CLINICS April 2012

I S S N - 1807-5932 printed versionI S S N - 1980-5322 online version

Scientifi c Journal of Hospital das ClínicasFaculdade de Medicina da Universidade de São Paulo

V o l u m e 6 7N u m b e r 4 - A p r i l / 2 0 1 2

CLINICSEditor

Mauricio Rocha-e-SilvaFaculdade de Medicina da Universidade de Sao Paulo

Sao Paulo, SP, Brazil

Editorial Board

Abhijit ChandraKing George’s Medical CollegeLucknow, India

Adamastor Humberto PereiraUniversidade Federal do Rio Grandedo SulPorto Alegre, RS, Brazil

Adauto CasteloUniversidade Federal de Sao PauloSao Paulo, SP, Brazil

Ademar LopesFundacao Antonio Prudente, Hospitaldo CancerSao Paulo, SP, Brazil

Alberto Azoubel AntunesFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Alexandre Roberto PreciosoFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Andrea SchmittUniversity of GoettingenGoettingen, Germany

Arnaldo Valdir ZumiottiFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Artur Brum-FernandesUniversite de SherbrookeQuebec, Canada

Carmita Helena Najjar AbdoFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Cesar Gomes VictoraFaculdade de Medicina da UniversidadeFederal de PelotasPelotas, RS, Brasil

Daniel Romero MunozFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Edmund NeugebauerWitten/Herdecke UniversityWitten, North Rhine - Westphalia,Germany

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

Ernest Eugene MooreUniversity of Colorado DenverDenver, CO, USA

Euclides Ayres CastilhoFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Fabio Biscegli JateneFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Francisco LaurindoFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Hiroyuki HirasawaChiba University School of MedicineChiba, Japan

Irismar Reis de OliveiraFaculdade de Medicina da UniversidadeFederal da BahiaSalvador, BA, Brasil

Irshad ChaudryUniversity of AlabamaBirmingham, AL, USA

Ivan CecconelloFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Ke-Seng ZhaoSouthern Medical UniversityGuangzhou, China

Laura Cunha RodriguesLondon School of Hygiene andTropical Medicine - Universityof LondonLondon, UK

Marcelo ZugaibFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Marco Martins AmatuzziFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Maria Aparecida Shikanai YasudaFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Mauro PerrettiWilliam Harvey Research InstituteLondon, UK

Michael Gregory SarrMayo ClinicRochester, MN, USA

Milton de Arruda MartinsFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Mitchell C. PosnerThe University of Chicago MedicalCenterChicago, IL, USA

Moyses SzkloJohns Hopkins Bloomberg School ofPublic HealthBaltimore, USA

Area Editors

Ana Maria de Ulhoa EscobarFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Anuar Ibrahim MitreFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Ashok AgarwalThe Cleveland Clinic FoundationCleveland, Ohio, USA

Berenice Bilharinho MendoncaFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Bruno ZilbersteinFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Carlos SerranoFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Carmen Silvia Valente BarbasFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Claudia Regina Furquim de AndradeFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Edmund Chada BaracatFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Eliete BouskelaUniversidade do Estado do Rio de JaneiroRio de Janeiro, RJ, Brazil

Emilia Inoue SatoUniversidade Federal de Sao PauloSao Paulo, SP, Brazil

Fulvio Alexandre ScorzaUniversidade Federal de Sao PauloSao Paulo, SP, Brazil

Geraldo BusattoFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Gustavo Franco CarvalhalFaculdade de Medicina da Pontifıcia UniversidadeCatolica do Rio Grande do SulPorto Alegre, Rio Grande do Sul, Brazil

Heitor Franco de Andrade Jr.Faculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Ivete Bedin PradoFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Joaquim Prado Moraes-FilhoFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Ludhmila Abrahao HajjarFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Luız Eugenio Garcez-LemeFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Lydia Masako FerreiraUniversidade Federal de Sao PauloSao Paulo, SP, Brazil

Maria Cecılia SolimeneFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Nelson WoloskerFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Newton Kara-JuniorFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Olavo Pires de CamargoFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Paulo HoffFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Raul CoimbraUniversity of California, San DiegoLa Jolla, CA, USA

Renato Delascio LopesUniversidade Federal de Sao PauloSao Paulo, SP, Brazil

Rubens Belfort Jr.Universidade Federal de Sao PauloSao Paulo, SP, Brazil

Ruth GuinsburgUniversidade Federal de Sao PauloSao Paulo, SP, Brazil

Ruy Jorge Cruz JuniorUniversity of PittsburghPittsburgh, PA, USA

Sergio Paulo BydlowskiFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Silvia Vanessa LourencoFaculdade de Odontologia da Universidade de Sao PauloSao Paulo, SP, Brazil

Simone AppenzellerUniversidade Estadual de CampinasCampinas, SP, Brazil

Sophie Francoise Mauricette DerchainFaculdade de Ciencias Medicas, UniversidadeEstadual de CampinasCampinas, SP, Brazil

Suely Kazue Nagahashi MarieFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Thelma Suely OkayFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Valeria AokiFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Navantino AlvesFaculdade de Ciencias Medicas de Minas GeraisBelo Horizonte, MG, Brazil

Noedir Antonio Groppo StolfFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Pedro Puech-LeaoFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Peter LibbyBrigham and Women’s HospitalBoston, Boston, MA, USA

Philip CohenUniversity of Houston Health Center

Houston, Texas, USA

Rafael Andrade-AlegreSanto Tomas HospitalRepublic of Panama, Panama

Ricardo Antonio RefinettiFaculdade de Medicina da Universidade Federal doRio de JaneiroRio de Janeiro, RJ, Brazil

Roberto ChiesaSan Raffaele HospitalMilan, Italy

Ronald A. AshersonNetcare Rosebank HospitalRosebank, Johannesburg, South Africa

Samir RasslanFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Tarcisio Eloy Pessoa de BarrosFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Valentim GentilFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Wagner Farid GattazFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Board of Governors

Alberto Jose da Silva DuarteAna Claudia Latronico XavierBerenice Bilharinho de MendoncaClarice TanakaClaudia Regina Furquim de AndradeCyro Festa NetoDalton de Alencar Fischer ChamoneDaniel Romero MunozEdmund Chada BaracatEduardo MassadEloisa Silva Dutra de Oliveira BonfaEuripedes Constantino MiguelFabio Biscegli JateneFlair Jose CarrilhoFrancisco Vargas SusoGerson ChadiGilberto Luis CamanhoIrene de Lourdes NoronhaIrineu Tadeu VelascoIvan CecconelloJorge Elias Kalil

Jose Antonio Franchini RamiresJose Antonio SanchesJose Eduardo KriegerJose Otavio Costa AulerJose Ricardo de Carvalho Mesquita AyresLenine Garcia BrandaoLuiz Augusto Carneiro D’AlbuquerqueLuiz Fernando OnuchicMagda Maria Sales Carneiro-SampaioManoel Jacobsen TeixeiraMarcelo ZugaibMarcos BoulosMarcus Castro FerreiraMaria Aparecida Shikanai YasudaMaria Irma Seixas DuarteMiguel SrougiMilton de Arruda MartinsNelson de LucciaNoedir Antonio Groppo StolfOlavo Pires de CamargoPaulo Andrade Lotufo

Paulo Hilario Nascimento SaldivaPaulo Marcelo Gehm HoffPedro Puech-LeaoRemo SusannaRicardo Ferreira BentoRoberto KalilRoberto ZatzRoger ChammasSamir RasslanSandra Josefina Ferraz Ellero GrisiSelma LancmanTarcısio Eloy Pessoa de BarrosUenis TannuriUmbertina Conti ReedValentim GentilVenancio Avancini Ferreira AlvesVicente OdoneWagner Farid GattazWerther Brunow de CarvalhoWilliam Carlos NahasWilson Jacob

Editorial Office: Rua Dr. Ovıdio Pires de Campos, 225 - 6˚ Andar CEP 05403-010 Sao Paulo/SP Tel.: +55-11-2661-6235

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Clinics. Sao Paulo: Scientific Journal of Hospital das Clınicas da Faculdade de Medicina da Universidade de Sao Paulo, 2005-

Monthly Periodical: January to December

ISSN 1807-5932 printed version

ISSN 1980-5322 online version

Formerly Revista do Hospital das Clınicas da FMUSP, 1946–2004.

1. Medicine-scientific production. 2. Medical Sciences I. Hospital das Clınicas da Faculdade de Medicina da Universidade de Sao Paulo.

CDD 610

Editorial DirectorKavita Kirankumar Patel-RolimFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Editorial AssistantNair GomesFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Daniela Aquemi HigaFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Ariane Maris GomesFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

CLINICS publishes peer-reviewed articles of interest to clinicians

and researchers in the medical sciences. CLINICS is registered

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All trials initiated after January 1, 2012 must be registered

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sponsors for each published article. Our 2012 prices are as

follows: original articles: R$ 1.850,00 (US$ 1,100.00); review

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Manuscripts are invited in the following categories:ORIGINAL STUDY: Complete original studies should be

submitted in this category. Three sections are offered: Basic,

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Title page:

N Title (up to 250 characters);

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Manuscript:

N Abstract: limited to 250 words and structured into:Objectives, Method, Results, Conclusions. Citationsor abbreviations (except internationally recognizedsuch as weights, measures, physical or chemical) arenot allowed. Authors are strongly recommended notto display numerical statistical information, butmerely to state what is significantly different (ornot) between described parameters.

N Keywords: 3–6 items from the Medical SubjectHeadings (Mesh) should be used.

N Introduction: should set the purpose of the study,give a brief summary (not a review) of previousrelevant works, and state what new advance has beenmade in the investigation. It should not include dataor conclusions from the work being reported. A finalsentence summarizing the novelty to be presented ispermissible.

N Materials and Methods: should briefly give clear andsufficient information to permit the study to berepeated by others. Standard techniques need onlybe referenced. Previously published methods may bebriefly described following the reference.

N Ethics: when reporting experiments on human sub-jects, indicate whether the procedures followed werein accordance with the ethical standards of theresponsible committee on human experimentation(institutional or regional) and with the HelsinkiDeclaration of 1975, as revised in 1983. Whenreporting experiments on animals, indicate whetherthe institution’s or a national research council’s guidefor, or any national law on the care and use oflaboratory animals was followed.

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PUBLICATION INFORMATION ANDEDITORIAL POLICIES

are complex medical problems (10).’’ Under excep-tional circumstances authors’ names may appear intext: Single author: ‘‘Einstein (11) proposed a newtheory …’’ Two authors: ‘‘Watson & Crick (12)reported on the structure of …’’ Three or moreauthors: ‘‘Smith et al (13) described …’’

N Reference List: Only citations that appear in the textshould be referenced. Unpublished papers, unlessaccepted for publication, should not be cited. Workaccepted for publication should be referred to as ‘‘inpress’’ and a letter of acceptance of the journal mustbe provided. Unpublished data should only be citedin the text as ‘‘unpublished observations’’, and a letterof permission from the author must be provided.Authors are responsible for the accuracy and com-pleteness of their references and for correct textcitation. CLINICS adopts the Vancouver format;references must be restricted to directly relevantpublished works, papers, or abstracts that have beenaccepted for publication. Usually the total number ofreferences should not exceed 35. For up to six authors,list all authors; for more than 6 authors, list first six,followed by ‘‘et al’’.

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N Manuscript: Abstract, keywords and text should bearranged as best suited to cover the subject beingreviewed. If appropriate, the manner of referencecollection should be described. The use of headings,subheadings, and paragraph titles is encouraged forsake of clarity. Abbreviations, acknowledgements,Tables and Figures as in original studies. The numberof references is at the discretion of the authors. Nopublication fee discount is allowed for spontaneouslysubmitted review articles accepted for publication.

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CLINICS

CONTENTS

Clinics 2012 67(4):301–407

EDITORIAL

Stomachs: does the size matter? Aspects of intestinal satiety, gastric satiety, hunger and gluttonySergio Santoro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301

CLINICAL SCIENCES

Predictors of the risk of falls among elderly with chronic atrial fibrillationAngela Cristina Silva dos Santos, Moacyr Roberto Cuce Nobre, Amit Nussbacher, Giselle Helena de Paula Rodrigues,

Otavio Celso Eluf Gebara, Joao Batista Carvalho Serro Azul, Mauricio Wajngarten . . . . . . . . . . . . . . . . . . . . . . . . 305

Chest computed tomography findings in severe influenza pneumonia occurring in neutropenic cancer

patientsRosana Souza Rodrigues, Edson Marchiori, Fernando A. Bozza, Melissa Tassano Pitrowsky, Eduardo Velasco,

Marcio Soares, Jorge I. F. Salluh. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313

Daily activities are sufficient to induce dynamic pulmonary hyperinflation and dyspnea in chronic

obstructive pulmonary disease patientsAntonio A.M. Castro, Claudia Kumpel, Rosana Chaves Rangueri, Maurıcio Dalcin Oliveira, Rodrigo Alves Dornelles,

Emerson Roberto Brito, Tania Maria Seki, Elias F. Porto . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319

The behavior and diagnostic utility of procalcitonin and five other inflammatory molecules in

critically ill patients with respiratory distress and suspected 2009 influenza a H1N1 infectionMariana Benevides Santos Paiva, Fernando Antonio Botoni, Antonio Lucio Teixeira Jr., Aline Silva de Miranda, Clara

Rodrigues Alves de Oliveira, Jamila de Oliveira Abrahao, Guilherme Marques Faria, Vandack Nobre . . . . . . . . . . . 327

Endophenotypes and serotonergic polymorphisms associated with treatment response in obsessive-

compulsive disorderFabio M. Corregiari, Marcio Bernik, Quirino Cordeiro, Homero Vallada . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335

Psychological crisis intervention for the family members of patients in a vegetative stateYa-Hong Li, Zhi-Peng Xu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341

Plasma kinetics of an LDL-like nanoemulsion and lipid transfer to HDL in subjects with glucose

intoleranceMarina P. Bertato, Carolina P. Oliveira, Bernardo L. Wajchenberg, Antonio C. Lerario, Raul C. Maranhao. . . . . . . 347

The impact of pretransplant donor-specific antibodies on graft outcome in renal transplantation: a

six-year follow-up study

ISSN-1807-5932

Elias David-Neto, Patricia Soares Souza, Nicolas Panajotopoulos, Helcio Rodrigues, Carlucci Gualberto Ventura, Daisa

Silva Ribeiro David, Francine Brambate Carvalhinho Lemos, Fabiana Agena, William Carlos Nahas, Jorge Elias Kalil,

Maria Cristina Ribeiro Castro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355

A proton nuclear magnetic resonance-based metabonomics study of metabolic profiling inimmunoglobulin a nephropathyWeiguo Sui, Liping Li, Wenti Che, Guimai Zuo, Jiejing Chen, Wuxian Li, Yong Dai . . . . . . . . . . . . . . . . . . . . . . . 363

BASIC RESEARCH

Influence of food restriction on lipid profile and spontaneous glucose levels in male rats subjected toparadoxical sleep deprivationTathiana Aparecida Alvarenga, Sergio Tufik, Gabriel Natan Pires, Monica Levy Andersen . . . . . . . . . . . . . . . . . . . 375

REVIEWS

Anesthesia-related mortality in pediatric patients: a systematic reviewLeopoldo Palheta Gonzalez, Wangles Pignaton, Priscila Sayuri Kusano, Norma Sueli Pinheiro Modolo, Jose Reinaldo

Cerqueira Braz, Leandro Gobbo Braz . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381

Castration-resistant prostate cancer: systemic therapy in 2012Fernando C. Maluf, Oren Smaletz, Daniel Herchenhorn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389

RAPID COMMUNICATION

DEFB1 gene 59 untranslated region (UTR) polymorphisms in inflammatory bowel diseasesValentina Zanin, Ludovica Segat, Anna Monica Bianco, Lara Padovan, Nathalia de Alencar Cunha Tavares,

Sergio Crovella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395

TECHNICAL NOTE

Assessment of organ transplants from donors with markers of hepatitis BEdson Abdala, Luis Sergio Fonseca de Azevedo, Vivian Iida Avelino-Silva, Sılvia Figueiredo Costa, Marlova

Luzzi Caramori, Tania Mara Varejao Strabelli, Lıgia Camera Pierrotti, Heloisa Helena Marques da Silva, Marta

Heloisa Lopes, Glaucia Fernanda Varkulja, Vera Aparecida Santos, Maria Aparecida Shikanai-Yasuda, Comissao de

Infeccao em Imunodeprimidos, Hospital das Clınicas da Faculdade de Medicina da USP. . . . . . . . . . . . . . . . . . . . . 399

CASE REPORT

Scurvy in an alcoholic malnourished cirrhotic man with spontaneous bacterial peritonitisAndre Luiz Maltos, Guilherme Vannucchi Portari, Joao Carlos Saldanha, Aderbal Garcia Bernardes Junior, Guilherme

Rocha Pardi, Daniel Ferreira da Cunha . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405

EDITORIAL

Stomachs: does the size matter? Aspects of intestinalsatiety, gastric satiety, hunger and gluttonySergio Santoro

Hospital Albert Einstein, Department of Surgery.

Many surgeons and most laypeople think that there is astrict correlation between the size of the stomach and thesize of the meals. The term ‘‘stomach reduction’’ became asynonym for weight loss surgeries, as if by reducing thestomach, all meals would automatically be small.

However, there is no strict correlation between the size ofthis organ and the size of meals. Some morbidly obesepatients have previously undergone total gastrectomy! If asimple stomach reduction treated obesity, the absence of astomach should cause impressive weight loss; however, thisprinciple is not always true. On the other hand, somepatients, despite having normal-sized stomachs, feel verysatisfied with three spoonfuls of food. How can thesephenomena be explained?

The stomach is a pouch with a hole. The rate of emptyingis even more important than the size of the pouch itself. Inaddition, a variety of different and complex mechanisms areinvolved in the initiation and termination of a meal.

The rate of gastric emptying to the bowel is veryimportant. It defines the amount of nutrients that may,through absorption, enter the blood stream, thereby alteringthe blood composition and threatening the homeostasis. Afast and intense nutrient absorption rapidly changes theblood composition (i.e., with elevations in sugars and lipids)and demands a quick and efficient metabolic answer.

Some refined and pre-digested elements of the moderndiet are free of fiber and completely ready for a fastabsorption. These elements have been called high-glycemicindex foods, ever since Jenkins (1) defined the concept of theglycemic index in 1980.

In the stomach, food is mixed with acidic gastric secretionsand proteolytic enzymes. Most microbes ingested with foodare killed by acid; therefore, any contamination risk isminimized. Large pieces of food are broken down, and theosmolarity of the content is adjusted. The food and drink thatpresent the right osmolarity will leave the stomach sooner,thereby explaining why one can drink a liter of sweet iced teamore easily and quickly than a liter of pure water.Additionally, the stomach adds the R factor and intrinsicfactor to allow vitamin B12 to be absorbed in the ileum.

Once these steps are complete, the stomach sends thechyme (i.e., the food mixed with digestive secretions) to the

small bowel, where it is immediately admixed with thebiliopancreatic juices that lead to near-complete digestion(mammals cannot digest fiber without the bacterial help offermentation, which occurs in the colon).

In the duodenum, the absorption of small particles isinitiated promptly, and the chyme keeps moving down-wards as the absorption process occurs. Endogenousglucose production is not interrupted at this stage (i.e., thepancreatic a-cells do not suspend glucagon production).Hypoglycemia may kill an animal in minutes, so theproximal bowel does not have the ‘‘authority’’ to give suchan extreme order, but it can produce a less powerful one(i.e., the proximal gut secretes glucose-dependent insulino-tropic polypeptide, an insulinotropic agent that cannotsuppress glucagon and the endogenous production ofglucose (2-4)). At this point, satiety is not supposed tooccur. For quite obvious reasons, the proximal gut is not thepoint at which food should trigger intense satiety or initiatethe suspension of glucagon production.

Nonetheless, when the distal portion of the small bowelreceives nutrients (meaning that a significant meal waseffectively consumed), the neuroendocrine L cells in themucosa produce hormones like glucagon-like peptide 1(GLP-1), oxyntomodulin and polypeptide YY (PYY), whichare typical postprandial hormones (5-7). These hormonespromote the transition from the fasting state to a postprandialstate. In the fasting state, there is hunger, high levels ofglucagon, endogenous glucose production, and lipolysis. In thetypical postprandial state, an intense production of insulin,blood clearance of glucose and lipids, lipogenesis, and adiminution in the gastric emptying and satiety progressivelyappear. They are opposite metabolic states.

GLP-1 inhibition of gastric emptying indeed outweighs itsinsulinotropic effects (8). In other words, the distal intestinestops the gastric emptying at a certain point (e.g., whenstimulated by nutrients). Therefore, the intestine defines thefunctional size of the stomach.

If we suddenly eat progressively more and more refinedfood, absorption becomes easier and more intense in theproximal gut, thereby reducing the distal stimulation.

As a consequence, there may be deficiencies in theproduction of distal gut hormones, like GLP-1 and PYY(which happens in obese and type 2 diabetic patients(9,10)), and much food may pass through stomach,regardless of its size. If we do not have the properstimulation of the distal gut, a simple reduction of thestomach may not work. Indeed, some post-surgical patientsleft with 30-mL stomach pouches can still eat enough toremain obese or at least to regain most of the lost weightafter post-operative adaptation.

Email: [email protected]

Tel.: 55-11-9137-0930

Copyright � 2012 CLINICS – This is an Open Access article distributed underthe terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided theoriginal work is properly cited.

No potential conflict of interest was reported.

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After the distal intestine is busy with digestion andabsorption, it secretes the distal gut hormones, whichimpede further gastric emptying. The typical hunger issuppressed in the brain, where there are receptors for guthormones. Elevated GLP-1 and insulin suppress theproduction of ghrelin (a hormone that provokes hungerand the behavior aimed at seeking food (11)).

Surprisingly, even then, superior animals (includinghumans) do not stop eating. At this stage, the stomach isnot emptying well anymore, yet the animal continues eatinguntil the stomach is very full. GLP-1 facilitates this storageprocess because it provokes a relaxation of the gastricfundus, therefore allowing the stomach to receive more food(12). At this point, an animal would not actively look forfood (i.e., the foraging behavior); however, if there is spacein the stomach and food available, the animal continueseating. The ‘‘intestinal satiety’’ has already been initiated,the typical hunger is gone, but ‘‘the gastric satiety’’ does notappear until the stomach is completely full.

It is not hunger that motivates the animal at this point, butgluttony. The distinction between intestinal and gastricsatiety was just created as an important didactic divisionfor general understanding.

Gluttony is not a sin. It is a wonderful instinct, developedover millions of years, for times of scarcity. A wild dog thatfinds food today is not certain he will find it againtomorrow. Creating reserves may cause some fullnesstoday, but it may save his life in the near future.

In summary, there are two different phases of alimenta-tion. Initially, there is hunger: the stomach empties easily(and its size does not much matter at this point), and theintestine is receptive. Later, the intestine is loaded, and thedistal gut hormones are produced to trigger a metabolicresponse. Gastric emptying is dramatically reduced, andGLP-1 relaxes the gastric fundus to allow further eating.Thus, the hunger is gone, but there is gluttony. Withgluttony, the animal keeps on eating until there the stomachis physically full, and the gas at the gastric fundus isprogressively expelled by eructation.

Therefore, after the intestinal satiety phase, it is normal tocontinue eating, so an animal can take along what the bowelcannot process immediately. The animal eats until thestomach is full. This gastric satiety phase complements theintestinal satiety phase. It is common to hear from patients:‘‘Doctor, I keep on eating even when I am not hungryanymore! I think it is anxiety!’’

Most obese patients present with an attenuated anddelayed intestinal satiety because they have a diminishedsecretion of distal gut hormones after meals. Therefore, thestopping point in gastric emptying is also delayed and thecentral satiety signals are jeopardized.

In this extreme scenario, impeding someone from eatingby simply tying the digestive tract with bands, narrowinganastomoses or reducing stomachs will create either a leanbut unhappy patient if he or she really cannot eat or apatient who is still fat if he or she can continue to eat.

So, does the size of the stomach matter at all? Yes, it matters.The development of this storage chamber that allows you to‘‘overeat’’ to create storage if the next meal fails is very adequateduring times of scarcity. If food scarcity is suddenly substitutedby food abundance and the next meal is always there,overeating may occur with every meal. To proportionallyreduce this organ adapts the individual to abundance. Theevolutionary data strongly support the idea that mechanisms of

food storage are found in those individuals exposed to scarcity(e.g., a camel stocks water, a frog does not).

In the current western world, there is abundance: the foodis refined, pre-digested and quickly absorbed in the upperbowel (i.e., high glycemic index foods are prevalent). Inthese circumstances, the intestinal satiety may come too late,and the gastric emptying is not properly diminished in time.In this case, the size of the stomach (although over-sized forthe times of abundance) will not matter greatly, and it willbe perfectly possible for someone with a minimal stomach,or even none, to be very fat.

Mechanical restriction and malabsorption were alwaysthe two pillars of classic bariatric surgery. However, it isnow clear that neither of these factors is responsible for themost beneficial effects of this type of surgery (13). Instead,they are a primary source of post-operative problems (14).

To reach ‘‘the two satieties’’ at the optimal time and in theabsence of mechanical restriction and malabsorption is aphysiological method of approaching the epidemics ofobesity and diabetes.

In recent years, the majority of researchers in the fieldrecognize that the most effective models of classic bariatricsurgery work because of the hormonal changes they provoke(13). As such, they started calling this surgical specialty‘‘Bariatric and Metabolic Surgery.’’ Nonetheless, the proceduresare the same, and they still contain restrictive and malabsorp-tive elements because they have been designed to include them.

New procedures, mainly developed in Brazil (15-20),were specifically designed to selectively cause the hormonaland metabolic corrections. By abolishing mechanical restric-tion (21) (through the means of minimal stomachs withnarrow anastomoses or bands) and also avoiding excludeddigestive segments and malabsorption, we may reach the‘‘Pure Metabolic Surgery.’’ It will be an evolution.

REFERENCES

1. Jenkins DJ, Wolever TM, Taylor RH, Ghafari H, Jenkins AL, Barker H,et al. Rate of digestion of foods and postprandial glycaemia in normaland diabetic subjects. Br Med J. 1980;281(6232):14-7, http://dx.doi.org/10.1136/bmj.281.6232.14.

2. Siegel EG, Creutzfeldt W. Stimulation of insulin release in isolated ratislets by GIP in physiological concentrations and its relation to islet cyclicAMP content. Diabetologia. 1985;28(11):857–61, http://dx.doi.org/10.1007/BF00291078.

3. Ross SA, Dupre J. Effects of ingestion of triglyceride or galactose on secretionof gastric inhibitory polypeptide and on responses to intravenous glucose innormal and diabetic subjects. Diabetes 1978;27(3):327–33.

4. Meier JJ, Gallwitz B, Siepmann N, Holst JJ, Deacon CF, Schmidt WE, et al.Gastric inhibitory polypeptide (GIP) dose-dependently stimulatesglucagon secretion in healthy human subjects at euglycaemia.Diabetologia 2003;46(6):798–801.

5. Bojanowska E. Physiology and pathophysiology of glucagon-likepeptide-1 (GLP-1): The role of GLP-1 in the pathogenesis of diabetesmellitus, obesity, and stress. Med Sci Monit. 2005;11(8):RA271-8.

6. Ballantyne GH. Peptide YY(1-36) and Peptide YY(3-36): Part I.Distribution, Release and Actions. Obes Surg. 2006;16:651-8, http://dx.doi.org/10.1381/096089206776944959.

7. Cohen MA, Ellis SM, Le Roux CW, Batterham RL, Park A, Patterson M,et al. Oxyntomodulin Suppresses Appetite and Reduces Food Intake inHumans. J Clin Endocrinol Metab. 2003;88(10):4696–701, http://dx.doi.org/10.1210/jc.2003-030421.

8. Nauck MA, Niedereichholz U, Ettler R, Holst JJ, Orskov C, Ritzel R, et al.Glucagon-like peptide 1 inhibition of gastric emptying outweighs itsinsulinotropic effects in healthy humans. Am J Physiol. 1997;273(5 Pt 1):E981-8.

9. Ranganath LR, Beety JM, Morgan LM, Wright JW, Howland R, Marks V.Attenuated GLP-1 secretion in obesity: cause or consequence? Gut.1996;38(6):916-9, http://dx.doi.org/10.1136/gut.38.6.916.

10. Lugari R, Dei Cas A, Ugolotti D, Finardi L, Barilli AL, Ognibene C, et al.Evidence for early impairment of glucagon-like peptide 1-inducedinsulin secretion in human type 2 (non insulin-dependent) diabetes.Horm Metab Res. 2002;34(3):150-4.

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11. Muccioli G, Tschop M, Papotti M, Deghenghi R, Heiman M, Ghigo E,et al. Neuroendocrine and peripheral activities of ghrelin: implications inmetabolism and obesity. Eur J Pharmacol 2002;440(2-3):235-54.

12. Schirra J, Wank U, Arnold R, Goke B, Katschinski M. Effects of glucagon-like peptide-1(7-36)amide on motility and sensation of the proximalstomach in humans. Gut. 2002;50(3):341-8, http://dx.doi.org/10.1136/gut.50.3.341.

13. Holst JJ. Postprandial Insulin Secretion After Gastric Bypass Surgery:The Role of Glucagon-Like Peptide 1. Diabetes 2011;60(9):2203-5.

14. Byrne TK. Complications of obesity surgery. Surg Clin North Am.2001;81(5):1181-93, http://dx.doi.org/10.1016/S0039-6109(05)70190-0.

15. Santoro S. Adaptive and Neuroendocrine procedures: A new pathway inBariatric and Metabolic Surgery. Obes Surg 2008;18(10):1343-45.

16. Milleo FQ, Campos AC, Santoro S, Lacombe A, Santo MA, Vicari MR,et al. Metabolic effects of an entero-omentectomy in mildly obese type 2Diabetes Mellitus patients after three years. Clinics 2011;66(7):1227-33.

17. Santoro S, Castro LC, Velhote MCP, et al. Sleeve Gastrectomy withTransit Bipartition. A Potent Intervention for Metabolic Syndrome andObesity. Ann Surg, in press.

18. Santoro S. Is the Metabolic Syndrome a Disease of the Foregut? Yes,Excessive Foregut. Ann Surg. 2008;247(6):1074-5.

19. de Paula AL, Macedo AL, Prudente AS, Queiroz L, Schraibman V, Pinus J.Laparoscopic sleeve gastrectomy with ileal interposition (‘‘neuroendo-crine brake’’)-pilot study of a new operation. Surg Obes Relat Dis.2006;2(4):464-7, http://dx.doi.org/10.1016/j.soard.2006.03.005.

20. Santoro S, Malzoni CE, Velhote MC, Milleo FQ, Santo MA, Klajner S, et al.Digestive Adaptation with Intestinal Reserve: A neuroendocrine-basedprocedure for morbid obesity. Obes Surg. 2006;16(10):1371-9, http://dx.doi.org/10.1381/096089206778663841.

21. Santoro S. Is sleeve gastrectomy a restrictive or an adaptive procedure?Reflections on the concepts of restriction and adaptation. Ann Surg.2010;252(5):892-3.

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CLINICAL SCIENCE

Predictors of the risk of falls among elderly withchronic atrial fibrillationAngela Cristina Silva dos Santos, Moacyr Roberto Cuce Nobre, Amit Nussbacher, Giselle Helena de Paula

Rodrigues, Otavio Celso Eluf Gebara, Joao Batista Carvalho Serro Azul, Mauricio Wajngarten

Faculdade de Medicina da Universidade de Sao Paulo, Heart Institute, Sao Paulo/SP, Brazil.

OBJECTIVES: Though elderly persons with chronic atrial fibrillation have more comorbidities that could limitindications for the chronic use of anticoagulants, few studies have focused on the risk of falls within this particulargroup. To evaluate the predictors of the risk of falls among elderly with chronic atrial fibrillation, a cross-sectional,observational study was performed.

METHODS: From 295 consecutive patients aged 60 years or older with a history of atrial fibrillation who wereenrolled within the last 2 years in the cardiogeriatrics outpatient clinic of the Instituto do Coracao do Hospital dasClınicas da Faculdade de Medicina da Universidade de Sao Paulo, 107 took part in this study. Their age was 77.9¡6.4years, and 62 were female. They were divided into two groups: a) no history of falls in the previous year and b) ahistory of one or more falls in the previous year. Data regarding the history of falls and social, demographic,anthropometric, and clinical information were collected. Multidimensional assessment instruments and ques-tionnaires were applied.

RESULTS: At least one fall was reported in 55 patients (51.4%). Among them, 27 (49.1%) presented recurrent falls,with body lesions in 90.4% and fractures in 9.1% of the cases. Multivariate logistic regression showed that self-reported difficulty maintaining balance, use of amiodarone, and diabetes were independent variables associatedwith the risk of falls, with a sensitivity of 92.9% and a specificity of 44.9%.

CONCLUSION: In a group of elderly patients with chronic atrial fibrillation who were relatively independent andable to attend an outpatient clinic, the occurrence of falls with recurrence and clinical consequences was high.Difficulty maintaining balance, the use of amiodarone and a diagnosis of diabetes mellitus were independentpredictors of the risk for falls. Thus, simple clinical data predicted falls better than objective functional tests.

KEYWORDS: Falls; Elderly Atrial Fibrillation; Questionnaires; Risk Assessment.

Santos ACS, Nobre MRC, Nussbacher A, Rodrigues GHP, Gebara OCE, Serro Azul JBC, et al. Predictors of the risk of falls among elderly with chronicatrial fibrillation. Clinics. 2012;67(4):305-311.

Received for publication on December 1, 2011; First review completed on December 7, 2011; Accepted for publication on December 22, 2011

E-mail: [email protected]

Tel.: 55 11 2661-5120

INTRODUCTION

Atrial fibrillation (AF) and falls are common among theelderly, which has increasing relevance as the life expec-tancy of the population rises.

Not only do structural and functional alterations of thecardiovascular system upon aging predispose patients toAF, but they also reduce the capacity of hemodynamictolerance to arrhythmia, which may lead to syncope and fall(1,2). The proportion of patients with relevant and highlyfall-related functional disabilities following ischemic strokerelated to AF may reach 59% (3).

Anticoagulation therapy is characterized by the strategyof preventing thromboembolic events in elderly personswith AF (4), but there is no consensus about the risk of itsuse among patients with a history of falls, particularly inrecurrent cases of falls.

In patients with AF who are at a high risk for falls, Gageet al. (5) showed that warfarin is related to mortalityassociated with intracranial hemorrhage, but not to theoccurrence of intracranial hemorrhage. Despite that, the useof anticoagulants was still beneficial owing to the high riskof embolic stroke in this group.

A meta-analysis of antithrombotic therapy in elderlypatients at risk for falls concluded that the propensity forfalling in elderly patients should not be an important factorwhen deciding whether a patient is a good candidate for theanticoagulation treatment for AF (6). However, in therandomized controlled trials evaluated in the meta-analysis,patients may have been monitored more intensely than isusual in clinical practice.




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Recently, in a study that included 4,093 patients olderthan 80 years who were carefully monitored by ananticoagulation clinic, the rate of bleeding was low,suggesting that age itself should not be considered acontraindication to treatment. However, a history of twoor more falls in a year was independently associated withbleeding risk, as assessed by Cox regression analysis (7).

Guidelines for the prevention of falls in the elderlyrecommend a series of instruments that should be appliedin individuals at risk of falling (8,9). However, a thoroughhistory, including questions about previous falls, medica-tions, and balance, could be used for fall risk assessment(10).

Thus, the issue of anticoagulation treatment of patientswith atrial fibrillation to prevent embolic stroke vis-a-vis therisk of falls has not been fully established in real-life elderlypatients. To evaluate predictors of falls among elderlypatients with chronic atrial fibrillation and indications foranticoagulant therapy, a cross-sectional, observational studywas designed.

METHODS

A cross-sectional, observational study design was used.The project was approved by the Scientific and EthicsCommittees of InCor-HCFMUSP (Scientific: PROTOCOLSDC 2775/06/020, approved on 20/04/2006, and Ethics:PROTOCOL No. 450/06, approved on 25/05/2006), and allparticipants agreed to participate and signed an InformedConsent Form.

Male and female patients aged 60 years or older with adiagnosis of AF from the cardiogeriatrics outpatient clinic ofthe Instituto do Coracao of the Hospital das Clınicas daFaculdade de Medicina da Universidade de Sao Paulo(InCor-HCFMUSP) were recruited from their regularambulatory visit or summoned through telephone callsfrom the outpatient clinic database registry of patients withAF from September 2006 to May 2007, in order of arrival.

The selected patients were divided into two groups: a)those with no history of falls in the previous year and b) thosewith a history of one or more falls in the previous year.

Data regarding the history of falls and social, demo-graphic, anthropometric, and clinical information werecollected. Furthermore, multidimensional assessmentinstruments and questionnaires were applied. The historyof falls included the occurrence, number, local, character-istics and consequences of falls (according to the definitionof Tinetti et al. (11) within the previous 12 months. Socialand demographic data included gender, age, marital status,education level, people living in the same dwelling,remuneration and professional activity. Anthropometricdata included weight, height, and body mass index (12).

Patients were further classified by whether they weresymptomatic, their eating habits, physical exercise habits,smoking status, abuse of alcohol, medications used, and thepresence of cardiovascular diagnoses and comorbidities.The physical examination, performed by the same physi-cian, included simplified and systematized assessments ofcranial pairs, ocular movements, pyramidal signals, muscletonus and strength, reflexes and motor coordination,sensitivity and proprioception, and the Romberg balancetest.

The following questionnaires and instruments validatedin Portuguese were used: Brazilian Functional Assessment

Questionnaire (BOMFAQ; Brazilian version of the OARS)(13,14), Mini Mental State Examination (MMSE) (15,16),Primary Care Evaluation for Mental Disorders (Prime MD)(17,18,19), and Health Assessment Questionnaire(HAQ)(20).

Sensorial assessment was performed by inquiring abouthearing conditions with the following question (21,22): ‘‘Doyou feel that you have experienced hearing loss?’’ Visualassessment included campimetry and the Snellen scale test(Herman Snellen, 1862) (23).

Balance and gait were assessed by applying the BergFunctional Balance Scale (24,25), Performance-OrientedMobility Assessment (POMA) (26,27), Balance POMA andGait POMA, and Timed Up & Go (TUG) (28).

Statistical analysisClassification variables were descriptively presented in

contingency tables comprising absolute (n) and relative (%)frequencies. The associations among variables wereassessed with the chi-square test, likelihood test, orFisher’s exact test. Quantitative variables were presenteddescriptively in tables, including mean and standarddeviation or median, minimum and maximum values.Means were compared using the Student’s t-test or theWilcoxon rank-sum test. Values of p,0.05 were consideredstatistically significant (29). Significant variables in theunivariate analysis were used in the regression modeladjustment to determine the sensitivity, specificity, esti-mated probability of falls and odds ratios. The logisticregression model was used to examine the relationshipsbetween independent variables for the risk of falling. Allsignificant variables (p#0.05) and the interactions betweenthem were used to obtain a selection for the multivariatemodel. We opted not to correct the critical value of p in thefunction of multiple comparisons because the non-signifi-cant variables were not included and were deemedirrelevant to those that remained in the model, whichmeant that the variables in the model did not requirecorrection. All significant variables in the univariateanalysis were adjusted in the logistic regression modeland related according to the ‘‘stepwise’’ procedure. Thevariables that remained in the model were independentpredictor variables.

RESULTS

From the approximately 3,000 patients/year enrolled inthe cardiogeriatrics outpatient clinic of the InCor-HCFMUSP, all 295 patients aged 60 years or older with ahistory of atrial fibrillation who were registered in the 24months prior to the period of clinical evaluation, which wasSeptember 2006 to May 2007, were eligible to participate inthe study. Between the date of registration and the date ofevaluation, 26 patients died, 97 were not available throughthe telephone numbers given to the institution, 11 patientsdid not show up on the day of evaluation, 15 could notparticipate due to living outside of Sao Paulo, 36 wereexcluded because they lacked the clinical conditionsrequired, and three refused to participate. Patients whowere not contacted by telephone after three attempts ondifferent days and times were excluded.

Ultimately, 107 patients took part in the full study(Figure 1).

Predictors of the Risk of FallsSantos ACS et al.

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They had an advanced age (77.9¡6.4 years) and apredominance of shared dwellings (81.3%) and femalegender (57.9%). Most patients reported cardiovascularsymptoms (82.2%), were sedentary (80.4%), and used fouror more medications (87%). The most frequent diagnoseswere arterial hypertension (91%), osteoarthritis (47.7%),dyslipidemia (46.7%), New York Heart Association classI/II heart failure (41%), obesity (30.8%), diabetes mellitus(18.7%), and heart failure class III (12.2%). The presence offour or more comorbidities was frequent (44.9%). Patientswere divided and compared according to the history of fallsin the previous year (Table 1).

Occurrence of falls: One fall was reported in 55 patients(51.4%). Of these, 49.1% presented recurrent falls, 32.7% fell

at home, and 28.9% fell on the street. The most frequentreasons for falling were trips (35.3%) and slips (23.5%).

Dizziness and vertigo caused falls only in 6% of thereports. The falls had consequences of body lesions in 90.4%and fractures in 9.1% (five cases). Sixty percent of thefractures were in the pelvis, and these led to hospitalizationin 7.7% (four cases) of the falling episodes.

Demographic data: There was no significant differenceregarding the occurrence of falls between the genders(54.8% in female patients and 46.7% in male patients).

Only 19% of the patients lived alone, and 6% were singleor had never lived with partners. However, there was nosignificant relationship between the occurrence of falls andmarital status, scholarship or income level, nor was there

Figure 1 - Study design.

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any association between the occurrence of falls and age orBMI.

Clinical data: There was a significant correlation betweenthe occurrence of falls and the presence of clinicalsymptoms (p = 0.015), mainly palpitations (p = 0.044), andtiredness (p = 0.001). There was no significant relationshipbetween falling and a sedentary lifestyle, smoking, oralcohol consumption.

Regarding the use of medications, 86.9% of the patientsused four or more medications, and only 2.8% (threepatients) did not use any medication. There was nosignificant correlation between the occurrence of falls andthe number of medications used.

There was a significant correlation between the occur-rence of falls and the use of anticonvulsants and benzodia-zepines (p = 0.0410) and amiodarone (p = 0.0046), despite theinfrequent use of these drugs.

Among the diagnoses, there were significant relationshipsbetween falling and congestive heart failure class III(p = 0.009) and systolic hypertension (p = 0.034).

Among the comorbidities, type 2 diabetes mellitus wasthe only condition with a significant correlation with theoccurrence of falls (p = 0.015). Decreased muscle strength inthe upper or lower limbs was also associated with theoccurrence of falls.

Questionnaires and instruments:There was a significant association between the occur-

rence of falls and the domain of daily life activities of theBOMFAQ (OARS), which revealed correlations betweenfalling and difficulty eating (p = 0.004), bathing withoutassistance (p = 0.026), getting to the bathroom in time(p = 0.009), preparing meals (p = 0.035), and trimming toe-nails (p = 0.049). Furthermore, low life satisfaction (p = 0.007),

Table 1 - Characteristics and results of the elderly population with AF.

Variable FALL TOTAL p-value

NO YES

Female 28 45.16% 34 54.84% 62 0.283 *

Age (years) 77. 07 + 6. 58 78. 46 + 6. 29 77. 86 + 6. 42 0.276 #

BMI (kg/m2) 28 (19 - 55) 26 (19 - 38) 27 (19 - 55) 0.444 ##

Visual deficiency 32 42.11% 44 57.89% 76 0.005 *

Hearing deficiency 19 45.24% 23 54.76% 42 0.029 **

Physical activity 10 45.45% 12 54.55% 22 0.705 *

Symptoms 39 43.33% 48 53.33% 90 0.015 *

Palpitations 25 41.67% 35 58.33% 60 0.044 *

Tiredness 28 37.33% 47 62.67% 75 0.001 *

Paresthesias 16 38.10% 26 61.90% 42 0.037 *

Cephalea 4 26.67% 11 73.33% 15 0.041 *

Syncopes 0 0.00% 5 100.00% 5 0.027 ***

Muscle strength

Hand grip 19 45.24% 30 71.43% 42 0.021 ***

Quadrıceps 17 40.48% 25 59.52% 42 0.001 **

CHF FC I/II 22 50.00% 22 50.00% 44 0.934 *

Dilated myocardiopathy 26 39.39% 40 60.61% 66 0.085 *

Mitral failure 11 55.00% 9 45.00% 20 0.586 *

Coronary failure 5 29.41% 12 70.59% 17 0.077 *

CHF FC III 2 15.38% 11 84.62% 13 0.009 *

Tricuspid failure 7 53.85% 6 46.15% 13 0.739 *

Aortic failure 6 85.71% 1 14.29% 7 0.112 ***

Previous AMI 1 16.67% 5 83.33% 6 0.205 ***

COMORBIDITIES

Obesity 17 51.52% 16 48.48% 33 0.731 *

Diabetes mellitus 5 25.00% 15 75.00% 20 0.015 *

Neuropathies 6 30.00% 14 70.00% 20 0.052 *

COPD 6 33.33% 12 66.67% 18 0.131 *

Depression 8 47.06% 9 52.94% 17 0.824 *

Thyropathies 8 47.05% 9 52.94% 17 0.487 **

Peripheral vascular disease 9 60.00% 6 40.00% 15 0.381 *

Rheumatological diseases 3 33.33% 6 66.67% 9 0.488 ***

Chronic renal failure 3 37.50% 5 62.50% 8 0.716 ***

Gastric disorders 1 14.29% 6 85.71% 7 0.113 ***

Prostatism 5 71.43% 2 28.57% 7 0.265 ***

Pulmonary hypertension 2 28.57% 5 71.43% 7 0.437 ***

Neoplasia 2 33.33% 4 66.67% 6 0.678 ***

Epilepsy 2 66.67% 1 33.33% 3 0.598 ***

Scores

HAQ 0.72 0-2.25) 1.20 (0-2.88) 0.97 (0-2.88) 0,002* ##

MMSE 25 (9-30) 23.98(7-30) 24.47(7-30) 0,1761 ##

Timed Up & Go 11 (6 - 30) 13 (8 - 56) 12 (6 - 56) 0.063 ##

Gait POMA 11 (5 - 13) 11 (2- 12) 11 (2 - 13) 0.148 ##

Balance POMA 16 (8 - 16) 15 (1 - 16) 16 (1 - 16) 0.018 ##

Berg Balance Scale 51 (20 - 56) 51 (15 - 56) 51 (15 - 56) 0.224 ##

*Chi-square test **Likelihood ratio test *** Fisher’s exact test.

# Student’s t-test ## Wilcoxon’s rank sum test.


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perception of poor health quality (p = 0.0001), a sense ofuselessness (p = 0.005), hearing difficulties in noisy environ-ments (p = 0.023), deglutition (p = 0.043), and balance diffi-culties (p = 0.0001) were also significantly associated withfalls.

The relationships between the occurrence of falls and theMini-mental questionnaire scores were not significant.However, the occurrence of falls was significantly asso-ciated with major depression due to physical illness,dysthymia and panic disorder according to the Prime MD,as well as with the HAQ scores.

There was a significant association between hearing orvision deficiencies and the occurrence of falls. From thebalance and gait tests, only the score of the Balance POMAtest revealed an association with the occurrence of falls.

Logistic regressionThe logistic regression model was used to examine the

relationships between independent variables on the risk offalling. All significant variables (p#0.05) and the interac-tions between them were used in the multivariate model.The following independent variables were identified bylogistic regression analysis: use of antiarrhythmics (amio-darone), difficulty maintaining balance as assessed by theBOMFAQ questionnaire, and the presence of diabetesmellitus. The sensitivity was 92.9%, and the specificity44.9%.

A patient with these three independent variables had a97% chance of falling within the next year (Table 2).Simulating possible results revealed that 1) patients takingamiodarone who had diabetes mellitus had an 86% chanceof falling within the next year; 2) those on amiodarone andpresenting balance difficulty, 89%; and 3) those withdiabetes and balance alterations, 85%. Patients not present-ing any of these variables had a 16.4% chance of fallingwithin a year (Table 3). The odds ratios for falls in one yearadjusted for all significant variables in the logistic regressionmodel indicated that the use of amiodarone increased therisk of falls in one year seven times (odds ratio = 6.952);diabetes mellitus increased the risk five times (oddsratio = 4.909); and self-reported difficulty maintaining bal-ance increased the risk six times (odds ratio = 5.862).

Similarly, the positive likelihood ratios showed that theincreased the risk of falling within the next year five times(positive likelihood ratio = 5). Positive likelihood ratio =sensitivity/1-specificity = 45/7 = 5; negative likelihoodratio = 1-sensitivity/specificity = 55/93 = 0.6.

DISCUSSION

The present study shows that use of amiodarone, thepresence of diabetes mellitus and self-reported difficultymaintaining balance (as assessed by the BOMFAQ ques-tionnaire) increase the likelihood of falls in elderly patients

with AF. Therefore, simple clinical data predict the risk offalls better than validated functional tests (30,31,32).

Furthermore, the high number of recurrences confirmsthe higher risk for patients with previous falls (33,34).

In the literature, there is no clear definition of whichpatients are at risk of falls.

The study population was relatively small, and thereforeinadequate to establish direct measures of risk prediction,but it did allow us to evaluate the relationships betweenmany variables and the occurrence of falls.

In contrast with other studies, age, gender (35,36), use ofanti-hypertensive drugs (37), and cognitive impairmentwere not associated with the occurrence of falls.

However, most of the variables considered as risk factorswere associated with the occurrence of falls, and it is knownthat the risk of falls rises consistently with the number ofassociated risk factors present (38).

Multivariate logistic regression showed that the use ofamiodarone, a diagnosis of diabetes and self-reporteddifficulty maintaining balance (BOMFAQ) were indepen-dent variables associated with the risk of falling. Theestimated probability of the risk of falling within a yearamong patients with AF who did not present these threeindependent variables was 16.3%, but it was almost sixtimes higher (97%) in the presence of all three variables.

Amiodarone is a highly effective drug, yet its use islimited in chronic AF (39). A prescription of amiodarone is amarker of disease severity associated with chronic AF,which could lead to a higher risk of falls by posturalhypotension, embolic events and/or other heart rhythmdisturbances. On the other hand, the increased risk of fallscould be directly related to side effects of the drug, such asretinal pigmentation, pulmonary fibrosis, thyroid dysfunc-tions, optical and peripheral neuropathy, fatigue, tremors,ataxia, sleep disorders, vertigo, and headaches.

In early reports, the frequencies of neurologic toxic effectsfrom amiodarone ranged from 27.5% to as high as 74% andcould have been related to a much higher daily dose(40,41,42). The frequency of adverse neurologic effects inlonger-term amiodarone clinical trials has been much lower,at less than 5% (43,44,45,46), as in the present study.

Diabetes could be associated with the risk of falls byseveral mechanisms, such as micro- and macroangiopathy(causing visual disturbances) and neuropathy (leading topostural hypotension and compromising proprioceptionand muscle strength).

Gerdhem et al. (10) concluded that for fall prediction inelderly women, the more time-consuming objective func-tional tests may be of limited importance compared to dataobtained from the clinical history. Similarly, the presentstudy emphasizes the value of clinical data in assessing therisk of falls in elderly patients with AF. Risk factors for fallsare numerous and vary among patients and populations.The results of the current study have important clinicalimplications, as they could simplify the evaluation and

Table 2 - Logistic regression of the independent variables of the risk of falls.

Variable

Estimated

parameter

Standard

error p-value Odds ratio Confidence interval (95%)

Antiarrhythmic drug (YES/NO) 1.939 0.906 0.032 6.952 1.177 41.053

Comorbid diabetes mellitus (YES/NO) 1.591 0.629 0.011 4.909 1.432 16.832

BOMFAQ: Difficulty maintaining balance walking? (YES/NO) 1.768 0.507 ,0.001 5.862 2.171 15.828

Constant -1.630 0.457 ,0.001 0.196


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improve the management of elderly patients with AF at riskof falling.

The generalization of the present findings to otherpopulations without or with chronic AF should beperformed with caution. This study was cross-sectional,and it did not compare the AF group with a non-AF groupto determine whether AF itself increases the occurrence offalls.

Furthermore, the studied population may represent aselect group with high functional status because a highnumber of patients were not able to participate in the study.Further prospective studies including a high number of AFand non-AF elderly patients, comparing the predictivevalue for falls of functional tests versus clinical data, areneeded to confirm the present findings.

In a group of elderly patients with chronic AF who wererelatively independent and able to attend an outpatientclinic, the occurrence of falls with recurrence and con-sequences was high. Self-reported difficulties maintainingbalance, the use of amiodarone and a diagnosis of diabetesmellitus were identified as independent predictors of therisk for falls. Thus, simple clinical data predicted falls betterthan objective functional tests.

AUTHOR CONTRIBUTIONS

Santos ACS was responsible for the study planning and execution, material

collection, and manuscript writing. Nobre MRC designed the study. Amit

Nussbacker and Serro Azul JB were responsible for the manuscript writing.

Rodrigues GHP and Gebara OCE were responsible for the material

collection. Pierri H was responsible for the study planning. Wajngarten M

designed and planned the study and was also responsible for the

manuscript writing and study supervision.

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Table 3 - Probability of the risk of a fall according to the simulation of combinations of independent variables.

Simulation

Variable combination

group Antiarrhythmia Diabetes mellitus Balance Estimated probability of fall Odds ratio

1 1 1 1 0.975126662 5.95

2 1 0 0 0.576711639 3.52

3 0 1 0 0.490315301 2.99

4 0 0 1 0.534614124 3.26

5 1 1 0 0.869927606 5.31

6 1 0 1 0.888720802 5.42

7 0 1 1 0.84937512 5.18

8 0 0 0 0.163861359 3.52


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CLINICAL SCIENCE

Chest computed tomography findings in severeinfluenza pneumonia occurring in neutropenic cancerpatientsRosana Souza Rodrigues,I,II Edson Marchiori,I Fernando A. Bozza,II,III Melissa Tassano Pitrowsky,IV Eduardo

Velasco,V Marcio Soares,II,IV Jorge I. F. SalluhII,IV

I Federal University of Rio de Janeiro, Rio de Janeiro/RJ, Brazil. II D’Or Institute for Research and Education, Rio de Janeiro/RJ, Brazil. III Instituto de Pesquisa

Clınica Evandro Chagas, FIOCRUZ, Rio de Janeiro/RJ, Brazil. IV Intensive Care Unit and Postgraduate Program, Instituto Nacional de Cancer, Rio de Janeiro/

RJ, Brazil. V Instituto Nacional de Cancer, Infectious Diseases Department, Rio de Janeiro/RJ, Brazil.

OBJECTIVE: To describe the chest computed tomography findings for severe influenza H1N1 infection in a series ofhospitalized neutropenic cancer patients.

METHODS: We performed a retrospective systematic analysis of chest computed tomography scans for eighthospitalized patients with fever, neutropenia, and confirmed diagnoses of influenza H1N1. The clinical data hadbeen prospectively collected.

RESULTS: Six of eight patients (75%) developed respiratory failure and required intensive care. Prolonged H1N1shedding was observed in the three mechanically ventilated patients, and overall hospital mortality in our series was25%. The most frequent computed tomography findings were ground-glass opacity (all patients), consolidation (7/8cases), and airspace nodules (6/8 cases) that were frequently moderate or severe. Other parenchymal findings werenot common. Five patients had features of pneumonia, two had computed tomography findings compatible withbronchitis and/or bronchiolitis, and one had tomographic signs of chronicity.

CONCLUSION: In this series of neutropenic patients with severe influenza H1N1 infection, chest computedtomography demonstrated mainly moderate or severe parenchymatous disease, but bronchiolitis was not acommon feature. These findings associated with febrile neutropenia should elicit a diagnosis of severe viralinfection.

KEYWORDS: H1N1; Viral pneumonia; Neutropenia; Cancer; Computed tomography.

Rodrigues RS, Marchiori E, Bozza FA, Pitrowsky MT, Velasco E, Soares M, et al. Chest computed tomography findings in severe influenza pneumoniaoccurring in neutropenic cancer patients. Clinics. 2012;67(4):313-318.

Received for publication on September 2, 2011; First review completed on October 24, 2011; Accepted for publication on December 12, 2011


Tel.: 55 21 2538 3541

INTRODUCTION

Respiratory infections are a frequent cause of fever inneutropenic patients with cancer and are associated withsignificant morbidity and mortality. In these patients, lowerrespiratory tract infections are usually attributed to bacterialand fungal agents (1). Respiratory viral infections are notfrequently considered as a diagnosis, except for cytomega-lovirus and herpesvirus in patients with bone marrowtransplants (2). However, influenza infection may occuramong cancer patients, especially during influenza season,with high rates of fatality.

From April through September 2009, during the fall/winter, the southern hemisphere experienced the first waveof the influenza A H1N1 (H1N1) virus, and by the end ofDecember 2009, over 1,600 H1N1-related deaths had beenreported in Brazil (3,4). This pandemic resulted in thehospitalization of severely ill cancer patients in Brazil aswell as in other geographic regions of the southern hemi-sphere (5). Despite the use of the H1N1 vaccine and otherefforts to control the pandemic, recent reports found that theH1N1 virus circulated in the northern hemisphere duringthe flu season of 2010-2011 (6).

Recently, several reports have highlighted pulmonaryimaging findings for the H1N1 virus (7-10). However, datadescribing the computed tomography (CT) findings ofpulmonary involvement in immunosuppressed patientsare scarce (11,12). In fact, little overall information onimaging findings related to viral infection in neutropenicpatients is available. To our knowledge, CT findings of theH1N1 virus in this group of patients have not been




313

described. The aim of this study was to describe the chestCT findings of confirmed H1N1 virus in a small series ofneutropenic febrile patients with hematologic malignanciesand solid tumors.

MATERIALS AND METHODS

PatientsWe retrospectively evaluated the chest CTs of eight

hospitalized neutropenic cancer patients with a confirmeddiagnosis of H1N1 between July 2009 and August 2009.Clinical data were prospectively collected using a standar-dized case report form that included demographic data,clinical presentation, comorbidities, use of immunosuppres-sive therapies, time course of acute illness, need forintensive care, oseltamivir treatment, advanced life support,and in-hospital mortality.

Cancer patients with a fever (.37.8 C), respiratoryinfluenza-like illness, neutrophil count below 1000/mm3 atpresentation, and who received a chest CT scan duringhospitalization were included. The H1N1 diagnosis wasconfirmed by at least one of the three following assays:indirect immunofluorescence (IFI), real-time RT-PCR(rRTPCR), or cell culture, and the diagnoses fulfilled theWorld Health Organization case definitions (13). Viralshedding was evaluated in three mechanically ventilatedpatients by collecting sequential respiratory samples atdifferent time points after the onset of illness (3). Theduration of viral shedding was considered to be the timefrom the initial onset of symptoms to the last H1N1-confirmed sample.

CT imagingChest CT was performed on a Philips Brilliance 6-slice CT

Scanner (Philips Systems, Netherlands). For eight patients(three with follow-up CT scans), 11 standard chest CTstudies were retrospectively reviewed. The imaging para-meters were as follows: 120 kVp, automatic mA adjustment,2-mm slice thickness, and 2.5 mm of reconstruction.Intravenous contrast was not used. The studies wereperformed using a standard (not high-resolution) algorithm.

CT analysisCT images were reviewed for consensus by two thoracic

radiologists with 12 and 21 years of experience inpulmonary imaging, respectively. The independent readersrecorded the presence of the following findings: consolida-tion, ground-glass opacities (GGOs), airspace nodules,centrilobular nodules, interlobular septal thickening, andperibronchovascular interstitial thickening, as defined bythe Fleischner Society (14). Bronchial wall thickening is notdefined in this glossary of terms, so we used the criteriaproposed by Shiley et al. (15). Among patients withcentrilobular nodules, the presence or absence of a tree-in-bud appearance was also noted. The lobes affected by thesefindings were recorded. The extent of consolidation, GGOs,and nodules was graded as mild (,25%), moderate (25-75%), or severe (.75%), depending on the percentage of thelobe affected (11). The findings were categorized accordingto their distribution as diffuse, central, peripheral, or havingno specific distribution, as well as by their predominance inthe upper, middle, or lower third of the lungs.

The three following patterns of disease were recordedbased on pulmonary findings and their distribution: (1) no

imaging findings associated with pulmonary infection; (2)direct findings of bronchitis and/or bronchiolitis character-ized by bronchial wall thickening and centrilobular opa-cities (including the tree-in-bud pattern); and (3) pre-dominant findings of pneumonia characterized by areas ofconsolidation and/or GGOs (15). The crazy-paving patternwas characterized by thickened septa superimposed on abackground of GGO. Fibrosis was characterized by irregularlinear opacities and bronchiectasis. Pleural effusion wasrecorded as small, moderate, or large.

RESULTS

The eight patients in our study population were fourwomen and four men whose ages ranged from 4 to 65 years(mean, 15.5 years). The included patients had no comorbid-ities other than hematological or solid malignancies. Themain clinical characteristics, neutrophil count, and out-comes are shown in Table 1. Hematologic cancer occurred in75% of the patients (n = 6). All patients had receivedcorticosteroid or chemotherapy in the previous 30 days.Six patients (75%) developed respiratory failure andrequired intensive care, and four of them needed invasivemechanical ventilation. The duration of H1N1 shedding inthe three mechanically ventilated patients was 23, 44, and 63days. All patients received antiviral therapy (oseltamivir),and four received double doses. Five patients receivedcorticosteroids during treatment of the viral infection.Overall hospital mortality was 25% (2/8 patients). The twodeaths were due to refractory hypoxia secondary topulmonary alveolar hemorrhage and acute respiratorydistress syndrome, respectively.

Initial CT was performed at a median of 5.5 days (range 4-32) after the onset of symptoms. GGO was seen in all cases(Figure 1) and was severe in three patients, moderate infour, and mild in one. The overall distribution of GGO wasdiffuse in five patients. In the remaining three patients, theGGO distribution was patchy. Consolidation (7/8 cases)and airspace nodules (6/8 cases) were also commonfindings. Consolidation was typically moderate with adiffuse and heterogeneous distribution in six patients(Figure 2) and a lobar (non-segmental) distribution in one.The extent of airspace nodules was mild in four of six cases,moderate in one case, and severe in one. The tree-in-budpattern, interlobular septal thickening, peribronchovascularinterstitial thickening, and airway wall thickening were eachseen in two patients on the initial scans (Figure 3). The tree-in-bud pattern was severe in one patient and mild inanother. It was most pronounced in the peripheral regionsand was associated with airway wall thickening.Interlobular septal thickening was severe and diffuse inone patient, while in another, it was moderate andpredominantly in the lower lobes. Peribronchovascularinterstitial thickening occurred preferentially in the lowerlobes and was intense in one patient and mild in another.The most frequent combination of findings was consolida-tion and GGO. This combination occurred in seven of eightpatients and was diffuse and heterogeneous in six patients(Figure 2).

Five patients had features of pneumonia, two had CTfindings compatible with bronchitis and/or bronchiolitis,and one had tomographic signs of chronicity (irregularlinear opacities and bronchiectasis). One patient withpneumonia had predominant findings of thickened septa

H1N1 CT findings in neutropenic patientsRodrigues RS et al.

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superimposed on a background of GGO and focal areas ofconsolidation creating a crazy-paving pattern (Figure 4). Ofthe five patients with pneumonia, lower-third predomi-nance was observed in four with a cranio-caudal distribu-tion. Of those five patients, peribronchial (central)distribution was noted in one and peripheral distributionwas noted in one, while three had no specific axialdistribution. In the two patients with bronchitis and/orbronchiolitis, the findings were diffuse (without predomi-nant cranio-caudal or axial distribution). Upper-third pre-dominance was noted in the patient with signs of chronicity.No patient had a normal CT appearance. The dominant CTfindings are presented in Table 2. Pleural effusion waspresent in only two patients and was small in both. Pleuraleffusion was unilateral in one patient and bilateral in theother.

Three patients underwent follow-up CT. CT scans of thepatient with the crazy-paving pattern in the first CT showedirregular linear opacities and ectatic bronchioles, as well asmild areas of consolidation and GGOs. The time betweenthe initial and follow-up CT was 25 days. The two otherpatients had pneumonia on initial CT. One patient showedcomplete resolution of the parenchymal abnormalities inthe second CT scan, which occurred 14 days later. In theother patient, the CT scans demonstrated increasing areasof consolidation and the partial resolution of GGOsand airspace nodules. The interval between the initial

examination and follow-up CT was 15 days. All threepatients survived.

DISCUSSION

We evaluated the chest CT findings and clinical featuresof severe pandemic H1N1 virus in eight neutropenicpatients. Our findings demonstrate that neutropenicpatients with H1N1 infection frequently present diffuseand moderate or severe parenchymatous involvement. Toour knowledge, this is the first study to evaluate radiologicfeatures in this population. Pneumonia is the most commoninfection in febrile neutropenic patients, and CT is theimaging method of choice for guiding the investigation ofthe lung parenchyma, with high sensitivity in the detectionof infiltrates. Viral infection, such as by influenza, parain-fluenza, adenovirus, or respiratory syncytial virus, is onecommon cause of lower respiratory tract infections inneutropenic patients (16). In our series, the most commonCT findings associated with pandemic influenza (H1N1)were GGO (all patients), consolidation (7 of 8 patients), or amixture of GGO and consolidation. Consolidation and GGOwere bilateral, diffuse, and heterogeneous in all but one

Table 1 - Summary of the clinical characteristics of the patients.

Patient

Sex/

age Fever Headache Malaise Cough

Purulent

sputum Hemoptoic Dyspnea

Fever

(days)

Respiratory

symptoms

(days)

Neutrophil count

-

Neutrophils/mm3 MV Outcome

1 M/7 Yes No Yes Yes Yes No Yes 2 2 198 No Discharged

2 M/10 Yes No No Yes No No No 3 4 38 Yes Died

3 F/8 Yes No No Yes No No No 4 4 22 No Discharged

4 F/15 Yes Yes No Yes No No Yes 1 1 27 No Discharged

5 F/4 Yes No No No No No Yes 10 2 2 No Discharged

6 F/65 Yes No Yes Yes Yes Yes Yes 2 2 950 Yes Discharged

7 M/7 Yes No Yes Yes No No Yes 1 1 3 Yes Discharged

8 M/8 Yes Yes Yes Yes No Yes Yes 8 8 284 Yes Died

Y: yes. N: no. MV: mechanical ventilation. No patient exhibited myalgia, arthralgia, conjunctivitis, coryza, sore throat, diarrhea, abdominal pain, nausea,

or vomiting.

Figure 1 - An 8-year-old girl with a confirmed diagnosis of H1N1and respiratory failure requiring non-invasive ventilation.Computed tomography scans acquired 4 days after the onsetof clinical symptoms show severe and diffuse ground-glassopacities without specific distribution in the lungs.

Figure 2 - A 10-year-old boy with a confirmed diagnosis of H1N1.Computed tomography images performed 4 days after the onsetof the symptoms demonstrate moderate peribronchovascularand subpleural consolidation predominant in the lower lobes.The boy developed respiratory failure and received mechanicalventilation for 19 days. The duration of viral shedding in thispatient was 23 days, and he died 30 days after the onset ofclinical symptoms.

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patient, who had lobar consolidation. Consolidation, GGO,or a mixture of the two are the findings most frequently seenin influenza infections, regardless of whether they arerelated to the H1N1 subtype (7,9,15,17,18). Airspace noduleswere the third most common finding in our study; however,they are not common in the literature as a viral pneumoniafinding, which is likely because some authors interpretairspace nodules as focal areas of consolidation (18,19). Thetree-in-bud pattern and airway wall thickening werepresent in only one-quarter of our patients, although thesefindings are frequently observed in viral infection, whetherof the H1N1 subtype or not (15,20). One patient in our studyshowed the crazy-paving pattern. This finding was pre-viously described in immunocompetent patients (21,22).Another patient showed signs of chronic disease withirregular linear opacities and bronchiectasis. A CT scanwas obtained for this patient 32 days after the onset of thesymptoms. Although imaging findings are not frequentlyseen in viral pneumonia, this patient had prolongedinfluenza shedding. As in previous studies, pleural effusionwas not a significant feature (20,23).

Although they occur with variable frequency, consolida-tion, GGO, bronchial wall thickening, and the tree-in-budpattern are the most frequent CT findings associated withinfection by the H1N1 virus, regardless of the host’simmune status. However, when we evaluated patterns,pneumonia was the most common (5 of 8 patients). In twopatients, the set of findings resembled organizing pneumo-nia, with GGO and consolidation predominantly distributedin the subpleural and basal regions and associated withperibronchovascular thickening. Direct signs of bronchioli-tis characterized by bronchial wall thickening and centri-lobular opacities (mainly exhibiting the tree-in-bud pattern)were not a frequent finding in our series (2 patients). Thenumber of cases of bronchiolitis, however, may be higherbecause we did not assess indirect signs of small airwaydisease, observed only at expiration scans (21,24). The tree-in-bud pattern was observed in two of three patients withhematologic malignancies and influenza infection studied

by Oikonomou et al. (20), and bronchial wall thickening wasthe most common finding in immunosuppressed patientswith H1N1 infection described in a study by Elicker et al.(12). In that study, the authors observed mainly bronchialinvolvement without diffuse and severe lung disease. Thesedifferences may be because our series comprised onlyhospitalized patients, most of whom were critically ill andprofoundly immunosuppressed. The source of immuno-suppression in our population was diverse; all patients hadrecently received corticosteroids and chemotherapy andwere neutropenic. Regarding neutropenia, it is interesting tonote that neutrophils play a key role in the pathogenesis ofacute lung injury (25), and their depletion could play aprotective role in different experimental pathologic condi-tions (26). However, recent data highlight the role ofneutrophil function in limiting the extent and severity oflung injury in experimental influenza infection (27,28).Studies demonstrate that neutrophils in influenza infectionact mainly to control the viral replication process andreduce the risk of severe lung injury (27,28). Interestingly,some of our neutropenic patients experienced not onlysevere lung disease but also prolonged viral sheddingdespite the use of oseltamivir.

Studies (21,29) have connected the duration of illness andimaging findings observed in the early stages of diffusealveolar damage with ground-glass opacities and consolida-tion. In later stages, typical findings of organization andfibrosis including interstitial fibrosis and bronchiolitisobliterans, with or without evidence of organizing pneu-monia, were noted. In most cases, pulmonary opacitiessecondary to H1N1 infection regress during convalescence.Even in cases with benign evolution, consolidations mayoccasionally progress to linear opacities (parenchymalbands) that likely represent organizing pneumonia. Airtrapping, which represents small-airway disease, may alsobe clinically and radiologically identified in certain cases.

Bacterial pneumonia is the most common cause ofhospitalization in immunosuppressed cancer patients withpulmonary symptoms. However, viral pneumonia andinvasive fungal infection should be excluded or treated if

Figure 3 - A 15-year-old girl with mild respiratory symptoms anda confirmed diagnosis of H1N1. A computed tomography scanperformed at the carina level shows centrilobular nodules and atree-in-bud pattern heterogeneously distributed through thelungs, as well as a pleural-based consolidation in the right lowerlobe. The girl recovered without the need for admission to theIntensive Care Unit.

Figure 4 - A 7-year-old boy with respiratory failure and with aconfirmed diagnosis of H1N1. A computed tomography scanperformed 5 days after the onset of symptoms shows severeground-glass opacity superimposed on a background of thick-ened septa charactering a crazy-paving pattern.


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they are suspected. Other differential diagnoses includealveolar hemorrhage, drug-induced pulmonary injury, andoncologic disease progression. Influenza pneumonia maypresent with a broad variety of CT patterns, none of whichare specific for diagnosis. Although the CT findings ofpulmonary H1N1 infection are frequently similar to theimaging findings of other diseases, extensive or diffuseGGO and consolidation patterns (mainly in a peribroncho-vascular or subpleural distribution) in an appropriateclinical and epidemiological context should suggest thepossibility of H1N1 infection. Less typical tomographicpresentations have a broad differential diagnosis. A specificdiscussion of each of these patterns and their causes arebeyond the scope of this work.

If the clinical setting or imaging studies are notcompatible with H1N1, or if the response to treatment isnot adequate, an alternative investigation must be initiated.Alveolar hemorrhage is suspected early in the course of thedisease, usually clinically. Drug-induced pulmonary injuryis also a possibility if a drug with this potential risk profilehas been used, and some clues in the CT scan may help withan accurate evaluation. Oncologic disease progression isalways a possibility; however, if clinical and CT data are notsuggestive, an invasive diagnosis such as lung biopsy isnecessary.

This study has certain limitations. It was a small seriesand an observational study that did not interfere withpatient care. Moreover, the observational design did notallow us to use specific imaging and clinical protocols,implying a variation in the time from initial symptoms tothe acquisition of chest CT scans.

In conclusion, in this series of neutropenic patients withsevere H1N1 viral infections, chest CT demonstrated thatpulmonary disease was characterized mainly by diffuse andmoderate or severe parenchymatous disease, and bronchio-litis was not a common feature. Viral infection should beconsidered in the differential diagnosis of febrile neutro-penic patients with these findings, especially during fluseason. Further studies should confirm this observation in alarger series of patients.


Rodrigues RS, Marchiori E, Bozza FA, Soares M, and Salluh JIF

conceived and designed the study, conducted and participated in data

analysis, and drafted the manuscript. Pitrowsky MT, Velasco E, Soares M,

and Salluh JIF were responsible for the acquisition of clinical data.

Rodrigues RS and Marchiori E were responsible for the collection of

radiological data. All authors read and approved the final version of the

manuscript.

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11. Marchiori E, Zanetti G, Hochhegger B, Iron KL. High-resolutioncomputed tomography findings in a patient HIV-positive with Swine-origin Influenza A (H1N1) virus-associated pneumonia. Brit J Radiol.2010;83(986):179, http://dx.doi.org/10.1259/bjr/93404758.

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14. Hansell DM, Bankier AA, MacMahon H, McLoud TC, Muller NL, Remy J.Fleischner Society: glossary of terms for thoracic imaging. Radiology.2008;246(3):697-722, http://dx.doi.org/10.1148/radiol.2462070712.

15. Shiley KT, Van Deerlin VM, Miller WT Jr. Chest CT features ofcommunityacquired respiratory viral infections in adult inpatients withlower respiratory tract infections. J Thorac Imaging. 2010;25(1):68-75,http://dx.doi.org/10.1097/RTI.0b013e3181b0ba8b.

Table 2 - Summary of the computed tomography findings of the eight initial computed tomography scans.

Patient GGO Consolidation

Airspace nodules/tree-

in-bud

Centrilobular

nodules

Septal

thickening

Peribronchovascular

thickening

Airway wall

thickening Pattern

1 +++ + - - +++ - - Pneumonia (**)

2 ++ ++ + - ++ ++ - Pneumonia(*)

3 +++ ++ + - - - - Pneumonia

4 ++ + ++ +++ - - +++ Bronchiolitis

5 ++ ++ + - - - - Pneumonia

6 +++ ++ ++ - - +++ - Pneumonia(*)

7 ++ + - - + ({) - - Fibrosis

8 + - + + - - +++ Bronchiolitis

(*) The findings were predominantly distributed in the subpleural and basal regions and were associated with peribronchovascular thickening; (**)

thickened septa superimposed on a background of ground-glass opacity (GGO) and focal areas of consolidation charactering a crazy-paving pattern; ({)

septal thickening was irregular and associated with irregular opacities and bronchiectasis.

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16. Heussel CP, Kauczor HU, Ullmann AJ. Pneumonia in neutropenic patients.Eur Radiol. 2004;14(2):256-71, http://dx.doi.org/10.1007/s00330-003-1985-6.

17. Karadeli E, Koc Z, Ulusan S, Erbay G, Demiroglu YZ, Sen N. Chestradiography and CT findings in patients with the 2009 pandemic (H1N1)influenza. Diagn Interv Radiol. 2011;17(3):216-22. doi: 10.4261/1305-3825.DIR.3337-10.1.

18. Kim EA, Lee KS, Primack SL, Yoon HK, Byun HS, Kim TS, et al. Viralpneumonias in adults: radiologic and pathologic findings. Rad-iographics. 2002;22:Spec NS137-149, http://dx.doi.org/10.1148/rg.226025060.

19. Aviram G, Bar-Shai A, Sosna J, Rogowski O, Rosen G, Weinstein I, et al.H1N1 influenza: initial chest radiographic findings in helping predictpatient outcome. Radiology. 2010;255(1):252-9, http://dx.doi.org/10.1148/radiol.10092240.

20. Oikonomou A, Muller NL, Nantel S. Radiographic and High-ResolutionCT Findings of Influenza Virus Pneumonia in Patients with HematologicMalignancies. AJR. 2003;181(2):507–11.

21. Marchiori E, Zanetti G, D’Ippolito G, Verrastro CGY, Meirelles GSP,Capobianco J, Rodrigues RS. Swine-origin influenza A (H1N1) viralinfection: thoracic findings on CT. AJR Am J Roentgenol. 2011 Jun;196(6):W723-8. Review, http://dx.doi.org/10.2214/AJR.10.5109.

22. Marchiori E, Zanetti G, D’Ippolito G. Crazy-paving pattern on HRCT ofpatients with H1N1 pneumonia. Eur J Radiol. 2011;80(2):573-5, http://dx.doi.org/10.1016/j.ejrad.2010.10.004.

23. Abbo L, Quartin A, Morris MI, Saigal G, Ariza-Heredia E, Mariani P, et al.Pulmonary imaging of pandemic influenza H1N1 infection: relationshipbetween clinical presentation and disease burden on chest radiographyand CT. Br J Radiol 2010;83(992):645-51.

24. Marchiori E, Zanetti G, Mano CM. Swine-origin Influenza A (H1N1)viral infection. Small airway disease. AJR 2010;195(10):W317.

25. Zemans RL, Colgan SP, Downey GP. Transepithelial migration ofneutrophils: mechanisms and implications for acute lung injury.Am J Respir Cell Mol Biol. 2009;40(5):519-35, http://dx.doi.org/10.1165/rcmb.2008-0348TR.

26. Grommes J, Soehnlein O. Contribution of neutrophils to acute lunginjury. Mol Med. 2011;17(3-4):293-307. doi: 10.2119/molmed.2010.00138.

27. Tate MD, Brooks AG, Reading PC. The role of neutrophils in the upperand lower respiratory tract during influenza virus infection of mice.Respir Res. 2008;9:57, http://dx.doi.org/10.1186/1465-9921-9-57.

28. Tate MD, Deng YM, Jones JE, Anderson GP, Brooks AG, Reading PC.Neutrophils ameliorate lung injury and the development of severedisease during influenza infection. J Immunol. 2009;183(11):7441-50,http://dx.doi.org/10.4049/jimmunol.0902497.

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CLINICAL SCIENCE

Daily activities are sufficient to induce dynamicpulmonary hyperinflation and dyspnea in chronicobstructive pulmonary disease patientsAntonio A.M. Castro,I Claudia Kumpel,II Rosana Chaves Rangueri,II Maurıcio Dalcin Oliveira,III Rodrigo

Alves Dornelles,III Emerson Roberto Brito,IV Tania Maria Seki,IV Elias F. PortoII

I Federal University of Sao Paulo (UNIFESP), Pulmonary Rehabilitation Center and Federal University of Pampa (Unipampa), Rio Grande do Sul/RS, Brazil.II Federal University of Sao Paulo (UNIFESP), Adventist University of Sao Paulo (Unasp), Pulmonary Rehabilitation Center, Sao Paulo/SP, Brazil. III Federal

University of Pampa, (Unipampa), Physical, Therapy, Rio Grande do Sul/RS, Brazil. IV Adventist University of Sao Paulo (Unasp), Physical Therapy, Sao Paulo/

SP, Brazil.

OBJECTIVE: The aim of this study was to measure dynamic lung hyperinflation and its influence on dyspneaperception in moderate and severe chronic obstructive pulmonary disease patients after performing activities ofdaily living.

METHODS: We measured inspiratory capacity, sensation of dyspnea, peripheral oxygen saturation, heart rate andrespiratory rate in 19 chronic obstructive pulmonary disease patients. These measurements were taken at rest andafter performing activities of daily living (e.g., going up and down a set of stairs, going up and down a ramp andsweeping and mopping a room).

RESULT: The inspiratory capacity of patients at rest was significantly decreased compared to the capacity of patientsafter performing activities. The change in inspiratory capacity was -0.67 L after going up and down a ramp, -0.46 Lafter sweeping and mopping a room, and -0.55 L after climbing up and down a set of stairs. Dyspnea perceptionincreased significantly between rest, sweeping and mopping, and going up and down a set of stairs. Dyspneaperception correlated positively with inspiratory capacity variation (r = 0.85) and respiratory rate (r = 0.37) andnegatively with peripheral oxygen saturation (r = -0.28).

CONCLUSION: Chronic obstructive pulmonary disease patients exhibited reductions in inspiratory capacity andincreases in dyspnea perception during commonly performed activities of daily living, which may limit physicalperformance in these patients.

KEYWORDS: COPD; Activities of daily living; Pulmonary hyperinflation; Exercise.

Castro AAM, Kumpel C, Rangueri RC, Oliveira MD, Dornelles RA, Brito ER, et al. Daily activities are sufficient to induce dynamic pulmonaryhyperinflation and dyspnea in chronic obstructive pulmonary disease patients. Clinics. 2012;67(4):319-325.

Received for publication on September 5, 2011; First review completed on September 21, 2011; Accepted for publication on December 12, 2011


Tel.: 11 5812-0459

INTRODUCTION

Chronic obstructive pulmonary disease (COPD) is a majorcause of morbidity and mortality worldwide. In COPDpatients, structural changes occur in the lung parenchyma,including remodeling, airway obstruction, destruction of thealveolar septa and cellular communication, increases in airspace volume distal to the terminal bronchioles, anddecreases in pulmonary elastic recoil. These changesinterfere with the structures that allow airways to remain

open during expiration (1-3). These disease-related changescause air trapping and difficulty in air removal, leading toairflow limitation and pulmonary hyperinflation, whichincreases functional residual capacity/total lung capacityand residual volume/total lung capacity ratios (4,5).

Dyspnea is the main symptom in COPD patients and thefirst motivation for individuals to seek medical help. As thedisease progresses, the intensity of dyspnea increases andleads to an anxiety state and a deterioration in quality of life.Many COPD patients experience dyspnea even duringactivities of daily living, such as walking and dressing.There are several mechanisms responsible for dyspneaduring exercise in COPD. Among them, the most significantincludes the ineffectiveness of respiratory muscle function,which leads to increased ventilation requirements anddynamic pulmonary hyperinflation (1,3,6). It is well knownthat increased ventilatory demand results in dynamic lung




319

hyperinflation, thus increasing the sensation of dyspneaduring exercise and significantly contributing to reductionsin physical performance (4). Dynamic hyperinflation occursin COPD patients and can be measured by a simple methodthat does not require sophisticated equipment and thatshould be part of routine evaluations of COPD patients.

It is well described that dynamic pulmonary hyperinfla-tion occurs in COPD patients during incremental exercisetests (5,7). It has also been shown that COPD patientsdevelop lung hyperinflation during simple activities, suchas lifting pots and storing them in overhead bins (8).However, whether this phenomenon occurs during theactivities of daily living that COPD patients can currentlyaccomplish has not yet been addressed. We hypothesize thatsome specific activities of daily living induce dynamicpulmonary hyperinflation in COPD patients. Thus, theobjective of this study was to measure dynamic pulmonaryhyperinflation and its influence on the sensation of dyspneain COPD patients with moderate to very severe airwayobstruction during the performance of activities of dailyliving.


This was a cross-sectional study that evaluated moderateto very severe COPD patients according to GOLD 2010 (9).The study was approved by the Ethics Committee ofUNIFESP (CEP No. 0353/03). We included 19 COPDpatients (13 males) with moderate to very severe degreesof airway obstruction, with a mean post-bronchodilatorforced expiratory volume in the first second (FEV1) of48.7¡7.1% predicted and a mean age of 64.1¡5.3 years.Demographic and pulmonary function data are displayed inTable 1.

The inclusion criteria were: COPD patients diagnosedwith moderate to very severe airway obstruction accordingto the GOLD classification (9) and clinical stability with noclinical exacerbation during the 30 days prior to thebeginning of the study. Furthermore, all participants were

required to sign an informed consent form. The exclusioncriteria were: inability to perform the slow vital capacitymaneuver, excessive perceived dyspnea during the test,arterial oxyhemoglobin saturation (SpO2)#80% (7) duringactivities and/or the need for oxygen supplementation. Allrecruited patients participated in the study, and nodropouts occurred.

ProtocolAll COPD patients enrolled in the study were part of the

cardiopulmonary rehabilitation program of the Sao PauloAdventist University Center Policlinic. All COPD patientswere taught in a simple manner to perform the proposedactivities of daily living (namely, walking up and down twosets of stairs for one minute, walking twice up and down aramp 40 m in length with an 11% incline and sweeping andmopping in two rooms that were 12 m2 each).

Heart rate (HR) was monitored with PolarH heart ratemonitors (model FS1, Sao Paulo, Brazil), respiratoryfrequency (f) was measured by observing the elevationand depression of the chest for a period of 60 seconds, andblood pressure was monitored with the patients in thesitting position with an aneroid sphygmomanometer(Glicomed Aneroide PremiumH, Sao Paulo, Brazil) and astethoscope (Littmann Classic IIH, Sao Paulo, Brazil) placeddirectly over the brachial artery, thereby allowing theexaminer to hear the Korotkoff sounds as the cuff deflated.SpO2 was monitored using a Nonin OnyxH pulse oximeter(model Onyx 9500, Minnesota, USA) placed on the finger ofthe patient, and the Borg scale (10) was used to verifydyspnea and fatigue in the patients’ legs (0 represented theslightest sensation of dyspnea and fatigue, and 10 repre-sented the maximum sensation). Patients were first taughtabout the Borg scale, and they were then asked toenumerate their dyspnea and lower-limb fatigue scoresduring the study.

Spirometry was performed using an EASY ONEHspirometer (model frontline, Boston, USA) in the forced(FVC) and slow (SVC) vital capacity modalities. Aftercalibration of the equipment, data such as name, age, sex,race, weight, height, smoking habits, and diagnosis ofasthma were recorded. Spirometry was accomplishedaccording to the ATS criteria (11). The forced vital capacity(FVC) modality was used to obtain the diagnosis and theseverity of disease for each patient. With the patient in thesitting position, he/she was asked to place the mouthpieceinside his/her mouth and to put on a nose clip to preventany air outflow leakage. Through a verbal stimulus, eachpatient was asked to inhale deeply to his/her limit and thento make a maximum expiration as quickly as possible. Theslow vital capacity (SVC) modality was used to obtain theinspiratory capacity (IC). For this measurement, patientswere first asked to breathe normally into the mouthpiece;then, when requested, a maximal inspiration followed by aslow maximal expiration was performed.

The activities of daily living (walking up and down twosets of stairs for one minute, walking twice up and down aramp 40 m in length with an incline of 11% and sweepingand mopping two rooms that each had an area of 12 m2)were performed in a random sequence. COPD patients wereinstructed not to use any bronchodilators before performingthe activities (four hours for short-term medications and12 hours for long-term medications).

Table 1 - Anthropometric, pulmonary function,hemodynamic and respiratory baseline characteristics ofthe COPD patients in this study.

Variables Pre-bronchodilator Post-bronchodilator

Age (years) 64.1¡5.3 -

BMI (kg/m2) 28.0¡3.0 -

BDI 8.0¡2.0 -

BODE 2.0¡1.0 -

Smoking history (pack-

years)

37.7¡13.1 -

FEV1 (%) 46.4¡9.1 48.7¡7.1

FVC (%) 71.2¡13.0 81.4¡11.2

FEV1/FVC (%) 65.5¡8.2 62.3¡6.8

IC (L) 2.4¡ 0.1 2.5¡0.1

IC (%) 70.8¡1.6 72.8¡1.3

Heart rate (bpm) 88.1¡10.3 -

Blood pressure (mmHg) 125.8¡13.5 -

Respiratory rate (rpm) 27.6¡3.9 -

SpO2 (%) 93.1¡4.2 -

Data are presented as the means¡standard deviations.

BMI = Body mass index; BDI = Baseline dyspnea index; BODE = Body mass

index, airway obstruction, dyspnea and exercise index; FEV1 = Forced

expiratory volume in 1 second; FVC = Forced vital capacity, FEV1/

FVC = forced expiratory volume in 1 second/forced vital capacity ratio;

IC = Inspiratory capacity; SpO2 = arterial oxyhemoglobin saturation.

Hyperinflation and daily activitiesCastro AAM et al.

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At the end of each activity and with the patient seated,heart rate, respiratory rate, blood pressure, and oxyhemo-globin saturation were measured. Additionally, patientswere asked to score the sensations of dyspnea and fatigue intheir lower limbs according to the Borg scale; afterward, aslow vital capacity maneuver was performed to obtain theirinspiratory capacities. The activities could either be per-formed on the same day or on a different day. If performedon the same day, an interval period between each activitywas set as at least 40 minutes or until the HR, f, IC, dyspnea,and fatigue parameters had returned to baseline values.

For the IC measurements, each patient used a nasal clipand breathed through a mouthpiece connected to the circuitof the KoKoH equipment (Koko Spirometer, PDS Instru-mentation, Lousville, Kentucky, USA), which numericallyand graphically recorded tidal volume values. Whenstability of the end-expiratory volume was reached, thepatient was asked to inspire up to his/her total pulmonarycapacity. A minimum of three maneuvers up to a maximumof eight were performed; to be considered reproducible, twocurves could not present a variation greater than 5% or0.15 L (12,13). Inspiratory capacity was registered as thevalue measured between the line of the end-expiratoryvolume and total pulmonary capacity. The higher value oftwo reproducible curves was considered for analysis.Measurement of inspiratory capacity was performed withintwo minutes after spirometry using the slow vital capacitymaneuver.

Pulmonary hyperinflation was considered when, after thespirometry, a reduction in inspiratory capacity of 10% and/or 0.15 L compared to the basal value was observed (14).

Up and down stairs (UDS)COPD patients were instructed to walk up and down two

sets of stairs totaling 20 steps (each step was 0.18 m high and0.33 m deep) at a comfortable speed over a period of oneminute. This time was chosen because we believe that this isthe time required for a healthy individual to perform thisactivity in his/her day-to-day life. If they wished, they couldstop climbing the stairs; however, the timer was not stopped.When the patient decided to continue with the activity untilthe end of the test period, the time at which the patientstopped and returned to complete the activity was noted.

Up and down a ramp (UDR)The patient was instructed to go up and down a ramp

40 m in length with an average slope of 11% twice at acomfortable pace (totaling 160 m in ascent and descent). Thebaseline time for task completion was not pre-determined.

Sweeping and mopping in two rooms with areas of12 m2 each (SM)

Prior to this activity, we spread an equivalent of 0.5 kg ofwet sand in each room. The patient was asked to sweep theroom floors with a broom and then mop them with a wetcloth in a squeegee. The activity was terminated when thepatient perceived that the rooms were clean. We recom-mended that the activity should be performed at acomfortable pace.

During each activity, each patient was accompanied byone investigator who asked him/her how he/she felt and ifhe/she was able to continue testing. The patient wasinformed that he/she could stop the test at any time.

The activities could be performed either on the same dayor on a different day; it was stipulated that either an intervalof 40 minutes of rest or enough time for the IC values,dyspnea and fatigue to return to baseline should pass beforeactivities recommenced. The time interval between activitieswas chosen according to previous publications from ourgroup (15,16).

Statistical analysisThe data are expressed as the means and standard

deviations. To analyze the normality of the data, we usedthe Kolmogorov-Smirnov test. We used analysis of variancewith repeated measurements (ANOVA with the Bonferronipost hoc test) to analyze IC, dyspnea, fatigue and SpO2 inthe three different activities (walking up and down theramp, sweeping and mopping and walking up and downthe stairs). Pearson’s correlation test was used to establishcorrelations between IC variability and dyspnea. Weconsidered p,0.05 to be statistically significant. The samplesize was calculated based on the outcome variable (IC) fromthe equation, namely the expected effect/standard devia-tion (E/S) ratio. In this study, the IC expected effect chosenwas 0.15 L (minimal clinical difference to diagnose pul-monary hyperinflation), and the sample standard deviationconsidered was the IC variability after performance of theactivities (e.g., 0.15 L). Therefore, for an a= 0.05 and astatistical power of b= 0.2, seventeen COPD patientswere needed to achieve outcomes significance.

RESULTS

There was a significant reduction in IC after theperformance of activities compared to resting IC values(p = 0.04). The changes in IC were 0.67¡0.15 L/- 19.1¡1.0 %(p = 0.003) after walking up and down the ramp,-0.46¡0.15 L/- 13.1¡0.9 % (p = 0.05) after sweeping andmopping and -0.55¡0.14 L/-15.7¡1.1 (p = 0.002) after walk-ing up and down the stairs. Six, one, and six COPD patientshad the greatest degree of pulmonary hyperinflation afterwalking up and down the ramp, mopping and walking up

Figure 1 - Boxplot of resting and post-activity (walking up anddown a ramp, sweeping/mopping and walking up and downstairs) inspiratory capacities in COPD patients. Data are presentedas medians¡maximum/minimum values.

CLINICS 2012;67(4):319-325 Hyperinflation and daily activitiesCastro AAM et al.

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and down the stairs, respectively. Six patients showed noloss of IC greater than 0.15 L (Figure 1).

Dyspnea perception correlated positively with the varia-tion in inspiratory capacity (r = 0.85, p = 0.0001) (Figure 2)and respiratory rate (r = 0.37, p = 0.04) and negatively withperipheral oxygen saturation (r = -0.28, p = 0.04). On average,the value of perceived dyspnea increased two-fold after thepatients had walked to the end of the ramp, swept andmopped the floors and completed the UDS test compared tothe resting values (p = 0.0036) (Figure 3). Likewise, theaverage value of lower-limb fatigue perception in the COPDpatients increased more than two-fold for the same activitiesbetween when they commenced and ended (p,0.005)(Figure 4).

SpO2 values at rest and after each activity revealed nosignificant decreases in any patient, and there was nodifference in SpO2 between the resting and post-activitystates (p = 0.65) (Figure 5). Likewise, blood pressure, heartand respiratory rates were not significantly increasedbetween the resting and post-activity periods (p = 0.33,p = 0.51 and p = 0.27, respectively).

DISCUSSION

The importance of this study resides in the fact that itinvestigated the dynamic pulmonary hyperinflation result-ing from activities of daily living in COPD patients. Thenovel finding is that activities that were never tested before(however commonly performed by COPD patients) wereshown to induce dynamic pulmonary hyperinflation. Thereare several studies in the literature comparing the increasesin metabolic demand for activities with a high energy cost,such as the 6-minute walking test (60 to 80% of oxygenconsumption) (10) and the endurance test (90% of oxygenconsumption) (13), and activities with decreased metabolicdemand, such as upper-limb exercises (e.g., the propriocep-tive neuromuscular facilitation [PNF] technique) (16) andactivities of daily living (e.g., erasing black boards, liftingpots and replacing lamps) (17). Nevertheless, when compar-ing these studies to our data, either the methods of activityassessment (e.g., accelerometer) (18) or the activitiesthemselves (19) were different. We believe that this is anextremely important issue to be studied, as there are several

Figure 2 - Scatterplot of the association between the sensation ofdyspnea and delta (post-pre) inspiratory capacity. Data arepresented as individual inspiratory capacity values.

Figure 3 - Boxplot of resting and post-activity (walking up anddown ramp, sweeping/mopping and up and down stairs)sensations of dyspnea in COPD patients. Data are presented asmedians¡maximum/minimum values.

Figure 4 - Boxplot of resting and post-activity (walking up anddown a ramp, sweeping/mopping and walking up and downstairs) lower-limb fatigue levels in COPD patients. Data arepresented as medians¡maximum/minimum values.

Figure 5 - Boxplot of resting and post-activity (walking up anddown a ramp, sweeping/mopping and walking up and downstairs) arterial oxyhemoglobin saturations in COPD patients. Dataare presented as medians¡maximum/minimum values.


CLINICS 2012;67(4):319-325

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activities (over 30) that our patients perform in their day-to-day lives that did not predispose the patients to anydeleterious effects that we are aware of.

For more than 30 years, IC has been measured to determinelung volume at end-expiration, usually after performing aphysical activity. Researchers use it with a high level ofconfidence by assuming that the total lung capacity does notchange from rest to peak exercise and stating that any ICreduction is associated with the increase in functional residualcapacity (FRC) (4). The slow vital capacity (SVC) is areproducible maneuver both at rest and at peak exercise (14).It has also been demonstrated to be a reliable maneuver thatprovides accurate data, such as the decrease in inspiratoryreserve volume, which is associated with increases in FRC (14).Additionally, measuring IC is a low-cost, noninvasive method.

Dynamic pulmonary hyperinflation is one factor thatcontributes to the reduction of the physical capacity ofpatients and correlates positively with oxygen consumptionat peak exercise (peak VO2) (4). It is already known that asmall lung capacity or ventilatory reserve can lower theblood oxygen supply, thereby reducing the aerobic capacityof the patient (4).

We analyzed IC behavior after each of the activitiesperformed in this study. We believe that most of thepulmonary hyperinflation occurred in patients after walk-ing up and down the ramp and the stairs. This was due to ahigher demand for physical effort and the use of largermuscle groups than those required in the sweeping andmopping activity. In their study, O’Donnell et al. (20)identified a strong correlation between IC and the distancewalked during the 6-minute walking test. The same authorsargued that the reduction of IC leads to an increase inventilation required by the large muscle groups recruited ina symptom-limit cycle ergometer test. In our study, we didnot measure minute ventilation and oxygen consumption;however, the activities performed by our patients alsoinvolved large muscle groups, and it is possible that theyrequired increased energy and oxygen consumption. Thiswas confirmed by the increased respiratory rate andmaximum oxygen consumption. This suggests that thedynamic pulmonary hyperinflation that took place in theseactivities occurred for the same reason. Many authors havedemonstrated the association of increased pulmonaryventilation and the development of dynamic pulmonaryhyperinflation during upper-limb exercises (10,14,20) inCOPD patients.

Despite the fact that the sweeping and mopping activitiesare not considered as intense as the other activitiesmentioned above, they also resulted in a significant ICreduction. Most of the muscles required for the completionof this activity are located in the upper limbs, which aretypically composed of muscle groups with less muscle massand lower maximal oxygen consumption compared to thelower-limb muscles. This proportionality has been demon-strated by Celli et al. (21), who showed that, after exercisewith a cycle ergometer, oxygen consumption was lower inthe upper limbs (9.3¡1.1 ml/kg) than in the lower limbs(30.8¡ 3.2 ml/kg). However, both the range of motion andthe degree of freedom of the shoulder girdle joint aresignificantly larger than those of the lower limbs. Thisallows the arms to perform movements that depart from themidline of the body. Therefore, proportionally, the force andtorque required for the execution of each arm move-ment increases significantly, resulting in greater oxygen

consumption (22). Porto et al. (15) were the first todemonstrate the development of dynamic lung hyperinfla-tion in COPD patients during exercises with diagonalmovements (PNF technique) that resemble the activities ofdaily living that involve the arms. The authors also showedthat the proportion of IC reduction in the upper-limbexercises was greater than in the lower limbs in patientswith COPD for the same metabolic demand (16,22). In ourstudy, we did not intend to compare the relative degrees ofIC reduction between the upper and lower limbs; however,we demonstrated the occurrence of dynamic pulmonaryhyperinflation during activities of daily living with thearms. Furthermore, the movements resemble many unsup-ported upper-limb exercises that are commonly used duringpulmonary rehabilitation sessions for COPD patients.

Our study revealed a strong correlation between both ICand Borg dyspnea and peak exercise. Similarly, Marin et al.(10) demonstrated that pulmonary hyperinflation leads to agreater degree of dyspnea, fatigue, and physical capacitylimitations. The sensation of dyspnea has been widelyreported in COPD patients during activities of daily living(23), physical exercises (24-26) and even breathing exercises(27). However, none of these authors demonstrated anassociation between dyspnea and dynamic pulmonaryhyperinflation in COPD patients when performing dailyactivities.

Dynamic hyperinflation occurred due to an acute lungadaptation to hyperpnea and enhanced metabolic require-ments. Once this overwhelmed stimulus was interrupted(i.e., during post-activity rest), the respiratory patternreturned to baseline, and the inspiratory capacity increased.Many studies in the literature exist regarding the reversibilityof dynamic hyperinflation with the use of bronchodilators(15,21,28). However, no data have yet been reportedregarding the spontaneous reversibility of dynamic hyperin-flation. Nevertheless, the tests that we have conducted in ourlaboratory confirm that dynamic hyperinflation is sponta-neously reversible and that it may require up to 10 minutesfor the inspiratory capacity to increase to its baseline valueafter the end of the exercise period (15,16).

The Borg scale was also used to measure lower-limbfatigue during these activities. For all activities, theperception of fatigue was greater than the sensation ofdyspnea. We did not assess muscle alterations in ourpatients, as this was not our goal; however, due to thesystemic characteristics of COPD, we can assume that thereis a reason for this difference that is associated with skeletalmuscle characteristics (29,30). It is likely that the skeletalmuscle alterations triggered early metabolic acidosis, whichmay have led to greater perceptions of fatigue reported byour patients. COPD is a systemic disease that also affects themusculoskeletal system, decreasing muscle mass due todisuse and leading to the development of many bioenergeticabnormalities (9). Maltais et al. (29) reported a reduction inthigh muscle mass in COPD patients and found that thisgroup of patients had higher lower-limb fatigue thansensations of dyspnea during physical activity, possiblydue to physical deconditioning. Many authors havereported changes in muscle mass (31,32) and bioenergetics,such as that related to severe myopathy (33), favoring earlyskeletal muscle fatigue in COPD patients.

Our patients were not hypoxemic at rest; after performingall of the activities in this study, SpO2 remained above 80%.Other studies (31,33) were conducted with patients who


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were not hypoxemic at rest, and they remained withinnormal SpO2 levels during exercise. Ramırez-Venegas et al.(34) did not identify any strong associations between eitherSpO2 or Borg dyspnea and the 12-minute walking test (r =-0.45, p,0.05). In our study, we found only a weakassociation between these variables (r = -0.28, p = 0.04),and there were no clinical differences between either SpO2

or Borg dyspnea and the activities of daily living performed.Despite the fact that the exercise type is strictly differentbetween the two parameters, SpO2 and Borg dyspneabehaved similarly in both studies. This suggests that thesensation of dyspnea is multifactorial and has a lowassociation with hemoglobin oxygen uptake. It is likely thatvascular outflow and pulmonary perfusion are crucial forthe development of these changes (35,36).

A possible limitation of our study is the fact that we didnot measure the static lung volume of our patients asresidual volume and total lung capacity. This might haverevealed additional data regarding lung function status forour patients. However, our focus was to evaluate dynamicchanges in lung volume, and the method used wasappropriate to address the problem. As this is one of thefirst studies to compare dynamic lung hyperinflation inpatients performing activities of daily living, it is difficult tocompare our results with those from other studies.

However, these findings have an important impact on thepulmonary rehabilitation of COPD patients in clinicalpractice. Based on our results, we can infer that the activitiesof daily living should be assessed in these patients, andpatients should be guided by a comprehensive multi-disciplinary team to provide them with better guidance.

We conclude that during activities of daily living, COPDpatients develop dynamic pulmonary hyperinflation, increaseddyspnea and lower-limb fatigue, which are the main causes ofphysical limitations.


Castro AAM, Kumpel C, Rangueri RC, and Porto EF were responsible for

the project conception, data collection, statistical analysis, and manuscript.

Oliveira MD, Dornelles RA, Brito ER, and Seki TM were responsible for

the data collection, data tabulation, and manuscript writing.

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15. Porto EF, Castro AM, Nascimento O, Oliveira RC, Cardoso F, Jardim JR.Modulation of operational lung volumes with the use of salbutamol inCOPD patients accomplishing upper limbs exercise tests. RespiratoryMedicine. 2009;103(2):251-7, http://dx.doi.org/10.1016/j.rmed.2008.08.018.

16. Porto EF, Castro AM, Velloso M, Nascimento O, Dal Maso F, Jardim JR.Exercises using the upper limbs hyperinflate COPD patients more thanexercises using the lower limbs at the same metabolic demand. MonaldiArch Chest Dis. 2009;71(1):21-6.

17. Velloso M, Stella SG, Cendon S, Silva AC, Jardim JR. Metabolic andventilatory parameters of four activities of daily living accomplishedwith arms in COPD patients. Chest. 2003;123(4):1047-53, http://dx.doi.org/10.1378/chest.123.4.1047.

18. Garcia-Rio F, Lores V, Mediano O, Rojo B, Hernanz A, Lopez-Collazo E,et al. Daily physical activity in patients with chronic obstructivepulmonary disease is mainly associated with dynamic hyperinflation.Am J Respir Crit Care Med. 2009;180(6):506-12, http://dx.doi.org/10.1164/rccm.200812-1873OC.

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22. Gigliotti F, Coli C, Bianchi R, Grazzini M, Stendardi L, Castellani C, et al.Arm exercise and hyperinflation in patients with COPD: effect of armtraining. Chest. 2005;128(3):1225-32, http://dx.doi.org/10.1378/chest.128.3.1225.

23. Langer D, Pitta F, Troosters T, Burtin C, Decramer M, Gosselink R.Quantifying physical activity in COPD: different measures for differentpurposes. Thorax. 2009;64(5):458-9.

24. O’Donnell DE, Bertley JC, Chau LK, Webb KA. Qualitative aspects ofexertional breathlessness in chronic airflow limitation. Am J Respir CritCare Med. 1997;155(1):109-15.

25. Bauldoff GS, Hoffman LA, Sciurba F, Zullo TG. Home-based, upper armexercise training for patients with chronic obstructive pulmonarydisease. Heart & Lung. 1996;25(4):288-94.

26. Celli BR, MacNee W. Standards for the diagnosis and treatment ofpatients with COPD: a summary of the ATS/ERS position paper. EurRespir J. 2004;23(6):932-46, http://dx.doi.org/10.1183/09031936.04.00014304.

27. Gosselink R. Breathing techniques in patients with chronic obstructivepulmonary disease (COPD). Chron Respir Dis. 2004;1(3):163-72, http://dx.doi.org/ --- Either ISSN or Journal title must be supplied.

28. Soriano JB, Vestbo J, Pride NB, Soriano JB, Kiri V, Maden C, et al.Survival in COPD patients after regular use of fluticasone propionateand salmeterol in general practice. Eur Resp J. 2002;20(4):819-25, http://dx.doi.org/10.1183/09031936.02.00301302.

29. Bernard S, Leblanc P, Whitton F, Carrier G, Jobin J, Belleau R, et al.Peripheral muscle weakness in patients with chronic obstructivepulmonary disease. Am J Respir Crit Care Med. 1998;158(2):629-34.

30. Man WD, Hopkinson NS, Harraf F, Nikoletou D, Polkey MI, Moxham J.Abdominal muscle and quadriceps strength in chronic obstructivepulmonary disease. Thorax. 2005;60(9):718-22, http://dx.doi.org/10.1136/thx.2005.040709.

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32. Vogiatzis I, Stratakos G, Simoes DC, Terzis G, Georgiadou O, Roussos C,et al. Effects of rehabilitative exercise on peripheral muscle TNF alpha,IL-6, IGF-I and MyoD expression in patients with COPD. Thorax. 2007;62(11):950-6, http://dx.doi.org/10.1136/thx.2006.069310.

33. Wagner PD. Skeletal muscles in chronic obstructive pulmonary disease:deconditioning, or myopathy? Respirology. 2006;11(6):681-6, http://dx.doi.org/10.1111/j.1440-1843.2006.00939.x.

34. Ramırez-Venegas A, Sanchez C, Regalado J, Sansores RH. Severity ofdyspnea during exercise: similarities and differences between patients

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35. Mispelaere D, Glerant JC, Audebert M, Remond A, Sevestre-Pietri MA,Jounieaux V. Pulmonary embolism and sibilant types of chronic obstructivepulmonary disease decompensations. Rev Mal Respir. 2002;19(4):415-23.

36. Vassaux C, Torre-Bouscoulet L, Zeineldine S, Cortopassi F, Paz-Dıaz H,Celli BR, et al. Effects of hyperinflation on the oxygen pulse as a markerof cardiac performance in COPD. Eur Respir J. 2008;32(5):1275-82,http://dx.doi.org/10.1183/09031936.00151707.


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CLINICAL SCIENCE

The behavior and diagnostic utility of procalcitoninand five other inflammatory molecules in critically illpatients with respiratory distress and suspected 2009influenza a H1N1 infectionMariana Benevides Santos Paiva,I Fernando Antonio Botoni,II Antonio Lucio Teixeira Jr.,II Aline Silva de

Miranda,II Clara Rodrigues Alves de Oliveira,I Jamila de Oliveira Abrahao,II Guilherme Marques Faria,II

Vandack NobreIII

I Universidade Federal de Minas Gerais, Faculdade de Medicina (UFMG), Hospital das Clınicas UFMG, Unidade Coronariana, Belo Horizonte/MG, Brazil.II Universidade Federal de Minas Gerais, Faculdade de Medicina (UFMG), Departamento de Clınica Medica, Belo Horizonte/MG, Brazil. III Faculdade de

Medicina da UFMG, Centro de Pos-Graduacao, Belo Horizonte/MG, Brazil.

OBJECTIVES: During the 2009 influenza A H1N1 pandemic, it became difficult to differentiate viral infections fromother conditions in patients admitted to the intensive care unit. We sought to evaluate the behavior and diagnosticutility of procalcitonin, C-reactive protein and four other molecules in patients with suspected 2009 Influenza AH1N1 infection.

METHODS: The serum levels of procalcitonin, C-reactive protein, tumor necrosis factor a, interferon c, interleukin1b, and interleukin 10 were tested on admission and on days 3, 5, and 7 in 35 patients with suspected 2009 H1N1infection who were admitted to two ICUs.

RESULTS: Twelve patients had confirmed 2009 influenza A H1N1 infections, 6 had seasonal influenza infections, and17 patients had negative swabs. The procalcitonin levels at inclusion and on day 3, and the C-reactive protein levelson day 3 were higher among subjects with 2009 influenza A H1N1 infections. The baseline levels of interleukin 1bwere higher among the 2009 influenza A H1N1 patients compared with the other groups. The C-reactive proteinlevels on days 3, 5, and 7 and procalcitonin on days 5 and 7 were greater in non-surviving patients.

CONCLUSION: Higher levels of procalcitonin, C-reactive protein and interleukin-1b might occur in critically illpatients who had a 2009 H1N1 infection. Neither procalcitonin nor CRP were useful in discriminating severe 2009H1N1 pneumonia. Higher levels of CRP and procalcitonin appeared to identify patients with worse outcomes.

KEYWORDS: Severe respiratory distress syndrome; C-reactive protein; Biomarker; Sensitivity; Specificity.

Paiva MB, Botoni FA, Teixeira Jr AL, Miranda AS, de Oliveira CRA, Abrahao JO, et al. The behavior and diagnostic utility of procalcitonin and fiveother inflammatory molecules in critically ill patients with respiratory distress and suspected 2009 influenza a H1N1 infection. Clinics. 2012;67(4):327-334.



Tel.: 55 31 3879-4685

INTRODUCTION

The 2009 influenza A H1N1 (2009 H1N1) pandemicgenerated a significant burden to health care services aroundthe world (1,2). This virus was first identified in Mexico and theUnited States (March-April 2009) and quickly spread world-wide (3,4). In Brazil, as in some other southern hemispherecountries, the outbreak started in June, lasted approximately 18weeks, and was responsible for at least 1,600 deaths (2,5).

The 2009 H1N1 infection caused a broad spectrum ofclinical syndromes, ranging from afebrile upper respiratoryillness to fulminating viral pneumonia and acute respiratorydistress syndrome (2,6-8). The real-time reverse transcrip-tase polymerase chain reaction (rRTPCR) analysis per-formed on respiratory secretions has a sensitivity of 98 to100% and a specificity of 100% in identifying the 2009 H1N1infection (9). However, with a turnaround time of 2-3 days,its results do not contribute to the initial therapeuticdecisions. The use of biomarkers in patients with suspectedor confirmed bacterial infections has been investigatedprimarily in the critical care setting. Procalcitonin (PCT)has been proven to be accurate in discriminating bacterialfrom viral infections (10,11). However, only a few studieshave evaluated the behavior of PCT in patients with severe




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2009 H1N1 pneumonia (12-15). It has been suggested thatthe immune response against the 2009 H1N1 virus mightdiverge from that observed against the seasonal influenzavirus (16). Interestingly, elevated levels of PCT have beenobserved in patients with viral 2009 H1N1 infection evenwhen the patients did not have a concomitant bacterialinfection (13,14).

In this study, we sought to evaluate the behavior and thediagnostic utility of the circulating serum levels of PCT inpatients with suspected 2009 H1N1 infection and severeacute respiratory illness. To further evaluate the immuneresponse in those individuals, we investigated the levels ofC-reactive protein (CRP), tumor necrosis factor a (TNF-a),interferon c (IFN-c), interleukin 1b (IL-1b) and interleukin10 (IL-10) in those patients.


Study setting and subjectsThis was a prospective observational study conducted

in two mixed medical and surgical intensive care units(ICUs) in two university hospitals in southeastern Brazil.From August to November 2009, all the patients aged $14years old that were admitted to one of the two participantICUs with suspected 2009 H1N1 infection were evaluatedfor potential eligibility. The following inclusion criteriawere used: (1) severe acute respiratory illness defined asdyspnea plus bilateral infiltrates on the x-ray; (2) suspi-cion of 2009 H1N1 infection and lack of any other obviousetiology to explain the respiratory symptoms; and (3) astay of at least 24 h in the ICU. The patients who fulfilledthe inclusion criteria were included in the study on thefirst day of ICU admission. The study was approved bythe Universidade Federal de Minas Gerais’ EthicCommittee, and written informed consent was obtainedfrom all the patients or the next of kin. To conduct thisstudy and write this report, we observed the Standard forReporting of Diagnostic Accuracy checklist and recom-mendations (17).

Study proceduresA dedicated fellow (MBSP) visited the participating ICUs

daily to follow the included subjects and to identify neweligible patients. Demographic, clinical, and laboratory datawere recorded at inclusion and then daily. The radiographicdiagnostics and microbiological examinations (i.e., culturesof urine, blood, blind bronchoalveolar lavage, and trachealaspirates) were performed at the discretion of the treatingphysicians. Severity and organ dysfunction at admissionwere defined based on the Acute Physiology and ChronicHealth Evaluation II (APACHE II) (18) and the Sepsis-Related Organ Failure Assessment (SOFA) scores (19). All-cause hospital mortality, ICU length of stay and hospitallength of stay were also recorded. No diagnostic ortherapeutic intervention was performed as part of the studyprotocol.

Identification of the 2009 H1N1 virusAll the included patients were submitted to the collection

of nasopharyngeal swabs or aspirates upon admission to theICU. These samples were tested for 2009 H1N1 in a publicreference laboratory according to the Centers for DiseaseControl (CDC) rRTPCR Protocol for the Detection andCharacterization of Swine Influenza (version 2009) (20).

PCT and CRP measurementPeripheral blood samples were collected in the morning

using vacuum tubes (BD Vacutainer SST II Plus plastictubes; Becton Dickinson Diagnostic Systems, Sao Paulo,Brazil). After centrifugation, the serum was stored at -80 Cuntil analyzed. The circulating plasma PCT and CRP levelswere measured at inclusion (baseline) and on days 3, 5, and7 following inclusion until the time of patient death or ICUdischarge. The PCT levels were measured using an enzyme-linked fluorescent immunoassay (PCT Vidas Brahms,bioMerieux, France) with an assay sensitivity of 0.05 mg/L,which was approximately fourfold higher than the meannormal levels. The circulating CRP levels were measuredusing dry chemistry with the Ektachem 950ICR System(Johnson & Johnson Clinical Diagnostics, Inc., Rochester,NY, USA). The detection limit for CRP was 7 mg/dL.Values above 10 mg/dL were considered abnormal.

Cytokine MeasurementsThe plasma levels of TNF-a, IFN-c, IL-10, and IL-1b were

measured using the enzyme-linked immunoassay (ELISA)sandwich (Duoset R & D Systems, Minneapolis, MN, USA)with pairs of marked antibodies. These cytokines weretested at inclusion and on days 3, 5, and 7 according to theICU length of stay.

Statistical analysisThe categorical variables are expressed as numbers and

percentages. The continuous variables are stated as themeans ¡ SD for normally distributed variables and asmedians and interquartile ranges (IQR) for non-normallydistributed variables. The comparability among the groupswas analyzed using the x2 tests (Yates’ test or Fisher’s exacttest), the two-sample t test, the Mann-Whitney U test or theKruskall-Wallis test. The ROC curves were built to establishthe accuracy of the inflammatory molecules tested in theidentification of patients infected by 2009 H1N1. Thepositive and negative predictive values were calculatedonce the best cutoffs for these molecules were defined. TheSpearman test was used to establish correlations betweenparameters. The data collected were analyzed using theSPSS software (SPSS 17.0; SPSS Inc., Chicago, United States).To compare the three studied groups regarding the behaviorof the CRP and PCT levels over time, i.e., for the fourmeasurements (baseline, day 3, day 5, and day 7), we fitteda linear mixed-effect model using R software (lme4 and nlmepacks). Therefore, the CRP and PCT levels were trans-formed to normally distributed variables with square rootand natural logarithm calculations, respectively. Signifi-cance was reported as a p-value of 0.05 or less.

RESULTS

Characteristics of the study populationForty-nine patients were assessed for eligibility. Five

patients did not meet the inclusion criteria: two patients hadan alternate diagnosis, and three patients stayed in the ICUfor less than 24 hours. Among the 44 remaining patients,nine were excluded from the final analysis: for eightpatients, we were unable to obtain the results for 2009H1N1 rRTPCR from the reference laboratory probably dueto inaccurate measurements, and in one patient, the resultsof the circulating levels of PCT upon admission wereunavailable.

Biomarkers in severe 2009 H1N1 infectionPaiva MB et al.

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Table 1 - Patients’ main characteristics.

Influenza A H1N1

(n = 12) Seasonal Influenza (n = 6) Negative rRTPCR (n = 17) p-valuea

Age (mean¡SD) 37.5 (21.6) 39.2 (11.8) 39.7 (22.0) 0.81

Sex (male, %) 6 (50) 1 (17) 7 (41) 0.39

Underlying medical condition (n, %) 8 (67) 3 (50) 10 (59) 0.78

Asthma 0 (0) 1 (17) 3 (18)

Chronic obstructive pulmonary disease 2 (17) 0 (0) 1 (6)

Diabetes 1 (8) 0 (0) 2 (12)

Chronic cardiovascular disease 2 (17) 1 (17) 2 (12)

Chronic renal disease 1 (8) 0 (0) 0 (0)

Immunosuppression 1 (8) 1 (17) 1 (6)

Pregnancy 2 (17) 0 (0) 3 (18)

Obesity 1 (8) 1 (17) 0 (0)

Apache 2 (median, IQR) 14.5 (11.0) 11.0 (5.0) 10.0 (8.0) 0.24

SOFA at admission (median, IQR) 6.0 (6.0) 3.0 (2.0) 2.0 (2.0) 0.006

PaO2/FiO2 at admission (median, IQR) 160 (127) 200 (119) 172 (136) 0.31

Positive cultures at admission (n, %)b 3 (25) 0 (0) 3 (18) 0.32

Blind-BAL or tracheal aspirate 2 (17) 0 (0) 2 (12)

Blood 2 (17) 0 (0) 1 (6)

Vasopressors (n, %) 11 (91.7) 3 (50) 8 (47.1) 0.04

Hydrocortisone (n, %) 7 (58.3) 2 (33.3) 9 (52.9) 0.60

Endotracheal intubation (n,%) 11 (91.7) 3 (50) 12 (70.6) 0.14

Mechanical ventilation (median days, IQR) 10 (11) 12 (29) 4 (23) 0.91

Acute kidney injury (n, %)c 8 (66.7) 2 (33.3) 3 (17.6) 0.03

Hemodialysis (n, %) 6 (50) 2 (33.3) 3 (17.6) 0.18

ICU LOS (median days, IQR) 12 (14) 17.5 (25) 7 (21) 0.65

Hospital LOS (median days, IQR) 16.5 (21) 28.5 (55) 18 (21) 0.85

Mortality (n, %) 5 (41.7) 0 (0) 6 (35.3) 0.18

LOS- length of stay; IQR- interquartile range; SOFA – sequential organ failure assessmentaFor comparison among the three groups, significant if , 0.05.bOne patient with H1N1 infection had both the respiratory secretion and the blood cultures positive for Staphylococcus aureus.cAccording to the AKIN criteria21

Figure 1 - The circulating levels of procalcitonin observed in the studied patients at the four points of measurement, according to therespective group.

CLINICS 2012;67(4):327-334 Biomarkers in severe 2009 H1N1 infectionPaiva MB et al.

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The demographic (i.e., age and gender) and the primarybaseline clinical characteristics (i.e., comorbidities, SOFAscore and APACHE score) were similar between theincluded and excluded patients (data not shown). TherRTPCR analysis was performed in a median of 6 (range: 0-16) days after the onset of symptoms. Twelve (34.3%)patients had confirmed 2009 H1N1 infections, 6 (17.1%)patients had seasonal influenza infections and 17 (48.6%)patients yielded swabs that were negative for the influenzavirus. The most frequent diagnoses among individuals withnegative rRTPCR were undefined pulmonary disease withbilateral infiltrates (5 patients), microbiologically confirmedcommunity-acquired pneumonia (3 patients), severe asthmaand pulmonary embolism (2 patients each). The patients’clinical characteristics are displayed in table 1. The SOFAscore measured at inclusion was higher among 2009 H1N1-infected individuals compared with the other groups; theuse of vasopressors and the occurrence of acute renal failurewere also higher among these patients (21).

Thirty-two (91.4%) patients had blood cultures obtainedat admission, and 16 (61.5% of those who underwenttracheal intubation) provided respiratory samples that weretested for bacterial agents. Positive blood culture resultswere observed in two patients of the 2009 H1N1 group andin one patient with negative rRTPCR results. Concerning therespiratory samples, two patients in the 2009 H1N1 group(both with S. aureus) and two patients in the negativerRTPCR group (1 with S. aureus and 1 with K. pneumoniae)had positive results.

Plasma levels of PCT and CRPThe circulating levels of PCT at inclusion and on day 3

were significantly higher among subjects with confirmedH1N1 infections (median 7.22 mg/L, IQR: 95.92) comparedwith the patients in the seasonal influenza group (0.28 mg/L,IQR: 1.39) and the patients without influenza (0.85 mg/L,IQR: 3.29); p = 0.005 and p = 0.015, respectively. Similarresults were observed when the circulating levels of CRPthat were tested on day 3 were compared among the threegroups: medians of 179 mg/dl (IQR: 274) in the 2009 H1N1group, 110 mg/dl (IQR: 119) in the seasonal influenzagroup, and 123 mg/dl (IQR: 106) in the patients without aninfluenza infection (p = 0.024). A smaller, although stillsignificant, difference was observed for both markers whena subgroup analysis excluding the three patients withbacteremia at inclusion was performed.

Regarding the changes in the levels of CRP and PCTduring the first seven days of follow-up in all the studiedpatients, the levels of both markers decreased with time(p = 0.003 for PCT and p = 0.079 for CRP). The trends wereindependently compared among the groups for eachmarker. For both markers, the patients in the 2009 H1N1group were primarily responsible for this trend. Whenthe entire period of time was considered, the CRP andPCT values were significantly lower in the seasonalinfluenza (p = 0.029 for CRP and p = 0.009 for PCT,respectively) and noninfluenza groups (p = 0.041 forCRP and p = 0.010 for PCT, respectively) compared withthe H1N1 patients.

Figure 2 - The circulating levels of C-reactive protein observed in the studied patients at the four points of measurement, according tothe respective group.


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Plasma levels of cytokinesExcept for the baseline levels of IL-1b, which were proven

to be significantly higher among the patients in the 2009H1N1 group (p = 0.014) compared with the other groups, noother difference was observed concerning the circulatinglevels of the tested cytokines. The AUC for IL-1b valuesindicating H1N1 infection was 0.79 (CI, 0.62 to 0.96); at acutoff point set at 30 pg/ml, the sensitivity was 91%, andthe specificity was 65%. The levels of IL-1b remainedsignificantly higher among patients infected with 2009H1N1 influenza when the subgroup with bacteremia wasexcluded from the analysis (p = 0.04).

Inflammatory molecules and outcomeWe further evaluated the time course of the tested

molecules in relation to in-hospital mortality. Eleven(31.4%) out of the 35 included patients died duringhospitalization. In the analysis that compared the CRP andPCT levels with the outcome, the CRP levels on days 3, 5 and7 (p = 0.047, 0.012 and 0.008, respectively) and the PCT levelson days 5 and 7 (p = 0.019 and 0.001, respectively) were

significantly higher in non-surviving patients. No studiedcytokine was associated with all-cause hospital mortality.

DISCUSSION

In this observational study on critically ill patients withsuspected 2009 H1N1 infection, we observed highercirculating levels of PCT and CRP among 2009 H1N1-infected individuals compared with patients with seasonalinfluenza and non-influenza-related respiratory distress.The IL-1b levels were also higher among 2009 H1N1subjects compared with the two other studied groups onadmission. The increased levels of PCT and CRP throughoutthe course of the disease were associated with highermortality.

Many studies have tested the role of PCT as a tool todifferentiate infectious and noninfectious systemic inflam-matory response syndrome (SIRS) (22-25) and to distinguishbetween bacterial and viral infections (10,11). Most of thestudies observed better results with PCT than with CRP andother markers in discriminating these conditions. Moreover,the PCT levels during the first days of antibiotic therapy

Figure 3 - The circulating levels of CRP during the first seven days of follow-up in the three studied groups.


331

seem to be an accurate marker for clinical response andoutcome (26). The higher levels of PCT presented by thepatients with 2009 H1N1 infection in the present studychallenges the notion that the levels of this marker wouldremain normal or only slightly elevated among patientswith viral conditions. As suggested by the SOFA scores, themost probable explanation of our results is the significantlygreater severity of the 2009 H1N1 subjects. Increased PCTconcentrations have already been shown to be associatedwith higher severity scores (i.e., SOFA score) in critical carepatients with sepsis (27). Both the PCT and CRP levels wereuseless in discriminating 2009 H1N1 infection from othercauses of respiratory distress in our study.

Although this possibility was uniformly present amongthe 35 studied subjects, we cannot rule out unidentifiedbacterial coinfection among the 2009 H1N1 patients as ahypothetical reason to explain their elevated CRP and PCTserum levels. Guervilly et al. observed that patients infectedwith this virus might present high PCT levels despite theabsence of bacterial coinfection (13). Similar results werereported by Cunha et al. in subjects with a definite orprobable 2009 H1N1 diagnosis (14).

Conversely, several authors have observed lower levels ofcirculating PCT among patients with an isolated 2009 H1N1infection compared with those with a bacterial or mixed(bacterial and viral) infection (12,28). In a multicenter, retro-spective study conducted in 23 French ICUs, Cuquemelle et al.investigated the initial circulating levels of PCT presented by52 patients admitted with confirmed 2009 H1N1 infection.They found that the PCT levels of 0.8 mg/L combined withalveolar pulmonary infiltrates are strongly suggestive ofbacterial coinfection (OR12.9; 95% CI 3.2–51.5) (15).

Regarding the tested cytokines, only the baseline levels ofIL-1b were higher among 2009 H1N1-infected subjects. TheIL-1b levels also maintained their association with H1N1infection in the analysis, excluding the patients withbacteremia. At a cutoff of 30 pg/ml, the IL-1 b levelsaccurately identified the patients infected with the virus.The levels of IL-1 b have been found to be elevated inpatients with severe 2009 H1N1 infections (29). It has beenshown that a proinflammatory response predominates inpatients with severe 2009 H1N1 infections (30). Thisinflammatory state might at least partially explain the highlevels of CRP and PCT observed.

Figure 4 - The circulating levels of PCT during the first seven days of follow-up in the three studied groups.


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In our study, the PCT levels tested on days 5 and 7 weresignificantly higher among non-survivors compared withsurvivors. It has been shown that the PCT trends, not thebaseline values, are associated with the outcome in intensivecare patients (22,31). Thus, the decrease in the PCT levels mighthave identified individuals with better outcomes. In otherwords, patients with persistently high PCT levels had highermortality. Elevated CRP levels have also been associated withpoor outcomes in intensive care patients (32) and in patientswith severe community-acquired pneumonia (33). We foundthat the circulating CRP levels tested on days 3, 5, and 7 alsoincreased in the non-survivors compared with the survivors.

This study has several limitations. First, our sample size wassmall, and the investigation was conducted in only twouniversity hospitals. Second, we were not able to obtain lowerrespiratory samples from some of the patients that underwentinvasive mechanical ventilation, which could have beenresponsible for a small number of negative RT-PCR results.The nasopharyngeal samples, however, were obtained in theacute phase of the disease and before the institution of antiviraltherapy in most cases. Additionally, the sensitivity of rRTPCRfor influenza in the nasopharyngeal swabs is between 97.8 and100%, and the specificity is 100% (9). Third, we were unable toobtain culture results from respiratory samples in 38.5% of thesubjects who underwent tracheal intubation. Finally, we didnot investigate Streptococcus pneumoniae or Legionella pneumo-phila urinary antigens.

In conclusion, in this prospective observational study, weobserved higher levels of PCT, CRP and IL-1b amongcritically ill 2009 H1N1-infected patients compared withsubjects with seasonal influenza infection and noninfluenzadiagnoses. Neither PCT nor CRP was useful in thediscrimination of severe 2009 H1N1 infection from othercauses of respiratory failure. The elevated levels of PCTand CRP might have been due to the greater severity ofcertain cases with a concomitant substantial inflammatoryresponse, although bacterial coinfection could not bedefinitively ruled out. The IL-1b values had a higherdiscriminative value for 2009 H1N1 infection, and theywere not correlated with positive blood or respiratorycultures. Overall, the PCT levels on days 5 and 7 and theCRP levels on days 3, 5, and 7 following admission wereassociated with all-cause hospital mortality.

ACKNOWLEDGMENTS

The authors thank the medical and nursing staff of the intensive care units

and the laboratory service at the Hospital das Clınicas da Universidade

Federal de Minas Gerais for their participation in the study. This study was

partially supported by Fundacao de Amparo a Pesquisa do Estado de

Minas Gerais (FAPEMIG) and Coordenacao de Aperfeicoamento de

Pessoal de Nıvel Superior (CAPES).


Paiva MB designed the study, supervised data collection, collected data,

and drafted the manuscript. Botoni FA participated in the study design and

data collection. Teixeira Jr AL and Miranda AS performed the

immunoassays. Oliveira CRA and Abrahao JO helped to draft the

manuscript. Faria GM helped with the data collection. Nobre V conceived

the study, participated in its design and coordination and helped to draft

the manuscript.

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CLINICAL SCIENCE

Endophenotypes and serotonergic polymorphismsassociated with treatment response in obsessive-compulsive disorderFabio M. Corregiari, Marcio Bernik, Quirino Cordeiro, Homero Vallada

Hospital das Clınicas da Faculdade de Medicina da Universidade de Sao Paulo, Instituto de Psiquiatria, Sao Paulo/SP, Brazil.

OBJECTIVES: Approximately 40-60% of obsessive-compulsive disorder patients are nonresponsive to serotoninreuptake inhibitors. Genetic markers associated with treatment response remain largely unknown. We aimed (1) toinvestigate a possible association of serotonergic polymorphisms in obsessive-compulsive disorder patients andtherapeutic response to selective serotonin reuptake inhibitors and (2) to examine the relationship between thesepolymorphisms and endocrine response to intravenous citalopram challenge in responders and non-responders toserotonin reuptake inhibitors and in healthy volunteers.

METHODS: Patients with obsessive-compulsive disorder were classified as either responders or non-responders afterlong-term treatment with serotonin reuptake inhibitors, and both groups were compared with a control group ofhealthy volunteers. The investigated genetic markers were the G861C polymorphism of the serotonin receptor 1Dbgene and the T102C and C516T polymorphisms of the serotonin receptor subtype 2A gene.

RESULTS: The T allele of the serotonin receptor subtype 2A T102C polymorphism was more frequent amongobsessive-compulsive disorder patients (responders and non-responders) than in the controls (p,0.01). The CCgenotype of the serotonin receptor subtype 2A C516T polymorphism was more frequent among the non-respondersthan in the responders (p,0.01). The CC genotype of the serotonin receptor subtype 1Db G681C polymorphism wasassociated with higher cortisol and prolactin responses to citalopram (p,0.01 and p,0.001, respectively) and with ahigher platelet-rich plasma serotonin concentration among the controls (p,0.05). However, this pattern was notobserved in the non-responders with the same CC genotype after chronic treatment with serotonin reuptakeinhibitors. This CC homozygosity was not observed in the responders.

KEYWORDS: Pharmacogenetics; Serotonin Receptor Subtype 1Db; Serotonin Receptor Subtype 2A; Citalopram;Challenge Test.

Corregiari FM, Bernik M, Cordeiro Q, Vallada H. Endophenotypes and serotonergic polymorphisms associated with treatment response in obsessive-compulsive disorder. Clinics. 2012;67(4):335-340.

Received for publication on December 3, 2011; First review completed on December 12, 2011; Accepted for publication on December 15, 2011


Tel.: 55 11 2661-6988

INTRODUCTION

Despite the efficacy of serotonin reuptake inhibitors (SRIs)in treating obsessive-compulsive disorder (OCD) (1), 40-60%of these patients are unresponsive to treatment (2).Individual genetic differences may play a role in determin-ing both clinical responses to medications and adverse sideeffects. However, few genetic factors have been identified,and the mechanisms underlying clinical failure remainlargely unknown (3).

One way to assess the brain serotonergic system isthrough the identification of endophenotypes with a varietyof tests, such as the neuroendocrine challenge test. In a

previous study (4), our group reported that cortisolresponse to citalopram in OCD patients who are responsiveto SRIs was comparable to the response of the controlsubjects who had no mental disorders. In contrast, OCDpatients who were therapeutically unresponsive afterseveral long-term SRI trials demonstrated a blunted cortisolresponse to citalopram.

The goal of the present study was (1) to investigatewhether there is an association of serotonergic polymorph-isms and treatment response to SRIs in OCD patients and (2)to examine the association between the selected polymorph-isms and endocrine responses to an acute intravenous (IV)citalopram challenge.

SUBJECTS AND METHODS

SamplesOutpatients who met the DSM-IV5 diagnostic criteria for

OCD and were between 18 and 65 years of age were classifiedaccording to their therapeutic response to long-term SRI




335

treatment (clomipramine, fluoxetine, fluvoxamine, citalo-pram, sertraline or paroxetine) as either non-responders(NRs) or responders (RPs) according to the followingcriteria:

– NR: no more than a 25% reduction in the Y-BOCS (6)score after at least: two different selective serotoninreuptake inhibitors (SSRIs) at a maximum or maximumtolerated dose for at least 8 weeks and (2) clomipramineat a maximum or maximum tolerated dose for at least 8weeks (the NRs in this study have received an average of13.27¡9.5 years of treatment).

– RP: a Y-BOCS score of less than 9 and a SheehanDisability Scale (7) score of less than 10 after treatmentwith an SRI and stable for at least 2 months.

We evaluated 239 OCD patients from our own service orreferred to us for specialized treatment. From these, 60patients met inclusion criteria. OCD patients who did notmeet the criteria for either NR or RP were excluded fromthis study. Patients with severe depression (HAMD-21.25),affective bipolar disorder, schizophrenia, schizoaffectivedisorder, active substance abuse or dependence wereexcluded. Patients currently taking fluoxetine wereexcluded because of the drug’s long half-life.

We selected 30 age- and gender-matched subjects whowere free of mental and medical disorders as a controlgroup (CN). The control subjects had never used psycho-tropic medications and were free from any medication for atleast one month.

The clinical characteristics of patients and controls arepresented elsewhere (4). All medications including SRIs(except for low-dose bedtime benzodiazepines, which weremaintained at a consistent dose throughout the study) weregradually tapered off, and the patients were observed over aone-week washout period before the challenge test. Femaleswere tested in the early follicular stage of their menstrualcycle (defined as the period between the third and tenth dayof the cycle).

Challenge test procedureChallenge test procedures began at 8:00 AM, after an

overnight fast. After a peripheral intravenous insertion and60 minutes of rest, blood sampling began at 9:00 AM. Thesamples were collected at 20-minutes intervals during 180minutes. The citalopram infusion began 20 minutes after thefirst blood sample had been collected. Twenty milligrams ofcitalopram were diluted in 250 ml of saline and infused over60 minutes. Subjects were tested in a reclining position andwere kept awake.

Plasma cortisol, prolactin and growth hormone (GH)levels were quantified by standard immunoradiometricassays at -20, 0 (onset of the citalopram infusion), 20, 40,60, 80, 100, 120, 140, and 160 minutes. The serotoninconcentration in platelet-rich plasma was measured throughhigh-performance liquid chromatography with a Shimadzu10Vp system in conjunction with a fluorescence detector(Shimadzu RF-10A XL). The platelets were counted on a cellcounter Ac T diff Coulter (Beckman Coulter) to normalizethe results.

Investigated polymorphismsThe investigated polymorphisms included the G861C

polymorphism (rs6296) of the serotonin receptor subtype

1Db (HTR1B) gene and T102C (rs6313) and C516T (rs6305)polymorphisms of the serotonin receptor subtype 2A(HTR2A) gene.

GenotypingGenomic DNA was extracted using a salting out method

(8). There were technical problems when genotyping threeresponders and three control subjects. Therefore, some ofthe gene variants were not analyzed for these samples.However, two resistant patients (excluded in the challengetest because of pairing issues) were included in the genefrequency analysis.

HTR1B (G861C). The G861C polymorphism of the genecoding for the serotonin receptor subtype 1Db wasgenotyped according to the procedures described byMundo et al. (9). The 548-bp PCR fragment was digestedwith Hinc II restriction enzyme. The alleles were detectedafter separation on an agarose gel, and the G allele was theundigested fragment.

HTR2A (T102C). Genotyping of the T102C polymorphismof the gene coding for the serotonin receptor subtype 2Awas performed using the primers and conditions describedby Warren al. (10). After amplification, the PCR productswere digested with Msp I. The fragment sizes were a single342-bp band for the 102T allele and two bands (216 bp and126 bp) for the 102C allele.

HTR2A (C516T). The primers and PCR conditions usedfor the analysis of the C516T polymorphism of the genecoding for the serotonin receptor subtype 2A weredescribed by Arranz et al. (11). The amplified DNAproducts from the patient and control samples weredigested with Sau 96I restriction enzyme (New EnglandBiolabs Inc., cat. R0165S). The C516 allele (digested PCRproduct) showed 109-bp and 87-bp fragments, whereas theT516 allele remained uncut.

To confirm the genotyping results for all the investigatedmarkers, we used a commercial 50–bp DNA ladder (GEHealthcare Life Sciences, product code: 27-4005-01). Afterthe restriction enzyme cleavage reaction, the fragments ofeach marker were separated electrophoretically on anagarose gel and visualized under a UV light. All of thegenotyping results were interpreted by two independentlytrained research technicians. The same person genotyped allsamples. The genotyping process was repeated when adisagreement arose (e.g., the T516C genotyping of twoheterozygous individuals was repeated during the analysis).

Statistical analysisThe prolactin and cortisol responses to citalopram were

measured as maximal percentage variation (max%D).Because of the null values observed at baseline, GHresponses were measured as the maximal variation overbaseline (maxD). The continuous variables were analyzedusing an analysis of variance (ANOVA) or a paired t-testwhen appropriate. Friedman, Mann-Whitney or Wilcoxonmatched tests were used when a non-parametric analysiswas required. A general linear procedure was used toperform a two-way ANOVA and post-hoc analysis.Categorical data were analyzed using a chi-square testand a chi-square partition. The significance level was 5%and based on two-tailed tests. A standard deviation wascalculated to indicate variability. A test for deviations fromthe Hardy-Weinberg equilibrium was performed using the

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CLINICS 2012;67(4):335-340

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HWE program (12). The analysis was performed usingEpiInfo version 6.0 and SPSS version 15.0.

ETHICSThe protocol was in accordance with Declaration of

Helsinki for research with human subjects and approvedby the Ethics Committee at the Hospital das Clınicas,University of Sao Paulo Medical School (CAPPesq). Allsubjects gave written, informed and free consent.

RESULTS

Hardy-Weinberg EquilibriumThe genotypic distributions of the HTR2A C516T, HTR2A

T102C, and HTR1B G681C polymorphisms were in Hardy-Weinberg equilibrium (HWE) in the CN (p.0.05). The casegroup was in HWE for all polymorphisms except for theHTR2A T102C polymorphism (p = 0.0001).

Frequency of Polymorphisms DistributionsThe patient and control polymorphism frequency data are

presented in Table 1. In the patients, the 102CC genotype isless frequent than in the CN (p,0.001 after a chi-squarepartition), and the heterozygosity frequency was higher(p = 0.02), with no difference in the 102TT genotype. Thefrequency of the T allele was higher in the patients than inthe CN (p,0.01). In Table 2, the allele frequency of eachDNA marker in the OCD patients group was subdividedinto responders versus non-responders for the SRIs. The516CC genotype was more frequent, and consequently,the heterozygosity frequency was reduced because therewere no patients with a 516TT genotype among the non-responders versus the responders (p,0.01). After a chi-square partition, CC homozygosity for the HTR1B G681Cpolymorphism was more frequent among non-respondersversus responders (p = 0.018).

Relationship among genetic polymorphisms, studygroups and endocrine/serotonin variables

Relationship with basal hormonal/serotonin levelsThe basal levels of serotonin, prolactin, growth hormone

(GH), and cortisol and the interactions with the investigatedpolymorphisms were compared among the three studygroups (NR, RP, and CN). No associations were foundamong the groups; polymorphisms; and prolactin, GH orcortisol basal levels. The CN showed the highest platelet-rich plasma serotonin concentration (p,0.001). The onlysignificant interaction was observed between the groupsand the HTR1B G681C polymorphism (p = 0.022; Table 3),with the highest serotonin level observed among the CChomozygous of the CN (558.01 ng/ml) and the lowest levelobserved among non-responders with the same genotype(74.68 ng/ml).

Relationship with endocrine response tointravenous citalopram

Prolactin, GH and cortisol responses to citalopram andinteractions with the investigated polymorphisms werecompared among the three groups (NR, RP, and CN).

As reported previously (4), the prolactin increase (as apercentage of the baseline) was greater in the CN than in theresponder and the non-responder groups (p = 0.017 for NRversus CN; p = 0.004 for RP versus CN). However, there wasno difference between the responder and the non-respondergroups.

The prolactin response was not associated with any allele orgenotype among the C516T HTR2A and T102C HTR2Apolymorphisms. However, the subjects with CC homozygos-ity for the G681C HTR1B polymorphism showed a moreintense prolactin response to citalopram (p,0.01 for both CC

Table 1 - Polymorphism frequency among patients andcontrols.

Genotypes Patients (RP + NR) (%) CN (%) p-value

HTR2A C516T

CC 48 (81.4) 26 (92.9) 0.16

CT 11 (18.6) 2 (7.1)

TT 0 (0) 0 (0)

HTR2A T102C

TT 17 (28.3) 6 (20.0) ,0.001

TC 43* (71.7) 14* (46.7)

CC 0** (0) 10** (33.3)

HTR1B G681C

CC 6 (10.2) 2 (6.9) 0.88

CG 24 (40.7) 12 (41.4)

GG 29 (49.2) 15 (51.7)

Alleles

HTR2A C516T 0.18

C 107 (90.7) 54 (96.4)

T 11 (9.3) 2 (3.6)

HTR2A T102C 0.007

T 77 (64.2) 26 (43.3)

C 43 (35.8) 34 (56.7)

HTR1B G681C 0.69

C 36 (30.5) 16 (27.6)

G 82 (69.5) 42 (72.4)

Note: RP = Responders; NR = Non-responders; CN = Controls. *p = 0.02 after

a chi-square partition for heterozygosity; **p,0.001 after a chi-square

partition for the CC genotype.

Table 2 - Polymorphism frequency among non-responders and responders.

Genotypes NR (%) RP (%) p-value

HTR2A C516T ,0.01

CC 30 (93.8) 18 (66.7)

CT* 2 (6.2) 9 (33.3)

HTR2A T102C 0.54

TT 8 (25.0) 9 (32.1)

TC* 24 (75.0) 19 (67.9)

HTR1B G 681 C 0.054

CC 6** (18.8) 0** (0)

CG 11 (34.4) 13 (48.1)

GG 15 (46.9) 14 (51.9)

HTR1B G 681 C 0.018

CC 6 (18.8) 0 (0)

GC or GG 26 (81.2) 27 (100)

Alleles

HTR2A C516T 0.01

C 62 (96.9) 45 (83.3)

T 2 (3.1) 9 (16.7)

HTR2A T102C 0.68

T 40 (62.5) 37 (66.1)

C 24 (37.5) 19 (33.9)

HTR1B G681C 0.16

C 23 (35.9) 13 (24.1)

G 41 (64.1) 41 (75.9)

Note: RP = Responders; NR = Non-responders; CN = Control. *No TT

genotype for HTR2A C516T and no CC genotype for HTR2A T102C were

observed among patients; **p,0.05 for the CC genotype after a chi-

square partition.

CLINICS 2012;67(4):335-340 5-HT polymorphisms and response to SSRIs in OCDCorregiari FM et al.

337

versus CG and CC versus GG genotype, Table 4). A significantgroup-genotype interaction was observed (p,0.001; Table 4)when the nine subgroups, corresponding to the combinationgroup-genotype, were compared. The CC genotype was notdetected among the responders. The control-CC genotypesubgroup showed the highest prolactin response, even

compared with the other two control subgroups (control-CGgenotype and control-GG genotype; p,0.001 after a Bonferronicorrection; Table 4).

The GH response to citalopram failed to show differencesamong the groups and was not associated with any of thestudied polymorphisms.

As reported previously, the peak secretion of cortisol asmeasured through maximal percentage variation was lesspronounced in the non-responders compared with theresponders (p = 0.015) and was smaller compared to theCN (p = 0.052). There was no significant difference for theCN vs. the responder group (p = 0.53) (4). The cortisolmaximal percentage variation was unrelated to the HTR2AC516T and T102C polymorphisms. A more intense cortisolresponse was observed in those subjects who were homo-zygous for the HTR1B G681C polymorphism (p = 0.011 foroverall comparison). A significant group-genotype interac-tion was observed (p = 0.001). The control-CC subgroupshowed the most intense cortisol response, higher than theother control group genotypes (p#0.003 for all comparisonsafter a Bonferroni correction; Table 5).

DISCUSSION

The present study investigates an association betweenDNA markers, the endocrine response to citalopram,peripheral serotonin concentration and the responsivitystatus in OCD patients and controls. As far as we know, thisis the first study to examine these associations in OCDpatients.

Few studies have investigated serotonergic polymorph-isms and SRI treatment responses in OCD patients. Denyset al. (13) reported that in paroxetine-treated patients, themajority of the responders were homozygote for the G alleleof the 1438 G/A polymorphism of the HTR2A gene.Cavallini et al. (14) found no association between theCys23Ser polymorphism of the HTR2C gene and atherapeutic response to clomipramine.

An important finding in our study, which was previouslyunreported, was the absence of CC homozygosity of the

Table 3 - Platelet-rich plasma serotonin levels in the threestudy groups, stratified by the G681C HTR1Bpolymorphism.

G681C HTR1B

Polymorphism

Genotype Mean (ng/ml) ¡Standard Deviation (%) N

NR C C 74.68¡49.13 5

C G 121.73¡79.37 11

G G 184.39¡162.59 14

Total 143.13¡127.12 30

RP C G 163.26¡128.90 13

G G 175.36¡124.81 12

Total 169.07¡124.44 25

CN C C 558.01¡80.12 2

C G 540.52¡220.26 12

G G 390.63¡117.03 15

Total 464.20*¡179.33 29

Total C C 212.78¡241.45 7

C G 276.32¡242.76 36

G G 257.20¡167.96 41

Total 261.69¡207.28 84

Note: RP = Responders; NR = Non-responders; CN = Control. The platelet-

rich plasma level of serotonin was statistically higher in the CN (*p,0.001;

F = 14.63) than in the other two groups. A significant interaction between

the groups and the G681C HTR1B polymorphism was observed (p = 0.022;

F = 3.383), with the highest serotonin level observed among the CC

homozygous of the CN and the lowest among the NR group with the

same genotype.

Table 4 - A comparison of the prolactin maximal percentof variation among the eight subgroups based on thethree groups of participants and the G681C HTR1Bpolymorphism genotypes.

Group

HTR1B G681C

Polymorphism Genotype Mean¡Standard Deviation (%) N

NR C C 20.53¡26.63 5

C G 14.91¡28.43 11

G G 18.25¡36.09 14

Total 17.41¡31.06 30

RP C G 23.96¡45.06 13

G G 9.75¡15.79 12

Total 17.14¡34.38 25

CN C C 317.48***¡206.23 2

C G 60.77¡101.17 12

G G 33.21¡38.09 15

Total 64.22*¡106.62 29

Total C C 105.38**¡168.98 7

C G 33.47¡67.39 36

G G 21.23¡33.11 41

Total 33.49¡70.84 84

Note: RP = Responders; NR = Non-responders; CN = Control. The CN

showed a higher prolactin response overall (*p,0.01 for CN versus both

RT and RP after a Bonferroni correction). Subjects with a CC homozygosity

showed a higher prolactin response overall (**p,0.01 for both CC versus

CG and CC versus GG comparisons yield significant results after Bonferroni

correction). The interaction between group and genotype was significant

(p,0.001). In the post-hoc analysis, the control-CC subgroup was the only

significantly different group and showed a higher prolactin response than

all other subgroups (***p,0.001 after a Bonferroni correction).

Table 5 - A comparison of the cortisol maximal percent ofvariation among the eight subgroups based on the threegroups of subjects and the HTR1B G681C polymorphismgenotypes.

Group

HTR1B G681C

Polymorphism

Genotype

Mean¡Standard

Deviation (%) N

NR C C -4.93¡8.76 5

C G 20.06¡51.04 11

G G 30.95¡71.75 14

Total 20.98¡58.14 30

RP C G 38.94¡66.79 13

G G 50.61¡65.96 12

Total 44.54¡65.26 25

CN C C 261.59*¡112.22 2

C G 51.92¡86.62 12

G G 48.83¡57.95 15

Total 64.78¡89.72 29

Total C C 71.21¡138.07 7

C G 37.50¡69.28 36

G G 43.24¡64.23 41

Total 43.11¡73.96 84

Note: RP = Responders; NR = Non-responders; CN = Control. *p#0.003 after

a Bonferroni correction compared with all other groups.


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HTR2A gene T102C polymorphism in both patient groups(responders and non-responders), whereas it was present inone-third of the CN (p,0.001). The T allele was more frequentamong patients than among controls (p,0.01). These findingssuggest that the presence of at least one T allele could be anOCD risk factor. The 102TT genotype was more frequentamong patients than among controls (28.3% vs. 20.3%,respectively), but this difference was not statistically sig-nificant after a chi-square partition (p = 0.39). The study maybe underpowered to confirm an excess of 102TT homozygousamong the patients. The fact that the HTR2A T102Cpolymorphism was not in HWE in the patients group maybe a concern. If deviations from HWE are present in thecontrol population, then one or more of the model assump-tions may have been violated or that a genotyping error hasoccurred. During our experiment, the genotyping processwas repeated when a disagreement arose between theindependently trained research technicians who interpretedthe data. However, a known homozygous sample was notused as a control, which would have guaranteed thegenotyping accuracy. Notably, the CN is in HWE (p = 0.78).Therefore, a genotyping error appears unlikely. In the presentstudy, the patient samples may not need to be in HWEbecause the patients were not randomly selected from thepopulation (15). In other words, a deviation from HWE in thecase subjects may indicate an association of a locus with OCD(16,17). Another reason for the lack of HWE could be ethnicdifferences between the subjects and consequent samplestratification. As noted, this reason could be the case in thecurrent study because we did not control for ethnic back-ground. Interestingly, the HTR2A T102C polymorphismlacked HWE, and this polymorphism was associated withthe disorder, suggesting that the association may be real.

Another novel finding was that an excess of CChomozygosity and the C allele of C516T polymorphism ofthe HTR2A gene was observed among the non-responders.This finding may indicate that having at least one T allelecould be a factor that indicates SRI responsiveness duringOCD treatment.

Notably, our previous study found no differences in theallele and genotype frequencies between OCD patients andthe control groups for the T102C HTR2A gene polymorphism(18). Those results suggested that the C516T variant of theHTR2A gene may be a genetic risk factor for OCD. Anotherstudy by our group found no association between the5HTTLPR and STin2 polymorphisms in the promoter regionof the serotonin transporter gene (SLC6A4), the G861Cpolymorphism (rs6296) of HTR1B, the T102C (rs6113) andC516T (rs6305) polymorphisms of HTR2A and the clomipra-mine response in OCD patients (19). In the present study,these divergences may be caused by differences in samplecharacteristics with the refractory patients in the RT groupand the highly responsive patients in the RP group.

Finally, CC homozygosity for the HTR1B gene G681Cpolymorphism showed another relevant finding regardingthe relationship between the endocrine response to citalo-pram and the investigated groups, despite a low tendencytoward gene frequency discrepancies between respondersand non-responders (p = 0.054). This genotype was presentin only 6 of the 32 non-responders and was absent from theresponders. The effect of this genotype on the hormonalresponse to IV citalopram, an indirect measure of serotoninfunctioning, is notable. The subgroup of the controls withthe CC genotype (n = 2) showed the highest prolactin and

cortisol response after a citalopram infusion and presentedthe highest platelet-rich plasma serotonin concentration. Incontrast, OCD patients with the same genotype (n = 6), all ofwhom were non-responders, presented the lowest cortisolresponses and serotonin concentrations (not statisticallysignificant with regard to other subgroups except for theControls-CC genotype). The difference in magnitude issurprising (prolactin response: CN-CC = 558.01¡80.12%;NR-CC = 74.68¡49.13%; cortisol response: CN-CC = 317.48¡206.23%; NR-CC = 20.53¡26.63%; p,0.001 after a Bon-ferroni correction for both comparisons). Interestingly,when we analyzed the frequency of the HTR1B CC homoz-ygosity alone in the OCD group, there was an excess of thisgenotype among the non-responders (p = 0.018; 6 of 32 NRs;0 of 27 RPs; see Table 2). These data should be interpretedcautiously because of the small subgroup size.

Nevertheless, we hypothesized what could account forthis genotype-group interaction and lead to a robust cortisolresponse to citalopram in the CC-Control subgroup andresponse in the CC-non responders subgroup. The 5-HT1Dbreceptor is localized chiefly in the 5-HT terminals in whichits stimulation inhibits the release of 5-HT. Therefore, the5-HT1Db receptor may be less active in the CC homozygousmembers of the CN, allowing a more intense post-synapticserotonergic stimulation after citalopram infusion. The 5-HT1Db receptor is also expressed, however, in the terminalsof nonserotonergic neurons, exerting heteroregulation ofneurotransmitter release and a postsynaptic receptor. Thelatter occurs mainly in the basal ganglia. Chronic SRIadministration desensitizes the 5-HT1Db receptors (20).This phenomenon, together with the desensitization of thesomatodendritic 5-HT1A receptor (21), leads to progressiveenhancement of 5-HT neurotransmission, correlating with atherapeutic response (22). Therefore, in CC homozygouspatients, the observed blunted response could be related tothe chronic SRI exposure. We infer that a higher acuteserotonin response after SRI treatment could lead to moreintense adaptation after chronic SRI exposure and conse-quently to a blunted cortisol response after a single dose ofcitalopram. Accordingly, disrupted 5-HT1Db acute regula-tion may cause other mechanisms to overcome an increasedserotonin transmission (e.g., post-synaptic receptor down-regulation or decreased serotonin synthesis), which maylead to an over-adaptation to chronic SRI exposure anddecreased SRI effectiveness. These hypotheses could beevaluated in future studies.

As acknowledged, it may be premature to determine thevalidity of the association of therapeutic response andgenetic polymorphisms because, as an alternative inter-pretation, they may be in linkage disequilibrium with otherproximal polymorphisms that could affect the therapeuticresponse.

Another limitation of this study was that the groups wereunmatched for ethnic origin. In populations of highly mixedethnicity, such as the Brazilian population, phenotypingexpressions of ethnicity, such as skin, hair and eyepigmentation, is not viable (23,24). The sample size maybe the main methodological limitation of this study. Becausethis was an exploratory study, we did not calculate thesample power a priori and instead studied all of theavailable subjects. It is worthwhile to note the uniquefeatures of this sample. OCD patients were selected by theirlong-term therapeutic responses; some of the resistantpatients have been in treatment for decades. Moreover, in

CLINICS 2012;67(4):335-340 5-HT polymorphisms and response to SSRIs in OCDCorregiari FM et al.

339

this study, the evaluated groups were extreme. Non-responders were unresponsive to at least three differentSRIs and remained symptomatic; responders met tightremission criteria; and controls (healthy volunteers) whoneither mental health nor major medical problems. Manyputative patients and volunteers were excluded. These strictcriteria may have increased the study power but decreasedthe external validity.

Because the data can be interpreted as false positive (at the0.01 level of significance), further investigations using a largersample size are necessary to confirm our results. Prospectivestudies with larger sample sizes and using selection criteriaaccording to the subjects’ genotype would be interesting. Inaddition, the examined polymorphisms may be in linkagedisequilibrium with yet to be identified markers or genes thatcontribute to OCD. Additionally, further research should beperformed to evaluate whether the endocrine response tocitalopram can be considered an endophenotype.

CONCLUSIONS

The conclusions of this study are as follows:

(1) The T allele of the 5-HTR2A T102C polymorphism wasmore frequent among OCD patients.

(2) The CC genotype of the HTR2A C516T polymorphismwas more frequent among OCD patients who werenonresponsive to several SRI trials than among the SRI-responsive patients.

(3) The CC genotype of the HTR1B G681C polymorphismwas associated with higher cortisol and prolactinresponses to citalopram and a higher platelet-richplasma serotonin concentration among health volun-teers. However, this pattern was not observed amongnonresponsive OCD patients with the same genotypeafter chronic SRI treatment.

ACKNOWLEDGMENTS

This research was supported by FAPESP (Fundacao de Amparo a Pesquisa

do Estado de Sao Paulo) grant number 99/00170-4. We thank Elisabete

Miracca for technical support.


Corregiari FM took part in the project design, clinical evaluation, challenge

tests execution and analysis, statistical analysis and contributed to the

discussion and manuscript. Bernik M took part in the project design,

clinical evaluation, challenge tests analysis, statistical analysis and

contributed to the discussion and manuscript. Cordeiro Q took part in

the project design, was responsible for the genotypings and contributed to

the discussion and manuscript. Vallada H took part in the project design,

was responsible for the genotypings and contributed to the discussion and

manuscript.

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18. Meira-Lima I, Shavitt RG, Miguita K, Ikenaga E, Miguel EC, Vallada H.Association analysis of the catechol-o-methyltransferase (COMT), ser-otonin transporter (5-HTT) and serotonin 2A receptor (5HT2A) genepolymorphisms with obsessive-compulsive disorder. Genes Brain Behav.2004;3(2):75-9, http://dx.doi.org/10.1046/j.1601-1848.2003.0042.x.

19. Miguita K, Cordeiro Q, Shavitt RG, Miguel EC, Vallada H. Associationstudy between genetic monoaminergic polymorphisms and OCDresponse to clomipramine treatment. Arq Neuropsiquiatr. 2011;69(2B):283-7, http://dx.doi.org/10.1590/S0004-282X2011000300003.

20. Chopin P, Moret C, Briley M. Neuropharmacology of 5-hydro-xytryptamine1B/D receptor ligands. Pharmacol Ther. 1994;62(3):385-405, http://dx.doi.org/10.1016/0163-7258(94)90051-5.

21. Blier P, de Montigny C, Chaput Y. Modifications of the serotonin systemby antidepressant treatments: implications for the therapeutic responsein major depression. J Clin Psychopharmacol. 1987;7(6 Suppl):24S-35S.

22. Mongeau R, Marsden CA. Effect of imipramine treatments on the 5-HT1A-receptor-mediated inhibition of panic-like behaviours in rats.Psychopharmacology (Berl). 1997;131(4):321-8, http://dx.doi.org/10.1007/s002130050299.

23. Silva MA, Cordeiro Q, Miracca EC, Guindalini C, Vallada H. Distributionof alleles of the VNTR polymorphism in the 39-untranslated region of theDAT1 gene (SLC6A3) in Sao Paulo/Brazil and its importance to geneticstudies of neuropsychiatric disorders in ethically admixed populations.Rev Med Chil. 2005;133(11):1392-3.

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CLINICAL SCIENCE

Psychological crisis intervention for the familymembers of patients in a vegetative stateYa-Hong Li,I Zhi-Peng XuII

I Department of Psychology, South-Central University for Nationalities, WuHan, People’s Republic of China. II Department of Neurology, Wuhan General

Hospital of Guangzhou Command, Wuhan, People’s Republic of China.

OBJECTIVES: Family members of patients in a vegetative state have relatively high rates of anxiety and distress. It isimportant to recognize the problems faced by this population and apply psychological interventions to help them.This exploratory study describes the psychological stress experienced by family members of patients in a vegetativestate. We discuss the effectiveness of a psychological crisis intervention directed at this population and offersuggestions for future clinical work.

METHODS: A total of 107 family members of patients in a vegetative state were included in the study. The interventionincluded four steps: acquisition of facts about each family, sharing their first thoughts concerning the event, assessmentof their emotional reactions and developing their coping abilities. The Symptom Check List-90 was used to evaluate thepsychological distress of the participants at baseline and one month after the psychological intervention. Differencesbetween the Symptom Check List-90 scores at the baseline and follow-up evaluations were analyzed.

RESULTS: All participants in the study had significantly higher Symptom Check List-90 factor scores than the nationalnorms at baseline. There were no significant differences between the intervention group and the control group atbaseline. Most of the Symptom Check List-90 factor scores at the one-month follow-up evaluation were significantlylower than those at baseline for both groups; however, the intervention group improved significantly more than thecontrol group on most subscales, including somatization, obsessive-compulsive behavior, depression, and anxiety.

CONCLUSION: The results of this study indicate that the four-step intervention method effectively improves themental health of the family members who received this treatment and lessens the psychological symptoms ofsomatization, obsessive-compulsive behavior, depression and anxiety.

KEYWORDS: Vegetative State; Family Members; Mental Health; Psychological Crisis Intervention.

Li YH, Xu ZP. Psychological crisis intervention for the family members of patients in a vegetative state. Clinics. 2012;67(4):341-345.

Received for publication on October 5, 2011; First review completed on October 29, 2011; Accepted for publication on December 21, 2011


Tel.: 0086-27-6784 2677

INTRODUCTION

Medical progress has increased the number of patientswho are able to survive severe acute brain injury in avegetative state. These patients will take more time andproceed through different stages before fully or partiallyrecovering awareness; alternatively, they may permanentlylose all brain functions. Many studies have shown thatpatients in a vegetative state suffer major and irreversiblebrain damage as a consequence of a traumatic or non-traumatic acute cerebral event (e.g., stroke, aneurysmruptures, intoxication, infections, etc.) (1). According to theAmerican Academy of Neurology, a vegetative state isdefined as ‘‘a condition of complete unawareness of the selfand the environment, accompanied by sleep-wake cycles,

with either complete or partial preservation of hypothala-mic and brain-stem autonomic functions’’ (2). This defini-tion means that patients in a vegetative state have somebasic reflexes and can usually breathe independently butcannot communicate, understand spoken language, orengage purposefully with the surrounding environment.There are no data concerning the number of patients in avegetative state in China; however, the annual incidence ofthis condition in the United States is estimated to bebetween 14,000 and 35,000 people (3). The life of expectancyof a patient in a vegetative state is approximately 2–5 years;however, surviving for more than 25 years is not unusual(4). Therefore, patient management raises a number ofmedical, ethical, and psychological concerns.

The families of patients in a vegetative state are affected bythe stress associated with this difficult situation. Familymembers develop a wide range of feelings and anxieties,including a sense of isolation, abandonment, fear regarding thefuture, guilt, and feelings of inadequacy concerning theproblems that they face. They may oscillate between acceptanceand denial and present a range of emotions, including sadness,exhaustion, weariness, excessive protection, aggression, anger,




341

and demand. These patients will often appear not to hear theinformation provided to them (5). Once a vegetative diagnosishas been established, the family members of the patient haverelatively high rates of anxiety and distress that adversely affecttheir health and quality of life. When these difficulties surpassan individual’s ability to cope, a psychological crisis may occurthat severely influences his or her physical and mental health(6). Thus, psychological crisis intervention plays an importantrole in maintaining mental and physical health. Throughpsychological crisis intervention, people learn to call on theirown resources and inner strength to recover psychologicalbalance and obtain new skills to cope with this psychologicalcrisis (7). In the last decade, early interventions, such aspsychological debriefing, have been increasingly used to treatpsychological crises. Crisis intervention became popular duringthe 1970s, and in 1983, Mitchell developed the Critical IncidentStress Debriefing (CISD) to prevent symptoms related totraumatic events in emergency responders (8). The CISD isdesigned to promote the emotional processing of traumaticevents through venting and normalizing reactions. The CISDalso prepares people for the experiences they may have in thefuture and generally involves a seven-phase treatment processwith the following steps:

a) Introduction: ground rules are established, confidenti-ality emphasized and participants urged to talk if theywish.

b) Facts: participants are asked to describe what happenedduring the incident from their own perspective. Thishelps to give a total picture of what happened.

c) Thoughts: participants describe their first thoughtsabout the event. The discussion becomes more personal.

d) Emotions: participants discuss their emotional reactions.

e) Assessment: physical and psychological symptoms arenoted and discussed.

f) Teaching or education: discussing stress reaction andresponses, coping strategies.

g) Re-entry: participants ask questions, team leaderssummarize what has occurred, advise participants thatthey can contact team members if they wish and drawthe debriefing to a close.

Living with and caring for patients that have beendiagnosed as being in a vegetative state can be a highlystressful experience for family members. These familymembers witness their loved ones transition from being indanger to undergoing active clinical treatment and finallyexhibiting symptoms that are identical to a coma. Thesuddenness of the final diagnosis of being in a vegetativestate usually directly affects the family members. If notmanaged and resolved appropriately, either by oneself orwith assistance, the stress experienced by the familymembers may lead to several psychological disorders,including posttraumatic stress disorder, depression, abuseof alcohol or other drugs, etc. Given their great emotionaldistress, the need for a psychological intervention amongthese sufferers is warranted. Few studies have investigatedthe psychological stress and the effects of a psychologicalintervention on the family members of patients in avegetative state. This study describes the efficacy of apsychological crisis intervention for this population andprovides evidence for additional psychological crisis inter-vention in clinical work.


ParticipantsThirty patients in a vegetative state admitted to long-stay

hospitals were selected. In accordance with the AmericanNeurology Academy (2), the vegetative state diagnosis andinclusion criteria were as follows: (a) no awareness ofoneself or the environment; (b) no directed behavioralresponses; (c) no expression or comprehension of language;(d) the presence of a sleep–waking cycle; and (e) thecomplete or partial preservation of hypothalamic andbrainstem function. The sample patients were healthyindividuals previously without any serious or life-threaten-ing problems such as heart attack, respiratory failure, coma,and had been previously expected to live normal lives.

The participants were family members of the patients in avegetative state and were consecutively admitted to theneurology department of a general hospital for treatment.The participants were clearly conscious and able to comm-unicate verbally without visual or auditory problems.Participants were excluded for having a previous psychia-tric illness, a history of brain injury, a serious familyaccident, or an acute psychological trauma which is a typeof damage to the psychology that occurs as a result of atraumatic event such as war, earthquakes, plane crashes,medical emergencies, etc. One hundred twenty-eight familymembers were eligible to participate in this study, and 89%of these members agreed to volunteer. Of the 114 partici-pants enrolled, 107 participants met the above inclusioncriteria and were included in data analyses.

We assigned the study participants to one of two groupsusing simple random sampling: the intervention group (54participants), who experienced a psychological intervention,or the control group (53 participants), who did not receive anintervention. The control and intervention designations (151ratio) were sealed in opaque envelopes that were intermixed.A research assistant assigned the participants to a group byopening an envelope when an eligible participant was intriage. The study and consent procedure were performed inaccordance with the Declaration of Helsinki. The hospitalethics committee approved this study.

InstrumentsAt the baseline and one-month follow-up examinations,

the Symptom Checklist-90-R (SCL-90-R) (9,10) evaluatedthe psychological stress as well as the effects of theintervention on participants. The SCL-90-R contains 90items concerning the participants’ psychosomatic symp-toms across the following nine domains: somatization,obsessive-compulsive disorder, interpersonal sensitivity,depression, anxiety, hostility, phobic anxiety, paranoidideation, and psychosis. All 90 items are rated on five-point Likert scales of distress that indicate the symptomoccurrence rate at a particular time. The SCL-90-R is par-ticularly useful for discriminating symptoms related todepression and anxiety.

The family members answered the questionnaire, andthe baseline data were collected prior to beginning thepsychological intervention. The family members wererequired to complete the questionnaire in thirty minutes.One month after the psychological intervention, a reevalua-tion was conducted during which the questionnaire wasadministered and retrieved for both groups.

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Psychological Crisis InterventionFollowing Mitchell’s seven-phase CISD process (11), a

brief single-session group intervention was established withthe participants. This intervention strategy has four steps: inthe facts stage, participants describe what happened duringthe vegetative state from their perspective, which helps toprovide the full picture of what happened; in the thoughtsphase, participants describe their first thoughts concerningthe event, and the discussion becomes more personal; in theassessment phase, participants discuss their emotionalreactions, and we note and discuss their physical andpsychological symptoms; and in the assisting stage, weencourage participants to reexamine their feelings of help-lessness and conduct the appropriate training to increasetheir coping abilities.

The intervention was held 48-72 hours after the patientwas diagnosed as being in a vegetative state. The interven-tion group participated in a psychological session that wasled by an occupational psychologist and lasted between 3and 4 hours. The family members of patients who wereinvolved in the same accident were randomly assigned intoseparate intervention debriefing group sessions containing3-10 participants.

Statistical AnalysesThe data are presented as the means and standard

deviations (SDs). Quantitative variables were investigatedusing SPSS 16.0. Descriptive statistics characterized theparticipant’s demographic characteristics, including educa-tion level and the relationship to the patient. A t-test wasconducted at the baseline and follow-up examinations forboth groups. Differences (g) between the SCL-90-R scores atthe baseline and follow-up evaluations were calculated foreach patient. A t-test also compared the differences betweenthe intervention and control groups. A p-value of ,0.05 wasconsidered as significant (two-tailed).

RESULTS

Participant characteristicsOne hundred seven participants were eligible to partici-

pate in this study. Of these participants, 61.7% were menand 39.3% were women. The average age was 55 years(range = 41-68 years), 89.7% of participants were marriedand 10.3% of participants were single. Fifty-nine partici-pants (55.1%) had a high school education, 25 participants(23.4%) had a middle school education (or less), and 23participants (21.5%) had a university degree. Of theparticipants who provided data regarding their relationshipto the patient, 25.2% were parents, 17.8% were spouses,29.9% were siblings and 27.1% were children (Table 1).

Between-group comparisons of SCL-90 factor scoresand the national norm

We chose the SCL-90 for this study because it evaluates abroad range of psychological symptoms in family membersof patients in a vegetative state. A single-sample t-test wasperformed using the SCL-90 factor scores for all partici-pants, and these scores were compared with the corre-sponding national norm (12). Table 2 shows that the factorscores of participants in both groups were significantlyhigher than those of the national norm (p,0.001). Therange of factor scores from highest to lowest correspondedto the categories of depression, phobia anxiety, anxiety,

somatization, paranoid ideation, hostility, interpersonalsensitivity, obsessive-compulsive behavior, and psychosis.Table 2 also compares the intervention and control groupswith regard to their SCL-90 factor scores at baseline. Therewas no difference at baseline between the intervention andcontrol groups with regard to their SCL-90 factor scores.

Comparisons between baseline and follow-upexaminations with regard to SCL-90 factor scoresfor both groups

Table 3 compares the SCL-90 factor scores at baseline tothose at the follow-up examination for both groups. TheSCL-90 factor scores of the intervention group weresignificantly lower at the follow-up evaluation comparedwith those observed at baseline (p,0.001). This differencewas also statistically significant (p,0.001) for the controlgroup except with regard to the somatization, obsessive-compulsive behavior, depression, and anxiety subscales.

Between-group comparisons with regard to themean difference of SCL-90 scores

Most SCL-90 factor scores at the follow-up evaluationwere significantly lower than those at baseline for bothgroups. There is not a definitive intervention effect becausetime may have influenced our results. To demonstrate adefinitive intervention effect, Table 4 reports the mean gbetween the SCL-90 scores for the baseline and follow-upexaminations for both groups. t-tests compared the mean gbetween the two groups; significant g values were found onthe following SCL-90 scales: somatization, obsessive-com-pulsive behavior, depression, and anxiety (p,0.05).

DISCUSSION

Loss is a situational crisis that poses a threat or challengeto an individual. When family members witnessed a lovedone’s loss of consciousness, a severe psychological stressresponse occurred, and their mental health was adverselyaffected. This study revealed psychological distress in thefamily members of patients in a vegetative state using theSCL-90-R. The SCL-90-R is a reliable, self-administered, and

Table 1 - Characteristics of the family members of patientsin a vegetative state (n = 107).

Characteristic N %

Gender

Male 65 61.7

Female 42 39.3

Age (years)

40-50 21 19.6

50-60 54 50.5

60-70 32 29.9

Marital Status

Married 96 89.7

Single 11 10.3

Education

Middle school or less 25 23.4

High school 59 55.1

College/university/graduate 23 21.5

Relationship to patient

Parent (mother/father) 27 25.2

Spouse (Husband/wife) 19 17.8

Sibling (brother/sister) 32 29.9

Child (son/daughter) 29 27.1

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validated instrument for assessing psychological andsymptomatic distress in both clinical and research settings(13). Our study demonstrated that the SCL-90 factor scoresof the family members of both groups were significantlyhigher than the national norm. Six SCL-90 factors exceededa score of two: depression, phobia anxiety, anxiety,somatization, paranoid ideation and hostility. Higher scoreson the SCL-90 indicate a higher level of psychologicaldistress. These results indicate that family members ofpatients in a vegetative state experience a range ofemotional reactions that include anxiety, depression, guilt,irritability, aggressiveness, and impulsivity. Many factorsmay contribute to these reactions, such as the loss of theirloved one, social circumstances, and the complex medicaland ethical decisions in which they become involved (14). Ina recent study, over 70% of family members of patients in avegetative state reported social isolation and low levels ofengagement in recreational activities (15). When there islittle expectation of recovery, many family members becomeupset and anxious because they do not want to abandonhope. Furthermore, the family members feel guilty becausetheir feelings for their loved ones change. These familymembers may be unable to mourn and adjust to their lossbecause the body remains alive.

Thus, the sudden unconsciousness of a loved one caused adramatic change in family members’ psychological function-ing. The need for urgent crisis intervention seems necessary.Crisis intervention refers to the methods used to offerimmediate short-term help to individuals who experience anevent that produces emotional, mental, physical, and beha-vioral distress. In many cases, it may be appropriate for the

teams who work with these families to encourage them to seekpsychological support from external sources. The CISD can bea valuable tool to assist others in coping with the physical orpsychological symptoms generally associated with exposureto trauma. Based on Mitchell’s CISD model, a brief four-stepintervention method was developed, and this study assessedits effectiveness. There was no difference between theintervention and control groups at baseline with regard totheir SCL-90 factor scores. Most of the SCL-90 factor scoreswere significantly lower at the follow-up evaluation than atbaseline for both groups. One month after the psychologicalintervention, the intervention group improved significantlymore than the control group with regard to most subscales,including somatization, obsessive-compulsive behavior,depression, and anxiety. This finding suggests that thepsychological and physical symptoms of family membersimprove with psychological intervention. Our findings sup-port our hypothesis that the four-step intervention effectivelyimproves the general psychological distress of family mem-bers of patients in a vegetative state with regard to reductionsin somatization, obsessive-compulsive behavior, depressionand anxiety. Psychological interventions must be providedthrough short-term crisis intervention methods. In this study,the intervention followed the four steps of self-introduction,expressing feelings and emotions, providing information, andassisting abreaction. In addition, our study showed that wedeveloped a reproducible, easily implemented, low-cost inter-vention to reduce psychological distress and the perception ofstress in family members of patients in a vegetative state.

Our study has several limitations. Our research recruiteda relatively small and heterogeneous sample across a

Table 2 - Between-group comparisons of SCL-90 psychological symptoms and the national norm (mean ¡ SD).

SCL-90 Factors

Intervention group

(n = 54)

Control Group

(n = 53)

National norm

(n = 1,388) p1-value p2-value p3-value

Somatization 2.33¡0.55 2.37¡0.61 1.37¡0.48 ,0.001 ,0.001 0.835

Obsessive-compulsive behavior 1.97¡0.44 1.92¡0.41 1.62¡0.57 ,0.001 ,0.001 0.733

Interpersonal sensitivity 2.03¡0.52 1.95¡0.49 1.65¡0.51 ,0.001 ,0.001 0.547

Depression 2.89¡0.57 2.92¡0.59 1.50¡0.59 ,0.001 ,0.001 0.874

Anxiety 2.61¡0.53 2.64¡0.55 1.39¡0.43 ,0.001 ,0.001 0.859

Hostility 2.01¡0.52 2.06¡0.57 1.48¡0.56 ,0.001 ,0.001 0.782

Phobia anxiety 2.81¡0.48 2.86¡0.51 1.23¡0.41 ,0.001 ,0.001 0.796

Paranoid Ideation 2.36¡0.50 2.33¡0.48 1.43¡0.57 ,0.001 ,0.001 0.891

Psychotic 1.96¡0.51 1.90¡0.47 1.29¡0.42 ,0.001 ,0.001 0.708

p1-value = Intervention group vs. national norm.

p2-value = Control group vs. national norm.

p3-value = Intervention group vs. control group.

Table 3 - Comparisons of SCL-90 factor scores at the baseline and follow-up examinations for both groups (mean ¡ SD).

SCL-90 Factors Intervention group Control Group

Baseline Follow-up p-value Baseline Follow-up p-value

Somatization 2.33¡0.55 1.71¡0.54 ,0.001 2.37¡0.61 2.25¡0.58 0.672

Obsessive-compulsive behavior 1.97¡0.44 1.69¡0.46 ,0.001 1.92¡0.41 1.94¡0.49 0.876

Interpersonal sensitivity 2.03¡0.52 1.62¡0.49 ,0.001 1.95¡0.49 1.65¡0.51 ,0.001

Depression 2.89¡0.57 2.03¡0.61 ,0.001 2.92¡0.59 2.95¡0.62 0.893

Anxiety 2.61¡0.53 1.97¡0.55 ,0.001 2.64¡0.55 2.66¡0.57 0.865

Hostility 2.01¡0.52 1.59¡0.54 ,0.001 2.06¡0.57 1.63¡0.46 ,0.001

Phobia anxiety 2.81¡0.48 1.79¡0.50 ,0.001 2.86¡0.51 2.02¡0.53 ,0.001

Paranoid Ideation 2.36¡0.50 1.74¡0.49 ,0.001 2.33¡0.48 1.88¡0.43 ,0.001

Psychotic 1.96¡0.51 1.52¡0.46 ,0.001 1.90¡0.47 1.55¡0.49 ,0.001

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CLINICS 2012;67(4):341-345

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relatively short period of time. Therefore, most of the SCLscores were still significantly different from populationnorms at the time of the follow-up observation. Additionalresearch should be conducted using a larger number ofpeople and with different lengths of follow-up. In addition,the four-step approach used in this research was notcompared with any alternative psychological therapies.Thus, we cannot conclude that this four-stepapproach ismore effective than other forms of psychological interven-tions; however, we can state that it is more effective thantypical interventions that do not include a psychologicalapproach. Furthermore, family members in the controlgroup had no intervention, and they completed thequestionnaire at the baseline and follow-up examinations.Therefore, the interventionist attended to all participants.Other factors, such as interventionist attention and partici-pant expectations, might have affected the measuredimprovement of participants. Despite these limitations, ourresults may have important implications for future psycho-logical crisis interventions.

ACKNOWLEDGMENTS

The authors thank all participants and their families as well as the staffs

at the Department of Psychology at South-Central University for

Nationalities and the Department of Neurology at Wuhan General

Hospital of Guangzhou Command for their invaluable assistance.


Li YH was responsible for project development, psychological crisis

intervention, clinical analyses, and manuscript writing. Xu ZP was

responsible for project development, clinical case collection and psycho-

logical testing.

REFERENCES

1. Wilson FC, Graham LE, Watson T. Vegetative and minimally consciousstates: serial assessment approaches in diagnosis and management.Neuropsychological Rehabilitation. 2005;15(3-4):431-41, http://dx.doi.org/10.1080/09602010543000091.

2. The Multi-Society Task Force on PVS. Medical aspects of the PersistentVegetative State (First of Two Parts). The New England Journal ofMedicine. 1994;330(21):1499-508.

3. The Quality Standards Subcommittee of the American Academy ofNeurology. Practice parameters: Assessment and management ofpatients in the persistent vegetative state. Neurology. 1995;45(5):859-60.

4. Chiambretto P, Rossi Ferrario S, Zotti AM. Patients in a persistentvegetative state: caregiver attitudes and reactions. Acta NeurologicaScandinavica. 2001;104(6):364-8, http://dx.doi.org/10.1034/j.1600-0404.2001.00107.x.

5. Tzidkiahu T, Sazbon L, Solzip P. Characteristic reactions of relatives ofpost-coma unawareness patients in the process of adjusting to loss. BrainInjure. 1994;8(2):159-65, http://dx.doi.org/10.3109/02699059409150967.

6. Caplan G. Principles of preventive psychiatry. New York: Basic Books.1964.

7. Bayliss, Corey M, Miller, Audrey K, Henderson, Craig E. PsychopathyDevelopment and Implications for Early Intervention. Journal ofCognitive Psychotherapy. 2010;24(2):71-80, http://dx.doi.org/10.1891/0889-8391.24.2.71.

8. Mitchell JT. When disaster strikes… the critical incident stress debriefingprocess. JEMS. 1983;8(1):36-9.

9. Derogatis LR, Cleary PA. Confirmation of the dimensional structure ofthe SCL-90: A study in construct validity. Journal of Clinical Psychology,33 :981-989. doi : 10.1002/1097-4679(197710)33 :4,981: :AID-JCLP2270330412.3.0.CO;2-0.

10. Wang XD, Wang XL, Ma H. Mental Health Assessment Scale (revisedversion). Beijing: Chinese Encyclopedia Press. 1999.

11. Mitchell JT. Stress. The history and future of critical incident stressdebriefings. JEMS. 1988;13(11):46-7, 49-52.

12. Jin H, Wu WY, Zhang MY. Initial analysis of SCL-90 with healthyChinese adults. Chinese Journal of Nervous and Mental Diseases.1986;12:260-3.

13. Derogatis LR, Rickels K, Rock AF. The SCL-90 and the MMPI: A step inthe validation of a new self-report scale. British Journal of Psychiatry.1976;128:280-9, http://dx.doi.org/10.1192/bjp.128.3.280.

14. Wilson FC, Harpur J, Watson T, Morrow JI. Vegetative state andminimally responsive patients-Regional survey, long-term case out-comes and service recommendations. NeuroRehabilitation. 2002;17(3):231-5.

15. Wilson BA, Gracey F, Bainbridge K. Cognitive recovery from "persistentvegetative state": psychological and personal perspectives. Brain Injury.2001;15(12):1083-92, http://dx.doi.org/10.1080/02699050110082197.

Table 4 - Between-group comparisons of the mean SCL-90difference score.

SCL-90 Factor Intervention g Control Group g p-value

Somatization -0.63 -0.12 0.028

Obsessive-

compulsive

behavior

-0.29 0.02 0.046

Interpersonal

sensitivity

-0.41 -0.35 0.612

Depression -0.86 0.03 0.002

Anxiety -0.62 0.02 0.019

Hostility -0.45 -0.46 0.893

Phobia anxiety -1.02 -0.84 0.095

Paranoid Ideation -0.63 -0.49 0.276

Psychotic -0.44 -0.38 0.604

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CLINICAL SCIENCE

Plasma kinetics of an LDL-like nanoemulsion and lipidtransfer to HDL in subjects with glucose intoleranceMarina P. Bertato,I,II Carolina P. Oliveira,I,II Bernardo L. Wajchenberg,I,II Antonio C. Lerario,I,II Raul C.

MaranhaoI,III

I Faculdade de Medicina da Universidade de Sao Paulo, Heart Institute (InCor), Sao Paulo/SP, Brazil. II Faculdade de Medicina da Universidade de Sao Paulo,

Endocrinology Service, Sao Paulo/SP, Brazil. III Universidade de Sao Paulo, School of Pharmaceutical Sciences, Sao Paulo/SP, Brazil.

OBJECTIVE: Glucose intolerance is frequently associated with an altered plasma lipid profile and increasedcardiovascular disease risk. Nonetheless, lipid metabolism is scarcely studied in normolipidemic glucose-intolerantpatients. The aim of this study was to investigate whether important lipid metabolic parameters, such as the kineticsof LDL free and esterified cholesterol and the transfer of lipids to HDL, are altered in glucose-intolerant patientswith normal plasma lipids.

METHODS: Fourteen glucose-intolerant patients and 15 control patients were studied; none of the patients hadcardiovascular disease manifestations, and they were paired for age, sex, race and co-morbidities. A nanoemulsionresembling a LDL lipid composition (LDE) labeled with 14C-cholesteryl ester and 3H-free cholesterol wasintravenously injected, and blood samples were collected over a 24-h period to determine the fractional clearancerate of the labels by compartmental analysis. The transfer of free and esterified cholesterol, triglycerides andphospholipids from the LDE to HDL was measured by the incubation of the LDE with plasma and radioactivitycounting of the supernatant after chemical precipitation of non-HDL fractions.

RESULTS: The levels of LDL, non-HDL and HDL cholesterol, triglycerides, apo A1 and apo B were equal in bothgroups. The 14C-esterified cholesterol fractional clearance rate was not different between glucose-intolerant andcontrol patients, but the 3H-freecholesterol fractional clearance rate was greater in glucose-intolerant patientsthan in control patients. The lipid transfer to HDL was equal in both groups.

CONCLUSION: In these glucose-intolerant patients with normal plasma lipids, a faster removal of LDE freecholesterol was the only lipid metabolic alteration detected in our study. This finding suggests that the dissociationof free cholesterol from lipoprotein particles occurs in normolipidemic glucose intolerance and may participate inatherogenic signaling.

KEYWORDS: Insulin Resistance; Cholesterol; Emulsions; Nanoparticles; Lipoproteins.

Bertato MP, Oliveira CP, Wajchenberg BL, Lerario AC, Maranhao RC. Plasma kinetics of an LDL-like nanoemulsion and lipid transfer to HDL in subjectswith glucose intolerance. Clinics. 2012;67(4):347-353.

Received for publication on October 7, 2011; First review completed on November 19, 2011; Accepted for publication on December 21, 2011


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INTRODUCTION

Insulin resistance, which leads to common disorders suchas glucose intolerance (GI), metabolic syndrome and type 2diabetes, affects glucose homeostasis and the regulation ofplasma lipids (1,2). Dyslipidemia that results from insulinresistance leads to the development of cardiovascular diseasenot only in overt type 2 diabetes patients but also in subjectswith GI (2). Interestingly, the odds ratio for cardiovasculardisease in GI patients approaches that estimated for type2 diabetes patients (3). Characteristically, dyslipidemia

associated with insulin resistance consists of hypertriglycer-idemia and decreased HDL cholesterol levels. LDL choles-terol levels are not usually elevated, and LDL plasma kineticsmay be normal in type 2 diabetes. Kinetic studies have beenperformed by radioactive or stable isotope labeling of apoB100, which is the only LDL protein (4-8). However,alterations in LDL metabolism may occur in the lipid partof the lipoprotein because insulin resistance causes changesin the LDL subfraction profile, with an increase in the moreatherogenic small-dense LDL subfraction (9).

LDL contains both unesterified and esterified cholesterol.Each of these forms of cholesterol has distinct physicalchemical and metabolic properties, and their status in theplasma has not been explored in metabolic diseases affectingplasma lipids. Esterified cholesterol is located in thelipoprotein core, wherefrom it can be dislocated only by theaction of transfer proteins. Unesterified cholesterol located inthe lipoprotein surface is a more unstable form of cholesterol.




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Cholesteryl ester transfer protein (CETP) and phospholi-pid transfer protein (PLTP) promote lipid transfer amonglipoprotein classes, including cholesterol, phospholipidsand triglycerides. These lipid transfers are crucial for theformation and metabolism of HDL in the plasma and arepart of the reverse cholesterol transport. Free cholesteroltransferred to HDL undergoes esterification by lecithin-cholesterol acyltransferase (LCAT) using apo A1, which isthe main HDL apolipoprotein, as a co-factor (10,11).

This study aimed to investigate whether insulin resistanceaffects the plasma kinetics of lipoprotein free and esterifiedcholesterol and the transfer of both cholesterol forms,phospholipids and triglycerides to HDL, even in theabsence of altered plasma lipids. GI patients with normallipid plasma levels were studied to determine whether theseparameters could indicate the existence of metabolicalterations. An artificial nanoemulsion that mimicked theLDL structure was used to probe the intravascularmetabolism of LDL cholesterol (12) and also as a lipiddonor to HDL (13) for the in vitro evaluation of the lipidtransfer.


SubjectsIn total, 14 GI individuals and 15 healthy controls, all

without clinical manifestations of cardiovascular disease,were recruited at the Medical School Hospital of theUniversity of Sao Paulo and from the an outpatient clinic ofthe Heart Institute, and they were paired for age, sex, raceand co-morbidities. All of the patients were submitted to anelectrocardiogram exercise stress test to evaluate the presenceof cardiovascular disease. A negative result indicated anabsence of cardiovascular disease. The electrocardiogramexercise stress test adopted was the Bruce protocol with aMAT 2100 treadmill coupled with an ML 800 Stress TestSystem (Fukuda Denshi Co., Ltd). The electrocardiographicrecordings of 15 simultaneous leads, with 12 classic deriva-tions of the Mason - Likar system and 3 leads of the Frankorthogonal system (X,Y,Z), were obtained.

All patients were submitted to an oral glucose tolerance test(OGTT) that was performed at 120 min after the patients drankthe glucose solution. According to the American DiabetesAssociation criteria (14), GI patients exhibited normal 2 hplasma glucose levels (7.8-11.1 mmol/L; n = 4) or impairedfasting glucose levels ($5.5 mmol/L; n = 10). The control grouphad 2 h plasma glucose ,7.8 mmol/L and fasting bloodglucose ,5.5 mmol/L.

None of the participants had liver or renal dysfunction asevaluated by clinical and laboratory criteria. None hadinflammatory, neoplastic or pulmonary disease, chronicrenal disease (creatinine .1.5 mg/dL), asthma, cardiovas-cular disease, peripheral arterial disease or a previousstroke. The patients were evaluated by cardiologists todetect issues related to coronary heart disease (e.g., angina,dyspnea, palpitations). No patient was taking metformin,glucocorticoids or any medication known to affect lipidmetabolism.

The inclusion criteria were as follows: age between 40 and70 years; total cholesterol ,6 mmol/L; LDL-C ,4 mmol/L;triglycerides ,2.2 mmol/L; normal blood pressure ortreated hypertension up to 130/85 mmHg; and thyroidfunction within the normal range or compensated withthyroid replacement therapy.

Cases and controls did not differ regarding the frequencyof arterial hypertension (8 in GI vs. 5 in controls, p = 0.27);family history of cardiovascular disease (7 vs. 5, p = 0.46);family history of type 2 diabetes (8 vs. 4, p = 0.13); currentsmoking (1 vs. 1, p = 1.00); hypothyroidism (2 vs. 4, p = 0.65);and current medications such as angiotensin-convertingenzyme inhibitors (5 vs. 3, p = 0.42), angiotensin II receptorblockers (1 vs. 2, p = 1.00), calcium channel blockers (1 vs. 3,p = 0.59), thiazides (6 vs. 3, p = 0.24) and levothyroxine (3 vs.4, p = 1.00). The characteristics of the study patients aredescribed in Table 1.

EthicsThe human experimental protocol was approved by the

institutional review boards of the Medical School Hospitalof the University of Sao Paulo and was in accordance withthe Helsinki Declaration of 1975, as revised in 1983. Allparticipants provided written informed consent.

Laboratory assaysBlood samples for the determination of laboratory para-

meters were collected after fasting for 12 h and on the sameday the kinetic study was performed. Commercial enzymaticcolorimetric methods were used for the determination of totalcholesterol, triglycerides and HDL-C. LDL cholesterol wascalculated using a direct method (kinetic automatized).Plasma apo A1 and apo B were assayed by turbidimetry(Roche/Hitachi, Roche Diagnostics, Mannheim, Germany),

Table 1 - Physical characteristics and current medicationsof the glucose-intolerant (GI) group and the controlgroup.

GI

n = 14

Control

n = 15 p-value

Age (years) 56¡6 53¡6 0.13

Sex (F/M) 9/5 8/7 0.71

Weight (kg) 78.5¡12 76.5¡8.7 0.63

BMI (kg/m2) 29.8¡4.4 27.6¡2.6 0.13

Waist circumference (cm) 99.7¡10.8 94.7¡12.7 0.26

HOMA2%b 111.9¡27.9 102.9¡34.1 0.44

HOMA2%S 80¡43.1 139.7¡74.7 0.01

HOMA2-IR 1.5¡0.6 0.9¡0.4 ,0.01

Total fat area (cm2) 475.2¡143.9 396.3¡97.7 0.10

Visceral fat area 171.5¡81.9 144.4¡48.1 0.29

Subcutaneous fat area 303.6¡101.7 251.8¡83.1 0.15

Visceral/subcutaneous ratio 0.61¡0.34 0.63¡0.30 0.86

Arterial hypertension 8 5 0.27

Familial history of CVD 7 5 0.46

Family history of type 2

diabetes

8 4 0.13

Current smoking 1 1 1.00

Hypothyroidism 2 4 0.65

Current medications

ACEi 5 3 0.42

ARB 1 2 1.00

Ca2+ channel blockers 1 3 0.59

Thiazides 6 3 0.24

Levothyroxine 3 4 1.00

Data are the means ¡ SD. Differences were assessed using Fisher’s exact

test, Student’s t-test and chi-squared test. HOMA2%b: insulin secretion by

pancreatic b cells; HOMA2%S: insulin sensitivity; HOMA2-IR: index of

insulin resistance (1/HOMAS); CVD: cardiovascular disease; ACEi:

angiotensin-converting enzyme inhibitor; ARB: angiotensin II receptor

blocker; SD: standard deviation. Analyses of abdominal fat area by CT

were performed with n = 14 for the GI group and n = 14 for the control

group.

Lipid metabolism in glucose intoleranceBertato MP et al.

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and apo E was measured by nephelometry. HbA1C wasmeasured by HPLC (National Glycohemoglobin Standar-dization Program (NGSP), USA; normal range 4.1 to 6%).Plasma insulin was measured by an enzyme-linked immuno-sorbent assay (AutoDELFIA, Perkin-Elmer Life Science,Wallac, Oy, Finland) with an intra-assay coefficient ofvariation of ,1.5%. The plasma glucose concentration wasmeasured using a hexokinase method.

Insulin resistanceThe fasting plasma glucose (mmol/L) and insulin (rmol/

L) values were used to evaluate insulin resistance via theHomeostasis Model Assessment (HOMA), which is amathematical model for defining insulin secretion bypancreatic b cells (HOMA2-%b) and insulin sensitivity(HOMA2-%S) as percentages of normal values and theindex of insulin resistance (HOMA2-IR). This model wasimplemented using the HOMA Calculator Version 2.2.2software, which was developed by The Oxford Center forDiabetes, Endocrinology & Metabolism (Diabetes Trial Unit,HOMA Calculator; available at http://www.dtu.ox.ac.uk)(15).

Computerized tomography of the abdomenThe abdominal fat distribution was evaluated by compu-

terized tomography (CT) of the abdomen without oral orintravenous contrast or any previous preparation of thepatients. The examination was performed at the Institute ofRadiology of the Medical School Hospital using a high-speed CT (GE). The initial image acquisition was used as the‘‘scout’’ (digital radiology) for the patient profile. A singleCT image with a thickness of 10 mm was obtained in themedian of the disc space of L4-L5, guided by the previouslyobtained ‘‘scout’’. The visceral and subcutaneous fat areaswere calculated in the CT image. The limits of the fatcomponent were calculated using the upper limit of -50Hounsfield units and the lower limit of -250 Hounsfieldunits as the attenuation values to determine the presence offat in the bound area. The ratio of the visceral andsubcutaneous fat areas was also calculated (16).

LDE preparationThe LDE was prepared from a lipid mixture composed of

40 mg cholesteryl oleate, 20 mg egg phosphatidylcholine,1 mg triolein and 0.5 mg cholesterol purchased from SigmaChemical, USA. 14C-cholesteryl oleate and 3H-cholesterolwere purchased from Perkin-Elmer, USA, and added to themixture. The emulsification of the lipids by prolongedultrasonic irradiation in aqueous media and the two-stepultracentrifugation of the crude emulsion with densityadjustment by the addition of KBr to obtain the LDE wereperformed as described by Maranhao et al. (17). The finallipid composition of the LDL-like LDE was 64% phospho-lipids, 33% cholesteryl oleate, 2% triacylglycerols and 1%cholesterol. The LDL-like LDE was dialyzed against a salinesolution and sterilized by passage through a 0.22-mm filterbefore injection into the patients. The entire LDE prepara-tion procedure was performed under laminar flow. Allglassware used in this study was made pyrogen-free byexposure to dried steam at 180 C for 2 h and sterilized bywet steam at 120 C for 30 min. All plastic materials weresterilized by ultraviolet light exposure.

Plasma kinetics study protocolAs previously indicated, the study patients were asked to

arrive at the laboratory at 7 a.m., after a 12-h fasting period.Blood samples were collected as described previously forthe determination of basal blood parameter levels. The LDElabeled with 14C-cholesteryl oleate (37kBq) and 3H-freecholesterol (74 kBq) was injected intravenously in a bolus of5-6 mg of total lipids in a 500-mL volume. Blood sampleswere obtained over 24 h (5 min, 1 h, 2 h, 4 h, 6 h, 8 h and24 h). The subjects were allowed to eat low-fat meals on theevening before the test day, after the first blood collectionand at about 1500 p.m. because low-fat meals do notinterfere with the plasma removal of LDE (17). The plasmasamples were separated by centrifugation, and 1 mL of eachsample was transferred to a counting vial containing 5 mLof scintillation solution. The samples were then counted in ascintillation counter (1600 TR model, Hewlett-Packard, PaloAlto, CA).

Estimation of the fractional clearance rate of theradioisotopes

The fractional clearance rates (FCRs) of 14C-cholesterylester and 3H-free cholesterol from the LDE were calculatedaccording to the method described by Couto et al. (18).FCR = (a1/b1+a2/b2)21, where a1, a2, b1 and b2 wereestimated from biexponential curves obtained from theremaining radioactivity found in the plasma after injectionand fitted by least squares procedure as y = a1e-b1t+a2e-b2t,where y is the curve of the radioactivity plasma decay as afunction of time (t); a indicates the linear coefficient and b isthe angular coefficient, which represents the FCR (h-1).Calculations were performed using ANACOMP computersoftware (19). The compartmental model is illustrated inFigure 1.

In vitro lipid transfer from LDE to HDLThe assay to estimate the lipid transfer of 3H-cholesteryl

oleate, 14C-phosphatidylcholine, 3H-triolein and 14C-freecholesterol from the LDE to HDL was performed asdescribed by Lo Prete et al. (13). The assay is based on a1-h incubation of the LDE with whole plasma, followed bythe measurement of the radioactivity in the supernatantafter the chemical precipitation of the non-HDL lipoproteinfractions and of the LDE. The safety of the radioactive doseintravenously injected into the patients was assuredaccording to the regulations of the International Comm-ission on Radiological Protection (20). The injected dose ineach experiment was 0.03 mSV.

Statistical analysisThe results are expressed as means ¡ standard deviations

(mean ¡ SD). Student’s t-test was used to compare themeans of the two groups. When the assumption of normal-ity was rejected, the non-parametric Mann-Whitney test wasused. To test the homogeneity between the proportions, thechi-squared or Fisher’s exact test was used. To study theinfluence of two factors on abdominal adiposity, a two-wayanalysis of variance was used. The coefficient of correlationwas determined to study the correlation between twovariables. Statistical analyses were performed using SPSS15.0 for Windows. Differences with p,0.05 were consideredto be significant.

CLINICS 2012;67(4):347-353 Lipid metabolism in glucose intoleranceBertato MP et al.

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RESULTS

GI patients and controls did not differ regarding the areaof total abdominal fat (p = 0.10) or visceral (p = 0.29) andsubcutaneous fat (p = 0.15) as evaluated by abdominal CT(Table 1).

There was no difference between GI patients and controlswith respect to b cell insulin secretion as indicated byHOMA2%b (p = 0.44) (Table 1). The fasting insulin concen-tration (p,0.01) and the insulin resistance index (p,0.01)were higher in the GI patients than in controls (Table 1 andTable 2). As expected, compared with controls, the GIsubjects had lower insulin sensitivity as expressed byHOMA2%S (p = 0.01).

The plasma biochemical parameters of the study groupsare shown in Table 2. The concentrations of total, LDL, non-HDL and HDL cholesterol and triglycerides were similar inGI patients and controls. Similarly, apo A1 and apo B didnot differ between the two groups. Apo E tended to belower in GI patients than in controls, but this difference didnot attain statistical significance (p = 0.06).

The plasma decay curves of both LDE labels are shown inFigure 2. The cholesteryl ester curves of GI patients andcontrol subjects were equal (Figure 2A), as confirmed by thecholesteryl ester FCR shown in Table 3. However, the freecholesterol curve (Figure 2B) was faster in GI patients thanin controls, as confirmed by a greater free cholesterol FCR(p = 0.04) (Table 3).

Table 4 shows the results of the in vitro transfer of the fourradioactive lipids from the donor LDE to the HDL fraction

after incubation of the LDE with whole plasma. GI patientsdid not show differences in the transfer of lipids comparedwith the control group.

Correlations were found between free cholesterol FCRand LDL cholesterol (r = -0.53, p = 0.04) and apo E (r = -0.55,p = 0.03); between phospholipid transfer to HDL and LDLcholesterol (r = 0.57, p = 0.03) and apo B (r = 0.64, p = 0.01);between triglyceride transfer to HDL and fasting insulin(r = -0.55, p = 0.03), insulin sensitivity (r = 0.53, p = 0.04) andinsulin resistance (r = -0.53, p = 0.04); and between freecholesterol transfer to HDL and LDL cholesterol (r = -0.55,p = 0.04), HDL cholesterol (r = 0.54, p = 0.04), plasma trigly-cerides (r = -0.76, p,0.01), apo B (r = -0.70, p,0.01) and apo E(r = -0.58, p = 0.02).

DISCUSSION

The stable isotope method traces the kinetics of theprotein component of lipoproteins. Alternatively, the radio-active nanoemulsion method used here traces the kinetics ofthe lipid components. When the LDE is injected into thebloodstream, it acquires apo E from the native lipoproteinsand binds to LDL receptors that recognize both apo B andapo E (17,21). Because apo E has greater affinity for the LDLreceptor than apo B, the LDE is removed more quickly fromthe plasma compartment than native LDL. This effect isadvantageous because it shortens the observation timerequired (17,21,22). This method was validated in severalexperimental (17,23) and clinical studies (12,18,24-26), inwhich the kinetic behavior of the nanoemulsion was similarto that expected from native LDL.

The interpretation of plasma removal data after a bolusinjection is straightforward. In addition, a single preparationcan be used in several subjects, and the radioactive dose isvery low, in accordance with the radiological protectioninternational standards.

Using the LDE approach, we showed that the cholesterylester moiety is cleared equally in GI and control patients.Because the cholesteryl ester is less prone to dissociate fromLDE particles (i.e., it can only be dissociated through CETPactivity), it may be used an approximate marker of theplasma removal of the nanoemulsion particles. This

Figure 1 - Compartmental model used to analyze the LDE 14C-cholesteryl ester (CE) and 3H-free cholesterol (FC). The modelconsists of four discrete compartments: two for 14C-CE and twofor 3H-FC. All compartments are in the intravascular space (1CE,2CE, 1FC and 2 FC). LDE 14C-CE and 3H-FC are injected intravenouslyin a bolus (arrow with asterisk) into compartments 1CE and 1FC,respectively. Fractions k1,0 CE and k1,0 FC of the labeled lipids areremoved to the extravascular space. Fractions k1,2 CE and k1,2 FCof the injected lipids are converted into compartments 2CE and2FC because of the incorporation of apolipoproteins available inthe plasma. Subsequently, the materials of those compartmentsare transferred to the extravascular space following the k2,0 CEand k2,0 FC routes. The samplings, represented by triangles,correspond to the indiscriminate combination of compartments1 and 2.

Table 2 - Serum biochemical parameters of the glucose-intolerant (GI) group and the control group.

GI

N = 14

Control

N = 15 p-value

Fasting glucose (mmol/

L)

5.61¡0.4 4.78¡0.2 ,0.01

2-h postload glucose

OGTT (mmol/L)

8.91¡0.7 5.83¡1.1 ,0.01

Fasting insulin (mU/mL) 11.9¡5.1 7.1¡3.7 ,0.01

HbA1C (%) 6.1¡0.5 5.6¡0.3 0.02

Cholesterol (mmol/L) 5.07¡0.6 4.82¡0.67 0.35

Non-HDL 3.67¡0.7 3.42¡0.5 0.34

LDL-C 3.02¡0.6 2.85¡0.5 0.47

HDL-C 1.37¡0.4 1.37¡0.5 0.99

Triglycerides (mmol/L) 1.44¡0.4 1.26¡0.4 0.27

Apolipoproteins (g/L)

A1 1.53¡0.29 1.5¡0.35 0.86

B 0.99¡0.24 0.91¡0.18 0.33

E 0.05¡0.01 0.04¡0.01 0.06

Data are the means¡SD. Student’s t-test. OGTT: oral glucose tolerance

test; SD: standard deviation.


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assumption is corroborated by the transfer assay (Table 4),which showed that the shift of cholesteryl esters from thenanoemulsion to HDL is small: 3.6%, compared withroughly 10% for free cholesterol and 22% for phospholipids.LDL removal with radiolabeled apo B from the plasma ofsubjects with GI has not been described in the literature. In

subjects with metabolic syndrome (4,27,28) or obesity (5),which are conditions in which insulin resistance ispresumptively involved, LDL apo B plasma removal wasfound to be slower than in control subjects. However, theparticipants of those studies had elevated LDL cholesteroland triglyceride levels (4,27,28) whereas our GI patients didnot.

An important finding of this study was the fact that thefree cholesterol component of the LDE was removed morequickly in GI patients than in controls. This finding suggeststhat in GI patients, the unesterified cholesterol maydissociate from the surface of the lipoprotein particles andwas independently and more rapidly cleared from the

Figure 2 - Decay curves of the LDE 14C-cholesteryl ester (a) and 3H-free cholesterol (b) obtained from the glucose-intolerant (GI) and thecontrol group. Double-labeled LDE was intravenously injected in a bolus, and blood samples were drawn in pre-established intervalsover 24 h for measurement of the radioactivity in the scintillation solution. Data are presented as the % of the radioactivity countingconsidering the first point as 100% and are expressed as the means¡SD. Black squares: GI group; white squares: control group.

Table 3 - Fractional clearance rate (FCR) and kineticparameters of LDE 14C-cholesteryl ester and 3H-freecholesterol in the glucose-intolerant group (GI) and inthe control group.

GI

n = 14

Control

n = 15 p-value

FCR 14C-CE 0.05¡0.01 0.05¡0.01 0.40

k 1.014C-CE 0.26¡0.18 0.24¡0.23 0.48

k 1.214C-CE 0.33¡0.17 0.59¡0.61 0.19

k 2.014C-CE 0.04¡0.01 0.06¡0.07 0.96

FCR 3H-FC 0.06¡0.02 0.04¡0.02 0.04

k 1.03H-FC 0.54¡0.35 0.66¡0.24 0.34

k 1.23H-FC 0.48¡0.19 0.44¡0.21 0.62

k 2.03H-FC 0.05¡0.10 0.02¡0.01 0.01

Data are expressed as the means¡SD. Student’s t-test and unpaired

Mann-Whitney test. FCR: fractional clearance rate; CE: cholesteryl ester;

FC: free cholesterol; k1,0: kinetic parameters for the removal of lipids from

the intravascular compartment; k1,2: kinetic parameters that receive

apolipoproteins from the intravascular compartment; k2,0: kinetic

parameters for the removal of the LDL-like nanoemulsion attached to the

apolipoproteins from the intravascular compartment; SD: standard

deviation.

Table 4 - In vitro lipid transfers from the LDE to the HDLfraction in the glucose-intolerant group (GI) and in thecontrol group (measured as the % of the totalradioactivity of each LDE lipid incubated with wholeplasma and recovered after 1 h in the HDL fraction).

LDE lipid transfer to

HDL

Gl

n = 14

Control

n = 15 p-value

Cholesteryl esters 3.5¡0.4 3.6¡0.6 0.63

Phospholipids 21.6¡1.1 22.2¡1.2 0.19

Triglycerides 7.0¡2.1 6.1¡1.3 0.15

Free cholesterol 9.2¡1.1 9.9¡2.0 0.29

Data are expressed as the means¡SD. Student’s t-test and unpaired

Mann-Whitney test.

SD: standard deviation.


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circulation. However, the cholesteryl esters persisted in thecore of the LDE particles.

In a previous study (29), we identified a similar kineticbehavior for LDE unesterified and esterified cholesterol innormocholesterolemic patients with coronary heart disease.Upon injection of the LDE, free cholesterol was removedfaster in diseased patients than in controls (29). Theassociation of this behavior with coronary heart diseasesuggests that free cholesterol may desorb from LDEparticles and deposit in the arteries (12). In this respect,after the injection of the LDE into patients with coronaryheart disease that were scheduled for revascularizationsurgery, there was proportionally more free than esterifiedcholesterol in the fragments of the vessels discarded duringthe surgery (18). This result supports the hypothesis thatafter shifting from LDE particles, free cholesterol precipi-tates in the vessels and will eventually disturb endothelialhomeostasis, which is a primordial triggering mechanism inatherogenesis.

The lipid transfer process is dependent upon the HDLcomposition and therefore may affect the adhesion ofproteins to the lipoprotein surface. Thus, the function ofHDL in not only reverse cholesterol transport but also otheratheroprotective functions, such as the antioxidant, anti-inflammatory, anti-apoptotic, vasodilatory, antithromboticand anti-infectious activity, may be affected by this process(30).

Low HDL cholesterol is a marker of metabolic syndromeand may also be diminished in GI patients (31), althoughnormal levels have also been described (32-35). In our GIpatients, HDL was within the normal range and did notdiffer from the levels in the control subjects. Furthermore,we found no difference between GI patients and the controlswith respect to the in vitro transfer of the four LDE labeledlipids to HDL. In GI, as in type 2 diabetes, the action ofCETP was found to be enhanced (31,33,37). However, otherfactors such as the normal HDL cholesterol concentrationmay have counteracted the eventual increase in the transferof cholesteryl esters and triglycerides that would befacilitated by CETP (10,30).

Note that in the abovementioned studies, the levels ofHDL cholesterol were lower in the experimental groupsthan in the controls whereas, in the present study, thisparameter did not differ between groups. However, PLTP,which transfers phospholipids and also facilitates thetransfer of free cholesterol, was described as normal in GIpatients (36).

An interesting finding of our study was the positivecorrelation between the transfer of free cholesterol and theHDL levels. In contrast, the three other lipids that are moredependent on transfer proteins for transport did notcorrelate with the HDL levels. This finding highlights theimportance of HDL for the reception of free cholesterol forsubsequent esterification. Because of the importance of thislipoprotein as the main esterification site, a deficiency infree cholesterol reception when HDL levels are low may bea major mechanism that links HDL cholesterol levels andatherosclerosis (13,37,38).

In conclusion, GI subjects without dyslipidemia or clinicalmanifestations of cardiovascular disease had normal in vitrotransfer of lipids to HDL and removal of the LDLnanoemulsion probe from the plasma. The latter findingmay account for their normal LDL cholesterol levels.Nonetheless, the GI patients exhibited an abnormal kinetic

behavior of free cholesterol that may be associated withatherogenesis. This finding is important for the under-standing of the overall role of insulin resistance in thepathophysiology of cardiovascular disease.

ACKNOWLEDGMENTS

This study was supported by Fundacao do Amparo a Pesquisa do Estado

de Sao Paulo (FAPESP), Sao Paulo, Brazil. Dr. Maranhao has a Research

Award from Conselho Nacional de Desenvolvimento Cientıfico e

Tecnologico (CNPq) Brasılia, Brazil. Dr. Bertato had a scholarship from

CNPq.


Bertato MP was responsible for the performance of the experiments, data

analysis and manuscript writing. Oliveira CP was responsible for the

patients selection, performance of experiments and data analysis.

Wajchenberg BL was responsible for the clinical protocol and interpreta-

tion of results. Lerario AC conceived the study and was also responsible for

the clinical protocol, and manuscript writing. Maranhao RC conceived the

study, and was also responsible for the results interpretation and

manuscript writing.

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CLINICAL SCIENCE

The impact of pretransplant donor-specificantibodies on graft outcome in renal transplantation:a six-year follow-up studyElias David-Neto,I Patricia Soares Souza,I Nicolas Panajotopoulos,II Helcio Rodrigues,II Carlucci Gualberto

Ventura,I Daisa Silva Ribeiro David,I,III Francine Brambate Carvalhinho Lemos,I Fabiana Agena,I William

Carlos Nahas,I Jorge Elias Kalil,II Maria Cristina Ribeiro CastroII

I Hospital das Clınicas da Faculdade de Medicina da Universidade de Sao Paulo, Renal Transplantation Service, Sao Paulo/SP, Brazil. II Hospital das Clınicas

da Faculdade de Medicina da Universidade de Sao Paulo, Laboratory of Immunology, Sao Paulo/SP, Brazil. III Hospital das Clınicas da Faculdade de

Medicina da Universidade de Sao Paulo, Division of Pathology, Sao Paulo/SP, Brazil.

OBJECTIVE: The significance of pretransplant, donor-specific antibodies on long-term patient outcomes is a subjectof debate. This study evaluated the impact and the presence or absence of donor-specific antibodies after kidneytransplantation on short- and long-term graft outcomes.

METHODS: We analyzed the frequency and dynamics of pretransplant donor-specific antibodies following renaltransplantation from a randomized trial that was conducted from 2002 to 2004 and correlated these findings withpatient outcomes through 2009. Transplants were performed against a complement-dependent T- and B-negativecrossmatch. Pre- and posttransplant sera were available from 94 of the 118 patients (80%). Antibodies weredetected using a solid-phase (LuminexH), single-bead assay, and all tests were performed simultaneously.

RESULTS: Sixteen patients exhibited pretransplant donor-specific antibodies, but only 3 of these patients (19%)developed antibody-mediated rejection and 2 of them experienced early graft losses. Excluding these 2 losses, 6 of14 patients exhibited donor-specific antibodies at the final follow-up exam, whereas 8 of these patients (57%)exhibited complete clearance of the donor-specific antibodies. Five other patients developed ‘‘de novo’’posttransplant donor-specific antibodies. Death-censored graft survival was similar in patients with pretransplantdonor-specific and non-donor-specific antibodies after a mean follow-up period of 70 months.

CONCLUSION: Pretransplant donor-specific antibodies with a negative complement-dependent cytotoxicitycrossmatch are associated with a risk for the development of antibody-mediated rejection, although survival ratesare similar when patients transpose the first months after receiving the graft. Our data also suggest that earlyposttransplant donor-specific antibody monitoring should increase knowledge of antibody dynamics and theirimpact on long-term graft outcome.

KEYWORDS: Renal Transplantation; Donor-Specific Antibodies; Solid-Phase Assay; Luminex; DSA.

David-Neto E, Souza PS, Panajotopoulos N, Rodrigues H, Ventura CG, David DSR, et al. The impact of pretransplant donor-specific antibodies on graftoutcome in renal transplantation: a six-year follow-up study. Clinics. 2012;67(4):355-361.

Received for publication on November 7, 2011; First review completed on December 21, 2011; Accepted for publication on December 21, 2011


Tel.: 55 11 2661-8089

INTRODUCTION

The introduction of solid-phase assays for the detection ofanti-HLA antibodies (ABs) as well as C4d staining for theevaluation of allograft biopsies has revolutionized thecurrent era of assessing acute and chronic donor-specificantibody-mediated rejection in clinical practice.

Preformed, donor-specific HLA ABs (DSA) are respon-sible for some renal allograft rejections. The detection ofthese ABs prior to transplantation is an important step in theassessment of a patient’s immunological risk and theexclusion of incompatible donors.

However, these new methods used for antibody recogni-tion are performed in special in vitro conditions that maynot accurately reflect conditions in vivo. Pre-Tx HLA-DSAare not necessarily harmful to the transplanted kidney, andthese ABs may also preclude the implant of a transplantableorgan due to a new technological barrier.

The relevance of pre-TX DSA in patients with a negativecrossmatch remains controversial (1-5). These discrepantresults may be due to the dynamics of DSA generation.




355

Following transplantation, the blood levels of DSA mayincrease, decrease or be completely cleared from therecipient’s blood post-transplantation, and these changesmay therefore impact graft outcomes.

A prospective trial to assess the impact of DSA dynamicson allograft outcome is a high-risk and potentially unethicalstudy.

Therefore, the current study examined previously frozenand stored sera from a retrospective population of renalpatients who had received transplants with a negativecomplement-dependent cytotoxicity crossmatch (CDC-XM).As the currently used assays were unavailable at the time ofserum collection, the current study examined these retro-spective samples using modern solid-phase assays.

This cross-sectional study analyzed the frequency of pre-Tx DSA and the presence/absence of these ABs followingTx using single antigen beads to assess the short- and long-term outcomes of MoDIFY trial participants who receivedtransplants between 2002 and 2004.

METHODS

PatientsMale and female patients between the ages of 18 and 65

years who had received a non-identical twin kidneyallograft and who presented an ELISA panel-reactiveantibody (PRA) level ,50% during a prospective, rando-mized and controlled trial of tacrolimus (TCL) (PrografH,Astellas, Deerfield, IL, USA) minimization (the MoDIFYstudy - Modification of Doses to Improve Function throughthe Years) (6) were eligible for the current trial. Subjectswere excluded if they had received a nonrenal organ orinduction with antilymphocyte preparations.

Subjects who fulfilled the inclusion criteria were rando-mized (2:1) to receive TCL at either an initial dose of 0.15 -0.25 mg/kg/day or a cyclosporine microemulsion (NeoralH,Novartis Pharma, USA) at an initial dose of 10 mg/kg/day.Drug doses were subsequently adjusted according to thewhole blood levels. For patients in the TCL groups, targetpre-dose (C0) levels were 10-15 ng/mL during the firstmonth of treatment but progressively decreased to 3-5 ng/mL at 6 months. In the CyA group, the dose was adjustedaccording to the CyA concentration during the second hour(C2) following the oral dose, and the target concentrationswere between 1,400 and 1,700 ng/mL during the first monthand between 800 and 1,000 ng/mL after 6 months. Allpatients received 1.5 to 2 g/day mycophenolate mofetil(MMF) (CellceptH, Hoffman La Roche). All groups receivedan initial dose of 0.5 mg/kg/day prednisone, which wastapered to 0.1 mg/kg/day prior to the third month oftreatment. TCL, CyA and MMF were administered in two12 h interval oral doses, typically at 8 a.m. and 8 p.m. Allpatients received induction with an anti-IL2R antibody,either basiliximab (SimulectH, Novartis Pharma) or daclizu-mab (ZenapaxH, Hoffman La Roche). The InstitutionalEthics Committee approved this study, which was alsoconducted in full compliance with the Guidelines for GoodClinical Practices.

Renal biopsiesAvailable biopsies from patients with acute allograft

dysfunction were reviewed according to the Banff 2007classification (7). C4d staining was performed using IHC(immunohistochemistry) from paraffin-embedded biopsies.

Staining of more than 10% of the PTC (peritubularcapillaries) was considered positive, as this has beenshown to correlate with graft loss and the presence ofDSA (8,9).

HLA typingAll donors and recipients were typed for HLA-A, -B and

-DR using low-resolution polymerase chain reactionsingle-strand polymorphisms (PCR-SSPs) (One Lambda,Canoga Park, CA). HLA-DP, -DQ and -Cw specificitieswere not examined in this study because these antigenswere not routinely typed in our region at that time. Donorsand recipients were also not typed for these loci because itwas not possible to re-type all of the donors for theseantigens.

Pretransplant crossmatchAll patients exhibited negative pretransplant CDC-XM

with antihuman globulin CDC (CDC-AHG-XM) for T cellsand long-incubation CDC for B cells at the time oftransplantation. The presence of IgM antibodies wasexcluded by testing for CDC-XM in the presence ofdithiothreitol (DTT).

Pre- and post-Tx serum collectionTwo serum samples from each patient were analyzed, one

pre-Tx and the other either after the last follow-up exam orprior to death or graft loss. Serum was collected from allMoDIFY study participants prior to Tx and was storedfrozen (-70˚ C). The second serum samples (post-Tx) werecollected under the following conditions: 1 - posttransplantserum was collected from active patients who agreed toparticipate at the time of providing informed consent; 2 -patients who returned for dialysis were invited to partici-pate; and 3 - the hospital laboratories were searched for serasamples that had been collected and frozen from patientswho had died with a functioning graft.

Panel-reactive antibodies (PRAs)PRA levels (% PRA) were measured using flow cytometry

for the detection of anti-HLA class I and II antibodies, asrecommended by the manufacturers.

Donor-specific antibodies (DSAs)All patients were tested for the presence of DSA in pre-

and posttransplant sera using a solid-phase assay consistingof a single HLA antigen-coated microspheres (LuminexH -One Lambda, Canoga Park, CA). Briefly, 5 ml of class I andclass II beads were mixed with 20 ml of the serum sample,and the mixture was incubated in the dark for 30 minutes atroom temperature. After 3 washes, 100 ml of 1:100 FITC goatantihuman IgG was added, and the samples were incubatedat the same conditions for 30 minutes. The microbeads werewashed twice and resuspended in 80 ml of fixing solution.Class I and II antibodies were distinguished using a singletest, and the tests were read on the LABScreenTM100Luminex machine (One Lambda, Inc.).

The immunodominant DSA (iDSA), i.e., the antidonorHLA antibody with the highest median fluorescent intensity(MFI), was used for the statistical analysis when more thanone antibody was detected. The charts display each of therecipients’ DSA-MFIs in addition to the iMFIs.

Renal allograft outcome in renal transplantDavid-Neto E et al.

CLINICS 2012;67(4):355-361

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DATA ANALYSIS

Delayed graft function was defined as the need for dialysisduring the first week after transplantation. Any cases with thedetection of Cytomegalovirus disease (syndrome or invasive)were classified as CMV-positive. Rejection was defined as anytreated or biopsy-proven acute rejection episode. Additionalvariables that were used for the statistical analysis includedthe following: recipient and donor age, gender, race, numberof HLA-A, -B and -DR incompatibilities, deceased/live donor,type of IL-2R AB administered, doses and blood levels of ISdrugs (TCL, CyA and MPA) at 1, 3, 6 and 12 months and 2, 3, 4and 5 years after Tx, serum creatinine level, eGFR (estimatedglomerular filtration rate) according to the MDRD equationand the urinary protein/creatinine from the same time-pointsas when the immunosuppressive drugs levels were measured.

Data are reported as the means¡SD, and the mediansand 95% C.I. are reported for nonparametric data. Data wereanalyzed during the first 6 months to identify an early

impact of DSA, and the data were re-analyzed to verify thelong-term impact of DSA on long-term allograft outcomesfor grafts that had survived the first 6 months. Coxproportional analysis evaluated the hazards of each avail-able variable to predict death-censored graft survival. ThePASW statistics 18 (SPSS) software was used for thestatistical analyses.

RESULTS

Study populationTwenty-four of the 118 patients in the Modify study did

not participate in this analysis. Fifteen patients lost theirgrafts during the first year due to vascular reasons and/orrenal rupture (n = 5), death (n = 8) or acute rejection (n = 2).No available post-Tx serum samples were available forthese patients. A post-Tx serum sample was also notavailable for one patient who died after 7 years of follow-up. Eight patients with functioning grafts at the time of

Figure 1 - Pre- and posttransplant MFIs of each of the detected DSAs in 11 patients who remained active until the last follow-up visit(figure 1a) and in 5 patients who lost their allografts or died (figure 1b). The dotted line represents the standard cut-off MFI value forthis method.

CLINICS 2012;67(4):355-361 Renal allograft outcome in renal transplantDavid-Neto E et al.

357

enrollment refused to participate in the study. Therefore, 94patients (80%) from the MoDIFY study population wereanalyzed.

Time of pre- and post-Tx sera collectionPre-Tx sera were collected and frozen at a median of 61

(-134 to 30) days prior to Tx.Post-Tx sera were collected from all 75 active patients at

our outpatient clinic at a mean of 1834 ¡ 389 daysposttransplant. Nine patients died with a functioning graftat a mean of 1080¡810 days after Tx, and post-Tx serumsamples were collected at a mean of -613 ¡ 623 days priorto death.

Ten patients lost their grafts and returned to dialysis.Serum samples were collected from two of these patients at38 and 222 days prior to graft loss. The remaining 8 caseswere recruited from dialysis facilities for the collection ofsera at a median of 836 [303–1,114] days after graft loss.

Donor-Specific Antibodies (DSA)Sixteen of the 94 study patients (17%) were transplanted

with DSA (pre-Tx DSA), and 78 patients (83%) weretransplanted without DSA (no DSA).

Table 1 describes the demographics and transplantcharacteristics of the study population.

The pre-Tx DSA and no DSA groups were similar atbaseline, although more patients in the pre-Tx DSA groupexhibited a PRA class I and II level above 0% as well as ahigher class I and II PRA percentage. The numbers ofbiopsy-proven AR (BPAR) and AMR cases were signifi-cantly higher in the pre-Tx DSA-positive group.

Patients with pre-Tx antibodiesTable 2 lists the DSA specificities, MFIs and outcomes of

patients who received transplants with pre-Tx DSA. Twelvepts (75%) exhibited anti-HLA class I antibodies, threepatients (19%) exhibited anti-class II antibodies, and onepatient exhibited (6%) both class I and II ABs.

Ten of these 16 (62%) patients did not develop acuterejection episodes (AR), whereas six (38%) patients devel-oped ARs, including one patient with a late AR episode onPO day 797. The biopsies revealed that only three (3%)patients in the pre-Tx DSA group developed the full clinicalpicture of antibody-mediated rejection (AMR) with C4d-positive staining in peritubular capillaries. No cases ofhyperacute rejection were observed. The cases with ARswere classified as AMR (n = 3), Banff Ib (n = 1), Banff IIa(n = 1), or clinically diagnosed but not biopsied (n = 1). Thethree cases of AMR were treated with thymoglobulin, andtwo of these cases were also treated with intravenousimmunoglobulin between 2002 and 2004 due to thediagnosis of ‘‘acute transplant vasculopathy’’ that suggesteda ‘‘humoral’’ component of the AR. Two of the threepatients with AMR lost their allografts. AMR only occurredin patients with class I DSA, as none of the three patientswith only class II DSA developed AMR.

The mean iMFI-DSA did not differ between pre-Tx DSApatients who developed AMR (n = 3) and those patients whodid not develop AMR (n = 13) (6106¡6600 vs 5423¡2910,p = NS, respectively, p = NS). The same results wereobserved when comparing patients who developed AR(AMR or not, n = 6) to those who did not develop AR (n = 10)(6609¡4948 vs 4917¡2511, respectively, p = NS). A ROC

curve analysis did not identify a cut-off MFI for pre-Tx DSAthat could predict the development of AMR.

Two patients, of the 14 pts with pre-Tx DSA who survivedthe first six months after transplantation, died with afunctioning graft (DwFG) at 23 and 72 months posttrans-plantation, and one patient lost his/her graft due to chronictransplant nephropathy at 49 months posttransplantation.

Profile of pre- and posttransplant DSA frequenciesand outcomes

The post-Tx DSA evaluation from the last follow-up visit,excluding the two patients who lost their allografts due toearly AMR, revealed that six of the 14 patients demon-strated DSA, whereas eight patients had completely clearedthe DSA (Figure 1A and 1B). Seven patients had clearedclass I DSA and one patient had cleared class II DSA.Changes in DSA specificities were observed in three cases(patients # 1, 13, and 16 in Table 2).

Patients who had completely cleared the DSA were notsignificantly different from patients who had not cleared theDSA in regards to all baseline variables, including the pre-Tx class I PRA level (22¡29 vs. 8¡14%, respectively,p = NS) and class II PRA level (26¡37 vs. 21¡ 29%,respectively, p = NS). The pre-Tx immunodominant MFI-DSA (iMFI-DSA) was not different among patients who hadcleared the DSA, as compared to patients who had not(6371¡2773 vs. 3503¡2607, respectively, p = 0.07). The oraldose and blood level of the IS drugs were not different at 1,3, 6, and 12 months or 2, 3, 4, and 5 years post-Tx betweenthese two groups.

Overall graft survival for pre-Tx DSA-positive patientswas lower than for patients without pre-Tx DSA, althoughthis difference was not statistically significant (LogRank = 0.21) (Figure 2). Death-censored graft survival wassimilar between the two groups, and renal function was also

Table 1 - Demographics and transplant characteristics ofthe study population.

No pre-Tx

DSA

Pre-Tx

DSA p-value

n 78 (83%) 16 (17%)

Gender (F/M) 33/45 10/6 NS

Race (white/no white) 55/23 11/5 NS

Age (years) 42¡12 40¡12 NS

Tx (1st/pre-Tx) 74/4 13/3 0.093

Donor (D/L) 39/39 13/3 0.028

PRA cl-I (+) 11 (15%) 10 (62%) ,0.001

% PRA cl-I 2¡7 18¡23 0.000

PRA cl-II (+) 8 (10%) 7 (44%) 0.003

% PRA cl-II 3¡11 26¡35 0.000

TCL/CyA (n = ) 53/25 11/5 NS

HLA-A mm (0/1/2) 14/37/27 0/9/7 NS

HLA-B mm (0/1/2) 10/42/26 0/7/9 NS

HLA-DR mm (0/1/2) 23/39/14 6/8/2 NS

AR (clinical) 16 (20%) 6 (38%) NS

BPAR 8 (10%) 5 (31%) 0.046

AMR 0 (0%) 3 (19%) 0.004

DGF

(in deceased donor)

24 (61%) 8 (50%) NS

CMV 17 (22%) 4 (25%) NS

Follow-up (mo) 69¡16 70¡15 NS

AR: clinically diagnosed acute rejection episodes; BPAR: biopsy-proven

acute rejection episodes; AMR: antibody-mediated rejection; DGF:

delayed graft function; CMV: cytomegalovirus disease; TCL: tacrolimus;

CyA: Cyclosporin-A.


CLINICS 2012;67(4):355-361

358

similar between patients with and without pre-TxDSA (64¡15 vs 58¡22 ml/min, respectively, p = NS).Additionally, the urinary P/Cr levels did not differ betweennon-pre-Tx DSA patients and pre-Tx DSA-positive patients(0.49¡0.83 vs. 0.86¡1.1, respectively, p = NS).

A Cox univariate analysis revealed that the only variablewith a risk for death-censored graft loss was the presence ofa positive post-Tx DSA, including patients with ‘‘de novo’’post-Tx DSA and patients with early graft loss due to AMR(HR: 5.1, 1.4-18.1, p = 0.012). The presence of pre-Tx DSA forclass I and/or II was not associated with a risk of death-censored graft loss (p = 0.297).

DISCUSSION

This cross-sectional study analyzed the impact of pre-TxDSA on the long-term outcomes of patients in a low-riskrenal transplant population.

The novel aspect of our study was the analysis of post-TxDSA, which revealed the dynamics of pre-Tx DSAresponses after transplantation.

The first finding of our study was that the majority ofpatients with low levels of pre-Tx DSA did not developAMR. However, occurrences of AMR were most often

severe and frequently led to early graft loss. The incidenceof AMR in our study (19%) was lower than that in previousstudies, particularly in patients with strongly positive DSAand a historic positive cross-match (10). One explanation forthis result could have been the low levels of DSA in ourpopulation, which were evidenced by the negative pre-TxCDC-XMs of the included patients. The AMR rates varybetween studies; in one large retrospective study, theprevalence of AMR was only 2%, and all patients withAMR had at least one strong donor-specific DSA with anMFI value greater than 6,000 (11).

However, the disparity in AMR rates between studies maybe due to the absence of protocol biopsies for the diagnosis ofsubclinical AMR. A cohort study of 54 DSA-positive kidneytransplant recipients demonstrated that 31% of patients metthe criteria for subclinical antibody-mediated rejection(SAMR) at the time of a three-month protocol biopsy (12).We used the BANFF-2007 classification, which requirespositive C4d staining for an AMR diagnosis. We alsoperformed C4d staining retrospectively using IHC. As IF ismore sensitive than IHC (13,14), we may have under-diagnosed cases of AMR due to the false-negative C4dstaining using IHC and the absence of protocol biopsies.

Table 2 - HLA-DSA and outcomes.

Pre-Tx Post-Tx

Patient

# cl - I MFI-Cl -I cl -II

MFI

cl -II

AR(Banff)

PO day

Day of serum

harvest

prior to death and

after loss cl-I MIF Cl-I cl -II MIF cl -II

Outcome

/cause/

Fup (months)

1 A33 3169 DR1 2913 AMR I

13PO

+1151 A33 10047 DR1 DR7 7727

4090

GL/AMR

2

2 B7 13666 -- -- AMR II

28PO

+1710 B7 1472 -- -- GL/AMR

4

3 A30 10122 -- -- AR clinical

13 PO

+600 -- -- -- -- GL/CAN

49

4 A31 800 -- -- No AR -324 A31 612 -- -- DwFG/

Suicide

23

5 B44 7322 -- -- No AR -134 B44 6584 -- -- DwFG/

Infection

72

6 A11 4583 -- -- No AR -- -- -- -- -- Active

62

7 B14 7173 -- -- No AR -- -- -- -- -- Active

62

8 A68 5215 -- -- No AR -- -- -- -- -- Active

62

9 A31 1050 -- -- No AR -- A31 871 -- -- Active

72

10 A23 8312 -- -- No AR -- -- -- -- -- Active

73

11 B51

A26

1485

1047

-- -- AMR I

27PO

-- -- -- -- -- Active

73

12 -- -- DR103 5297 No AR -- -- -- -- -- Active

76

13 -- -- DR16

DR1

1048

5721

No AR -- A31 1215 -- -- Active

77

14 A2

A29

8782

3148

-- -- Ib

797 PO

-- -- -- -- -- Active

79

15 A11 2429 -- -- IIa

6PO

-- A11 2325 -- -- Active

81

16 -- -- DR4 3703 No AR -- A68 886 DR53 14377 Active

84

class I and class II: Donor-specific anti-HLA antibodies anti-class I and anti-class II; MFI: median fluorescent intensity; AMR: antibody-mediated rejection;

GL: graft loss; DwFG: death with a functioning graft; CAN: chronic allograft nephropathy; Fup: follow-up.


359

However, our results also suggest that patients with lowlevels of pre-Tx DSA rarely develop AMR, which suggeststhe possibility of an association between a low concentrationof pre-Tx DSA and a negative CDC-XM. This rationale isconsistent with previous data demonstrating an associationbetween assay results and, graft loss (15). A retrospectiveanalysis of transplanted patients who had a negative CDC-XM but positive results from a solid-phase assay or FCXMrevealed that most patients neither developed AMRepisodes nor exhibited impaired graft survival (5).However, two of the three patients who developed AMRlost their grafts. Unfortunately, it was not possible to retestthe pre-Tx sera against the donor lymphocytes to performFCXM in our population.

Whether pre-Tx DSA-MFI levels can predict the develop-ment of AMR is an additional topic of debate. Theprevalence of AMR increases with increasing pre-Tx DSA-MFIs, and a peak serum MFI between 465 and 1500 in thepeak serum is associated with a 25-fold increase in therelative risk for the development of AMR (10). However, wecould not detect a pre-Tx MFI cutoff value that could predictthe occurrence of AMR. The reasons for these discrepantresults remain unclear, although the examination of onlypre-Tx serum, rather than serum from the peak of theresponse, may provide one explanation.

Another important finding of our study was related to thediscordance between previous studies as to the impact ofpre-Tx DSA levels (not detected by CDC-XM) on transplantoutcome. Some authors have observed worse outcomes,whereas others have not (5,10,16). A retrospective study wasperformed on 113 kidney transplant recipients with nega-tive prospective T- and B-cell CDC-XM at the time oftransplant, and these patients were screened for thepresence of circulating anti-HLA antibodies and DSA usingthe Luminex assay. One-year allograft survival rates weresimilar between the Luminex pre-Tx DSA-positive andDSA-negative groups (17). Patients with DSA who did notexperience AMR exhibited the same graft survival rates aspatients without DSA (10), and the results of the currentstudy are in line with these previous findings.

The major finding of the current study was that differencesin long-term graft survival among individuals with pre-TxDSA may be due to the dynamics of the DSA response. This is

the first study to analyze pre- and post-Tx DSA data for thesame patient population and correlate these dynamics withlong-term patient outcome. A large effort was made to collectblood from patients who returned to dialysis and to searchfor frozen serum samples collected from patients prior todeath. We acknowledge that, in many cases, the post-Txserum samples were collected in a cross-sectional analysismany years following transplantation and that this factormay explain the long-term profile of low-level pre-Tx DSA.

Patients who either completely cleared the DSA post-transplant or who demonstrated decreased levels of DSAexhibited similar long-term outcomes as compared to DSA-negative patients. Therefore, the simple presence of pre-TxDSA does not impact transplant outcome, and it seems thatit is critical for outcome that the DSA level remains above acertain cutoff value post-transplantation.

Patients with DSA who survived the first six months post-transplant presented a similar long-term outcome aspatients without DSA. These results are consistent withthose of previous studies, although these studies failed toidentify the reasons behind the observed improved out-come. It is possible that a mean 6-year follow-up period maynot be sufficient for drawing definitive conclusions (5,10).

In summary, this study revealed that the presence of pre-Tx DSA was initially associated with the occurrence ofAMR, and this condition may lead to graft loss in low-riskrenal patient populations transplanted with a negative CDCcrossmatch. Our data also suggest that early posttransplantDSA monitoring can improve the understanding of DSAdynamics. Moreover, pre-Tx DSAs are likely only proble-matic when these concentrations remain positive aftertransplantation and above a certain threshold. However, alarger post-Tx analysis of pre-Tx DSA-positive patientswould be necessary to prove this hypothesis.


David-Neto E was responsible for the manuscript writing and the

intellectual and scientific content of the study. Agena F, Souza PS,

Ventura CG and Rodrigues H were responsible for the generation,

collection and assembly of the data. Panajotopoulos N, Lemos FC, Castro

Table 3 - Causes of graft loss and death with afunctioning graft prior to 6 months and at the finalfollow-up visit post-Tx.

Non-DSA group DSA group p-value

Pts at risk n = 78 16

Early Losses (#6

mo)

3 (3.8%) 2 (12.5%) 0.056

due to ARE 1 (1.3%) 2 (12.5%) 0.056

due to DwFG 2 (2.5%) -- (0%)

Pts at risk n = 75 14

Late Losses (. 6

mo)

11 (14.6%) 3 (21.4%) 0.74

CAN 5 (6.6%) 1 (7.1%) NS

DwFG 5 (6.6%) 2 (14.3%) NS

Other 1 (1.4%) -- (0%) NS

DSA: donor-specific antibody; PTS: patients; ARE: acute rejection episodes;

DwFG: death with a functioning graft; CAN: chronic allograft

nephropathy.

Figure 2 - Death-censored graft survival curves for the pre-Tx DSAgroup as compared to the non-DSA group.


CLINICS 2012;67(4):355-361

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MCR, Nahas WC and Kalil JE critically revised the intellectual content of

the manuscript. David DSR performed the revision of renal biopsies.

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2. Gupta A, Iveson V, Varagunam M, Bodger S, Sinnott P, ThuraisinghamRC. Pretransplant donor-specific antibodies in cytotoxic negative cross-match kidney transplants: are they relevant? Transplantation.2008;85(8):1200-4, http://dx.doi.org/10.1097/TP.0b013e31816b1c37.

3. Lefaucheur C, Loupy A, Hill GS, Andrade J, Nochy D, Antoine C, et al.Preexisting donor-specific HLA antibodies predict outcome in kidneytransplantation. J Am Soc Nephrol 2010;21(8):1398-406.

4. Patel AM, Pancoska C, Mulgaonkar S, Weng FL. Renal transplantation inpatients with pretransplant donor-specific antibodies and negative flowcytometry crossmatches. Am J Transplant. 2007;7(10):2371-7, http://dx.doi.org/10.1111/j.1600-6143.2007.01944.x.

5. Vlad G, Ho EK, Vasilescu ER, Colovai AI, Stokes MB, Markowitz GS,et al. Relevance of different antibody detection methods for theprediction of antibody-mediated rejection and deceased-donor kidneyallograft survival. Hum Immunol. 2009;70(8):589-94, http://dx.doi.org/10.1016/j.humimm.2009.04.018.

6. Pereira L, Castro M, Ventura C, Reis F, Sumita N, Sato MI, et al. TheMODIFY Study in renal transplantation (Modification of doses to improvefunction through the years). Am J Transplant. 2005;11(Suppl 5):466.

7. Solez K, Colvin RB, Racusen LC, Haas M, Sis B, Mengel M, et al. Banff 07classification of renal allograft pathology: updates and future directions.Am J Transplant. 2008;8(4):753-60, http://dx.doi.org/10.1111/j.1600-6143.2008.02159.x.

8. David-Neto E, Prado E, Beutel A, Ventura CG, Siqueira SA, Hung J, et al.C4d-positive chronic rejection: a frequent entity with a poor outcome.Transplantation. 2007;84(11):1391-8, http://dx.doi.org/10.1097/01.tp.0000288807.52520.5e.

9. Kedainis RL, Koch MJ, Brennan DC, Liapis H. Focal C4d+ in renalallografts is associated with the presence of donor-specific antibodiesand decreased allograft survival. Am J Transplant. 2009;9(4):812-9,http://dx.doi.org/10.1111/j.1600-6143.2009.02555.x.

10. Lefaucheur C, Suberbielle-Boissel C, Hill GS, Nochy D, Andrade J,Antoine C, et al. Clinical relevance of preformed HLA donor-specificantibodies in kidney transplantation. Contrib Nephrol. 2009;162:1-12,http://dx.doi.org/10.1159/000170788.

11. Rafiq MA, de Boccardo G, Schroppel B, Bromberg JS, Sehgal V, DinavahiR, et al. Differential outcomes in 3 types of acute antibody-mediatedrejection. Clin Transplant. 2009;23(6):951-7, http://dx.doi.org/10.1111/j.1399-0012.2009.01036.x.

12. Loupy A, Suberbielle-Boissel C, Hill GS, Lefaucheur C, Anglicheau D,Zuber J, et al. Outcome of subclinical antibody-mediated rejection inkidney transplant recipients with preformed donor-specific antibodies.Am J Transplant. 2009;9(11):2561-70, http://dx.doi.org/10.1111/j.1600-6143.2009.02813.x.

13. Seemayer CA, Gaspert A, Nickeleit V, Mihatsch MJ. C4d staining of renalallograft biopsies: a comparative analysis of different staining techni-ques. Nephrol Dial Transplant. 2007;22(2):568-76.

14. Nadasdy GM, Bott C, Cowden D, Pelletier R, Ferguson R, Nadasdy T.Comparative study for the detection of peritubular capillary C4d depositionin human renal allografts using different methodologies. Hum Pathol.2005;36(11):1178-95, http://dx.doi.org/10.1016/j.humpath.2005.08.002.

15. Ho EK, Vasilescu ER, Colovai AI, Stokes MB, Hallar M, Markowitz GS,et al. Sensitivity, specificity and clinical relevance of different cross-matching assays in deceased-donor renal transplantation. TransplImmunol. 2008;20(1-2):61-7, http://dx.doi.org/10.1016/j.trim.2008.09.013.

16. Lachmann N, Terasaki PI, Budde K, Liefeldt L, Kahl A, Reinke P, et al.Anti-human leukocyte antigen and donor-specific antibodies detected byLuminex posttransplant serve as biomarkers for chronic rejection of renalallografts. Transplantation. 2009;87(10):1505-13, http://dx.doi.org/10.1097/TP.0b013e3181a44206.

17. Aubert V, Venetz JP, Pantaleo G, Pascual M. Low levels of humanleukocyte antigen donor-specific antibodies detected by solid phase assaybefore transplantation are frequently clinically irrelevant. Hum Immunol.2009;70(8):580-3, http://dx.doi.org/10.1016/j.humimm.2009.04.011.


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CLINICAL SCIENCE

A proton nuclear magnetic resonance-basedmetabonomics study of metabolic profiling inimmunoglobulin a nephropathyWeiguo Sui,I Liping Li,I,II Wenti Che,I Guimai Zuo,I Jiejing Chen,I Wuxian Li,III Yong DaiI,IV

I 181st Hospital Guangxi, Central Laboratory, Laboratory of Metabolic Diseases Research, Guangxi Province, China. II Guangxi Normal University, the Life

Science College, Guangxi Province, China. III Key Laboratory of Laboratory Medical Diagnostics of Education Ministry, Chongqiong Medical University,

Chongqing, China. IV Clinical Medical Research Center, the Second Clinical Medical College of Jinan University (Shenzhen People’s Hospital), Shenzhen,

Guangdong Province, China.

OBJECTIVES: Immunoglobulin A nephropathy is the most common cause of chronic renal failure among primaryglomerulonephritis patients. The ability to diagnose immunoglobulin A nephropathy remains poor. However, renalbiopsy is an inconvenient, invasive, and painful examination, and no reliable biomarkers have been developed foruse in routine patient evaluations. The aims of the present study were to identify immunoglobulin A nephropathypatients, to identify useful biomarkers of immunoglobulin A nephropathy and to establish a humanimmunoglobulin A nephropathy metabolic profile.

METHODS: Serum samples were collected from immunoglobulin A nephropathy patients who were not usingimmunosuppressants. A pilot study was undertaken to determine disease-specific metabolite biomarker profiles inthree groups: healthy controls (N = 23), low-risk patients in whom immunoglobulin A nephropathy was confirmed asgrades I-II by renal biopsy (N = 23), and high-risk patients with nephropathies of grades IV-V (N = 12). Serum sampleswere analyzed using proton nuclear magnetic resonance spectroscopy and by applying multivariate patternrecognition analysis for disease classification.

RESULTS: Compared with the healthy controls, both the low-risk and high-risk patients had higher levels ofphenylalanine, myo-Inositol, lactate, L6 lipids ( = CH-CH2-CH = O), L5 lipids (-CH2-C = O), and L3 lipids (-CH2-CH2-C = O)as well as lower levels of b-glucose, a-glucose, valine, tyrosine, phosphocholine, lysine, isoleucine, glycerolpho-sphocholine, glycine, glutamine, glutamate, alanine, acetate, 3-hydroxybutyrate, and 1-methylhistidine.

CONCLUSIONS: These metabolites investigated in this study may serve as potential biomarkers of immunoglobulin Anephropathy. Point scoring of pattern recognition analysis was able to distinguish immunoglobulin A nephropathypatients from healthy controls. However, there were no obvious differences between the low-risk and high-riskgroups in our research. These results offer new, sensitive and specific, noninvasive approaches that may be of greatbenefit to immunoglobulin A nephropathy patients by enabling earlier diagnosis.

KEYWORDS: Immunoglobulin A Nephropathy; Metabonomics; Biomarkers; Proton Nuclear Magnetic ResonanceSpectroscopy; Orthogonal Partial Least-Squares Discriminant Analysis.

Sui W, Li L, Che W, Zuo G, Chen J, Li W, Dai Y. A proton nuclear magnetic resonance-based metabonomics study of metabolic profiling inimmunoglobulin a nephropathy. Clinics. 2012;67(4):363-373.

Received for publication on November 28, 2011; First review completed on November 28, 2011; Accepted for publication on December 26, 2011


Tel.: 86 0755-25626750

INTRODUCTION

Immunoglobulin A nephropathy (IgAN) is the mostcommon primary glomerulopathy worldwide; it is amesangial proliferative glomerulonephritis characterizedby diffuse mesangial deposition of immunoglobulin A (1).

The underlying mechanism of IgAN is poorly understood,and the outcomes of patients vary greatly. Variations in themethods used to treat IgAN patients account for less thanhalf of the variability in outcomes (2). Determining anaccurate diagnosis poses great challenges. Multiple observa-tional cohort studies have identified several risk factorsassociated with the progression of IgAN (3,4). Five clinicalfeatures, namely high proteinuria, hypertension, impairedrenal function, hypoproteinemia, and hyperuricemia, areindependent predictors of an unfavorable renal outcome (5).However, the threshold above which the risk develops is stilldebated. IgAN presents with variable clinical symptoms andpathological patterns, making it difficult to predict the risk of




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progression to end-stage renal failure in individuals. Despitethe fact that the progression of IgAN is generally slow, up to15-40% of patients with IgAN develop end-stage renaldisease within 20 years (6). As a "gold standard," renalbiopsy is currently the primary diagnostic tool for renaldisease, as well as for treatment and prognosis. Therefore,renal biopsy has been quite helpful in the understanding ofthe different types of human renal diseases and has alsoprovided insight into their pathogenesis (7). Nevertheless,renal biopsy requires an inconvenient, invasive, and painfulexamination that is lacking in sensitivity, specificity andreproducibility and that suffers from inappropriate categor-ization and can even result in some complications (4,8).Additionally, general physicians find it difficult to performbiopsies. Thus, although both the clinical and histologicfeatures of IgAN are highly variable (9), a novel andconvenient strategy for predicting diagnosis from a biopsy-based pathological evaluation has not been well established(4). Neither a biomarker nor a biomarker profile is generallyaccepted in clinical practice. Therefore, identifying definite,as well as sensitive, biomarkers for early diagnosis would beof great significance for making diagnostic and treatmentdecisions.

Metabonomics is a well-established field in systemsbiology, which refers to the study of individual metabolicprofiles and to their changes over time due to disease, toxicityand nutritional, environmental, and genetic alterations (10-12). In a process that is known as ‘‘metabolic fingerprinting,’’global metabolite profiles of complex samples are used toidentify potential biomarkers, and this process can providenew and unexpected insights into biological processes (13).Nuclear magnetic resonance (NMR) spectroscopy appears tobe a cost-effective, useful technique for providing routinecare and screening (14) and is well suited for the analysis ofdifferent types of biological fluids for the evaluation ofindividual metabolomes (15). Thus, proton-NMR (H-NMR)spectroscopic analysis allows for the simultaneous detection,identification, and quantification of hundreds of low-mole-cular-weight metabolites (max. 20 kDa) within a biologicalmatrix (16) for the detection of dynamic changes in globalmetabolism and, specifically, of noninvasive blood markers,which represents a novel and robust method for assessingorgan response to pathophysiologic stimuli (17-18). Due to itsusefulness in evaluating systemic responses to any subtlemetabolic perturbation, 1H NMR-based metabonomics hasbeen extensively applied for the diagnosis and evaluation ofcancer (19-21), diabetes (22), neurological diseases (23), heartdisease (17), and cerebral infarction (24). To date, no NMR-based metabolomic studies of IgAN have been reported.

In the present work, we investigated the perturbedmetabolic pattern in serum derived from IgAN patientsand identified potential biomarkers of this disease usinghigh-resolution NMR spectroscopy coupled with multi-variate statistical analysis. The integration of metabolic datacould provide a systematic approach for the study ofmetabolic profiles associated with IgAN and could facilitatea detailed examination of the underlying molecularmechanisms of the disease. We sought to determinewhether we could identify the metabolic phenotypes andcharacterize the metabolic changes related to the degree ofIgAN, whether we could distinguish valuable biomarkersfor disease diagnosis, and most importantly, whetherunderstanding the pathogenetic process of IgAN on asystemic, metabolic level could hold the key to designing

therapies to arrest the development of IgAN and prevent itslater manifestations. The results highlight the possibility for"metabolic fingerprinting" to become a new diagnosticmethod with improved sensitivity and specificity.


SubjectsBefore starting this study, the patients and controls were

informed of the study protocol and signed written consentforms, which were approved by the Regional EthicsCommittee of the 181st Hospital Guilin, Guangxi.

For this study, 35 IgAN patients and 23 age- and sex-matched healthy controls were recruited from theNephrology Department of the 181st Hospital Guilin,Guangxi, China, from August 2010 to December 2010. Allpatients with biopsy-proven primary IgAN were reviewed.The subjects were classified into three groups: a controlgroup consisting of 23 healthy subjects, a low-risk groupconsisting of twenty-three IgAN patients with diseases ofgrades I-III based on renal biopsies stained for IgAN-A, and ahigh-risk group composed of twelve IgAN patients withdiseases of grades IV-V based on biopsies stained for IgAN-B.The inclusion criteria for IgAN were (1) patient age rangingfrom 18 to 60 years and evidence of predominant mesangialIgA deposits from a renal biopsy, and (2) absence ofcorticosteroid or immunosuppressive therapy in the previous10 weeks in both the patients and controls. The exclusioncriteria were (1) presence of a secondary cause of IgAN, suchas systemic lupus erythematous, Schonlein-Henoch purpura,chronic liver disease, malignancies, active peptic-ulcer dis-ease and other autoimmune disorders; (2) diagnosis ofdiabetes mellitus, acute interstitial nephritis, Alport’s syn-drome, acute myocardial infarction or stroke; (3) evidence orsuspicion of severe uncontrolled hypertension (systolic bloodpressure $220 mmHg and/or diastolic blood pressure$120 mmHg); and (4) history of alcoholism, smoking or oralcontraception within the previous six months.

Approximately 4 mL of peripheral venous blood wascollected in separate biochemistry tubes from the IgAN patientsand healthy volunteers in the morning following a 12-h fastingperiod. The blood was allowed to clot for 60 min at roomtemperature and was centrifuged at 20006g for 10 min. Thesupernatants were then divided into 0.5-mL polyethylene tubesand were stored at –80 C until the NMR analysis wasperformed.

Clinical chemistry and histopathologyClinical chemistry analyses of serum samples were

conducted, using an automatic chemistry analyzer (MOL-300, Heal Force, China) with enzymatic methods usingcommercially available kits, for the measurement of severalserum parameters, including proteinuria, alanine amino-transferase, aspartate aminotransferase, total protein, albu-min, globulin, glucose, urea nitrogen, creatinine, uric acid,cholesterol, triglyceride, HDL cholesterol, and LDL choles-terol. The values were expressed as the means ¡ SDs.Statistical comparisons were performed using SPSS 11.5software (SPSS Inc., Chicago, IL, USA), and differences wereconsidered significant when p,0.05.

Renal tissue was obtained from all patients by percuta-neous renal biopsy. Histopathological changes in kidneytissue were assessed in at least 20 randomly selected tissuesections from each group under study. Sections of 2-3 mm in

Metabonomics profiling in immunoglobulin A nephropathySui W et al.

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thickness were paraffin-embedded and stained with Mayer’shematoxylin and eosin for direct immunofluorescenceobservations. The pathological diagnosis of IgAN was madeaccording to the previous World Health Organizationdiagnostic system and its reference standard of clinical typebased on Lee’s classification scheme (45).

Proton nuclear magnetic resonance spectroscopyFor the NMR experiments, the serum samples were

thawed at room temperature and centrifuged at 30006g for5 minutes at 4 C. The supernatants were then adjusted topH 7.4 with sodium phosphate buffer solutions and mixedwith 100 mL of deuterium oxide solution to calibrate theNMR spectrometer. Finally, an aliquot of the preparedmixture (500 mL) was transferred to a 5-mm NMR tube(Wilmad, Buena, NJ, USA).

The 1H NMR spectra were acquired using a Varian UnityINOVA-600 MHz NMR spectrometer (Varian Inc., PaloAlto, CA, USA) operating at a 599.93-MHz frequency anda temperature of 300 K. The spectra were acquired usinga conventional presaturation pulse sequence with aNOESYPR 1-D solvent suppressor (RD-90 -t1-90 -tm-90 -ACQ). Standard two-dimensional (2-D) total correlationspectroscopy (TOCSY) NMR spectra were also acquired forspectral assignment purposes in the selected samples. 1H-1HTOCSY experiments were performed using the tntocsypulse sequence. For each sample, the free-induction decayswere collected with 128 transients into 32K data pointsusing a spectral width of 8000 Hz with a relaxation delay of2.1 s, an acquisition time of 1 s and a mixing time of 100 ms.All of the spectra were multiplied by an exponentialweighting function corresponding to a line-broadening of1.0 Hz prior to Fourier transformation (25).

Data reduction and multivariate patternrecognition analysis

All of the NMR spectra were phased, baseline-corrected,and data-reduced to 225 integrated regions of d0.002 inwidth, corresponding to the region of d0.5 to d9.0, usingTopSpin software (Varian 3.0, Bruker Biospin, Germany).The 1H-NMR spectra of the serum samples were referencedto the internal lactic acid CH3 resonance at 1.33 ppm. Then,the region of water resonance (d4.37-6.88) was excluded toeliminate the baseline effects of an imperfect water signal.The remaining bins were integrated and normalized for usein further analysis. The normalized data were imported intothe SIMCA-P software package, version 11.0 (Umetrics AB,Umea, Sweden).

Unsupervised principal components analysis (PCA),supervised projection to partial least squares-discriminantanalysis (PLS-DA), and orthogonal partial least squares-discriminant analysis (OPLS-DA) were performed for classdiscrimination and biomarker identification. First, PCA,based on a mean center-scaling model, was performed toexamine the intrinsic variation in the dataset. The data werevisualized with the score plots of the first two principalcomponents (PC1 and PC2) to provide the most efficienttwo-dimensional representation of the information (21). Asan extension of PLS-DA and featuring integrated orthogonalsignal correction (OSC), OPLS-DA can remove variabilitynot relevant to class separation. OPLS-DA was used tomaximize the covariance between the measured data (Xvariable, peak intensities in NMR spectra) and the responsevariable (Y variable, predictive classifications) (21) and to

simultaneously remove uncorrelated variations between theX and Y variables or X variables that were orthogonal to Yvariables (26). A scores plot combining the reliability andcorrelation from the OPLS-DA model helped to identifydifferential metabolites among the groups (27). The relevantmetabolites for each separation model and their differencesamong the groups are shown as coefficient of variationplots. With a significance level of 0.05, a correlationcoefficient (Corr(t,X)) of ¡0.497 was adopted as a cutoffvalue to select the variables that were most correlated withthe OPLS-DA discriminant scores. The correlation coeffi-cients were calculated using Pearson linear correlationcoefficients incorporated into MATLAB R2007a software(MathWorks, Inc., Natick, MA, USA).

Both the PLS-DA and OPLS-DA models were based on aunit variance scaling strategy. A 10-fold cross-validationwas employed to obtain Q2 and R2 values. R2 is defined asthe proportion of variance in the data explained by themodels and indicates goodness of fit, while Q2 is defined asthe proportion of variance in the data predictable by themodel and indicates predictability (24). To further validatethe quality of the PLS-DA model and to investigate thepredictability of the OPLS-DA model, permutation tests,consisting of random permutation class membership andthe performance of 200 iterations, were conducted (28).These tests compared the goodness of fit of the originalmodel with the goodness of fit of several models based ondata in which the order of the Y observations wererandomly permuted while the X matrix was kept intact(29). p-values were generated by an unpaired t-test, and a p-value of ,0.05 was considered statistically significant.

RESULTS

Clinical chemistry analysis and histopathologyThe percentages of IgAN patients with microscopic

hematuria, hypertension, hyperlipidemia, and chronic renalfailure were 97.01, 27.74, 13.26, and 20.32%, respectively. Atotal of 80.44% of long-term hypertension patients (bloodpressure .140/90 mmHg) were required to undergo anti-hypertensive therapy to maintain normotension before under-going renal biopsy. The demographic features and body fluidparameters determined by biochemical analyses of all of thepatients and healthy volunteers are shown in Table 1. Nosignificant differences in age, sex distribution, BMI, or systolicand diastolic blood pressures were found among the threegroups. The serum levels of total protein, albumin andglobulin were lower in the IgAN patients than in the healthycontrols, whereas the urea nitrogen, creatinine, and uric acidlevels were higher in IgAN patients than in the healthycontrols. Furthermore, proteinuria was significantly higher inthe IgAN-B patients compared with the IgAN-A patients.

In the renal biopsy, both the IgAN-A and IgAN-B groupswere similar with regard to mesangial proliferation, cellularcrescents, global and segmental glomerulosclerosis, inter-stitial fibrosis and tubular atrophy, with some IgAN-Bpatients displaying a predominance of active/proliferativelesions and chronic/sclerosing lesions. As shown in Table 2,the most common IgAN pathological category in 35 caseswas the mesangial proliferative type (35 cases; 57.14%), andthe second most common was focal segmental glomerulo-sclerosis (FSGS) (12 cases; 34.29%). Membranous glomer-ulonephritis (MGN) (2 cases; 5.71%) and sclerosisglomerulonephritis (SGN) (1 case; 2.86%) were the next

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most common disease types. The classes of the IgANpathological grades were predominantly II, III, and IV,while grades I and V were rare. This result may haveoccurred because grade I corresponded to the earliest stageof kidney disease and was hidden in the clinic due tountimely treatment or insufficient medical attention, andgrade V was the most severe stage of renal disease thatalways progressed to renal failure, often requiring dialysistreatment in the clinic. Therefore, these patients rarelyunderwent biopsy.

Qualitative proton nuclear magnetic resonanceanalysis of serum samples

Figure 1 depicts the typical 1H CPMG spectra of serumsamples obtained from healthy individuals and from IgAN-A and IgAN-B patients. The 1H-NMR signals of all commonmetabolites, including amino acids, organic acids, andcarbohydrates, were assigned according to previous pub-lications (30). The spectral profiles were generally similar,making accurate visual comparison difficult, but somerelative intensity differences could be observed among thethree groups. As shown in Figure 1, predominantly

endogenous compounds were identified from the spectracomprising numerous lipoprotein fractions, some of whichwere derived from the glycerol of lipids, glycerolpho-sphocholine, low-density lipoprotein (L), and very low-density lipoprotein (VLDL). The sharp peaks arose fromsugar signals, including n-acetyl glycoprotein, a-glucose,and b-glucose. There were also a large number of aminoacid signals, such as methylhistidine, alanine, glutamine,glutamate, glycine, isoleucine, leucine, lysine, phenylala-nine, tyrosine, and valine, as well as signals from acetate,citrate, creatine, lactate, phosphocholine, pyruvate, acetone,creatinine, myo-Inositol, and methylamine.

Metabonomic analysis of human serum in controlsand IgAN patients

Unsupervised PCA was applied to examine the intrinsicvariation in the serum data sets. Figure 2 shows the two-dimensional PCA scores plots based on the 1H CPMG NMRspectra of the serum obtained from different groups. Moresamples were present in the 95% confidence interval of themodeled variation. We found that the control samples werevery homogeneous and that their distributions of PCA scoreplots were relatively close. The IgAN patients and healthycontrols, although scattered, were located in differentclusters, demonstrating a relatively clear distinction, whilethe score plots showed slight separations between theIgAN-A and IgAN-B groups and the serum (R2X = 79.5%;Q2Y = 0.760) models.

PLS-DA, which is more focused on detecting variationthan the PCA approach, was further performed to investi-gate subtle metabolic differences among the groups. Theparameters used to assess modeling quality, including R2X,R2Y, and Q2Y, are displayed under each picture in Figure 3.The model parameters for the explained variation, R2, andthe predictive capability, Q2, were significantly higher(R2, Q2.0.5) in the serum, indicating excellent models.

Table 1 - Demographic and clinical patient characteristics.

IgAN-A IgAN-B Controls

Number of samples 23 12 23

Age (years)a 31.46¡9.31 36.00¡7.34 35.61¡11.35

Age range (years) 21-47 22-47 21-51

Sex (F/M) 1 10/13 6/6 12/11

BMI (kg/m2) a 22.35¡2.22 24.60¡2.41 23.40¡3.26

DBP (mmHg) a 76.76¡9.05 79.42¡9.32 74.4¡4.81

SBP (mmHg) a 125.82¡12.94 126.88¡16.28 125.30¡11.31

Proteinuria (g/day) b 0.95¡0.39 2.13¡1.44 0.04¡0.04

Alanine aminotransferase (U/L) a 21.28¡19.32 22.71¡21.59 17.48¡11.96

Aspartate aminotransferase (U/L) a 23.63¡12.44 23.84¡11.39 18.13¡11.30

Total protein (g/L) c 61.36¡12.26 59.63¡8.84 70.83¡7.82

Albumin (g/L) b 37.06¡8.00 32.94¡11.65 44.43¡6.34

Globulin (g/L) c 24.29¡7.29 23.28¡8.53 25.73¡4.19

Fasting glucose (mmol/L) a 4.82¡0.70 5.04¡0.51 4.92¡0.38

Urea nitrogen (mmol/L) b 6.04¡5.36 7.29¡6.30 4.56¡1.13

Creatinine (mmol/L) b 109.71¡49.27 123.73¡47.09 87.42¡13.46

Uric acid (mmol/L) b 369.56¡80.76 427.84¡90.39 230.16¡113.76

Total cholesterol (mmol/L) a 5.61¡2.07 4.62¡1.26 4.53¡0.84

Triglycerides (mmol/L) a 1.65¡0.83 1.45¡0.77 1.14¡0.31

HDL cholesterol (mmol/L) c 1.68¡0.61 1.75¡0.42 1.43¡0.53

LDL cholesterol (mmol/L)c 2.62¡2.02 2.61¡1.95 2.26¡0.56

Data are presented as the means ¡ SDs.1Categorical variables are expressed in percentages.aThere was no significant difference in demographic data among the IgAN-A, IgAN-B and control groups (p.0.05).bSignificantly higher between the IgAN and control groups, as well as between the IgAN-A and IgAN-B groups (p,0.001).cSignificantly lower between the IgAN and control groups, as well as between the IgAN-A and IgAN-B groups (p,0.05).

Table 2 - Brief summary of histologic changes in 35 casesof IgAN according to Lee9s classification system.

Pathology category I II III IV V

Mesangial proliferative Slight 3 6 1 0 0

Moderate 0 4 2 2 0

Severe 0 0 1 1 0

FSGS 0 0 6 4 2

MGN 0 0 0 2 0

SGN 0 0 0 0 1

Total 3 10 10 9 3

The comparisons between all pathological types resulted in significance

levels of p,0.01.


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The PLS-DA scores plot (Figure 3a) showed that the clusterof IgAN patients was located further away from the clusterof healthy controls (R2X = 28.5%, R2Y = 0.836, Q2Y = 0.724),indicating that the metabolic profile of the IgAN patientswas quite different from that of the healthy controls.Furthermore, Figure 3(b) shows a clear distinction betweenthe IgAN-A patients and healthy controls (R2X = 32.1%,R2Y = 0.866, Q2Y = 0.741). Similarly, Figure 3(c) reveals thescores plot of the IgAN-B patients and healthy controls,which were also located in different clusters, demonstratingwell-pronounced discrimination (R2X = 28.0%, R2Y = 0.906,Q2Y = 0.671). In Figure 3(d), some samples from IgAN-Apatients are relatively mixed into the cluster of IgAN-Bsamples, which indicates that the metabolic profiles of thosegroups were quite similar (R2X = 25.4%, R2Y = 0.573, Q2Y =-0.160). The goodness of fit (R2 and Q2) of the original PLS-DA models and of the cluster of 200 Y-permutated modelscan be visualized in validation plots (Figure 3, left panel).The validation plots clearly show that the original PLS-DAmodels were valid, as the Q2 regression line had a negativeintercept, and all of the permuted R2 values on the left werelower than the original points on the right.

An OPLS-DA model was constructed in which one PLScomponent and one orthogonal component were calculatedusing the spectral data scaled to that used by Pareto (31).OSC filters were developed to remove the strong structuredvariation in X that was not correlated with Y. That is, OSCfilters removed the structured Y-orthogonal variation fromX in such a way that the filters could be applied to furtherdata (32). The OPLS-DA models used to compare thespectral data between the groups were built using the NMRdata as the X matrix and group information (i.e., control orIgAN patient groups) as the Y matrix (31). In the OPLS-DAmodel score plots, a clear differentiation between healthyindividuals and IgAN patients (Figure 4a, left panel) can beseen. Additionally, a significant biochemical distinctionbetween the IgAN-B group and control group (Figure 4b,left panel) is evident, as well as between the IgAN-A groupand control group (Figure 4c, left panel). Some degree of

separation between the IgAN-A patients and IgAN-Bpatients can also be observed (Figure 4d, left panel).

The OPLS-DA loading plots (Figure 4, middle and rightpanels) were generated to identify the metabolites responsiblefor the differentiation in the score plots. The color map showsthe significance of metabolite variations between the twoclasses. Peaks in the positive direction indicate metabolites thatare more abundant in the groups in the positive direction ofthe first principal component (t[1]P). Consequently, metabo-lites that are more abundant in the groups in the negativedirection of the first primary component are presented aspeaks in the negative direction. In addition, the signals in thespectrum are associated with the significance of metabolites incharacterizing the NMR data for a given group and the color-scaling map on the right-hand side of each loading plot. Thiswas a function of variable importance in OPLS-DA loadingplots (Figure 4) providing information about the magnitude ofthe importance of the metabolites in explaining the scores. Forinstance, red indicates a more significant contribution to theseparation between the groups than blue. Based on the resultsof the OPLS-DA loading plot, in which the targeted profile canbe observed, the serum of IgAN patients was characterized byhigher levels of lactate, myo-Inositol, phenylalanine, and L6( = CH-CH2-CH = ), L5 (-CH2-C = O) and L3 (-CH2-CH2-C = O)lipids, as well as by lower levels of b-glucose, a-glucose, valine,tyrosine, phosphocholine, lysine, isoleucine, glycerolphospho-choline, glycine, glutamine, glutamate, alanine, acetate, 3-hydroxybutyrate, and 1-methylhistidine, compared withhealthy individuals (Figure 4a, middle and right panels).Compared with the control group, the IgAN-A and IgAN-Bgroups had very similar levels of metabolic changes. Increasedlevels of pyruvate, phenylalanine, lactate, L5 lipids (CH2-C = O), creatinine, and creatine and decreased levels of glycinewere marked in the IgAN-A patients, while obvious differ-ences were not observed in the IgAN-B group (Figure 4b, c,middle and right panels). Overall, the high-risk patients hadrelatively higher or lower statistically significant differences in24 variable metabolites than the low-risk patients. Intriguingly,it was difficult to differentiate the low-risk group from the

Figure 1 - 600-MHz 1H NMR spectra (d0.4-4.7 and d5.2-9.0) of serum obtained from the (A) control, (B) IgAN-A and (C) IgAN-B groups.The region of d5.2-9.0 (in the dashed box) was magnified 8 times compared with the corresponding region of d0.4-4.7 for the purposeof clarity. Key: 1-MH: 1-Methylhistidine; Ace: Acetate; Acet: Acetone; Ala: Alanine; Ci: Citrate; Cr: Creatine; Cn: Creatinine; GL: Glycerolof lipids; Gln: Glutamine; Glu: Glutamate; Gly: Glycine; GPC: Glycerolphosphocholine; Ileu: Isoleucine; L1: LDL&VLDL, CH3-(CH2)n-; L2:LDL&VLDL, CH3-(CH2)n-; L3: -CH2-CH2-C = O; L4: -CH2-CH = CH-; L5: -CH2-C = O; L6: = CH-CH2-CH = ; L7: -CH = CH-; Lac: Lactate; Leu:Leucine; Lys: Lysine; MA: Methylamine; m-I: myo-Inositol; NAG: N-acetyl glycoprotein signals; PC: Phosphocholine: Phe: Phenylalanine;Py: Pyruvate; Tyr: Tyrosine; Val: Valine; a-Glc: a-Glucose; b-Glc: b-Glucose.


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high-risk group using OPLS-DA score plots (Figure 4d, middleand right panels).

External validation was performed to test the reliability ofthe PLS-DA model. For our applications, the sensitivity,specificity and classification rate of the established modelsin correctly assigning diagnostic groups are shown inTable 3.

To further investigate the significance of these metabo-lites, a combination of statistical approaches was necessary(33). Thus, according to the cutoff value of the correlationcoefficient, a total of 24 variables proposed as candidatebiomarkers, contributing to the determination of themetabolic profiles for each model, are summarized inTable 4, along with the relative concentrations of themetabolites in the various groups.

DISCUSSION

IgAN is a complex trait and a significant cause of renalinsufficiency in different cohort populations. Manyresearchers have elucidated the causal mechanisms under-lying IgAN in various fields. Nakamaki et al. (33) suggestedthat endothelin-1 is a major disease-promoting factor inrenal disease. It has been documented that the deposition ofpolymeric IgA triggers glomerular immuno-inflammatoryinjury and that subsequent proteinuria may further inducetubulointerstitial lesions, thereby contributing to the pro-gression of IgA nephropathy (34). Suzuki et al. (35)described the characteristics of IgG autoantibodies in theabnormally glycosylated IgA1 secreted by immortalized Bcells derived from patients with sporadic forms of IgAnephropathy, which may offer new insights into diseasepathogenesis and may lead to new methods of diagnosis,monitoring, and therapy for patients with IgA nephropathy.Many IgAN studies have been published, but most havesuffered from small sample sizes and methodologicalproblems, and none of the results have been convincinglyvalidated. Therefore, the molecular basis of IgA nephro-pathy is still not fully understood.

In our study, NMR-based metabolomic analysis technol-ogy was used for the first time to identify the characteristicsof metabolite profiles from the serum samples of IgANpatients and from age- and sex-matched healthy controls.Using a comprehensive NMR screening process, we aimedto assess the potential of the technique as a diagnostic toolfor the disease and to identify the characteristics ofmetabolites. The discovery of distinctive patterns in themetabolite profiles associated with different renal condi-tions could be of great diagnostic significance. The PCAscore plots achieved good separation between the IgANpatients and healthy controls, and the IgAN-A and IgA-Bsamples manifested similar global differences in metabolicprofiles. The results support the hypothesis that themetabolic fingerprinting of serum could be useful indistinguishing IgAN patients from healthy individuals.Upon testing of these results, the OPLS-DA models showedconsiderable R2 and Q2 values. Importantly, the resultsshowed high sensitivity and specificity for diagnosingpatients with IgAN. However, this technique was notsensitive enough to differentiate between high and lowdisease risk. This finding suggests that the diseases mayshare, to some extent, the same metabolic pathway in theirpathogenesis. Nevertheless, potential biomarkers, includingb-glucose, a-glucose, valine, tyrosine, pyruvate, phenylala-nine, phosphocholine, myo-Inositol, lysine, lactate, L6 lipids( = CH-CH2-CH = ), L5 lipids (-CH2-C = O), L3 lipids (-CH2-CH2-C = O), isoleucine, glycerolphosphocholine, glycine,glutamine, glutamate, creatinine, creatine alanine, acetate,3-hydroxybutyrate and 1-methylhistidine, as detected inthis study, might explain the pathogenesis of IgAN andwarrant further investigation in a systemic review.

Similar to how genes and proteins are normally asso-ciated with specific pathways and processes, so aremetabolites. Changes in the relative concentrations ofcertain ‘‘universal’’ metabolites, such as glucose, lactate,and citrate, reflect changes in cell viability (apoptosis), levelsof oxygenation (anoxia, ischemia, and oxidative stress), localpH, and general homeostasis (36). Among these metabolites,

Figure 2 - PCA scores plotted based on 1H CPMG NMR spectra of serum obtained from the different groups. Left panel: C: controlgroup, black box (&); IgAN group, red dot (N). Right panel: C: control group, black box (&); A: IgAN-A group, red dot (N); B: IgAN-Bgroup, blue diamond (X).


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we observed significantly elevated levels of lactate andpyruvate in the serum of IgAN-A patients, while neithercould be detected in IgAN-B patients. Graham et al. (37)suggested that lactate arises from a shift toward anaerobic

glycolysis in potentially viable cells that continue tometabolize glucose under local hypoxic conditions.Alternatively, high levels of lactate may arise frominflammatory and phagocytic cell infiltration in tissue.Additionally, these are types of energy metabolism pro-cesses, especially anaerobic glycolysis. Because an increasein anaerobic cell respiration occurs under renal damage,there is an increased concentration of lactate and pyruvatein IgAN-A serum due to anaerobic glycolysis of serumglucose. In combination with a lower level of glucose, thisresult indicates up-regulated glycolysis. Furthermore, pos-sibly due to the reduced utilization of pyruvate in the Krebscycle, citrate levels also decrease. Glucose and pyruvate canobviously be quite informative with regard to cell functionor cell stress and, therefore, organ function.

IgAN causes perturbations in the Krebs cycle, as well asincreased protein degradation from cell necrosis, leading toaltered concentrations of free amino acids in serum. In thisstudy, the significantly decreased amounts of valine,tyrosine, lysine, isoleucine glycine, glutamate, and alaninemay be associated with reduced translation from citrate, a-ketoglutarate, succinyl-CoA and fumarate, which are inter-mediates of the Krebs cycle. Such results may indicate thatprotein is degraded into amino acids to regulate biologicalfunctions, such as gene transcription, cell cycle progression,and inflammatory and autoimmunity responses. The resultsare also indicative of alterations in energy metabolism aswell as impairment in mitochondrial function (38). Thisfinding is consistent with our report (Table 1) of decreasedlevels of total protein, albumin, and globulin in serumclinical chemistry assays. The lower level of glutamine mayresult from the inadequacy of a-ketoglutarate, which fluxesout of the mitochondria and is converted into glutamate inthe cytosol.

Other types of metabolites are specifically associated withtissue remodeling, muscle atrophy and myofibrillar break-down (e.g., methylhistidine, creatine, creatinine, and gly-cine). Changes in the levels of these metabolites can provideimportant information regarding the extent of tissue repairor tissue damage (39,40). Thus, an elevation in creatine/creatinine levels is indicative of damage to the renalparenchyma or of kidney necrosis, which is also inaccordance with clinical chemistry assays. Moreover, thelower concentrations of 1-methylhistidine, which is meta-bolized into histidine, might be closely related to proteinenergy wasting, inflammation and oxidative stress (41).

Phosphorylcholine is a precursor of glycerolphosphor-ylcholine, which is an essential component of membranestructures. The decreased serum levels of phosphorylcho-line in both the IgAN-A and IgAN-B patients might berelated to the augmented utilization of glycerolphosphor-ylcholine in impaired cells and organelle membranes. Morerecent evidence has demonstrated that the proportion ofPC/GPC can be used as a measurement of enhanced cellproliferation in cancerous tissues (42).

Myo-Inositol, L6 lipids ( = CH-CH2-CH = ), L5 lipids(-CH2-C = O), and L3 lipids (-CH2-CH2-C = O) are involvedin lipid metabolism. In our study, clinical chemistry assaysindicated slightly higher levels of free fatty acids and nosignificant differences in levels of triglycerides or totalcholesterol between the IgAN and control groups, as well asbetween the IgAN-A and IgAN-B groups. NMR spectrashowed enhanced serum levels of VLDL/LDL and unsatu-rated lipids in the IgAN patients. The enhanced lipid levels

Figure 3 - Cross-validation of partial least squares-discriminantanalysis (PLS-DA) models obtained from 200 permutation tests.PLS-DA score plots showing the separation between (a) controlsand IgAN patients, (b) controls and IgAN-A patients, (c) controlsand IgAN-B patients, and (d) IgAN-A and IgAN-B patients.


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Figure 4 - OPLS-DA score plots (left panel) derived from 1H NMR spectra of serum and corresponding coefficient loading plots (middleand right panels) obtained from different groups. Keys for the assignments are shown in Figure 1.


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can most likely be attributed to the lower activity of thelipoprotein lipase. Additionally, the increase in lipidmetabolism is enhanced in response to kidney injury whenIgAN occurs (21). The increase in acetate, which is the endproduct of lipid metabolism, further supports the above-mentioned hypothesis. Furthermore, our work demon-strated that the concentrations of serum 3-hydroxybutyratein the renal tissue of IgAN patients were significantly lowerthan those in the renal tissue of controls, which may confirmthe shift in energy metabolism toward the formation andutilization of ketone bodies.

In summary, the systemic changes in endogenousmetabolites from IgAN patients mainly influenced energy,amino acid, lipid, and nucleotide metabolism. As shown inFigure 5, the number of metabolic pathways involved inIgAN highlights the complexity of the metabolic response toIgAN. Metabolite profiling, obtained from 1H-NMR-basedmetabonomic analysis of IgAN serum samples, clearlydistinguished IgAN patients from healthy controls, whichmay be a minimally invasive method for detecting IgANbefore symptoms develop, suggesting that the utilization of

metabonomics in serum may assist in early diagnosis, moreappropriate therapy and better clinical management.

Although we identified disease-specific metabolite pro-files, the future use of metabolite biomarkers in the clinicalenvironment requires optimization to improve their accu-racy. The limitations of this study must also be recognized(43,44). First, metabolic investigations can be helpful for thedifferentiation of healthy controls from IgAN patients,but they are not sensitive enough to be used to drawconclusions about the dynamic processes of high andlow risk for this disease. Moreover, different histologicalclassifications subjectively present selection bias to a certaindegree when screening patients. Histological classificationmust be validated in different cohorts. Furthermore, thisstudy was not specifically designed to answer the question ofwhether medical treatment consisting of diuretics, angioten-sin-converting enzyme inhibitors and/or angiotensin IIreceptor blockers, n-3 polyunsaturated fatty acids, andhypolipidemic agents should be administered to IgANpatients. We did not evaluate the associations betweentherapeutic interventions and metabolite changes in this

Table 3 - Sensitivity, specificity and classification rate calculated for the cross-validated OPLS-DA model applied to thespectra of serum samples from healthy controls and patients with IgAN.

IgAN vs. control IgAN-A vs. control IgAN-B vs. control IgAN-A vs. IgAN-B

Sensitivity (%) 88.6 92.3 95.4 -

Specificity (%) 97.1 100 100 -

Classification rate (%) 93.1 97.0 98.2 -

Table 4 - Summary of the 1H NMR-detected statistically significant changes in the relative levels of serum metabolites forIgAN patients compared with the healthy controls.

Metabolites ra

C-IgAN C- A C- B A-B

b-Glucose: 3.25 (ddb), 3.41 (t), 3.46 (m), 3.49 (t), 3.90 (dd), 4.65 (d) -0.784 -0.859 -0.797 -

a-Glucose: 3.42 (t), 3.54 (dd), 3.71 (t), 3.73 (m), 3.84 (m), 5.23 (d) -0.812 -0.843 -0.871 -

Valine: 0.99 (d), 1.04 (d) -0.816 -0.799 -0.811 -

Tyrosine: 6.89 (d), 7.19 (d) -0.619 -0.638 -0.652 -

Pyruvate: 2.37 (s) - 0.426 - -

Phenylalanine: 7.32 (d), 7.37 (t), 7.42 (dd) 0.436 0.473 - -

Phosphocholine: 3.21 (s), 3.35 (s) -0.791 -0.747 -0.798 -

myo-Inositol: 3.65 (dd) 0.513 0.671 0.672 -

Lysine: 1.72 (m), 1.90 (m), 3.02 (t), 3.76 (m) -0.577 -0.546 -0.560 -

Lactate: 1.33 (d), 4.11 (q) 0.445 0.611 - -

L6 lipid, = CH-CH2-CH = : 2.78 (br) 0.422 0.555 0.617 -

L5 lipid, -CH2-C = O: 2.24 (br) 0.433 0.514 - -

L3 lipid, -CH2-CH2-C = O: 1.58 (br) 0.527 0.610 0.561 -

Isoleucine: 0.94 (t), 1.01 (d) -0.650 -0.604 -0.644 -

Glycerolphosphocholine: 3.22 (s), 3.36 (s) -0.876 -0.858 -0.878 -

Glycine: 3.56 (s) -0.524 -0.477 - -

Glutamine: 2.14 (m), 2.45 (m), 3.78 (t) -0.762 -0.796 -0.707 -

Glutamate: 2.10 (m), 2.35 (m), 3.78 (t) -0.762 -0.751 -0.697 -

Creatinine: 3.04 (s), 4.06 (s) - 0.506 - -

Creatine: 3.04 (s), 3.93 (s) - 0.417 - -

Alanine: 1.48 (d) -0.451 - - -

Acetate: 1.92 (s) -0.519 -0.454 -0.563 -

3-Hydroxybutyrate: 1.18 (d) -0.834 -0.823 -0.843 -

1-Methylhistidine: 7.05 (s), 7.75 (s) -0.692 -0.674 -0.608 -

OPLS-DA coefficients derived from the NMR data of metabolites in serum obtained from different groups.aCorrelation coefficients and positive and negative signs indicate positive and negative correlations in the concentrations, respectively. The correlation

coefficient of |r|. 0.404 was used as the cutoff value for statistical significance based on the discrimination significance at the level of p = 0.05 and df

(degree of freedom) = 22 (for C-IgAN and C-A), and it was |r|. 0.553 at the level of p = 0.05 for df = 11 (for C-B and A-B). ‘‘-’’ means that the correlation

coefficient |r| is less than the cutoff value.bMultiplicity: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; m, multiplet.


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cohort of patients. Finally, given our relatively small samplesize, especially the IgAN-B sample size, our observations stillremain to be verified in larger studies. Further studies withlarger numbers of patients and controls, including otherdistinctive types of IgAN diseases, will be crucial tovalidating this model and to assuring its use in routinepractice. Additionally, to explore the potential of this model,follow-up work is required, which should include long-termstudies of large numbers of patients with early IgANdiagnoses. It is also necessary to validate these biomarkersin another cross-sectional study with a separate population,and it would be of interest to perform a longitudinal study todetermine the predictive power of this model with regard todisease progression.

In conclusion, this study showed that one potential use ofmetabonomics might be the development of a moresensitive, reliable and successful method of diagnosis thanthe invasive puncture procedure for distinguishing IgANpatients from healthy controls. Nevertheless, the model ofthe high-risk IgAN group versus the low-risk group was notperfect; that is, the 1H NMR-based metabonomic analysis ofserum samples revealed some characteristic metabolicfeatures, but not processes, of the pathological changesassociated with IgAN. Further NMR-based metabonomicstudies of various IgAN models could provide insight intothe molecular mechanisms involved in the pathogenesis ofIgAN and could offer invaluable information that could beused in the development of a novel diagnostic techniqueand better IgAN treatments.

ACKNOWLEDGMENTS

This project was funded by grant support received from the Natural

Science Foundation of Guangxi, China, which is gratefully acknowledged

(fund number 2010GXNSFA013273). We are also thankful to the patients

and healthy volunteers who participated in this study.


Sui W collected data and wrote the paper. Li L performed the research and

wrote the paper. Che Wand Chen J analyzed the data. Zuo G and Li W

revised the paper. Dai Y designed the research protocol and revised the

paper.

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BASIC RESEARCH

Influence of food restriction on lipid profile andspontaneous glucose levels in male rats subjected toparadoxical sleep deprivationTathiana Aparecida Alvarenga, Sergio Tufik, Gabriel Natan Pires, Monica Levy Andersen

Universidade Federal de Sao Paulo, Departamento de Psicobiologia, Sao Paulo/SP, Brazil.

OBJECTIVES: The purpose of this study was to determine the paired consequences of food restriction andparadoxical sleep deprivation on lipid profile and spontaneous glucose levels in male rats.

METHOD: Food restriction began at weaning, with 6 g of food being provided per day, which was subsequentlyincreased by 1 g per week until reaching 15 g per day by the eighth week. At adulthood, both rats subjected tofood restriction and those fed ad libitum were exposed to paradoxical sleep deprivation for 96 h or weremaintained in their home-cage groups.

RESULTS: Animals subjected to food restriction exhibited a significant increase in high-density lipoprotein levelscompared to animals that were given free access to food. After the paradoxical sleep deprivation period, the food-restricted animals demonstrated reduced concentrations of high-density lipoprotein relative to their respectivecontrols, although the values for the food-restricted animals after sleep deprivation were still higher than those forthe ad libitum group. The concentration of low-density lipoproteins was significantly increased in sleep-deprivedanimals fed the ad libitum diet. The levels of triglycerides, very low-density lipoproteins, and glucose in food-restricted animals were each decreased compared to both ad libitum groups.

CONCLUSION: These results may help to illustrate the mechanisms underlying the relationship between sleepcurtailment and metabolism and may suggest that, regardless of sleep deprivation, dietary restriction can minimizealterations in parameters related to cardiovascular risk.

KEYWORDS: Cardiovascular Diseases; Cholesterol; Food Deprivation; Sleep Deprivation; Triglycerides.

Alvarenga TA, Tufik S, Pires GN, Andersen ML. Influence of food restriction on lipid profile and spontaneous glucose levels in male rats subjected toparadoxical sleep deprivation. Clinics. 2012;67(4):375-380.

Received for publication on October 4, 2011; First review completed on November 19, 2011; Accepted for publication on December 5, 2011


Tel.: 55 11 2149-0155

INTRODUCTION

Although many studies have demonstrated the impor-tance of sleep duration for overall health, sleep loss is aninevitable consequence of societal pressure. In addition tosleep curtailment, other sleep disorders, such as obstructivesleep apnea (OSA) and insomnia, have become markedlyprevalent (1). For instance, sleep apnea has importantclinical consequences, including hypertension (2) andincreased risks for cardiovascular and endocrine diseases.Patients with OSA experience recurrent sleep disruptionsand a significant decrease in rapid eye movement (REM)sleep (3). Moreover, it is well-known that in patients withOSA, most apnea events occur during REM sleep.Therefore, cerebrovascular function may be most affected

during REM sleep, due to the high demand for energy bythe brain during that sleep stage (4).

In rats, sleep deprivation induced by the platformtechnique causes numerous awakenings, which predomi-nantly affect the REM stage of sleep. Therefore, we reasonedthat this procedure could mimic sleep fragmentation due torepeated awakenings and could therefore be a useful toolfor investigating the effects of sleep loss on bloodparameters. In a previous study, our group assessed theeffects of sleep loss on blood parameters associated withcardiovascular risk in male rats. These data indicated thatsleep-deprived rats have significantly increased high-den-sity lipoprotein (HDL) and low-density lipoprotein (LDL)levels compared to controls. Sleep deprivation has also beenshown to decrease triglyceride and very-low-density lipo-protein (VLDL) levels significantly (5).

However, one study demonstrated that a reduction in caloricintake is related to lifespan extension because several physio-logical functions were improved under conditions of foodrestriction (FR) (6). In rodents, the effects of FR are extensive andinclude a significant reduction in risk factors associated with thedevelopment cardiovascular and neurodegenerative diseases




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(7-10). Broderick et al. (11,12) reported enhanced contractileproperties of the heart, improved blood-flow regulation andresistance to cerebral ischemia in rats maintained on an 8-monthFR diet (45% reduction) compared to ad libitum-fed animals.Similarly, 30-40% of FR rodents had significantly fewermyocardial infarcts over a 10-month period (13). More recently,Venkatachalam et al. (14) reported decreases in inflammationand oxidative damage to heart cells following occlusion of theleft anterior descending coronary artery in FR rats compared tosimilarly treated ad libitum-fed rats.

Although several studies have examined the behavioralalterations in FR rats, no study has addressed the specificeffects of FR associated with lack of sleep on lipid profileand spontaneous glucose levels. Thus, the aim of this studywas to investigate the consequences of both FR and sleepdeprivation on the blood parameters associated withcardiovascular disease risk in male rats.


SubjectsThirty-eight male Wistar rats were bred and raised in the

animal facility of the Center for Development of ExperimentalModels for Medicine and Biology (CEDEME) at UniversidadeFederal de Sao Paulo. The animals were housed in a colonythat was maintained at 22 C on a 12:12-h light-dark cycle(lights on at 07:00 h), and animals were allowed free access towater inside of standard polypropylene cages. The experi-mental protocol was approved by the Ethical Committee ofUNIFESP (CEP N. 05/434) and was conducted under therecommendations of the European Convention for theProtection of Vertebrate Animals used for Experimental andother Scientific Purposes, as well as those of the BrazilianSociety of Science in Laboratory Animals.

Food restriction (FR)From the time of weaning (at 30 days) onward, rats were fed

6 g of chow daily. This amount of food was increased by 1 gper week until it reached 15 g by the eighth week. The animalswere kept in separate cages during feeding and remainedseparated until all food was completely ingested. The protocolthat was used for the calculation of chow consumption was incompliance with our previous studies (15-17). The animalswere distributed into the following two groups:

a) those fed an ad libitum diet (AD, n = 19) and allowed toeat and drink ad libitum from the time of weaningthroughout the experiment, or

b) those placed under food restriction (FR, n = 19) and fedaccording to the dietary restriction protocol describedabove. For this group, water was offered ad libitum, andthe FR rats were fed at approximately 19:00 h.Previously, we demonstrated that there were nostatistically significant effects of feeding time on thesleep-wake patterns of diurnal periods (15).

Protocol designsAfter eight weeks of FR or AD, the animals were

randomly distributed into the following groups:

a) AD-CTRL (n = 9): AD rats maintained in the home cage.

b) AD-PSD (n = 10): AD rats subjected to 96 h of PSD.

c) FR-CTRL (n = 9): FR rats maintained in the home cage.

d) FR-PSD (n = 10): FR rats subjected to 96 h of PSD.

Paradoxical Sleep Deprivation (PSD)The rats were distributed into the following two additional

groups: the control (CTRL) or the PSD group. The animals inthe PSD group were subjected to 96 h of PSD using themodified multiple platform method. We used a duration of96 h based on the results of previous studies (18,19). Six ratswere placed inside of one tiled water tank (143641630 cm),which contained 12 circular platforms that were 6.5 cm indiameter, and the water was filled up to 1 cm from the uppersurface of the platform. The rats could therefore move aroundinside of the tank by jumping from one platform to another.When they reached the paradoxical phase of sleep, muscleatonia set in, and the animals fell into the water and woke up.Throughout the study, the experimental room was main-tained at a controlled temperature (22¡1 C) and light-darkcycle (lights came on at 07:00 h and were turned off at19:00 h). Food was provided freely to the AD group in theform of chow pellets, and water bottles were made accessibleon a grid located on top of the tank. The water in the tank waschanged daily throughout the PSD period. The FR rats werefed at approximately 19:00 h in individual cages and werereturned to the water tank after they had eaten all of the foodpellets. Lastly, cage-control groups (AD and FR) weremaintained in separate cages in the same room as theexperimental rats during the sleep-deprivation procedure. Byhousing both groups in the same room, we maintainedcontrol over differences in housing conditions between thetwo groups.

Blood samplingFollowing the behavioral tests, the PSD and CTRL rats

were brought to an adjacent room and decapitated.Although decapitation is aesthetically questionable, it is anextremely fast and efficient method that does not causeadditional pain or distress to the animals. Moreover,decapitation induces an immediate disappearance of thecorneal reflex and promotes minimal physiological altera-tions in the tissue. For the purpose of this study, this methodwas used to provide the amount of blood required for theassays. Blood samples were immediately collected andstored individually. Blood was collected in glass tubes andcentrifuged at 3018.4g for 15 min at room temperature.Cholesterol and triglyceride concentrations were measuredusing an automated colorimetric method (ADVIA 16/50,BAYER Diagnostics Corporation, NY, USA). Duplicateserum aliquots were also assessed. These assays wereperformed using the standard automated procedures ofour clinical laboratory.

Statistical analysesThe data were analyzed using a one-way ANOVA test

followed by a Tukey test for comparisons between groups.Values are expressed as the means ¡ SEM. The level ofsignificance was set at p,0.05.

RESULTS

Total CholesterolAn analysis of the total cholesterol concentrations

revealed significant differences between the groups(F(3.33) = 8.38, p,0.0002), as shown in Figure 1A. FollowingPSD, the FR rats demonstrated significantly lower choles-terol levels did than the respective CTRL group (p,0.01).

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TriglyceridesTriglyceride levels were substantially reduced in the AD-

PSD and all FR groups compared to the AD-CTRL group(Figure 1B). Indeed, sleep deprivation significantly reducedtriglyceride levels in both FR and AD animals compared tothe respective non-sleep-deprived controls (p,0.0001).

Cholesterol fractionsAs shown in Figure 2A, high-density lipoprotein (HDL)

levels were higher among animals in the sleep-deprived groupsand the FR-CTRL group relative to those in the AD-CTRLgroup (F(3,33) = 55.19, p,0.0001). In contrast, the very-low-density lipoprotein (VLDL) levels followed an opposite pattern(Figure 2B), as the FR-CTRL group had lower levels than theAD-CTRL group. The sleep-deprived animals exhibited evengreater decreases compared to the CTRL groups (p,0.05). Withregard to the low-density lipoprotein (LDL) levels, only animalsin the AD-PSD group exhibited an increase in comparison to theother groups evaluated (Figure 2C, p,0.0001).

GlucoseAs shown in Figure 3, all groups demonstrated decreased

glucose levels in comparison to the AD-CTRL groupindependently of sleep deprivation (p,0.001). Moreover,the FR-PSD animals had reduced glucose levels in compar-ison to those in the FR-CTRL group (p,0.001).

DISCUSSION

These results demonstrate that long-term FR can alterbiochemical blood parameters related to cardiovascularrisks independently of PSD. The glucose, triglyceride, andVLDL cholesterol concentrations were similarly reduced byFR, and the reductions in these parameters were maintainedwhen PSD was combined with FR. In addition, LDLcholesterol levels were higher only among animals in theAD-PSD group. However, FR was found to increase HDLcholesterol levels. Moreover, these changes were reversedby combining FR with PSD, indicating that selective sleeploss can override the beneficial effects of low caloric intake.However, animals in the FR-PSD group maintained higherlevels of the tested parameters than did those in the AD-PSDgroup.

The beneficial effects of low caloric intake, which includeimprovements in cardiovascular function, have been widelyreported in the literature (7,8,14). However, to the best ofour knowledge, this study is the first to focus on whetherthe positive effects of FR can be maintained when combinedwith sleep loss. This study showed that for some para-meters, such as HDL, triglyceride and glucose levels, FRmay reduce the potential damage caused PSD. Of note,HDLs were present at higher concentrations in the FRgroups compared to the ad libitum groups, even when FRwas combined with PSD.

Figure 1 - Effects of food restriction (FR) or ad libitum diet (AD), either paired or unpaired with paradoxical sleep deprivation (PSD), onthe total cholesterol (panel A) and triglyceride levels (panel B) in male rats. *Significantly different from the AD-CTRL group, ¥Significantly different from the AD-PSD group and # Significantly different from the respective control group.

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It has been documented that HDL concentrations areinversely associated with cardiovascular disease risks (20).Pharmacological interventions that increase HDL concentra-tions typically also improve other lipid parameters. Forexample, the contribution of increased HDL levels is noteasily distinguishable from the contributions of decreasedLDL and/or triglycerides levels (21). The low glucose, VLDLand triglyceride levels observed in the FR groups may be

protective against an adverse challenge, such as PSD, as aconsequence of FR. One example of the beneficial effects of alow caloric intake is the reduction of oxidative stress (22).Moreover, FR leads to alterations in energy balance, such asreduced energy expenditure, as a result of decreased oxygenconsumption (23,24) or mitochondrial adaptation (25).

Studies have demonstrated sleep loss affects several bloodparameters in rats, and previous studies from our group

Figure 2 - Concentrations of HDL (panel A), VLDL (panel B) and LDL cholesterol (panel C) in male ad libitum (AD)-fed or food-restricted(FR) rats subjected to normal sleep (CTRL) or paradoxical sleep deprivation (PSD). Data are expressed as the means ¡ SEM.*Significantly different from the AD-CTRL group, ¥ Significantly different from the AD-PSD group, { Significantly different from the FR-CTRL group and # Significantly different from the respective control group.


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(5,26-28) have isolated the influence of PSD on cardiovascularparameters. In general, these findings indicated increases intotal cholesterol and the HDL fraction of cholesterol in maleand female rats after sleep loss. In these studies, ratsmaintained on a normal diet had increased levels oftriglycerides, VLDL cholesterol and glucose compared tothe other groups. However, during periods of PSD, the levelsof HDL and LDL cholesterol increased, whereas those oftriglycerides and VLDL cholesterol decreased. It is possiblethat these results may have been due to reduced foodconsumption during the sleep deprivation procedure, whichwas accompanied by weight loss. It has also been reportedthat sleep deprivation can cause hyperphagia and weight loss(29,30). Therefore, one might hypothesize that the differencesobserved in the triglyceride and cholesterol levels after PSDwere the result of greater food intake. However, during PSD,the impaired capacity to increase food intake in response toincreased energy expenditure contributes to an energy deficitin rats (30). Data from animals with normal food intake levelsindicate that total cholesterol levels remain unchanged aftersleep deprivation, whereas the levels of HDL and LDLincrease. The heterogeneity of these responses argues againsta simple effect of increased food consumption on thesemeasures.

Recently, there has been growing concern regarding thepotential effects of dietary restriction on several healthparameters. Most of this attention has been the result ofsocietal interest in longevity, health and welfare. In parti-cular, dietary restriction has been reported as being the onlyintervention that can promote increased longevity, reduce theincidence of disease and slow processes related to aging (31).

It is important to note that the sleep fragmentationinduced by PSD evaluated in the current study did notnegatively alter all of the parameters related to cardiovas-cular risk. In addition, the FR data provide additionalevidence regarding the mechanisms underlying an increasein life expectancy. Finally, our data suggest that indepen-dently of sleep deprivation, dietary restriction can minimizechanges in the parameters related to cardiovascular risk.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the assistance of Waldemarks Leite.

This work was supported by grants from the Associacao Fundo de

Incentivo a Pesquisa (AFIP), the Fundacao de Amparo a Pesquisa do

Estado de Sao Paulo (FAPESP-CEPID no. 98/14303-3 to ST.) and

Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq)

to Andersen ML and Tufik S. Alvarenga TA and Pires GN are recipients of

scholarships by FAPESP (no 11/12325-6 and 10/14768-0).


Alvarenga TA was responsible for design development, conducting

experiments, statistical analysis, data interpretation, bibliographic review

and manuscript writing. Tufik S was responsible for data interpretation and

writing the review. Pires GN was responsible for data interpretation,

statistical analysis and manuscript writing. Andersen ML was responsible

for design development, data interpretation, writing the review and

checking grammar and spelling.

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REVIEW

Anesthesia-related mortality in pediatric patients: asystematic reviewLeopoldo Palheta Gonzalez,I Wangles Pignaton,II Priscila Sayuri Kusano,II Norma Sueli Pinheiro Modolo,II

Jose Reinaldo Cerqueira Braz,II Leandro Gobbo BrazII

I Universidade do Estado do Amazonas (UEA), Manaus/AM, Brazil. II Universidade Estadual Paulista, Botucatu Medical School (UNESP), Department of

Anesthesiology, Botucatu/SP, Brazil.

This systematic review of the Brazilian and worldwide literature aimed to evaluate the incidence and causes ofperioperative and anesthesia-related mortality in pediatric patients. Studies were identified by searching EMBASE(1951-2011), PubMed (1966-2011), LILACS (1986-2011), and SciElo (1995-2011). Each paper was revised to identify theauthor(s), the data source, the time period, the number of patients, the time of death, and the perioperative andanesthesia-related mortality rates. Twenty trials were assessed. Studies from Brazil and developed countriesworldwide documented similar total anesthesia-related mortality rates (,1 death per 10,000 anesthetics) anddeclines in anesthesia-related mortality rates in the past decade. Higher anesthesia-related mortality rates (2.4-3.3per 10,000 anesthetics) were found in studies from developing countries over the same time period. Interestingly,pediatric perioperative mortality rates have increased over the past decade, and the rates are higher in Brazil (9.8per 10,000 anesthetics) and other developing countries (10.7-15.9 per 10,000 anesthetics) compared with developedcountries (0.41-6.8 per 10,000 anesthetics), with the exception of Australia (13.4 per 10,000 anesthetics). The majorrisk factors are being newborn or less than 1 year old, ASA III or worse physical status, and undergoing emergencysurgery, general anesthesia, or cardiac surgery. The main causes of mortality were problems with airwaymanagement and cardiocirculatory events. Our systematic review of the literature shows that the pediatricanesthesia-related mortality rates in Brazil and in developed countries are similar, whereas the pediatricperioperative mortality rates are higher in Brazil compared with developed countries. Most cases of anesthesia-related mortality are associated with airway and cardiocirculatory events. The data regarding anesthesia-relatedand perioperative mortality rates may be useful in developing prevention strategies.

KEYWORDS: Anesthesia; Cardiac Arrest; Mortality; Perioperative; Pediatric; Review.

Gonzalez LP, Pignaton W, Kusano PS, Modolo NSP, Braz JRC, Braz LG. Anesthesia-related mortality in pediatric patients: a systematic review. Clinics.2012;67(4):381-387.



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INTRODUCTION

Studies have revealed that there is a higher perioperativemortality rate in children compared with adults (1-4).Within the pediatric population, perioperative mortality ismore frequent in neonates and infants compared with olderchildren (2,3,5-8).

An analysis of overall perioperative mortality and anesthe-sia-related mortality in particular may help determine whichchildren are at higher risk and may help guide planning toimprove the safety profile of perioperative techniques.

Large-scale and national studies of anesthesia-relatedmortality in children have been performed in a number ofcountries (3,5,9-11). In Brazil, there have been three studies

on pediatric perioperative mortality (12-14), but no nation-wide study has been undertaken.

The aim of the present review was to perform asystematic review of the Brazilian and worldwide literatureto evaluate the incidence and causes of perioperative andanesthesia-related mortality in pediatric patients.

METHODS

We performed a text word search strategy to identifycross-sectional studies of perioperative and/or anesthesia-related mortality in children using the following words:‘‘cardiac arrest’’, ‘‘mortality’’ and ‘‘anesthesia’’ (classifiedaccording to MeSH terms). Studies were obtained fromEMBASE (1951-2011), the US National Library of Medicine(PubMed, 1966-2011), LILACS (1986-2011), and the ScientificElectronic Library Online (SciElo, 1995-2011). We also usedthe ‘‘related articles’’ function on PubMed and the refer-ences cited in the studies found in the database searches.The present review was completed in October 2011. Therewas no language restriction since the publications includedan abstract written in English and if the studies used theoriginal data to suggest a perioperative cardiac arrest and/or




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mortality rate that was related to anesthetic management in apediatric patient population (at least 5,000 cases of anestheticuse) over a defined period. Some studies included pediatricand adult patients; however, we only reported the mortalityincidence of the pediatric population (the mortality rate = thenumber of deaths x 10,000/ the number of children who wereanesthetized). Studies were excluded if the anesthetic manage-ment was limited to a particular technique, surgery, associateddisease state, or within a specific pediatric age group. Otherstudies on anesthesia-related cardiac arrest and/or mortalitythat offered additional relevant information were alsoexamined.

Mortality-triggering factors were assigned to one of thefollowing groups: surgery-related, related to patient diseaseor condition, and anesthesia-related. Three reviewers inde-pendently screened the titles that were identified in theliterature search, extracted the data, and analyzed the results.Discrepancies in the results were resolved by discussion. Astandard form was used to extract the following information:the author(s), the data source, the time period, the number ofpediatric patients, the time of death (i.e., in the operatingroom, during recovery from anesthesia, during the first 12 or24 postoperative hours, and within seven or eight post-operative days), the perioperative mortality rate (including

all mortality-triggering factors), the anesthesia-related mor-tality rate, the type of anesthesia, the American Society ofAnesthesiologists (ASA) physical status, the patients’ ages,and the factors that contributed to mortality.

RESULTS

The database queries identified 20 articles that reportedperioperative and anesthesia-related cardiac arrest ormortality data in children: 17 of these articles were frominternational investigators, and three were from Brazilianinvestigators (Tables 1 and 2).

A comparison of the data reported by investigators fromdeveloped countries worldwide between 1961 and 2000with papers published between 2001 and 2011 demon-strated that anesthesia-related mortality rates declined from0.2-2.9 per 10,000 anesthetics (9,10,15-17) for the forty-yearperiod ending in 2000 (Table 1) to 0.0-0.69 per 10,000anesthetics (6,8,18-20) for the ten-year period ending in 2011(Table 2). In Brazil, one study performed during the latterperiod reported an anesthesia-related mortality rate of 0.0per 10,000 anesthetics (14), which was similar to the ratesreported by the studies in developed countries (Table 2).The highest anesthesia-related mortality rates (2.4 to 3.3 per

Table 1 - Mortality incidence in pediatric patients who underwent anesthesia between 1961 and 2000.

Investigators and

Year of Publication

Time Period and

Data Source

Number of Patients and

Time of Death Age

Mortality Incidence per 10,000

Anesthetics

Perioperative

Anesthesia-

related

Rackow et al.

(1961) (15)

1947-1956

Teaching hospital

USA

34,499

Deaths in OR and PACU

#12 y 4.9 2.9

Keenan & Boyan

(1985) (16)

1969-1983

Teaching hospital

USA

12,712

Deaths within 8 days

#11 y NR 1.57

Tiret et al.

(1988) (9)

1978-1982

Group of 440 hospitals

France

40,240

Deaths within 24 h

#14 y NR 0.2

Cohen et al.

(1990) (21)

1982-1987

Teaching hospital

Canada

29,220


#16 y

,1 mo

1-12 mo

1-5 y

6-10 y

11-16 y

3.8

83.1

7.9

3.0

1.4

1.8

NR

Aubas et al.

(1991) (17)

1983-1987

Teaching hospital

France

16,207


#14 y

0-4 y

5-14 y

NR 1.23

2.3

0

Conceicao & Costa

(1995) (12)

1980-1993

Teaching hospital

Brazil

30,028

Deaths within 24 h

#12 y 0.33 NR

Cicarelli et al.

(1998) (13)

1995

Teaching hospital

Brazil

7,392

Deaths within 24 h

#12 y 5.4 NR

Morray et al.

(2000) (10)

1994-1997

Group of hospitals

USA, Canada

1,089,200 (estimated)


#18 y NR 0.36

OR = operating room; PACU = postanesthesia care unit; NR = not reported.

Pediatric mortality in anesthesiaGonzalez LP et al.

CLINICS 2012;67(4):381-387

382

10,000 anesthetics) were observed in the studies that wereperformed in the developing countries (7,11) between 2001and 2011 (Table 2).

An analysis of the data that were reported by theinvestigators from the developed countries showed perio-perative mortality rates ranging from 3.8 to 4.9 per 10,000

Table 2 - Mortality incidence in pediatric patients who underwent anesthesia between 2001 and 2011.

Investigators and Year

of Publication

Time Period and

Data Source

Number of Patients and

Time of Death Age

Mortality Incidence per 10,000

Anesthetics

Perioperative

Anesthesia-

related

Biboulet et al.

(2001) (18)

1989-1995

Teaching hospital

France

23,832

Deaths within 12 h

Excluded: ASA V patients

#14 y

0-4 y

5-14 y

NR 0.42

0

0.84

Morita et al.

(2001)* (3)

1999

Group of hospitals

Japan

732,788 (adults and children)


,1 mo

,12 mo

,5 y

,18 y

42.75

2.95

2.54

1.7

0

0

0

0.17

Tay et al.

(2001) (22)

1997-1999

Teaching hospital

Singapore

10,000


NR 3.0 0

Morita et al.

(2002)* (5)

2000

Group of hospitals

Japan

910,757 (adults and children)


,1 mo

,12 mo

,5 y

,18 y

18.86

4.6

1.26

1.57

0

0

0.21

0.14

Newland et al.

(2002) (19)

1989-1999

Teaching hospital

USA

16,051

Deaths within 24 h

#20 y NR 0

Murat et al.

(2004) (20)

2000-2002

Teaching hospital

France

24,165

Deaths within 24 h

Excluded: neurosurgery and cardiac

surgery

#16 y

0-,1 y

1-7 y

8-16 y

0.41

2.71

0

0

0

Braz et al.

(2006) (14)

1996-2004

Teaching hospital

Brazil

15,263


#17 y 9.8 0

Flick et al.

(2007) (6)

1998-2005

Teaching hospital

USA

92,881


,18 y

0-30 days

31 days-,1 y

1-3 y

4-9 y

10-,18 y

6.8

144.7

19.2

5.7

3.1

2.1

0.21

0

0

0

0.39

0.26

Ahmed et al.

(2009) (23)

1992-2006

Teaching hospital

Pakistan

20,216

Deaths in OR

Excluded: cardiac surgery

#18 y

0-1 y

.1-18 y

3.46

11.43

1.26

0.49

2.28

0

Bharti et al.

(2009) (7)

2003-2008

Teaching hospital

India

12,158

Deaths within 48 h

Excluded: eye and cardiac surgeries

#17 y

0-,1 y

1-,4 y

4 -,10 y

10-17 y

10.7

18.5

6.3

6.5

9.1

3.3

4.7

0

2.5

0

Bunchungmongkol et al.

(2009) (11)

2003-2004

Group of hospitals

Thailand

25,098

Deaths within 24 h

#15 y

0-1 y

.1-8 y

.8-15 y

15.9

35.1

9.4

12.2

2.4

3.7

0.9

3.3

van der Griend et al.

(2011) (8)

2003-2008

Teaching hospital

Australia

101,885

Deaths within 24 h

#18 y

0-30 days

31 days-,1 y

1-,4 y

4-,10 y

10-18 y

13.4

180.1

32.2

6.6

3.4

5.9

0.69

3.51

0.80

1.11

0.31

0.36

OR = operating room; PACU = postanesthesia care unit; NR = not reported.*Japanese text: the information was obtained from the English abstract (the number of children anesthetized was not reported in the abstract).

CLINICS 2012;67(4):381-387 Pediatric mortality in anesthesiaGonzalez LP et al.

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anesthetics (15,21) from 1961-2000 (Table 1) to 0.41-13.4 per10,000 anesthetics (6,8,20) from 2001-2011 (Table 2). In thedeveloping countries, the perioperative mortality rates from2001-2011 ranged from 3.0 to 15.9 per 10,000 anesthetics(7,11,22,23) (Table 2). A comparison of the Brazilian datathat was reported between 1961 and 2000 with data from2001-2011 showed that perioperative mortality rates rangedfrom 0.33 to 5.4 per 10,000 anesthetics (12,13) during theformer period to 9.8 per 10,000 anesthetics (14) during thelatter period (Tables 1 and 2, respectively).

In studies from both developing (7,11,23) and developed(3,5,6,8,17,21) countries, the mortality rates were higher innewborns and children under one year of age comparedwith older children.

An ASA physical status of III or greater (6-9,11,14,19) andemergency surgery (6,7,14,23,24) were risk factors forpediatric perioperative mortality. Studies from developingcountries verified anesthesia-related mortality in ASAphysical status I-II children (7,11). Although one study (7)found that female children had a greater risk of cardiacarrest and another study (11) found that male children had agreater risk of cardiac arrest, other studies have shown thatgender is not a risk factor for mortality in children (6,14). Insome studies (14,23), the patient’s condition or diseaserepresented the major perioperative mortality-triggeringfactor, with surgery and anesthesia being the next mostimportant factors. Interestingly, other studies (8,25,26) haveindicated that complications associated with cardiac proce-dures are the major cause of pediatric perioperativemortality.

Regarding the type of anesthesia, higher rates ofperioperative cardiac arrest and mortality have beenreported in patients who underwent general anesthesiacompared with patients who underwent neuraxial anesthe-sia (7,10,14,23).

Many studies have found that respiratory and cardiovas-cular causes contribute equally to the cases of cardiac arrestand mortality that occur in children during anesthesia(16,27). In some studies, respiratory causes of cardiac arrestand mortality in children were more frequent thancardiovascular causes (7,11,14,20,23,25). In other studies,however, cardiovascular effects were reported to be themajor factor that contributed to anesthesia-related mortality(10,19,24,28,29). The administration of anesthetic drugs andinadequate blood management during hemorrhage andanemia were the most frequent causes of cardiovasculareffects (10).

DISCUSSION

There is no consensus in the literature regarding thedefinition of anesthesia-related mortality (30). Attributingdeath to anesthesia is difficult and often relies on thesubjective interpretation of various definitions. The NorthAmerican Pediatric Perioperative Cardiac Arrest (POCA)Registry classifies cardiac arrest as anesthesia-related ifanesthesia personnel or the anesthetic process played atleast some role (the role can range from minor to major) inthe cause of the cardiac arrest (10). The Australian and NewZealand College of Anaesthetists Mortality Committee hasclassified anesthesia-related mortality into 3 categories:category 1 includes cases in which it is reasonably certainthat death was caused by anesthesia or other factors underthe control of the anesthetist, category 2 is used when there

is some doubt as to whether death was entirely attributableto the anesthesia or other factors under the control of theanesthetist, and category 3 applies when death was causedby both surgical and anesthetic factors (31). The existence ofthese differences shows that there is an urgent need for aconsensus when defining ‘‘anesthesia-related mortality’’(30). Further improvements will depend on internationalmultispecialty efforts to standardize the terminology (32).

Other substantial differences in methodology make itdifficult to compare anesthesia-related mortality ratesamong various studies. In the studies in which periopera-tive and anesthesia-related mortality in children have beenexamined (3,5-23), the definition of deaths in whichanesthesia was the primary cause or a contributing causehave varied widely among the investigators, as did the timewindow that was considered to be the perioperative period.The perioperative period has been defined as intraoperativeonly (23), intraoperative through the recovery from anesthe-sia (6,10,14,15,17,21,22), the first 12 postoperative hours (18),the first 24 postoperative hours (8,9,11-13,19,20), two post-operative days (7), and 7-8 postoperative days (3,5,16).Although the majority of the studies of anesthesia-relatedmortality are cross-sectional (3,5-9,11-23), several studies arebased on voluntary declarations of critical incidents (10).Furthermore, the mortality rate may depend on the surgicalpediatric population. Several studies have examined alltypes of surgery (3,5,6,8,10-17,21,22), whereas other studieshave excluded procedures in ASA V patients (18), cardiacsurgery (7,20,23), eye surgery (7), or neurosurgery (20).Some studies have included children up to 12 years of age(12,13,15), whereas other studies have also includedchildren from 14 to 17 (3,5-7,9,11,14,17,18,20,21) or from 18to 20 years of age (8,10,19,23).

Despite these differences in methodology, studiesundertaken in a variety of countries, including Brazil,have suggested that anesthesia-related mortality rates inchildren are lower today than they were 50 years ago. Thepioneering study by Rackow et al. (15), which covered theperiod from 1947-1956, reported an anesthesia-relatedmortality rate in children of 2.9 per 10,000 anesthetics.Most studies that have been published over the pastdecade have reported an overall pediatric anesthesia-related mortality rate of less than 1 per 10,000 anesthetics,which represents a threefold improvement (6,8,18-20,22,23). One Brazilian study has confirmed this trend(i.e., zero anesthesia-related deaths per 10,000 anesthetics)(14). Multiple factors, including safer anesthetic agents,better monitoring devices and the development of aspecialized pediatric environment, have contributed tothis improvement. However, surveys of anesthesia-relatedmortality conducted in certain developing countriesbetween 2001 and 2011 have reported higher anesthesia-related mortality rates (i.e., 2.4 to 3.3 per 10,000 anes-thetics) (7,11). According to Lagasse (33), the highermortality rates that have been reported in these studiescould either represent real differences in anesthesia safetyor differences in the tools that were used to measureanesthesia-related mortality.

In contrast to the apparent decrease in anesthesia-relatedmortality in children over the last decade, the majority ofstudies conducted in developed (6,8,15,21) and developingcountries (7,11,22,23), including Brazil (14), have suggestedthat perioperative pediatric mortality has not decreasedin the last decade. Worldwide, even higher perioperative


CLINICS 2012;67(4):381-387

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(3,5-8,11,20,23) and anesthesia-related mortality rates (7,11)have been reported for neonates and infants. Prematurity,congenital heart disease, congenital neurological disease,and other congenital defects place neonates and infants at ahigher anesthesia risk compared with older children andadults (10). The incidence of respiratory events duringanesthesia is also higher in younger children (20). Thiseffect may be due to the relatively narrow infant airwayand the higher incidence of respiratory tract infections inyoung children (34,35). The findings of increased risk forchildren who are younger than 1 year of age (especiallychildren younger than 1 month) indicate the need forgreater caution when caring for children who are under 1year of age (30).

Poor ASA physical status ($ III) and emergency surgeryhave been reported as risk factors for pediatric perioperativemortality and are the only predictive factors of mortalityafter cardiac arrest (8,10). Morita et al. (3) found that mostincidents of perioperative cardiac arrest and death inneonates can be attributed to underlying comorbiditiesrather than causes related to the anesthesia. Children withheart disease exhibit higher rates of perioperative cardiacarrest and mortality when undergoing cardiac (8,25,26) ornoncardiac surgery (8,36).

In addition, with the advent of sophisticated extracorpor-eal cardiorespiratory support systems and specific medicaltherapies, children with highly complex medical problemswho were once considered unfit for anesthesia are increas-ingly submitted to surgery and other procedures, includingmedical imaging (8). Pre-existing morbidities, such assepsis, are also important factors in perioperative cardiacarrest in children both in Brazil (14) and in other developingcountries (7). Flick et al. (6) have reported an overallperioperative mortality rate for children in the United Statesof 6.8:10,000 anesthetics. In the Flick et al. study, themortality rate associated with cardiac surgery was 115.5 per10,000 anesthetics, whereas the mortality rate associatedwith noncardiac surgery was 1.6 per 10,000 anesthetics. Arecent study conducted in Australia, performed by van derGriend et al. (8), reported a high overall perioperativemortality rate in children (13.4 per 10,000 anesthetics). Forcardiac surgery, the associated mortality rate was 127.1 per10,000 anesthetics, and the rate for noncardiac surgery was8.2 per 10,000 anesthetics. The same authors proposed a newdefinition of anesthesia-related death that includes all casesin which it is more likely than not that factors that arerelated to anesthesia and are under the control of theanesthesiologist influenced the timing of death. Thisdefinition does not imply that care was inadequate ornegligent. The use of this definition may explain the higherpediatric mortality rates reported by van der Griend et al.(8) with a recent study in the USA (6). Indeed, it seems likelythat the exclusion of cardiac surgery patients (7,20,23) orchildren with a poor ASA physical status (18) has a sig-nificant impact on the estimated pediatric perioperativemortality rate. There were no anesthesia-related deaths inchildren without significant comorbidities (8). These resultsimply that the anesthesia-related mortality rate in healthychildren is not a great indicator for assessing the quality ofcare. The results also imply that when seeking routineconsent for anesthesia and when discussing anesthesia risk,the risk of death may not be a concern for children who donot have significant comorbidities. This statement, however,

requires some caution because anesthesia-related mortalitystill occurs in ASA physical status I-II children (7,11).

The POCA Registry was formed by the ASA in 1994 (10).This registry includes 63 participating institutions in theUSA and Canada, most of which are tertiary referralcenters. Each of the institutions in the registry submits astandardized data form for each case of cardiac arrest thatoccurs in a child younger than 18 years old during theintraoperative period or while the patient is in thepostanesthesia care unit. The POCA Registry has docu-mented that the medication-related cardiac arrest rate inchildren decreased from 37% between 1994 and 1997 to18% between 1998 and 2004 (24). This decrease wasattributed to the increased use of sevoflurane, which is ananesthetic that has been reported to have a lower potentialthan halothane for inducing bradycardia (37) and myocar-dial depression (38) in infants and children. According tothe POCA Registry, cardiovascular causes were the mostcommon causes of cardiac arrest and were associatedwith 41% of all cardiac arrests in the period between 1998and 2004. Hypovolemia from blood loss and hyperka-lemia from the transfusion of stored blood were themost common cardiovascular causes of cardiac arrest.Respiratory causes accounted for 27% of all cardiac arrests,with airway obstruction from laryngospasm being the mostcommon cause. In the present report, mortality afteranesthesia-related cardiac arrest was 28%, which wassimilar to the 26% that was initially reported by thePOCA Registry (10). As acknowledged by Morray et al.(10), however, the POCA Registry’s assessment of thefrequency of cardiac arrest in children during and aftersurgery has several methodological limitations. The mostsignificant is the potential for underreporting in thisvoluntary system. Selection bias is also likely, and moresensitive cases may not always be reported.

Mortality due to ventilation problems has decreased sincethe 1990s because of advanced respiratory monitoring andthe introduction of laryngeal mask airway devices forneonates and children. Interestingly, cardiovascular eventshave joined ventilatory problems as the major risk sourcesfor anesthesia-related pediatric cardiac arrest and death indeveloped countries (10,19,29). In the 2000s, respiratoryevents were still the main cause of anesthesia-attributablecardiac arrests in Brazil (14), other developing countries(7,11,23), and even in the USA (6).

In a study of 532 cases from the ASA’s Pediatric ClosedClaims, the proportion of claims due to death/braindamage and respiratory-related damaging events, particu-larly those associated with inadequate ventilation/oxyge-nation, decreased between 1973 and 2000 (29). However,claims due to death (47%) and brain damage (21%)constituted the largest proportion of the claims made inthe 1990s. During the last period, cardiovascular events(26%) joined respiratory events (23%) as the major sourcesof liability. This trend may be related to the increased useof pulse oximetry and end-tidal carbon dioxide monitoringand to the introduction of sevoflurane. The Jimenez et al.study also reported that an ASA physical status of III-IVwas associated with an increased frequency of claims dueto death and brain damage compared with claims for lesssevere injuries.

The incidence of perioperative cardiac arrest and mortal-ity in children is higher during general anesthesia comparedwith neuraxial anesthesia (7,10,14,23). Morray et al. (10)


385

examined data from the POCA Registry and observed thatin most cases of anesthesia-related cardiac arrest, onlygeneral anesthesia was administered (88%). This findingmay not be surprising because many pediatric high-risksurgeries, including cardiac, thoracic, and neurosurgicalprocedures, are performed under general anesthesia. Inaddition, improved knowledge of neuraxial block physiol-ogy and the use of newer, safer local anesthetics incombination with the routine use of oxygen according topulse oximetry values have decreased major complicationsduring neuraxial anesthesia.

A monitoring period in the postanesthesia care unit isnow mandatory following all types of anesthesia. For high-risk children, continued monitoring in a pediatric intensivecare unit may reduce anesthetic-related mortality. Theinability to provide or the failure to use these facilitiesmay increase anesthesia-related mortality rates.

Pediatric anesthesia requires trained pediatric anesthe-siologists or trainees who are supervised by a consultantpediatric anesthesiologist. Anesthesia-related mortality maydiffer between centers that primarily include staff who aretrained in pediatric anesthesiology and centers with fewerspecifically trained staff (8). In addition, continuing educa-tion for pediatric anesthesiologists is highly recommended.Indeed, one study demonstrated that the poor practicalapplication of techniques rather than a lack of knowledgecan lead to critical incidents (39).

The present review had some methodological weak-nesses. Because of the temporal limitations of the databases,it was difficult to verify that all of the relevant studies thatpredated the EMBASE (1951) and Medline databases (1966),and even certain studies from after these dates, wereconsidered.

Our systematic review of the literature confirms thatpediatric anesthesia-related mortality rates in Brazil over thelast decade are similar to those in developed countries.Higher pediatric anesthesia-related mortality rates over thesame time period have been found in some studies fromdeveloping countries. Pediatric perioperative mortality rateshave not decreased in the previous decade and are higher inBrazil and other developing countries compared withdeveloped countries. The major risk factors for anesthesia-related mortality are age (i.e., newborns or infants less than1 year old have the greatest risk), ASA III or worse physicalstatus, emergency surgery, general anesthesia, and cardiacsurgery. Airway management problems and cardiocircula-tory events accounted for the majority of the cases ofanesthesia-related mortality in children with comorbidities.These data may be useful in developing preventionstrategies to prevent anesthesia-related deaths in pediatricpatients.


Gonzalez LP and Braz JRC were responsible for the study design, the data

analysis, and manuscript writing. Pignaton W, Kusano PS, and Modolo

NSP were responsible for the data analysis and the approval of the final

version of manuscript. Braz LG was responsible for the study design, data

analysis, manuscript writing, and archiving of the study files.

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14. Braz LG, Braz JR, Modolo NS, do Nascimento P, Brushi BA, CarvalhoLR. Perioperative cardiac arrest and its mortality in children. A 9-yearsurvey in a Brazilian tertiary teaching hospital. Paediatr Anaesth.2006;16(8):860-6.

15. Rackow H, Salanitre E, Green LT. Frequency of cardiac arrest associatedwith anesthesia in infants and children. Pediatrics. 1961;28:697-704.

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19. Newland MC, Ellis SJ, Lydiatt CA, Peters KR, Tinker JH, Romberger DJ,et al. Anesthetic-related cardiac arrest and its mortality: a report covering72,959 anesthetics over 10 years from a US teaching hospital.Anesthesiology. 2002;97(1):108-15, http://dx.doi.org/10.1097/00000542-200207000-00016.

20. Murat I, Constant I, Maud’huy H. Perioperative anaesthetic morbidity inchildren: a database of 24,165 anaesthetics over a 30-month period.Paediatr Anaesth. 2004;14(2):158-66.

21. Cohen MM, Cameron CB, Duncan PG. Pediatric anesthesia morbidity andmortality in the perioperative period. Anesth Analg. 1990;70(2):160-7.

22. Tay CL, Tan GM, Ng SB. Critical incidents in paediatric anaesthesia: an auditof 10 000 anaesthetics in Singapore. Paediatr Anaesth. 2001;11(6):711-8.

23. Ahmed A, Ali M, Khan M, Khan F. Perioperative cardiac arrests inchildren at a university teaching hospital of a developing country over 15years. Paediatr Anaesth. 2009;19(6):581-6.

24. Bhananker SM, Ramamoorthy C, Geiduschek JM, Posner KL, DominoKB, Haberkern CM, et al. Anesthesia-related cardiac arrest in children:update from the Pediatric Perioperative Cardiac Arrest Registry. AnesthAnalg. 2007;105(2):344-50, http://dx.doi.org/10.1213/01.ane.0000268712.00756.dd.

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27. Salem MR, Bennett EJ, Schweiss JF, Baraka A, Dalal FY, Collins VJ.Cardiac arrest related to anesthesia. Contributing factors in infants andchildren. JAMA. 1975;233(3):238-41.

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29. Jimenez N, Posner KL, Cheney FW, Caplan RA, Lee LA, Domino KB. Anupdate on pediatric anesthesia liability: a closed claims analysis. AnesthAnalg. 2007;104(1):147-53, http://dx.doi.org/10.1213/01.ane.0000246813.04771.03.

30. Deshpande JK. Cause and effect or conjecture? A call for consensus ondefining "anesthesia-related mortality’’. Anesth Analg. 2011;112(6):1259-61.

31. Gibbs N (Ed.). Safety of anesthesia. A review of anesthesia – relatedmortality reporting in Australia and New Zealand 2003 – 2005.Melbourne: Australian and New Zealand College of Anesthetists, 2009.

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34. Parnis SJ, Barker DS, Van Der Walt JH. Clinical predictors of anaestheticcomplications in children with respiratory tract infections. PaediatrAnaesth. 2001;11(1):29-40.

35. Tait AR, Malviya S. Anesthesia for the child with an upper respiratorytract infection: still a dilemma? Anesth Analg. 2005;100(1):59-65, http://dx.doi.org/10.1213/01.ANE.0000139653.53618.91.

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37. Lerman J, Sikich N, Kleinman S, , Yentis. The pharmacology ofsevoflurane in infants and children. Anesthesiology. 1994;80(4):814-24,http://dx.doi.org/10.1097/00000542-199404000-00014.

38. Holzman RS, van der Velde ME, Kaus SJ, Body SC, Colan SD, SullivanLJ, et al. Sevoflurane depresses myocardial contractility less thanhalothane during induction of anesthesia in children. Anesthesiology.1996;85(6):1260-7, http://dx.doi.org/10.1097/00000542-199612000-00006.

39. Arbous MS, Meursing AE, van Kleef JW, de Lange JJ, Spoormans HH,Touw P, et al. Impact of anesthesia management characteristics on severemorbidity and mortality. Anesthesiology. 2005;102(2):257-68, http://dx.doi.org/10.1097/00000542-200502000-00005.


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REVIEW

Castration-resistant prostate cancer: systemictherapy in 2012Fernando C. Maluf,I Oren Smaletz,II Daniel HerchenhornIII

I Hospital Sao Jose, Beneficencia Portuguesa de Sao Paulo, Servico de Oncologia Clınica, Sao Paulo/SP, Brazil. II Hospital Israelita Albert Einstein,

Department of Oncology, Sao Paulo/SP, Brazil. III Instituto Nacional de Cancer, Department of Clinical Oncology, Brazil.

Prostate cancer is the most common non-cutaneous neoplasm in the male population worldwide. It is typicallydiagnosed in its early stages, and the disease exhibits a relatively indolent course in most patients. Despite thecurability of localized disease with prostatectomy and radiation therapy, some patients develop metastatic diseaseand die. Although androgen deprivation is present in the majority of patients with metastatic prostate cancer, a stateof androgen resistance eventually develops. Castration-resistant prostate cancer, defined when there is progression ofdisease despite low levels of testosterone, requires specialized care, and improved communication between medicaland urologic oncologists has been identified as a key component in delivering effective therapy. Despite beingconsidered a chemoresistant tumor in the past, the use of a prostate-specific antigen has paved the way for a newgeneration of trials for castration-resistant prostate cancer. Docetaxel is a life-prolonging chemotherapy that has beenestablished as the standard first-line agent in two phase III clinical trials. Cabazitaxel, a novel taxane with activity incancer models resistant to paclitaxel and docetaxel, is the only agent that has been compared to a chemotherapycontrol in a phase III clinical trial as a second-line therapy; it was found to prolong the overall survival of patients withcastration-resistant prostate cancer previously treated with docetaxel when compared to mitoxantrone. Other agentsused in this setting include abiraterone and sipuleucel-T, and novel therapies are continually being investigated in anattempt to improve the outcome for patients with castration-resistant prostate cancer.

KEYWORDS: Drug Therapy; Antineoplastic Agents; Prostate Neoplasms.

Maluf FC, Smaletz O, Herchenhorn D. Castration-resistant prostate cancer: systemic therapy in 2012. Clinics. 2012;67(4):389-394.

Received for publication on October 6, 2011; First review completed on October 18, 2011; Accepted for publication on December 11, 2011


Tel.: 55 113505-6611

INTRODUCTION

Prostate cancer is the most common non-cutaneousneoplasm in the male population worldwide (1). The vastmajority of cases are diagnosed in the early stages (2), and thedisease exhibits a relatively indolent course in most patients(3). In the United States, prostate cancer remains the mostcommon malignancy in men (2), despite the recent trend ofdecreasing mortality from the disease (4). Likely as a result ofthe early diagnosis through prostate-specific antigen (PSA)testing, the clinical behavior of prostate cancer, and the age ofpatients with this disease, there is a large difference betweenincidence and mortality rates from prostate cancer in theUnited States and Europe (2,5). Recently, prostate cancer hasbecome the most common cancer in Brazil, surpassing breastcancer with an estimated 52,000 new cases each year (6).

Despite the indolent course of the disease and thecurability of localized disease with prostatectomy andradiation therapy, some patients develop metastatic disease,frequently involving the bones and other organs (7). Oncemetastatic disease is diagnosed, the likelihood of dying fromprostate cancer surpasses death from other causes (8). For

these patients, treatment is performed with a palliativeintent, often involving androgen deprivation throughpharmacological or surgical orchiectomy. As a general rule,androgen deprivation is present in 80% to 90% of patientswith metastatic prostate cancer. These patients have amedian progression-free survival (PFS) ranging from 12 to30 months after treatment is initiated (9,10). However, astate of androgen independency eventually emerges, his-torically leading to a median overall survival (OS) of only 8to 16 months from the time of its appearance (9,10). Theterms ‘androgen-independent,’ ‘hormone-refractory’, and‘castration-resistant’ have been used interchangeably overthe years – not without some controversy (11) – to denotethe progression of disease despite castration levels oftestosterone (12). However, many recent studies and guide-lines in metastatic disease have used the term castration-resistant prostate cancer (CRPC) (13-16), which will be usedin the following review, based on the available therapeuticmodalities for patients whose disease progresses after theuse of standard hormone therapy.

DEFINING THE CASTRATION-RESISTANT STATE

Although most patients with metastatic prostate cancerinitially respond to androgen deprivation due to testoster-one dependence in prostate cancer cells, and despite the factthe secondary hormonal manipulations are active in some



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patients (17), prostate tumor cells eventually acquire thecapacity to survive and proliferate in an androgen-depletedenvironment (7,18). Mechanisms that underlie the transitionfrom an androgen-sensitive to an androgen-resistant phe-notype have been elucidated to some extent, and a variety ofcellular pathways are implicated in this phenomenon(7,9,18-20). As a result, androgen-receptor mutations andalterations in the androgen-signaling cascade are consideredto be responsible for the androgen-withdrawal responsethat is observed in a minority of patients being treated withantiandrogens (21).

In clinical practice, it is important to identify the patientswith metastatic prostate cancer that require treatment asopposed to those whose disease is only manifested by arising serum PSA level (22). Likewise, it is important todetermine when an initially sensitive disease is no longerresponsive to androgen deprivation, and improved com-munication between medical and urologic oncologists hasbeen identified as a key component in achieving this goal(23). There is anecdotal evidence that many patientscontinue to receive hormone therapy, despite the failure ofprevious treatments, before being referred to a medicaloncologist. For practical purposes, it is useful to considerpatients as having progressive CRPC if their diseaseprogresses during androgen-deprivation therapy, includingthe withdrawal of antiandrogens, and if at least 4-8 weekshave elapsed after the withdrawal of antiandrogens (24,25)The progression can be confirmed by a PSA elevation alone,a bone scan with measurable disease, or clinical progression(symptomatic progression). It should be noted that patientswith CRPC may benefit from continued androgen depriva-tion, as androgen-sensitive clones are thought to play a rolein disease progression after discontinuing hormone therapy(18).

FIRST-LINE TREATMENT OF CRPC

Historical development of and assessment ofresponse to chemotherapy

Until the late 1980s, prostate cancer was considered achemoresistant tumor (26). Several authors noted that theresponse rates to the available agents were typically low butalso varied widely in different studies (26-28). In addition,authors postulated that the documentation of responses inprostate cancer was complicated by a lack of establishedcriteria to assess the effects of the drugs, as nearly 80% ofpatients with CRPC have no measurable soft-tissue lesions(29). Thus, the response rates could only be determined inthe minority of patients with measurable disease before thePSA era. In the early 1990s, PSA became widely availableand was used in clinical trials as a measure of response(7,30). In 1999, a consensus conference suggested that adecline in PSA of at least 50% could represent a partialresponse in clinical trials as long as there was confirmationat least four weeks later and no clinical or radiographicevidence of disease progression (31). The use of PSA hasallowed for a new generation of trials of CRPC treatments,and PSA responses have been used as surrogates forobjective responses in this setting for early drug develop-ment (31). However, the PSA response has not beenvalidated as a surrogate for OS in androgen-sensitivedisease or in CRPC, and OS remains the most relevantendpoint in phase III clinical trials (32,33). In addition, othertime-dependent endpoints, such as PFS and the time to

tumor progression (TTP), have been increasingly used inclinical trials (12), and recent data from nearly 600 patientswith CRPC suggest that PSA progression is able to predictOS in CRPC (25). Of note, PSA levels are not independentpredictors of OS in CRPC when other clinical or laboratoryparameters are considered (34).

Several chemotherapeutic agents, such as the anthracy-clines, alkylating agents, antimetabolites, platinums, andtopoisomerase inhibitors, have been assessed in numerousphase II clinical trials over the years (26,27). In a review of 26different drugs before the PSA era, the average responserate was only 8.7%, but the combination of vinblastine plusestramustine was regarded as promising (27). This combi-nation was assessed in randomized trials, but the results atthe time did not establish a reference regimen, and toxicityremained a concern in the setting of palliative therapy fortypically elderly men (35,36). In combination, both mitox-antrone and low-dose prednisone had displayed modestsingle-agent activity and good tolerability in phase II clinicaltrials (37,38). In randomized trials, mitoxantrone and acorticosteroid relieved pain and improved the quality of lifemore frequently than with the same corticosteroid alone (39-41). Therefore, before the advent of docetaxel, mitoxantroneeventually became the reference chemotherapeutic agent forthe treatment of patients with CRPC (24). However, thisapproach was not associated with gain in OS (approxi-mately 12 months) or gains in quality of life, and improvedregimens were sought.

Docetaxel as the standard of care in the first-linetreatment of CRPC

After the demonstration of its single-agent activity (42,43),docetaxel was assessed in two phase III clinical trialspublished in 2004 (24,44). In the first study, 1,006 patientswith CRPC were randomized to receive mitoxantrone(12 mg/m2 every three weeks), docetaxel (75 mg/m2 everythree weeks), or weekly docetaxel (30 mg/m2), all combinedwith prednisone (5 mg twice daily) (24). As the primaryendpoint of the study, OS improved in both docetaxel armscompared to mitoxantrone; however, these improvementswere statistically significant when docetaxel was adminis-tered every three weeks (hazard ratio [HR] = 0.76; p = 0.009),but not weekly (HR = 0.91; p = 0.36). The median OS timeswere 16.5 months with mitoxantrone, 18.9 months withdocetaxel every three weeks, and 17.4 months with weeklydocetaxel. The secondary endpoints of predefined reduc-tions in pain (22% vs. 35% vs. 31%), PSA response (32% vs.45% vs. 48%), and improvements in quality of life (13% vs.22% vs. 23%) were all significantly superior for bothdocetaxel schedules. However, docetaxel led to moreadverse events than mitoxantrone – mainly neutropenia.In the second study, 674 eligible patients with CRPC wererandomized to receive estramustine (280 mg three timesdaily on days 1 through 5), docetaxel (60 mg/m2 on day 2),and dexamethasone (in three divided doses before doc-etaxel), or mitoxantrone (12 mg/m2) plus prednisone (5 mgof twice daily), both regimens given every three weeks (44).The median OS, the primary endpoint, was longer withdocetaxel and estramustine than with mitoxantrone (17.5months vs. 15.6 months; HR = 0.80; p = 0.02). Likewise, theTTP (median of 6.3 months vs. 3.2 months) and decline inPSA (50% vs. 27%) significantly favored the docetaxel-containing regimen, but it was also more frequentlyassociated with adverse events. The results of these two

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studies established docetaxel as the new standard agent forfirst-line treatment of CRPC, and the toxicity of this agentwas considered acceptable. However, the role of estramus-tine remained uncertain, and docetaxel every three weeksplus prednisone was accepted as a reference for futurestudies and for clinical practice.

SECOND-LINE TREATMENT OPTIONS FOR CRPC

CabazitaxelCabazitaxel, a novel member of the taxane class of

antimicrotubule agents, demonstrated effectiveness in pre-clinical models resistant to paclitaxel and docetaxel (45,46).Furthermore, cabazitaxel is able to cross the blood-brainbarrier, a potential advantage in the treatment of somemalignancies (47). On the basis of phase I and II clinicaltrials, the dose of cabazitaxel recommended for futurestudies ranged from 20 to 25 mg/m2, and the effects wereobserved in docetaxel-refractory prostate cancers and inother tumors (46,48). Thus, a phase III clinical trial waslaunched with the aim of comparing cabazitaxel tomitoxantrone in docetaxel-refractory CRPC (13). In thisstudy, 755 patients were treated with prednisone (10 mgdaily) and randomized to receive either cabazitaxel (25 mg/m2) or mitoxantrone (12 mg/m2) every three weeks, withOS as the primary endpoint. The median OS was 15.1months in the cabazitaxel group and 12.7 months in themitoxantrone group (HR = 0.70; p,0.0001). Cabazitaxel wasassociated with a higher incidence of adverse events thanmitoxantrone. The most common toxicities associated withcabazitaxel were neutropenia, anemia, and diarrhea. Ofnote, peripheral neuropathy was uncommon and typicallymild or moderate in severity (13). The results of this phaseIII clinical trial led to the approval of cabazitaxel for thesecond-line treatment of CRPC in many countries, includingBrazil. To date, cabazitaxel is the only agent that has beencompared with a chemotherapy control in a phase IIIclinical trial in this disease setting. Although not based onphase III data in the second-line setting, mitoxantrone waschosen as a control arm in this study due to its frequent usein the community practice. Recently after the Food andDrug Administration (FDA) approved cabazitaxel on June2010, the drug was approved in Brazil by the NationalAgency of Health Surveillance (ANVISA) to be adminis-tered in association with prednisone or prednisolone in thetreatment of docetaxel-refractory metastatic CPRC. Inaddition to the United States, cabazitaxel had beenapproved for marketing in Israel, Curacao and in theEuropean Union, along with Iceland, Lichtenstein andNorway.

AbirateroneAbiraterone is a selective inhibitor of androgen biosynth-

esis through its action on cytochrome P450 17 (17a-hydroxylase-17,20-lyase), the key enzyme in androgen andestrogen biosynthesis (49). Based on the evidence that CRPCremains sensitive to androgens derived from the adrenalgland or by endocrine synthesis, preclinical studies havesuggested that abiraterone is effective in CRPC (19).Moreover, a phase I/II clinical trial has found a PSAresponse in nearly two-thirds of the 42 patients withchemotherapy-naıve CRPC (50). In a phase III trial,abiraterone (1000 mg/day) was compared to placebo, bothcombined with prednisone (5 mg twice daily), in 1196

patients with docetaxel-refractory metastatic CRPP (51). Theprimary OS endpoint was significantly different betweenthe two groups: there was a 35% reduction in the risk ofdeath (HR = 0.65; p,0.0001) and a median OS of 14.8 monthswith abiraterone vs. 10.9 months with placebo. Secondaryefficacy endpoints, such as PFS, TTP, and PSA responses,consistently favored the abiraterone group, and the toxi-cities of this agent were primarily hypokalemia and fluidretention. Based on the results of this study, the FDAapproved abiraterone in combination with prednisone as atreatment for docetaxel-refractory metastatic CPRC patientsin April 2011.

Sipuleucel-TImmunological mechanisms likely influence the behavior

of prostate cancer and other malignancies. Sipuleucel-T, atype of therapeutic cancer vaccine, is able to elicit activeimmunologic cellular responses by autologous peripheral-blood mononuclear cells that have been activated ex vivowith a recombinant fusion protein consisting of prostaticacid phosphatase and the immune-cell activator granulo-cyte–macrophage colony-stimulating factor (14). The use ofsipuleucel-T involves harvesting peripheral blood mono-nuclear cells from the patient, culturing them with thefusion protein, and then infusing the antigen-presentingcells back into the patient. The combined analysis of tworelatively small randomized trials have shown that sipu-leucel-T produced a survival benefit in 225 patients withCRPC compared to those treated with placebo; it had anacceptable toxicity profile consisting mostly of chills, fever,and headache (52). Thus, a third placebo-controlled phaseIII clinical trial was launched, and 512 patients wererandomized in a 2:1 ratio to receive sipuleucel-T or placebointravenously every two weeks for a total of three infusions(14). The study revealed a 22% relative reduction in the riskof death with the use of sipuleucel-T (HR = 0.78; p = 0.03),which represented a 4.1-month improvement in the medianOS (25.8 months vs. 21.7 months in the placebo group).However, the TTP was similar in the two study groups; thisrelatively uncommon finding in medical oncology (i.e.,survival prolongation with no accompanying delay intumor progression) has been identified as surprising andworthy of further investigation, likely related to themechanism of action of the vaccine, which could changethe natural history of disease progression (52). Nevertheless,sipuleucel-T has been approved by the FDA for thetreatment of patients with asymptomatic or minimallysymptomatic CRPC.

OTHER AGENTS

Novel antitumor therapiesSeveral novel agents are currently being investigated for

the treatment of patients with CRPC (19). MDV3100 is anandrogen-receptor antagonist with no agonistic activity thatallows for the exploitation of the continued reliance onandrogen-receptor signaling by CRPC. In a phase I/IIclinical trial conducted in the United States, antitumoractivity was noted at all dosage levels with a PSA responsein 56% of 140 patients with CRPC (53). Such promisingresults have prompted the continued development of thisagent in larger trials. Bevacizumab, the anti-vascularendothelial growth factor antibody, has been added tostandard docetaxel therapies but has not been shown to

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improve the OS in patients with CRPC, despite animprovement in PFS; it was also associated with a highermorbidity and mortality than docetaxel alone in a largephase III clinical trial published in abstract form (54).

Bone-targeting agentsThere are several potential explanations for the marked

predisposition of prostate cancer to metastasize preferen-tially to bones (55). Both osteoblasts and osteoclasts appearto play a critical role in the interactions between prostatecancer cells and bone, and the receptor activator of nuclearfactor kappa-B ligand (RANKL) is a key element inosteoclastogenesis, bone resorption, and chemoattractionof tumor cells (56). The prominent role of bone metastases inthe natural history of metastatic prostate cancer hasprompted investigators to target this site of metastasis inhopes of palliating symptoms and prolonging survivalthrough the use of androgen ablation, bisphosphonates,radiopharmaceuticals, focal radiation, chemotherapy, andtargeted agents (55). Successful results from these effortshave included the demonstration that zoledronic acidreduces the rate of skeletal-related events in patients withCRPC (57) and that radiopharmaceuticals may have a roleas a consolidation therapy in patients treated with anti-tumor agents (58). More recently, targeting RANKL with themonoclonal antibody denosumab has been found to bemore efficacious than treatment with zoledronic acid interms of skeletal-related events in patients with CRPC, asobserved by a significant delay in the time to first andsubsequent on-study skeletal-related events (HR 0.82;p = 0.004) compared to zoledronic acid. Additionally, themedian time to the first on-study skeletal-related event was20.7 months for the denosumab group compared with 17.1months for the zoledronic acid group (59).

Bone-seeking agentsAlthough the use of bone-seeking radiopharmaceuticals is

currently approved for the palliation of bone pain, arandomized phase II study using sequential ketoconazoleplus doxorubicin and strontium-89 showed a prolongedsurvival (16.8 to 27.7 months) compared to chemotherapyalone (58). More recently, a phase I study evaluated theeffect of samarium-153 ethylenediamine tetramethylene-phosphonate (153Sm-EDTMP) administered repetitively ina docetaxel chemotherapy regimen in patients with castra-tion-resistant metastatic prostate cancer (60). The resultsfrom this study demonstrated that this combination led to agreater than 50% decline in prostate-specific antigen with-out significant bone marrow toxicity.

Endothelin receptor antagonistsThe endothelin pathway has been implicated in promot-

ing osteoblastic activity, a feature of bone metastases inprostate cancer (61). Two selective endothelin-A antagonistshave been evaluated in this population: atrasentan andzibotentan. Data from a study in 809 CRPC patientsdemonstrated that atrasentan (10 mg/day) did not reducethe TTP compared to placebo, although a significantdecrease in the molecular markers of disease progressionwas observed. The same result for the TTP was observedwith zibotentan, despite the improvement in OS, in a phaseII clinical trial. These preliminary findings highlight theneed for further evaluation in this group of patients.

CalcitriolThere is no consensus among clinical oncologists on the

utility of calcitriol in the treatment of CRPC, althoughpreclinical data have demonstrated it has potent antitumoractivity with antiproliferative, antiangiogenic, and apopto-sis-induction effects. Calcitriol also appears to synergisti-cally act with dexamethasone.

In 2010, Chadha et al. (62) published the results of a phaseII clinical trial using intravenous calcitriol and dexametha-sone in patients with CRPC. The results from this studyindicated that this combination did not produce a clinical orPSA response. More recently, a phase III study evaluatedthe efficacy of docetaxel plus high-dose calcitriol comparedto docetaxel plus prednisone (ASCENT 2) in 953 patientswith CRPC; it reported a higher number of deaths, shorteroverall survival and shorter duration of treatment in theASCENT arm compared to the control arm. These findingsled to the termination of the trial, and the clinicaldevelopment of the formulation of calcitriol used in thisstudy was discontinued (63,64).

CONCLUSIONS

The introduction of docetaxel in 2004 began a new era inthe management of CRPC. Over the past few years,additional progress has been made in the development ofnovel agents with activity against docetaxel-refractorydisease and with the potential to improve docetaxel-basedfirst-line therapies in the near future. At present, cabazitaxelis the only agent that has been compared to a chemotherapycontrol in a phase III clinical trial in patients with docetaxel-refractory CRPC, and sipuleucel-T is available in somecountries for asymptomatic patients. Abiraterone appears tobe a new treatment option for docetaxel-refractory CPRCpatients. Over the next few years, results from studies onthese and other novel agents will likely increase thetherapeutic arsenal used to treat CRPC.

Conflicts of interest: We certify the financial support bySanofi-aventis and disclose our interest statement. FernandoC. Maluf is a consultant for Janssen Cilag and Sanofi-aventis. Daniel Herchenhorn is an investigator in theTROPIC trial. Oren Smaletz is a recipient of honoraria as aspeaker and consultant for Sanofi-aventis and receivesresearch funding from Janssen-Cilag. All authors aremembers of the advisory board of Sanofi-Aventis. Thiswork represents the opinions of the authors.


Maluf FC conceived and designed the study, and was also responsible for

the manuscript writing, and final approval of the manuscript. Smaletz O

and Herchenhorn D were responsible for the manuscript writing and final

approval of manuscript.

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RAPID COMMUNICATION

DEFB1 gene 59 untranslated region (UTR)polymorphisms in inflammatory bowel diseasesValentina Zanin,I Ludovica Segat,II Anna Monica Bianco,II Lara Padovan,II Nathalia de Alencar Cunha

Tavares,III Sergio CrovellaIV

I University of Trieste, Institute for Maternal and Child Health – IRCCS ‘‘Burlo Garofolo’’, Trieste, Italy. II Institute for Maternal and Child Health – IRCCS

‘‘Burlo Garofolo’’- Trieste, Italy. III Federal University of Pernambuco, Laboratory of Immunopathology Keizo Asami (LIKA), Recife/PE, Brazil. IV Institute for

Maternal and Child Health - IRCCS ‘‘Burlo Garofolo’’, Trieste, University of Trieste, Italy.


Tel.: 39 0403785422

INTRODUCTION

Inflammatory bowel diseases (IBDs) are multifactorialdisorders resulting from an abnormal immune responsedriven by the presence of normal luminal flora. IBDs areassociated with the production of nonspecific mediators ofinflammation that initiate inflammatory processes andtissue destruction (1-3). Crohn’s disease (CD) and ulcerativecolitis (UC) are the two main forms of IBDs.

An accepted hypothesis for IBD pathogenesis is that anuncorrected balance between the host defenses and thecommensal microbiota in the gut of CD and UC patientsmight promote the disease by causing bacterial invasion,inflammation and loss of tolerance (4). A key role in the hostdefense is performed by the intestinal epithelium, whichacts as a physical barrier that limits the access of entericmicrobes that are able to produce endogenous antimicrobialpeptides (AMPs) (5).

Human defensins, which are classified as a-defensins (HDs)and b-defensins (HBDs) based on the arrangement of threedisulfide bridges (6), are antimicrobial peptides that representa wide spectrum of activity against pathogens. Human b-defensin-1 (hBD-1), the first described b-defensin, is character-ized by antimicrobial, chemotactic and immuno-enhancingactivities (7-9). The hBD-1 protein, which is encoded by theDEFB1 gene (8p23.1), is constitutively expressed by epithelialcells of a wide variety of tissues, but its expression can varybetween individuals and can be modified during theinflammatory process. A decrease in hBD-1 expression wasreported in the mucosa of CD and UC patients (10-12).

Impaired production of defensins appears to contribute tothe pathogenesis of IBDs (13,14), and a correlation betweenDEFB1 expression and single-nucleotide polymorphisms(SNPs) present in the regulatory region of the gene hasbeen reported (15,16). Therefore, we analyzed the possibleassociation of 59 untranslated region (UTR) DEFB1 SNPs,namely c.-52G.A (rs1799946) c.-44C.G (rs1800972) and c.-20G.A (rs11362), with the susceptibility to inflammatory

bowel diseases in a group of Italian IBD patients (CD andUC) and healthy control patients.

MATERIAL AND METHODS

Patients and controlsWe enrolled 145 patients with inflammatory bowel disease

(93 males, mean age at diagnosis 37.18¡14.11; 52 females,mean age at diagnosis 37.36¡16.35). Among the IBDpatients, 108 suffered from Crohn’s disease (mean age atdiagnosis 36.52¡14.01) and 37 suffered from ulcerativecolitis (mean age at diagnosis 38.24¡15.66). A total of 130healthy adult blood donors (63 M/67F, mean age 31,3¡12.36)with no history of IBDs and from the same ethnic origin as theIBD patients were recruited and used as controls.

The IRCCS Burlo Garofolo Ethical Committee approvedthe study (prot n. CIB 15/07, 03/03/2008).

The diagnosis of Crohn’s or UC disease was established byfollowing the European Crohn’s and Colitis Organisation(ECCO) Consensus guidelines for IBDs (https://www.ecco-ibd.eu/) (17,18) and the protocols of the InflammatoryBowel Disease and Functional Bowel Disorders Review Group(http://www.cochrane.uottawa.ca/ibd/default.htm) (19).

DEFB1 SNP genotypingGenomic DNA was extracted from peripheral whole blood

with the EZ1 DNA blood kit (Qiagen, Milan, Italy) followingthe manufacturer’s instructions. The three polymorphisms(rs1799946, rs1800972 and rs11362) in the 59UTR region of theDEFB1 gene were genotyped by direct sequencing with BigDye Terminator chemistry and an ABI 3130 DNA Sequencer(Applied Biosystems, Foster City, CA) using primers andprotocols previously described in the literature(20,21). Thesequences were analyzed with the 4peaks software (http://www.mekentosj.com/science/4peaks).

Statistical analysesThe allele and genotype frequencies were calculated by

direct gene counting and compared by the Fisher exact testusing 2X2 and 3X2 contingency tables. The haplotypefrequencies were obtained according to the EM algorithmusing the Arlequin software (22) (version 3.11). The oddsratios (O.R.) and 95% confidence intervals (C.I.) were alsocalculated. The Bonferroni correction for multiple tests wasapplied when required, and only corrected p,0.05 wereconsidered to be statistically significant. The R package(www.r-project.org) was used for all the statistical analyses.




395

RESULTS

No statistically significant differences were observed inthe distribution of the genotypic or allelic frequencies of theDEFB1 c.-52G.A and c.-44C.G SNPs among the CD, UCand healthy control subjects (Table 1). For the DEFB1 c.-20G.A SNP, no significant differences in the allelic orgenotype frequencies were observed between the UCpatients and the healthy controls. A slight difference wasobserved in the allelic distribution with a higher frequencyof the c.-20G.A G allele in the healthy subjects than in theCD patients (62% vs. 53%; uncorrected p = 0.04; O.R. = 0.676;95% C.I. = 0.461-0.992). Smaller differences were alsoobserved for the other genotypes. These differences, how-ever, did not retain statistical significance after Bonferronicorrection. The three DEFB1 SNPs were observed to be inlinkage disequilibrium (p,0.05, D’.0.7) in the CD, UC orcontrol subjects and combined into three major haplotypes(G/C/A; A/C/G; G/G/G) with a frequency .0.05 andthree minor haplotypes (A/G/G; G/C/G; A/C/A) with afrequency ,0.05 (Table 2). No statistically significantdifferences were observed when comparing the CD andUC patients with the controls.

For 73 of the 108 CD patients, a classification of the diseaseaccording to the bowel-disease anatomical localization was

also possible: 25 patients (34%) presented ileal CD, 30 (41%)presented ileocolonic CD and 18 (25%) presented colonic CD.The DEFB1 c.-52G.A G allele was more frequent in thepatients with ileal localization than in the healthy controlpatients (78% vs. 58%; uncorrected p = 0.010, O.R. = 2.552; 95%C.I. = 1.215-5.781), which was also observed with the GGgenotype (64% versus 30%, uncorrected p = 0.002; O.R. = 4.106;95% C.I. = 1.554-11.521).

The C allele of the DEFB1 c.-44C.G SNP was morefrequent in the patients with colonic localization than inthe healthy subjects (94% vs. 78%; uncorrected p = 0.037;O.R. = 4.773; 95% C.I. = 1.113-20.473), which was alsoobserved with the CC genotype (89% vs. 59%; uncorrectedp = 0.017; O.R. = 5.633; 95% C.I. = 1.242-52.507).

The DEFB1 c.-20G.A G allele was more frequent in thecontrol patients (62%) than in the colonic patients, (44%)(uncorrected p = 0.046; O.R. = 0.485; C.I. = 0.224-1.038), whichwas also observed with the GG genotype (p = 0.03, O.R. = 0.222;95% C.I. = 0.024-1.013).

However, the statistical significances of all the compar-isons were lost after applying Bonferroni’s correction.

When analyzing the haplotype frequencies, we observeda different distribution of the three major haplotypesbetween CD colonic patients and healthy control patients(uncorrected p = 0.039). This difference was even greater

Table 1 - Allele and genotype count (and frequencies) of 59UTR DEFB1 single-nucleotide polymorphisms in Crohn’sdisease patients, ulcerative colitis patients and healthy controls. The Crohn’s disease patients are also stratifiedaccording to the anatomical localization of the disease. The p-values ,0.05 (uncorrected), odds ratios (ORs) and 95% CIsare also reported.

DEFB1

SNPs CD Patients

UC

patients

Healthy

Controls

Ileal CD

n.25

Ileo-colonic

CD n.30

Colonic CD

n.18

total CD

n.108

total UC

n.37

total HC

n.30

-52G.A

Alleles

G 39/50 (0.78) # 34/60 (0.57) 21/36 (0.58) 137/216 (0.63) 44/74 (0.60) 151/260 (0.58)

A 11/50 (0.22) 26/60 (0.43) 15/36 (0.42) 79/216 (0.37) 30/74 (0.40) 109/260 (0.42)

Genotypes

GG 16/25 (0.64) ## 9/30 (0.30) 6/18 (0.33) 44/108 (0.41) 14/37 (0.38) 39/130 (0.30)

GA 7/25 (0.28) 16/30 (0.53) 9/18 (0.50) 49/108 (0.45) 16/37 (0.43) 73/130 (0.56)

AA 2/25 (0.08) 5/30 (0.17) 3/18 (0.17) 15/108 (0.14) 7/37 (0.19) 18/130 (0.14)

-44C.G

Alleles

C 39/50 (0.78) 49/60 (0.82) 34/36 (0.94) $ 179/216 (0.83) 59/74 (0.80) 203/260 (0.78)

G 11/50 (0.22) 11/60 (0.18) 2/36 (0.06) 37/216 (0.17) 15/74 (0.20) 57/260 (0.22)

Genotypes

CC 14/25 (0.56) 19/30 (0.63) 16/18 (0.89) $$ 71/108 (0.66) 22/37 (0.60) 76/130 (0.59)

CG 11/25 (0.44) 11/30 (0.37) 2/18 (0.11) 37/108 (0.34) 15/37 (0.40) 51/130 (0.39)

GG 0/25 0/30 0/18 0/108 0/37 3/130 (0.02)

-20G.A

Alleles

G 22/50 (0.44) 36/60 (0.60) 16/36 (0.44)* 114/216 (0.53)** 43/74 (0.58) 162/260 (0.62)

A 28/50 (0.66) 24/60 (0.40) 20/36 (0.56) 102/216 (0.47) 31/74 (0.42) 98/260 (0.38)

Genotypes

GG 4/25 (0.16) 9/30 (0.30) 2/18 (0.11)*** 25/108 (0.23) 12/37 (0.33) 47/130 (0.36)

GA 14/25 (0.56) 18/30 (0.60) 12/18 (0.67) 64/108 (0.59) 19/37 (0.51) 68/130 (0.52)

AA 7/25 (0.28) 3/30 (0.10) 4/18 (0.22) 19/108 (0.18) 6/37 (0.16) 15/130 (0.12)

# Ileal CD vs. HC, p = 0.01, O.R. = 2.552 (1.215-5.781);

## Ileal CD vs. HC, p = 0.002, O.R. = 4.106 (1.554-11.521);

$ Colonic CD vs. HC, p = 0.037, O.R. = 4.773 (1.113-20.473);

$$ Colonic CD vs. HC, p = 0.017, O.R. = 5.633 (1.242-52.507);*Colonic CD vs. HC, p = 0.046, O.R. = 0.485 (0.224-1.038);**CD vs. HC, p = 0.04, O.R. = 0.676 (0.461-0.992);***Colonic CD vs. HC, p = 0.03, O.R. = 0.222 (0.024-1.013).

DEFB1 polymorphisms in inflammatory bowel diseasesZanin V et al.

CLINICS 2012;67(4):395-398

396

when the minor haplotypes were also included in theanalysis (uncorrected p,0.001). (Table 2).

DISCUSSION

To our knowledge, only one study has previously investi-gated the role of DEFB1 SNPs in Crohn’s disease (23). Thefrequencies of the DEFB1 SNPs in CD patients reported byKocsis et al. are comparable to the frequencies reported by ourstudy. Moreover, similar to our study, the authors failed toobserve any association between the c.-52G.A and c.-20G.ADEFB1 SNPs and the overall susceptibility to CD. For the c.-44C.G SNP, they observed a different distribution betweenthe CD and control patients with an increased frequency of thec.-44C.G GG genotype in the control patients (12%) com-pared with the CD patients (4%). This difference was notobserved in our study; however, none of the CD patients inour study presented the c.-44C.G GG genotype, whereasthree (2%) of the control patients, which were matched for sex,age and ethnicity, presented the genotype.

In addition, Kocsis et al. (23) reported several significantassociations between the DEFB1 SNPs and the anatomicallocalization of Crohn’s disease (ileal, ileocolonic or colonic).

For the DEFB1 c.-44C.G SNP, Kocsis et al. observed asignificant increase in the CC genotype in ileocolonicpatients and to a lesser extent in colonic but not ilealpatients compared with healthy control patients. Our resultsindicate an increased frequency of the CC genotype inthe colonic patients (89% vs. 59% in control patients).Additionally, a slight increase in the CC genotype frequencywas observed in the ileocolonic patients (63% vs. 59% incontrol patients), but not in ileal.

Kocsis et al. (23) also showed that the GG genotype (c.-20G.A SNP) was more frequent in control subjects than inpatients with colonic CD. Our results appear to confirmthese findings. Because colonic CD localization has beenassociated with impaired hBD-1 expression and because thec.-20G.A A allele appears to be associated with reducedlevels of DEFB1 expression, it can be hypothesized that thispolymorphism might cause lower hBD-1 expression incolonic epithelial cells.

For c.-52G.A, Kocsis et al. (23) did not report anydifference between patients with different localization of CDand controls. Conversely, we observed that the GG

genotype was more frequent in the ileal CD patients (64%)than in the controls (30%), but the difference was notstatistically significant.

The constitutive expression of hBD-1, which is known tobe a constitutively expressed peptide, has already beenreported in CD and UC patients. Moreover, stressful eventscould regulate DEFB1 mRNA levels, (24-27) which could berelevant for the intestinal mucosa. The presence of 59UTRDEFB1 SNPs may be able to explain interindividual changesin DEFB1 expression, and these differences may be able toaccount for different susceptibility to diseases (such as CD).However, the mechanism by which the same DEFB1 SNPcan be associated with the development of the disease in aspecific anatomical region and not in others remainsunknown. Variations in hBD-1 production among differentintestinal traits of CD and UC patients are more likely toresult from the actions of other genes or factors involved inthe regional regulation of the hBD-1 peptide (24-27) ratherthan as a direct consequence of a single SNP or acombination of SNPs to form haplotypes.

We are aware that the loss of statistical significance of thep-values after correcting for multiple tests comparisons,which is possibly due to the limited number of samplesanalyzed, is likely the major weakness of this study.However, it is noteworthy that our results confirm thefindings previously reported by another research group(23), which indicate that DEFB1 may a contributing factor tosusceptibility to Crohn’s disease.

ACKNOWLEDGMENTS

Grants RC03/04 from IRCCS Burlo Garofolo Trieste (Italy) and APQ-

1130-2.02/08 (FACEPE, Recife, Brazil) supported this study. SC is the

recipient of a fellowship grant from the European Project ‘‘Talents for an

International House’’ within the framework of the 7th Research &

Development Framework Programme PEOPLE – Marie Curie Actions –

COFUND (Co-Funding of Regional, National and International

Programmes).


Zanin V, Bianco AM and Padovan L collected the biological samples and

extracted the DNA. Crovella S designed the study. Crovella S and Segat L

redacted the manuscript and performed statistical analysis. Tavares CN

and Zanin V performed the DEFB1 genotyping.

Table 2 - Haplotype counts (and frequencies) of 5’UTR DEFB1 polymorphisms in Crohn’s disease patients, ulcerativecolitis patients and healthy controls. The Crohn’s disease patients are also stratified according to the anatomicallocalization of the disease. The p-values (uncorrected) ,0.05 are also indicated.

DEFB1 Haplotypes

-52G.A/-44C.G/-20G.A CD patients UC patients

Healthy

Controls

Ileal CD

n.25

Ileo-colonic

CD n.30

Colonic CD

n.18

total CD

n.108

total UC

n.37

total HC

n.30

Major (freq.0.05)

G/C/A 28/50 (0.56) 23/60 (0.38) 19/36 (0.53)#* 100/216 (0.46) 30/74 (0.41) 112/260 (0.43)

A/C/G 11/50 (0.22) 25/60 (0.42) 13/36 (0.36)#* 76/216 (0.35) 28/74 (0.39) 99/260 (0.38)

G/G/G 11/50 (0.22) 11/60 (0.18) 1/36 (0.02)#* 36/216 (0.17) 14/74 (0.19) 50/260 (0.19)

Minor (freq,0.05)

A/G/G 0 0 1/36 (0.02)* 1/216 (0.004) 1/74 (0.01) 0

G/C/G 0 0 1/36 (0.02)* 1/216 (0.004) 0 0

A/C/A 0 1/60 (0.01) 1/36 (0.02)* 2/216 (0.009) 1/74 (0.01) 0

# Colonic CD vs. HC major haplotypes, p = 0.039.*Colonic CD vs. HC major+minor haplotypes, p = 0.0004595.

CLINICS 2012;67(4):395-398 DEFB1 polymorphisms in inflammatory bowel diseasesZanin V et al.

397

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12. Wehkamp J, Harder J, Weichenthal M, Mueller O, Herrlinger KR,Fellermann K, et al. Inducible and costitutive-defensis are differentiallyexpressed in Crohn’s disease and ulcerative colitis. Inflammatory BowelDisease. 2003;9(4):215-23.

13. Ramasundara M, Leach ST, Lemberg DA, Day AS. Defensins andinflammation: the role of defensins in inflammatory bowel disease.J Gastroenterol Hepatol. 2009;24(2):202-8, http://dx.doi.org/10.1111/j.1440-1746.2008.05772.x.

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15. Milanese M, Segat L, Crovella S. Transcriptional effect of DEFB1 gene 59

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CLINICS 2012;67(4):395-398

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TECHNICAL NOTE

Assessment of organ transplants from donors withmarkers of hepatitis BEdson Abdala,I Luis Sergio Fonseca de Azevedo,II Vivian Iida Avelino-Silva,III,IV Sılvia Figueiredo Costa,III

Marlova Luzzi Caramori,V Tania Mara Varejao Strabelli,VI Lıgia Camera Pierrotti,IV Heloisa Helena Marques

da Silva,VII Marta Heloisa Lopes,III Glaucia Fernanda Varkulja,VIII Vera Aparecida Santos,IX Maria Aparecida

Shikanai-Yasuda,III Comissao de Infeccao em Imunodeprimidos, Hospital das Clınicas da Faculdade de

Medicina da USPI Hospital das Clınicas da Faculdade de Medicina da Universidade de Sao Paulo (HCFMUSP), Liver Transplant Service, Sao Paulo/SP, Brazil. II Hospital das

Clınicas da Faculdade de Medicina da Universidade de Sao Paulo (HCFMUSP), Kidney Transplant Unit, Urology Division, Sao Paulo/SP, Brazil. III Faculdade

de Medicina da Universidade de Sao Paulo (FMUSP), Department of Infectious and Parasitic Diseases, Sao Paulo/SP, Brazil. IV Hospital das Clınicas da

Faculdade de Medicina da Universidade de Sao Paulo (HCFMUSP), Clinical Division of Infectious Diseases, HCFMUSP Sao Paulo/SP, Brazil. V Instituto do

Coracao do Hospital das Clınicas da Faculdade de Medicina da Universidade de Sao Paulo (INCOR-HCFMUSP), Lung Transplant Unit, Sao Paulo/SP, Brazil.VI Instituto do Coracao do Hospital das Clınicas da Faculdade de Medicina da Universidade de Sao Paulo (INCOR-HCFMUSP), Heart Transplant Area, Sao

Paulo/SP, Brazil. VII Hospital das Clınicas da Faculdade de Medicina da Universidade de Sao Paulo (ICr-HCFMUSP), Instituto da Crianca, HCFMUSP Sao Paulo/

SP, Brazil. VIII Hospital das Clınicas da Faculdade de Medicina da Universidade de Sao Paulo (ICHC-HCFMUSP), Infection Control Committee of the Instituto

Central, Sao Paulo/SP, Brazil. IX Hospital das Clınicas da Faculdade de Medicina da Universidade de Sao Paulo (ICHC-HCFMUSP), Division of the Central

Laboratory, Sao Paulo/SP, Brazil.


Tel.: 55 11 3061-7048

Organ transplantation (TX) is currently a therapeuticalternative in the management of end-stage or lethaldiseases and, in some cases, for improving the quality oflife and reducing the complications of chronic conditions.The scarcity of organ grafts or donors due to the limitednumber of deceased donors or to a lack of compatible livingdonors is one of the main limitations for the execution oforgan TX. In Brazil, there is a great disparity between thenumber of patients on waiting lists and TX procedures,especially for liver and kidney TX. As a result of thisinequality, grafts from donors who are considered marginalor not ideal have been used, including those with higherrisk of failure following TX or those with potentiallytransmissible infections, involving donors with positiveserologic markers for the hepatitis B virus (HBV).

Donors who are positive for HBV markers have beenroutinely used in some TX centers. Four aspects should beconsidered for the assessment of the risk-benefit ratio of thisprocedure: 1. donor serologic profile, 2. recipient serologicprofile, 3. TX variety and 4. the use of preventive therapywith human-specific HBV immunoglobulin and/or antiviraldrugs. To establish recommendations, our group helddiscussions based on the data available in the medicalliterature and on the accumulated experience of the TX unitsat Hospital das Clınicas da Faculdade de Medicina daUniversidade de Sao Paulo. The definitions of serologicprofiles for donors and recipients are described in Tables 1and 2, respectively. The following transplants have beenevaluated: liver, kidney, heart, lung, and hematopoieticstem cell transplants (HSCT). The IDSA (Infectious Diseases

Society of America) rating system was used to assess thequality of evidence for graft acceptance and to identify theappropriate preventive strategy.

1. Liver TX

HBsAg-positive donor: few reports are available, underparticular circumstances, and there is a high risk of ‘‘denovo’’ HBV infection.

HBc-Ab-positive donor:

N HBsAg-positive recipient: regardless of the donor’sserologic profile, this recipient must be administeredcombined prophylaxis with HBIG and an antiviral drug.The risk of viral reactivation does not seem to be increasedby the involvement of an HBc-Ab-positive donor.

N Recipients who are positive for HBc-Ab and HBs-Ab:there are no reports of ‘‘de novo’’ HBV infection, with orwithout prophylaxis.

N Recipients with isolated HBc-Ab or a history of vaccina-tion: the risk of ‘‘de novo’’ HBV is reduced in case seriesthat described lamivudine and/or HBIG prophylaxis.


Table 1 - Definitions of HBV serologic profiles – Donors.

HBc-Ab* HBs-Ab** HBsAg*** Definition

+ - + Positive HBsAg

+ - -

+ + -

Positive HBc-Ab

*anti-core hepatitis B antibody,**anti-hepatitis B surface antigen antibody,***hepatitis B surface antigen.


399

N Naıve recipient: group at highest risk; case series thatdescribed lamivudine and HBIG prophylaxis havedemonstrated a reduction in risk.

2. Kidney TX

The largest case series of donors with positive serologicmarkers for HBV were published in kidney TX. However,some studies describe only the post-TX clinical course andfail to assess HBV serology.

HBsAg-positive donor: among 48 donors, ‘‘de novo’’ HBVinfection has been detected in three recipients with variablepre-TX serologic profiles, while liver enzyme elevation hasbeen detected in nine recipients.

HBc-Ab-positive donor:

N HBsAg-positive recipient: when lamivudine is used aspreventive therapy, the clinical evolution is similar to thatof recipients whose donors have negative HBV serology.

N Recipients with isolated HBc-Ab: without any prophy-laxis, the seroconversion of HBsAg has been observed in0.5% of recipients after TX. No reports have describedseroconversion under lamivudine prophylaxis.

N Vaccinated or Naıve recipient: after TX, without anyprophylaxis, seroconversion of HBc-Ab occurs in 2% ofcases, and seroconversion of HBsAg occurs in less than0.5% - without any impact on the clinical course. Noreports have described seroconversion under lamivu-dine prophylaxis.

3. Heart TX

There are two case series on heart TX that reportlamivudine prophylaxis in some recipients. Only one caseof ‘‘de novo’’ HBV infection is reported in a naıve recipientwith an HBsAg-positive donor.

4. Lung TX

In two small case series of HBc-Ab-positive donors, nocases of recipient post-TX seroconversion were described.Among the two case series, one involved lamivudineprophylaxis.

5. Hematopoietic Stem Cell Transplant

In HSCT, the most relevant consideration is the risk ofreverse seroconversion (loss of HBs-Ab after TX).

HBsAg-positive donor: in a study of HBsAg-negativerecipients with a historical control group, the risk of ‘‘denovo’’ HBV was significantly reduced with lamivudineprophylaxis.

HBc-Ab-positive donor: when the bone marrow donor isnaturally immunized (HBc-Ab and HBs-Ab positive), thereis an unmistakable reduction in the risk of reverseseroconversion.

Assessment of organ transplants from donors withhepatitis B-positive markers

Recommendations:

A. General recommendations

1. Potential TX recipients should receive HBV vaccina-tion before TX, if possible (AII).

2. HSCT donors should receive HBV vaccinationbefore TX, if possible (BII).

3. HSCT recipients who are negative for HBsAg mustbe directed to HBV vaccination, starting six monthsafter TX (BIII).

4. Liver TX recipients who are HBsAg positive must beprescribed an antiviral drug and HBIG as preventivetherapy, regardless of the donor’s serologic profile,due to the risk of post-TX recurrence (AII).

5. TX recipients (except for those undergoing liver TX)who are HBsAg positive must be prescribed anantiviral drug, due to the risk of viral replicationfollowing TX (AII).

6. HSCT donors who are HBsAg positive with activeviral replication (positive polymerase chain reaction)must start on antiviral treatment before TX (BIII).

B. Specific recommendations:

1. Liver TX (B1).

2. Kidney TX (B2).

3. Heart and Lung TX (B3).

4. Hematopoietic Stem Cell Transplantation TX (B4).

Table 2 - Definitions of HBV serologic profiles –Recipients.

HBc-Ab* HBs-Ab** HBsAg*** Definition

- - - Naıve

+ - + Positive HBsAg

- + - Isolated HBs-Ab/Vaccinated

+ - - Isolated HBc-Ab

+ + - HBc-Ab and HBs-Ab

*anti-core hepatitis B antibody,**anti-hepatitis B surface antigen antibody,***hepatitis B surface antigen.

B1. Liver TX: Specific Recommendations

Donor Recipient

HBsAg + HBc-Ab+ HBs-Ab+ HBc-Ab- HBs-Ab+ Isolated HBc-Ab+ Naıve

HBsAg+ No (DII) No (DII) No (DII) No (DII) No (EI)

HBc-Ab+ Yes (BII) Yes (BII) Yes (BIII) Yes (BIII) No (DII)

Prophylaxis LAM + HBIG* (AII) LAM (BIII)** LAM (BIII)** LAM (BIII)** _

LAM = lamivudine*HBIG for one year, lamivudine indefinitely; **For at least one year.

Obs.: lamivudine may be replaced by an alternative antiviral.

Transplantation involving donors with Hepatitis BAbdala E et al.

CLINICS 2012;67(4):399-404

400

ACKNOWLEDGMENTS

We thank the Clinical Directors from Hospital das Clınicas da Faculdade

de Medicina da Universidade de Sao Paulo (Prof. Jose Otavio Costa Auler

Junior, Prof. Tarcısio Eloi Pessoa de Barros Filho and Prof. Eloısa Bonfa)

for all their support.


Abdala E wrote the manuscript (Portuguese), supervised the revision that

addressed the recommendation for liver transplantation, participated in the

discussion of the final recommendations and revised the English version of

the text. Azevedo LSF presented the kidney transplantation recommenda-

tion and discussed the final recommendations. Avelino-Silva VI presented

the liver transplantation recommendation and translated the text into

English. Costa SF, Strabelli TMV, Caramori ML presented the

recommendation for bone marrow, heart and lung transplantation,

respectively, and discussed the final recommendations. Pierrotti L,

Marques da Silva HH, Lopes MH, Varkulja GF, Santos VA participated

in the discussion of the recommendations. Shikanai-Yasuda MA

coordinated the presentations and discussion of the recommendations,

helped revise the final recommendations and to prepare the manuscript for

submission.

Conflicts of interests: Edson Abdala - clinical research with Bristol.

Heloisa Helena de Souza Marques - clinical research with Boehringer

Ingelheim. Tania Mara Varejao Strabelli - speaker of Novartis, works with

Novartis, clinical research with Merck

REFERENCES

1. Akalin E, Ames S, Sehgal V, Murphy B, Bromberg JS. Safety of usinghepatitis B core antibody or surface antigen-positive donors in kidney orpancreas transplantation. Clin Transplant. 2005;19(3):364-6, http://dx.doi.org/10.1111/j.1399-0012.2005.00350.x.

2. Barcena R, Moraleda G, Moreno J, Martın MD, de Vicente E, Nuno J, et al.Prevention of de novo HVB infection by the presence on anti-HBs intransplanted patients receiving core antibody-positive livers.World J Gastroenterol. 2006;12(13):2070-4.

3. De Feo TM, Grossi P, Poli F, Mozzi F, Messa P, Minetti E, et al. Kidneytransplantation from anti-HBc+ donors: results from a retrospectiveItalian study. Transplantation. 2006;81(1):76-80, http://dx.doi.org/10.1097/01.tp.0000189930.89031.1b.

4. Fong TL, Bunnapradist S, Jordan SC, Cho YW. Impact of hepatitis B coreantibody status on outcome of cadaveric renal transplantation: analysisof United network of organ sharing database between 1994 and 1999.Transplantation. 2002;73(1):85-9, http://dx.doi.org/10.1097/00007890-200201150-00016.

5. Gallegos-Orozco JF, Vargas HE. Should antihepatitis B virus corepositive or antihepatitis C virus core positive subjects be accepted as

B4. Hematopoietic Stem Cell Transplantation: Specific Recommendations

Donor Recipient

HBsAg+HBc-Ab+HBs-Ab+

HBc-Ab-

HBs-Ab+Isolated

HBc-Ab+ Naıve

HBsAg+ Yes (BII) Yes (BII) Yes (BII) Yes (BII) Special circumstances

(CIII)

Prophylaxis LAM (AII)* LAM (AII)** LAM (AII)** LAM (AII)** LAM (AII)**

HBc-Ab+ HBs-Ab- Yes (BII) Yes (BII) Yes (BII) Yes (BII) Yes (BII)

Prophylaxis LAM (AII)* *** *** *** ***

HBc-Ab+ HBs-Ab+ Yes (AII) Yes (AII) Yes (AII) Yes (AII) Yes (AII)

Prophylaxis LAM (AII) # - - -

*Indefinitely; **For at least one year; ***Request donor PCR – If positive, treat as if donor HBsAg+; if negative, treat as if donor HBc-Ab+ and HBs-Ab+;

#Follow up with serologic assessment every three months.


B2. Kidney TX: Specific Recommendations

Donor Recipient


HBc-Ab-

HBs-Ab+Isolated

HBc-Ab+ Naıve

HBsAg+ Exceptional

circumstances* (CIII)

Exceptional


Exceptional


Exceptional


No (DIII)

Prophylaxis LAM (AII)** LAM (BIII)*** LAM (BIII)*** LAM (BIII)*** -

HBc-Ab+ Yes (BII) Yes (BII) Yes (BII) Yes (BII) No (DIII)

Prophylaxis LAM (AII)** LAM (CIII)**** LAM (CIII)**** LAM (CIII)**** _

*Exceptional circumstances, defined by the TX group; **Indefinitely; ***For at least one year; ****Optional use.


B3. Heart and Lung TX: Specific Recommendations

Donor Recipient


HBc-Ab-

HBs-Ab+Isolated

HBc-Ab+ Naıve

AgHBs+ Exceptional


Exceptional


Exceptional


Exceptional


No (DIII)

Prophylaxis LAM (AII)** LAM (BIII)*** LAM (BIII)*** LAM (BIII)*** -

Anti-HBc+ Yes (BII) Yes (BII) Yes (BII) Yes (BII) No (DIII)

Prophylaxis LAM (AII)** LAM (CIII)*** LAM (CIII)*** LAM (CIII)*** -

*Exceptional circumstances, defined by the TX group; **Indefinitely; ***For at least one year.


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organ donors for liver transplantation? J Clin Gastroenterol.2007;41(1):64-74.

6. Avelino-Silva VI, D’Albuquerque LA, Bonazzi PR, Song AT, Miraglia JL,De Brito Neves A, et al. Liver transplant from Anti-HBc-positive,HBsAg-negative donor into Hbs-Ag-negative recipient: is it safe? Asystematic review of the literature. Clin Transplant. 2010;24(6):735-46,doi: 10.1111/j.1399-0012.2010.01254.x.

7. Gross PA, Barrett TL, Dellinger EP, Krause PJ, Martone WJ, McGowan JEJr, et al. Purpose of quality standards for infectious diseases: InfectiousDiseases Society of America. Clin Infect Dis. 1994;18(3):421, http://dx.doi.org/10.1093/clinids/18.3.421.

8. Hartwig MG, Patel V, Palmer SM, Cantu E, Appel JZ, Messier RH, et al.Hepatitis B core antibody positive donors as a safe and effectivetherapeutic option to increase available organs for lung transplantation.Transplantation. 2005;80(3):320-5, http://dx.doi.org/10.1097/01.tp.0000165858.86067.a2.

9. Hui CK, Lie A, Au WY, Ma SY, Leung YH, Zhang HY, et al. Effectivenessof prohylatic anti-HBV therapy in allogeneic hematopoietic stem celltransplantation with HbsAg positive donors. Am J Transplant.2005;5(6):1437-45, http://dx.doi.org/10.1111/j.1600-6143.2005.00887.x.

10. Idilman R, Ustun C, Karayalcin S, Aktemel A, Turkyilmaz AR, Ozcan M,et al. Hepatitis b virus vaccination of recipients and donors of allogeneicperipheral blood stem cell transplantation. Clin Transplant.2003;17(5):438-43, http://dx.doi.org/10.1034/j.1399-0012.2003.00070.x.

11. Ko WJ, Chou NK, Hsu RB, Chen YS, Wang SS, Chu SH, et al. Hepatitis Bvirus infection in heart transplant recipient in a hepatitis B endemic area.J Heart Lung Transplant. 2001;20(8):865-75, http://dx.doi.org/10.1016/S1053-2498(01)00280-7.

12. No authors listed. Screening of donor and recipient prior to solid organtransplantation. Am J Transplant. 2004;4(Suppl 10):10-20.

13. Pinney SP, Cheema FH, Hammond K, Chen JM, Edwards NM, ManciniD. Acceptable recipients outcomes with the use of hearts from donorswith hepatitis-B core antibodies. J Heart Lung Transplant. 2005;24(1):34-7, http://dx.doi.org/10.1016/j.healun.2003.09.036.

14. Prakoso E, Strasser SI, Koorey DJ, Verran D, McCaughan GW. Long-termlamivudine monotherapy prevents development of hepatitis B virusinfection in hepatitis B surface-antigen negative liver transplantrecipients from hepatitis B core-antibody-positive donors. ClinTransplant. 2006;20(3):369-73, http://dx.doi.org/10.1111/j.1399-0012.2006.00495.x.

15. Rubin RH, Schaffner A, Speich R. Introduction to the Immuno-compromised Host Society Consensus Conference on Epidemiology,Prevention, Diagnosis, and Management of Infections in Solid-OrganTransplant Recipients. Clin Infect Dis. 2001;33(Suppl1):S1-S4, http://dx.doi.org/10.1086/320896.

16. Shitrit AB, Kramer MR, Bakal I, Morali G, Ben Ari Z, Shitrit D.Lamivudine prophylaxis for hepatitis B virus infection after lungtransplantation. Ann Thorac Surg. 2006;81(5):1851-2, http://dx.doi.org/10.1016/j.athoracsur.2005.12.026.

17. [No authors listed]. Preface to the 1997 USPHS/IDSA guidelines for theprevention of opportunistic infections in persons infected with humanimmunodeficiency virus. USPHS/IDSA Prevention of OpportunisticInfections Working Group. US Public Health Service/Infectious DiseasesSociety of America. Clin Infect Dis. 1997;25(Suppl3):S299-S312.

APPENDIX

USO DE DOADORES COM MARCADORSOROLOGICO PARA VIRUS DA HEPATITE B EMTRANSPLANTES

O transplante (tx) consiste, atualmente, em alternativaterapeutica para doencas terminais e fatais ou, em algunscasos, para melhoria da qualidade de vida e diminuicao dosriscos de complicacoes de doencas cronicas. Uma dasprincipais limitacoes atuais para a realizacao de tx e aescassez de orgaos ou de doadores, por dificuldades nacaptacao de doadores falecidos ou pela indisponibilidade dedoadores vivos compatıveis. Ha, no Brasil, um grandenumero de pacientes em lista de espera, especialmente paratx de fıgado e de rim, em relacao ao numero deprocedimentos realizados. Como consequencia destas limit-acoes, doadores considerados nao ideais tem sido utiliza-dos, incluindo-se aqueles cujo enxerto apresenta maior riscode nao funcionamento apos o transplante e doadores comrisco potencial de transmissao de alguma infeccao. Entreestes ultimos encontram-se aqueles com marcador sorolo-gico para o vırus da hepatite B (VHB).

Doadores com algum marcador sorologico para VHB temsido utilizados como rotina por alguns grupos. A relacaorisco-benefıcio desta medida e dependente de quatrofatores: 1. Estado sorologico do doador, 2. Estado sorologicodo receptor, 3. Tipo de transplante e 4. Uso de profilaxiacom imunoglobulina especıfica e/ou droga antiviral. Com o

objetivo de estabelecer uma recomendacao em nosso meio,realizamos discussoes baseadas nos dados disponıveis naliteratura e na experiencia dos grupos de transplante doHospital das Clınicas da Faculdade de Medicina daUniversidade de Sao Paulo. As definicoes dos perfissorologicos possıveis para doador e receptor encontram-senas Tabelas 1 e 2, respectivamente. Foram analisados osseguintes transplantes: fıgado, rim, coracao, pulmao etransplante de celulas-tronco hematopoieticas (TCTH).Para a determinacao dos nıveis de evidencia para aceitar oenxerto e indicar profilaxia foi utilizado o sistema da IDSA(Infectious Diseases Society of America).

1. Transplante de Fıgado

Doador AgHBs positivo: existem poucos relatos, sobcircunstancias especiais. Risco de desenvolvimento dehepatite B ‘‘de novo’’ muito alto.

Doador Anti-HBc positivo:

N Receptor AgHBs positivo: este receptor recebe, inde-pendentemente do estado sorologico do doador,profilaxia combinada com imunoglobulina especıfica(HBIG) e antiviral. Aparentemente, o risco de reativa-cao nao aumenta nos casos de doadores Anti-HBcpositivos.

N Receptor Anti-HBc e Anti-HBs: nao ha casos dehepatite B ‘‘de novo’’ descritos, com ou sem profilaxia.

Tabela 1 - Definicoes para perfis sorologicos para o vırusda hepatite B – Doadores.

Anti-HBc* Anti-HBs** AgHBs*** Definicao

+ - + AgHBs positivo

+ - -

+ + -

Anti-HBc positivo

*Anticorpo anti-core vırus hepatite B, ** anticorpo anti-antıgeno de

superfıcie do vırus da hepatite B, ***Antıgeno de superfıcie do vırus da

hepatite B.

Tabela 2 - Definicoes para perfis sorologicos para o vırusda hepatite B – Receptor.

Anti-HBc Anti-HBs AgHBs Definicao

- - - Naıve

+ - + AgHBs positivo

- + - Anti-HBs isolado/Vacinado

+ - - Anti-HBc isolado

+ + - Anti-HBc e Anti-HBs

*Anticorpo anti-core vırus hepatite B, ** anticorpo anti-antıgeno de

superfıcie do vırus da hepatite B, ***Antıgeno de superfıcie do vırus da

hepatite B.


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N Receptor vacinado ou Anti-HBc isolado: a ocorrenciade hepatite B ‘‘de novo’’ e menor nas series de casosem que foi utilizada profilaxia com lamivudina e/ouHBIG.

N Receptor naive: grupo de maior risco. Series de casossob profilaxia com lamivudina e HBIG demonstraramdiminuicao deste risco.

2. Transplante de Rim

As maiores series de doadores com marcador sorologicopara VHB sao em tx de rim. Entretanto, em algumas delas edescrita apenas a evolucao clınica pos-transplante, semavaliacao sorologica.

Doador AgHBs positivo: em um total de 48 doadores,detectou-se hepatite B ‘‘de novo’’ em 3 receptores (perfissorologicos pre-tx variados) e aumento de enzimas hepati-cas em 9.

Doador Anti-HBc positivo:

N Receptor AgHBs positivo: a evolucao e semelhante aoscaos de doadores sem marcador sorologico para oVHB, utilizando-se profilaxia com lamivudina.

N Receptor Anti-HBc isolado: soroconversao doAgHBs apos o tx de 0,5% sem profilaxia. Nao harelatos de soroconversao sob profilaxia com lami-vudina.

N Receptor vacinado ou naive: apos o tx, sem uso deprofilaxia, soroconversao do Anti-HBc de 2% doscasos, e do AgHBs de menos de 0.5% - sem alteracaona evolucao clınica. Utilizando-se profilaxia comlamivudina, nao ha descricao de soroconversao.

3. Transplante de Coracao

Existem duas series de casos em tx de coracao, utilizando-se profilaxia com lamivudina em alguns casos. Ha apenasum relato de hepatite B ‘‘de novo’’, em um receptor naivecom doador AgHBs positivo.

4. Transplante de Pulmao

Duas series pequenas com doadores Anti-HBc positivosnao demonstraram soroconversao do receptor apos o tx.Lamivudina profilatica foi utilizada em uma delas.

5. Transplante de Celulas-Tronco Hematopoieticas (TCTH)

Em TCTH, a consideracao mais significativa e o risco desoroconversao reversa (perda do Anti-HBs pos-tx).

Doador AgHBs positivo: estudo com controle historico,em receptores AgHBs negativos, demonstrou diminuicaosignificativa do risco de hepatite B ‘‘de novo’’ com profilaxiacom lamivudina.

Doador Anti-HBc positivo: existe um evidente benefıciosobre o risco de soroconversao reversa quando o doa-dor e naturalmente imunizado (Anti-HBc e Anti-HBspositivos).

Uso de doadores com marcador sorologico paravırus da hepatite B em transplantes

Recomendacoes:

A. Gerais

1. Todo candidato a transplante de orgao solido deve serencaminhado para vacinacao contra o VHB antes dotransplante, se possıvel (AII).

2. Todo doador de celulas-tronco hematopoieticas deve serencaminhado para vacinacao contra o VHB antes dotransplante, se possıvel (BII).

3. Todo receptor de celulas-tronco hematopoieticas que forAgHBs negativo deve ser encaminhado para vacinacaocontra o VHB, a partir de 6 meses do transplante (BIII).

4. Todo receptor de transplante de fıgado que for AgHBspositivo deve receber profilaxia com droga antiviral eHBIG, independentemente do estado sorologico dodoador, pelo risco de recidiva pos-transplante (AII).

5. Todo receptor de transplante (exceto fıgado) que forAgHBs positivo deve receber profilaxia com drogaantiviral, pelo risco de replicacao apos o transplante(AII).

6. Todo doador de celulas-tronco hematopoieticas que forAgHBs positivo e estiver com replicacao viral (PCRpositivo) deve iniciar tratamento com antiviral antes dotransplante (BIII).

B. Especıficas

1. Transplante de Fıgado: Recomendacoes Especıficas (B1).

2. Transplante de Rim: Recomendacoes Especıficas (B2).

3. Transplantes de Coracao e Pulmao: RecomendacoesEspecıficas (B3).

4. Transplante de Celulas Tronco-Hematopoieticas: Reco-mendacoes Especıficas (B4).

B1. Transplante de Fıgado: Recomendacoes Especıficas

Doador Receptor

AgHBs + Anti-HBc+ Anti-HBs+ Anti-HBc- Anti-HBs+ Anti-HBc+ isolado Naive

AgHBs+ Nao (DII) Nao (DII) Nao (DII) Nao (DII) Nao (EI)

Anti-HBc+ Sim (BII) Sim (BII) Sim (BIII) Sim (BIII) Nao (DII)

Profilaxia LAM + HBIG* (AII) LAM (BIII)** LAM (BIII)** LAM (BIII)** _

*HBIG por 1 ano, lamivudina indefinidamente; **Por pelo menos 1 ano.

Obs.: outro antiviral pode ser utilizado, no lugar da lamivudina.

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B3. Transplantes de Coracao e de Pulmao: Recomendacoes Especıficas

Doador Receptor

AgHBs +Anti-HBc+Anti-HBs+

Anti-HBc-

Anti-HBs+ Anti-HBc+ isolado Naive

AgHBs+ Situacoes excepc.* (CIII) Situacoes excepc.* (CIII) Situacoes excepc.* (CIII) Situacoes excepc.* (CIII) Nao (DIII)

Profilaxia LAM (AII)** LAM (BIII)*** LAM (BIII)*** LAM (BIII)*** -

Anti-HBc+ Sim (BII) Sim (BII) Sim (BII) Sim (BII) Nao (DIII)

Profilaxia LAM (AII)** LAM (CIII)**** LAM (CIII)**** LAM (CIII)**** _

*Situacoes excepcionais, definidas pelo grupo de transplante; **Indefinidamente; ***Por pelo menos 1 ano; ****O Nucleo sugere o uso, por pelo menos 1 ano.


B4. Transplantes de Celulas Tronco-Hematopoieticas: Recomendacoes Especıficas

Doador Receptor

AgHBs +Anti-HBc+Anti-HBs+

Anti-HBc-

Anti-HBs+ Anti-HBc+ isolado Naive

AgHBs+ Sim (BII) Sim (BII) Sim (BII) Sim (BII) Situacoes especiais (CIII)

Profilaxia LAM (AII)* LAM (AII)** LAM (AII)** LAM (AII)** LAM (AII)**

Anti-HBc+ Anti-

HBs-

Sim (BII) Sim (BII) Sim (BII) Sim (BII) Sim (BII)

Profilaxia LAM (AII)* *** *** *** ***

Anti-HBc+ Anti-

HBs+Sim (AII) Sim (AII) Sim (AII) Sim (AII) Sim (AII)

Profilaxia LAM (AII) # - - -

*Indefinidamente; **Por pelo menos 1 ano; ***fazer PCR do doador – se positivo, tratar como doador AgHBs+, se negativo, tratar como doador Anti-HBc+e Anti-HBs+; #Acompanhar com sorologia a cada 3 meses.


B2. Transplante de Rim: Recomendacoes Especıficas

Doador Receptor

AgHBs + Anti-HBc+ Anti-HBs+ Anti-HBc- Anti-HBs+ Anti-HBc+ isolado Naive

AgHBs+ Situacoes excepc.* (CIII) Situacoes excepc.* (CIII) Situacoes excepc.* (CIII) Situacoes excepc.* (CIII) Nao (DIII)

Profilaxia LAM (AII)** LAM (BIII)*** LAM (BIII)*** LAM (BIII)*** -

Anti-HBc+ Sim (BII) Sim (BII) Sim (BII) Sim (BII) Nao (DIII)

Profilaxia LAM (AII)** LAM (CIII)**** LAM (CIII)**** LAM (CIII)**** _

*Situacoes excepcionais, definidas pelo grupo de transplante; **Indefinidamente; ***Por pelo menos 1 ano; ****Uso opcional.



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CASE REPORT

Scurvy in an alcoholic malnourished cirrhotic manwith spontaneous bacterial peritonitisAndre Luiz Maltos,I Guilherme Vannucchi Portari,III Joao Carlos Saldanha,II Aderbal Garcia Bernardes

Junior,I Guilherme Rocha Pardi,I Daniel Ferreira da CunhaI

I Federal University of Triangulo Mineiro, Department of Internal Medicine, Uberaba/MG, Brazil. II Federal University of Triangulo Mineiro, Special

Pathology Division, Uberaba/MG, Brazil. III Federal University of Triangulo Mineiro, Faculty of Nutrition, Uberaba/MG, Brazil.


Tel.: 55 34 33185335

INTRODUCTION

Decreased serum levels of vitamin C are quite common inelderly hospitalized patients (1), especially those who aremalnourished (2) or those with recent hip fractures (3).Although full-blown scurvy is currently uncommon inindustrialized societies, the literature has described sporadiccases of scurvy in psychiatric patients and in malnourished,alcoholic, homeless patients (4). Scurvy has also beenreported in cases where reductions in serum vitamin Clevels occur concomitantly with activation of the immunesystem and increased circulating interleukins, as occurs inHIV-infection, malignancies and sepsis (5,6).

Whereas patients with alcoholic cirrhosis often haveseveral forms of malnutrition, including obesity, pellagra,protein-energy malnutrition, and folic acid or thiaminedeficiency, in a literature search of the MEDLINE databaseusing terms such as cirrhosis, liver failure or liverinsufficiency, vitamin C or ascorbic acid, and scurvy, wefound only two cases of scurvy associated with hepaticdisease: a 15-year-old girl awaiting combined kidney andliver retransplantation (7) and a malnourished alcoholicpatient with mild fatty liver, without clinical or ultrasoundevidence of cirrhosis or portal hypertension (8).

In this paper, we report the case of a chronicallymalnourished patient with alcoholic cirrhosis (Child-Pughclass B) who developed spontaneous bacterial peritonitis,kwashiorkor-like acute malnutrition, a full-blown picture ofscurvy, and refeeding syndrome despite his low-energydiet.

CASE DESCRIPTION

A 38-year-old white male with alcoholic cirrhosis diag-nosed 3 years prior was admitted to the University Hospitalwith a 30-day history of anorexia, asthenia, ascites, andedema of the legs. He also complained of fever, diarrhea (3-4episodes per day), and a productive cough over the last twodays. The patient was taking 100 mg of spironolactone/day.At the time of the physical examination, the patient did not

have a fever (axillary temperature: 37 C) and was cachectic,pale (+++), with rales in both lungs, which were moreintense on the left side. The patient had tense ascites, spiderangiomas, and caput medusae, in addition to palmarerythema, and leg and scrotal edema. His skin was dry,with ecchymosis around both knees, and he had thinninghair and lesions resembling pellagra. Neurologic examina-tion revealed that the patient was alert without intellectualor motor impairments (West-Haven hepatic encephalopathycriteria: 0).

The laboratory data at admission were as follows: bloodhemoglobin: 6.2 g/dL; hematocrit: 20.3%; red blood cells:2,160,000/mm3; mean corpuscular volume: 94 fL; red celldistribution width: 16%; white blood cells: 18,080, with aneutrophil count of 16,091/mm3, and 1,446 band forms/mm3; platelets: 265,000/mm3; C-reactive protein: 128 mg/dL; aspartate aminotransferase: 34 IU/L; alanine amino-transferase: 25 IU/L; lactate dehydrogenase: 435 IU/L;serum total protein: 4.1 g/dL; albumin 1.07 g/dL; interna-tional normalized ratio: 1.35; prothrombin activity: 68%;serum total bilirubin: 1.1 mg/dL; serum sodium: 136 mEq/L; potassium: 4.3 mEq/L; calcium: 7.2 mg/dL; phosphorus:3.8 mg/dL; magnesium: 1.9 mg/dL; urea: 36 mg/dL; crea-tinine: 0.54 mg/dL; fasting blood glucose: 86 mg/dL; serumiron: 15.5 mg/dL, total iron-binding capacity: 74.5 mg/dL;folic acid: 5.5 ng/mL; vitamin B12: .1,000 pg/mL. Asciticfluid analysis revealed the following: pH 8.0; density: 1,010;cells: 950/mm3, with a neutrophil count of 646/mm3;glucose: 95 mg/dL; lactate dehydrogenase: 931 IU/L; albu-min: 0.16 g/dL (serum/ascitic liquid albumin gradient:0.91, suggestive of exudate). Cytopathological examinationrevealed no neoplastic cells, and the bacterial culture wasnegative. The abdominal ultrasound revealed a normalgallbladder and biliary tract and a homogeneous atrophicliver. Abdominal computed tomography revealed a calci-fied pancreas, voluminous ascites, and mild left pleuraleffusion. Esophagogastroduodenoscopy revealed esopha-geal varices and mild gastritis.

After the diagnosis of liver cirrhosis (Child-Pugh class B)and spontaneous bacterial peritonitis, the patient wastreated with IV ceftriaxone (1 g b.i.d.), oral spironolactone(100 mg/day), IV albumin (1.5 g/kg on the 1st day and1.0 g/kg on the 3rd day), furosemide (20 mg IV, b.i.d.),intramuscular phytomenadione (10 mg), oral vitamin Bcomplex, and packed blood cells.

The Nutrology Support Team was consulted and foundsigns of chronic malnutrition, including a low body mass




405

index, 17.9 kg/m2 (body weight: 48.8 kg) and signs ofreduced corporal fat and muscle reserves (arm circumfer-ence: 15.5 cm; triceps skin fold: 3 mm). In addition toatrophic musculature in both thighs and lower legs,extensive bilateral subcutaneous edema with the presenceof ecchymosis, corkscrew hairs, and perifollicular hemor-rhage, mainly around the knees, were noted (Figure 1). Adiagnosis of kwashiorkor (acute protein malnutrition) andscurvy was made, and because the patient was anorecticand consumed a diet that was low in energy and nutrients,enteral tube feeding was initiated. A skin biopsy and ablood sample to measure plasma ascorbic acid wereobtained from the patient before the continuous IV infusionof 1.5 g of vitamin C/day was started.

The patient maintained a daily positive body waterbalance, experienced diarrhea, and developed serum elec-trolyte abnormalities, including hyponatremia (sodium:130 mEq/L), hypokalemia (potassium: 2.99 mEq/L), hypo-magnesemia (magnesium: 1.1 mg/dL), and hypophospha-temia (phosphorus: 1.98 mg/dL). A diagnosis of refeedingsyndrome was made based on these electrolyte imbalances;the enteral nutrition that the patient was receiving (contain-ing 1600 kcal (32 kcal/body weight) was tapered, and theelectrolyte abnormalities were treated.

Despite hemodynamic support and medication treatment,the patient had a poor clinical evolution and died of septicshock 10 days after admission to the hospital. His initialserum level of vitamin C was ,0.2 mg/dL, which increasedto normal values (1.3 mg/dL) on the 4th day of IV vitamin Csupplementation. The skin biopsy showed atrophic, hyper-keratosis epidermis. On the dermis, there were several hairfollicles plugged with keratin, hemorrhage, and dermalatrophy, which are suggestive of scurvy (Figure 2).

DISCUSSION

Although full-blown vitamin C deficiency is currentlyuncommon, sporadic cases of scurvy have been described,mainly among psychiatric, alcoholic, and HIV-AIDSpatients or in children with bizarre food-intake habits(4,5,8). Although low ascorbic acid levels in leukocytes (ameasure of tissue stores) are common in patients withalcoholic cirrhosis (9), this is the first reported case of scurvyin an alcoholic, malnourished man with cirrhosis andspontaneous bacterial peritonitis.

In contrast to the typical features of scurvy in children,such as subperiosteal hematomas and gingival hypertrophy(10), adults with scurvy often manifest with weight loss,purpura, and other dermatologic lesions such as follicularhyperkeratosis and corkscrew hairs; in our case, thediagnosis was confirmed based on low serum vitamin Clevels and a skin biopsy exhibiting the typical microscopicfeatures of scurvy (6).

The presence of desquamative erythematous dermatitissuggestive of pellagra, anemia, and low albumin serumlevels, as observed in our patient, suggests insufficient foodintake and a chronic deficiency of vitamins, includingascorbic acid (11). Moreover, cirrhotic patients are at riskof vitamin deficiencies because of the reduced intake,malabsorption, diminished storage capacity, and increasedrenal excretion, in addition to increased tissue vitaminconsumption due to infection or sepsis (9).

While the low albumin serum levels could be attributed toliver failure, this condition could also be caused by infection(12), as is the case for spontaneous bacterial peritonitis.Moreover, as often occurs in malnourished, vitamin A-deficient children, who can develop acute anatomicallesions such as xerophthalmia or keratomalacia duringmeasles infection (13), it is also possible that a patient with achronic vitamin C deficiency could develop the signs andsymptoms of scurvy during an acute systemic inflammatoryresponse, as was reported in a hospitalized patient withmetastatic renal-cell carcinoma treated with high-dose ofinterleukin-2 (14). We also described the case of an AIDSpatient with increased serum levels of C-reactive proteinand multiple opportunistic infections, including neurotox-oplasmosis and acute pneumonia, who developed a full-blown case of scurvy (15). These findings are consistent withreports of an association between low serum vitamin Clevels with the presence of an acute-phase response inintensive care units (16). It is noteworthy that in a criticallyill patient, the drastic depletion of ascorbate is a direct resultof the acute oxidative stress that is intrinsic to the systemicinflammatory response (16). Although our patient died ofsepsis, it is noteworthy that high doses of vitamin Csupplementation have been associated with better prog-noses in critical care patients with low serum levels of

Figure 1 - Ecchymosis, perifollicular hemorrhage, and corkscrewhairs in the right knee.

Figure 2 - Scurvy: red cell extravasations around vessels in theupper dermis, dermal and epidermal atrophy and parakeratosis,and mild chronic perivascular inflammation.

Scurvy in an alcoholic cirrhotic manMaltos AL et al.

CLINICS 2012;67(4):405-407

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ascorbic acid because it helps to restore endothelial nitricoxide synthase, the expression of which is impaired in septicpatients (16). In particular, high doses (3 g or more,administered parenterally) of ascorbic acid are required toachieve normal plasma levels in septic patients (18).

Refeeding syndrome can occur in patients with marasmusor kwashiorkor, alcoholism, or anorexia nervosa; afterprolonged fasting; in the malnourished elderly; in cancerpatients; and in obese patients after duodenal switchsurgery (17). Our findings suggest that even when foodintake is increased in a gradual and careful way, alcoholiccirrhotic patients with infection can develop electrolyteabnormalities and refeeding syndrome.

The limitations of this study include the measurements ofserum ascorbic acid, instead of the intracellular leukocytecontent of this vitamin. However, the skin biopsy showedcharacteristic features of scurvy, and although this patienthad liver cirrhosis, the platelet count and the prothrombinactivity, which were within normal values, suggest that theskin hemorrhage could not be ascribed to liver failure.

The description of this case of scurvy in a malnourishedman with cirrhosis and infection should alert gastroenter-ologists to perform a careful skin examination, with ascorbicacid measurements when this exam is available.

ACKNOWLEDGMENTS

This study was supported by the following: Fundacao de Amparo a

Pesquisa do Estado de Minas Gerais - FAPEMIG (process n˚3311/06) and

Conselho Nacional de Pesquisa – CNPq (process n˚ 402832/2005-1).


Cunha DF, Maltos AL, Bernardes Junior AG, Pardi GR designed the

study, analyzed the data and wrote the paper. Saldanha JC conducted the

histopathological analysis and analyzed the results. Portari GV carried out

the vitamin C analysis and analyzed the results.

REFERENCES

1. Vannucchi H, da Cunha DF, Bernardes MM, Unamuno MR. Serum levels ofvitamin A, E, C and B2, carotenoid and zinc in hospitalized elderly patients.

Rev Saude Publica. 1994;28(2):121-6, http://dx.doi.org/10.1590/S0034-89101994000200005.

2. Cunha DF, Cunha SF, Unamuno MR, Vancucchi H. Serum levelsassessment of vitamin A, E, C, B2 and carotenoids in malnourished andnon-malnourished hospitalized elderly patients. Clin Nut. 2001;20(2):167-70, http://dx.doi.org/10.1054/clnu.2000.0378.

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CLINICS 2012;67(4):405-407 Scurvy in an alcoholic cirrhotic manMaltos AL et al.

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