Horm Res Paediatr. 2011;76: (suppl 4): abst 11. 22. Fleury Y, Van Melle G, Woringer V, Gaillard RC, Portmann L. Sex-dependent variations and timing of thyroid growth during puberty. J Clin Endocrinol Metab. 2001;86:750-4. 23. Macchia PE. Recent advances in understanding the molecular basis of primary congenital hypothyroidism. Mol Med Today. 2000;6:36-42. 24. Rubio IGS, Knobel M, Nascimento AC, Santos CL, Toniolo JV, Medeiros-Neto G. Hipotireoidismo congênito: recentes avanços em genética molecular. Arq Bras Endocrinol Metab. 2002;46:391-401. 25. De Felice M, Di Lauro R. Thyroid development and its disorders: genetic and molecular mechanisms. Endocr Rev. 2004;25:722-46. 26. Castanet M, Polak M, Bonaïti-Pellié C, Lyonnet S, Czernichow P, Léger J. Nineteen years of national screening for congenital hypothyroidism: familial cases with thyroid disgenesis suggest the involvement of genetic factors. J Clin Endocrinol Metabolism. 2001;86:2009-14. 27. Castanet M, Polak M, Léger J. Familial forms of thyroid dysgenesis. Endocr Dev. 2007;10:15-28. 28. Ramos HE, Nesi-França S, Boldarine VT, Pereira RM, Chiamolera MI, Camacho CP, et al. Clinical and molecular analysis of thyroid hypoplasia: a population-based approach in Southern Brazil. Thyroid. 2009;19:61-8. 29. Moreno JC, Visser TJ. New phenotypes in thyroid dyshormonogenesis: hypothyroidism due to DUOX2 mutations. Endocr Dev. 2007;10:99-117.

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and characteristics and, when the gland is not visualized, it allows suggesting thyroid dysgenesis. According to this classification patients may be referred to molecular study of thyroid dysgenesis or dyshormonogenesis in the first year of life, without the need for treatment interruption, which offers more comfort to the patient, allowing the physician to attain an accurate diagnosis and offer adequate genetic counseling.

original article

IL-6, TNF-α, and IL-10 levels/ polymorphisms and their association with type 2 diabetes mellitus and obesity in Brazilian individuals Kathryna Fontana Rodrigues1, Nathalia Teixeira Pietrani1, Adriana Aparecida Bosco2, Fernanda Magalhães Freire Campos3, Valéria Cristina Sandrim4, Karina Braga Gomes3

ABSTRACT Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brasil 2 Instituto de Ensino e Pesquisa, Santa Casa de Belo Horizonte, Belo Horizonte, MG, Brasil 3 Faculdade de Farmácia, UFMG, Belo Horizonte, MG, Brasil 4 Instituto de Biociências, Universidade Estadual Paulista Júlio de Mesquita Filho (Unesp), Botucatu, SP, Brasil 1

Correspondence to: Karina Braga Gomes Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Minas Gerais Av. Antônio Carlos, 6627 31270-901 – Belo Horizonte, MG, Brasil karinabgb@gmail.com

Objective: This study aimed to investigate the association of plasma TNF-α, IL-6, and lL-10 levels and cytokine gene polymorphisms [TNF-α (-308 G→A), IL-6 (-174 C→G) and IL-10 (-1082 A→G, -819 T→C and -592 A→C)] in type 2 diabetes mellitus (T2DM) and obese patients. Subjects and methods: One hundred and two T2DM patients and 62 controls were included in this study. Cytokine plasma levels were measured by the Cytometric Bead Array method. Genotyping was carried out by the polymerase chain reaction. Results: IL-6 levels were significantly different between T2DM patients and controls. Interestingly, IL-6 levels were higher in T2DM patients with BMI > 30 kg/m2 compared with other patients and obese controls. The genotype and allele frequencies were similar between patients and controls. In the T2DM group, the SNP IL-10 -819 T/C showed a difference between the cytokine level and genotypes: IL-10 level in the TT genotype was significantly higher when compared to CC genotype. Conclusions: These results suggest an association between IL-6 levels and obesity, and IL-10 levels and the SNP -819 T/C in T2DM. Knowledge of these variants in T2DM might contribute to a better understanding of the role of inflammation in the etiology and progression of this disease. Arch Endocrinol Metab. 2017;61(5):438-46 Keywords Type 2 diabetes mellitus; polymorphisms; interleukin-6; interleukin-10; tumor necrosis factor-alpha

Received on May/3/2016 Accepted on Nov/25/2016 DOI: 10.1590/2359-3997000000254

INTRODUCTION

ype 2 diabetes mellitus (T2DM) is a heterogeneous group of metabolic disorders characterized by chronic hyperglycemia and represents a significant global health problem (1). According to the International Diabetes Federation (IDF), diabetes mellitus is a major metabolic disease affecting approximately 415 million people worldwide and it is expected to reach 642 million in 2040 (2). The pathogenesis of insulin resistance and T2DM has been associated with a subclinical chronic inflammation and activation of the immune system; however, what triggers this inflammation is still unclear (3,4). Some studies have shown that T2DM patients have higher levels of inflammatory markers such as interleukin-6 438

(IL-6), C reactive protein (CRP), plasminogen activator inhibitor-1 (PAI-1), tumor necrosis factor-α (TNF-α), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) (5-10). Furthermore, it is known that obesity, especially the visceral type, is an independent risk factor for T2DM development (11). In fact, adipose tissue is an endocrine organ that co-regulates wholebody metabolism. It is able to produce a variety of cytokines (TNF-α, IL-6, IL-1β) and other bioactive products, such as leptin, resistin, and monocyte chemoattractant protein-1 (MCP-1/CCL2) (12,13). Adipose tissue in an obese individual is characterized by the presence of pro-inflammatory immune cells (CD8+ T lymphocytes, IFN-γ+ Th1 cells, B cells, mast Arch Endocrinol Metab. 2017;61/5

Cytokines, type 2 diabetes, and obesity

SUBJECTS AND METHODS Ethical aspects This study was approved by the Ethics Committee of the Federal University of Minas Gerais (Minas Gerais, Brazil)-ETIC 0062.0.203.000-11-and Santa Casa Hospital (Minas Gerais, Brazil)-059/2011; in accordance with the Helsinki Declaration. Informed consent was obtained from all subjects.

Study design This cross-sectional study was conducted with 102 patients with clinical and laboratory diagnosis of T2DM (19 men and 83 women) and 62 non-diabetic controls (12 men and 50 women); both groups were aged from 32 to 70 years and matched by gender, age, and body mass index (BMI) in a 2:1 case/control proportion, according to the sample calculation based on the mean values for each cytokines level obtained from a small Arch Endocrinol Metab. 2017;61/5

sample of the groups (power = 0.95; significance level = 0.05). The patients were selected from the Clinic of Endocrinology (Santa Casa Hospital, Minas Gerais, Brazil), and the controls were selected from the local community between June 2012 and September 2013. T2DM diagnosis was based on the American Diabetes Association (ADA) criteria (1). The controls showed normal levels of fasting glucose (60-99 mg/dL) and no use of hypoglycemic drugs. Were excluded subjects older than 70 years, pregnant, with cancer, autoimmune disease, recent history of cardiovascular disease (heart attack, stroke, thrombosis in the last five years), and current or recent infections and/or inflammatory processes.

Clinical and laboratorial data Clinical (gender, age, BMI, waist circumference, waist-hip ratio, T2DM onset, and hypertension), and laboratorial data (fasting glucose, HbA1c, and postprandial glucose) were obtained for all of the T2DM patients through interviews and medical records. The criteria used for determining hypertension were: systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 80 mmHg, or use of antihypertensive drugs, and comply with the criteria adopted by the ADA (1). Clinical data (gender, age, BMI, waist circumference, and waist-hip ratio) for the controls were obtained through interviews. The fasting glucose in the control group was measured in serum samples after eight hours fasting. The serum samples were centrifuged at 1,100 x g for 20 min at 25ºC and the assays performed immediately. The tests were performed using the enzyme-colorimetric method, BTR 811 spectrophotometer (Biotron, Minas Gerais, Brazil), and Glicose-PP kit (Gold Analisa, Minas Gerais, Brazil), following the manufacturer’s instructions. The concentrations of fasting glucose were expressed in mg/dL. Serum samples were used for quantification high sensitivity C reactive protein (hs-CRP). They were centrifuged at 1,100 x g for 20 min at 25ºC and stored at -80ºC until analysis. The tests were performed using the immunoturbidimetric method, System Vitros Chemistry 5.1 FS (Ortho Clinical Diagnostics, New York, USA), and hsCRP VITROS Chemistry Products (Ortho Clinical Diagnostics, New York, USA), following the manufacturer’s instructions. All samples were assayed at the same time. The concentrations of hs-CRP were expressed in mg/L. 439

cells, neutrophils, and M1 macrophages) attracted by chemokines secreted from stressed adipocytes in response to lipid overload (14). The expression of pro- and anti-inflammatory cytokines may be modulated by single nucleotide polymorphisms (SNPs) located in the regulatory regions of genes (15,16). Some studies have investigated the association among TNF-α, IL-6, and IL-10 gene polymorphisms with metabolic diseases (17-25). Despite these reports examining the association of inflammation markers and SNPs in cytokine genes, much controversy remains as to their role in diabetes occurrence (26-34). In this study, we evaluated the role of cytokines in T2DM and obesity by measuring plasma TNF-α, IL-6, and IL-10 levels. We also investigated whether these levels are modulated by polymorphisms located in the regulatory regions of genes (TNF-α (-308 G/A, rs1800629), IL-6 (-174 C/G, rs1800795), and IL10 (-1082 G/A, rs1800896; -819 T/C, rs1800871; and -592 A/C, rs1800872)). Higher IL-6 levels were found in T2DM patients and our results suggest that obesity acts synergistically with T2DM by modulating the increase of this cytokine. Although this study failed to demonstrate that these polymorphisms could modulate TNF-α, IL-6, and IL-10 plasma levels, the IL-10 -819 T/C polymorphism seems to influence IL10 levels in T2DM.

Cytokines, type 2 diabetes, and obesity

Determination of cytokine plasma levels Samples collected in EDTA were centrifuged at 1,100 x g for 20 min at 25ºC to obtain plasma, which was stored at -80ºC until analysis. Data acquisition and analysis were performed in an LSR FortessaTM flow cytometer (BD Biosciences Pharmingen, California, USA) using FCAP Array Software version 1.0.1. TNF-α, IL-6, and IL-10 levels were determined by the Cytometric Bead Array (CBA) method using Human Enhanced Sensitivity Flex Set Systems (BD Biosciences Pharmingen, California, USA), following the manufacturer’s instructions. All samples were assayed at the same time. The concentrations of each cytokine were expressed in fg/mL.

Cytokine gene polymorphism analysis Genomic DNA was extracted from whole blood collected in EDTA using Biopur Mini Spin Kit (Biometrix, São Paulo, Brazil). The polymorphisms were determined using the Cytokine Genotyping Tray Kit (One Lambda Inc., California, USA), which employs Polymerase Chain Reaction-Sequence Specific Primers (PCR-SSP), followed by electrophoresis in 2.5% agarose gel stained with GelGreen Stain (Biotium Inc., California, USA). In order to evaluate the reproducibility rate of genotyping, 10% of the samples in both groups were randomly selected to be re-genotyped. The results showed 100% agreement. The polymorphisms analyzed in the present study were: TNF-α (-308 G/A, rs1800629), IL-6 (-174 C/G, rs1800795), IL-10 (-1082 G/A, rs1800896; -819 T/C, rs1800871; and -592 A/C, rs1800872).

Statistical analysis Deviations from Hardy-Weinberg equilibrium (HWE) were tested using an exact test (available at: http:// genepop.curtin.edu.au/genepop_op1.html). All of the statistical analyses were performed with Statistical Package of the Social Sciences (SPSS) version 17.0. An analysis of normality was performed by Shapiro-Wilk test. Data are presented as “mean ± (standard deviationSD)” (parametric variables), “median (interquartile range-IQR)” (non-parametric variables), or “percentage of total (categorical variables)”. Comparisons between the two groups were made with Student’s t-test for parametric variables and the Mann-Whitney test for non-parametric variables. Comparisons of non-parametric variables between three groups were performed with the Kruskal-Wallis test. When differences were detected, they were compared 440

in pairs by the Mann-Whitney method, followed by Bonferroni’s Correction. The comparison of categorical variables was performed using the chi-square test (χ2). Differences in genotype and allele frequencies between the groups (T2DM patient and control) were tested by Pearson’s χ2-test or Fisher’s Test. IL-10 haplotype estimation was conducted by PHASE software version 2.1. We excluded haplotypes whose frequencies were less than 5%. The differences in the haplotype frequencies between the groups were tested by χ2-test. Linear regression analysis was performed for evaluating the confounding influence of variables in cytokines plasma levels. Gender, age, BMI, waist circumference, waist-hip ratio, and fasting glucose were considered as independent variables. Spearman’s correlations were computed only in the T2DM patient group to assess correlations between cytokine levels, anthropometric, and laboratorial data. A p-value < 0.05 was considered statistically significant.

RESULTS Clinical and laboratorial characteristics of T2DM patients and controls are summarized in Table 1. Groups were matched by gender, age, and BMI (p > 0.05 for all). Waist circumference and waist-hip ratio were higher in T2DM patients than in controls (p < 0.0001 for both). When analyzing cytokine levels in two groups, IL-6 levels were higher in the diabetic patients (p = 0.001), and no significant differences were observed for TNF-α (p = 0.332) and IL-10 (p = 0.317). Diabetic patients on treatment with insulin (n = 18, 17.65%), oral antidiabetic drugs (n = 17, 16.67%) or insulin plus oral antidiabetic drugs (n = 67, 65.68%) show similar levels of cytokines compared with each other (p > 0.05 for allSupplementary Material-SM1). We have evaluated cytokines levels according to BMI categories (BMI < 25 – lean, 25 ≤ BMI < 30 – overweight, and BMI ≥ 30 kg/m2 – obese) within each group (T2DM patient or control) and between these groups (Table 2). In T2DM group, only IL-6 levels were different between patients (p = 0.001) when compared to those with BMI < 25 kg/m2 versus BMI ≥ 30 kg/m2 (p = 0.001). Comparison among 25 ≤ BMI < 30 kg/m2 versus BMI ≥ 30 kg/m2 was not significant after Bonferroni’s correction (p > 0.02). No differences were found in the cytokine levels considering only control group. Finally, when comparing T2DM patients versus controls, IL-6 levels were higher in Arch Endocrinol Metab. 2017;61/5

Cytokines, type 2 diabetes, and obesity

obese patients than in obese controls (p = 0.019). No other associations were found for TNF-α and IL-10 levels and BMI categories. We performed an analysis of genotype and allele frequencies of polymorphisms for cytokine genes TNF-α, IL-6, and IL-10. All polymorphisms were under Hardy-Weinberg equilibrium (p > 0.025). We found no differences between genotype and allele frequencies when compared T2DM and control groups (p > 0.05 for all; Supplementary Material-SM2). Additionally, no difference regarding the IL-10 haplotype analyses was observed between these groups (data not shown). Aiming to evaluate whether polymorphisms in TNF-α, IL-6, and IL-10 genes modulate cytokine Table 1. Clinical and laboratorial characteristics in T2DM patients and controls

plasma levels, we compared their levels and genotypes considering all the individuals (T2DM patients and controls) and performed an analysis to predict their inheritance pattern. However, no differences were found (p > 0.05 for all; Table 3). Interestingly, when these comparisons were applied only in the T2DM patient group (Table 4), IL-10 -819 T/C polymorphism showed a difference between cytokine levels and genotypes (p = 0.021): IL-10 levels in the TT genotype (256 (207) fg/mL] were significantly higher than in the CC genotype (182 (57) fg/mL] (p = 0.006). No differences were found for IL-6 levels and -174 C/G polymorphism after Bonferroni’s correction (p > 0.02) and TNF-α levels and -308 G/A polymorphism (p > 0.05). Regarding the inheritance pattern, only IL-10 -592 A/C polymorphism showed significant differences in IL-10 levels (p = 0.039) when compared to the CC genotype (182 (57) fg/mL] and AC+AA genotypes (192 (115) fg/mL], suggesting a recessive model.

T2DM (n = 102)

Control (n = 62)

18.6 / 81.4

19.3 / 81.7

0.908

Age (years)

56 (12)

53 (18)

0.358

BMI (kg/m2)

BMI < 25

22.6 (3.7)

23.4 (2.8)

0.503

25 ≤ BMI < 30

28.3 (3.0)

27.6 (1.9)

0.215

Cytokine (fg/mL)

BMI ≥ 30

37.8 (9.2)

39.3 (11.0)

0.850

TNF-α

Parameters Gender (Male/ Female) %

Waist circumference (cm)

108.1 ± 16.8

96.9 ± 16.2

< 0.0001*

1.0 ± 0.1

0.9 ± 0.1

< 0.0001*

T2DM onset (%)a

≤ 10 years

40.2

> 10 years

56.9

Waist-hip ratio

Hypertension (Yes/No) % Fasting glucose (mg/dL) Post-prandial glucose (mg/dL)

Not applicable

Table 2. Cytokines levels in T2DM patients and controls according to BMI categories

126.5 (79.0)

85.3 (10.3)

< 0.0001*

T2DM (n = 102)

Control (n = 62)

p’

BMI < 25

86 (48)

90 (75)

0.786

92 (48)

86 (68)

0.748

25 ≤ BMI < 30

IL-6

BMI ≥ 30

92 (42)

112 (58)

0.061

0.922

0.294

BMI < 25

452 (412)

412 (492)

0.422

731 (917)

532 (439)

0.458 0.019*

25 ≤ BMI < 30

92.2 / 7.8

BMI (kg/m2)

IL-10

BMI ≥ 30

1118 (1506)

663 (534)

0.001*

0.356

BMI < 25

232 (102)

192 (48)

0.172

25 ≤ BMI < 30

188 (71)

169 (64)

0.947

BMI ≥ 30

185 (55)

190 (37)

0.473

0.085

0.353

203.0 (118.0)

HbA1c (%)

8.9 ± 1.9

hs-CRP (mg/L)

3.5 (6.6)

2.7 (2.6)

0.094

BMI: body mass index; TNF-α: tumor necrosis factor-alpha; IL-6: interleukin-6; IL-10: interleukin-10.

TNF-α (fg/mL)

91 (42)

95 (64)

0.332

Cytokine levels: no normal variable; the data are shown as “median (IQR)”.

IL-6 (fg/mL)

805 (993)

476 (516)

0.001*

IL-10 (fg/mL)

194 (67)

185 (57)

0.317

* p < 0.05 was considered statistically significant (comparison within the groups: T2DM or control). * p’ < 0.05 was considered statistically significant (comparison between the groups: T2DM versus control).

Normal variables (waist circumference, waist-hip ratio, and HbA1c): the data are shown as “mean ± SD”. No normal variables (age, BMI, fasting glucose, post-prandial glucose, hs-CRP, TNF-α, IL-6, and IL-10): the data are shown as “median (IQR)”. Categorical variables (gender, T2DM onset, and hypertension): the data are shown as “percentage of total”.

Mann-Whitney test with Bonferroni’s Correction for IL-6 levels (T2DM group):

Missing data for three patients.

* p < 0.05 was considered statistically significant.

Arch Endocrinol Metab. 2017;61/5

BMI: body mass index; HbA1c: glycated hemoglobin; hs-CRP: high sensitivity C reactive protein; TNF-α: tumor necrosis factor-alpha; IL-6: interleukin-6; IL-10: interleukin-10.

* p1 – p3: were considered statistically significant if p < 0.02. p1: [BMI < 25 (T2DM) versus 25 ≤ BMI < 30 (T2DM)] = 0.334. p2: [BMI < 25 (T2DM) versus BMI ≥ 30 (T2DM)] = 0.001*. p3: [25 ≤ BMI < 30 (T2DM) versus BMI ≥ 30 (T2DM)] = 0.024.

441

Cytokines, type 2 diabetes, and obesity

Table 3. Cytokines levels according to genotypes considering all the individuals (T2DM patients and controls) Polymorphism

Cytokine (fg/mL) GG (n = 125)

95 (55)

GA (n = 38)

90 (63)

TNF-α (-308 G/A) (rs1800629)

IL-6 (-174 C/G) (rs1800795)

0.705

AA (n = 1)

47.5

CC (n =10)

1381 (2892)

†

CG (n = 50)

565 (754)

GG (n = 104)

674 (770)

GG (n = 20)

204 (55)

GA (n = 73)

182 (63)

AA (n = 71)

185 (65)

TT (n = 20)

214 (136)

TC (n = 69)

182 (69)

IL-10 (-1082 G/A) (rs1800896)

IL-10 (-819 T/C) (rs1800871)

IL-10 (-592 A/C) (rs1800872)

0.361

0.278

0.215

CC (n = 75)

193 (55)

AA (n = 21)

208 (135)

AC (n = 68)

182 (68)

CC (n = 75)

193 (55)

0.409

DISCUSSION

TNF-α: tumor necrosis factor-alpha; IL-6: interleukin-6; IL-10: interleukin-10. Cytokine levels: no normal variable; the data are shown as “median (IQR)”. p < 0.05 was considered statistically significant. †

data only one individual.

Table 4. Cytokines levels according to genotypes in T2DM patients group Cytokine (fg/mL)

Polymorphism TNF-α (-308 G/A) (rs1800629)

IL-6 (-174 C/G) (rs1800795)

GG (n = 78)

95 (45)

GA (n = 23)

85 (44)

AA (n = 1)

47.5†

CC1 (n = 7)

1901 (3167)

CG2 (n = 26)

496 (746)

GG (n = 69)

878 (945)

IL-10 (-1082 G/A) (rs1800896)

IL-10 (-819 T/C) (rs1800871)

IL-10 (-592 A/C) (rs1800872)

GG (n = 12)

202 (62)

GA (n = 47)

178 (68)

AA (n = 43)

199 (87)

TT1 (n = 13)

256 (207)

TC (n = 40)

184 (90)

CC3 (n = 49)

182 (57)

AA (n = 14)

249 (212)

AC (n = 39)

185 (91)

CC (n = 49)

182 (57)

p’

0.137

pa = 0.047 pb = 0.210

0.026*

pc = 0.023 0.181

pa = 0.066

0.021*

pb = 0.006* pc = 0.207

0.055

* p < 0.05 was considered statistically significant. * p’ < 0.02 was considered statistically significant (Mann-Whitney test with Bonferroni’s Correction). pa: genotype 1 versus genotype 2. pb: genotype 1 versus genotype 3. pc: genotype 2 versus genotype 3. †

data only one individual.

442

We investigated the correlation between cytokine plasma levels and anthropometric and laboratorial data in the T2D patients group. IL-6 levels showed a significant positive correlation with BMI (r = 0.314, p = 0.002), waist circumference (r = 0.318, p = 0.002), hs-CRP (r = 0.452, p < 0.0001), and IL-10 levels (r = 0.336, p = 0.001). No other significant correlation was observed between cytokines levels (TNF-α and IL10) and these parameters. Furthermore, fasting glucose and hs-CRP showed a significant negative and positive correlations, respectively, with BMI (r = -0.365, p < 0.0001; r = 0.476, p < 0.0001) and waist circumference (r =- 0.278, p = 0.005; r = 0.442, p < 0.0001). Finally, the linear regression analysis did not show an independent association between gender, age, BMI, waist circumference, waist-hip ratio, and fasting glucose with TNF-α, IL-6, and IL-10 levels (p > 0.05 for all).

This study evaluated the importance of TNF-α, IL6, and IL-10 levels and their association with gene polymorphisms in T2DM disease and the obesity. Clinical and laboratorial characteristics of T2DM patients and controls showed that T2DM patients have higher waist circumferences and waist-hip ratios when compared to the controls. These results are consistent with the knowledge that not only obesity, but mainly the distribution of body fat (mostly upper body obesity), influence glucose metabolism and are independent risk factors for developing T2D (11). Among the cytokines levels measured, only IL-6 levels were higher in the T2DM group. IL-6 is a multifunctional cytokine and is secreted by many types of cells, mainly T cells, macrophages, endothelial cells, smooth muscle cells, adipocytes, and hepatocytes (35). Furthermore, IL-6 regulates/stimulates production of cell adhesion molecules, chemotactic mediators, and acute phase protein, and mediates the release of other cytokines that amplify the inflammatory response (3537). Similar to our result, other studies have shown that T2DM individuals have higher circulating IL-6 levels when compared to non-diabetic controls (38-43). The increased levels of IL-6 and other inflammatory markers (IL-1β, CRP) emerge as early predictors of T2DM, preceding its clinical onset (44). Obesity is also associated with a state of low-grade inflammation (11,13) and elevated levels of IL-6, which has been commonly described in obese diabetic patients Arch Endocrinol Metab. 2017;61/5

or only in obese individuals (38,45,46). We performed an analysis in order to investigate the influence of BMI in cytokine levels. For the control group, no differences were found. However, IL-6 levels in obese T2DM patients (BMI ≥ 30 kg/m2) were higher than lean (BMI < 25 kg/m2) and overweight (25 ≤ BMI < 30 kg/m2) patients (tendency), although the linear regression analysis has not showed an independent association between these parameters. Moreover, obese T2DM patients presented higher IL-6 levels when compared to obese controls. These results suggest that higher BMI values in T2DM are associated with increased IL-6 levels, but some other variable appears to act synergistically, since this association was not independent. When compared to the control group, T2DM seems to act synergistically with obesity to promote an increase in IL-6 levels. Although no difference was found in TNF-α and IL-10 levels between the groups (T2DM patients and controls) and the BMI categories, these cytokines have been associated with T2DM. TNF-α is a pro-inflammatory cytokine produced by a variety of cell-types, mainly macrophages, lymphocytes, and adipocytes (13). Some studies found higher TNF-α levels in T2DM patients when compared with non-diabetic controls (39,40,43). IL-10 is an anti-inflammatory cytokine that plays an important role in the regulation of the immune system leading to decreased cytokine production, reducing tissue factor expression, inhibiting matrix-degrading metalloproteinase, and promoting the phenotypic switching of lymphocytes to the Th2 phenotype (47). This cytokine is produced by T-cells, B-cells, monocytes, and macrophages, and it is estimated that 75% of the variation in IL-10 production is genetically determined (47). An important study showed that IL10 level was lower in subjects with impaired glucose tolerance or T2DM when compared with subjects with normal glucose tolerance and showed an inverse correlation with BMI (48). Conversely, Al-Shukaili and cols. (49) found higher IL-10 levels in T2DM patients when compared with healthy controls. Taken together, it is not clear whether higher IL-10 levels confer protection against T2DM development by decreased pro-inflammatory cytokines production, or increased IL-10 levels in T2DM result in a compensatory response against the elevation of pro-inflammatory mediators, primarily TNF-α and IL-6. No polymorphism showed a difference in allele and genotype frequencies when compared to T2DM Arch Endocrinol Metab. 2017;61/5

patients and the control group. Therefore, no polymorphism in this study was associated with T2DM. However, the association of these polymorphisms with T2DM remains unclear. In 2011, Feng and cols. (26) in a meta-analysis did not find a significant association between TNF-α -308 G/A polymorphism and T2DM risk when considering Caucasian and Asian populations. In contrast, in 2014, a meta-analysis conducted by Zhao and cols. (27) indicated that TNF-α -308A allele could be a risk factor for the development for T2DM in Asian subjects. Similarly, Golshani and cols. (28) found that TNF-α -308 GA+AA genotypes are associated with higher risk for T2DM development in an Iranian population. According to Qi and cols. (29), IL-6 -174 C/G polymorphism is not associated with the risk of T2DM development; however, a recent study (23) shows a significant association between T2DM and IL-6 -174G allele. Finally, four recent meta-analyses evaluated the association between IL10 gene polymorphisms (-1082 G/A, -819 T/C, and -592 A/C) and the risk of T2DM development. The -819 T/C and -592 A/C polymorphisms did not show an association with the disease in these studies (30-33). However, Li and cols. (31) and Hua and cols. (30) found an association between the -1082GA genotype and -1082G allele, respectively, with T2DM. Additionally, a case-control study conducted by Bai and cols. (34) found higher risk for T2DM development associated with -1082 GA+GG and -592 AC+AA genotypes. These conflicting results are probably related to the sample size and different genetic background of the populations. Larger scale genome studies are required to further evaluate these associations. The plasma levels of IL-6 were significantly different between -174 C/G polymorphism genotypes, but this difference was not maintained after Bonferroni’s correction. The patients with IL-10 -819TT genotype showed higher IL-10 levels than patients with -819CC genotype. Previous studies (50,51) showed that, not only the -819T allele, but also the ATA haplotype (-1082A, -819T, -592A) are related to lower transcriptional activity and, consequently, lower IL10 levels. However, no further study was carried out on diabetic patients to prove the association between transcriptional activity of IL-10 gene and IL-10 serum/ plasma levels and -819 T/C polymorphism. IL-6 levels were positively correlated with BMI, waist circumference, hs-CRP, and IL-10 levels. Similarly, hs-CRP also showed a significant positive correlation 443

Cytokines, type 2 diabetes, and obesity

with BMI and waist circumference. Obesity, especially visceral obesity, is characterized by the increased size of adipocytes and recruitment of immune cells (mainly macrophages), which display a pro-inflammatory profile. These cells are responsible for the increased production of inflammatory mediators and acute phase proteins, such as IL-6 and CRP (12). Considering these events, it is expected that IL-6 and hs-CRP levels are positively correlated with each other and with anthropometric parameters that express weight gain (BMI) and the increase in upper body fat (waist circumference) in the T2DM group. Finally, the positive correlation between IL-6 and IL-10 levels showed that, in T2DM, the increase of pro-inflammatory mediators may cause a compensatory increase in anti-inflammatory cytokines to control subclinical inflammation. The small sample size, owing to the strict selection criteria for patients and controls, and absence of functional analysis of polymorphisms can be considered as the main limitations of this study, since the effect of the polymorphisms on protein activity was not evaluated. Indeed, the statistical power became lower when the total number of the participants in each group was classified according to BMI categories, but as cytokine levels are associated with adiposity, this selection was necessary in order to avoid a bias in the results. Therefore, further studies with a much larger sample exploring other populations (different genetic background) and others clinical characteristics as a practice of physical activities, are needed to better understand the role of these polymorphisms in the subclinical inflammation in T2DM. In conclusion, few studies have evaluated inflammatory markers and cytokines genes polymorphisms in T2DM Brazilian patients. Considering the increased number of diabetic patients in Brazil and the population’s genetic background, improved knowledge on the markers that contribute to the etiology and progression of T2DM are important for prevention, diagnosis, and follow-up of this disease. Taken together, our results show that IL-6 and IL10 levels and the SNP -819 T/C in IL-10 gene are associated with the subclinical inflammation in the T2DM. Moreover, the association between IL-6 levels and obesity in T2DM indicates that weight control may be an action adopted for preventing inflammatory status in T2DM. Acknowledgments: the authors thank Fundação de Amparo à Pesquisa do Estado de Minas Gerais (Fapemig), Coordenação de Aper444

feiçoamento de Pessoal de Nível Superior (Capes), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/Brazil), and Pró-Reitoria de Pesquisa da Universidade Federal de Minas Gerais (PRPq/UFMG) for financial support. VCS and KBG are grateful to CNPq Research Fellowship. Disclosure: no potential conflict of interest relevant to this article was reported.

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35. Barton BE. The biological effects of interleukin 6. Med Res Rev. 1996;16(1):87-109. 36. Kamimura D, Ishihara K, Hirano T. IL-6 signal transduction and its physiological roles: the signal orchestration model. Rev Physiol Biochem Pharmacol. 2003;149:1-38. 37. Fisman EZ, Tenenbaum A. The ubiquitous interleukin-6: a time for reappraisal. Cardiovasc Diabetol. 2010;9:62. 38. Hansen D, Dendale P, Beelen M, Jonkers RA, Mullens A, Corluy L, et al. Plasma adipokine and inflammatory marker concentrations are altered in obese, as opposed to non-obese, type 2 diabetes patients. Eur J Appl Physiol. 2010;109(3):397-404. 39. Goyal R, Faizy AF, Siddiqui SS, Singhai M. Evaluation of TNF-α and IL-6 Levels in Obese and Non-obese Diabetics: Pre- and Postinsulin Effects. N Am J Med Sci. 2012;4(4):180-4. 40. Mirza S, Hossain M, Mathews C, Martinez P, Pino P, Gay JL, et al. Type 2-diabetes is associated with elevated levels of TNF-alpha, IL-6 and adiponectin and low levels of leptin in a population of Mexican Americans: a cross-sectional study. Cytokine. 2012;57(1):136-42. 41. Guzel S, Seven A, Kocaoglu A, Ilk B, Guzel EC, Saracoglu GV, et al. Osteoprotegerin, leptin and IL-6: association with silent myocardial ischemia in type 2 diabetes mellitus. Diab Vasc Dis Res. 2013;10(1):25-31. 42. Hang H, Yuan S, Yang Q, Yuan D, Liu Q. Multiplex bead array assay of plasma cytokines in type 2 diabetes mellitus with diabetic retinopathy. Mol Vis. 2014;20:1137-45. 43. Daniele G, Guardado Mendoza R, Winnier D, Fiorentino TV, Pengou Z, Cornell J, et al.The inflammatory status score including IL-6, TNF-α, osteopontin, fractalkine, MCP-1 and adiponectin underlies whole-body insulin resistance and hyperglycemia in type 2 diabetes mellitus. Acta Diabetol. 2014;51(1):123-31. 44. Spranger J, Kroke A, Möhlig M, Hoffmann K, Bergmann MM, Ristow M, et al. Inflammatory cytokines and the risk to develop type 2 diabetes: results of the prospective population-based European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam Study. Diabetes. 2003;52(3):812-7. 45. Lukic L, Lalic NM, Rajkovic N, Jotic A, Lalic K, Milicic T, et al. Hypertension in obese type 2 diabetes patients is associated with increases in insulin resistance and IL-6 cytokine levels: potential targets for an efficient preventive intervention. Int J Environ Res Public Health. 2014;11(4):3586-98. 46. Andrews M, Soto N, Arredondo-Olguín M. Association between ferritin and hepcidin levels and inflammatory status in patients with type 2 diabetes mellitus and obesity. Nutrition. 2015;31(1):51-7. 47. Scarpelli D, Cardellini M, Andreozzi F, Laratta E, Hribal ML, Marini MA, et al. Variants of the interleukin-10 promoter gene are associated with obesity and insulin resistance but not type 2 diabetes in caucasian italian subjects. Diabetes. 2006;55(5): 1529-33. 48. Blüher M, Fasshauer M, Tönjes A, Kratzsch J, Schön MR, Paschke R. Association of interleukin-6, C-reactive protein, interleukin-10 and adiponectin plasma concentrations with measures of obesity, insulin sensitivity and glucose metabolism. Exp Clin Endocrinol Diabetes. 2005;113(9):534-7. 49. Al-Shukaili A, Al-Ghafri S, Al-Marhoobi S, Al-Abri S, Al-Lawati J, Al-Maskari M. Analysis of inflammatory mediators in type 2 diabetes patients. Int J Endocrinol. 2013;2013:976810. 50. Turner DM, Williams DM, Sankaran D, Lazarus M, Sinnott PJ, Hutchinson IV. An investigation of polymorphism in the interleukin-10 gene promoter. Eur J Immunogenet. 1997;24(1):1-8. 51. Crawley E, Kay R, Sillibourne J, Patel P, Hutchinson I, Woo P, et al. Polymorphic haplotypes of the interleukin-10 5’ flanking region determine variable interleukin-10 transcription and are associated with particular phenotypes of juvenile rheumatoid arthritis. Arthritis Rheum. 1999;42(6):1101-8.

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18. Bouhaha R, Baroudi T, Ennafaa H, Vaillant E, Abid H, Sassi R, et al. Study of TNFalpha -308G/A and IL6 -174G/C polymorphisms in type 2 diabetes and obesity risk in the Tunisian population. Clin Biochem. 2010;43(6):549-52.

Cytokines, type 2 diabetes, and obesity

SUPPLEMENTARY MATERIAL Table SM1. Cytokines levels according to types of pharmacological treatment in T2DM group Types of treatment Cytokine (fg/mL) TNF-α

Oral antidiabetic drugs (n = 17)

Insulin (n = 18)

Insulin plus oral antidiabetic drugs (n = 67)

94 (51)

74 (35)

92 (51)

0.440

IL-6

911 (1546)

729 (877)

783 (1518)

0.530

IL-10

187 (63)

180 (42)

194 (78)

0.511

Table SM2. Distributions of genotypes and alleles frequencies in T2DM patients and controls Polymorphisms TNF-α (-308 G/A) (rs1800629)

IL-6 (-174 C/G) (rs1800795)

IL-10 (-1082 G/A) (rs1800896)

IL-10 (-819 T/C) (rs1800871)

IL-10 (-592 A/C) (rs1800872)

T2DM (n = 102) (%)

Control (n = 62) (%)

Genotypes

78 (76.5)

47 (75.8)

23 (22.6)

15 (24.2)

1 (0.9)

0.965

Alleles

179 (87.8)

109 (87.9)

25 (12.2)

15 (12.1)

Genotypes

7 (6.8)

3 (4.8)

26 (25.5)

24 (38.7)

69 (67.7)

35 (56.5)

Alleles

40 (19.6)

30 (24.2)

164 (80.4)

94 (75.8)

Genotypes

12 (11.8)

8 (12.9)

47 (46.1)

26 (41.9)

43 (42.1)

28 (45.2)

Alleles

71 (34.8)

42 (33.9)

133 (65.2)

82 (66.1)

Genotypes

13 (12.8)

7 (11.3)

40 (39.2)

29 (46.8)

49 (48.0)

26 (41.9)

Alleles

66 (32.4)

43 (34.7)

138 (67.6)

81 (65.3)

Genotypes

14 (13.7)

7 (11.3)

39 (38.3)

29 (46.8)

49 (48.0)

26 (41.9)

Alleles

67 (32.8)

43 (34.7)

137 (67.2)

81 (65.3)

0.966

0.348

0.326

0.864

0.863

0.674

0.665

0.742

0.733

TNF-α: tumor necrosis factor-alpha; IL-6: interleukin-6; IL-10: interleukin-10. p < 0.05 was considered statistically significant.

446

Arch Endocrinol Metab. 2017;61/5

original article

Use of prophylactic oral calcium after total thyroidectomy: a prospective study Erwin Langner1, Alfio José Tincani2, André del Negro2

ABSTRACT Objective: The aim of this study was to evaluate the use of prophylactic oral calcium after total thyroidectomy in the prevention of symptomatic hypocalcemia, and to develop a rational strategy of oral calcium supplementation following this type of surgery. Subjects and methods: Prospective study including 47 patients undergoing total thyroidectomy from January 2007 to February 2012. The patients were allocated to one of the following groups: I (no postoperative calcium) or II (oral calcium 3 g per day). Oral calcium was started at the first postoperative day and administered until the sixth postoperative day. The patients were followed up for a minimum of 6 months and evaluated with a minimum of five measurements of ionized calcium: preoperative, 16 hours after surgery, seventh postoperative day, and at postoperative days 90 (PO90) and 180 (PO180). The cohort included three men and 44 women, of whom 24 (51.9%) had benign thyroid disease, and 23 had suspected or confirmed malignant disease. Results: When compared with Group II, Group I had significantly higher rates of postoperative biochemical hypocalcemia at PO1 and PO180, and of symptomatic hypocalcemia at PO1, PO7, and PO90. Other data were not significantly different between the groups. Conclusion: We conclude that postoperative calcium supplementation effectively prevents symptomatic and biochemical hypocalcemia after total thyroidectomy, and can be safely used after this procedure. The presented strategy of oral calcium supplementation may be implemented in a viable manner. Arch Endocrinol Metab. 2017;61(5):447-54

Limeira, SP, Brasil Universidade Estadual de Campinas (Unicamp), SP, Brasil

1 2

Correspondence to: Erwin Langner Rua Santa Cruz, 754, sala 114 13480-041 – Limeira, SP, Brasil erwin.langner@gmail.com Received on May/4/2016 Accepted on Oct/10/2016 DOI: 10.1590/2359-3997000000286

INTRODUCTION

he correct execution of any surgical procedure depends on the knowledge of possible complications associated with such procedure. Thyroid surgeries are no exception to this rule. One of the postoperative complications of this type of surgery is hypoparathyroidism, which has a 0.6% to 17% (1) incidence in its permanent form and 1.6% to 87% (2-4) in its transient form. This complication has been a matter of concern to surgeons since the first thyroidectomies have been performed in the contemporary age (5,6). The proper surgical management of thyroid diseases requires a familiarity with the locoregional anatomy, including the morphology, syntopy, vascularization, and embryology of the thyroid and parathyroid glands. The role of a meticulous surgical technique is well established in the literature, including the dissection of the superior and recurrent laryngeal nerves, careful dissection of the parathyroid glands, and ligation of the peripheral thyroid arteries as the main preventive Arch Endocrinol Metab. 2017;61/5

measures against postoperative complications such as hypoparathyroidism and associated symptoms (1,7). Other causes may contribute to postoperative hypocalcemia, such as surgery extension, surgeon’s experience, resection of one or more of the parathyroid glands, glandular lesions caused by suction in the operatory field and hemodilution (2,8-11), central compartment neck dissection, and reoperations. In a classical description of the normal parathyroid glands, they are described as varying between one and six in number (a study by Hojaij has described the presence of four glands in 78.56% of 56 patients) (5,12) and as having a kidney-like shape, location in the posterior aspect of the thyroid gland, measurement of 3-8 mm and weight of 15-30 mg, yellow-brownish color, and irrigation by delicate branches of the inferior thyroid artery (1,5,13). Considerable anatomic variants of these glands may be found, and in 1998, Hojaij (5) reported the finding of mediastinal, intrathyroidal, and subcapsular parathyroids in 21.4%, 5.4%, and 14.3% of the cases, respectively. 447

Keywords Calcium, prophylactic, thyroidectomy, study, prospective

Prophylactic calcium after thyroidectomy

Unlike temporary hypocalcemia, permanent (or definitive) hypocalcemia lasts for more than 6 months after surgery (3). Both types of hypocalcemia are uncomfortable complications due to their clinical presentation, including the occurrence of the Chvostek’s and Trousseau’s signs, paresthesia, carpopedal spasm, tetany at various levels, electrocardiographic changes, seizures, and behavioral changes. Patients with symptomatic hypoparathyroidism may require prolonged hospitalization, which significantly increases their treatment costs (14). The frequency of postoperative hypocalcemia is significantly greater after total thyroidectomy. In a report of 119,000 thyroid surgeries, Baldassare and cols. (15) found a hypocalcemia rate of 1.9% after partial thyroidectomy and 9% after total thyroidectomy compared with 23.4% after total thyroidectomy and selective bilateral neck dissection. Several authors (2,16-19) have proposed ways to reduce the occurrence of hypocalcemia, studying predisposing factors and proposing strategies to reduce its incidence and symptoms. The latter includes proposals to prevent hypocalcemia with calcium replacement using calcium carbonate, as reported by Moore in 1994 (20), with effervescent preparations of other types of calcium, as reported by Bellantone and cols. (21), or calcitriol (vitamin D), as described by Bellantone and cols. (21) and Tartaglia and cols. in 2005 (22). Recently, Docimo and cols. (23) have reported the preoperative and postoperative use of calcium and calcitriol, with a 10% incidence of biochemical hypocalcemia and 6% of symptomatic hypocalcemia when administered for 3 days before and 14 days after surgery. An analysis of these studies reveals that the preoperative administration of calcium preparations prevents symptomatic hypocalcemia, particularly in its severe forms, with no significant difference between the administration of calcium alone or calcium combined with calcitriol (21). The objectives of this study were to perform a prospective evaluation of the use of oral calcium supplementation after total thyroidectomy and demonstrate its efficacy in preventing symptomatic hypoparathyroidism, in addition to evaluating a viable strategy for the use of oral calcium supplementation after total thyroidectomy.

SUBJECTS AND METHODS This study was performed using data of patients examined and operated on by the same surgical team, 448

coordinated by the main author, in the city of Limeira (São Paulo, Brazil), after approval by the Ethics Committee in Research at all the hospitals and the Ethics Committee in Research at the State University of Campinas (Unicamp) under the protocol number 1014/2010. A total of 47 patients undergoing total thyroidectomy from January 2007 to August 2012 were studied by sequential analysis. All patients underwent a routine preoperative evaluation that included the measurement of serum electrolytes, cell blood count, coagulation tests, chest X-ray, and an electrocardiogram with evaluation by a cardiology specialist, if necessary. We measured the patients’ serum ionized calcium and thyroid-stimulating hormone (TSH) levels preoperatively and recorded the following parameters: age, gender, surgery date, prior diagnosis, and presence or absence of a thyroid hormone disorder. All patients were informed about the procedures in this study by a Statement of Informed Consent, which was signed and approved by the Ethics Committee in Research at the involved institutions. The research was performed with the researchers’ resources. The exclusion criteria included partial thyroidectomy of any type, partial or total resections of parathyroid tissue, and extended thyroidectomy with neck dissection. One patient was excluded due to laryngeal invasion detected during surgery and was then treated by partial laryngectomy, while another patient was excluded due to the execution of bilateral neck dissection. Overall, 15 patients were lost to follow-up and were excluded from the analysis, and one patient died 30 days after surgery with a diagnosis of thyroid lymphoma. After surgery, the patients were divided into two groups according to their postoperative treatment: Group I: 27 patients who did not receive calcium treatment after surgery, except in cases of symptomatic hypocalcemia or detection of ionized calcium below 0.8 mmol/L at 16 hours after surgery. Group II: 20 patients who received treatment with postoperative oral effervescent calcium (Sandoz FF®) 3 g daily for 6 days after surgery. The patients were evaluated before and after the procedure and were followed up for a minimum of 6 months. According to the protocol, the patients were followed up with at least five measurements of ionized calcium: before surgery, 16 hours after surgery, on the seventh postoperative day, and 90 and 180 days after surgery. Arch Endocrinol Metab. 2017;61/5

Prophylactic calcium after thyroidectomy

RESULTS A total of 47 patients were analyzed, including three men (6.4%) and 44 women (93.6%), with a mean age of 52.1 years (standard deviation [SD] 12.8 years, median of 52 years). Total thyroidectomy was performed in 24 patients (51.9%) for treatment of a benign disease and in 23 (48.1%) for treatment of a suspected or previously confirmed malignant disease. Overall, 27 patients (78.7%) presented a normal thyroid function at the time of the surgical indication, six (12.8%) presented hyperthyroidism, and four (8.5%) presented previous hypothyroidism. Data from the two groups are summarized in Table 1. All patients underwent total thyroidectomy; 33 patients showed no signs and symptoms of hypocalcemia (70.2%) while 14 others (29.8%) presented mild symptoms of hypocalcemia. No severe symptoms of hypocalcemia were observed. Biochemical hypocalcemia occurred in six patients (12.8%) in the preoperative evaluation, in 23 patients (48.9%) on the first postoperative day, in 17 patients (36.2%) on the seventh postoperative day, in 15 Arch Endocrinol Metab. 2017;61/5

patients (31.9%) 90 days after surgery, and in nine patients (19.2%) 180 days after the procedure, while seven patients (14.9%) still had hypoparathyroidism at the end of this study. Among the six patients with preoperative hypocalcemia, four maintained their status of biochemical hypocalcemia at the first postoperative day (66.6%), two at the seventh postoperative day (33.3%), four at 90 days after the procedure (66.6%), and two cases at 180 days after the surgery (33.3%), while two patients with preoperative hypocalcemia (both in Group I, in which no postoperative calcium was administered) presented all measurements below 1.1 mmol/L). These data are shown in Table 2.

Table 1. General characteristics of the 47 patients included in the study n

93.6

6.4

Sex

Age N

Mean

52.1

Standard deviation

12.8

Median

52.0

Indication CPG

38.3

MNG

6.4

34.0

Sca

6.4

8.5

High

8.5

Low

4.3

Normal

87.2

70.2

Yes

29.8

Hyper

12.8

Hypo

8.5

Normal

78.7

TSH

Postop hypocalc

Hypo/hyperthyroidism

F: female gender; M: male gender; n: sample size; indication: surgery indication; CPG: compressive goiter; MNG: multinodular goiter; GD: Graves’ disease; PC: papillary carcinoma; SCa: clinical or cytological suspicion of cancer; FT: follicular tumor; TSH: previous thyroid stimulating hormone; Postop hypocalc: postoperative hypocalcemia; hypo/hyperthyroidism: previous hypothyroidism or hyperthyroidism.

449

Administration of oral calcium was maintained in the presence of symptomatic hypocalcemia or persistent serum calcium measurements below 0.8 mmol/L after the sixth postoperative day, until normal calcium measurements were obtained. We considered as hypocalcemia the occurrence of serum calcium levels under 1.1 mmol/L, and as severe hypocalcemia the occurrence of levels below 0.8 mmol/L. Calcium levels between 1.1 and 1.4 mmol/L were considered normal. The presence and intensity of signs and symptoms of hypocalcemia were recorded in a dedicated form and classified into three groups: absence of symptoms, mild symptoms (paresis or Chvostek’s sign), and severe symptoms (Trousseau’s sign, carpopedal spasm, tetany or cardiac signs and symptoms). The presence and duration of the hypocalcemia after treatment, presence or absence of side effects, and number and characteristics of the parathyroid glands observed during surgery were recorded. The data of the two groups were analyzed and compared using Fisher’s chi-square test, analysis of variance (ANOVA) for repeated measures, and Wilk’s test using the software SAS, v. 9.2 (SAS Institute, Inc, Cary, NC, USA). The significance level (p) was set at 0.05.

Prophylactic calcium after thyroidectomy

Table 2. Postoperative progression of serum calcium levels (mmol/L) Values (in mmol/L)

0.8 to 1.1

12.8

Normal

87.2

0.8 to 1.1

48.9

Normal

51.1

< 0.8

4.3

0.8 to 1.1

31.9

Normal

62.8

Preop

POi

PO7

hypocalcemia are resumed in Table 4. The correlation between the occurrence of hypocalcemia and symptoms in both groups, evaluated using ANOVA for repeated measures, is shown in Table 5. Table 3. Incidence of postoperative hypocalcemia according to study groups Group

Values (in mmol/L)

Preop

PO90 0.8 to 1.1

31.9

Normal

68.1

PO180 < 0.8

6.4

0.8 to 1.1

12.8

Normal

80.8

Preop: preoperative; Poi: immediate postoperative; PO7: 7 days after surgery; PO90: 90 days after surgery; PO180: 180 days after surgery.

0.2205

0.8 to 1.1

18.5%

5.0%

Normal

81.5%

95.0%

0.8 to 1.1

63.0%

30.0%

Normal

37.0%

70.0%

< 0.8

3.7%

5.0%

0.8 to 1.1

40.7%

20.0%

Normal

55.6%

75.0%

0.8 to 1.1

40.7%

20.0%

Normal

59.3%

80.0%

< 0.8

7.4%

5.0%

0.8 to 1.1

22.2%

0.0%

Normal

70.4%

95.0%

POi

0.0254*

PO7

0.2912

PO90

0.1315*

PO180

The prevalence of biochemical hypocalcemia in both groups and the statistical comparison according to serum calcium level are described in Table 3. The progression of the calcium levels according to the study group and presence or absence of symptomatic

0.0409

Fisher’s test / * Chi-square test. Preop: preoperative; Poi: immediate postoperative; PO7: 7 days after surgery; PO90: 90 days after surgery; PO180: 180 days after surgery.

Table 4. Progression of serum calcium levels according to group/symptoms IV.1 – Progression of serum calcium levels according to groups Group I (n = 27) Measurement

Group II (n = 20) Time Effect

Group Effect

Mean

Standard deviation

Median

Mean

Standard deviation

Median

Preop

1.16

0.07

1.20

1.22

0.09

1.22

POi

1.09

0.09

1.09

1.16

0.08

1.16

< 0.0001

0.0013

PO7

1.10

0.11

1.12

1.17

0.16

1.21

0.0087

0.0028

PO90

1.12

0.12

1.14

1.17

0.14

1.21

0.0448

0.0088

PO180

1.13

0.17

1.14

1.20

0.12

1.23

0.4771

0.0056

Time Effect

Group Effect

0.0001

< 0.0001

IV.2 – Progression of serum calcium according to symptoms. Asymptomatic (n = 33)

Measurement

Symptomatic (n = 14)

Mean

Standard deviation

Median

Mean

Standard deviation

Median

Preop

1.20

0.09

1.20

1.17

0.08

1.20

POi

1.16

0.07

1.16

1.04

0.07

1.06

< 0.0001

0.0005

PO7

1.17

0.09

1.18

1.02

0.17

1.05

0.0007

0.0016

PO90

1.18

0.11

1.20

1.05

0.14

1.06

0.0077

0.0022

PO180

1.19

0.12

1.22

1.08

0.19

1.13

0.2490

0.0102

ANOVA for repeated measures. Preop: preoperative; Poi: immediate postoperative; PO7: 7 days after surgery; PO90: 90 days after surgery; PO180: 180 days after surgery.

450

Arch Endocrinol Metab. 2017;61/5

Prophylactic calcium after thyroidectomy

Table 5. Hypocalcemia and presence of symptoms in both study groups Measurement Preop

Group I, asymptomatic

Group I, symptomatic (n = 10)

Group II, asymptomatic (n = 16)

Group II, symptomatic (n = 4)

(n = 17)

Median

Mean

Median

Mean

Median

Mean

Median

1.17

0.07

1.19

1.16

0.08

1.20

1.23

0.09

1.24

1.19

0.08

1.18

0.0620

POi

1.14

0.07

1.12

1.02

0.07

1.03

1.18

0.08

1.17

1.08

0.06

1.08

< 0.0001

PO7

1.14

0.06

1.15

1.03

0.15

1.01

1.21

0.10

1.22

1.00

0.26

1.07

0.0009

PO90

1.14

0.11

1.16

1.08

0.12

1.08

1.22

0.08

1.23

0.97

0.17

0.97

0.0007

PO180

1.17

0.16

1.18

1.05

0.16

1.13

1.22

0.05

1.23

1.14

0.27

1.19

0.0678

DISCUSSION Although the parathyroid glands were first described in Indian rhinoceros by Owen in 1852 (cited by Thompson) (26), the relationship between hypocalcemia after total thyroidectomy and resection or injury to the parathyroid glands was only established in 1891, with the first report of tetany after thyroidectomy occurring in 1877 (4). At that same year, Sandström (27,28) started to observe the parathyroid glands in animals and after dissecting 50 human bodies, described in 1880 the anatomy, number, and shape of these glands. Sandström suggested the name of the parathyroid gland while recognizing the independence of its anatomic structure in relation to the thyroid gland. The association between the parathyroid glands and tetany was recognized after 1890 according to Thompson (26) and as cited by Hojaij (5). In 1907, Pool (29) and Hojaij (5) coined the term tetania paratireopriva. Also in 1907, Halsted and Evans (30), as well as Reeve and Thompson (31), after dissecting 20 human bodies, confirmed the need for prevention of parathyroid injury during thyroid surgery and identified one delicate arterial bunch for each gland, derived from the inferior thyroid artery in 90% of the patients. Many authors, including Lahey in 1926 (32), Milzner in 1927 (6), Murley and Peters in 1961 (33), and Croyle and Oldroyd em 1978 (34), reported a 10 to 24% incidence of parathyroid resections in thyroidectomy, which led Loré and Pruet in 1983 (35) and Shaha and cols. in 1991 (36) to suggest a meticulous examination of the surgical specimen for identification of parathyroid glands potentially removed during surgery, with the intention of surgically reimplanting them. Careful dissection, preservation of the parathyroid glands, and peripheral vascular ligation of the thyroid arteries with minimum damage to the parathyroid Arch Endocrinol Metab. 2017;61/5

irrigation, associated with preservation and eventual parathyroid reimplantation, remain today as time-honored surgical procedures to prevent hypoparathyroidism after thyroid surgery. The prevalence of female patients in this study, as well as their mean and median age, are consistent with data found in the literature and caused by the larger prevalence of thyroid disorders in female patients when compared with male ones. Some authors (37-39) correlate the decrease in serum calcium levels at the first postoperative day as a prognostic factor of the occurrence of postoperative hypocalcemia after total thyroidectomy. This correlation is more precise with measurement of ionized calcium, which may be safely used for research in hypoparathyroidism (18) since ionized calcium is not affected by variations in protein concentrations as occurring with total calcium. Other factors identified as responsible for decreasing calcium levels after total thyroidectomy include intraoperative hemodilution, which explains the occurrence of hypocalcemia in other extracervical surgeries with similar extension (2,8), and the hungry bone syndrome, in which a normal parathyroid function is maintained (9). Clark and Duh (40) suggested in 1989 that the parathyroid glands located above the thyroid gland have a higher risk of intraoperative injury due to the longer extension of their vascular pedicles, which have to be dissected during the procedure. In this study, the first postoperative calcium measurement was performed at 16 hours after the surgery. This decision followed the findings by Bentrem and cols. (18) in 2001, who reported a 94.5% ability to predict postoperative hypocalcemia when calcium is measured at this time point. Marohn and LaCivita (17) in 1995 measured serum calcium levels at 8, 14, and 451

ANOVA. SD: standard deviation; Preop: preoperative; Poi: immediate postoperative; PO7: 7 days after surgery; PO90: 90 days after surgery; PO180: 180 days after surgery.

Prophylactic calcium after thyroidectomy

20 hours after thyroidectomy and concluded that the levels decrease in most cases, reaching their lowest at 14 hours after surgery. In the present study, biochemical hypocalcemia was observed in the first postoperative day in 48.9% of the patients, reaching 63% in patients in Group I and 30% in those in Group II (p = 0.0254) (Table 3). A significant difference in calcium measurement was observed between the two groups 180 days after surgery; of nine patients with hypocalcemia (19.2%), eight belonged to Group I and one to Group II (p = 0.0409). The incidence of permanent hypocalcemia (14.9%) was consistent with that in the literature (17%) (1). Other measurements (preoperative [PO] 7, PO90) showed no statistical differences between the study groups, with rates of hypocalcemia of 12.8% before surgery, 36.2% at 7 days after surgery, and 31.9% at 90 days after the procedure, as reported in Table 2. The incidence of preoperative hypocalcemia (12.8%) suggested a need for routine measurements of calcium, although the possibility of measuring serum calcium levels is not always available in the preoperative protocols in services performing thyroidectomy among us. The progression of patients with preoperative hypocalcemia, with incidence greater than the mean in the PO1, PO90, and PO180, suggests an ability to predict hypoparathyroidism after total thyroidectomy. The incidence of postoperative biochemical hypocalcemia was 63% on the first postoperative day without the use of prophylactic calcium, which is consistent with data from the literature (2,4,19,24). Patients in Group II received 3 g of oral calcium daily for a minimum of 6 days, following the procedure reported by Bellantone and cols. (21) in 2002. These authors measured the levels of serum calcium on the first, second, third, and seventh postoperative days, and reported a significant decrease in biochemical hypocalcemia at PO2 and PO3 and a significant decrease in symptomatic hypocalcemia in all study groups. Some authors (37) have associated the occurrence of symptomatic compressive goiter to a significant risk of postoperative hypocalcemia, while others have reported that the risk of hypocalcemia is greater after surgery performed for malignant tumors (15,41). Dedivitis and cols. (24) observed in a prospective study no significant difference in postoperative hypocalcemia according to the indication for thyroidectomy. Similarly, the present study found no significant correlation between surgical indication and the incidence of postoperative hypocalcemia. 452

We observed no significant differences in the postoperative progression of calcium levels according to gender, due to the low number of male patients, although the data suggest the occurrence of a higher calcemia in men. The prevalence of symptoms was clearly related to the occurrence of low serum calcium levels, which is consistent with the calcium physiology. A significant greater rate of symptomatic hypocalcemia was observed in patients who did not receive calcium at the PO1, PO7, and PO90, while in other measurements we observed no significant differences, as shown in Table 5. The data suggest an effective prevention of symptomatic hypocalcemia with the use of oral calcium, which leads to safe and early hospital discharge, according to the literature on this topic (20-22). Outpatient thyroid surgery with same-day discharge is still avoided by the majority of the authors (42). Lo Gerfo (42) in 1998 defended the implementation of surgery on an outpatient basis, while Clark and Ituarte (42) opposed to it. Schwartz (42) concluded that outpatient surgery yields no financial benefit to the patients, despite reducing costs in 13-30%. A discharge on the first postoperative day, if associated with the prevention of hypoparathyroidism, is considered safe by various authors (42,43), and with marked reduction of costs, an advantage due to more efficient techniques of prevention and control of bleeding, pain, and postoperative hypocalcemia, resulting in a 32 to 56% reduction in hospital-associated costs (44). In a Colombian report, Sanabria and cols. (45) studied the use of prophylactic calcium and vitamin D, analyzing its cost-benefit and reporting its effectiveness. The total cost of the treatment remains below US$ 2 a day, while calcium measurements due to symptomatic hypocalcemia cost US$ 3.86 and the additional hospital daily fee costs US$ 33.12. In this report, we observed a significant difference in the incidence of hypocalcemia between the study groups after 180 days of surgery, suggesting the efficacy of the use of prophylactic oral calcium in the prevention of permanent hypoparathyroidism. In contrast, Pattou and cols. (2) in 1998 showed that low calcium levels have a high predictive value of hypoparathyroidism in patients not receiving calcium after thyroidectomy. In the present study, all patients were operated on by the same surgical team in Limeira (SĂŁo Paulo, Brazil), and coordinated by the main author. They all underwent standard thyroidectomy with careful Arch Endocrinol Metab. 2017;61/5

Prophylactic calcium after thyroidectomy

dissection of the parathyroid glands and peripheral ligation of the thyroid arteries near the thyroid capsule, which prevents hypoparathyroidism, as demonstrated by Thomusch and cols. (7) in an extensive multivariate analysis with 5846 consecutive patients. In conclusion, in this study, surgical indication showed no relationship with the incidence of postoperative hypocalcemia. We observed a lower incidence of permanent hypoparathyroidism after the use of the suggested regimen, prediction of postoperative hypocalcemia with early measurement of postoperative calcium, prediction of hypocalcemia after total thyroidectomy with preoperative calcium measurement, and ability to discharge the patient early and safely with a minimum of 24-hour of hospitalization. A careful surgical technique, including peripheral ligation of the thyroid arteries and meticulous dissection of the parathyroid glands, remains the best approach to preventing hypocalcemia after total thyroidectomy. The use of prophylactic oral calcium after total thyroidectomy significantly reduced the incidence of laboratory and symptomatic hypocalcemia and may be implemented in a simple, efficient, and safe manner. The strategy shown in this study may be reproduced in a viable and rational way.

10. Burnett HF, Mabry CD, Westbrook KC. Hypocalcemia after thyroidectomy: mechanisms and management. South Med J. 1977;70(9):1045-8.

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

20. Moore FD Jr. Oral calcium supplements to enhance early hospital discharge after bilateral surgical treatment of the thyroid gland or exploration of the parathyroid glands. J Am Coll Surg. 1994;178(1):11-6.

1. Kahky MP, Weber RS. Complications of surgery of the thyroid and parathyroid glands. Surg Clin North Am. 1993;73(2):307-21. 2. Pattou F, Combemale F, Fabre S, Carnaille B, Decoulx M, Wemeau JL, et al. Hypocalcemia following thyroid surgery: incidence and prediction of outcome. World J Surg. 1998;22(7):718-24. 3. Tredici P, Grosso E, Gibelli B, Massaro MA, Arigoni C, Tradati N. Identification of Patients at High Risk for Hypocalcemia After Total Thyroidectomy. Acta Otorhinolaryngol Ital. 2011;31(3):144-8. 4. Araújo Filho VJF, Machado MTAS, Sondermann A, Carlucci Jr D, Moysés RA, Ferraz AR. Hipocalcemia e hipoparatireoidismo clínico após tireoidectomia total. Rev Col Bras Cir. 2004;31(4):233-5. 5. Hojaij FC. Contribuição à Anatomia Cirúrgica das Glândulas Paratireóides. São Paulo, 1998. 166p. Tese (Doutorado) – Faculdade de Medicina da Universidade de São Paulo. 6. Millzner RJ. The occurrence of parathyroids on the anterior surface of the thyroid gland. J Am Assoc. 1927;88:1053-5. 7. Thomusch O, Machens A, Sekulla C, Ukkat J, Brauckhoff M, Dralle H. The impact of surgical technique on postoperative hypoparathyroidism in bilateral thyroid surgery: a multivariate analysis of 5846 consecutive patients. Surgery. 2003;133(2):180-5. 8. Demeester-Mirkine N, Hooghe L, Van Geertruyden J, de Maertelaer V. Hypocalcemia after thyroidectomy. Arch Surg. 1992;127:854. 9. See ACH, Soo KC. Hypocalcemia Following Thyroidectomy for Thyrotoxicosis. Br J Surg. 1997;84(1):95-7. Arch Endocrinol Metab. 2017;61/5

12. Hojaij F, Vanderlei F, Plopper C, Rodrigues CJ, Jácomo A, Cernea C, et al. Parathyroid gland anatomical distribution and relation to anthropometric and demographic parameters: a cadaveric study. Anat Sci Int. 2011;86(4):204-12. 13. Gardner E, Gray DJ, O’Rahilly R. Anatomia – Estudo Regional do Corpo Humano. 4th. Rio de Janeiro: Guanabara Koogan S.A., 1988, p. 679-80. 14. Shaha AR, Jaffe BM. Parathyroid preservation during thyroid surgery. Am J Otolaryngol. 1998;19(2):113-7. 15. Baldassare RL, Chang DC, Brumond KT, Bouvet M. Predictors of hypocalcemia after thyroidectomy: results from the nationwide inpatient sample. ISRN Surg. 2012;2012:838614. 16. Ghaheri BA, Liebler SL, Andersen PE, Schuff KG, Samuels MH, Klein RF, et al. Perioperative parathyroid hormone levels in thyroid surgery. Laryngoscope. 2006;116(4):518-21. 17. Marohn MR, LaCivita KA. Evaluation of total/near-total thyroidectomy in a short-stay hospitalization: safe and costeffective. Surgery. 1995;118(6):943-7. 18. Bentrem DJ, Rademaker A, Angelos P. Evaluation of serum calcium levels in predicting hypoparathyroidism after total/ near-total thyroidectomy or parathyroidectomy. Am Surg. 2001;67(3):249-51. 19. Chia SH, Weisman RA, Tieu D, Kelly C, Dillmann WH, Orloff LA. Prospective study of perioperative factors predicting hypocalcemia after thyroid and parathyroid surgery. Arch Otolaryngol Head Neck Surg. 2006;132(1):41-5.

21. Bellantone R, Lombardi CP, Raffaeli M, Boscherini M, Alesina PF, De Crea C, et al. Is routine supplementation therapy (calcium and vitamin D) useful after total thyroidectomy? Surgery. 2002;132(6):1109-12. 22. Tartaglia F, Giuliani A, Sgueglia M, Biancari F, Juvonen T, Campana FP. Randomized study on oral administration of calcitriol to prevent symptomatic hypocalcemia after total thyroidectomy. Am J Surg. 2005;190(3):424-9. 23. Docimo G, Tolone S, Pasquali D, Conzo G, D’Alessandro A, Casalino G, et al. Role of pre and post-operative oral calcium and vitamin D supplements in prevention of hypocalcemia after total thyroidectomy. G Chir. 2012;33(11-12):374-8. 24. Dedivitis RA, Pfuetzenreiter Jr EG, Nardi CEM, de Barbara ECD. Estudo prospectivo da queda da calcemia após cirurgia da tireoide. Rev Bras Cir Cabeça Pescoço. 2009;38(2):72-5. 25. Owen R. On the Anatomy of the Indian Rhinoceros. Trans Zool Soc Lond. 1862;4:31-58. 26. Thompson NW. The history of hyperparathyroidism. Acta Chir Scand. 1990;156:5-21. 27. Breimer L, Sourander P. The discovery of the parathyroid glands in 1880: triumph and tragedy of Ivar Sandström. Bull Hist Med. 1981 Winter;55(4):558-63. 28. Sandström I. On a New Gland in Man and several Mammals – Glandulae Parathyreoidae. Upsala Läkareförenings Förhandlinger. 1880;15:441-71. 29. Pool EH. Tetany Paratireopriva. Ann Surg. 1907;46:507-40.

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30. Halsted WS, Evans HM. The Parathyroid Glandules. Their Blood Supply and their Preservation in Operation upon the Thyroid Gland. Ann Surg. 1907;46(4):489-506.

39. Adams J, Andersen P, Everts E, Cohen J. Early postoperative calcium levels as predictors of hypocalcemia. Laryngoscope. 1998;108(12):1829-31.

31. Reeve T, Thompson NW. Complications of thyroid surgery: how to avoid them, how to manage them, and observations on their possible effect on the whole patient. World J Surg. 2000;24(8):971-5.

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32. Lahey FH. The transplantation of parathyroids in partial thyroidectomy. Surg Gynecol Obstet. 1926;62:508-9. 33. Murley RS, Peters PM. Inadvertent parathyroidectomy. Proc R Soc Med. 1961;54:487-9. 34. Croyle PH, Oloroyd JJ. Incidental parathyroidectomy during thyroid surgery. Ann Surg. 1978;44:559-63. 35. Loré JM, Pruet CW. Retrieval of the parathyroid glands during thyroidectomy. Head Neck Surg. 1983;5(3):268-9. 36. Shaha AR, Burnett C, Jaffe BM. Parathyroid autotransplantation during thyroid surgery. J Surg Oncol. 1991;46(1):21-4. 37. Nahas ZS, Farrag TY, Lin FR, Belin RM, Tufano RP. A safe and costeffective short hospital stay protocol to identify patients at low risk for the development of significant hypocalcemia after total thyroidectomy. Laryngoscope. 2006;116(6):906-10.

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Arch Endocrinol Metab. 2017;61/5

original article

Serum nesfatin-1 levels are decreased in pregnant women newly diagnosed with gestational diabetes Esra Nur Ademoglu1, Suheyla Gorar2, Muge Keskin3, Ayse Carlioglu4, Rifki Ucler5, Husamettin Erdamar6, Cavit Culha3, Yalcin Aral3

ABSTRACT Objective: To investigate serum nesfatin-1 levels at 24-28 weeks of pregnancy in women newly diagnosed with gestational diabetes and determine the association of nesfatin-1 with several metabolic parameters. Subjects and methods: Forty women newly diagnosed with gestational diabetes at 24-28 weeks of pregnancy and 30 healthy pregnant women matched in age and gestational week were included in this cross-sectional study. Serum nesfatin-1 levels were analyzed using ELISA, and the relationship between nesfatin-1 and several metabolic parameters were assessed. Results: Serum nesfatin-1 levels were found to be lower in women with gestational diabetes compared to the pregnant women in the control sample (p = 0.020). Multiple linear regression analysis revealed that nesfatin-1 was lower in participants with gestational diabetes independently from gestational age, BMI, HOMA-IR, fasting plasma glucose, and age. In correlation analysis, the only variable that was found to have a statistically significant correlation with nesfatin-1 was gestational age (p = 0.015, r = 0.30). Conclusion: Lower nesfatin-1 levels in women with gestational diabetes compared to the control group at 24-28 weeks of gestation draws attention to nesfatin-1 levels in gestational diabetes and motivates further research in this area. Arch Endocrinol Metab. 2017;61(5):455-9 Keywords Gestational diabetes; nesfatin-1; insulin resistance

1 Department of Endocrinology and Metabolism, Bolu University, Bolu, Turkey 2 Department of Endocrinology and Metabolism, Antalya Education and Research Hospital, Antalya, Turkey 3 Department of Endocrinology and Metabolism, Ankara Education and Research Hospital, Ankara, Turkey 4 Department of Endocrinology and Metabolism, Erzurum Education and Research Hospital, Erzurum, Turkey 5 Department of Endocrinology and Metabolism, Van Yuzuncu Yil University Faculty of Medicine, Van, Turkey 6 Department of Biochemistry, Turgut Ozal University Faculty of Medicine, Ankara, Turkey

Correspondence to: Suheyla Gorar Antalya Education and Research Hospital, Department of Endocrinology and Metabolism sgorar@hotmail.com Received on June/17/2016 Accepted on July/4/2016

INTRODUCTION

estational diabetes mellitus (GDM) is defined as glucose intolerance of variable degree that begins or is first recognized during pregnancy. Worldwide, the incidence of GDM is gradually increasing every year (1), varying from 3% to 14%. Hyperglycemia during pregnancy is associated with the development of preeclampsia, fetal macrosomia, emergency cesarean section, birth trauma, and neonatal hypoglycemia. GDM is important not only due to its complications but also its conversion rates to Type 2 diabetes mellitus (DM) as high as 2.6% to 70% over a period of 6 weeks to 28 years (2). Nesfatin-1 is a newly discovered hormone derived from nucleobindin-2 (NUCB2) and has been thought to be involved in the regulation of appetite and various metabolic conditions. It is prominently expressed in several regions of the hypothalamus and exists in the Arch Endocrinol Metab. 2017;61/5

general circulation. Nesfatin-1 is also produced in the peripheral tissues, including adipocytes, gastric mucosa, and pancreatic beta-cells of both humans and rats. One of the most important functions of nesfatin-1 is reducing food intake. It causes loss of appetite, less frequent hunger, and a sense of fullness (3-6). It has been demonstrated that pancreatic beta cells colocalize with nesfatin/NUCB2 in the islets of both mice and rats, indicating the possible involvement of nesfatin-1 in the regulation of insulin secretion from pancreatic beta cells (7). It has been shown that fasting nesfatin-1 levels were significantly lower in type 2 diabetic patients, but its effects on those who have newly been diagnosed with GDM are unknown (8). In this study, we aimed to examine serum nesfatin-1 concentrations and to investigate whether they have any correlation with insulin resistance, and to examine 455

DOI: 10.1590/2359-3997000000288

Decreased nesfatin-1 in gestational diabetes

other metabolic parameters in patients who had newly been diagnosed with gestational diabetes.

SUBJECTS AND METHODS Forty women newly diagnosed with GDM at 24-28 weeks of pregnancy and 30 healthy pregnant women matched in terms of age and gestational age was enrolled in the study. Diagnosis of GDM was based on 2015 American Diabetes Association recommendations (9). A two-step approach to screening with a 1-h 50-g glucose load test (GLT) followed by a 3-h 100-g OGTT for those who screened positive was used in examining all individuals. Pregnant women who had glucose levels higher than 7.8 mmol/l one hour after a 50-g glucose-screening test were referred to a 3-hour, 100-g oral glucose tolerance test and Carpenter/Coustan thresholds were used. GDM was diagnosed if two or more of the following plasma glucose levels were exceeded: fasting ≥ 5.1 mmol/l, 1-hour ≥ 10 mmol/l, 2-hour ≥ 8.6 mmol/l, and 3-hour ≥ 7.8 mmol/l (9). Exclusion criteria for the study were known pregestational diabetes, liver or renal dysfunction, acute or chronic inflammatory diseases, pre-eclampsia, use of anti-inflammatory drugs, and smoking. The body mass index was calculated for all participants [BMI: body weight (kg)/squared height (m²)]. The ethical committee of Ankara Education and Research Hospital approved the study. A written informed consent was obtained from all the participants. Blood sampling was conducted in the morning following one night of fasting and centrifuged immediately. Serum samples were stored at −80ºC until the time of analysis. Serum nesfatin-1 concentrations were analyzed with ELISA kits from USCN Life Science Instruments (Wuhan, China). In this assay system, the intra-assay and inter-assay coefficient of variation are always under 10%. This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific for human NES1 was pre-coated onto a microplate. A competitive inhibition reaction was launched between biotin-labeled human NES1 and unlabeled human NES1 (standards or samples) with the pre-coated antibody specific for human NES1. After incubation, the unbound conjugate was washed off. Next, avidin conjugated to horseradish peroxidase (HRP) was added to each microplate well and incubated. The amount of bound HRP conjugate was reverse proportional to the concentration of NES1 456

in the sample. After addition of the substrate solution, the intensity of color developed was reverse proportional to the concentration of NES1 in the sample. Serum glucose was assayed with a hexokinase method using an Olympus AU 2700 analyzer (Olympus UK, London, UK), and hemoglobin A1c (HbA1c) was measured with high-performance liquid chromatography (HLC723 G7 HPLC systems; Tosoh Corporation., Tokyo, Japan). Serum insulin was determined using a sandwich enzyme-linked immunosorbent assay (ELISA) (Dynex DSX full automatic ELISA analyzer, USA). Insulin resistance (IR) was calculated for each patient using the homeostasis model assessment insulin resistance index (HOMA-IR), fasting plasma glucose (mmol/l) × fasting insulin (mIU/mL)/22.5. In all statistical analyses, SPSS software version 15.0 was used. Distributions were tested for normality using the Shapiro-Wilk test. The normally distributed variables were analyzed with the Student’s t-test, and the data variables that did not show a normal distribution were compared with the Mann-Whitney U-test. Bivariate correlations between nesfatin-1 and several parameters were analyzed by Pearson correlation test for normally distributed variables. Spearman correlation test was used for nonnormally distributed variables. Multiple regression analysis was used to exclude the possible confounding effect of other variables on the result of each correlation analysis. P values less than 0.05 were considered statistically significant for all statistical analyses. The data for continuous variables were presented as mean ± standard deviation.

RESULTS Demographical characteristics and biochemical values of women with GDM and healthy controls are shown in Table 1. The participants with GDM and the control group were similar in terms of mean age (29.6 ± 5.3 vs. 27.8 ± 6.0 years, respectively). Serum fasting glucose, 1-h glucose after 50-g glucose screening test, 1-h glucose, 2-h glucose, and 3-h glucose after 100-g oral glucose tolerance test, HbA1c, and BMI were higher in women with GDM than those in the control group (p < 0.05). Fasting insulin and HOMA-IR were similar between the two groups. Serum nesfatin-1 concentrations were significantly found to be lower in the participants with GDM compared to the controls (7.9 ± 2.8 vs. 11.2 ± 7.7 ng/mL, respectively, p = 0.020, Table 1). Arch Endocrinol Metab. 2017;61/5

Decreased nesfatin-1 in gestational diabetes

Table 1. Demographical characteristics and biochemical values of controls and women with gestational diabetes

Standardized coefficient (β)

p value

Gestational age

0.021

0.553

Age

0.014

0.197

BMI

0.032

0.048a

0.018a

Nesfatin-1

-0.026

0.014a

62.4 ± 38.2

0.441

HOMA-IR

-0.038

0.423

2.0 ± 1.7

0.074

Fasting plasma glucose

0.008

0.054

Controls (n = 30)

Gestational diabetes (n = 40)

p value

Age, year

27.8 ± 6.0

29.6 ± 5.3

0.242

Gestational age (week)

25.9 ± 1.5

26.2 ± 1.8

0.443

BMI (kg/m2)

28.2 ± 1.5

31.0 ± 5.5

Fasting insulin (pmol/l)

55.2 ± 38.0 1.4 ± 0.7

HOMA-IR

Table 2. Multiple linear regression analysis including nesfatin-1, gestational age, BMI, age, fasting plasma glucose, and HOMA-IR

50 g OGTT

β coefficients and p values are given.

Glucose 1 h (mmol/l)

6.7 ± 1.7

10.3 ± 2.2

0.000a

Dependent variable; gestational diabetes. Independent variables; nesfatin-1, gestational age, BMI (body mass index), age, fasting plasma glucose, and HOMA-IR (homeostasis model assessment insulin resistance index).

100 g OGTT Glucose 0 h (mmol/l)

4.0 ± 0.4

4.8 ± 0.8

0.001a

Glucose 1 h (mmol/l)

9.1 ± 0.4

11 ± 1.4

0.000a

Glucose 2 h (mmol/l)

7.6 ± 1.9

9.4 ± 2.3

0.000a

Glucose 3 h (mmol/l)

5.7 ± 0.9

6.2 ± 2.2

0.004a

(%)

4.8 ± 1.0

5.9 ± 1.1

Nesfatin-1 (ng/mL)

11.2 ± 7.7

7.9 ± 2.8

HbA1c (mmol/mol)

Table 3. The correlation analyses of nesfatin-1 with some parameters in women with gestational diabetes

0.020a

BMI: body mass index; HOMA-IR: homeostasis model assessment insulin resistance index. The difference between controls and gestational diabetes was statistically significant (p < 0.05).

50,00 *

0.19

0.09

BMI

0.46

0.12

Fasting insulin

0.14

0.25

Fasting blood glucose

0.80

-0.04

HOMA-IR

0.22

-0.21

1-h glucose (50 g OGTT)

0.18

-0.23

HbA1c

0.308

-0.13

Gestational age

0.015a

0.30

BMI: body mass index; HOMA-IR: homeostasis model assessment index. a

30,00 20,00

p value Age

The correlation between nesfatin 1 and gestational age was statistically significant in

gestational diabetes (p < 0.05).

DISCUSSION

10,00 ,00 Controls

Gestational diabetes

Figure 1. Distribution of serum nesfatin-1 concentration in controls and in gestational diabetes. 97 x 54 mm (300 x 300 DPI).

Multiple linear regression analysis revealed that nesfatin-1 was lower in women with GDM independent of gestational age, BMI, age, fasting plasma glucose, and HOMA-IR (Table 2). Nesfatin-1 levels had a statistically significant positive correlation with gestational age (r = 0.30, p = 0.015, Table 3). There was not any correlation between nesfatin-1 and BMI, HOMA-IR, fasting glucose, 1h-glucose after 50-g glucose screening test, and HbA1c (p > 0.05). Arch Endocrinol Metab. 2017;61/5

In the present study, we demonstrated that serum nesfatin-1 levels at 24-28 weeks of gestation are significantly lower in women newly diagnosed with GDM compared to healthy pregnant women. In 2010, Aydın S. first described the presence of nesfatin-1 in breast milk (10). In that study, nesfatin-1 was investigated in serum, milk, and colostrum of lactating women who were diagnosed with GDM and in healthy lactating women. Nesfatin-1 was demonstrated to be lower in the serum of lactating women diagnosed with GDM than that of the control participants. It was suggested that the breast tissue was likely to be a source of nesfatin-1 just like the central nervous system, adipocytes, gastric endocrine cells, and pancreatic beta cells. It was also pointed out that this may thus be important for growth, energy regulation, and maturation of the gastrointestinal system in neonates. 457

Nesfatin-1 (ng/mL)

40,00

Statistically significant (p < 0.05).

Decreased nesfatin-1 in gestational diabetes

Clinical significance of nesfatin-1 in various metabolic diseases like obesity, Type 2 DM, and insulin resistance was demonstrated in several studies in the literature, but its effects on GDM are unknown. The literature reports only one study about the nesfatin-1 levels in gestational diabetes. In this study, Aslan and cols. assessed both serum and cord blood apelin and nesfatin-1 levels in pregnant women with GDM and in healthy pregnant women (11). Cord blood nesfatin-1 and apelin-36 levels were comparable between women with GDM and controls, serum apelin-36 concentrations were found to be higher, and serum nesfatin-1 concentrations were found to be lower in women with GDM compared to controls. The designs differ between our study and that study in some aspects, including the time of nesfatin-1 sampling and patient selection. In the study of Aslan and cols., the study group was composed of pregnant women who were being followed with diagnosis of GDM and whose blood glucose levels were strictly controlled, whereas our study included pregnant women who were newly diagnosed with GDM so that none of them were on restricted diets or using insulin therapy. Additionally, Aslan and cols. did not point out whether the women diagnosed with GDM were using an insulin regimen or only following a specific diet. They measured nesfatin-1 levels at the time of birth before placenta delivery in patients with GDM and in controls. Differently, we measured nesfatin-1 at 2428 weeks of pregnancy in control and study groups. In 2012, Boutsikou and cols. measured cord blood nesfatin-1 and insulin concentrations in 40 large (9 born from diabetic and 31 from non-diabetic mothers) and 20 appropriate for gestational age pregnancies (12). Cord blood nesfatin-1 concentrations were shown to be significantly lower in large gestational age pregnancies compared to appropriate for gestational age pregnancies. Furthermore, fetal nesfatin-1 concentrations were found to be elevated in infants born from mothers with GDM compared to those born from mothers without GDM. Serum nesfatin-1 levels in obesity and Type 2 DM reported in the literature was rather controversial. In 2010, Tsuchiya and cols. reported that fasting concentrations of nesfatin-1 were significantly lower in persons with high BMI compared to non-obese participants (13). However, in contrast to this study, Ramanjaneya and cols. found increased nesfatin-1 levels in obese states in both rodents and humans 458

in their study (14). In a recent study, Gonzalez and cols. demonstrated that nesfatin-1/NUCB2 mRNA expression in the pancreatic islets is markedly increased in Goto-Kakizaki rats, a model with Type 2 DM, characterized by impaired insulin secretion and visceral fat accumulation (15). Involvement of nesfatin-1 in the regulation of insulin secretion remains largely unknown. In 2013, Nakata and cols. reported that nesfatin-1 exerts its metabolic effects partly via promoting insulin release by Ca+2 influx through L-Type Ca+2 channels independently of protein kinase A (PKA) and phospholipase A2 (PLA2) so that dysregulation of nesfatin-1 might be implicated in metabolic disorders, particularly in Type 2 DM (16). In 2010, Su and cols. reported that intravenous injection of nesfatin-1 suppresses hyperglycemia in ob/ ob mice by enhancing insulin secretion (17). In another study, Li and cols. demonstrated that fasting nesfatin-1 levels were found to be decreased in persons with Type 2 DM compared to healthy participants and persons with Type 1 DM (8). Additionally, it was pointed out that although this result is unclear, nesfatin-1 may be a causal factor in diabetic hyperphagia. In 2012, in contrast to the study of Li and cols., Zhang and cols. demonstrated elevated levels of nesfatin-1 in participants with Type 2 DM and with impaired glucose tolerance (IGT) compared to controls (18). It was shown that plasma nesfatin-1 was positively correlated with BMI, HbA1c, fasting blood glucose, fasting plasma insulin, and HOMA-IR. In a recent study, Ding and cols. revealed that Type 2 DM patients with peripheral arterial disease (PAD) exhibited marked lower serum nesfatin-1 concentrations than those without PAD. They hypothesized that serum nesfatin-1 levels were inversely correlated with the development and severity of PAD in Type 2 DM patients (19). Previous studies reveal conflicting data about the correlations of nesfatin-1 with metabolic parameters. For instance, Zhang and cols. showed that nesfatin-1 was positively correlated with BMI, HbA1c, fasting blood glucose, fasting insulin, and HOMA-IR in participants with newly diagnosed Type 2 DM (18). In contrast to that study, Deniz and cols. revealed a negative correlation between nesfatin-1 and BMI, fasting blood glucose, and HOMA-IR in patients with polycystic ovary syndrome, an endocrine disorder commonly presenting with obesity and insulin resistance as well as hyperandrogenemia and hirsutism (20). Ding and cols. also revealed a negative correlation between nesfatin-1 and BMI in their study Arch Endocrinol Metab. 2017;61/5

Decreased nesfatin-1 in gestational diabetes

(19). In the present study, we did not find any correlation between nesfatin-1 and fasting glucose, 1-h glucose after 50-g glucose screening test, 1-h, 2-h, and 3-h glucose after 100-g oral glucose tolerance test, HbA1c, HOMA-IR, and BMI. We found a significant positive correlation between nesfatin-1 and gestational age, contrary to the results of the study by Aslan and cols., as they had observed a negative correlation between nesfatin-1 and gestational age (11). Because few data are currently present in the literature, the circulating levels of nesfatin-1 and its correlation with several parameters in GDM are not well-known and might require further investigation. Measurement of serum nesfatin-1 consecutively during the gestation may provide more evidence about the role of nesfatin-1 in GDM in future research. In conclusion, we have demonstrated for the first time reduced nesfatin-1 levels at 24-28 weeks of pregnancy in women newly diagnosed with gestational diabetes. Although the significance of this result is yet unclear, it is important because it draws attention to nesfatin-1 levels in GDM and may shed light on further research in this area. Funding statement and any grants or fellowships: nothing. Disclosure: no potential conflict of interest relevant to this article was reported.

REFERENCES 1. Metzger BE, Gabbe SG, Persson B, Buchanan TA, Catalano PA, Damm P, et al. International association of diabetes and pregnancy study group recommendations on the diagnosis and classification of hyperglycemia in pregnancy. Diabetes Care. 2010;33:676-82. 2. Kim C, Newton KM, Knopp RH. Gestational diabetes and the incidence of type-2 diabetes: a systematic review. Diabetes Care. 2002;25:1862-8. 3. Oh-I S, Shimizu H, Satoh T, Okada S, Adachi S, Inoue K, et al. Identification of nesfatin-1 as a satiety molecule in the hypothalamus. Nature. 2006;443:709-12.

5. Foo KS, Brauner H, Ostenson CG, Broberger C. Nucleobindin-2/ nesfatin in the endocrine pancreas: distribution and relationship to glycaemic state. J Endocrinol. 2010;204:255-63. 6. Stengel A, Tache Y. Nesfatin-1–role as possible new potent regulator of food intake. Regul Pept. 2010;163:18-23. 7. Gonzalez R, Tiwari A, Unniappan S. Pancreatic beta cells colocalize insulin and pronesfatin immunoreactivity in rodents. Biochem Biophys Res Commun. 2009;381:643-8. 8. Li QC, Wang HY, Chen X, Guan HZ, Jiang ZY. Fasting plasma levels of nesfatin-1 in patients with Type 1 and Type 2 diabetes mellitus and the nutrient-related fluctuation of nesfatin-1 level in normal humans. Regul Pept. 2010;159:72-7. 9. Standards of medical care in diabetes--2015: summary of revisions. Diabetes Care. 2015;38 Suppl:S4. 10. Aydın S. The presence of the peptides apelin, ghrelin and nesfatin-1 in the human breast milk, and the lowering of their levels in patients with gestational diabetes mellitus. Peptides. 2010;31:2236-40. 11. Aslan M, Celik O, Celik N, Turkcuoglu I, Yılmaz E, Karaer A, et al. Cord blood nesfatin-1 and apelin-36 levels in gestational diabetes mellitus. Endocrine. 2012;41:424-9. 12. Boutsikou T, Briana DD, Boutsikou M, Kafalidis G, Piatopoulou D, Baka S, et al. Cord blood nesfatin-1 in large for gestational age pregnancies. Cytokine. 2013;61:591-4. 13. Tsuchiya T, Shimizu H, Yamada M, Osaki A, Oh-I S, Ariyama Y, et al. Fasting concentrations of nesfatin-1 are negatively correlated with body mass index in non-obese males. Clin Endocrinol. 2010;73:484-90. 14. Ramanjaneya M, Chen J, Brown JE, Hallschmid M, Patel S, Kern W, et al. Identification of nesfatin-1 in human and murine adipose tissue: a novel depot-specific adipokine with increased levels in obesity. Endocrinology. 2010;151:3169-80. 15. Gonzalez R, Reingold BK, Gao X, Gaidhu MP, Tsushima RG, Unniappan S. Nesfatin-1 exerts a direct, glucose-dependent insulinotropic action on mouse islet β- and MIN6 cells. J Endocrinol. 2011;208:R9-16. 16. Nakata M, Manaka K, Yamamoto S, Mori M, Yada T. Nesfatin-1 enhances glucose-induced insulin secretion by promoting Ca2+ influx through L-Type channels in Mouse islet β-cells. Endocrine J. 2011;58:305-13. 17. Su Y, Zhang J, Tang Y, Bi F, Liu JN. The novel function of nesfatin-1: anti-hyperglycemia. Biochem Biophys Res Commun. 2010;391:1039-42. 18. Zhang Z, Li L,Yang M, Liu H, Boden G,Yang G, et al. Increased Plasma Levels of Nesfatin-1 in Patients with Newly Diagnosed Type 2 Diabetes Mellitus. Exp Clin Endocrinol Diabetes. 2012;120:91-5. 19. Ding S, Qu W, Dang S, Xie X, Xu J, Wang Y, et al. Serum nesfatin-1 is reduced in type 2 diabetes mellitus patients with peripheral arterial disease. Med Sci Monit. 2015;21:987-91. 20. Deniz R, Gurates B, Aydin S, Celik H, Sahin I, Baykus Y, et al. Nesfatin-1 and other hormone alterations in polycystic ovary syndrome. Endocrine. 2012;42:694-9.

4. Stengel A, Goebel M, Wang L, Rivier J, Kobelt P, Mönnikes H, et al. Central nesfatin-1 reduces dark-phase food intake and gastric

emptying in rats: differential role of corticotropin-releasing factor 2 receptor. Endocrinology. 2009;150:491-9.

Arch Endocrinol Metab. 2017;61/5

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original article

Novel immunoassay for TSH measurement in rats

1 Laboratório de Endocrinologia Molecular e Translacional, Divisão de Endocrinologia, Departamento de Medicina, Universidade Federal de São Paulo (Unifesp), São Paulo, SP, Brasil 2 Divisão de Biologia Molecular, Departamento de Bioquímica, Escola Paulista de Medicina, Unifesp, São Paulo, SP, Brasil 3 Grupo Fleury, São Paulo, SP, Brasil 4 Departamento de Ciências Biológicas, Unifesp, Diadema, SP, Brasil

* These authors contributed equally to this work. Correspondence to: Thalita G. Alves Laboratory of Molecular and Translational Endocrinology (LEMT/Unifesp) Rua Pedro de Toledo, 669, 11º andar 04039-032 – São Paulo, SP, Brasil tha.alves@gmail.com Received on Oct/06/2016 Accepted on June/11/2017 DOI: 10.1590/2359-3997000000293

Thalita G. Alves1,2*, Maria Clara de C. Melo1,3*, Teresa S. Kasamatsu1, Kelen C. Oliveira1, Janaina Sena de Souza1, Rodrigo Rodrigues da Conceição1, Gisele Giannocco1,4, Magnus R. Dias-da-Silva1, Maria Izabel Chiamolera1,3, José Gilberto Vieira1,3

ABSTRACT Measuring thyroid hormones is an important aspect for the study of metabolism and for monitoring diseases in both human and animal models. The traditional method for hormone measurement in rats is the radioimmunoassay (RIA). However, the RIA is associated with some practical disadvantages, including the use of radioactive material, the need for specialized equipment and expert staff, the short shelf-life of kits according to the half-life of the radioisotope and high costs. The objective of this study was to develop a new cost-effective method for measuring TSH levels in rats that avoids the use of radioactive material. We developed an in-house competitive immunoassay using a reference standard, polyclonal antibody produced in rabbits and biotinylated antigen. This method was tested in 64 Wistar rats that were divided into a control group (n = 41) and a group with hypothyroidism (n = 23). Our assay demonstrated an analytical sensitivity of 0.24 ng/mL (n = 12) and an intra-assay coefficient of variation (CV) of 8.9% for sera with TSH levels of 1.5 ng/mL and 13.2% for sera with TSH levels of 17.5 ng/mL (n = 14). The inter-assay CV was 13.5% for sera with TSH levels of 1.4 ng/ mL and 14.5% for TSH levels of 18.2 ng/mL (n = 5). The analysis of mean TSH levels in control rats (5.06 ± 0.5701) and hypothyroid rats (51.09 ± 5.136) revealed a statistically significant difference (p < 0.001) between the groups. This method showed good sensitivity, can be automated and is low-cost compared with RIA. Our method offers a viable alternative for TSH measurement in rats. Arch Endocrinol Metab. 2017;61(5):460-3 Keywords Immunoassay; rats; TSH

INTRODUCTION

ormone measurement is an important component for the study of metabolism, analysis of diseases and as a parameter of health assessments. Of the different hormone assays, immunological assays have become standard due to several advantages: they offer great specificity and potentially high sensitivity and present with ease of use and wide-ranging applications (1). Immunological assays are largely used for measuring biologically active compounds present in low concentrations, such as hormones, proteins, drugs and microorganisms. In this context, the most commonly used techniques are radioactively labeled competitive and non-competitive immunoassays (RIA, IRMA), which are highly sensitive and precise but are also associated with several disadvantages. Among the drawbacks, the use of radioactive tracers is of most concern because they present a health hazard, require special attention for handling, well-trained staff

460

and well-defined waste storage. Moreover, because radioisotopes have a limited half-life, kits have limited shelf lives, and radioactive counting can be timeconsuming and requires expensive instrumentation. Because of these issues, alternative immunoassays (competitive or non-competitive) have been developed using alternative labels such as enzymes (EIA, ELISA), luminescent compounds, fluorescent probes (IFMA, FIA) and metals. Assays that use alternative tracers offer similar or better sensitivity than that of IRMA and RIA. Importantly, avoidance of radiation is a major advantage. Several immunoassays for measuring hormones have been described, and the most commonly used technique for the measurement of rat serum is the radioimmunoassay (RIA), which is associated with the disadvantages described above (2). In addition, commercial kits are costly, and their shelf life depends on the half-life of the radioisotope. Therefore, Arch Endocrinol Metab. 2017;61/5

New rat TSH fluoroimmunoassay

MATERIALS AND METHODS Assay development We developed an in-house competitive immunoassay using rat TSH extracted from pituitary gland as a standard, polyclonal antibody produced in rabbits against rat TSH and biotinylated TSH from the same source (National Institute of Diabetes & Digestive & Kidney Diseases, NIDDK). Rat thyroid-stimulating hormone was prepared at a concentration of 50 µg/mL, after which it was biotinylated using the EZ-link-SulfoNHS-LC-Biotin kit according to the manufacturer’s protocol (Thermo Scientific). A six-value curve was constructed from serial dilution of standard rat TSH in bovine fetal serum at the following concentrations: 100, 25, 6.25, 1.56, 0.39 and 0 ng/mL. The standards, rat sera used as controls and test samples were incubated with a rabbit polyclonal antibody provided by NIDDK. After 24 h of incubation at 4°C, biotinylated TSH was added to tubes at a dilution of 1:1000. After another 24 h incubation at 4°C, samples were transferred to a microtiter plate (Fluoronunc, Nunc, Roskilde, Denmark) that was previously adsorbed with an antirabbit IgG monoclonal antibody (D4P4) produced by our laboratory (monoclonal antibody, 2 µg/well). After a 4-h incubation at room temperature and subsequent washing (Tris-HCl 50 mM with 0.5% BSA and 0.1% gamma-globulin), a solution of streptavidin-europium was added (200 µL/well). Finally, after incubation for 30 min at room temperature, plate was washed 12 times with buffer, and an enhancement solution was added (200 µL/well, Delfia Enhancement Solution, Perkin-Elmer, Turku, Finland). The plate was then read by time-synchronized fluorometry in a Victor3 timeresolved fluorometer (Perkin-Elmer, Turku, Finland). The amount of analyte was calculated based on the standard curve, and the results are expressed in ng/mL.

Animals To test our method, we measured the serum TSH levels of 64 adult male Wistar rats (200-250 g) from the animal facility of the Institute of Pharmacology of Unifesp (Infar). Rats were kept under standard conditions for temperature (25 ± 1°C) and light/dark Arch Endocrinol Metab. 2017;61/5

cycle (12/12 hours per day). Drinking water and food were provided ad libitum. Animals were divided into two experimental groups: a control group and a group with hypothyroidism. The hypothyroidism group consisted of 23 animals that were subjected to total thyroidectomy followed by methimazole treatment for 20 days. The control group consisted of 41 rats subjected to sham surgery.

Statistical analysis All results are presented as mean values ± standard error of the mean. Statistical analysis was performed in Graph Pad Prism® version 5 software for Windows. First, the Shapiro-Wilk normality test was performed to verify normality of the distribution of the two sample groups. Non-parametric distribution was observed in the two groups, and thus a Mann-Whitney test was used to compare control and hypothyroidism groups. For all analyses, a significance threshold of p < 0.05 was assumed.

Ethics All procedures were performed in accordance with the Brazilian College of Animal Experimentation and were approved by the Bioethical Commission of the Universidade Federal de São Paulo (Unifesp) (protocol number 5097101316).

RESULTS Analytical sensitivity was determined using TSH stripped fetal bovine serum (FBS). FBS was measured multiple times, from which we calculated mean and standard deviation. Sensitivity was defined by the concentration corresponding to the mean - 2 standard deviations of the latter measurements. A 95% confidence interval was obtained, and the limit was defined by doubling the standard deviation. The new assay showed sensitivity of 0.24 ng/mL (n = 12). Intra-assay CV was 8.9% for sera with TSH levels of 1.5 ng/mL and 13.2% for sera with TSH levels of 17.5 ng/mL (n = 14). Inter-assay CV was 13.5% for sera with TSH levels of 1.4 ng/mL and 14.5% for sera with TSH levels of 18.2 ng/mL (n = 5). The values in the control rats ranged between 0.81 and 19.50 ng/mL, whereas the values in hypothyroid rats ranged between 15 and > 100 ng/mL. 461

development of new assays that do not use radioisotopes is needed.

New rat TSH fluoroimmunoassay

Linearity of our assay was tested by serial dilution of a hypothyroid rat serum (36 ng/mL), which produced a value of R2 = 0.97. The analysis of mean TSH obtained from control rat sera (5.06 ± 0.5701) and hypothyroid rat sera (51.09 ± 5.136) showed statistically significant differences (p < 0.001) between the groups. To verify cross-reaction with the antibody used in our test, sera from different species were tested: FBS, mouse, rabbit, horse and primate. Rabbit serum interferes significantly in the assay, although sheep and horse also have some interference, generating a lower reading signal. Then, we used standard TSH to create curves using these different matrices, to identify if assay sensitivity could be improved. Curves in FBS and in buffer with 5% BSA are the most sensitive, although FBS remains the best matrix since, in buffer, the curve is slightly shifted to the right, indicating lower analytical sensitivity. Horse and sheep sera are much less sensitive (data not shown). As a recovery test we used three sera samples with different TSH concentrations and added 50 ng of the standard in the two matrices.

Figure 1. Mean TSH levels (± SEM) measured using a novel fluoroimmunoassay in 41 control and 23 rats with hypothyroidism. Dashed line indicates the lower limit of detection of the assay.

DISCUSSION Measurement of TSH levels is useful for clinical evaluation of human patients, as small changes in free thyroid hormone levels lead to greater changes in TSH levels. Serum TSH is considered the single most reliable diagnostic test for all common forms of hypothyroidism and hyperthyroidism. Animal research has played a vital part in medical and biomedical research and has led to important scientific discoveries in many fields, 462

including endocrinology (3). Therefore, more sensitive and specific tests in animal field could increase the frequency of testing and, consequently, detection of thyroid conditions, with the possibility of detecting degrees of thyroid dysfunction in research animals. In this study, we developed a new solid-phase fluorometric immunoassay for measuring TSH in rats that does not use radioactive material. The lack of radioactive material offers a considerable advantage because handling radioactive material presents a health hazard for researchers, and the assay shelf life is not constrained by the half-life of the radioisotope. Our method shows good sensitivity, can be automated, and with its low operational cost offers considerable advantages compared with traditional RIA methods. Additionally, as NIDDK reagents are the same as those used in the traditional RIA, following recommendations in ATA Guidelines for thyroid hormone investigation in rodents and cell models (2), this assay can be compared with available data in the literature. However, our method has some limitations. In recovery test, all samples recovered above the expected (data not shown). One reason that should be taken in consideration is the fact that the standard originates from rat pituitary. Unfortunately, there is no source of rat serum TSH to use as the standard for assembling the immunoassay, forcing us to use pituitary TSH. The antibody used in the assay was also produced against pituitary TSH, and, probably, recognizing it more specifically and with greater affinity than serum TSH. For this reason, the assay shows high recovery. This is a limitation of experimental design, since all the assay reagents are derived from pituitary TSH, that has a different immunogenic structure than rat serum TSH. More studies measuring TSH levels in rats with hyperthyroidism should be conducted. Nevertheless, the method described here offers an effective alternative for TSH measurement in rats. Acknowledgements: the authors are grateful to Angela Faria, Gilberto Furusawa, Ilda Kunii and Teresa Kasamatsu for administrative and technical support. We also thank Dr. A. F. Parlow for his contributions to the hormone measurements over the course of the several studies of our group and for providing reliable reagents. Funding information: Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp) Grant number 2013/26851-7 (to Chiamolera, MI). Disclosure: no potential conflict of interest relevant to this article was reported. Arch Endocrinol Metab. 2017;61/5

New rat TSH fluoroimmunoassay

REFERENCES 1. HemmilĂ¤ I. Fluoroimmunoassays and immunofluorometric assays. Clin Chem. 1985;31(3):359-70.

2. Bianco AC, Anderson G, Forrest D, Galton VA, Gereben B, Kim BW, et al. American Thyroid Association Guide to investigating thyroid hormone economy and action in rodent and cell models. Thyroid. 2014;24(1):88-168.

3. Bahn Chair RS, Burch HB, Cooper DS, Garber JR, Greenlee MC, Klein I, et al. Hyperthyroidism and other causes of thyrotoxicosis: management guidelines of the American Thyroid Association and American Association of Clinical Endocrinologists. Thyroid. 2011;21(6):593-646.

Arch Endocrinol Metab. 2017;61/5

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original article

Serum ghrelin levels in papillary thyroid carcinoma Bekir Ucan1, Mustafa Sahin2, Muhammed Kizilgul1, Mustafa Ozbek1, Seyda Ozdemir3, Mustafa CalÄąskan1, Erman Cakal1

ABSTRACT SBU Diskapi Yildirim Beyazit Training and Research Hospital, Department of Endocrinology and Metabolism, Ankara, Turkey 2 Ankara University, School of Medicine, Department of Endocrinology and Metabolism, Ankara, Turkey 3 SBU Diskapi Yildirim Beyazit Training and Research Hospital, Department of Biochemistry, Ankara, Turkey 1

Correspondence to: Bekir Ucan Diskapi Hospital, I-rfan Bastug Caddesi, Ankara uzm.dr.bekir@hotmail.com Received on Aug/17//2016 Accepted on May/15/2017 DOI: 10.1590/2359-3997000000290

Objective: Ghrelin plays a role in several processes of cancer progression, and numerous cancer types express ghrelin and its receptor. We aimed to investigate serum levels of ghrelin in patients with papillary thyroid carcinoma (PTC) and its association with the prognostic factors in PTC. Materials and methods: We enrolled 54 patients with thyroid cancer (7 male, 47 female) and 24 healthy controls (6 male, 18 female) in the study. We compared demographic, anthropometric, and biochemical data, and serum ghrelin levels between the groups. Serum ghrelin levels were measured using as enzyme-linked immunosorbent assay. Results: Ghrelin levels were similar between the groups, but plasma ghrelin levels were significantly higher in tumors larger than 1 cm diameter compared with papillary microcarcinomas. Serum ghrelin levels also correlated with tumor size (r = 0.499; p < 0.001). Body mass index, thyroid-stimulating hormone, and HOMA-IR levels were similar between the groups. There were no statistically significant differences regarding average age and other prognostic parameters including lymph node invasion, capsule invasion, multifocality and surgical border invasion between patients with microcarcinoma and tumors larger than 1 cm. Conclusion: In our study, no significant difference in serum ghrelin levels was determined between patients with papillary thyroid cancer and healthy controls however, serum ghrelin levels were higher in tumors larger than 1 cm compared to in those with thyroid papillary microcarcinoma. Arch Endocrinol Metab. 2017;61(5):464-9 Keywords Papillary thyroid carcinoma, ghrelin, tumor size

INTRODUCTION

hrelin, a 28-amino acid peptide hormone, was discovered in 1999 as the endogenous ligand for the growth hormone secretagogue/ghrelin receptor (GHS-R) (1). Ghrelin has well-documented systemic actions including stimulation of gastrointestinal system motility; gastric acid secretion; regulation of sleep, taste sensation, and reward-seeking behavior; modulation of glucose metabolism; suppression of brown fat thermogenesis; regulation of stress and anxiety; prevention of muscle atrophy; and improvement of cardiovascular functions such as vasodilatation and cardiac contractility (2). Ghrelin was implicated in several processes of cancer progression including cell proliferation, cell migration and invasion, angiogenesis, and apoptosis, probably via an autocrine/paracrine mechanism. Pituitary adenomas, gut carcinoids, endocrine pancreatic, ovarian, endometrial, testicular, adrenocortical, prostate, renal, lung, and breast cancer were demonstrated to express ghrelin. Some reports demonstrated that ghrelin may have an inhibitory

464

effect in the proliferation of some cancer types, including thyroid, prostate, and breast cancer, and small cell lung carcinoma (3). Thyroid follicular/ parafollicular and thyroid carcinoma cells also express ghrelin (4). The effect of thyroid hormone status on serum ghrelin concentrations was investigated in some studies. Malandrino and cols. observed that plasma ghrelin levels were significantly higher in patients with Hashimotoâ&#x20AC;&#x2122;s thyroiditis after the levothyroxine treatment maintaining euthyroid state (5). Ruchala and cols. demonstrated ghrelin levels varied depending on hyperthyroid, hypothyroid or euthyroid state in the same patients (6). Biyikli and cols. demonstrated lower ghrelin levels in patients with euthyroid hashimoto thyroiditis (7). Circulating ghrelin levels were demonstrated as higher in a range of cancer types including colon cancer (8), prostate carcinoma (9), ovarian carcinoma (10), and hepatocellular carcinoma (11) however, no studies have evaluated serum ghrelin levels in papillary thyroid carcinoma. We aimed to investigate serum levels of ghrelin Arch Endocrinol Metab. 2017;61/5

Ghrelin levels in thyroid cancer

MATERIALS AND METHODS Study population Fifty-four patients (7 male, 47 female) with PTC and 24 age-, sex-, and body mass index (BMI)-matched controls (6 male, 18 female) were included in the study. The mean age of the patients was 42.4 ± 10.1 years and the control group was 42.5 ± 8.9 years. Ethics committee approval and written informed consent of participants were obtained before the study. Histopathologic documents confirmed the diagnosis of PTC. Blood was collected from patients with thyroid cancer before surgery and from healthy individuals as the controls. Subjects with other cancers and autoimmune disorders, hypertension, hepatic or renal dysfunction, diabetes mellitus, or any other inflammatory or medical condition were excluded.

Clinical, biochemical, and hormonal measurements Weight, height, waist circumference (WC), hip circumference (HC), and systolic and diastolic blood pressure (BP) were measured. WC was determined by measuring the narrowest point between the costal margin and iliac crest at the end of a normal expiration. BMI was calculated as weight (kg)/height (m)2. After an 8-12 hour overnight fast, venipuncture was performed between 8:00 a.m. and 9:00 a.m. and blood samples were collected into plain tubes. Blood samples were centrifuged at 2500 g for 15 min within 30 min of collection, and serum samples were stored at –80°C until required for analysis. Serum levels of glucose, insulin, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), thyroid-stimulating hormone (TSH), free T4 (fT4), antithyroglobulin (Anti-Tg) and antithyroid peroxidase (Anti-TPO) were also measured. The normal range for fT4 was 0.74-1.52 ng/dL. TSH levels ranging between 0.55-4.78 mIU/L was considered normal and normal ranges for anti-Tg and anti-TPO are 0-40 IU/mL and 0-35 IU/mL, respectively.

Determination of ghrelin in plasma Blood samples were collected into EDTA-containing tubes and then aprotinin (Phoenix Pharmaceuticals, California, USA) was added immediately. The blood was centrifuged at 1600 x g for 15 minutes; after Arch Endocrinol Metab. 2017;61/5

separation of the plasma, it was stored at −80°C until the ghrelin assessment. Measurements of ghrelin were performed in an EPOCH system (BioTek Instruments, Inc, USA) using a com mercially available enzymelinked immunosorbent assay (ELISA) kit (Phoenix Pharmaceuticals, California, USA) following the manufacturers’ instructions. The test range of the ghrelin ELISA kit was 0-100 ng/mL. The specimens were run together in the same experiment.

Statistical analysis The descriptive values for the data obtained are expressed as mean ± SD, numbers, and percentage frequencies. Normality was tested using the Kolmogorov-Smirnov test. In addition, the differences between the groups were analyzed using the appropriate Chi-square test. The relationships between individual numeric properties were reviewed using Pearson’s correlation analysis in the patient and control groups. p ≤ 0.05 was used as the level of statistical significance and IBM SPSS 20.0 was used to process the calculations.

RESULTS Fifty-four patients with PTC (mean age: 42.4 ± 10.1 years) and 24 age-, sex-, and BMI matched controls (mean age: 42.5 ± 8.9 years) were enrolled in the study. BMI, WC, TSH levels and insulin resistance (HOMA-IR) were similar between the groups. There was no statistically significant difference in ghrelin levels between patients and controls (p > 0.05) (Table 1). The mean 25-hydroxyvitamin D3 levels were similar between both groups (p > 0.05) (Table 1). Plasma ghrelin levels were significantly higher in tumors larger than 1 cm diameter when compared with papillary microcarcinomas (p = 0.011) (Table 2). There were no statistically significant differences regarding average age and other prognostic parameters including lymph node invasion, capsule invasion, multifocality and surgical border invasion between patients with microcarcinoma and tumors larger than 1 cm (Table 2). Plasma ghrelin levels were correlated with tumor size (r = 0.499; p < 0.001) (Figure 1). Ghrelin levels were not correlated with other parameters including insulin level, fasting plasma glucose, waist circumference, BMI, TSH, 25-hydroxyvitamin D3 level, and age. The proportion of Anti-Tg and Anti-TPO positivity between group with tumor < 1 cm versus group with tumor > 1 cm were similar 465

in patients with papillary thyroid carcinoma (PTC) and its association with the prognostic factors in PTC.

Ghrelin levels in thyroid cancer

Table 1. Clinical and laboratory characteristics of patients with thyroid cancer compared with controls Controls n = 24

Patients n = 54

Age (years)

42.5 ± 9

42.4 ± 10

BMI (kg/m²)

28.6 ± 2.5

29.1 ± 5.6

Waist (cm)

96.3 ± 12.1

96.4 ± 12.8

Waist/hip ratio

0.90 ± 0.04

0.91 ± 0.07

NS NS

Gender Male

Female

44.1 (26-144)

46.9 (28-141)

Ghrelin (ng/mL) Fasting glucose, mg/dL

85 ± 10

87 ± 20

Insulin (mIU/L)

12.7 (5.7-19.7)

11.3 (9.8-12.7)

HOMA-IR (%)

2.96 (0.9-5)

2.5 (2.1-2.9)

Total cholesterol, mg/dL

193 ± 39

207 ± 41

Plasma triglycerides, mg/dL

136 ± 17

145 ± 10

NS NS

High density lipoprotein cholesterol (mg/dL)

48 ± 10

52 ± 11

Low-density lipoprotein cholesterol (mg/dL)

118 ± 33

120 ± 39

Calcium (mg/dL)

9.6 ± 0.5

9.2 ± 0.7

0.035

Serum phosphorus (mg/dL)

3.4 ± 0.4

3.6 ± 0.8

Vitamin D3 (ng/mL)

16.6 ± 6

14.2 ± 7

TSH (mIU/L)

1.8 (1.4-2.2)

2.3 (1.8-2.8)

Anti-TPO (IU/mL)

104 (22-186)

135 (20-292)

Anti-Tg (IU/mL)

45 (8.5-82)

45 (22.1-67.8)

BMI: body mass index; Anti-Tg: antithyroglobulin; Anti-TPO: antithyroid peroxidase; TSH: thyroid stimulating hormone T4 thyroxine; HOMA-IR: Homeostasis model assessment for insulin resistance.

DISCUSSION Ghrelin was initially isolated from the stomach and demonstrated to robustly stimulate hormone secretion from the anterior pituitary (1). It is well-documented that ghrelin and its receptor, the growth hormone secretagogue receptor (GHSR), are expressed in a broad array of normal tissues and cancer types, and they are considered to function as autocrine/paracrine growth factors. Ghrelin stimulates proliferation in a range of cancer cells, including human hepatoma (12), human erythroleukemia (13), adrenocortical carcinoma (14), pancreatic adenocarcinoma (15), colorectal cancer (16), choriocarcinoma (17), prostate cancer (18), breast cancer (19), and endometrial cancer cell lines (20). Ghrelin regulates apoptosis in cancer. It was shown that ghrelin treatment inhibited apoptosis in endometrial cancer (20), pheochromocytoma (21), and adrenocortical carcinoma cell lines (22). Ghrelin stimulates cell migration and invasion in pancreatic cell lines (15), colorectal cell lines (16), and astrocytoma cells (23). Nikolopoulos and cols. observed that patients with colon cancer had significantly higher levels of 466

total serum ghrelin. Patients with end-stage disease and patients with poorly differentiated tumors had statistically significantly higher serum total ghrelin levels (8). In contrast to this finding, a study demonstrated that serum ghrelin levels were lower in patients with colorectal carcinoma compared with healthy controls, and patients with end-stage disease had lower ghrelin levels (24). Serum levels of active ghrelin were significantly higher in patients with prostate (18), ovarian (10), and hepatocellular carcinoma (11). Major hyperghrelinemia was observed in end-stage well-differentiated neuroendocrine carcinomas (25). Corbetta and cols. indicated that plasma ghrelin concentrations in patients with gastroenteropancreatic tumors were similar to healthy controls (26). Lin and cols. demonstrated that ghrelin could activate Snail function, thus promoting renal cell carcinoma metastasis, and was associated with unfavorable prognosis (27). Altogether, although there have been a few studies with conflicting results, most studies demonstrated that several cancer tissues expressed and released ghrelin, and it played a role in cancer progression. Arch Endocrinol Metab. 2017;61/5

R2 Linear = 0.499

Plasma ghrelin (pg/mL)

1600,00 1400,00 1200,00 1000,00 800,00 600,00 400,00 ,00

1,00 2,00 3,00 4,00 5,00 Maximum tumor diameter (cm)

6,00

Figure 1. Correlation of tumor size with plasma ghrelin levels.

Table 2. Comparison of clinical features and prognostic factors according to tumor size Group with tumor size < 1 cm

Group with tumor size > 1 cm

p-value

41.66 ± 10.52

43.07 ± 9.91

0.615

Yes

Age (years) Gender

0.224

Central lymph node positivity 0.573

Capsule invasion Yes

Yes

0.273

Multifocality 0.073

Lymphadenopathy 0.668

Surgical border invasion Yes

Ghrelin (ng/mL)

374.63 ± 105.66

507.26 ± 237.48

0.011

Tumor Size (cm)

0.56 ± 0.26

2.34 ± 1.19

< 0.0001

TSH (mIU/L)

1.61 (0.01-9.90)

1.11 (0.02-28.80)

0.572

FT4 (ng/dL)

1.09 (0.82-1.44)

1.27 (0.50-1.97)

0.169

Anti TPO (IU/mL)

39.00 (1.141300.00)

33.30 (0.801000.00)

0.916

Anti-Tg (IU/mL)

30.00 (10.00336.00)

20.00 (0.90500.00)

0.375

Arch Endocrinol Metab. 2017;61/5

0.149

Ghrelin expression in the thyroid is related to follicular and parafollicular cells. The association between ghrelin and thyroid cancer has been investigated in a few preclinical and clinical studies. Thyroid carcinomas (medullar, follicular, and papillary) in rats were demonstrated to express ghrelin (28). Ghrelin and GHSR were expressed in human thyroid carcinoma cells (4,29). Volante and cols. also showed that cell proliferation of thyroid carcinoma cells was inhibited by ghrelin (29). Another study demonstrated that although ghrelin alone did not stimulate cell proliferation in thyroid cell lines, it augmented the effects of thyroid stimulating hormone on cell proliferation (30). A recent study demonstrated that ghrelin expression was similar in patients with medullary cancer, papillary cancer, and nodular goiter (31). Karaoglu and cols. indicated that ghrelin tissue levels were lower in papillary carcinoma cells compared with non-cancerous thyroid tissues (32). Morpurgo and cols. reported that ghrelin levels were similar in patients with medullary thyroid cancer and healthy controls (33). We found that ghrelin levels were similar in patients with PTC and controls; however, ghrelin levels were higher in patients with tumor size larger than 1cm compared with papillary microcarcinoma. The prognostic importance of tumor size in PTC is well documented (34). Endogenous ghrelin stimulates the release of GH, which regulates IGF-1 concentrations (35,36). IGF-1 has mitogenic and antiapoptotic properties (37). Thyroid carcinoma cell lines contain IGF-1 receptors (38). GH might also exert a mitogenic effect by directly inducing c-myc expression (39). In light of this information, we aimed to investigate serum ghrelin levels in papillary thyroid cancer and its association with prognostic factors. We found no difference in ghrelin levels; however, serum ghrelin levels were correlated with tumor size, which is known as an important prognostic factor. Additionally, decreased vitamin D deficiency and increased HOMA-IR index have been found associated with thyroid cancer (40). Therefore, we evaluated levels of vitamin D3 and the HOMA-IR index. However, we found no relationship between ghrelin and these parameters. To the best of our knowledge, this is the first study to investigate serum ghrelin levels in patients with PTC. There are some minor limitations to this study such as the relatively small sample size and its single-center design. The presence of thyroiditis could have some influence in ghrelin levels however, it wasn’t analyzed. Additionally, we did not investigate ghrelin receptor 467

Ghrelin levels in thyroid cancer

expression and its association with plasma ghrelin levels, which would have increased the strength of our study. In conclusion, no significant difference in serum ghrelin levels was determined between patients with papillary thyroid cancer and healthy controls however, serum ghrelin levels were higher in patients with papillary thyroid cancer with larger tumor size. Further prospective studies investigating ghrelin expression and its association with serum ghrelin levels could be helpful to clarify this issue. Acknowledgement: none.

13. De Vriese C, Grégoire F, De Neef P, Robberecht P, Delporte C. Ghrelin is produced by the human erythroleukemic HEL cell line and involved in an autocrine pathway leading to cell proliferation. Endocrinology. 2005;146(3):1514-22. 14. Barzon L, Pacenti M, Masi G, Stefani AL, Fincati K, Palù G. Loss of growth hormone secretagogue receptor 1a and overexpression of type 1b receptor transcripts in human adrenocortical tumors. Oncology. 2005;68(4-6):414-21. 15. Duxbury MS, Waseem T, Ito H, Robinson MK, Zinner MJ, Ashley SW, et al. Ghrelin promotes pancreatic adenocarcinoma cellular proliferation and invasiveness. Biochem Biophys Res Commun. 2003;309(2):464-8.

Funding statement: none.

16. Waseem T, Javaid-Ur-Rehman, Ahmad F, Azam M, Qureshi MA. Role of ghrelin axis in colorectal cancer: a novel association. Peptides. 2008;29(8):1369-76.

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

17. Rak-Mardyła A, Gregoraszczuk E. Effect of ghrelin on proliferation, apoptosis and secretion of progesterone and hCG in the placental JEG-3 cell line. Reprod Biol. 2010;10(2):159-65.

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12. Murata M, Okimura Y, Iida K, Matsumoto M, Sowa H, Kaji H, et al. Ghrelin modulates the downstream molecules of insulin signaling in hepatoma cells. J Biol Chem. 2002;277(7):5667-74.

10. Markowska A, Ziółkowska A, Jaszczyn8ska-Nowinka K, Madry R, Malendowicz LK. Elevated blood plasma concentrations of active ghrelin and obestatin in benign ovarian neoplasms and ovarian cancers. Eur J Gynaecol Oncol. 2009;30(5):518-22. 11. Ataseven H, Bahcecioglu IH, Kuzu N, Yalniz M, Celebi S, Erensoy A. The levels of ghrelin, leptin, TNF-alpha, and IL-6 in liver cirrhosis and hepatocellular carcinoma due to HBV and HDV infection. Mediators Inflamm. 2006;2006(4):78380.

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18. Jeffery PL, Herington AC, Chopin LK. Expression and action of the growth hormone releasing peptide ghrelin and its receptor in prostate cancer cell lines. J Endocrinol. 2002;172(3):R7-11. 19. Jeffery PL, Murray RE, Yeh AH, McNamara JF, Duncan RP, Francis GD et al. Expression and function of the ghrelin axis, including a novel preproghrelin isoform, in human breast cancer tissues and cell lines. Endocr Relat Cancer. 2005;12(4):839-50. 20. Fung JN, Seim I, Wang D, Obermair A, Chopin LK, Chen C. Expression and in vitro functions of the ghrelin axis in endometrial cancer. Horm Cancer. 2010;1(5):245-55. 21. Yang M, Hu S, Wu B, Miao Y, Pan H, Zhu S. Ghrelin inhibits apoptosis signal-regulating kinase 1 activity via upregulating heat-shock protein 70. Biochem Biophys Res Commun. 2007;359(2):373-8. 22. Delhanty PJ, van Koetsveld PM, Gauna C, van de Zande B, Vitale G, Hofland LJ, et al. Ghrelin and its unacylated isoform stimulate the growth of adrenocortical tumor cells via an anti-apoptotic pathway. Am J Physiol Endocrinol Metab. 2007;293(1):302-9. 23. Dixit VD, Weeraratna AT, Yang H, Bertak D, Cooper-Jenkins A, Riggins GJ, et al. Ghrelin and the growth hormone secretagogue receptor constitute a novel autocrine pathway in astrocytoma motility. J Biol Chem. 2006;281(24):16681-90. 24. D’Onghia V, Leoncini R, Carli R, Santoro A, Giglioni S, Sorbellini F, et al. Circulating gastrin and ghrelin levels in patients with colorectal cancer: correlation with tumour stage, Helicobacter pylori infection and BMI. Biomed Pharmacother. 2007;61(23):137-41. 25. Walter T, Chardon L, Hervieu V, Cohen R, Chayvialle JA, Scoazec JY, et al. Major hyperghrelinemia in advanced well-differentiated neuroendocrine carcinomas: report of three cases. Eur J Endocrinol. 2009;161(4):639-45. 26. Corbetta S, Peracchi M, Cappiello V, Lania A, Lauri E, Vago L, et al. Circulating ghrelin levels in patients with pancreatic and gastrointestinal neuroendocrine tumors: identification of one pancreatic ghrelinoma. J Clin Endocrinol Metab. 2003;88(7):3117-20. 27. Lin TC, Liu YP, Chan YC, Su CY, Lin YF, Hsu SL et al. Ghrelin promotes renal cell carcinoma metastasis via Snail activation and is associated with poor prognosis. J Pathol. 2015;237(1):50-61. 28. Raghay K, García-Caballero T, Nogueiras R, Morel G, Beiras A, Diéguez C, et al. Ghrelin localization in rat and human thyroid and parathyroid glands and tumours. Histochem Cell Biol. 2006;125(3):239-46. Arch Endocrinol Metab. 2017;61/5

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29. Volante M, Allia E, Fulcheri E, Cassoni P, Ghigo E, Muccioli G, et al. Ghrelin in fetal thyroid and follicular tumors and cell lines. Am J Pathol 2003;162(2):645-54. 30. Park YJ, Lee YJ, Kim SH, Joung DS, Kim BJ, So I, et al. Ghrelin enhances the proliferating effect of thyroid stimulating hormone in FRTL-5 thyroid cells. Mol Cell Endocrinol. 2008;285(1-2):19-25. 31. Gurgul E, Kasprzak A, Blaszczyk A, Biczysko M, Surdyk-Zasada J, Seraszek-Jaros A, et al. Ghrelin and obestatin in thyroid gland-immunohistochemical expression in nodular goiter, papillary and medullary cancer. Folia Histochem Cytobiol. 2015;53(1):19-25.

patients surgically treated at one institution during 1940 through 1989. Surgery. 1993;114(6):1050-7. 35. Jeffery PL, Herington AC, Chopin LK. The potential autocrine/ paracrine roles of ghrelin and its receptor in hormone-dependent cancer. Cytokine Growth Factor Rev. 2003;14(2):113-22. 36. Clemmons DR. The relative roles of growth hormone and IGF-1 in controlling insulin sensitivity. J Clin Invest. 2004;113(1):25-7. 37. Khandwala HM, McCutcheon IE, Flyvbjerg A, Friend KE. The effects of insulin-like growth factors on tumorigenesis and neoplastic growth. Endocr Rev. 2000;21(3):215-44. 38. Yashiro T, Ohba Y, Murakami H, Obara T, Tsushima T, Fujimoto Y, et al. Expression of insulin-like growth factor receptors in primary human thyroid neoplasms. Acta Endocrinol (Copenh). 1989;121(1):112-20.

33. Morpurgo PS, Cappiello V, Verga U, Vicentini L, Vaghi I, Lauri E, et al. Ghrelin in human medullary thyroid carcinomas. Clin Endocrinol (Oxf). 2005;63(4):437-41.

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34. Hay ID, Bergstralh EJ, Goellner JR, Ebersold JR, Grant CS. Predicting outcome in papillary thyroid carcinoma: development of a reliable prognostic scoring system in a cohort of 1779

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32. Karaoglu A, Aydin S, Dagli AF, Cummings DE, Ozercan IH, Canatan H, et al. Expression of obestatin and ghrelin in papillary thyroid carcinoma. Mol Cell Biochem. 2009;323(1-2):113-8.

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469

original article

Screening tests for distal symmetrical polyneuropathy in Latin American patients with type 2 diabetes mellitus Nicolás Gómez-Banoy1, Virginia Cuevas1, Fernando Soler1, Maria Fernanda Pineda1, Ismena Mockus1

ABSTRACT Laboratorio de Lípidos y Diabetes, Facultad de Medicina, Universidad Nacional de Colombia, Bogotá, Colombia 1

Correspondence to: Nicolás Gómez-Banoy Carrera 45, No. 45-03 Ciudad Universitaria, Facultad de Medicina, Piso 2, Oficina 204 Bogotá, Colombia ngomezb@unal.edu.co Received on Out/12/2016 Accepted on Apr/6/2017 DOI: 10.1590/2359-3997000000283

Objective: This cross sectional study intended to evaluate two bedside tests (Neuropad and VibraTip) as screening tools for distal symmetrical polyneuropathy (DSPN) in Latin American patients with type 2 diabetes mellitus (T2D). Subjects and methods: Ninety-three Colombian patients diagnosed with T2D were recruited. Anthropometric variables, glycemic control parameters, lipid profile and renal function were assessed for each patient. DSPN was defined by a Michigan Neuropathy Screening Instrument (MNSI) clinical score greater than 2. Both Neuropad and Vibratip tests were applied to each patient. Contingency analyses were performed to evaluate the diagnostic power of both tools. Results: The prevalence of DSPN determined clinically by MNSI was 25.8%. DSPN in these patients was associated with age, worsening renal function, and insulin treatment. The sensitivity and specificity of the Neuropad test for DSPN was 66.6% and 63% respectively. Its negative predictive value (NPV) was 84.6%. The VibraTip test exhibited a sensitivity of 54.1% and specificity of 91.3%, with a NPV of 85.1%. Conclusion: Neuropad and VibraTip are reliable screening tools for DSPN in Latin American population. VibraTip presents a considerable diagnostic power for DSPN in this population. Further studies regarding the cost-effectiveness of these tools in clinical practice are needed. Arch Endocrinol Metab. 2017;61(5):470-5 Keywords Type 2 diabetes; diabetic neuropathy; complications; diagnostic test

INTRODUCTION

iabetic neuropathy (DN) is a common microvascular complication of type 2 diabetes mellitus (T2D). It is estimated that DN affects 26-47% of diabetes patients in the United States, and it comprises a wide variety of clinical syndromes (1). Specifically, distal symmetrical polyneuropathy (DSPN) is the most common presentation of DN, and it accounts for 50% of neuropathies associated to diabetes (2). In Latin America, the prevalence of DN has been reported around 55% in some countries (3), and in Colombia it is present in 68% of hospitalized T2D patients (4). The diagnosis of DSPN is primarily clinical, involving a detailed medical history and neurological examination. The Toronto Consensus Panel on Diabetic Neuropathy defined specific diagnostic criteria based on various signs and symptoms (5), however standardized tests and questionnaires have been developed and are frequently used in clinical practice. One of this is the Michigan Neuropathy Screening Instrument (MNSI), which consists of a self-administered questionnaire and a 5-item physical examination. This tool has been widely

470

validated (6,7) and used to determine the presence of peripheral diabetic neuropathy in various longitudinal studies (8). In order to improve the diagnosis and screening of peripheral neuropathy in T2D patients, new tests have been developed in the last years (9). Among these, Neuropad and VibraTip are characterized by being bedside, simple and accessible tools to evaluate the presence of small fiber and large fiber dysfunction respectively. Neuropad is a test designed to measure sudomotor dysfunction in the foot through a cobalt II salt-impregnated patch applied to the soles’ skin. The reaction from the water produced in the sweat glands and the mentioned chemical will gradually change the color of the patch from blue to pink. The Neuropad has been evaluated extensively in European T2D patients as a reliable screening test for DSPN (10). The VibraTip is a handheld device designed to test vibration perception by producing stimulus of 128 Hz; it has been validated as a useful test for diabetic neuropathy screening in European patients with T2D (11). Due to the potential usefulness of both Neuropad Arch Endocrinol Metab. 2017;61/5

Screening tests for diabetic neuropathy

SUBJECTS AND METHODS Patients The patients considered for this study were outpatients belonging to the “Program for the Prevention of Diabetes Complications” of the Lipids and Diabetes Laboratory, Faculty of Medicine, National University of Colombia. The study was conducted in the premises of the National University of Colombia, once approved by the institutional Ethics Committee in accordance with the Helsinki Declaration. All patients were diagnosed with T2D based on the American Diabetes Association (ADA) criteria (12). Exclusion criteria included diagnosed or established neuropathy from other etiology (according to clinical history), active neoplastic or autoimmune disease, acute exacerbation of chronic disease, pregnancy, and age under 18.

Physical examination and general tests During a period of two months, each patient received their usual follow-up medical exam, during which anthropometric variables, body mass index (BMI) (kg/m2) and waist circumference (cm) were determined. Blood pressure (mmHg) was determined clinically with a mercury sphygmomanometer. Results from their routine metabolic and lipid profile were assessed: plasma glucose (mg/dL), triglycerides (mg/dL), total cholesterol (mg/dL), HDL cholesterol (mg/dL), and serum creatinine (mg/dL). LDL cholesterol was calculated using the Friedewald formula (13). Haemoglobin A1c (HbA1c) was reported in NGSP units (%) and in IFCC units (mmol/mol). All laboratory exams were determined from blood samples taken after an 8 hour fast, in a range of 1 to 3 days previous to medical consultation. Estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation as recommended by current guidelines (14). On the same day of medical examination, the objectives and procedures of the study were explained to each patient, and a signed informed consent form Arch Endocrinol Metab. 2017;61/5

was obtained from all participants. Patients were then asked to answer the MNSI 15 – item symptom-based questionnaire. According to previous validation studies, if the summing of abnormal answers was greater than 7, the questionnaire was considered positive for DSPN (15). Afterwards, participants underwent neurological examination of both feet based on the MNSI clinical test, involving: 1) inspection to detect deformities, dry skin, calluses, infection, 2) inspection to detect ulcerations, 3) grading of ankle reflexes, 4) assessment of vibration perception at great toe, and 5) 10-g monofilament testing. Each foot was examined individually. Previous validation studies established a maximum score of 8 for the MNSI (15); however, they did not include the 10-g monofilament test. Here, we established a maximum possible score of 10, in accordance with recent epidemiological studies (8,16). In this analysis, the presence of DSPN was defined by a score greater than 2 (> 2) in the clinical examination component of MNSI (8).

Neuropad and VibraTip tests The Neuropad and VibraTip tests were performed by two qualified primary-care physicians blinded to results of the MNSI. For Neuropad testing, patients were asked to remove their footwear and socks 10 minutes before applying a Neuropad patch to each of their soles, in a callus-free area between first and second metatarsal head. A test was considered positive for DSPN if the patch remained completely blue or had a patchy appearance 10 minutes after application in one or both soles. For the VibraTip test, the physician touched the hallux of each foot twice with the device. In one of the touches, a vibratory stimulus would be applied. Then, the patient was asked in which of the two he/she felt a vibration. A test was considered positive for DSPN if the patient failed to detect the vibratory stimuli in one or both feet (11).

Statistical analysis Data are presented as means ± standard deviation (SD). Statistical Analyses were conducted using the Prism 6.0e software for Mac (GraphPad Software, Inc). Patients were divided into two groups for comparison: subjects with clinical DSPN (MNSI > 2) and subjects without DSPN (MNSI ≤ 2). A student’s T test was used to compare the means of important variables in both groups. To compare proportions between both 471

and VibraTip in outpatient clinical practice, and the limited data regarding the validity of these tests in Latin American patients, we sought to evaluate the diagnostic performance of Neuropad and VibraTip for DSPN in Colombian patients with T2D.

Screening tests for diabetic neuropathy

groups, a Chi-square test was performed. Measures of diagnostic performance (sensitivity, specificity, negative and positive predictive values) were calculated as previously described (17). All tests were two-tailed. A p value of less than 0.05 was considered significant.

RESULTS DSPN prevalence and clinical characteristics of the population Of the 120 eligible patients belonging to the “Program for the Prevention of Diabetes Complications”, 93 matched with the inclusion and exclusion criteria and were included in the present study. Table 1 shows the demographic and clinical characteristics of the participants. Fifty-two men and 41 women participated. Mean age at the time of the study was 70.8 ± 7.7 years. The prevalence of neuropathy was 25.8% (24 out of 93 patients presented a MNSI clinical score > 2). Men presented with a prevalence of 30.7% whereas women had 19.5%, with no statistical difference between both groups (p = 0.218). Regarding the MNSI symptom-based questionnaire,

only 4.3% of patients had a score greater than 7. The proportion of patients with evidence of CKD (eGFR < 60) was 21.5%. The duration of T2D in the overall population was 10 ± 8.2 years. Patients with DSPN presented slightly longer T2D duration than patients without DSPN, but the difference was not significant (p = 0.27). Also, the presence of DSPN was associated with increasing age (p < 0.001), increased creatinine values (p < 0.001), lower eGFR (p < 0.001), and higher urinary albumin excretion (p < 0.05). In terms of medication usage, DSPN was associated with insulin treatment (p < 0.01), and usage of angiotensin receptor blockers (ARB) (p < 0.05).

Diagnostic performance of Neuropad and VibraTip A total of 41 patients presented an abnormal Neuropad test. In patients without clinical DSPN (MNSI ≤ 2) 36.2% presented abnormal Neuropad test, whereas in patients presenting clinical DSPN (MNSI > 2) the proportion was 66.6% (p < 0.05). Table 2 depicts the diagnostic utility of the Neuropad test for DSPN. The specificity and sensitivity are very similar, and it presents a high negative predictive value (NVP).

Table 1. Clinical characteristics of total T2D patients and patients with and without neuropathy All patients (n = 93)

Patients with neuropathy (MNSI > 2)

Patients without neuropathy (MNSI ≤ 2)

Age (years)

70.8 ± 7.7

75.8 ± 7.3

69.1 ± 7

< 0.001

T2D duration (years)

10.0 ± 8.2

11.5 ± 7.5

9.4 ± 8.4

0.27

Hemoglobin A1C (%)

7.3 ± 1.4

7.5 ± 1.1

7.2 ± 1.5

0.376

Fasting plasma glucose (mg/dL)

130 ± 43

129 ± 35

130 ± 45

0.9241

Triglycerides (mg/dL)

149 ± 55

151 ± 50

148 ± 56

0.829

Total cholesterol (mg/dL)

172 ± 36

170 ± 27

173 ± 38

0.705

HDL cholesterol (mg/dL)

41.9 ± 7.2

40.8 ± 5.8

42.3 ± 7.6

0.392

LDL cholesterol (mg/dL)

100.5 ± 31.9

98.6 ± 21.5

101.1 ± 34.7

0.738

Creatinine (mg/dL)

1.0 ± 0.2

1.1 ± 0.3

0.9 ± 0.2

< 0.001

eGFR (ml/min/1.73 m2)

72 ± 14.5

63.1 ± 14.8

75.1 ± 13

< 0.001

Urinary albumin (mg/L)

54.9 ± 135.6

106.3 ± 225

37 ± 77

< 0.05

27.8 ± 3.5

27.5 ± 3.4

27.9 ± 3.6

0.647

63,4%

54.2%

66.6%

0.273

BMI (kg/m2) Subjects treated with metformin (%) Subjects treated with insulin (%)

Subjects treated with sulphonylureas (%)

29%

50%

21.7%

< 0.01

19.4%

16.6%

20.3%%

0.698

Subjects treated with DPP-4 inhibitor (%)

15.1%

12.5%

15.9%

0.684

Subjects treated with ACE inhibitor (%)

18.3%

16.6%

18.8%

0.316

Subjects treated with ARB (%)

46.2%

66.6%

39.1%

< 0.05

Subjects treated with statins (%)

64.5%

66.6%

63.7%

0.798

Data are expressed in Mean ± SD. P values depict differences between patients with and without neuropathy. T2D: type 2 diabetes; HDL: High Density Lipoprotein; LDL: Low Density Lipoprotein; eGFR: estimated Glomerular Filtration Rate; BMI: Body Mass Index; DPP-4: Dipeptidyl Peptidase 4; ACE: Angiotensin Converting Enzyme; ARB: Angiotensin Receptor Blocker.

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Diagnostic performance of individual diagnostic tools Table 4 shows the diagnostic power of each individual component of the MNSI. Interestingly, the 10-g monofilament shows the highest sensitivity, followed by the 128- Hz-tuning fork. Table 2. Diagnostic performance of Neuropad for detection of diabetic neuropathy Reference Test 5-item MNSI

Sensitivity

Specificity

PPV

NPV

66,6%

63.8%

39%

84.6%

128-Hz-tuning fork

39%

82.9%

64%

63.2%

10-g monofilament

24.3%

94.2%

76.9%

61.2%

Ankle reflex

60.9%

71.2%

62.5%

69.8%

VibraTip

29.2%

86.4%

63.1%

60.8%

PPV: positive predictive value; NPV: negative predictive value. MNSI: Michigan Neuropathy Screening Instrument.

Table 3. Diagnostic performance of VibraTip for detection of diabetic neuropathy Reference Test

Sensitivity

Specificity

PPV

NPV

54.2%%

91.3%

68.4%

85.1%

128-Hz-tuning fork

73.6%

85.1%

56%

91.7%

10-g monofilament

42.1%

93.2%

61.5%

86.2%

Ankle reflex

89.4%

68.9%

42.5%

96.2%

Neuropad

63.1%

60.8%

29.2%

86.5%

5-item MNSI

PPV: positive predictive value, NPV: negative predictive value. MNSI: Michigan Neuropathy Screening Instrument.

Table 4. Performance of all diagnostic tools for detection of diabetic neuropathy (MNSI > 2) Reference Test

Sensitivity

Specificity

PPV

NVP

128-Hz-Tuning Fork

70.8%

88.4%

68%

89.7%

10-g monofilament

54.2%

100%

86.3%

Ankle reflex

75%

68.1%

45%

88.7%

Neuropad

66.7%

63.8%

39%

84.6%

VibraTip

54.2%

91.3%

68.4%

85.1%

PPV: positive predictive value; NPV: negative predictive value. MNSI: Michigan Neuropathy Screening Instrument. Arch Endocrinol Metab. 2017;61/5

DISCUSSION In the present study we aimed to study the diagnostic performance of two new screening tools for diabetic DSPN, Neuropad and Vibratip. Our work demonstrates that both Neuropad and Vibratip are reliable tests for the screening of clinical DSPN measured by the MNSI. Additionally, Vibratip showed a better diagnostic performance than Neuropad. The prevalence of diabetic neuropathy measured by the MNSI was 25.8% in our population, which is low compared to other studies in Colombian and Latino patients (3,4). A cross-sectional study in Mexico reported prevalence as high as 69% of DSPN using the 5-item clinical MNSI (18). Similar to our results, studies in European population have shown 28.8% of DSPN prevalence in T2D patients using the 5-item clinical MNSI (16), and 30.6% using a modified version of the clinical MNSI (19). Regarding the MNSI questionnaire, previous studies have shown that it underestimates the prevalence of diabetic neuropathy, with values bellow 5% (7,19), similar to our study (4.3%). For this reason, the clinical component of the MNSI was used to determine the presence of DSPN. One of the potential reasons for the low neuropathy prevalence in our population is that these patients belong to a specialized screening program for diabetes complications and are carefully followed and treated to achieve adequate glycemic control (mean HbA1c of 7.3). Furthermore, there is a low incidence of other micro-vascular complications, such as diabetic nephropathy (21.5%). Consistently, the patients presenting DSPN were significantly older, showed worsening renal markers, and presented a more advanced metabolic disease (insulin treatment). Neuropad is an adhesive patch utilized to determine skin hydration status in the soles of the foot; this way, it measures the degree of sudomotor dysfunction and, indirectly, small fiber functionality (20). This test has been widely validated in European countries, where it has been utilized as a screening test for DSPN due to its high sensitivity values (65.1%-100%), moderate specificity (32-78.5%), and high negative predictive values (63-100%) (9). A recent study evaluated the diagnostic performance of Neuropad in Latin American patients with T2D, finding a sensitivity of 77.8% and a NPV of 63.8% for DSPN measured by a sign-based scale (Michigan Neuropathy Disability Score). Interestingly, they found a better correlation between Neuropad and cardiovascular autonomic neuropathy (CAN) than with 473

Regarding the VibraTip test, a total of 19 patients presented an abnormal result. In participants without clinical neuropathy 8.7% had an abnormal VibraTip test, whereas the proportion in the neuropathy group was 54.2% (p < 0.05). Table 3 shows the diagnostic performance of VibraTip. Importantly, it has a high specificity value.

Screening tests for diabetic neuropathy

DSPN (21). Similarly, in our study we found sensitivity values (66.6%) comparable to those reported in the literature, and, importantly, Neuropad presented a high NPV (84.6%) for DSPN. Taken together, our findings confirm the utility of Neuropad as a reliable screening test for DSPN in Latin American population. One of the potential advantages of this tool is that its results may be witnessed and understood by patients; this may aid the physician in promoting self-consciousness and adequate foot care habits. This is especially important, since patient education alone does not seem to lead to clinically relevant reductions in ulcer and amputation incidence (22). On the other hand, VibraTip is a portable, small device used to assess vibration perception on the hallux by delivering a stimulus of 128 Hz. It is mainly used to evaluate large fiber functionality (9). The advantages of the VibraTip are its small size, low cost, and convenience for rapid neurological evaluation in the outpatient context. A study in European patients assessing the diagnostic performance of VibraTip showed moderate to high sensitivity and specificity values (79% and 82%) when compared to vibration perception threshold (VPT) ≥ 25 Volts (V) using a Neurosthesiometer as gold standard for DPSN (23). Another previous study reported a 100% sensitivity and 96.6% specificity of VibraTip compared to VPT ≥ 25 V and the Neuropathy Disability Score (NDS) ≥ 6 (11). The values considered as thresholds in this study were considerably higher than the standard validated diagnostic values and denoted severe DSPN; thus, interpretation of their conclusions is limited. The comparison of our results to the mentioned studies is very difficult since crucial clinical characteristics of patients were not mentioned. Our study showed VibraTip presents a high specificity (91.3%), higher than the study by Bracewell and cols. (23), which may confer this test an important diagnostic power. It also displays a high NVP, which reinforces its potential role as screening test for DSPN. The National Institute for Health and Care Excellence (NICE) evaluated the scientific evidence and cost-effectiveness of VibraTip and considered it is a technology that shows potential to improve the detection of DSPN in diabetes patients, but more research is needed to assess its diagnostic accuracy (24). To the best of our knowledge, this is the first study to evaluate the diagnostic performance of VibraTip in Latin American patients with T2D. Furthermore, our study provides a complete characterization of T2D 474

patients that may benefit from this tool. Our results suggest VibraTip may be reliable tool to screen for DSPN, and it may even have the diagnostic power to replace instruments as the 128-Hz tuning fork. Its ease to use, portability and ability to produce a consistent vibratory stimulus are advantages that may be beneficial in the outpatient context. We consider that the use of new tools for the early diagnosis of DSPN is necessary in the everyday detection of DSPN, and VibraTip seems an attractive candidate. For the implementation of VibraTip and Neuropad in middle to low-income countries, a crucial factor to take into account is their cost. Compared to other widespread screening tools such as the 10-g monofilament and the tuning fork, VibraTip is cheaper in terms of the cost per device and the estimated cost per-examination (24). Studies assessing the cost-effectiveness of Neuropad as a screening tool for DSPN are lacking; however, its non-reusable nature might implicate larger costs for public health systems. Our study presents potential limitations that need to be addressed before interpreting our results. First, the sample size is relatively small belonging to a single center; multi-centered, large cohort studies in other Latin American countries are certainly needed to confirm our findings in this population, especially regarding the VibraTip. Second, the patients from our study are elderly (mean age of 70.8 years), and it is possible that the diagnostic performance of the addressed tools changes in younger population. Third, it would be interesting to assess the cost-effectiveness of both VibraTip and Neuropad in middle and lowincome countries. Future public health and economic studies addressing this issue are certainly needed. In conclusion, VibraTip and Neuropad are simple, bedside tests for the screening of DSPN in Latin American T2D patients. VibraTip presents a high diagnostic performance for DSPN, and constitutes a promising candidate for the early diagnosis of this entity in our population. Acknowledgements: the authors wish to thank the clinical laboratory of UNISALUD, Bogotá, Colombia, for sample processing, and Miss Yamile Vargas for her collaboration with administrative tasks. Funding statement: this work was supported and funded by the National University of Colombia (Grant: QUIPU Number: 201010023501). Disclosure: no potential conflict of interest relevant to this article was reported. Arch Endocrinol Metab. 2017;61/5

Screening tests for diabetic neuropathy

REFERENCES 1. Barrett AM, Lucero MA, Le T, Robinson RL, Dworkin RH, Chappell AS. Epidemiology, public health burden, and treatment of diabetic peripheral neuropathic pain: a review. Pain Med. 2007;8 Suppl 2:S50-62. 2. Kaku M, Vinik A, Simpson DM. Pathways in the diagnosis and management of diabetic polyneuropathy. Curr Diab Rep. 2015;15(6):609. 3. Lazo Mde L, Bernabé-Ortiz A, Pinto ME3, Ticse R, Malaga G, Sacksteder K, et al. Diabetic peripheral neuropathy in ambulatory patients with type 2 diabetes in a general hospital in a middle income country: a cross-sectional study, PLoS One. 2014;9(5):e95403.

12. American Diabetes Association. Standards of medical care in diabetes-2015 abridged for primary care providers. Clin Diabetes. 2015;33(2):97-111. 13. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972;18(6):499-502. 14. Andrassy KM. Comments on ‘KDIGO 2012 Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease’. Kidney Int. 2013;84:622-3. 15. Feldman EL, Stevens MJ, Thomas PK, Brown MB, Canal N, Greene DA. A practical two-step quantitative clinical and electrophysiological assessment for the diagnosis and staging of diabetic neuropathy. Diabetes Care. 1994;17(11):1281-9.

4. Alayón AN, Altamar-López D, Banquez-Buelvas C, Barrios-López K [Chronic complications, hypertension and obesity in diabetic patients living in Cartagena, Colombia]. Rev Salud Publica (Bogota). 2009;11(6):857-64.

16. Timar B, Timar R, Gaita# L, Oancea C, Levai C, Lungeanu D. The Impact of Diabetic Neuropathy on Balance and on the Risk of Falls in Patients with Type 2 Diabetes Mellitus: A Cross-Sectional Study. PLoS One. 2016;11(4):e0154654.

5. Dyck PJ, Albers JW, Andersen H, Arezzo JC, Biessels GJ, Bril V, et al.; Toronto Expert Panel on Diabetic Neuropathy. Diabetic polyneuropathies: update on research definition, diagnostic criteria and estimation of severity. Diabetes Metab Res Rev. 2011;27(7):620-8.

17. Pepe MS. The statistical evaluation of medical tests for classification and prediction. Oxford University Press, Oxford; New York, 2003.

6. Moghtaderi A, Bakhshipour A, Rashidi H. Validation of Michigan neuropathy screening instrument for diabetic peripheral neuropathy. Clin Neurol Neurosurg. 2006;108(5):477-81.

19. Salvotelli L, Stoico V, Perrone F, Cacciatori V, Negri C, Brangani C, et al., Prevalence of neuropathy in type 2 diabetic patients and its association with other diabetes complications: The Verona Diabetic Foot Screening Program. J Diabetes Complications. 2015;29(8):1066-70.

7. Herman WH, Pop-Busui R, Braffett BH, Martin CL, Cleary PA, Albers JW, et al.; DCCT/EDIC Research Group. Use of the Michigan Neuropathy Screening Instrument as a measure of distal symmetrical peripheral neuropathy in Type 1 diabetes: results from the Diabetes Control and Complications Trial/ Epidemiology of Diabetes Interventions and Complications. Diabet Med. 2012;29(7):937-44. 8. Lee CC, Perkins BA, Kayaniyil S, Harris SB, Retnakaran R, Gerstein HC, et al., Peripheral Neuropathy and Nerve Dysfunction in Individuals at High Risk for Type 2 Diabetes: The PROMISE Cohort. Diabetes Care. 2015;38(5):793-800. 9. Papanas N, Ziegler D. New vistas in the diagnosis of diabetic polyneuropathy. Endocrine. 2014;47(3):690-8. 10. Papanas N, Boulton AJ, Malik RA, Manes C, Schnell O, Spallone V, et al. A simple new non-invasive sweat indicator test for the diagnosis of diabetic neuropathy. Diabet Med. 2013;30(5): 525-34.

20. Papanas N, Papatheodorou K, Papazoglou D, Kotsiou S, Maltezos E. A prospective study on the use of the indicator test Neuropad® for the early diagnosis of peripheral neuropathy in type 2 diabetes. Exp Clin Endocrinol Diabetes. 2011;119(2):122-5. 21. Mendivil CO, Kattah W, Orduz A, Tique C, Cárdenas JL, Patiño JE. Neuropad for the detection of cardiovascular autonomic neuropathy in patients with type 2 diabetes. J Diabetes Complications. 2016;30(1):93-8. 22. Dorresteijn JA, Kriegsman DM, Assendelft WJ, Valk GD. Patient education for preventing diabetic foot ulceration. Cochrane Database Syst Rev. 2012;10:CD001488. 23. Bracewell N, Game F, Jeffcoate W, Scammell BE. Clinical evaluation of a new device in the assessment of peripheral sensory neuropathy in diabetes. Diabet Med. 2012;29(12):1553-5. 24. Willits I, Cole H, Jones R, Dimmock P, Arber M, Craig J, Sims A. ViibraTip for Testing Vibration Perception to Detect Diabetic Peripheral Neuropathy: A NICE Medical Technology Guidance. Appl Health Econ Health Policy. 2015;13(4):315-24.

11. Bowling FL, Abbott CA, Harris WE, Atanasov S, Malik RA, Boulton AJ. A pocket-sized disposable device for testing the integrity of sensation in the outpatient setting. Diabet Med. 2012;29(12):1550-2.

18. Ibarra CT, Rocha Jde J, Hernández RO, Nieves RE, Leyva RJ. [Prevalence of peripheral neuropathy among primary care type 2 diabetic patients]. Rev Med Chil. 2012;140(9):1126-31.

Arch Endocrinol Metab. 2017;61/5

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original article

Endothelial dysfunction in children with type 1 diabetes mellitus 1 Programa de Pós-Graduação em Ciências Médicas, Universidade de Brasília (UnB), Campus Universitário Darcy Ribeiro, Brasília, DF, Brasil 2 Hospital Universitário de Brasília, Campus Universitário Darcy Ribeiro, Brasília, DF, Brasil 3 Departamento de Ciências Farmacêuticas, Faculdade de Ciências da Saúde, UnB, Campus Universitário Darcy Ribeiro, Brasília, DF, Brasil 4 Escola de Ciências e Tecnologia, Departamento de Matemática, Matemática e Aplicações, Universidade Nova de Lisboa, Quinta da Torre, Caparica, Lisboa, Portugal 5 Metodologias Aplicadas, Laboratório de Metodologias Aplicadas às Doenças Infecciosas, UnB, Campus Universitário Darcy Ribeiro, Brasília, DF, Brasil

Correspondence to: Yanna Karla de Medeiros Nóbrega Departamento de Ciências Farmacêuticas, Faculdade de Ciências da Saúde, Universidade de Brasília, Campus Universitário Darcy Ribeiro 70900-910 – Brasília, DF, Brasil yannanobrega@gmail.com

Antonella Márcia Mercadante de Albuquerque do Nascimento1,2,3, Inês Jorge Sequeira4, Daniel França Vasconcelos2, Lenora Gandolfi1,5, Riccardo Pratesi1,5, Yanna Karla de Medeiros Nóbrega1,3,5

ABSTRACT Objective: The purpose of this study was to verify the presence of endothelial dysfunction and initial structural atherosclerotic changes in children with Type 1 diabetes mellitus (T1DM). Subjects and methods: The study population comprised 31 diabetic children aged 6 to 12 years, divided into two subgroups according to the duration of the T1DM diagnosis: subgroup 1, with less than 5 years elapsed since diagnosis, and subgroup 2, with more than 5 years elapsed since diagnosis. The control group comprised 58 age-matched healthy children. Ultrasonographic techniques were used to measure the flow-mediated dilatation (FMD) of the brachial artery and the intima-media thickness (IMT) of the carotid arteries. Results: Children with T1DM with longer disease duration showed significantly decreased mean values of FMD compared with those in the control group. No significant differences between the groups were found in relation to IMT. The FMD percentage presented a moderate negative correlation with glycated hemoglobin (HbA1c) and fasting glucose levels. Conclusion: Our findings suggest that endothelial dysfunction may be already present in children with 5 years or more elapsed since diagnosis, even in the absence of atherosclerotic structural changes. The decreased vasodilation response correlated with hyperglycemia. Arch Endocrinol Metab. 2017;61(5):476-83 Keywords Type 1 diabetes mellitus; endothelial dysfunction; atherosclerosis; flow-mediated dilation; carotid intima-media thickness

Received on Out/30/2015 Accepted on Feb/1/2017 DOI: 10.1590/2359-3997000000271

INTRODUCTION

ype 1 diabetes mellitus (T1DM) is currently one of the most common chronic diseases in childhood, displaying a high frequency of complications involving microvascular and macrovascular abnormalities (1). Metabolic abnormalities characterizing T1DM, such as hyperglycemia and increased circulating free fatty acids, trigger molecular mechanisms that contribute to the development of endothelial dysfunction. These mechanisms include a decreased bioavailability of nitric oxide, increased oxidative stress, disturbances in intracellular signal transduction, and activation of advanced glycation end products (AGEs) (2). Endothelial dysfunction, thought to be an early event in the structural changes of the atherosclerotic process,

476

is characterized by abnormalities in the regulation of the lumen of the vessels, resulting in a blunted vasodilatory response (3). This vascular endothelial impairment results in increased production of inflammatory cytokines and augmented expression of cellular adhesion molecules and several other biologically active substances that contribute to the induction of a proinflammatory and prothrombotic state (4). These mechanisms represent an important step in the development of the initial atherosclerotic changes and subsequent complications in patients with T1DM (5). Although the macrovascular complications of T1DM do not usually disclose major clinical manifestations during childhood and adolescence, evaluation with a reliable and noninvasive highArch Endocrinol Metab. 2017;61/5

Endothelial dysfunction in type 1 diabetes

SUBJECTS AND METHODS Subjects The study group comprised 31 children aged 6 to 12 years (mean age 8.36 ± 1.81 years) divided into two subgroups according to the time elapsed since the diagnosis of T1DM: subgroup 1, diagnosed within the previous 5 years (mean time from diagnosis 2.73 ± 1.11 years) and subgroup 2, diagnosed more than 5 years before the study (mean time from diagnosis 6.11 ± 1.28 years). A total of 58 healthy children aged 6-12 years (mean 6.9 ± 1.77 years) comprised the control group. The diabetic children were recruited among patients attending the Pediatric Endocrinology Outpatient Clinic at the Brasilia University Hospital and the Pediatric Clinic of the Brasilia Children’s Hospital. Children in the control group were selected among dependents of the hospital’s employees. In order to minimize the influence of other factors on the endothelial function Arch Endocrinol Metab. 2017;61/5

excluding those due to T1DM, we followed the criteria of inclusion and exclusion described in Table 1. The study was conducted according to the Declaration of Helsinki, and the Ethics Committee of the University of Brasilia School of Medicine approved the study protocol. Written informed consent was obtained from the legal guardians of the children.

Study design and sample size calculation The sample size was calculated with the software Epi Info, version 3.5.3. A minimum sample size of 66 children was obtained, considering a maximum error of 5% and a 95% confidence interval. The final sample comprised 31 patients with T1DM and 58 healthy controls, totaling 89 children.

Table 1. Inclusion and exclusion criteria Inclusion criteria for the T1DM children group Absence of microvascular complications* Inclusion criteria for the control group Normal weight and height for age** Fasting glucose < 100 mg/dL HbA1c ≤ 5.6% hsCRP < 0.3 mg/dL Exclusion criteria for both groups Onset of puberty*** Arterial hypertension**** Total cholesterol ≥ 200 mg/dL HDL cholesterol < 45 mg/dL LDL cholesterol ≥ 130 mg/dL VLDL cholesterol ≥ 41mg/dL Triglycerides ≥ 100 mg/dL Hemoglobin < 11 g/dL Hematocrit < 33% Family history of primary dyslipidemia Family history of premature death from CVD or stroke Presence of acute infectious conditions Chronic diseases, lasting 3 months or more, excluding T1DM Continuous use of drugs, except insulin T1DM: type 1 diabetes mellitus; HbA1c: glycated hemoglobin; hsCRP: high sensitive C-reactive protein; CVD: cardiovascular disease. *All children underwent a detailed physical examination by a pediatric endocrinologist (that included assessment of blood pressure and skin sensitivity), specific serum and urinary biochemical tests (including a complete renal function and microalbuminuria), and complete ophthalmic evaluation. ** According to CDC growth charts (Kuczmarski and cols., 2002 [30]) adapted for Brazilian children (Silva and cols., 2010 [31]). *** Children not presenting Tanner stage M1P1 (females) or G1P1 (males) were excluded. **** Blood pressure was measured with suitable cuffs and classified according to systolic and diastolic curves, specific for each age, gender, and height, in agreement with the curves of the Second Task Force on Blood Pressure Control in Children (1996). High blood pressure was diagnosed when the percentiles of systolic and/or diastolic readings were above the 95th percentile for age and sex, on at least three occasions. If the readings were between the 90th and 95th percentiles, the blood pressure was considered to be normal-high, and when below the 90th percentile, it was considered normal.

477

resolution ultrasound equipment allows an early diagnosis of endothelial dysfunction through the study of flow-mediated dilation (FMD) of the brachial artery. Additionally, the measurement of the carotid intima-media thickness (IMT) allows the detection of initial atherosclerotic alterations (6). FMD is defined as a reactive vasodilatation of the brachial artery after hyperemic stimulation (7) and when decreased, has a predictive value for future cardiovascular disease (8). Similarly, increased carotid artery width measured with the IMT technique is a risk factor for future atherosclerosis or suggestive of an already established atherosclerosis and an atherosclerotic process affecting peripheral, coronary, and femoral arteries. IMT is considered a strong predictor of vascular disease in high-risk individuals, such as those with T1DM (9). The evaluation of children with T1DM using the FMD technique and IMT measurement at an early preclinical stage of the disease could allow the implementation of strategies to prevent or at least reduce the cardiovascular morbidity and mortality in diabetic children. Consequently, the aim of our study was to evaluate children with T1DM without evidence of microvascular or macrovascular complications for the presence of endothelial dysfunction and early vascular structural abnormalities applying the techniques of FMD and IMT.

Endothelial dysfunction in type 1 diabetes

Ultrasound studies The ultrasonographic studies were performed with ultrasound equipment model ACUSON X 300 (Siemens HG, Munich, Germany), equipped with a vascular software for two-dimensional (2D) and color images, color and spectral Doppler, internal monitor for echocardiography, and a high-frequency (8.9 MHz) vascular transducer (VF13-5). All images were obtained and evaluated by the same examiner and processed manually with an ultrasonic compass. Each child underwent both ultrasound techniques sequentially, always in the afternoon, in dimmed light, and at room temperature (23°C).

Brachial artery FMD Neither the consumption of high-fat foods, vitamin C, and stimulants beverages (e.g. caffeine), nor intense physical activity during the previous 24 hours was allowed before the FMD evaluation. All tests were preceded by blood pressure measurement after 10 minutes of rest, using a pneumatic sphygmomanometer with a cuff appropriate for the child’s upper limb size. Electrodes were applied for continuous electrocardiographic monitoring. In accordance with protocols suggested by Corretti and cols. and Barac and cols. (10,11), the children were placed comfortably in a supine position with the sphygmomanometer cuff placed on their left forearm over the brachial artery, 5 to 15 cm above the antecubital fossa. Initially, a baseline image of the left brachial artery in a longitudinal plane was obtained, and its quality was further optimized in a 2D mode. The image was subsequently expanded and recorded for 1 minute to allow later measurement of the larger artery diameter at rest (at the end of diastole, which coincides with the R wave of the QRS complex of the continuous ECG). The cuff was then inflated 50 mmHg above the systolic pressure (10,11) during 4 minutes (8). New images of the brachial artery were obtained 1, 3, 5, and 9 minutes after cuff deflation. After that, new images were obtained allowing measurement of the maximal diameter and the flow of the brachial artery. The arterial lumen diameter was defined as the distance between the intima of the far and near vessel walls. The dilatation was calculated by subtracting the lumen diameter at baseline from the maximal lumen diameter after ischemia and dividing the result by 478

the lumen diameter at baseline (12). Results were expressed as a percentage FMD (%).

Carotid artery IMT All measurements were performed according to the protocol described by Järvisalo and cols. and Bots and cols. (13,14). Images of the right and left common carotid arteries were obtained in 2D mode with the subjects in a supine position with their heads lateralized, and the transducer positioned on the contralateral vessel at an approximate angle of 45°, about 1 to 2 cm proximal to the bifurcation of the carotid artery. The images of each artery were subsequently magnified to allow the measurement of the IMT of the posterior wall, at the end of the diastole (at the onset of the QRS complex). The IMT was defined as the distance between the lumen-intima and media-adventitia ultrasound interfaces of the carotid artery measured in mm (12).

Statistical analysis The results are shown as mean ± standard deviation. Statistical analyses were performed using the software GraphPad Prism, version 5.0 (GraphPad Software, San Diego, California, USA). Student’s t test or permutation tests (Kruskal-Wallis) were applied for comparisons with the control group. One-way ANOVA was used when applicable, and Dunnett’s post-test was used when diabetic children were compared with the children in the control group. To quantify the association between two variables, Pearson’s correlation coefficient was used. The data were considered statistically significant when p ≤ 0.05.

RESULTS Characteristics of the groups The demographic and laboratory profile of the diabetic and control groups are shown in Table 2. Both groups were similar regarding gender and age. The laboratory profiles showed statistical differences in levels of triglycerides and VLDL cholesterol between the groups, although the results were still within the normal reference values. As expected, the levels of glycated hemoglobin (HbA1c) and fasting glucose were significantly higher in the diabetic group. Arch Endocrinol Metab. 2017;61/5

Endothelial dysfunction in type 1 diabetes

In order to establish if the disease duration influenced the results above, the diabetic group was further divided into two subgroups. The first subgroup comprised 22 children diagnosed with diabetes less than 5 years before the study (T1DM < 5, 14 males and 8 females, mean age 8.64 ± 1.79 years). The second group comprised 9 children diagnosed with diabetes more than 5 years before the study (T1DM ≥ 5, 5 males and 4 females, mean age 10.1 ± 1.27 years). When compared with the control group, both subgroups (T1DM < 5 and T1DM ≥ 5) continued to show significant p values for HbA1c (8.83 ± 1.56%, p = 0.015 and 9.57 ± 1.79%, p = 0.018, respectively) and fasting glucose (194.14 ± 103.94 mg/dL, p = 0.0002 and 170.11 ± 89.11 mg/dL, p = 0.002, respectively) when compared with the control group. Ultrasound studies Results obtained with the brachial artery FMD technique are shown in Table 3.

No significant differences were found in the percentage increase in the arterial FMD (%) between the control group (11.45 ± 2.86%) and subgroup T1DM < 5 (10.21 ± 2.70%, p = 0.078), but a significant difference was observed when the control group was compared with the subgroup T1DM ≥ 5 (7.04 ± 1.38%, p = 0.0001). The timing of the maximal dilation after stimulation did not change with the division into subgroups. There was no significant difference in the percentage of increase in FMD between genders (data not shown), both in the control group and diabetics subgroups in the four repeated measurements performed after post-occlusive reactive hyperemia. Similarly, no significant differences between groups were observed in the analysis of parameters related to the brachial artery, including systolic peak (SP), pulsatility index (PI), resistivity index (RI), maximum acceleration time (AT max), end diastolic speed (ED), and the ratio peak systolic velocity/peak diastolic velocity (S/D). The results from the measurements of the carotid arteries obtained with the IMT technique are shown in Table 4.

Table 2. Laboratory and demographic data of the study population

Female

Diabetics groups Controls (n = 58)

T1DM (n = 31)

T1DM < 5 (n = 22)

T1DM ≥ 5

P value

24 (41.4%)

12 (38.7%)

8 (36.4%)

4 (44.4%)

0.8068

8.36 (±1.81)

9.06 (±1.77)

8.64 (±1.79)

10.11 (±1.27)

0.0817

Controls

T1DM total

T1DM < 5

T1DM ≥ 5

Hemoglobin (mg/dL)

13.65 (±0.84)

13.70 (±0.90)

13.54 (±0.81)

14.10 (±1.03)

Age Laboratory parameters

Diabetic groups

P value 0.8060*

Hematocrit (%)

40.26 (±2.97)

40.30 (±2.32)

39.87 (±2.25)

41.36 (±2.26)

0.9402*

Glycated hemoglobin (%)

5.30 (±0.27)

9.04 (±1.64)

8.83 (±1.56)

9.57 (±1.79)

< 0.0001* 0.015** 0.018***

Total cholesterol (mg/dL)

166.95 (±22.14)

165.42 (±16.92)

165.27 (±17.53)

165.78 (±16.33)

0.7172*

63.80 (±16.7)

60.23 (±20.26)

57.95 (±18.35)

65.78 (±24.62)

0.0198*

Triglycerides (mg/dL) HDL cholesterol (mg/dL)

51.47 (±9.70)

53.87 (±8.66)

51.59 (±6.73)

59.44 (±10.64)

0.2357*

LDL cholesterol (mg/dL)

101.95 (±18.06)

99.84 (±17.09)

102.59 (±16.71)

93.11 (±17.05)

0.5884*

VLDL cholesterol (mg/dL)

12.60 (±3.33)

14.40 (±4.84)

11.59 (±3.69)

13.22 (±4.79)

0.0179*

Fasting glucose (mg/dL)

84.40 (±9.03)

187.16 (±99.01)

194.14 (±103.94)

170.11 (±89.11)

< 0.0001* 0.0002** 0.002***

*P values were obtained by comparing the mean (± standard deviation [SD]) values obtained in each group, applying Student’s t test. ** P values were obtained by comparing the mean (±SD) values obtained in the control group versus those in the T1DM < 5 subgroup. *** P values were obtained by comparing the mean (±SD) values obtained in the control group versus those in the T1DM ≥ 5 subgroup applying Student’s t test, chi-square test, and Kruskal-Wallis test. All results are expressed as mean ± SD values. Anthropometric data: percentiles (ranging from 50-85%) were normal for age and weight both in the diabetic and control groups. The mean body mass index (BMI) for the group of diabetic children was 16.9 kg/m2 and for nondiabetic children was 17.2 kg/m2. The mean weights in both groups were 31.5 kg and 33.4 kg, respectively, and the mean heights were 133.6 cm and 135.4 cm, respectively. Arch Endocrinol Metab. 2017;61/5

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Demographic parameters

Endothelial dysfunction in type 1 diabetes

Table 3. Ultrasonographic parameters showing the maximal percentage flow-mediated dilation (FMD) in the diabetic and control groups Diabetic groups

Parameters analyzed

Controls

T1DM total

Mean brachial artery baseline diameter (mm)

2.59 (±0.13)

Max FMD (%)

11.45 (±2.86)

1 (±1.13)

Max FMD time (min)

P value

T1DM < 5

T1DM ≥ 5

2.59 (±0.13)

2.58 (±0.13)

2.60 (±0.13)

0.887*

9.29 (±2.79)

10.21 (±2.70)

7.04 (±1.38)

0.0008* 0.078** 0.0001***

1(±1.69)

1(±1.81)

1 (±1.41)

0.753*

T1DM: type 1 diabetes mellitus; FMD: flow-mediated dilation; max FMD (%): maximal FMD (%); max FMD time: time to the occurrence of the maximal FMD. * P values were obtained by comparing the mean (± standard deviation [±SD]) values in the control group with those in the entire T1DM group, with the application of the Kruskal-Wallis test. ** P values were obtained by comparing the mean (±SD) values in the control group with those in the T1DM < 5 subgroup. *** P values were obtained by comparing the mean (±SD) values in the control group with those in the T1DM ≥ 5 subgroup, with the application of the Kruskal-Wallis test. All results are expressed in mean (±SD) values.

Table 4. Carotid artery intima-media thickness (IMT) measurements by the ultrasound technique in the diabetic and control groups Parameters

Diabetic groups

P value

Controls

T1DM total

T1DM < 5

T1DM ≥ 5

IMT LCA mean

0.55 (± 0.04)

0.56 (± 0.06)

0.55 (± 0.05)

0.57 (± 0.08)

0.762* 0.737** 0.208***

IMT RCA mean

0.55 (± 0.05)

0.56 (± 0.04)

0.54 (± 0.05)

0.359* 0.185** 0.805***

Max IMT LCA and RCA (mm)

0.61 (± 0.06)

0.61 (± 0.05)

0.60 (± 0.04)

0.63 (± 0.07)

0.826* 0.628** 0.252***

IMT: intima-media thickness; LCA: left carotid artery; RCA: right carotid artery; Max: maximal. * P values correspond to the comparison of the mean (± standard deviation [SD]) values in the entire T1DM group versus those in the control group. ** P values correspond to the comparison of the mean (±SD) values in the T1DM < 5 subgroup and those in the control group. *** P values correspond to the comparison of the mean (±SD) values in the T1DM ≥ 5 subgroup with those in the control group. The permutation test of Kruskal-Wallis was applied in the calculations. All results are expressed in mean (±SD) values.

Correlation between the variables FMD (%) and HbA1c

Pearson’s linear correlation coefficient, which evaluates the correlation between two measurable variables, was applied to determine the presence of a relationship between the FMD (%) and serum levels of HbA1c (Figure 1), and FMD (%) and fasting glucose (Figure 2). The test revealed a moderately negative correlation between FMD (%) and HbA1c (r = -0.36, p = 0.0025) and between FMD (%) and fasting glucose (r = -0.36, p = 0.0024) (Figure 2). As these correlations were negative, the coefficient suggested that the higher the level of HbA1c and fasting glucose, the lower the FMD (%), confirming our hypothesis.

DISCUSSION Macrovascular and microvascular complications are the main causes of morbidity and mortality in patients 480

with diabetes mellitus, and their main trigger may be represented by dysregulation of the modulatory function of the vascular endothelium (15). It is an accepted fact that T1DM predisposes to premature atherosclerotic artery disease and there is evidence that the endothelial function impairment precedes the establishment of this pathological process (16). The endothelial cells actively adjust the vascular tonus and reactivity in physiological and pathological conditions in response to mechanical stimuli and neurohumoral mediators with the release of a variety of factors leading to vascular constriction or relaxation. Additionally, these cells synthesize substances with a regulatory function on inflammation and hemostasis (3). Since the action of these cells can affect one or more functions simultaneously or sequentially, a gold-standard test to evaluate the presence of endothelial dysfunction has not yet been established (3). However, the endothelial function has been generally evaluated by changes in Arch Endocrinol Metab. 2017;61/5

blood flow or measurement of blood vessel diameter in response to mechanical or chemical stimulation, measured invasively (by coronary catheterization) or noninvasively (by ultrasound technique) (3). The latter was the method used in our study. r = -0.36, p = 0.0025

% FMD

20 15 10 5 0 0

HbA1c

Figure 1. Pearson’s linear correlation scatter plot of the percentage of maximal flow-mediated dilatation (FMD %) versus glycated hemoglobin (HbA1c) levels.

r = -0.36, p = 0.0024

% FMD

20 15 10 5 0 0

100

200

300

400

Glucose (fasting)

Figure 2. Pearson’s linear correlation scatter plot of the percentage of maximal flow-mediated dilation (FMD %) versus fasting glucose levels.

Our results revealed significant differences in relation to fasting glucose and HbA1c levels between the diabetic patients’ groups (T1DM < 5 and T1DM ≥ 5) and the control group (Table 2), reflecting a suboptimal glycemic control in diabetic patients independent of the time from diagnosis. A lasting hyperglycemic state can affect the endothelial function due to alteration of molecular regulatory mechanisms of the nitric oxide synthesis and/or degradation (4) and the onset of oxidative stress inducing a deleterious effect on the endothelial cells (17). This process involves four metabolic pathways, with activation of protein kinase C (PKC), the formation of AGEs, overactivity of the hexosamine pathway, and increased Arch Endocrinol Metab. 2017;61/5

flux of glucose through the polyol (sorbitol) pathway (1). Thus, the imbalance between the production of endothelium-derived factors impairs the vascular tonus and other physiological properties of the vascular endothelium (3), favoring vasoconstriction (18) and an inflammatory state (4), perpetuated by the long-lasting hyperglycemia (19). The results of Pearson’s correlation coefficients corroborate these claims in our population, suggesting a moderate association between decreased vasodilation and increased serum levels of HbA1c and fasting glucose (Figures 1 and 2, respectively). On reviewing the FMD technique protocol, we found a disagreement among several authors regarding the degree of occlusion to be applied. Corretti and cols. (10) and Barac and cols. (11) advocate inflating the blood pressure cuff to ≥ 50 mmHg above the patient’s systolic pressure, while Shivalkar and cols. (8) recommend inflating the cuff to 100 mmHg above the systolic pressure. In our pediatric population, we chose to inflate the cuff to 50 mmHg above the systolic pressure, considering that there was a consensus in this regard between two of the authors and because this pressure was more comfortable for the children. Another controversial point was the duration of the occlusion. Corretti and cols. (10) favored an occlusion duration of 5 minutes since changes in the vessel diameter are similar after 5 or 10 minutes and an occlusion for 5 minutes is more easily tolerated. However, in our experience, this recommendation was difficult to be carried out in our children, who showed considerable intolerance toward remaining with the cuff inflated 50 mmHg above the systolic pressure throughout the 5 minutes. Based in a previous study (8) that applied an occlusion duration of 4 minutes to adult patients obtaining good results, and considering that our patients were aged 6 to 12 years, we adopted this 4-minute occlusion time. This duration was well tolerated by our children and did not affect the test since the increase in the brachial artery diameter after cuff release occurs within 30 seconds to 5 minutes, and these changes in diameter are similar after 5 and 10 minutes (10). The maximal dilatation response in our diabetic and control children was obtained 1 minute after the hyperemic stimulus (Table 3). Previous studies have suggested that the maximal increase in arterial diameter occurs approximately 60 seconds after cuff release (20-22). Our results are in accordance with those obtained by other authors, and the baseline brachial 481

Endothelial dysfunction in type 1 diabetes

artery diameter was similar in all our groups (Table 3), thus eliminating any bias related to the baseline artery size since arteries with larger diameters show lower percentages of maximal dilation and vice versa (7). In spite of FMD abnormalities in the T1DM ≥ 5 subgroup when compared with controls (Table 3), we could not find in our study IMT abnormalities in any of the diabetic subgroups (Table 4), which suggests the inexistence of atherosclerotic structural alterations that usually evolve after the advent of endothelial dysfunction. Assessing our results and comparing our FMD and IMT findings with data reported in a previous study, we detected similar results in a group of diabetic children with a mean age of 14 years and a disease duration of 6 years. These children also failed to show vascular complications, although they presented impaired endothelial function assessed by FMD when compared with a control group (23). Similarly, a decreased FMD response was detected in a Finnish population with T1DM without microvascular complications, aged 7 to 14 years and with a mean disease duration of 4.4 ± 2.9 years. However, differently from our results, the subgroup with endothelial dysfunction in that study had significantly higher values of carotid IMT than the subgroup without endothelial dysfunction and the control group; no statistically significant differences were observed in the brachial artery baseline diameter, disease duration or HbA1c levels among diabetic children with and without endothelial dysfunction (6). In another study, Singh and cols. (24) also found no differences in carotid artery IMT between diabetic patients and control individuals, although the FMD detected was significantly lower in the diabetic group. These authors concluded that although the endothelial dysfunction appears during the first decade of T1DM onset, increased carotid IMT only occurs after a more extended time. In another, more recent study, Eltayeb and cols. (25) assessed the endothelial function and myocardial alterations in pediatric patients with T1DM aged 5 to 16 years, without microvascular complications, and with a duration of 1-4 years from the initial diagnosis. These authors also reported a maximal FMD significantly lower and carotid IMT significantly higher in the diabetic group when compared with the control group. Similarly, a Turkish T1DM study conducted by Çiftel and cols. (27) in a group of children aged 7 to 16 years with a disease duration of at least 5 years from the 482

diagnosis and no microvascular diabetic complications or additional cardiovascular risk showed a lower FMD and higher carotid IMT when comparing diabetic subjects with controls. A recent Italian longitudinal study by Bruzzi and cols. (28) demonstrated in a group of children and adolescents aged 5 to 18 years and with a mean time elapsed from diagnosis of 4 years, that after a mean follow-up of 3 years, the endothelial function deteriorates sharply and that the male gender is considered a negative predictor of impaired FMD over time. Additionally, these authors reported that the maintenance of blood glucose levels near the normal range apparently does not avert the evolution of diabetic vasculopathy, suggesting the influence of intrinsic (genetic) and extrinsic (nutritional factors, physical inactivity) factors in the establishment of the endothelial damage. The endothelial vasomotor activity is generally higher in females than males. Various hypotheses have been proposed to explain this fact: smaller body surface area, lower body mass index, and lower baseline diameter of the brachial artery in females when compared with males. Additionally, in prepubertal and adult subjects, estrogen can improve the endothelial function (29). No gender difference was detected in the vasodilatation response both in the T1DM and in the control group, probably due to similar baseline brachial artery diameters and lack of hormonal influence, since all children in our study were in the prepubertal stage. This study is relevant for assessing the presence of endothelial dysfunction in T1DM children from an early age to the prepubertal stage, with a wide range of disease duration, and by noninvasive methods, further evidencing an association of diabetes with early structural atherosclerotic alterations. However, the study has some limitations. It does not clarify if the assessment of the endothelial function by the FMD technique and the laboratory determination of adhesion molecules and other inflammatory factors are clinically useful tools to stratify the cardiovascular risk in T1DM population groups or individual children. It also does not determine if an improvement in endothelial function would decrease the risk of development of future vascular atherosclerotic complications. In conclusion, our study suggests that endothelial dysfunction without structural changes suggestive of atherosclerosis may appear in diabetic children with a history of 5 or more years of disease duration. In these Arch Endocrinol Metab. 2017;61/5

Endothelial dysfunction in type 1 diabetes

Acknowledgement: we wish to thank Prof. Dr. Katie Barbosa da Cruz and the Brasilia University Hospital endocrinology team, and Dr. Maristela Stephen Barbosa of the Brasilia Children Hospital, who kindly referred their patients for this study. We would also like to thank the entire staff of the Laboratory of Clinical Analysis of the Brasilia University Hospital, especially Sandra Ferreira and Maria Rosana Oliveira Borges Souza, Fábio Barros Almeida, and Jorge Mário Lopes da Silva, who collaborated on the collection and transport of the samples.

11. Barac A, Campia U, Panza JA. Methods for evaluating endothelial function in humans. Hypertension. 2007;49:748-60. 12. Giménez M, Gilabert R, Lara M, Conget I. Preclinical arterial disease in patients with type 1 diabetes without other major cardiovascular risk factors or micro-/macrovascular disease. Diab Vasc Dis Res. 2011;8(1):5-11. 13. Järvisalo MJ, Jartti L, Nänto-Salonen K, et al. Increased aortic intima-media thickness: a marker of preclinical atherosclerosis in high-risk children. Circulation. 2001;104(24):2943-7. 14. Bots ML, Hofman A, Grobbee DE. Increased common carotid intima-media thickness. Adaptive response or a reflection of atherosclerosis? Findings from the Rotterdam Study. Stroke. 1997;28(12):2442-7. 15. De Vriese AS, Verbeuren TJ, Van de Voorde J, et al. Endothelial dysfunction in diabetes. Br J Pharmacol. 2000;130(5):963-74. 16. Di Carli MF, Janisse J, Grunberger G, Ager J. Role of chronic hyperglycemia in the pathogenesis of coronary microvascular dysfunction in diabetes. J Am Coll Cardiol. 2003;41(8):1387-93. 17. Siegelaar SE, Holleman F, Hoekstra JBL, DeVries JH. Glucose variability; does it matter? Endocr Rev. 2010;31(2):171-82. 18. Drexler H. Endothelial dysfunction: clinical implications. Prog Cardiovasc Dis. 1997;39(4):287-324. 19. King GL.The role of hyperglycaemia and hyperinsulinemia in causing vascular dysfunction in diabetes. Ann Med. 1996;28:427-32.

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

20. Vogel RA. Measurement of endothelial function by brachial artery flow-mediated vasodilation. Am J Cardiol. 2001;88(2A):31E-34E.

REFERENCES

21. Uehata A, Lieberman EH, Gerhard MD, Anderson TJ, Ganz P, Polak JF, et al. Noninvasive assessment of endothelium-dependent ﬂow-mediated dilation of the brachial artery. Vasc Med. 1997;2(2):87-92.

1. Giannini C, Mohn A, Chiarelli F, Kelnar CJH. Macrovascular angiopathy in children and adolescents with type 1 diabetes. Diabetes Metab Res Rev. 2011;27(5):436-60. 2. Creager MA, Lüscher TF, Cosentino F, Beckman JA. Diabetes and vascular disease: pathophysiology, clinical consequences and medical therapy: party l. Circulation. 2003;108(12):1527-32. 3. Calles-Escandon J, Cipolla M. Diabetes and endothelial dysfunction: a clinical perspective. Endocr Rev. 2001;22(1):36-52. 4. Beckman JA, Creager MA, Libby P. Diabetes and atherosclerosis: epidemiology, pathophysiology, and management. JAMA. 2002;287(19):2570-81. 5. Sheetz MJ, King GI. Molecular understanding of hyperglycemia’s adverse effects for diabetic complications. JAMA. 2002;288(20):2579-88. 6. Järvisalo MJ, Raitakari M, Toikka JO, Putto-Laurila A, Rontu R, Laine S, et al. Endothelial dysfunction and increased arterial intima-media thickness in children with type 1 diabetes. Circulation. 2004;109(14):1750-5. 7. Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller OI, Sullivan ID, et al. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet. 1992;340(8828):1111-5. 8. Shivalkar B, Dhondt D, Goovaerts I, Van Gaal L, Bartunek J, Van Crombrugge P, et al. Flow mediated dilatation and cardiac function in type 1 diabetes mellitus. Am J Cardiol. 2006;97(1):77-82. 9. O’Leary DH, Polak JF, Kronmal RA, Manolio TA, Burke GL, Wolfson SK Jr. Cardiovascular health study collaborative research group. Carotid artery intima and media thickness as a risk factor for myocardial infarction and stroke in older adults. N Engl J Med. 1999;340(1):14-22. 10. Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbonneau F, Creager MA, et al. Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery. J Am Coll Cardiol. 2002;39(2):257-65. Arch Endocrinol Metab. 2017;61/5

22. Corretti MC, Plotnick GD, Vogel RA. Technical aspects of evaluating brachial artery vasodilatation using high-frequency ultrasound. Am J Physiol. 1995;268:H1397-404. 23. Wiltshire EJ, Gent R, Hirte C, Pena A, Thomas DW, Couper JJ. Endothelial dysfunction relates to folate status in children and adolescents with type 1 diabetes. Diabetes. 2002;51:2282-6. 24. Singh TP, Groehn H, Kazmers A. Vascular function and carotid intimal-medial thickness in children with insulin-dependent diabetes mellitus. J Am Coll Cardiol. 2003;41(4):661-5. 25. Eltayeb AA, Ahmad FA, Sayed DM, Osama AM. Subclinical vascular endothelial dysfunction and myocardial changes with type 1 diabetes mellitus in children and adolescents. Pediatr Cardiol. 2014;35:965-74. 26. From AM, Scott CG, Chen HH. Changes in diastolic dysfunction in diabetes mellitus overtime. Am J Cardiol. 2009;103(10):1463-6. 27. Çiftel M, Ertuğ H, Parlak M, Akçurin G, Kardelen F. Investigation of endothelial dysfunction and arterial stiffness in children with type 1 diabetes mellitus and the association with diastolic dysfunction. Diab Vasc Dis Res. 2014;11(1):19-25. 28. Bruzzi P, Predieri B, Patianna VD, Salvini A, Rossi R, Modena MG, et al. Longitudinal evaluation of endothelial function in children and adolescents with type 1 diabetes mellitus: a long-term follow-up study. Pediatr Int. 2014;56(2):188-95. 29. Mizia-Stec K, Gasior Z, Mizia M, Haberka M, Holecki M, Zwolińska W, et al. Flow-mediated dilation and gender in patients with coronary artery disease: arterial size influences gender differences in flow-mediated dilation. Echocardiography. 2007;24(10):1051-7. 30. Kuczmarski RJ, Ogden CL, Guo SS, Grummer-Strawn LM, Flegal KM, Mei Z, et al. 2000 CDC Growth Charts for the United States: methods and development. Vital Health Stat 11. 2002;(246):1-190. 31. Silva DA, Pelegrini A, Petroski EL, Gaya AC. Comparison between the growth of Brazilian children and adolescents and the reference growth charts: data from a Brazilian project. J Pediatr (Rio J). 2010;86(2):115-20.

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children, a decreased vasodilation response correlates with a higher degree of hyperglycemia. As the decreased reactivity of the brachial artery seems to improve with the modification of risk factors and drug treatment to reduce the cardiovascular risk, the acknowledgement of the presence of endothelial dysfunction in diabetic children allows the adoption of preventive strategies to mitigate the consequences of T1DM in this population, contributing to the reduction of the morbidity and mortality of the disease in adulthood.

original article

Effects of energetic restriction diet on butyrylcholinesterase in obese women from southern Brazil – A longitudinal study Willian dos Santos1, Luciane Viater Tureck1,2, Louise Farah Saliba1, Caroline Schovanz Schenknecht1, Débora Scaraboto1, Ricardo Lehtonen R. Souza1, Lupe Furtado-Alle1

ABSTRACT Laboratório de Polimorfismos e Ligação, Departamento de Genética, Universidade Federal do Paraná (UFPR), Curitiba, PR, Brasil 2 Departamento de Educação, Universidade Tecnológica Federal do Paraná (UTFPR), Curitiba, PR, Brasil 1

Correspondence to: Luciane Viater Tureck Rua Francisco H. dos Santos, 210 Centro Politécnico/Setor de Ciências Biológicas, sala 43 81531-970 – Curitiba, PR, Brasil Caixa postal 19071 luviater@gmail.com Received on Apr/11/2016 Accepted on Jan/13/2017 DOI: 10.1590/2359-3997000000268

Objective: Butyrylcholinesterase (BChE) activity has been associated with obesity, lipid concentrations, and CHE2 locus phenotypes. This, the aim of this study was to evaluate the effects of an energetic restriction diet intervention on anthropometrical and biochemical variables and on absolute and relative BChE activity in CHE2 C5+ and CHE2 C5- individuals. Subjects and methods: One hundred eleven premenopausal obese women from Southern Brazil participated in an energetic restriction diet intervention (deficit of 2500 kJ/day) for 8 weeks. Their anthropometric and biochemical parameters were evaluated before and after the intervention. Plasma BChE activity was measured, and BChE bands in plasma and CHE2 locus phenotypes were detected by electrophoresis. Results: The dietetic intervention decreased anthropometric and biochemical parameters as well as absolute BChE activity and relative activity of the G4 band. The CHE2 C5+ phenotype presented a different effect when compared with the CHE2 C5- phenotype. The CHE2 C5+ phenotype showed an effect in absolute BChE activity and in the relative activity of the G4 form, maintaining higher BChE activity regardless of the metabolic changes. Conclusion: In our study, 8 weeks was not sufficient time to lower the body mass index to normal, but it was enough to significantly reduce the absolute BChE activity, which became similar to the levels in nonobese individuals. CHE2 C5+ individuals were resistant to the decrease in BChE activity compared to CHE2 C5- individuals. This shows that the diet did not affect the CHE2 and G4 fraction complex and that the products of the CHE2 locus in association with BChE have a role in energy metabolism, maintaining high levels of enzymatic activity even after dietary intervention. Arch Endocrinol Metab. 2017;61(5):484-9 Keywords Obesity; dietetic intervention; butyrylcholinesterase activity; BChE molecular forms; lipid metabolism

INTRODUCTION

uman butyrylcholinesterase (BChE; EC 3.1.1.8) is an esterase encoded by the BCHE gene (3q26.1-q26.2) (1), synthesized in the liver, and distributed to several parts of the organism (2). Despite the fact that BChE’s main function and its natural substratum remain unknown, it has been related to lipid metabolism and has been associated with body mass index (BMI), waist-hip ratio, waist circumference, weight, and cholesterol and triglyceride levels (3-8). Plasmatic BChE is found in 5 different molecular structures: G1 (monomers), G1-A (monomers linked to albumin), G2 (dimers), G3 (trimers), and G4 (tetramers) (9). Chemically, G2 is formed by a disulfide bond between 2 G1 monomers (10), and G4 is formed by 2 G2 dimers bonded by noncovalent interactions (11).

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The organization and stabilization of G4 is mediated by lamellipodin proline rich peptides (12-14). In addition to these 4 basic BChE molecular forms, a protein can bind to the G4 tetramers and originate the C5 band, determining 2 phenotypes: CHE2 C5+ and CHE2 C5-, characterized by the presence and the absence of the extra C5 band, respectively (15). The frequency of the C5 band in a population sample of Southern Brazil was 10.3% (16). Although the protein associated with G4 to form the C5 band is not known yet, its coding gene has been mapped to chromosome 2q33-35 (CHE2 locus). CHE2 C5+ individuals show mean BChE activity approximately 30% higher than CHE2 C5- individuals (15,17). Despite the fact that the higher enzyme activity is related to weight, obesity, and BMI, CHE2 C5+ individuals presented a lower BMI and weight when compared with CHE2 Arch Endocrinol Metab. 2017;61/5

Diet restriction effect on butyrylcholinesterase

SUBJECTS AND METHODS Subjects This study was previously approved by the ethical committee of the Pontifical Catholic University of Paraná (PUC-PR), under registration 0005306/11. Women living in Curitiba and neighboring cities were invited to participate in this study via local radio and television ads, with the aim to reduce weight. Initially, we had a group of 199 obese women; at the end of the study, 111 completed the dietetic intervention. Only this group was statistically analyzed. All the women in the study had a BMI ≥ 30, being an inclusion criterion, along with being 20 years old or older, being in their reproductive period (not in menopause), not being pregnant, and not lactating. The women were excluded when they were already participating in a diet; using weight control medicaments; had a confirmed diagnosis of diabetes I, noncontrolled hypertension, hypothyroidism, or renal chronic disease; had undergone stomach reduction surgery; had been a vegetarian; and did not have the availability to attend the meetings at PUC-PR on Saturdays. The women who agreed to participate and who were in agreement with the study criteria read, accepted, and signed the terms of informed consent (IC). The 111 women who started and concluded the study had predominantly Euro-Brazilian ancestry (selfdeclared). Their ages ranged from 20 to 50 years (all in the premenopausal stage), and the predominant age range was 30-39 years (45% of participants). During the study, 70.4% of the women were employed (19). In the preintervention phase, 56% of the participants were class I obese (30-34.9 kg/m2), 24.8% were class II obese (35-39.9 kg/m2), and 19.2% were class III obese (≥ 40 kg/m2) (19).

and after the dietetic intervention. The diet comprised 9 weeks of energetic restriction, and the eating habits before the intervention were documented in an interview, during which participants were instructed to follow the diet. It was made an adequacy of the intake of macronutrients before the intervention to the current recommendations (20). The nutritional orientation was set up to provide a deficit of 2500 kJ/day, and the diet models were developed from the adapted protocol of diet intervention of Nugenob (21) and with the recommendations of the American Dietetic Association (22). The nutritional percentage of each macronutrient was recommended as follows: the energy from fat was in the range of 20% to 35%, from carbohydrates in the range of 45% to 65%, and from proteins in the range of 10% to 35% (23).

Biochemical and anthropometric parameters Blood samples were collected after the agreement of participation and instruction of 8 to 12 hours of fasting at 2 instances: before and after the dietetic intervention. Biochemical parameters, total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), and triglycerides (TG) were obtained by standard automated methods. Weight and height were measured with an accuracy of 0.1 kg and 0.1 cm, respectively. BMI, waist circumference (WC), and abdominal circumference (AC) were also measured. The abdominal-stature ratio (ASR) and waist-stature ratio (WSR) were obtained from these measurements.

BChE activity analysis and detection of BChE bands Plasma BChE activity was measured using the protocol of Dietz and cols. (24) modified by Evans and Wroe (25) CHE2 locus phenotypes were identified by acid agar gel electrophoresis (pH: 6.40) (26). The detection of BChE bands in plasma was made according to Boberg and cols. (27). The relative intensity (RI) of each band was measured by KODAK 1D Image Analysis Software, and the relative activity (RA) of each band was the result of multiplication of the total BChE plasma activity by the RI of each band. Samples without any detectable BChE band were excluded from the analysis.

Study design and energetic restriction diet

Statistics

The study design was longitudinal, or a quasi-experimental intervention, measured using an analysis of variables before

The phenotype frequencies of CHE2 C5- and CHE2 C5+ were obtained by direct counting. The comparisons

Arch Endocrinol Metab. 2017;61/5

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C5- individuals, suggesting that the presence of the C5 complex has a protective effect against obesity (15,18). Considering the evidence connecting BChE with obesity, the aim of this work is to investigate the effects of an energetic restriction diet on total and relative BChE activity in relation to the CHE2 C5 phenotype in obese women from Southern Brazil.

Diet restriction effect on butyrylcholinesterase

between means (biochemical and anthropometric parameters and BChE activity) were performed by t test (parametric variables) or by Mann-Whitney test (nonparametric and independent variables). Multiple regression analysis was performed to verify the interaction between variables. The probability value for the comparative tests was considered significant at p < 0.05 (5%).

RESULTS The anthropometric variables WC, AC, and BMI suffered a significant decrease after the intervention, as did the mean plasma absolute BChE activity (Table 1). HDL-C was the only fraction that showed a significant decrease; the values of the other biochemical variables (TG, TC, and LDL-C) showed no significant changes (Table 1). Multiple regression analysis was performed to verify the independent effect of BChE molecular forms (G1, G1-A, G2, G3, and G4), before and after dietetic intervention, on the lipid profile (TC, TG, HDL-C,

and LDL-C) and anthropometrical parameters (BMI, WSR, and ASR). Before the intervention, G2 acted independently in the determination of TC levels and WSR. After the intervention, G2 showed an independent effect on ASR. G1-A showed an independent effect on WSR before the intervention and on TC levels after. G1 showed an independent effect on WSR before the intervention (Table 2). The phenotype frequencies of CHE2 C5- and CHE2 C5+ were, respectively, 90.84% ± 2.53 and 9.16% ± 2.52. The comparison of mean absolute BChE activity between CHE2 C5+ and CHE2 C5phenotypes before the intervention did not show a difference in the sample of studied women. After the intervention, only the CHE2 C5- phenotype suffered a significant decrease, and CHE2 C5+, besides not showing a decrease, presented approximately 23% higher activity than CHE2 C5- (Table 3). Before the intervention, there was no difference between the RA of G4 of CHE2 C5+ and of CHE2 C5- individuals. After the intervention, CHE2 C5+ individuals maintained their RA of G4, while it

Table 1. Means (± SE) of the parameters before and after the dietetic intervention and their comparisons (P) Parameters

Before

After

Weight (kg)

90.76 ± 14.62

88.93 ± 15.19

0.29

Waist circumference (cm)

95.70 ± 9.88

91.36 ± 13.24

0.0008a

109.44 ± 11.55

102.05 ± 10.84

< 0.0001a

BMI (kg/m²)

35.14 ± 5.32

33.81 ± 6.04

< 0.0001a

TC (mg/dL)

192.94 ± 37.98

192.72 ± 38.09

0.32

HDL-C (mg/dL)

52.29 ± 12.78

47.68 ± 10.99

< 0.0001a

LDL-C (mg/dL)

112.63 ± 30.79

119.34 ± 33.67

0.09

TG (mg/dL)

140.25 ± 62.48

143.3 ±74.5

0.44

5.1 ± 1.53

4.88 ± 1.04

0.01a

Abdominal circumference (cm)

BChE absolute activity (KU/L)

BChE: butyrylcholinesterase; BMI: body mass index; HDL-C: high density lipoprotein cholesterol; LDL-C: low density lipoprotein cholesterol; TC: total cholesterol; TG: triglyceride. a Significant values.

Table 2. Results from multiple regression analysis Dependent variables

Before intervention

After intervention

Lipid profile and anthropometrical parameters

Independent variables considered

G1, G1-A, G2, G3, and G4

Confirmed effect

G2 on TC and WSR

0.26 and 0.51, respectively

0.02 and 0.04, respectively

G1 on WSR

0.56

0.003

G1-A on WSR

0.58

0.001

G2 on ASR G1-A on TC

0.35 0.28

0.001 0.01

ASR: abdominal-stature ratio; TC: total cholesterol; WSR: waist-stature ratio.

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decreased in CHE2 C5- individuals (Table 4). The analysis of total BChE activity also showed that the CHE2 C5- phenotype was the only one affected by the dietetic intervention (Table 3), reinforcing that there is a different biochemical interaction in the CHE2 C5+ phenotype regarding the effects of an energetic restriction diet intervention. On the other hand, the RA did not show a significant decrease after the intervention or a difference between the CHE2 phenotypes for the G2, G1-Alb, and G1 bands (Table 4), suggesting that the dietetic intervention was not effective on these specific bands. Table 3. Comparison of BChE activity (± SD) between CHE2 C5- and CHE2 C5+ phenotypes before and after dietary intervention (a) and between the same phenotype in response to dietary intervention (b) Phenotype

After

(P)b

CHE2 C5-

5.03 ± 1.56

4.78 ± 1.0

0.037*

CHE2 C5+

5.74 ± 2.14

5.89 ± 1.24

0.69

0.13

0.002*

(P) *

Before

Significant values.

Table 4. Comparison (P) of BChE bands’ relative activity (KU/L) before and after dietetic intervention and between CHE2 C5+ and CHE2 C5phenotypes CHE2 C5-

CHE2 C5+

(P)

G4 (before)

3.36SD1.25

3.91SD1.86

0.24

G4 (after)

3.07SD0.81

3.89SD1.0

0.006a

0.02

0.97

G2 (before)

0.55SD0.35

0.56SD0.32

0.93

G2 (after)

0.58SD0.32

0.72SD0.42

0.22

(P)

0.37

0.24

G1-A (before)

0.72SD0.38

0.79SD0.39

0.6

G1-A (after)

0.71SD0.44

0.73SD0.35

0.92

0.78

0.37

G1 (before)

0.46SD0.34

0.36SD0.29

0.41

G1 (after)

0.44SD0.32

0.44SD0.27

0.99

0.52

0.38

(P)

(P) a

Significant values.

DISCUSSION Anthropometrically, this 8-week diet was efficient, leading to a reduction of WC, AC, and BMI, however, not enough to decrease the BMI to the normal range, but it was sufficient to decrease the mean plasma absolute BChE activity significantly. These results suggest that there is a significant influence of the diet intake on plasma BChE activity, supporting the role of BChE in lipid metabolism and obesity (15,18,28-30). Arch Endocrinol Metab. 2017;61/5

It has been suggested that the role of BChE in lipid metabolism could be the hydrolysis of choline esters, which are results of the nonesterified fatty acid metabolism and liver lipogenesis (28-30). Considering that in obesity the increased lipogenesis from carbohydrates leads to hyperlipidemia and to increased BChE activity, it could also explain why the energetic restriction diet, due to the decreased availability of carbohydrates in the organism, decreased BChE activity. In our study, after the dietary intervention, the BChE activity of the obese women became similar to the described mean activity of nonobese women from Southern Brazil (4.68 ± 1.51 KU/L) (31). The fact that the HDL-C levels decreased after the intervention can be explained by a possible lack of physical exercise (PE) during the dietetic intervention, although this is only a nontestable hypothesis since we do not have systematized data that allow inference of the level of physical activity of the women who composed our sample. The lack of PE may cause an excessive formation of ammonia that leads to fatigue and therefore an even greater decrease of PE habits (32) Possibly, other parameters (TG, TC, and LDL-C) would need a longer or a stronger intervention to decrease their values, in contrast to Silva and cols. (33), who demonstrated a significant reduction of biochemical parameters after a 12-week (PE) intervention in obese adolescents. Considering that both interventions decreased BChE activity, it is possible that a combination of PE and diet could be ideal (33). The relations confirmed by multiple regression analysis between molecular forms of BChE and anthropometrical parameters and TC before (G2 on TC, β = 0.26 and p = 0.02) and after (G1-A on TC, β = 0.28 and p = 0.01) the intervention are mainly due to its relation with lipid metabolism, BMI, and WC, as related before and in other studies (34-38). As for the BChE molecular form’s individual effect on enzymatic activity, our results are different from the decreased RA of all BChE bands observed by Silva and cols. (33), probably due to the higher effect and duration of PE intervention. In the present study, only the RA of the G4 band showed reduction after the intervention, probably because the G4 band is the most abundant and therefore responsible for most of the plasma BChE activity. The phenotype frequencies of CHE2 C5- and CHE2 C5+ are in accordance with the frequency of CHE2 C5+ 487

Diet restriction effect on butyrylcholinesterase

in a population sample from Southern Brazil (10.3%; SD = 0.60; p = 0.89) (16). Previously, the phenotype CHE2 C5+ was associated with increased BChE activity but with a lower weight (16) and BMI (18). The resistance of CHE2 C5+ individuals to BChE activity decreased after the intervention compared to CHE2 C5- individuals, which is in accordance with other works, which showed increased BChE activity in the CHE2 C5+ phenotype (15,18) and also suggest that the intervention did not affect the complex G4 associated with the CHE2 protein, highlighting the importance of understanding the biochemical interactions of the CHE2 C5+ phenotype and BChE activity. Among the limiting factors of our study was the relatively small sample size, which diminished the power of the study and may have contributed to the nonidentification of effects, especially of smaller magnitudes. Another limiting factor may be that we did not combine dietary intervention with an exercise program to maximize some results. However, our study presents original and specific data about the CHE2 C5 phenotype’s influence on the dietetic intervention’s effect on total and relative BChE activity. Other studies are still required for a full understanding of the interaction between the CHE2 locus products and BChE concerning the response to metabolic changes. These data helped to increase the knowledge about the role of BChE in obesity, showing how much the levels of enzyme activity are influenced by diet, independent of BMI entering in normal threshold. In conclusion, after the 8-week diet, a decrease in the BMI, WC, AC, and HDL-C was observed, establishing a relation between the dietetic intervention and the decrease of plasma absolute BChE activity. However, CHE2 C5+ individuals were resistant to this decrease in enzyme activity, maintaining a high level of the RA of G4, which provides the major portion of BChE’s active form, suggesting that the CHE2 locus holds a strong biochemical relation with increased BChE activity, maintaining its elevated level even after the energetic restriction.

Acknowledgements: Diagnósticos do Brasil (DB) clinical laboratory performed the automated measurements of biochemical parameters. Financing statement: grants and scholarships were received by Araucaria Foundation and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes). Disclosure: no potential conflict of interest relevant to this article was reported. 488

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and the response to a weight loss diet intervention in adult women. Genet Mol Biol. 2014;37(1):15-22. 20. Board, NAoSIoMFaN. Dietary Reference Intakes For Energy, Carbohydrates, Fiber, Fat, Fatty Acids, Cholesterol, Protein and Amino acids. Washington DC, The National Academies Press (NAP), (2002/2005). 21. Petersen M, Taylor MA, Saris WH, Verdich C, Toubro S, Macdonald I. Randomized, multi-center trial of two hypo-energetic diets in obese subjects: high- versus low-fat content. Int J Obes. 2006;30(3):552-60. 22. Seagle HM, Strain GW, Makris A, Reeves RS, American Dietetic Association: Position of the American Dietetic Association: weight management. J Am Diet Assoc. 2009;109(2):330-346. 23. Macronutrients, ARotPo, SoURLo, et al. Dietary Reference Intakes For Energy, Carbohydrates, Fiber, Fat, Fatty Acids, Cholesterol, Protein and Amino acids. Washington DC, The National Academies Press (NAP), 2005. 24. Dietz AA, Rubinstein HM, Lubrano T, Hodges LK. Improved method for the differentiation of cholinesterase variants. Am J Genet. 1972;24:58-64. 25. Evans RT, Wroe J. Is serum cholinesterase activity a predictor of succinyl choline sensitivity? An assessment of four methods. Clin Chem. 1978;24:1762-6. 26. Van Ros G, VervootT. Frequencies of the “atypical” and C5 variants of serum cholinesterase in Zairians and Belgians. Detection of the C5 variant by agar gel electrophoresis with an acid buffer. Ann Soc Belg Med Trop. 1973;53:633-44. 27. Boberg DR, Furtado-Alle L, Souza RLR, Chautard-Freire-Maia EA. Molecular forms of butyrylcholinesterase and obesity. Genet Mol Biol. 2010;33:452-4.

38. Furtado-Alle L, Andrade FA, Nunes K, Mikami LR, Souza RLR, Chautard-Freire-Maia EA. Association of variants of the -116 site of the butyrylcholineterase BCHE gene to enzyme activity and body mass index. Chem Biol Inter. 2008;175:115-8.

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29. Chu MI, Fontaine P, Kutty KM, Murphy D, Redheendran R. Cholinesterase in serum and low density lipoprotein of hyperlipidemic patients. Clin Chim Acta. 1978;85:55-9. 30. Kutty KM, Huang SN, Kean KT. Pseudocholinesterase in obesity: hypercaloric diet induced changes in experimental obese mice. Experimentia. 1981;37:1141-2. 31. Guimarães LO, de Andrade FA, Bono GF, Setoguchi TE, Brandão MB, Chautard-Freire-Maia EA, et al. Gestational diabetes mellitus (GDM) decreases butyrylcholinesterase (BChE) activity and changes its relationship with lipids. Genet Mol Biol. 2014;37(1):1-6. 32. Franco LDP, Campos JADB, Demonte A. Teor lipídico da dieta, lipídios séricos e peso corporal em ratos exercitados. Ver Nutr. 2009;22(3):359-66. 33. Silva IMW, Leite N, Boberg D, Chaves TJ, Eisfeld GM, Eisfeld GM. Effects of physical exercise on butyrylcholinesterase in obese adolescents. Genet Mol Biol. 2012;35(4):741-2. 34. Alcântara VM, Oliveira LC, Réa RR, Suplicy HL, Chautard-FreireMaia EA. Butyrylcholinesterase activity and metabolic syndrome in obese patients. Clin Chem Lab Med. 2005;43(3):285-8. 35. Randell EW, Mathews MS, Zhang H, Seraj JS, Sun G. Relationship between serum butyrylcholinesterase and metabolic syndrome. Clin Biochem. 2005;38(9):799-805. 36. Souza RL, Mikami LR, Maegawa OB, Chautard-FreireMaia EA. Four new mutations in the BCHE gene of human butyrylcholinesterase in a Brazilian blood donor sample. Mol Genet Metab. 2005;84(4):349-53. 37. Iwasaki T, Yoneda M, Nakajima A, Terauchi Y. Serum butyrylcholinesterase is strongly associated with adiposity, the serum lipid profile and insulin resistance. Intern Med. 2007;46(19):1633-9.

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489

review

The clinical genetics of phaeochromocytoma and paraganglioma P. T. Kavinga Gunawardane1, Ashley Grossman1,2

ABSTRACT Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, UK 2 Green Templeton College, University of Oxford, UK 1

Correspondence to: Ashley Grossman Neuroendocrine Tumour Unit Royal Free Hospital London NW3 2QG UK ashley.grossman@ocdem.ox.ac.uk Received on Sept/23/2017 Accepted on Sept/26/2017 DOI: 10.1590/2359-3997000000299

Phaeochromocytoma and paraganglioma are rare catecholamine-producing tumours, recognised to have one of the richest hereditary backgrounds of all neoplasms, with germline mutations seen in approximately 30% of patients. They can be a part of genetic syndromes such as MEN 2 or Neurofibromatosis type 1, or can be found as apparently sporadic tumours. Germline mutations are almost always found in syndromic patients. Nonetheless, apparently sporadic phaeochromocytoma too show high germline mutation rates. Early detection of a genetic mutation can lead to early diagnosis of further tumours via surveillance, early treatment and better prognosis. Apart from this, the genetic profile has important relevance for tumour location and biochemical profile, and can be a useful predictor of future tumour behaviour. It also enables family screening and surveillance. Moreover, recent studies have demonstrated significant driver somatic mutations in up to 75% of all tumours. Arch Endocrinol Metab. 2017;61(5):490-500 Keywords

Phaeochromocytoma; paraganglioma; genetics

INTRODUCTION

haeochromocytomas and paragangliomas are uncommon tumours originating from the neural crest-derived chromaffin cells of the adrenal medulla and sympathetic/parasympathetic ganglia respectively. The highest prevalence of phaeochromocytoma is in the fourth and fifth decades, while its incidence is equal in men and women. Malignant phaeochromocytoma is defined by the presence of distant metastases in nonchromaffin tissues, which only account for about 15-20% of lesions (1). Although a majority of these catecholamine secreting tumours are by definition non-malignant, most of them secrete an excess of one or more catecholamines: epinephrine (adrenaline), norepinephrine (noradrenaline) or dopamine, which gives rise to a wide array of clinical complications, including resistant hypertension, tachyarrhythmia and cardiomyopathy. The genetic nature of these catecholamine secreting tumours has been an area of extensive research interest over the last few decades, and as a result multiple genes have been identified in association with phaeochromocytoma as well as paraganglioma. Therefore, in contrast to conventional 490

teaching of a 10% familial tendency (“the 10% rule”), phaeochromocytoma has now been shown to have a much higher genetic tendency with more than one third of patients harbouring a disease-causing germline mutation (2). As these tumours are recognised to have one of the richest hereditary backgrounds among all neoplasms, most authorities and guidelines currently recommend genetic testing of all patients for the presence of disease-causing mutations (3).

CLINICAL IMPLICATIONS OF GENETIC TESTING Genetic analysis in phaeochromocytoma is an extremely useful tool in clinical practise as accumulating data have shown genetics to be equally valuable not only for screening but also for diagnosis and prognostication of hereditary phaeochromocytoma. Firstly, differentiation between the benign and malignant nature of a phaeochromocytoma can be a challenge to the managing physician. Genetic evaluation can be of assistance in this situation, where one can predict a higher tendency towards the development of malignant disease with metastases in patients harbouring certain mutations (e.g. mutations of SDHB lead to metastatic disease in 40% or more of Arch Endocrinol Metab. 2017;61/5

Genetics of phaeochromocytomas

PATHOGENESIS: GENETIC GERMLINE HETEROGENEITY The pathogenesis of the hereditary nature of phaeochromocytoma can be described in two main clusters (6). The first cluster contains pseudohypoxiadriven tumours including VHL, SDH, EGLN1 and HIF2A mutant tumours. The second cluster contained the kinase signalling subgroup including the RET, NF1, TMEM 127 and MAX mutant tumours. The feature common to all cluster 1 tumours is the activation of HIFs. Hypoxia inducible factors (HIFs) are transcription factors induced as a physiological response to cellular hypoxia. In the presence of VHL, SDH, EGLN1 and HIF2A mutations, HIFs are induced and stabilised, pointing the cell towards a pseudo-hypoxic state. Pseudohypoxia occurs when HIF pathways are constitutively activated, regardless of oxygen levels. This cellular pseudohypoxia leads to epigenetic modifications in HIF target genes affecting multiple cellular processes including apoptosis, angiogenesis, proliferation, migration, and invasion. The second cluster of genes cause catecholamine secreting tumours by way of affecting the kinase signalling pathways. Activation of RET proto-oncogene in MEN 2 and inactivation of NF1 leads to activation of RAS/MAPK and PI3/AKT signalling pathways. Similarly, TMEM127 mutation activates the mTOR pathway while MAX mutation too has been established to affect the downstream mTOR pathway via the MYCMAX- MXD1 network. However, the pathogenesis of phaeochromocytoma may not be quite as simple, where there can be significant Arch Endocrinol Metab. 2017;61/5

overlap due to high degree of redundancy and cross-talk between constituents of these pathways. For example, mTOR can activate HIF, while MYC cooperates with HIF2α in oncogenesis (6). Furthermore, there is increasing evidence that SDH and related mutations can lead to the build-up of succinate which can act as an oncometabolite causing marked changing in patterns of gene methylation.

FAMILIAL SYNDROMES ASSOCIATED WITH PHAEOCHROMOCYTOMA/PARAGANGLIOMA Multiple endocrine neoplasia-2 (MEN 2) Multiple endocrine neoplasia-2 is one of the earliest syndromes to have been associated with phaeochromocytoma and is caused by an activating (gain-of-function) germline mutation in the RET proto-oncogene located on chromosome 10q11.2. This proto oncogene encodes a transmembrane receptor tyrosine kinase involved in the regulation of cell proliferation and apoptosis (7). Sipple first described an association between thyroid cancer and phaeochromocytoma in 1961 and since then this familial constellation of pathology has been studied extensively, including the identification of the underlying germline mutation. Clinically, there are three main subtypes of MEN 2; 1) MEN2A is characterised by medullary thyroid cancer in 95% of patients, phaeochromocytoma in 40-50% and primary hyperparathyroidism in 20%-30%; 2) MEN2B accounts for approximately 5% of MEN syndromes and has medullary thyroid cancer in 100%, phaeochromocytoma in 50% of cases, a Marfanoid body habitus, and multiple mucosal ganglioneuromas; however, it is not associated with hyperparathyroidism. 3); the third group is the rarest RET proto-oncogene associated MEN2 which represents familial medullary thyroid cancer alone (8,9). Identification of phaeochromocytoma is vital in these patients with MEN2 to avoid perioperative hypertensive crisis during thyroidectomy for medullary thyroid carcinoma. The genetic defect in MEN 2 is inherited in an autosomal dominant pattern with high penetrance. In MEN 2, clinical heterogeneity has been noted due to mutations in several codons in the RET gene: the great majority of MEN 2A (now changed simply to MEN2) are associated with a mutation at codon 634, exon11 which codes for the extra-cellular domain of RET, while 491

affected patients, or less commonly seen MAX and FH mutations) (4,5). In fact, germline genetic forms of phaeochromocytoma are often multiple, extra-adrenal and recurrent; consequently, regular surveillance and strict follow-up is recommended for better prognosis of such patients. Secondly, establishing certain hereditary syndromes with associated tumours with a high malignant potential (e.g. patients with MEN 2 – 100% potential to develop medullary carcinoma of thyroid) can lead to early diagnosis and treatment of other malignant syndromic manifestations in patients and relatives. Finally, identification of germline mutations of phaeochromocytoma can lead to early diagnosis and treatment, offering better prognosis to family members via screening and surveillance.

Genetics of phaeochromocytomas

for MEN 2B (now MEN3) the dominant mutation lies in codon 918, exon 16 which codes for part of the intracellular domain. In MEN 2A the RET mutation occur in the extracellular domain of the RET and causes ligandindependent activation of PI3K–AKT, RAS, p38 MAPK and JUN N-terminal kinase pathways, resulting in the stimulation of cell growth, differentiation and survival. On the other hand, MEN2B-related mutations target a few codons affecting the catalytic site of the kinase, leading to loss of substrate specificity only. Therefore, it has been established that the subtle changes in the clinical presentation and molecular outcome is due to these genetic variations in the mutations (10). Phaeochromocytomas seen in MEN 2 are frequently bilateral, adrenal in localisation and almost always benign (11) with the rate of malignant transformation being between 1 to 5%. However, it has been reported that children with phaeochromocytoma diagnosed with MEN2B have a higher risk of harbouring a malignant phaeochromocytoma compared to children with MEN2A or sporadic phaeochromocytoma (12). The biochemical phenotype is also rather different in patients with phaeochromocytoma associated with MEN2. They commonly overexpress phenylethanolamine N-methyltransferase which is an enzyme that converts norepinephrine to epinephrine, leading to hypersecretion of epinephrine in large amounts. This is consistent with increased levels of metanephrine, which is a catecholamine O-methylated metabolite of epinephrine, detected in plasma and excreted in urine in these patients (13). Interestingly, only half of the patients with MEN2A harbouring a phaeochromocytoma present with it, which might be explained by earlier presentation with medullary carcinoma of the thyroid or family screening (8). Genetic identification is also important as children born with the codon 634 mutation are advised to undergo total thyroidectomy before the age of 5 years, while with 918 mutations thyroidectomy in the first year is recommended. With other mutations, it is suggested that the specific published data on such families are explored for prognosis and therapeutic options.

NEUROFIBROMATOSIS TYPE 1 (NF1) NF1 or von Recklinghausen’s disease is another autosomal dominant disorder, characterized by neurofibromas, café-au-lait spots, freckling, Lisch 492

nodules, phaeochromocytoma and paraganglioma: 0.1 to 5.7% of patients with the NF1 gene present with solitary and benign phaeochromocytomas (14). NF1 is due to an inactivating mutation in the tumour suppressor gene NF1, located on chromosome 17q11.2. The NF1 gene encodes a large, 327 kDa protein called neurofibromin, belonging to a family of GTPase-activating proteins (GAP). This protein downregulates a cellular proto-oncogene, p21-RAS. RAS is a major oncogene in human malignancies. It is well known to regulate cell growth and differentiation, and activates a number of signalling pathways including the stem cell factor, mTOR, and MAP kinase pathways. mTOR is a crucial downstream signal of both RAS and RET pathways, and is aberrantly activated in NF1deficient malignant peripheral nerve sheath tumours, phaeochromocytomas and paragangliomas (15). Fifty per cent of phaeochromocytomas in NF1 are familial while the rest are due to de novo mutations (16). Familial NF1 shows “complete penetrance”, where the individual carrying the mutation will be almost always affected by it. However, it is highly variable in its “expression”, indicating that the severity of disease of the affected individuals can vary marked within families (17). Since the cloning of the NF1 gene in 1990, numerous constitutional mutations of patients have been described (Upadhyaya and Cooper 1998, NNFF International NF1 Genetic Mutation Analysis Consortium, Human Gene Mutation Database Cardiff) including cytogenetically visible translocations, intronic rearrangements affecting splicing, deletions, duplications, insertions; and many different point mutations and substitutions (18). Although many mutations have been identified in association with NF1 there is still no conclusive evidence to correlate the genotype with the phenotype or predict clinical risk factors with certain mutations (19). The diagnosis of NF1 is based on multiple cutaneous and bony lesions (Table 1). Patients with NF1 have an increased frequency to develop both benign and malignant tumours. Optic path gliomas are the predominant type of central nervous system tumours. Patients can also develop astrocytomas, brain stem gliomas, insulinomas and soft tissue sarcomas. Phaeochromocytoma is a rare but important manifestation of NF1 which usually presents in fourth or fifth decade, by which time most patient would have developed some form of a cutaneous manifestation Arch Endocrinol Metab. 2017;61/5

Genetics of phaeochromocytomas

Table 1. NIH diagnostic criteria for neurofibromatosis type 1 (20) Two or more of the following clinical features must be present: Six or more café-au-lait macules of more than 5 mm in greatest diameter in pre-pubertal individuals, and more than 15 mm in greatest diameter in post-pubertal individuals Two or more neurofibromas of any type or one plexiform neurofibroma Freckling in the axillary or inguinal regions Optic glioma Two or more iris hamartomata (Lisch nodules) Distinctive bony lesion such as sphenoid dysplasia, or thinning of the long bone cortex with or without pseudo-arthrosis A first-degree relative (parent, sibling, or offspring) with NF1 based on the above criteria

VON HIPPEL-LINDAU (VHL) SYNDROME VHL is a rare (incidence of 1:36,000 in the general population) autosomal-dominant inherited syndrome associated with the development of a variety of benign and malignant tumours. Families and individuals with VHL have been divided into types 1 and 2, based on their likelihood of developing phaeochromocytoma. Patients with type 1 VHL have a low likelihood of developing phaeochromocytoma, although they are at a higher risk of developing other VHL-associated tumours. Families with type 2 disease are at an increased risk of developing phaeochromocytoma. Type 2 is again divided into 3 groups: 2A phaeochromocytoma with low incidence of renal cell carcinoma (RCC), 2B phaeochromocytoma with high incidence of RCC, 2C only develop phaeochromocytoma as apparent sporadic tumours. These sub classifications are used as a guide and are not by any means absolute. In general, mutations which lead to complete loss of function tend not to be associated with phaeochromocytomas. VHL-related lesions occur at a wide range of ages with the retinal lesions commencing at a very young age. Patients need to be screened for CNS haemangioblastoma, retinal angioma, clear cell renal cell carcinoma, pancreatic neuroendocrine tumours (which are seen in around 10%) and middle ear tumours regularly. Haemangioblastomas are the most common lesions associated with VHL disease, affecting 60 to 84%, typically occurring in the cerebellum or spinal cord (22). The incidence of development retinal Arch Endocrinol Metab. 2017;61/5

capillary haemangioblastomas increases with age where 70% of VHL patients will harbour multifocal, bilateral lesions by the age of 60 (23). Almost all RCC in VHL are clear cell carcinoma with a mean age of onset of 44 years. VHL is caused by a heterozygous germline mutation on the VHL tumour suppressor gene on chromosome 3p25.5 and contains three exons. The VHL gene encodes two proteins, pVHL30, pVHL19. They are “substrate recognition components” which target HIF1α and HIF2α for proteasomal-mediated degradation. Therefore, loss of function of VHL leads to inappropriate accumulation of HIF and subsequent activation of the hypoxic response, promoting angiogenesis, glycolysis and proliferation. This explains the predisposition for patients to develop vascular and other types of tumours in VHL syndrome (24). In VHL syndrome, catecholamine-secreting tumours develop in 10-20% with a mean age of presentation of 30 years (25). They are more frequently benign, intraadrenal and bilateral. However, rarely mediastinal, abdominal and pelvic sympathetic paragangliomas as well as head and neck parasympathetic paragangliomas have also been reported (26). Interestingly, patients harbouring the VHL mutation have a lower incidence of hypertension and have specifically elevated normetanephrine, in contrast to patients with MEN-2 and NF, who show elevated metanephrine levels (13,27).

FAMILIAL CATECHOLAMINE-HYPERSECRETING TUMOURS IN SUCCINATE DEHYDROGENASE (SDH) GENE MUTATION SDH is an enzyme complex on the inner mitochondrial membrane with 4 subunits, SDHA, SDHB, SDHC, and SDHD. This enzyme complex catalyses the important oxidation of succinate to fumarate in the Kreb cycle with the reduction of ubiquinone to ubiquinol via the mitochondrial respiratory chain. The four subunits of the enzyme complex are encoded by four SDH genes – SDHA, SDHB, SDHC, and SDHD. SDHA and SDHB are the hydrophilic subunits responsible for the catalytic process of the SDH enzyme complex. SDHA is a flavoprotein and SDHB is an ironsulphur protein. SDHC and SDHD, on the other hand, are hydrophobic and act as the two anchorage proteins. Apart from these four proteins a fifth factor, succinate dehydrogenase complex-assembly factor 2 (SDHAF2), essential for the proper function of the SDHA subunit 493

of NF1. Mostly phaeochromocytoma in NF1 are benign and unilateral; however, they can occasionally be bilateral or extra-adrenal and up to 12% of these phaeochromocytomas can be malignant (14,21).

Genetics of phaeochromocytomas

(cofactor of flavin adenine dinucleotide), has now been recognised. SDHAF2 is encoded by SDHAF2 gene which, similar to genetic defects in other SDH gene defects can cause familial catecholamine-hypersecreting tumours. Apart from catecholamine secreting tumours, genetic defects in the SDH complex less frequently gives rise to renal cell carcinomas and gastrointestinal stromal tumours (GISTs), and more recently to pituitary adenomas (28-31). The two main functions of SDH are the oxidative dehydrogenation of succinate to fumarate in the tricarboxylic acid cycle (TCA) cycle and the reduction of ubiquinone in the electron transport chain during ATP synthesis. Therefore, the SDH enzyme complex plays a vital role in the initial deprotonation step, where electrons are derived from succinate oxidation via FAD. After the electrons have been liberated from the oxidation of succinate, they are tunnelled along the Fe-S relay to an awaiting ubiquinone molecule. The common feature in all SDH mutations is the inactivation of the SDH complex which leads to the accumulation of succinate and increase in oxygen free radical production. Succinate affects HIF stability through its effects on post-translational regulation of HIFα subunits, an essential step for the recognition of HIF for proteasome-mediated degradation. Therefore, accumulation of succinate and an increase in oxygen free radical production in SDH inactivation leads to stabilisation of HIF. Through similar mechanisms as in VHL, stabilisation of HIF-α activate multiple hypoxia-dependent pathways leads to epigenetic modifications in HIF target genes (DNA and histone hypermethylation). These genes that are affected by hypermethylation have been implicated in many vital effects on cellular processes including apoptosis, angiogenesis, energy metabolism, proliferation, migration, and invasion of tumour cells (32). Thus, HIF-α stabilisation in SDH mutations cause subsequent epigenetic modifications giving rise to multiple benign and malignant tumour pathology including phaeochromocytomas and paragangliomas. Interestingly, both DNA demethylation and histone demethylation associated with an SDH mutation can be corrected by the addition of the methylase inhibitor, decitabine. These findings support a potential reversible hypermethylation process in patients with an SDH mutation, suggesting a possible therapeutic pathway. Moreover, over the last several years, new molecules to inhibit HIF2α have been developed, especially in the 494

treatment of clear cell carcinoma of the kidney (33). PT2385 is one such molecule and it binds to a HIF-2α unique protein pocket in the PAS-B domain, and thus, prevents the HIF-2α-ARNT dimerization and the formation of an active HIF-2 transcription complex. The development of these molecules (PT2385 and PT2399) have may provide a therapeutic opportunity to perhaps successfully treat pharmacologically several types of cancers which currently have limited therapeutic options (e.g. patients with SDHB-related metastatic phaeochromocytoma/paraganglioma) (34). In addition, previous evidence suggested that SDHdeficient cells rely on lactate dehydrogenase A (LDHA) for regeneration of NAD+ or pyruvate carboxylase for the uptake of extracellular pyruvate and increased aspartate synthesis, both raising the possibility that LDH inhibition might be selectively toxic to SDH-loss cells. Development of these molecules may give the possibility of non-cytotoxic metabolite for the treatment of SDH-loss in phaeochromocytoma/ paragangliomas.

SDHD MUTATION Fifteen percent of phaeochromocytoma and paraganglioma are associated with germline SDH mutations. Inactivating mutations in the SDHD gene, autosomal-dominantly acquired, give rise to familial parasympathetic head and neck paragangliomas. They can also give rise to sympathetic extra-adrenal paragangliomas and rarely unilateral phaeochromocytoma. The head-and-neck paragangliomas are usually bilateral or multifocal. Although, the paragangliomas can be recurrent they are rarely malignant (< 5%) (35). Intriguingly, SDHD mutations are highly penetrant and show maternal genomic imprinting (36). Thus, almost all tumours are only seen in the children of male-affected parents, and the mutation is inactivated if inherited from the maternal side (although it will still be genetically transmitted).

SDHB MUTATION Germline mutations of SDHB gene are inherited as autosomal dominant with the presence of sympathetic extra-adrenal paragangliomas, followed by adrenal phaeochromocytomas and parasympathetic head and neck paragangliomas (37,38). Typically, they originate from extra-adrenal locations in the abdomen, thorax and the pelvis and are usually solitary tumours Arch Endocrinol Metab. 2017;61/5

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SDHC MUTATION SDHC mutation is located on chromosome 1q21 and is similarly inherited in an autosomal dominant pattern. However, it is rare and gives rise to benign head-and-neck paragangliomas as well as sympathetic paragangliomas and phaeochromocytomas; these can be multiple (40).

SDHA MUTATION SDHA gene mutation was initially thought to cause Leigh syndrome, a neurodegenerative syndrome associated with subacute necrotising encephalomyelopathy with developmental delay and psychomotor regression. However, recently germ-line mutations in SDHA were detected in patients with both sympathetic and parasympathetic paragangliomas (41).

OTHER GENES RELATED TO PHAEOCHROMOCYTOMAS AND PARAGANGLIOMAS TMEM 127 TMEM127 is a tumour suppressor gene (four exons, chromosome 2q11) linked with mTOR (mammalian target of rapamycin) kinase pathway which has recently been associated with the development of phaeochromocytoma. Since the original report, more than 30 mutations have been identified in TMEM127. Although all variants were detected in germline DNA, less than 20% of patients carrying a TMEM127 mutation report a family history of phaeochromocytomas, suggesting low penetrance of the mutant alleles (42). TMEM127 encodes for a transmembrane protein which localizes to the plasma membrane and multiple components of the endosome machinery, including early, late and recycling endosome, Golgi complex and lysosome. Once mutated, it is mostly located in the cytoplasm, suggesting the localization of TMEM127 to endomembrane pools is important for its tumour suppressor function (42).

SDHAF2 MUTATION

MAX Mutation

Inactivating mutations in the SDHAF2 gene has recently been recognised to cause a rare type of familial paraganglioma syndrome which causes head-andneck paragangliomas, exclusively in children of fathers carrying the defective gene. This point towards a maternal imprinting and is inherited in an autosomal dominant manner. The mean age of presentation is 30 years and studies suggest that screening for SDHAF2 is important in patients with head-andneck paragangliomas with either a family history of head-and-neck paraganglioma, young age of onset or multiple tumours in whom SDHB, SDHC, and SDHD gene testing was negative (36).

The MAX gene is located on chromosome 14q23 and encodes for MAX protein. MAX is a low abundance basic helix–loop–helix (bHLH) leucine zipper domaincontaining protein that is predominantly found in complex with the MYC transcription factor. MYC is a common oncogene in many human cancers and MYC– MAX heterodimers bind to E-box sequences in the promoters that binds to genes encoding proteins with a wide range of cellular functions, including metabolism, growth and angiogenesis (43). Moreover, MAX can bind to other transcription factors such as MXD1, MNT and MGA which can repress the transcription of target genes, ultimately leading to the inhibition of cell

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with a significantly high malignant potential (30%) (4,38). Therefore, all patients with metastatic phaeochromocytoma or paragangliomas should undergo SDHB mutation testing at the very least. The typical age of presentation of paragangliomas due to SDHB mutations is 30 years. However, they can present at any age, including in childhood. Moreover, an SDHB mutation has a poor genotype and phenotype correlation due to low penetrance and high variable expression, where even identical mutations give rise to different types of tumours in location, behaviour and severity (4). The predominant biochemical phenotype of an SDHB mutation is hypersecretion of dopamine alone or dopamine and norepinephrine. Therefore, increased levels of 3-methoxytyramine, which is a product of dopamine metabolism, could help biochemically identify SDHB or other likely malignant tumours (13). Apart from biochemistry, immunohistochemistry for SDHB too can aid in the discrimination between SDHB and other mutations. If the phaeochromocytoma or the paraganglioma is due to an SDHB mutation, staining the tumour for SDHB will be negative with a sensitivity of 100% and specificity of 84% for any type of SDH mutation (39).

Genetics of phaeochromocytomas

growth and promotion of terminal differentiation (44). Therefore, MAX can function as both a suppressor and activator of genes involved in many oncogenic pathways. Thus, a balance between MAX complexes with MYC and MAX complexes with MYC repressors dictates the output of transcription of E box-containing genes as a result of either activation or repression (43). Although the mechanism in which a MAX mutation causes phaeochromocytoma remains unclear, recent studies show that partial deletion and reintroduction of MAX results in cell growth arrest supporting the role of MAX repressing the oncogenic effects of MYC on paraganglial cells (44). MAX associated catecholamine-secreting tumours can be either adrenal or extra-adrenal. Adrenal tumours are often bilateral (67%) with a possible association with malignant behaviour. Therefore, mutations in the MAX gene should be sought in patients with familial, bilateral or apparently sporadic phaeochromocytoma/ paraganglioma (45).

OTHER GENES The actual mutation load of individual phaeochromocytomas and paragangliomas is unknown. Multiple novel germline mutations have been associated with the development of phaeochromocytoma. A few well recognized ones are HIF2A (also known as EPAS1), KIF1B and EGLN1. KIF1B is a rare germline mutation which causes phaeochromocytoma and neuroblastoma. Located on chromosome 1p36.22, KIF1B belongs to the kinesin family encoding a protein that induces apoptosis. KIF1B acts in a prolyl hydroxylase domain‐containing protein-3 (PHD3) dependent apoptosis pathway that occurs physiologically in sympathetic lineage precursor cells during development (46). Another rare germline mutation causing phaeochromocytoma together with congenital erythrocytosis is the EGLN1 mutation. EGLN1 (egl-nine-homolog-1) gene, also termed PHD2, is located on chromosome 1q42.1, encodes a prolyl hydroxylase, which has a crucial function in the oxygendependent proline hydroxylation of the HIF-α pathway. Therefore, through similar pseudohypoxic mechanisms as in SDH, EGLN1 mutations can give rise to familial paraganglioma (47). Loss of function of fumarate hydratase (FH), which catalyses the conversion of fumarate to malate, 496

has been demonstrated to cause accumulation of the precursor metabolite, fumarate, Fumarate shares structural similarities with succinate. Similar to succinate accumulation in SDH mutations, fumarate accumulation in FH activates the pseudo-hypoxia driven pathways to give rise to catecholamine secreting tumours (48). Similar to succinate and fumarate accumulation, which leads to enzymatic inhibition of multiple α-KGdependent dioxygenases in the Krebs cycle, a new germline mutation in MDH2 (malate dehydrogenase 2) has been found to cause phaeochromocytoma/ paraganglioma (with possible metastasis). This mutation causes a deletion in the tumour suppressor gene prompting a stable silencing of MDH2 expression. It has been suggested that suppression of MDH2 leads to accumulation of malate which, similar to succinate, inhibits the HIFα pathway. This mutation was found in patients neuroblastomas, as well as in malignant phaeochromocytoma and paraganglioma (49). Mechanisms underlying phaeochromocytoma are astonishingly diverse, with both inherited and somatic drivers influencing tumorigenesis through a broad range of biological pathways. Apart from germline mutations, recent studies have attempted to locate somatic mutations in the phaeochromocytoma/ paragangliomas. Somatic mutations of the HRAS gene, which is one of the most frequently disordered genes in many malignancies was isolated in phaeochromocytoma by exome sequencing (50). These mutations target the signal downstream of the RAS-MAPK pathway. Identification of somatic mutations is useful specially in the differentiation between malignant and benign phaeochromocytoma, which can be quite challenging to the managing physician. Another well-known somatic mutation is the HIF2α mutation. This somatic gain-of-function mutation targets the HIF2α-stabilising prolyl sites, Pro531, affecting the conformation of HIF2α. This conformational change induces downstream targets leading to tumour growth. Interestingly, despite the somatic nature, patients with HIF2α mutation were found to develop somatostatinomas and 50% developed early onset or congenital polycythaemia. It seems probable that this is due to germline mosaicism (51). Interestingly, ophthalmic complications are also being recognised in this syndrome. Recent data have revealed that DNA translocation and fusion genes act as a component of phaeochromocytoma Arch Endocrinol Metab. 2017;61/5

Genetics of phaeochromocytomas

tumorigenesis. Moreover, certain germ-line mutations as well as somatic mutations and fusion genes can be used as markers/predictors of aggressive disease-free survival (ADFS), the time until the occurrence of distant metastases, local recurrence, or positive regional lymph nodes. Apart from germ-line mutations such as SDHB, certain somatic mutations including ATRX and MAML3 fusion gene were shown to predict clinical outcome in patient with phaeochromocytoma (52). Certain of these gene products seem to be involved in the b-catenin pathway, indicating a separate sub-group of this type of tumour. Currently around 75% of all phaeochromocytomas and paragangliomas show either a clear germline or a likely somatic driver mutation. Finally, deep exome-sequencing studies have revealed very low frequency germline gain-offunction mutations in histone methylators such as H3F3A and H3K9; this is an area of intense research and undoubtedly more will be learnt with clinical applicability in the near future.

APPROACH TO GENETIC TESTING IN CLINICAL PRACTICE With the rising number of genes identified in association with phaeochromocytoma/paraganglioma, routine testing for all known germ-line has in the past been expensive and Relevant genetic testing

Family history of syndrome

Relevant genetic testing

With metastasis

SDHB

Adrenal

No metastasis

SDHD, SDHC, VHL, MAX, NF1

↑metanephrine

RET

NF1, VHL, TMEM127, MAX

↑normetanephrine

VHL

SDHB, SDHD, SDHC, MAX

↑methoxytyramine

SDHB, SDHD, SDHC

↑normetanephrine

SDHB, SDHD, VHL, SDHC, MAX

↑methoxytyramine

SDHD, SDHB, SDHC

↑normetanephrine

SDHD

↑methoxytyramine

SDHD, SDHC, SDHB

Pheochromocytoma/ paraganglioma

Syndromic features

time-consuming. It is also important to remember that the majority of these tumours are still sporadic and may not carry a germline mutation. Therefore, the suggestion has been to employ various predictors to suggest a screening process for genetic testing: based on many studies, germline mutations are common in patients with early onset disease (< 45 years), bilateral phaeochromocytoma, extraadrenal disease (e.g. head and neck paraganglioma), multifocal, recurrent or malignant disease and a positive family history of phaeochromocytoma. Therefore, patients with these features were considered for genetic testing (25,53). Then, depending on certain feature associated with different mutations one could decide on the order of genes to be tested (Figure 1). This decisionmaking process could be guided by several other factors including presence of syndromic clinical features on clinical evaluation, positive family history of syndromic features (e.g. a family member with medullary thyroid carcinoma suggest possible MEN2), tumour location, type of catecholamine produced by the tumour and histological evaluation. For patients with sporadic phaeochromocytoma (without family history or syndromic feature), decision making can be aided by several tumour characteristics such as tumour location, biochemical phenotype and histopathology. A summary of the indicative factors is given below:

SDHC, VHL, TMEM 127, RET

Head and neck

Figure 1. Decisional flow-chart for genetic testing in patients with a proven phaeochromocytoma/paraganglioma. Arch Endocrinol Metab. 2017;61/5

497

Genetics of phaeochromocytomas

LOCATION OF THE TUMOUR Considering the location of the tumour; intra-adrenal tumours suggest possible RET, VHL, NF1, TMEM 127, MAX or rarely KIF1B mutations. In addition, bilateral phaeochromocytomas are mostly found with these same mutations (11,12,14,21,26,42,45,53). On the other hand, SDH mutations cause intra-adrenal tumours less commonly; 25% of SDHB-related tumours are phaeochromocytomas while the frequency of intraadrenal tumours in SDHD, SDHA and SDHC are even lower (4,7,36,37,41,53). Most of the extra-adrenal tumours are due to mutations in SDH genes (4,7,36,37,40,53). Apart from which, extra-adrenal tumours were also found in rare EGLN1 mutation (47). Although, rare extra adrenal tumours can also be found in VHL, TMEM 127, NF1, and RET mutations as well (11,12,14,21,26,42,53). Of the extra-adrenal tumours, head-and-neck paragangliomas hold a special importance as they have a high possibility of carrying an underlying genetic mutation. Of the SDH mutations, SDHD-related tumours are commonly seen in the head and neck region and are usually multiple. Head and neck tumour are also seen in SDHB and SDHC mutations; however, they are much less common (7,31,53). An even rarer cause for head-and-neck paraganglioma is the SDHAF2 mutation, which should be considered if SDHD, SDHB, and SDHC testing is negative (36). Due to high rate of an underlying genetic defects, head and neck paraganglioma negative for all SDH mutations can be tested for VHL and TMEM127 (although the possibility is very rare). Sympathetic paragangliomas, which are large, solitary tumours located in abdomen, thorax and pelvis, are often due to SDHB mutations, while SDHC and SDHA can rarely be causal (7,31,36,38,40,41,53).

BIOCHEMICAL PHENOTYPE Metabologenomics is another area that can also shed some light on the underlying genetic defect. Depending on the mutation, tumours show distinct differences in metabolic pathways that relate to or even directly impact clinical presentation. Therefore, the biochemical phenotype can be an important tool when deciding on the order of genetic testing in patients. Patient with catecholaminesecreting tumours due to RET and NF1 mutations secrete high levels of metanephrine, indicating epinephrine production in the tumour, while patients with mutations in the VHL gene exhibited an increased production of 498

normetanephrine, indicating norepinephrine production. On the other hand, SDH mutations, especially SDHB and SDHD mutations, frequently show elevated levels of methoxytyramine (an indicator of dopamine production and often malignancy) (13).

HISTOPATHOLOGICAL DIFFERENTIATION Finally, histopathological differentiation can be a useful tool when planning genetic screening in phaeochromocytoma. The presence of malignant features can suggest certain genetic defects; phaeochromocytomas and especially extra-adrenal paragangliomas of malignant nature are associated mostly with SDHB mutations (in 30% patients) (4,3638,40). Malignant phaeochromocytomas can also be not infrequently seen with several mutations including MAX (25%) and NF1 (12%) mutations (7,14,21,45,53). Malignant tumours are rare (< 5%) in RET, VHL, SDHD, SDHC, SDHAF2 and TMEM127 mutations. Immunohistochemistry can add to this by negative staining in SDHB and SDHA mutations (39,41).

GENE PANEL SCREENING Most recently, it has become clear that with the large number of possible genetic disturbances, simple algorithmic screening has become slow and resource intensive, and a number of groups have shown the utility of simultaneous screening for a whole panel of genes, independent of any other background information (except where there is clear evidence of a patientâ&#x20AC;&#x2122;s syndromic or family diagnosis). Such panel screening was initially with Sanger sequencing, and indeed using this approach we identified germline mutations in a series of patients with phaeochromocytomas in 25% of patients, including 15% of patients with unilateral sporadic non-recurrent phaeochromocytomas (54). Similarly, Brito and cols. in a meta-analysis identified germline mutations in 5% of gene-panelled sporadic unilateral tumours (55). With next-generation sequencing (NGS), this approach should probably be the assessment of choice in all patients presenting with phaeochromocytomas and paragangliomas (56).

FINAL REMARKS Phaeochromocytomas and paragangliomas have been paradigm shifters in genetic studies, being the first Arch Endocrinol Metab. 2017;61/5

Genetics of phaeochromocytomas

human tumour model recognised to carry a genetic defect in a metabolic enzyme (SDHD) two decades ago. Since then numerous genetic and epigenetic changes have been discovered in association with these tumours, opening up novel avenues for early and correct diagnosis, appropriate treatment and better prognosis for patients. These discoveries benefit not only the patient but also family members as positive genetic screening can lead to early diagnosis through regular surveillance. In conclusion, the era of NGS has opened up new avenues of rapid and successful diagnosis and effective screening. Disclosure: no potential conflict of interest relevant to this article was reported.

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45. Comino-Méndez I, Gracia-Aznárez FJ, Schiavi F, Landa I, LeandroGarcía LJ, Letón R, et al. Exome sequencing identifies MAX mutations as a cause of hereditary pheochromocytoma. Nat Genet. 2011;43(7):663-7. 46. Schlisio S, Kenchappa RS, Vredeveld LC, George RE, Stewart R, Greulich H, et al. The kinesin KIF1Bbeta acts downstream from EglN3 to induce apoptosis and is a potential 1p36 tumor suppressor. Genes Dev. 2008;22(7):884-93. 47. Ladroue C, Carcenac R, Leporrier M, Gad S, Le Hello C, GalateauSalle F, et al. PHD2 mutation and congenital erythrocytosis with paraganglioma. N Engl J Med. 2008;359(25):2685-92. 48. Castro-Vega LJ, Buffet A, De Cubas AA, Cascón A, Menara M, Khalifa E, et al. Germline mutations in FH confer predisposition to malignant pheochromocytomas and paragangliomas. Hum Mol Genet. 2014;23(9):2440-6. 49. Cascón A, Comino-Méndez I, Currás-Freixes M, de Cubas AA, Contreras L, Richter S, et al. Whole-exome sequencing identifies MDH2 as a new familial paraganglioma gene. J Natl Cancer Inst. 2015;107(5). pii: djv053. 50. Crona J, Delgado Verdugo A, Maharjan R, Stålberg P, Granberg D, Hellman P, et al. Somatic mutations in H-RAS in sporadic pheochromocytoma and paraganglioma identified by exome sequencing. J J Clin Endocrinol Metab. 2013;98(7):E1266-71. 51. Pacak K, Jochmanova I, Prodanov T, Yang C, Merino MJ, Fojo T, et al. New syndrome of paraganglioma and somatostatinoma associated with polycythemia. J Clin Oncol. 2013;31(13):1690-8. 52. Fishbein L, Leshchiner I, Walter V, Danilova L, Robertson AG, Johnson AR, et al. Comprehensive Molecular Characterization of Pheochromocytoma and Paraganglioma. Cancer Cell. 2017;31(2):181-93. 53. Mannelli M, Castellano M, Schiavi F, Filetti S, Giacchè M, Mori L, et al. Clinically guided genetic screening in a large cohort of italian patients with pheochromocytomas and/or functional or nonfunctional paragangliomas. J Clin Endocrinol Metab. 2009;94(5):1541-7. 54. Sbardella E, Cranston T, Isidori AM, Shine B, Pal A, JafarMohammadi B, et al. Routine genetic screening with a multi-gene panel in patients with pheochromocytomas. Endocrine. 2017 May 5. doi: 10.1007/s12020-017-1310-9. 55. Brito JP, Asi N, Bancos I, Gionfriddo MR, Zeballos-Palacios CL, Leppin AL, et al. Testing for germline mutations in sporadic pheochromocytoma/paraganglioma: a systematic review. Clin Endocrinol (Oxf). 2015;82(3):338-45. 56. NGS in PPGL (NGSnPPGL) Study Group, Toledo RA, Burnichon N, Cascon A, Benn DE, Bayley JP, Welander J, et al. Consensus Statement on next-generation-sequencing-based diagnostic testing of hereditary phaeochromocytomas and paragangliomas. Nat Rev Endocrinol. 2017;13(4):233-47.

43. Blackwood EM, Lüscher B, Eisenman RN. Myc and Max associate in vivo. Genes Dev. 1992;6:71-80.

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Mechanisms involved in hearing disorders of thyroid ontogeny: a literature review Caio Leônidas Oliveira de Andrade1, Gabriela Carvalho Machado1, Luciene da Cruz Fernandes2, Jamile Morais de Albuquerque2, Luciana Lyra Casais-e-Silva3, Helton Estrela Ramos4, Crésio de Aragão Dantas Alves5

ABSTRACT Endocochlear, retrocochlear and/or central origin hearing damage may be related to the absence of appropriate levels of thyroid hormone during morphogenesis and/or auditory system development. Hearing disorders related to the thyroid are not well studied, despite speculation on the pathophysiological mechanisms. The objective of this review was to characterize the main pathophysiological mechanisms of congenital hypothyroidism and to evaluate the relationship with central and peripheral hearing disorders. We conducted a literature review using the databases MedLine, LILACS, Cochrane Library, SciELO, Institute for Scientific Information (ISI), Embase, and Science Direct between July and September on 2016. We identified the studies that address hearing disorder mechanisms on the congenital hypothyroidism. Congenital hypothyroidism may have clinical and subclinical manifestations that affect the auditory system and may be a potential risk factor for hearing impairment. Hearing impairment can severely impact quality-of-life, which emphasizes the importance of monitoring and evaluating hearing during the clinical routine of these patients. Arch Endocrinol Metab. 2017;61(5):501-5 Keywords Thyroid gland; thyroid diseases; congenital hypothyroidism; hearing disorders; hearing loss

1 Programa de Pós-graduação dos Processos Interativos dos Órgãos e Sistemas (PPgPIOS), Instituto de Ciências da Saúde (ICS), Universidade Federal da Bahia (UFBA), Salvador, BA, Brasil 2 Departamento de Fonoaudiologia, Instituto de Ciências da Saúde, Universidade Federal da Bahia (UFBA), Salvador, BA, Brasil 3 Laboratório de Neuroimunoendocrinologia e Toxinologia, Departamento de Biorregulação, Instituto de Ciências da Saúde (ICS), UFBA, Salvador, BA, Brasil 4 Departamento de Biorregulação, Instituto de Ciências da Saúde (ICS), UFBA, Salvador, BA, Brasil 5 Faculdade de Medicina, Unidade de Endocrinologia Pediátrica, Universidade Federal da Bahia (UFBA), Salvador, BA, Brasil

Correspondence to: Caio Leônidas Oliveira de Andrade Av. Reitor Miguel Calmon, SN 40110-100 – Salvador, BA, Brasil caioleonidas@gmail.com Received on Feb/26/2017 Accepted on May/30/2017

INTRODUCTION

he development of the auditory system depends on the presence of proper levels of thyroid hormone (TH) (1). Several proteins and the synthesis of multiple enzymes require the normal function of the thyroid gland, and hormones are necessary for the structural formation of the middle and inner ear (2) as well as the central auditory system (3). Therefore, it is possible that congenital hypothyroidism may lead to auditory damage with endocochlear origin, retrocochlear origin and/or central parts of the auditory system (3). The THs play an important role in the morphogenesis, development and maturation of the auditory pathway. Thus, congenital hypothyroidism (CH) can be a potential risk factor for hearing impairment (HI) (4) if the hormones decrease or are absent during the development of the peripheral and central auditory system structures (5).

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While the HI incidence in CH individuals is currently unknown, studies suggest it may affect 20% of carriers (5-7). The rate of hearing disorders in CH patients is approximately 100-fold higher than the euthyroid population and occurs in approximately 1 per 1000 births (6). Although the CH auditory aspects have been investigated in different experimental models involving both in humans and animals, the pathophysiological mechanisms have not been well explored and are not fully elucidated. This lack of information makes it difficult to comprehend all the processes involved in the possible hearing disorders that this disease may cause. The aim of this literature review was to evaluate the relationships between CH and both peripheral and central hearing disorders. We focused on the pathophysiological mechanisms involved with these disorders. 501

DOI: 10.1590/2359-3997000000292

Hearing disorders and thyroid ontogeny

MATERIAL AND METHODS Identification and selection of studies The literature search was conducted using the following electronic databases: MedLine, LILACS, Cochrane Library, SciELO, Institute for Scientific Information (ISI), Embase, and Science Direct. The databases were consulted between July and September 2016. The databases were mined for literature that specifically focused on pathophysiological processes of congenital hypothyroidism and hearing in human and animal models. The following keywords and descriptors were used during the search and were combined in a number of sequences in English, Portuguese and Spanish languages: hypothyroidism, congenital hypothyroidism, thyroid hormone, thyroid gland, thyroid ontogeny versus auditory hearing maturation, cochlear function, middle olivocochlear system, central auditory processing, hearing loss, and hearing test. The selected studies were chosen based on their title and abstract description. The desired outcomes were structural, physiological, and/or biochemical disorders of the auditory system due to impaired function of the thyroid gland. Papers were excluded from the analysis if they addressed hearing disorders in syndromic cases associated with hypothyroidism or other hypothyroidism conditions that were not caused by abnormalities due to the formation or function of the thyroid gland.

LITERATURE REVIEW

Congenital hypothyroidism CH is related to defective TH action due to decreased or absent hormones. CH is the most common metabolic dysfunction in newborn infants. CH affects 1:3000 to 1:4000 births worldwide (8) and 1:2500 births in regions of Brazil (9). CH etiology is clinically classified as either permanent (80-90%) or transitory (10-20%) (10). The causes of CH are broadly categorized into dyshormonogenesis in 15% of cases and thyroid dysgenesis (TD) in 85% of cases (11-14). Dyshormonogenesis is caused by autosomal recessive mutations of key molecules regulating thyroid hormone synthesis, and thyroid hormone production fails in a structurally sound thyroid gland (15). Conversely, TD is caused by a wide range of different structural malformations in the thyroid that result in a wide variety of 502

different CH phenotypes (16-18). TD is subcategorized into the following classes: 1) thyroid agenesis, which is the most severe form and has a complete lack of thyroid tissue (i.e., both lobes); 2) thyroid hemiagenes, which has one of the thyroid lobes completely missing; 3) thyroid hypoplasia, which is characterized as a smaller gland in the normal position; and 4) thyroid ectopia, which involves an abnormal positioning but the gland rests along the migratory path of the primordium. It is known that 5% of thyroid dysgenesis cases are associated with mutations of the genes responsible for the development of the thyroid follicular cells (e.g., NKX2.1, FOXE1, PAX8, and TSHR) and display a complex pathogeny (18,19). Untreated CH can result in a profound impairment of the somatic growth and central nervous system functional differentiation because THs are essential for metabolic development, growth, and homeostasis.

Endocochlear mechanisms of congenital hypothyroidism Animal model studies demonstrated that thyroxin (T4) plays an important role in the development of embryonic inner ear. In CH cases with maturation of the sensory epithelium, the inner ear is injured, which suggests there are periods of sensitivity to THs in the developing cochlea (20). In humans the critical time for hearing maturation corresponds approximately to the gap between the embryonic period and the first year of postnatal life (21) (Figure 1). The cellular function of THs, specifically the active form triiodothyronine (T3), is mediated by the thyroid hormone receptor (TR). TR is a binding transcription factor that changes target gene expression (22). The action of T3 on the cochlear sensory cells is partially caused by differential expression of the TH receptor isoforms receptors present in the developing cochlea: α (THRA) and β (THRB) (23). The expression pattern suggests the cochlea is a direct site of action for THs, which can explain several findings of morphological abnormalities on the spiral organ in hypothyroid rodents (20,24,25). A delay of THs supply before hearing function development starts results in permanent defects on the cochlea. The deficits of THs can also lead to permanent decreases of the β-tectorin protein levels in the tectorial membrane, which is associated with tectorial membrane structural abnormalities and cochlear function (26). The outer hair cells (OHC) are highly sensitive to THs serum levels (26). In cases with low hormone Arch Endocrinol Metab. 2017;61/5

Hearing disorders and thyroid ontogeny

Postnatal development Foetal/Infant tyrold hormone

Maternal tyrold hormone Auditory system Structural morphogenesis maturation

Functional maturation

Auditory responses Foetal thyrold First trimester

Second trimester

Third trimester

Conception  Otic placode  Cochlear duct  Statoacoustic ganglion  Spiral ganglion  Ossicles

 Organ of cortil  Hair cells  Brainstem/ midbrain auditory nuclel  Auditory cortex in cerebrum  Innervation

Infancy (months/years)

Birth  Myelination  Synaptic maturation  Middle ear cavitation  Outer ear canal opening

 Pulmonary oxygenation

Figure 1. The role of T3 in human auditory system formation and development. In the foetal period, the primary auditory responses and the hearing sensitivity progressively matures until early childhood. During the first quarter, the embryo depends totally on the mother’s thyroid hormones, which are produced in small amounts during the second half of gestation. After birth occurs, there is an increase in the T4 and T3 levels in the newborn. Adapted from Ng and cols., 2013 (23).

levels in the beginning of hearing function, the OHC are poorly differentiated from the other cells in the cochlea. This reduces the number of organelles in the cytoplasm, including ribosomes, endoplasmic reticulum, and mitochondria (27). It is also possible to verify an insufficient formation and changes in microtubule stability with the rise of filamentous actin expression, which increases the stiffness and decreases the cell membrane mass. These changes directly affect the cochlear amplification process (28). The patients with hypothyroidism show reduced SLC26A4 gene expression. This gene encodes the prestin protein that functions as the motor of the OHC and regulates the cochlear amplification process (29). The reduction in prestin and decreased amplification decrease its distribution in the OHCs membrane (30). Additionally, the K+ channel encoded by KCNQ4 is responsible for endolymphatic potential formation and is also significantly decreased in these conditions (31). Cumulatively, these factors together with an insufficient opening of the cochlea fluid spaces (inner spiral sulcus, tunnel of Corti, and Nuel’s space) affect the development of cochlear micromechanics (32) and damage both the passive and active cochlea mechanisms (33). Arch Endocrinol Metab. 2017;61/5

There are also descriptions of abnormalities in numerous afferent dendrites and growth delays of the efferent terminals under the OHCs (34). These findings confirm the hypothesis that the absence or decrease of THs can cause harmful effects to the peripheral auditory system and cochlear function.

Retrocochlear/central mechanisms on congenital hypothyroidism Previous studies conducted in animal models focused on central nervous system (CNS) development and how the decrease or absence of THs leads to clinical signs suggesting stagnation of normal CNS maturation in CH cases (34). These findings show an abnormality on the myelination process and subtraction of the axonal projections of the anterior commissure and corpus callosum (35). The abnormalities decrease the pyramidal neurons and cause irregular localization of the corpus callosum neurons. Additionally, there are reduced numbers of microtubules in the neural cytoplasm, changes to the distribution of apical dendrites of the pyramidal neurons (36), and a delay in the cholinergic axons arrival to the hippocampus (37). Prior studies of the superior auditory pathway have shown reduced levels of the metabolic activity marker deoxyglucose in the following regions: the cochlear nucleus, superior olivary complex, lateral lemniscus nucleus, inferior colliculus, medial geniculate body, and auditory cortex. These data suggest the entire auditory pathway is sensitive to insufficient TH serum levels (38). A possible explanation for these findings may be associated with the reduced expression of the type 2 deiodinase enzymes, which convert the T4 into T3 hormone in individuals affected by hypothyroidism and reduce the amount of T3 for the auditory centres (39). Studies of the regions located closer to the spiral organ show changes on the spiral ganglion that cause smaller neurons than found in euthyroid people (40). The morphology of neurons from the medial olivocochlear tract is altered in CH cases. However, there are no changes to the neuron population and distribution of this tract. If the neurons do not make proper synaptic contact with the OHC (34), then they can contact other cochlear structures (27,32). Recent evidence indicates the medial olivocochlear tract innervation is more severely affected in the cases with hypofunctional thyroid glands because it remains at an immature stage compared to lateral olivocochlear tract innervation (34). 503

Foetal development

Hearing disorders and thyroid ontogeny

Audiological findings of congenital hypothyroidism

REFERENCES

CH causes heterogeneity in the disturbances of the auditory structures. Thus, several audiological findings are possible. However, the audiometric disturbances are frequently described as having the following features: sensorineural, bilateral, symmetric, mainly in high frequencies, in a varying degree, and are often mild to moderate severity (6,7,41-46). Conductive hearing loss and tympanometric abnormalities in addition to acoustic middle ear reflex have also been described in several studies (5,6,7,4143). However, these conditions are found less frequently and are restricted to cases that are linked to any syndrome. Once the TH is essential for auditory nervous system neuromaturation, there is evidence indicating a relationship between the presence of symptoms and central auditory processing disorders in CH cases (47). The electroacoustic tests, such as otoacoustics emissions (OAE), responsible for high frequency sensibility and selectivity show varied results. Therefore, it is possible to see an expressive abnormality of the OAE (43), signal amplitude reduction (44), and an increase in the number of ears classified by the equipment as “fail” due to pre-clinical cochlear susceptibility (48). The tests used to accurately investigate the neurophysiology of the auditory pathway in CH include the brainstem auditory evoked potentials (BAEP) analysis. The test results show diverse findings, such as prolongation of absolute latency of the I (42,49), III and V (50) waves and increased interpeak interval latency for I-III (50), I-IV (49), and I-V (42). These results suggest there are several alteration sites.

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CONCLUSION In considering the reviewed content, it has been shown that hypothyroidism, especially in its congenital form, is a potential risk factor for hearing impairment. It can affect hearing from the peripheral structures to central areas that may also lead to inappropriate auditory development. These defects can affect the comprehension and acquisition of acoustic information. Inappropriate auditory development can lead to scholarly, cognitive, language, behavioural and/or social emotional problems. Therefore, it is critical to monitorand evaluate hearing as part of the clinical routine of these patients. Disclosure: no potential conflict of interest relevant to this article was reported. 504

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case report

Use of cinacalcet and sunitinib to treat hypercalcaemia due to a pancreatic neuroendocrine tumor Hernan Valdes-Socin1, Matilde Rubio Almanza2, Mariana Tomé FernándezLadreda3, Daniel Van Daele4, Marc Polus4, Marcela Chavez5, Albert Beckers1

Service d’ Endocrinologie. CHU de Liège, Belgium 2 Servicio de Endocrinología y Nutrición, Hospital Universitari i Politècnic La Fe, Valencia, Spain 3 Unidad de Gestión Clínica de Endocrinología y Nutrición, Hospital Universitario de Valme, Área de Gestión Sanitaria Sur de Sevilla, Spain 4 Service de Gastroentérologie, CHU de Liège, Belgium 5 Department of Medicine, Division of Hematology, CHU de Liège, Liège, Belgium 1

Correspondence to: Hernan Valdes-Socin Service d’Endocrinologie, CHU de Liège Rue de l’Hôpital B35 (4000) – Liège Belgium hg.valdessocin@chu.ulg.ac.be

SUMMARY Neuroendocrine tumors (NETs) can secrete hormones, including ectopic secretions, but they have been rarely associated with malignant hypercalcemia. A 52-year-old man with a history of diabetes mellitus was diagnosed with a pancreatic tumor. A pancreatic biopsy confirmed a well-differentiated pancreatic NET (pNET). The patient subsequently developed liver metastasis and hypercalcemia with high 1,25 OH vitamin D and suppressed parathyroid hormone (PTH) levels. Hypercalcemia was refractory to chemotherapy, intravenous saline fluids, diuretics, calcitonin and zoledronate. Cinacalcet administration (120 mg/day) resulted in a significant calcium reduction. Hypocalcemia was observed when sunitinib was added three months later and cinacalcet was stopped. Subsequently, the calcium and PTH levels normalized. After six months, we observed 20% shrinkage of the pancreatic tumor and necrosis of a liver metastasis. Cinacalcet is an allosteric activator of the calcium receptor agonist, and it is used for severe hypercalcemia in patients with primary (benign and malignant) hyperparathyroidism. In this patient, cinacalcet demonstrated a calcium lowering effect, normalized hypophosphatemia, and improved the clinical condition of the patient. The mechanism through which cinacalcet improved PTH-rp mediated hypercalcemia is still unclear, but studies have suggested that a potential mechanism is the activation of calcitonin secretion. Sunitinib is an oral multi-targeted tyrosine kinase inhibitor used to treat advanced pNETs. The hypocalcemic effects of sunitinib have not been previously described in a patient with pNET. Here, we report for the first time the successful combination of cinacalcet and sunitinib in the treatment of a pNET patient presenting with malignant hypercalcemia. Arch Endocrinol Metab. 2017;61(5):506-9

Received on Feb/21/2016 Accepted on June/20/2016 DOI: 10.1590/2359-3997000000291

INTRODUCTION

ypercalcemia of malignancy (HCM) develops as a paraneoplastic process in several types of cancers, such as lymphoma, breast cancer, and lung cancer (1), but it has rarely been described in neuroendocrine tumors (NETs) (1-3). In the setting of neuroendocrine tumors (NETs), HCM is associated with advanced disease, poor prognosis, and decreased survival rates (4,5). The most common cause of HCM is the tumor secretion of parathyroid hormone related peptide or PTH-rp (PTH-rp-oma) (2,5). Pancreatic NETs are typically pluripotent, and they have the ability to produce several types of hormones, including PTH-rp and calcitonin (2-6). When feasible, primary tumor surgery should be performed to normalize hypercalcemia. Unfortunately, the conventional hypocalcemic treatment options (e.g., bisphosphonates, corticosteroids, diuretics, hyper hydration) do not seem

506

to improve patient survival in these cases. Here, we report, for the first time, the successful combination of cinacalcet and sunitinib in a pNET patient presenting with malignant hypercalcemia. In this case, in the setting of pancreatic NET, hypercalcemia associated with hypophosphatemia and suppressed PTH levels were highly likely due to PTH-rp secretion, although serum PTH-rp levels could not be measured.

CASE REPORT A 52-year-old-man with a history of diabetes mellitus and smoking presented with abdominal pain and asthenia for one year. Abdominal computed tomography (CT) revealed a 15 cm mass involving the pancreas and the retroperitoneum, with splenic and hepatic carcinomatosis. The histology of the pancreatic lesion showed a well-differentiated pNET positive for Arch Endocrinol Metab. 2017;61/5

Cinacalcet and sunitinib for pNET hypercalcemia

Arch Endocrinol Metab. 2017;61/5

tomography (PET) scan revealed a 20% shrinkage of the pancreatic tumor and necrosis of a metastasis in the VII hepatic segment following the cinacalcet and sunitinib treatment. Currently the patient is alive and still being treated with sunitinib. According to the RECIST (Response Evaluation Criteria in Solid Tumors) criteria, the patient has had stable disease for the last four years. Calcium (Mmol/L) 3.8 3.6 3.4 3.2 3 2.8 2.6 2.4 2.2 2

Cinacalcet Cinacalcet + Sunitinib Sunitinib

3 2.87 2.79

2.65

2.48 2.36 2.12

7/2012

10/2012

12/2012

7/2013

12/2015

Figure 1. Serum calcium levels at the time of diagnosis, during the cinacalcet treatment (Mimpara© 120 mg/day PO), and during the combined treatment with Mimpara (120 mg/day) and sunitinib (Sutent© 35.7 mg/day). Cinacalcet was stopped when the calcium levels reached 2.12 mmol/L. The red line represents the upper normal calcium levels. The patient, whose calcium parameters remain normal, is currently being treated with Sutent only.

DISCUSSION HCM occurs in approximately 30% of all cancer patients. HCM may be caused by different mechanisms, including the production of parathyroid hormonerelated peptide (PTH-rp), which is the most frequent cause (1-4), and/or 1,25 OH vitamin D by tumor cells. In addition, ectopic PTH secretion can rarely occur (1-4). NETs often produce ectopic hormone secretions, but they are rarely associated with HCM (2,5). In this case, an osteolytic lesion and a PTHdependent mechanism of hypercalcemia were excluded. Unfortunately, we could not determine the PTH-rp levels of our patient, and no tumor specimen was available for PTH-rp studies. Therefore, it remains unproven whether PTH-rp hypersecretion was (at least partially) responsible for the hypercalcemia in this case. Several treatment options are available to manage hypercalcemia in cancer patients (5-9). The extracellular volume must be restored with intravenous saline fluids because patients are frequently volume depleted (1). In addition, high diuretic doses can increase urinary calcium excretion levels (7). In addition, gallium nitrate, calcitonin, and hemodialysis have been used to treat 507

AE1/AE3, synaptophysin, and CD56. Chromogranin immunostaining was negative. The Ki-67 labelling index was 2%. Somatostatin-receptor scintigraphy (octreoscan) detected areas of pathologic uptake in the liver and pancreas, whereas bone scintigraphy did not reveal any skeletal metastatic deposits. Hypercalcemia was diagnosed with calcium and ionized calcium levels of 3.54 mmol/L (2.15-2.6) and 1.55 mmol/L (1.141.3), respectively. Other bone metabolism abnormalities included hypophosphatemia levels of 0.42 mmol/L (0.74-1.51), PTH levels of < 4 ng/mL (12-58), 1-25 OH vitamin D levels of 100 pg/mL (< 85), 25 OH vitamin D levels of 9 ng/ml (> 30) and calcitonin (as a tumor maker) levels of 1116 ng/mL (< 10). The chromogranin A level was 26.9 UI/L (< 23). Urine analysis showed hypercalciuria and hyperphosphaturia. A bone density analysis showed mild cortical and femoral osteopenia. Because surgery was not feasible, the patient underwent treatment with several cycles of streptozotocin-adriamycin and FOLFOX. The tumor mass and calcium levels were partially controlled (2.61 mmol/L), whereas the PTH concentration remained low (19 ng/mL). Three months later, the patient’s calcitonin levels were 29 ng/mL, and his calcium level increased again (2.94 mmol/L), whereas his PTH level was < 2 pg/mL. Treatment with octreotide LAR 30 mg sc every 4 weeks was introduced for 3 months, without any remarkable clinical impact. Hypercalcemia (total calcium 3.17 mmol/L) was refractory to intravenous saline fluids, diuretics, recombinant calcitonin, and zoledronate. Therefore, compassionate treatment with oral cinacalcet (120 mg/day) was attempted. The patient’s calcium level gradually decreased from 3.17 to 2.87 mmol/L and later to 2.65 mmol/L (Figure 1). The phosphatemia normalized from 0.42 mmol/L to 0.84 mmol/L during the cinacalcet treatment. PTH, 1,25 OH vitamin and calcitonin levels, as well as the tumor size, remained unchanged. After a significant clinical improvement following a three months of cinacalcet treatment (Mimpara© 120 mg per day), sunitinib was added (Sutent© 35.7 mg per day) for tumor control (Figure 1). Both drugs were well tolerated, without any side effects. One month after beginning the combined treatment, the patient’s calcium level decreased to 2.12 mmol/L. Because of the hypocalcemia, the PTH level increased to 78 pg/mL, requiring discontinuation of cinacalcet. The calcitonin and 1,25 OH vitamin D levels normalized, whereas the positron emission

Cinacalcet and sunitinib for pNET hypercalcemia

cancer-related hypercalcemia (3). Glucocorticoids are frequently used to treat hypercalcemia in hematological malignancies. Intravenous bisphosphonates can be effective in treating HCM, and they have demonstrated extended durations of action and low rates of acute phase reaction symptoms (1-3). Denosumab, a human monoclonal antibody binding RANKL that inhibits osteoclast maturation and activation, is used to treat osteoporosis. Denosumab has been approved to treat HCM in the United States of America (USA), and it should be used in bisphosphonate resistant cases. It has been used to treat bisphosphonate-refractory HCM (8). Cinacalcet is an oral drug that acts as a calcimimetic by activating the calcium-sensing receptor (CaSR). It is used to treat secondary hyperparathyroidism in chronic kidney disease and for severe hypercalcemia in patients with primary hyperparathyroidism who are not suitable candidates for parathyroidectomy (9). It is also indicated for the treatment of hypercalcemia in patients with parathyroid carcinoma. CaSR binding by cinacalcet results in decreased PTH secretion and synthesis, and it inhibits kidney 1,25 vitamin D synthesis and calcium reabsorption; in addition, it seems to have an anabolic effect on bone formation (9). Possibly because of high levels of a putative PTH-rp like peptide, the synthesis of 1,25 vitamin D was not affected in the above described patient during the cinacalcet treatment; however, it normalized when the patient was administered sunitinib. In murine models of Leydig cell and colon tumors, cinacalcet attenuated hypercalcemia without affecting the synthesis of PTR-rp mRNA by the tumor. This effect occurred in a dose-dependent manner independently from the PTH-rp administration in parathyroidectomized animals (10). Furthermore, these studies suggest that cinacalcet mediates the reduction in calcium levels, at least partially, by stimulating the release of calcitonin by C-cells. Another possible mechanism yet to be investigated could be the calcium lowering effect of cinacalcet through enhanced renal excretion of calcium (10). Hypophosphatemia is also related to HCM, namely the decrease of renal reabsorption of phosphates, likely due to a PTH-rp-induced effect. In our case report, cinacalcet normalized low phosphates levels, as previously described in mouse models of HCM treated with cinacalcet. This effect has been attributed to an inhibitory effect of calcimimetics on phosphaturic hormones such as FGF-23 (9,10). 508

To the best of our knowledge, the hypocalcemic effects of cinacalcet have never been documented in pancreatic neuroendocrine tumor patients. The clinical case we have described is the second case in the literature in which cinacalcet was used successfully to treat refractory HCM. A previous report described a 57-year-old male with hypercalcemia and a pulmonary tumor secreting PTH-rp (11). In that case, the patient’s calcium and PTH-rp levels decreased during combined chemotherapy and cinacalcet monotherapy. When hypercalcemia recurred after the fourth chemotherapy cycle, cinacalcet monotherapy induced a consistent decline in PTH-rp levels, thus preventing a further increase in serum calcium levels (11). Sunitinib is an oral, multi-target, tyrosine kinase inhibitor used to treat GIST, advanced renal cell carcinomas, and advanced pancreatic neuroendocrine tumors. A calcium-lowering effect of this drug was observed in a case of metastatic renal cell carcinoma and paraneoplastic hypercalcaemia (12). The authors observed that there was no reported case of paraneoplastic hypercalcemia recovery with targeted therapy until their report. We are unaware of any report, thus far, that describes the hypocalcemic effect of sunitinib in pNET patients. In the clinical case reported here, the combined use of cinacalcet and sunitinib decreased the patient’s serum calcium levels to low-normal levels and normalized the PTH concentration. Although chemotherapy initially controlled the calcium levels at upper normal levels, the combined treatment induced a strong calcium lowering effect twice. After the commencement of combined cinacalcet + sunitinib treatment, the calcium levels were significantly lower compared to when the patient was undergoing cytotoxic chemotherapy. Moreover, the combined cinacalcet + sunitinib treatment resulted in a dramatic drop of calcium levels (i.e., hypocalcaemia) and induced the occurrence of secondary hyperparathyroidism.

CONCLUSIONS This case report describes the unusual association of malignant pNET-associated hypercalcemia, high 1,25 OH vitamin D, and high calcitonin levels. Several treatments options for the management of hypocalcaemia have been unsuccessfully attempted, finally leading to the compassionate use of cinacalcet. Arch Endocrinol Metab. 2017;61/5

Cinacalcet and sunitinib for pNET hypercalcemia

Cinacalcet demonstrated a definite calcium lowering effect and improved the clinical condition of the patient. Thus, we believe that cinacalcet can enrich the pharmacological armamentarium for the treatment of HCM. Moreover, sunitinib helped to normalize the patient’s calcium and calcitonin levels, with modest tumor shrinkage. The precise mechanism of the calcium lowering effect of sunitinib remains to be elucidated. In conclusion, here, we report for the first time the successful use of cinacalcet and sunitinib in the management of pNET-associated HCM. Acknowledgments: the authors are grateful to Dr. L. Rostomyan (CHU de Liège) and Dr. G.L. Tamagno (Mater Misericordiae University Hospital – University College Dublin) for revising the manuscript. Disclosure: no potential conflict of interest relevant to this article

3. Shah RH, Martínez D. Pancreatic neuroendocrine tumor associated with humoral hypercalcemia of malignancy and carcinoid tumor: a case report and review of the literature. Pancreas. 2013;42(3):549-51. 4. Rosner MH, Dalkin AC. Onco-nephrology: the pathophysiology and treatment of malignancy-associated hypercalcemia. Clin J Am Soc Nephrol. 2012;7(10):1722-9. 5. Basso U, Maruzzo M, Roma A, Camozzi V, Luisetto’ G, Lumachi F. Malignant Hypercalcemia. Curr Med Chem. 2011;18(23):3462-7. 6. Milanesi A,Yu R, Wolin EM. Humoral hypercalcemia of malignancy caused by parathyroid hormone-related peptide-secreting neuroendocrine tumors. Report of six cases. Pancreatology. 2013;13(3):324-6. 7. LeGrand SB, Leskuski D, Zama I. Narrative review: furosemide for hypercalcemia: an unproven yet common practice. Ann Intern Med. 2008;149(4):259-63. 8. Boikos SA, Hammers HJ. Denosumab for the Treatment of Bisphosphonate-Refractory Hypercalcemia. J Clin Oncol. 2012;30(29):e299. 9. Nemeth EF, Shoback D. Calcimimetic and calcilytic drugs for treating bone and mineral-related disorders. Best Pract Res Clin Endocrinol Metab. 2013;27(3):373-84.

was reported.

10. Colloton M, Shatzen E, Wiemann B, Starnes C, Scully S, Henley C, et al. Cinacalcet attenuates hypercalcemia observed in mice bearing either Rice H-500 Leydig cell or C26-DCT colon tumors. Eur J Pharmacol. 2013;712(1-3):8-15.

REFERENCES

11. Bech A, Smolders K, Telting D, de Boer H. Cinacalcet for Hypercalcemia caused by pulmonary squamous cell carcinoma producing parathyroid hormone-related Peptide. Case Rep Oncol. 2012;5(1):1-8.

1. Reagan P, Pani A, Rosner, MH. Approach to diagnosis and treatment of hypercalcemia in a patient with malignancy. Am J Kidney Dis. 2014 ;63(1):141-7.

12. Karaca H, Lale A, Dikilitas M, Ozkan M, Er O. Recovery of paraneoplastic hypercalcemia by sunitinib treatment for renal cell carcinoma: a case report and review of the literature. Med Oncol. 2010;27(3):1023-6.

2. Valdes-Socin H, Niaourou V, Vandeva S, Bosquée L, Beckers A. Paraneoplastic endocrine syndromes: diagnosis and management. Rev Med Suisse. 2009;5(214):1668-74.

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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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