UNIVERSIDADE ESTADUAL DE CAMPINAS

FACULDADE DE ODONTOLOGIA DE PIRACICABA

THAYSE RODRIGUES DE SOUZA

RELAÇÃO ENTRE A ATIVIDADE DA ANIDRASE CARBÔNICA VI,

ALFA-AMILASE SALIVAR, CAPACIDADE TAMPÃO, FLUXO

SALIVAR E CÁRIE DENTAL EM CRIANÇAS

RELATIONSHIP AMONG SALIVARY CARBONIC ANHYDRASE VI

ACTIVITY, ALPHA-SALIVARY AMYLASE, BUFFERING CAPACITY,

SALIVARY FLOW RATE AND DENTAL CARIES IN CHILDREN

Piracicaba

2016

THAYSE RODRIGUES DE SOUZA

RELAÇÃO ENTRE A ATIVIDADE DA ANIDRASE CARBÔNICA VI,

ALFA-AMILASE SALIVAR, CAPACIDADE TAMPÃO, FLUXO

SALIVAR E CÁRIE DENTAL EM CRIANÇAS

RELATIONSHIP AMONG SALIVARY CARBONIC ANHYDRASE VI

ACTIVITY, ALPHA-SALIVARY AMYLASE, BUFFERING CAPACITY,

SALIVARY FLOW RATE AND DENTAL CARIES IN CHILDREN

Piracicaba

2016

Tese apresentada à Faculdade de Odontologia de

Piracicaba da Universidade Estadual de Campinas

como parte dos requisitos exigidos para a obtenção

do título de Doutora em Odontologia, na Área de

Odontopediatria.

Thesis presented to the Piracicaba Dental School of

the University of Campinas in partial fulfillment of

the requirements for the degree of Doctor in

Dentistry in Pediatric Dentistry Area.

Orientador: Profa. Dr

a. Marinês Nobre dos Santos Uchôa

ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA

TESE DEFENDIDA PELA ALUNA THAYSE RODRIGUES DE

SOUZA E ORIENTADA PELA PROFa. DR

a MARINÊS

NOBRE DOS SANTOS UCHÔA.

DEDICATÓRIA

À Deus por me guiar nos diversos caminhos que se abriram para mim... Por me

iluminar nas decisões mais difíceis... Por ser minha fortaleza, refúgio e morada espiritual e

por sempre estar comigo em qualquer lugar que eu vá.

AGRADECIMENTOS ESPECIAIS

Aos meus pais Ana Dalva e Antônio Rodrigues por terem me dado a oportunidade de

estudar e sempre me guiarem a este caminho... Por sempre me apoiarem em minhas decisões

e pelo carinho e amor sustentadores. Obrigada não só por me dar a vida, mas principalmente

por me ensinar a vivê-la.

Ao meu querido esposo Jorge Leão pelo amor, carinho, incentivo e companheirismo

fundamental... Por sua mão sempre estendida a me ajudar, por ser parte de mim, parte de

quem eu sou e por tornar meus dias imensamente felizes.

À minha orientadora, Profa. Dra. Marinês Nobre dos Santos Uchôa, por ter me

aceitado como sua orientanda, pela edificante orientação, por me proporcionar mais uma

experiência da pesquisa científica e sempre acreditar em minha dedicação e empenho, por

todos os ensinamentos e compreensão quando mais precisei.

Às crianças que fizeram parte dessa pesquisa... Meus amores, vocês foram

fundamentais e me deram incentivo a cada dia... Obrigada pelo olhar de pureza, felicidade e

carinho que me passavam em cada dia de coleta.

À Força Aérea Brasileira nas pessoas do Coronel Médico Laerte Lobato de Moraes,

diretor do Hospital de Aeronáutica de Belém e Tenente-Coronel Luiz Fernando da Costa

Tavares, chefe da Divisão Odontológica, pela compreensão e apoio no seguimento de meu

curso de doutorado.

À minha irmã Thalyta Souza e às famílias Souza, Rodrigues e Leão pelo enorme

carinho apoio e por sempre torcerem pelas minhas conquistas.

AGRADECIMENTOS

À Universidade Estadual de Campinas, na pessoa do reitor Prof. Dr. José Tadeu

Jorge, à Faculdade de Odontologia de Piracicaba FOP-UNICAMP, na pessoa do seu diretor

Prof. Dr. Guilherme Elias Pessanha Henriques, à Comissão de Pós-Graduação da FOP-

UNICAMP na pessoa da presidente Profa. Dr

a. Cínthia Pereira Machado Tabchoury e da

Coordenadora do Programa de Pós-Graduação em Odontologia Profa. Dr

a. Juliana Trindade

Clemente Napimoga, pela participação dessa conceituada instituição no meu crescimento

científico.

À Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) pelo apoio

financeiro concedido durante o desenvolvimento dessa tese.

Às Profas

. Dras

., Fernanda Miori Pascon, Maria Beatriz Duarte Gavião, Regina

Maria Puppin Rontani do Departamento de Odontologia infantil, por terem me recebido tão

bem quando cheguei à faculdade, por todos os conhecimentos passados, tanto os conteúdos

relacionados à Odontopediatria quanto àqueles relacionados ao ensino e à pesquisa.

A todos os professores do Programa de Pós-Graduação em Odontologia da FOP-

UNICAMP e aqueles professores convidados a ministrar diversas aulas que engrandeceram

nossos conhecimentos científicos principalmente no campo do ensino e pesquisa.

Aos professores colaboradores, Prof. Dr. Sérgio Line e Prof. Dr. Marcelo Marques

pelo desenvolvimento do protocolo de visualização da atividade da enzima anidrase carbônica

VI que possibilitou a execução desse projeto de tese.

Ao técnico do laboratório de Odontopediatria, Marcelo Corrêa Maistro, pelo auxílio

fundamental nas etapas laboratoriais da pesquisa.

À Secretaria Municipal de Educação do município de Piracicaba por ter permitido a

realização da pesquisa.

Às diretoras das creches visitadas por terem me acolhido tão bem na ocasião da coleta

de dados e amostras da pesquisa.

À Professora Dra. Thais Manzano Parisotto pela ajuda fundamental, pelas

orientações e por sempre estar disposta a ajudar na execução da pesquisa.

À Professora Dra. Cínthia Pereira Machado Tabchoury e Prof. Dra. Maria Beatriz

Duarte Gavião membros da banca de pré-qualificação pelas sugestões para execução desse

trabalho.

Ao Prof. Dr. Natanael Barbosa e Prof. Dr. Milton Duarte, do Departamento de

Cariologia da Universidade Federal de Alagoas- Faculdade de Odontologia pelo exemplo de

pesquisadores, por terem me iniciado na pesquisa científica na graduação em Odontologia,

tendo sido fundamentais na escolha dos caminhos no ensino e pesquisa. Ao Prof. Dr. Luiz

Alcino Monteiro Gueiros e Prof. Dr. Jair Carneiro Leão do Departamento de

Estomatologia da Universidade Federal de Pernambuco- Faculdade de Odontologia, meus

orientadores do mestrado pelos conhecimentos passados e que foram fundamentais para a

subida de mais um degrau em minha formação acadêmica.

À Maria Elisa dos Santos, Eliane Melo Franco de Souza, Érica A. Pinho Sinhoreti

e Raquel Q. M. Cesar Sacchi e Roberta C. Morales dos Santos, pela ajuda e atenção nas

etapas administrativas e a todos os funcionários da FOP-UNICAMP, pela colaboração.

Às amigas Lívia Pagotto e Fabiana Furtado por terem me acolhido tão bem na

cidade na ocasião de minha chegada pela amizade e companhia diária.

Às amigas Lívia Pagotto e Bruna Raquel por o auxílio em etapas da pesquisa.

À amiga Andréia Alves, pela mão estendida não só para aprender, mas também

ajudar. Obrigada pela companhia nas muitas horas esperando as bandas da anidrase e pela

amizade.

Aos colegas e amigos: Maria Carolina S. Marquezin, Marina S. Leme, Bárbara

Lucas, Ana Bheatriz M. Montes, Filipe Martins, Alexsandra S. Iwamoto, Ariany B.

Carvalho, Bruna R. Zancopé, Lívia P. Rodrigues, Luciana T. Inagaki, Vanessa

Benetello, Micaella Cardoso, Natalia Martins, Darlle Araújo, Thais Varanda e Lenita

Lopes pelo convívio e amizade durante essa importante etapa.

Aos amigos da Força Aérea Brasileira em especial às Tenentes Patrocínio, Paola,

Flávia Carvalho, Camilla Pinto, Camila Rocha, Kobayashi, Luciane Bertoldi, Cibelle, Glauce

Vaz , Thayanna e Valéria pela amizade e carinho.

Às amigas Cíntia Priscila, Talita França e Samantha Mendonça pela amizade e

torcida.

EPÍGRAFE

“Bom mesmo é ir a luta com

determinação, abraçar a vida com paixão,

perder com classe e vencer com ousadia,

porque o mundo pertence a quem se atreve (e

tem fé) e a vida é muito para ser

insignificante.” Augusto Branco

RESUMO

As enzimas anidrase carbônica VI (AC VI) e α-amilase estão presentes na saliva. AC

VI é responsável por catalisar a principal reação tamponante da cavidade bucal. A enzima α-

amilase é responsável pela formação da película, biofilme e no metabolismo do amido. Não

há relatos na literatura que tenham investigado longitudinalmente a relação entre a AC VI e

cárie dental ou transversalmente a atividade de α-amilase logo após um desafio cariogênico. A

tese foi apresentada em dois Capítulos. Os objetivos do Capítulo 1 foram: Determinar o fluxo

salivar estimulado (FSE), capacidade tampão (CT) e a atividade de AC VI na saliva de

crianças com cárie e livres de cáries antes e após o bochecho com solução de sacarose a 20%

e investigar a relação entre essas variáveis e a cárie dental longitudinalmente após um ano e

no Capítulo 2: Investigar a atividade de α-amilase na saliva de crianças com cárie e livres de

cáries antes e após o bochecho com uma solução de sacarose a 20% e sua relação com FSE,

CT e a cárie dental transversalmente. No Capítulo 1 foram alocadas 47 crianças de 48 a 78

meses de idade, divididos em três grupos após cálculo do incremento de cárie após um ano:

grupo livre de cárie (LC, n=10), grupo com cárie (C, n=20) e grupo de cárie paralisada (CP,

n=17). No Capítulo 2, 38 crianças de 48 a 77 meses de idade, divididas em dois grupos: com

cárie (C, n=20) e livres de cárie (LC, n=18). A atividade da AC VI foi quantificada por

zimografia. O FSE foi expresso em mL/min. A CT foi medida pelo método de Ericsson por

meio de um eletrodo de pH conectado a um peagâmetro. A análise de α-amilase foi realizada

por ensaio enzimático. Os dados de AC VI foram submetidos ao teste de Wilcoxon e

Kruskall-Wallis para comparações pareadas dos valores antes e depois do bochecho e

comparação entre grupos respectivamente. Os dados de FSE e CT foram submetidos aos

testes acima mencionados nos dois Capítulos. Os dados da atividade de α-amilase foram

submetidos aos testes T de Student pareado e independente. Foi realizado também análise de

correlação de Spearman (α=0.05). Os resultados do Capítulo 1 mostraram que a atividade de

AC VI apresentou um decréscimo significativo após o bochecho nos grupos LC no baseline e

após um ano e no grupo CP somente após um ano (p= 0.037, p=0.028 e p=0.027,

respectivamente). Não se observou mudanças na atividade de AC VI no grupo CL antes e

depois do bochecho nos dois períodos do estudo. A atividade de AC VI antes do bochecho no

baseline exibiu correlação negativa significativa como índice de cárie no baseline antes e

depois do bochecho e após um ano antes do bochecho no grupo C (r=-0.609, p=0.004 e r=-

0.516, p=0,020, r= -0.545, p=0.013, respectivamente). Uma correlação negativa significativa

foi encontrada entre o índice de cárie nos dois tempos do estudo e CT após o bochecho após

um ano (r=-0.345, p=0.017 e r=-0.303, p=0.038, respectivamente). Os resultados do Capítulo

2 mostraram que o grupo C exibiu um aumento significativo na atividade de α-amilase após o

bochecho diferindo significativamente do grupo LC (p=0.024 e p=0.019). Observou-se nos

dois Capítulos aumento do FSE após o bochecho e diminuição dos valores de CT após o

bochecho com sacarose. Conclui-se que a atividade de AC VI exerce possível participação no

controle de pH bucal após um desafio cariogênico, principalmente em crianças com cárie.

Sugere-se ainda, uma possível participação da α-amilase como facilitadora do processo de

cárie devido ao aumento de sua atividade quando as crianças com cárie foram submetidas a

um desafio cariogênico.

Palavras-chave: Anidrase carbônica VI, fluxo salivar, capacidade tampão da saliva, cárie

precoce da infância.

ABSTRACT

The carbonic anhydrase VI (CAVI) and α-amylase (SAA) enzymes are present in

saliva. AC VI is responsible for catalyzing the main reaction buffering the oral cavity. SAA is

associated with the pellicle and biofilms formation and starch metabolism. There are no

reports in the literature that have longitudinally investigated the relationship between AC VI

and dental caries and a cross-sectional study to investigate the SAA activity after a cariogenic

challenge. The objectives of the Chapter 1 of this thesis were: Determine the stimulated

salivary flow (SSFR), buffer capacity (BC) and CA VI activity in the saliva of children with

caries and caries-free before and after rinsing with a sucrose solution to 20% and to

investigate the relationship of these variables with dental caries in a longitudinal study of one

year of follow-up. And of the Chapter 2: Investigate the SAA activity in saliva of children

with caries and caries-free before and after rinsing with a sucrose solution at 20% and its

relationship with SSFR, BC and dental caries in a cross-sectional study. Were allocated to the

study of Chapter 1 47 children 48-78 months age, divided into three groups after calculation

of caries increment after one year: caries free group (CF), caries lesion group (CL) and

arrestment caries group (AC). And in Chapter 2, 38 children aging 48-77 months old, divided

into two groups: caries lesion group (CL) and caries free group (CF). The activity of CA VI

was quantified by zymography. The SSFR was expressed in mL/min. The BC was measured

by Ericsson’s method. The SAA activity was analyzed by the enzyme kinetic assay. Wilcoxon

test and the Kruskal-Wallis test for paired comparisons of the values of CAVI before and after

the rinses and comparison between groups respectively. To SSFR and BC data were

employed the tests mentioned above in the two Chapters. The Student t test paired and

independent were employed to the SAA data. It was also performed Spearman correlation

analysis (α = 0.05). The results of chapter 1 show that CA VI activity significantly decreased

after the cariogenic challenge at the CF group in baseline and follow-up and at AC group only

at the follow-up (p= 0.037, p=0.028 e p=0.027, respectively). No change in CA VI activity

was found at the two periods of the study in CL group. Salivary CA VI activity before rinse at

the baseline shows also a negative correlation with dental caries at the baseline before and

after rinse and at the follow-up before the rinse in the CL group (r=-0.609, p=0.004 e r=-

0.516, p=0,020, r= -0.545, p=0.013, respectively). A negative correlation was found between

dental caries at baseline as well at follow-up and BC after rinse at follow-up (r=-0.345,

p=0.017 e r=-0.303, p=0.038 respectively). The results of Chapter 2 shows that the CL group

exhibited a significant increase on SAA activity after rinse (p=0.001), and significantly

differed from CF group (p=0.033). The results of the two Chapters show a significant increase

and decrease of SSFR and BC respectively after the sucrose rinse solution in both groups. It is

concluded that the AC VI activity possible participates on the oral pH control after a

cariogenic challenge, particularly in children with caries. It is also suggested possible

involvement of SAA as a facilitator of the decay process due to the increase of its activity

when the children were submitted to a cariogenic challenge in the group of children with

caries.

KEY-WORDS: Carbonic anhydrase VI, salivary flow, salivary buffer capacity, early childhood

caries.

LISTA DE ILUSTRAÇÕES

FIGURA 1. Exame clínico para avaliação do índice de cárie em pré-escolares do

município de Piracicaba-SP (Capítulo 1 e 2)

91

FIGURA 2. Cárie precoce da infância (Capítulo 1 e 2) 92

FIGURA 3. Forma de coleta de saliva estimulada (capítulos 1 e 2) 93

FIGURA 4. Bochecho com solução de sacarose 20% (Capítulos 1 e 2) 94

FIGURA 5. Material utilizado na coleta de saliva (Capítulos 1 e 2) 95

FIGURA 6. Metodologia de avaliação da capacidade tampão (Capítulos 1 e 2) 95

FIGURA 7. Metodologia de avaliação da atividade do fluxo salivar estimulado

(Capítulos 1 e 2)

96

FIGURA 8. Metodologia de avaliação da atividade da enzima Anidrase Carbônica VI

(Capítulo 1)

97

FIGURA 9. Metodologia de avaliação da atividade da enzima α-amilase salivar

(Capítulo 2)

99

LISTA DE ABREVIATURAS E SIGLAS

CPI Cárie precoce da infância

AC VI Anidrase carbônica VI

AC II Anidrase carbônica II

CPOD Índice de cariados, perdidos e obturados

FSE Fluxo salivar estimulado

CT Capacidade tampão

CA VI Carbonic anhydrase VI

SSFR Stimulated salivary flow rate

CF Caries free group

CL Caries lesions group

AC Arrested caries group

CO2 Gás carbônico

HCO3-

Íon Bicarbonato

WHO+ECL World Health Organization diagnostic

criteria and the early caries lesions

IQR Interquatile range

dmfs+ ECL Decayed, missing and filled surfaces plus

early caries lesions

CA II Carbonic anhydrase II

AC II Anidrase Carbônica II

SAA Salivary α-amylase

GtF B Glucosiltransferase B

SUMÁRIO

1. INTRODUÇÃO 17

2. ARTIGOS 23

2.1 Artigo: Relationship among dental caries and salivary carbonic anhydrase VI

activity, buffer capacity and flow rate – A longitudinal study in children

23

2.2 Artigo: Sucrose increases salivary α-amylase activity in saliva of children- A

cross-sectional study

46

3. DISCUSSÃO 69

4. CONCLUSÃO 77

REFERÊNCIAS 78

APÊNDICE – Produção bibliográfica da aluna 85

ANEXOS 86

Anexo 1 - Certificado do Comitê de Ética em Pesquisa da FOP- UNICAMP 86

Anexo 2 - Autorização da Secretaria Municipal de Saúde de Piracicaba-SP para

realização da pesquisa

87

Anexo 3 - Ficha clínica utilizada na coleta de dados 88

Anexo 4 - Declaração 89

Anexo 5- Confirmação de envio do artigo para publicação – Caries Research 90

17

1 INTRODUÇÃO

A cárie precoce da infância (CPI), uma apresentação agressiva da cárie dental, tem

início com lesões de manchas brancas nas faces vestibulares de incisivos decíduos superiores

ao longo da margem gengival (AAPD, 2008). A doença em crianças é associada a fatores

como, hábitos alimentares inapropriados, alto consumo de carboidratos, medidas de higiene

bucal deficientes e baixo poder socioeconômico (Parisotto et al., 2010). Se não tratada, a

doença pode destruir a dentição decídua, causar dor e desconforto, infecção aguda,

insuficiências nutricionais, problemas de fala e aprendizagem (AAPD, 2008, Parisotto et al.,

2010).

A prevalência da CPI é alta e sua severidade aumenta com a idade. Além disso, uma

pesquisa longitudinal recentemente realizada demonstrou que pré-escolares com CPI

apresentaram risco 17 e 24 vezes maiores de desenvolverem novas lesões de manchas brancas

ativas e de apresentarem lesões de cárie cavitadas, respectivamente (Parisotto et al., 2012).

Levantamentos epidemiológicos evidenciaram também, que no Brasil a doença apresenta-se

como um problema de saúde pública (Ferreira et al., 2007, Moimaz et al., 2016). No último

relatório de saúde bucal, Projeto SB Brasil 2010 (Ministério da Saúde), apenas 46,6% das

crianças brasileiras aos cinco anos de idade apresentou-se livre de cárie na dentição decídua e

43,5% aos 12 anos, já na dentição permanente (Ministério da Saúde, 2010).

A cárie dental é uma doença biofilme-sacarose dependente resultado do desequilíbrio

do biofilme no meio ambiente bucal o que contribue assim para a agregação e metabolismo

bacteriano na superfície dos dentes (Marsh, 2009, Sheiham e James, 2015). Neste aspecto, a

saliva é um fator de proteção fundamental que participa do processo de cárie tanto na dentição

decídua quanto na permanente (Laine et al., 2014). A saliva tem em sua composição vários

mecanismos de defesa, que incluem imunoglobulinas (IgA, IgG e IgM), proteínas aglutinantes

e várias enzimas (lactoferrina, lisozima, e peroxidades) oriundas do plasma e de células

acinares, que interferem no crescimento microbiano (Kivela et al., 1999a, Gao et al., 2016).

Não apenas a composição da saliva, mas também fatores como o fluxo salivar e a

capacidade tampão são extremamente importantes na dinâmica do processo de cárie (Cunha-

Cruz et al., 2013). O fluxo salivar é o parâmetro salivar mais importante neste processo, pois a

atividade cariostática ou eficácia de praticamente todos os outros parâmetros salivares

(capacidade tampão salivar, agentes antimicrobianos) dependem do fluxo salivar (Lagerlof e

Oliveby, 1994, Tenovuo, 1997, Laine et al., 2014).

18

O fluxo salivar normal é um fator altamente protetor contra a cárie, uma vez que

geralmente está associado ao pH e à capacidade tampão salivar elevados, pois provoca um

aumento de todos os componentes salivares. Por outro lado, há uma correlação mais fraca

entre uma baixa capacidade tampão da saliva e o aumento do índice de cárie (Leone e

Oppenheim, 2001). No entanto, já foi demonstrada uma clara relação inversa entre a

capacidade tampão salivar e suscetibilidade à cárie (Ericsson, 1959). Estudos previamente

realizados mostraram que crianças com cárie apresentavam baixos valores de capacidade

tampão (Bhayat et al., 2013, Kuriakose et al., 2013). No entanto, a presença de baixos valores

de capacidade tampão ainda não é considerada fator de risco para a ocorrência da doença cárie

(Gao et al., 2016).

Para evitar que o pH diminua a um nível crítico, a saliva contém mecanismos

tamponantes específicos (Llena-Puy, 2006). A capacidade tampão da saliva envolve três

sistemas tamponantes que são o bicarbonato, o fosfato e as proteínas salivares, de forma que

esses três sistemas trabalham em diferentes intervalos de pH. Enquanto que a atividade

tampão ótima dos sistemas bicarbonato e fosfato ocorre em valores de pKa 6.1-6.3 e 6.8-7.2,

respectivamente, o sistema de proteínas salivares atua de forma efetiva em valores de pKa em

torno de 4,0 (Bardow et al., 2000, Cheaib et al., 2012). No entanto, a concentração destas

macromoléculas na saliva é baixa, e em condições normais, estas, não são muito importantes

como substâncias tampão na saliva (Fejerskov e Kidd, 2007).

O sistema tampão mais importante em condições de estimulação salivar é o sistema

bicarbonato, que é responsável por 70 a 90% da capacidade tampão da saliva total. Baseia-se

no equilíbrio do ↑CO2 + H2O ↔ H2CO3↔ HCO3-

+ H+ onde a concentração de bicarbonato

tende a aumentar com a estimulação do fluxo salivar (Lilienthal, 1955, Izutsu, 1981, Bardow

et al., 2000). Uma característica importante e exclusiva deste sistema é a conversão do gás

carbônico do estado dissolvido para o estado volátil. Quando o ácido é adicionado essa

conversão de estados aumenta a eficácia da neutralização, não havendo acúmulo de produtos

finais, mas a completa remoção de ácido, o que é conhecido como “fase tampão”(Kivela et

al., 1999a). Esta reação na cavidade oral e no trato alimentar alto é catalisada pela enzima

anidrase carbônica VI (AC VI) que está presente na saliva (Kivela et al., 1999a, Kimoto et al.,

2006).

As anidrases carbônicas são metaloenzimas de zinco que participam da manutenção da

homeostase do pH em vários tecidos e fluidos biológicos do corpo humano catalisando a

19

reação de hidratação reversível do dióxido de carbono, CO2 + H2O ↔ HCO3-

+ H+ (Sly e Hu,

1995, Pastorekova et al., 2004). Dentre as 16 isoenzimas isoladas de mamíferos, pelo menos

duas (AC II e AC VI) estão envolvidas na fisiologia salivar, uma vez que expressas nas

glândulas salivares de humanos, participam da regulação do pH no meio bucal (AC VI) e da

secreção de bicarbonato na saliva (AC II) (Kadoya et al., 1987, Parkkila et al., 1990, Supuran

e Scozzafava, 2007). Em humanos, a AC VI é produzida unicamente pelas células acinares

serosas das glândulas parótidas e submandibulares e é secretada na saliva, seguindo o ritmo

circadiano, com baixa concentração durante o sono, aumentando rapidamente ao acordar e

após a primeira refeição (Parkkila et al., 1990, Parkkila et al., 1995). Tal secreção é muito

semelhante à da enzima α-amilase salivar, e uma correlação positiva foi encontrada entre o

nível de atividade de α-amilase salivar e a concentração de AC VI, sugerindo-se que as duas

enzimas poderiam ser secretadas pelos mesmos grânulos e mecanismos secretórios (Parkkila

et al., 1995, Kivela et al., 1999b).

O papel fisiológico da AC VI salivar tem sido esclarecido nos últimos anos (Leinonen

et al., 1999, Kivela et al., 1999b, Kivela et al., 2003, Kimoto et al., 2006, Frasseto et al.,

2012). Pesquisas previamente realizadas demonstraram que a AC VI salivar pode ser

considerada uma proteína anti-cárie na saliva (Leinonen et al., 1999, Kimoto et al., 2006).

Quando da exposição do biofilme à sacarose, ocorre uma queda do pH no intervalo de poucos

minutos, o que pode levar à dissolução do mineral do esmalte. Esse femômeno continua

ocorrendo até que o pH retorne ao valor acima do pH crítico do esmalte (Dawes, 2008). O

mecanismo pelo qual esta isoenzina atua no controle do pH, sugere que a AC VI liga-se a

película de esmalte e facilita a neutralização ácida pelo bicarbonato salivar (Leinonen et al.,

1999). No biofilme dental, a AC VI fica situada em sítios ideais para catalisar a reação

reversível de conversão de bicarbonato salivar e íons de hidrogênio fornecidos por bacterias

cariogênicas, em dióxido de carbono e água (HCO3 + H+ ↔ CO2 + H2O) (Leinonen et al.,

1999, Kimoto et al., 2006). O estudo de Kimoto et al. (2006) evidenciou a presença desta

enzima no biofilme dental, sendo mostrado uma diminuição do pH do biofilme, quando a

cavidade bucal era submetida a um bochecho com solução de acetazolamida, inibidor

específico da enzima AC VI. Esses autores sugerem que pelo mecanismo catalisador exercido

pela enzima, a AC VI seja capaz de prover uma maior neutralização dos ácidos do biofilme

dental.

20

A literatura aponta que ao catalisar o sistema tampão mais importante da cavidade

bucal, o mecanismo de ação de AC VI protege a superfície dental pela neutralização dos

ácidos nesse micro ambiente. Pesquisas nessa área tem indicado ainda resultados

inconclusivos. Algumas têm indicado uma correlação negativa entre a concentração salivar

AC VI e a experiência de cárie (Szabo, 1974, Kivela et al., 1999b). O estudo realizado por

Szabó (1974) mostrou que a saliva de crianças de 7 a 14 anos de idade e livres de cárie,

expressava uma maior concentração da AC VI do que aquela de crianças com cárie.

Posteriormente, Kivela et al. (1999b) mostraram também que baixas concentrações de AC VI

na saliva pareciam estar associadas a um aumento na prevalência de cárie, particularmente em

adultos jovens com a higiene bucal negligenciada. Por outro lado, ao investigar a atividade da

AC VI antes e após um bochecho de sacarose a 20%, Frasseto et al. (2012), observaram que a

variação da atividade da isoenzima foi significativamente maior na saliva de pré-escolares

com cárie quando comparada aqueles livres de cárie. Esses autores observaram também uma

correlação negativa entre a variação da atividade da isoenzima e o índice de cárie.

Encontraram ainda, maior atividade da enzima antes do bochecho no grupo com cárie

(p=0.051). Os resultados de Ozturk et al. (2008) e Yarat et al. (2011), não mostraram

diferença significativa na concentração de AC VI entre grupos com e sem cárie, no entanto,

Ozturk et al. (2008) encontraram uma correlação negativa significativa entre a concentração

de proteínas total e o índice CPOD de adultos jovens, sugerindo a diminuição na concentração

de proteínas protetoras na saliva de indivíduos com cárie.

Resultados contraditórios também são encontrados na literatura. Culp et al. (2013),

encontraram marcada contribuição da deleção do gene que transcreve a AC VI na redução de

cáries em ratos. Por outro lado Li et al. (2015) encontraram a presença significativa do

genótipo polimórfico do gene rs17032907, transcritor de AC VI em indivíduos com

susceptibilidade à cárie. Ainda, a análise da literatura relacionada à AC VI evidencia que, com

exceção da pesquisa realizada por Frasseto et al. (2012) e Aidar et al. (2013) que analisaram a

atividade de AC VI, todas determinaram apenas a concentração da AC VI na saliva ou

biofilme. No entanto, uma alta concentração de AC VI na saliva ou biofilme não

necessariamente significa que toda isoenzima presente nestes meios esteja ativa e assim, possa

exercer o seu efeito. Além disso, não se tem conhecimento de pesquisas longitudinais que

tenham investigado a atividade da AC VI no início e na progressão da cárie dentária em

crianças. Dessa forma, a determinação da atividade de AC VI na saliva pode fornecer

21

evidências adicionais dos efeitos desta isoenzima na dinâmica do processo de cárie no que

concerne o seu início e progressão.

Um dos componentes mais abundantes da saliva é a enzima α-amilase, que é

produzida e secretada pelas células epiteliais acinares das glândulas salivares, principalmente

as glândulas parótidas. A enzima exerce na saliva atividade hidrolítica, responsável pela

quebra inicial de amido em carboidratos de baixo peso molecular, que são substratos

fermentados por várias espécies de bactérias presentes na cavidade bucal (Rogers et al.,

2001). Linhas de evidência apontam para a participação da α-amilase na formação do biofilme

dental, uma vez que esta enzima é um constituinte abundante da película adquirida

(Scannapieco et al., 1989, Douglas, 1990, Scannapieco et al., 1995, Vacca-Smith et al., 1996,

Rogers et al., 1998, Rogers et al., 2001, Hannig et al., 2004). Estes autores sugeriram também

que a enzima pode modular a colonização bacteriana no biofilme, pois atua na película

adquirida como um receptor de alta afinidade para espécies de estreptococos que são

colonizadores iniciais dos tecidos dentais, incluindo S. gordonii, S. mitis, S. parasanguis, S.

crista, S. salivarius e S. sanguis. No biofilme, esta enzima facilita a hidrólise do amido e

forneceria glicose adicional para o metabolismo de microorganismos em estreita proximidade

com a superfície do dente (Scannapieco et al., 1993, Vacca-Smith et al., 1996, Rogers et al.,

2001). Ainda, essa ligação da α-amilase com microorganismos orais em solução, contribui

também para a depuração bacteriana (clearance) da cavidade oral (Scannapieco et al., 1993).

Tem sido demonstrado que a presença de amido aumenta o potencial cariogênico da sacarose

e o biofilme formado a partir dessa combinação exibiria diferenças em sua composição e

estrutura, resultando na síntese de maior quantidade de glucosiltransferase B e polissacarídeos

insolúveis. Isto aumentaria também a aderência de bactérias cariogênicas como S. mutans e

levaria, consequentemente, a maior perda mineral durante os desafios cariogênicos (Ribeiro et

al., 2005, Duarte et al., 2008).

A maioria dos estudos encontrados na literatura investiga a quantidade de proteínas

totais e sua relação com a ocorrência de cárie (Kargul et al., 1994, Dodds et al., 1997,

Tulunoglu et al., 2006, Roa et al., 2008, Preethi et al., 2010). Poucos estudos investigam

especificamente a relação da enzima α-amilase com a ocorrência de cáries, sendo a literatura

ainda inconclusiva (Fiehn et al., 1986, Liang et al., 1999, de Farias e Bezerra, 2003, Bardow

et al., 2005, Vitorino et al., 2006, Shimotoyodome et al., 2007, Bhalla et al., 2010, Kejriwal et

al., 2014, Singh et al., 2015). Os estudos que avaliam a saliva de crianças são escassos na

22

literatura (de Farias e Bezerra, 2003, Bhalla et al., 2010, Grychtol et al., 2015, Singh et al.,

2015). Embora a enzima seja responsável pela quebra do amido, há também na literatura

relato que pode haver um sinergismo entre a atividade de α-amilase e a presença de sacarose,

de forma que a atividade da enzima no biofilme seria maior naquele formado na presença de

sacarose (Dodds e Edgar, 1986). No entanto, não há na literatura estudos em crianças que

tenham avaliado a atividade da enzima imediatamente após um desafio cariogênico

considerando crianças com cárie e livres de cárie.

Além da participação dessa enzima como mediador no processo de cárie, também é

descrito na literatura a possível participação da mesma na capacidade tampão realizada por

proteínas, de modo que a α-amilase seria responsável por 35% da capacidade tampão de

proteínas em faixa de pH de 4 a 5 (Cheaib e Lussi, 2013).

A partir do que foi exposto, torna-se relevante investigar como as enzimas α-amilase e

AC VI se comportariam na saliva de crianças com cáries submetidos a um desafio

cariogênico. Crianças com cárie estão sujeitas a modificações bioquímicas e microbiológicas

importantes na saliva e no biofilme, decorrentes da alta exposição à sacarose, bem como a

composição de proteínas da saliva e taxa de formação e aparência ultraestrutural da película

difere entre dentes decíduos e permanentes (Nobre dos Santos et al., 2002, Parisotto et al.,

2010, Grychtol et al., 2015). O estudo dos componentes salivares individualmente irá guiar o

estudo da influência destes na comunidade microbiana de biofilmes e sua participação no

processo de cárie (Nyvad, 2013). A modificação da atividade destas enzimas, ao ser o meio

bucal exposto à sacarose também deve ser pesquisada em virtude de ser esse o principal

substrato bacteriano, grande causador da cárie dental (Sheiham e James, 2015). Portanto os

objetivos desta tese foram no Capítulo 1, investigar o comportamento da enzima AC VI, fluxo

salivar, capacidade tampão antes e após um desafio cariogênico em crianças com cárie dental

em um estudo longitudinal, e no Capítulo 2, Investigar o comportamento da enzima α-

amilase, fluxo salivar e capacidade tampão antes e após um desafio cariogênico em crianças

com cárie dental em um estudo transversal. Os capítulos serão apresentados em formato

alternativo segundo a Resolução CCPG 001/2015 e encontram-se nas normas de publicação

das revistas Archives of Oral Biology e Caries Research respectivamente.

23

2 ARTIGOS

2.1 Relationship between dental caries and salivary carbonic anhydrase VI activity,

buffer capacity and flow rate – A longitudinal study in children

Artigo submetido ao periódico Archives of Oral Biology (Anexo 5)

Souza TRa, Zancopé BR

a, Parisotto TM

b , Rocha Marques M

c, Nobre-dos-Santos M

d*

a DDS, MS, student of Department of Pediatric Dentistry, Piracicaba Dental School,

University of Campinas, Piracicaba- SP, Brazil

b DDS, MS, PhD of the Laboratory of Microbiology and Molecular Biology, Sao Francisco

University Dental School, Bragança Paulista, SP, Brazil.

c DDS, MS, Professor of Department of Morphology, Piracicaba Dental School, University of

Campinas, Piracicaba- SP, Brazil.

dDDS, MS, PhD, Professor of the Department of Pediatric Dentistry, Piracicaba Dental

School, University of Campinas, Piracicaba- SP, Brazil.

Running Title: Salivary carbonic anhydrase VI and dental caries in children.

*Corresponding Author: Prof. Marinês Nobre dos Santos, Av. Limeira, 901 Zip Code: 13414-

903, Piracicaba-SP, Brazil, email: nobre@fop.unicamp.br, phone number: +55-19-21065290,

Fax:+55-19-21065218

mailto:nobre@fop.unicamp.br

24

Abstract

Objective: To investigate the relationship among dental caries and salivary carbonic

anhydrase VI (CA VI) activity, buffering capacity (BC) and stimulated salivary flow rate

(SSFR) in 48 to 78 month-old children. Design: After dental examination and caries diagnosis

of 47 children, saliva was collected to evaluate SSFR, BC and CA VI activity before and after

a 20% sucrose rinse at baseline and after one year of follow-up. Children were divided into

three groups: caries free children (CF), children presenting caries lesions (CL), and children

with arrested caries (AC). Presence of clinically visible biofilm in the upper incisors was

verified. The activity of CA VI was quantified by zymography. The SSFR was expressed in

mL/min and BC was measured using the Ericsson method. Wilcoxon and Kruskall-Wallis

tests were used for comparisons. The Spearman correlation analysis was used for comparison

between dental caries and independent variables and between BC and CA VI activity (α =

0.05). Results: At baseline, CA VI activity decreased significantly after the cariogenic

challenge in CF children (p=0.037). No change in this parameter was noted for CL group at

baseline and follow-up (p=0.825 and p=0.232, respectively). At follow-up, CA VI activity

decreased significantly only at CF and AC group (p=0.028 and 0.027 respectively). The SSFR

significantly increased after cariogenic challenge in all groups at baseline (p

25

Introduction

Dental caries is a dynamic process caused by acids produced by bacteria inside a

adherent biofilm that causes many cycles of demineralization and remineralization

(Featherstone, 2008). The disease is one of the most common chronic disease of childhood, a

serious public health problem in both developing and industrialized countries (Colak,

Dulgergil, Dalli, & Hamidi, 2013). Among the several factors involved in the multifactorial

etiology of dental caries, dietary sugars were recognized as the major cause of caries process

because they provide a substrate for cariogenic oral bacteria to flourish and to generate

enamel-demineralizing acids (Sheiham & James, 2015).

In the caries dynamic process, saliva is a protective factor against hard tissue loss and

is essential for the maintenance of oral health. Saliva contains inorganic compounds and

multiple proteins that affect conditions in the oral cavity and locally on the tooth surfaces. In

addition, its neutralizing and remineralizing properties are important for healthy tooth

structures (Dawes, 2003). In this regard, factors as the salivary flow rate and the buffering

capacity act as protective in the carious process and have direct influence on the evaluation of

the caries risk (Leone & Oppenheim, 2001; Tenovuo, 1997). To prevent the pH from

decreasing to a critical level, saliva contains specific buffer mechanisms such as bicarbonate,

phosphate and some protein systems, which have a buffering effect that neutralizes acids that

oral cavity is exposed (Fejerskov & Kidd, 2007).

The main buffering system in stimulated saliva is the carbonic acid/bicarbonate buffer

that is based on the equilibrium HCO-3

+ H+ ↔H2CO3↔ CO2 + H2O, and is catalyzed by the

isoenzyme carbonic anyhydrase VI (Breton, 2001). This enzyme is part of a group of

isoenzymes that participate in a variety of physiological processes on the body that involve

pH regulation, CO2 and HCO3-

transport, ion transport, and water and electrolyte balance by

catalyzing the reversible reaction described above (Kivela, Parkkila, Parkkila, Leinonen, &

Rajaniemi, 1999a). CA VI is the only secreted isoenzyme of the CA family. It is secreted into

saliva by serous acinar cells of the human parotid and submandibular glands (Parkkila et al.,

1990). The presence of the enzyme was proved and quantified at the saliva and the presence

of CA VI in the biofilm and its ability to connect to it and keep its activity in this place also

was suggested. In this regard, early investigations suggested that in this site the enzyme

catalyze the conversion of salivary bicarbonate and microbe-delivered hydrogen ions to

26

carbon dioxide and water (Leinonen, Kivela, Parkkila, Parkkila, & Rajaniemi, 1999; Parkkila,

Parkkila, Vierjoki, Stahlberg, & Rajaniemi, 1993). Therefore, by this mechanism, this

enzyme would protect teeth by catalyzing the most important buffer system in the oral cavity,

thus accelerating the removal of acid (H+) from the local microenvironment of the tooth

surface (Kivela, Parkkila, Parkkila, & Rajaniemi, 1999b). Later, it was suggested the role of

CA VI in regulating dental biofilm pH (Kimoto, Kishino, Yura, & Ogawa, 2006).

The participation of CA VI on the caries process is not completely elucidated and

literature shows conflicting results. Most of studies that investigated the role of CA VI on

dental caries just determined the salivary concentration of CA VI, however, a high

concentration of this isoenzyme in saliva does not necessarily mean that all enzyme is active

in the middle (Aidar et al., 2013). Studies show a negative correlation between the CA VI

salivary levels and caries and raised the hypothesis that CA VI present in saliva protected

enamel surfaces from caries. (Kivela et al., 1999b; Szabo, 1974). However, a previous

investigation found no evidence of the relationship between the concentration of the

isoenzyme and dental caries (Ozturk et al., 2008). Later evidence demonstrated that the

activity of CA VI was higher in saliva of preschool children with caries, highlighting the

relevance of the isoenzyme being active in those subject who are frequently expose to

cariogenic challenges (Frasseto et al., 2012). Although some of the previously cited studies

have shown that CA VI isoenzyme is present in saliva, the results of its relationship with

caries are conflicting and needs to be further investigated. Studies evaluating the behavior of

this isoenzyme over time have not been reported in the literature. Thus, the aim of this follow-

up study was to investigate the relationship among dental caries and salivary carbonic

anhydrase VI activity, buffering capacity and stimulated salivary flow rate in 48 to 78 month-

old children.

Materials and methods

Ethical Considerations

This study was approved by the Ethics Committee in Research of Piracicaba Dental

School University of Campinas (UNICAMP) under protocol no. 014/2012. The Secretaria

Municipal de Saúde of Piracicaba city of the State of Sao Paulo selected the two urban

nurseries that could be used on the research. The procedures were explained to the parents of

the subjects involved, and an informed written consent was obtained prior to the investigation.

27

At baseline and follow-up evaluations, children received a kit containing a toothbrush,

fluoride toothpaste (1100 ppm F) and oral hygiene instructions. In addition, children who

needed dental treatment were referred to receive comprehensive dental care at the Pediatric

Dentistry Department of Piracicaba Dental School-University of Campinas.

Subjects

Three hundred children attending public pre-schools in the fluoridated (0.7 ppm F)

urban area of Piracicaba, São Paulo state, were invited to take part in this study. At baseline,

104 children of both genders 53 (50.97 %) girls and 51 (49.03 %) boys of low socioeconomic

level aging 48 to 78 months were allocated for the study. After a one-year of follow-up, 47

children (27 boys and 20 girls), mean age 72.3 months, remained in the cohort (55.8% of

dropout rate) (Fig.1). This occurred because most of children, who were seven years old at

follow-up, moved from their original pre-school and could not be found. After clinical

examination, children were divided into three groups:

Caries free children, CF group (n=10): decayed, missing and filled surfaces

plus early caries lesion=0 (dmft+ ECL), children who were caries-free at the

beginning of the study and remained caries-free after one year;

Children presenting caries lesions, CL group (n=20): dmft+ECL ≥1. Children

who had one or more caries lesions at the beginning of the study and continued

to develop caries after one year;

Children with arrested caries or that had negative caries increment after one

year, AC group (n=17).

Children with and without caries lesions were included in the study. The exclusion

criteria of the study were children with systemic diseases, those who were under antibiotic

therapy or taking medications for central nervous system diseases, children presenting

communication or neuromotor difficulties as well as those with severe fluorosis, dental

hypoplasia, children who refused the procedures or whose parents refused to sign the

informed consent document were also excluded.

28

Calibration of the Examiner, Clinical examination and Caries Assessment

The examination considered all components of the World Health Organization

diagnostic criteria and the early caries lesions (WHO+ECL) (Assaf, de Castro Meneghim,

Zanin, Tengan, & Pereira, 2006). Dental examinations of each child were performed at

baseline and after 1 year from the start of the study by only one examiner (T.R.S.) after

calibration following cross-infection control measures. At first, clinical slides were used to

train the examiner regarding the use of the WHO + ECL criteria. A clinical training session,

using a gold standard for criteria, was held to achieve an acceptable level of agreement before

the intraexaminer reliability assessment. The entire time spent on the calibration process (eg,

theoretical discussions, training, and calibration exercises) was 30 hours. Intraexaminer

reliability (Kappa calculation) regarding all components of the diagnostic criteria was

assessed by re-examination of approximately 10% of children (both at baseline and at follow-

up), with a 1-week-interval period. Kappa values at baseline and follow-up for the tooth

surfaces were 0.82 and 0.80, respectively.

The examination was carried out with a focusable flashlight, a mirror and a ball-ended

probe. Gauze was employed in order to dry or clean teeth, favoring the identification of early

caries lesions. The units of evaluation used in the clinical examinations were d, m, f and s

(decayed, missing and filled surfaces). Findings were recorded by a dental assistant.

Presence of visible biofilm examination

The presence of visible biofilm was observed on buccal surfaces of the four upper

incisors by visual examination (Alaluusua & Malmivirta, 1994) and recorded in the clinical

record as 0 for no visible biofilm and 1 for presence of visible biofilm.

Salivary Flow Rate and Buffering Capacity Determination

To avoid influence of the circadian rhythms, saliva samples were collected in the

morning between 9 and 11 a.m., 2h after eating, drinking or chewing gum. Before sampling,

children were left to relax for 5min. Each allocated children was instructed to chew a piece of

parafilm weighing approximately 0.18g Parafilm® (Sigma Chemical Company, Missouri,

USA) and to deposit whole saliva in a Falcon® tube (BD Biosciences, California, USA) for 5

min as previously described (Dawes & Kubieniec, 2004). If the secretion rate was low, the

29

collection was continued further for a maximum of 10 min and saliva was deposited in a

sterile graduated ice-cooled container to prevent sample warming (Kirstila, Hakkinen,

Jentsch, Vilja, & Tenovuo, 1998). All subjects were instructed to swallow at time zero. SSFR

was calculated by measuring the total volume of saliva and dividing it by the collection time,

and was expressed as mL/min (Ericsson & Hardwick, 1978). After the first saliva collection, a

second collection of stimulated saliva was performed 5 minutes after a rinse with 5 ml of a

20% sucrose solution for 1 min (Frasseto et al, 2012). This procedure was performed to

determine the effects that exposure of the oral environment to a cariogenic challenge would

have on the salivary flow and buffering capacity, as well as on the activity of the CA VI

isoenzyme.

After collection, saliva samples were immediately transported to the laboratory in a

box containing ice sealed with plastic film to prevent the carbon dioxide elimination. BC of

saliva was determined by Ericsson method (Ericsson, 1959). Thus, 0.5 mL of saliva was

placed in a tube with 1.5 mL of HCl (0.005 mol/L), the tube was shaken mixed for 30 seconds

using a vortex (AP 56, Phoenix) and a waiting period of 20 min was adopted for carbon

dioxide elimination and the solution pH was measured. Buffering capacity was assessed using

an electronic pH meter (Orion Analyzer Model 420A, USA).

After calculating the SSFR, and BC, saliva samples were centrifuged at 5.000 rpm for

10 min at 4oC, and stored in 2.0 mL microtubes, and were frozen at –40°C for later

determination of CA VI activity.

Quantification of CA VI Activity in Saliva

The determination of CA VI activity was performed by the zymography method using

a modified protocol (Aidar et al., 2013; Kotwica et al., 2006). After being thawed, 100 μL of

saliva was added to 100 μL of Tris buffer. The solution was stirred before being placed on

acrylamide gel at 30% and bisacrylamide at 0.8%. After that, 10 μL of this sample was placed

in each channel of the gel, which remained for 1h: 50min at 140 V and at 4°C. After

electrophoresis, the gel was stained with 0.1% bromothymol blue for 10 min. CA VI activity

was observed after immersing the gel in distilled deionized water saturated with CO2. The

gels were photographed, and images were quantified using the Image J software (Collins,

2007) was used to calculated the luminescence in area of the band, which expressed CA VI

activity in numerical values (pixels/area).

30

Statistical analysis

The dependent variable was dental caries. The independent variables were: SSFR, BC

and CA VI activity, before and after a sucrose rinse as well as presence of biofilm visible at

the upper incisors. Data normality was checked using the Shapiro-Wilk test. Descriptive

analysis by inferential statistics was performed and percentages, medians and interquatile

ranges (IQR) were calculated for quantitative data of each independent variable before and

after a 20% sucrose solution rinse (SSFR, BC and CAVI activity). Comparisons inside each

group at baseline and follow-up before and after sucrose rinse were performed using the

Wilcoxon test. Comparisons between the three groups were done using the Kruskal-Wallis

test. Association between biofilm presence and dental caries at baseline and follow-up was

determined using Fisher test. The Spearman correlation coefficient was calculated between

SSFR, BC and CAVI activity and caries index at baseline and follow-up, also between BC

and CA VI activity. We considered the 5% level of significance. Data were analyzed using the

Statistical Package for Social Science 13.0 (SPSS Inc., IL, USA).

Results

The means and standard deviations of numbers of surfaces affected by caries at

baseline and at follow-up in the studied population was 4.2 ± 4.8 and 5.3 ± 6.2 respectively

(p=0.005, Wilcoxon test). The 1-year caries increment was 1.1 ± 2.5. In the CL group and AC

group the mean numbers at baseline and follow-up were 6.5 ± 5.6 and 9.9 ± 6.5 / 3.94 ± 3.3

and 3.0 ± 3.2 respectively (p

31

noticed the same decreases in BC after sucrose rinse (p= 0.047, p= 0.003, p=0.001, p

32

to a cariogenic challenge whether children were caries-free, had only arrested caries or had

caries.

At baseline and follow-up, the CF group as well as the AC group at the follow-up

showed significant decreases in the CA VI activity after sucrose rinse. These results were

expected and can partially be explained if we consider that caries-free children as well as

those having arrested caries are less frequently submitted to pH drops as a consequence of low

acid production after the cariogenic challenge which decreased enzyme activity, since the acid

is also a substrate for the reaction. Carbonic anhydrase VI catalyzes the reaction of HCO-3

+

H+ ↔H2CO3↔ CO2 + H2O in both directions and it is possible that it may neutralize the

media (Leinonen et al., 1999). This result is in accordance with the negative correlation

between the BC before rinse and activity CA VI after rinse and at the baseline and the supply

of H+ ions as a substrate for the reaction catalyzed by CA VI. On the other hand, early

investigations found no association between salivary pH, BC and CA VI concentration in

saliva (Kivela et al., 1997; Parkkila et al., 1993). However, it is important to notice that it is

known the CA VI isoenzyme catalyzes the reaction that balances pH in the oral cavity after a

cariogenic challenge, so the results of this study was expected.

Another result of this study was that at the two periods of study, the CL group (dmft >

0) showed no change in CA VI activity after sucrose rinse. Children having caries are

frequently exposed to high daily sugar consumption and it is known that this sugar

consumption pattern is significantly correlated with early childhood caries (Nobre dos Santos,

Melo dos Santos, Francisco, & Cury, 2002; Parisotto et al., 2010). In the presence of a sugar-

rich diet and a greater acid formation by metabolism of dental biofilm microbiota in this

group, there is a possibility that salivary CA VI activity remained unchanged after the sucrose

rinse to provide a higher protection against dental caries. The suggested mechanism would be

that that in these individuals salivary CA VI would neutralize greater amounts of acid mainly

in the form of latic, acetic, formic and propionic produced by the microbial metabolism in the

mouth and over dental surfaces. This acid neutralization would be accomplished via

conversion of salivary bicarbonate and microbe-delivered hydrogen ions to carbon dioxide

and water catalyzed by salivary CA VI (Leinonen et al., 1999). In line with this assumption,

in this group, we found a moderate negative correlation between CA VI activity before as

well as after sucrose rinse and dental caries (Table 4). A further explanation for this finding,

could be that if the CO2 + H2O ↔ H+ + HCO

3– reaction is fueled by HCO

3- provided by

33

salivary CA II supply and H+ delivery by the microbial metabolism of carbohydrates, the

reaction would work in a reverse way by neutralizing the salivary pH and this fact may have a

role in children with caries. And this was confirmed by the essential feature of this buffer

system under the conditions prevailing in the oral cavity is the phase conversion of carbon

dioxide from a dissolved state into a volatile gas (Kivela et al., 1999a). In the other side, in

caries-free subjects and in those who had arrested caries at baseline and at follow-up, after the

cariogenic challenge, there was a significant reduction in the CA VI activity probably as

consequence of a low acid production of in the oral environment since these individuals are

less frequently exposed to cariogenic carbohydrates and consequently, to regular pH falls in

saliva and dental biofilm (Nobre dos Santos et al., 2002, Parisotto et al., 2010). In this way,

acid buffering in the oral environment provided by CA VI activity would not be so necessary

in these individuals. In this regard, previous investigations suggested that the isoenzyme

participates not only in preventing caries development by always maintaining the pH of oral

cavity at a level higher than the critical one, but also appears to be active during the

occurrence of a cariogenic challenge in individuals with the disease already installed.

(Frasseto et al., 2012; Leinonen et al., 1999).

Our study, did not notice any difference in the results of CA VI activity among groups

neither before nor after the cariogenic challenge. Regarding before rinse data our results are in

accordance with Ozturk et al. (2008) who also did not find any difference in CA VI

concentration between caries and caries-free young adults. However, different findings were

obtained by Frasseto et al. (2012). These authors found a higher CA VI activity in the caries

group than in the caries-free group before sucrose rinse (p=0.0516). Concerning CA VI

activity after sucrose rinse, our results are in line with Frasseto et al. (2012).

Another result of this study was that at baseline and at follow-up, there was no

difference among groups concerning the variation of CAVI activity. Our data are not in line

with the results found by Frasseto et al. (2012), who detected that variation of CA VI activity

was significantly higher in the CL group than in the CF group. A possible explanation for

these findings could be the large inter-individuals variation of CA VI concentration and

activity as pointed out in several studies (Frasseto et al., 2012; Kivela, Laine, Parkkila, &

Rajaniemi, 2003; Parkkila, Parkkila, & Rajaniemi, 1995).

Based on the findings regarding the CA VI behavior in the oral environment, the

isoenzyme should not be interpreted as a factor that favors the decay process, but as protective

34

salivary protein acting in an attempt neutralize the pH of acid produced as previously

demonstrated by Kimoto et al. (2006). This mechanism would be most important especially in

subjects having caries to whom the enzyme would be more active after a cariogenic challenge,

as a catalyst agent in the buffering reaction of bicarbonate in saliva. The findings of this study

suggest that the CA VI behavior did not change with pH drop in the oral cavity at CL group.

In line with this thought, the recent data of a genetic study that suggest that salivary CA VI

plays an important role in protecting teeth from caries (Li, Hu, Zhou, Xie, & Zhang, 2015).

The results of the present study also showed a moderate negative correlation between

CA VI activity before rinse and dental caries at the baseline as well as at follow-up in the CL

group (Table 5). There is a possibility that in these subjects the higher enzyme activity would

act better to control oral pH under normal conditions before and after the cariogenic

challenge, in the oral cavity. These results are in agreement with Kivela et al. (1999b), who

claimed that this correlation with CA VI concentration was most significant in subjects with

poor oral hygiene. In line with this assumption, our results showed a significant association

between dental caries and biofilm presence. In the other side, our results differed from those

obtained by Ozturk et al. (2008) and Frasetto et al., (2012) who did not find any correlation

between dental caries and CA VI concentration and activity respectively.

Saliva is believed to be one of the most important host factors and an essential

mediator controlling the speed and direction of the cariogenic pathway (Gao, Jiang, Koh, &

Hsu, 2016). Our study also showed that at baseline the SSFR increased significantly after

sucrose rinse in the three groups. The results are in line Frasseto et al. (2012). However, at

follow-up this change was noted only in the AC group. These findings can be explained if we

consider the mechanical and gustatory stimulation promoted by rinse and the stimulus of the

salivary glands provided by sucrose (Proctor, 2016). These results also are in accordance with

found by Dawes & Kubieniec (2004). We did not found any difference among groups

regarding SSFR at baseline and follow-up and any correlation between caries and SSFR at

baseline or at follow-up. In line with this assumption, previous studies have shown that in

individuals with normal salivary flow rates, the relationship between salivary flow and caries

has little or no predictive value for the occurrence of disease (Lenander-Lumikari &

Loimaranta, 2000).

Salivary pH and buffering capacity are known to be central factors protecting teeth

from caries and could be considered a moderate risk factor for its prevalence and incidence

35

(Gao et al., 2016; Kivela et al., 1999b). Concerning BC, we noticed a significant decrease in

all groups at baseline and follow-up after sucrose rinse. This result is in line with those

obtained by Frasseto et al. (2012) for biofilm pH after sucrose rinse in caries and caries-free

children. Moreover, we also found a significant negative correlation between BC after sucrose

rinse and dental caries at baseline as well as at follow-up. For baseline data, similar results

were found by Kivella et al. (1999b) and are in line with previous studies (Kuriakose,

Sundaresan, Mathai, Khosla, & Gaffoor, 2013; Ruiz Miravet, Montiel Company, & Almerich

Silla, 2007; Singh et al., 2015; Yildiz, Ermis, Calapoglu, Celik, & Turel, 2016). However,

these authors did not perform sucrose rinse in their investigation. We did not find any

difference among groups concerning BC. For baseline data, these results are in agreement

with previous investigations (Peres et al., 2010; Yarat et al., 2011).

In summary, this study suggests that the enzyme CA VI provides a protective role

when the oral cavity environment is submitted to cariogenic challenge. In addition, a low CA

VI activity showed to correlate with caries prevalence before cariogenic challenge mainly in

caries children. Our findings demonstrate the importance of this enzyme as a participant of the

mouth physiology in controlling saliva after cariogenic challenges. In conclusion, this study

demonstrated that CA VI isoenzyme remains active in saliva of children with caries after

cariogenic challenge with sucrose and suggests the participation of CA VI on the BC of

saliva.

Funding

The study was supported by FAPESP (2012/02516-1 and 2012/15834-1).

Competing Interests

The authors reported no conflict of interest. The authors alone were responsible for the

content and the writing of the paper.

Ethical Approval

The protocol was approved by the local Bioethics Committee of Piracicaba

Dental School, University of Campinas, Piracicaba, SP, Brazil (Protocols #014/2012).

36

Acknowledgements

This paper was based on a thesis submitted by the first author to Piracicaba Dental

School, University of Campinas, in partial fulfillment of the requirements for a DDS degree in

Dentistry (Pediatric Dentistry area). This study was supported by FAPESP (2012/02516-1 and

2012/15834-1). We thank the Secretary of Education and Health of Piracicaba-SP/Brazil for

collaborating with this research. We specially thank the volunteers and their parents for

participating in this research.

37

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41

Figure captions

Fig. 1. Subjects allocation and disposition. * The division of groups was done after the

follow-up period of study. The comparisons at the baseline were done with the disposition of

groups adopted at the end of the study to all comparisons.

Fig. 2. Stimulated salivary flow rate before (BR) and after rinse (AR) at the baseline (T0) and

follow-up (T1) in caries free, arrestment caries and caries group.

42

Fig. 3. Buffer capacity before (BR) and after rinse (AR) at the baseline (T0) and follow-up

(T1) in caries free, arrestment caries and caries group.

43

Tables

Table 1. Medians and interquartile ranges (IQR) of CA VI activity before and after a 20%

sucrose solution rinse and its variation (Δ) at baseline.

Groups Before rinse After rinse p value* Δ CA VI

All children (n=47) 0.69 (0.73) 0.40 (0.50) 0.015 -0.05 (0.39)

CF (n=10) 0.89 (0.58) 0.50 (0.50) 0.037 -0.11 (0.43)

CL (n=20) 0.51 (0.74) 0.44 (0.47) 0.825 0.01(0.28)

AC (n=17) 0.71 (0.68) 0.29 (0.44) 0.076 -0.19 (0.51)

p** 0.25 0.81 0.252

CF: caries free group. CL: caries lesion group. AC: arrestment caries group. Δ CA VI: variation of CA VI activity, difference

between CAVI activity after rinse and before rinse at the baseline and follow-up. IQR: Interquatile range. p values derived

from Wilcoxon* and Kruskal-Wallis** test.

Table 2. Medians and interquartile ranges (IQR) of CA VI activity before and after a 20%

sucrose solution rinse and its variation (Δ) at follow-up.

Groups Before rinse After rinse p value* Δ CA VI

All children (n=47) 0.42 (0.69) 0.26 (0.36) 0.03 -0.09 (0.42)

CF (n=10) 0.28 (0.34) 0.24 (0.32) 0.028 -0.11 (0.2)

CL (n=20) 0.36 (0.8) 0.38 (0.49) 0.232 -0.07 (0.31)

AC (n=17) 0.46 (0.68) 0.23 (0.36) 0.027 -0.16 (0.5)

p** 0.98 0.532 0.715

CF: caries free group. CL: caries lesion group. AC: arrestment caries group. Δ CA VI: variation of CA VI activity, difference

between CAVI activity after rinse and before rinse at the baseline and follow-up. IQR: Interquatile range. p values derived

from Wilcoxon* and Kruskal-Wallis** test.

44

Table 3. Spearman correlation coefficients (r) and probabilities of statistical significance (p)

between dental caries and independent variables.

Variables Dental Caries

Baseline Follow-up

r p value r p value

SSFR BR baseline -0.009 0.951 -0.082 0.585

SSFR AR baseline -0.054 0.717 -0.111 0.459

SSFR BR Follow-up -0.110 0.461 -0.213 0.151

SSFR AR Follow-up -0.107 0.475 -0.223 0.132

BC BR baseline -0.055 0.712 -0.152 0.306

BC AR baseline -0.161 0.281 -0.239 0.105

BC BR Follow-up -0.183 0.218 -0.198 0.183

BC AR Follow-up -0.345 0.017 -0.303 0.038

CAVI BR baseline -0.305 0.037 -0.286 0.051

CAVI AR baseline -0.201 0.175 -0.106 0.478

CAVI BR Follow-up 0.77 0.605 0.075 0.614

CAVI AR Follow-up 0.169 0.256 0.137 0.359

Δ CAVI baseline 0.164 0.272 0.268 0.068

Δ CAVI Follow-up 0.079 0.597 0.056 0.707

SSFR: stimulated salivary flow rate. BC: buffer capacity. CAVI: carbonic anhydrase activity. Δ CAVI: variation of CAVI

activity. BR: before rinse. AR: after rinse.

Table 4. Spearman correlation coefficients (r) and probabilities of statistical significance (p)

between dental caries and CA VI activity in the caries lesion group.

Variables Dental caries

Baseline Follow-up

r p value r p value

CA VI / Before rinse -0.609 0.004 -0.545 0.013

CA VI / After rinse -0.516 0.020 -0.382 0.096

CAVI: carbonic anhydrase VI activity.

45

Table 5. Spearman correlation coefficients (r) and probabilities of statistical significance (p)

between means of BC at baseline and CA VI activity before and after rinse at baseline and

follow-up.

Correlation analysis variable

Baseline Follow-up

r p value r p value

BC BR x CA VI BR -0.112 0.453 -0.366 0.011

BC BR x CAVI AR -0.397 0.006 -0.089 0.553

BC AR x CA VI BR -0.043 0.774 -0.378 0.009

BC AR x CAVI AR -0.095 0.527 -0.110 0.462

BC: buffer capacity. CAVI: Carbonic anhydrase VI activity. BR: Before rinse. AR: After rinse.

46

2.2 Sucrose increases salivary α-amylase activity in saliva of children: a cross-sectional

study

Artigo submetido ao periódico Caries Research (Anexo 6)

Souza TR1, Rodrigues LP

1, Parisotto TM

2, Nobre-dos-Santos M

3*

1 DDS, MS, student of Department of Pediatric Dentistry, Piracicaba Dental School,

University of Campinas, Piracicaba- SP, Brazil

2 DDS, MS, PhD of the Department of Pediatric Dentistry, Piracicaba Dental

School, University of Campinas, Piracicaba-SP, Brazil

3DDS, MS, PhD, professor of the Department of Pediatric Dentistry, Piracicaba Dental

School, University of Campinas, Piracicaba- SP, Brazil.

Short Title: Salivary amylase activity and dental caries

Corresponding Author: Prof. Marinês Nobre dos Santos, Av. Limeira, 901 Zip Code: 13414-

903, Piracicaba-SP, Brazil, email: nobre@fop.unicamp.br, phone number: +55-19-21065290,

Fax:+55-19-21065218

mailto:nobre@fop.unicamp.br

47

Declaration of Interests

The authors deny any conflicts of interest related to this study.

__________________________

Marinês Nobre dos Santos

48

Abstract

Objective: To investigate the influence of a cariogenic challenge on the salivary amylase

activity (SAA) and the relationship among dental caries, SAA, stimulated salivary flow rate

(SSFR) and buffering capacity (BC) in children. Subjects and Methods: After dental

examination and caries diagnosis 38 children aging 48 to 77 months-old were divided into

two groups: caries free group (CF, n=18) and caries lesion group (CL, n=20). Saliva samples

were collected before and after a 20% sucrose mouth rinse. The activity of SAA was

quantified by enzyme kinetic assay. The SSFR was expressed in mL/ min. The BC was

electronically measured with a pH meter. Wilcoxon and Mann Whitney tests were applied for

comparisons of SSFR and BC data. Independent T test and paired T test were used for SAA

data. Correlations between caries and independent variables were performed using the

Spearman correlation analysis. Results: After sucrose rinse, SSFR significantly increased

(p=0.03 for CF and p=0.038 for CL) and BC significantly decreased (p= 0.009 for CF and

p=0.005 for CL) in both groups. CL group exhibited a significant increase in SAA activity

after sucrose rinse (p=0.024). In this group, after sucrose rinse, SAA activity was significantly

higher than in CF group (p=0.019). We found a positive correlation between caries and SAA

(r= 0.317, p= 0.052). Conclusion: These results suggest that a cariogenic challenge with

sucrose increases the SAA activity in saliva of children having caries.

Key Words: alpha-amylase, children, caries, saliva

49

Introduction

Scientific evidence suggested that dental caries is a biofilm-sugar-dependent disease

[Sheiham and James, 2015], but other factors are involved on its development such as dietary

habits, microorganisms count, oral hygiene and socioeconomic factors [Chaffee et al., 2015;

Parisotto et al., 2015]. Moreover, the protective functions of saliva, like clearance promoted

by salivary flow and pH stability, mainly due to bicarbonate and phosphate buffer systems as

well as the salivary proteins [Dodds et al., 2005] are considered important factors in

modulating the caries process development [Fejerskov and Kidd, 2007].

Among salivary proteins, α-amylase (46-60 kDa) is one of the most plentiful

components in human saliva. It is mainly secreted by the parotid-gland and accounts for 10–

20% of the total protein content [Arhakis et al., 2013]. This enzyme has a biological function

of hydrolytic activity and it is responsible for the initial break down of starch to low

molecular fermentable carbohydrates, such as glucose and maltose, which are fermentable

substrates for many oral bacterial species like S. mutans, the major pathogen of dental caries,

non-mutans streptococci and Actinomyces [Rogers et al., 2001]. Furthermore, early

investigation suggested a possible participation of α-amylase on the protein buffering capacity

with a positive correlation between salivary protein buffering capacity and the amylase

concentration [Cheaib and Lussi, 2013].

The presence of dietary sugars as a fundamental causes of dental caries not only in

children but for all life should be considered [Sheiham and James, 2015]. Fermentable

carbohydrates such mainly as sucrose it also serves as a substrate for the synthesis of

extracellular and intracellular polysaccharides in dental plaque and are considered caries

predictors [Paes Leme et al., 2006; Parisotto et al., 2010]. The combination of sucrose and

starch produces biofilms with more biomass and acidogenicity, and a higher content of water-

insoluble polysaccharides and highest mineral loss and lactobacillus count [Ribeiro et al.,

2005; Duarte et al., 2008]. Also it was demonstrated that the presence of starch hydrolysates

increases the glucan production by GtF B in vitro [Vacca-Smith et al., 1996]. Several lines of

evidence indicated that since the SAA is an abundant constituent of the acquired enamel

pellicle it may modulate bacterial colonization by binding to hydroxyapatite and acting as an

adherence receptor for amylase binding bacteria to the tooth surface. Moreover, this protein

binds with high affinity to a number of the oral streptococci that are early colonizers of the

tooth, including Streptococcus gordonii, S. mitis, S. parasanguis, S. crista and S. salivarius. In

50

solution this binding also contributes to bacterial clearance from oral cavity [Douglas, 1983;

Rogers et al., 2001].

Studies indicate that particularly S. gordonii has in its surface amylase-binding protein

(20-kDa protein) coding by an amylase-binding protein A gene and amylase may also serve as

an adherence receptor with high-affinity for these amylase-binding bacteria [Rogers et al.,

2001]. Bound to bacteria in biofilm, α-amylase may facilitate dietary starch hydrolysis to

provide additional glucose and maltose for metabolism by plaque microorganisms in close

proximity to the tooth surface. This causes local pH to fall below a critical value resulting in

demineralization of tooth tissues [Rogers et al., 2001]. In this way, in presence of α-amylase

the pH fall produced by cultures of streptococci in vitro incubated with cooked starch is

around 3.9 to 4.4, values that were closer to that observed for the metabolism of glucose,

sucrose and maltose for the same bacteria, which suggests that cooked starch is potentially

more acidogenic in presence of the enzyme [Aizawa et al., 2009]. Moreover, previous work

showed that α-amylase activity was higher in dental plaque of adults subjects on a high

sucrose supplemented diet than of subjects on the low one [Dodds and Edgar, 1986]. In

addition, starch fermentation may be enhanced by exposure of plaque to sucrose. This may be

explained by a synergistic effect between starch and sucrose or the fact that sweetened starch

is more retentive than sucrose alone. Thus, a synergistic effect between sucrose and starch

may be due to the enhanced fermentation of starch by plaque-bound α-amylase with a

subsequent increase in caries activity [Scannapieco et al., 1993].

Regarding the relationship between salivary α-amylase and dental caries, the activity

of this protein was demonstrated to be higher in saliva of 4 and 8 years old children with

active caries as compared with the caries-free one [Singh et al., 2015]. In the same way, was

found that parotid saliva samples of caries rampant group had a significantly higher level of

α-amylase than saliva of the caries-resistant children [Balekjian et al., 1975]. In addition,

higher concentrations of α-amylase were detected in saliva of caries susceptible young adults

[Vitorino et al., 2006]. In another side, other authors did not find any difference in levels of

the enzyme in comparison between caries free and caries subjects [de Farias and Bezerra,

2003; Shimotoyodome et al., 2007].

In face of the controversial findings published in the literature, regarding the

relationship between α-amylase and caries, the role of salivary α-amylase in children should

be further investigated [de Farias and Bezerra, 2003]. Furthermore, studies investigating t