RELAÇÃO ENTRE A ATIVIDADE DA ANIDRASE CARBÔNICA...
100
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
Transcript of RELAÇÃO ENTRE A ATIVIDADE DA ANIDRASE CARBÔNICA...
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: [email protected], phone number: +55-19-21065290,
Fax:+55-19-21065218
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<0.05). At the
follow-up SSFR was higher only at the AC group (p=0.001). BC decreased in all groups after
cariogenic challenge at the baseline and follow-up (p<0.05) and values before rinse at
baseline were negatively correlated to CA VI activity after rinse. At baseline, we found a
moderate negative correlation between the salivary CA VI activity before and after sucrose
rinse and dental caries in CL group (p=0.004 and 0.020 respectively). At follow-up, the same
trend was noted only before sucrose rinse (p=0.013). Conclusion: In summary, this study
demonstrated the participation of CA VI on the BC of saliva and suggest that children who
had caries at baseline and continued to develop caries CA VI activity remains active after
cariogenic challenge as a protection mechanism.
Key Words: carbonic anhydrase VI, children, caries
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<0.005, Wilcoxon test). We also found a positive association
between biofilm presence and caries at baseline and follow-up (p=0.037 and p=0.066
respectively for Fisher test).
Data of figure 2 demonstrate that at baseline after sucrose rinse, the SSFR significantly
increased in the three investigated groups (p = 0.030; p=0.024 and p=0.002 for the CF group,
CL group and AC group respectively). The same trend of result was found for the whole
sample (p <0.001). At follow-up, results showed that the SSFR significantly increased only
for the whole sample as well as for the AC group (p= 0.010 and p= 0.001, respectively).
Data of figure 3 demonstrate that at the baseline in all groups as well as in the whole
sample, BC significantly decreased after sucrose rinse (p= 0.005, p= 0.001, p= 0.005 and
p<0.001 for CF, CL, AC group and for the whole sample, respectively. At follow-up, we
31
noticed the same decreases in BC after sucrose rinse (p= 0.047, p= 0.003, p=0.001, p<0.001
respectively for the same).
Table 1 shows that at baseline, a significant decrease in CA VI activity occurred after
sucrose rinse for the whole sample as well as for the CF group (p= 0.015 and p= 0.037
respectively). On the other side, at this time no change in this parameter was noted for the CL
group (p= 0.825). At follow-up, after sucrose rinse, the CA VI activity was significantly lower
for the whole sample, as well as for the CF and AC groups (p=0.03, p=0.028 and p=0.027
respectively). However, no change in CA VI activity was found for the CL group (p=0.232)
(Table 2). We could not find any difference among groups related to CA VI activity and its
variation at baseline and follow-up (Table 1 and 2).
Correlations between dental caries and variable characteristics at baseline and follow-
up are shown in Table 3. The results demonstrate that at baseline, variables that showed a
significant negative correlations with dental caries were BC after rinse (r = -0.345 and p =
0.017) and CA VI activity before rinse (r = -0.305 and p = 0.037). At follow-up, BC after
sucrose rinse was the only variable that showed a negative correlation with caries. (r = -0.308
and p = 0.038).
Table 4 shows that for the CL group, there is a significant negative moderate
correlation between dental caries and CA VI activity before and after sucrose rinse at baseline
(r = -0.609 p = 0.004 and r=-0.516 p=0.020 respectively). At follow-up a significant negative
correlation between dental caries and CA VI activity before sucrose rinse was also found (r =
-0.545 p = 0.013).
Table 5 reveals the correlations between BC and CA VI activity before and after
sucrose rinse at baseline and follow-up. A negative correlation between BC before sucrose
rinse and CA VI activity before sucrose rinse was detected at follow-up (r=-0.366, p=0.011)
but not at baseline. This table also reveals a negative correlation between BC after sucrose
rinse and CA VI activity before sucrose rinse only at follow-up (r=-0.378, p=0.009).
Discussion
Our study investigated for the first the time behavior of CA VI activity in whole
saliva of children before and 5 minutes after a cariogenic challenge in caries-free children, as
well as in those with caries and with arrested caries after one year of follow-up. The results of
the present study showed that the CA VI activity exhibits a different behavior when submitted
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
Referências Bibliográficas
Aidar, M., Marques, R., Valjakka, J., Mononen, N., Lehtimaki, T., Parkkila, S., & Line, S. R.
(2013). Effect of genetic polymorphisms in CA6 gene on the expression and catalytic
activity of human salivary carbonic anhydrase VI. Caries Res, 47(5), 414-420.
Alaluusua, S., & Malmivirta, R. (1994). Early plaque accumulation--a sign for caries risk in
young children. Community Dent Oral Epidemiol, 22(5 Pt 1), 273-276.
Assaf, A. V., de Castro Meneghim, M., Zanin, L., Tengan, C., & Pereira, A. C. (2006). Effect
of different diagnostic thresholds on dental caries calibration - a 12 month evaluation.
Community Dent Oral Epidemiol, 34(3), 213-219.
Breton, S. (2001). The cellular physiology of carbonic anhydrases. JOP, 2(4 Suppl), 159-164.
Colak, H., Dulgergil, C. T., Dalli, M., & Hamidi, M. M. (2013). Early childhood caries
update: A review of causes, diagnoses, and treatments. J Nat Sci Biol Med, 4(1), 29-
38.
Collins, T. J. (2007). ImageJ for microscopy. Biotechniques Physiologist, 43, 25-30.
Dawes, C. (2003). What is the critical pH and why does a tooth dissolve in acid? J Can Dent
Assoc, 69(11), 722-724.
Dawes, C., & Kubieniec, K. (2004). The effects of prolonged gum chewing on salivary flow
rate and composition. Arch Oral Biol, 49(8), 665-669.
Ericsson, Y. (1959). Clinical investigations of the salivary buffering action. Acta Odontol
Scand, 17, 131–165.
Ericsson, Y., & Hardwick, L. (1978). Individual diagnosis, prognosis and counselling for
caries prevention. Caries Res, 12 Suppl 1, 94-102.
Featherstone, J. D. (2008). Dental caries: a dynamic disease process. Aust Dent J, 53(3), 286-
291.
Fejerskov, O., & Kidd, E. (2007). Dental caries - The disease and its clinical management.
São Paulo: Santos.
Frasseto, F., Parisotto, T. M., Peres, R. C., Marques, M. R., Line, S. R., & Nobre Dos Santos,
M. (2012). Relationship among salivary carbonic anhydrase VI activity and flow rate,
biofilm pH and caries in primary dentition. Caries Res, 46(3), 194-200.
Gao, X., Jiang, S., Koh, D., & Hsu, C. Y. (2016). Salivary biomarkers for dental caries.
Periodontol 2000, 70(1), 128-141.
38
Kimoto, M., Kishino, M., Yura, Y., & Ogawa, Y. (2006). A role of salivary carbonic
anhydrase VI in dental plaque. Arch Oral Biol, 51(2), 117-122.
Kirstila, V., Hakkinen, P., Jentsch, H., Vilja, P., & Tenovuo, J. (1998). Longitudinal analysis
of the association of human salivary antimicrobial agents with caries increment and
cariogenic micro-organisms: a two-year cohort study. J Dent Res, 77(1), 73-80.
Kivela, J., Laine, M., Parkkila, S., & Rajaniemi, H. (2003). Salivary carbonic anhydrase VI
and its relation to salivary flow rate and buffer capacity in pregnant and non-pregnant
women. Arch Oral Biol, 48(8), 547-551.
Kivela, J., Parkkila, S., Metteri, J., Parkkila, A. K., Toivanen, A., & Rajaniemi, H. (1997).
Salivary carbonic anhydrase VI concentration and its relation to basic characteristics
of saliva in young men. Acta Physiol Scand, 161(2), 221-225.
Kivela, J., Parkkila, S., Parkkila, A. K., Leinonen, J., & Rajaniemi, H. (1999a). Salivary
carbonic anhydrase isoenzyme VI. J Physiol, 520 Pt 2, 315-320.
Kivela, J., Parkkila, S., Parkkila, A. K., & Rajaniemi, H. (1999b). A low concentration of
carbonic anhydrase isoenzyme VI in whole saliva is associated with caries prevalence.
Caries Res, 33(3), 178-184.
Kotwica, J., Ciuk, M. A., Joachimiak, E., Rowinski, S., Cymborowski, B., & Bebas, P.
(2006). Carbonic anhydrase activity in the vas deferens of the cotton leafworm -
Spodoptera littoralis (Lepidoptera: Noctuidae) controlled by circadian clock. J Physiol
Pharmacol, 57 Suppl 8, 107-123.
Kuriakose, S., Sundaresan, C., Mathai, V., Khosla, E., & Gaffoor, F. M. (2013). A
comparative study of salivary buffering capacity, flow rate, resting pH, and salivary
Immunoglobulin A in children with rampant caries and caries-resistant children. J
Indian Soc Pedod Prev Dent, 31(2), 69-73.
Leinonen, J., Kivela, J., Parkkila, S., Parkkila, A. K., & Rajaniemi, H. (1999). Salivary
carbonic anhydrase isoenzyme VI is located in the human enamel pellicle. Caries Res,
33(3), 185-190.
Lenander-Lumikari, M., & Loimaranta, V. (2000). Saliva and dental caries. Adv Dent Res, 14,
40-47.
Leone, C. W., & Oppenheim, F. G. (2001). Physical and chemical aspects of saliva as
indicators of risk for dental caries in humans. J Dent Educ, 65(10), 1054-1062.
39
Li, Z. Q., Hu, X. P., Zhou, J. Y., Xie, X. D., & Zhang, J. M. (2015). Genetic polymorphisms
in the carbonic anhydrase VI gene and dental caries susceptibility. Genet Mol Res,
14(2), 5986-5993.
Nobre dos Santos, M., Melo dos Santos, L., Francisco, S. B., & Cury, J. A. (2002).
Relationship among dental plaque composition, daily sugar exposure and caries in the
primary dentition. Caries Res, 36(5), 347-352.
Ozturk, L. K., Furuncuoglu, H., Atala, M. H., Ulukoylu, O., Akyuz, S., & Yarat, A. (2008).
Association between dental-oral health in young adults and salivary glutathione, lipid
peroxidation and sialic acid levels and carbonic anhydrase activity. Braz J Med Biol
Res, 41(11), 956-959.
Parisotto, T. M., Steiner-Oliveira, C., Duque, C., Peres, R. C., Rodrigues, L. K., & Nobre-dos-
Santos, M. (2010). Relationship among microbiological composition and presence of
dental plaque, sugar exposure, social factors and different stages of early childhood
caries. Arch Oral Biol, 55(5), 365-373.
Parkkila, S., Kaunisto, K., Rajaniemi, L., Kumpulainen, T., Jokinen, K., & Rajaniemi, H.
(1990). Immunohistochemical localization of carbonic anhydrase isoenzymes VI, II,
and I in human parotid and submandibular glands. J Histochem Cytochem, 38(7), 941-
947.
Parkkila, S., Parkkila, A. K., & Rajaniemi, H. (1995). Circadian periodicity in salivary
carbonic anhydrase VI concentration. Acta Physiol Scand, 154(2), 205-211.
Parkkila, S., Parkkila, A. K., Vierjoki, T., Stahlberg, T., & Rajaniemi, H. (1993). Competitive
time-resolved immunofluorometric assay for quantifying carbonic anhydrase VI in
saliva. Clin Chem, 39(10), 2154-2157.
Peres, R. C., Camargo, G., Mofatto, L. S., Cortellazzi, K. L., Santos, M. C., Nobre-dos-
Santos, M., . . . Line, S. R. (2010). Association of polymorphisms in the carbonic
anhydrase 6 gene with salivary buffer capacity, dental plaque pH, and caries index in
children aged 7-9 years. Pharmacogenomics J, 10(2), 114-119.
Proctor, G. B. (2016). The physiology of salivary secretion. Periodontol 2000, 70(1), 11-25.
Ruiz Miravet, A., Montiel Company, J. M., & Almerich Silla, J. M. (2007). Evaluation of
caries risk in a young adult population. Med Oral Patol Oral Cir Bucal, 12(5), E412-
418.
Sheiham, A., & James, W. P. (2015). Diet and Dental Caries: The Pivotal Role of Free Sugars
Reemphasized. J Dent Res, 94(10), 1341-1347.
40
Singh, S., Sharma, A., Sood, P. B., Sood, A., Zaidi, I., & Sinha, A. (2015). Saliva as a
prediction tool for dental caries: An in vivo study. J Oral Biol Craniofac Res, 5(2), 59-
64.
Szabo, I. (1974). Carbonic anhydrase activity in the saliva of children and its relation to caries
activity. Caries Res, 8(2), 187-191.
Tenovuo, J. (1997). Salivary parameters of relevance for assessing caries activity in
individuals and populations. Community Dent Oral Epidemiol, 25(1), 82-86.
Yarat, A., Ozturk, L. K., Ulucan, K., Akyuz, S., Atala, H., & Isbir, T. (2011). Carbonic
anhydrase VI exon 2 genetic polymorphism in Turkish subjects with low caries
experience (preliminary study). In Vivo, 25(6), 941-944.
Yildiz, G., Ermis, R. B., Calapoglu, N. S., Celik, E. U., & Turel, G. Y. (2016). Gene-
environment Interactions in the Etiology of Dental Caries. J Dent Res, 95(1), 74-79.
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: [email protected], phone number: +55-19-21065290,
Fax:+55-19-21065218
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 the
51
behavior of salivary α-amylase immediately after a cariogenic challenge with 20% sucrose
solution and its relationship with salivary buffer capacity (BC), stimulated salivary flow rate
(SSFR) and caries in children are highly necessary in face to be sucrose the main substrate at
the caries process and that causes major biochemical and physiological changes during the
process of biofilm formation, which enhance its caries-inducing properties [Sheiham and
James, 2015]. This would provide further evidence of the effect of this enzyme in the
dynamics of dental caries process and the effect that a sucrose rinse would have on SAA
activity. Considering the above, the aims of this study were to investigate the influence of a
cariogenic challenge on the salivary amylase activity as well as to examine the relationship
among dental caries, SAA, SSFR and BC in children.
Materials and Methods
Ethical Considerations
The present investigation was approved by Piracicaba Dental School/ University of
Campinas Ethic Committee in Research (Protocol 014/ 2012) and was in full accordance with
the World Medical Association Declaration of Helsinki. The procedures were explained to the
parents of the involved subjects, and an informed written consent was obtained prior to
investigation.
Sample
The study was conducted with children of Piracicaba city, state of São Paulo, Brazil a
city with water fluoridation (0.7 ppm F). Children who participated in the study received a
new toothbrush and dentifrice, as well as oral health preventive instructions.
A convenience sample of thirty eight children, aging 48 to 77 months age, from both
genders were selected and divided into two groups: Group I (CF, n=18), caries-free children,
being 11 girls and 7 boys, mean age; and Group II (CL, n=20) children with caries, being 9
girls and 11 boys mean age. There were no differences between gender and age in the selected
groups (p>0.05).
Children with syndromes or chronic systemic diseases, severe fluorosis, dental
hypoplasia, using braces, under antibiotic therapy or taking medications for central nervous
system diseases, and having communication or neuromotor difficulties were excluded from
the study. Children whose parents refused to sign the informed terms of consent and those
52
who did not collaborate with the necessary procedures for the clinical examinations were also
excluded from the study.
Calibration of the Examiner
Calibration was assessed by reexaminations of 10% of the children with a 1-week
interval to avoid dental examiner memorization. The intra-examiner reliability was calculated
using Kappa statistics and recorded as 0.82.
Oral examination
Dental examination was performed by only one examiner, using the visual/tactile
method based on diagnostic criteria of the World Health Organization with additional
measurement of the early caries lesions and following rigorously strict cross-infection control
measures [Assaf et al., 2006]. The examination was carried out using a head focusable flash-
light, a mirror and a ball-ended probe to remove debris to enhance visualization and confirm
questionable findings. Gauze was employed in order to dry or clean teeth, favoring the
identification of early caries lesions.
Saliva samples collection
Two samples of stimulated saliva was collected from each children: The first one
before and the second one after five minutes of a 20% sucrose solution rinse to determine the
effect of sucrose on the salivary flow rate, the BC as well as on SAA. To control the circadian
rhythm, 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 approximately 0.18 g of Parafilm®
(Sigma Chemical Company, Missouri, USA) for 5 min as previously described and the
produced saliva was deposited in a sterile graduated ice- cooled container to prevent sample
warming [Kirstila et al., 1998; Dawes and Kubieniec, 2004]. SSFR was calculated by
measuring the total volume of saliva and dividing it by the collection time for each child, and
expressed in milliliters per minute. The samples were codified, stored in sealed tubes in an
icebox at 0o C, and immediately transported for the biochemical assays.
53
Buffering capacity determination
Salivary buffering capacity was assessed according to a modified method of Ericsson
[Ericsson, 1959], by adding 1.5 mL of 5 mM HCl to a tube containing 0.5 mL of stimulated
saliva. Then, the tube was shaken for 30 seconds and opened to release CO2 dissolved in the
saliva and after a waiting period of 20 minutes, the pH was determined using a pH electrode
connected to a pH meter (Orion Analyzer Model 420A, USA).
Amylase activity determination
For analysis of α-amylase activity, saliva samples were initially centrifuged at 5000
rpm for 10 min at 4o C. The SAA activity was performed by enzyme kinetic assay with
spectrophotometry technique using Amylase SL Elietch Kit (Eli Tech, Seppim S.A.SEES,
France) according to manufacturer instructions. Saliva samples were diluted in a saline
solution 1:9 (0.9%) NaCl and the reactions were processed at 37oC for 2 min of incubation
and 3 min of reading was performed with absorbance reading at 450 nm in a
spectrophotometry (Evolution 260, Thermo Scientific, USA). Data were expressed as U/mL.
Statistical analysis
Statistical analysis was performed using SPSS version 13.0 (SPSS Inc., IL, USA).
Data distribution of was checked for normality using the Shapiro-Wilk test. Differences inside
each group (before and after sucrose rinse) regarding SSFR and BC values were analyzed by
Wilcoxon test, and differences among groups were analyzed using U Mann- Whitney test. For
results of SAA activity the comparison within groups were analyzed by paired T test, and the
comparison among groups were analyzed by independent T test. The level of significance was
regarded as p<0.05. Correlations between dental caries and independent variables were
determined using the Spearman correlation coefficient (α = 0.05).
54
Results
Medians and interquartile ranges of variables SSFR and BC are presented in Table 1
and 2. Table 1 shows that the two groups of children presented a significant increase in the
SSFR after sucrose rinse (p=0.03 for CF group and p=0.038 for CL group), however, no
difference was found between the two analyzed groups. Table 2 shows a statistically
significant decrease in BC after sucrose rinse for the two analyzed groups (p= 0.009 for CF
group and p=0.005 for CL group). As for the SSFR, we did not find any difference between
analyzed groups.
Means and standard deviations of the SAA are presented at Table 3. It was noticed a
significant increase in α-amylase activity after sucrose rinse only in the CL group (p=0.024).
In addition, we found that after the sucrose rinse, α-amylase activity was significantly higher
in CL than in CF group (p=0.019). However, no difference between groups was detected
before sucrose rinse (p=0.955). The correlations between dental caries and analyzed variables
are shown in Table 4. This table shows a positive correlation between caries and activity of
salivary α-amylase after sucrose rinse (p=0.052). Mean and standard deviation of dental caries
in the investigated children was 4.03± 5.58.
Discussion
The present study investigated for the first time the effect that a high cariogenic
challenge (20% sucrose rinse) would have on behavior of salivary α-amylase activity in
children with or without caries. Literature shows studies that evaluate the amount of total
protein and its relationship with caries. Some of them have found a greater amount of total
protein in saliva of individuals with caries experience [Tulunoglu et al., 2006; Preethi et al.,
2010]. Others found no difference in total protein of saliva of groups with and without decay
[Shimotoyodome et al., 2007; Roa et al., 2008]. Studies involving the participation of children
to investigate the relationship between caries and amylase activity are scarce [de Farias and
Bezerra, 2003; Bhalla et al., 2010; Grychtol et al., 2015; Singh et al., 2015].
Our results showed that after sucrose rinse, the α-amylase activity significantly
increased only in the CL group. Moreover, after cariogenic challenge the α-amylase activity
was significantly higher in CL group than in the CF group. In the same way, Dodds and Edgar
[1986] found a higher amylase activity in biofilm of adults who had a high sucrose diet and a
rinse with starch during the period of study in comparison with control adults. These authors
55
have shown that the cariogenic potential of starch in plaque appears to be affected by the
level of sucrose in the diet.
The finding that exposure of oral environment to a cariogenic challenge increased the
activity of the enzyme only in caries lesion group, leads us to suggest that the subject sucrose
exposure influences the enzyme activity. This finding is relevant because it highlights the
importance of sucrose presence as a synergistic agent to the SAA that has starch as substrate.
A possible explanation for this mechanism could be that in the presence of sucrose, the starch
metabolism would increase in consequence of a higher amylase activity and thus provide
more starch hydrolysis to cariogenic bacteria. Possibly, the caries lesion group had higher
enzyme concentration in saliva and biofilm and when these subjects were submitted to a
cariogenic challenge, it may have increased the enzyme activity. In line with this assumption,
after sucrose rinse, we found a positive correlation between dental caries and amylase activity.
Another result of our study was that before sucrose rinse, the caries-free and caries
lesion group showed no difference in the salivary α-amylase activity. These finding is in line
with early work previously reported [Dodds and Edgar, 1986; de Farias and Bezerra, 2003;
Bhalla et al., 2010], but these authors did not use a rinse with sucrose to after evaluate the
enzyme activity. In the other side, a higher amylase activity in saliva was found in literature in
caries active than in caries-free subjects [Singh et al., 2015]. The diversity of findings on
literature could be explained by various methologies employed for this analyze [Fiehn et al.,
1986; Liang et al., 1999; de Farias and Bezerra, 2003; Vitorino et al., 2006; Shimotoyodome
et al., 2007; Grychtol et al., 2015]. The enzyme kinetic method is the standard technique for
measuring α-amylase activity [Rohleder and Nater, 2009].
Despite of these results, until now there is not sufficient evidence to establish any
salivary proteins as a biomarker for this disease and is still is uncertain how salivary
components, and in this case salivary amylase, behaves in the caries dynamic process [Gao et
al., 2016].
Were evaluated the SSFR and BC. The salivary flow rate and composition are well
recognized as important protective factors that modify the caries process and plays an
important role in the assessment of caries risk [Leone and Oppenheim, 2001]. Regarding the
SSFR, results of our study found no difference among groups before and after sucrose rinse.
In addition, no correlation between SSFR and caries experience was evidenced. These results
56
corroborated with the previous findings [Leonor et al., 2009] who evaluated stimulated
salivary flow rate in 7 years-old children of and found that the caries index was not
significantly associated with the high or low SSFR patterns.
Concerning the caries risk assessment the salivary flow rate and BC parameters have
good specificity and low sensitivity [Leone and Oppenheim, 2001]. As expected, we found a
significant increase in salivary flow rate in both groups after sucrose rinse, that could be
explained by stimulation the salivary glands caused by chewing and presence of sucrose rinse
[Proctor, 2016]. This result is in line with previous investigations [Dodds et al., 2005;
Frasseto et al., 2012]. In addition, we did not detect any correlation between salivary flow rate
and caries, in agreement to results of a systematic review [Leone and Oppenheim, 2001]. The
reason as why we chose to evaluate the SSFR instead of the non stimulated salivary flow rate
was mainly because despite of unstimulated salivary flow be more effective on clearance, this
saliva is mainly composed by contribution of submandibular glands. Parotid saliva increases
dramatically during stimulation, and it is main role is to produce copious, highly buffered,
fluid to protect against extrinsic insult (for instance, acid) [Dodds et al., 2005]. Thus, the BC
is more evidenced in the SSFR, as well as the presence of amylase is more manifested in the
parotid secretion when the secretion doubles [Jenkins, 1978; Hannig et al., 2005].
The mean of salivary flow rates observed in the present study were above the levels
considered strong indicators for caries risk (below 0.5mL/min to 7 years old), however, in
children, the SSFR has been estimated to range from 0.1 and 6 mL / min showing a wide inter
- and intra-individual variations as we found in this study [Leonor et al., 2009]. Regarding BC
the results of the present study showed a statistically significant decrease in BC after sucrose
rinse for the CL as well as for the CF group. BC was determinate five minutes after the
cariogenic challenge. After this time, was demonstrated that saliva and biofilm exhibit the
greatest decreased in pH in reason the acid production. This decrease and subsequent increase
in reason of buffer mechanisms in pH is termed the “Stephan curve” [Stephan and Miller,
1943; Dawes, 2008]. We found no correlation between caries and BC, agreeing with other
authors [Faine et al., 1992; Wiktorsson et al., 1992; O'Sullivan and Curzon, 2000; Leone and
Oppenheim, 2001].
In summary, the results of this study suggest that a rinse with a 20% sucrose solution
increases activity of SAA in saliva mainly in children having caries and that this salivary
protein plays a role in tooth decay in these subjects. Additionally, this study allow to highlight
57
the importance of oral biochemistry in the caries process, showing that the identification of
other factors, that are not yet established in the literature, can influence the carious process.
The identification of subjects at risk of developing aggressive caries could conceivably be
done through compositional analysis of saliva. We further emphasize the potential of salivary
molecules to exhibit influence on the caries process, and the participation of salivary α-
amylase in hydrolysis of dietary starch into fermentable carbohydrates by cariogenic bacteria.
However, further researches using larger sample size to confirm these findings are needed. In
the same way, longitudinal investigations to assess whether changes in the subject caries
profile would be accompanied by alterations in enzyme activity after follow-up. We also
suggest investigations to evaluate how the enzyme behaves in dental biofilm in the presence
of sucrose or in subjects under a high sucrose exposure.
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). 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. The study was supported by FAPESP
(2012/02516-1 and 2012/15834-1).
58
References
Aizawa S, Miyasawa-Hori H, Nakajo K, Washio J, Mayanagi H, Fukumoto S, Takahashi N:
Effects of alpha-amylase and its inhibitors on acid production from cooked starch by
oral streptococci. Caries Res 2009;43:17-24.
Arhakis A, Karagiannis V, Kalfas S: Salivary alpha-amylase activity and salivary flow rate in
young adults. Open Dent J 2013;7:7-15.
Assaf AV, de Castro Meneghim M, Zanin L, Tengan C, Pereira AC: Effect of different
diagnostic thresholds on dental caries calibration - a 12 month evaluation. Community
Dent Oral Epidemiol 2006;34:213-219.
Balekjian AY, Meyer TS, Montague ME, Longton RW: Electrophoretic patterns of parotid
fluid proteins from caries-resistant and caries-susceptible individuals. J Dent Res
1975;54:850-856.
Bhalla S, Tandon S, Satyamoorthy K: Salivary proteins and early childhood caries: A gel
electrophoretic analysis. Contemp Clin Dent 2010;1:17-22.
Chaffee BW, Cheng J, Featherstone JD: Baseline caries risk assessment as a predictor of
caries incidence. J Dent 2015;43:518-524.
Cheaib Z, Lussi A: Role of amylase, mucin, iga and albumin on salivary protein buffering
capacity: A pilot study. J Biosci 2013;38:259-265.
Dawes C: Salivary flow patterns and the health of hard and soft oral tissues. J Am Dent Assoc
2008;139 Suppl:18S-24S.
Dawes C, Kubieniec K: The effects of prolonged gum chewing on salivary flow rate and
composition. Arch Oral Biol 2004;49:665-669.
de Farias DG, Bezerra AC: Salivary antibodies, amylase and protein from children with early
childhood caries. Clin Oral Investig 2003;7:154-157.
Dodds MW, Edgar WM: Effects of dietary sucrose levels on pH fall and acid-anion profile in
human dental plaque after a starch mouth-rinse. Arch Oral Biol 1986;31:509-512.
Dodds MW, Johnson DA, Yeh CK: Health benefits of saliva: A review. J Dent 2005;33:223-
233.
Douglas CW: The binding of human salivary alpha-amylase by oral strains of streptococcal
bacteria. Arch Oral Biol 1983;28:567-573.
59
Duarte S, Klein MI, Aires CP, Cury JA, Bowen WH, Koo H: Influences of starch and sucrose
on streptococcus mutans biofilms. Oral Microbiol Immunol 2008;23:206-212.
Ericsson H: Clinical investigations of salivary buffering action. Acta Odontol Scand
1959;17:131-165.
Faine MP, Allender D, Baab D, Persson R, Lamont RJ: Dietary and salivary factors
associated with root caries. Spec Care Dentist 1992;12:177-182.
Fejerskov O, Kidd E: Dental caries - the disease and its clinical management. São Paulo,
Santos, 2007.
Fiehn NE, Oram V, Moe D: Streptococci and activities of sucrases and alpha-amylases in
supragingival dental plaque and saliva in three caries activity groups. Acta Odontol
Scand 1986;44:1-9.
Frasseto F, Parisotto TM, Peres RC, Marques MR, Line SR, Nobre Dos Santos M:
Relationship among salivary carbonic anhydrase vi activity and flow rate, biofilm ph
and caries in primary dentition. Caries Res 2012;46:194-200.
Gao X, Jiang S, Koh D, Hsu CY: Salivary biomarkers for dental caries. Periodontol 2000
2016;70:128-141.
Grychtol S, Viergutz G, Potschke S, Bowen WH, Hoth-Hannig W, Leis B, Umanskaya N,
Hannig M, Hannig C: Enzymes in the in-situ pellicle of children with different caries
activity. Eur J Oral Sci 2015.
Hannig C, Hannig M, Attin T: Enzymes in the acquired enamel pellicle. Eur J Oral Sci
2005;113:2-13.
Jenkins GN: The physiology and biochemistry of the
mouth, ed 4th. Oxford, Blackwell Scientific Publications, 1978.
Kirstila V, Hakkinen P, Jentsch H, Vilja P, Tenovuo J: Longitudinal analysis of the
association of human salivary antimicrobial agents with caries increment and
cariogenic micro-organisms: A two-year cohort study. J Dent Res 1998;77:73-80.
Leone CW, Oppenheim FG: Physical and chemical aspects of saliva as indicators of risk for
dental caries in humans. J Dent Educ 2001;65:1054-1062.
Leonor SP, Laura SM, Esther IC, Marco ZZ, Enrique AG, Ignacio MR: Stimulated saliva
flow rate patterns in children: A six-year longitudinal study. Arch Oral Biol
2009;54:970-975.
Liang H, Wang Y, Wang Q, Ruan MS: Hydrophobic interaction chromatography and
capillary zone electrophoresis to explore the correlation between the isoenzymes of
60
salivary alpha-amylase and dental caries. J Chromatogr B Biomed Sci Appl
1999;724:381-388.
O'Sullivan EA, Curzon ME: Salivary factors affecting dental erosion in children. Caries Res
2000;34:82-87.
Paes Leme AF, Koo H, Bellato CM, Bedi G, Cury JA: The role of sucrose in cariogenic
dental biofilm formation--new insight. J Dent Res 2006;85:878-887.
Parisotto TM, Steiner-Oliveira C, Duque C, Peres RC, Rodrigues LK, Nobre-dos-Santos M:
Relationship among microbiological composition and presence of dental plaque, sugar
exposure, social factors and different stages of early childhood caries. Arch Oral Biol
2010;55:365-373.
Parisotto TM, Stipp R, Rodrigues LK, Mattos-Graner RO, Costa LS, Nobre-Dos-Santos M:
Can insoluble polysaccharide concentration in dental plaque, sugar exposure and
cariogenic microorganisms predict early childhood caries? A follow-up study. Arch
Oral Biol 2015;60:1091-1097.
Preethi BP, Reshma D, Anand P: Evaluation of flow rate, ph, buffering capacity, calcium,
total proteins and total antioxidant capacity levels of saliva in caries free and caries
active children: An in vivo study. Indian J Clin Biochem 2010;25:425-428.
Proctor GB: The physiology of salivary secretion. Periodontol 2000 2016;70:11-25.
Ribeiro CC, Tabchoury CP, Del Bel Cury AA, Tenuta LM, Rosalen PL, Cury JA: Effect of
starch on the cariogenic potential of sucrose. Br J Nutr 2005;94:44-50.
Roa NS, Chaves M, Gomez M, Jaramillo LM: Association of salivary proteins with dental
caries in a colombian population. Acta Odontol Latinoam 2008;21:69-75.
Rogers JD, Palmer RJ, Jr., Kolenbrander PE, Scannapieco FA: Role of streptococcus gordonii
amylase-binding protein in adhesion to hydroxyapatite, starch metabolism, and
biofilm formation. Infect Immun 2001;69:7046-7056.
Rohleder N, Nater UM: Determinants of salivary alpha-amylase in humans and
methodological considerations. Psychoneuroendocrinology 2009;34:469-485.
Scannapieco FA, Torres G, Levine MJ: Salivary alpha-amylase: Role in dental plaque and
caries formation. Crit Rev Oral Biol Med 1993;4:301-307.
Sheiham A, James WP: Diet and dental caries: The pivotal role of free sugars reemphasized. J
Dent Res 2015;94:1341-1347.
61
Shimotoyodome A, Kobayashi H, Tokimitsu I, Hase T, Inoue T, Matsukubo T, Takaesu Y:
Saliva-promoted adhesion of streptococcus mutans mt8148 associates with dental
plaque and caries experience. Caries Res 2007;41:212-218.
Singh S, Sharma A, Sood PB, Sood A, Zaidi I, Sinha A: Saliva as a prediction tool for dental
caries: An in vivo study. J Oral Biol Craniofac Res 2015;5:59-64.
Stephan RM, Miller BF: A quantitative method for evaluating physical and chemical agents
which modify production of acids in bacterial plaques on human teeth. Dent Res
1943;22:45-51.
Tulunoglu O, Demirtas S, Tulunoglu I: Total antioxidant levels of saliva in children related to
caries, age, and gender. Int J Paediatr Dent 2006;16:186-191.
Vacca-Smith AM, Venkitaraman AR, Quivey RG, Jr., Bowen WH: Interactions of
streptococcal glucosyltransferases with alpha-amylase and starch on the surface of
saliva-coated hydroxyapatite. Arch Oral Biol 1996;41:291-298.
Vitorino R, de Morais Guedes S, Ferreira R, Lobo MJ, Duarte J, Ferrer-Correia AJ, Tomer
KB, Domingues PM, Amado FM: Two-dimensional electrophoresis study of in vitro
pellicle formation and dental caries susceptibility. Eur J Oral Sci 2006;114:147-153.
Wiktorsson AM, Martinsson T, Zimmerman M: Salivary levels of lactobacilli, buffer capacity
and salivary flow rate related to caries activity among adults in communities with
optimal and low water fluoride concentrations. Swed Dent J 1992;16:231-237.
62
Legends
Table 1. Medians and interquartile ranges of stimulated salivary flow rate (mL/min) before
and after a 20% sucrose rinse.
Table 2. Medians and interquartile ranges of salivary buffering capacity before and after a
20% sucrose rinse.
Table 3. Means and standard deviations of salivary α-amylase (U/mL) before and after a 20%
sucrose rinse.
Table 4. Spearman correlation coefficients and probabilities of statistical significance between
dental caries and independent variables before and after a 20% sucrose rinse .
Figure 1. Salivary activity of α-amylase in the caries lesion group before and after rinse with a
20% sucrose solution.
Figure 2. Salivary activity of α-amylase after a 20% sucrose rinse, comparison between
caries-free and caries lesion group.
63
Tables
Table 1.
Groups Before rinse After rinse p*
Caries-free (n=18) 0.75 (0.58) 0.87 (0.45) 0.030
Caries lesion (n=20) 0.69 (0.9) 0.88 (0.73) 0.038
p** 0.851 0.637
p values from Wilcoxon* and U Mann-Whitney** test.
64
Table 2.
Groups Before rinse After rinse p*
Caries-free (n=18) 5.04 (1.35) 4.05 (1.04) 0.009
Caries lesion (n=20) 4.55 (1.48) 3.9 (0.84) 0.005
p** 0.654 0.393
p values from Wilcoxon* and U Mann-Whitney** test.
65
Table 3.
Groups Before rinse After rinse p*
Carie-free(n=18) 1999.02 ±717.13 1934.56 ± 528.62 0.688
Caries lesion (n=20) 1985.19 ± 783.75 2387.37 ± 605.55 0.024
p** 0.955 0.019
p values from Paired T test * and Independent T Test** test.
66
Table 4.
Variables
r p
SSFR Before rinse -0.075 0.657
SSFR After rinse -0.192 0.310
BC Before rinse -0.064 0.703
BC After rinse -0.209 0.209
SAA Before rinse 0.058 0.729
SAA After rinse 0.317 0.052
SSFR: stimulated salivary flow rate. BC: buffer capacity. SAA: salivary α-amylase activity.
67
Illustrations
Figure 1.
(U/mL)
BR AR
p = 0,024
68
Figure 2.
(U/mL)
CF CL
p = 0,019
69
3 DISCUSSÃO
A saliva é um importante fluido biológico, têm em sua composição várias proteínas
que participam da proteção dos tecidos orais (Leone e Oppenheim, 2001, Dodds et al., 2005).
Fatores como o fluxo salivar e a capacidade tampão influem na avaliação do risco de cárie,
bem como a presença de componentes orgânicos e inorgânicos na saliva participam do
processo da doença (Leone e Oppenheim, 2001, Van Nieuw Amerongen et al., 2004, Chaffee
et al., 2015). É descrito na literatura que a secreção salivar e seus componentes poderiam ser
usados no diagnóstico de indivíduos cárie susceptíveis, situação em que as moléculas
presentes na saliva refletiriam a atividade bacteriana e na susceptibilidade à doença cárie
desde o seu início até sua progressão (Lagerlof e Oliveby, 1994).
Diferenças na composição salivar no que tange a concentração de proteínas entre
indivíduos livres de cárie e cárie-susceptíveis podem prover uma explicação diferente e mais
satisfatória para a diferença na susceptibilidade inter-indivídual à cárie, que irão confirmar
diferenças genéticas encontradas e irão ser somadas com os vários fatores associados ao
desenvolvimento da doença. Embora fatores genéticos relacionados à estrutura do esmalte
dental ou a microbiota bucal afetem a susceptibilidade à cárie, eles não podem explicar o
porquê de indivíduos livres de cárie neutralizarem ácidos mais eficientemente (Levine, 2011).
No entanto, apesar dos avanços em pesquisas relacionadas a proteínas presentes na saliva, não
há ainda na literatura evidências suficientes que relacione qualquer componente salivar ao
aumento da susceptibilidade à cárie dental (Leone e Oppenheim, 2001).
Foram estudadas nesta tese, a atividade, antes e depois de um bochehco com sacarose,
de duas importantes enzimas presentes na saliva, a AC VI e a α-amilase salivar, por meio de
um estudo longitudinal e transversal, respectivamente. Esta tese abordou aspectos
modificadores do processo de cárie e que influem consequentemente na atividade da
comunidade bacteriana do biofilme participando da acidificação e neutralização do meio
ambiente oral e assim influindo na ocorrência de progressão da cárie dental. Demonstrou-se
pelos resultados desta tese e pela discussão dos mesmos, que estas enzimas desempenham
papéis relevantes na dinâmica do processo de cárie.
O Capítulo 1 desta tese abordou a enzima AC VI, presente na saliva e no biofilme
dental. É sugerido na literatura que esta enzima participa da regulação do pH no meio bucal
70
catalisando o sistema tampão mais importante da cavidade bucal (ácido-carbônico/
bicarbonato) e assim agiria na proteção da superfície dental pela neutralização dos ácidos na
saliva e biofilme dental, o que seria fator de importância primária para a manutenção da
homeostase bucal. No entanto, resultados divergentes são encontrados na literatura. A maioria
dos estudos que envolvem a avaliação desta enzima na saliva e/ou biofilme analisa a
concentração da enzima por meio de técnicas diversas dentre elas, métodos de Krebs,
Roghton, Lowry e Verpoorte, técnicas imunofluorométricas, de imunocoloração,
imunobloting, imunoistoquímica, zimografia, técnicas colorimétricas e imunofluorescência.
Somente Frasseto et al. (2012) avaliaram a atividade da enzima. Deve-se considerar o fato de
que nem toda a concentração enzimática disponível em determinado meio estará
necessariamente ativa. Fatores como a concentração do substrato, o pH do meio, íons e
moléculas inibidores enzimáticos podem modular ou regular a atividade enzimática de forma
negativa ou positiva (Aidar et al., 2013). Na forma negativa a fração ativa da enzima ficaria
inibida deixando de exercer sua ação catalítica. Pelo fato de a fração ativa ser aquela a exercer
a função catalítica, também avaliou-se neste estudo a atividade enzimática de AC VI. Alguns
resultados da literatura apontam para a associação significativa entre uma baixa concentração
de AC VI na saliva e o aumento do índice de cárie (Szabo, 1974, Kivela et al., 1999b). A
literatura mostra ainda que a enzima está presente e ativa na película adquirida e que controla
o pH no biofilme (Leinonen et al., 1999, Kimoto et al., 2006). No entanto, ainda é incerto se a
atividade da enzima influi no processo de cárie em virtude de haver apenas um estudo na
literatura que avaliou a relação da atividade enzimática de AC VI e a prevalência de cárie em
crianças em um estudo transversal (Frasseto et al., 2012).
Nosso estudo, mostrado no Capítulo 1, avaliou o comportamento da atividade de AC
VI na saliva total de crianças antes e imediatamente após um desafio cariogênico, simulado
por meio de um bochecho com solução de sacarose a 20%. Os dados resultantes foram
correlacionados com medidas de fluxo salivar estimulado (FSE), capacidade tampão (CT) e
índice de cárie. Todas as análises foram repetidas após um ano de seguimento. Este foi o
primeiro estudo empregando esse delineamento experimental. Após um ano, os grupos foram
comparados segundo o status de cárie atual. Os resultados mostraram que a enzima
pesquisada exibe comportamento diferente quando submetida a um desafio cariogênico de
acordo com o índice de cárie. Após o desafio cariogênico, a atividade de AC VI no grupo
livre de cárie nos dois tempos do estudo e no grupo de crianças que tiveram cárie paralisada
após um ano (quando apresentou incremento de cárie negativo ou zero) exibiu um decréscimo
71
em sua atividade. Provavelmente, esse decréscimo pode explicado pelo fato de que após o
bochecho as crianças exibiram valores de pH mais altos e então houve menor disponibilização
de substrato para a reação catalisada pela enzima em questão. Consequentemente, a atividade
de AC VI foi menor. Estes dados estão de acordo com a correlação negativa encontrada entre
os valores de capacidade tampão antes do bochecho e a atividade de AC VI após o bochecho.
Outros autores não encontraram correlação entre a capacidade tampão e a concentração de AC
VI na saliva (Parkkila et al., 1993, Kivela et al., 1997). No entanto, a literatura aponta uma
ação da enzima como catalisadora da reação tampão mais importante da cavidade bucal
(Kivela et al., 1999a) e, portanto seria esperada associação entre essas variáveis. Ainda, deve-
se salientar que os autores acima mencionados investigaram a concentração da isoenzima.
Observou-se também que no grupo com cárie, os valores de atividade de AC VI
mantiveram-se elevados após o bochecho nos dois períodos do estudo. Isto pode ser explicado
pelo fato de que o decréscimo no pH bucal após o desafio cariogênico nestes indivíduos seria
de maior magnitude, já que é evidenciado na literatura que crianças com CPI em torno da
faixa etária estudada são significativamente mais expostas diariamente aos açucares da dieta
(Nobre dos Santos et al., 2002). Assim, devido aos desafios cariogênicos frequentes e quedas
constantes do pH bucal em razão da presença de uma microbiota cariogênica estabelecida,
haveria maior disponibilidade de íons H+, derivados do metabolismo bacteriano, bem como de
íons HCO3-
, derivados das glândulas salivares pela ação da enzima AC II devido ao aumento
do fluxo salivar. Portanto, haveria mais substrato para a reação catalisada por AC VI, na
tentativa de neutralizar o meio ácido. A partir da situação encontrada, podemos verificar que a
enzima neste momento (após o bochecho) não exibiu decréscimo em sua atividade neste
grupo, o que seria importante no processo de cárie, pois a enzima estaria mais ativa durante a
ocorrência do desafio cariogênico, um papel chave na tentativa de retorno do pH a valores
normais num momento considerado crítico. No entanto, não foram encontradas diferenças
relacionadas aos valores de atividade de AC VI entre os grupos, o que já foi observado na
literatura também por outros autores (Ozturk et al., 2008). Nossos resultados diferem daqueles
encontrados por de Frasseto et al. (2012) que observaram uma maior atividade da AC VI no
grupo com cárie antes do desafio cariogênico (p=0.0516). Esses autores, no entanto, também
não avaliaram a capacidade tampão nem reavaliaram os indivíduos após um ano para
confirmar os achados encontrados. Ainda no Capítulo 1, encontramos uma correlação
negativa entre a atividade da enzima AC VI antes e depois do bochecho e a prevalência de
cárie no baseline no grupo de crianças com cárie. Este resultado pode indicar que a
72
manutenção de níveis altos de atividade da enzima em condições normais estaria relacionada
a uma menor prevalência de cárie em crianças, em razão da manutenção de valores elevados
de pH favorecidos pela ação da enzima. Esses dados corroboram os achados de Kivela et al.
(1999b), que encontraram uma correlação negativa significativa entre a concentração da
enzima e a cárie e em indivíduos com higiene oral pobre. Já Ozturk et al. (2008) não
encontraram nenhuma correlação entre essas variáveis. Resultados contrários ao de Frasseto
et al. (2012) os quais encontraram uma atividade significativamente maior em crianças com
cárie. Observou-se ainda uma correlação negativa entre a capacidade tampão antes do
bochecho e atividade de AC VI após o bochecho o que nos leva a considerar, que a enzima
estudada desempenha um papel protetor no processo de cárie.
Os resultados deste capítulo sugerem que a enzima é responsável pela manutenção da
homeostase do pH da cavidade bucal como agente protetor, catalisando o sistema tampão
mais importante em situações de estimulação salivar. Atua, portanto, de sobremaneira quando
o meio bucal é submetido a um desafio cariogênico, principalmente no grupo de indivíduos
com cárie por apresentarem mais frequentemente baixos valores de pH e consequentemente,
por haver no meio maior concentração de íons H+. Resultados estes que concordam com a
corrente da literatura que acredita que a enzima atua constantemente para equilibrar as
oscilações de pH que acontecem frequentemente na cavidade bucal.
O Capítulo 2 desta tese abordou a relação da enzima α-amilase e a cárie dental em
crianças. Esta enzima pode promover um papel importante no processo de cárie como
facilitadora da colonização e metabolismo de espécies de estreptococos na película dental,
levando a formação do biofilme dental e consequentemente a cárie dental. Por ser um
constituinte da película adquirida, esta enzima atua como receptor para a adesão de micro-
organismos a superfície dental, onde hidrolisam o amido, dando origem a carboidratos de
baixo peso molecular que são metabolizados em ácidos por bactérias cariogênicas (Rogers et
al., 2001). Os resultados relacionados a esta enzima evidenciaram que após a exposição do
ambiente oral a um desafio cariogênico, sua atividade mostrou-se significativamente maior
somente no grupo com cárie. Isso confirmou alguns achados da literatura sobre a possível
participação desta enzima no processo de cárie e a sua ação como influenciadora deste
processo em indivíduos com a doença já instalada (Vitorino et al., 2006, Singh et al., 2015).
Observou-se também que os resultados do Capítulo 2 corroboraram com achados
semelhantes na literatura que indicavam uma maior atividade da enzima no biofilme de
73
adultos quando o meio bucal foi exposto à dieta altamente sacarogênica (Dodds e Edgar,
1986). Scannapieco et al. (1993) explana em sua revisão de literatura que haveria um efeito
sinérgico entre sacarose e amido devido ao metabolismo de bactérias cariogênicas em
biofilmes nos quais a enzima α-amilase estivesse presente com um aumento subsequente da
atividade de cárie. Tem sido demonstrado que a combinação amido e sacarose é mais
cariogênica que a sacarose sozinha, por ser esta combinação responsável por produzir
biofilmes com maior massa e acidogenicidade, maior conteúdo de polissacarídeos insolúveis e
alta contagem de lactobacilos e por promover consequentemente maior perda mineral no
tecido dental (Ribeiro et al., 2005, Duarte et al., 2008).
No presente estudo utilizou-se um bochecho com sacarose a 20% e mediu-se a
atividade da enzima 5 minutos após o desafio cariogênico. Possivelmente, as crianças do
grupo cárie que tinham uma alta concentração da enzima na saliva quando submetida a um
desafio cariogênico, exibiram um aumento da atividade da enzima, dados que relacionam o
aumento da atividade da enzima com a presença da sacarose e com o status dental do
indivíduo neste Capítulo. Assim, o aumento da atividade da enzima em indivíduos com o
processo de cárie instalado, favoreceria a progressão do processo de cárie por promover
carboidratos de baixo peso molecular para o metabolismo bacteriano no biofilme derivados do
metabolismo do amido, fato que foi comprovado por Vacca-Smith et al. (1996). Estes autores
evidenciaram um aumento da produção de glucanos pela enzima glucosiltransferase B em
presença de hidrolisados do amido. Este mecanismo seria acelerado na presença de sacarose
no ambiente bucal devido ao possível sinergismo existente entre amido e sacarose,
evidenciada há tempos por Dodds e Edgar (1986).
Os achados deste Capítulo nos levam a acreditar que a maior atividade de α-amilase
salivar em indivíduos com cárie favorece o processo de cárie por fornecer substrato para o
metabolismo de bactérias cariogênicas, produzindo assim polissacarídeos insolúveis
contribuindo para o aumento da biomassa do biofilme e ácidos orgânicos o que culminaria na
desmineralização dos tecidos dentais. Ainda, esta enzima estaria em posição privilegiada na
película e biofilme dental e estaria mais ativa em indivíduos com cárie após serem submetidos
a um bochecho com sacarose a 20%.
74
Os resultados da atividade da enzima antes do bochecho corroboram com os resultados
de alguns autores que também não encontraram diferenças significativas na atividade da
enzima entre os grupos com e sem cárie (de Farias e Bezerra, 2003, Bhalla et al., 2010,
Grychtol et al., 2015). No entanto, similarmente ao nosso estudo o grupo cárie exibiu uma
maior atividade da enzima, embora não significativa (de Farias e Bezerra, 2003, Grychtol et
al., 2015). Com o desafio cariogênico, a atividade da enzima exibiu um comportamento
diferente dos valores apresentados antes do bochecho. Encontramos um aumento significativo
da atividade da enzima no grupo com cárie. Nossos resultados corroboraram com os
resultados de outros autores (Fiehn et al., 1986, Vitorino et al., 2006, Singh et al., 2015). No
entanto, nenhum desses estudos utilizou um bochecho com solução de sacarose antes das
análises para simular um desafio cariogênico e avaliar a dinâmica da atividade da enzima
antes e depois do bochecho que traduziria o que ocorre constantemente na cavidade bucal e
que indicasse a participação da sacarose na atividade da amilase, já que esta enzima está
presente na saliva e no biofilme dental.
Apesar de não existir evidência suficiente que comprove a relação entre qualquer
componente salivar como biomarcador para cárie dentária (Martins et al., 2013), o presente
estudo realizado mostrou que as enzimas estudadas (anidrase carbônica VI e α-amilase
salivar) exercem papel relevante no processo de cárie. Portanto, torna-se importante para o
entendimento da doença cárie no que tange o seu início, desenvolvimento e susceptibilidade
individual, entender o papel de cada componente salivar, em especial as proteínas da saliva
de crianças, principalmente naquelas acometidas por formas mais severas da doença. Estudos
que envolvam uma amostra maior, fazem-se necessários para comprovar os resultados aqui
descritos. Adicionalmente, um estudo longitudinal que avalie a a atividade da enzima α-
amilase, também se faz necessário para avaliar se os efeitos do tempo e da mudança do índice
de cárie modificariam a atividade desta enzima, bem como a inclusão da análise da atividade
da enzima no biofilme dental. De semelhante relevância, os fatores genéticos associados com
a expressão fenotípica destas proteínas e que também podem estar envolvidos na secreção
destas, que não foram estudados e que podem ter influenciado os resultados do presente
estudo devem ser investigados.
Abordou-se ainda em ambos os capítulos desta tese a análise do FSE e da CT, dois
parâmetros importantes para a avaliação do risco de cárie (Leone e Oppenheim, 2001). No
Capítulo 1 nosso estudo mostrou que o FSE aumentou significativamente após o desafio
75
cariogênico na amostra total pesquisada e nos grupos avaliados. Estes resultados concordam
com aqueles obtidos por Frasseto et al. (2012). No follow-up, apenas no grupo de crianças
com cárie paralisada observou-se este comportamento. O aumento do fluxo salivar após o
bochecho pode ser explicado tanto pelo estímulo gustatório promovido pela exposição do
meio bucal à sacarose bem como ao estímulo mecânico promovido pela ação de bochecho
(Proctor, 2016).
Nós observamos após um ano aumento significativo nos valores de FSE, isso pode ser
explicado pelo desenvolvimento e maturação das glândulas salivares com a idade reportado
em outros estudos (Tukia-Kulmala e Tenovuo, 1993, Torres et al., 2006, Leonor et al., 2009).
Também observou-se uma grande variação inter-individual dos valores de FSE o que também
concorda com achados prévios (Tukia-Kulmala e Tenovuo, 1993, Tenovuo, 1997).
Adicionalmente, não foram encontradas diferenças nos valores de FSE entre os grupos
avaliados, dados que corroboram com os resultados de outros autores, no entanto, estes
autores não avaliaram esta variável após um período de seguimento (Frasseto et al., 2012,
Yildiz et al., 2016). Ainda nesse estudo, não encontramos correlação entre o FSE e o índice de
cárie nem no baseline nem após um ano, resultados semelhantes aos encontrados por Leone et
al. (2001) em uma revisão sistemática.
O fluxo salivar pode ser considerado um dos fatores mais importantes na avaliação do
risco de cárie, pois a atividade cariostática ou a eficácia de praticamente todos os outros
fatores dependem do fluxo salivar (Gao et al., 2016). Os estudos têm mostrado que em
indivíduos com fluxo salivar normal, a avaliação do fluxo salivar tem pouco ou nenhum valor
preditivo de cárie (Lenander-Lumikari e Loimaranta, 2000, Gao et al., 2016). Por outro lado,
quando expresso em valores baixos é um forte indicador de aumento do risco de cárie, pois
nesses indivíduos as propriedades mecânicas, de limpeza e capacidade tampão na cavidade
bocal estarão comprometidas.
Quando avaliados os valores de CT, não encontramos nessa amostra diferença
significativa entre os grupos avaliados, dados que corroboram com os estudos de Peres et al.
(2010) e Yarat et al. (2011), no entanto, foi encontrado nos três grupos um decréscimo
significativo nos valores de CT nos dois tempos do estudo, após o desafio cariogênico,
similarmente ao que foi observado no estudo transversal de Frasseto et al. (2012).
Observamos ainda uma correlação significativa entre a CT após o bochecho no follow-up e o
índice de cárie no baseline e follow-up. Resultados similares foram também encontrados por
76
Frasseto et al. 2012 no baseline, único estudo que empregou um desafio cariogênico. No
entanto, esses autores obtiveram essa correlação entre o pH do biofilme e a cárie dental.
Estudos previamente realizados encontraram relação inversa entre a presença de cárie a
capacidade tampão, no entanto, sem que tenha sido empregado o desafio cariogênico para
aferição da capacidade tampão (Kivela et al., 1999b, Ruiz Miravet et al., 2007, Kuriakose et
al., 2013, Singh et al., 2015, Yildiz et al., 2016).
No Capítulo 2 também não encontramos diferenças entre os grupos quanto aos valores
de FSE e CT. Encontramos um aumento significativo do FSE e uma diminuição significativa
na CT nesta amostra após o desafio cariogênico.
Abordamos nos dois Capítulos a análise da saliva, uma vez que tal fluido biológico se
mostra de fundamental importância no entendimento do processo de cárie. No campo da
pesquisa é um material que facilmente pode ser obtido, de coleta indolor e têm em sua
composição uma série de proteínas que podem influir no processo de cárie, além de que a
partir da saliva pode ser extraído o DNA do indivíduo. Portanto, a atenção tem se voltado
cada vez mais para pesquisas envolvendo a coleta de saliva, a avaliação das proteínas que a
compõe e a expressão dessas na célula por meio da avaliação do DNA o que pode fornecer
informações importantes sobre o metabolismo do biofilme e avaliação de indivíduos
susceptíveis à cárie. Ainda, estudos em crianças são importantes, pois a composição de
aminoácidos, taxa de formação e aparência ultraestrutural da película difere entre dentes
decíduos e permanentes (Grychtol et al., 2015). A investigação crescente dos componentes
salivares permitirá a melhor compreensão de todos os componentes envolvidos no processo
de cárie, seja como elementos protetores ou elementos de risco para o desenvolvimento da
doença.
Esta tese evidencia a participação e importância das proteínas AC VI e α-amilase
salivar no processo de cárie. A primeira agiria como catalisador da reação tamponante mais
importante da cavidade bucal e a segunda em virtude de sua atividade aumentada após um
desafio cariogênico teria ação hidrolítica sobre o amido na cavidade bucal. Sua presença na
película e biofilme ligada a bactérias cariogênicas facilitaria a disponibilização de substrato
para bactérias cariogênicas, o que seria fundamental no processo de cárie.
77
4 CONCLUSÕES
A enzima AC VI exerce possível participação no controle de pH bucal após um
desafio cariogênico com solução de sacarose a 20%, principalmente em crianças com
cárie. A correlação negativa entre índice de cárie e atividade de AC VI evidenciada no
grupo com cárie sugere o efeito protetor da enzima no processo de cárie por sua
participação no mecanismo tamponante da saliva em crianças com a doença já
instalada;
A enzima α-amilase exibiu aumento de sua atividade após desafio cariogênico com
bochecho com solução de sacarose a 20% em crianças com cárie. A atividade da
enzima também foi significativamente maior após o bochecho em crianças com cárie,
o que sugere possível participação da enzima como facilitador do processo de cárie
devido ao aumento de sua atividade quando as crianças foram submetidas a um
desafio cariogênico;
As enzimas AC VI, α-amilase, CT e FSE podem ser componentes importantes no
processo da doença cárie.
78
REFERÊNCIAS *
1. AAPD. Policy on early childhood caries (ECC): classifications, consequences, and
preventive strategies. Pediatr Dent. 2008;30(7 Suppl):40-3.
2. Aidar M, Marques R, Valjakka J, Mononen N, Lehtimaki T, Parkkila S, et al. Effect of
genetic polymorphisms in CA6 gene on the expression and catalytic activity of human
salivary carbonic anhydrase VI. Caries Res. 2013;47(5):414-20.
3. Bardow A, Hofer E, Nyvad B, ten Cate JM, Kirkeby S, Moe D, et al. Effect of saliva
composition on experimental root caries. Caries Res. 2005 Jan-Feb;39(1):71-7.
4. Bardow A, Moe D, Nyvad B, Nauntofte B. The buffer capacity and buffer systems of
human whole saliva measured without loss of CO2. Arch Oral Biol. 2000 Jan;45(1):1-12.
5. Bhalla S, Tandon S, Satyamoorthy K. Salivary proteins and early childhood caries: A
gel electrophoretic analysis. Contemp Clin Dent. 2010 Jan;1(1):17-22.
6. Bhayat A, Ahmad MS, Hifnawy T, Mahrous MS, Al-Shorman H, Abu-Naba'a L, et al.
Correlating dental caries with oral bacteria and the buffering capacity of saliva in children in
Madinah, Saudi Arabia. J Int Soc Prev Community Dent. 2013 Jan;3(1):38-43.
7. Chaffee BW, Cheng J, Featherstone JD. Baseline caries risk assessment as a predictor
of caries incidence. J Dent. 2015 May;43(5):518-24.
8. Cheaib Z, Ganss C, Lamanda A, Turgut MD, Lussi A. Comparison of three strip-type
tests and two laboratory methods for salivary buffering analysis. Odontology. 2012
Jan;100(1):67-75.
9. Cheaib Z, Lussi A. Role of amylase, mucin, IgA and albumin on salivary protein
buffering capacity: a pilot study. J Biosci. 2013 Jun;38(2):259-65.
10. Cunha-Cruz J, Scott J, Rothen M, Mancl L, Lawhorn T, Brossel K, et al. Salivary
characteristics and dental caries: evidence from general dental practices. J Am Dent Assoc.
2013 May;144(5):e31-40.
11. Dawes C. Salivary flow patterns and the health of hard and soft oral tissues. J Am
Dent Assoc. 2008 May;139 Suppl:18S-24S.
12. de Farias DG, Bezerra AC. Salivary antibodies, amylase and protein from children
with early childhood caries. Clin Oral Investig. 2003 Sep;7(3):154-7.
13. Dodds MW, Edgar WM. Effects of dietary sucrose levels on pH fall and acid-anion
profile in human dental plaque after a starch mouth-rinse. Arch Oral Biol. 1986;31(8):509-12.
* De acordo com as normas da UNICAMP/FOP, baseadas na padronização do International Committee of Medical Journal Editors - Vancouver Group. Abreviatura dos periódicos em conformidade com o PubMed.
79
14. Dodds MW, Johnson DA, Mobley CC, Hattaway KM. Parotid saliva protein profiles
in caries-free and caries-active adults. Oral Surg Oral Med Oral Pathol Oral Radiol Endod.
1997 Feb;83(2):244-51.
15. Dodds MW, Johnson DA, Yeh CK. Health benefits of saliva: a review. J Dent. 2005
Mar;33(3):223-33.
16. Douglas CW. Characterization of the alpha-amylase receptor of Streptococcus
gordonii NCTC 7868. J Dent Res. 1990 Nov;69(11):1746-52.
17. Duarte S, Klein MI, Aires CP, Cury JA, Bowen WH, Koo H. Influences of starch and
sucrose on Streptococcus mutans biofilms. Oral Microbiol Immunol. 2008 Jun;23(3):206-12.
18. Ericsson H. Clinical investigations of salivary buffering action. Acta Odontol Scand.
1959 Mar 19;17:131-65.
19. Fejerskov O, Kidd E. Dental caries - The disease and its clinical management. São
Paulo: Santos; 2007.
20. Ferreira SH, Beria JU, Kramer PF, Feldens EG, Feldens CA. Dental caries in 0- to 5-
year-old Brazilian children: prevalence, severity, and associated factors. Int J Paediatr Dent.
2007 Jul;17(4):289-96.
21. Fiehn NE, Oram V, Moe D. Streptococci and activities of sucrases and alpha-amylases
in supragingival dental plaque and saliva in three caries activity groups. Acta Odontol Scand.
1986 Feb;44(1):1-9.
22. Frasseto F, Parisotto TM, Peres RC, Marques MR, Line SR, Nobre Dos Santos M.
Relationship among salivary carbonic anhydrase VI activity and flow rate, biofilm pH and
caries in primary dentition. Caries Res. 2012;46(3):194-200.
23. Gao X, Jiang S, Koh D, Hsu CY. Salivary biomarkers for dental caries. Periodontol
2000. 2016 Feb;70(1):128-41.
24. Grychtol S, Viergutz G, Potschke S, Bowen WH, Hoth-Hannig W, Leis B, et al.
Enzymes in the in-situ pellicle of children with different caries activity. Eur J Oral Sci. 2015
Aug 28.
25. Hannig C, Attin T, Hannig M, Henze E, Brinkmann K, Zech R. Immobilisation and
activity of human alpha-amylase in the acquired enamel pellicle. Arch Oral Biol. 2004
Jun;49(6):469-75.
26. Izutsu KT. Theory and Measurement of the buffer value of bicarbonate in saliva. J
Theor Biol. 1981 Jun 7;90(3):397-403.
80
27. Kadoya Y, Kuwahara H, Shimazaki M, Ogawa Y, Yagi T. Isolation of a novel
carbonic anhydrase from human saliva and immunohistochemical demonstration of its related
isozymes in salivary gland. Osaka City Med J. 1987 Jul;33(1):99-109.
28. Kargul B, Yarat A, Tanboga I, Emekli N. Salivary protein and some inorganic element
levels in healthy children and their relationship to caries. J Marmara Univ Dent Fac. 1994
Sep;2(1):434-40.
29. Kejriwal S, Bhandary R, Thomas B, Kumari S. Estimation of levels of salivary mucin,
amylase and total protein in gingivitis and chronic periodontitis patients. J Clin Diagn Res.
2014 Oct;8(10):ZC56-60.
30. Kimoto M, Kishino M, Yura Y, Ogawa Y. A role of salivary carbonic anhydrase VI in
dental plaque. Arch Oral Biol. 2006 Feb;51(2):117-22.
31. Kivela J, Laine M, Parkkila S, Rajaniemi H. Salivary carbonic anhydrase VI and its
relation to salivary flow rate and buffer capacity in pregnant and non-pregnant women. Arch
Oral Biol. 2003 Aug;48(8):547-51.
32. Kivela J, Parkkila S, Metteri J, Parkkila AK, Toivanen A, Rajaniemi H. Salivary
carbonic anhydrase VI concentration and its relation to basic characteristics of saliva in young
men. Acta Physiol Scand. 1997 Oct;161(2):221-5.
33. Kivela J, Parkkila S, Parkkila AK, Leinonen J, Rajaniemi H. Salivary carbonic
anhydrase isoenzyme VI. J Physiol. 1999a Oct 15;520 Pt 2:315-20.
34. Kivela J, Parkkila S, Parkkila AK, Rajaniemi H. A low concentration of carbonic
anhydrase isoenzyme VI in whole saliva is associated with caries prevalence. Caries Res.
1999b May-Jun;33(3):178-84.
35. Kuriakose S, Sundaresan C, Mathai V, Khosla E, Gaffoor FM. A comparative study of
salivary buffering capacity, flow rate, resting pH, and salivary Immunoglobulin A in children
with rampant caries and caries-resistant children. J Indian Soc Pedod Prev Dent. 2013 Apr-
Jun;31(2):69-73.
36. Lagerlof F, Oliveby A. Caries-protective factors in saliva. Adv Dent Res. 1994
Jul;8(2):229-38.
37. Laine MA, Tolvanen M, Pienihakkinen K, Soderling E, Niinikoski H, Simell O, et al.
The effect of dietary intervention on paraffin-stimulated saliva and dental health of children
participating in a randomized controlled trial. Arch Oral Biol. 2014 Feb;59(2):217-25.
81
38. Leinonen J, Kivela J, Parkkila S, Parkkila AK, Rajaniemi H. Salivary carbonic
anhydrase isoenzyme VI is located in the human enamel pellicle. Caries Res. 1999 May-
Jun;33(3):185-90.
39. Lenander-Lumikari M, Loimaranta V. Saliva and dental caries. Adv Dent Res. 2000
Dec;14:40-7.
40. Leone CW, Oppenheim FG. Physical and chemical aspects of saliva as indicators of
risk for dental caries in humans. J Dent Educ. 2001 Oct;65(10):1054-62.
41. Leonor SP, Laura SM, Esther IC, Marco ZZ, Enrique AG, Ignacio MR. Stimulated
saliva flow rate patterns in children: A six-year longitudinal study. Arch Oral Biol. 2009
Oct;54(10):970-5.
42. Levine M. Susceptibility to dental caries and the salivary proline-rich proteins. Int J
Dent. 2011;2011:953412.
43. Liang H, Wang Y, Wang Q, Ruan MS. Hydrophobic interaction chromatography and
capillary zone electrophoresis to explore the correlation between the isoenzymes of salivary
alpha-amylase and dental caries. J Chromatogr B Biomed Sci Appl. 1999 Mar 19;724(2):381-
8.
44. Lilienthal B. An analysis of the buffer systems in saliva. J Dent Res. 1955
Aug;34(4):516-30.
45. Llena-Puy C. The role of saliva in maintaining oral health and as an aid to diagnosis.
Med Oral Patol Oral Cir Bucal. 2006 Aug;11(5):E449-55.
46. Marsh PD. Dental plaque as a biofilm: the significance of pH in health and caries.
Compend Contin Educ Dent. 2009 Mar;30(2):76-8, 80, 3-7; quiz 8, 90.
47. Martins C, Buczynski AK, Maia LC, Siqueira WL, Castro GF. Salivary proteins as a
biomarker for dental caries--a systematic review. J Dent. 2013 Jan;41(1):2-8.
48. Ministério da Saúde. Pesquisa Nacional de
Saúde Bucal - Resultados Principais. Brasil: Ministério da Saúde; 2010.
49. Moimaz SA, Borges HC, Saliba O, Garbin CA, Saliba NA. Early Childhood Caries:
Epidemiology, Severity and Sociobehavioural Determinants. Oral Health Prev Dent.
2016;14(1):77-83.
50. Nobre dos Santos M, Melo dos Santos L, Francisco SB, Cury JA. Relationship among
dental plaque composition, daily sugar exposure and caries in the primary dentition. Caries
Res. 2002 Sep-Oct;36(5):347-52.
82
51. Ozturk LK, Furuncuoglu H, Atala MH, Ulukoylu O, Akyuz S, Yarat A. Association
between dental-oral health in young adults and salivary glutathione, lipid peroxidation and
sialic acid levels and carbonic anhydrase activity. Braz J Med Biol Res. 2008
Nov;41(11):956-9.
52. Parisotto TM, Santos MN, Rodrigues LK, Costa LS. Behavior and progression of early
carious lesions in early childhood: a 1-year follow-up study. J Dent Child (Chic). 2012 Sep-
Dec;79(3):130-5.
53. Parisotto TM, Steiner-Oliveira C, Duque C, Peres RC, Rodrigues LK, Nobre-dos-
Santos M. Relationship among microbiological composition and presence of dental plaque,
sugar exposure, social factors and different stages of early childhood caries. Arch Oral Biol.
2010 May;55(5):365-73.
54. Parkkila S, Kaunisto K, Rajaniemi L, Kumpulainen T, Jokinen K, Rajaniemi H.
Immunohistochemical localization of carbonic anhydrase isoenzymes VI, II, and I in human
parotid and submandibular glands. J Histochem Cytochem. 1990 Jul;38(7):941-7.
55. Parkkila S, Parkkila AK, Rajaniemi H. Circadian periodicity in salivary carbonic
anhydrase VI concentration. Acta Physiol Scand. 1995 Jun;154(2):205-11.
56. Parkkila S, Parkkila AK, Vierjoki T, Stahlberg T, Rajaniemi H. Competitive time-
resolved immunofluorometric assay for quantifying carbonic anhydrase VI in saliva. Clin
Chem. 1993 Oct;39(10):2154-7.
57. Pastorekova S, Parkkila S, Pastorek J, Supuran CT. Carbonic anhydrases: current state
of the art, therapeutic applications and future prospects. J Enzyme Inhib Med Chem. 2004
Jun;19(3):199-229.
58. Preethi BP, Reshma D, Anand P. Evaluation of Flow Rate, pH, Buffering Capacity,
Calcium, Total Proteins and Total Antioxidant Capacity Levels of Saliva in Caries Free and
Caries Active Children: An In Vivo Study. Indian J Clin Biochem. 2010 Oct;25(4):425-8.
59. Proctor GB. The physiology of salivary secretion. Periodontol 2000. 2016
Feb;70(1):11-25.
60. Ribeiro CC, Tabchoury CP, Del Bel Cury AA, Tenuta LM, Rosalen PL, Cury JA.
Effect of starch on the cariogenic potential of sucrose. Br J Nutr. 2005 Jul;94(1):44-50.
61. Roa NS, Chaves M, Gomez M, Jaramillo LM. Association of salivary proteins with
dental caries in a Colombian population. Acta Odontol Latinoam. 2008;21(1):69-75.
83
62. Rogers J, Palmer AR, Kolenbrander P, Scannapieco F. Role of Streptococcus gordonii
amylase-binding protein A in adhesion to hydroxyapatite, starch metabolism, and biofilm
formation 1. Infect Immun. 2001;69(11):7046-56.
63. Rogers JD, Haase EM, Brown AE, Douglas CW, Gwynn JP, Scannapieco FA.
Identification and analysis of a gene (abpA) encoding a major amylase-binding protein in
Streptococcus gordonii. Microbiology. 1998 May;144 ( Pt 5):1223-33.
64. Rogers JD, Palmer RJ, Jr., Kolenbrander PE, Scannapieco FA. Role of Streptococcus
gordonii amylase-binding protein A in adhesion to hydroxyapatite, starch metabolism, and
biofilm formation. Infect Immun. 2001 Nov;69(11):7046-56.
65. Ruiz Miravet A, Montiel Company JM, Almerich Silla JM. Evaluation of caries risk in
a young adult population. Med Oral Patol Oral Cir Bucal. 2007 Sep;12(5):E412-8.
66. Scannapieco F, Torres G, Levine M. Salivary alpha-amylase: role in dental plaque and
caries formation. Crit Rev Oral Biol Med. 1993;4(3-4):301-7.
67. Scannapieco FA, Bergey EJ, Reddy MS, Levine MJ. Characterization of salivary
alpha-amylase binding to Streptococcus sanguis. Infect Immun. 1989 Sep;57(9):2853-63.
68. Scannapieco FA, Torres GI, Levine MJ. Salivary amylase promotes adhesion of oral
streptococci to hydroxyapatite. J Dent Res. 1995 Jul;74(7):1360-6.
69. Sheiham A, James WP. Diet and Dental Caries: The Pivotal Role of Free Sugars
Reemphasized. J Dent Res. 2015 Oct;94(10):1341-7.
70. Shimotoyodome A, Kobayashi H, Tokimitsu I, Hase T, Inoue T, Matsukubo T, et al.
Saliva-promoted adhesion of Streptococcus mutans MT8148 associates with dental plaque
and caries experience. Caries Res. 2007;41(3):212-8.
71. Singh S, Sharma A, Sood PB, Sood A, Zaidi I, Sinha A. Saliva as a prediction tool for
dental caries: An in vivo study. J Oral Biol Craniofac Res. 2015 May-Aug;5(2):59-64.
72. Sly WS, Hu PY. Human carbonic anhydrases and carbonic anhydrase deficiencies.
Annu Rev Biochem. 1995;64:375-401.
73. Supuran CT, Scozzafava A. Carbonic anhydrases as targets for medicinal chemistry.
Bioorg Med Chem. 2007 Jul 1;15(13):4336-50.
74. Szabo I. Carbonic anhydrase activity in the saliva of children and its relation to caries
activity. Caries Res. 1974;8(2):187-91.
75. Tenovuo J. Salivary parameters of relevance for assessing caries activity in individuals
and populations. Community Dent Oral Epidemiol. 1997 Feb;25(1):82-6.
84
76. Torres SR, Nucci M, Milanos E, Pereira RP, Massaud A, Munhoz T. Variations of
salivary flow rates in Brazilian school children. Braz Oral Res. 2006 Jan-Mar;20(1):8-12.
77. Tukia-Kulmala H, Tenovuo J. Intra- and inter-individual variation in salivary flow
rate, buffer effect, lactobacilli, and mutans streptococci among 11- to 12-year-old
schoolchildren. Acta Odontol Scand. 1993 Feb;51(1):31-7.
78. Tulunoglu O, Demirtas S, Tulunoglu I. Total antioxidant levels of saliva in children
related to caries, age, and gender. Int J Paediatr Dent. 2006 May;16(3):186-91.
79. Vacca-Smith AM, Venkitaraman AR, Quivey RG, Jr., Bowen WH. Interactions of
streptococcal glucosyltransferases with alpha-amylase and starch on the surface of saliva-
coated hydroxyapatite. Arch Oral Biol. 1996 Mar;41(3):291-8.
80. Van Nieuw Amerongen A, Bolscher JG, Veerman EC. Salivary proteins: protective
and diagnostic value in cariology? Caries Res. 2004 May-Jun;38(3):247-53.
81. Vitorino R, de Morais Guedes S, Ferreira R, Lobo MJ, Duarte J, Ferrer-Correia AJ, et
al. Two-dimensional electrophoresis study of in vitro pellicle formation and dental caries
susceptibility. Eur J Oral Sci. 2006 Apr;114(2):147-53.
82. Yildiz G, Ermis RB, Calapoglu NS, Celik EU, Turel GY. Gene-environment
Interactions in the Etiology of Dental Caries. J Dent Res. 2016 Jan;95(1):74-9.
85
APÊNDICE
Produção bibliográfica da aluna Thayse Rodrigues de Souza durante o Mestrado.
1. Souza TR, Silva IHM, Carvalho AT, Gomes VB, Duarte AP, Leão JC, Gueiros LAM.
Juvenile Sjögren Syndrome: Distinctive Age, Unique Findings. Ped Dent. 2012 Sep;
34 (5):427-30.
2. Leão Filho JC, Braz AK, Souza TR, de Araújo RE, Pithon MM, Tanaka OM. Optical
coherence tomography for debonding evaluation: an in-vitro qualitative study. Am J
Orthod Dentofacial Orthop. 2013 Jan; 143(1):61-8.
3. Souza TR, Carvalho AT, Duarte AP, Porter SR, Leão JC, Gueiros LAM. Th1 and Th2
polymorphisms in Sjogren’s syndrome and rheumatoid arthritis. J Oral Pathol Med.
2014;43: 418-26.
86
ANEXOS
ANEXO 1 – Certificado do Comitê de Ética em Pesquisa da FOP- UNICAMP
87
ANEXO 2- Autorização da Secretaria Municipal de Saúde de Piracicaba-SP para
realização da pesquisa
88
ANEXO 3- Ficha clínica utilizada na coleta de dados
89
ANEXO 4- Declaração
Declaração
As cópias dos documentos de minha autoria já submetidos para publicação em revistas
científicas sujeitos a arbitragem, que constam da minha tese de Doutorado intitulada
“RELAÇÃO ENTRE A ATIVIDADE DA ANIDRASE CARBÔNICA VI, ALFA-AMILASE
SALIVAR, CAPACIDADE TAMPÃO, FLUXO SALIVAR E CÁRIE DENTAL EM
CRIANÇAS” não infringem os dispositivos da Lei nº 9.610/98, nem o direito autoral de
qualquer editora.
Piracicaba, 05 de Maio de 2016.
Autora: Thayse Rodrigues de Souza Leão
RG: 567954
Orientadora: Marinês Nobre dos Santos Uchôa
RG: 416.641
90
ANEXO 5- Confirmação de envio do artigo para publicação – Caries Research
91
ILUSTRAÇÕES
FIGURA 1. Exame clínico para avaliação do índice de cárie em pré-escolares do município de
Piracicaba-SP (Capítulo 1 e 2)
92
FIGURA 2. Cárie precoce da infância (Capítulo 1 e 2)
93
FIGURA 3. Forma de coleta de saliva estimulada (capítulos 1 e 2).
94
FIGURA 4. Bochecho com solução de sacarose 20% (Capítulos 1 e 2).
95
FIGURA 5. Material utilizado na coleta de saliva (Capítulos 1 e 2).
FIGURA 6. Metodologia de avaliação da capacidade tampão.
96
FIGURA 7. Metodologia de avaliação da atividade do fluxo salivar estimulado.
97
FIGURA 8. Metodologia de avaliação da atividade da enzima Anidrase Carbônica VI.
A: Bancada preparada para o preparo das soluções que dariam origem aos géis.
B: Preparo das amostras utilizadas na corrida eletroforética: Amostras centrifugadas por 10
minutos a 5000 rpm a 4º C.
C: Preparo das amostras adicionando 100 µl da amostra e 100µl do tampão da amostra.
D: Componentes utilizados do kit de eletroforese Mini protein tetra cell, Bio-Rad
E: Limpeza e montagem dos vidros no suporte, colocação do gel de corrida e de separação e
em seguida o pente.
F: Remoção do pente, visualização das lacunas onde serão colocadas as amostras preparadas
para a corrida.
G: Pipetagem de 10 µl de amostra preparada em cada lacuna.
H: Lacunas preenchidas.
I: Montagem do gel na cuba.
J: Colocação do tampão de corrida até a linha demarcada.
K: Cuba preparada e colocada em geladeira a -4°C.
L: Conexão da cuba ao aparelho.
M: Ajuste de voltagem, 140 Volts e tempo 1h:50min
N, O, P: Após a corrida realizada remoção do gel, feita marcação para identificar sequência
das amostras.
Q: Colocação cuidadosa do gel em recipiente contendo 250 ml de corante de azul de
bromotimol 0.1%.
98
R: Recipiente colocado em máquina agitadora por 10 min em velocidade 02.
S: Preparo de solução contendo 250 ml de água deionizada e 30g de gelo seco deixada por 10
min.
T, U: Imersão do gel em recipiente contendo água deionizada e gelo seco.
V: Posicionamento do gel. Fotografia com câmera Samsung Digital Variplan zoom 4.0 –
72mm, 18x.
99
FIGURA 9. Metodologia de avaliação da atividade da enzima Alfa-amilase salivar.
A: Centrífuga utilizada.
B: Amostras diluídas.
C: Espectofotômetro.
D: Cubetas para espectofotometria.
