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6067632 RX REVISTA ARQUIVOS GASTRO ED. 01/2012

ISSN 0004-2803 ISSN 1678-4219 on-line Coden ARQGA

ARQUIVOS DE GASTROENTEROLOGIA Número 1 | Janeiro/Março 2012 | Volume 49

Arquivos de Gastroenterologia

ARCHIVES OF GASTROENTEROLOGY Publication of the Brazilian Institute for Studies and Research in Gastroenterology and others Specialities - IBEPEGE

Founded in 1964 by Prof. Dr. José Fernandes Pontes

ORGÃO DE DIVULGAÇÃO Publication

CBCD

Colégio Brasileiro de Cirurgia Digestiva

PAT R O C Í N I O

SBNPE

Sociedade Brasileira de Nutricção Parenteral e Enteral

SOBED

FBG

Federação Brasileira de Gastroenterologia

SBM D

Sociedade Brasileira de Motilidade Digestiva

Sociedade Brasileira de Endoscopia Digestiva

SBH

Sociedade Brasileira de Hepatologia


No Tratamento da Sii1,2 1, 2, 3 l a n i m o d b a r O alívio da do

O principal sintoma da Sii:

O brometo de pinavériodiminuiu

DOR 92% 4,5

a duração da dor de várias horas para 7 alguns minutos.

O brometo de pinavério diminui a dor em até

dos casos.

5

USO ADULTO Apresentações e composição: Comprimido revestido de 50 mg em embalagens com 30 unidades. Comprimido revestido de 100 mg em embalagens com 10, 30 e 60 unidades. Indicações: Tratamento sintomático da dor ou desconforto abdominal, dos distúrbios da freqüência ou consistência das evacuações (constipação ou diarréia) e da distensão abdominal, decorrentes dos transtornos funcionais do intestino (SII). Tratamento sintomático das dores decorrentes dos transtornos funcionais das vias biliares. Preparação de enemas opacos. Contra indicações: hipersensibilidade conhecida aos componentes da fórmula. Precauções e advertências: É desaconselhável a utilização do brometo de pinavério durante a gravidez. Além disso, deve-se observar a presença de bromo, cuja administração no final da gravidez pode causar alterações neurológicas no recém-nascido (hipotonia, sedação). Em função da falta de estudos, recomenda-se não utilizar o brometo de pinavério durante a lactação. Interações medicamentosas: os estudos realizados não demonstraram interações medicamentosas com nenhum dos seguintes tratamentos concomitantemente prescritos: hipoglicemiantes orais, anticoagulantes e digitálicos. Reações adversas: foram relatados, raramente, alguns casos de alterações digestivas leves e casos isolados de reações cutâneas, algumas do tipo alérgico. Raramente pode haver agravamento da constipação e epigastralgia. Em casos de erupção cutânea, é conveniente interromper a administração do medicamento. Posologia: recomenda-se a administração de 1 comprimido de 50 mg, 3 ou 4 vezes ao dia, ou 1 comprimido de 100 mg, 2 vezes ao dia (manhã e noite). Excepcionalmente, a posologia pode ser aumentada para 6 comprimidos de 50 mg ou 3 comprimidos de 100 mg ao dia. Os comprimidos devem ser deglutidos inteiros, sem mastigar, com um pouco de água, imediatamente antes ou durante as refeições. Na preparação de enemas opacos, a posologia é de 2 comprimidos diários de 100 mg ou 4 comprimidos diários de 50 mg, nos 3 dias anteriores ao exame. MS - Registro MS – 1.0639.0254. SICO_NSPC_1209

Contraindicações: hipersensibilidade conhecida aos componentes da fórmula. Interações medicamentosas: os estudos realizados não demonstraram interações medicamentosas com nenhum dos seguintes tratamentos concomitantemente prescritos: hipoglicemiantes orais, anticoagulantes e digitálicos. Referências bibliográficas: 1) Guslandi M. Profilo farmacologico clinico del pinaverio bromuro. Minerva Med. 1994;85:179-85. 2) Poynard T, Regimbeau C, Benhamou Y. Meta-analysis of smooth muscle relaxants in the treatment of irritable bowel syndrome. Aliment Pharmacol Ther. 2001;15(3):355-61. 3) Siilif*®[Bula]. São Paulo: Nycomed Pharma. 4) Cloarec D et al. Efficacite du Dicetel 100 mg au coursdes troubles fonctionnels intestinaux: résultats d’une étude réalisée em pratique gastroentérologique. Rev Fr Gastroenterol. 1997;33(324-325):1107-35. 5) Corazziari E et al. Consensus report: clinical trial guidelines for pharmacological treatment of irritable bowel syndrome. Aliment Pharmacol Ther. 2003;18(6):569-80. 6) Cloarec D et al. Efficacité du pinaverium: 100mg, deux foius par jour, dans le traitement sumptomatique du syndrome de l’intestin irritable. Ann Gastroenterol Hepatol. 1997;33(4):181-4. 7) Awad R, Dibildox M, Ortiz F. Irritable bowel syndrome treatment using pinaverium bromide as a calcium channel blocker. Acta Gastroenterol Latinoam. 1995;25(3):137-44. Material destinado exclusivamente a profissionais habilitados a prescrever e/ou dispensar medicamentos. Fev/2012 Nycomed Pharma Ltda. Rua do Estilo Barroco, 721 - 04709-011 - São Paulo - SP. Mais informações poderão ser obtidas diretamente com o nosso Departamento Médico ou por meio de nossos representantes. *Marca depositada. A PERSISTIREM OS SINTOMAS, O MÉDICO DEVERÁ SER CONSULTADO.

Linha Gastro


40mg

*

pantoprazol magnésico di-hidratado Alívio dos sintomas diurnos e noturnos da DRGE.1,2 POTÊ N

CIA 1

24

HORAS PO

NC

1-3

pantoprazol

magnésico

não possui GENÉRICO

IA 1

OU SIMILARES

4


Comparado ao pantoprazol sódico, Tecta demonstrou em 4 semanas:1 t Taxas superiores de cicatrização da esofagite.1

t Níveis elevados de alívio dos sintomas.1

Taxa de cicatrização da esofagite1†

71,6%

77,9%

pantoprazol sódico 40mg 1x ao dia

40mg

*

pantoprazol magnésico di-hidratado

1x ao dia

n= 532

4 semanas

4 semanas

†População PP Adaptado de: Hein J. 2011


40mg

*

pantoprazol magnésico di-hidratado

Melhora dos sintomas em 4 semanas

5

ReQuest (Reflux Questionaire) TM

7

n=3306

6

escore

5 4 3 2 1 0

Base

2

3

Queixas de acidez Queixas de abdome superior / estômago

4

5

6

Queixas de abdome interior / digestivo

7

14

21

28

Distúrbios do sono Bem-estar geral

Náusea Adaptado de: Lopez LH, et al. 2006

Tecta 40mg, 1x ao dia, reduziu significantemente

os escores de sintomas associados à DRGE.

5


Melhorou o bem-estar a geral a partir da 1 tomada.

5

Após 4 semanas, a melhora do escore dos sintomas foi de

74% a 81% quando comparada às queixas iniciais. 5


pantoprazol

magnésico

não possui GENÉRICO

OU SIMILARES

40mg

*

4

pantoprazol magnésico di-hidratado

3

Posologia na DRGE: 1 comprimido de 40mg, 1x ao dia

Contraindicação: Tecta não deve ser usado em casos de hipersensibilidade conhecida ao pantoprazol ou aos demais componentes da fórmula. Interação medicamentosa: Tecta , assim como outros medicamentos da mesma classe, não deve ser coadministrado com atazanavir/nelfinazir. ®

®

USO ORAL USO ADULTO ACIMA DE 18 ANOS Apresentações e composição: Comprimidos gastrorresistentes de 40 mg. Embalagens com 2, 15, 28,30, ou 60 comprimidos. Indicações: TECTA® 40 mg está indicado para o tratamento das esofagites de refluxo moderada ou grave e dos sintomas de refluxo gastroesofágico. Também é indicado para tratamento intermitente de sintomas de acordo com a necessidade (on demand). Contra-indicações: TECTA® não deve ser usado em casos de hipersensibilidade conhecida ao pantoprazol ou aos demais componentes da fórmula. TECTA®, assim como outros IBPs, não deve ser coadministrado com atazanavir/nelfinazir (vide Advertências e Precauções/Interações Medicamentosas). TECTA® não deve ser administrado em terapia combinada para erradicação do Helicobacter pylori a pacientes com disfunção hepática ou renal moderada a grave, uma vez que não existe experiência clínica sobre a eficácia e a segurança da terapia combinada nesses pacientes. Este medicamento é contra-indicado na faixa etária de 0 a 18 anos. Categoria B de risco na gravidez: Este medicamento não deve ser utilizado por mulheres grávidas sem orientação médica ou do cirurgião-dentista. Advertências e Precauções: Quando prescrito dentro de uma terapia combinada, as instruções de uso de cada uma dos fármacos devem ser seguidas. Na presença de qualquer sintoma de alarme (como significante perda de peso não intencional, vômitos recorrentes, disfagia, hematêmese, anemia ou melena) e quando houver suspeita ou presença de úlcera gástrica, deve ser excluída a possibilidade de malignidade, já que o tratamento com pantoprazol pode aliviar os sintomas e retardar o diagnóstico. Casos os sintomas persistam apesar de tratamento adequado, investigações adicionais devem ser consideradas. Gravidez e lactação: Categoria B de risco na gravidez. Este medicamento não deve ser utilizado por mulheres grávidas sem orientação médica ou do cirurgião-dentista. TECTA® não deve ser administrado em gestantes e lactantes, a menos que absolutamente necessário, uma vez que a experiência clínica sobre seu uso em mulheres nestas condições é limitada. Estudos de reprodução em animais demonstraram uma fetotoxicidade leve com doses acima de 5 mg/kg. Os dados disponíveis sobre o uso de pantoprazol em lactantes são limitados. A excreção do pantoprazol no leite humano foi detectada em caso isolado após uma única dose oral de 40 mg. A relevância clínica desta descoberta não é conhecida. TECTA® só deve ser utilizado durante a gravidez e a lactação quando o benefício para a mãe for considerado maior que o risco potencial ao feto ou à criança. Pacientes idosos: Não é necessária nenhuma adaptação posológica para pacientes idosos. TECTA® pode ser utilizado por pessoas com mais de 65 anos. Em voluntários idosos, a ASC e a Cmax (concentração máxima) aumentam discretamente, quando comparadas às de indivíduos jovens; porém, estes aumentos não são clinicamente significativos. Não se recomenda nenhum ajuste posológico baseado na idade. A dose diária em pacientes idosos, via de regra, não deve ultrapassar os regimes posológicos recomendados. Pacientes pediátricos: A segurança e eficácia do emprego de TECTA® não foram estabelecidas em menores de 18 anos, portanto o seu uso não está indicado para pessoas menores de 18 anos. Insuficiência renal: para paciente com disfunção renal leve a moderada não é necessário ajuste posológico; a dose diária não deve ultrapassar os regimes posológicos recomendados. Nos casos de insuficiência renal grave o paciente deve ser cuidadosamente monitorado. Em pacientes com função renal reduzida (p. ex., pacientes em diálise), nenhum ajuste de dose é necessário. Assim como para indivíduos sadios, a meia-vida do pantoprazol é curta. Somente pequenas quantidades de pantoprazol são dialisáveis. Embora a meia-vida do principal metabólito tenha sido moderadamente aumentada para 2-3 h, a excreção é ainda rápida e, portanto não ocorre acúmulo. Insuficiência hepática: não é recomendado ajuste posológico para paciente com disfunção hepática leve a moderada. Em caso de redução intensa da função hepática a dose deve ser ajustada para 1 comprimido de 40 mg a cada dois dias. Em pacientes com insuficiência hepática grave, devem ser regularmente monitoradas as enzimas hepáticas durante o tratamento com TECTA®; se houver aumento nos valores enzimáticos, o tratamento deve ser descontinuado. A meia-vida aumentou para 7 e 9 horas, a ASC aumentou em um fator de 5 a 7 e a Cmax aumentou em um fator de 1,5 em pacientes com cirrose hepática em comparação com indivíduos sadios após a administração de 40 mg de pantoprazol sódico. Efeitos na capacidade de dirigir e operar máquinas: não há efeitos conhecidos na capacidade de dirigir e operar máquinas. Interações medicamentosas: O conteúdo de magnésico em um comprimido de TECTA® não é clinicamente significante (1,268 g a cada comprimido de 40 mg). Assim, não são esperadas diferenças nas interações medicamentosas entre o pantoprazol magnésico e o pantoprazol sódico. Como os demais membros de sua classe, TECTA® pode alterar a absorção de medicamentos cuja biodisponibilidade seja dependente do pH do suco gástrico, como o cetoconazol e itraconazol. Isso se aplica também a medicamentos ingeridos pouco tempo antes de TECTA®. Assim como outros medicamentos da mesma classe, não deve ser coadministrado com atazanavir/nelfinazir, pois a absorção desses antirretovirais é pH dependente, podendo ocorrer uma redução substancial na biodisponibilidade dos mesmos (ver Contra-Indicações). Pantoprazol é extensivamente metabolizado no fígado. Inicialmente sofre desmetilação e oxidação a sulfonas pelas subenzimas CYP2C19 e CYP3A4 do citocromo P 450 (Fase I do metabolismo). Como conseqüência da baixa afinidade do pantoprazol e de seus metabólitos, o hidroxipantoprazol e o hidroxipantoprazol sulfona pelas enzimas do citocromo P 450, seu potencial de interação na Fase I é limitado, o que permite que o fármaco saia rapidamente do retículo endoplasmático e seja transferido subsequentemente para o citoplasma para ser conjugado com sulfato, na Fase II do metabolismo. Esta baixa afinidade resulta em predominância do metabolismo no sistema de conjugação (Fase II) que, ao contrário do sistema P 450, não é saturável e consequentemente não-interativa. Esta etapa independe do sistema enzimático citocromo P 450. A interação entre pantoprazol e outras substâncias metabolizadas na Fase I do metabolismo não pode, em princípio, ser excluída. Nos estudos sobre interações medicamentosas conduzidos até o momento, onde foram analisados os substratos de todas as famílias do citocromo P450 envolvidas no metabolismo de fármacos no homem, verificou-se que pantoprazol não afeta a farmacocinética ou a farmacodinâmica da carbamazepina, cafeína, diazepam, diclofenaco, digoxina, etanol, glibenclamida, metoprolol, naproxeno, nifedipina, fenitoína, piroxicam, teofilina, e contraceptivos orais. TECTA® não aumenta a excreção urinária dos marcadores de indução, ácido D-glucarídico e 6 ß-hidroxicortisol. Da mesma forma, os fármacos investigados não influenciaram a farmacocinética do pantoprazol. Embora, em estudos clínicos farmacocinéticos não tenha sido observada nenhuma interação durante a administração concomitante à femprocumona ou à varfarina, foram observados no período de pós-comercialização alguns casos isolados de alterações no INR (tempo de protrombina do paciente/média normal do tempo de protrombina) nessas situações. Consequentemente, em pacientes que estão sendo tratados com anticoagulantes cumarínicos, é recomendada a monitoração do tempo de protrombina/INR após o início, término ou durante o uso irregular de pantoprazol. Não existe interação na administração concomitante com antiácidos. De maneira geral, o tratamento diário com qualquer medicamento bloqueador de ácido por um longo tempo (p. ex., mais que três anos) pode levar a uma má absorção da cianocobalamina (vitamina B12). Estudos de interação farmacocinética em humanos, administrando-se pantoprazol simultaneamente aos antibióticos claritromicina, metronidazol e amoxicilina não demonstraram nenhuma interação clinicamente significativa. Ingestão com alimentos: O consumo de alimentos não interfere com as ações do TECTA® no organismo. Interferência em testes de laboratório: Em alguns poucos casos isolados, detectou-se alterações no tempo de coagulação durante o uso de pantoprazol. Desta forma, em pacientes tratados com anticoagulantes cumarínicos, recomenda-se a monitoração do tempo de coagulação após início, final ou durante o tratamento com pantoprazol. Reações adversas: O perfil de segurança do TECTA® não deve diferir do observado com o pantoprazol sódico, uma vez que ambos contêm o mesmo princípio ativo – o pantoprazol “livre” dissociado (ânion pantoprazol, íons Mg ou Na). Embora o pantoprazol (a substância ativa) seja muito bem tolerado, a maioria dos eventos adversos observados tem sido leve e transitória, não apresentando nenhuma relação consistente com o tratamento. Assim, podem ocorrer as seguintes reações adversas com o uso do produto: Reações comuns (ocorrem entre 1% e 10% dos pacientes que utilizam este medicamento): dor abdominal, diarréia, constipação, flatulência, cefaléia. Reações incomuns (ocorrem entre 0,1% e 1% dos pacientes que utilizam este medicamento): náusea/vômito, vertigem, distúrbios visuais (visão turva), reações alérgicas como prurido e exantema. Reações raras (ocorrem entre 0,01% e 0,1% dos pacientes que utilizam este medicamento): boca seca, artralgia. Reações muito raras (ocorrem em menos de 0,01% dos pacientes que utilizam este medicamento): depressão, leucopenia, trombocitopenia, edema periférico, dano hepatocelular grave levando a icterícia com ou sem insuficiência hepática, reações anafiláticas, incluindo choque anafilático, aumento nos níveis de enzimas hepáticas (transaminases, γ-GT), aumento nos níveis de triglicerídios, elevação da temperatura corporal, mialgia, nefrite intersticial, reações dermatológicas graves como síndrome de Stevens Johnson, eritema multiforme, síndrome de Lyell, fotossensibilidade, urticária e angioedema. Atenção: este produto é um medicamento novo e, embora as pesquisas tenham indicado eficácia e segurança aceitáveis, mesmo que indicado e utilizado corretamente, podem ocorrer eventos adversos imprevisíveis ou desconhecidos. Nesse caso, notifique os eventos adversos pelo Sistema de Notificações em Vigilância Sanitária NOTIVISA, disponível em http://www8.anvisa.gov.br/notivisa/frmCadastro.asp, ou para a Vigilância Sanitária Estadual ou Municipal. Posologia e modo de usar: A posologia habitualmente recomendada é de 1 comprimido de 40 mg ao dia, antes, durante ou após o café da manhã, a menos que seja prescrito de outra maneira pelo seu médico. A duração do tratamento fica a critério médico e dependente da indicação. Na maioria dos pacientes, o alívio dos sintomas é rápido e um período de tratamento de 4 a 8 semanas é, em geral suficiente. TECTA® é para uso exclusivamente oral e os comprimidos devem ser ingeridos inteiros com um pouco de líquido. Na doença de refluxo gastroesofágico: Tratamento da esofagite de refluxo - 1 comprimido de 40 mg ao dia em um período de 4 semanas. Nos casos com esofagite não cicatrizada ou com sintomas persistentes é recomendado um período adicional de 4 semanas. Os sintomas recorrentes poderão ser controlados administrando-se 1 comprimido de TECTA® 40 mg ao dia, quando necessário (“on demand”), de acordo com a intensidade dos mesmos. A mudança para terapia contínua deve ser considerada nos casos em que os sintomas não puderem ser devidamente controlados sob terapia “on demand”. Em casos isolados de esofagite por refluxo, a dose diária pode ser aumentada para 2 comprimidos ao dia, particularmente nos casos de pacientes refratários a outros medicamentos antiulcerosos. MS – 1.0639.0256. MEDICAMENTO SOB PRESCRIÇÃO.TC40_1004_0611_VPS. Referências bibliográficas: 1) Hein J. Comparison of the efficacy and safety of pantoprazole magnesium and pantoprazole sodium in the treatment of gastro-oesophageal reflux disease: a randomized, double-blind, controlled, multicentre trial. Clin Drug Investig. 2011. doi: 2011;31(9):655-64. 2) Morales-Arambula M, et al. Nighttime GERD: prevalence, symptom intensity and treatment response to a 4 week treatment with 40 mg of pantoprazole magnesium O.D.. A report from the GERD Mexican Working Group. Gastroenterology. 2009; 136(5 supl 1):A428. 3) Tecta® [Bula]. São Paulo: Nycomed Pharma. 4) Agência Nacional de Vigilância Sanitária (BR). Resolução – RE nº 5.630, de 03 de dezembro de 2010. Concessão de registro de medicamento novo, revalidação de registro, retificação de publicação. Diário Oficial [da República Federativa do Brasil]. Brasília, 06 dez 2010; n. 232, p. 100-1. 5) Lopez LH, et al. Effectiveness of pantoprazole magnesium dihydrate in the treatment of symptoms in gastro-oesophageal reflux disease. Gut. 2006; 55 (Suppl 5): A275, WED-G-143. Material destinado exclusivamente a profissionais habilitados a prescrever e/ou dispensar medicamentos. Fev/2012.

A PERSISTIREM OS SINTOMAS, O MÉDICO DEVERÁ SER CONSULTADO.

Nycomed Pharma Ltda. Rua do Estilo Barroco, 721 - 04709-011 - São Paulo - SP. Mais informações poderão ser obtidas diretamente com o nosso Departamento Médico ou por meio de nossos representantes. *Marca depositada.

Linha Gastro


A GASTROENTEROLOGIA É NOSSA PAIXÃO.

Eficaz na profilaxia das lesões por AINES1 90% dos pacientes em uso crônico de AINES tratados com Pantozol 20mg uma vez ao dia, durante 6 meses, mantiveram a remissão dos sintomas gástricos1. ®

Mais segurança para pacientes polimedicados2-4 Via metabólica diferente que não influencia o efeito antiplaquetário do clopidogrel5 Mínimos riscos de interações medicamentosas, se comparado aos demais IBPs2-6

Posologia na gastrite: 1 comprimido, 1 vez ao dia7


Contraindicação: Pantozol® não deve ser usado por indivíduos que apresentem alergia conhecida aos componentes da fórmula. Interação medicamentosa: Pantozol®, assim como outros medicamentos da mesma classe, não devem ser coadministrado com atazanavir. Pantozol® Comprimidos Revestidos 20mg (pantoprazol sódico sesquiidratado). USO ADULTO. Apresentação e composição: embalagens com 2,7, 14, 28, 42 e 56 comprimidos gastro-resistentes. Indicações: Alívio dos sintomas por problemas no estômago e no início do intestino que dependem da secreção do ácido produzido pelo estômago. Gastrites ou gastroduodenites agudas ou crônicas e dispepsias não-ulcerosas. Tratamento da doença por refluxo gastroesofágico sem esofagite, das esofagites leves e na manutenção de pacientes com esofagite de refluxo cicatrizada, prevenindo as recidivas. Prevenção das lesões agudas que ocorrem no revestimento do estômago e do início do intestino, induzidas por medicamentos como os antiinflamatórios não-hormonais. Contra-indicações: Pantozol® 20mg não deve ser usado por indivíduos que apresentem alergia (hipersensibilidade) conhecida ao pantoprazol ou aos demais componentes da fórmula. Assim como outros medicamentos da mesma classe, não deve ser administrado ao mesmo tempo com atazanavir (medicamento usado no tratamento da infecção por HIV). Este medicamento é contra-indicado na faixa etária de 0 a 12 anos. Precauções e advertências: Na presença de qualquer sintoma de alarme (p. ex. significante perda de peso não intencional, vômitos recorrentes, disfagia, hematêmese, anemia ou melena) e quando houver suspeita ou presença de úlcera gástrica, deve ser excluída a possibilidade de malignidade, já que o tratamento com pantoprazol pode aliviar os sintomas e retardar o diagnóstico. Casos os sintomas persistam apesar de tratamento adequado, investigações adicionais devem ser consideradas. Em pacientes com disfunção hepática grave (insuficiência hepática), as enzimas hepáticas devem ser regularmente monitoradas durante o tratamento com Pantozol®, se houver aumento nos valores enzimáticos, o tratamento deve ser descontinuado. Assim como os outros inibidores da secreção ácida, Pantozol® pode reduzir a absorção da vitamina B12 (cianocobalamina) devido à hipocloridria ou acloridria. Esse fato deve ser considerado em terapia por tempo prolongado em pacientes com reserva baixa de vitamina B12 ou com fatores de risco de absorção reduzida de vitamina B12. O aumento do risco deve ser avaliado de acordo com fatores individuais, assim como idade (acima de 65 anos), histórico de gastrite, úlcera duodenal ou sangramento da parte superior gastintestinal. Em terapia de longo prazo, especialmente quando o tratamento exceder 1 ano, os pacientes devem ser mantidos sob acompanhamento regular. Pacientes que não responderem ao tratamento após 4 semanas deverão ser investigados. Este medicamento não deve ser utilizado por mulheres grávidas sem orientação médica ou do cirurgião-dentista. Pantozol® não deve ser administrado em gestantes e lactantes, a menos que absolutamente necessário, uma vez que a experiência clínica sobre seu uso em mulheres nestas condições é limitada. Pantozol® pode ser utilizado por pessoas com mais de 65 anos, porém a dose de 40mg ao dia não deve ser ultrapassada. Não é necessária nenhuma adaptação posológica. Não há efeitos conhecidos na habilidade de dirigir e operar máquinas. Em alguns poucos casos isolados, detectou-se alterações no tempo de protrombina (INR). Desta forma, em pacientes tratados com anticoagulantes cumarínicos, recomenda-se monitoração do tempo de protrombina (INR) após início, final ou durante o tratamento com pantoprazol. Interações medicamentosas: Pantozol® pode alterar a absorção de medicamentos cuja biodisponibilidade seja dependente do pH do suco gástrico, como por exemplo o cetoconazol. Isso se aplica, também, aos medicamentos ingeridos pouco tempo antes de Pantozol®. IBPs, incluindo o pantoprazol, não devem ser coadministrados com atazanavir. Pantoprazol é extensivamente metabolizado no fígado. Inicialmente ocorre desmetilação e oxidação a sulfonas pelas sub enzimas CYP2C19 e CYP3A4 do citocromo P 450 (Fase I do metabolismo). Como consequência da baixa afinidade do pantoprazol e de seus metabólitos, o hidroxipantoprazol e o hidroxipantoprazol sulfona pelas enzimas do citocromo P 450, seu potencial de interação na Fase I é limitado, o que permite que a droga saia rapidamente do retículo endoplasmático e seja subsequentemente transferida para o citoplasma para ser conjugada com sulfato, na Fase II do metabolismo. Esta baixa afinidade resulta em predominância do metabolismo no sistema de conjugação (Fase II) que, ao contrário do sistema P 450, não é saturável e consequentemente não interativa. Esta etapa independe do sistema enzimático citocromo P 450. A interação entre pantoprazol e outras substâncias metabolizadas na Fase I do metabolismo não pode, em princípio, ser excluída. Nos estudos sobre interações medicamentosas conduzidos até o momento, onde foram analisados os substratos de todas as famílias do citocromo P450 envolvidas no metabolismo de drogas no homem, verificou-se que pantoprazol não afeta a farmacocinética ou a farmacodinâmica da carbamazepina, cafeína, diazepam, diclofenaco, digoxina, etanol, glibenclamida, metoprolol, naproxeno, nifedipina, fenitoína, piroxicam, teofilina, e contraceptivos orais. A ingestão de antiácidos não interfere na absorção do pantoprazol. Pantozol® não aumenta a excreção urinária dos marcadores de indução, ácido D-glucarídico e 6 ß-hidroxicortisol. Da mesma forma, as drogas investigadas não influenciaram a farmacocinética do pantoprazol. Embora, em estudos clínicos farmacocinéticos, não tenha sido observada, nenhuma interação durante a administração concomitante à femprocumona ou à varfarina, foram observados, no período de pós comercialização, alguns casos isolados de alterações no INR (tempo de protrombina do paciente/ média normal do tempo de protrombina) ISI nessas situações. Consequentemente, em pacientes que estão sendo tratados com os anticoagulantes cumarínicos, é recomendada a monitorização do tempo de protrombina / INR, após o início, término ou durante o uso irregular de pantoprazol. Não existe interação na administração concomitante de antiácidos. Estudos de interação farmacocinética em humanos, nos quais o pantoprazol foi administrado simultaneamente aos antibióticos claritromicina, metronidazol e amoxicilina, não demonstraram nenhuma interação clinicamente siginificante. Reações adversas: Distúrbios gastrointestinais: ocasionalmente foram relatados casos de dor abdominal, diarréia, constipação ou flatulência. Houve raros relatos de náusea e vômito. Distúrbios do sistema nervoso: ocasionalmente pode ocorrer o aparecimento de cefaléia. Têm sido relatados raros casos de vertigem ou distúrbios visuais (visão turva). Distúrbios do tecido cutâneo e subcutâneo: reações alérgicas como prurido e exantema foram ocasionalmente relatados. Urticária e angioedema foram relatados em casos isolados. Distúrbios hepato-biliares: aumento de enzimas hepáticas (transaminases, y - GT) em casos isolados. Em casos raros ocorreu dano hepatocelular severo conduzindo à icterícia com ou sem insuficiência hepática. Distúrbios músculo-esqueléticos e do tecido conjuntivo: mialgia, em casos isolados, que regrediu com o término do tratamento. Distúrbios psiquiátricos: depressão mental, em casos isolados, que regrediu com o término do tratamento. Distúrbios gerais: febre e edema periférico, em casos isolados, que regrediram com o término do tratamento. Distúrbios metabólicos: aumento de triglicérides em casos individuais. Distúrbios no sistema imune: o tratamento com pantoprazol pode levar, em casos isolados, à reações anafiláticas, incluindo o choque anafilático. Posologia: Recomenda-se 1 comprimido de Pantozol® 20mg ao dia. A duração do tratamento fica à critério médico e dependente da indicação. Na maioria dos pacientes, o alívio dos sintomas é rápido, sendo atingido dentro de 2 a 4 semanas. No tratamento da esofagite a cicatriazação pode ser alcançada em 4 semanas. Os sintomas recorrentes poderão ser controlados administrando-se 1 comprimido de Pantozol® 20mg ao dia, quando necessário (“on demand”). A mudança para terapia contínua deve ser considerada nos casos em que os sintomas não puderem ser devidamente controlados sob terapia “on demand”. Em pacientes com insuficiência renal, a dose diária de 40mg não deve ser ultrapassada. Em caso de redução intensa da função hepática, a dose de 1 comprimido de 20mg ao dia não deve ser ultrapassada. Quando administrado por longos prazos, recomenda-se a manutenção da dose de 1 comprimido de Pantozol® 20mg ao dia, aumentando-se para 40mg de pantoprazol ao dia em casos de reincidência. Após a melhora, a dose pode ser reduzida novamente para 20mg de pantoprazol. Em terapia de longo prazo, especialmente quando o tratamento exceder 1 ano, os pacientes devem ser mantidos sob acompanhamento regular. Pantozol® pode ser administrado antes, durante ou após o café da manhã. O prazo de validade está impresso na embalagem do produto. MEDICAMENTO SOB PRESCRIÇÃO. Registro MS - 1.0639.0182 PT20_0207_0909. Material destinado exclusivamente a profissionais habilitados a prescrever e/ou dispensar medicamentos. 6064872 PZ PROTEÇÃO AINES 2012 - Fev/2012 Referências Bibliográficas: 1) Regula J et al. Prevention of NSAID-associated gastrointestinal lesions: a comparison study pantoprazole versus omeprazole. Am J Gastroenterol. 2006;101:1-9. 2) Blume H et al. Pharmacokinetic drug interaction profiles of proton pump inhibitors. Drug Saf. 2006;29(9):769-84. 3) Steinijans VW et al. Lack of pantoprazole drug interactions in man. Inter J Clin Pharmacol Ther. 1994;32(8):385-99. 4) Meyer UA. Metabolic interactions of the proton-pump inhibitors lansoprazole, omeprazole and pantoprazole with other drugs. Eur J Gastroenterol Hepatol. 1996;8(suppl 1):S21-5. 5) Juhász M, Herszényi L, Tulassay Z. Current standings of the proton pump inhibitor and clopidogrel co-therapy: review on an evolving field with the eyes of the gastroenterologist. Digestion. 2010;81(1):10-5. 6) Maton PN, Burton ME. Clinician’s manual on drug interactions in gastroenterology. London: Life Science, 2000. 7) Pantozol® [Bula]. São Paulo: Nycomed Pharma. A PERSISTIREM OS SINTOMAS, O MÉDICO DEVERÁ SER CONSULTADO.

Nycomed Pharma Ltda. Rua do Estilo Barroco, 721 04709-011 - São Paulo - SP. Mais informações poderão ser obtidas diretamente com o nosso Departamento Médico ou por meio de nossos representantes.

Linha Gastro


ISSN 0004-2803 ISSN 1678-4219 – on-line Coden ARQGA

CONTENTS Introduction of the Editor 1580

EDITORIAL

Another link in the chain Joel FAINTUCH, Ricardo Guilherme VIEBIG, 1

ORIGINAL ARTICLES 1581

Diagnosis of adult-type hypolactasia/lactase persistence: genotyping of single nucleotide polymorphism (SNP C/T-13910) is not consistent with breath test in Colombian Caribbean population Evelyn Mendoza TORRES, Lourdes Luz Varela PRIETO, José Luiz Villareal CAMACHO, Daniel Antonio Villanueva TORREGROZA, 5

1582

Effect of HFE gene polymorphism on sustained virological response in patients with chronic hepatitis C and elevated serum ferritin Silvia COELHO-BORGES, Hugo CHEINQUER, Fernando Herz WOLFF, Nelson CHEINQUER, Luciano KRUG, Patricia ASHTON-PROLLA, 9

1583

Assessment of portal venous index as a non-invasive method for diagnosing liver fibrosis in patients with chronic hepatitis C Haroldo Luiz Oliva Gomes ROCHA, Angélica Lemos Debs DINIZ, Valéria Ferreira de Almeida e BORGES, Frederico Chaves SALOMÃO, 14

1584

Nutritional assessment in patients with cirrhosis Sabrina Alves FERNANDES, Lilian BASSANI, Flávia Feijó NUNES, Maria Eugênia Deutrich AYDOS, Alexandro Vaesken ALVES, Cláudio Augusto MARRONI, 19

1585

Investigation of nutritional risk factors using anthropometric indicators in hospitalized surgery patients Vânia Aparecida LEANDRO-MEHRI, José Luiz Braga de AQUINO, 28

1586

Immunohistochemical evaluation of p53 and Ki-67 proteins in colorectal adenomas Walysson Alves Tocantins de SOUSA, Lusmar Veras RODRIGUES, Raimundo Gerônimo da SILVA Jr., Fernando Lopes VIEIRA, 35

1587

Common bile duct stones. Analysis of the videolaparoscopic surgical treatment Marco Aurelio SANTO, Carlos Eduardo DOMENE, Daniel RICCIOPPO, Lian BARREIRA, Flavio Roberto TAKEDA, Henrique Walter PINOTTI, 41

1588

Prevalence of upper digestive endoscopy and gastric histopathology findings in morbidly obese patients Judite DIETZ, Jane Maria ULBRICH-KULCYNSKI, Katia Elisabete Pires SOUTO, Nelson Guardiola MEINHARDT, 52

1589

Helicobacter pylori eradication does not influence gastroesophageal reflux disease: a prospective, parallel, randomized, open-label, controlled trial Lino RODRIGUES Jr., Cintya Miler de FARIA, Stephan GEOCZE, Luiz CHEHTER, 56

1590

Esophageal manometry findings and degree of acid exposure in short and long Barrett’s esophagus Laura HELMAN, Beatriz Nunes BICCAS, Eponina M. O. LEMME, Paula NOVAIS, Viviane FITTIPALDI, 64

ARCHIVES OF GASTROENTEROLOGY

v. 49 – no.1 – Jan./Mar. 2012


PEDIATRIC GASTROENTEROLOGY 1591

Portal vein thrombosis in children and adolescents: 20 years experience of a pediatric hepatology reference center Priscila Menezes FERRI, Alexandre Rodrigues FERREIRA, Eleonora Druve Tavares FAGUNDES, Shinfay Maximilian LIU, Mariza Leitão Valadares ROQUETE, Francisco José PENNA, 69

EXPERIMENTAL GASTROENTEROLOGY 1592

Hepatic hyperplasia and damages induces by zearalenone Fusarium mycotoxins in Balb/C mice Pronobesh CHATOPADHYAY, Anurag PANDEY, Asshwani K. CHAURASIA, Addesh UPADHYAY, Sanjev KARMAKAR, Lokendra SINGH, 77

1593

Noise-induced gastric lesions: a light and electron microscopy study of the rat gastric wall exposed to low frequency noise Jorge FONSECA, José Martins-dos-SANTOS, Pedro OLIVEIRA, Nuno LARANJEIRA, Artur AGUAS, Nuno CASTELO-BRANCO, 82

REVIEW 1594

Metabolic syndrome and risk factors for non-alcoholic fatty liver disease Mônica Rodrigues de Araújo SOUZA, Margareth de Fátima Formiga de Melo DINIZ, José Eymard Moraes de Medeiros-FILHO, Maria Salete Trigueiro de Araújo, 89

ARCHIVES OF GASTROENTEROLOGY

ISSN 0004-2803 ISSN 1678-4219 – on-line Coden ARQGA

v. 49 – no.1 – Jan./Mar. 2012


ISSN 0004-2803 ISSN 1678-4219 – on-line Coden ARQGA

CONTEÚDO Notas do Editor

Editorial 1580

mais um elo na corrente Joel FAINTUCH, Ricardo Guilherme VIEBIG, 1

ARTIGOS ORIGINAIS 1581

Diagnóstico de hipolactasia tipo adulto/persistência da lactase: a genotipagem do polimorfismo de um único nucleotídeo (SNP C/T-13910) não é consistente com o teste de hidrogênio na população do Caribe Colombiano Evelyn Mendoza TORRES, Lourdes Luz Varela PRIETO, José Luiz Villareal CAMACHO, Daniel Antonio Villanueva TORREGROZA, 5

1582

Efeito do polimorfismo do gene HFE sobre a resposta viral sustentada em portadores de hepatite crônica C com ferritina sérica elevada Silvia COELHO-BORGES, Hugo CHEINQUER, Fernando Herz WOLFF, Nelson CHEINQUER, Luciano KRUG, Patricia ASHTON-PROLLA, 9

1583

Emprego do índice venoso portal como método não-invasivo no diagnóstico de fibrose hepática em pacientes portadores de hepatite C crônica Haroldo Luiz Oliva Gomes ROCHA, Angélica Lemos Debs DINIZ, Valéria Ferreira de Almeida e BORGES, Frederico Chaves SALOMÃO, 14

1584

Avaliação nutricional de pacientes cirróticos Sabrina Alves FERNANDES, Lílian BASSANI, Flavia Feijó NUNES, Maria Eugênia Deutrich AYDOS, Alexandro Vaesken ALVES, Cláudio Augusto MARRONI, 19

1585

Investigação de fatores de risco nutricional por meio de indicadores antropométricos em pacientes cirúrgicos hospitalizados Vânia Aparecida LEANDRO-MEHRI, José Luiz Braga de AQUINO, 28

1586

Avaliação imunoistoquímica das proteínas p53 e Ki-67 em adenomas colorretais. Walysson Alves Tocantins de SOUSA, Lusmar Veras RODRIGUES, Raimundo Gerônimo da SILVA Jr, Fernando Lopes VIEIRA, 35

1587

Coledocolitíase. Análise do tratamento videolaparoscópico Marco Aurelio SANTO, Carlos Eduardo DOMENE, Daniel RICCIOPPO, Lian BARREIRA, Flavio Roberto TAKEDA, Henrique Walter PINOTTI, 41

1588

Prevalência de achados gástricos endoscópicos e histopatológicos em pacientes obesos Judite DIETZ, Jane Maria ULBRICH-KULCYNSKI, Katia Elisabete Pires SOUTO, Nelson Guardiola MEINHARDT, 52

1589

Erradicação do Helicobacter pylori não influencia a doença do refluxo gastroesofágico: estudo prospectivo paralelo, randomizado, aberto e controlado Lino RODRIGUES Jr, Cintya Miler de FARIA, Stephan GEOCZE, Luiz CHEHTER, 56

1590

Alterações manométricas e intensidade do refluxo no esôfago de Barrett curto e longo Laura HELMAN, Beatriz Nunes BICCAS, Eponina M. O. LEMME, Paula NOVAIS, Viviane FITTIPALDI, 64

ARquIVos de GASTROENTEROLOGia

v. 49 – no.1 – Jan./Mar. 2012


ISSN 0004-2803 ISSN 1678-4219 – on-line Coden ARQGA

GASTROENTEROLOGIA PEDIÁTRICA 1591

Trombose de veia porta em crianças e adolescentes: experiência de 20 anos de um serviço de referência em hepatologia pediátrica Priscila Menezes FERRI, Alexandre Rodrigues FERREIRA, Eleonora Druve Tavares FAGUNDES, Shinfay Maximilian LIU, Mariza Leitão Valadares ROQUETE, Francisco José PENNA, 69

GASTROENTEROLOGIA EXPERIMENTAL 1592

Hiperplasia e danos hepáticos induzidos por micotoxinas do gênero Fusarium-zearalenone em camundongos bab/c Pronobesh CHATOPADHYAY, Anurag PANDEY, Asshwani K. CHAURASIA, Addesh UPADHYAY, Sanjev KARMAKAR, Lokendra SINGH, 77

1593

Lesões gástricas induzidas pelo ruído: um estudo por microscopia óptica e eletrônica da parede gástrica do rato exposta a ruído de baixa frequência Jorge FONSECA, José Martins-dos-SANTOS, Pedro OLIVEIRA, Nuno LARANJEIRA, Artur AGUAS, Nuno CASTELO-BRANCO, 82

REVISÃO 1594

Síndrome metabólica e fatores de risco para a doença hepática gordurosa não-alcoólica Mônica Rodrigues de Araújo SOUZA, Margareth de Fátima Formiga de Melo DINIZ, José Eymard Moraes de Medeiros-FILHO, Maria Salete Trigueiro de Araújo, 89

ARquIVos de GASTROENTEROLOGia

v. 49 – no.1 – Jan./Mar. 2012


EDITOR FUNDADOR/FOUNDING EDITOR Editor Científico/Scientific Editor Editor Executivo / Editor-in-chief EDITORES ASSISTENTES / ASSISTANT

José Fernandes Pontes (IBEPEGE, São Paulo) Mounib Tacla (IBEPEGE, São Paulo) Ricardo Guilherme Viebig (IBEPEGE, São Paulo) Fernando Pardini (IBEPEGE), Ivan Cecconello (CBCD) Alberto Queiroz Farias (SBH), Flávio A. Quilici (FBG) Paulo Roberto Arruda Alves (SOBED), Maria do Carmo Friche Passos (SBMD)

EDITORES ASSOCIADOS/ASSOCIATE EDITORS Adávio de Oliveira e Silva (USP, São Paulo) Adérson Omar C.A. Damião (USP, São Paulo) Alfredo J. Afonso Barbosa (UFMG, Belo Horizonte, MG) Aloísio Souza Felipe Silva (HU, São Paulo) Angelo A. Mattos (UFRS, Porto Alegre, RS) Ary Nasi (USP, São Paulo) Ben-Hur Ferraz Neto (PUC, Sorocaba, SP) Carlos Walter Sobrado (USP, São Paulo) Claudemiro Quireze Jr. (UFGO, Goiânia, GO) Claudia P. Marques de Oliveira (USP, São Paulo) Dan L. Waitzberg (USP, São Paulo) Dulce Reis Guarita (USP, São Paulo) Edna Frasson de Souza Montero (UNIFESP, São Paulo) Edna Strauss (USP, São Paulo) Fabio Guilherme Campos (USP, São Paulo) Fabio P. Lopasso (USP, São Paulo) Fauze Maluf Filho (USP, São Paulo) Flair José Carrilho (USP, São Paulo) Gaspar de Jesus Lopes Filho (UNIFESP, São Paulo) Heitor Rosa (UFGO, Goiânia, GO) Jacques Waisberg (FMABC, Santo André, SP) João Gomes Netinho (FM São José do Rio Preto, SP) Joaquim Prado P. de Moraes Filho (USP, São Paulo) Joel Faintuch (USP, São Paulo) José Celso Ardengh (USP, Ribeirão Preto, SP) José Eduardo Monteiro da Cunha (USP, São Paulo) José Marcio Neves Jorge (USP, São Paulo) Julio Cezar Uili Coelho (UFPR, Curitiba, PR)

Luiz Augusto Carneiro D’Albuquerque (USP, São Paulo) Luiz Fernando Corrêa Zantut (USP, São Paulo) Marcel Autran C. Machado (USP, São Paulo) Marcelo Averbach (Hospital Sírio Libanês, São Paulo, SP) Marcelo Gil Cliquet (PUC, Sorocaba, SP) Marcelo Eidi Nita (USP, São Paulo) Marco Aurelio Santo (USP, São Paulo) Mario Guimarães Pessoa (USP, São Paulo) Mauro Batista de Morais (UNIFESP, São Paulo) Milton M. Barbosa Costa (UFRJ, Rio de Janeiro) Nora Manoukian Forones (UNIFESP, São Paulo) Osvaldo Malafaia (UFPR, Curitiba, PR.) Paula Poletti (Hospital do Coração, São Paulo) Paulo Lisboa Bittencourt (Hospital Português, Salvador, BA) Paulo Sakai (USP, São Paulo) Renata Pugliese (Instituto da Criança - USP, São Paulo) Roberto Carlos Burini (UNESP, Botucatu, SP.) Roberto Oliveira Dantas (USP, Ribeirão Preto, SP.) Sender Jankiel Miszputen (UNIFESP, São Paulo) Sergio Carlos Nahas (USP, São Paulo) Sonia Penteado (USP, São Paulo) Suzane Kioko Ono-Nita (USP, São Paulo) Ulysses Fagundes Neto (UNIFESP, São Paulo) Ulysses Ribeiro Jr. (USP, São Paulo) Valter Nilton Felix (USP, São Paulo) Venâncio Avancini Ferreira Alves (USP, São Paulo) Yu Kar Ling Koda (Inst. da Criança – USP, São Paulo)

CONSULTORES - BRASIL Angelita Habr-Gama (USP, São Paulo), Arthur B. Garrido Jr. (USP, São Paulo), Cervantes Caporossi (UFMT, Cuiabá, MT), Desidério Roberto Kiss (USP, São Paulo), Edmundo Machado Ferraz (UFPE, Recife, PE), Helio Moreira (UFGO, Goiânia, GO.), João Batista Marchesini (UFPR, Curitiba, PR.), Joaquim Gama Rodrigues (USP, São Paulo), Luiz Rohde (UFRS, Porto Alegre, RS.), Marcel Cerqueira César Machado (USP, São Paulo), Maria Aparecida C. A. Henry (UNESP, Botucatu, SP.), Paulo Roberto Ott Fontes (FFFCMPA, Porto Alegre, RS.), Renato Bonardi (UFPR, Curitiba, PR.), Samir Rasslam (USP, São Paulo), Sérgio Brenner (UFPR, Curitiba, PR.), William Abrão Saad (USP, São Paulo), William Saad Hossne (UNESP, Botucatu, SP.)

CONSULTANT - INTERNATIONAL Prof. Dr. med. Peter Malfertheiner (OTTO-von-Guericke-Universität, Magdeburg, Germany); Prof. Francis Megraud (INSERM - U853, Bordeaux, France). Daniel Sifrim, MD, PhD (Barts and The London School of Medicine and Dentistry, London, UK); Steven Wexner MD, PhD (Cleveland Clinic Florida, Weston, FL, USA); Mark Scott, MD, PhD (Royal London Hospital, London, UK); Etsuro Yazaki (Barts and The London School of Medicine and Dentistry, London, UK). A revista ARQUIVOS de GASTROENTEROLOGIA é indexada nas seguintes Bases de Dados / The journal ARCHIVES of GASTROENTEROLOGY is abstracted and/or indexed in: EMBASE/Excerpta Medica, Hygiene and Communicable Diseases (CAB Abstracts), LILACS, PUBMED/MEDLINE, Periódica: Índice de Revistas Latinoamericana en Ciencias, Tropical Diseases Bulletin (CAB Abstracts). On-line texto completo/Full texts: http://www.scielo.br/ag.htm

Expediente / Editorial Offices

Secretaria Executiva/Executive Secretary Maria Cecilia P. Pinheiro Rosangela Evangelista dos Santos

Redação e Administração / Correspondence Rua Dr. Seng, 320 – Bela Vista – 01331-020 – São Paulo, SP – Brasil Tel./Fax: (0xx11) 3147-6227 e-mail: secretariaarqgastr@hospitaligesp.com.br

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Publicação Trimestral/Quarterly Publication Annual Subscription Rate: US$ 70.00/year – Single copies: US$ 20.00

ARQUIVOS DE GASTROENTEROLOGIA / ARCHIVES OF GASTROENTEROLOGY

Official Publication of: BRAZILIAN INSTITUTE FOR STUDIES AND RESEARCH IN GASTROENTEROLOGY AND OTHER SPECIALITIES Alcides Felix Terrivel (Representative) BRAZILIAN COLLEGE OF DIGESTIVE SURGERY Cleber Dario Pinto Kruel (President) BRAZILIAN DIGESTIVE MOTILITY SOCIETY Eponina Maria Lemme (President) BRAZILIAN FEDERATION OF GASTROENTEROLOGY José Galvão Alves (President) BRAZILIAN SOCIETY of HEPATOLOGY Raymundo Paraná (President) BRAZILIAN SOCIETY of DIGESTIVE ENDOSCOPY Sérgio Luiz Bizinelli (President)


INSTRUCTIONS TO AUTHORS The journal ARQUIVOS de GASTROENTEROLOGIA (Archives of Gastroenterology) a quarterly journal is the Official Publication of the Instituto Brasileiro de Estudos e Pesquisas de Gastroenterologia – IBEPEGE (Brazilian Institute for Studies and Research in Gastroenterology), Colégio Brasileiro de Cirurgia Digestiva – CBCD (Brazilian College of Digestive Surgery), Sociedade Brasileira de Motilidade Digestiva – SBMD (Brazilian Digestive Motility Society) and of the Federação Brasileira de Gastroenterologia – FBG (Brazilian Federation of Gastroenterology), is dedicated to the publishing of scientific papers by national and foreign researchers who are in agreement with the aim of the journal as well as with its editorial polices. Scientific papers sent for publication should be unpublished and intended exclusively for ARQUIVOS de GASTROENTEROLOGIA. Papers for publication should be submitted in triplicate typed double-spaced (authors are advised to keep a copy for their own files) on the white bond paper ISO A4 (210 x 297 mm) with margins of at least 2.5 cm (1 in) and all pages numbered consecutively, beginning with the tittle page. Each manuscript submitted to ARQUIVOS de GAS­TRO­ ENTEROLOGIA should be arraged as follows: 1) title; 2) author(s) name(s); 3) the departament and institution where the work was performed; 4) the name, telephone number, FAX number, electronic address and postal correspondence address of author to whom galley proofs and requests for reprints should be sent; 5) acknowledgement of grants and other financial support; 6) structured abstract – the papers should be sent with abstract in English (200 words at least); abbreviations, footnotes and references should be avoided; 7) key words (3 to 10). Whenever possible, use terms of Medical Subject Headings (MESH) list from MEDLINE; 8) introduction; 9) literature; 10) material; 11) method; 12) results; 13) discussion; 14) conclusions; 15) references – arranged in alphabetical order of author’s last name (or the name of the first author, in case of more than one). Abbreviations of journals should conform to those used in INDEX MEDICUS. The references are identified in the text by arabical numerals in parenthesis. The style of the references follow the format of the ‘Vancouver style”: Uniform requirements for manuscripts submitted to biomedical journals of the International Committee of Medical Journal Editors (ICMJE), complete text in: Ann Intern Med. 1997;126:36-47; N Eng J Med. 1991;324:424-8 or

in Canadian Medical Association site: http://www.cma.ca/mwc/ uniform.htm. The references are identified in the text by arabical numerals in parenthesis. Exemples: Journal article (list all authors and do not use “et al.”): Ribeiro Jr U, Cecconello 1, Safatle-Ribeiro AV, Zilberstein B, Pinotti HW. Squamous cell carcinoma of the esophagus and multiple primary tumors of the upper aerodigestive tract. Arq Gastroenterol. 1999;36:195-200. Books and other monographs (list all authors/editors and do not use “et al.”): Castell DO, Richter JE, editors. The esophagus. 3. ed. Philadelphia: Lippincott Williams & Wilkins; 1999. Chapter in a book (list all authors and do not use “et al.”): Cohen RV, Roll S, Schaffa TD. Hernioplastia incisional videolaparoscópica. Rio de Janeiro: Reichmann & Affonso; 1999. p. 127-31. Dissertations and thesis: Cecconello 1. Contribuição ao conhecimento e histopatologia do colédoco. [Dissertação de mestrado]. São Paulo: Instituto Brasileiro de Estudos e Pesquisas de Gastroenterologia – IBEPEGE; 1979, Published proceedings paper (list all authors and do not use “et al.”): Nasi A, Cenatti A, Falcão A, Cecconello 1, Sallum RAA, Pinotti HW. Evaluation of lower esophageal sphincter pressure by two variant techniques in patents with endoscopic reflux esophagitis [abstract]. In: Meeting abstracts of the Esophagus ’98: 7th World Congress of the International Society for Diseases of the Esophagus; 1998; Montreal, Canada. Can J Gastroenterol. 1998;12Suppl.B:93B.[Abstract 278]. TABLES - Number tables in arabic numbers and supply a legend for each. Explanatory matter should be placed in footnotes as well as nonstandard abbreviation that are used. Do not use internal horizontal or vertical rules. ILLUSTRATIONS – Photographs, graphics and drawings should be sent sharp, glossy, black-and-white photographic prints, usually 127 mm x 178 mm. Each illustration should have a label pasted on its back indicating its number, the first author’s name and the article’s title. Ilustrations in colour only if the author pays for the extra cost.

MANUSCRIPT SUBMISSION Manuscripts must be submitted using the Online Submission system available at: http://submission.scielo.br/index.php/ag/index accessing the link Online Submission. The author responsible for the submission must be a system user prior the submission. All steps of the editorial process will occur using the system. When submitting a manuscript the author must digitally assign that the article is no being considered for publication at the same time in no other journal. This procedure avoids the need of sending the letters of Authorship Agreement and Copyright Transferral.


INSTRUÇÕES AOS AUTORES A revista trimestral, ARQUIVOS de GASTROENTEROLOGIA, é órgão

ordem alfabética em relação ao sobrenome do autor (ou do primeiro,

oficial do Instituto Brasileiro de Estudos e Pesquisas de Gastroenterologia

no caso de vários), de acordo com o “estilo Vancouver”: Uniform

– IBEPEGE, do Colégio Brasileiro de Cirurgia Digestiva - CBCD, da

requirements for manuscripts submitted to biomedical journals of the

Sociedade Brasileira de Motilidade Digestiva – SBMD e da Federação

International Committee of Medical Journal Editors (ICMJE), cujo

Brasileira de Gastroenterologia - FBG. Destina-se a publicar trabalhos

texto completo pode ser consultado em: Ann Intern Med. 1997;126:36-

científicos de pesquisadores nacionais ou estrangeiros que se enquadrem

47; N Engl J Med. 1997;336:309-15 ou no site da Canadian Medical

no objetivo da Revista e em suas exigências redatoriais. Os textos devem ser

Association: http://www.cma.ca/mwc/uniform.htm. Os títulos dos

redigidos em inglês.

periódicos devem ser referidos de forma abreviada de acordo com

Os trabalhos científicos enviados para publicação devem ser inéditos e destinar-se exclusivamente a ARQUIVOS de GAS­TROENTEROLOGIA. Toda matéria relacionada à investigação humana e à pesquisa animal deve

a List of Journals Indexed in INDEX MEDICUS. As citações no texto devem ser por números índices, correspondendo às respectivas referências bibliográficas.

ter aprovação prévia da Comissão de Ética da instituição onde o trabalho foi

Exemplos:

realizado, de acordo com as recomendações da Declaração de Helsinque (1964

Artigos de periódicos (cite todos os autores; não use “et al.”):

e suas versões posteriores de 1975, 1983 e 1989), as Normas Internacionais de

Ribeiro Jr U, Cecconello I, Safatle-Ribeiro AV, Zilberstein B, Pinotti HW.

Proteção aos Animais e a Resolução no 196/96 do Conselho Nacional de Saúde

Squamous cell carcinoma of the esophagus and multiple primary tumors of

sobre pesquisa envolvendo seres humanos. São aceitos estudos de natureza

the upper aerodigestive tract. Arq Gastroenterol. 1999;36:195-200.

original, clínicos ou cirúrgicos, técnicas e estudos de epidemiologia. Artigos de revisão e atualização só são aceitos à convite do Conselho Editorial. Não se publicam Relatos de caso. Os estudos submetidos a publicação nos ARQUIVOS de GASTROENTEROLOGIA são enviados a três pareceristas de reconhecida competência no tema abordado. O anonimato é garantido durante todo o processo de julgamento. A decisão sobre aceitação é tomada pela Comissão Editorial. Os originais (manuscritos) para publicação devem ser enviados em triplicata (recomenda-se ao autor guardar consigo uma quarta via

Livros e outras monografias (cite todos os autores/editores; não use “et al.”): Castell DO, Richter JE, editors. The esophagus. 3.ed. Philadelphia: Lippincott Williams & Wilkins; 1999. Capítulo de livro (cite todos os autores e não use “et al.”): Cohen RV, Roll S, Schaffa TD. Hernioplastia incisional videolaparoscópica. In: Goldenberg S, editor. Avanços em cirurgia laparoscópica. Rio de Janeiro: Reichmann & Affonso; 1999. p.127-31.

para seu arquivo), digitados em uma só face, com espaço duplo, em

Dissertação e tese:

fonte Times New Roman ou Arial, tamanho 12, em papel branco

Cecconello I. Contribuição ao conhecimento e histopatologia do colédoco.

tamanho ISO A4 (210 × 297 mm), com margens de, pelo menos,

[Dissertation]. São Paulo: Instituto Brasileiro de Estudos e Pesquisas de

2,5 cm, numerando-se as páginas consecutivamente, iniciando na

Gastroenterologia – IBEPEGE; 1979.

página de identificação. Todo artigo para publicação deverá constar de: 1) Página de identificação, que deve conter: título do artigo (com tradução em inglês); nome(s) completo(s) do(s) autor(es) com sua principal filiação acadêmica ou profissional. Para estudos originais admite-se até um máximo de seis autores, nos demais apenas três; nome do departamento e instituição onde o trabalho foi realizado; nome, endereço completo, telefone, número do Fax e e-mail do autor para quem deverão ser enviadas as provas tipográficas e pedidos de separatas; agradecimentos (quando pertinentes); 2) Página de resumos e descritores, contendo resumos estruturados – um em português, outro

Trabalho de evento (publicado) (cite todos os autores e não use “et al.”): Nasi A, Cenatti A, Falcão A, Cecconello I, Sallum RAA, Pinotti HW. Evaluation of lower esophageal sphincter pressure by two variant techniques in patients with endoscopic reflux esophagitis [abstract]. In: Meeting abstracts of the Esophagus ’98: 7th World Congress of the International Society for Diseases of the Esophagus; 1998; Montreal, Canada. Can J Gastroenterol. 1998;12 Suppl.B:93B. [Abstract 278]. TABELAS – Devem ser apresentadas em folhas separadas do texto numeradas com algarismos arábicos na ordem em que forem citadas, contendo legendas;

em inglês (com pelo menos 200 palavras cada um); descritores (de 3

explicações dos símbolos devem figurar no rodapé.

a 10), sempre que possível, extraídos do Medical Subject Headings

ILUSTRAÇÕES – Fotos, gráficos e desenhos devem ser enviados em preto

(MESH) da National Library of Medicine ou do vocabulário Descritores

e branco, com dimensões 127 mm × 178 mm, limitados a quantidade de seis.

em Ciências da Saúde, que pode ser consultado no site www.bireme.

Devem conter identificação no verso, com a orientação, o número de ordem

br; 3) introdução; 4) literatura; 5) material; 6) método; 7) resultados; 8)

no texto, o nome do autor e o título do artigo. O custo da reprodução de

discussão; 9) conclusões; 10) referências bibliográficas – relacionadas na

ilustrações coloridas correrão por conta do autor do artigo.

Submissão de artigos Os artigos deverão ser submetidos pelo Sistema de Submissão Online disponível no site: http://submission.scielo.br/index.php/ag/index acessando o link submissão online. O autor responsável pela submissão deverá cadastrar-se previamente no sistema. Toda a tramitação das etapas do processo editorial será realizada por meio deste sistema. Ao submeter o manuscrito, o autor deverá firmar eletronicamente que o artigo não está sendo submetido paralelamente a outro periódico. Este procedimento elimina a necessidade do envio de cartas de responsabilidade de Autoria e Transferência de Direitos Autorais.


INTRODUCTION OF THE EDITOR The editorial trajectory of the ARCHIVES of GASTROENTEROLOGY is an undeniable example of strict adherence to standard of norms, rules and principles which should govern the medical scientific publication and research thinking, through various research vehicles (tests, experiments, reviews, comments), specifically in the multiplicity of different gastroenterologic science approaches. Since its first issues, a pattern of publications already structured in a multi and interdisciplinary design and formulation could be noticed, consequently joining the various currents of research and scientific thinking related to the specialty of Gastroenterology. The journal profile was a projection of the research approach, the thinking, thought-provoking and restless behavior of its intellectual mentor, Prof. José Fernandes Pontes, leading it like a trademark of his personality and excellence that permeated all the editorial development of the ARCHIVES. The holistic approaches that lent to the evaluation, research and discussion of clinical observations of patients with digestive disorders, integrating them to emotional dynamics phenomena, hitherto Behavioral and Science properties, thus encouraging psychoanalytic a broader look and integrator to the current science methodology, were at least, adventurous. The ARCHIVES followed step by step, and always updating, the complex transformations and acquisitions experienced by Brazilian and international Gastroenterology over the past 50 years, provided by the recognized standard of excellence, with professional editorial board and high levels of scientific publishing, always with strict respect to the stringent mechanisms and ethical precepts that govern the complex process of feasibility and publication of scientific works. This publication enters this century adapted and restructured to the demands and technological innovations for informatics, renewing and rebuilding it graphically, getting a privileged position compared to the criteria adopted for classification of periodicals, reflecting and promoting its International

indexing in databases of recognized reputation and, consequently, giving great penetration in international scientific and academic community. Loyal to its principles to adapt its focus on multiple currents, integrating thoughts and aspects of national scientific research, the ARCHIVES provided and embraced, with its mature and competent editorial structure, the representative and reputable societies and entities engaged in various segments of study and research, such as: Brazilian College of Digestive Surgery, Brazilian Society of Gastrointestinal Motility, Brazilian Federation of Gastroenterology, Brazilian Society of Hepatology and Brazilian Society of Digestive Endoscopy, and became the Official Representative Journal, strengthening and extending through diverse publications, leading to significant editorial acquisitions. This present edition celebrates and inaugurates, with great pride, a new partnership, with one more remarkable and prestigious scientific entity of the country, the Brazilian Society of Clinical Nutrition, another pillar that holds and integrates the multi-faceted fabric of research and clinical practice of Gastroenterology, also holder of great experience and representativeness in the editorial field, through regular publication of its magazine. For the ARCHIVES, this embrace meets and solidifies its goal of expansion and enrichment, based on cooperation, because they will certainly become more “well nourished” with their important contributions, based on the research and medical practice of human nutrition, which, consequently, will enrich readers, the entities already aggregated and represented by them, and all of our scientific community and editorial. The ARCHIVES of GASTROENTEROLOGY join to the Brazilian Society of Clinical Nutrition, welcomes and invites: “ Take your place, the meal is served”.

Fernando PARDINI Assistant Editor

NOTA DO EDITOR A trajetória editorial da revista ARQUIVOS de GASTROENTEROLOGIA é um incontestável exemplo de padrão de cumprimento rigoroso de normas, regras e princípios que devem nortear a publicação da pesquisa e pensamento científico da medicina, através de vários veículos de investigação (ensaios, experimentos, revisões, observações), especificamente na multiplicidade de diferentes enfoques da ciência gastroenterológica. Desde suas primeiras edições observou-se um padrão de publicações já estruturado numa concepção e formulação multi e interdisciplinar, portanto agregadora, das várias correntes de pesquisa e pensamento científico relacionadas à especialidade da Gastroenterologia. Projetava-se neste caráter de abordagem das pesquisas, o pensamento irriquieto, instigante e aglutinador de seu mentor intelectual, Prof. José Fernandes Pontes, marca registrada de personalidade e excelência que permeou todo o desenvolvimento editorial dos ARQUIVOS. Foram, no mínimo, ousadas as abordagens holísticas que emprestava à avaliação, investigação e discussão de observações clínicas de pacientes com distúrbios digestivos, integrando-os aos fenômenos de sua dinâmica emocional, até então propriedades das Ciências Comportamental e Psicanalítica, incentivando, assim, um olhar mais abrangente e integrador à metodologia científica vigente. Os ARQUIVOS acompanharam passo a passo, atualizando-se sempre, as complexas transformações e aquisições experimentadas pela gastroenterologia brasileira e internacional ao longo dos últimos cinquenta anos, proporcionadas pelo reconhecido padrão de excelência profissional e científica de seus Corpos Editoriais, sempre atentos aos rigorosos mecanismos e preceitos éticos que norteiam o complexo processo de viabilização e publicação dos trabalhos científicos. Adentram o presente século adaptados e reestruturados às exigências e inovações tecnológicas da informática, renovam-se e repaginam-se graficamente, obtendo posição privilegiada frente aos critérios adotados para classificação de periódicos, repercutindo e favorecendo sua indexação internacional em

Bases de Dados de reconhecida reputação e, conseqüentemente, grande penetração na comunidade acadêmica e científica internacional. Fieis a seus princípios básicos de adequar seu enfoque integrador às várias correntes, pensamentos e vertentes da pesquisa científica gastroenterológica nacional, os ARQUIVOS se disponibilizaram e abraçaram, com sua competente e madura estrutura editorial, as representativas e idôneas Sociedades e Entidades atuantes nos vários segmentos de estudo e pesquisa, como: Colégio Brasileiro de Cirurgia Digestiva, Sociedade Brasileira de Motilidade Digestiva, Federação Brasileira de Gastroenterologia, Sociedade Brasileira de Hepatologia e Sociedade Brasileira de Endoscopia Digestiva, das quais se tornou Órgão Oficial de Divulgação, robustecendo e ampliando através de publicações diversificadas, seu extenso e significativo acervo editorial. Esta presente edição dos ARQUIVOS comemora e inaugura, com muito orgulho, nova parceria com mais uma notável e prestigiada entidade científica do país, a Sociedade Brasileira de Nutrição Clínica, outro eixo em que se sustenta e integra a malha multifacetada da investigação médica e da prática clínica da Gastroenterologia, detentora, também de grande experiência e representatividade no campo editorial, através de publicação regular de sua revista. Para os ARQUIVOS este abraço atende e solidifica seu objetivo de expansão e enriquecimento baseado na cooperação, pois certamente tornar-se-ão mais “nutridos” com suas importantes contribuições advindas da investigação e prática médica da Nutrição Humana, o que, por conseguinte, enobrecerá os leitores, as entidades já agregadas e representadas por eles, e toda nossa comunidade científica e editorial. Os Arquivos de Gastroenterologia irmana-se à Sociedade Brasileira de Nutrição Clínica, lhe saúda e convida : “ Ocupe seu lugar, a mesa está posta “.

Fernando PARDINI Editor Assistente


EDITORIAL

ARQGA/1580

ANOTHER LINK IN THE CHAIN Faintuch J, Viebig RG. Another link in the chain. Arq Gastroenterol. 49(1):1-2. HEADINGS – Scientific and technical publications. Manuscripts, medical. Editorial policies. Nutrition, public health.

In 1840 a modest publication appeared in the United Kingdom, the Provincial Medical and Surgical  Journal, nowadays the famous British Medical Journal. By that time medical activity already faced professional challenges and dilemmas. One editorial emphasized that the best way of advancing in the debate was to regularly publish all medically relevant issues and problems(1). Almost two centuries later the doubt is not anymore why to publish, but what, where, when, and with which standard of excellence. Nowadays everyone feels threatened by the devouring sphynx and its new riddle of “publish or perish”. Whoever is not in the media becomes anonymous or almost so, and media for doctors are preferentially renowned and respected publications with high impact, thus able to convey status, credibility and wide repercussion to submitted studies. One may identify here two encroaching circles, one virtuous and the other vicious. From one side there are limited and vulnerable communications, with unsophisticated methodology and unconvincing results. Journals that agree to publish them are exposing themselves to criticism and may in the future only receive similarly fragile submissions, with the risk of progressive irrelevance. The opposite is true as well. Elite publications with first class articles attract the best authors and protocols in a rich-gets-richer effect, therefore tending to become even more prestigious and powerful. It becomes obvious that everybody should quest for excellence and escape the abyss. However even councillor Acacio, created by the writer Eça de Queiroz, would lough at the mediocrity of this conclusion. One needs more than good intentions to reach such aim. Almost all current advices extol the importance of cooperative efforts and the union of forces. Solitary genius investigators still exist, conducting innovative and pioneering studies year after year. By the same token isolated journals with small author circles sometimes maintain top quality. In most circumstances however, there is no alternative for a broad and solid supporting base. Albert Einstein succeeded in deducting some of the most fundamental laws and equations of the 1 2

universe nearly without human or material help. He equipped himself with pencil, paper and mathematical formulas, during the free time on his job in Bern at the “Eidgenossisches Institut fur Geistiges Eigentum”, the Swiss agency for patents and intellectual property. In contrast, modern science is hardly viable without ambitious, complicated and expensive protocols and experimental designs. Generation of meaningful knowledge is becoming less and less probable in the absence of collaborative teams, resources, disciplines and technologies(2). The Brazilian Society of Clinical Nutrition was born in the remote year of 1975, and the Journal has been regularly published for more than three decades. Nevertheless, the time has come for greater proximity with a more robust and well established publication, which has successfully tackled big challenges of indexation and circulation and will overcome many more, namely the ARCHIVES of GASTROENTEROLOGY. It is therefore in the spirit of friendship and fraternity that the meal arrives to the gastrointestinal tube, or better, that nutrition seeks lodgement under the welcoming roof of Gastroenterology. There are reasons to believe that a true synergy will follow, with the whole growing bigger than the sum of its parts. From one side the Archives already accept contributions geared toward nutrition, obesity, bariatric surgery and correlated subjects. From the other side, many professionals in the area of enteral and parenteral nutrition are physically or intellectually housed in services of gastroenterology or digestive surgery, bearing witness to their appreciation of these specialties. Toasts, particularly alcoholic ones, have become politically incorrect in the XXI century, however there is no way of completely avoiding them. May this event be a harbinger of a luminous future, and may the ARCHIVES of GASTROENTEROLOGY, now encompassing Clinical Nutrition, march from triumph to triumph, for the pride of those who subscribe its pages and abide by its respected editorial line. Joel FAINTUCH1 Ricardo Guilherme VIEBIG2

Associate Editor Editor-in-chief

v. 49 – no.1 – jan./mar. 2012

Arq Gastroenterol

1


Faintuch J, Viebig RG. Another link in the chain

Faintuch J, Viebig RG. Mais um elo na corrente. Arq Gastroenterol. 49(1):1-2. DESCRITORES – Publicações científicas técnicas. Manuscritos médicos. Políticas editoriais. Nutrição em saúde pública.

REFERENCES 1.

2

Anonymous. Necessity of publishing a condensed analysis of parliamentary evidence. Prov Med Surg J (1840). 1841;1:271-2.

Arq Gastroenterol

2.

Scobba V. PS1-36. The collaborative research library: creative strategies for the advancement of scholarship. Clin Med Res. 2011;9:167-8.

v. 49 – no.1 – jan./mar. 2012


EDITORIAL

ARQGA/1580

MAIS UM ELO NA CORRENTE Faintuch J, Viebig RG. Mais um elo na corrente. Arq Gastroenterol. 49(1):3-4. DESCRITORES – Publicações científicas técnicas. Manuscritos médicos. Políticas editoriais. Nutrição em saúde pública.

Em 1840 vinha à luz na Inglaterra um modesto periódico, o Provincial Medical and Surgical Journal, hoje o famoso British Medical Journal. Já naquela quadra a profissão médica se debatia com dilemas e desafios profissionais, e um editorial chama a atenção que a melhor forma de se progredir no debate era mediante a publicação regular dos problemas e questões de interesse médico(1). Quase dois séculos mais tarde, a dúvida deixou de ser porque publicar, mas o quê, onde, como e com qual padrão de excelência. Sim, porque na atualidade todos são assombrados pela esfinge devoradora do “publish or perish”. Quem não está na mídia está no anonimato ou quase, e mídia para nós esculápios são de preferência veículos conhecidos e respeitados, de alto impacto, que confiram solidez, credibilidade e ampla repercussão ao estudo submetido. Há dois círculos se entrechocando neste contexto, um virtuoso e outro vicioso. De uma parte situam-se as comunicações limitadas e vulneráveis, com metodologia singela e resultados pouco convincentes. As revistas que se dispõem a divulgá-las correm o risco de arranhar sua imagem, e no futuro somente conseguir submissões igualmente frágeis, com a possibilidade de mergulhar na irrelevância. O oposto é igualmente verdadeiro. Ótimas publicações com artigos de primeira linha atraem os melhores autores e protocolos, tendendo a se fortalecer e consagrar cada vez mais. Torna-se cristalino que todos devem buscar a excelência e escapar do abismo, porém até o conselheiro Acácio de Eça de Queiroz riria da mediocridade desta conclusão. São indispensáveis mais que boas intenções para se atingir tal desiderato. Quase todas as propostas modernas enfatizam o esforço colaborativo e a união de forças. Ainda existem pesquisadores solitários de grande genialidade, que ano após ano conduzem investigações pioneiras e inovadoras, assim como revistas isoladas com escasso elenco de autores que conseguem se manter próximas do topo. Para a maioria, entretanto, não há como escapar da ampliação cada vez maior da base de sustentação. Albert Einstein conseguiu deduzir algumas das leis e equações mais fundamentais do universo quase 1 2

sem ajuda material ou humana. Muniu-se apenas de lápis, papel e fórmulas matemáticas nas horas vagas do seu emprego em Berna no “Eidgenossisches Institut fur Geistiges Eigentum”, a repartição de patentes e propriedade intelectual suíça. Contrariamente a ciência moderna não prescinde de métodos e desenhos experimentais cada vez mais ambiciosos, complicados e dispendiosos, e sem a reunião de recursos, equipes, disciplinas e tecnologias será cada vez mais improvável a geração de conhecimentos significativos(2). A Sociedade Brasileira de Nutrição Clínica nasceu no longínquo ano de 1975, e sua revista vem à luz ininterruptamente há mais de três décadas. No entanto é chegado o momento de se aproximar de uma congênere mais robusta e bem alicerçada, que já triunfou em numerosos desafios de circulação e indexação e vencerá em muitos mais, quais sejam os ARQUIVOS de GASTROENTEROLOGIA. É, portanto, sob a égide do irmanamento e da fraternidade que a refeição se achega ao tubo gastrointestinal, ou melhor, que a Nutrição busca guarida sob o acolhedor teto da Gastroenterologia. Há razões para acreditar que ocorrerá verdadeira sinergia, onde o todo superará a soma das partes. De um lado os ARQUIVOS já regularmente inserem textos voltados para nutrição, obesidade, cirurgia bariátrica e temas correlatos. De outro muitos profissionais da nutrição parenteral e enteral estão física e intelectualmente domiciliados em serviços de gastroenterologia e cirurgia do aparelho digestivo, demonstrando apreço pela área. Os brindes particularmente alcoólicos tornaram-se politicamente incorretos no século XXI, porém não há como fugir do lugar comum. Que este evento sirva de marco para um futuro cada vez mais auspicioso, e que os ARQUIVOS de GASTROENTEROLOGIA, agora abrangendo a área de Nutrição Clínica, marchem de sucesso em sucesso, para orgulho de todos aqueles que subscrevem suas páginas e endossam sua respeitada linha editorial. Joel FAINTUCH1 Ricardo Guilherme VIEBIG2

Editor Associado Editor executivo

v. 49 – no.1 – jan./mar. 2012

Arq Gastroenterol

3


Faintuch J, Viebig RG. Mais um elo na corrente.

Faintuch J, Viebig RG. Another link in the chain. Arq Gastroenterol. 49(1):3-4. HEADINGS – Scientific and technical publications. Manuscripts, medical. Editorial policies. Nutrition, public health.

REFERÊNCIAS 1.

4

Anonymous. Necessity of publishing a condensed analysis of parliamentary evidence. Prov Med Surg J (1840). 1841;1:271-2.

Arq Gastroenterol

2.

Scobba V. PS1-36. The collaborative research library: creative strategies for the advancement of scholarship. Clin Med Res. 2011;9:167-8.

v. 49 – no.1 – jan./mar. 2012


ARTIGO ORIGINAL / ORIGINAL ARTICLE

ARQGA/1581

DIAGNOSIS OF ADULT-TYPE HYPOLACTASIA/LACTASE PERSISTENCE: genotyping of single nucleotide polymorphism (SNP C/T-13910) is not consistent with breath test in Colombian Caribbean population Evelyn MENDOZA TORRES, Lourdes Luz VARELA PRIETO, José Luis VILLARREAL CAMACHO and Daniel Antonio VILLANUEVA TORREGROZA

ABSTRACT – Context - Genotyping of single nucleotide polymorphism (SNP C/T-13910) located upstream of the lactase gene is used to determine adult-type hypolactasia/lactase persistence in North-European Caucasian subjects. The applicability of this polymorphism has been studied by comparing it with the standard diagnostic methods in different populations. Objective - To compare the lactose hydrogen breath test with the genetic test in a sample of the Colombian Caribbean population. Methods - Lactose hydrogen breath test and genotyping of SNP C/T-13910 were applied to 128 healthy individuals (mean age 35 ± 1). A positive lactose hydrogen breath test was indicative of hypolactasia. Genotyping was done using polymerase chain reaction/restriction fragment length polymorphism. The kappa index was used to establish agreement between the two methods. Results - Seventy-six subjects (59%) were lactose-maldigesters (hypolactasia) and 52 subjects (41%) were lactose-digesters (lactase persistence). The frequencies of the CC, CT and TT genotypes were 80%, 20% and 0%, respectively. Genotyping had 97% sensitivity and 46% specificity. The kappa index = 0.473 indicates moderate agreement between the genotyping of SNP C/T-13910 and the lactose hydrogen breath test. Conclusion - The moderate agreement indicates that the genotyping of the SNP C/T-13910 is not applicable to determine adult-type hypolactasia/lactase persistence in the population participating in this study. HEADINGS - Lactose intolerance. Lactase. Polymorphism, single nucleotide. Breath tests. Colombia. Caribbean region.

INTRODUCTION

Lactase-florizine hydrolase (E.C. 3.2.1.23/62), commonly known as lactase, is the intestinal enzyme that allows lactose digestion to yield its absorbable constituent monosaccharides, glucose and galactose(25). This ability persists throughout adulthood only in a minority of human subjects, resulting in the lactase persistence (LP) phenotype, while lactase enzyme is significantly reduced after weaning in most adults worldwide, resulting in lactase non-persistence or adulttype hypolactasia (ATH) phenotype(27). Both conditions are genetically determined and their frequencies vary in human populations according to geographical and ethnic variations(7). The direct method to diagnose ATH is to assay lactase activity in intestinal biopsies(4). However, a non invasive method, the hydrogen breath test after lactose

ingestion, allows to indirectly determine the phenotype with high sensitivity and specificity(1, 24). With the discovery of phenotype-genotype association in the past few years, a direct genetic diagnostic method becomes relevant. In fact, genotyping of the single nucleotide polymorphism (SNP) C/T-13910, located upstream of the lactase gene (LCT), has shown that CC-genotype is associated with ATH phenotype, whereas the TC and TT-genotypes are associated with LP phenotype in Europeans of Caucasian origin(6, 14, 23). Taking into account the relevance of the genetic test as a predictor of ATH in some Europeans populations and the European contribution to the ethnic admixture of the Colombian Caribbean population, the purpose of this study was to compare the lactose hydrogen breath test (LHBT) with the genetic test in order to ascertain the applicability of genotyping to diagnose LP/ATH in a Colombian Caribbean population.

Universidad Libre Seccional Barranquilla, Barranquilla, Colombia. Correspondence: Dr. Daniel Villanueva Torregroza – Universidad Libre Seccional Barranquilla – Departamento de Medicina, Kilometro 7, Vía a Puerto Colombia, Barranquilla, Colombia. E-mail: danielvillanueva@unilibrebaq.edu.co; danvito@hotmail.com

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Mendoza Torres E, Varela Prieto LL, Villarreal Camacho JL, Villanueva Torregroza DA. Diagnosis of adult-type hypolactasia/lactase persistence: genotyping of single nucleotide polymorphism (SNP C/T-13910) is not consistent with breath test in Colombian Caribbean population

Subjects A group of 128 healthy individuals, men and women (mean age 35 ± 1; range 17–69 years) without family ties among them, born in the Colombian Caribbean were selected. Their parents and grandparents were also born in this geographical zone. None of the individuals were smokers, showed digestive disease, had undergone abdominal surgery nor had undergone antibiotic treatment within the last 3 months preceding the study. The subjects gave their informed consent. The study was approved by the Ethics Committee of Universidad Libre of Barranquilla, Colombia. Lactose hydrogen breath test (LHBT) The LHBT was performed after ingestion of 250 mL of 10% lactose. Ten-hours of fasting were required before testing. The breath hydrogen level was detected by Microlyzer Quintron Model 12i plus (Quintron Instrument Company Inc., Milwaukee, USA) at baseline and after the ingestion of lactose every hour over a period of 3 hours. The LHBT was considered positive if the hydrogen production was equal to or above 20 ppm compared to baseline in any measurement during the 3 hours after the ingestion of lactose. A positive LHBT showed that the subject was lactose maldigester (hypolactasia)(10, 20). Genotyping of the SNP C/T-13910 DNA was obtained from venous blood using the Wizard® Genomic DNA Purification Kit (Promega, USA). Polymorphism identification was done by Polymerase Chain Reaction/Restriction Fragment Length Polymorphism (PCR/RFLP). The fragment was amplified using the primers: 5’-GCTGGCAATACAGATAAGATAATGGA-3’ and 5’CTGCTTTGGTTGAAGCGAAGAT-3’(18). Reaction was carried out in a total volume of 20 µL, with Tris HCl buffer 1X pH 9.0; MgCl2 1.5 mM; dNTPs 0.2 mM; Taq polymerase 1.5U and 1 µM for each primer (Invitrogen®, USA). PCR conditions were: 95°C for 10 min (denaturation cycle), followed by 35 cycles of 95°C for 1 min, 59°C for 1 min and 72°C for 1 min, and a final elongation cycle at 72°C for 8 min. PCR product was digested with Hinf I (New England Biolabs) generating fragments of 177 and 24 bp. Amplification and digestion products were run in an 8% polyacrylamide gel electrophoresis. Fragments were visualized with ethidium bromide stain. Samples that only presented a 201 bp (C) fragment or a 177 bp (T) fragment were interpreted as CC and TT genotype, respectively. While samples that presented two fragments of 201 bp and 177 bp, were interpreted as the CT genotype. The 24 bp fragment was not visualized. Statistical analysis Allelic and genotypic frequencies, as well as Hardy-Weinberg equilibrium and linkage disequilibrium were determined with Arlequin version 3.1 software(8). The agreement between the genotyping of the SNP C/T-13910 and the LHBT was assessed

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by Kappa statistics. All of the analyses were done with a confidence interval of 95%. RESULTS

LHBT analysis Seventy-six of the 128 subjects (59%) had positive and 52 (41%) had negative LHBT. There was a significant difference (P = 0.0005) of hydrogen averages between lactose-digester and lactose-maldigester subjects. Figure 1 shows breath hydrogen average during testing time.

90 80 Breath Hydrogen (ppm)

METHODS

70 60 50 40 30 20 10

0

20

40

60

80 100 120 140 160 180 200 Time (minutes)

Lactose digester subjects (n = 52) Lactose maldigester subjects (n = 76) FIGURE 1. Hydrogen in breath test

Genotyping results The frequencies of the CC, CT and TT genotypes were 80%, 20% and 0%, respectively. The frequency of the -13910*C allele was 90% and the frequency of the -13910*T allele was 10%. The population in this study was in Hardy-Weinberg equilibrium for the SNP C/T-13910 (P = 0.3667). Correlation between the LHBT and the genotyping of the SNP C/T-13910 The results of genotyping compared with lactose digestion are presented in Table 1.

TABLE 1. Genotyping of SNP C/T-13910 compared with the lactose digestion evaluated by the LHBT CT (lactase persistence) Lactose digester (n = 24) 92% n = 26 Lactose maldisgester (n = 2) 8% CC (ATH) Lactose digester (n = 28) 27% n = 102 Lactose maldigester (n = 74) 73%

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Mendoza Torres E, Varela Prieto LL, Villarreal Camacho JL, Villanueva Torregroza DA. Diagnosis of adult-type hypolactasia/lactase persistence: genotyping of single nucleotide polymorphism (SNP C/T-13910) is not consistent with breath test in Colombian Caribbean population

Seventy-four of 102 individuals (73%) with CC genotype were lactose maldigesters and 24 of 26 individuals (92%) with CT genotype were lactose digesters. Using the LHBT as the standard method, genotyping of C/T-13910 had 97% sensitivity and 46% specificity. The positive predictive value of genotyping was 73% and the negative predictive value was 92%. The Kappa index for the agreement between the genotyping of C/T-13910 and the HBT was 0.473, suggesting a moderate agreement. DISCUSSION

In this study, no close correlation between the CT genotype and the digesters (LP) or between CC genotype and the maldigesters (ATH) was found. The genetic test showed high sensitivity (97%) but low specificity (46%) and poor positive predictive value (71%). These last data suggest that the probability that a subject with a positive hypolactasia result actually has this genetic condition is not high, which means that the test is susceptible to false positives and is inconsistent compared to the LHBT for the ATH diagnosis in the studied population. The results of this study differ from those of Di Stefano et al.(5), Hogenauer et al.(10), Krawczyk et al.(13), Nagy et al.(19), and Pohl et al.(22), carried out on European populations which showed strong agreement between LHBT and the genetic studies. It is possible that the discrepancy can be due to the genetic heterogeneity of the Colombian Caribbean population that arose from the crossing of different races as opposed to the studies with Europeans with Caucasian descent only(26). Racial difference has implications on the genotypes that are reflected on the distribution of lactase phenotypes. In Europeans, the most common phenotype (lactase persistence) is strongly associated with the dominant and most prevalent -13910*T allele due to strong positive selection; but the ATH phenotype is rare and it is associated with the recessive and less prevalent -13910*C allele(2, 12). In people of mixed ancestry, the genetic influence of their racial background is reflected. Thus, the low frequency of the -13910T allele found in the present study most likely represents the European contribution to the mixture of the three main ethnicities of the Colombian Caribbean people, whereas the high frequency of -13910C allele would represent the contribution from the other two ethnicities (Amerindians and Africans)(17). The above hypothesis agrees with the Torniainen et al.(29) and Mattar et al.(16) studies. The former found in South Africa and Ghana that both -13910*T and -14010*C alleles reflected the contribution from Europeans and Africans in mixed ancestry

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subjects, respectively; the latter study reported that the lactase persistence allele (-13910*T) in mestizos subjects, product of white and African-Brazilian interbreeding, is present not only as a consequence of the European contribution but also of the ancestral contribution of Africans who have that allele(9, 16, 28) . This is not the case for African Caribbean Colombians, because the blacks brought as slaves from Africa during the colonial period came mainly from Western Africa where the -13910*T allele is scarce(18, 29). The importance of the ethnic origin, when doing genotype-phenotype correlation studies is evident with the results recently published in Brazil by Bulhões et al.(3) and Mattar et al.(15) who reported perfect correlation between the SNP C/T-13910 and lactose digestion. They got this result because the subjects in their study were Brazilians of Caucasian descendence. On the other hand, the low prevalence of -13910*T allele found in Colombian Caribbean population would indicate that some different lactase persistence associated allele is present in the population studied. Indeed, the C/G-13907, T/C-13913, G/C-14010 and T/G-13915 polymorphisms, all of them located upstream of the LCT gene, have been identified in some African and Middle Eastern populations(7, 11, 21, 28). This finding shows that the genotyping of C/T-13910, valid as a diagnostic resource for Northern Europeans, does not apply worldwide(28). It would be interesting to verify if those polymorphisms found in African populations are associated with LP/ATH in the Colombian Caribbean population, because of its African ancestry. Likewise, since the heterogeneity has implications on the lactase genotypes, new studies about the true contribution of Blacks, Spanish and Amerindians to the genetic structure of the population being studied are necessary. In our study, no subjects had lactose intolerance symptoms such as diarrhea or abdominal distention at the time of the LBHT test. It would be interesting to study a group of symptomatic Colombian Caribbean subjects and correlate the lactose test with the -13910 polymorphism. CONCLUSION

In a sample of the Colombian Caribbean population, no close correlation between the genotyping of the SNP C/T-13910 polymorphism and the LHBT was found. From these findings we conclude that the genetic test is not reliable to diagnose ATH/lactase persistence in Colombian Caribbean subjects.

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Mendoza Torres E, Varela Prieto LL, Villarreal Camacho JL, Villanueva Torregroza DA. Diagnosis of adult-type hypolactasia/lactase persistence: genotyping of single nucleotide polymorphism (SNP C/T-13910) is not consistent with breath test in Colombian Caribbean population

Mendoza Torres E, Varela Prieto LL, Villarreal Camacho JL, Villanueva Torregroza DA. Diagnóstico de hipolactasia tipo adulto/persistência da lactase: a genotipagem

do polimorfismo de um único nucleotídeo (SNP) C/T-13910 não é consistente com o teste de hidrogênio na população do Caribe Colombiano. Arq Gastroenterol. 2011;49(1):5-8. RESUMO – Contexto - A genotipagem do SNP C/T-13910 localizado corrente acima do gene da lactase é usada para determinar hipolactasia e persistência da lactase tipo adulto em indivíduos caucasianos do Norte da Europa. A aplicabilidade deste polimorfismo tem sido estudada em comparação com métodos padronizados de diagnóstico em diferentes populações. Objetivo - Comparar o teste de hidrogênio expirado após a ingestão de lactose com o teste genético em uma mostra da população do Caribe Colombiano. Métodos - O teste de hidrogênio expirado após a ingestão de lactose  e a genotipagem do SNP C/T-13910 foram aplicados em 128 sujeitos sadios (idade media 35 ± 1). O teste de hidrogênio positivo foi indicativo de hipolactasia. A genotipagem foi feita pelo método “polymerase chain reaction/restriction fragment length polymorphism”. O índice Kappa foi utilizado para estabelecer a concordância entre os dois métodos. Resultados - Setenta e seis indivíduos (59%) foram mau digestores da lactose (hipolactasia) e 52 outros (41%) foram digestores da lactose (persistência da lactase). As frequências dos genotipos CC, CT e TT foram 80%, 20% e 0%, respectivamente. A genotipagem mostrou 97% da sensibilidade e 46% da especificidade. O índice kappa: 0,473 indicou moderada concordância entre os dois métodos. Conclusão - A moderada concordância indica que a genotipagem do SNP C/T-13910 nao é aplicável para determinar hipolactasia tipo adulto/persistência da lactase na população estudada. DESCRITORES – Intolerância à lactose. Lactase. Polimorfismo de um único nucleotídeo. Testes respiratórios. Colômbia. Região do Caribe.

References 1. 2. 3.

4. 5. 6. 7.

8. 9. 10. 11.

12. 13. 14. 15.

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Arola H, Koivula T, Jokela H, Jauhiainen M, Keyriläinen O, Ahola T, Uusitalo A, Isokoski M. Comparison of indirect diagnostic methods for hypolactasia. Scand J Gastroenterol. 1988;23:351-7. Bersaglieri T, Sabeti PC, Patterson N, Vanderploeg T, Schaffner SF, Drake JA, Rhodes M, Reich DE, Hirschhorn JN. Genetic signatures of strong recent positive selection at the lactase gene. Am J Hum Genet. 2004;74:1111-20. Bulhões AC, Goldani HA, Oliveira FS, Matte US, Mazzuca RB, Silveira TR. Correlation between lactose absorption and the C/T-13910 and G/A-22018 mutations of the lactase-phlorizin hydrolase (LCT) gene in adult-type hypolactasia. Braz J Med Biol Res. 2007;40:1441-6. Dahlqvist A. Assay of intestinal disaccharidases. Scand J Clin Lab Invest. 1984;44:169-72. Di Stefano M, Terulla V, Tana P, Mazzocchi S, Romero E, Corazza GR. Genetic test for lactase non-persistence and hydrogen breath test: is genotype better than phenotype to diagnose lactose malabsorption? Dig Liver Dis. 2009;41:474-9. Enattah NS, Sahi T, Savilahti E, Terwilliger JD, Peltonen L, Järvelä I. Identification of a variant associated with adult-type hypolactasia. Nat Genet. 2002;30:233-7. Enattah NS, Jensen TG, Nielsen M, Lewinski R, Kuokkanen M, Rasinpera H, El-Shanti H, Seo JK, Alifrangis M, Khalil IF, Natah A, Ali A, Natah S, Comas D, Mehdi SQ, Groop L, Vestergaard EM, Imtiaz F, Rashed MS, Meyer B, Troelsen J, Peltonen L. Independent introduction of two lactase-persistence alleles into human populations reflects different history of adaptation to milk culture. Am J Hum Genet. 2008;82:57-72. Excoffier L, Laval G, Schneider S. Arlequin (version 3.0): an integrated software package for population genetics data analysis. Evol Bioinform Online. 2007;1:47-50. Gonçalves VF, Carvalho CM, Bortolini MC, Bydlowski SP, Pena SD. The phylogeography of African Brazilians. Hum Hered. 2008;65:23-32. Högenauer C, Hammer HF, Mellitzer K, Renner W, Krejs GJ, Toplak H. Evaluation of a new DNA test compared with the lactose hydrogen breath test for the diagnosis of lactase non-persistence. Eur J Gastroenterol Hepatol. 2005;17:371-6. Ingram CJ, Elamin MF, Mulcare CA, Weale ME, Tarekegn A, Raga TO, Bekele E, Elamin FM, Thomas MG, Bradman N, Swallow DM. A novel polymorphism associated with lactose tolerance in Africa: multiple causes for lactase persistence? Hum Genet. 2007;120:779-88. Ingram CJ, Mulcare CA, Itan Y, Thomas MG, Swallow DM. Lactose digestion and the evolutionary genetics of lactase persistence. Hum Genet. 2009;124:579-91. Krawczyk M, Wolska M, Schwartz S, Gruenhage F, Terjung B, Portincasa P, Sauerbruch T, Lammert F. Concordance of genetic and breath tests for lactose intolerance in a tertiary referral centre. J Gastrointestin Liver Dis. 2008;17:135-9. Kuokkanen M, Enattah NS, Oksanen A, Savilahti E, Orpana A, Järvelä I. Transcriptional regulation of the lactase-phlorizin hydrolase gene by polymorphisms associated with adult-type hypolactasia. Gut. 2003;52:647-52. Mattar R, Monteiro MdoS, Villares CA, dos Santos AF, Carrilho FJ. Single nucleotide polymorphism C/T(-13910), located upstream of the lactase gene, associated with adult-type hypolactasia: validation for clinical practice. Clin Biochem. 2008;41:628-30.

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16. Mattar R, Monteiro MS, Villares CA, Santos AF, Silva JM, Carrilho FJ. Frequency of LCT-13910C>T single nucleotide polymorphism associated with adult-type hypolactasia/lactase persistence among Brazilians of different ethnic groups. Nutr J. 2009;8:46-8. 17. Mendoza E, Hernandez AC, Wilches R, Varela LL, Villarreal JL, Barrera LA, Villanueva DA. Genotype frequencies of C/T-13910 and G/A-22018 polymorphisms in a Colombian Caribbean population do not correspond with lactase persistence prevalence reported in the region. Colomb Med. 2010;41:290-4. 18. Mulcare CA, Weale ME, Jones AL, Connell B, Zeitlyn D, Tarekegn A, Swallow DM, Bradman N, Thomas, MG. The T allele of a single-nucleotide polymorphism 13.9 kb upstream of the lactase gene (LCT) (C-13.9kbT) does not predict or cause the lactase-persistence phenotype in Africans. Am J Hum Genet. 2004;74:1102-10. 19. Nagy D, Bojácsi-Szabó E, Várkonyi Á, Csányi B, Czibula Á, Bede O, Tari B, Raskó I. Prevalence of adult-type hypolactasia as diagnosed with genetic and lactose hydrogen breath tests in Hungarians. Eur J Clin Nutr. 2009;63:909-12. 20. Ojetti V, La Mura R, Zocco MA, Cesaro P, De Masi E, La Mazza A, Cammarota G, Gasbarrini G, Gasbarrini A. Quick test: a new test for the diagnosis of duodenal hypolactasia. Dig Dis Sci. 2008;53:1589-92. 21. Olds LC, Ahn JK, Sibley E. 13915*G DNA polymorphism associated with lactase persistence in Africa interacts with Oct-1. Hum Genet. 2011;129:111-3. 22. Pohl D, Savarino E, Hersberger M, Behlis Z, Stutz B, Goetze O, Eckardstein AV, Fried M, Tutuian R. Excellent agreement between genetic and hydrogen breath tests for lactase deficiency and the role of extended symptom assessment. Br J Nutr. 2010;104:900-7. 23. Ridefelt P, Hakansson LD. Lactose intolerance: lactose tolerance test versus genotyping. Scand J Gastroenterol. 2005;40:822-6. 24. Romagnuolo J, Schiller D, Bailey RJ. Using breath tests wisely in a gastroenterology practice: an evidence-based review of indications and pitfalls in interpretation. Am J Gastroenterol. 2002;97:1113-26. 25. Sahi T. Genetics and epidemiology of adult-type hypolactasia. Scand J Gastroenterol Suppl. 1994;202:7-20. 26. Salzano FM, Bortolini MC. The evolution and genetics of Latin American populations. Cambridge, UK: Cambridge University Press; 2002. 27. Swallow DM. Genetics of lactase persistence and lactose intolerance. Ann Rev Genet. 2003;37:197-219. 28. Tishkoff SA, Reed FA, Ranciaro A, Voight BF, Babbitt CC, Silverman JS, Powell K, Mortensen HM, Hirbo JB, Osman M, Ibrahim M, Omar SA, Lema G, Nyambo TB, Ghori J, Bumpstead S, Pritchard JK, Wray GA, Deloukas P. Convergent adaptation of human lactase persistence in Africa and Europe. Nat Genet 2007;39:31-40. 29. Torniainen S, Parker MI, Holmberg V, Lahtela E, Dandara C, Jarvela I. Screening of variants for lactase persistence/non-persistence in populations from South Africa and Ghana. BMC Genet. 2009;10:31.

Received 11/4/2011. Accepted 5/8/2011.

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ARTIGO ORIGINAL / ORIGINAL ARTICLE

ARQGA/1582

EFFECT OF HFE GENE POLYMORPHISM ON SUSTAINED VIROLOGICAL RESPONSE IN PATIENTS WITH CHRONIC HEPATITIS C AND ELEVATED SERUM FERRITIN Silvia COELHO-BORGES1,2, Hugo CHEINQUER1,2, Fernando Herz WOLFF2,3,4, Nelson CHEINQUER2, Luciano KRUG5 and Patricia ASHTON-PROLLA6,7

ABSTRACT – Context - Abnormal serum ferritin levels are found in approximately 20%-30% of the patients with chronic hepatitis C and are associated with a lower response rate to interferon therapy. Objective - To determine if the presence of HFE gene mutations had any effect on the sustained virological response rate to interferon based therapy in chronic hepatitis C patients with elevated serum ferritin. Methods - A total of 44 treatment naïve patients with histologically demonstrated chronic hepatitis C, all infected with hepatitis C virus genotype non-1 (38 genotype 3; 6 genotype 2) and serum ferritin above 500 ng/mL were treated with interferon (3 MU, 3 times a week) and ribavirin (1.000 mg, daily) for 24 weeks. Results - Sustained virological response was defined as negative qualitative HCV-RNA more than 24 weeks after the end of treatment. Serum HCV-RNA was measured by qualitative in house polymerase chain reaction with a limit of detection of 200 IU/mL. HFE gene mutation was detected using restriction-enzyme digestion with RsaI (C282Y mutation analysis) and BclI (H63D mutation analysis) in 16 (37%) patients, all heterozygous (11 H63D, 2 C282Y and 3 both). Sustained virological response was achieved in 0 of 16 patients with HFE gene mutations and 11 (41%) of 27 patients without HFE gene mutations (P = 0.002; exact Fisher test). Conclusion - Heterozigozity for H63D and/or C282Y HFE gene mutation predicts absence of sustained virological response to combination treatment with interferon and ribavirin in patients with chronic hepatitis C, non-1 genotype and serum ferritin levels above 500 ng/mL. HEADINGS – Polymorphysm, genetic. Hepatitis C, chronic. Ferritins. Interferons.

INTRODUCTION

The Brazilian Public Health System provides antiviral treatment free of cost for selected patients with chronic hepatitis C virus (HCV) infection. Current government guidelines indicates 48 weeks of pegylated interferon (PEG-IFN) plus ribavirin (RBV) for patients with HCV genotype 1 and 24 weeks of conventional interferon (IFN) plus RBV for those with HCV genotypes 2 or 3(7). Major predictive factors for sustained virological response (SVR) to IFN based treatment are well established for genotype 1 infected patients, including liver fibrosis stage, viral load, hepatic steatosis, body mass index, insulin resistance and IL-28 polymorphism(14,  19). However, predictive factors for SVR are not so clearly defined for genotypes 2 or 3, since recent studies showed that viral load, insulin resistance

and IL-28 polymorphism failed to predict SVR to IFN based therapy in these patients(30). Feder et al.(13), in 1996, discovered the HFE gene and two of its polymorphisms, C282Y and H63D, which were clearly associated with higher prevalence of elevated serum ferritin, transferrin saturation, and genetic hemochromatosis. It is well known that 20%-30% of patients with chronic hepatitis C have serum markers of iron overload, but not an increased prevalence of HFE polymorphisms(4, 5, 10). Elevated serum ferritin has long been recognized as a marker of poor response to antiviral treatment in chronic HCV infected patients(3, 12, 16), however the mechanism underlying this effect is not clear and the role of HFE polymorphisms has not yet been established. Interestingly, there is intriguing evidence of an association between HFE polymorphism and higher SVR rates(8, 31), but this issue has not been specifically investigated among

All authors had access to the data, were actively involved in its analysis and interpretation, and approved the final manuscript. The authors declare no conflict of interest concerning the present article. 1 Graduate Program in Medicine: Gastroenterology - Faculdade de Medicina, Universidade Federal do Rio Grande do Sul (UFRGS); 2 Gastroenterology Department, Hospital de Clínicas de Porto Alegre; 3 Graduate Program in Epidemiology - Faculdade de Medicina, UFRGS; 4 National Institute of Science and Technology for Health Technology Assessment (IATS) CNPq/Brazil; 5Amplicon – Molecular Biology Laboratory; 6 Medical Genetics Service, Hospital de Clínicas de Porto Alegre; 7 Genetics Department, UFRGS, Porto Alegre, RS, Brasil. Correspondence: Dr. Fernando Herz Wolff – Rua Dr. Freire Alemão, 351/703 – 90450-060 – Porto Alegre, RS, Brazil. E-mail: fhwolff@gmail.com; silvia.cb@terra.com.br

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Coelho-Borges S, Cheinquer H, Wolff FH, Cheinquer N, Krug L, Ashton-Prolla P. Effect of hfe gene polymorphism on sustained virological response in patients with chronic hepatitis c and elevated serum ferritin

HCV patients with high serum ferritin, which are recognized as having difficult to treat disease. Thus, the aim of the present study was to verify the prevalence of HFE gene polymorphisms and its impact on SVR rates among patients with HCV genotypes 2 or 3 and elevated serum ferritin treated with IFN/RBV in the setting of the Brazilian Public Health System. The finding of a reliable predictor of treatment outcome in the present study could help to improve government guidelines and refine treatment strategies, saving costs and avoiding futile therapy. METHODS

Study design A historical cohort study was conducted to evaluate the association between HFE gene polymorphisms and the response rate to IFN/RBV, using a cross-sectional design. The study was conducted in accordance with the ethics principles of the Declaration of Helsinki and was consistent with Good Clinical Practice guidelines, being approved by appropriate Institutional Review Boards. Written informed consent was obtained from all patients. All authors had access to the data, were actively involved in its analysis and interpretation, and approved the final manuscript. Patients Forty-four patients were recruited from the Viral Hepatitis Outpatient Clinic of Hospital de Clínicas de Porto Alegre, RS, Brazil a tertiary care institution in Porto Alegre, South of Brazil. Key inclusion criteria were: age equal or above 18 years old, detectable HCV RNA by polymerase chain reaction (PCR; Roche Amplicor; lower limit of detection: 50 IU/mL), liver biopsy consistent with the diagnosis of chronic hepatitis C 1 year or less before treatment start, baseline serum ferritin above 500 ng/mL, HCV genotype 2 or 3 infection, complete treatment with IFN 3 MU 3 times a week plus RBV 1000 mg daily for 24 weeks with compliance equal or above 80% of the IFN/RBV dose and intended duration (80/80/80 rule), with a valid PCR result after 6 months of follow-up to establish the occurrence or not of SVR. Demographic data was collected at baseline. Biochemical and haematological parameters were assessed at baseline and every 4 weeks during treatment, and 24 weeks after the end of therapy. Patients with cirrhosis were eligible provided that they had compensated liver disease (Child-Pugh A). Main exclusion criteria were: co-infection with human immune deficiency virus and/or hepatitis B virus, liver disease attributable to a cause other than HCV infection, ultrasound compatible with hepatocellular carcinoma, and suboptimal IFN/RBV treatment (less than 80% of the expected IFN/RBV dose and/ or less than 80% of the intended treatment duration). The study was approved by the ethics committee of the Hospital de Clínicas de Porto Alegre (2004). Exposure and outcomes HFE gene mutations were determined using restriction fragment lenght polymorphism. Patients were considered

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exposed if heterozygous or homozygous for C282Y and/or H63D HFE polymorphisms. Primary outcomes were the rate of end of treatment virological response (EoTVR) and SVR between exposed and non-exposed, defined as undetectable HCV RNA by qualitative PCR assay at the end of treatment and after 24-weeks of follow-up, respectively. Co-primary outcome was the degree of liver fibrosis between exposed and non-exposed, assessed by an experienced pathologist using Metavir score, with cirrhosis considered as Metavir F3 or F4. Hepatic iron deposition was estimated using standard Perls iron stain protocol. Statistical Methods Patients were grouped as positive or negative for HFE gene mutations. Differences in baseline characteristics and outcomes between patients with and without HFE gene mutations were examined by Fisher’s exact test (categorical variables), and Kruskall-Wallis (continuous variables). All statistical tests were two-sided and a P-value below 0.05 was considered significant. The Statistical Package for the Social Sciences (SPSS 13.0, Chicago, IL) was used for statistical analyses. RESULTS

Among the 44 included patients, 40 (90.9%) were male, all were caucasians, with a mean age of 48.4 ± 7.7 years (range 26-63). Thirty-eight were HCV genotype 3 (86.4%) and 6 genotype 2 (13.6%). Metavir fibrosis score was distributed as follows: F0 (n = 2); F1 (n = 8); F2 (n = 1); F3 (n = 5) and F4 (n = 28). Distribution of iron in the liver was restricted to Kuppffer cells, ranging from absent to moderate deposition, with no histological diagnosis of hemochromatosis. Overall, mean baseline serum ferritin was 1.097 ng/mL (standard deviation ± 552; range 500-2.865). Mean baseline serum transferrin saturation was 50.5% (standard deviation ± 17.7; range 25%-86%). All patients had elevated baseline serum ALT, with a mean value of 229.6 UI/mL (standard deviation ± 120.3; range 55-516). HFE gene mutations were detected in 16 patients (36%), with the following distribution: heterozygous H63D (n = 11); heterozygous C282Y (n = 2), and compound heterozygous H63D plus C282Y (n = 3). No patient was homozygous for any of the studied mutations. EoTVR and SVR were observed in 22 (50%) and 11 (25%) of the 44 treated patients, respectively. Comparison of demographic and disease characteristics between patients with versus without HFE gene mutations are depicted in Table 1. DISCUSSION

In this study we found an overall SVR rate of 25% among patients with genotype 2 and 3. Remarkably, none of the 16 patients with HFE polymorphisms achieved SVR as opposed to 39% observed among the 28 individuals without this genetic marker. Although our SVR rates were lower than the 64%-79% reported in the IFN/RBV registration trials for HCV genotypes 2 and 3(15, 27) it was similar to that obtained in other Brazilian

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TABLE 1. Characteristics of the 44 patients according to HFE gene polymorphisms status With HFE polymorphisms Without HFE polymorphisms (n = 16) (n = 28) Mean age ± SD (years) 50 ± 6 48 ± 8 Male gender, n (%) 15 (94) 25 (89) HCV genotype 3, n (%) 14 (88) 24 (86) Baseline serum ALT ± SD (UI/mL) 221 ± 88 239 ± 138 Baseline serum ferritin ± SD (ng/dL) 1.171 ± 587 1.060 ± 546 Baseline serum TS ± SD (%) 57 ± 18 46 ± 17 Cirrhosis (metavir F3/F4), n (%) 12 (75) 21 (75) EOT virological response, n (%) 4 (25) 18 (64) Sustained virological response, n (%) 0 11 (39)

P-value 0.33 0.54 0.62 0.66 0.68 0.05 1.0 0.03 0.003

SD: standard deviation; HCV: hepatitis C virus; ALT: alanine aminotransferase; TS: transferrin saturation; EOT: end of treatment

studies that included real life cohorts. Indeed, a retrospective analysis of 173 patients with genotype 2 or 3 treated with conventional interferon alpha and ribavirin in the Public Health System of the State of Rio Grande do Sul, Brazil, showed an overall SVR rate of only 39%(1). The lower SVR found in the present study could also be related to some characteristics of our cohort, namely the fact that 75% of the patients presented with advanced fibrosis and that only those with elevated serum ferritin at baseline were included. The reason for including only patients with serum ferritin levels above 500 ng/dL was to select genotypes 2 and 3 patients with really difficult to treat disease. Since transferring saturation was not available for all patients, serum ferritin level above 500 ng/dL was also the best available marker for iron overload, despite the fact that, in this situation, less than 10% of patients are expected to have a genetic iron overload disorder(20). Individuals with treatment compliance outside the 80/80/80 rule were excluded to avoid introducing confounding factors such as dose reduction or treatment interruption, which could bias any potential relationship between SVR and HFE gene polymorphisms. There was significantly higher serum transferring saturation among individuals with HFE polymorphism, despite no difference in serum ferritin levels. However, the absence of SVR observed in the group of individuals with this genetic marker was probably not directly related to iron overload, since most studies, so far, failed to report a significant difference in hepatic iron concentration between HCV patients with or without SVR after IFN/RBV treatment(23, 25, 29, 33). Moreover, therapeutic phlebotomies generally did not increase SVR, indicating that reduction of body iron stores was not associated with treatment outcome(11, 21). It is possible that ferritin, as an acute phase reactant, behave as a marker of more active and advanced liver disease, instead of representing body iron content. In this regard, it has been reported that patients with chronic hepatitis C and high serum ferritin have significantly more liver inflammation and fibrosis compared to those with normal serum ferritin values(2, 3, 35). This finding could explain the fact that the majority of our patients were cirrhotic. The recent finding of more rapid fibrosis progression among patients with chronic hepatitis C

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infected with HCV genotype 3 could also explain the high percentage of those with advanced disease in our cohort, since almost 90% of our patients had this genotype(9). There was no patient with hemochromatosis in the present study, further supporting the fact that the mere presence of high serum ferritin in HCV infected patients does not necessarily correlates with condition. Data on the possible relationship between carriage of the HFE gene mutations and response to IFN based therapy have been controversial(5, 22, 24, 32, 35, 37). Previous studies showed an association between H63D HFE polymorphism and higher SVR rates in patients with chronic hepatitis C(6).This relationship could be due to the fact that the HFE gene is located on the short arm of chromosome 6, close to the major histocompatibility complex (MHC). In this regard, it is well known that MHC genes encode the human leukocyte antigens, which are important in antigen presentation and regulation of CD8 + and CD4 + T cells(18, 34). Previous studies have shown an impact of MHC gene variants and response to therapy in chronic HCV infection(17, 28, 31, 36). Our finding contradicts the previously described association between H63D mutation and higher SVR(6). The most seemingly explanation for the negative effect of HFE on SVR seen in the present study might be related to the unique characteristics of our cohort, comprised exclusively of individuals infected with genotype non-1 with elevated serum ferritin levels. It is also possible that HFE mutations, when associated with markers of iron overload such as serum ferritin and/or transferrin saturation, behave differently in respect to its gene expression profile. If our results prove to be correct in future studies with larger samples and a prospective design, it could have a major impact on current Brazilian Guidelines, which still indicates IFN/RBV as first line therapy for HCV patients infected with genotypes 2 or 3. Indeed, PEG-IFN/RBV is only offered as initial therapy to HCV genotype 1 carriers and as second line therapy in HCV non-1 genotypes after failure of IFN/RBV. The finding of HFE polymorphism as a strong predictor of non-response in our sample should lead to further studies in order to better clarify if the negative impact of HFE on SVR of patients with genotypes 2 or

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3 and high serum ferritin could be neutralized using more potent therapy. The limitations of the present study have to be addressed, and are mainly related to the relative small sample size, retrospective design and absence of other predictors of response to therapy, such as viral load and IL-28B. Nevertheless, it is the only study conducted so far in this

patient population chronically infected with HCV genotypes 2 and 3 with high serum ferritin. Moreover, both viral load and IL-28B do not seem to have a major impact on SVR in the majority of non-1 HCV genotypes(26). Finally, we believe the relationship between HFE mutations and absence of SVR in this cohort merits future research to clarify this seemingly important association.

Coelho-Borges S, Cheinquer H, Wolff FH, Cheinquer N, Krug L, Ashton-Prolla P. Efeito do polimorfismo do gene HFE sobre a resposta viral sustentada em portadores de hepatite crônica C com ferritina sérica elevada. Arq Gastroenterol. 2011;49(1):9-13. RESUMO – Contexto - Níveis séricos anormais de ferritina são encontrados em aproximadamente 20%-30% dos pacientes com hepatite crônica C e estão associadas a uma baixa taxa de resposta à terapia com interferon. Objetivo - Avaliar a associação entre a presença de mutações do gene HFE e a taxa de resposta virológica sustentada ao interferon em pacientes portadores de hepatite crônica C com ferritina sérica elevada. Métodos - Um total de 44 pacientes, virgem de tratamento, infectado pelo vírus da hepatite C de genótipos não-1 (38 genótipo 3; 6 genótipo 2) e ferritina sérica acima de 500 ng/mL foi tratado com interferon (3 MU, três vezes por semana) e ribavirina (1000 mg/dia) por 24 semanas. Resposta virológica sustentada foi definida como HCV-RNA indetectável 24 semanas após o fim do tratamento. Foi utilizado técnica de reação em cadeia da polimerase em tempo-real com limite de detecção de 200 UI /mL. Resultados - Mutações do gene HFE foram detectadas por “restriction-enzyme digestion” com RsaI (análise de mutação C282Y) e BclI (análise de mutação H63D) em 16 pacientes (37%), todos heterozigotos (11 H63D, 2 C282Y e 3 ambos). Resposta virológica sustentada foi alcançada em 0 de 16 pacientes com mutações do gene HFE e 11 (41%) dos 27 pacientes sem mutações do gene HFE (P = 0,002; teste exato de Fisher). Conclusão - A heterozigose para os genes H63D e/ou C282Y HFE está associada à redução significativa da taxa de resposta virológica sustentada ao tratamento com interferon e ribavirina em pacientes com hepatite crônica C, genótipo não-1 e com níveis séricos de ferritina acima de 500 ng/mL. DESCRITORES – Polimorfismo genético. Hepatite C crônica. Ferritinas. Interferons.

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21. Herrera JL. Iron depletion is not effective in inducing a virologic response in patients with chronic hepatitis C who failed to respond to interferon therapy. Am J Gastroenterol. 1999;94:3571-5. 22. Hézode C, Cazeneuve C, Coué O, Roudot-Thoraval F, Lonjon I, Bastie A, Duvoux C, Pawlotsky JM, Zafrani ES, Amselem S, Dhumeaux D. Liver iron accumulation in patients with chronic active hepatitis C: prevalence and role of hemochromatosis gene mutations and relationship with hepatic histological lesions. J Hepatol. 1999;31:979-84. 23. Hofer H, Osterreicher C, Jessner W, Penz M, Steindl-Munda P, Wrba F, Ferenci P. Hepatic iron concentration does not predict response to standard and pegylated-IFN/ribavirin therapy in patients with chronic hepatitis C. J Hepatol. 2004;40:1018-22. 24. Kazemi-Shirazi L, Datz C, Maier-Dobersberger T, Kaserer K, Hackl F, Polli C, Steindl PE, Penner E, Ferenci P. The relation of iron status and hemochromatosis gene mutations in patients with chronic hepatitis C. Gastroenterology. 1999;116:127-34. 25. Lebray P, Zylberberg H, Hue S, Poulet B, Carnot F, Martin S, Chretien Y, Pol S, Caillat-Zuckman S, Bréchot C, Nalpas B. Influence of HFE gene polymorphism on the progression and treatment of chronic hepatitis C. J Viral Hepat. 2004;11:175-82. 26. Mangia A, Thompson AJ, Santoro R, Piazzolla V, Tillmann HL, Patel K, Shianna KV, Mottola L, Petruzzellis D, Bacca D, Carretta V, Minerva N, Goldstein DB, McHutchison JG. An IL28B polymorphism determines treatment response of hepatitis C virus genotype 2 or 3 patients who do not achieve a rapid virologic response. Gastroenterology. 2010;139:821-7. 27. Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, Goodman ZD, Koury K, Ling M, Albrecht JK. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet. 2001;358:958-65. 28. Neumann-Haefelin C, Spangenberg HC, Blum HE, Thimme R. Host and viral factors contributing to CD8+ T cell failure in hepatitis C virus infection. World J Gastroenterol. 2007;13:4839-47. 29. Pianko S, McHutchison JG, Gordon SC, Heaton S, Goodman ZD, Patel K, Cortese CM, Brunt EM, Bacon BR, Blatt LM. Hepatic iron concentration does not influence response to therapy with interferon plus ribavirin in chronic HCV infection. J Interferon Cytokine Res. 2002;22:483-9.

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30. Rauch A, Kutalik Z, Descombes P, Cai T, Di Iulio J, Mueller T, Bochud M, Battegay M, Bernasconi E, Borovicka J, Colombo S, Cerny A, Dufour JF, Furrer H, Günthard HF, Heim M, Hirschel B, Malinverni R, Moradpour D, Müllhaupt B, Witteck A, Beckmann JS, Berg T, Bergmann S, Negro F, Telenti A, Bochud PY; Swiss Hepatitis C Cohort Study; Swiss HIV Cohort Study. Genetic variation in IL28B is associated with chronic hepatitis C and treatment failure: a genome-wide association study. Gastroenterology. 2010;138:1338-45. 31. Rhodes SL, Erlich H, Im KA, Wang J, Li J, Bugawan T, Jeffers L, Tong X, Su X, Rosen HR, Yee LJ, Liang TJ, Yang H; Virahep-C Study Group. Associations between the human MHC and sustained virologic response in the treatment of chronic hepatitis C virus infection. Genes Immun. 2008;9:328-33. 32. Smith BC, Gorve J, Guzail MA, Day CP, Daly AK, Burt AD, Bassendine MF. Heterozygosity for hereditary hemochromatosis is associated with more fibrosis in chronic hepatitis C. Hepatology. 1998;27:1695-9. 33. Souza RM, Freitas LA, Lyra AC, Moraes CF, Braga EL, Lyra LG. Effect of iron overload on the severity of liver histologic alterations and on the response to interferon and ribavirin therapy of patients with hepatitis C infection. Braz J Med Biol Res. 2006;39:79-83. 34. Ten Elshof AE, Brittenham GM, Chorney KA, Page MJ, Gerhard G, Cable EE, Chorney MJ. Gamma delta intraepithelial lymphocytes drive tumor necrosis factoralpha responsiveness to intestinal iron challenge: relevance to hemochromatosis. Immunol Rev. 1999;167:223-32. 35. Thorburn D, Curry G, Spooner R, Spence E, Oien K, Halls D, Fox R, McCruden EA, MacSween RN, Mills PR. The role of iron and haemochromatosis gene mutations in the progression of liver disease in chronic hepatitis C. Gut. 2002;50:248-52. 36. Tseng KC, Chang CK, Chou AL, Hsieh YH, Tseng CA, Lai NS. Prognostic effect of human leukocyte antigen class I and II alleles on chronic hepatitis C patients treated by pegylated interferon-alfa plus ribavirin in Taiwan. Hepatogastroenterology. 2010;57:456-61. 37. Tung BY, Emond MJ, Bronner MP, Raaka SD, Cotler SJ, Kowdley KV. Hepatitis C, iron status, and disease severity: relationship with HFE mutations. Gastroenterology.2003;124:318-26.

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Received 25/4/2011. Accepted 10/8/2011.

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ARTIGO ORIGINAL / ORIGINAL ARTICLE

ARQGA/1583

ASSESSMENT OF PORTAL VENOUS INDEX AS A NON-INVASIVE METHOD FOR DIAGNOSING LIVER FIBROSIS IN PATIENTS WITH CHRONIC HEPATITIS C Haroldo Luis Oliva Gomes ROCHA1, Angélica Lemos Debs DINIZ2, Valéria Ferreira de Almeida e BORGES1 and Frederico Chaves SALOMÃO3

ABSTRACT – Context - Hepatitis C is an important cause of chronic liver disease worldwide. The grading of hepatic fibrosis in chronic hepatitis C is important for better clinical management. However, until now, liver biopsy is the only test accepted for this purpose, despite their contraindications and complications. New methods for non-invasive assessment of hepatic fibrosis are under investigation. One proposal is the Doppler ultrasound, as a non-invasive, widely available and inexpensive. Objective - To compare Doppler parameters of portal vein in patients with chronic hepatitis C with a healthy control group and to correlate these parameters with fibrosis degree obtained by liver biopsy. Methods - Fifty patients with chronic hepatitis C submitted to liver biopsy and 44 healthy controls had Doppler of the portal vein performed, with the calculation of the portal venous index. We conducted a comparison between the averages of the two groups of portal venous index. For the correlation between portal venous index and fibrosis was employed the Spearman test. Results - There was a difference between the average portal venous index between controls (0.33 ± 0.07) and patients (0.23 ± 0.09) with P<0.001. No difference was observed between the portal venous index in patients with chronic hepatitis C who have significant fibrosis or not. The correlation between the portal venous index and fibrosis degree was reverse and moderate (r =-0.448 P<0.001). The area under the ROC curve was 78.4% (95% CI: 68.8% to 88%). The cutoff for the portal venous index was 0.28 with sensitivity of 73.5% and specificity of 71.1%. Conclusion - The portal venous index was useful in distinguishing healthy patients from patients with CHC. However, there was no significant difference in the quantification of degree of fibrosis. HEADINGS – Hepatitis C, chronic. Liver cirrhosis. Portal vein, ultrasonography. Ultrasonography

INTRODUCTION

Hepatitis C virus (HCV) infection is a global health problem. It is estimated that about 3% of the world’s population is infected with HCV and that 3 million people are infected each year. Eighty percent of cases will become chronic carriers. Of these patients, 10% to 20% will develop cirrhosis and 1% to 5% will develop hepatocellular carcinoma(13, 14). The current recommended treatment for chronic hepatitis C (CHC) is pegylated interferon with ribavirin. However, this treatment is costly and has potential adverse effects. Treatment indications are, in most cases, the degree of liver inflammation and fibrosis, with liver biopsy being the testing method of choice(23). Liver biopsy, however, has its limitations since it does not diagnose cirrhosis in

10% to 30% of cases, and it results in the misclassification of fibrosis by at least 1 grade in 20% to 30% of cases. In addition, there are certain contraindications and adverse events. Some have reported mortality rates after liver biopsy at 3 in 10,000 procedures(4, 19, 22). Given the importance of assessing liver fibrosis in hepatitis C patients, coupled with the limitations of biopsy, a non-invasive assessment of liver fibrosis based on laboratory tests(19) and imaging(22) needs to be developed. Imaging methods such as ultrasound, computerized tomography and magnetic resonance imaging can provide useful information on morphological changes in the diseased liver(21, 22). Elastography is a new method that is the most effective at differentiating between cirrhosis and non-cirrhosis and is considered to be a quick and easy, non-invasive procedure for diagnosing cirrhosis.

No external support relevant to this submission. The authors have no conflicts of interest to declare. From the Departamento de Diagnóstico por Imagem, Universidade Federal de Uberlândia, MG, Brazil. 1 Serviço de Gastroenterologia, Hospital de Clínicas, Universidade Federal de Uberlândia (HC-UFU); 2 Pós-graduação - Ciências da Saúde, UFU; 3 Departamento de Anatomia Patológica – UFU, Uberlândia, MG, Brasil Correspondence: Dr. Haroldo Luís Oliva Gomes Rocha – Rua Quinze de Novembro, 365/1500 – Centro – 38400-214 – Uberlândia, MG, Brasil. E-mail: haroldoluisrocha@ yahoo.com.br

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Rocha HLOG, Diniz ALD, Borges VFA, Salomão FC. Assessment of portal venous index as a non-invasive method for diagnosing liver fibrosis in patients with chronic hepatitis c

Elastography has been adopted as an alternative to liver biopsy in patients with formal contraindications(7). However, it is not yet available for small-scale providers. Ultrasound is widely available throughout the world thanks to its low cost, ease of use, non-invasive nature, and wide acceptance by patients. It provides useful information on the morphology of the hepatic parenchyma, as well as on hemodynamics changes, using pulsed color Doppler velocimetry(18, 24). The objective of this study was to compare the portal vein velocities and venous pulsatility indexes (VPIs) obtained in patients with CHC versus a healthy control group, and to correlate these parameters with grade of fibrosis obtained from transcutaneous biopsy. METHODS

A prospective observational study that included 50 patients with CHC and 44 healthy controls was conducted between April 2009 and October 2010. The Ethics Committee of Federal University of Uberlândia, MG, Brazil, approved the project (Protocol No. 005/09). For ethical reasons, only patients with HCV underwent liver biopsy. All patients signed the informed consent form. The inclusion criteria were the presence of serology and panel reactive antibodies that were positive for HCV and age between 18 and 70 years. The control group had the same inclusion criteria, except for the presence of HCV. Patients with alcohol intake >140 g/week for males and >70 g/week for females were excluded(10), as were those using medications known to be hepatotoxic or medications causing hemodynamic changes that could cause changes in liver Doppler flow data. Patients with any blood clotting disorder, heart disease with signs of heart failure, nephropathy with modification of renal texture during the ultrasound examination and pregnant or lactating women were also excluded. All 94 volunteers were examined by two-dimensional ultrasonography with Doppler, using a 2 MHz–5 MHz multifrequency convex transducer (Voluson 730 PRO V, General Electric, Milwaukee, USA). The subjects fasted for 10 hours to 12 hours and then in the morning underwent an ultrasound exam in a supine position with arms outstretched alongside the head. All ultrasounds were performed by the same experienced examiner. All segments of the liver were examined and patients that were found to have vascular malformations, cysts, and focal parenchymal lesions were excluded. A liver biopsy was performed in the outpatient clinic using intercostal access guided by ultrasound, using a TruCut automatic needle (16 ga x 15 cm). Biopsies that contained at least five portal tracts were considered representative and those with 10 or more portal tracts were considered ideal. The slides were examined by a single experienced pathologist who had no knowledge of the patients’ clinical and laboratory reports or ultrasound data. Classifications from the Clube Brasileiro de Hepatologia of the Sociedade Brasileira de Patologia(12) and METAVIR(3) were used for histological assessment. For clinical application, the HCV patients were divided into two groups by grade of fibrosis: the first had no significant

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fibrosis, consisting of patients with F0 and F1 fibrosis (n = 17); the second had significant fibrosis, consisting of patients with F2, F3, and F4 fibrosis (n = 33). The ultrasound parameters were obtained within a week after performing the biopsy. The portal vein Doppler velocimetry was performed during apnea, at the beginning of inspiration to avoid changes caused by deep inspiration. Spectral analysis of the portal vein flow velocity waveform (FVW) was recorded for at least 5 seconds of suspended inspiration(16). The measurement point for the portal vein FVW was in the extrahepatic portion, adjacent to the hepatic hilum (Figure 1). The Doppler sample volume was set to between 4 mm and 8 mm, with a pulse repetition frequency of less than 3.3 KHZ and FVW recorded with an angle of less than 60º. Maximum and minimum portal vein velocities were recorded and photo documented for each volunteer (Figure 1), after obtaining at least three FVWs with homogeneous patterns. The VPI was obtained for all patients and controls by subtracting Vmin from Vmax and dividing the remainder of Vmax [VPI = (Vmax - Vmin) / Vmax] in a single wave for each patient.

FIGURE 1. On the left, a portal vein Doppler result showing strong pulsatility of the portal vein (VPI = 0.47) and, on the right, low pulsatility (VPI = 0.18)

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Rocha HLOG, Diniz ALD, Borges VFA, Salomão FC. Assessment of portal venous index as a non-invasive method for diagnosing liver fibrosis in patients with chronic hepatitis c

Descriptive statistics were used to characterize the sample. Continuous variables were expressed as mean ± standard deviation, and categorical variables were expressed as absolute (n) and relative (%). The distribution of the data was analyzed using the Lilliefors normality test. For variables that followed a normal distribution, comparisons were made between the means of the two samples using Student’s t test. For variables that did not follow a normal distribution, the Mann-Whitney test was used. The Kruskal-Wallis test and Student-Newman-Keuls test were used for comparisons between VPI samples for the three patient groups. The correlation analyses were performed using the Spearman coefficient. The receiver operating characteristic (ROC) was also calculated for the parameters with the greatest degree of significance based on the above tests. All tests were considered significant at P<0.05. The statistical analyses were performed using Statistical Package for the Social Sciences (SPSS for Windows, version 17.0, Chicago, IL). RESULTS

The groups were matched for sex and age. The CHC patient group had an average age of 45 ± 11 years. There was no difference between the mean age of the study group and the control group, which was 41 ± 11 years (P = 0.06). There were 29 men (58%) in the study group compared to 18 male volunteers (40.9%) in the control group (P = 0.098), as shown in Table 1. Most patients (74%) were infected with HCV genotype 1. The average viral load was of 2,742,812 ± 4,001,501 copies and 6.09 ± 0.59 log. The aspartate transaminase (AST), alanine transaminase (ALT), and gamma glutamyl transpeptidase (GGT) liver enzymes were higher in the CHC carrier group compared to the levels of the control group (P<0.001; Table 1).

Of the patients who underwent biopsy, 5 (10%) had F0 fibrosis, 12 (24%) had F1 fibrosis, 22 (44%) had F2 fibrosis,  9 (18%) had F3 fibrosis and only 2 (4%) had F4 fibrosis. All HCV patients had some degree of liver inflammation indicated during the pathology exam: 31 (62%) showed A1 activity, 16 (32%) showed A2 activity and 3 (6%) had A3 activity. Hydropic degeneration was found in 94% of patients, with ballooning in 44% of patients and hemosiderosis in 12%. Only 12 patients (24%) had some degree of steatosis on biopsy: 8 with grade I steatosis and only 4 with moderate or severe steatosis. The portal vein flow velocity waveform was more pulsatile in the control group than in patients with HCV. The mean values for VPI were 0.33 ± 0.07 in the control group and 0.23 ± 0.09 in patients with HCV (P<0.001; Table 1). The correlation between VPI and the degree of fibrosis was moderate and inverse (r = –0.448, P<0.001). There was a significant difference between the mean VPI (greater) for the control group compared to the means of the HCV patient groups that were subdivided into insignificant fibrosis (F0 and F1) and significant fibrosis (F2, F3 and F4), with P = 0.001 and P<0.001, respectively (Figure 2). But when comparing the values for VPI between the two CHC patient groups — without significant fibrosis (F0 and F1) and with significant fibrosis (F2, F3, F4) — we found no significant differences (P = 0.715). In order to define the cutoff parameter for VPI for the diagnosis of liver fibrosis in HCV patients, we used the ROC curve. The area under the curve was 78.4% (CI 95%: 68.8%–88%). The ideal cutoff point for the VPI was calculated to be 0.28, with sensitivity equal to 73.5% and specificity equal to 71.1% (Figure 3).

Age (years) Males (%) Platelet count (n) Albumin(g/dL) Total bilirubin(mg/dL) AST* ALT* GGT* ALP* Vmax (cm/s) Vmin (cm/s) Portal venous index

HCV (n = 50) 45.32 ± 11.14 29 (58%) 212,170 ± 56,921 4.43 ± 0.37 0.77 ± 0.41 1.32 ± 0.78 1.9 ± 1.32 2.39 ± 1.92 0.69 ± 0.19 24.88 ± 6.31 16.50 ± 4.26 0.33 ± 0.07

Control (n = 44) 41.98 ± 11.09 18 (40.90%) 251,054 ± 45,230 4.62 ± 0.41 0.81 ± 0.4 0.4 ± 0.1 0.45 ± 0.21 0.48 ± 0.3 0.57 ± 0.19 23.37 ± 5.38 17.77 ± 3.75 0.23 ± 0.09

P-value 0.060 0.098 <0.001 0.570 0.650 <0.001 <0.001 <0.001 0.070 0.214 0.128 <0.001

AST = aspartate aminotransferase; ALT = alanine aminotransferase; GGT = gamma glutamyl transferase; ALP = alkaline phosphatase; HCV = hepatitis C virus; Vmax = portal vein maximum velocity; Vmin = portal vein minimum velocity * Liver enzymes were described in relation to their upper normal limit

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Portal Vein Pulsatility Index

0.60 TABLE 1. Clinical, laboratory, and ultrasound characteristics of groups with HCV and control groups, expressed as mean ± standard deviation, except for categorical variables, which are expressed in absolute and percentage frequencies, where indicated

0.581 0.544 0.487

0.50 0.40 0.30 0.20 0.10 0.00 Control

F0 and F1 F2,F3 and F4 Fibrosis Grade by biopsy

FIGURE 2. Box plot graph of portal vein pulsatility index (VPI) according to the degree of fibrosis at histology. Assessment results using the KruskalWallis test and post hoc analysis using the Student-Newman-Keuls test, with respective degrees of significance shown in bars. Numbers next to the black dots reflect outliers, and the numbers next to the graphs show the median, with the standard deviation of each group

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Rocha HLOG, Diniz ALD, Borges VFA, Salomão FC. Assessment of portal venous index as a non-invasive method for diagnosing liver fibrosis in patients with chronic hepatitis c

1.0

Sensivity

0.8

0.6

0.4 0.2

0.0 0.0

0.2

0.4 0.6 1 - Specificity

0.8

1.0

FIGURE 3. ROC curve graph for sensitivity plotted against 1-VPI specificity for the diagnosis of fibrosis on biopsy

DISCUSSION

HCV infection is a major cause of chronic liver disease throughout the world(25). The assessment of the degree of liver fibrosis in HCV patients is important for better clinical management. Until now, liver biopsy has been the gold standard for defining the degree of liver fibrosis and providing treatment, despite its contraindications and adverse effects(19). New methods for non-invasive assessment of liver fibrosis are being investigated(19). Among these methods, ultrasound with Doppler has been the subject of several publications(5, 17, 26) . These methods include portal vein maximum velocity (Vmax), portal vein minimum velocity (Vmin), portal blood flow volume, congestive index, and portal venous index (VPI). VPI is defined as a parameter that evaluates portal venous pulsatility and its major advantage is it does not depend on the angulation obtained in the Doppler sample, which makes it more reproducible. The relative lack of studies in the literature looking at VPI as a parameter in the assessment of fibrosis in patients with CHC(2, 16) was the motive for this study. It is known that HCV can lead to various degrees of liver inflammation and fibrosis, with modification of the usual architecture of the liver, associated with sinusoidal vascular distortion. These changes may lead to changes in intra- and extra-hepatic vascular impedance, which theoretically can be detected by the Doppler examination of the portal vein, hepatic vein and hepatic artery. A large number of Doppler ultrasound parameters have been studied in order to identify hemodynamic changes in the liver, but there is no consensus on which option is best in identifying the grade of liver fibrosis(6, 8, 15). The numerous hemodynamic changes present in livers with established cirrhosis is unquestionable. In these cases there is an increase of intrahepatic vascular resistance and a

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reduction of portal blood flow, which are changes that lead to compensatory increase in hepatic artery flow. In addition, intrahepatic shunts may develop between branches of the portal vein, hepatic vein, and hepatic artery. Porto-systemic collateral vessels may increase resistance to portal blood flow, and their formation precedes the clinical manifestations of hypertension per se(20). At issue is to what extent fibrosis, inflammation, and steatosis may modify the arterial and venous hepatic vascular patterns before cirrhosis is established, and what would be the ideal non-invasive method for diagnosing and grading fibrosis levels. Given that Doppler ultrasound is a method that aids in the diagnosis of hepatic hemodynamic changes, we chose to evaluate VPI of the portal vein in this group of patients with HCV, given the small number of studies in the literature that calculate VPI in grading liver fibrosis(1, 2). Galix et al.(11) Barakat(2), and Balci et al.(1) described the following reference values for VPI in normal patients: 0.39 ± 0.10, 0.48 ± 0.31 and 0.32 ± 0.06, respectively. In this study, the mean VPI for the control group was 0.33 ± 0.07, which is in agreement with most of the authors cited above. The isolated use of portal vein velocities was not sufficient to discriminate between healthy patients and fibrotic patients, which is in agreement with the literature. Lim et al.(17) compared portal vein Vmax between patients with mild and moderate hepatitis and cirrhosis, but did not observe significant differences. The same occurred in the Bernatik et al.(5) study comparing Vmax for grades I to IV patients, which failed to yield significant results. Our study agrees with the above research in that we found no significant difference in Vmax and Vmin between groups of normal patients and patients with CHC. Changes in VPI were initially described in patients with congestive heart failure. Later it was found that patients with chronic liver disease also exhibited changes in portal vein flow patterns(2). As shown in the current study, the portal vein flow velocity waveform is less pulsatile in patients with CHC compared to healthy controls, after quantification using VPI and defining significant differences between the two groups. In previously published work, this same difference was also observed(2). Our study, however, found no significant difference between groups of patients with significant or insignificant fibrosis. Barakat’s(2) 2002 study classified patients with chronic liver disease according to the Child-Pugh clinical criteria and found no significant difference between VPI values, independent of severity. After constructing a ROC curve, we obtained a cutoff of 0.28 for VPI with good sensitivity and specificity. Data published in the literature suggest a normal range for VPI between 0.20 and 0.50(2). In our study, only two (4%) patients had F4 fibrosis. This fact is explained by the rigorous criterion used in conducting the biopsy; that is, any clotting disorder was considered a contraindication for performing the procedure. The small number of patients with cirrhosis may explain the absence of VPI values difference among groups of patients with CHC. It is known that the main changes occur in cases with severe fibrosis and cirrhosis, where there is a parenchymal and

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Rocha HLOG, Diniz ALD, Borges VFA, Salomão FC. Assessment of portal venous index as a non-invasive method for diagnosing liver fibrosis in patients with chronic hepatitis c

vascular remodeling(20). Further studies will be important to analyse the portal venous index pattern in patients with severe liver fibrosis and cirrhosis. However, these patients most often are categorized as cirrhotic through imaging and laboratory examinations, which commences a special treatment protocol, and they do not require biopsy to start drug therapy(9).

CONCLUSIONS

The portal venous index was useful in differentiating healthy subjects from patients with fibrosis due to chronic HCV. However, there was no significant difference regarding the grade of fibrosis in the patient group.

Rocha HLOG, Diniz ALD, Borges VFA, Salomão FC. Emprego do índice venoso portal como método não-invasivo no diagnóstico de fibrose hepática em pacientes portadores de hepatite C crônica. Arq Gastroenterol. 2012;49(1):14-8. RESUMO – Contexto - A hepatite C é uma importante causa de hepatopatia crônica no mundo. A avaliação do grau de fibrose hepática na hepatite C crônica é importante para o melhor manejo clínico. Entretanto, até o momento, a biopsia hepática é o único teste aceito para esta finalidade, apesar de suas contra-indicações e complicações. Novos métodos para avaliação não invasiva de fibrose hepática estão sendo pesquisados. Uma proposta é o ultrassom com Doppler, por ser um método não-invasivo, amplamente disponível e de baixo custo. Objetivo - Comparar os parâmetros Doppler da veia porta de portadores de hepatite C crônica com um grupo controle sadio e correlacionar estes parâmetros com os graus de fibrose obtidos por biopsia hepática. Métodos - Cinquenta pacientes portadores de hepatite C crônica com biopsia hepática e 44 controles sadios foram submetidos ao Doppler da veia porta, com cálculo do índice venoso portal. Realizou-se a comparação entre as médias do índice venoso portal dos dois grupos. Para a correlação entre o índice venoso portal e os graus de fibrose empregou-se o teste de Spearman. Resultados - Houve diferença entre a média do índice venoso portal entre os controles (0,33 ± 0,07) e os doentes (0,23 ± 0,09) com P<0,001. Não foi observada diferença do índice venoso portal entre os pacientes com hepatite C crônica que tem ou não fibrose significante. A correlação entre o índice venoso portal e o grau de fibrose foi inversa e moderada (r = -0,448 P<0,001). A área sob a curva ROC foi de 78,4% (IC 95%: 68,8%-88%). O ponto de corte para o índice venoso portal foi de 0,28 com sensibilidade de 73,5% e especificidade de 71,1%. Conclusão - O índice venoso portal foi útil em diferenciar pacientes sadios de pacientes portadores de hepatite C crônica. No entanto, não se encontrou diferença significante na quantificação dos graus de fibrose. DESCRITORES – Hepatite C crônica. Cirrose hepática. Veia porta, ultrassonografia. Ultrassonografia Doppler.

References 1. 2. 3. 4. 5. 6. 7.

8.

9.

10. 11. 12.

13.

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Balci A, Karazincir S, Sumbas H, Oter Y, Egilmez E, Inandi T. Effects of diffuse fatty infiltration of the liver on portal vein flow hemodynamics. J Clin Ultrasound. 2008;36:134-40. Barakat M. Portal vein pulsatility and spectral width changes in patients with portal hypertension: relation to the severity of liver disease. Br J Radiol. 2002;75:417-21. Bedossa P, Poynard T. An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group. Hepatology. 1996;24:289-93. Bedossa P, Dargère D, Paradis V. Sampling variability of liver fibrosis in chronic hepatitis C. Hepatology. 2003;38:1449-57. Bernatik T, Strobel D, Hahn EG, Becker D. Doppler measurements: a surrogate marker of liver fibrosis? Eur J Gastroenterol Hepatol. 2002;14:383-7. Borges VFA, Diniz ALD, Cotrim HP, Rocha HLOG, Salomão FC. Dopplerfluxometria da veia hepática em pacientes com esteatose hepática não alcoólica. Radiol Bras. 2011;44:1-6. Coco B, Oliveri F, Maina AM, Ciccorossi P, Sacco R, Colombatto P, Bonino F, Brunetto MR. Transient elastography: a new surrogate marker of liver fibrosis influenced by major changes of transaminases. J Viral Hepat. 2007; 14:360-9. Colli A, Cocciolo M, Riva C, Martinez E, Prisco A, Pirola M, Bratina G. Abnormalities of Doppler waveform of the hepatic veins in patients with chronic liver disease: correlation with histologic findings. AJR Am J Roentgenol. 1994;162:833-7. de Araújo ES, Mendonça JS, Barone AA, Gonçales FL Jr, Ferreira MS, Focaccia R, Pawlotsky JM. Consensus of the Brazilian Society of Infectious Diseases on the management and treatment of hepatitis C. Braz J Infect Dis. 2007;11:446-50. Farrell GC, George J, Pauline de la M Hall P, McCullough AJ, editors. Fatty liver disease: NASH and related disorders. Malden, MA: Blackwell; 2005. p.1-319. Gallix BP, Taourel P, Dauzat M, Bruel JM, Lafortune M. Flow pulsatility in the portal venous system: a study of Doppler sonography in healthy adults. AJR Am J Roentgenol. 1997;169:141-4. Gayotto LCC, Comitê SBP/SBH. Visão histórica e consenso nacional sobre a classificação das hepatites crônicas - projeto do Clube de Patologia Hepática da Sociedade Brasileira de Patologia aprovado pela Sociedade Brasileira de Hepatologia. GED Gastroenterol Endosc Dig. 2000;19:137-140. Global Burden of Hepatitis C Working Group. Global burden of disease (GBD) for hepatitis C. J Clin Pharmacol. 2004;44:20-9.

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14. Global surveillance and control of hepatitis C. Report of a WHO Consultation organized in collaboration with the Viral Hepatitis Prevention Board, Antwerp, Belgium. J Viral Hepat. 1999;6:35-47. 15. Iliopoulos P, Vlychou M, Margaritis V, Tsamis I, Tepetes K, Petsas T, Karatza C. Gray and color Doppler ultrasonography in differentiation between chronic viral hepatitis and compensated early stage cirrhosis. J Gastrointestin Liver Dis. 2007;16:279-86. 16. Karabulut N, Kazil S, Yagci B, Sabir N. Doppler waveform of the hepatic veins in an obese population. Eur Radiol. 2004;14:2268-72. 17. Lim AK, Patel N, Eckersley RJ, Kuo YT, Goldin RD, Thomas HC, Cosgrove DO, Taylor-Robinson SD, Blomley MJ. Can Doppler sonography grade the severity of hepatitis C-related liver disease? AJR Am J Roentgenol. 2005;184:1848-53. 18. Martínez-Noguera A, Montserrat E, Torrubia S, Villalba J. Doppler in hepatic cirrhosis and chronic hepatitis. Semin Ultrasound CT MR. 2002;23:19-36. 19. Myers RP. Noninvasive markers of liver fibrosis: playing the probabilities. Liver Int. 2008;28:1328-31. 20. Ridolfi F, Abbattista T, Marini F, Vedovelli A, Quagliarini P, Busilacchi P, Brunelli E. Contrast-enhanced ultrasound to evaluate the severity of chronic hepatitis C. Dig Liver Dis. 2007;39:929-35. 21. Ronot M, Asselah T, Paradis V, Michoux N, Dorvillius M, Baron G, Marcellin P, Van Beers BE, Vilgrain V. Liver fibrosis in chronic hepatitis C virus infection: differentiating minimal from intermediate fibrosis with perfusion CT. Radiology. 2010;256:135-42. 22. Searle J, Mendelson R, Zelesco M, Sanford J, Cheng W, McKinstry C, Ramsay D. Non-invasive prediction of the degree of liver fibrosis in patients with hepatitis C using an ultrasound contrast agent. A pilot study. J Med Imaging Radiat Oncol. 2008;52:130-3. 23. Strader DB, Wright T, Thomas DL, Seeff LB; American Association for the Study of Liver Diseases. Diagnosis, management, and treatment of hepatitis C. Hepatology. 2004;39:1147-71. 24. Tchelepi H, Ralls PW, Radin R, Grant E. Sonography of diffuse liver disease. J Ultrasound Med. 2002;21:1023-32. 25. Verna EC; Brown RS Jr. Hepatitis C virus and liver transplantation. Clin Liver Dis. 2006;10:919-40. 26. Walsh KM, Leen E, MacSween RN, Morris AJ. Hepatic blood flow changes in chronic hepatitis C measured by duplex Doppler color sonography: relationship to histological features. Dig Dis Sci. 1998;43:2584-90. Received 24/7/2011. Accepted 19/10/2011.

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ARTIGO ORIGINAL/ ORIGINAL ARTICLE

ARQGA/1584

NUTRITIONAL ASSESSMENT IN PATIENTS WITH CIRRHOSIS Sabrina Alves FERNANDES, Lilian BASSANI, Flávia Feijó NUNES, Maria Eugênia Deutrich AYDOS, Alexandro Vaesken ALVES and Cláudio Augusto MARRONI ABSTRACT – Context - Malnutrition in cirrhotic patients with end-stage disease is common, and the degree of nutritional debilitation can play an important role in the pathogenesis of complications and cause a negative impact on prognosis. However, it involves difficulties and controversies regarding the identification of the best nutritional assessment method. Objective - To identify a method that provides a safe and effective nutritional diagnosis. Methods - Cross-sectional study with 129 cirrhotic patients. Anthropometric measurements, subjective global assessment, hand grip strength and bioelectrical impedance. Results - Through phase angle of bioelectrical impedance analysis (BIA) method, significant associations with Child-Pugh (P = 0.008), age group and gender were observed. The ROC (receiver operator characteristic) curve was generated to determine the best cutoff point of the phase angle of cirrhotic patients, serving as one of the reference parameters for the nutritional assessment with bioimpedance in this study, considering the classification through Child-Pugh score as the reference standard for the clinical conditions of patients with cirrhosis. Conclusions - The assessment through bioelectrical impedance presented a statistically significant correlation with Child-Pugh score. The identification of phase angle of 5.44º is the new parameter suggested for the classification of the nutritional conditions of cirrhotic patients. HEADINGS – Liver cirrhosis. Electrical impedance. Malnutrition. Nutrition assessment.

INTRODUCTION

Protein caloric malnutrition is a syndrome considered as progressive loss of both lean body mass (protein) and adipose tissue (calorie). Significant changes in the metabolism of protein, carbohydrates and lipids appear simultaneously the consumption of muscular and lipid compartments to satisfy a higher energetic demand(10). This clinical condition is common in patients with chronic hepatopathy and affects 20% of the patients with compensated cirrhosis and more than 60% of these patients with severe hepatic dysfunction(21, 22). Several factors are present in the development of malnutrition in cirrhotic. Inadequate oral ingestion resulting from anorexia, dysgeusia, early satiety, nauseas and vomits associated with hepatopathy, use of drugs and their adverse effects(12, 24, 25, 26). We should also consider as contributing factors the poor absorption and deficient intestinal digestion, which are more accentuated in patients with bile duct diseases with cholestasis, for whom steatorrhea is common(25, 29). Malnutrition is a relevant factor when determining the progress of hepatic disease, as it affects the

storage of nutrients, contributes to hypoalbuminemia resulting from impaired hepatic synthesis and intensifies the hydroelectrolytic unbalance determined by renal alterations, characteristics of this disease state (23, 25, 28, 29). However, it involves difficulties and controversies regarding the identification of the best nutritional assessment method considered as gold standard, that is a low cost, loyal and easy-to-apply method that does not affect the final result. Due to these characteristics, which negatively interfere in the nutritional status of the cirrhotic patient and which are part of the natural history of the disease, it is necessary to identify when malnutrition begins its course. This preventive measure applies primarily to patients on the liver transplant list, who will thus have a better quality of life until the time of the transplant. Therefore, the present study has the purpose of identifying amongst the available methods, of low cost and easily reproducible, which one offers a safe and efficient nutritional diagnosis, which will help the clinical practice and allow an early intervention in cirrhotic patients.

The three authors involved participated sufficiently in the work to take public responsibility for the content of the paper and must approve of the final version of the manuscript. There is no conflict of interest involved. Postgraduate Program of Hepatology, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS, Brasil. Correspondence: Dr. Sabrina Alves Fernandes – Rua Marcilio Dias, 918 – apt. 08 – Menino Deus – 90130-000 – Porto Alegre, RS, Brazil. E-mail: sabrinaafernandes@ gmail.com

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Fernandes SA, Bassani L, Nunes FF, Aydos MED, Alves AV, Marroni CA. Nutritional assessment in patients with cirrhosis

METHODS

A prospective cross-sectional study was conducted between April 2007 and January 2008 with 129 cirrhotic patients, with diagnosis based on histological evidences, clinical and laboratorial or imaging diagnosis (presence of hepatic irregularities due to portal hypertension and hepatic insufficiency) and evidences of chronic hepatic disease. The clinical evidences of cirrhosis were defined with the presence of portal hypertension or hepatic insufficiency. The study excluded patients with hepatocellular carcinoma, poor intestinal absorption, acquired immunodeficiency syndrome, chronic renal insufficiency, use of enteral diet, neuromuscular alterations in upper limbs, chronic pancreatitis, chronic diarrhea and psychic and/or cognitive alterations. The patients with inclusion criteria were classified according to the disease severity through Child-Pugh score. They were invited to participate in the study after they agreed with it and signed the Informed Consent Term, approved by the ethics committee of the Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), RS, Brazil. Protocol The clinical, anthropometric, subjective global assessments was performed through dynamometry and bioelectrical impedance during a routine doctor’s visit, in the morning shift, with the same duration and performed by the same appraiser. Anthropometry Weight was measured using a Filizola 100 g resolution scale and height with a wall-mounted stadiometer. The body mass index (BMI) was calculated using the formula proposed by Quetelet(40), BMI = weight/height2; and the parameters of OMS 1995(42) were considered for the nutritional state classification. The triceps skinfold (TSF) and upper arm circumference (UAC) were measured using mathematical formulas to calculate the arm muscle circumference (AMC)(11). Measurement of TSF was measured using a Cescorf skinfold caliper. The UAC was measured with a graduated, non-retracting, flexible measuring tape. Subjective global assessment The subjective global assessment (SGA) was performed following the protocol of Detsky et al.(9). It analyzed history data of ponderal loss, reduced daily caloric ingestion, gastrointestinal symptoms, functional capacity and physical signs of malnutrition (reduction of subcutaneous tissue and/ or muscular mass, edema, ascites). The patients were classified as: well nourished A, with moderate malnutrition B and severe malnutrition C, according to the method scores. Dynamometry A mechanical hand grip dynamometer of adjustable handle was used to assess the hand grip strength (HGS) (Baseline® Smedley Spring, made in New York, USA). The

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patient sits on a chair without armrest and with the plantar region on the floor; the appraiser adjusts the dynamometer handle to place the patient’s hand comfortably, that, during the test, should be kept far from the body and chair. After the dynamometer adjustment to the correct position, the patient compresses the handle with as much strength as possible with the non-dominant hand. Three assessments were made, with an interval of more than 30 seconds between them, and the pointer was zeroed after each attempt. The result of highest value from the three assessments was utilized as criterion-result, and later the nutritional state was classified along with the patient’s age and gender, according to the reference parameter of Álvares da Silva et al.(4). Bioelectrical impedance For the assessment of nutritional state, Biodynamics, model 450, Seattle, WA, USA was utilized. The patient remains in dorsal decubitus position, with hands and legs parallel to the body. One electrode is placed on the dorsal hand, at the middle finger level, and one in the wrist joint, both on the right side. Another pair of electrodes is placed on the dorsal foot, at the middle toe level, and in the ankle joint, also on the right side. The electrical current used in the measurement is 800 A and 50 kHz, which enables to measure resistance and reactance and obtain the phase angle (PA) value. The PA derives from two segments of corporal composition, calculated as follows: PA = tangent arc (Xc /R) x 180/ 3.1416, proposed by Barbosa-Silva et al.(7). The PA result enables to classify the patient according his/her nutritional state. The patients were classified according to two reference parameters: the first is named bioelectrical impedance analysis (BIA), based on the reference parameters of the study conducted by Barbosa-Silva et al.(8), which validated cutoff points for the method, according to the age and gender of a population of inpatients with different diseases from an American hospital; the second one, BIA cirrhosis, named by the author of this study, is related to the population of cirrhotic patients included in this study and presents the value of 5.44º as cutoff point. Statistical analysis A descriptive analysis was performed through mean values and standard deviation for continuous variables of symmetric distribution, or median value and interquartile amplitude for continuous variables of asymmetric distribution, and absolute and relative frequency for categorical variables. Chi-square test of McNemar was used in the comparison of the nutritional state between the techniques. Kappa coefficient of agreement was used to assess the agreement between the classifications of BIA. The assessment of the association between the reference values of HGS and BIA and severity, disease etiology and demographic characteristics used the t-Student test or the analysis of variance (ANOVA), one way for continuous variables of symmetric distribution, and Mann-Whitney or Kruskal-Wallis test for continuous variables of asymmetric distribution. For

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multiple comparisons, Tukey test (symmetric distribution) or Mann-Whitney test (asymmetric distribution) was used. Pearson’s chi-square test was used to assess the association between the categorical variables. The significance level of 5% was considered and the analyses were performed using SPSS (Statistical Package for the Social Sciences) version 13.0. Ethical considerations Secrecy was maintained about the identity and data from all patients submitted to the assessment protocol (number: 06/135). The study was conducted after the approval of the Ethics Committee, according to the Declaration of Helsinki of 1975, revision of 1983. RESULTS

The sample is composed of 129 patients, mean age of 55 years (± 11.4 years, ranging from 20 to 78 years), 70 (54.3%) male and 59 (45.7%) female.

The clinical characteristics of the cirrhotic patients included in the study are described in Table 1. The anthropometric evaluation (Table 2) shows that weight, height and AMC found in the male population are higher than those found in the female population (P≤0.001), but, when analyzing the TSF, the female population presents higher values than those of the male population (<0.001). On the other hand, no difference was observed between the genders when the parameters of BMI and UAC were analyzed. In the analysis by subjective global assessment (SGA), only 20.2% of the analyzed patients were classified as malnourished, with no statistically significant difference in the anthropometry, as observed in Figure 1. In the HGS assessment through dynamometry, new decreasing values as the age increased were found in male patients, with a similar tendency observed in the female patients above 40-49 years old, as described in Table 3. The male patients of ≥60 years of age and the female patients of all ages showed significantly less strength (kg) when compared to male patients below 50 years of age.

TABLE 1. Characterization of the sample constituted of cirrhotic patients Characteristics Age (years) – Mean (SD) Gender – n (%) Male Female Child – n (%) A B C Etiology – n (%) Alcohol Virus B Virus C Virus B + alcohol Virus C + alcohol Cryptogenic Autoimmune Others

Total Sample (n = 129)

Age group (years) 40-49 50-59 (n = 25) (n = 35) 45.5 (2.58) 53.8 (2.94)

55.1 (11.4)

20-39 (n = 12) 31.4 (5.81)

70 (54.3) 59 (45.7)

8 (66.7) 4 (33.3)

15 (60.0) 10 (40.0)

21 (60.0) 14 (40.0)

26 (45.6) 31 (54.4)

91 (70.5) 28 (21.7) 10 (7.8)

11 (91.7) 1 (8.3) 0 (0.0)

15 (60.0) 6 (24.0) 4 (16.0)

23 (65.7) 7 (20.0) 5 (14.3)

42 (73.7) 14 (24.6) 1 (1.8)

33 (25.6) 4 (3.1) 56 (43.4) 1 (0.8) 11 (8.5) 5 (3.9) 5 (3.9) 14 (10.9)

1 (8.3) 0 (0.0) 1 (8.3) 0 (0.0) 1 (8.3) 2 (16.7) 2 (16.7) 5 (41.7)

8 (32.0) 1 (4.0) 11 (44.0) 0 (0.0) 1 (4.0) 0 (0.0) 1 (4.0) 3 (12.0)

9 (25.7) 2 (5.7) 18 (51.4) 0 (0.0) 3 (8.6) 1 (2.9) 1 (2.9) 1 (2.9)

15 (26.3) 1 (1.8) 26 (45.6) 1 (1.8) 6 (10.5) 2 (3.5) 1 (1.8) 5 (8.8)

≥60 (n = 57) 65.2 (4.35)

TABLE 2. Descriptive analysis of the anthropometric data by gender Gender Variables Weight (kg) Height (cm) BNU TSF UAC AMC

Total sample (n = 129) Mean (SD) 74.4 (14.9) 164 (9.19) 27.6 (5.16) 18.4 (9.24) 30.4 (4.47) 24.6 (3.31)

Male (n = 70) Mean (DP) 78.5 (15.6) 170 (6.49) 27.1 (4.73) 14.7 (7.93) 30.1 (4.43) 25.4 (3.37)

Female (n = 59) Mean (DP) 69.6 (12.6) 157 (7.00) 28.2 (5.61) 22.8 (8.80) 30.7 (4.54) 23.5 (2.95)

P-value1 0.001 <0.001 0.229 <0.001 0.478 0.001

BMI: body mass index; TSF: tricipital skin fold; UAC: underarm circumference; AMC: arm muscle circumference. 1Value obtained by Student’s t-test for independent samples

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Regarding the gender, both populations presented lower strength for those above 60 years of age, according to Table 3. In the HGS method and in the phase angle values, the age groups of male patients presented decreasing values, which does not occur with the female patients. Women and patients of ≥60 years of age presented a significantly lower phase angle when compared to men (P = 0.041) and individuals below 50 years of age (P = 0.001) (Table 4). We observed that only male patients of ≥60 years of age and female patients between 50 and 59 years of age presented a significantly lower phase angle when compared to the other patients (Table 4). Figure 1 does not show statistical difference between the different anthropometric methods, except for the BMI, which was similar to TSF only.

The SGA detected a significantly higher number of malnourished patients than the BMI, with the similar result in the other anthropometric parameters. The HGS method detected the higher number of malnourished patients in the studied population, with significant difference when compared to the other methods (P = 0.001). The percentage of malnourished patients found with BIA method presents a statistically significant difference (P = 0.001) when compared to the percentages obtained with the other methods, except for the HGS (P = 0.013), which detected a higher number of malnourished patients. Table 5 shows that 129 patients were analyzed through BIA and 127 through HGS, 2 patients could not be analyzed through HGS: 1 due to gouty arthritis and 1 for not presenting the minimum strength to manifest pain when trying to compress the grip. TABLE 3. Evaluation through HGS by age and gender Cirrhotic men

Age group

Mean (SD)

-2SD

20–39

41.4 (10.2)

40–49

33.3 (11.2)

50–59 ≥60

Cirrhotic women Mean (SD)

-2SD

21.0

19.8 (4.5)

10.8

10.9

22.2 (9.08)

4.04

30.3 (7.53)

15.2

16.0 (7.27)

1.46

27.6 (11.2)

5.2

15.8 (8.34)

0.00

TABLE 4. Evaluation through phase angle by age group and gender

BMI = body mass index; TSF = tricipital skin fold; AMC = arm muscle circumference; UAC = underarm circumference; SGA = subjective global assessment; BIA = bioelectrical impedance analysis; HGS = hand grip strength. a, b, c, d Equal letters do not differ through McNemar chi-square test

FIGURE 1. Evaluation of the percentage of malnourished individuals according to different methods of nutritional assessment

Cirrhotic men

Cirrhotic women

Age group

Mean (SD)

-2SD

Mean (SD)

-2SD

20–39

8.40 (2.62)

3.16

6.57 (1.13)

4.31

40–49

6.35 (1.17)

4.01

7.39 (4.32)

0.00

50–59

6.52 (2.04)

2.44

5.47 (1.62)

2.23

≥60

5.63 (1.11)

3.41

7.18 (4.73)

0.00

TABLE 5. Association of the disease etiology with malnutrition assessed through HGS and BIA HGS (127) BIA (129) Malnourished Nourished Malnourished Nourished Etiology (n = 88; 69.3%) (n = 39; 30.7%) (n = 44; 34.1%) (n = 85; 65.9%) n (%) n (%) n (%) n (%) Virus C 42 (47.7) 13 (33.3) 22 (50.0) 34 (40.0) Alcohol 21 (23.9) 12 (30.8) 7 (15.9) 26 (30.6) Others 11 (12.5) 2 (5.1) 7 (15.9) 7 (8.2) Virus C + alcohol 7 (8.0) 4 (10.3) 5 (11.4) 6 (7.1) Cryptogenic 3 (3.4) 2 (5.1) 1 (2.3) 4 (4.7) Autoimmune 3 (3.4) 2 (5.1) 1 (2.3) 4 (4.7) Virus B 1 (1.1) 3 (7.7) 0 (0.0) 4 (4.7) Virus B + alcohol 0 (0.0) 1 (2.6) 1 (2.3) 0 (0.0) HGS = hand grip strength; BIA = bioelectrical impedance analysis

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Table 5 also shows that the number of malnourished patients evaluated through HGS is proportional to the number of malnourished patients evaluated through BIA, and that the percentage of malnourished patients through HGS is similar to the percentage of malnourished patients through BIA. The highest prevalence related to the cirrhosis etiology in malnourished patients, according to HGS and BIA, is virus C, whose indexes are 42 (47.7%) patients through HGS and 22 (50%) through BIA. The second cause verified was alcohol, with 21(23.9%) through HGS and only seven (15.9%) through BIA. Through phase angle of BIA, a significant association with Child-Pugh (P = 0.008) was observed. The patients classified as Child-Pugh C presented significantly lower phase angle than those classified as Child-Pugh A and B. For this reason, the patients clinically sorted as A and B were grouped (Table 6). Besides Child-Pugh, a significant association was observed between phase angle, age group, and gender. HGS did not present significant relation with Child-Pugh, keeping the relation with age group and gender, as already established as a reference parameter. Figure 2 shows the analysis and association between HGS and BIA methods, which presented a higher number of malnourished patients, with the disease classification of Child-Pugh A, B and C. The analysis showed that BIA is the method that presents a statistically significant relation between Child-Pugh C and malnutrition. Figure 3 shows that the ROC curve, generated to determine the best cutoff point of the phase angle of cirrhotic patients, serving as one of the reference parameters for the nutritional assessment with BIA in this study, considering the ChildPugh classification as the reference standard for the clinical conditions of patients with cirrhosis. Was used the ROC curve to identify the cutoff point for malnutrition through the phase angle. Then, the patients

presenting values below 5.44º were classified as malnourished and the patients with 5.44º or more were classified as nourished. As illustrated in Figure 3, sensitivity and specificity were compared using BIA and HGS, with values of 68.9%-70.0% and 49.2%-56%, respectively. The comparison between the values found of patients classified as malnourished, using the two denominations of BIA, i.e., malnourished through BIA, denomination from the reference parameters of Barbosa-Silva et al.(8), and BIA Cirrhosis, named by the author of this study, showed the agreement of 80.6% of the cases, as illustrated in Figure 4, which is statistically significant (Kappa = 0.566, P<0.001). Figure 4 illustrates the agreement of 70.5% of the malnutrition cases using the two reference parameters: BIA

FIGURE 2. Association between the methods of nutritional assessment and Child-Pugh BIA = bioelectrical impedance analysis; HGS = hand grip strength; HGS: P = 1.000; BIA: P = 0.031

TABLE 6. Association between the values measured through HGS and phase angle with gender, age group, and Child-Pugh of the patients HGS Phase angle Variables n Mean (SD) P-value Median (p25-p75) P-value Gender – n (%) 6.21 (5.61-6.89) 0.0413 Male 70 31.2 (10.8) <0.0011 Female 59 17.2 (8.19) 5.45 (4.97-6.65) Age Group – n (%) 0.0012 7.29 (6.52-8.11)a 0.0014 20 – 39 12 34.2 (13.6)a 40 – 49 24 29.1 (11.6)a 6.59 (5.54-7.09)b 50 – 59 35 24.6 (10.2)ab 5.96 (4.97-6.69)bc 5.62 (4.95-6.35)c 56 21.3 (11.3)b ≥60 Child – n (%) 6.03 (5.34-6.81)a 0.0084 A 91 24.7 (11.2) 0.5102 a B 27 26.6 (14.3) 5.78 (5.19-6.96) C 9 21.3 (11.7) 4.32 (3.72-5.82)b HGS = hand grip strength 1 Student’s t-test for independent samples; 2 Analysis of variance (ANOVA), one-way; 3 Mann-Whitney test; 4 Kruskal-Wallis test; a,b,c Equal letters do not differ through Tukey’s (parametric) test or Mann-Whitney (non-parametric) test

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1.0 Phase angle Dynamomete Reference line

Sensitivit

0.8 0.6 0.4 0.2 0.0 0.0

0.2

0.4 0.6 1 - Specificity

0.8

1.0

Phase angle Area under the curve: 0.794; CI95%:0.64-0.95 Cutoff point: <5.44: malnourished; ≥5.44: well nourished; sensitivity: 68.9% and specificity: 70.0% HGS Area under the curve: 0.565; CI95%:0.38-0.75 Cutoff point: <23: malnourished; ≥23: well nourished; sensitivity: 49.2% and specificity: 56%

FIGURE 3. Receiver operator characteristic (ROC) curve to determine the cutoff point for malnutrition for the phase angle and HGS considering Child-Pugh divided into group 1 = A or B and group 2 = C, as reference standard for the clinical state of cirrhosis.

BIA: bioelectrical impedance analysis

FIGURE 4. Agreement between BIA and BIA cirrhosis

and BIA Cirrhosis, which disagree in only 29.5% of the cases. The study found equivalence of 85.9% of the cases of nourished patients, with disagreement of 14.1%. DISCUSSION

This study was conducted at an outpatient clinic, with most patients clinically classified as Child-Pugh A and B;

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the small group classified as Child-Pugh C might affect data accuracy. Another limitation of this study was the failure to include a control group. The malnutrition percentages of 11.6% obtained through TSF and 13.2% through AMC disagree with the percentages of Abbott et al.(1) and Alberino et al.(2), which show 54% of malnourished patients when combining the two methods, and of Merli et al.(27), which suggest AMC as an indicator of severe malnutrition in patients in the end stage of cirrhosis. The prevalence of overweight was identified in the studied population, when analyzing the BMI, which may present a strong relation with the clinical conditions of the patient, due to water retention and ascites. Through SGA, 20.2% of the patients in this study were classified as malnourished; in the study conducted by Gottschall et al.(14) with a similar population, the index achieved 38%. SGA presents sensitivity of 22% in patients with cirrhosis and underestimates the nutritional state of this population in 57%, with overestimation of 6%(13). Paradoxically, some studies suggest that the benefits of SGA for the nutritional state progress of candidates for kidney transplantation, while other studies showed that SGA detects malnutrition in only 25% of the cases(13, 16, 30, 32). However, it should be noted that SGA is an instrument composed of quantitative and qualitative variables, subject to varied interpretations, as it is a partially subjective method. In agreement with the literature, the method that presented the highest proportion of malnourished patients was HGS, with 69.3% of the cases. Álvares-da-Silva et al.(6) observe that the method presents the capacity to detect 100% of the malnutrition cases and, according to Norman et al.(31), identifies the nutritional risk before the serious state of malnutrition. In the evaluation of cirrhotic patients hospitalized, HGS presents the best relation with the nutritional state progress of the cirrhotic patient, as in the study, 89% of the malnourished patients were classified as Child-Pugh B and C(34). This study demonstrated that HGS does not present statistically significant relation with the Child-Pugh classification, described here as a parameter for the clinical condition of the patient with chronic hepatic disease. Knowing that patients classified as Child C are malnourished by definition(5), the study observed the highest percentage of malnourished patients (93.2%), classified through HGS as Child A and B, and only 6.8% as Child C, reinforcing the non-relation with Child-Pugh. The study used the reference parameters for HGS, validated by Álvares-da-Silva et al.(3), with healthy individuals. A discrepancy was observed when comparing it to the values found in the population of cirrhotic patients, a factor that might have contributed to overestimated proportion of cirrhotic patients. Regarding the utilization of BIA, a study with cirrhotic patients, divided into patients with and without ascites, Pirlich et al.(35) report that this is not the clinical sign of

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greatest negative impact for the corporal composition determination. The authors suggest that specific values and equations to cirrhotic patients should be created as a reference parameter for the utilization of bioelectrical impedance assessment. Lee and Gallagher(19) compare BIA to other high-cost methods, such as bone densitometry, computed tomography and magnetic resonance, in an attempt to identify a low-cost effective method to assess the human corporal composition. This study concludes that BIA presents poor accuracy in individual or group assessments, but effective for a specific population, i.e., individuals without corporal alterations. Lehnert et al.(20) suggest the use of this method in a serial form, identifying and following the cirrhotic patient’s nutritional state evolution. The meta-analysis of Kyle et al. (17, 18) describes that BIA is not an adequate method to patients that present many alterations in the corporal geometric composition, illustrating it with individuals with ascites. The same meta-analysis suggests the use of BIA in a segmental form. Another hypothesis studied is the indication of BIA for nutritional assessment, using the phase angle as a reference value for the method and analyzing the cellularity of the individual impacted by the disease. Schwenk et al. (38) suggest the phase angle as a global marker for malnourished individuals infected with HIV and discuss that the phase angle reflects the integrity and vitality of the cell membrane. Gupta et al.(15) demonstrate that the phase angle is more powerful indicator of survival than the traditional parameters of nutritional assessment, presenting a cutoff point for phase angle of 5.0º, for patients with pancreatic cancer. Studies demonstrated the correlation of the phase angle as a good prognostic indicator in severe clinical situations(33, 36). Another study that also evaluated patients with chronic hepatic diseases, presented the cutoff point of 5.4º for the phase angle(39). Coincidently, our study found a similar cutoff point of 5.44º to characterize malnutrition. Norman et al. (31) describe in their study of BIA evaluation in nutritional diagnosis, the phase angle as a poor indicator of survival. BIA, through the phase angle, was the only method that presented a statistically significant relation with Child-Pugh. However, due to the limitation of this study in terms of low number of patients sorted as Child-Pugh C and age group of 20-39 years, it was not possible to validate a specific reference parameter for this population, dividing them by age, gender and Child-Pugh. However, this limitation does not invalidate our findings with regard to the values of phase angle and to the

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agreement with the patient’s clinical condition by ChildPugh score. We show this agreement through the results of phase angle, by obtaining the same cutoff point as studies by L. Scheunemann et al.(37) and Wagner et al.(41). Given this fact, the phase angle is a strong prognostic marker regardless of the pathology, since this method evaluates cell capacitance, i.e., the integrity and functionality of the individual’s cell, unlike other methods routinely used for nutritional diagnosis, which are usually based on the patient’s body weight. In our case, this parameter is error-prone. Based on such data, the total 34.1% of malnourished patients were found through BIA. The study conducted by Barbosa-Silva et al.(8) was used as the reference parameter, which validates the reference values for the phase angle, for the population of patients from an American hospital, dividing them by age and gender. These results agree with the findings of our study, classifying malnutrition in 33.3% of the sample through BIA cirrhosis, using the phase angle of 5.44º, with agreement of 80.6% between them. Although no gold standard is said to exist for the assessment of the nutritional state of cirrhotic patients, we suggest future studies using BIA, as it showed a low margin of error, when compared to the other methods. BIA was the only method to present a significant relation with Child-Pugh, age group and gender for chronic hepatic diseases. Figueiredo et al.(13) suggest that the four-compartment model of assessment, provided by a CT scan, might be sensitive in detecting malnutrition even in early stages of the disease. However, this method is costly and of low reproducibility for assessing the nutritional status of  patients and, therefore, it was not used in our research, as it did not fulfill one of the purposes of the study. CONCLUSIONS

The assessment of the nutritional status of cirrhotic patients and the comparison with different methods show marked discrepancies, with the percentage of malnourished patients ranging from 5.4% to 68.2%. HGS and BIA showed a very high variation, with malnutrition percentages of 68.2% to 34.1%, respectively. In the comparison to Child-Pugh score, BIA was the only method that showed to be statistically significant. The identification of the phase angle of 5.44º is the new parameter suggested for the classification of the nutritional state of the cirrhotic patient, which can be named BIA Cirrhosis. However, more studies are needed to confirm these data, particularly expanding the number of cases of patients rated as Child-Pugh C.

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Fernandes SA, Bassani L, Nunes FF, Aydos MED, Alves AV, Marroni CA. Avaliação nutricional de pacientes cirróticos. Arq Gastroenterol. 2012;49(1):19-27. RESUMO – Contexto - A desnutrição em pacientes cirróticos com doença em estágio final é comum, e o grau de debilitação nutricional pode desempenhar papel importante na patogênese de complicações e causar impacto negativo no prognóstico. No entanto, envolve dificuldades e controvérsias sobre a identificação do melhor método de avaliação nutricional. Objetivo - Identificar um método que ofereça diagnóstico nutricional seguro e eficiente. Métodos - Estudo transversal avaliou 129 pacientes com cirrose hepática. Foram realizadas medidas antropométricas, avaliação subjetiva global, dinamometria e bioimpedância elétrica. Resultados - Através do ângulo de fase do método BIA, associações significativas com Child-Pugh (P = 0,008), faixa etária e sexo foram observadas. A curva ROC (receiver operator characteristic) foi realizada para determinar o melhor ponto de corte do ângulo de fase de pacientes cirróticos, servindo como um dos parâmetros de referência para a avaliação nutricional com bioimpedância neste estudo, considerando a classificação por pontuação Child-Pugh como o padrão de referência para as condições clínicas dos pacientes com cirrose. Conclusões - A avaliação por meio de bioimpedância elétrica apresentou correlação estatisticamente significativa com o escore de Child-Pugh. A identificação do ângulo de fase de 5,44º é o novo parâmetro sugerido para a classificação do estado nutricional de pacientes cirróticos. DESCRITORES – Cirrose hepática. Impedância elétrica. Desnutrição. Avaliação nutricional.

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41. Wagner D, Adunka C, Kniepeiss D, Jakoby E, Schaffellner S, Kandlbauer M, Fahrleitner-Pammer A, Roller RE, Kornprat P, Müller H, Iberer F, Tscheliessnigg KH. Serum albumin, subjective global assessment, body mass index and the bioimpedance analysis in the assessment of malnutrition in patients up to 15  years after liver transplantation. Clin Transplant. 2011;3:1-5. 42. World Health Organization. Physical status: the use and interpretation of anthropometry. Geneva: WHO, 1995. (WHO - technical report series 854).

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Received 8/10/2009. First review completed on 27/10/2010. Accepted 10/8/2011.

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ARTIGO ORIGINAL / ORIGINAL ARTICLE

ARQGA/1585

INVESTIGATION OF NUTRITIONAL RISK FACTORS USING ANTHROPOMETRIC INDICATORS IN HOSPITALIZED SURGERY PATIENTS Vânia Aparecida LEANDRO-MERHI1 and José Luiz Braga de AQUINO2 ABSTRACT – Context - The investigation of risk factors associated with nutritional status could contribute for better knowledge of the malnutrition. Objective - To investigate the incidence of malnutrition and its possible association with many parameters that assess nutritional status and to identify the associated risk factors. Methods - The nutritional status was assessed in 235 hospitalized patients. Malnutrition was defined as present when the patient presented at least two anthropometric criteria below the normal range and habitual energy intake below 75% of the energy requirement (HEI/ER<75%). Gender, age, type of disease, recent weight change and dental problems were investigated as possible associated risk factors. The chi-square and Mann-Whitney tests were used to compare the data and univariate and multiple logistic regressions were used to identify the factors associated with malnutrition. The odds ratio (OR) and confidence interval (CI) of 95% were calculated with the significance level set at 5% (P<0.05). Results – One-fifth (20%) of the patients were malnourished on admission to the hospital and 27.5% reported recent weight loss. Malnutrition (P<0.0001) was greater in patients with malignant diseases. The only variables significantly associated with malnutrition according to univariate logistic regression were recent weight loss (P = 0.0058; OR = 2.909; IC95% = 1.362; 6.212) and malignant disease (P = 0.0001; OR = 3.847; IC95% = 1.948; 7.597). When multiple regression was used in the model which included type of disease, malignant disease was shown to increase the chance of malnutrition fourfold (P = 0.0002; OR = 3.855; IC95% = 1.914; 7.766). When disease was excluded, recent weight loss also increased malnutrition fourfold (P = 0.0012; OR = 3.716; IC95% = 1.677; 8.236). Conclusion - Patients with a history of recent weight loss and those with malignant diseases are more susceptible to malnutrition. HEADINGS – Nutritional status. Malnutrition. Postoperative period. Anthropometry. Risk factors. Inpatients.

INTRODUCTION

Malnutrition in hospitalized patients is still considered highly prevalent in most hospitals, with hospital malnutrition rates ranging from 15% to 60%(1, 11, 18, 35, 39) depending on the type of hospital, region, studied population and methods routinely used to investigate nutritional status(2, 3, 11, 28, 32). These data have been used in an attempt to establish diagnostic and nutritional intervention strategies that reduce the impact of poor nutritional status and its implications on the disease, morbidity, mortality, length of hospital stay and, especially, to reverse the high hospital malnutrition rates(1, 11, 35, 39). The investigation of risk factors associated with nutritional status right after admission could contribute for better knowledge and identification of this situation, allowing better control of strategies for the primary

prevention of this condition. Generally, the diagnosis of malnutrition is based on objective measurements of nutritional status, which may include assessment of energy intake, weight loss, anthropometric data, biochemical data and body composition(2, 11, 28, 39). Many methods have been developed and used for the assessment and diagnosis of hospital malnutrition, such as anthropometric measurements, biochemical tests, subjective global assessment (SGA), nutritional risk index (NRI), nutritional risk screening (NRS), and a combination of these. However, literature diverges on what is considered the gold standard among these various methods. Some studies indicate that the SGA has greater sensitivity for identifying patients at nutritional risk(9), others suggest the use of the NRS(17, 19) or the NRI(27, 37) to reflect the risk of malnutrition regardless of the severity of the disease, and still others claim that the

The authors declare that they have no competing interests. Vania Aparecida Leandro Merhi and José Luiz Braga de Aquino were involved in the protocol and study design, analysis, carried out the statistical analysis, writing of the article and critically reviewed the article. Pontifícia Universidade Católica de Campinas (PUC-Campinas), Campinas, SP, Brasil. 1 Escola de Nutrição, PUC-Campinas; 2 Escola de Medicina, PUC-Campinas, Campinas, SP, Brasil; Correspondence: Dr. Vânia A. Leandro-Merhi. E-mail: valm@dglnet.com.br

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Leandro-Merhi VA, Aquino JLB. Investigation of nutritional risk factors using anthropometric indicators in hospitalized surgery patients

biochemical and anthropometric markers are good predictors of nutritional status in hospitalized patients(3, 29, 39). The objective of the present study was to investigate the incidence of malnutrition at admission and its possible association with many parameters that assess nutritional status and to identify the associated risk factors in hospitalized patient. METHODS

This study was done from January to December 2010 and included 235 hospitalized patients of both genders (46.4% females and 53.6% males), staying at the surgery ward of Hospital e Maternidade Celso Pierro of the Pontifical Catholic University of Campinas, state of São Paulo, Brazil. This is a tertiary-care university hospital that, in its routine, includes cases of high risk such as politrauma and complex cancer surgeries. The study began after approval from the hospital administration and local Research Ethics Committee, protocol number 743/09. This study is part of a bigger project called “Nutritional status of the hospitalized patient and its relationship with disease, clinical and surgical variables and length of hospital stay.” The inclusion criteria were: age equal to or greater than 18 years, having undergone nutritional assessment within the first 48 hours after hospital admission, length of hospital stay and disease recorded in the medical records of the institution. Anthropometric indicators The following anthropometric indicators were measured: current weight; height; arm circumference; triceps skinfold thickness (TST) and calf circumference (CC). From these measurements the body mass index (BMI), mid-arm muscle circumference (MAMC), arm muscle area (AMA), and arm fat area (AFA) were calculated. AC and AFA were classified according to the parameters established by Frisancho(13) and MAMC and AMA were classified according to Frisancho(12). The TST were assessed as determined by Heymsfield et al.(15). The anthropometric parameters established by Burr and Phillips(5) were used for the elderly. BMI was obtained by dividing weight by the square of the height and classified according to the WHO’s criteria(40) for adults up to 60 years of age: underweight if BMI ≤18.4 kg/m2; normal weight if 18.5 ≤BMI ≤24.9 kg/ m2; pre-obese if 25.0 ≤BMI ≤29.9 kg/m2 and obese if BMI ≥30.0 kg/m2. For the elderly (≥60 years of age), BMI was classified as determined by Lipschitz(24), who gives the following cut-off points: underweight if BMI ≤22 kg/m2; normal weight if 22 <BMI <27 kg/m 2; and overweight if BMI ≥27 kg/m2. Recent weight change was classified as weight maintenance for patients who reported not losing or gaining weight before admission, weight loss for those who reported losing weight shortly before admission and weight gain for those who reported gaining weight shortly before admission. The average weight loss was declared by the patients.

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Investigation of the habitual energy intake (HEI) The habitual energy intake (HEI) was assessed based on the habitual food intake when the patient was admitted and was calculated by the software NutWin®(38), as previously described in previous papers(8, 20, 22, 30). The energy adequacy of the habitual energy intake in relation to the energy requirement (ER) was then calculated (%HEI/ER). The ER represents the total energy expenditure of the individual and was calculated by the Harris-Benedict equation(14). Assessment of malnutrition Malnutrition was assessed after the nutritional status with the objective parameters described above. In this study, the criterion to malnutrition was proposed based on anthropometric nutritional status parameters. Malnutrition was defined as present when the patient presented at least two anthropometric criteria below the normal range and habitual energy intake below 75% of the energy requirement (HEI/ER<75%)(26). The anthropometric criteria were: BMI <18.5 kg/m2 for adults and <22 kg/m2 for the elderly and AC, TST, MAMC, AMA or AFA below the 10th percentile (<P10). Assessment of the risk factors associated with malnutrition Gender, age, type of disease, recent weight change, presence or absence of dental problems, number of drugs prescribed during hospital stay and length of hospital stay were investigated as possible risk factors to malnutrition. The diseases were classified into two categories: benign diseases and malignant diseases (neoplasms). We considered patients with malignant diseases those who had carcinoma in general, and patients with benign diseases those who had other clinical conditions such as the digestive tract diseases and annexed glands, vascular diseases, urological diseases and others. Statistical analysis At first, a descriptive analysis of the data was done by determining the mean, standard deviation and proportion of the studied variables. The chi-square test or Fisher’s exact test, when necessary, were used to compare the proportions. The Mann-Whitney test was used to compare the continuous or ordinal measurements between two groups. Univariate and multiple logistic regressions were used to identify the factors associated with malnutrition. The odds ratio (OR) and confidence interval (CI) of 95% were calculated(6, 16) and the significance level was set at 5% (P<0.05). The data were analyzed by the software Statistical Analysis System (SAS)(34). RESULTS

Of the 235 studied patients, 126 (53.6%) were males and 109 (46.4%) were females; 60% were in the 18 to 59 years age range, 20.8% were in the 60 to 69 years age range and 19.2% were 70 years old or older. Most (75.7%) had benign diseases and 24.3% had malignant diseases. About half (50.6%) the patients reported having dental problems (use of prosthesis, missing teeth, etc.). Almost one-third (27.5%) of the patients reported losing weight before

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Leandro-Merhi VA, Aquino JLB. Investigation of nutritional risk factors using anthropometric indicators in hospitalized surgery patients

admission, 24.9% reported gaining weight and 47.6% reported no weight change in the few months before admission (6 months). The average weight lost before admission was 9.45 ± 5.95 kg and the mean weight gained was 6.45 ± 5.0 kg.

55 50 45 40 35 % 30 25 20 15 10 5 0

Benign disease Malignant disease (b)

(c)

(d) (e)

(a)

BMI AC TST MAMC AMA AFA HEI/ER <18.5 adults <P10 <P10 <P10 <P10 <P10 <75% <22.0 elderly

Malnutrition

Criteria BMI: body mass index; AC: arm circumference; TST: triceps skinfold thickness; MAMC: mid-arm muscle circumference; AMA: arm muscle area; AFA: arm fat area; HEI/ER>75%: habitual energy intake

FIGURE 1. Percentage distribution of the criteria used to define malnutrition and of the risk assessed in each disease group. There was a significant difference between the groups for the criteria: (a) P-value<0.0001 (chi-square); (b) P-value = 0.0020 (chi-square); (c) P-value = 0.0090 (chisquare); (d) P-value = 0.0003 (chi-square) and for assessed malnutrition (e) P-value<0.0001 (chi-square)

The mean age of the entire sample was 53.0 ± 17.9 years; the mean BMI was 25.0 ± 5.4 kg/m2; mean CC was 32.2 ± 4.4 cm; mean AC was 28.5 ± 4.5 cm; mean TST was 17.9 ± 9.2 mm; mean MAMC was 228.6 ± 36.0 mm; mean AMA was 42.6 ± 13.1 cm2; mean AFA was 23.6 ± 13.9 cm2; and mean length of hospital stay was 7.7 ± 9.4 days. Note that 20% of the patients were already at malnutrition at admission and 45% of the patients had a HEI below 75% of their requirement. Figure 1 shows the percentage distribution of the criteria used for the definition of malnutrition in each disease group and also of the assessed malnutrition by type of disease. A significant difference was found between the groups for the following criteria: BMI (P<0.0001); AC (P = 0.0020); MAMC (P = 0.0090); AMA (P = 0.0003) and mainly for malnutrition (P<0.0001), evidencing that malnutrition was greater in patients with malignant diseases. When the characteristics of the population were compared by type of disease (Table 1), a statistically significant difference was found between the two types of disease regarding gender (P = 0.0014), age group (P = 0.0004), recent weight change (P = 0.0005) and death (P = 0.0319). When the numerical variables were compared between the two types of diseases (Table 2), a significant difference was found for nearly all studied indicators, except for calf circumference CC, HEI and %HEI/ER. Univariate and multiple logistic regressions were then used to analyze the risk factors associated with malnutrition. Table 3 shows the factors associated with malnutrition analyzed using a univariate logistic regression model. The model evidenced that the only significant variables for malnutrition were recent weight loss (P = 0.0058; OR = 2.909; CI95% = 1.362; 6.212) and malignant disease (P = 0.0001; OR = 3.847; CI95% = 1.948; 7.597). Additional data are shown in Table 3.

TABLE 1. Comparison of the characteristics of the population by type of disease Types of disease Benign disease Malignant disease n (%) n (%)

Variables Gender female male Age group (years) 18-59 60-69 ≥70 Dental problems yes no Recent weight change weight gain weight loss no change in weight Death yes no *Chi-square test;

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93 (52.3) 85 (47.7)

16 (28.1) 41 (71.9)

119 (66.8) 33 (18.5) 26 (14.6)

22 (38.6) 16 (28.1) 19 (33.3)

84 (47.2) 94 (52.8)

35 (61.4) 22 (38.6)

44 (24.9) 38 (21.5) 95 (53.7)

14 (25.0) 26 (46.4) 16 (28.6)

2 (1.1%) 176 (98.9%)

4 (7.0%) 53 (93%)

P-value 0.0014*

0.0004*

0.0618*

0.0005*

0.0319 **

**Fisher’s exact test

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Leandro-Merhi VA, Aquino JLB. Investigation of nutritional risk factors using anthropometric indicators in hospitalized surgery patients

TABLE 2. Comparison of the studied nutritional indicators by type of disease Indicators n Age (years) benign disease 178 malignant disease 57 Length of hospital stay (days) benign disease 178 malignant disease 57 Current weight (kg) benign disease 171 malignant disease 56 Body mass index (kg/m2) benign disease 171 malignant disease 56 Calf circumference (cm) benign disease 153 malignant disease 53 Arm circumference (cm) benign disease 178 malignant disease 57 Triceps skinfold thickness (mm) benign disease 178 malignant disease 57 Mid-arm muscle circumference (mm) benign disease 178 malignant disease 57 Arm muscle area (cm2) benign disease 178 malignant disease 57 Arm fat area (cm2) benign disease 178 malignant disease 57 HEI/ER<75% benign disease 178 malignant disease 57 Number of drugs taken during stay benign disease 142 malignant disease 48

X ± SD

Median

P-value

50.2 ± 18.0 61.9 ± 14.7

52.5 64.0

<0.0001

7.4 ± 9.9 8.6 ± 7.1

4.0 7.0

0.0053

69.1 ± 14.4 62.0 ± 15.0

68.0 59.9

0.0043

25.6 ± 5.2 23.4 ± 5.7

24.9 22.8

0.0015

32.4 ± 4.4 31.5 ± 4.6

32.0 31.0

NS

29.1 ± 4.3 26.7 ± 4.5

29.0 26.0

<0.0001

18.8 ± 9.1 15.3 ± 9.0

17.0 14.0

0.0039

231.9 ± 35.0 218.0 ± 37.3

232.1 215.4

0.0092

43.8 ± 12.9 38.9 ± 12.9

42.9 36.7

0.0092

25.0 ± 13.9 19.9 ± 12.9

21.7 15.9

0.0005

87.4 ± 36.9 77.3 ± 32.8

81.1 74.5

NS

6.0 ± 3.4 7.5 ± 3.9

5.0 7.0

0.0132

* Mann-Whitney test

TABLE 3. Factors associated with malnutrition analyzed by univariate logistic regression Variables Gender M x F Age group 60-69 x 18-59 years Age group ≥70 x 18-59 years Recent weight change: gain x not Recent weight change: loss x not Dental problems: yes x no Disease: malignant and benign Age Amount of weight lost or gained

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P-value 0.1189 0.7621 0.3811 0.2298 0.0058 0.1727 0.0001 0.3988 0.1161

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Odds ratio 1.691 1.134 1.431 1.670 2.909 1.571 3.847 1.008 1.040

Confidence interval (95%) 0.874; 3.272 0.501; 2.562 0.642; 3.192 0.723; 3.854 1.362; 6.212 0.821; 3.006 1.948; 7.597 0.990; 1.026 0.990; 1.091

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Leandro-Merhi VA, Aquino JLB. Investigation of nutritional risk factors using anthropometric indicators in hospitalized surgery patients

TABLE 4. Multiple logistic regression model for the study of malnutrition estimated by the stepwise selection process with and without the type of disease Variables 1st model * Intercept Malignant x benign disease 2nd model ** Intercept RWC: gain x not RWC: loss x not

Estimate

Standard error

P-value

OR

CI (95%)

-1.7707 1.3495

0.2208 0.3573

<0.0001 0.0002

3.855

1.914;7.766

-1.8563 0.5126 1.3127

0.2776 0.4268 0.4061

<0.0001 0.2298 0.0012

1.670 3.716

0.723; 3.854 1.677; 8.236

RWC: recent weight change; OR: odds ratio; CI: confidence interval

In an attempt to identify the best model for malnutrition analysis (Table 4), a multiple logistic regression analysis was done for studying malnutrition estimated by the stepwise selection process, with or without type of disease. In the model with type of disease, the presence of malignant disease increased the chance of malnutrition fourfold (P = 0.0002; OR = 3.855; CI95% = 1.914; 7.766). When type of disease was left out, recent weight loss also increased malnutrition almost fourfold (P = 0.0012; OR = 3.716; CI95% = 1.677; 8.236). DISCUSSION

First of all, it is important to point out that the present study is part of an area of research that has been conducted about the nutritional status of hospitalized patients as shown in previous works(21, 22, 25). Despite the existence of many published studies(1, 11, 18, 29, 39) discussing the nutritional status of hospitalized patients, the strength of this study was to use univariate and multiple logistic regression analyses to investigate which factors were associated with risk. In this study, 20% of the patients presented malnutrition and this diagnosis was done shortly after admission to the hospital, as well as the investigation of the other nutritional indicators, which may suggest that these patients were already at nutritional risk at admission and the findings of the study are consistent with many published studies(11, 29, 39). In a recent study, Filipovic et al.(11), compared nutritional assessment methods in 299 patients with gastrointestinal diseases and found some degree of malnutrition in 45.7% of their sample when they used the SGA and 63.9% when they used the NRI. In another multicentric study, Amaral et al.(1) found 36% of their sample to be at risk of malnutrition when the NRS was used and 9.7% to be malnourished when anthropometry was used. Other studies in European hospitals have shown a prevalence of malnutrition ranging from 10% to 50%, depending on the group of studied patients(33). In a British study, Stratton et al.(36) reported that the prevalence of malnutrition in hospitalized patients ranged from 19% to 60%, and a study in German hospitals revealed a malnutrition rate of 27.4% according to the SGA(29). Recently, a study with hospitalized patients in Turkey(18) found 15% of the patients to be at nutritional risk. Other studies(21, 22) carried out in Brazil, with hospitalized surgery patients using anthropometrical

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indicators to assess the nutritional status, showed malnutrition rates between 11.4% and 14.1%. These findings corroborate the data found in this present study. Another study(26), also conducted in Brazil but with medical clinic inpatients, found that 45.3% of the patients were underweight according to their BMI. Other contemporary studies have also reported prevalence of 30% to 50%(23, 35). This evidences the different malnutrition rates found by different studies, using different instruments. This situation implies on the need to use many nutritional assessment parameters in an attempt to diagnose hospital malnutrition. This fact was considered in the present study which used more than one indicator to investigate malnutrition with objective parameters and associated factors. Other studies(10, 21, 22, 25, 39) also show malnutrition with objective parameters of nutritional assessment in hospitalized patients. This fact reinforces the importance of submitting the patient to nutritional assessment right after admission in order to implement intervention strategies early to improve the nutritional status of the patient, improve his or her clinical course and reduce the length of hospital stay(7). However, a study which compared the accuracy of commonly used nutritional assessment parameters found that the adopted methods were considered weak predictive factors of clinical outcomes, death, infection and length of hospital stay(3). The data found in the present study show that when nutritional indicators are compared by type of disease (Table 2) depletion is greater in those with malignant diseases, except for CC and HEI/ER <75%. Habitual food intake is a weak tool to evaluate energy intake to compare with energy needs and this was a limitation of this study. For the same sample, other factors were higher age (P = 0.0004), recent weight loss (P = 0.0005) and death (P = 0.0319) (Table 1), in addition to greater malnutrition (P<0.0001) (Figure 1) found in those with malignant diseases. With specific regard to inpatients of a surgery ward, many studies have already documented malnutrition that these patients present in the postoperative period when they had a poor nutritional prognosis in the preoperative period, especially patients who had lost weight shortly before surgery. Literature has shown that malnutrition in this population is a significant risk factor for postoperative complications, especially after abdominal surgeries(4, 10, 18, 31).

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Leandro-Merhi VA, Aquino JLB. Investigation of nutritional risk factors using anthropometric indicators in hospitalized surgery patients

The present study also found that 46.4% of the patients with malignant diseases and 21.5% of those with benign diseases lost weight during their hospital stay. This is a worrisome fact since literature states that isolated weight loss or weight loss in combination with other assessment parameters has been considered the main indicator of poor nutritional status(4). Univariate analysis revealed that weight change and presence of malignant disease are significant factors for malnutrition. Multiple regression analysis then revealed that the risk was greater in patients who had lost weight recently (P = 0.0012; OR = 3.716; CI95% = 1.677; 8.236) or had malignant diseases (P = 0.0002; OR = 3.855; CI95% = 1.914; 7.766): they were almost 4 times more likely to present malnutrition. This was a study on anthropometric nutritional status indicators of hospitalized patients. The results showed that malignant disease is a determinant in the depletion of body tissues, regardless of gender and age. This confirms the pertinence of using anthropometry for assessing the

nutritional status of hospitalized patients and this study reinforces the need of paying attention to patients who report weight loss at admission. This is often ignored by the medical and health professional teams during initial assessment. Using univariate and multiple logistic regressions, this study has shown that recent weight loss is the main factor, together with malignant disease, that facilitates the development of malnutrition. CONCLUSION

In the conditions of this study, the analyzed data demonstrated that the main factors associated with malnutrition were recent weight loss and malignant disease. ACKNOWLEDGMENTS

Research Support Fund of the Pontifícia Universidade Católica de Campinas, SP, Brazil.

Leandro-Merhi VA, Aquino JLB. Investigação de fatores de risco nutricional por meio de indicadores antropométricos em pacientes cirúrgicos hospitalizados. Arq Gastroenterol. 2012;49(1):28-34. RESUMO – Contexto - A investigação de fatores de risco associados ao estado nutricional pode contribuir para o melhor conhecimento da desnutrição. Objetivo - Investigar a incidência de risco nutricional com a associação de vários parâmetros de avaliação nutricional e identificar os fatores de risco relacionados. Método - Foi avaliado o risco nutricional em 235 pacientes hospitalizados com doenças benignas e malignas, sendo o sexo, a faixa etária, o tipo de doença, a alteração de peso recente e os problemas dentários, investigados como possíveis fatores de risco associados. Para a comparação dos dados, foi utilizado o teste Qui ao quadrado e Mann-Whitney e para identificar os fatores associados ao risco nutricional foi utilizada a análise de regressão logística univariada e múltipla, sendo calculado o odds ratio (OR) e o intervalo de confiança (IC) de 95%, com P<0,05. Resultados Verificou-se 20% dos pacientes com risco nutricional na admissão hospitalar e 27,5% referiram perda de peso recente, com diferença significativa entre os grupos nos parâmetros avaliados e para o risco nutricional de desnutrição (P<0,0001), maior naqueles com doenças malignas. Na regressão logística univariada, as únicas variáveis significativas para o risco nutricional foram a perda de peso recente (P = 0,0058; OR = 2,909; IC95% = 1,362; 6,212) e a doença maligna (P = 0,0001; OR = 3,847; IC95% =1 ,948; 7,597). Posteriormente, na regressão múltipla, no modelo com o tipo de doença, foi comprovado que a doença maligna elevou a chance de risco nutricional em 4 vezes (P = 0,0002; OR = 3,855; IC95% = 1,914; 7,766). Excluindo-se a doença, comprovou-se que a perda de peso recente elevou o risco nutricional também em quase 4 vezes (P = 0,0012; OR = 3,716; IC95% = 1,677; 8,236). Conclusão - Pacientes que perderam peso recentemente e aqueles com doença maligna apresentaram mais chances de desenvolver risco nutricional. DESCRITORES – Estado nutricional. Desnutrição. Período pós-operatório. Antropometria. Fatores de risco. Pacientes internados.

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18. Korfali G, Gündoğdu H, Aydmtug S, Bahar M, Besler T, Moral AR, Oğuz M, Sakarya M, Uyar M, Kihçturğay S. Nutritional risk of hospitalized patients in Turkey. Clin Nutr. 2009;28:533-7. 19. Kyle UG, Kossovsky MP, Karsegard VL, Pichard C. Comparison of tools for nutritional assessment and screening at hospital admission: a population study. Clin Nutr. 2006;25:409-17. 20. Leandro-Merhi VA, Portero-Mclellan KC, Bernardi JLD, Frenhani PB, de Camargo JG, de Aquino JL. Dental and gastrointestinal changes as indicators of nutritional depletion in elderly inpatients. J Eval Clin Pract. 2010;16:873-7. 21. Leandro-Merhi VA, de Aquino JL, de Camargo JG, Frenhani PB, Bernardi JLD, Portero-Mclellan KC. Clinical and nutritional status of surgical patients with and without malignant diseases: cross-sectional study. Arq Gastroenterol. 2011;48:58-61. 22. Leandro-Merhi VA, de Aquino JL, Sales Chagas JF. Nutrition status and risk factors associated with length of hospital stay for surgical patients. JPEN J Parenter Enteral Nutr. 2011;35:241-8. 23. Liang X, Jiang ZM, Nolan MT, Efron DT, Kondrup J. Comparative survey on nutritional risk and nutritional support between Beijing and Baltimore teaching hospitals. Nutrition. 2008;24:9696-76. 24. Lipschitz DA. Screening for nutritional status in the elderly. Prim Care. 1994;22:55-67. 25. McLellan KCP, Bernardi JLD, Jacob P, Soares CSR, Frenhani PB, Leandro-Merhi VA. Estado nutricional e composição corporal de pacientes hospitalizados: reflexos da transição nutricional. Rev Bras Promoç Saúde. 2010;23:25-33. 26. Mimiran P, Hosseinpour-Niazi S, Mehrabani HH, Kavian F, Azizi F. Validity and reliability of a nutrition screening tool in hospitalized patients. Nutrition. 2010;27:647-52. 27. Naber TH, Schermer T, de Bree A, Nusteling K, Eggink L, Kruimel JW, Bakkeren J, van Heereveld H, Katan MB. Prevalence of malnutrition in nonsurgical hospitalized patients and its association with disease complications. Am J Clin Nutr. 1997;66:1232-9. 28. Pereira Borges N, D’Alegria Silva B, Cohen C, Portari Filho PE, Medeiros FJ. Comparison of the nutritional diagnosis, obtained through different methods and indicators, in patients with cancer. Nutr Hosp. 2009;24:51-5. 29. Pirlich M, Schütz T, Norman K, Gastell S, Lübke HJ, Bischoff SC, Bolder U, Frieling T, Güldenzoph H, Hahn K, Jauch KW, Schindler K, Stein J, Volkert D, Weimann A, Werner H, Wolf C, Zürcher G, Bauer P, Lochs H. The German hospital malnutrition study. Clin Nutr. 2006;25:563-72.

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30. Portero-McLellan KC, Staudt C, Silva FR, Delbue Bernardi JL, Baston Frenhani P, Leandro-Merhi VA. The use of calf circumference measurement as an anthropometric tool to monitor nutritional status in elderly inpatients. J Nutr Health Aging. 2010;14:266-70. 31. Putwatana P, Reodecha P, Sirapo-ngam Y, Lertsithichai P, Sumboonnanonda. Nutrition screening tools and the prediction of postoperative infectious and wound complications: comparison of methods in presence of risk adjustment. Nutrition. 2005;21:691-7. 32. Raslan M, Gonzalez MC, Dias MCG, Paes-Barbosa FL, Cecconello I, Waitzberg DL. Aplicabilidade dos métodos de triagem nutricional no paciente hospitalizado. Rev Nutr. 2008;21:553-61. 33. Rasmussen HH, Kondrup J, Staun M, Ladefoged K, Kristensen H, Wengler A. Prevalence of patients at nutritional risk in Danish hospitals. Clin Nutr. 2004;23:1009–15. 34. SAS System for Windows (Statistical Analysis System), versão 9.2. Cary, NC, USA: SAS Institute Inc; 2002-2008. 35. Sorensen J, Kondrup J, Prokopowicz J, Schiesser M, Krähenbbühl L, Meier R, Liberda M, EuroOOPS study group. EuroOOPS: an international, multicentre study to implement nutritional risk screening and evaluate clinical outcome. Clin Nutr. 2008;27:340-9. 36. Stratton RJ, Hackston A, Longmore D, Dixon R, Price S, Stroud M, King C, Elia M. Malnutrition in hospital outpatients and inpatients: prevalence, concurrent validity and ease of use of the ‘malnutrition screening tool universal tool’ (‘MUST’) for adults. Br J Nutr. 2004;92:799-808. 37. Sungurtekin H, Sungurtekin U, Hanci V, Erdem E. Comparison of two nutrition assessment techniques in hospitalized patients. Nutrition. 2004;20:428-32. 38. Universidade Federal de São Paulo. Escola Paulista de Medicina. Programa de Apoio a Nutrição (NUTWIN) – programa de computador, versão 1.5. São Paulo: UNIFESP/ EPM; 2002. 39. Westergren A, Wann-Hansson C, Borgdal EB, Sjolander J, Stromblad R, Klevsgard R, Axelsson C, Lindholm C, Ulander K. Malnutrition prevalence and precision in nutritional care differed in relation to hospital volume-a cross-sectional survey. Nutr J. 2009;8:20. 40. World Health Organization. Obesity: preventing and managing the global epidemic - Report of a WHO consultation on obesity. Geneva: WHO, 1998. Received 14/4/2011. Accepted 11/8/2011.

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ARTIGO ORIGINAL / ORIGINAL ARTICLE

ARQGA/1586

IMMUNOHISTOCHEMICAL EVALUATION OF p53 AND Ki-67 PROTEINS IN COLORECTAL ADENOMAS Walysson Alves Tocantins de SOUSA, Lusmar Veras RODRIGUES, Raimundo Gerônimo da SILVA Jr. and Fernando Lopes VIEIRA ABSTRACT – Context - The appearance of adenomas and their progression to adenocarcinomas is the result of an accumulation of genetic changes in cells of the intestinal mucosa inherited or acquired during life. Several proteins have been studied in relation to the development and progression of colorectal cancer, including tumor protein p53 (p53) and antigen identified by monoclonal antibody Ki-67 (Ki-67). Objective - To evaluate the expression of p53 and Ki-67 in colorectal adenomas and correlate the observed levels with clinical and pathologic findings. Method - The sample consisted of 50 adenomatous polyps from patients undergoing colonoscopy. After performing polypectomy, polyps were preserved in a formalin solution with 10% (vol./vol.) phosphate buffer, submitted for routine preparation of sections and slides and stained with hematoxylin and eosin. For each adenoma we then performed immunohistochemistry to detect specific p53 and Ki-67 proteins using a streptavidin-biotin-peroxidase enzyme immunoassay. Results - p53 was detected in 18% of the adenomas. The average Ki-67 protein index (i.Ki-67) was 0.49. A statistically significant difference was observed in p53 (P = 0.0003) and Ki-67 (P = 0.02) expression between adenomas with low- and high-grade dysplasia, particularly for p53. The expression of Ki-67 was greater in rectal adenomas than in colic adenomas (P = 0.02). No relationship was found between the expression of the two proteins in the sample. Conclusion - The p53 protein is expressed in a proportion of adenomas, while the Ki-67 protein was expressed in all adenomas. The expression of p53 was higher in adenomas with high-grade dysplasia. The expression of Ki-67 was higher in rectal adenomas and in adenomas with high-grade dysplasia. HEADINGS – Colorectal neoplasm. Tumor suppressor protein p53. Ki67-antigen. Immunohistochemistry.

INTRODUCTION

Colorectal cancer is among the most frequent types of malignancy. In terms of incidence, it is the third most common cancer worldwide in both sexes(7). Among the key risk factors associated with the presence of adenomas is a factor closely linked to the development of malignant neoplasms. The chances of cure and survival are greater when detected at an early stage. Thus, the management of adenomas has an important role in the prevention of colorectal cancer, making it necessary to conduct studies to determine new prognostic factors related to development and progression. The cell cycle is controlled by several genes, the main function of which is the synthesis of proteins that promote perfectly organized cell multiplication. These genes are basically divided into two groups: genes that stimulate cell proliferation, called protooncogenes, and genes that inhibit cell proliferation, called tumor suppressor genes(21, 28). The appearance of

adenomas and their progression to adenocarcinomas is the result of an accumulation of genetic changes, in cells of the intestinal mucosa that have been inherited or acquired during life. Several proteins have been studied in relation to the development and progression of colorectal cancer, including tumor protein p53 (p53) and antigen identified by monoclonal antibody Ki-67 (Ki-67). In humans, the p53 tumor suppressor gene is located on the locus of subdivision 13 of the short arm of chromosome 17 (17p13). Composed of 11 exons interspersed by 10 introns(8, 14), it encodes a nuclear phosphoprotein – molecular mass equivalent to 53 kDa – that contains a sequence of 393 amino acids(13). The main function of p53 is to mediate the cell response when DNA is damaged, assisting in the maintenance of genomic stability(26, 33). Mutation of the p53 gene (also known as TP53) promotes an accumulation of this protein in the cell nucleus and extends its half-life, making it detectable by immunohistochemistry(6, 19)

No conflicts of interest. Financial disclosures: none reported. Department of Surgery, Federal University of Ceará, Fortaleza, Brazil. Correspondence: Dr. Walysson Alves Tocantins de Sousa – Avenida Lindolfo Monteiro, 2801 – apt. 402 – Fátima – Teresina, PI, Brasil. E-mail: wtocantins@yahoo.com.br

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Sousa WAT, Rodrigues LV, Silva Jr RG, Vieira FL. Immunohistochemical evaluation of p53 and Ki-67 proteins in colorectal adenomas

The expression of the Ki-67 protein in humans is closely associated with cell proliferation, and this is detected during the cell-cycle phases G1, S, G2 and mitosis, and absent in the G0 phase. Thus, Ki-67 is an excellent marker for proliferating cells(22). The aim of this study was to evaluate the expression of p53 and Ki-67 in colorectal adenomas and correlate the observed levels with clinical and pathologic findings. METHODS

The study was conducted at the Department of Coloproctology at Walter Cantídio University Hospital, Federal University of Ceará, Fortaleza, CE, Brazil and at the Digestive Endoscopy Service at Santa Maria Hospital in Teresina, PI, Brazil after having been approved by the local ethics committee (protocol 593/2009). Sample The sample consisted of 50 adenomas from 44 patients who underwent routine screening colonoscopy and were diagnosed with colorectal adenomatous polyps. Twentyfive adenomas were from 23 female patients and 25 from 21 male patients. The average age of the patients was 62.7 years (41–89 years). Inclusion criteria To be included in the study, individuals had to have histopathologic confirmation of colorectal adenoma. Exclusion criteria Patients with current or previous malignant neoplasms, intestinal polyposis, prior radiotherapy or inflammatory bowel disease were excluded. Endoscopy Patients underwent complete colonoscopy after mechanical preparation of the colon with a 10% (vol./vol.) mannitol solution. All examinations were performed by the same professional. All polyps were resected using a diathermy loop and measured with the aid of a caliper. The polyps were preserved in a formalin solution with 10% (vol./vol.) phosphate buffer. Histopathology The material was sent to the pathology laboratory for macroscopic analysis and preparation for routine histological sectioning and staining with hematoxylin and eosin. The histological type and grade of dysplasia were subsequently determined(27). All cases were evaluated independently by two pathologists for confirmation of histopathologic findings. Immunohistochemistry An immunohistochemical analysis was carried out to detect the expression of the p53 and Ki-67 proteins, using a streptavidin–biotin immunoenzymic method. This included

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the following steps: (1) deparaffinization at 60ºC (20 min); (2) deparaffinization at room temperature (30  min); (3) hydration with alcohol and water; (4) antigen retrieval: heating the slides in a solution of 0.01 mol/L citric acid (pH 6.0) in a microwave oven (maximum power) for p53 (10 min), and in a pressure cooker for Ki-67 (5 min); (5) blockage of endogenous peroxidase in two baths of 3% (vol./vol.) H2O2 (10 min each) and rinsing with water and phosphate buffered saline (PBS); (6) incubation in a moist chamber at 4ºC (18 h) with monoclonal mouse antibodyspecific primary anti-human MIB-1 (Dako®, Carpinteria, CA, USA) at a titration of 1:100 for Ki-67 and monoclonal mouse anti-human DO-7 (Dako®, Carpinteria, CA, USA) at a titration of 1:800 for p53; (7) incubation with secondary biotinylated antibody(kit LSAB Dako®, Carpinteria, CA, USA) in a moist chamber at 25ºC (30 min) followed by rinsing in PBS; (8) incubation with the streptavidin–biotin–peroxidase complex in a moist chamber (37ºC) for 30 min followed by rinsing in PBS; (9) disclosure by immersion in a solution of 60 mg% diaminobenzidine in PBS containing 0.06% (vol./ vol.) hydrogen peroxide followed by rinsing in distilled water; and (10) counterstaining in Harris hematoxylin, dehydration in alcohol and mounting in Entellan with coverslips. The positive nuclei present a typical brownish hue for each of the markers. Quantitative analysis of p53 and Ki-67 The expression of p53 and Ki-67 was quantified with an automated system consisting of a Nikon light microscope coupled to a color video camera (Digital Camera Samsung SCC-131, Seoul, South Korea) with image acquisition by microcomputer (Pentium IV, 80 GB hard disk, 3.0 GHz processor, 1024 MB RAM and graphics card for Windows XP®). Cell counts positive and negative for the expression of p53 and Ki-67 were performed under ×400 magnification for the most intensely stained samples. After scanning, 500 cells were counted in random fields of the mucosal epithelium using the image analysis program Image J®. Fairly obvious dark-brown staining of cell nuclei was considered positive. The calculation of the index of expression was performed using the following formula: i = (number of immunostained nuclei / number of nuclei counted). Samples were considered positive for p53 when at least 10% of the cells presented this staining(10, 17). The cell proliferation index for Ki-67 (i.Ki-67) was calculated with the same formula and was expressed in absolute values. Statistical analysis The results were expressed as mean, standard deviation (SD), standard error (SE), minimum and maximum values for numerical variables and frequency for nominal variables. Nominal variables were tested for statistical significance with Fisher’s exact test. Numerical variables were submitted to Student’s t test or Pearson’s linear correlation. The level of statistical significance was set at P<0.05. The statistics software Bioestat 5.0® was used for statistical analysis.

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RESULTS

Location The polyps were distributed as follows: 15 (30%) were located in the rectum and 35 (70%) in the colon. Synchronous polyps Five patients had synchronous polyps with a total of 11 adenomas (Table 1). One patient had 3 adenomas. The remainder had 2 each. Macroscopic findings The size of the polyps ranged from 3 mm to 25 mm (average: 6.5 ± 4.7 mm). Polyps measured <10 mm in 36 cases (72%) and ≥10 mm in 14 cases (28%) (Tables 1 and 2).

The expression of p53 in relation to sex was positive in three men and six women (P = 0.46) (Table 1). Polyps <10 mm were p53-positive in five cases (13.8%). Polyps ≥10 mm were p53-positive in four cases (28.5%) (P =  0.41) (Table 1). As for location, two (13.3%) of the polyps located in the rectum and seven (20%) of the polyps located in the colon were positive for p53 (P = 0.7) (Table 1). Concerning the degree of dysplasia, one out of 33 lowgrade specimens and eight out of 17 high-grade specimens were positive for p53 (P = 0.0003) (Table 1).

Age (years)

P=0.42

Microscopic findings With the exception of one case of tubulo-villous adenoma, all other cases were histologically classified as tubular adenoma. Regarding the degree of dysplasia, 33 (66%) were classified as low grade and 17 (34%) as high grade (Tables 1 and 2).

90

Immunohistochemistry Nine specimens (18%) were considered positive for p53 (Table 1). The average (±SD) expression of Ki-67 was 0.49 ± 0.17 (Table 2). Specimens positive for p53 came from patients aged 65.2  ±  9.1 years (range: 52–80 years). The corresponding figures for p53-negative specimens were 62.2 ± 10.9 years (41–89 years) (P = 0.42) (Figure 1).

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FIGURE 1. Box-plot of patients’ age according to p53 protein expression

TABLE 1. Expression of p53 protein in adenomas Total Sex n (%) n (%) Male Female p53+ 9 (18) 3 (12) 6 (24) 41 (82) 22 (88) 19 (76) p53− Total 50 (100) 25 (100) 25 (100) P-value P = 0.46

Size n (%) <10 mm ≥10 mm 5 (13.8) 4 (28.5) 31 (86.2) 10 (71.5) 36 (100) 14 (100) P = 0.41

a

Site n (%) Rectum Colon 2 (13.3) 7 (20) 13 (86.7) 28 (80) 15 (100) 35 (100) P = 0.7

80 70

50 40 p53+

p53-

Dysplasia n (%) Low Higha 1 (3) 8 (47) 32 (97) 9 (53) 33 (100) 17 (100) P = 0.0003

Synchronous polyp n (%) No Yes 8 (20.5) 1 (9) 31 (79.5) 10 (91) 39 (100) 11 (100) P = 0.66

The difference between two groups was statistically significant

TABLE 2. Expression of Ki-67 protein in adenomas Variable n Mean Male 25 0.51 Sex Female 25 0.46 15 0.57 Rectuma Site Colon 35 0.45 <10 mm 36 0.49 Size 14 0.47 ≥10 mm Low 33 0.45 Dysplasia Highb 17 0.56 Total 50 0.49

SD 0.19 0.14 0.19 0.15 0.19 0.11 0.16 0.17 0.17

SE 0.04 0.03 0.05 0.02 0.03 0.03 0.02 0.04 0.02

Minimum 0.24 0.19 0.28 0.19 0.19 0.29 0.19 0.29 0.19

Median 0.44 0.49 0.61 0.42 0.46 0.46 0.41 0.56 0.46

Maximum 0.95 0.71 0.95 0.9 0.95 0.62 0.85 0.95 0.95

P-value P = 0.35 P = 0.02 P = 0.7 P = 0.02

The difference between the two groups was statistically significant

a, b

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Sousa WAT, Rodrigues LV, Silva Jr RG, Vieira FL. Immunohistochemical evaluation of p53 and Ki-67 proteins in colorectal adenomas

As for synchronous polyps (n = 11), one case was positive for p53. Eight of the 39 single polyps were p53 positive (P =��0.66) (Table 1). When the association between p53 and Ki-67 expression was evaluated, i.Ki-67 was 0.53 ± 0.1 for p53-positive polyps and 0.48 ± 0.18 for p53-negative polyps (P = 0.4) (Figure 2). Pearson’s linear correlation was used to test the association between age and Ki-67 expression. The resulting coefficient was r = −0.04 (P = 0.67) (Figure 3). On average, Ki-67 expression was 0.46 ± 0.14 in women and 0.51 ± 0.19 in men (P = 0.35) (Table 2). With regard to size, Ki-67 expression was 0.49 ± 0.19 for polyps <10 mm and 0.47 ± 0.11 for polyps ≥10 mm (P = 0.7) (Table 2).

i.Ki67

P=0.4 1.1 0.8 0.6 0.4 0.2 0.0 p53+

p53-

FIGURE 2. Box-plot of the expression of Ki-67 protein according to p53 protein expression

1.1 0.9 0.7 i.Ki-67 0.5 0.3 1.1 40

50

60

70

80

90

Age FIGURE 3. Scatter plot between age and i.Ki-67

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Ki-67 expression was 0.57 ± 0.19 in rectal polyps and 0.45 ± 0.15 in colonic polyps (P = 0.02) (Table 2). Concerning the degree of dysplasia, the average Ki-67 expression index was 0.45 ± 0.16 for polyps with low-grade dysplasia and 0.56 ± 0.17 for polyps with high-grade dysplasia (P = 0.02) (Table 2). DISCUSSION

Knowledge of factors associated with the genesis and progression of colorectal cancer is of great importance in the development of strategies for prevention and treatment. Polypectomy currently constitutes the best strategy for preventing colorectal cancer(2). The identification of parameters reflecting the biological behavior of tumors, correlated with the severity and degree of evolution, is an important determinant of prognosis and improvement in cancer therapy. Despite recent progress in the study of the molecular mechanisms of cancer development and progression, few biological markers of prognostic value have been identified. The p53 protein, sometimes referred to as the ‘guardian of the genome’, has been extensively researched in various types of cancers. Mutations are found in about 50% of all human cancers and more than 50 types of tumor(12). The association of the protein Ki-67 is important when measuring cellular proliferative activity that may be exacerbated in neoplastic cells or in cells that have lost the ability to selfregulate. A high cell-proliferation index suggests a more aggressive behavior on part of the neoplasm and may have important prognostic implications(22). The average age of the study population was 63 years and the sex distribution was fairly equal, matching findings in the literature showing the prevalence of polyps in patients aged >50 years, with no sex preference(9, 11, 18, 32). The data also support the claim that patients with adenomas and colorectal cancer have a similar age distribution (generally >50 years)(1, 11, 21). In our study, nine specimens tested positive for p53 (18% of all cases). Watatani et al.(31) analyzed the expression of p53 in adenomas and found variable rates of expression: initial adenomas, 12%; synchronous adenomas with carcinomas, 20%; metachronous adenoma after initial adenoma, 8%; and metachronous adenoma after carcinoma, 30%(27). Other authors have found positivity rates between 10% and 60%(3, 17, 24, 30) The rate of p53 expression depends on the criteria used in the research method. In this study, only those cases in which the expression index was above 10% were considered positive. The choice of this cut-off point to classify the expression of p53 is owing to the fact that this figure shows the highest correlation between the immunohistochemical detection and mutation points of the P53 gene(5, 23). The cellular levels of p53 are known to be very low under normal conditions because of the short half-life of the protein (5-45 min). Thus, detection by immunohistochemistry is not possible(6). However, mutations in the P53 gene lead to the production of a p53 protein with greater stability and that may be detected immunohistochemically(19).

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Sousa WAT, Rodrigues LV, Silva Jr RG, Vieira FL. Immunohistochemical evaluation of p53 and Ki-67 proteins in colorectal adenomas

When relating the expression of p53 to clinical and pathologic characteristics (sex, age, location, size and degree of dysplasia), a statistically significant difference was found for the degree of polyp dysplasia, matching findings from earlier studies showing a positive relation between the degree of tumor dysplasia and immunohistochemical positivity for p53(19, 24, 29, 31). The relationship between p53 and the clinical and pathologic characteristics of adenomas is rarely explored in studies on this topic. In addition to a study by Vernillo et al.(29) Voskuil et al.(30) evaluated the immunohistochemical expression of p53 in sporadic adenomas and in adenomas from families with hereditary non-polyposis colorectal cancer (HNPCC). The study included intergroup (sporadic x hereditary) and intragroup comparisons. Sporadic adenomas >10 mm were associated with a higher frequency of p53 positivity. No other aspects, including the degree of dysplasia, were statistically significant. Likewise, no relationship was found between the presence of synchronous adenomas and the detection of the p53 protein. The only p53-positive case of synchronous adenoma came from a patient with two adenomas, of which only one was positive. The factor responsible for such differences in positivity in the same patient remains unknown. The two adenomas also differed in size and degree of dysplasia: the p53-positive adenoma was >10 mm and presented high-grade dysplasia. Fearon and Vogelstein(4) suggested a model for the evolution of colorectal tumors. The adenoma-to-carcinoma sequence is the result of an accumulation of genetic mutations that involve a different sequence: the mutation of the APC gene, DNA hypomethylation, activation of the K-ras protein, mutation of DCC (deleted in colorectal carcinoma) protein and, finally, mutation of the p53 protein. According to these authors, this last step is characteristic of carcinomas and rare in adenomas at any stage. In this study, 18% positivity in mutated p53 protein can not be regarded as unusual, although it is evident that the expression of p53 increases as the adenoma accumulates more dysplasia. There was greater expression of Ki-67 protein in adenomas with high-grade dysplasia compared with low-grade dysplasia, matching results published by Radovanovic-Dinic et al.(20). The expression of Ki-67 was also higher in adenomas located in the rectum compared with the colon. To our knowledge, earlier studies contain no references to Ki-67 expression in relation to adenoma location. Thus, our results suggest that neoplastic cells in the rectal mucosa have a higher power of cell proliferation. This would explain the peculiar behavior of rectal cancer in relation to the location

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of colonic adenomas and also the higher frequency of malignancy in this segment of the large intestine. As for the other parameters evaluated for the protein Ki67 (sex, age and size), no significant differences were found. Similar results were reported by Vernillo et al.(29). In another study, Radovanovic-Dinic et al.(20) found a higher expression of Ki-67 in adenomas of diameter larger than 10 mm. In fact, the proliferative activity of cells in the colorectal mucosa and throughout the gastrointestinal tract is constant, but increases under special circumstances, such as in inflammatory and neoplastic conditions(15, 16, 24, 25). In a study of patients with ulcerative colitis, Shinozaki et al.(25) found a higher expression of Ki-67 in areas of dysplastic mucosa compared with areas without dysplasia. Muneyuki et al.(15) conducted an evaluation of the expression of several proteins in tumors of the small intestine, in which i.Ki-67 was significantly higher in tumor areas than in areas of cancer-free mucosa. Sheikh et al.(24) evaluated the expression of Ki-67 in colorectal adenomas and observed an invariably stronger expression in areas with higher dysplasia. In a study on p53 and Ki-67 in gastrointestinal stromal tumors, Neves et al.(16) found higher values of Ki-67 in patients with tumors of medium and high risk. There was no statistically significant relationship between the expression of p53 and Ki-67, in support of the results published by Vernillo et al.(29) who found no association between p53 and Ki-67 in a group of 68 adenomas. The loss of function of p53 and its suppressive activity may have led to uncontrolled cellular activity with consequent accumulation of Ki-67, but further studies would be needed to confirm this association as most other studies in this area have assessed proteins individually rather than testing possible correlations between them. In addition, other factors and proteins act concurrently, either stimulating or suppressing cell proliferation. The determination of more prognostic factors related to the development of colorectal cancer is of fundamental importance for primary prevention programs. Thus, the expression of p53 and Ki-67 may be useful as prognostic factors for adenomas in association with other known histopathologic features. In conclusion, the p53 protein is expressed in a proportion of adenomas, while the Ki-67 protein is expressed in all adenomas. There is greater expression of p53 in adenomas with high-grade dysplasia, while the expression of Ki-67 is higher in rectal adenomas and adenomas with high-grade dysplasia. No other parameters correlated with protein expression.

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Sousa WAT, Rodrigues LV, Silva Jr RG, Vieira FL. Avaliação imunoistoquímica das proteínas p53 e Ki-67 em adenomas colorretais. Arq Gastroenterol. 2012;49(1):35-40. RESUMO – Contexto - O aparecimento de adenomas intestinais e sua progressão para adenocarcinoma é o resultado do acúmulo de mutações genéticas da mucosa intestinal, herdadas ou adquiridas durante a vida. Dessa forma, várias proteínas têm sido estudadas em relação ao desenvolvimento e progressão do câncer colorretal, incluindo as proteínas p53 e Ki-67. Objetivo - Avaliar a expressão das proteínas p53 e Ki-67 em adenomas colorretais, suas relações com características clinicopatológicas e avaliar a relação entre as duas proteínas. Método - A amostra consistiu de 50 pólipos adenomatosos encontrados em pacientes submetidos a exames colonoscópicos. Após a realização de polipectomia, os pólipos eram conservados em solução tamponada de formalina a 10% e submetidos a rotina de preparo de cortes e lâminas e coloração pela hematoxilina-eosina para confirmação da natureza adenomatosa. Realizou-se imunoistoquímica específica para as proteínas p53 e Ki-67 pelo método imunoenzimático da streptoavidinabiotina-peroxidase para cada adenoma. Resultados - A proteína p53 foi positiva em 18% dos adenomas e a proteína Ki-67, expresso como índice (i.Ki-67), obteve média de 0,49. Houve diferença estatisticamente significante na expressão de p53 (P = 0,0003) e Ki-67(P = 0,02) entre os adenomas com alto e baixo grau de displasia, sendo maior no primeiro grupo. Encontrou-se, ainda maior expressão da proteína Ki-67 nos adenomas retais em relação aos de localização cólica (P = 0,02). Não houve relação entre a expressão das duas proteínas, na amostra. Conclusão - A proteína p53 é expressa em parte dos adenomas enquanto Ki-67 é expressa na sua totalidade. A expressão de p53 foi maior nos adenomas com alto grau de displasia. A expressão de ki-67 foi maior nos adenomas retais e com alto grau de displasia. DESCRITORES – Neoplasias colorretais. Proteína supressora de tumor p53. Antígeno Ki67. Imunoistoquímica.

References 1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

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Brenner H, Hoffmeister M, Haug U. Should colorectal cancer screening start at same age in European countries? Contributions from descriptive epidemiology. Br J Cancer. 2008;99:532-5. Citarda F, Tomaselli G, Capocaccia R, Barcherini S, Crespi M; Italian Multicenter Study Group. Efficacy in standard clinical pratice of colonoscopic polipectomy in reducing colorectal cancer incidence. Gut. 2001;48:812-5. Enomoto T, Kuranami M, Kakita A. Variations in the expression of plateletderived endothelial cell growth factor in human colorectal polyps. Surg Today. 2000;30:711-7. Fearon ER, Volgelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990;61:759-67. Grizzle WE, Myers RB, Manne U, Srivastava S. Immunohistochemical evaluation of biomarkers in prostatic and colorectal neoplasia. In: Hanausek M, Walaszek Z, editors. Methods in molecular medicine-tumor marker protocols. Totowa: Humana Press; 1998. p.143-60. Horiuchi H, Kawamata H, Omotehara F, Fujii S, Fujimori T, Kuroda Y. Negative immunohistochemical staining of p53 protein does not always reflect wild-type p53 gene in cancer cells. J Gastroenterol. 2004;39:801-3. Instituto Nacional do Câncer. Estimativa 2010 – Incidência de câncer no Brasil. Rio de Janeiro: INCA, 2010. Available at: http://www.inca.gov.br Isobe M, Emmanuel BS, Givol D, Oren M, Crocen CM. Localization of gene for human p53 tumor antigen to band 17p13. Nature. 1986;320:84-5. Loeve F, Boer R, Zauber AG, Van Ballegooijen M, Van Oortmarssen GJ, Winamer SJ, Habbema JD. National polyp study data: evidence for regression of adenomas. Int J cancer. 2004;111: 633-9. Lustosa SA, Logullo A, Artigiani R, Saad SS, Goldenberg A, Matos D. Analysis of the correlation between p53 and bcl-2 expression with staging and prognosis of the colorectal adenocarcinoma. Acta Cir Bras. 2005;20:353-7. Mansmann U, Crispin A, Henschel V, Adrion C, Augustin V, Birkner B, Munte A. Epidemiology and quality of control of 245 000 outpatient colonoscopies. Dtsch Arztebl Int. 2008;105:434-40. Matos LL, Machado LN, Sugiyama MM, Bozzetti RM, Pinhal AS. Technology applied to the detection of tumor markers. Arq Med ABC. 2005;30:19-25. May P, May E. Twenty years of p53 research: structural and functions aspects of the protein. Oncogene. 1999;18:7621-738. Miller C, Mohandas T, Wolf D, Prokocimer M, Rotter V, Koeffler HP. Human p53 gene localized to short arm of chromosome 17. Nature. 1986;319:783-4. Muneyuki T, Watanabe M, Yamanaka M, Isaji S, Kawarada Y, Yatani R. Combination analysis of genetic alterations and cell proliferation in small intestinal carcinomas. Dig Dis Sci. 2000;45:2022-8. Neves LR, Oshima CT, Artingiani-Neto R, Yanaguibashi G, Lourenço LG, Forones NM. Ki67 and p53 in gastrointestinal stromal tumors – GIST. Arq Gastroenterol. 2009;46:116-20. Nomura M, Watari J, Yokota K, Saitoh Y, Obara T, Kohgo Y. Morphogenesis of nonpolypoid colorectal adenomas and early carcinomas assessed by cell proliferation and apoptosis. Virchows Arch. 2000;437:17-24.

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18. Petroianu A, Alberti LR, de Lima DC, Hauter HL, Rodrigues KC, Mendes JC. [Colonoscopic findings in asymptomatic people]. Arq Gastroenterol. 2009;46:173-8. 19. Porcelli B, Frosi B, Arezzini L, Marinello E, Vernillo R, De Martino A, Vatti R, Minacci C. Expression of p185 and p53 in benign and malignant colorectal lesions. Histochem J. 2001;33:51–7. 20. Radovanovic-Dinic B, Nagorni A, Katic V, Satmenkovic I, Zlatic A. An immunohistochemical study of Ki-67 in colorectal adenoma. Med Arh. 2009;63:16-8. 21. Santos Jr JCM. Ano-rectal-colic cancer. Current Issues II - Colorectal cancer Risk factors and prevention. Rev Bras Coloproctol. 2007;27:459-73. 22. Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol. 2000;182:311-22. 23. Shanmugam C, Katkoori VR, Jhala NC, Grizzle WE, Siegal GP, Manne U. p53 Nuclear accumulation and Bcl-2 expression in contiguous adenomatous components of colorectal adenocarcinomas predict aggressive tumor behavior. J Histochem cytochem. 2008;56:305-12. 24. Sheikh RA, Min BH, Yasmeen S, Teplitz R, Tesluk H, Ruebner BH, Tobi M, Hatfield J, Fligiel S, Lawson MJ. Correlation of Ki-67, p53, and Adnab-9 immunohistochemical staining and ploidy with clinical and histopathologic features of severely dysplastic colorectal adenomas. Dig Dis Sci. 2003;48:223-9. 25. Shinozaki M, Watanabe T, Kubota Y, Sawada T, Nagawa H, Muto T. High proliferative activity is associated with diysplasia in ulcerative colitis. Dis Colon Rectum. 2000;43:34-9. 26. Soussi T, Caron de Fromentel C, May P. Structural aspects of p53 protein in relation to gene evolution. Oncogene. 1990;5:5945-52. 27. Sugai T, Habano W, Uesugi N, Jiao Y, Nakamura S, Sato K, Chiba T, Ishii M. Molecular validation of the modified Vienna classification of colorectal tumors. J Mol Diagn. 2002;4:191-200. 28. Vermeulen K, Van Bockstaele DR, Berneman ZN. The cell cycle: a review of regulation, desregulation and therapeutic targets in cancer. Cell Prolif. 2003; 36:131-49. 29. Vernillo R, Lorenzi B, Banduccci T, Minacci C, Vindigni C, Lucenti Fei A, Lorenzi M. Immunohistochemical expression of p53 and ki67 in colorectal adenomas and prediction of malignancy and development of new polyps. Int J Biol Markers. 2008;23:89-95. 30. Voskuil DW, Kampman E, van Geloof W, Grubben M, Kok F, Van Muijen G, Nagengast F, Vasen H, Van’t Veer P. No major difference in K-ras and p53 abnormalities in sporadic and hereditary nonpolyposis colorectal adenomas. Dig Dis Sci. 2000;45:2187-94. 31. Watatani M, Ieda S, Kuroda K, Inui H, Nishimura K, Yasutomi M. Comparison of p53 and bcl-2 expression in initial, synchronous, and metachronous colorectal adenomas. Surg Today. 1999:29:707-12. 32. Williams AR, Balasooriya BA, Day DW. Polyps and cancer of the large bowel: a necropsy study in Liverpool. Gut. 1982;23:835-42. 33. Yonish-Rouach E. A question of lie or death: the p53 tumor suppressor gene. Pathol Biol. 1997;45:815-23. Received 1/6/2011. Accepted 23/8/2011.

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ARTIGO ORIGINAL / ORIGINAL ARTICLE

ARQGA/1587

COMMON BILE DUCT STONES. Analysis of the videolaparoscopic surgical treatment Marco Aurelio SANTO1, Carlos Eduardo DOMENE1, Daniel RICCIOPPO1, Lian BARREIRA1, Flavio Roberto TAKEDA1 and Henrique Walter PINOTTI2

ABSTRACT – Context - About 9% of the Brazilian population has gallstones and the incidence increases significantly with aging. The choledocholithiasis is found around 15% of these patients, and a third to half of these cases presented as asymptomatic. Once the lithiasis in the common bile duct is characterized through intraoperative cholangiography, the laparoscopic surgical exploration can be done through the transcystic way or directly through choledochotomy. Objective - To evaluate the results and outcomes of the laparoscopic treatment of common bile duct lithiasis. Methods - Seventy consecutive patients were evaluated. They prospectively underwent the treatment of the lithiasis in the common bile duct and the exploration ways were compared according to the following parameters: criteria on their indication, success in the clearance, surgical complications. It was verified that about ½ of the choledocholithiasis carriers did not show any expression of predictive factors (clinical antecedents of jaundice and/or acute pancreatitis, compatible sonographic data and the pertaining lab tests). The laparoscopic exploration through the transcystic way is favored when there are no criteria for the practice of primary choledochotomy, which are: lithiasis in the proximal bile duct, large (over 8 mm) or numerous calculi (multiple calculosis). Results - The transcystic way was employed in about 50% of the casuistic and the choledochotomy in about 30%. A high success rate (around 80%) was achieved in the clearance of the common bile duct stones through laparoscopic exploration. The transcystic way, performed without fluoroscopy or choledochoscopy, attained a low rate of success (around 45%), being 10% of those by transpapilar pushing of calculi less than 3 mm. The exploration through choledochotomy, either primary or secondary, if the latter was performed after the transcystic route failure, showed high success rate (around 95%). When the indication to choledochotomy was primary, the necessity for choledochoscopy through choledochotomy to help in the removal of the calculi was 55%. However, when choledochotomy was performed secondarily, in situations where the common bile duct diameter was larger than 6 mm, the use of choledochoscopy with the same purpose involved about 20% of the cases. There was no mortality in this series. Conclusion - The laparoscopic exploration of the common bile duct was related to a low rate of morbidity. Therefore, the use of laparoscopy for the treatment of the lithiasis in the common bile duct depends on the criteria for the choice of the best access, making it a safe procedure with very good results. HEADINGS – Choledocholithiasis. Laparoscopy. Cholangiography.

INTRODUCTION

The occlusion of the biliary tract is the most important phenomenon in biliary illness. It can be said that with the compromise of the main biliary tract, affections initially of the gallbladder, a local process, convert into systemic disease(39). Calculosis of the hepatocholedochus constitutes a particularly important aspect of biliary lithiasis, not only due to its high incidence, but also for the severity of its

complications and for the various treatment options(55). Acute suppurative cholangitis and acute biliary pancreatitis are conditions in which the major cause are bile duct stones, and both are potential lethal conditions in its severe forms(81). Epidemiological studies indicate that approximately 10% to 15% of the adult population in United States of America contains calculi in the gallbladder, estimating approximately one million cases diagnosed yearly(58). In Brazil, a study demonstrates prevalence of gallbladder

No conflicts of interest. 1 Departmento de Gastroenterologia, Faculdade de Medicina da Universidade de São Paulo, SP, Brasil; 2 In Memoriam. Correspondence: Dr. Daniel Riccioppo - Rua Doutor Barachisio Lisboa, 88 - 05441-090 - São Paulo, SP, Brasil. E-mail: danriccioppo@uol.com.br

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lithiasis in 9.3% of the general population(13). This coefficient rises from 2.4% to 27.5% when comparing the age groups of 20 to 29 and 70 and over, respectively. In women over 50 years of age this value is superior to 20%. The historical incidence of choledocholithiasis has been estimated at approximately 15%, with reports varying from 7 to 22%(3, 6, 14, 54, 55). The lower current incidence, approximately 7%, varying from 3% to 10% of those afflicted by gallbladder lithiasis, is explained based on larger availability of diagnosis and precocious referral to surgical therapeutics, especially laparoscopic cholecystectomy(3, 7, 21). Perhaps, the less frequent application of intraoperative cholangiography in the videolaparoscopic era may underestimate this occurrence(47, 67, 78). A German study found that only 6% of institutions perform routine transoperative cholangiography during laparoscopic cholecystectomy, while 7 times more hospitals do not(53). In Brazil, in a multicenter study to evaluate the results obtained with laparoscopic cholecystectomy in 33,563 cases, lithiasis in the main biliary tract was diagnosed in approximately 1,400 cases, or 4.2%(72). Despite attempts in literature to use predictors to perform selective intraoperative cholangiography(20), approximately 30% to 50% of the common bile duct stones cases present themselves in an unsuspicious manner, given the low pinpointing capacity of the commonly used tests in the preoperative evaluation of patients programmed for cholecystectomy. This emphasizes the role of routine intraoperative cholangiography as the only way to establish diagnosis in these circumstances(35, 49, 65). Classically, the surgery by laparotomy for access to the main biliary tract constituted the standard treatment for choledocholithiasis, preceding choledocholithotomy and external biliary drainage(80). In the 70’s, the introduction of the retrograde endoscopic cholangiography and subsequently papillotomy permitted a therapeutic alternative, especially in those patients that had previously been subject to cholecystectomy(12, 17, 23, 44). With the advent of videolaparoscopic surgery, the endoscopic approach became an important complementary procedure, recommended in the preoperative, when used for the diagnosis of choledocholithiasis, as well as in the postoperative period(9, 32, 34, 46). The central reasons for this behavior were the absence of satisfactory techniques for the laparoscopic exploration of the biliary tract or the restriction of its practice to a few centers, as well as the surgeon’s desire to avoid conversion to laparotomy(3, 30, 46, 69). As experience with the laparoscopic access accumulated, associated with technological developments that made available more delicate instruments and enabled choledochoscopy with a flexible device, the surgical exploration of the biliary tracts through this access became a reality(61, 66). The possibility of complete extraction of the calculi in a single operative procedure, avoiding manipulation of the papilla by endoscopic procedures and its consequences, progressively turned this technique in a more utilized procedure(4, 24, 76). Upon deciding on the usage of videolaparoscopic surgical exploration, it is fundamental to establish the strategy, which includes analysis of local conditions and interpretation of

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the intraoperative cholangiography images, to decide the type of access to the main biliary tract, either through the transcystic access or directly through choledochotomy(25, 36). Then, the objective of the present study is to evaluate the outcome of videolaparoscopic surgical exploration of the biliary tract, for treatment of common bile duct stones, taking into consideration the transcystic and the transcholedochus accesses. This evaluation consists of the analysis of the following parameters: criteria for indication, success in the clearance of biliary lithiasis, and surgical complications. METHODS

The study included 70 consecutive patients that were submitted, in a prospective manner, to videolaparoscopic cholecystecomy and treatment of common bile duct stones. They were admitted into the Division of Clinical Surgery II – Department of Digestive Tract Surgery of the Hospital das Clínicas, University of São Paulo Medical School, São Paulo, SP, Brazil, over a period of 6 years. The total number of patients with common bile duct stones corresponds to 3.53% of the global casuistic of 1,979 patients submitted to videolaparoscopic cholecystectomy for cholelithiasis in the same period. Intraoperative cholangiography was performed in 90.95% of the total number of patients. The study was approved by the Ethics and Research Commission of the Department of Gastroenterology of the University of São Paulo Medical School and all patients having been properly informed about the videolaparoscopic surgical procedure, especially in what concerns the possible conversion to an open intervention. The mean age was 51.6 years, varying from 20 to 85 years. Fifty-three patients were female and 17 male, corresponding to 75.7% and 24.3%, respectively. The mean weight was 65.9 kilograms, varying from 44 to 100 kilograms. The mean height was 1.59 meters, varying from 1.41 to 1.82 meters. The choice of the surgical procedure to be adopted was based on criteria dependent on the interpretation of the intraoperative cholangiography. Depending on radiological criteria, indication of primary choledochotomy or transcystic access was done. When diameter of the calculi present in the main biliary tract was larger than 8 mm (in the smallest axis when deformed), or larger than 2 to 3 times the diameter of the cystic duct, when location of calculi was in the main biliary tract proximally to the insertion of the cystic duct (common hepatic duct), or in cases of multiple calculosis, the approach by choledochotomy was preferred. Indication of exploration through transcystic access was preferential when choledocholithiasis that does not present with any indication for exploration through primary choledochotomy, as described above. Special circumstance, for which previously defined conduct can not be established, constitutes the finding of impacted calculus close to the duodenal papilla. In this situation, when dilation of the main biliary tract can not be observed, normally because of small calculus, the transcystic access can be attempted, aiming for its distal transpapillary displacement.

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On the other hand, when biliary dilation is already present, usually by larger calculi, other therapeutic approaches can be endeavored, including transcholedochus exploration. Surgical technique for exploration of the main biliary tract The instrumental exploration of the biliary tract, either through the transcystic or transcholedochus accesses, requires the puncture of the abdominal wall with a metallic cannula with a diameter of 2.5 mm, enabling the introduction of specific instruments into the abdominal cavity for extraction of calculi. The optimal position of this cannula is the middistance between the epigastric and right hypochondriac trocars, projecting itself perpendicularly over the biliary tract. When performing the choledochoscopy, an additional 5 mm trocar is introduced in that region. The instrumental manipulation through transcystic access is performed through an incision in the cystic duct made for the intraoperative cholangiography. The adequate dilation of the cystic duct is necessary, normally obtained by the passage of a Fogarty® (Baxter®) catheter, available in the diameters 4, 5 and 6 Fr, which, after insufflation of the balloon, is slowly tractioned until its exteriorization in the cystic duct orifice, repeating the maneuver several times until the degree of dilation necessary for the unrestricted introduction of the catheter is obtained. In some situations the widening of the orifice can be achieved through the prolongation of the longitudinal and distal incisions of the cystic duct. Through the transcystic access there are two techniques for the manipulation of the main biliary tract. With mechanic extraction with capture, the complete extraction of calculi is obtained through capture with basket type probes (Figure 1). In transpapillary forced migration, clearance is obtained by the migration of calculi through transpapillary displacement, utilizing anti-spasmodic drug by intravenous infusion (scopolamine bromide, 20 mg) to relax the sphincter of Oddi, followed by the rapid instillation of saline through the transcystic plastic catheter used for intraoperative cholangiography, or through the actual push by means of the Fogarty catheter (4, 5 or 6 Fr catheter, with a number chosen based on the gauges of the cystic duct and common bile duct) or basket type probe. This therapeutic method is only attempted in circumstances in which the choledochus is not dilated, containing calculi with diameter up to 3 mm, preferably isolated in the distal portion. Through a choledochotomy, success of the clearance can be obtained by means of three techniques of manipulation of the main biliary tract. To achieve this, it is necessary to expose the anterior portion of the choledochus through the dissection of the peritoneal layer that covers this area, with medial displacement of the hepatic artery, or its branches, in some situations. The transverse choledochotomy is used, with the approximate extension of the largest diameter of the calculi shown by cholangiography. After section of the biliary tract and outflow of its contents, a small gauze plug, whose extremity is tied to a cotton thread, which facilitates its removal, is inserted through the proximal orifice (Figure 2).

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This maneuver prevents the migration of cranial calculi or fragments when manipulating the distal segment of the choledochus.

FIGURE 1. Capture of calculi with “basket” type probe introduced through the cystic duct

FIGURE 2. Transverse choledochotomy and the insertion of the gauze plug in the proximal segment

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FIGURE 3A. Mixed technique for calculi extraction through choledochotomy – extrusion of calculi by fluid turbulence

The transcholedochus exploration is performed primarily, following criteria for indication already mentioned, or secondarily, following an unsuccessful transcystic exploration. For this condition it is necessary that the size of the choledochus be greater than 6 mm. In order to assess the implications of the indications for determination of efficiency of this technique, the success rate among patients with primary and those with secondary indication for choledochotomy was compared. The results of application of each type of technique were also compared in relation to the groups that realized the choledochotomy primarily or secondarily. Different techniques also can be mixed, involving the use of the following procedures, alone or combined. Immediately after the choledochotomy, the rapid infusion of saline, usually through the plastic catheter used for intraoperative cholangiography inserted through the cystic duct, allows the mobilization and subsequent cleaning of calculi, with removal through the opening in the choledochus (Figure 3A). Next, additional mobilization is achieved with the Fogarty catheter, also allowing the removal of calculi (Figure 3B). Finally, by basket type probe approach, calculi capture and extraction through choledochotomy is attempted (Figure 3C). In some situations, not all three procedures are necessary. To perform a direct visualization of the common bile duct, a flexible choledochoscope is used, with a caliber of 5 mm, coupled to a second video monitor; it contains an internal work channel of 2 mm, through which you can insert pressure clamps and Fogarty or basket type probes. Thus, the visibility inside the main biliary tract, proximal and distally, may be required for the extraction of calculi (choledochoscopy for extraction), or simply to control the clearance after surgical manipulation

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FIGURE 3B. Mixed technique for calculi extraction through choledochotomy – traction of the catheter after inflation of the balloon and dragging of calculi

FIGURE 3C. Mixed technique for calculi extraction through choledochotomy – capture of calculi with “basket” type probe

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(choledochoscopy for control) (Figure 4). Manipulation probes can be inserted via the choledochoscope’s work channel or directly through the choledochotomy, in this case allowing for the use of larger caliber probes. Choledochoscopy is most commonly used after failure of the mixed technique.

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FIGURE 4. Choledochoscopy of the distal segment

FIGURE 5. External drainage of the biliary tract through the Kehr drain

The external drainage of the biliary tract after transcystic exploration is used exceptionally, only upon the poor drainage of the contrast to the duodenum even after intravenous infusion of the anti-spasmodic (scopolamine bromide, 20 mg). When necessary, it is done by the plastic catheter used for intraoperative control cholangiography, secured by double clamping with metallic clip or non absorbable thread and exteriorized in the right flank of abdomen. On the other hand, in transcholedochus exploration, in a routinely fashion, it is followed by the external biliary drainage through a Kehr drain, having both branches of the transversal loop at about 2 cm each. The long branch of the drain can be exteriorized directly through choledochotomy, or through the cystic duct. In the latter situation, the surgical suture of  the choledochus becomes easier, feasible with separate stitches or continuous suture with 5-0 absorbable thread, having the long branch fixed to the cystic stump with no absorbable thread, avoiding strangulation (Figure 5). Subsequently, the intraoperative control cholangiography is performed with injection of contrast material into the Kehr drain, evaluating the success in the clearing of lithiasis, proper positioning of the drain, any eventual leakage through the surgical suture of the choledochus and drainage of  the contrast material to the duodenum. The long branch of the drain is exteriorized to the right hypochondrium region, usually at the point of insertion of the metal cannula through which the probes to manipulate the biliary tract are inserted, which coincides with the area of insertion of the trocar when the choledochoscopy is performed. The external biliary drainage can also be performed by means of a plastic catheter inserted through the cystic duct.

In special circumstances, where local conditions permit safe suture of the choledochus and intraoperative control cholangiography demonstrates success in clearing the lithiasis, as well as the satisfactory drainage of the contrast material to the duodenum, the external drainage may be abdicated, simply by removing the plastic catheter and occluding the cystic duct.

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RESULTS

The indication for transcystic exploration occurred in 37 patients. The choledochus presented normal caliber in 20 of these patients, being dilated, with caliber above 6 mm, in the remaining 17. All calculi were found in the distal choledochus, and in 4 patients were smaller than 3 mm and positioned close to the duodenal papilla, not determining, however, dilation of the biliary tract. The indication for primary transcholedochus exploration occurred in 20 patients. All of the patients in this groups exhibited choledochus caliber superior to 6 mm. In 12 patients the indication was exclusively resulting of large calculi. However, the indication because of proximal localization of calculi occurred in 3 patients. Multiple calculosis induced this procedure in 2 patients. Three patients submitted primarily to choledochotomy displayed, in the intraoperative cholangiography, image of an impacted calculus in the duodenal papilla. The endoscopic treatment was opted for in nine patients, in which five were in the preoperative period and four in the postoperative, without any surgical intervention of the biliary tract in the course of the cholecystectomy.

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Santo MA, Domene CE, Riccioppo D, Barreira L, Takeda FR, Pinotti HW. Common bile duct stones: analysis of the videolaparoscopic surgical treatment

In two patients, given the presence of undiagnosed great dilation of the biliary tract associated with multiple calculosis, the implementation of a biliary bypass through choledochalduodenal anastomosis was opted for. In two patients in whom intraoperative cholangiography revealed image suggestive of lithiasis, with small calculus close to the duodenal papilla, together with a choledochus of small caliber, raising questions as to the real presence of the calculus, expectant conduct was adopted, without instrumental manipulation of the biliary tract, but with external drainage of the same through a plastic catheter exteriorized by the cystic duct itself. Success in the clearance of the biliary tract Among the 37 patients that underwent transcystic exploration, success was achieved in clearing of the biliary tract in 16 patients (43.2%), out of which four were submitted to transpapillary displacement through forced transcystic migration due to the presence of calculi <3 mm and choledochus without dilation. In 21 patients, after unsuccessful attempts through transcystic manipulation, direct manipulation of the biliary tract through the choledochotomy was opted for in 11 patients, whereas postoperative endoscopic therapy was attempted in 8 patients, and conversion to laparotomy in 2 patients. Among the 31 patients undergoing videolaparoscopic transcholedochus surgical exploration, 20 of them primarily and 11 secondarily after failure of transcystic exploration, success was obtained in clearing the biliary tract in 29 patients (93.5%). In 2 patients among the 20 with primary indication to choledochotomy, conversion to laparotomy was required. The use of mixed technique was successful in 7 patients (35%) and choledochoscopy was necessary in 11 patients (55%). Among the 11 patients with secondary indication for choledochotomy, success with the mixed technique occurred in 9 patients (81.8%) and choledochoscopy was necessary in 2 patients (18.2%). Comparing the two groups of indication for choledochotomy, it was observed that the choledochoscopy was most needed in patients with primary indication, while in those in which the transcholedochus exploration was performed secondarily the mixed technique was sufficient to resolve the majority of cases (P = 0.024). Drainage of the biliary duct Among the 16 patients that underwent exclusive transcystic exploration, the external biliary drainage was used in 7 of them. The drain used was the same plastic catheter of the intraoperative cholangiography (intra-cath), exteriorized through the cystic duct and secured by two metallic clips or thread. For those that underwent choledochotomy, in 5 cases primary suturing without external biliary drainage was performed. In the other 24, transcystic drainage via intracath was used in 10 patients, a Kehr’s drain exteriorized through the cystic duct in 9, and the remaining 5 patients also received a Kehr’s drain, however exteriorized by the choledochotomy itself.

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Complications There were no deaths in this casuistic. Among the patients who underwent transcystic surgical exploration there was one case of perforation of the choledochus, retro-duodenal portion, whose detection was evidenced by leakage of the contrast material during the control intraoperative cholangiography post instrumental manipulation with basket type probe. It was then opted to precede the conversion to laparotomy with identification of the lesion in the posterior face of the choledochus, applying suture with absorbable thread (two separate sutures). Subsequently, transverse choledochotomy was performed on the supraduodenal portion, with the introduction of a Kehr’s drain, exteriorized by the cystic duct, and followed by suture of the choledochus with separate sutures with absorbable thread. There were also three cases of hyperamylasemia and another case of premature displacement of the external biliary drain (transcystic intra-cath) in the 4th postoperative day, not resulting in any clinical complications. In patients that underwent transcholedochus exploration, there were three cases with complications. In one case, in the 1st postoperative day, the patient developed toxemia and output of biliary secretion through the Penrose drain, being re-submitted to laparoscopy with discovery of choleperitoneum and fistula of the suture of the choledochus; this was followed by cleaning of the cavity, closing of the point of leakage, drainage of the cavity and maintenance of biliary drainage, with satisfactory postoperative evolution. In one patient, premature displacement of external biliary drain was observed, evolving with biliary fistula and satisfactory clinical control. In one case, 3 days after the scheduled withdrawal of the biliary drain (21st postoperative day), the patient presented pain in right hypochondrium, the ultrasound examination revealing presence of sub-hepatic collections, and the patient was maintained under clinical control with satisfactory evolution. There were five cases of transient hyperamylasemia without clinical manifestation. DISCUSSION

The biliary lithiasis with unsuspicious presentation corresponded to about one-third of our casuistic, with patients that showed any signs, either clinical, laboratory or sonographical, of the impairment of biliary tract. Previous studies showed high incidence of unsuspicious lithiasis in the main biliary tract (about 40% of patients with choledocholithiasis) and highlighted the conduct of routine use of intraoperative cholangiography to widen the possibility for this diagnosis; in addition, this method has other advantages, such as the study of the anatomy of the biliary tract(51, 65). On the other hand, some authors advocate that unsuspicious biliary lithiasis is infrequent, especially in individuals under 60 years of age(41, 42). Attempt to validate scores obtained from the combined analysis of various factors were made, based on the belief that these indexes were useful to support the indication of preoperative endoscopic evaluation or to whether or not employ the intraoperative cholangiography(5, 38, 40, 42, 71).

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In a study of 959 cholecystectomies with intraoperative cholangiography, 46 patients (4.8%) had filling deffects in choledocus or common hepatic duct. In these cases, the cholangiogram catheter was held in place, and cholangiogram was repeated subsequently, and after 48 hours 26% of these patients had normal cholangiogram. Another 26% had normal exam at 6 weeks. According to this results the incidence of choledocholithiasis with potential for long-term morbidity is less than 2.5%(15). However the authors do not focus the potential risk of early postoperative complications related to choledocholithiasis. High definition and elevated accuracy examinations, such as magnetic resonance cholangiography and endoscopic ultrasonography, would establish, preoperatively, the diagnosis of choledocholithiasis in almost all cases, even in non-dilated ducts(1, 22, 28). But their high cost impedes their common use(7, 69). Choice of technical procedure The complexity of the therapeutic approach of choledocholithiasis is evidenced by the many therapeutic propositions. In 1993, a consensus meeting sponsored by the National Institute of Health of the USA supported all forms of well-established treatment of lithiasis in the main biliary tract: laparotomy, laparoscopy and endoscopy, including the expected conduct in special circumstances, highlighting the role and perspective that the laparoscopic access had acquired(37). A multi-centric study conducted in Brazil demonstrates this complexity(72). The endoscopic approach, pre or postoperative, represented the main form of treatment for choledocholithiasis, as it was used in about 55% of cases, while videolaparoscopy was used in about 33% of cases, and laparotomy to approximately 12%. This scenario implies that the treatment of these patients should be individualized, considering the patient’s clinical conditions, the characterization and timing of diagnosis of the lithiasis, the surgical experience, and resources of the institution(7, 21, 27, 50). Recognizing limits is the basis for working together and establishing the actual indications for different methods of treatment of biliary calculi. The criteria to select the transcystic or transcholedochus access are based on analysis of cholangiography characteristics of the biliary tract and calculi present. The transcystic exploration is, in principle, always used, reserving choledochotomy for special circumstances or upon failure of the former(8, 63). The transcholedochus approach is preferential when large gallstones are present. The transcystic extraction of big calculi, larger than 8 mm or disproportionate to the caliber of the cystic duct, involves great risk of laceration of the cystic-choledochus junction. The difficult access to calculi proximal to the insertion of the cystic duct through the cystic duct itself, also excluding the transcystic path as real option. Finally, the large possibility of residual calculosis after transcystic exploration of the main biliary tract occupied by numerous calculi, and the difficulty in controlling the

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clearance, requires transcholedochus access as the primary option in multiple calculosis. In our series of 70 patients, the videolaparoscopic exploration was indicated in 57 cases, or 81.5% of total. The transcystic route represented 52.8% of patients, while the transcholedochus route, 28.7%. In a multi-institutional series of 226 patients, videolaparoscopy through the transcystic route was used in 83% of cases, and transcholedochus in the other 17%(6). A choledochus caliber larger than 6 mm is a secure guarantee for the implementation of choledochotomy, particularly with regard to the instrumental manipulation of biliary tract, with inherit risk for lacerations and mainly because of the possibility of stenosis after suture of the choledochus(67, 69). The preference for transverse incision is made on the basis of lesser compromise of choledochus blood irrigation as well as the smaller possibility of narrowing after suture of the choledochus. The longitudinal incision of the choledochus is preferred by some surgeons because it allows easy access for the insertion of the choledochoscope, and it can be more easily extended(7, 60). The direct approach of the biliary tract should be avoided when intense inflammatory process is found in the hepatic hilum due to difficulties in dissection and possibility of bleeding from larger vessels(36, 62, 63). Literature data supports the idea of advantages in treatment of cholelithiasis and common bile duct stones in a one-stage procedure(74). The endoscopic sphincterotomy up to 48 hours after cholecystectomy is associated with acute pancreatitis within 30% of procedures and up to 0.6% of patients will develop pancreatic necrosis. This serious complication is related to mortality rate of 0.4%. The postoperative endoscopic approach, anyway, is established as an option for the treatment of choledocholithiasis, is a procedure commonly used in clinical practice, constituting as an alternative to conversion to laparotomy, although adding considerable cost and risk(31, 52, 75). Some favorable factors for postoperative endoscopic exploration are admitted, such as: small non-obstructive calculi, thin biliary tract and therefore with the possibility of stenosis due to surgical manipulation, and patients at risk for prolonged operations. The presence of intense inflammatory process in the gallbladder, extending to the region of the hepatic hilum, for bringing bigger technical difficulties and possible complications of the surgical operation, is also a favorable circumstance for postoperative endoscopic approach. One of the major concerns about the postoperative endoscopic therapy is its potential failure and consequences in this phase. The external biliary drainage through a transcystic catheter in order to facilitate the endoscopic procedure is often used(60, 69, 78). The best placement of this catheter is intra-choledochus, not surpassing the duodenal papilla. The fear of catheters surpassing the papilla comes from the risk of obstruction of the pancreatic duct and consequent acute pancreatitis. But it is admitted that the risk of pancreatitis is low, based on experience of placement of stents by endoscopy.

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Santo MA, Domene CE, Riccioppo D, Barreira L, Takeda FR, Pinotti HW. Common bile duct stones: analysis of the videolaparoscopic surgical treatment

The indication for biliary bypass is favored when severe compromise of the choledochus is present, evidenced by large biliary dilation and severe difficulty in transpapillar emptying in intraoperative cholangiography, especially if associated with multiple calculi(29). One should not, however, indicate it too often, since the intensity of the inflammatory process, preferably chronic, present in the wall of choledochus is not linked to their degree of dilation and therefore do not constitute irreversible changes(10). The possibility of achieving safe choledochus-duodenal anastomosis by videolaparoscopy made it the most used technique for biliary bypass(19, 26), being employed in two patients in our casuistic, precisely because they presented large dilations of the choledochus, one of which with multiple calculi. The minimum choledochus caliber highlighted, to perform safe anastomoses, in order to ensure adequate permeability and avoid the complications arising from possible stenosis, is 16 mm(29). A prospective study found recurrent choledocholithiasis rate of 10.1% in 169 patients who were previously treated with choledocholithotomy and T-tube drainage (including laparoscopic approach) or endoscopic sphincterotomy, for common bile duct stones, and monitored for a mean period of 9.6 years. In this same study the 44 patients who undergone choledochoduodenostomy for the purpose of preventing choledocholithiasis recurrence were followed for the same period have no recurrence(45). The possibility of false-positive images, as well as the possibility of spontaneous migration of small calculi, supports the expected conduct in the situation of doubt in diagnosis. Surgical exploration is not carried on in these cases, neither transcystic nor transcholedochus, with the convenience of external biliary drainage through a transcystic catheter to allow subsequent re-evaluation by postoperative control cholangiography. In two patients, in whom this practice was established in our study, the postoperative control cholangiography did not confirm choledocholithiasis. In the case of diagnostic doubt, the external biliary drainage may be the best option because it allows the possible postoperative diagnostic confirmation, and if so, helps the additional endoscopic procedure(31). The risk to treating calculi less than 3 mm must be balanced with the risk of arising complications(80). Treatment in such cases should only be indicated in those with history of acute biliary pancreatitis. The risk of exploratory procedures of the biliary tract, either by laparoscopy or endoscopic, may be higher than the expectant conduct, and more studies are necessary to define the safety of this practice in these conditions. However, many cases of acute pancreatitis are results of “probably insignificant” calculi abandoned in the biliary tract(43). Success in clearing the biliary tract The success rate in our casuistic using the transcystic access was around 50%. The technical method of forced migration was attempted, given that the criteria for indication including small calculi and no biliary dilation, in approximately 10% of cases, achieving success in all of them.

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A wide study brings us an overview of this point. Although they have obtained a success rate around 80%, about 40% of these successes were achieved by the technical method of forced migration or irrigation, denoting the presence of small calculi, or even suspected, in a significant contingent of patients(6). The use of a 3 mm scope via transcystic duct allows the surgeon to perform a more complete and direct visual exploration of the common duct system, increasing the chances of successful treatment(2). However, it remains the exception limitations of this approach even with the use of scope, where success will also depend fundamentally on the size of calculi and diameter of the cystic duct. The transcholedochus exploration achieved, in our study, overall success rates of around 93%, being 90% and 100% in those undergoing choledochotomy primarily and secondarily, respectively. No significant difference between rates of success was found, showing that even with more complex circumstances, primarily indicative of transcholedochus approach, satisfactory success rates are achieved. A review of studies of exploration by choledochotomy, gathered 25 patients in which the success rate was of 96%(60). In our series the endoscopic therapy was successful in all patients with whom it was employed, either in pre or postoperative phase. However, the success rate with this procedure has been described to be around 90%. If presented with the impossibility of endoscopic removal of the calculi, especially with large calculi, a biliary stent can be employed to avoid impaction and maintain the duct pervious until the definitive solution is reached(70). However, in isolated studies, not related to reference centers, there are lower rates of success(16, 18). The results of endoscopic procedures vary according to the experience of the endoscopic team and selection of patients, indicating the success rate of clearance of 65% in some series, with report of a success rate of 56% and 91% during pre and postoperative procedures, respectively(64). In a randomized study comparing laparoscopic exploration to postoperative endoscopic treatment, a success rate of 75% after the first intervention in both procedures was identified. With the completion of two or three endoscopic attempts, the success achieved for this group was around 93%(67). Approximately 10% to 15% of patients require more than one endoscopic session to complete clearance of choledocholithiasis(48). It is also important to consider the possibility of residual calculi, approximately 3% to 10%, after the endoscopic procedures(11, 57). When the postoperative endoscopic therapy does not achieve success and as an alternative to reoperation, other conservative methods can be used, such as extra-corporeal lithotripsy(73). In a review about this theme, the authors addressed an interesting point of view. In pre-minimally invasive surgery era, there was any study showing advantages in endoscopic sphincterotomy before open cholecystectomy over conventional open choledocholithotomy(79). It seems that this discussion was started due to technical difficulties encountered in the early experience with laparoscopy.

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Santo MA, Domene CE, Riccioppo D, Barreira L, Takeda FR, Pinotti HW. Common bile duct stones: analysis of the videolaparoscopic surgical treatment

Complications The biliary fistulas are complications of bile duct closure; they may be the result of unclenching of the clip for occlusion of the cystic duct, or directly from the suture of the choledochus, clinically manifesting itself by bile leakage and formation of intra-abdominal collections. The formation of intra-abdominal collection is possible, if the cavity drainage is insufficient, being the computerized tomography as the best method for diagnosis. Clinical support treatment is sufficient as an adequate solution for most cases, provided that no maintaining factors exist, such as difficulty in biliary drainage. In these situations, additional treatment should be established, in particular the endoscopic approach(56). The early biliary fistula, which results in choleperitoneum with acute abdomen, should be treated surgically(77). In our series one case of biliary fistula occurred, originating from the suture of the choledochus, expressed in the first postoperative day by leakage of bile through the drain cavity. Given the association of clinical signs of toxemia and peritoneal irritation, re-laparoscopy was indicated, with favorable postoperative evolution. However, the occurrence of biliary fistula after laparoscopic exploration of the biliary tract is small, below 2%. Originating from the cystic duct, fistula was reported in 1.3%, arising from the detachment of the clip for occlusion of the cystic duct, treated conservatively(59). The possibility of premature displacement of the external biliary drain is not uncommon, occurring in about 2% of cases(7, 67). The occurrence of these complications after the

scheduled removal of the drain, usually around the 21st day after surgery, is exceptional and a possible downstream biliary obstruction should be suspected. The premature displacement of the biliary drain occurred in four patients in our study. In two patients there were no postoperative complications. Another patient, after removal of the transcystic Kehr’s drain, developed a fistula manifested by leakage of bile and sub-hepatic collection, kept under clinical control, with favorable evolution. In one patient there was premature displacement of the transcystic intra-cath (secured with two clips), evolving with a small exteriorized fistula. In general, fistulas arising from inadvertent movement of the biliary drain are favorable, controlled conservatively(59), including eventual endoscopic evaluation with performance of a papillotomy for biliary decompression(33). However, in certain situations, there is a need for surgical re-intervention, which can also be performed by laparoscopy(8, 36, 68). CONCLUSION

The lithiasis in the main biliary tract was presented in an unsuspicious way by about one-third of the cases. The intra-operative cholangiography characterizes the calculi affection in the biliary tract and supports the decision of the conduct, especially in the choice of transcystic or transcholedochus routes. The videolaparoscopic surgical exploration of the biliary tract for treatment of choledocholithiasis achieves high success rates, around 80%, and is associated with low morbidity.

Santos MA, Domene CE, Riccioppo D, Barreira L, Takeda FR, Pinotti HW. Coledocolitíase. Análise do tratamento videolaparoscópico. Arq Gastroenterol. 2012;49(1):41-51. RESUMO – Contexto - Aproximadamente 9% da população brasileira apresenta colecistolitíase e esta incidência aumenta significativamente com o envelhecimento. A coledocolitíase é encontrada em torno de 15% destes pacientes, e de um terço a metade destes casos apresenta-se de maneira assintomática. Uma vez que a litíase do ducto biliar comum é caracterizada através de colangiografia intra-operatória, a exploração cirúrgica laparoscópica pode ser feita através da via transcística, ou diretamente através de coledocotomia. Objetivo - Avaliar os resultados do tratamento laparoscópico da coledocolitíase. Métodos - Setenta pacientes foram avaliados prospectivamente. Todos foram submetidos ao tratamento da coledocolitíase por videolaparoscopia, e as formas de exploração foram comparadas de acordo com os seguintes parâmetros: os critérios de sua indicação, o sucesso no clareamento da via biliar, complicações cirúrgicas. A exploração laparoscópica transcística foi favorecida quando não há critérios para a prática de coledocotomia primária, sendo estes litíase do ducto biliar proximal, cálculos grandes (mais de 8 mm) ou calculose múltipla. Resultados - Verificouse que cerca de um terço dos doentes com coledocolitíase não mostraram qualquer expressão de fatores preditivos (antecedentes clínicos de icterícia e/ou pancreatite aguda, suspeita ultrassonográfica ou alterações de testes laboratoriais relacionados à coledocolitíase). A exploração transcística foi empregada em cerca de 50% dos casos e a coledocotomia em cerca de 30%. A depuração da via biliar através da laparoscopia foi alcançada em 80% dos casos. A exploração transcística, realizada sem fluoroscopia ou coledocoscopia, atingiu baixa taxa de sucesso (em torno de 45%), sendo 10% desses casos com passagem transpapilar de cálculos com menos de 3 mm. A exploração por coledocotomia, primária ou secundária, quando esta foi realizada após o insucesso da via transcística, mostrou alta taxa de sucesso (em torno de 95%). Quando a indicação para coledocotomia era primária, a necessidade de visualização direta através da coledocoscopia para ajudar na remoção dos cálculos foi de 55%. No entanto, quando foi realizada coledocotomia secundariamente, nas situações em que o diâmetro do ducto biliar comum foi maior do que 6 mm, o uso de coledocoscópio com o mesmo propósito foi necessário em cerca de 20% dos casos. Não houve mortalidade nesta série. Conclusão - A exploração laparoscópica da via biliar principal está relacionada com baixa taxa de morbidade. Portanto, o uso da laparoscopia para o tratamento da coledocolitíase depende dos critérios para a escolha do melhor acesso, tornando-se procedimento seguro, com resultados bastante satisfatórios. DESCRITORES – Coledocolitíase. Laparoscopia. Colangiografia.

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56. Morgenstern L, Berci G, Pasternak EH. Bile leakage after biliary tract surgery. A laparoscopic perspective. Surg Endosc. 1993;7:432-8. 57. Neuhaus H, Feussner H, Ungeheuer A, Hoffmann W, Siewert JR, Classen M. Prospective evaluation of the use of endoscopic retrograde cholangiography prior to laparoscopic cholecystectomy. Endoscopy. 1992;24:745-9. 58. NIH Consensus conference. Gallstones and laparoscopic cholecystectomy. JAMA. 1993;269:1018-24. 59. Paganini AM, Lezoche E. Follow-up of 161 unselected consecutive patients treated laparoscopically for common bile duct stones. Surg Endosc. 1998;12:23-9. 60. Perissat J, Huibregtse K, Keane FB, Russell RC, Neoptolemos JP. Management of bile duct stones in the era of laparoscopic cholecystectomy. Br J Surg. 1994;81:799-810. 61. Petelin JB. Laparoscopic approach to common duct pathology. Surg Laparosc Endosc. 1991;1:33-41. 62. Petelin JB. Laparoscopic approach to common duct pathology. Am J Surg. 1993;165:487-91. 63. Phillips EH, Rosenthal RJ, Carrol BJ, Fallas MJ. Laparoscopic trans-cystic duct common bile duct exploration. Surg Endosc. 1994;8:1389-94. 64. Phillips EH, Liberman M, Carrol BJ, Fallas MJ, Rosenthal RJ, Hiatt JR. Bile duct stones in the laparoscopic era. Is the preoperative sphincterotomy necessary? Arch Surg. 1995;130:880-6. 65. Pinotti HW, Domene CE, Volpe P, Santo MA, Onari P. Formação do cirurgião em cirurgia laparoscópica do aparelho digestivo. Experiência de 1.818 intervenções sem acidentes e sem mortalidade. Rev Assoc Med Bras. 1999;45:337-41. 66. Quattlebaum JK Jr, Flanders HD. Laparoscopic treatment of common bile duct stones. Surg Laparosc Endosc. 1991;1:26-32. 67. Rhodes M, Sussman L, Cohen L, Lewis MP. Randomised trial of laparoscopic exploration of common bile duct versus postoperative endoscopic retrograde cholangiography for common bile duct stones. Lancet. 1998;351:159-61. 68. Robinson G, Hollinshead J, Falk G, Moulton J. Technique and results of laparoscopic choledochotomy for the management of bile duct calculi. Aust N Z J Surg. 1995;65:347-9. 69. Rosenthal RJ, Rossi RL, Martin RF. Options and strategies for the management of choledocholithiasis. World J Surg. 1998;22:1125-32.

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70. Sakai P, Artifon ELA, Ishioka S. Uso de endoprótese biliar plástica na coledocolitíase. GED Gastroenterol Endosc Dig. 1999;18:233-6. 71. Santucci L, Natalini G, Sarpi L, Fiorucci S, Solinas A, Morelli A. Selective endoscopic retrograde cholangiography and preoperative bile duct stone removal in patients scheduled for laparoscopic cholecystectomy: a prospective study. Am J Gastroenterol. 1996;91:1326-30. 72. Savassi-Rocha PR, Ferreira JT, Diniz MT, Sanches SR. Laparoscopic cholecystectomy in Brazil: analysis of 33,563 cases. Int Surg. 1997;82:208-13. 73. Schwab G, Pointner R, Wetscher G, Glaser K, Foltin E, Bodner E. Treatment of calculi of the common bile duct. Surg Gynecol Obstet. 1992;175:115-20. 74. Shojaiefard A, Esmaeilzadeh M, Ghafouri A, Mehrabi A. Various techniques for the surgical treatment of common bile duct stones: a meta review. Gastroenterol Res Pract. 2009 Aug 6. doi:10.1155/2009:840208. 75. Sjer AE, Boland DM, van Rijn PJ, Mohamad S. A decade of washing out common bile duct stones with papillary balloon dilatation as a one-stage procedure during laparoscopic cholecystectomy. Surg Endosc. 2010;24:2226–30. 76. Smith PC, Clayman RV, Soper NJ. Laparoscopic cholecystectomy and choledochoscopy for the treatment of cholelithiasis and choledocholithiasis. Surgery. 1992;111:230-3. 77. Speranzini MB, Castillo-Netto JM, Lima Jr SE. Defeitos na drenagem cirúrgica do colédoco. Rev Col Bras Cir. 1993;20:283-8. 78. Strasberg SM, Callery MP, Soper NJ. Laparoscopic surgery of the bile ducts. Gastrointest Endosc Clin N Am. 1996;6:81-105. 79. Targarona EM, Bendahan GE. Management of common bile duct stones: controversies and future perspectives. HPB (Oxford). 2006;6:140-3. 80. Way LW, Admirand WH, Dunphy JE. Management of choledocholithiasis. Ann Surg. 1972;176:347-59. 81. Yeom DH, Oh HJ, Son YW, Kim TH. What are the risk factors for acute suppurative cholangitis caused by common bile duct stones? Gut Liver. 2010;4:363-7.

Arq Gastroenterol

Received 26/7/2011. Accepted 31/8/2011.

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ARTIGO ORIGINAL / ORIGINAL ARTICLE

ARQGA/1588

PREVALENCE OF UPPER DIGESTIVE ENDOSCOPY AND GASTRIC HISTOPATHOLOGY FINDINGS IN MORBIDLY OBESE PATIENTS Judite DIETZ1, Jane Maria ULBRICH-KULCYNSKI2, Katia Elisabete Pires SOUTO3 and Nelson Guardiola MEINHARDT4

ABSTRACT – Context - The prevalence of obesity has been increasing in modern society. Roux-en-y gastric bypass is a bariatric surgery that involves the exclusion of significant part of the stomach. Atrophy, intestinal metaplasia and gastric cancer have been associated with infection by Helicobacter pylori. Objectives - To evaluate the presence of endoscopy findings and histological changes in morbid obese patients for the presence of inflammatory cells, inflammatory activity, lymphoid hyperplasia, H. pylori infection, atrophy and intestinal metaplasia in the gastric mucosa. Methods - Upper digestive endoscopy and gastric histopathological were studied in 126 obese patients in the preoperative evaluation for bariatric surgery. Results - Upper digestive endoscopy abnormalities were diagnosed in 73/126 (57.9%) patients. In three patients (2.4%) the upper gastrointestinal endoscopy diagnosed gastric ulcer and one patient (0.8%) had duodenal ulcer. The histopathological from gastric biopsies of these obese patients showed 65.1% of mucosa inflammation, inflammatory activity in 50.0%, infection by H. pylori in 53.2%, lymphoid hyperplasia in 50.0% and atrophy and/ or intestinal metaplasia in 16.7%. Conclusions - In present study, with routine preoperative upper gastrointestinal endoscopy and histopathological examination, were detected 57.9% patients with endoscopy abnormalities, high prevalence of infection by H. pylori (53%) and 16.7% of gastric atrophy and/or intestinal metaplasia. HEADINGS – Obesity, morbid. Gastric bypass. Endoscopy gastrointestinal. Helicobacter infections.

INTRODUCTION

Obesity is considered to be a growing problem in modern society and its prevalence has been increasing in both developed and developing countries(3). Most types of bariatric surgery include resection, sutures or partial stomach exclusion. The most frequent bariatric surgery is the Roux-en-Y gastric bypass (RYGBP), which involves partial exclusion of the stomach(3). Mucosal alterations after vertical banded RYGBP have not been clearly evaluated because the excluded stomach is not easily reached by conventional endoscopy(18, 21, 39). Infection by the Helicobacter pylori (H. pylori) causes inflammation of the gastric mucosa, which can develop atrophy, intestinal metaplasia, dysplasia and cancer(3, 6, 27). The eradication of the H. pylori leads to a regression of the inflammatory process of the gastric mucosa, the same does not happen in the more advanced stages, with the presence of atrophy and intestinal metaplasia(8, 13, 24, 28, 40).

The present study aims to evaluate the prevalence of endoscopic findings and gastric histopathological alterations in morbidly obese patients. The histopathological alterations comprise presence of inflammation cells, inflammatory activity, presence of lymphoid hyperplasia, infection by the H. pylori, atrophy and intestinal metaplasia in the gastric mucosa. METHODS

Consecutive study conducted in outpatient service with 126 morbidly obese patients [body mass index (BMI) = ≥40 kg/m2] or obese patients (with BMI between 35-40 kg/m2) with indication for bariatric surgery by significant co-morbidities. Upper gastrointestinal endoscopy (UGI) and gastric biopsies was required in the preoperative period. The patients had not undergone UGI or treatment to eradicate H. pylory previously. The study was approved by the Ethics Committee of Hospital Conceição, Porto Alegre, RS, Brazil, and all patients gave informed consent prior to their inclusion in the study. The upper endoscopies were realized by

Financial support: no. The authors declare that they have no conflicts of interest Hospital Nossa Sra. da Conceição (HNSC), Class III Obesity Care Center, Porto Alegre, RS, Brazil. 1 Division of Endoscopy, HNSC; 2 Department of Pathology, Universidade Federal do Rio Grande do Sul (UFRGS); 3 Division of Endocrinology, HNSC; 4 Division of Bariatric Surgery, HNSC, Porto Alegre, RS, Brazil. Correspondence: Dr. Judite Dietz - Rua Augusto L. Lima, 129 - ap. 701 - 90470-120 - Porto Alegre, RS, Brazil. E-mail: judi60@gmail.com

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Dietz J, Ulbrich-Kulcynski JM, Souto KEP, Meinhardt NG. Prevalence of upper digestive endoscopy and gastric histopathology findings in morbidly obese patients

the same gastroenterologist with a video-endoscope Fujinon series 400. Four gastric biopsies were carried out, two in the body and two in the antrum, always in the greater gastric curvature. Los Angeles and Sydney classification were used in the endoscopic analysis of esophagitis and gastritis(1, 8). Alterations of the gastric mucosa were classified by the pathologist as an inflammatory process through the presence of lymphocytic and plasmocytic cells and inflammatory activity through the presence of neutrophils. Lymphoid hyperplasia, presence of H. pylori, epithelial atrophy and intestinal metaplasia were other parameters analyzed. RESULTS

Female population predominated (104 patients - 82.5%). The mean age was 42.08 years and the mean BMI was 51.2 kg/m² (standard deviation ± 9.34). Upper digestive diseases (UGI) were diagnosed in 73 patients (57.9%) through endoscopy, being esophagitis and/or hiatal hernia in 31 cases (24.6%); exsudative/enanthematous gastritis in 6 patients (4.8%); exsudative/enanthematous erosive gastritis in 38 patients (30.2%); gastric ulcer in 3 (2.4%); duodenal ulcer in 1 obese (0.8%). In 53 (42.1%) patients the endoscopy was normal (Table 1). Some patients had more than one lesion at UGI. TABLE 1. Upper digestive endoscopic findings (n = 126) Diagnosis Esophagitis and/or hiatal hernia Exudative/enanthematous gastritis Erosive gastritis Gastric ulcer Duodenal ulcer Normal endoscopy

Patients Percentage 31 24.6% 6 4.8% 38 30.2% 3 2.4% 1 0.8% 53 42.1%

The prevalence of inflammatory cells in the gastric mucosa was found in 82 (65.1%) patients, inflammatory activity in 63 (50.0%), lymphoid hyperplasia in 63 (50.0%), infection by H. pylori in 67 (53.2%), epithelial atrophy and/or intestinal metaplasia in 21 (16.7%) and normal gastric histopathological in 33 (26.2%) patients (Table 2). TABLE 2. Histological analysis of gastric mucosa (n = 126) Diagnosis Patients Percentage Mucosa inflammation 82 65.1% Inflammatory activity 63 50.0% Lymphoid hyperplasia 63 50.0% Helicobacter pylori 67 53.2% Epithelial atrophy and/or intestinal metaplasia 21 16.7% Normal 33 26.2%

Five (8.5%) of the 59 patients with negative results for H. pylori had gastric intestinal metaplasia or epithelial atrophy. In these obese patients, the gastric histopathological parameters studied was not statistical different (P<0.05%) in the patients with stomach or duodenum abnormalities at

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UGI, comparatively with patients without any gastroduodenal abnormalities at UGI (Table 3), except the parameter inflammatory activity (Table 3). The diagnosis of inflammatory activity in gastric mucosa was greater in the group of normal UGI when compared with patients with UGI findings (P = 0.04) (Table 3). DISCUSSION

The prevalence of obese individuals in USA (defined as BMI>30 kg/m2) has increased from 15.3% to 23.9% in the period of 1995 to 2005 and the prevalence of morbidly obese individuals (BMI>40 kg/m2) was of 4.8% in 2005(4). The most performed bariatric procedure is the RYGBP, which involves the exclusion of a significant part of the stomach. Lesions of the excluded stomach have been reported and the diagnosis of this possibility is difficult, onerous, not always available and, as a result of that, gastric diseases, such as ulcers and malignant neoplasms in the excluded stomach may not be diagnosed(18, 21, 39). H. pylori is associated to gastric diseases, such gastritis, ulcer, epithelial atrophy, intestinal metaplasia, lymphoma, adenocarcinoma(9, 31). The prevalence of infection caused by the H. pylori in Latin America is around 60%, varying from 30% to 90%(5). H. pylori causes inflammation of the gastric mucosa in all infected individuals(10). This inflammation consists initially in recruitment of neutrophils, followed by lymphocytes, with later epithelial damage(15). The chronic inflammatory process increases the turnover of epithelial cells and apoptosis (cellular death), evolving to epithelial atrophy and intestinal metaplasia(9). The development of gastric cancer involves several stages and can start with chronic gastritis, atrophy, intestinal metaplasia, dysplasia and finally invasive cancer(6). The World Health Organization concluded, in 1994, for the existence of epidemiological and histological evidences to classify the H. pylori as carcinogenic, group 1(16). Studies have shown the increased incidence of gastric cancer in the population infected by the H. pylori, as well as a positive correlation between intestinal metaplasia and stomach cancer(27, 31, 37). Eradication of the H. pylori leads to a regression of the inflammation, reduction of cellular turnover, increase in the  acid secretion, which are important factors for the prevention of gastric cancer(9, 14, 22, 24). Some studies have concluded for the irreversibility of the carcinogenic process, even with the eradication of the H. pylori, basically when intestinal metaplasia of the stomach was present(13, 14, 20). The incidence of stomach cancer is still now a frequent causes in the group of world malignant neoplasm, being an important cause of mortality(17). Few cases of gastric cancer in the bypassed stomach have been described after RYGBP for morbid obesity(18, 36). Factors such as difficulty of access to the excluded stomach and the reduced number of patients with adequate follow-up after bariatric surgery can interfere in this analysis. The rationale for performing UGI in the candidates to bariatric surgery is to detect and treat lesions that might

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TABLE 3. Obese patients with endoscopic abnormalities in stomach and/or duodenum versus obese with normal stomach and/or duodenum at endoscopy. Gastric histopathological analysis

Mucosal inflammation Inflammatory activity Lymphoid hyperplasia Helicobacter pylori Epithelial atrophy and/or intestinal metaplasia Normal

Abnormal stomach/duodenum (n = 46) n % 48 60.0 35 43.8 40 50.0 41 51.3 15 18.8 22

Normal stomach/duodenum (n = 80) n % 34 73.9 28 60.9 23 50.0 26 56.5 6 13.0

27.5

11

23.9

Odds ratio (95%CI)

P-value

0.52 (0.23-1.17) 0.50 (0.23-1.04) 1.00 (0.48-2.06) 0.80 (0.38-1.67) 1.53 (0.55-4.28)

0.08 0.04 0.57 0.35 0.28

1.20 (0.52-2.78)

0.41

CI = confidence interval

potentially lead to complications in the immediate postoperative period, or result in diseases in the months or years following RYGBP surgery(25, 26). Eradication of the H. pylori as a routine in the preoperative of bariatric surgery is still debated and scarce researches were performed in regards the histology of gastric mucosa(2, 7, 11, 23, 25, 26, 30, 33, 34, 35). Mong et al.(25) studied 272 patients who were candidates to bariatric surgery, in which 33 (12%) showed lesions in the upper digestive tract. Endoscopic findings were: esophagitis in 3.7%; Barrett’s esophagus in 3.7%; gastric ulcer and/or erosive gastritis in 3.2%; duodenal ulcer in 0.7% and gastric carcinoid tumor in 0.3%. Muñoz et al.(26) diagnosed UGI abnormalities in 46% in the obese patients, one of them with gastric cancer. Dutta et al.(11) found prevalence of gastric ulcer of 2.9%, identified histologically gastritis in 23.7% and H. pylori in 6.9% in the obese patients, finding that was not different from the control (non-obese) patients. Safatle-Ribeiro et al.(33) examined the excluded stomach in 40 patients by enteroscopy in an average of 78 months after surgery. In this study, an inflammatory process in the gastric mucosa was diagnosed in 100% of the cases, atrophy in 14%, intestinal metaplasia in 11.4 % and infection by the H. pylori in

20%. All cases of H. pylori infection in the excluded stomach also had the same infection in the gastric functional pouch. A review of the literature revealed that the preoperative prevalence of H. pylori ranges from 6.9% to 61.3% in obese patients undergoing bariatric surgery(2, 11, 12, 19, 23, 29, 30, 32, 38). In the present study, there were 73 (57.9%) cases with abnormalities in upper digestive endoscopy and 53 (42.1%) patients with normal UGI. In 42 cases (33.3%) gastroduodenal abnormalities were diagnosed, like exudative/ enanthematous gastritis, erosive gastritis, gastric or duodenal ulcers. Approximately 50% of the obese patients showed inflammatory activity and infection by the H. pylori and 16% had atrophy and/or intestinal metaplasia in gastric mucosa. The absence of H. pylori infection does not rule out gastric metaplasia or atrophy. The prevalence of H. pylori infection and gastric atrophy and/or intestinal metaplasia in obese patients with gastric or duodenal lesions at endoscopy, comparatively with obese patients with normal stomach or duodenal at UGI, was not statistical different (P<0.05). The long-term follow-up of patients submitted to RYGBP may probably clarify if histopathological abnormalities of the gastric mucosa diagnosed prior surgery could interfere in the postoperative complications or in risk of gastric cancer.

Dietz J, Ulbrich-Kulcynski, Souto KEP, Meinhardt NG. Prevalência de achados gástricos endoscópicos e histopatológicos em pacientes obesos. Arq Gastroenterol. 2012;49(1):52-5. Resumo – Context - A prevalência de obesidade tem aumentado significativamente nos últimos anos. Bypass gástrico em Y-de-Roux, uma das técnicas cirúrgicas realizadas no tratamento da obesidade, envolve exclusão de parte do estômago. Atrofia, metaplasia intestinal e câncer gástrico têm sido associados com infecção pelo Helicobacter pylori. Objetivos - Avaliar a prevalência de achados endoscópicos e alterações histopatológicas em pacientes obesos mórbidos em relação à presença de células inflamatórias, atividade inflamatória, hiperplasia linfóide, infecção pelo H. pylori, atrofia e metaplasia intestinal na mucosa gástrica. Métodos - Achados na endoscopia digestiva alta e histopatologia gástrica foram analisados em 126 pacientes obesos na avaliação pré-operatória de cirurgia bariátrica. Resultados - Anormalidades endoscópicas do trato digestivo superior foram diagnosticadas em 73/126 pacientes, com três pacientes (2,4%) apresentando úlcera gástrica e um paciente (0,8%) com úlcera duodenal. A histopatologia das biopsias gástricas dos obesos estudados revelou 65,1% de processo inflamatório na mucosa, atividade inflamatória em 50,0%; infecção pelo H. pylori em 53,2%, hiperplasia linfóide em 50,0%, atrofia e/ou metaplasia intestinal em 16,7%. Conclusões - No presente estudo, através de endoscopia digestiva alta e histopatologia de biopsias gástricas, foram detectados 57,9% de pacientes com anormalidades endoscópicas, prevalência de infecção pelo H. pylori em 53% e atrofia e/ou metaplasia intestinal em 16,7%. DESCRITORES – Obesidade mórbida. Derivação gástrica. Endoscopia gastrointestinal. Infecções por helicobacter.

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Dietz J, Ulbrich-Kulcynski JM, Souto KEP, Meinhardt NG. Prevalence of upper digestive endoscopy and gastric histopathology findings in morbidly obese patients

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23. Madan AK, Speck KE, Hiler ML. Routine preoperative upper endoscopy for laparoscopic gastric bypass: is it necessary? Am Surg. 2004;70:684-6. 24. Malfertheiner P, Sipponen P, Naumann M, Moayyedi P, Mégraud F, Xiao SD, Sugano K, Nyrén O, Lejondal H. pylori-Gastric Cancer Task Force. Helicobacter pylori eradication has the potential to prevent gastric cancer: a state-of-the-art critique. Am J Gastroenterol. 2005;100:2100-15. 25. Mong C, Van Dam J, Morton J, Gerson L, Curet M, Banerjee S. Preoperative endoscopic screening for laparoscopic Roux-en-Y gastric bypass has a low yield for anatomic findings. Obes Surg 2008;18:1067-73. 26. Muñoz R, Ibáñez L, Salinas J, Escalona A, Pérez G, Pimentel F, Guzmán S, Boza C. Importance of routine preoperative upper GI endoscopy: why all patients should be evaluated? Obes Surg. 2009;19:427-31. 27. Nomura A, Stemmermann GN, Chyou PH, Kato I, Perez-Perez GI, Blaser MJ. Helicobacter pylori infection and gastric carcinoma among Japanese Americans in Hawaii. N Engl J Med. 1991;325:1132-6. 28. Ohata H, Kitauchi S, Yoshimura N, Mugitani K, Iwane M, Nakamura H, Yoshikawa A, Yanaoka K, Arii K, Tamai H, Shimizu Y, Takeshita T, Mohara O, Ichinose M. Progression of chronic atrophic gastritis associated with Helicobacter pylori infection increases risk of gastric cancer. Int J Cancer. 2004;109:138-43. 29. O’Mahony R, Erim T, Szomstein S, Rosenthal R. Prevalence of Helicobacter pylori infection among patients undergoing bariatric surgery. Surg Obes Relat Dis. 2006;2(3):303. 30. Papasavas PK, Gagné DJ, Donnelly PE, Salgado J, Urbandt JE, Burton KK, Caushaj PF. Prevalence of Helicobacter pylori infection and value of preoperative testing and treatment in patients undergoing laparoscopic Roux-en-Y gastric bypass. Surg Obes Relat Dis. 2008;4:383-8. 31. Parsonnet J, Friedman GD, Vandersteen DP, Chang Y, Volgeman JH, Orentreich N, Sibley RK. Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med. 1991;325:1127-31. 32. Renshaw AA, Rabaza JR, Gonzalez AM, Verdeja JC. Helicobacter pylori infection in patients undergoing gastric bypass surgery for morbid obesity. Obes Surg. 2001;11:281-3. 33. Safatle-Ribeiro AV, Kuga R, Iriya K, Ribeiro U Jr, Faintuch J, Ishida RK, Corbett CE, Garrido AB Jr, Ishioka S, Sakai P. What to expect in the excluded stomach mucosa after vertical banded Roux-en-Y gastric bypass for morbid obesity. J Gastrointest Surg. 2007;11:133-7. 34. Sauerland S, Angrisani L, Belachew M, Chevallier JM, Favretti F, Finer N, Fingerhut A, Garcia Caballero M, Guisado Macias JA, Mittermair R, Morino M, Msika S, Rubino F, Tacchino R, Weiner R, Neugebauer EA; European Association for Endoscopic Surgery. Obesity surgery: evidence-based guidelines of the European Association for Endoscopic Surgery (EAES). Surg Endosc. 2005; 19:200-21. 35. Sharaf RN, Weinshel EH, Bini EJ, Rosenberg J, Sherman A, Ren CJ. Endoscopy plays an important preoperative role in bariatric surgery. Obes Surg. 2004; 14:1367-72. 36. Trincado MT, del Olmo JC, García Castaño J, Cuesta C, Blanco JI, Awad S, Carbajo MA. Gastric pouch carcinoma after gastric bypass for morbid obesity. Obes Surg. 2005;15:1215-7. 37. Uemura N, Okamoto S, Yamamoto S, Matsumura N, Yamaguchi S, Yamakido M, Taniyama K, Sasaki N, Schlemper RJ. Helicobacter pylori infection and the development of gastric cancer. N Engl J Med. 2001;345:784-9. 38. Vanek VW, Catania M, Triveri K, Woodruff RW Jr. Retrospective review of the preoperative biliary and gastrointestinal evaluation for gastric bypass surgery. Surg Obes Relat Dis. 2006;2:17-22;discussion 22-3. 39. Voellinger DC, Inabnet WB. Laparoscopic Roux-en-Y gastric bypass with remnant gastrectomy for focal intestinal metaplasia of the gastric antrum. Obes Surg. 2002;12:695-8. 40. Wong BC, Lam SK, Wong WM, Chen JS, Zheng TT, Feng RE, Lai KC, Hu WH, Yuen ST, Leung SY, Fong DY, Ho J, Ching CK, Chen JS, China Gastric Cancer Study Group. Helicobacter pylori eradication to prevent gastric cancer in a high-risk region of China: a randomized controlled trial. JAMA 2004; 291:187-94.

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Received 31/1/2011. Accepted 1/9/2011.

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ARTIGO ORIGINAL / ORIGINAL ARTICLE

ARQGA/1589

Helicobacter pylori ERADICATION DOES NOT INFLUENCE GASTROESOPHAGEAL REFLUX DISEASE: a prospective, parallel, randomized, open-label, controlled trial Lino RODRIGUES Jr., Cintya Miler de FARIA, Stephan GEOCZE and Luiz CHEHTER

ABSTRACT – Context - Helicobacter pylori has been associated with worsening of gastroesophageal reflux disease (GERD). Objective To evaluate the effect of H. pylori eradication in GERD patients. Methods - We conducted a prospective, randomized, controlled trial performing symptom evaluation, endoscopy, histology, manometry and esophageal pH testing on GERD patients. Patients infected with H. pylori were randomized to: 1) eradication treatment plus proton pump inhibitors treatment, or 2) proton pump inhibitors alone. Patients not infected constituted a negative control group. After 3 months, patients were re-evaluated by symptom assessment, endoscopy, histology and manometry. Results - GERD treatment resulted in significantly higher lower esophageal sphincter pressure, as measured by mean expiratory pressure, in H. pylori negative patients. There was significantly lower proportion of hypotensive waves and significantly higher proportion of normotensive waves in non-eradicated patients. All symptom scores were significantly reduced in the post-treatment period compared to baseline, to values that were similar among the three groups, in the post-treatment period. In the post-treatment period, erosive esophagitis was significantly less frequent on those not eradicated. Conclusion - Manometric, clinical and endoscopic data showed no benefit in eradicating H. pylori in GERD. Our data supports the hypothesis that H. pylori eradication does not influence GERD. HEADINGS – Helicobacter infections. Gastroesophageal reflux.

INTRODUCTION

Since the publication of the discovery of Helicobacter pylori (Hp) in 1984(21), its presence has been associated with various diseases, such as peptic ulcer disease (PUD), gastric cancer and mucosal-associated lymphoid tissue lymphoma(3, 19, 27, 29). Gastroesophageal reflux disease (GERD) is a multifactorial disease whose mechanisms include lower esophageal sphincter (LES) hypotension, LES transient relaxation, esophageal/gastric dysmotility and altered gastric juice composition, among others(8, 23). GERD is very prevalent and often co-exists with Hp infection. Whether Hp infection worsens, attenuates or does not influence GERD is not fully demonstrated yet. Epidemiological studies have shown similar prevalences of GERD in both healthy and Hp infected subjects(26, 28). However, Hp-derived antritis is associated with hypergastrinemia(37) with consequent gastric hyperacidity which may aggravate GERD(17). Additionally, cardia inflammation, associated to Hp-

derived pangastritis, can increase the frequency of LES transient relaxation either via local or vagus-mediated pathways(10). Patients with esophagitis have higher prevalence of cytotoxin producing (TOX+) Hp strains, which may lead to increased direct mucosal damage(11, 34). Hp infection also alters proton pump inhibitors (PPI) treatment efficacy. Labenz et al.(15) demonstrated that Hp-positive patients reached higher pH levels during omeprazole treatment than non-infected patients. On the other hand, hypergastrinemia associated with Hp infection can increase LES pressure, while atrophic gastritis found in Hp pangastritis leads to hypochloridria, which may attenuate GERD(22). Additionally, it was found a higher incidence of erosive esophagitis (EE) in patients whose Hp infection was eradicated(16) and a lower risk of GERD complications, like Barrett’s esophagus and esophageal cancer, in patients infected with CagA + Hp strains(5, 36). In light of these findings, we conducted a prospective trial to evaluate the effects of Hp eradication on GERD patients treated with PPI.

The authors have no conflict of interest to declare. Disciplina de Gastroenterologia da Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brasil. Correspondence: Dr. Lino Rodrigues-Júnior – Rua Botucatu, 720 – 2º andar – 04023-900 – São Paulo, SP, Brasil. E-mail: linorj@gmail.com

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Rodrigues Jr L, Faria CM, Geocze S, Chehter L. Helicobacter pylori eradication does not influence gastroesophageal reflux disease: a prospective, parallel, randomized, open-label, controlled trial

METHODS

This was a prospective, parallel, randomized, open-label, controlled trial. Patients who satisfy selection criteria were submitted to symptom evaluation, upper gastrointestinal endoscopy (UGE), histological evaluation and esophageal manometry and 24h pH testing. Hp positive patients were randomized to eradication treatment or PPI treatment. Those not infected constituted a negative control group. Three moths after baseline evaluation, patients were re-evaluated by symptom assessment, UGE, histology and esophageal manometry. Written informed consent was obtained from all individuals before any study procedure was performed. This study was approved by the Institutional Review Board of the Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil, under record number 127600 and complies with all national and international ethical standards in clinical research. Patients GERD patients at least 18 years old, with typical symptoms for at least 6 months before enrollment, confirmed by UGE and/or ambulatory pH study were included in the trial. We excluded patients with history of complicated PUD, hepatobiliary diseases, diabetes, adrenal and thyroid dysfunction, Chagas’ and Crohn’s disease, connective tissue diseases; those with previous eradication of Hp, previous upper digestive tract surgery, presence of active PUD, neoplasia, eosinophilic esophagitis or grade IV esophagitis; pregnant or lactating women; those using antibiotics, bismuth-containing medications, antacids, H2-receptor antagonists, PPIs, prokinetics, steroidal or non-steroidal anti-inflammatory drugs (NSAID), xanthines, antidepressants, benzodiazepines, chemotherapeutic agents, calcium channel blockers, anticholinergics or hormone replacement therapy within 30 days from baseline measurements or those unable to follow the protocol requirements. Symptom assessment Symptoms were assessed for frequency and severity. Frequency was coded as follows: 0 = asymptomatic; 1  =  less than twice/week, 2 = 2 to 4 times/week, and 3 = more than 4 times/week. Severity was coded as follows: 0  =  asymptomatic; 1 = symptoms with spontaneous resolution; 2 = symptoms that resolve with symptomatic treatment (antacids), and 3 = symptoms that persist despite of symptomatic treatment. For each symptom, a score was calculated multiplying frequency by severity. Heartburn and regurgitation scores were added to define the Typical Symptoms Score (TySS). All symptom scores were added to define the Total Symptoms Score (ToSS). Symptom assessment was performed by the same investigator before (baseline), and 2 months after treatment was completed. Symptom improvement was defined as at least 50% decrease in symptom scores.

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Upper gastrointestinal endoscopy UGE was performed in all patients. EE presence and severity were assessed using Savary-Miller classification. Hp status was evaluated in fragments collected from the corpus and antrum, by both urease and histology methods. Histology examinations were performed after staining with Giemsa. Patients were defined as Hp-positive if either urease or histology test was positive. Presence and severity of gastritis were evaluated according to the updated endoscopic division of the Sydney system(9). Hp eradication was defined as the histological absence of bacteria and a negative urease test result 3 months after eradication treatment. Manometry All medications that alter esophageal motility were discontinued 7 days prior to manometry. Medtronics and Synetics hardware/software were used. Conventional stepwise pull-through technique was used to define position and pressure of LES and upper esophageal sphincter. Esophageal body peristalsis was assessed after 10 wet swallows 3 cm above LES. Quantitative evaluation was performed according to Dalton and Castell(6), Cargill(4), and Mittal et al.(24). LES resting pressure was calculated both as maximum expiratory pressure (MEP) and mean respiratory pressure (MRP). MRP was defined as the arithmetic mean of the mean respiratory amplitudes in the four radial channels (in mm Hg), measured within the high pressure zone, immediately before the pressure inversion point (PIP), relatively to gastric baseline. MEP was defined as the arithmetic mean of the expiratory values in the four radial channels (in mm Hg), measured within the high pressure zone, immediately before the PIP, relatively to gastric baseline. All tracings were analyzed by the same investigator. 24h esophageal pH study Antisecretory and prokinetic drugs were not allowed 2 weeks prior to pH study, as well as antacids 12 hours before the pH study, which was performed according to standard technique(7), using Medtronics and Synetics hardware/software. In patients with extra-esophageal symptoms, two-channel catheters were used, in order to detect supra-esophageal reflux. Patients were asked to behave normally, eating, working and sleeping in their regular patterns. All tracings were analyzed by the same investigator. Symptom association was evaluated using the symptom association probability (SAP), and was defined as positive if SAP was equal or greater than 80%. GERD confirmation by pH study required abnormal acid exposure in distal or proximal channels or a positive SAP. Groups and treatment assignment After baseline assessments, Hp-positive patients were randomized in a 1:1 ratio to receive open-label eradication treatment or lansoprazole by one of the investigators. Randomization codes were generated by a computer program; concealed allocation was achieved by using sequentially numbered opaque envelopes prepared by one of the authors not involved in patient randomization and assessment.

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Rodrigues Jr L, Faria CM, Geocze S, Chehter L. Helicobacter pylori eradication does not influence gastroesophageal reflux disease: a prospective, parallel, randomized, open-label, controlled trial

All patients received non-pharmacological recommendations for GERD. Those randomized for eradication received 1-week triple therapy (amoxicillin 2 g/d, clarithromycin 1 g/d and lansoprazole 60 mg/d), followed by lansoprazole 30  mg/d for 7 additional weeks. Patients randomized to lansoprazole received lansoprazole 60 mg/d for 1 week followed by 30 mg/d for 7 additional weeks. Hp-negative patients received lansoprazole 60 mg/d for 1 week followed by 30 mg/d for 7 additional weeks. Treatment compliance was evaluated by pill count and was required to be greater than 80% during the study. Adherence to non-pharmacological recommendations was evaluated by the investigator at the post-treatment visit and classified as total (followed all recommendations), partial (followed some recommendations) and absent (followed none). One month after the end of treatment, patients underwent symptomatic and endoscopic assessment and esophageal manometry. Based on post-treatment Hp status, patients were classified into: group 1 (eradicated) – patients randomized to eradication treatment that were negative after 3 months; group 2 (non-eradicated) – patients randomized to receive lansoprazole plus patients randomized to the eradication in whom treatment was unsuccessful; and group 3 (negative control) – patients originally Hp negative. Statistical analysis Demographic data was tabulated for each group. Simple randomization was used, with a 1:1 ratio. Nominal variables were summarized by absolute (n) and relative (%) frequency. Continuous variables were summarized as means or medians ± standard deviation (SD). Categorical variables were analyzed using Pearson’s chi-square (or Fisher’s exact, when appropriate). Two continuous paired variables were tested with Wilcoxon signed-rank test. When

three or more continuous variables were evaluated, the Kruskal-Wallis one-way analysis of variance was used. McNemar’s test was applied for dichotomous, categorical variables and the Spearman’s rank correlation coefficient was used as a measure of statistical dependence between two variables. The primary evaluation was the final (post-treatment) manometric values from the eradicated group (group 1) compared to the non-eradicated (group 2) and negative control (group 3) scores. Sample size was calculated by UNIFESP’s statistical department, assuming α = 0.05 (two-sided) and (1-β) = 80%. Data was analyzed with Statistical Package for Social Sciences (SPSS Inc, Chicago, USA) version 17.0. RESULTS

Thirty-two patients were included in the trial. Nineteen (59.4%) were Hp positive and 13 were Hp negative (40.6%). Eleven Hp positive patients received triple therapy and eight Hp positive received lansoprazole treatment. Nine patients were negative after 3 months (eradication rate = 81.8%) and entered group 1. Group 2 was composed by eight Hp positive patients treated with lansoprazole plus two patients treated with triple therapy that remained positive (eradication failures). Group 3 was composed by 13 Hp negative subjects. All patients who received at least one dose of study medication were analyzed, composing an intention-to-treat analysis set. The three groups were comparable at baseline regarding demographic variables. GERD severity was similar among the groups at baseline, as the groups had similar esophagitis rates, DeMeester scores and ToSS, atypical symptoms and hiatal hernia presence. Manometric parameters were also similar at baseline between the three groups (Table 1).

TABLE 1. Baseline characteristics by group Variable Males Age (years)§ Alcohol use BMI (kg/m2)§ Presence of hiatal hernia Presence of erosive esophagitis Presence of atypical symptoms Total symptom score§ DeMeester composite score§ LES pressure - MRP (mm Hg) § LES pressure - MEP (mm Hg) § LES length (cm) § Diaphragmatic crura pressure (mm Hg) § Abnormal peristalsis §

Group 1 (eradicated) n (%) 6 (66.7) 37.40 (12.5) 3 (33.3) 30.92 (10.06) 1 (11.1) 5 (55.6) 5 (50.0) 13.56 (7.30) 26.57 (18.86) 19.9 (12.5) 10 (11.27) 2.94 (0.63) 28.71 (10.63) 2 (22.2%)

Group 2 (non-eradicated) Group 3 (negative control) n (%) n (%) 7 (70.0) 9 (69.2) 40.4 (7.5) 44.8 (9.8) 2 (20.0) 5 (38.5) 24.80 (3.28) 25.96 (2.86) 0 (00.0) 2 (15.4) 5 (50.0) 9 (69.2) 7 (53.8) 5 (55.6) 12.90 (4.89) 14.92 (6.17) 17.28 (10.48) 18.03 (14.99) 13.48 (6.05) 15.22 (5.88) 3.93 (5.97) 3.91 (3.95) 3.05 (0.86) 3.00 (0.50) 26.08 (5.92) 36.85 (17.46) 1 (10.0%) 0 (00.0%)

P-value 0.987 0.245 0.631 0.130 0.445 0.624 0.969 0.725 0.335 0.237 0.121 0.937 0.131 0.212

expressed as mean (standard deviation). BMI = body mass index

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Rodrigues Jr L, Faria CM, Geocze S, Chehter L. Helicobacter pylori eradication does not influence gastroesophageal reflux disease: a prospective, parallel, randomized, open-label, controlled trial

LES Resting LES pressures, when measured by MRP were not affected by treatments in any group. Furthermore, posttreatment values for all groups were also similar. However, when measured by MEP, group 3 showed a small but statistically significant rise in pressure after lansoprazole treatment compared to baseline (6.2 ± 4.9 vs 3.9 ± 3.9, respectively, P = 0.033). Esophageal peristalsis Amplitude and duration of contractile waves at distal esophagus were not altered neither by eradication nor lansoprazole treatment in all groups. Post-treatment values were also similar between the groups. However, patients who remained Hp positive (group 2) had a smaller proportion of hypotensive waves (23.6% ± 41.8% vs 35.3% ± 43.0%, P = 0.043) and a higher proportion of normotensive waves (75.4% ± 41.3% vs 55.2% ± 44.6%, P = 0.012) after lansoprazole treatment compared to baseline values. Upper esophageal sphincter parameters were similar in all groups. Manometric parameters are summarized in Table 2. Clinical evaluation Among all patients, 87.5% had symptom improvement with treatment (77.8%, 90.0% and 92.3% in the groups 1, 2 and 3, respectively; P = 0.574). Every individual symptom score was significantly reduced in the post-treatment period compared to baseline measurements (Table 3). ToSS and TySS were also significantly reduced from baseline for each group (Figure 1). In the post treatment period, ToSS and TySS reached similar values between the three groups (ToSS, P = 0.411; TySS, P = 0.276).

Body mass index (BMI) pre and post-treatment did not differ (Table 4), as well as adherence to non-pharmacological recommendations (total in 66.7%, 60.0 and 71.9%, for groups 1, 2 and 3, respectively, P = 0.394). Ten (31.3%) patients reported side-effects with treatments, 9 (81.8%) of 11 patients who received eradication treatment and 1 (4.8%) of 21 who received lansoprazole treatment (P = 0.000). From those receiving eradication treatment: six (63.6%) had transient diarrhea, one (9.1%) had a cutaneous rash, one (9.1%) had dizziness and one (9.1%) had dysgeusia. From those receiving lansoprazole treatments only one (4.8%) had headache. All reported side-effects were mild in severity, did not interfere with treatment continuation and resolved spontaneously after treatment completion. Endoscopic and histological evaluation EE was present in 59.4% of all patients and did not differ between groups at baseline. Three months after treatment EE was present in significantly smaller proportion in group 2. Endoscopic gastritis presence also did not differ at baseline and was present at similar frequencies in the post-treatment periods (Table 4). Detailing of the EE grade according to period and group is presented in Table 5. According to histology, gastritis was present at baseline in all Hp positive subjects. There was no significant difference in gastritis patterns (pangastritis in 100% and 90% in groups 1 and 2, respectively, P = 0.526). After Hp eradication, gastritis frequency became similar to the Hp negative group (Table 4). There was no association between LES pressure and gastritis pattern (P = 0.851 using MEP and P = 0.682 using MRP). There was also no correlation between gastritis pattern and symptom improvement [correlation coefficient (r) = 0.234; P = 0.197].

TABLE 2. Manometric features pre and post-treatment, by groups Variable Lower esophageal sphincter Length (cm) Pressure, MRP (mm Hg) Pressure, MEP (mm Hg) Esophageal body Amplitude, distal (mm Hg) Duration, distal (s) % Hypertensive % Normotensive % Hypotensive Upper esophageal sphincter Length (cm) Pressure (mm Hg)

Group 1 (eradicated) mean (SD) pre post 2.9 (0.6) 19.9 (12.5) 10.0 (11.2)

2.4 (1.2) 15.9 (4.8) 6.5 (1.4)

Group 2 (non-eradicated) Group 3 (negative control) mean (SD) mean (SD) pre post pre post

P-value*

3.0 (0.7) 13.5 (6.0) 3.9 (6.0)

2.9 (1.2) 15.9 (6.6) 6.8 (3.8)

3.0 (0.5) 15.2 (5.9) 3.9 (3.9)

2.8 (1.6) 16.2 (5.9) 6.2 (4.9)

NS NS 0.033‡

109.3 (47.1) 105.9 (51.7) 4.6 (0.7) 5.2 (1.3) 8.9 (19.0) 25.3 (38.2) 60.5 (34.3) 46.1 (36.3) 30.6 (37.8) 28.6 (38.5)

85.3 (47.1) 4.5 (0.9) 9.4 (29.7) 55.2 (44.6) 35.3 (43.0)

91.9 (51.7) 4.7 (0.9) 0.9 (2.9) 75.4 (41.3) 23.6 (41.8)

115.0 (96.3) 4.4 (0.9) 16.2 (37.3) 64.4 (39.8) 20.3 (29.4)

88.9 (34.6) 4.7 (1.2) 10.1 (20.1) 64.7 (27.2) 25.2 (29.8)

NS NS NS 0.012§ 0.043†

3.8 (0.8) 69.1 (26.2)

2.9 (0.8) 71.8 (35.6)

3.0 (0.9) 64.1 (35.1)

3.1 (0.7) 76.1 (35.1)

3.1 (1.2) 61.0 (18.5)

NS NS

3.4 (1.0) 76.7 (25.0)

SD = standard deviation. * Whenever P values for all comparisons (pre vs post-treatment in each group and inter-groups post-treatment values) are >0.05, NS is indicated. If any comparison is significant, the P value for that comparison is indicated; ‡ Group 3, pre vs post; § Group 2, pre vs post; † Group 2, pre vs post.

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Rodrigues Jr L, Faria CM, Geocze S, Chehter L. Helicobacter pylori eradication does not influence gastroesophageal reflux disease: a prospective, parallel, randomized, open-label, controlled trial

(All patients)

30,0

30,0

25,0

25,0

20,0

20,0

15,0

15,0

10,0

10,0

n = 32 P < 0,001

5,0 0,0

Pre

n=9 P = 0,023

5,0 0,0

Post Group 2 (Non-eradicated)

25,0

25,0

20,0

20,0

15,0

15,0

Pre

Post Group 3 (Negative control)

10,0

10,0 n = 10 P = 0,007

5,0 0,0

Group 1 (Eradicated)

Pre

n = 13 P < 0,001

5,0 0,0

Post

Pre

Post

FIGURE 1. Comparison of pre and post total symptom score. Means are represented as thick horizontal bars TABLE 3. Symptom scores pre and post-treatment for all patients Variable Symptom scores* ToSS TySS Heartburn Regurgitation Bloating Belching Sialorrhea *

All groups pre

post

13.91 (6.02) 8.19 (3.61) 5.38 (2.86) 2.81 (1.86) 2.03 (2.06) 1.50 (1.57) 0.88 (1.41)

4.34 (5.18) 2.97 (3.52) 1.84 (2.50) 1.13 (1.45) 0.38 (0.79) 0.38 (0.87) 0.25 (0.76)

P-value < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.001 0.01

Values expressed as means (standard deviation). ToSS = total symptom score; TySS = typical symptom score

TABLE 4. Endoscopic, histological and clinical variables pre and post-treatment, by group Variable Erosive esophagitis (%) Endoscopic gastritis (%) Histological gastritis (%) BMI‡

Group 1 (eradicated) pre post 55.6 66.7 44.4 30.0 100.0 77.2 30.9 (10.1) 30.4 (11.0)

Group 2 (non-eradicated) pre post 50.0 20.0 40.0 25.0 100.0 100.0 24.8 (3.3) 24.9 (4.0)

Group 3 (negative control) pre post 69.2 69.2 69.2 45.0 46.2 78.1 26.7 (6.3) 25.9 (8.0)

P-value* 0.040§ NS 0.001† NS

Whenever P values for all comparisons (pre vs post-treatment in each group and inter-groups post-treatment values) are >0.05, NS is indicated. If any comparison is significant, the P value for that comparison is indicated; § Post-treatment: Group 2 vs Groups 1 and 3; † Pre-treatment, Group 3 vs Groups 1 and 2; ‡ Body mass index, expressed as mean (standard deviation)

*

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Rodrigues Jr L, Faria CM, Geocze S, Chehter L. Helicobacter pylori eradication does not influence gastroesophageal reflux disease: a prospective, parallel, randomized, open-label, controlled trial

TABLE 5. Frequency of erosive esophagitis by period and group, according to Savary-Miller Period Pre-treatment

Post-treatment

*

Group Eradicated Non-eradicated Negative control Total Eradicated Non-eradicated Negative control Total

Absent n (%) 4 (44.4) 5 (50.0) 4 (30.8) 13 (40.6) 3 (33.3) 8 (80.0) 4 (30.8) 15 (46.9)

Grade 1 n (%) 5 (55.6) 5 (50.0) 8 (61.5) 18 (56.3) 6 (66.7) 2 (20.0) 8 (61.5) 16 (50.0)

Grade 2 n (%) 0 (0.0) 0 (0.0) 1 (7.7) 1 (3.1) 0 (0.0) 0 (0.0) 1 (7.1) 1 (3.1)

P-value*

NS

0.040§

Whenever P values for all comparisons are >0.05, NS is indicated. If any comparison is significant, the P value for that comparison is indicated; § for absence versus presence: Group 2 vs Groups 1 and 3

DISCUSSION

Baseline characteristics (clinical, endoscopic, manometric and pHmetric) between Hp positive and negative subjects were comparable. Manes et al.(20) also found no significant difference between Hp positive and negative subjects, regarding manometric variables. They evaluated, however, only baseline differences, rather than pre and post-treatment differences. In our study, manometric data analysis showed a small but significant rise in LES pressure, when measured by MEP, in Hp negative subjects treated with PPI. However, there was no difference on the post-treatment LES pressures between Hp positive (non-eradicated) and Hp negative (either eradicated or originally negative). That is in accordance with findings by Sarnelli et al.(30), who found similar LES resting pressures in negative patients and those whose infection was eradicated. Although these authors found a significantly longer acid clearance time in pH monitoring in eradicated subject, compared to negative controls, esophageal distal wave amplitudes were similar in the two groups(30). We found similar results for both amplitude and duration of peristaltic waves on distal esophagus, as well as for the proportion of hypotensive, normotensive and hypertensive waves. However, when comparing pre and post treatment data, a significant decrease in the mean percentage of hypotensive and rise in the mean percentage of normotensive waves in subjects that were Hp positive and were not eradicated was also noted. Both effects were not present in eradicated patients and one may suggest that those benefits were masked by the effect of the eradication itself. However the effect was also not present in negative controls, which makes it unlikely that eradication exerted a detrimental effect on esophageal peristalsis. In this study, conventional manometry was used. Although measurements were performed consistently at the same period of the day, circadian variations of measured parameters can not be excluded. The use of ambulatory manometry in future studies may provide useful. GERD treatment was effective in reducing disease symptoms: all symptom scores improved significantly from pre to post-treatment, and they improved similarly between all

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groups, for every score calculated. At week 12, there were no significant differences on both total and typical scores. These findings are consistent with previous randomized controlled trials that showed no GERD benefit on treating Hp(18). The present trial had a limited duration of only 12 weeks; however, the clinical findings here reported seem to be maintained throughout 1 year of observation, as demonstrated by a similar randomized double-blind placebocontrolled trial(25). Additionally, the relatively small sample size studied may have interfered with the statistical power of the measurements. Nevertheless, statistical significance was reached for the primary comparison and clinical scores. H. pylori has been associated with augmentation of the effect of PPIs on intragastric pH(13, 33) and its eradication may restore full gastric secretory capacity with lower pH values achieved with omeprazole treatment(4). In our study, however, endoscopic resolution of EE was achieved in only 53% of the treated patients. It is known that subjects with atypical symptoms need PPI regimens of longer duration. The fact that 53.1% of our subject sample had atypical symptoms may explain why the EE healing rate was lower that expected in our sample. Furthermore, EE healing was achieved in a significantly greater proportion on patients whose infection was not eradicated. Hackelsberger et al.(12) have found lower reflux grades in Hp-positive patients with GERD compared with Hp-negative ones. Likewise, Sekiguchi et al.(32) also found a greater proportion of Hp presence in patients with mild reflux esophagitis, compared with those with severe reflux esophagitis. Another study achieved results similar to ours: Schenk at al.(31) studied prospectively GERD patients and found that Hp-negative subjects had more severe esophagitis and significantly higher prevalence of Barrett’s esophagus, however failing to show significant difference on PPI dosage needed to keep patients asymptomatic or esophagitis-free. Drug effect seems unlikely to explain differences found, since clarithromycin effect on augmenting LES pressure and esophageal wave amplitude(2) is not expected to last after 3 months. Additionally, there is no sound evidence that lansoprazole has a direct effect in LES pressure or peristalsis, apart from

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Rodrigues Jr L, Faria CM, Geocze S, Chehter L. Helicobacter pylori eradication does not influence gastroesophageal reflux disease: a prospective, parallel, randomized, open-label, controlled trial

indirect influence due to gastrin changes, which is known to modulate LES pressure in healthy subjects(1). Although gastrin levels were not measured, we would not expect any PPI effect one month after PPI discontinuation, since even with delayed release formulations of PPIs, gastrin levels return to baseline values within 1 week of drug discontinuation(38). We found no correlation between gastritis pattern and LES pressures. Additionally, post-treatment LES pressures between all groups did not differ, which makes it unlikely that it could explain any clinical or endoscopic difference between groups. Conversely, subjects that remained Hp positive had a better peristaltic profile with greater proportion of normotensive waves and fewer hypotensive waves which may explain the smaller esophagitis rates in this group, since investigations on pathophysiology of reflux esophagitis using combined high resolution manometry and pHmetry, like the one by Ikawari et al.(13), have demonstrated that esophageal clearance is the key factor on esophagitis healing. Preservation of acid secretion on those Hp-negative or restoration of secretion on those eradicated may lead to lower rates of esophagitis healing, compared to Hp-positive patients, whose secretory capacity remains impaired(16). Besides that putative link, other factors may confound this relationship, like timing of measurements, changes on BMI, occurrence of acid rebound amongst others. The gastritis pattern may modify GERD symptomatic response to Hp eradication: Hp infection causing corpus gastritis may interfere with gastric acid production, while eradicating Hp could restore secretory capacity and trigger GERD. Additionally, PPI treatment in Hp-positive patients may lead to strong corpus gastritis(14), lowering acid output substantially. In our study we did not find any correlation between gastritis pattern and GERD symptomatic response. There was also no significant difference in the gastritis pattern between groups 1 and 2, so we do not expect that this could have accounted for the endoscopic findings. Similarly, there was no significant difference in weight variation (pre minus post) between the three groups. Likewise, adherence to non-

pharmacological recommendations may not have interfered in the results, since a similar proportion of patients in each group followed all recommendations given. We can not infer on acid rebound, since pH testing was carried out only at baseline, which constitutes one of the limitations of this study. Changes in the feeding patterns besides the standard recommendations for GERD treatment were not measured in this study. Selective alterations on these variables could interfere with the endoscopic findings and may have contributed to the discrepancy observed with the clinical data. GERD is a multi-factorial disease and other factors not measured in this study like mucosal barrier permeability, gastric emptying, submucosal blood flow and epithelial function may be implicated. Other underlying mechanisms may still be discovered, since known parameters can not explain disease course in all patients. Our prospective trial shows that the treatment of Hp infection is effective in 81.8% of patients, a result similar to what other Brazilian groups reported previously in trials with larger samples(35). Treatment is well tolerated with most side-effects being mild and self-limited in nature. In conclusion, our study supports the hypothesis that Hp eradication does not influence GERD. Symptom resolution after treatment was not influenced by eradication. Statistically significant manometric differences were demonstrated only in the non-eradicated and Hp-negative groups, with endoscopic healing significantly more frequent in patients whose Hp infection was not eradicated. Clinical, endoscopic and manometric data collected in this study showed no benefit in eradicating Hp in GERD. ACKNOWLEDGMENTS

We would like to thank UNIFESP’s Disciplina de Gastroenterologia Cirúrgica - Motility Laboratory (SEFIDI), especially Dr. Edison Yanagita, for providing the equipment for motility testing; UNIFESP’s Departamento de Patologia, especially Dr. Ricardo Artigiani for performing histology evaluations, and Medley S/A for donating the study medication.

Rodrigues Jr L, Faria CM, Geocze S, Chehter L. Erradicação do Helicobacter pylori não influencia a doença do refluxo gastroesofágico: estudo prospectivo, paralelo, randomizado, aberto e controlado. Arq Gastroenterol. 2012;49(1):56-63. RESUMO – Contexto - Existem trabalhos associando a erradicação do Helicobacter pylori à piora da doença do refluxo gastroesofágico (DRGE). Objetivo - Avaliar o efeito da erradicação do H. pylori em pacientes com DRGE. Métodos - Estudo prospectivo, randomizado, controlado em que se avaliaram clínica, endoscopia digestiva alta, histologia, manometria e pHmetria de pacientes com DRGE. Pacientes infectados pelo H. pylori foram randomizados para: 1) erradicação da infecção seguida de tratamento com inibidor de bomba protônica, ou 2) tratamento com inibidor de bomba protônica apenas. Os não-infectados constituíram grupo-controle negativo. Após 3 meses, os pacientes foram reavaliados. Resultados - A pressão do esfíncter inferior do esôfago, medida pela pressão expiratória máxima, foi significativamente maior em pacientes H. pylori negativos. Houve redução significativa na proporção de ondas hipotensivas e aumento significativo na proporção de ondas normotensivas nos pacientes que permaneceram Helicobacter pylori positivos. Todos os escores de sintomas foram reduzidos significativamente em comparação ao período inicial, para valores semelhantes, entre os três grupos, no pós-tratamento. Esofagite erosiva foi significativamente menos frequente no período pós-tratamento no grupo não-erradicado. Conclusão - Os achados manométricos, clínicos e endoscópicos não mostram benefício em se erradicar a infecção em pacientes com DRGE. Este estudo apoia a hipótese de que a erradicação do H. pylori não influencia a DRGE. DESCRITORES – Infecções por helicobacter. Refluxo gastroesofágico.

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Allescher HD, Stoschus B, Wünsch E, Schusdziarra V, Classen M. Effect of human gastrin-17 with and without acid suppression on human esophageal motility. Z Gastroenterol. 1995;33:385-91. Bortolotti M, Pandolfo N, La Rovere G, Giovannini M, Miglioli M. Effect of clarithromycin on esophageal motility. Dis Esophagus. 2000;13:231-3. Buckley M, Culhane A, Drumm B, Keane C, Moran AP, O’Connor HJ, Collins J, Kelleher D, McAvinchey D, Sloan J, O’Morain C. Guidelines for the management of Helicobacter pylori-related upper gastrointestinal diseases. Irish Helicobacter Pylori Study Group. Ir J Med Sci. 1996;165:1-11. Cargill G. How can the contribution of intra-abdominal pressure, crural diaphragm, and smooth muscle sphincter be distinguished in LESP and in LES movements? In: Giuli R, editor. O.E.S.O - The esophagogastric junction. Paris: John Libbey Eurotext; 1998. p. 19-23. Chow WH, Blaser MJ, Blot WJ, Gammon MD, Vaughan TL, Risch HA, PerezPerez GI, Schoenberg JB, Stanford JL, Rotterdam H, West AB, Fraumeni JF Jr. An inverse relation between cagA+ strains of Helicobacter pylori infection and risk of esophageal and gastric cardia adenocarcinoma. Cancer Res. 1998;58:588-90. Dalton CB, Castell DO. Measurements and interpretations. In: Castell DO, Castell JA, editors. Esophageal motility testing. Norwalk, Connecticut: Appleton & Lange; 1994. p. 61-80. Demeester TR, Johnson LF, Joseph GJ, Toscano MS, Hall AW, Skinner DB. Patterns of gastroesophageal reflux in health and disease. Ann Surg. 1976;184:459-70. Dent J. Gastro-oesophageal reflux disease. Digestion. 1998;59:433-45. Dixon MF, Genta RM, Yardley JH, Correa P. Classification and grading of gastritis. The updated Sydney System. International Workshop on the Histopathology of Gastritis, Houston 1994. Am J Surg Pathol. 1996;20:1161-81. Genta RM, Huberman RM, Graham DY. The gastric cardia in Helicobacter pylori infection. Hum Pathol. 1994;25:915-9. Gisbert JP, Pajares JM, Losa C. Helicobacter pylori and gastroesophageal reflux disease: friends or foes? Hepatogastroenterology. 1999;46:1023-9. Hackelsberger A, Schultze V, Gunther T, et al. H. pylori prevalence in reflux esophagitis - a case-control study [abstract]. Gastroenterology. 1997;112(Suppl):A137. Iwakiri K, Kawami N, Sano H, Tanaka Y, Umezawa M, Kotoyori M, Hoshihara Y, Sakamoto C. Mechanisms of excessive esophageal acid exposure in patients with reflux esophagitis. Dig Dis Sci. 2009;54:1686-92. Kuipers EJ, Uyterlinde AM, Peña AS, Hazenberg HJ, Bloemena E, Lindeman J, Klinkenberg-Knol EC, Meuwissen SG. Increase of Helicobacter pylori-associated corpus gastritis during acid suppressive therapy: implications for long-term safety. Am J Gastroenterol. 1995;90:1401-6. Labenz J, Tillenburg B, Peitz U, Idström JP, Verdú EF, Stolte M, Börsch G, Blum AL. Helicobacter pylori augments the pH-increasing effect of omeprazole in patients with duodenal ulcer. Gastroenterology. 1996;110:725-32. Labenz J, Blum AL, Bayerdörffer E, Meining A, Stolte M, Börsch G. Curing Helicobacter pylori infection in patients with duodenal ulcer may provoke reflux esophagitis. Gastroenterology. 1997;112:1442-7. Labenz J, Malfertheiner P. Helicobacter pylori in gastro-oesophageal reflux disease: causal agent, independent or protective factor? Gut. 1997;41:277-80. Laine L, Sugg J. Effect of Helicobacter pylori eradication on development of erosive esophagitis and gastroesophageal reflux disease symptoms: a post hoc analysis of eight double blind prospective studies. Am J Gastroenterol. 2002;97:2992-7. Malfertheiner P, Mégraud F, O’Morain C, Bell D, Bianchi Porro G, Deltenre M, Forman D, Gasbarrini G, Jaup B, Misiewicz JJ, Pajares J, Quina M, Rauws E. Current European concepts in the management of Helicobacter pylori infection-the Maastricht Consensus Report. The European Helicobacter Pylori Study Group (EHPSG). Eur J Gastroenterol Hepatol. 1997;9:1-2.

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20. Manes G, Esposito P, Lioniello M, Bove A, Mosca S, Balzano A. Manometric and pH-metric features in gastro-oesophageal reflux disease patients with and without Helicobacter pylori infection. Dig Liver Dis. 2000;32:372-7. 21. Marshall BJ, Warren JR. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet. 1984;1:1311-5. 22. McCallum RW, Walsh JH. Relationship between lower esophageal sphincter pressure and serum gastrin concentration in Zollinger-Ellison syndrome and other clinical settings. Gastroenterology. 1979;76:76-81. 23. McNamara D, O’Morain C. Gastro-oesophageal reflux disease and Helicobacter pylori: an intricate relation. Gut. 1999 Jul;45:I13-7. 24. Mittal RK, Rochester DF, McCallum RW. Sphincteric action of the diaphragm during a relaxed lower esophageal sphincter in humans. Am J Physiol. 1989;256(1 Pt 1):G139-44. 25. Moayyedi P, Bardhan C, Young L, Dixon MF, Brown L, Axon AT. Helicobacter pylori eradication does not exacerbate reflux symptoms in gastroesophageal reflux disease. Gastroenterology. 2001;121:1120-6. 26. Newton M, Bryan R, Burnham WR, Kamm MA. Evaluation of Helicobacter pylori in reflux oesophagitis and Barrett’s oesophagus. Gut. 1997;40:9-13. 27. NIH Consensus Conference. Helicobacter pylori in peptic ulcer disease. NIH Consensus Development Panel on Helicobacter pylori in Peptic Ulcer Disease. JAMA. 1994;272:65-9. 28. O’Connor HJ, Cunnane K. Helicobacter pylori and gastro-oesophageal reflux disease - a prospective study. Ir J Med Sci. 1994;163:369-73. 29. Sainz R, Borda F, Domínguez E, Gisbert JP. [Helicobacter pylori infection. The Spanish consensus report. The Spanish Consensus Conference Group]. Rev Esp Enferm Dig. 1999;91:777-84. 30. Sarnelli G, Ierardi E, Grasso R, Verde C, Bottiglieri ME, Nardone G, Budillon G, Cuomo R. Acid exposure and altered acid clearance in GERD patients treated for Helicobacter pylori infection. Dig Liver Dis. 2003;35:151-6. 31. Schenk BE, Kuipers EJ, Klinkenberg-Knol EC, Eskes SA, Meuwissen SGM. H. pylori, GERD and the efficacy of omeprazole therapy [abstract]. Gastroenterology. 1997;112(Suppl):A282. 32. Sekiguchi T, Shirota T, Horikoshi T, et al. Helicobacter pylori infection and severity of reflux [abstract]. Gastroenterology. 1996;110(Suppl):A755. 33. Simpson RJ, Graham SM, Florida-James GD, Connaboy C, Clement R, Jackson AS. Perceived exertion and heart rate models for estimating metabolic workload in elite British soldiers performing a backpack load-carriage task. Appl Physiol Nutr Metab. 2010;35:650-6. 34. Tee W, Lambert JR, Dwyer B. Cytotoxin production by Helicobacter pylori from patients with upper gastrointestinal tract diseases. J Clin Microbiol. 1995;33:1203-5. 35. Vakily M, Wu J, Atkinson SN. Lack of electrocardiographic effect of dexlansoprazole MR, a novel modified-release formulation of the proton pump inhibitor dexlansoprazole, in healthy participants. J Clin Pharmacol. 2009;49:1447-55. 36. Vicari JJ, Peek RM, Falk GW, Goldblum JR, Easley KA, Schnell J, Perez-Perez GI, Halter SA, Rice TW, Blaser MJ, Richter JE. The seroprevalence of cagApositive Helicobacter pylori strains in the spectrum of gastroesophageal reflux disease. Gastroenterology. 1998;115:50-7. 37. Wu JC, Sung JJ, Ng EK, Go MY, Chan WB, Chan FK, Leung WK, Choi CL, Chung SC. Prevalence and distribution of Helicobacter pylori in gastroesophageal reflux disease: a study from the East. Am J Gastroenterol. 1999;94:1790-4. 38. Zhang W, Wu J, Atkinson SN. Effects of dexlansoprazole MR, a novel dual delayed release formulation of a proton pump inhibitor, on plasma gastrin levels in healthy subjects. J Clin Pharmacol. 2009;49:444-54.

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Received 8/4/2011. Accepted 1/9/2011.

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ARTIGO ORIGINAL / ORIGINAL ARTICLE

ARQGA/1590

ESOPHAGEAL MANOMETRY FINDINGS AND DEGREE OF ACID EXPOSURE IN SHORT AND LONG BARRETT’S ESOPHAGUS Laura HELMAN1, Beatriz Nunes BICCAS2, Eponina M. O. LEMME3, Paula NOVAIS4 and Viviane FITTIPALDI5

ABSTRACT – Context - Barrett’s esophagus (BE) is characterized by intestinal metaplasia in the distal esophagus and is classified as short-segment (<3 cm – SSBE) or long-segment (>3 cm - LSSBE). It is suggested that LSSBE is associated with more severe esophageal motor abnormalities and increased acid exposure time than SSBE. Objective - To evaluate the prevalence of esophageal manometriy abnormalities and acid exposure times in patients with SSBE and LSSBE. Methods - Barrett’s esophagus patients identified by upper endoscopy and confirmed by histopathology were, retrospectively, reviewed and divided into two groups: SSBE and LSBE. Demographic data, symptom duration, prevalence of hiatal hernia, lower esophagus sphincter basal pressure, prevalence of esophageal body abnormalities and acid exposure times were evaluated. Results - Forty-six patients with SSBE (24 males - 52.2%, mean age of 55.2 years) and 28 patients with LSBE (18 males - 64.3%, mean age of 50.5 years). Mean symptom duration was 9.9 years for SSBE and 12.9 years for LSSBE. Hiatal hernia was present in 84.2% of SSBE, 96.3% of LSBE; average lower esophagus sphincter pressure in SSBE 9.15 mm Hg, in LSBE 6.99 mm Hg; lower esophagus sphincter hypotension in SSBE was 65.9%, in LSSBE 82.1%; aperistalsis in SSBE 6.5%, LSSBE 3.6%; mild/moderate ineffective esophageal motility in SSBE 34.8%, LSBE 46.4%; severe moderate ineffective esophageal motility in SSBE 10.9%, LSBE 7,1%; nutcracker esophagus/segmental nutcracker esophagus in SSBE 8.6%, LSBE 0%; normal body in SSBE 39.1%, in LSBE 42.9%, no statistical difference for any of these values (P<0.05). Average % total time pH<4 in SSBE 9.12, LSBE 17.27 (P<0.000); % time pH<4 upright in SSBE 11.91; LSBE 24.29 (P=0.003); % time pH<4 supine in SSBE 10.86, LSBE 33.26 (P = 0.000). Conclusion - There was no difference between the prevalence of motor disorders in patients with SSBE and LSSBE. Acid reflux in upright and supine positions was more intense in LSBE. HEADINGS – Barrett esophagus. Gastroesophageal reflux. Manometry. Esophageal motility disorders.

INTRODUCTION

METHODS

Gastro-esophageal reflux disease (GERD) can present in erosive and, nonerosive forms and as complications. Barrett’s esophagus (BE) is a complication, which has attracted much attention because of its malignancy potencial. It is characterized by the presence of intestinal metaplasia in the distal esophagus(20). This metaplasic segment is called short Barrett’s esophagus (SSBE) when its length is less than 3 cm and long Barrett’s esophagus (LSBE), when it is 3 cm long or longer(14). BE pathophysiology is multiple. The main factors are chronic and prolonged acid and/or bile reflux. When compared to controls, BE patients present more pronounced motor abnormalities such as severe LES hypotension and esophageal body hypomotility(10, 11, 12). The aim of this study was to compare SSBE and LSBE with regard to demographic and esophageal manometriy characteristics and esophageal pH monitoring.

The charts of patients with BE who underwent esophageal manometry and pH monitoring from March 1992 to March 2010 were reviewed. All were diagnosed with BE on the basis of endoscopic and biopsy findings described below and were divided into SSBE and LSBE groups. Endoscopy The examinations were performed on a fasting patient, after topical anesthesia of the oropharynx with lidocaine and under sedation. A sliding hiatal hernia was defined when the esophagogastric junction was displaced more than 2 cm above the diaphragmatic impression. Suspected BE was diagnosed when a salmon-pink epithelium similar to the gastric mucosa (columnar  epithelium) was found above the gastro-esophageal junction. The diagnosis was confirmed histopathology when goblet cells (intestinal metaplasia) were found in this mucosa. SSBE was diagnosed when the metaplasia was less than

Unidade de Esôfago, Serviço de Gastroenterologia, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, RJ, Brasil. Correspondence: Dr. Laura Helman – Praia de Botafogo, 252 – ap. 602 bloco A – Botafogo – 22250-040 – Rio de Janeiro, RJ, Brasil. E-mail: helman@ufrj.br

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3 cm long; LSBE was diagnosed when the metaplastic area was equal or longer than 3 cm(14). Esophageal manometry All patients underwent esophageal manometry using a water-perfused polyvinyl catheter with eight lumens and an external diameter of 4.5 mm, with four distal ports arranged radially at the same level and the four other ports spaced 5 cm apart from each other (Medtronic, Synectics, Sweden or Alacer Corp, SP, Brazil). Each opening was connected to an external pressure transducer perfused with distilled water using a pneumo-hydraulic capillary infusion system. The intraluminal pressure was recorded on a polygraph, digitized and transferred to a computer. After local anesthesia with 2% viscous lidocaine applied to the nasopharynx, the catheter was passed into the stomach per the nares and withdrawn through the lower esophageal sphincter (LES) using the station pull-through technique at 1 cm increments every 20 seconds. The lower esophageal sphincter pressure (LESP) was defined as the difference between the end-expiratory gastric baseline pressure and the highest end-expiratory pressure just distal to the respiratory inversion point. It was calculated by the averaging the reading from the four radial ports and the relaxation was assessed after six wet swallows. Esophageal body contractions were then evaluated by the four ports (spaced 5 cm apart) after the patient took 10 wet swallows at 20-second intervals. Appropriate software was used for interpretation. For diagnosis of primary motility disorders the International Classification of Abnormalities(18) was used, when appropriate to adjust the normal values employed in the Esophagus Unit of Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro, RJ, Brazil, which derived from a study of healthy volunteers as described in the chart (Figure 1)(9). MOTILITY ABNORMALITY IEM (ineffective esophageal motility)

DES (diffuse esophageal spasm)

NE (nutcracker esophagus) LES † hypotension LES † hypertension Non-specific motor disorders

MANOMETRIC FINDINGS >20% of low amplitude contractions (<30 mm Hg) or non-transmitted contractions in the distal esophagus during 10 wet swallows ≥20% of simultaneous contractions in the distal esophagus during 10 wet swallows Mean distal amplitude of esophageal contractions >140 mm Hg LESP ‡ >10 mm Hg LESP ‡ >32 mm Hg Increased-duration (>6 sec)/ and/or triple-peak or retrograde contractions in more than 20% of esophageal body swallows

† = Lower esophageal sphincter; ‡ = Lower esophageal sphincter pressure

FIGURE 1. Esophageal motility abnormalities

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The term segmental nutcracker esophagus (SegNE) was used when the mean amplitude of distal esophagus segments (3 or 8 cm above the LES) was >165 mm Hg(1). Ineffective esophageal motility (IEM) is defined in the distal esophagus when at least 30% of 10 wet swallows exhibit any combination of the following abnormalities: (1) distal esophageal peristaltic wave amplitude <30 mm Hg,  (2)  simultaneous contractions with amplitudes <30 mm Hg, (3) failed peristalsis in which the peristaltic wave does not traverse the entire length of the distal esophagus, or (4) absent peristalsis (18). The number of ineffective contractions (peristaltic failure and/or the number of low amplitude waves in the distal esophagus) observed were used to quantify the alterations that constitute IEM. IEM was rated as mild to moderate when 30%-80% of contractions were ineffective and severe when this change was present in more than 80% of wet swallows(17). Esophageal 24h pH monitoring Intraesophageal pH monitoring was performed using a portable digital system (Synectics Medical, MK III, Stockholm, Sweden or Alacer Corp, SP, Brazil or Sigma, MG, Brazil) coupled to an antimony catheter positioned 5 cm above the upper limit of the LES previously defined by manometry and connected to an external reference electrode. Proton pump inhibitors (PPIs) were discontinued for 10 days, H2-receptor antagonists for 72 hours and prokynetics for 24 hours before the test. Patients were instructed to carry out their usual daily activities, on a liberal diet and avoiding only carbonated drinks and citrus fruits. A diary was kept of food and fluid intake, symptoms, and time spent in the supine and upright positions. A reflux episode was defined when esophageal pH dropped below 4 for at least 15 seconds. Patients with pH less than 4 for more than 4.5% of the total time recorded, 7% of the time in upright position and 2.5% of the time in supine position were considered as having increased esophageal acid exposure(12). Statistical analysis The duration of symptoms, prevalence of hiatal hernia and manometric and pH monitoring data (total, upright and supine) for the SSBE and LSBE groups were compared. Statistical tests included Student’s t- and χ2 and the significance level was P<0.05. RESULTS

Seventy-four patients met the inclusion criteria for the study, with 46 placed in the SSBE group (24 males, 62.2%) and 28 in the LSBE group (18 males, 64.6%) P = 0.308. Ages ranged from 26 to 79 years, mean 55.22 ± 13.56 years (median 54 years) in the SSBE group and 30 to 86 years, mean 51.75 ± 12.56 years (median 50.5 years) in the LSBE group (P = 0.218). The duration of symptoms (obtained for 31/46 patients in the SSBE group and for 18/28 in the LSBE group) was

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5-30 years (mean, 9.88 ± 8.76 years, median 6 years) and 1-40 years (mean 12.94 ± 11.66, median 10 years), respectively, (P = 0.484). The prevalence of hiatal hernia was high in both groups (Table 1) and there was no statistically significant difference between them (P = 0.224). Table 2 shows esophageal manometry LES data for the two groups. Esophageal body manometry data is shown in Table 3. Aperistalsis and IEM were found in patients of both groups. Nutcracker esophagus (two patients) and segmental nutcracker esophagus (one patient) were found only in SSBE group. These esophageal body abnormalities were found in 28/46 (60.87%) of the SSBE group and in 16/28 (57.14%) of the LSBE group, none of them were statistically different between the groups. Esophageal pH was monitored in 29 of the 46 SSBE patients and in 15 of the LSBE 28 patients for 24h. The results of the three variables, % total time pH <4, % time pH <4 upright and % time pH <4 supine, can be seen in Table 4.

TABLE 1. Hiatal hernia prevalence   SSBE † n = 46 Hiatal hernia 32/38 (84.2%)

LSBE ‡ n = 28 26/27 (96.3%)

P-value P = 0.224

† = Short segment Barrett esophagus ‡ = Long segment Barrett esophagus

TABLE 2. LES manometric data (n = 73)   SSBE † n = 46 LSBE ‡ n = 27 P-value Mean LE SP § 9.15 mm Hg ± 7.06 6.99 mm Hg ± 8.28 P = 0.066 LES§ 29/44 (65.9%) 23/28(82.1%) P = 0.180 hypotension † = short segment Barrett esophagus ‡ = long segment Barrett esophagus § = lower esophageal sphincter

TABLE 3. Esophageal body manometry data SSBE † n = 46 LSBE‡ n = 28 Aperistalsis 3 (6.5%) 1 (3.6%) Mild/moderate 16 (34.8%) 13 (46.4%) IEM § Severe IEM § 5 (10.9%) 2 (7.1%) NE/Seg NE ¶ 4 (8.6%) 0 (0%) Normal body 18 (39.1%) 12 (42.9%)

P-value P = 1.00 P = 0.338 P = 0.703 P = 0.291 P = 0.810

† = short segment Barrett esophagus ‡ = long segment Barrett esophagus § = ineffective esophageal motility ¶ = nutcracker esophagus/segmental nutcracker esophagus

TABLE 4. pH monitoring data SSBE n = 29 LSBE n = 15 P-value % total time pH <4 9.12 ± 9.61 17.27 ± 18.84 P<0.000* % time pH <4 upright 11.91 ± 17.28 24.29 ± 16.85 P = 0.003* % time pH <4 supine 10.86 ± 18.77 33.26 ± 24.58 P = 0.000* * = statistically significant difference between the two groups

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DISCUSSION

The exact pathophysiology that leads to the development of Barrett’s epithelium remains to be fully elucidated(5). Possible explanations for intestinal metaplasia include the duration of reflux disease, the composition of the refluxate, and/or underlining genetic traits(11). The most accepted theory is that this process takes place within a short period of time during which Barrett’s epithelium reaches its maximum length, with little progression or regression happening thereafter(2, 21). The other theory is based on progressive stepwise growth, starting with intestinal metaplasia at the esophagogastric junction. With the weakening of the LES and continuous acid exposure, this would progress to the distal esophagus and in time would lead to a complete loss of LES function and consequently to the progression of metaplasia to more proximal esophageal segments. The development of Barrett’s epithelium would therefore be a dynamic phenomenon, with its extent determined by the severity of functional abnormalities(4, 12). Among BE risk factors, the male gender is a constant in both groups. The average age is in the fifties, with no statistical difference between groups. Several other studies confirm these findings(1, 4, 5, 10, 11, 12) and present a profile of patients who are Caucasian men mainly, over 50 with longstanding heartburn(20). The mean age similarity between the two groups is one of the arguments against the possibility of progression in the length of Barrett’s mucosa. If there were such a progression, the SSBE group would be younger than the LSBE group, thus, there must be other mechanisms at work in the development of Barrett’s metaplasia(10). As this was a retrospective study, information about symptom onset time, which would characterize the duration of the disease, could not be obtained for all patients, but there was no statistical difference between groups. Other authors, however, have indicated a longer duration of symptoms in LSBE(12). The presence of hiatal hernia is a frequent feature of patients with GERD especially in a complicated form like Barrett esophagus. In this sample, hiatal hernia was observed in 84.2% of patients with SSBE and in 96.3% of those with LSBE. The literature shows a 30% to 75% prevalence of hiatal hernia for the SSBE group with 72% to 100% for the LSBE group(4, 5, 8). One study evaluated the size of hiatal hernias and found more voluminous hernias in LSBE(5). Nowadays, hiatal hernia is among the risk factors for Barrett’s esophagus. A study(7) found a similar prevalence of hiatal hernia among patients with BE (including SSBE and LSBE), erosive disease and non-erosive reflux disease. However, the study also showed a higher prevalence of larger hernias in BE. In evaluating LES, it was found that the mean pressure drop was more pronounced in the LSBE group with a tendency toward a statistically significant difference. This is also mentioned by some authors(4, 10, 21), but not by others(11, 12).

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LES hypotension (LES pressure below 10 mm Hg) was found in 65.9% of SSBE and 82.1% of LSBE patients, with no statistically significant difference. In those series in which this aspect was different, there were values of 65.9% and 73.3% for the SSBE and values of 94.1% and 93.8% for the LSBE patients(11, 12) with statistical difference between groups. Due to the retrospective nature of this study, wave amplitude and wave velocity were not evaluated in both groups. It is questioned whether these data may have relevance. Ineffective esophageal motility (IEM) was defined by Leite et al.(8), as the presence of distal low amplitude peristaltic waves or peristaltic failure in at least 30% of swallows and is the principal motor disorder found in GERD(21). In this study, IEM was categorized as mild/moderate when low amplitude or failure occurred in 80% of swallows and as severe when it occurred in more than 80% of swallows(17). The relationship between length of Barrett’s esophagus and severity of IEM could not be confirmed because there was no difference between the groups. The average amplitude of distal esophageal body waves decreased, with no difference between the SSBE and LSBE groups as many authors have already described(4, 6, 10, 12). However, this observation can not be made in this study. High amplitude distal peristaltic waves, which characterize NE, may be associated with non-cardiac chest pain and dysphagia(15). Some studies have shown that NE is associated with reflux in 30% to 40% of patients diagnosed by pH monitoring; erosive esophagitis is a rare finding(15, 16). In this study we found NE associated with 8.6% of SSBE patients and none of the LSBE patients. In a study by Csendes et al. (4) of 80 NE patients, 22 (27.5%) had esophageal SSBE; none had LSBE. The pH monitoring to quantify acid reflux in patients with a confirmed diagnosis of BE, especially LSBE, is not routine. With SSBE, the test is ordered more frequently. The reason for this is that some patients do not exhibit typical reflux symptoms and unlike what happens with LSBE, many do not show abnormal reflux. pH monitoring was

performed on 29 of the 46 patients with SSBE and in 15 of 28 of the LSBE patients, without the use of anti-secretory drugs. Several studies show that BE patients have greater esophageal acid exposure than other GERD groups and control subjects(3, 19). More pronounced acid reflux in patients with LSBE than SSBE was also identified by several other authors(4, 11, 12, 21). In the present study we found that not only the percentage of total time, but also the percentages of time in upright and supine positions were also higher in the LSBE group(5, 8, 10). Thus, a direct correlation between BE length and acid exposure could be shown. Duration of acid exposure may not be the only factor contributing to the length of Barrett’s esophagus. Duodenal reflux may also be an important factor in its appearance and reaching its maximum length. Two studies that monitored the presence of bilirubin in the lower esophagus by spectrophotometry showed no difference between the SSBE and LSBE groups(4, 11). A study using pH-impedance in patients with mild erosive GERD, non-erosive GERD and BE, concluded that the nonerosive GERD and BE patients presented higher exposure to non-acid reflux (pH>4) in the supine position, in addition to increased acid exposure in both upright and supine positions. This suggests that nocturnal non-acid exposure could play a role in BE pathogenesis(7). However, these data need to be interpreted with caution, another study using both pH-impedance and bilimetry on patients who had typical GERD symptoms refractory to proton pump inhibitor therapy or atypical symptoms revealed that non-acid reflux and bilirubin reflux are distinct phenomena that require distinct evaluation techniques(13). pH-impedance cannot describe the composition of the refluxate, only its physical status and pH. Therefore, the finding of increased non-acid reflux in BE may not correlate with the amount of bile reflux. More studies are needed to clarify this point. In conclusion, there was no difference between the prevalence of motor disorders in patients with SSBE and LSBE. Acid reflux was more intense in BE in total time, upright and supine positions.

Helman L, Biccas BN, Lemme EMO, Novais P, Fittipaldi V. Alterações manométricas e intensidade do refluxo no esôfago de Barrett curto e longo. Arq Gastroenterol. 2012;49(1):64-8. RESUMO – Contexto - O esôfago de Barrett (EB) se caracteriza pela presença de metaplasia intestinal no esôfago distal, quando menor que 3 cm é chamado Barrett curto (EBC) e com 3 cm ou mais Barrett longo (EBL). Sugere-se que o EBL cursa com mais alterações motoras esofagianas e com refluxo mais intenso que o EBC. Objetivo - Avaliar a prevalência de alterações manométricas e a intensidade do refluxo gastroesofágico à pHmetria em pacientes com EBC e EBL. Métodos - Estudo retrospectivo de pacientes com endoscopia digestiva alta e comprovação histopatológica de EB, divididos em dois grupos: EBC e EBL. Foram avaliados os dados demográficos, o tempo de doença, prevalência de hérnia hiatal, dados obtidos à esofagomanometria e pHmetria. Resultados - EBC 46 pacientes (24 masculino 52,2% e média de idade de 55,22 anos), EBL 28 pacientes (18 masculino 64,3% e média de idade 50,5 anos); tempo de sintomas: EBC 9,88 anos e EBL 12,94 anos; hérnia de hiato: EBC 84,2%, EBL 96,3%; pressão média do esfíncter inferior do esôfago: EBC 9,15 mm Hg, EBL 6,99 mm Hg; hipotensão do esfíncter inferior do esôfago: EBC 65,9%, EBL 82,1%; motilidade esofagiana ineficaz (MEI) leve/moderado: EBC 34,8%, EBL 46,4%; MEI acentuado: EBC 5 10,9%, EBL 7,1%; aperistalse: 6,5%, EBL 3,6%; esôfago em quebra-nozes: EBC 8,6%, EBL 0%; corpo normal: EBC 39,1%, EBL 42,9%, sem diferença estatística para qualquer desses valores (P<0,05). Médias de pHmetria: % de tempo total com pH <4: EBC (29/46) 9,12% EBL (15/28) 27,27% P<0,000; % de tempo ereto com pH<4: EBC 11,91%, EBL 24,29% P = 0,003; % de tempo supino com pH <4: EBC 10,86% EBL 33,26% P = 0,000. Conclusões - Não houve diferença entre a prevalência das alterações motoras em pacientes com EBC e EBL. O refluxo ácido, tanto em posição ereta como em posição supina, foi mais intenso no EBL. DESCRITORES – Esôfago de Barrett. Refluxo gastroesofágico. Manometria. Transtornos da motilidade esofágica.

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References 1.

Achem SR, Kolts BE, Burton L. Segmental versus diffuse nutcracker esophagus: an intermittent motility pattern. Am J Gastroenterol. 1993;88:847-51. 2. Cameron AJ, Lomboy CT. Barrett’s esophagus: age, prevalence, and extent of columnar epithelium. Gastroenterology. 1992;103:1241-5. 3. Champion G, Richter JE, Vaezi MF, Singh S, Alexander R. Duodenogastroesophageal reflux: relationship to pH and importance in Barrett’s esophagus. Gastroenterology. 1994,107:747-54. 4. Csendes A, Smok G, Quiroz J, Burdiles P, Rojas J, Castro C, Henríquez A. Clinical, endoscopic, and functional studies in 408 patients with Barrett’s esophagus, compared to 174 cases of intestinal metaplasia of the cardia. Am J Gastroenterol. 2002;97:554-60. 5. Fass R, Hell RW, Garewal HS, Martinez P, Pulliam G, Wendel C, Sampliner RE. Correlation of esophageal acid exposure with Barrett’s oesophagus length. Gut. 2001;48:310-3. 6. Frazzoni M, Manno M, De Micheli E, Savarino V. Pathophysiological characteristics of the various forms of gastro-oesophageal reflux disease. Spectrum disease or distinct phenotypic presentations? Dig Liver Dis. 2006;38:643-8. 7. Gutschow CA, Bludau M, Vallböhmer D, Schröder W, Bollschweiler E, Hölscher AH. NERD, GERD, and Barrett’s esophagus: role of acid and non-acid reflux revisited with combined pH-impedance monitoring. Dig Dis Sci. 2008;53:3076-81. 8. Leite LP, Johnston BT, Barrett J, Castell JA, Castell DO. Ineffective esophageal motility (IEM): the primary finding in patients with nonspecific esophageal motility disorder. Dig Dis Sci. 1997;42:1859-65. 9. Lemme EMO, Domingues GR, Silva LFD, Firman CG, Pantoja JAS. Esophageal manometry: preliminary values in health adult volunteers. GED Gastroenterol Endosc Dig. 2001;20:29-35. 10. Loughney T, Maydonovitch CL, Wong RK. Esophageal manometry and ambulatory 24-hour pH monitoring in patients with short and long segment Barrett’s esophagus. Am J Gastroenterol. 1998:93:916-9. 11. Oberg S, Ritter MP, Crookes PF, Fein M, Mason RJ, Gadensytätter M, Brenner CG, Peters JH, DeMeester TR. Gastroesophageal reflux disease and mucosal injury with emphasis on short-segment Barrett’s esophagus and duodenogastroesophageal reflux. J Gastrointestinal Surg. 1998;2:547-52.

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12. Oberg S, DeMeester TR, Peters JH, Hagen JA, Nigro JJ, DeMeester SR, Theisen J, Campos GM, Crookes PF. The extent of Barrett’s esophagus depends on the status of the lower esophageal sphincter and the degree of esophageal acid exposure. J Thorac Cardiovasc Surg. 1999;117:572-80. 13. Pace F, Sangaletti O, Pallotta S, Molteni P, Porro GB. Biliary reflux and non-acid reflux are two distinct phenomena: a comparison between 24-hour multichannel intraesophageal impedance and bilirubin monitoring. Scand J Gastroenterol. 2007;42:1031-9. 14. Sharma P, Morales TG, Sampliner RE. Short segment Barrett’s esophagus--the need for standardization of the definition and of endoscopic criteria. Am J Gastroenterol. 1998;93:1033-6. 15. Silva LF, Lemme EM. [Nutcracker esophagus: clinical evaluation of 97 patients]. Arq Gastroenterol. 2000;37:217-23. 16. Silva LF, de Oliveira Lemme EM. Are there any differences between nutcracker esophagus with and without reflux. Dysphagia. 2007;21:198-205. 17. Simrén M, Silny J, Holloway R, Tack J, Janssens J, Sifrim D. Relevance of ineffective oesophageal motility during oesophageal acid clearance. Gut. 2003; 52:784-90. 18. Spechler SJ, Castell DO. Classification of oesophageal motility abnormalities. Gut 2001;49:145-51. 19. Vaezi MF, Richter JE. Complicated Barrett’s esophagus: role of acid and bile. Am J Gastroenterol. 1994;89:1630. 20. Wang KK, Sampliner RE; Practice Parameters Committee of the American College of Gastroenterology. Updated guidelines 2008 for the diagnosis, surveillance and therapy of Barrett’s esophagus. Am J Gastroenterol. 2008; 103:788-97. 21. Zentilin P, Conio M, Mele MR, Mansi C, Pandolfo N, DulbEcco P, Gambaro C, Tessieri L, Iiritano E, Bilardi C, Biagini R, Vigneri S, Savarino V. Comparison of the main oesophageal pathophysiological characteristics between shortand long-segment Barrett’s oesophagus. Aliment Pharmacol Ther. 2002; 16:893-8. Received 23/8/2011. Accepted 27/9/2011.

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GASTROENTEROLOGIA PEDIÁTRICA/ PEDIATRIC GASTROENTEROLOGY

ARQGA/1591

PORTAL VEIN THROMBOSIS IN CHILDREN AND ADOLESCENTS: 20 years experience of a pediatric hepatology reference center Priscila Menezes FERRI1, Alexandre Rodrigues FERREIRA2, Eleonora Druve Tavares FAGUNDES3, Shinfay Maximilian LIU4, Mariza Leitão Valadares ROQUETE5 and Francisco José PENNA6

ABSTRACT – Context - Portal vein thrombosis refers to a total or partial obstruction of the blood flow in this vein due to a thrombus formation. It is an important cause of portal hypertension in the pediatric age group with high morbidity rates due to its main complication - the upper gastrointestinal bleeding. Objective - To describe a group of patients with portal vein thrombosis without associated hepatic disease of the Pediatric Hepatology Clinic of the Hospital das Clínicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil with emphasis on diagnosis, presentation form and clinical complications, and the treatment of portal hypertension. Methods - This is a descriptive study of a series of children and adolescents cases assisted from January 1990 to December 2010. The portal vein thrombosis diagnosis was established by ultrasound. Results - Of the 55 studied patients, 30 (54.5%) were male. In 29 patients (52.7%), none of the risk factors for portal vein thrombosis was observed. The predominant form of presentation was the upper gastrointestinal bleeding (52.7%). In 20 patients (36.4%), the initial manifestation was splenomegaly. During the whole following period of the study, 39 patients (70.9%) showed at least one episode of upper gastrointestinal bleeding. The mean age of patients in the first episode was 4.6 ± 3.4 years old. The endoscopic procedure carried out in the urgency or electively for search of esophageal varices showed its presence in 84.9% of the evaluated patients. The prophylactic endoscopic treatment was performed with endoscopic band ligation of varices in 31.3% of patients. Only one died due to refractory bleeding. Conclusions - The portal vein thrombosis is one of the most important causes of upper gastrointestinal bleeding in children. In all non febrile children with splenomegaly and/or hematemesis and without hepatomegaly and with normal hepatic function tests, it should be suspect of portal vein thrombosis. Thus, an appropriate diagnostic and treatment approach is desirable in an attempt to reduce morbidity and mortality. HEADINGS – Venous thrombosis. Portal vein. Hypertension, portal. Child. Adolescent.

INTRODUCTION

Portal vein thrombosis (PVT) refers to a total or partial obstruction of the blood flow in this location, secondary to a thrombus formation. These thrombus may extend until the liver, affecting intra-hepatic vases, or reach splenic and/or mesenteric veins(6, 34). The first PVT case was described in 1868 by Balfour and Stewart(5), in a patient with splenomegaly, ascites and esophageal varices. Recently, the number of diagnoses is increasing, possibly due to the greater availability of diagnostic methods, specially the Doppler ultrasound(6, 25, 47, 50). PVT incidence in the general population is estimated in 1% after the study carried out by Ogren et al.(31) in autopsies. In adults, factors that lead to hypercoagulability are the main cause of PVT not associated with liver cirrhosis or cancer(34). Among the local risk factors, situations that increase the intra-abdominal inflammatory response as

acute or chronic pancreatitis, diverticulitis, appendicitis, inflammatory bowel diseases, liver abscesses, cholangitis and surgeries have been described(12, 15, 31, 33, 34). In children and adolescents, the main identified causes are: direct injury of the vein (omphalitis and umbilical vein catheterization) and sepsis with abdominal focus(29, 41, 50). Others possible causes are abdominal trauma, surgical trauma, cysts and tumors in the porta hepatis, neonatal sepsis, dehydration, cardiovascular malformations and exchange transfusion(6, 41, 50, 52). Most of the cases remain idiopathic(32, 35, 40). PVT is important in the pediatric age group because it is one of the most frequent causes of portal hypertension, with high morbidity rates due to its main complication - the upper gastrointestinal bleeding. Approximately 79% of the children diagnosed with PVT will show at least one episode of upper gastrointestinal bleeding during their lives(2, 41). In

The authors have no conflicts of interests to declare. Departamento de Gastroenterologia Pediátrica do Hospital das Clínicas da Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brasil. Correspondence: MD Priscila Menezes Ferri - Rua São Romão, 197/202 - 30.330.120 - Santo Antônio, Belo Horizonte, MG, Brazil. E-mail: pmferri.liu@gmail.com

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the literature there are few reports on PVT in children and adolescents, being some of them also studies about casuistic descriptions and others about specific characteristics of the disease as thrombophilia(1, 17, 20, 32, 43, 45, 48, 53, 54, 55). Due to the scarcity of studies in the pediatric age group and the importance of the theme, this study`s objective is to describe the clinical characteristics, diagnosis, evolution and treatment of the portal hypertension in children and adolescents with diagnosis of portal vein thrombosis assisted at the Pediatric Hepatology Clinic of the Hospital das Clínicas of Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brazil. METHODS

Patients This is a descriptive study of a series of children and adolescents cases that were diagnosed with PVT and were assisted at the Pediatric Hepatology Clinic of the Hospital das Clínicas of UFMG from January 1990 to December 2010. The PVT diagnosis was established by ultrasound where cavernomatous transformation of the portal vein was observed. The exclusion of associated hepatic disease was carried out through clinical, laboratorial and radiological evaluation. The following variables were evaluated: gender, age at the diagnosis, birth conditions (weight and complications in the  perinatal period, such as infections, use of umbilical catheter and omphalitis history), form of disease presentation, alterations at the physical exam, disease complications, laboratory tests and approach of the secondary prophylaxis of the upper gastrointestinal bleeding due to esophagogastric varices. When hepatomegaly was observed on clinical examination, patients were tested for the presence of chronic liver disease that could be the cause of this finding. The evaluation included viral hepatitis, autoimmune hepatitis, alpha-1-antitrypsin deficiency, Wilson’s disease among others, through laboratory tests. If these diseases were discarded and the patient had liver enzymes (AST, ALT, GGT, ALP) and bilirubin within the reference values, liver biopsy was not performed. Until the time of completion of this series, the full assessment of the hypercoagulability tests was not available in the laboratory service of Hospital das Clínicas. Thus, it was not part of the purpose of this study to evaluate hypercoagulability due to incomplete data. Endoscopic treatment All patients with upper gastrointestinal bleeding, after the emergency approach, were elective referred to perform a secondary endoscopic prophylaxis – sclerotherapy or endoscopic band ligation (EBL). Endoscopic treatment was carried out in the Endoscopy Unit of the Alfa Institute of Gastroenterology of the Hospital das Clínicas (UFMG), always in a same week day, by two pediatric endoscopists who were simultaneously present during the exam. Varices were classified according to the Japanese classification (Japanese Research Society for Portal Hypertension, 1980), using as parameters the varices’ diameter and shape. They were classified in three grades: grade I (small caliber) — small, nontortuous varices; grade II (medium caliber) — slightly

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wide and tortuous varices, occupying less than one third of the esophageal lumen; grade III (large caliber) — nodular varices, similar to rosary beads, occupying more than one third of the esophageal lumen. Gastric varices were classified as esophagogastric varices with extension along the lesser curvature of the stomach (GEV1S type); esophagogastric varices extending along the greater curvature towards the gastric fundus (GEV2S type); isolated varices in the gastric fundus (IGV1S); or varices located in the gastric antrum, corpus or pylorus (IGV2S). The gastropathy classification of the portal hypertension was mild, when a mosaic pattern of light degree without presence of red spots was observed; severe, when the mosaic pattern was superimposed with red spots, or if some other red spot was present; or gastric antral vascular ectasia which is an endoscopic and histopathological entity characterized by clusters of red spots arranged in a linear pattern or diffuse lesions. The latter may be observed in others conditions than in the portal hypertension. Sclerotherapy was carried out in children younger than 2 years old or in those who technically was not possible the passage of the apparel with the band ligation kit, with a transparent teflon® injector (diameter 23) Wilson-cook trademark, using the technique of free hand. The injection was performed intra and para vasal and the sclerosing agent used was ethamolin 3%. The amount injected ranged from 1.0 mL to 1.5 mL per varix, according to the size of it and with a total maximum volume of 10 mL. An injection was given in each varix, starting just above the gastroesophageal junction and, then proximally with intervals of 2 cm of distance. All found varices underwent sclerotherapy. EBL was carried out through the multiband ligator. It started near to the gastroesophageal junction and ascended cranially with a distance of 5 cm. In each session, each varix was tied using an elastic band and all found varices were treated. This technique was not used in children younger than 2 years due to technical reason. In patients who were no longer technically possible the accomplishment of the EBL, the sclerotherapy was performed for variceal eradication. Patients underwent endoscopy every 3 weeks until the variceal eradication. After the eradication, endoscopy was carried out with a 3 months interval during the first 6 months, later a halfyearly interval and, if no varices reappeared, annual controls or, at any moment, if bleeding. Ethical issues The project was approved by the Ethical in Research Committee of Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil, in April 2009 (Committee on Ethics and Research ETIC protocol 079/09). Parents or responsible and the children/ adolescents were informed of the importance of the research, its objectives, safety on the tests’ conduction and warranty of secret of data, through the informed consent term. Statistical analysis Data were analyzed through the software Excel 2007® (Microsoft) and SPSS 15® (SPSS Inc.) and were evaluated through the calculation of simple frequency. The continuous variables

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without normal distribution are showed through medians and interquartiles range 25%–75% (IR 25%–75%). RESULTS

Fifty-five children with diagnosis of PVT, without associated liver disease were evaluated, 30 patients were male (54.5%) and 25 female (45.5%). The age at diagnosis ranged from 1 month to 13.8 years old, with a mean age of 3.7 years, median of 2.6 years (IR 1–5.5) and standard deviation of 3.5 years. The time of the patients’ follow-up had a mean of 5.5 years, a standard deviation of 4.5 years, median of 5.2 (IR 1.8–7.9) and ranged from 1 month to 19.9 years. Initial clinical manifestation The main initial clinical manifestations are showed in Table 1. TABLE 1. Initial clinical manifestation observed in 55 patients Number of patients and Clinical manifestation percentage Upper gastrointestinal bleeding 29 (52.7%) Splenomegaly 20 (36.4%) Occasional finding 6 (10.9%)

In the cases of occasional finding, one patient was under investigation due to recurrent epistaxis, three under investigation of chronic abdominal pain, one was evaluated due to the prolonged prothrombin time in a routine exam, and one was under investigation of acute abdominal pain. Risk factors The main risk factors observed are showed in Table 2. Association between more than one factor was observed in 10 patients (18.2%). The associations were between umbilical catheterization and neonatal sepsis in seven patients (12.7%), umbilical catheterization, neonatal sepsis and hepatic abscess in one patient (1.8%) and abdominal surgery due to enterocolitis condition, with neonatal sepsis and umbilical catheterization in another two patients (3.6%). TABLE 2. Risk factors involved in the PVT etiology Risk factor n (%) Umbilical catheterization 20 (36.4%) Neonatal sepsis 9 (16.4%) Abdominal infection 2 (3.6%) Cardiovascular malformation 3 (5.5%) Coagulation disorder 1 (1.8%) Abdominal surgery 1 (1.8%) Unknown 1 (1.8%) Absent 28 (50.9%)

In 18 patients the indication of umbilical catheterization was due to prematurity and in two patients it occurred due to other complications of the neonatal period. Isolated abdominal infection was reported in one patient whose diagnosis was acalculous cholecystitis. Coagulation disorder was reported in one patient (antithrombin III and v. 49 – no.1 – jan./mar. 2012

protein S deficiencies), and it was confirmed by family study. This patient was referred from another institution already diagnosed with antithrombin III and protein S deficiencies to monitor the context of portal vein thrombosis, mainly because of recurrent episodes of upper gastrointestinal bleeding. One patient, with previous diagnosis of Blackfan-Diamond syndrome, showed during the follow-up thrombosis of superior mesenteric and splenic veins and required anticoagulation. Another patient did not have a record of previous history, because the adoptive family did not have such information. Findings in the clinical and laboratorial exams During the physical exam carried out at the first consultation it was observed splenomegaly in 49 patients (89.1%). Hepatomegaly  was associated with splenomegaly in eight cases (14.6%). None of the patients with hepatomegaly showed alterations in the assessment of liver function or liver enzymes and bilirubin levels were also normal. Chronic liver disease was not observed in any of these patients. Visible collateral circulation in the abdomen was present in six patients (10.9%), all with concomitant splenomegaly. Ascites was an isolated finding in just one patient (1.8%). Five patients (9.1%) did not show alterations in the clinical exam. Out of these, however, two were splenectomized. The splenectomy was performed in other services, before the PVT diagnosis, due to the massive splenomegaly and severe hypersplenism. In the laboratorial evaluation, nine patients (16.4%) showed elevation of hepatic enzymes (aspartate aminotransferase and/ or alanine aminotransferase) at some moment of the followup. These patients’ results were on average 1.6 times above the reference value of AST and 1.7 times of ALT. Hemoglobin, International Normalized Ratio (INR ), platelet count, albumin and gamma-glutamyltransferase (GGT) in the patients’ first and last consultation were also evaluated according to Table 3. TABLE 3. Results of the laboratorial evaluation Standard Amplitude Evaluated item Mean Median deviation (IR) Albumin (g/dL) 2.6–5.1 4.1 4.0 0.63 1st consultation (3.8–4.4) Albumin (g/dL) 2.6–5.6 4.1 4.1 0.54 Last consultation (3.7–4.3) GGT 2–97 21.5 17.5 15.61 1st consultation (14–17.5) GGT 1–131 28.4 22.5 23.20 Last consultation (28–22.5) Hemoglobin (g/dL) 3,6–14.1 10.5 10.7 2.40 1st consultation (8.3–12.5) Hemoglobin (g/dL) 5.1–15.5 11.8 11.9 2.08 Last consultation (10.6–13.1) 37000–380000 Platelets (/mm3) 150660 135000 84164.2 1st consultation (68000–200000) Platelets (/mm3) 39000–312000 117344 92500 71047.9 Last consultation (68000–180000) INR 0.56–2.30 1.25 1.21 0.27 1st consultation (1.00–1.40) INR 0.95–2.50 1.31 1.25 0.29 Last consultation (1.10–1.40)

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Six patients (10.9%) showed hypoalbuminemia at the first consultation, and in three of them the levels normalized during the follow-up, remaining low in three patients (5.5%). Thrombocytopenia due to hypersplenism (platelets <150.000/mm3) was detected in 28 patients (50.9%) at the first consultation and six other patients developed thrombocytopenia along the follow-up, totaling 34 patients (61.8%) at the last consultation. Prothrombin activity lower than 70% was observed in 19 patients (34.5%) at the first consultation, and 11 (57.9%) of them persisted with this alteration at the last consultation. Ultrasound needed to be repeated in our service in 22 patients (40%), because at the first exam of these patients the cavernomatous transformation of the portal vein was not identified.

TABLE 4. Esophageal varices observed at the first endoscopy Varices classification n (%) Small caliber esophageal varices 9 (16.4%) Medium caliber esophageal varices 22 (40%) Large caliber esophageal varices 14 (25.5%) No description of findings 8 (14.5%) Patients without the first UGIE 2 (3.6%) UGIE – Upper gastrointestinal endoscopy

Episodes of upper gastrointestinal bleeding (UGIB) During the follow-up, 39 patients (70.9%) had at least one episode of upper gastrointestinal bleeding. The number of bleedings of these patients showed a median of 2 (IR 1-3). Thirty-one patients (79.5% of those who had bled) required at least one red blood cells transfusion during acute bleeding episode. The age at which occurred the first bleeding showed a mean of 4.6 ± 3.4 years, median of 4 (QI 2-6) and 75% of these patients bled before 6 years old. Among the 39 patients who bled, 10 of them (25.6%) showed recurrent bleeding during or after the secondary prophylaxis. Endoscopic approach and secondary prophylaxis of UGIB The esophageal varices profile observed at the first upper gastrointestinal endoscopy (UGIE) is described in Table 4. Patients without the first UGIE had recently begun the follow-up and they did not carry out such exam yet. Given this, out of the 53 evaluated patients, 45 (84.9%) showed description of esophageal varices at the first endoscopy. Others findings described in the endoscopic follow-up are showed in Table 5. Thirty-two patients underwent secondary endoscopic prophylaxis. Fourteen patients (43.8%) underwent treatment with sclerotherapy, 10 patients (31.3%) with variceal band ligation and 8 patients (25%) required association of both treatments. In patients who underwent secondary prophylaxis, the number of sessions for eradication of the esophageal varices had a mean of 4.9 ± 2.2, median of 4 (IR 4-6), ranging from 2 to 11 sessions. Twelve out of the 32 patients (37.5%) showed recurrence of esophageal varices during the endoscopic followup. The median number of bleedings was one episode, with an IR interval from 1 to 1.3. Relapse occurred with median of 13.5 months after eradication (IR 12-36). Esophageal stenosis due to endoscopic treatment occurred in five patients (15.6%). Four out of these patients underwent sclerotherapy separately and in the last one this procedure was associated with EBL. This complication was treated with endoscopic dilatations.

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TABLE 5. Endoscopic findings in the patients’ follow-up Gastric varices Gastropathy of portal hypertension GEV 1S* 8 patients (14.6%) Mild 41 patients (74.6%) GEV2S# 21 patients (38.2%) IGV1S& 2 patients (3.6%) Severe 1 patient (1.8%) No patient IGV2S§ Total 31 patients (56.4%) Total 42 patients (76.4%) GEV1S : esophagogastric varices extending along the lesser curvature of the stomach GEV2S: esophagogastric varices extending along the greater curvature towards the gastric fundus & IGV1S: isolated varices in the gastric fundus § IGV2S: varices located in the gastric antrum, corpus or pylorus * #

Surgical treatment Eight patients (14.5%) underwent surgical intervention. Two underwent splenectomy in others services. Azygoportal disconnection due to UGIB of difficult control was performed in others two patients. Another patient required this procedure in the urgency due to persistent bleeding. Splenic artery ligation was performed in two patients and splenorenal shunt was performed in another due to hypersplenism. Only one patient died due to acute bleeding with no possibility of control. DISCUSSION

Portal vein thrombosis is not frequent in the pediatric age group, however, it is an important cause of portal hypertension and shows as main morbidity the upper gastrointestinal bleeding (21, 34, 41, 48). As observed in the present study, PVT does not show gender predominance and its more common initial manifestations in children are upper gastrointestinal bleeding and splenomegaly, which corroborates results reported in the literature(1, 6, 41, 47, 50, 53). In the present casuistic, upper gastrointestinal bleeding was the main initial clinical manifestation in 52.7% of patients, followed by splenomegaly in 36.4%. These data are similar to the ones reported by Gürakan et al.(20) whose casuistic of 12 patient, showed the occurrence of UGIB in 50% of patients. On the other hand, Abd El-Hamid et al.(1) and Weiss et al.(53) reported a greater proportion of patients with splenomegaly as the initial finding (63% and 43.3%, respectively). Thus, the possibility of PVT should be raised and a Doppler ultrasound exam should be requested in children and adolescents with UGIB or isolated finding of splenomegaly during the clinical exam.

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It should also be emphasized that a clinical situation of hypersplenism, manifested through thrombocytopenia associated with splenomegaly, is also frequently observed in pediatrics cases and, most of the time, the patients are referred firstly to the hematologist(6, 41, 44, 50). Today, the PVT etiology is considered multifactorial, with association of prothrombotic and local trigger factors found even in the pediatric age group(6, 10, 21, 23, 29, 33, 34, 37, 47, 52, 53). The causes of PVT may be grouped into three categories: direct injury of the portal vein with subsequent thrombus formation; vascular malformation which includes stenosis or even atresia of the portal vein; and hypercoagulable conditions which favor the thrombus formation(6, 15, 19, 22, 39, 49, 50, 52). Cases that do not fit in these groups are denominated as idiopathic PVT and, due to the great percentage of these cases, recent studies, mainly in adults, have focused on the evaluation of new etiological factors(7, 16, 17, 22, 23). In the pediatric age group, the few available literature has been demonstrating that approximately 50% of the cases still remained with an unknown etiology (1, 2, 20, 53). This fact was also observed in the present study. Umbilical catheterization, omphalitis and neonatal sepsis are the most common causes in children(2, 25, 40, 42). The prevalence reported in two other studies was lower than the one observed in this study. Abd El-Hamid et al.(1) reported 12% of patients with umbilical catheterization and 7% with neonatal sepsis and Weiss et al.(53) reported six patients (20%) with catheterization history and two (6.7%) with omphalitis among the 30 patients evaluated. On the other hand, Alvarez et al.(2) observed 44 patients among the 108 evaluated (41%) with previous history of possible injury of the portal vein due to catheterization, omphalitis and surgery. This fact may be associated with the greater number of patients with prematurity history among the patients evaluated in the present study (32.7% of patients). Data on prematurity was not reported in the other studies. No cases of omphalitis were observed in the present study. Others possible causes are: abdominal trauma, surgical trauma, cysts and tumors in the portal hepatis, neonatal sepsis, congenital malformations and, more recently described, thrombophilias(6, 9, 17, 20, 21, 30, 41, 50, 52). In spite of the lower prevalence, these factors are also reported in the pediatric age group. Abd El-Hamid et al.(1) observed that in a sample of 108 patients with PVT, 8% had abdominal infection and 24% had congenital malformations. Alvarez et al.(2) reported that 19% of the 108 patients had several congenital malformations. In the present study, such factors were also observed, indicating, thus, the importance  of a rigorous evaluation of the patient. The association of  factors observed in 10 patients (18.2%) may be explained by the multifactorial nature of PVT(6, 33, 34, 47, 52). Therefore, the approach of patients with possible diagnosis of PVT should be performed through a complete anamnesis, careful physical exam and laboratorial and radiological propedeutics. Anamnesis should mainly evaluate the presence of possible trigger factors, the patient’s previous history, presence of others comorbidities and the family history.

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Splenomegaly related to portal hypertension, even when not detected as the first manifestation, is present in most of patients(6, 41). In children, its presence occurred in 63% to 92% of the patients in the few casuistic reported in the literature(1, 2, 16, 20, 53). The results of this study corroborate the others. Laboratorial exams, in general, showed normal values of aminotransferases, albumin and coagulation tests. Albumin may be decreased and associated with ascites, for a short period, after episodes of UGIB(36, 51). On the other hand, patients with long-term portal hypertension secondary to PVT may show ascites without trigger factors(36). This same group may also show reduced level of albumin, prolonged prothrombin time and increased levels of aminotransferases. The evaluation of adults, children and adolescents with prolonged evolution of the PVT showed that up to a fifth of these patients may develop such alterations(36). The hepatic dysfunction may be attributed to the prolonged reduction of the portal blood flow and/or the development of portal biliopathy, which would indicate that PVT has a progressive character(8, 11, 18, 36). No case of portal biliopathy, which has a progressive and silent manifestation, was observed in the present study. Attention should be given to the elevation of alkaline phosphatase and GGT. The portal biliopathy usually manifest mainly in the adulthood age(11, 39). Hypoalbuminemia was observed in 10.9% of patients at the diagnosis and, during the clinical follow-up, 5.5% maintained this alteration. This fact may be occurred due to the presence of UGIB episodes, but it may be also due to the possible hepatic dysfunction associated with PVT. The reduction of prothrombin activity was observed in 34.5% of patients, which was also reported in the literature, and it has been attributed to increased consumption of coagulation factors due to the portal obstruction and the formation of portosystemic shunts and to the reduced hepatic blood flow(18, 23, 26). Hypersplenism leading to thrombocytopenia was detected in 61.8% of patients, and, in spite of the reduction in the number of platelets, they are functionally normal(4). Two patients had severe thrombocytopenia associated with esophageal varices, which led to surgical intervention. The procedure used was ligation of the splenic artery, with good results. Two other patients, evaluated in other services, underwent splenectomy, however the diagnosis of PVT was given after. Therefore, it is important to include the PVT in the differential diagnosis of afebrile splenomegaly so that an appropriate therapeutic approach may be accomplished in these cases, since splenectomy is not indicated in the cases of portal hypertension secondary to PVT. For the PVT diagnosis, the ultrasound exam, performed out of the Hospital das Clínicas’ service, needed to be repeated in 40% of patients. The first examination of these patients had not shown cavernomatous transformation. Doppler ultrasound is considered effective, less invasive and less expensive, so, it is the method of first choice for investigation(6, 20, 25, 41, 50, 52). Its sensitivity and specificity are, however, examiner-dependent. Therefore, PVT may not be

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diagnosed correctly by an inexperienced examiner, which may lead to an inadequate approach to the patient, and, also, to a delay in the control of complications. The presence of neovascularization in the thrombus area (cavernoma) occurs in the chronic cases(6, 20, 41, 50, 52) Abd El-Hamid et al.(1), Weiss et al.(53) and Alvarez et al.(2) reported percentage of UGIB similar to the one observed in the present study. Such percentage ranged from 69% to 79% in those studies. The mean and median of age in the present sample at the first episode were 4.6 and 4, respectively, (IR 2-6) very close to the ones reported by Abd El-Hamid et al.(1) (mean = 4.6 year old). In the study of Zargar et al.(56) the mean age was 6.4 years. When the endoscopic and/or drug secondary prophylaxis are not established, or it is unsuccessful, the chance of new bleeding is higher(10, 28, 56). In the present study, the mean number of UGIB episodes was the same as the one reported by Abd El-Hamid et al.(1). The UGIB treatment is based on the initial hemodynamic stabilization of the patient, cardiopulmonary resuscitation when necessary and evaluation of the need for a red blood cells transfusion, which was required in 79.5% of patients at least once(41). Abd El-Hamid et al.(1) reported the need for blood transfusion in 49 patients (45.4%). Even patients who did not bleed had esophageal varices at the first endoscopic exam. So, it is important to monitor all patients with PVT with gastrointestinal endoscopy to assess the presence of varices and to program the best approach of the endoscopic prophylaxis when it is necessary(45). The prevention of recurrence of UGIB secondary to bleeding from esophageal varices is fundamental for the follow-up of patients with portal hypertension. The adults’ treatment includes the use of drugs such as propranolol, endoscopic measures (sclerotherapy or EBL) and portosystemic shunts(10, 13, 56). As there are few studies in pediatrics, the treatment for this age group has been based on extrapolations of treatment in adults and in experience reported in the few pediatric casuistics found in the literature(10, 27, 28, 46, 56). Thirty-two patients (58.2%) underwent secondary endoscopic prophylaxis in our service. EBL has become the choice procedure, because studies have shown that this is a safe (lower incidence of complications) and effective procedure (shorter interval time to varices eradication, lower number of sessions and lower rebleeding ratio) for the secondary prophylaxis in children(10, 28, 46, 56). However, sclerotherapy still has room in the treatment of younger children, mainly in those who it is not possible the accomplishment of EBL due to technical limitations. Maksoud-Filho et al.(27), in

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recent study, showed that sclerotherapy is effective and safe for the treatment of varices in children. Among the major complications of endoscopic treatment, esophageal stenosis is the most common. It occurs most often associated with sclerotherapy, but it may also result from EBL(10, 28, 56). Abd El-Hamid et al.(1) reported complications in 34% of the 108 patients and stenosis and ulcers were the most commonly complications. The difference of prevalences observed between the study of Abd El-Hamid et al.(1) and the present study may be related to the most frequent use of sclerotherapy in that other casuistic. Surgical treatment is indicated in cases of recurrent bleeding even after appropriate endoscopic treatment, in massive splenomegaly and/or severe hypersplenism, in the retardation of children growth and in the symptomatic portal biliopathy(3, 14, 41, 52). The appearance of new surgical techniques has been improving the results obtained with shunts in children(3, 14, 24, 26). Today, there still preference to the distal splenorenal shunt in most services, but the Rex shunt (mesenteric left bypass) has emerged as the most appropriate technique and the one of choice(3, 13, 23). Our service has no experience with this technique, so this was not performed in any patient. Only one patient (1.8%) died due to acute bleeding episode. Mortality due to variceal bleeding in patients with PVT, but without cirrhosis, is of approximately 2% to 5% in adults. In children, the prognostic is usually better due to the low incidence of associated diseases, with a survival rate in 10 years higher than 70%(51). The patients assisted at the Hospital das Clínicas of UFMG showed characteristics similar to those evaluated in other pediatric samples and they had access to an appropriate clinical and endoscopic prophylactic treatment. However, there still is a lack of experience with the accomplishment of shunts, mainly the Rex shunt, which has become a treatment option in the PVT cases. As it is a non frequent condition and its diagnosis depends on the knowledge of the disease, it is possible that the number of cases referred to the reference service is lower than the one actually observed in the population. PVT is one of the most important causes of UGIB in children and the etiological factors are absent in up to 50% of patients. In every child with afebrile splenomegaly and/or hematemesis, without hepatomegaly and with normal hepatic function tests, we should suspect of PVT. Thus, an appropriate diagnosis and therapy is desirable in an attempt to reduce morbidity and mortality and improve quality of life of these patients.

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Ferri PM, Ferreira AR, Fagundes EDT, Liu SM, Roquete MLV, Penna FJ. Portal vein thrombosis in children and adolescents: 20 years experience of a pediatric hepatology reference center

Ferri PM, Ferreira AR, Fagundes EDT, Liu SM, Roquete MLV, Penna FJ. Trombose de veia porta em crianças e adolescentes: experiência de 20 anos de um serviço de referência em hepatologia pediátrica. Arq Gastroenterol. 2012;49(1):69-76. RESUMO – Contexto - Trombose da veia porta refere-se a uma obstrução total ou parcial do fluxo de sangue nesta veia devido à formação de um trombo. É uma causa importante da hipertensão porta na faixa etária pediátrica, com taxas elevadas de morbidade devido a sua principal complicação – a hemorragia digestiva alta. Objetivo - Descrever o grupo de crianças e adolescentes com trombose de veia porta sem doença hepática associada do Ambulatório de Hepatologia Pediátrica do Hospital das Clínicas da UFMG, Belo Horizonte, MG, Brasil, com ênfase no diagnóstico, forma de apresentação, complicações clínicas e na abordagem da hipertensão porta. Métodos - Trata-se de estudo descritivo de uma série de casos de crianças e adolescentes atendidos de janeiro de 1990 a dezembro de 2010. O diagnóstico de trombose de veia porta foi estabelecido por ultrassonografia. Resultados - Dos 55 pacientes analisados, 30 (54,5%) eram do gênero masculino. Em 29 pacientes (52,7%) não foi identificado nenhum fator de risco para trombose de veia porta. A forma de apresentação predominante foi hemorragia digestiva alta (52,7%). Em 20 pacientes (36,4%), a manifestação inicial foi esplenomegalia. Durante todo o período de seguimento, 39 pacientes (70,9%) apresentaram, pelo menos, um episódio de hemorragia digestiva alta. A média de idade dos pacientes neste primeiro episódio foi de 4,6 ± 3,4 anos. O exame endoscópico, seja realizado na urgência ou eletivamente para pesquisa de varizes esofágicas, mostrou sua presença em 84,9% dos pacientes avaliados. O tratamento endoscópico profilático foi realizado com ligadura elástica de varizes em 31,3% dos pacientes. Apenas um evoluiu para óbito devido a sangramento refratário. Conclusões - A trombose de veia porta é uma das causas mais importantes de hemorragia digestiva alta em crianças. Deve-se suspeitar de trombose de veia porta em toda criança com esplenomegalia afebril e/ou hematêmese, sem hepatomegalia e com testes de função hepática normais. Desta forma, uma abordagem diagnóstica e terapêutica adequada é desejável na tentativa de reduzir a morbimortalidade. DESCRITORES – Trombose venosa. Veia porta. Hipertensão portal. Crianças. Adolescentes.

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29. Northup PG, Sundaram V, Fallon MB, Reddy KR, Balogun RA, Sanyal AJ, Anstee QM, Hoffman MR, Ikura I, Caldwell SH. Hypercoagulation and thrombophilia in liver disease. J Thromb Haemost. 2008;6:2-9. 30. Odièvre M, Pigé G, Alagille D. Congenital abnormalities associated with extrahepatic portal hypertension. Arch Dis Child. 1977;52:383-5. 31. Ogren M, Bergqvist D, Björck M, Acosta S, Eriksson H, Sternby NH. Portal vein thrombosis: prevalence, patient characteristics and lifetime risk: a population study based on 23,796 consecutive autopsies. World J Gastroenterol. 2006;12:2115-9. 32. Pinto RB, Silveira TR, Bandinelli E, Röhsig L. Portal vein thrombosis in children and adolescents: the low prevalence of hereditary thrombophilic disorders. J Pediatr Surg. 2004;39:1356-61. 33. Ponziani FR, Zocco MA, Campanale C, Rinninella E, Tortora A, Di Maurizio L, Bombardieri G, De Cristofaro R, De Gaetano AM, Landolfi R, Gasbarrini A. Portal vein thrombosis: insight into physiopathology, diagnosis, and treatment. World J Gastroenterol. 2010;16:143-55. 34. Primignani M. Portal vein thrombosis, revisited. Dig Liver Dis. 2010;42:163-70. 35. Pugliese RP, D´Amico EA, Rocha TR, Bydlowski SP, Venturinelli ML, Cardoso RA, Miura IK, Chamone DA, Porta G. Risk factors in children and adolescents with portal vein thrombosis (PVT) and portal hypertension. Hepatology. 1998;28:551A. 36. Rangari M, Gupta R, Jain M, Malhotra V, Sarin SK. Hepatic dysfunction in patients with extrahepatic portal venous obstruction. Liver Int. 2003;23:434-9. 37. Rosendaal FR. Venous thrombosis: a multicausal disease. Lancet. 1999;353:1167-73. 38. Sakha SH, Rafeey M, Tarzamani MK. Portal venous thrombosis after umbilical vein catheterization. Indian J Gastroenterol. 2007;26:283-4. 39. Sarin SK, Agarwal SR. Extrahepatic portal vein obstruction. Semin Liver Dis. 2002;22:43-58. 40. Sarin SK, Sollano JD, Chawla YK, Amarapurkar D, Hamid S, Hashizume M, Jafri W, Kumar A, Kudo M, Lesmana LA, Sharma BC, Shiha G, Janaka de Silva H. Consensus on extra-hepatic portal vein obstruction. Liver Int. 2006;26:512-9. 41. Schettino GC, Fagundes ED, Roquete ML, Ferreira AR, Penna FJ. Portal vein thrombosis in children and adolescents. J Pediatr (Rio J). 2006;82:171-8. 42. Schwartz DS, Gettner PA, Konstantino MM, Bartley CL, Keller MS, Ehrenkranz RA, Jacobs HC. Umbilical venous catheterization and the risk of portal vein thrombosis. J Pediatr. 1997;131:760-2. 43. Seixas CA, Hessel G, Siqueira LH, Machado TF, Gallizoni AM, AnnichinoBizzacchi JM. Study of hemostasis in pediatric patients with portal vein thrombosis. Haematologica. 1998;83:955-6.

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44. Senders Aguirre B, Garcia Sanz MC, Casa Morcillo A. Trombosis de la vena porta. Ana Esp Pediatr. 2001;55:565-8. 45. Sharma S, Kumar SI, Poddar U, Yachha SK, Aggarwal R. Factor V Leiden and prothrombin gene G20210A mutations are uncommon in portal vein thrombosis in India. Indian J Gastroenterol. 2006;25:236-9. 46. Shneider B, Emre S, Groszmann R, Karani J, McKiernan P, Sarin S, Shashidhar H, Squires R, Superina R, de Ville de Goyet J, de Franchis R. Expert pediatric opinion on the Report of the Baveno IV consensus workshop on methodology of diagnosis and therapy in portal hypertension. Pediatr Transplant. 2006;10:893-907. 47. Sogaard KK, Astrup LB, Vilstrup H, Gronbaek H. Portal vein thrombosis; risk factors, clinical presentation and treatment. BMC Gastroenterol. 2007;7:34. 48. Uttenreuther-Fischer MM, Vetter B, Hellmann C, Otting U, Ziemer S, Hausdorf G, Gaedicke G, Kulozik AE. Paediatric thrombo-embolism: the influence of non-genetic factors and the role of activated protein C resistance and protein C deficiency. Eur J Pediatr. 1997;156:277-81. 49. Valla DC, Condat B. Portal vein thrombosis in adults: pathophysiology, pathogenesis and management. J Hepatol. 2000;32:865-71. 50. Wang JT, Zhao HY, Liu YL. Portal vein thrombosis. Hepatobiliary Pancreat Dis Int. 2005;4:515-8. 51. Webb LJ, Sherlock S. The aetiology, presentation and natural history of extrahepatic portal venous obstruction. Q J Med. 1979;48:627-39. 52. Webster GJ, Burroughs AK, Riordan SM. Review article: portal vein thrombosis -- new insights into aetiology and management. Aliment Pharmacol Ther. 2005;21:1-9. 53. Weiss B, Shteyer E, Vivante A, Berkowitz D, Reif S, Weizman Z, Bujanover Y, Shapiro R. Etiology and long-term outcome of extrahepatic portal vein obstruction in children. World J Gastroenterol. 2010 ;16:4968-72. 54. Yachha SK, Aggarwal R, Sharma BC, Misra RN, Aggarwal A, Naik SR. Functional protein C and anti-cardiolipin antibody in children with portal vein thrombosis. Indian J Gastroenterol. 2001;20:47-9. 55. Yamada RM, Antunes MM, Cardoso SR, Servidoni MF, Hessel G. [Portal vein thrombosis in children: clinical and laboratory study of 26 cases]. Arq Gastroenterol. 1999;36:49-53. 56. Zargar SA, Javid G, Khan BA, Yattoo GN, Shah AH, Gulzar GM, Singh J, Rehman BU, Din Z. Endoscopic ligation compared with sclerotherapy for bleeding esophageal varices in children with extrahepatic portal venous obstruction. Hepatology. 2002;36:666-72. Received 25/5/2011. Accepted 5/9/2011.

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GASTROENTEROLOGIA EXPERIMENTAL / EXPERIMENTAL GASTROENTEROLOGY

ARQGA/1592

HEPATIC HYPERPLASIA AND DAMAGES INDUCES BY ZEARALENONE Fusarium Mycotoxins in BALB/c Mice Pronobesh CHATOPADHYAY, Anurag PANDEY, Asshwani K. CHAURASIA, Addesh UPADHYAY, Sanjev KARMAKAR and Lokendra SINGH Abstract – Context - Zearalenone is a mycoestrogen and considered a mycotoxin. Objective - To establish whether zearalenone produced hepatotoxicity via oral administration. Methods - Zearalenone was orally administered at a dose of 50 μg, 100 μg and 200 μg ZEN/ body weight/daily, respectively, for 14 days to three groups of BALB/c mice. Diagnostic modalities used to evaluate hepatic damage and impaired hepatic function pre- and post zearalenone administration included hepatic marker enzyme activity, pentobarbital sleeping time, cytochrome P-450 activities and histopathologic evaluation of liver. Results - Significant histopathologic changes viz. sinusoidal congestion, cytoplasmic vacuolization, hepatocellular necrosis and neutrophil infiltration were observed after evaluating of liver section from each group after accumulated zearalenone exposure. Further, zearalenone exposure increased activities of alanine transaminase, aspartate transaminase and lipid peroxides whereas activities of tissue glutathione and cytochrome P450 were decreased as compared to control mice. Zearalenone also increased the sleeping time and decreased sleeping latency after pentobarbital through intraperitoneal route as compared to control mice which indicates that the impairment of hepatic metabolizing enzymes by zearalenone. Conclusion - Zearalenone is a potential hepatotoxin by oral route. Headings – Zearalenone. Hyperplasia. Liver, pathology. Mice.

INTRODUCTION

Zearalenone (6-[10-hydroxy-6-oxo-trans-1-undecenyl]Bresorcyclic acid lactone) (ZEN), is a non-steroidal mycoestrogen that activates the estrogen receptors (ERs), ESR1 and ESR2, where it acts as an agonist and partial antagonist to estradiol(5, 8, 10). ZEN also reported that facilitates carcinogenesis, reproductive toxicity(10) and suppress immunity(9). ZEN contaminated of cereals and grains and related products causes food and feed-borne intoxications (myco-toxicoses) in man and livestock. This mycotoxins produces by various members of the genus Fusarium, including deoxynivalenol (mostly produced by F. graminearum and F. sporotrichoides) and zearalenone (produced by F. graminearum and F. culmorum among others) which are major concern in health. Most of its biological activities of ZEN are attributed to the agonist effect on the ER and certain biological reactions of zearalenone that can not be explained by its estrogenic activity(5). Estrogens taken by women for birth control purposes are implicated as a cause for development of both benign and malignant hepatic neoplasms(11, 12). In addition to endogenous steroidal estrogens, a variety of compounds with estrogenic

activity also are found in the environment because a number of drugs, insecticides, and natural food products representing many different structures can act like estrogens by products by first phase metabolic transformation after ingestion. Therefore estrogenic activities of ZEN have possibilities to extend the other biological effects - hepatotoxicity. The aim of the research was to examine the toxic influence of ZEN on liver through estimating mycotoxin influence on markers evaluating hepatic damage. Methods

Animals and housing conditions All the born in the laboratory breeding colony of Central Facility of Animal House, Defence Research Laboratory Tezpur, Assam, India, and maintained under temperature controlled rooms at animal house with 12h alternating light and dark cycles. Food was obtained from Pranav Agrotech, Delhi, India. All experimental protocols using animals were performed according to the “Principles of Laboratory Animal care” (NIH publication 85-23, revised 1985) and approved by Institutional Use and Care Committee.

Division of Pharmaceutical Technology, Defence Research Laboratory. Correspondence: Dr. Pronobesh Chatopadhyay - Post Bag No: 2, Tezpur-784001, Assam, India. E-mail: chattopadhyay.drdo@gmail.com

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Study design Twenty-four Balb/c mice were divided into control group (I) (n = 6) were given 0.9% saline (5 mL/kg, p.o), ZEN treated groups viz. Groups II, III and IV were given 50 μg,100 μg, 200 μg ZEN/ body weight /daily, respectively, by oral route for 14 days (n = 6). After 14 days mice were euthanized by ether inhalation and sacrificed. Blood samples were obtained from the right ventricle via a left anterior thoracotomy and separated serum. The serum samples were stored at -20°C until used for ALT and AST assays. A portion of liver lobes were fixed in buffered 10% formalin, embedded in paraffin, and used for histopathology by using hematoxylin and eosin (H-E) staining. Another portion of lobe was stored at -20°C for enzymatic assay. Preparation of mice of liver microsome Liver microsome was measured as described by previously(18). Briefly, 100 mg liver was excised and homogenized with a loosefitting Teflon pestle in 900 mL of 50 mM L-1 Tris-HC1buffer, pH 7.4, containing 0.3 M L-1 sucrose, 10 mM L-1 DTT, and 10  mM L-1 EDTA. The homogenate was centrifuged at 20,000 x g for 15 min. The supernatant solution was centrifuged at 10,000 x g for 60 min. The microsomal fraction obtained was suspended in a homogenizing medium without DTT and re-centrifuged at 10,000 x g for 60 min. The resulting microsomal fraction was suspended in 0.1 ML-1 phosphate buffer pH 7.4, containing 1 mM L-1 EDTA and volume was adjusted to 10 mL with phosphate buffer. Estimation of hepatic marker enzymes Serum was used for the assay of alanine transaminase (ALT) and aspartate transaminase (AST) using assay kits (Transasia, Mumbai, India) according to the manufacturer’s instructions. Determination of cytochome P450 (CYP 450) Hepatic cytochome P450 was measured as described by previously(16). Briefly 1 mL supernatant of liver microsomes was taken and 10 mg of sodium dithionate was added. Air was bubbled for 10 seconds and then centrifuged for 5 minutes at 3000 x g. Five hundred µl (500 µl) supernatant was mixed with 5 mL Tris HCl buffer (pH 7.4) and absorbance was measured at 450 nm wavelengths using excitation coefficient of 91 mol L-1cm -1 for A 450. Light microscopy analysis Six µm stains of formalin –fixed and paraffin-embedded were taken and stained with H-E. Slide was examined by a pathologist who had no prior knowledge of the treatment groups. Histopathology of liver was evaluated by using a scoring system (Table 1) as described previously(19). Two types of eosinophilic hepatocellular changes are presumed to be apoptotic in origin viz. round and detached from surrounding hepatocytes (classical Councilman bodies) and shrunken compared to adjacent hepatocytes, but still firmly attached were counted in 5 to 20 fields from each liver to count at least 100 stellate –Abs. The changes in stellate cells were calculated by using a scoring system.

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TABLE 1. Scale of histopathology scoring Histopathology Assessment Steatosis (the percentage of liver cells (containing) 5% to 25% of cells containing fat 26% to 50% of cells containing fat 51% to 75% of cells containing fat >75% of cells containing fat Inflammation and necrosis One focus/ lobule Two or more foci/ lobules Apoptosis Stellate cells If nucleus fragmented present or absent If nucleus Pyknotic (or those in intact nuclei) If nucleus Pyknotic, fragmented or absent If adjacent inflammation present If located in an acidophilic domain If nodular hyperplasia If atrophy present If necrosis present If apoptosis present

Score 1+ 2+ 3+ 4+ 1+ 2+ 3+ 1+ 2+ 3+ 4+ 5+ 6+ 7+ 8+ 9+

Determination of lipid peroxides (LPO) Hepatic LPO was measured as method described(7). To 100 µl separated microsomes in 0.1(M) phosphate buffer saline, 1 mL of 28% trichloroacetic acid was added and centrifuged at x 2000 g at 4oC for 20 minutes. One milliliter of supernatant was separated and 900 µl of 1% thiobarbituric acid was added and volume was adjusted to 3 mL by using phosphate buffer (pH 7.0), heated in water bath for 60 min and cooled in ice bath. The absorbance was measured at 532 nm. The lipid peroxidation was calculated on the basis of the molar extinction coefficient (1.56 x 105) of malondialdehyde. Tissue glutathione (GSH) assays The hepatic reduced GSH level was determined by the method of Ellman(2). Briefly, after 0.2 g liver tissues were homogenized in 4 mL of 0.02 M EDTA Na2 (using an all glass homogenizer in an ice bath). In 2.5 mL tissue homogenates (aliquots) were mixed with 2.0 mL of distilled water and 0.2 mL of 50% TCA. All tubes were shaken intermittently for 10-15 min and centrifuged for 15 min at approximately 3000 × g. Two milliliter of 0.4 M Tris buffer (pH 8.9) and 0.1 mL of 0.01 M 5,5’dithiobis- 2-nitrobenzoic acid (DTNB) were added to 2.0 mL of tissue supernatant, and the sample was shaken. The absorbance was read within 5 min of the addition of DTNB at 412 nm against a reagent blank with no homogenate. GSH levels were calculated using standard curve prepared by known amounts of GSH (Aldrich Chemical Co. Ltd, Germany). The concentration of GSH was expressed as mg/g tissue.

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Pentobarbital sleeping time The pentobarbital sleeping time test was performed using pentobarbital. After 14 days treatment with ZEN animals received pentobarbital (concentration of 3 g/100 mL of solution) through intraperitoneal injection at a dosage of 50 mg/kg of mice body weight (or 0.0017 mL/g of animal weight) and time was measured in minutes, from the loss of position reflex to its gaining reflexes. Statistical analysis Data are expressed as mean ± S.D. The statistical significance between data means was determined by Student’s t-test. P-values P<0.05 were considered as significant RESULTS

Effect of ZEN on hepatic marker enzymes and cytochrome P450 ZEN increases significantly (P<0.01) AST, ALT levels whereas cytochrome P450 levels decreases as compared to controlled mice. Activities of ALT and AST were 55.23 ± 7.1 and 52.19 ± 9.40 I.UL-1, respectively, in controlled mice which increased to 87.96 ± 4.64 and 70.33 ± 3.59.UL-1, respectively, in ZEN treated (200 µg/kg body weight) mice. Microsomal, cytochrome P450 levels in the control mice were 0.66 ± 0.032 nML-1 mg-1 proteins which decreased to 0.29 ± 0.017 nML-1 mg-1 proteins after treatment for 14 days with ZEN 200 µg/kg body weight (Table 2).

Effect of ZEN on LPO, GST, sleeping time and sleep latency After treatment with ZEN significantly (P<0.01) increased LPO levels and GST levels were significantly decreased (P<0.01) as compared to control group. Activities of LPO and GST were 0.7 ± 0.05 nML-1 MDA mg-1 of protein and 52.40 ± 4.90 mM L-1 of 1-chloro 2,4-dinitrobenzene (CDNB) conjugated min-1 100 mg-1 protein in control groups and after treatment with ZEN (200 µg/kg body weight) increased to 5.22 ± 0.80 nML-1 MDA mg-1 of protein whereas GST decreased to 36.20 ± 3.77 mM L-1 of 1-chloro 2,4-dinitrobenzene (CDNB) conjugated min-1 100 mg-1 protein. Microsomal, cytochrome P450 levels in the control mice were 0.66 ± 0.032 nML-1 mg-1 proteins which decreased to 0.29 ± 0.017 nML-1 mg-1 proteins after treatment for 14 days with ZEN 200 µg/kg body weight (Table 3). Pentobarbital along with ZEN increased significantly (P<0.01) sleeping time as compared to control mice (Table 4). Effect of ZEN on liver histopathology Liver histopathology was evaluated based on sinusoidal congestion, cytoplasmic vacuolization, hepatocellular necrosis, and neutrophil infiltration (Table 5). Histopathology examination of liver sections of control group showed normal cellular architecture with distinct hepatic cells, sinusoids spaces and central vein (Figure 1a). ZEN treated group showed that disarrangement and degeneration of normal hepatic cells with intense centri-lobular necrosis extending to mid– zone and sinusoidal hemorrhages and dilation.

TABLE 2. The effect of ZEN on ALT, AST, cytochrome P450 levels after 14 days treatment a Groups ALT Control mice (Group I) 55.23 ± 7.10 ZEN treated (Group II) (50 µg/kg body weight) 62.11 ± 5.32* ZEN treated (Group III) (100 µg/kg body weight) 70.33 ± 6.91* ZEN treated (Group IV) (200 µg/kg body weight) 87.36 ± 4.64*

AST 52.19 ± 9.40 60.73 ± 4.47* 64.77 ± 5.06* 70.33 ± 3.59* a

Cytochrome P450 0.66 ± 0.032 0.50 ± 0.047 0.33 ± 0.053* 0.29 ± 0.017*

b

Results are expressed as mean ± SD (n = 6). *Statically different (P<0.01) from control. aExpressed in I.U. L-1, bExpressed as nML-1 protein

TABLE 3. The effect of ZEN on GST and LPO after 14 days oral administration Groups Control mice (Group I) ZEN treated (Group II) (50 µg/kg body weight) ZEN treated (Group III) (100 µg/kg body weight) ZEN treated (Group IV) (200 µg/kg body weight)

GST 52.40 ± 4.90 43.27 ± 6.66 40.16 ± 2.75 36.20 ± 3.77 a

LPO 0.7 ± 0.05 2.36 ± 0.77 3.56 ± 0.82 5.22 ± 0.80 a

Results are expressed as mean ± SD (n = 6). *Statically different (P<0.01) from control. aExpressed as nM L-1 of 1-cloro 2,4-dinitrobenzene (CDNB) conjugated min-1 100 mg-1 protein for GST; b Expressed as nML-1 MDA mg-1 of protein

TABLE 4. The effect of ZEN on sleeping time and sleep latency after 14 days oral administration of pentobarbital Groups Duration of sleep time Control mice + Phenobarbitone (Group I) 80.02 ± 2.77 ZEN treated + Phenobarbitone (Group II) (50 µg/kg body weight) 91.54 ± 3.56* ZEN treated + Phenobarbitone (Group III) (100 µg/kg body weight) 107.89 ± 9.23* ZEN treated + Phenobarbitone (Group IV) (200 µg/kg body weight) 125.64 ± 11.54*

Sleep latency 2.84 ± 0.88 2.35 ± 0.54* 2.33 ± 0.47 1.97 ± 0.75*

Results are expressed as mean ± SD (n = 6). *Statically different (P<0.01) from control. Sleep time, sleep latency were expressed in minutes

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TABLE 5. The effect of ZEN on histopathology of liver after 14 days oral administration Groups Histopatological features a. Nucleus fragmented or absent b. Nucleus Pyknotic (intact nucleus) c. Nucleus Pyknotic fragmented or absent d. Adjacent inflammation present e. Located in an acidophilic domain f. Nodular hyperplasia g. Atrophy

A

C

B

D

Control groups Round AB Stellate AB 5/105 (5%) 15/400 (4%) 4/110 (4%) 13/200 (7%) Not significant 1/125 (2%) Not significant Not significant 2/108 (2%) 6/330 (2%) 4/125 (3%) 17/220 (8%) 4/116 (3%) 4/155 (3%)

FIGURE 1. Showing histopathology of liver sections after staining with H-Eosin (H-E, x.400). (A) Liver section of control mice showing normal architecture of liver with central vein. (B) Liver section of ZEN (50 μg/kg body weight) administered showing sinusoidal congestion and cytoplasmic vacuolization. (C) Liver section of ZEN (100 μg/kg body weight) administered showing hepatocellular necrosis and neutrophil infiltration. (D) Liver section of ZEN (200 μg/kg body weight) administered showing severe nodular hyperplasia, pyknotic nucleus and necrosis

There was chronic inflammatory cells infiltrate in the portal tracks. There was also extensive hepatocellular necrosis, sinusoidal congestion, and neutrophil infiltration. Shrunken acidophilic hepatocytes were both round and detached from surrounding hepatocytes (round – Abs) and stellate – shaped firmly attached to adjacent hepatocytes (stellate – Abs). In 44% of round – Abs, the nucleus was fragmented or absent and among those with an intact nucleus, 80% had nuclear pyknosis stellate. Stellate – Abs often clustered in acidophilic domain usually without significant lymphoid infiltrate or necrosis. Round- Abs were usually not clustered and seen more often with an adjust lymphocytes infiltrate. Also apoptotic bodies, in the form of round or stellate – Abs, were seen (Figure 1b). Discussion

In this present investigation ZEN showed hepatotoxicity in BALB/c mice which characterized by increasing of hepatic marker enzymes (ALT, AST), decreasing of microsomal

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ZEN treated groups Round AB Stellate AB 77/189 (41%) 59/510 (12%) 40/70 (57%) 190/620 (31%) 102/140 (72%) 240/550 (44%) 87/109 (80%) 222/487 (46%) 104/188 (55%) 98/175 (56%) 103/127 (81%) 104/125 (83%) 105/160 (66%) 69/104 (66%)

cytochrome P450 and histopathology study showed that ZEN decreased in the number of perfused sinusoids and hepatocellular hypoxia. Zearalenone is resorcylic acid lactones and functionally is mycoestrogen which found in contaminating grain. Mycoestrogen increased incidence of adenomas (pituitary, liver) was detected in one species after a 2-yr oral carcinogenicity study(11). In present investigation ZEN shows the potential hepatotoxicant may due to estrogenic property. Thus, the impaired hepatic function caused after administration of ZEN, that might be a reason for activation of hepatocytes led to increase hepatic marker enzymes, as seen in the present work. The pathophysiology of hepatic injury is complex and it is thought that hepatotoxin activates hepatic cells and subsequently causes free radical- mediated tissue injury and by series of chain reactions produces Lipid peroxidation (LPO)(17) (Figure 1). Ayed et al.(1) reported the genotoxicity of ZEN and concluded that biotransformation of ZEN involved only partial detoxification and remaining metabolites are relatively toxic. Another study of Frizzell et al.(3) reported that ZEN and its metabolites showed potential endocrine disruptors by altering hormone production. Further, ZEN ingestion of animals from contaminated feed decreased the TNF-α synthesis and IL-8 synthesis(14). Another study of Marin et al.(13) reported similar immunosuppressive effects of ZEN and its derivatives (alpha-ZOL, beta-ZOL, ZAN) in swine. Thus, ZEN showed multiple side effects and our observation reported first time hepatotoxicity of ZEN. Formation of LPO leads to many pathological changes in tissue including liver necrosis(6). The consequences of lipid peroxidation may be manifested as alternation in membrane integrity of membrane- associated functions in sub-cellular organelles. Reduced glutathione (GSH) is known to function as an antioxidant and a physiological reservoir for cysteine and is involved in DNA synthesis, protein synthesis regulation, and detoxification, etc. Cellular GSH deficiency affects the mitochondrial GSH pool and the cytosolic GSH pool. Mitochondrial GSH is important for the detoxification of ROS generated by the respiratory chain, conjugation of xenobiotics, maintenance of thiol-containing proteins, and regulation of the mitochondrial membrane potential(15) (Table 5).

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Chatopadhyay P, Pandey A, Chaurasia AK, Upadhyay A, Karmakar S, Singh L. Hepatic hyperplasia and damages induces by zearalenone fusarium mycotoxins in balb/c mice

In our present study indicates that in parallel increased AST and ALT, increased levels of LPO and decreased GSH activity occurred liver injury by ZEN. Another point to be considered in this context is that there are increased sleeping times of pentobarbital with combination of ZEN. This result indicates that ZEN impaired metabolism of pentobarbital by hepatic damage with inhibits the activity of CYP450. Previous study shows that ZEN activates the nuclear receptor PXR and induces the expression of the drug-metabolizing enzyme CYP3A4, a hepatic monooxygenase involved in the metabolism of about 60% of clinically used drugs(4). Further, in our present investigation decreased of CYP450 activities correlates the increasing sleeping time of pentobarbital. Thus, it is likely that ZEN inhibits CYP450 activities by hepatotoxicity.

In summary, the present study provides the first functional anatomical evidence that phenobarbitone sleep time increase may predispose the liver to significant oxidative injury and subsequently hepatic damage, CYP450 deactivation by ZEN. Although the detailed mechanisms concerning the deactivation of CYP450 are not fully understood, the current findings present important insights into the potential detrimental effect of oxidative stress and impaired hepatic function of ZEN. AcknowledgementS

This work has been supported by a grant of Defence Research Development Organization (DRDO), Ministry of Defence, Government of India.

Chatopadhyay P, Pandey A, Chaurasia AK, Upadhyay A, Kamarkar S, Singh L. Hiperplasia e danos hepáticos induzidos por micotoxinas do gênero Fusarium-zearalenone em camundongos bab/c. Arq Gastroenterol. 2012;49(1):77-81. RESUMO – Contexto - Zearalenone é um micoestrógeno e considerado como micotoxina. Objetivo - Avaliar se o Zearalenone produz hepatotoxicidade por administração via oral. Métodos - Zearalenone foi administrada por via oral em doses de 50 µg, 100 µg e 200 µg/peso corporal/dia/14 dias, respectivamente, para três grupos de camundongos BAB/C. Modalidades diagnósticas usadas para avaliar o dano hepático e comprometimento da função hepática pré- e pós-administração de Zearalenone incluíram atividade enzimática de marcadores hepáticos, tempo de sono por pentobarbital, atividade do citocromo P-450 e avaliação histopatológica hepática. Resultados - Alterações histopatológicas significantes como congestão sinusoidal, vacuolização citoplasmática, necrose hepatocelular e infiltração neutrofílica foram observadas após avaliação histológica de cada grupo após exposição acumulada de Zearalenone. Além disto, a exposição à Zearalenone incrementou a atividade das enzimas alanina transaminase e aspartato transaminase e peróxidos lipídicos, ao passo que as atividades teciduais de glutationa e citocromo P-450 diminuiram, quando comparadas com camundongos-controle. Zearalenone também aumentou o tempo de sono e diminuiu a latência do sono após a administração de pentobarbital por via intra-abdominal, quando comparados com camundongos-controle, o que indica o comprometimento das enzimas do metabolismo hepático por ela. Conclusão - Zearalenone é uma potente hepatotoxina quando administrada por via oral. DESCRITORES – Hiperplasia. Zearalenona. Fígado, patologia. Camundongos.

REFERENCES 1.

Ayed Y, Ayed-Boussema I, Ouanes Z, Bacha H. In vitro and in vivo induction of chromosome aberrations by alpha- and beta-zearalenols: comparison with zearalenone. Mutat Res. 2011 [in press]. 2. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959;82:70–7. 3. Frizzell C, Ndossi D, Verhaegen S, Dahl E, Eriksen G, Sorilie M, Ropstad E, Muller M, Elliott Ct, Connolly L. Endocrine disrupting effects of zearalenone, alphaand beta-zearalenol at the level of nuclear receptor binding and steroidogenesis. Toxicol Lett. 2011;206:210-7. 4. Guengerich FP. Cytochrome P-450 3A4: regulation and role in drug metabolism. Annu Rev Pharmacol Toxicol. 1999;39:1–17. 5. Hidy PH, Baldwin RS, Greasham RL, Keith CL, McMullen JR. Zearalenone and some derivatives: production and biological activities. Adv Appl Microbiol. 1977;22:59–82. 6. Jaeschke H, Farhood A. Neutrophil and kupffer cell-induced oxidant stress and ischemic–reperfusion injury in rat liver. Am J Physiol. 1991;260:G355-G62. 7. Jordan RA, Schenkman JB. Relationship between malondialdehyde production and arachidonate consumption during NADPH supported microsomal lipid peroxidation. Biochem Pharmacol. 1982;31:1393-400. 8. Katzenellenbogen BS, Korach KS. A new actor in the estrogen receptor dramaenter ER-beta. Endocrinology. 1997;138:861–2. 9. Kilakivi-Clarke L, Cho E, Onojafe I, Raygada M, Clarke R. Maternal exposure to genistein during pregnancy increases carcinogen–induced mammary tumorgenesis in female rat offspring. Oncol Rep. 1999;6:1089-95. 10. Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT, van

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der Burg B, Gustafsson JA. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology. 1998;139:4252–63. 11. La Vecchia C, Negri E, Parazzini F. Oral contraceptives and primary liver cancer. Br J Cancer. 1989;59:460–1. 12. Mant JW, Vessey MP. Trends in mortality from primary liver cancer in England and Wales 1975-92: influence of oral contraceptives. Br J Cancer. 1995;72:800–3. 13. Marin DE, Taranu I, Burlacu R, Tudor DS. Effects of zearalenone and its derivatives on the innate immune response of swine. Toxicon. 2010;56:956-63. 14. Marin DE, Taranu I, Burlacu R, Manda G, Motiu M, Neagoe I, Dragomir C, Stancu M, Calin L. Effects of zearalenone and its derivatives on porcine immune response. Toxicol In Vitro. 2011;25:1981-8. 15. Marubayashi S, Dohi K, Ochi K, Kawaski T. Role of free radicals in ischemic rat liver cell injury: prevention of damage by alpha-tocopherol administration. Surgery. 1986;99:184-2. 16. Omura T, Sato R. The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem. 1964;239;2370-8. 17. Salah-Abbès J, Abbès S, Ouanes Z, Houas Z, Abdel-Wahhab MA, Bacha H, Oueslati R. Tunisian radish extract (Raphanus sativus) enhances the antioxidant status and protects against oxidative stress induced by zearalenone in Balb/c mice. J Appl Toxicol. 2008;28:6-14. 18. Schenkman JB, Cinti DL. Preparation of microsomes with calcium. Methods Enzymol. 1978;52:83-9. 19. Shimamatsu K, Wanless IR. Role of ischemia causing apoptosis, athropy and nodular hyperplasia in human liver. Hepatology. 1997;26:343-50.

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Received 1/9/2011. Accepted 20/9/2011.

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GASTROENTEROLOGIA EXPERIMENTAL/ EXPERIMENTAL GASTROENTEROLOGY 82

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NOISE-INDUCED GASTRIC LESIONS: a light and electron microscopy study of the rat gastric wall exposed to low frequency noise Jorge FONSECA1, José MARTINS-dos-SANTOS1, Pedro OLIVEIRA1, Nuno LARANJEIRA1, Artur AGUAS2 and Nuno CASTELO-BRANCO3 ABSTRACT – Context - Only a few studies evaluated the digestive alterations caused by low frequency noise (LFN) and most focused only on mucosal alterations. Objectives - To investigate the morphological injury of LFN-exposed gastric wall, beyond the epithelial layer. Methods - Wistar rats were exposed to low frequency noise (LFN), during increasing periods, 1 to 13 weeks. A control group was kept in silence. Gastric specimens were studied using: (i) light microscopy with hematoxylin-eosin and immunostaining for collagens; (ii) transmission electron microscopy; (iii) morphometry allowing statistical analysis. Results - Submucosa of all LFN-exposed animals exhibit increased thickness with fibrous proliferation. Transmission electron microscopy showed massive collagen deposition. Immunostaining identified collagen IV as responsible for the increased thickness. Morphometry allowed the demonstration of a significant difference of thickness between control and exposed groups. Vascular alterations included: i) intima proliferation and thickening, rupture of the internal elastic lamina, thrombotic changes; ii) thickening of the media; iii) after 9 weeks of LFN-exposure, we found new formed vessel presenting tortuous and twisted. There is a significant difference of arterial wall thickness between control and exposed groups. Conclusions - Deeper layers of gastric wall undergo alterations, including fibrosis of the submucosa caused by collagen IV deposition, an early marker of neoangiogenesis. Vascular alterations included thickening and thrombotic phenomena, but also images of newly formed vessels. This study suggests that, at least in the stomach, LFN-induced fibrosis could be linked with neoangiogenesis. HEADINGS – Gastric mucosa, physiopathology. Noise effects. Microscopy, electron. Rats.

Introduction

In our modern urban societies, noise is a well recognised environmental hazard. For legal purposes, the sound description uses a unit called A-decibel, dB(A). As the human ear has a reduced sensitivity to sound in the frequencies under 500 Hz, a sound analyser is used, with a special filter that “mimics” the human ear evaluation of sound (A-weighting). Although useful to prevent auditory diseases, dB(A) underestimates low frequency noise (LFN) and dB(A) measurements are inappropriate to understand noise systemic effects. In order to evaluate these noiseinduced systemic effects the “linear-weighting” must be used. Using “linear-weighting”, expressed in linear decibel, dB(L), every sound frequency in the spectrum of 10–20 000 Hz has the same sound attenuation, a physical process similar to the one that occurs when sound reaches organs and tissues. As dB(A) measurements are used for legal evaluations, environmental LFN is usually higher than measured and frequently overlooked. The scientific community has long recognized the under evaluation of LFN(21). World Health Organization repeatedly addressed the

public authorities and recommended the use of more accurate noise measurements(3). Most of the published medical research on noise-induced diseases is related to hearing disorders. Nevertheless, nonauditory effects of noise have been reported for decades, including gastrointestinal (GI) clinical complaints and gastric lesions. Gastric lesions were found after chronic(1) and acute(5) noise aggression, both in humans(29) and animals(1, 29). A study performed among boiler-plant workers reported GI complaints, including heartburn, stomach cramps, nausea and diarrhea(13). After the implementation of a hearing protection program, these GI complaints increased, suggesting that the hearing protection, decreasing the audible exposure to noise, also decreased the avoidance of noise. This would increase the exposition to noise sources, which could be harmful through non-auditory pathways. In the last three decades, most of these non-auditory effects of noise were related to the systemic effects of LFN. These included neurologic(31, 34, 41, 42, 48, 49), cardiac (7, 33) , vascular(17, 35) and respiratory disorders(10, 16, 44, 45, 46). Lesions of the parotid gland(47) and periodontium of LFN-exposed rats were recently reported(38). Regardless of the organs and tissues studied, the LFN-induced

1 Instituto Superior de Ciências da Saúde Egas Moniz (CiiEM), Almada, Portugal; 2Instituto de Ciências Biomédicas Abel Salazar – Universidade do Porto (UMIB), Porto, Portugal; 3 Center for Human Performance, Alverca, Portugal. Correspondence: Dr. Jorge Fonseca. E-mail: jorgedafonseca@hotmail.com

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Fonseca J, Martins-dos-Santos J, Oliveira P, Laranjeira N, Aguas A, Castelo-Branco N. Noise-induced gastric lesions: a light and electron microscopy study of the rat gastric wall exposed to low frequency noise

morphologic alterations seem to occur with significant fibrosis(9, 17, 35, 47) and with major vascular lesions(17, 35, 47). LFN-induced disorders include clinically identified digestive symptoms(6, 8). Using LFN-exposed Wistar rats, we disclosed massive cell death leading to diffuse gastric erosions(14, 19). Using the same experimental model, we proved that the duodenal mucosa also developed superficial erosions(15). Nevertheless, these upper GI lesions were restricted to the superficial mucosal layer. It was never reported any clinical or experimental study addressing the thickness of the gut wall, where important morphologic alterations could be expected. The goal of the present study was to investigate the morphological injury of the gastric wall, beyond the epithelial layer, using light microscopy (LM), and transmission electron microscopy (TEM). METHODS

Animals We used adult Wistar rats weights ranging from 210 g to 292 g, exposed to continuous LFN, during increasing periods: Group 1 – 1 week (168 hours) - 10 rats Group 2 – 3 weeks (504 hours) - 10 rats Group 3 – 5 weeks (840 hours) - 10 rats Group 4 – 9 weeks (1512 hours) - 10 rats Group 5 – 13 weeks (2184 hours) - 10 rats Each group had 5 males and 5 females. A control group (group C), of 15 rats (7 males and 8 females – weights from 209 g to 292 g), had the same experimental conditions, but was kept in silence. They were housed in groups of 2 or 3 rats per cage, in which they could move around. They had light cycles of 12 hours day/12 hours night. All animals were fed with standard rat food, had unrestrained access to water, and were treated according with the EU directive on Animal Protection for Experimental and Scientific Purposes (86/609/EEC). Low frequency noise exposure – experimental protocol The rat cages were placed in front of a noise generator, consisting of a sub-woofer Magnat xtc 1200, in an isolated compartment measuring 217 × 211 × 195cm. A computer Compaq (Pentium 133 MHz) reproduced a “white noise” previously recorded. The sound signal was amplified with a QSC amplifier and frequencyfiltered, creating an acoustic environment rich in low frequency components. The noise exposure protocol was previously described(14). Noise level and the spectral analysis, similar in the entire compartment, were analysed by a digital real time analyser, near the position of the LFN-exposed rats. Levels were above 90 dB in the frequencies ranging from 50 to 500 Hz. Light microscopy Rats were sacrificed with a lethal intraperitoneal injection of pentobarbital. After removing a small fragment for electron microscopy, the stomach was removed using a procedure similar to the one described by Nakamura et al.(43). Sections were stained with haematoxylin-eosin. Immunostaining for collagens I, IV and V was performed using the peroxidase technique.

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Transmission electron microscopy For TEM, the specimens were washed in phosphate buffer saline, fixed at room temperature in an aldehyde mixture made up of 4% formaldehyde, 1.25% glutaraldehyde and 10 nmol/L Ca Cl2 in 0.05 mol/L cacodylate buffer. The samples were dehydrated in ethanol and cut with a LKB microtome. The preparations were stained with uranyl acetate - lead citrate and examined in a JEOL 100C transmission electron microscope. Morphometry Morphometry was performed using the OPTIMAS software that allows an easy measurement of lengths and areas. In each rat, three artery images, stained with haematoxylin-eosin, were evaluated. In each artery we defined the lumen area, the wall area and the wall/lumen ratio (W/L ratio). Globally, we studied 45 arteries of control group rats and 30 arteries in each one of the 5 LFN-exposed groups. With the same software, thickness of gastric submucosa was measured in the proximal rat gastric wall (forestomach), where wall thickness is rather regular. Statistic evaluation was performed using the SPSS 13.0 program. For the W/L ratio we used the Scheffé “Post-hoc” test. For submucosal thickness evaluation we used the Welch and Brown-Forsythe tests and the Tamhane and Dunett tests. RESULTS

We disclosed several morphologic changes on the gastric wall of LFN-exposed rats. Gastric submucosa Observed with LM, submucosa of all LFN-exposed animals exhibit increased thickness, more striking in the rats exposed to noise for longer periods. This increased thickness was caused by fibrous tissue proliferation that frequently invaded the muscular layer. The TEM images revealed the collagen proliferation responsible for enlargement of the submucosa (Figure 1). Staining for collagens revealed that, in control

FIGURE 1. TEM images disclosing the collagen (C) proliferation responsible for the submucosal increased thickness (x 2000)

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rats, collagen I (Figure 2a) and collagen V (Figure 3a) were very abundant and collagen IV was hardly found and just in perivascular topography (Figure 4a). The submucosa of LFN-exposed rats revealed an almost complete disappearance of collagen I (Figure 2b) and collagen V (Figure 3b). In contrast, collagen IV was greatly increased (Figure 4b) and appeared to be responsible for the increase of gastric wall thickness. Morphometry confirmed the increasing width of the submucosa (Table 1), starting in the earlier exposure periods. The differences between the submucosal thickness of control group and each one of the noise-exposed groups were statistically significant (P<0.05). With progressive exposure the submucosa increased its thickness. Gastric arteries Gastric vessels and most remarkably submucosal arteries reveal significant alterations after a minimum

of 3 weeks of LFN-exposure (groups 2 to 5). These alterations included: i) focal proliferation and thickening of the intima, with rupture of the internal elastic lamina, sometimes associated with thrombotic changes (Figure 5), ii) regular thickening of the media layer presenting eosinofilia and vacuolization (Figure 5) iii) in groups 4 and 5 (9 or 13 weeks of LFN-exposure), blood vessels on greater number, arteries and also veins were tortuous, convoluted and twisted (Figure 6), sometimes overlapping. These vascular changes were also present in other layers, namely in the adventitia. Morphometric evaluation confirmed the increasing W/L ratio, an indirect assessment of increasing arterial wall width exhibiting a progressive W/L ratio (Table 2). Group 1 had a W/L ratio similar to control group. Groups 3, 4 and 5 displayed a W/L ratio greater then control group and the Scheffé “Post-hoc” test demonstrated a statistically significant difference (P<0.005).

FIGURE 2. Immunostaining for collagen I (x 200): very abundant in control rat (Figure 2a) and very reduced in the gastric wall of a rat exposed to LFN during 13 weeks (Figure 2b)

FIGURE 3. Immunostaining for collagen V (x 200): abundant in control rat (Figure 3a) and almost disappeared in the gastric wall of a rat exposed to LFN during 13 weeks (Figure 3b)

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FIGURE 5. Artery of a rat exposed to LFN during 9 weeks (x 200): thickening of the intima and the media, eosinofilia and vacuolization of the media and rupture of the internal elastic lamina (arrows)

FIGURE 4. Immunostaining for collagen IV (x 200): sparse in control rat (x 200), located only in perivascular topography (Figure 4a) and abundant in gastric wall of a rat exposed to LFN during 5 weeks (x 100) causing the enlargement of the submucosa (Figure 4b) TABLE 1. Variation of the submucosal thickness according with the LFN-exposure period Number of Exposure Submucosal rats period thickness (micra) Group C 15 ø 86 Group 1 10 1 week 145 Group 2 10 3 weeks 152 Group 3 10 5 weeks 163 Group 4 10 9 weeks 177 Group 5 10 13 weeks 217 TABLE 2. Variation of the wall/lumen ratio (W/L ratio) according with the LFN-exposure period Number of rats / Exposure W/L ratio Number of arteries period Group C 15 / 45 ø 3,75 (SD: 2,32) Group 1 10 / 30 1 week 3,95 (SD:1,96) Group 2 10 / 30 3 weeks 6,00 (SD:3,77) Group 3 10 / 30 5 weeks 8,41 (SD:3,32) Group 4 10 / 30 9 weeks 6,81 (SD:3,37) Group 5 10 / 30 13 weeks 8,96 (SD:4,29)

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FIGURE 6. Blood vessels in greater number, arteries and also veins (arrow) tortuous, convoluted and twisted of a rat exposed to LFN during 9 weeks (x 100) DISCUSSION

Reports had long linked non-auditory effects of noise with digestive complaints(13) and GI lesions(1, 29). Also, the spectra of the clinical disorders caused by LFN includes gastric and gut symptoms(8, 11). Using the Wistar rat model, we had previously proved that the proximal gut suffered with LFN-exposure developing diffuse gastric(14, 19) and duodenal(15) erosions caused by considerable epithelial cell lesion and death(14, 19). These epithelial cell lesions were similar to lesions previously found in other LFN-exposure tissues, namely the rat pleura(44, 46) and the bronchi(10, 16, 45). These lesions were also similar to ulcerative lesions caused by whole-body vibration (WBV), a vibratory phenomenon comparable to noise. Most of the clinical manifestations of the “WBV disease” are related with bone and joint trauma(4, 5) causing debilitating back pain(27, 28, 30) but digestive complaints are also frequent(39, 53). Lesions were found in a large group of WBV-exposed workers(55), using endoscopy and biopsies. Nakamura et al.(43), with an experimental

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model, produced WBV-induced gastric ulcers and proved that those ulcers were caused by direct mechanical effect of gastric vibration. Nevertheless, previous human and animal studies on LFN or WBV focused only the gastric mucosa and did not provide any understanding on the effects of vibration in deeper layers of the stomach. Looking deeper into the gastric wall, the present study disclosed two main types of alterations of submucosa and other layers: vascular alterations and fibrosis. We found proliferation and thickening of the intima and the media, with rupture of the internal elastic lamina and thrombotic changes. These lesions were similar to those disclosed in other vessels, arteries, veins and lymphatics exposed to this kind of noise(17, 35, 47). Arterial lesions in the gastric submucosa were also similar to those reported in the “hand-arm vibration syndrome” that is a well known occupational disease related with the use of vibrating tools(37). Although joint (40, 56) and neural(22, 32, 51, 52) lesions are important, the syndrome seems to have a vascular pathogenesis. Clinically, it is characterised by recurrent vasospasm similar to Reynaud phenomenon(2, 12, 18, 36, 54). Biopsies of these patients revealed thickening, fibrosis and destruction of elastic fibres(57) analogous to the lesions found in the vessels of LFN-exposed rats in the present and previous studies(17, 35, 47). The resemblance of vascular lesions occurring in LFN-exposed and directly transmitted vibration is expectable since the physical phenomenon underlying directly transmitted vibration is the same of noise and sound. Only the auditory system allows the perception of air borne vibration as sound or noise. Reaching other organs, air borne vibration is similar to directly transmitted vibration. These mechanical forces could be responsible for the destruction of elastic fibres seen in the arteries of “hand-arm vibration syndrome” patients and could cause some of the lesions observed in LFN-exposed arteries, as the rupture of the internal elastic lamina. The other important alteration disclosed was the massive proliferation of fibrous tissue. From previous reports in other organs, we already knew that LFN-exposure was related with major fibrosis(9, 17, 20, 33, 35, 47, 50). In our model, we disclose the presence of fibrous tissue enlarging the gastric submucosa of all exposed animals. Morphometric analysis proved that submucosal thickness of noise-exposed groups is significantly larger than submucosal thickness of control group. The same mechanical forces that cause fragmentation of the elastic fibres of the arterial wall may be the cause of collagen production. Ingber(23, 24, 25, 26) has proved that mechanical forces applied to individual cells could change cell reactions to biochemical stimuli or even induce entirely different cellular responses. Mechanical forces resulting from tissue vibration may be the initial stimulus

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for collagen production. Although we could not clearly identify the triggering sign of the fibrotic process, the nature of the collagen production was enlightened. The use of specific collagen immunostaining identified collagen IV as the main contributor to this increased fibrous tissue. It is known that collagen IV appears early after an aggression, associated with neoangiogenesis, and later in the repairing process new formed vessels become visible and collagen I replaces collagen IV(58). In our model, the relationship between collagen IV and angiogenesis points towards a pathophysiologic linkage between these vascular lesions and the fibrosis. In the first weeks of LFN-exposure, vessels, most strikingly arteries, start showing thickening and thrombotic lesions. Simultaneously fibrotic changes start early: the thickness of the submucosa of 1 week exposed rats is significantly larger than the submucosa of control rats. Unlike other experimental models in which the aggression is time limited(58), in our model the LFNexposure is continuous. Consequently, the fibrotic process is constantly induced and thickness increases with exposure. In the rats exposed for longer periods, group 4 (9 weeks) and group 5 (13 weeks) we observed simultaneously two features: as the persistent LFN-exposure is constantly stimulating the production of new collagen we found recently formed collagen IV, representative of the early stages of angiogenesis, but also an increased number of new vessels, frequently tortuous, convoluted and twisted, the end result of the neoangiogenesis pathway developing during 9 or 13 weeks. In the gastric wall exposed to LFN, fibrosis is due to collagen IV production and related with the neoangiogenesis process. CONCLUSIONS

In the present study we proved that LFN-induced gastric lesions were not limited to the mucosa. Deeper layers, mainly the submucosa, also suffer important morphologic alterations. These included vascular lesions, namely thickening of the arterial walls with thrombotic phenomena. Fibrotic changes of the submucosa were caused by increased collagen IV production as part of a neoagiogenesis pathway resulting in new vessels formation after longer LFN-exposure periods. Although the presence of vascular lesions and fibrosis were long known features of LFN-exposed tissues, this study established a link between these two phenomena. At least in the gastric wall, the present data points to the collagen IV production occurring during a neoangiogenesis pathway as an important feature of the LFN-induced fibrosis. Further studies addressing hollow organs are needed, in order to evaluate the importance of neoangiogenesis in structures exposed to this kind of noise.

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Fonseca J, Martins-dos-Santos J, Oliveira P, Laranjeira N, Aguas A, Castelo-Branco N. Lesões gástricas induzidas pelo ruído: um estudo por microscopia ótica e eletrônica da parede gástrica do rato exposta a ruído de baixa frequência. Arq Gastroenterol. 2012;49(1):82-8. RESUMO – Contexto - Só um reduzido número de estudos avaliou as alterações digestivas causadas pelo ruído de baixa frequência (RBF) e, na sua maioria, focados nas alterações da mucosa. Objetivo - Avaliar as lesões morfológicas induzidas na parede gástrica pelo RBF, para além da camada mucosa. Métodos - Ratos Wistar foram expostos a RBF, por períodos crescentes, desde 1 a 13 semanas. Um grupo controle foi mantido em silêncio. Fragmentos da parede gástrica foram estudados com recurso a: (i) microscopia ótica usando hematoxilina-eosina e imunomarcação para colágenos; (ii) microscopia eletrônica de transmissão; (iii) morfometria, permitindo a colheita de dados para análise estatística. Resultados - Observaram-se espessamento e proliferação de tecido fibroso na submucosa de todos os animais expostos aos RBF. A microscopia eletrônica de transmissão mostrou marcada deposição de colágeno. A imunomarcação identificou o colágeno IV como responsável pelo espessamento da submucosa. A morfometria permitiu demonstrar que a diferença de espessura da submucosa entre o grupo controle e os grupos de animais expostos era estatisticamente significativo. As alterações vasculares incluíram: (i) proliferação e espessamento da íntima, com rotura da lâmina elástica interna e fenômenos trombóticos; (ii) espessamento da média; (iii) após 9 semanas de exposição ao RBF, observaram-se vasos neoformados tortuosos e contorcidos. A espessura dos vasos dos animais expostos ao ruído é significativamente maior que a espessura no grupo controle. Conclusões - As camadas profundas da parede gástrica sofrem alterações que incluem fibrose causada pela deposição de colágeno IV, marcador precoce de neoangiogênese. As alterações vasculares incluem espessamento e fenômenos trombóticos e vasos neoformados. O presente estudo sugere que, pelo menos no estômago, a fibrose associada ao RBF pode estar ligada à neoangiogênese. DESCRITORES – Mucosa gástrica, fisiopatologia. Efeitos do ruído. Microscopia eletrônica. Ratos.

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36. Matoba T, Ishitake T. Cardiovascular reflexes during vibration stress. Kurume Med J. 1990;37(suppl):S61-71. 37. McGeoch KL, Lawson IJ, Burke F, Proud G, Miles J. Diagnostic criteria and staging of hand-arm vibration syndrome in the United Kingdom. Ind Health. 2005;43:527-34. 38. Mendes J, Martins dos Santos J, Oliveira P, Branco NA. Low-frequency noise effects on periodontium of the Wistar rat – a light microscopy study. Eur J Anat. 2007;11:27-30. 39. Miyashita K, Morioka I, Tanabe T, Iwata H, Takeda S. Symptoms of construction workers exposed to whole-body vibration and local vibration. Int Arch Occup Environ Health. 1992;64:347-51. 40. Miyashita K, Mivamoto K, Kuruda M, Takeda S, Iwata H. Hand-arm vibration exposure and the development of vibration syndrome. Nagoya J Med Sci. 1994;57:43-8. 41. Moller H. Physiological and psychological effects of infrasound in humans. J Low Freq Noise Vib. 1984;3:1-17. 42. Moller H. Annoyance of audible infrasound. J Acoust Soc Am. 1985;78:s32. 43. Nakamura H, Katoh A, Nohara S, Nakamura H, Okada A. Experimental studies on the pathogenesis of the gastric mucosal lesions induced by whole-body vibration. Environ Res. 1992;58:220-9. 44. Oliveira MJ, De Sousa Pereira A, Aguas AP, Grande NR, Monteiro E, Serrano I, Castelo Branco NA. Effects of low frequency noise upon the reaction of pleural milky spots to mycobacterial infection. Aviat Space Environ Med. 1999;70:A137-54. 45. Oliveira MJ, Pereira AS, Guimarães L, Freitas D, Carvalho AP, Grande NR, Águas AP. Chronic exposure of rats to cotton-mill-room noise changes the cell composition of the tracheal epithelium. J Occup Environ Med. 2002;44:1135-42. 46. Oliveira MJ, Pereira AS, Ferreira PG, Grande NR, Aguas AP, Guimarães L, Freitas D, Carvalho AP. Reduction of rat pleural microvilli caused by noise pollution. Exp Lung Res. 2003;29:445-54. 47. Oliveira PM, Pereira da Mata AD, Martins dos Santos JA, da Silva Marques DN, Branco NC, Silveira JM, Correia da Fonseca JC. Low-frequency noise effects on the parotid gland of the Wistar rat. Oral Dis. 2007;13:468-73.

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48. Persson Waye K. Effects of low frequency noise on sleep. Noise Health. 2004;6:87-91. 49. Pimenta MG, Martinho Pimenta AJ, Castelo Branco MS, Silva Simoes JM, Castelo Branco NA. ERP P300 and brain magnetic resonance in patients with vibroacoustic disease. Aviat Space Environ Med. 1999;70:A107-14. 50. Reis Ferreira JM, Couto AR, Jalles-Tavares N, Castelo Branco MS, Castelo Branco NA. Air flow limitation in patients with vibroacoustic disease. Aviat Space Environ Med. 1999;70:A63-9. 51. Sakakibara H, Hirata M, Hashiguchi T, Toibana N, Koshiyama H, Zhu SK, Yamada S. Digital nerve conduction velocity for evaluation of peripheral nerve impairments in vibration syndrome. Cent Eur J Publ Health. 1995;3:52-3. 52. Sakakibara H, Yamada S. Vibration syndrome and autonomic nervous system. Cent Eur J Publ Health. 1995;3:11-4. 53. Seidel H, Heide R. Long-term effects of whole-body vibration: a critical survey of the literature. Int Arch Occup Environ Med 1986;58:1-26. 54. Stoyneva Z, Lyapina M, Tzevetkov D, Vodenicharov E. Current pathophysiological views on vibration induced Raynaud’s phenomenon. Cardiovasc Res. 2003;57:615-24. 55. Sukharevskaia TM, Nepomniashchikh GI, Bobrova SV, Belov IIu, Aidagulova SV, Lapii GA. Clinical endoscopic and pathomorphologic study of the stomach in vibration disease. Med Tr Prom Ekol. 1999:(6):16-9. 56. Sutinen P, Koskimies K, Aalto H, Stark J, Pyykko I. Shoulder and elbow musculoskeletal disorder in forest workers exposed to hand-arm vibration. In: Proceedings of the Eighth International Conference on Hand-Arm Vibration; 1998; Umea, Sweden, 1998. p.193-8. 57. Takeuchi T, Futatsuka M, Imanishi H, Yamada S. Pathological changes observed in the finger biopsy of patients with vibration-induced white finger. Scand J Work Environ Health. 1986;12:280-3. 58. Teles-Grilo ML, Leite-Almeida H, Martins dos Santos J, Oliveira C, Boaventura P, Grande NR. Differential expression of collagens type I and type IV in lymphangiogenesis during the angiogenic process associated with bleomycininduced pulmonary fibrosis in rat. Lymphology. 2005;38:130-5. Received 25/9/2011. Accepted 10/10/2011.

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REVISÃO/ REVIEW

ARQGA/1594

METABOLIC SYNDROME AND RISK FACTORS FOR NON-ALCOHOLIC FATTY LIVER DISEASE Mônica Rodrigues de Araújo SOUZA, Margareth de Fátima Formiga de Melo DINIZ, José Eymard Moraes de MEDEIROS-FILHO and Maria Salete Trigueiro de ARAÚJO ABSTRACT – Context - Non-alcoholic fatty liver disease (NAFLD), hepatic manifestation of metabolic syndrome, has been considered the most common liver disease nowadays, which is also the most frequent cause of elevated transaminases and cryptogenic cirrhosis. The greatest input of fatty acids into the liver and consequent increased beta-oxidation contribute to the formation of free radicals, release of inflammatory cytokines and varying degrees of hepatocytic aggression, whose histological expression may vary from steatosis (HS) to non-alcoholic steatohepatitis (NASH). The differentiation of these forms is required by the potential risk of progression to cirrhosis and development of hepatocellular carcinoma. Objective - To review the literature about the major risk factors for NAFLD in the context of metabolic syndrome, focusing on underlying mechanisms and prevention. Method - PubMed, MEDLINE and SciELO data basis analysis was performed to identify studies describing the link between risk factors for metabolic syndrome and NAFLD. A combination of descriptors was used, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, metabolic syndrome and risk factors. At the end, 96 clinical and experimental studies, cohorts, meta-analysis and systematic reviews of great impact and scientific relevance to the topic, were selected. Results - The final analysis of all these data, pointed out the central obesity, type 2 diabetes, dyslipidemia and hypertension as the best risk factors related to NAFLD. However, other factors were highlighted, such as gender differences, ethnicity, genetic factors and the role of innate immunity system. How these additional factors may be involved in the installation, progression and disease prognosis is discussed. Conclusion - Risk factors for NAFLD in the context of metabolic syndrome expands the prospects to 1) recognize patients with metabolic syndrome at high risk for NAFLD, 2) elucidate pathways common to other co-morbidities, 3) determine risk factors associated with a worse prognosis, 4) develop therapeutic strategies with goal of reducing risk factors, 5) apply acquired knowledge in public health policies focusing on preventive strategies. HEADINGS – Metabolic syndrome X. Fatty liver, non-alcoholic. Risk factors.

INTRODUCTION

Clinical importance of non-alcoholic fatty liver disease NAFLD has grown in recent years, mainly in consequence of the obesity epidemics, sedentary habits and high calorie diet adopted by people of Western countries, reflecting the increase in cardiovascular and endocrine-metabolic diseases(30). It has been considered the most common liver disease and the most frequent cause of elevated aminotransferase and cryptogenic cirrhosis(19, 20, 21). The prevalence of NAFLD has been estimated in 2.8% to 88%, depending on population and investigative methods(2, 7, 9, 19, 21, 48). NAFLD refers to fat accumulation, mainly triglycerides, in hepatocytes so that it exceeds 5% of the liver weight(2). Available data from clinical, experimental and epidemiological studies describe the NAFLD as the hepatic manifestation of metabolic syndrome (MS)(59). Main risk factors associated with metabolic syndrome are abdominal obesity, insulin resistance, diabetes and dyslipidemia(73).

Interestingly, NAFLD can be described in non-obese and non-diabetic patients(50). The first Brazilian guidelines for diagnosis and treatment, based on systematic reviews, defines metabolic syndrome as a complex disorder represented by a set of cardiovascular risk factors, usually related to central fat deposition and insulin resistance. Its importance should be highlighted from an epidemiological standpoint, responsible for increased general and cardiovascular mortality estimated at 1.5 times and 2.5 times, respectively(29). Patients with NAFLD exhibit a higher mortality rate than general population(1). The most frequent causes of death are represented by liver-related diseases, malignant neoplasms and cardiovascular disease(83). Although not part of the diagnostic criteria for metabolic syndrome, several clinical and pathophysiological conditions are often associated with it, such as polycystic ovary syndrome, acanthosis nigricans, fatty liver disease, microalbuminuria, prothrombotic states, pro-inflammatory states and endothelial dysfunction and hyperuricemia(8, 62).

Universidade Federal da Paraíba (UFPB), PB, Brasil. Correspondence: Dr. Mônica Rodrigues de Araújo Souza - Rua Rita Sabino de Andrade, 354 – Bessa – 58036-610 – João Pessoa, PB, Brasil. E-mail: mrsmonicca@gmail.com

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NAFLD’s pathogenic mechanisms are still under investigation; however, triglycerides accumulation within hepatocytes, result of insulin resistance, is considered the first step in the pathogenic model proposed and widely accepted so far. Oxidative stress resulting from mitochondrial oxidation of fatty acids and expression of inflammatory cytokines, have been considered secondary causal factors for liver damage, fibrosis and inflammation(25). Histological evaluation remains the most important method of distinguishing steatosis from advanced forms of NAFLD, i.e. non-alcoholic steatohepatitis (NASH) and fibrosis. Included NAFLD lesions are steatosis, lobular and portal inflammation, hepatocyte injury as ballooning and apoptosis, and fibrosis(10). In the latter are included inflammatory phenomena with elevated aminotransferase levels(4), often associated with increased fat intake or changes in metabolic processes, with involvement of the mitochondrial beta-oxidation of fatty acids, generating reactive oxygen species and increasing the production of inflammatory cytokines. These, in turn, represent important role in activation of stellate cells, inducing liver fibrogenesis, predisposing to cirrhosis and hepatocellular carcinoma(21); however, risk factors for this progression have not been fully established(43). METHODS

A PubMed, MEDLINE and SciELO data basis analysis was performed to identify studies describing the link between risk factors for MS and NAFLD. Search terms included “Non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, metabolic syndrome AND risk factors”. Additional searches were also made for each of the individual methods described, e.g. NAFLD and obesity, diabetes, hypertension, genetic factors, etc. Selected articles referenced in these publications were also examined. Using the search strategy described above, approximately 130 articles were considered, limited to those published in English, Spanish and Portuguese language but not date-restricted. Following review, 96 articles met the selection criteria and were included, clinical and experimental studies, cohorts, meta-analysis and systematic reviews of great impact and scientific relevance to the topic. Obesity Obesity is a major public health problem in the U.S. and its prevalence has increased significantly over the past 30 years. Obesity is a global epidemic with more than 1 billion overweight adults and at least 300 million obese patients worldwide(17). Information collected from 1999 to 2002 shows that more than one third of American adult population is obese (27.6% men and 33.2% women) and among children, one in six is overweight(6). In previous reports, other authors have reported NAFLD prevalence around 57% to 74% and 22% to 58% of obese children and adults, respectively(71, 81). A cross-sectional population-based study was carried out in a sample of 1720 adults between 20 to 59 years of age, residents in an urban area of Brazil. The prevalence of central

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obesity was 50.5% (95% CI: 46.6-54.4) among men and 38.9% (95% CI 34.4-43.5) among women. In the adjusted analysis, central higher prevalence of obesity was observed in women aged 50 to 59 years(27). Steatohepatitis, however, can be found in 40% to 100% obesity cases in adults(81) and 15% to 25% in children(70). Considering obesity as a growing epidemic condition, the prevalence and impact of NAFLD continues to increase, making NASH potentially the most common cause of advanced liver disease in coming decades(85). A clinical series in Korean patients revealed that the body mass index (BMI) was useful in distinguishing between NASH and simple steatosis. In this study, 28.9 BMI was suggested as a threshold for NASH. It was proposed that abdominal fat is directly associated with disease state(73). Waist circumference and waist/hip ratio were independent determinants of hepatic necro-inflammation degree(77). A 158 cm2 of visceral fat area was considered an independent predictor of risk disease(78). Dorsocervical lipo-hypertrophy has also been strongly linked to NASH(15). A positive correlation was found between adiposity indexes (BMI, serum cholesterol and LDL levels) and advance stage fibrosis(73). Obesity and ethnicity Demographic analysis of obese populations added impressive evidence of how ethnic variations can influence extent and NASH incidence. Steatohepatitis is common among Hispanic populations, probably due to the high obesity level in this ethnic group(9). Despite the high prevalence of other risk factors, such as type 2 diabetes, NASH prevalence in African-American population is not so high, this could explain the significantly lower chance to developing serious liver disease, compared to caucasian patients(40). In two series of patients with NASH and another one with cryptogenic cirrhosis, African-Americans accounted for only 1% and 0.6% of those groups, respectively(11). Solga et al.(79) presented a study in which steatohepatitis was completely absent in obese African-Americans. It is speculated that both genetic and environmental factors (eg. eating habits) may be related to decreased incidence of liver disease in this ethnic population. Little information is available about the NASH prevalence in Western countries. A multicenter epidemiological Brazilian study involving 1280 patients with NAFLD described obesity prevalence in 44.7% of the studied sample(23). In another Brazilian study, the prevalence of hepatic steatosis among obese adolescents was estimated at 70% and 40% for males and females, respectively(24). In an American school-bases sample obese adolescent boys have an increased prevalence of fatty liver compared with obese adolescent girls(75). Hypoxia, obesity and sleep apnea The obstructive sleep apnea (OSA) is a commonly condition associated with obesity. It is characterized by chronic intermittent breathing cessation during sleep due to airway obstruction, occurring in 2% to 4% overall population. Tanné et al.(80) reported higher prevalence and severity of NASH in patients with severe OSA, suggesting that intermittent

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hypoxia may play a role in the NASH pathogenesis. Although intermittent hypoxia does not cause direct liver damage in non-obese animals, necro-inflammation and fibrosis were observed in livers of animals that already had steatosis, suggesting that hypoxia may serve as an additional insult in the progression from simple steatosis to more advanced stages of the disease(74, 94). Data derived from a PubMed-based meta-analysis of recent cost effectiveness, standards of practice, and epidemiological studies of OSA, which were ranked using a hierarchical strength of recommendation taxonomy, presents evidence supporting the value of diagnosing and treating OSA in reducing morbidity and mortality, improving comorbid disease processes, and improving patient quality of life(68). Birth weight The association between an adverse early life environment and increased susceptibility to later-life metabolic disorders such as obesity, type 2 diabetes and cardiovascular disease was described by the developmental origins of health and disease hypothesis. A systematic review summarizes the relationship of both low birth weight and catch-up-growth with some aspects of a later metabolic syndrome. The majority of the studies in children, adolescents and adults born small for gestational age suggested that insulin resistance could represent the prelude to other metabolic disorders(66). Meta-analysis of 224 studies estimated the association between birth weight and MS. All but two studies reported an inverse relationship between birth weight and MS. A comparison between low birth weight versus normal birth weight subjects showed the random effects odds ratio for metabolic syndrome was 2.53 (95% CI: 1.57;4.08), indicating that low birth weight increases the risk of metabolic syndrome in adults(76). Employing a rat model of maternal high fat (MHF) nutrition, it was recently reported that offspring born to exposed mothers are small at birth and develop a postnatal phenotype that closely resembles that of the human metabolic syndrome. Livers of offspring born to MHF mothers also display a fatty phenotype reflecting hepatic steatosis and characteristics of non-alcoholic fatty liver disease. In this study it was hypothesised that a MHF diet leads to altered regulation of liver development in offspring; a derangement that may be detectable during early postnatal life(32). Gender and age There are clear differences in the amount and distribution of body fat between men and women. The first usually store fat in the upper body, specifically around the organs in the abdominal cavity: the visceral fat. However women, with less body fat, tend to store in the subcutaneous tissue. Gender also influences the lipids circulation (triglycerides and fatty acids), lipoproteins and cholesterol(73, 84, 90). The reasons for the difference in fat accumulation in men and women are not completely understood; however, evidence suggests that lipid metabolism may play an important role in the differences observed(73). At the cellular level, there are distinctions between men and women in activity and

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metabolism of lipids. A Japanese study showed that the particle size of triglyceride and cholesterol were higher in men than women, with evidence of metabolic syndrome(51). Comparing by age group, the hepatic lipase activity in men is twice that observed in women, which probably contributes to an atherogenic lipid profile with lower HDL cholesterol and increased levels of dense LDL particles in men(12). Gender distinctions in hepatic lipase activity have been attributed to the suppression of androgenic steroids and visceral adiposity(73). Studies in rats and mice have shown worsening of biochemical and histopathological patterns in male rodents(49). However, researches towards defining gender role in NASH in humans are scarce and limited and the available information is contradictory. An American study including 365 adults revealed NASH prevalence in 90% of men and 30.8% of women. Males were also significantly associated with incidence of diabetes and metabolic syndrome(3). Studies conducted in pediatric patients also reported that males are at greater risk of obesity and associated liver disease(54). In contrast to the evidence described for the Western population, a series of 193 Japanese patients had NASH proven by histopathology predominantly in women aged above 55 years(91). In Brazil, mean age of patients with NAFLD was 49.8 ± 13.59 years, of which 53.3% were male(23, 76). On the other hand, several reports with different ethnic groups can not identify the difference between men and women, excluding the possibility of inferring any conclusions about the role of gender as a risk factor for NASH(73). Regarding age, it is known that aging brings restrictions on physical mobility, which in the context of metabolic syndrome, contributes to maintenance or worsening of abdominal obesity, hyperglycemia, hyperlipidemia and hypertension(69). The prevalence of metabolic syndrome among Americans over 70 years was estimated at around 42%(37). In another study NAFLD was associated with increased risk of cardiovascular disease especially among older patients with elevated C-reactive protein levels(16). Diabetes Insulin resistance and hyperinsulinemia are the most closely associated laboratory findings with the presence of fatty liver disease in a large series of patients, even in thin subjects with normal glucose tolerance(8, 41, 59). NAFLD prevalence is increased in individuals with impaired glucose tolerance and those with newly diagnosed diabetes at the proportion of 43% to 62%, respectively(48). In a prospective study of 100 patients with type 2 diabetes, the incidence of hepatic steatosis was 49%, confirming this strong independent risk factor for NAFLD(41). In Brazil, the prevalence of metabolic syndrome and diabetes among patients with NAFLD was estimated at 41.3% and 22.7%, respectively(23). The association of both conditions is related with more aggressive disease and increasing mortality(34, 93). Targher et al.(82) described increased prevalence of NAFLD and its association with cardiovascular disease in diabetic patients, independent of other confounding factors.

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Frequency and risk factors associated with NAFLD in patients with type 2 diabetes mellitus was studied. Almost half of patients with type 2 diabetes mellitus were found to have NAFLD, and they have more elevated BMI, as well as higher levels of aminotransferases, γ-GT, uric acid, TNF-α, insulin and HOMA-IR than subjects without NAFLD(36). A possible pathogenic link between diabetes and NASH involves advanced glycation end products. These products constitute a large variety of substances formed from aminocarbonyl interactions of non-enzymatic nature, among reducing sugars or oxidized lipids and proteins, aminophospholipids or nucleic acids(64). The formation of these adducts occurs at high rates in diabetes mellitus type 2, compared with healthy controls or patients with simple steatosis(47). The interaction of these products with the cell surface associated receptor has been related to induction of oxidative stress and as well as the increased fibrogenic potential stellate cells cultures(35). The interaction of these advanced glycation products and cell surface receptors resulted in intermediate reactive oxygen species, which has important role in the disease pathogenesis in diabetic(73). Hypertension Hypertension, especially systolic hypertension is also an independent NAFLD predictor (29). The presence of multiple metabolic disorders like diabetes mellitus, obesity, dyslipidemia and hypertension are associated with serious illness, potentially progressive of liver(61). Cotrim et al.(23) confirmed these data when reporting 64% hypertension prevalence among NAFLD patients steatohepatitis. Hsiao et al.(46) demonstrated that presence of severe hepatic steatosis was significantly correlated with the prevalence and degree of hypertension, serum glucose levels and triglycerides. On the other hand, a study on non-obese and non-diabetic patients with primary hypertension has shown that hepatic steatosis prevalence can double as compared with control group(31). A meta-analysis compared individuals with metabolic syndrome and without revealed an increased mortality from all causes (relative risk [RR] 1.35; 95% confidence interval [CI], 1.17-1.56) and cardiovascular disease (RR 1.74; 95% CI, 1.29-2.35); as well as an increased incidence of cardiovascular disease (RR 1.53; 95% CI, 1.26-1.87), coronary heart disease (RR 1.52; 95% CI, 1.37-1.69) and stroke (RR 1.76; 95% CI, 1.37-2.25). Relative risk of cardiovascular disease associated with metabolic syndrome was higher in women compared with men and higher in studies that used World Health Organization definition compared with studies that used Adult Treatment Panel III definition(38). Li et al.(55) investigated by meta-analysis the association between MS and risk of stroke. Compared to individuals without MS, patients had a 1.6-fold increased risk of stroke (95% CI, 1.48-1.75). The relative risk of stroke associated with MS was 2.2 in the studies using the World Health Organization definition and 1.6 in those using Adult Treatment Panel III definition, but difference was not statistically significant.

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Abnormalities in left ventricular geometry and diastolic function have been described in patients with NAFLD as well as a more severe coronary artery disease characterized by vulnerable plaques, though observed in small cohort studies(83). Dyslipidemia Increased serum triglycerides and low-density lipoproteins (LDL), combined with decreased high-density lipoproteins (HDL), represent a threat for cardiovascular disease development. Evidences suggest that NAFLD is linked to the increased incidence of cardiovascular disease, both in non-diabetic and type 2 diabetic patients(52). The presence of dyslipidemia (hypercholesterolemia, hypertriglyceridemia, or both) defined by the National Institute of Health (NIH)(65) was reported in 20% to 80% cases associated with NAFLD(6, 28, 57). Hypertriglyceridemia is present in 64% of patients with hepatic steatosis with low HDL-C levels reported in 30%-42% of cases(60). Cotrim et al.(23), reported the presence of hyperlipidemia in 66.8% of patients with NAFLD in Brazil. Recent studies have shown NAFLD strongly associated with increased risk of cardiovascular disease, there being independent association between hepatic steatosis, carotid atherosclerotic plaques and endothelial dysfunction(86, 87). Considering all liver related causes, cardiovascular disease represents the major survival risk of patients with NASH(33). On the other hand, McKimmie et al.(63) did not find independent association between hepatic steatosis and cardiovascular disease in a subset of participants in Diabetes Heart Study. They suggested that hepatic steatosis is more a secondary phenomenon than a direct mediator of cardiovascular disease. Evidence of endocrine system role in the pathogenesis of non-alcoholic fatty liver disease Evidence shows that chronic activation of hypothalamicpituitary-adrenal axis is present in metabolic syndrome, like patients with Cushing’s syndrome that also have similar characteristics of metabolic syndrome and mild increase of aminotransferase levels being commonly seen in patients with adrenal insufficiency as well as adult patients with growth hormone deficiency who develop the phenotype of metabolic syndrome with obesity and dyslipidemia(14). Insulin resistance, type 2 diabetes, sleep apnea syndrome, cardiovascular disorders and non-alcoholic liver disease are common conditions in women with polycystic ovary syndrome, being non-alcoholic fatty liver disease twice as common in postmenopausal women in which hormone replacement therapy reduces the risk of steatosis. Similarly, it seems appropriate to consider polycystic ovary syndrome as the ovarian manifestation of metabolic syndrome(5). Hypoandrogenism in males and hyperandrogenism in females are also related to hepatic steatosis, obesity and insulin resistance(42). Meta-analysis of available cross-sectional data suggests that MS can be associated to male hypogonadism, although only few randomized controlled trials have been

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reported. Testosterone replacement therapy seems to improve metabolic control, as well as central obesity(22). Thyroid hormones play essential role in mobilization and degradation of lipids and fatty acid oxidation so that hypothyroidism has been associated with non-alcoholic steatohepatitis(56). There is evidence that replacement of thyroid hormones regulates lipid metabolism(96). However, it is unclear whether thyroid dysfunction plays a role in the NASH pathogenesis. Liangpunsakul and Chalasani(56) conducted a case-control study to investigate this association. In this study, the prevalence of hypothyroidism in NAFLD patients was estimated at 15%, significantly higher than in controls (7.2%, P<0.001). By multivariate analysis, hypothyroidism prevalence in NASH group was significantly higher than in control group. The association between thyroid diseases and hepatocellular carcinoma in men and women was studied(44). Authors found a significant association between increased risk of hypothyroidism and hepatocellular carcinoma in women, regardless establishing hepatocellular carcinoma risk factors. However, experimental investigations are needed for complete evaluation of the association between these two diseases. Changes of the endocrine system should be considered in the context of non-alcoholic and cryptogenic fatty liver disease. The prospect of endocrine system participation may help in therapeutic approaches in the future(57). Genetic factors Not all patients with risk factors for metabolic syndrome develop hepatic steatosis and among them, not all evolve to steatohepatitis or cirrhosis. Genetic polymorphisms can explain these variations(26). Weltman et al.(88, 89) working on experimental and human models of steatohepatitis, had previously described the overexpression of cytochrome P450 CYP2E1, with generation of excess oxygen free radicals. Increased CYP2E1 expression induced by alcohol, ketone and free fatty acids was associated with overproduction of oxygen free radicals and increased oxidative stress. In NAFLD, genetic factors are not fully known; however, it was suggested that steatosis development may be associated with genes involved in insulin resistance coding for proteins involved in lipid metabolism, oxidative stress, inflammation (cytokines and adipokines) and fibrogenesis(18 ,45) . Other genetic polymorphisms have been reviewed, such as angiotensinogen and TGF-β1 associated with liver fibrosis in obese patients(32). Chalasani et al.(13) in a pilot study, identified genetic variations associated with histological parameters in patients with NAFLD biopsy-proven. NAFLD histological activity score was associated with single nucleotide polymorphism (SNP) rs2645424 on chromosome 8, degree of fibrosis was associated with SNP rs343062 on chromosome 7 and lobular inflammation was associated with single nucleotide polymorphism in chromosomes 10, 11 and 12. If future studies validate these results, then testing should be clinically useful, since histologic findings are associated with NAFLD

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prognosis. Mutations in HFE gene of hemochromatosis and NASH have been described, mainly by establishing connections between increased iron stores in the liver and development of hepatic fibrosis. George et al.(39) studying 51 patients with NASH, demonstrated that 31 had mutation in HFE gene being considered heterozygous for hemochromatosis. Authors argue that iron overload, even light, can act synergistically in promoting fibrosis observed in NASH. However, posterior studies(18, 92) found no association between increased iron and fibrosis, questioning its importance as NASH risk factor. After having systematically reviewed the most studied single nucleotide polymorphisms and metabolic syndrome associations, it was found evidence for an association with the MS for eight alleles, mostly located in genes involved in lipid metabolism(70). Thus, genetic studies are needed to identify new genes involved in the NAFLD development. Orthotopic liver transplantation (OLT) There are few published studies that have specifically reported the development of individual components of metabolic syndrome following OLT. The prevalence of post-transplant metabolic syndrome and its individual components has been found to be higher post-OLT versus a comparable population without this surgical procedure. The rate of metabolic syndrome in liver transplant recipients is more than twice that reported for general population(53). Pre-transplant risk factors include immunosuppression, higher age at transplant, male gender, history of smoking, pre-transplant body mass index, preOLT diabetes, etiology of the underlying liver disease that resulted in OLT and an increased donor body mass index(67). Recent data suggests that prospective studies are required to determine the significance and management of post-transplant metabolic syndrome(53) and NAFLD. Smoking Smoking history was associated with advanced liver fibrosis in a large multicenter cohort of NAFLD patients(95). The results indicate that smoking may enhance progression of NAFLD partly through its effect on insulin resistance. This was the first study to show that cigarette smoking is associated with increased fibrosis severity in human NALFD, suggesting it may accelerate disease progression. These results may support a formal recommendation of smoking cessation in patients with NAFLD. CONCLUSION

NAFLD may affect any age and seems to be different among different ethnic groups. Prevalence is significantly higher in white man than in white women. Differences in body fat distribution and body composition among ethnic groups may partially explain racial differences in prevalence. Central obesity phenotype is associated with increased intra-abdominal fat, therefore, patients with central obesity are characteristically insulin resistant, and more commonly present with NAFLD compared to patients having lower-body obesity. Besides central obesity, type 2 diabetes, dyslipidemia

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and hypertension are risk factors for development of NAFLD, represents more than a clinical diagnosis, being a pre–morbid condition with high rate of cardiovascular, renal, and liver risks. Environmental and lifestyle-related factors such as reduced physical activity and high-fat diets are well-known influences for development of insulin resistance-associated comorbidities and NAFLD. Genetic predisposition for the development of central obesity and type 2 must be considered. Recent findings have advanced in the field of genetic and

immune response (innate immunity) in NASH pathogenesis, although family studies and studies specifically addressing the genetic susceptibility for NAFLD development are lacking. Prospects remain to: 1) elucidate pathways common to other co-morbidities, 2) establish more accurate and less invasive diagnostic tools, 3) determine risk factors related to outcomes of poor prognosis, 4) develop therapeutic strategies aiming to reduce risk factors, 5) apply the acquired knowledge in public health policies focusing on preventive strategies.

Souza MRA, Diniz MFFM, Medeiros-Filho JEM, Araújo MST. Síndrome metabólica e fatores de risco para a doença hepática gordurosa não-alcoólica. Arq Gastroenterol. 2012;49(1):89-96. RESUMO – Contexto - A doença hepática gordurosa não alcoólica (DHGNA) vem sendo considerada a manifestação hepática da síndrome metabólica e a hepatopatia mais frequente da atualidade, sendo também a causa mais frequente de aumento das transaminases e de cirrose criptogênica. O maior aporte de ácidos graxos ao fígado e consequente aumento da beta-oxidação concorrem para formação de radicais livres, liberação de citocinas inflamatórias e graus variáveis de agressão hepatocítica, cuja expressão histológica pode variar da esteatose hepática (EH) à esteatohepatite nãoalcoólica (EHNA), cuja diferenciação se faz necessária pelo risco potencial de progressão para cirrose e desenvolvimento do carcinoma hepatocelular. Objetivo - Revisar a literatura sobre os principais fatores de risco para a DHGNA no contexto da síndrome metabólica, com foco nos mecanismos subjacentes e nas estratégias de prevenção. Método - Foi realizada pesquisa bibliográfica no PubMed para identificar estudos que descrevessem a associação entre os fatores de risco para síndrome metabólica e a DHGNA, utilizando-se a combinação de descritores: “Non-alcoholic fatty liver disease, Non-alcoholic steatohepatitis, metabolic syndrome and risk factors”. Foram selecionados 96 estudos clínicos, experimentais, de cohort, metanálises e revisões sistemáticas de maior impacto e relevância científica para o tema. Resultados - A análise das informações consolidou a obesidade central, diabetes melitus tipo 2, dislipidemia e hipertensão como os fatores de risco mais bem relacionados à DHGNA. Entretanto, outros fatores foram destacados, como diferenças entre gêneros, etnia, fatores genéticos e o papel da imunidade inata, como estes fatores adicionais que podem estar implicados na instalação, progressão e prognóstico da doença. Conclusão - O conhecimento dos fatores de risco para a DHGNA no contexto da síndrome metabólica amplia os caminhos para: 1) reconhecer pacientes com risco elevado para a doença; 2) elucidar vias comuns a outras comorbidades; 3) determinar fatores de risco relacionados a pior prognóstico; 4) desenvolver estratégias terapêuticas com o objetivo de reduzir fatores de risco; 4) aplicar os conhecimentos adquiridos nas políticas públicas de saúde com foco em estratégias preventivas. DESCRITORES – Síndrome X metabólica. Fígado gorduroso não-alcoólico. Fatores de risco.

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11. Caldwell SH, Harris DM, Patrie JT, Hespenheide EE. Is NASH underdiagnosed among African Americans? Am J Gastroenterol. 2002;96:1496-1500. 12. Carr MC, Ayyobi AF, Murdoch SJ, Deeb SS, Brunzell JD. Contribution of hepatic lipase, lipoprotein lipase, and cholesteryl ester transfer protein to LDL and HDL heterogeneity in healthy women. Arterioscler Thromb Vasc Biol. 2002;22:667-73. 13. Chalasani N, Guo X, Loomba R, Goodarzi MO, Haritunians T, Kwon S, Cui J, Taylor KD, Wilson L, Cummings OW, Chen YD, Rotter JI. Genome-wide association study identifies variants associated with histologic features of nonalcoholic fatty liver disease. Gastroenterology. 2010;139:1567-76. 14. Chanson P, Salenave S. Metabolic syndrome in Cushing’s syndrome. Neuroendocrinology. 2010;92(Suppl 1):96-101. 15. Cheung O, Kapoor A, Puri P, Sistrun S, Luketic VA, Sargeant CC, Contos MJ, Shiffman ML, Stravitz RT, Sterling RK, Sanyal AJ. The impact of fat distribution on the severity of nonalcoholic fatty liver disease and metabolic syndrome. Hepatology. 2007;46:1091-100. 16. Chiang CH, Huang CC, Chan WL, Chen JW, Leu HB. The severity of nonalcoholic fatty liver disease correlates with high sensitivity C-reactive protein value and is independently associated with increased cardiovascular risk in healthy population. Clin Biochem. 2010;43:1399-404. 17. Chiang DJ, Pritchard MT, Nagy LE. Obesity, diabetes mellitus, and liver fibrosis. Am J Physiol Gastrointest Liver Physiol. 2011;300:G697-702. 18. Chitturi S, Weltman M, Farrell GC, McDonald D, Kench J, Liddle C, Samarasinghe D, Lin R, Abeygunasekera S, George J. HFE mutations, hepatic iron, and fibrosis:ethinic-specific association of NASH with C82Y but not with fibrotic severity. Hepatology. 2002;36:142-9. 19. Clark JM, Brancati FL, Diehl AM. The prevalence and etiology of elevated aminotransferase levels in the United States. Am J Gastroenterol. 2003;98:960-7.

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20. Clark JM, Diehl AM. Nonalcoholic fatty liver disease: an underrecognized cause of cryptogenic cirrhosis. JAMA. 2003;289:3000-4. 21. Clark JM. The epidemiology of nonalcoholic fatty liver disease in adults. J Clin Gastroenterol. 2006;40(3 Suppl 1):s5-10. 22. Corona G, Monami M, Rastrelli G, Aversa A, Tishova Y, Saad F, Lenzi A, Forti G, Mannucci E, Maggi M. Testosterone and metabolic syndrome: a meta-analysis study. J Sex Med. 2011;8:272-83. 23. Cotrim HP, Parise ER, Oliveira CP, Leite N, Martinelli A, Galizzi J, Silva R de C, Mattos A, Pereira L, Amorim W, Ivantes C, Souza F, Costa M, Maia L, L, Pessoa M, Oliveira F. Nonalcoholic fatty liver disease in Brazil. Clinical and histological profile. Ann Hepatol. 2011;10:33-37. 24. Dâmaso AR, Tock L, Tufik S, Prado WL, Stella SG, Fisberg M, Cintra IP, Caranti DA, Siqueira KO, Nascimento CM, Oyama LM, Lederman HM, Cristofalo D, Antunes HK, Comparoni A, Santos LC, Meloo MT. Tratamento multidisciplinar reduz o tecido adiposo visceral, leptina, grelina e a prevalência de esteatose hepática não alcoólica (NAFLD) em adolescentes obesos. Rev Bras Med Esporte. 2006;12:263-67. 25. Day CP, James OF. Steatohepatitis: a tale of two “hits”? Gastroenterology. 1998;114:842. 26. Day CP. The potential role of genes in nonalcoholic fatty liver disease. Clin Liver Dis. 2004;8:673-91. 27. de Sousa TF, Nahas MV, Silva DA, Del Duca GF, Peres MA. Factors associated with central obesity in adults from Florianópolis, Santa Catarina: a population based-study. Rev Bras Epidemiol. 2011;14:296-309. 28. Diehl AM, Goodman Z, Ishak KG. Alcohollike disease in nonalcoholics. A clinical and histological comparison with alcohol-induced liver injury. Gastroenterology. 1988;94:1056-62. 29. Diretriz Brasileira de Diagnóstico e Tratamento da Síndrome Metabólica I [Internet]. Arq Bras Cardiol. 2005;84:3-28. 30. Dixon JB, Bhathal PS, O’Brien PE. Nonalcoholic fatty liver disease: predictors of nonalcoholic steatohepatitis and liver fibrosis in the severely obese. Gastroenterology. 2001;121:91-100. 31. Donati G, Stagni B, Piscaglia F, Venturoli N, Morselli-Labate AM, Rasciti L, Bolondi L. Increased prevalence of fatty liver in arterial hypertensive patients with normal liver enzymes: role of insulin resistance. Gut. 2004;53:1020-3. 32. Dowman JK, Tomlinson JW, Newsome PN. Pathogenesis of non-alcoholic fatty liver disease. QJM. 2010;103:71-83. 33. Dudley KJ, Sloboda DM, Connor KL, Beltrand J, Vickers MH. Offspring of mothers fed a high fat diet display hepatic cell cycle inhibition and associated changes in gene expression and DNA methylation. PLoS One. 2011;6:e21662. 34. Ekstedt M, Franzén LE, Mathiesen UL, Thorelius L, Holmqvist M, Bodemar G, Kechagias S. Long-term follow-up of patients with NAFLD and elevated liver enzymes. Hepatology. 2006;44:865-73. 35. Fehrenbach H, Weiskirchen R, Kasper M, Gressner AM. Up-regulated expression of the receptor for advanced glycation end products in cultured rat hepatic stellate cells during transdifferentiation to myofibroblasts. Hepatology. 2001;34:943-952. 36. Ferreira VSG, Pernambuco RB, Lopes EP, Morais CN, Rodrigues MC, Arruda MJ, Silva LM, Vilar L. Frequency and risk factors associated with non-alcoholic fatty liver disease in patients with type 2 diabetes mellitus. Arq Bras Endocrinol Metab. 2010;54;362-8. 37. Ford ES, Giles WH, Dietz WH. Prevalence of the metabolic syndrome among US adults: findings from the third National Health and Nutrition Examination Survey. JAMA. 2002;287:356-9. 38. Galassi A, Reynolds K, He J. Metabolic syndrome and risk of cardiovascular disease: a meta-analysis. Am J Med. 2006;119:812-9. 39. George DK, Goldwurm S, MacDonald GA, Cowley LL, Walker NI, Ward PJ, Jazwinska EC, Powell LW. Increased hepatic iron concentration in nonalcoholic steatohepatitis is associated with increased fibrosis. Gastroenterology. 1998;114:311-8. 40. Giday SA, Ashiny Z, Naab T, Smoot D, Banks A. Frequency of nonalcoholic fatty liver disease and degree of hepatic steatosis in African-American patients. J Natl Med Assoc. 2006;98:1613-5. 41. Gupte P, Amarapurkar D, Agal S, Baijal R, Kulshrestha P, Pramanik S, Patel N, Madan A, Amarapurkar A, Hafeezunnisa. Non-alcoholic steatohepatitis in type 2 diabetes mellitus. J Gastroenterol Hepatol. 2004;19:854-8. 42. Hagymási K, Reismann P, Rácz K, Tulassay Z. [Role of the endocrine system in the pathogenesis of non-alcoholic fatty liver disease]. Orv Hetil. 2009;150:2173-81. 43. Hashizume H, Sato K, Takagi H, Hirokawa T, Kojima A, Sohara N, Kakizaki S, Mochida Y, Shimura T, Sunose Y, Ohwada S, Mori M. Primary liver cancers with nonalcoholic steatohepatitis. Eur J Gastroenterol Hepatol. 2007;19:827-4. 44. Hassan MM, Kaseb A, Li D, Patt YZ, Vauthey JN, Thomas MB, Curley SA, Spitz MR, Sherman SI, Abdalla EK, Davila M, Lozano RD, Hassan DM, Chan W, Brown TD, Abbruzzese JL. Association between hypothyroidism and hepatocellular carcinoma: a case-control study in the United States. Hepatology. 2009;49:1563-70.

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45. Hijona E, Hijona L, Arenas JI, Bujanda L. Inflammatory mediators of hepatic steatosis. Mediators Inflamm. 2010;2010:837419. 46. Hsiao PJ, Kuo KK, Shin SJ, Yang YH, Lin WY, Yang JF, Chiu CC, Chuang WL, Tsai TR, Yu ML. Significant correlations between severe fatty liver and risk factors for metabolic syndrome. J Gastroenterol Hepatol. 2007;22:2118-23. 47. Hyogo H, Yamagishi S, Iwamoto K, Arihiro K, Takeuchi M, Sato T, Ochi H, Nonaka M, Nabeshima Y, Inoue M, Ishitobi T, Chayama K, Tazuma S. Elevated levels of serum advanced glycation end products in patients with non-alcoholic steatohepatitis. J Gastroenterol Hepatol. 2007;22:1112-19. 48. Jimba S, Nakagami T, Takahashi M, Wakamatsu T, Hirota Y, Iwamoto Y, Wasada T. Prevalence of nonalcoholic fatty liver disease and its association with impaired glucose metabolism in Japanese adults. Diabet Med. 2005;22:1141-5. 49. Kashireddy PR, Rao MS. Sex differences in choline-deficient diet-induced steatohepatitis in mice. Exp Biol Med (Maywood). 2004;229:158-62. 50. Kim HJ, Kim HJ, Lee KE, Kim DJ, Kim SK, Ahn CW, Lim SK, Kim KR, Lee HC, Huh KB, Cha BS. Metabolic significance of nonalcoholic fatty liver disease in nonobese, nondiabetic adults. Arch Intern Med. 2004;164:2169-75. 51. Kobayashi J, Maruyama T, Watanabe H, Kudoh A, Tateishi S, Sasaki T, Murano S, Watanabe M. Gender differences in the effect of type 2 diabetes on serum lipids, pre-heparin plasma lipoprotein lipase mass and other metabolic parameters in Japanese population. Diabetes Res Clin Pract. 2003;62:39-45. 52. Kotronen A, Yki-Järvinen H. Fatty liver: a novel component of the metabolic syndrome. Arterioscler Thromb Vasc Biol. 2008;28:27-38. 53. Laish I, Braun M, Mor E, Sulkes J, Harif Y, Ben Ari Z. Metabolic syndrome in liver transplant recipients: prevalence, risk factors, and association with cardiovascular events. Liver Transpl. 2011;17:15-22. 54. Lavine JE, Schwimmer JB. Nonalcoholic fatty liver disease in the pediatric population. 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Uma Dose Diária1-3

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Eficácia da mesalazina com a comodidade da tecnologia MMX*1-3 N com ão ex a te iste g cno ené r log ia M ico MX * 5

O que é a TECNOLOGIA MMX* ? 4

Componentes hidrofílicos Comprimidos gastrorresistentes (Eudragit-S®)

1 2 3

Manutenção 6,7 MESACOL MMX 2400mg/dia ®

Componentes lipofílicos

*

Indução à remissão 2,8,9 MESACOL® MMX* 4800mg/dia

1x ao dia

1x ao dia

Contraindicação: Mesacol® MMX* não é recomendado em casos de hipersensibilidade a salicilatos. Interação Medicamentosa: A administração da mesalazina pode potencializar a toxicidade do metotrexato. Mesacol® MMX* - mesalazina - Uso adulto - Acima de 18 anos - Apresentações e composição: Comprimidos revestidos de liberação prolongada, com 1,2 g de mesalazina cada. Embalagens com 10 e 30 unidades. Indicações: antiinflamatório de ação local no tratamento da colite ulcerativa ativa leve a moderada, na fase aguda (indução da remissão) e na manutenção da remissão. Contra-indicações: Este medicamento não deve ser usado por pacientes com história de hipersensibilidade aos salicilatos (que inclui o ácido acetilsalicílico), à mesalazina, à sulfassalazina ou a qualquer dos componentes da fórmula; pacientes com insuficiência hepática e/ou renal graves; pacientes com úlcera gástrica e duodenal ativa; pacientes com tendência elevada a sangramento. Este medicamento é contraindicado para menores de 18 anos. Precauções e advertências: As mesmas precauções e advertências relacionadas com o uso de preparações contendo mesalazina ou pró-drogas de mesalazina devem ser consideradas para Mesacol® MMX*. Assim como todos os salicilatos, a mesalazina deve ser utilizada com cautela em pacientes com história de úlcera gástrica ou duodenal, por pacientes asmáticos (em função das reações de hipersensibilidade), com disfunção renal ou hepática (leve a moderada), ou com história de miocardite ou pericardite. Mesalazina não é recomendada para pacientes com disfunção renal grave e deve-se ter cautela com pacientes com níveis sangüíneos aumentados de uréia ou com proteinúria. A mesalazina é rapidamente excretada pelos rins, principalmente o seu metabólito ácido N-acetil-5-aminossalicílico. Em ratos, altas doses da mesalazina, administradas por via intravenosa, causaram toxicidade tubular e glomerular. Em caso de aparecimento de disfunção renal durante o tratamento deve-se suspeitar de nefrotoxicidade induzida pela mesalazina. Nestes casos é recomendado monitorar a função renal, especialmente no início do tratamento. Durante tratamento prolongado, é também necessário monitorar regularmente a função renal (creatinina sérica). Ainda não está estabelecida a segurança do produto em crianças. Gravidez e lactação: Mesacol® MMX* está classificado na Categoria B de risco de fármacos destinados ao uso em grávidas. O produto, a princípio, não deve ser empregado em gestantes e lactantes, exceto quando absolutamente necessário. A segurança de Mesacol® MMX* para uso durante a gravidez ou a amamentação ainda não foi estabelecida, mas sabe-se que a mesalazina atravessa a placenta e é excretada pelo leite materno em pequenas quantidades. Estudos pré-clínicos não revelaram evidência de efeitos teratogênicos ou de toxicidade fetal oriundos da mesalazina, nem na evolução da gestação ou no desenvolvimento perinatal e pós-natal. A pequena experiência de uso da mesalazina em outras formulações durante a gravidez não mostrou efeito prejudicial ao feto; entretanto, a mesalazina deve ser usada com cautela durante a gravidez e somente quando os benefícios para a mãe forem superiores aos riscos potenciais ao feto. Baixas concentrações de mesalazina e de seu metabólito N-acetilado foram detectadas no leite materno, mas o significado clínico desta evidência ainda não foi determinado. Portanto, deve-se ter cautela na administração da mesalazina a lactantes. Categoria B de risco na gravidez – Este medicamento não deve ser utilizado por mulheres grávidas sem orientação médica ou do cirurgião-dentista. Pacientes pediátricos: Devido à falta de dados sobre a administração da mesalazina em altas doses na população pediátrica, Mesacol® MMX* não é recomendado para pacientes menores de 18 anos. Pacientes idosos: Não existe experiência suficiente sobre o uso de Mesacol® MMX* em pacientes com idade acima de 65 anos. No entanto, não foram identificadas diferenças entre o uso em pacientes mais jovens e em idosos com outras formulações de mesalazina. Pacientes com insuficiência renal: não são disponíveis informações sobre o uso em pacientes com insuficiência renal leve ou moderada. O medico deverá avaliar a relação risco/benefício para o seu uso. Pacientes com insuficiência hepática: não são disponíveis informações sobre o uso em pacientes com insuficiência hepática leve ou moderada. O medico deverá avaliar a relação risco/benefício para o seu uso. Dirigir e operar máquinas: É improvável que o uso deste medicamento tenha qualquer efeito na capacidade de dirigir veículos ou de usar máquinas. Interações medicamentosas: Nenhum estudo formal de interação foi conduzido. Não são disponíveis informações sobre interações entre Mesacol® MMX* e outros fármacos. Entretanto, existem relatos de interação entre a mesalazina (outras formulações) e outros medicamentos. O uso concomitante da mesalazina com agentes sabidamente nefrotóxicos, inclusive com os anti-inflamatórios não-hormonais (AINHs – como aspirina, ibuprofeno, diclofenaco, etc.) e azatioprina, pode aumentar o risco de reações renais; o potencial para discrasias sangüíneas da azatioprina e da 6-mercaptopurina pode ser aumentado; a ação hipoglicemiante das sulfoniluréias pode ser intensificada; a atividade anticoagulante dos derivados cumarínicos (varfarina) pode ser reduzida; a toxicidade do metotrexato pode ser potencializada; o efeito uricosúrico da probenecida e da sulfimpirazona pode ser diminuído, assim como a ação diurética da furosemida e da espironolactona e a ação tuberculostática da rifampicina. Em tese, a administração concomitante de anticoagulantes orais deve ser feita com cautela. Substâncias como a lactulose, que diminuem o pH do cólon, podem reduzir a liberação da mesalazina dos comprimidos revestidos de Mesacol® MMX*. Reações adversas: A maioria das reações adversas relatadas com Mesacol® MMX* foi transitória, e de intensidade leve a moderada. Foram descritas as seguintes reações adversas, distribuídas em grupos de freqüências: Reação comum (> 1/100 e < 1/10): Gastrintestinal: Flatulência e Náusea. Sistema Nervoso: Cefaléia. Estas reações ocorreram em menos de 3% dos pacientes, independente da dose administrada. Reação incomum (> 1/1.000 e < 1/100): Gastrintestinal: vômito, dor abdominal, distensão abdominal, diarréia, dispepsia, pancreatite, colite e pólipo retal. Hepatobiliar: aumento das transaminases, anormalidades no teste da função hepática. Sistema nervoso: tontura, sonolência, tremores. Cardiovascular: taquicardia, hipertensão e hipotensão arterial. Respiratório: dor faringolaríngea. Ouvido e labirinto: otalgia. Pele e tecido subcutâneo: acne, alopécia, prurigo, urticária, exantema, prurido. Sangue e linfa: Redução do número de plaquetas. Musculosqueléticas: artralgia, lombalgia. Gerais: Astenia, fadiga, pirexia, edema da face. Posologia e modo de usar: Mesacol® MMX* é para uso exclusivo por via oral. Para o tratamentoda colite ulcerativa leve a moderada, a dose usual para adultos acima de 18 anos é de 2.400 mg a 4.800 mg (2 a 4 comprimidos) ao dia, administrada em dose única, de preferência sempre à mesma hora de cada dia, acompanhada de uma refeição. Caso o paciente esteja tomando a dose mais elevada (4.800 mg/dia), deve ser reavaliado após 8 (oito) semanas de tratamento. Não apresentando mais sintomas, pode-se prescrever uma dose diária de 2.400 mg (2 comprimidos) para prevenir a recorrência de novas crises da doença (manutenção da remissão). A duração recomendada é de 8 semanas consecutivas, salvo critério médico diferente. Este medicamento não deve ser partido, mastigado ou dissolvido. A PERSISTIREM OS SINTOMAS, O MÉDICO DEVERÁ SER CONSULTADO. MEDICAMENTO SOB PRESCRIÇÃO. Registro MS - 1.0639. 0248 MEMX_0109_1210_VP. Referências bibliográficas: 1) Mesacol® MMX* [Bula]. São Paulo: Nycomed Pharma. 2) Lakatos PL. Use of new once-daily 5-aminosalicylic acid preparations in the treatment of ulcerative colitis: Is there anything new under the sun? World J Gastroenterol 2009; 15(15):1799-1804. 3) D’Haens GD, et al. Once daily MMX mesalazine for the treatment of mild-to-moderate ulcerative colitis: a phase II, dose-ranging study. Aliment Pharmacol Ther. 2006;24:1087-97. 4) Hu MY, Peppercorn MA. MMX mesalamine: a novel high-dose, once-daily 5-aminosalicylate formulation for the treatment of ulcerative colitis. Expert Opin Pharmacother. 2008;9(6):1049-58. 5) Lista de genéricos. Guia da Farmácia. 2011;18(220 Supl):2-32. 6) Prantera C, et al. A new oral delivery system for 5-ASA: preliminary clinical findings for MMx. Inflamm Bowel Dis 2005;11:421–7. 7) Osterman MT, Lichtenstein GR. Reformulation of an aminosalicylate: an example of the importance of pill burden on medical compliance rates. Methods Find Exp Clin Pharmacol. 2009;31(1):41-6. 8) Kamm MA, et al. Randomised trial of once- or twice-daily MMX mesalazine for maintenance of remission in ulcerative colitis. Gut. 2008;57(7):893-902.9) Kruis W, et al. Once daily versus three times daily mesalazine granules in active ulcerative colitis: a double-blind, double dummy, randomized, non-inferiority trial. Gut. 009;58(2):233-40. Material destinado exclusivamente a profissionais habilitados a prescrever e/ou dispensar medicamentos. MESACOL® é Marca Registrada da Nycomed Pharma Ltda., MMX* é Marca Depositada de Giuliani S.p.A. Nycomed Pharma Ltda. Rua do Estilo Barroco, 721 - 04709-011 - São Paulo - SP. Mais informações poderão ser obtidas diretamente com o nosso Departamento Médico ou por meio de nossos representantes. *Marca depositada.

Linha Gastro


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Apresentação com JVTWYPTPKVZ3,5** t01 DPNQSJNJEPEFNH02WF[FTBPEJB3 USO ADULTO Apresentações e composição: Comprimido revestido de 50 mg em embalagens com 30 unidades. Comprimido revestido de 100 mg em embalagens com 10, 30 e 60 unidades. Indicações: Tratamento sintomático da dor ou desconforto abdominal, dos distúrbios da freqüência ou consistência das evacuações (constipação ou diarréia) e da distensão abdominal, decorrentes dos transtornos funcionais do intestino (SII). Tratamento sintomático das dores decorrentes dos transtornos funcionais das vias biliares. Preparação de enemas opacos. Contra indicações: hipersensibilidade conhecida aos componentes da fórmula. Precauções e advertências: É desaconselhável a utilização do brometo de pinavério durante a gravidez. Além disso, deve-se observar a presença de bromo, cuja administração no final da gravidez pode causar alterações neurológicas no recém-nascido (hipotonia, sedação). Em função da falta de estudos, recomenda-se não utilizar o brometo de pinavério durante a lactação. Interações medicamentosas: os estudos realizados não demonstraram interações medicamentosas com nenhum dos seguintes tratamentos concomitantemente prescritos: hipoglicemiantes orais, anticoagulantes e digitálicos. Reações adversas: foram relatados, raramente, alguns casos de alterações digestivas leves e casos isolados de reações cutâneas, algumas do tipo alérgico. Raramente pode haver agravamento da constipação e epigastralgia. Em casos de erupção cutânea, é conveniente interromper a administração do medicamento. Posologia: recomenda-se a administração de 1 comprimido de 50 mg, 3 ou 4 vezes ao dia, ou 1 comprimido de 100 mg, 2 vezes ao dia (manhã e noite). Excepcionalmente, a posologia pode ser aumentada para 6 comprimidos de 50 mg ou 3 comprimidos de 100 mg ao dia. Os comprimidos devem ser deglutidos inteiros, sem mastigar, com um pouco de água, imediatamente antes ou durante as refeições. Na preparação de enemas opacos, a posologia é de 2 comprimidos diários de 100 mg ou 4 comprimidos diários de 50 mg, nos 3 dias anteriores ao exame. MS - Registro MS – 1.0639.0254. SICO_NSPC_1209.

Contraindicações: hipersensibilidade conhecida aos componentes da fórmula. A PERSISTIREM OS SINTOMAS, O MÉDICO DEVERÁ SER CONSULTADO. Nycomed Pharma Ltda. Rua do Estilo Barroco, 721 - 04709-011 - São Paulo - SP. Mais informações poderão ser obtidas diretamente com o nosso Departamento Médico ou por meio de nossos representantes. *Marca depositada.


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Eficácia da mesalazina na RCUI com a comodidade do MMX* 1-3

O que é a TECNOLOGIA MMX*? Comprimidos gastrorresistentes (Eudragit-S®)

1

Componentes hidrofílicos

2 3

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24 meses

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Eficácia da mesalazina na RCUI

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Eficácia da mesalazina com a comodidade do MMX*1-3

Manutenção1,8,9

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Posologia na Doença Diverticular13,15

1 comprimido 800mg, 2x ao dia Durante sete dias consecutivos a cada mês.


Contraindicação: Mesacol® MMX* não é recomendado em casos de hipersensibilidade  a salicilatos. Interação Medicamentosa: A administração da mesalazina pode potencializar a toxicidade do metotrexato. Mesacol® MMX* - mesalazina - USO ADULTO ACIMA DE 18 ANOS. USO ORAL - Apresentações e composição: Comprimidos revestidos de liberação prolongada, com 1,2 g de mesalazina cada. Embalagens com 10 e 30 unidades. Indicações: antiinflamatório de ação local no tratamento da colite ulcerativa ativa leve a moderada, na fase aguda (indução da remissão) e na manutenção da remissão. Contra-indicações: Este medicamento não deve ser usado por pacientes com história de hipersensibilidade aos salicilatos (que inclui o ácido acetilsalicílico), à mesalazina, à sulfassalazina ou a qualquer dos componentes da fórmula; pacientes com insuficiência hepática e/ou renal graves; pacientes com úlcera gástrica e duodenal ativa; pacientes com tendência elevada a sangramento. Este medicamento é contra-indicado para menores de 18 anos. Precauções e advertências: As mesmas precauções e advertências relacionadas com o uso de preparações contendo mesalazina ou pró-drogas de mesalazina devem ser consideradas para Mesacol® MMX*. Assim como todos os salicilatos, a mesalazina deve ser utilizada com cautela em pacientes com história de úlcera gástrica ou duodenal, por pacientes asmáticos (em função das reações de hipersensibilidade), com disfunção renal ou hepática (leve a moderada), ou com história de miocardite ou pericardite. Mesalazina não é recomendada para pacientes com disfunção renal grave e deve-se ter cautela com pacientes com níveis sangüíneos aumentados de uréia ou com proteinúria. A mesalazina é rapidamente excretada pelos rins, principalmente o seu metabólito ácido N-acetil-5-aminossalicílico. Em ratos, altas doses da mesalazina, administradas por via intravenosa, causaram toxicidade tubular e glomerular. Em caso de aparecimento de disfunção renal durante o tratamento deve-se suspeitar de nefrotoxicidade induzida pela mesalazina. Nestes casos é recomendado monitorar a função renal, especialmente no início do tratamento. Durante tratamento prolongado, é também necessário monitorar regularmente a função renal (creatinina sérica). Ainda não está estabelecida a segurança do produto em crianças. Gravidez e lactação: Mesacol® MMX* está classificado na Categoria B de risco de fármacos destinados ao uso em grávidas. O produto, a princípio, não deve ser empregado em gestantes e lactantes, exceto quando absolutamente necessário. A segurança de Mesacol® MMX* para uso durante a gravidez ou a amamentação ainda não foi estabelecida, mas sabe-se que a mesalazina atravessa a placenta e é excretada pelo leite materno em pequenas quantidades. Estudos pré-clínicos não revelaram evidência de efeitos teratogênicos ou de toxicidade fetal oriundos da mesalazina, nem na evolução da gestação ou no desenvolvimento perinatal e pós-natal. A pequena experiência de uso da mesalazina em outras formulações durante a gravidez não mostrou efeito prejudicial ao feto; entretanto, a mesalazina deve ser usada com cautela durante a gravidez e somente quando os benefícios para a mãe forem superiores aos riscos potenciais ao feto. Baixas concentrações de mesalazina e de seu metabólito N-acetilado foram detectadas no leite materno, mas o significado clínico desta evidência ainda não foi determinado. Portanto, deve-se ter cautela na administração da mesalazina a lactantes. Categoria B de risco na gravidez – Este medicamento não deve ser utilizado por mulheres grávidas sem orientação médica ou do cirurgião-dentista. Pacientes pediátricos: Devido à falta de dados sobre a administração da mesalazina em altas doses na população pediátrica, Mesacol® MMX* não é recomendado para pacientes menores de 18 anos. Pacientes idosos: Não existe experiência suficiente sobre o uso de Mesacol® MMX* em pacientes com idade acima de 65 anos. No entanto, não foram identificadas diferenças entre o uso em pacientes mais jovens e em idosos com outras formulações de mesalazina. Pacientes com insuficiência renal: não são disponíveis informações sobre o uso em pacientes com insuficiência renal leve ou moderada. O medico deverá avaliar a relação risco/benefício para o seu uso. Pacientes com insuficiência hepática: não são disponíveis informações sobre o uso em pacientes com insuficiência hepática leve ou moderada. O medico deverá avaliar a relação risco/benefício para o seu uso. Dirigir e operar máquinas: É improvável que o uso deste medicamento tenha qualquer efeito na capacidade de dirigir veículos ou de usar máquinas. Interações medicamentosas: Nenhum estudo formal de interação foi conduzido. Não são disponíveis informações sobre interações entre Mesacol® MMX* e outros fármacos. Entretanto, existem relatos de interação entre a mesalazina (outras formulações) e outros medicamentos. O uso concomitante da mesalazina com agentes sabidamente nefrotóxicos, inclusive com os anti-inflamatórios não-hormonais (AINHs – como aspirina, ibuprofeno, diclofenaco, etc.) e azatioprina, pode aumentar o risco de reações renais; o potencial para discrasias sangüíneas da azatioprina e da 6-mercaptopurina pode ser aumentado; a ação hipoglicemiante das sulfoniluréias pode ser intensificada; a atividade anticoagulante dos derivados cumarínicos (varfarina) pode ser reduzida; a toxicidade do metotrexato pode ser potencializada; o efeito uricosúrico da probenecida e da sulfimpirazona pode ser diminuído, assim como a ação diurética da furosemida e da espironolactona e a ação tuberculostática da rifampicina. Em tese, a administração concomitante de anticoagulantes orais deve ser feita com cautela. Substâncias como a lactulose, que diminuem o pH do cólon, podem reduzir a liberação da mesalazina dos comprimidos revestidos de Mesacol® MMX*. Reações adversas: A maioria das reações adversas relatadas com Mesacol® MMX* foi transitória, e de intensidade leve a moderada. Foram descritas as seguintes reações adversas, distribuídas em grupos de freqüências: Reação comum (> 1/100 e < 1/10): Gastrintestinal: Flatulência e Náusea. Sistema Nervoso: Cefaléia. Estas reações ocorreram em menos de 3% dos pacientes, independente da dose administrada. Reação incomum (> 1/1.000 e < 1/100): Gastrintestinal: vômito, dor abdominal, distensão abdominal, diarréia, dispepsia, pancreatite, colite e pólipo retal. Hepatobiliar: aumento das transaminases, anormalidades no teste da função hepática. Sistema nervoso: tontura, sonolência, tremores. Cardiovascular: taquicardia, hipertensão e hipotensão arterial. Respiratório: dor faringolaríngea. Ouvido e labirinto: otalgia. Pele e tecido subcutâneo: acne, alopécia, prurigo, urticária, exantema, prurido. Sangue e linfa: Redução do número de plaquetas. Musculosqueléticas: artralgia, lombalgia. Gerais: Astenia, fadiga, pirexia, edema da face. Posologia e modo de usar: Mesacol® MMX* é para uso exclusivo por via oral. Para o tratamentoda colite ulcerativa leve a moderada, a dose usual para adultos acima de 18 anos é de 2.400 mg a 4.800 mg (2 a 4 comprimidos) ao dia, administrada em dose única, de preferência sempre à mesma hora de cada dia, acompanhada de uma refeição. Caso o paciente esteja tomando a dose mais elevada (4.800 mg/dia), deve ser reavaliado após 8 (oito) semanas de tratamento. Não apresentando mais sintomas, pode-se prescrever uma dose diária de 2.400 mg (2 comprimidos) para prevenir a recorrência de novas crises da doença (manutenção da remissão). A duração recomendada é de 8 semanas consecutivas, salvo critério médico diferente. Este medicamento não deve ser partido, mastigado ou dissolvido. MEDICAMENTO SOB PRESCRIÇÃO. Registro MS - 1.0639. 0248 MEMX_0109_1210_VP.

Contraindicação: Mesacol® não é recomendado em casos de hipersensibilidade  a salicilatos. Interação Medicamentosa: A administração da mesalazina pode potencializar a toxicidade do metotrexato.

Mesacol® - mesalazina - Uso adulto - Apresentações e composição: Comprimido revestido 400 mg e 800 mg de mesalazina. Embalagens com 30 unidades. Uso oral. Supositório 250 mg e 500 mg de mesalazina. Embalagens com 10 unidades. Uso anorretal. Indicações: indicado como antiinflamatório de ação local no tratamento de doenças inflamatórias intestinais, na fase aguda e na prevenção ou redução das recidivas destas enfermidades: retocolite ulcerativa inespecífica (RCUI) (tanto a colite como a proctite ulcerativa) e doença de Crohn. Também indicado para o tratamento sintomático da Doença Diverticular do cólon, associado ou não com terapia à base de antibióticos como ampicilina/sulbactam ou rifaximina. Contraindicações: Hipersensibilidade a salicilatos e aos componentes da fórmula. Insuficiências hepática e renal graves, com uma taxa de filtração glomerular menor que 20 ml/ min, úlcera gástrica e duodenal ativa; tendência elevada a sangramento. Crianças abaixo de 2 anos. Precauções e advertências: Assim como todos os salicilatos, a mesalazina deve ser utilizada com cautela em pacientes com úlceras gástricas ou duodenais e por pacientes asmáticos (em função das reações de hipersensibilidade). Não é recomendado para os pacientes com a função renal prejudicada e deve-se ter cautela com pacientes cujos níveis sangüíneos de uréia ou proteinúria estejam aumentados. A mesalazina é rapidamente excretada pelos rins, principalmente o seu metabólito ácido N-acetil-5-aminosalicílico. Em ratos, altas doses da mesalazina, administradas por via IV, causaram toxicidade tubular e glomerular. Em caso de aparecimento de disfunção renal durante o tratamento deve-se suspeitar de nefrotoxicidade induzida pela mesalazina. Nestes casos é recomendado monitorar a função renal, especialmente no início do tratamento. Durante tratamento prolongado, é também necessário monitorar regularmente a função renal. Em casos isolados, devido à alteração do trânsito e/ou pH intestinal, pode ocorrer a eliminação do comprimido de Mesacol® nas fezes, sem ocorrer sua completa desintegração. Nestes casos a terapia deve ser reavaliada. O produto, a princípio, não deve ser empregado em gestantes e lactantes, exceto quando absolutamente necessário. O risco teórico de kernicterus relacionado à sulfapiridina (parte da molécula da sulfassalazina) é evitado com Mesacol®. Estudos pré-clínicos não revelaram evidência de efeitos teratogênicos ou de toxicidade fetal oriundos da mesalazina. A pequena experiência de uso da mesalazina durante a gravidez não mostrou efeito prejudicial ao feto; entretanto, a mesalazina deve ser usada com cautela durante a gravidez e somente quando os benefícios para a mãe forem superiores aos riscos potenciais ao feto. Baixas concentrações de mesalazina e de seu metabólito N-acetilado foram detectadas no leite materno, mas o significado clínico desta evidência ainda não foi determinado. Portanto, deve-se ter cautela na administração da mesalazina à lactantes. Ainda não está estabelecida a segurança do produto em crianças. O produto contém lactose e deve ser evitado por pacientes com intolerância a esta substância. A diminuição da contagem e função dos espermatozóides observada com a sulfassalazina parece não estar associada à mesalazina. Podem ocorrer reações de hipersensibilidade cardíaca (miocardite e pericardite). Usar com cautela em pacientes que têm predisposições a essas condições’’. Em pacientes idosos existe o risco de ocorrer discrasias sanguíneas. Em pacientes com doenças tromboembólicas ou outros fatores de risco, recomenda-se monitoramento dos parâmetros hematológicos. Em pacientes com doença renal moderada e grave foi reportado o falência hepática. Dessa forma, recomenda-se cautela no uso do Mesacol nesses pacientes. Pacientes com hipersensibilidade à sulfasalazina devem tomar Mesacol com cautela e observação médica por risco de reação cruzada. Interações medicamentosas: A ação hipoglicemiante das sulfoniluréias pode ser intensificada, assim como a hemorragia gastrointestinal causada por cumarínicos. A administração oral da mesalazina pode potencializar a toxicidade do metotrexato. O efeito uricosúrico da probenecida e sulfimpirazona pode ser diminuído, assim como a ação diurética da furosemida e da espironolactona. A ação tuberculostática da rifampicina também pode ser diminuída. Em tese, a administração concomitante de anticoagulantes orais deve ser feita com cautela. Substâncias como a lactulose, que diminuem o pH do cólon, podem reduzir a liberação da mesalazina dos comprimidos revestidos de Mesacol®. Reações adversas: As reações adversas ocorrem em uma pequena proporção de pacientes que, previamente, não toleraram a sulfassalazina, tais como náuseas, diarréia, vômitos, dor abdominal, cefaléia e flutuações do humor.Têm sido relatadas reações de hipersensibilidade, como exantema alérgico, febre, broncoespasmo, lúpus eritematoso, rashes e artralgia. Estes efeitos ocorrem independentemente da dose administrada. Pode haver aumento dos níveis de metahemoglobina. Mesacol® pode estar associado com a exacerbação dos sintomas da colite nos pacientes que tiveram previamente problemas com a sulfassalazina. Foram relatados casos de pancreatite, miocardite, pericardite, nefrite intersticial, síndrome nefrótica e insuficiência renal com o tratamento por via oral; geralmente estes sintomas regridem com a suspensão do tratamento. Há raros relatos de reações alérgicas pulmonares, pneumonia eosinofílica, hepatite e discrasias sangüíneas, tais como leucopenia, neutropenia, trombocitopenia e anemia aplástica. Posologia e modo de usar: Comprimido: A dose recomendada para adultos é de 800 a 2400 mg por dia, igualmente dividida a critério médico na dependência da gravidade do caso. Nos casos mais graves a posologia pode ser aumentada para 4.800 mg ao dia. De forma geral recomenda-se as seguintes posologias para adultos em doses divididas diariamente: Colite ulcerativa: Indução da remissão: dose de 2.400 - 4.800 mg; Manutenção da remissão: dose de 1.200 – 2.400 mg podendo ser aumentada para 4.800 mg. Doença de Crohn: Manutenção da remissão: dose de 2.400 mg. Doença Diverticular Sintomática: 800 mg duas vezes ao dia durante sete dias consecutivos a cada mês. Os comprimidos não devem ser mastigados, mas sim ingeridos inteiros, com um pouco de líquido. Não há dose recomendada para crianças. Supositório de 250 e 500 mg: Os supositórios são utilizados para tratamento da proctite e da proctosigmoidite. A dose recomendada para adultos é de 1 – 2 supositórios de 500 mg ou 2 a 4 de 250 mg, até 3 vezes ao dia, após a defecação. A dose depende da gravidade da doença, e pode ser diminuída assim que houver melhora dos sintomas. Na colite ulcerativa grave generalizada, afetando o reto ou retosigmóide, e em casos de resposta lenta à terapia oral, recomenda-se 1 – 2 supositórios de 500 mg, pela manhã e à noite, como adjunto da terapia oral. Não há dose recomendada para crianças. Com a remissão da sintomatologia clínica, preconiza-se como dose de manutenção, na dependência da resposta individual, um supositório de 250 mg ao dia em dias alternados ou mais espaçadamente. MEDICAMENTO SOB PRESCRIÇÃO. Registro MS - 1.0639.0200 (Comprimido revestido 400 mg e supositório 250 mg), MS – 1.0639.0248 (Comprimido revestido 800 mg e supositório 500 mg). MESACOL® é Marca Registrada da Nycomed Pharma Ltda., MMX* é Marca Depositada de Giuliani S.p.A. Referências bibliográficas 1) Mesacol® MMX* [Bula]. São Paulo: Nycomed Pharma. 2) Lakatos PL. Use of new once-daily 5-aminosalicylic acid preparations in the treatment of ulcerative colitis: Is there anything new under the sun? World J Gastroenterol 2009; 5(15):1799-1804. 3) D’Haens GD, et al. Once daily MMX mesalazine for the treatment of mild-to-moderate ulcerative colitis: a phase II, dose-ranging study. Aliment Pharmacol Ther. 2006;24:1087-97. 4) Kamm MA et al. Once-daily, highconcentration MMX mesalamine in active ulcerative colitis. Gastroenterology.2007;132:66—75. 5) Tenjarla S et al. Release of 5-aminosalicylate from na MMX meslaminr tablet during transit throught a simulated gastrointestinal tract system. Adv Ther. 2007;24(4):82640. 6) Brunner M, Assandri R,Kletter K,et al. Gastrointestinal transit and 5-ASA release from a new mesalazine extended-release formulation. Aliment Pharmacol Ther 2003;17:395–402. 7) Prantera C, Viscido A, Biancone L et al. A new oral delivery system for 5-ASA: preliminary clinical findings for MMX. Inflamm Bowel Dis 2005;11:421–7. 8) Kamm MA et al. Randomised trial of once- or twice-daily MMX mesalazine for maintenance of remission in ulcerative colitis. Gut. 2008;57(7):893-902. 9) Prantera C, et al. Clinical trial: ulcerative colitis maintenance treatment with 5-ASA: a 1-year, randomized multicentre study comparing MMX with Asacol. Aliment Pharmacol Ther. 2009; 30: 908–918. 10) Kruis W et al. Once daily versus three times daily mesalazine granules in active ulcerative colitis: a double-blind, double-dummy, randomized, non-inferiority trial. Gut. 2009;58(2):233-40. 11) Lichtenstein GR et al. Effect of once or twice-daily MMX mesalamine (SPD476) for the induction of remission of mild to moderately active ulcerative colitis. Clin Gastroenterol Hepatol. 2007;5:95-102. 12) Kane S, et al. Medication nonadherence and the outcomes of patients with quiescent ulcerative colitis. Am J Med. 2003;114(1):39-43. 13) Mesacol® [Bula]. São Paulo: Nycomed Pharma. 14) Agência Nacional de Vigilância Sanitária (BR). RE nº 3.065, de 23 de julho de 2009. Concessão de registro de medicamento novo, revalidação de registro, refitificação de publicação. Diário Oficial [da República Federativa do Brasil]. Brasília, 27 jul 2009; n. 141, p. 4-5. [cited 2011 dec 15]. http://www.in.gov.br/imprensa/visualiza/index.jsp?jornal=1010&pagina=5&data=27/07/2009. 15) Comparato et al. quality of life in uncomplicated symptomatic diverticular disease: is it another good reason for treatment? Dig Dis. 2007;25:252-59. Material destinado exclusivamente a profissionais habilitados a prescrever e/ou dispensar medicamentos. Fev/2012 Nycomed Pharma Ltda. Rua do Estilo Barroco, 721 - 04709-011 - São Paulo - SP. Mais informações poderão ser obtidas diretamente com o nosso Departamento Médico ou por meio de nossos representantes.

Linha Gastro

A PERSISTIREM OS SINTOMAS, O MÉDICO DEVERÁ SER CONSULTADO.


No Tratamento da Sii1,2 1, 2, 3 l a n i m o d b a r O alívio da do

O principal sintoma da Sii:

O brometo de pinavériodiminuiu

DOR 92% 4,5

a duração da dor de várias horas para 7 alguns minutos.

O brometo de pinavério diminui a dor em até

dos casos.

5

USO ADULTO Apresentações e composição: Comprimido revestido de 50 mg em embalagens com 30 unidades. Comprimido revestido de 100 mg em embalagens com 10, 30 e 60 unidades. Indicações: Tratamento sintomático da dor ou desconforto abdominal, dos distúrbios da freqüência ou consistência das evacuações (constipação ou diarréia) e da distensão abdominal, decorrentes dos transtornos funcionais do intestino (SII). Tratamento sintomático das dores decorrentes dos transtornos funcionais das vias biliares. Preparação de enemas opacos. Contra indicações: hipersensibilidade conhecida aos componentes da fórmula. Precauções e advertências: É desaconselhável a utilização do brometo de pinavério durante a gravidez. Além disso, deve-se observar a presença de bromo, cuja administração no final da gravidez pode causar alterações neurológicas no recém-nascido (hipotonia, sedação). Em função da falta de estudos, recomenda-se não utilizar o brometo de pinavério durante a lactação. Interações medicamentosas: os estudos realizados não demonstraram interações medicamentosas com nenhum dos seguintes tratamentos concomitantemente prescritos: hipoglicemiantes orais, anticoagulantes e digitálicos. Reações adversas: foram relatados, raramente, alguns casos de alterações digestivas leves e casos isolados de reações cutâneas, algumas do tipo alérgico. Raramente pode haver agravamento da constipação e epigastralgia. Em casos de erupção cutânea, é conveniente interromper a administração do medicamento. Posologia: recomenda-se a administração de 1 comprimido de 50 mg, 3 ou 4 vezes ao dia, ou 1 comprimido de 100 mg, 2 vezes ao dia (manhã e noite). Excepcionalmente, a posologia pode ser aumentada para 6 comprimidos de 50 mg ou 3 comprimidos de 100 mg ao dia. Os comprimidos devem ser deglutidos inteiros, sem mastigar, com um pouco de água, imediatamente antes ou durante as refeições. Na preparação de enemas opacos, a posologia é de 2 comprimidos diários de 100 mg ou 4 comprimidos diários de 50 mg, nos 3 dias anteriores ao exame. MS - Registro MS – 1.0639.0254. SICO_NSPC_1209

Contraindicações: hipersensibilidade conhecida aos componentes da fórmula. Interações medicamentosas: os estudos realizados não demonstraram interações medicamentosas com nenhum dos seguintes tratamentos concomitantemente prescritos: hipoglicemiantes orais, anticoagulantes e digitálicos. Referências bibliográficas: 1) Guslandi M. Profilo farmacologico clinico del pinaverio bromuro. Minerva Med. 1994;85:179-85. 2) Poynard T, Regimbeau C, Benhamou Y. Meta-analysis of smooth muscle relaxants in the treatment of irritable bowel syndrome. Aliment Pharmacol Ther. 2001;15(3):355-61. 3) Siilif*®[Bula]. São Paulo: Nycomed Pharma. 4) Cloarec D et al. Efficacite du Dicetel 100 mg au coursdes troubles fonctionnels intestinaux: résultats d’une étude réalisée em pratique gastroentérologique. Rev Fr Gastroenterol. 1997;33(324-325):1107-35. 5) Corazziari E et al. Consensus report: clinical trial guidelines for pharmacological treatment of irritable bowel syndrome. Aliment Pharmacol Ther. 2003;18(6):569-80. 6) Cloarec D et al. Efficacité du pinaverium: 100mg, deux foius par jour, dans le traitement sumptomatique du syndrome de l’intestin irritable. Ann Gastroenterol Hepatol. 1997;33(4):181-4. 7) Awad R, Dibildox M, Ortiz F. Irritable bowel syndrome treatment using pinaverium bromide as a calcium channel blocker. Acta Gastroenterol Latinoam. 1995;25(3):137-44. Material destinado exclusivamente a profissionais habilitados a prescrever e/ou dispensar medicamentos. Fev/2012 Nycomed Pharma Ltda. Rua do Estilo Barroco, 721 - 04709-011 - São Paulo - SP. Mais informações poderão ser obtidas diretamente com o nosso Departamento Médico ou por meio de nossos representantes. *Marca depositada. A PERSISTIREM OS SINTOMAS, O MÉDICO DEVERÁ SER CONSULTADO.

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6067632 RX REVISTA ARQUIVOS GASTRO ED. 01/2012

ISSN 0004-2803 ISSN 1678-4219 on-line Coden ARQGA

ARQUIVOS DE GASTROENTEROLOGIA Número 1 | Janeiro/Março 2012 | Volume 49

Arquivos de Gastroenterologia

ARCHIVES OF GASTROENTEROLOGY Publication of the Brazilian Institute for Studies and Research in Gastroenterology and others Specialities - IBEPEGE

Founded in 1964 by Prof. Dr. José Fernandes Pontes

ORGÃO DE DIVULGAÇÃO Publication

CBCD

Colégio Brasileiro de Cirurgia Digestiva

PAT R O C Í N I O

SBNPE

Sociedade Brasileira de Nutricção Parenteral e Enteral

SOBED

FBG

Federação Brasileira de Gastroenterologia

SBM D

Sociedade Brasileira de Motilidade Digestiva

Sociedade Brasileira de Endoscopia Digestiva

SBH

Sociedade Brasileira de Hepatologia


Arquivos de Gastroenterologia v49n1