CENTRO DE PESQUISA EM CIÊNCIAS DA SAÚDE · ANEXO A – Termo de consentimento Livre e Esclarecido...
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Londrina 2015 CENTRO DE PESQUISA EM CIÊNCIAS DA SAÚDE Mestrado Profissional em Exercício Físico na Promoção da Saúde MARIO CARLOS WELIN BALVEDI SUPLEMENTAÇÃO DE L-ARGININA E DESEMPENHO MUSCULAR – EVIDÊNCIAS CIENTÍFICAS
Transcript of CENTRO DE PESQUISA EM CIÊNCIAS DA SAÚDE · ANEXO A – Termo de consentimento Livre e Esclarecido...
Londrina 2015
CENTRO DE PESQUISA EM CIÊNCIAS DA SAÚDE Mestrado Profissional em Exercício Físico na Promoção da Saúde
MARIO CARLOS WELIN BALVEDI
SUPLEMENTAÇÃO DE L-ARGININA E DESEMPENHO
MUSCULAR – EVIDÊNCIAS CIENTÍFICAS
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Londrina 2015
SUPLEMENTAÇÃO DE L-ARGININA E DESEMPENHO
MUSCULAR – EVIDÊNCIAS CIENTÍFICAS
Relatório Técnico apresentado ao Centro de Pesquisa em Ciências da Saúde, Universidade Norte do Paraná, Unidade Piza, como requisito para obtenção do título de Mestre Profissional em Exercício Físico na Promoção da Saúde.
Orientador: Prof. Dr. Andreo Fernando Aguiar
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MÁRIO CARLOS WELIN BALVEDI
SUPLEMENTAÇÃO DE L-ARGININA E DESEMPENHO
MUSCULAR – EVIDÊNCIAS CIENTÍFICAS
Relatório Técnico apresentado ao Centro de Pesquisa em Ciências da Saúde, Universidade Norte do Paraná, Unidade Piza, como requisito para obtenção do título de Mestre Profissional em Exercício Físico na Promoção da Saúde, conferido e aprovado pela Banca Examinadora:
BANCA EXAMINADORA
____________________________________ Prof. Dr. Andreo Fernando Aguiar
Universidade Norte do Paraná (Orientador)
____________________________________ Prof. Dr. Cosme Franklim Buzzachera
Universidade Norte do Paraná
____________________________________ Prof. Dr. Leandro Ricardo Altimari
(Membro Externo)
____________________________________ Prof. Dr. Dartagnan Pintos Guedes
(Coordenador do Curso)
Londrina, _____de ___________de _____.
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BALVEDI, Mário Carlos Welin. Suplementação de L-arginina e Desempenho Muscular – Evidências Científicas. 73f. Relatório Técnico. Mestrado Profissional em Exercício Físico na Promoção da Saúde. Centro de Pesquisa em Ciências da Saúde. Universidade Norte do Paraná, Londrina. 2015.
RESUMO
O presente relatório técnico apresenta uma descrição detalhada das fases de elaboração do guia prático intitulado: Suplementação de L-arginina e Desempenho Muscular – Evidências Científicas. Adicionalmente, apresentamos a produção científica submetida ao American Journal of Clinical Nutrition, que expressa a interface entre as atividades de pesquisa e conteúdo prático apresentado no guia. A elaboração do guia prático foi realizada em 4 etapas: (1) Seleção de artigos científicos, (2) Definição dos tópico do guia prático, (3) Arte final do guia prático, e (4) Solicitação do International Standard Book Number (ISBN). Com base no conteúdo dos artigos científicos selecionados, os seguintes tópicos foram descritos: (i) Nossa proposta, (ii) L-arginina: definições e funções, (iii) Fontes alimentares de L-arginina, (iv) Mecanismos de ação da L-arginina sobre a função endotelial durante o exercício físico, (v) Efeitos ergogênicos da L-arginina - Evidências científicas, (vi) Possíveis efeitos colaterais e contraindicações, (vii) Doses utilizadas, (viii) Nosso estudo, e (ix) Considerações finais. Subsequentemente, um profissional de design gráfico foi contratado para concluir os processos de criação, diagramação e arte final do guia prático. O material produzido neste trabalho será posteriormente submetido à análise editorial, visando à solicitação do número ISBN, e subsequente divulgação do produto. Este guia prático foi elaborado para atender as características peculiares do curso de Mestrado Profissional em Exercício Físico na Promoção da Saúde da presente IES, cujo escopo principal é apresentar um produto técnico para auxiliar na atuação profissional direcionada ao mercado de trabalho. A compilação de informações contidas em ambas as partes deste relatório técnico, poderá contribuir substancialmente para o entendimento dos reais benefícios da suplementação de L-arginina sobre a saúde endotelial e muscular de diferentes populações, auxiliando, assim, profissionais da área da saúde na tomada de decisão em relação ao uso deste suplemento no contexto prático e clínico. Palavras-chave: guia prático, L-arginina, exercício físico, desempenho muscular, suplemento.
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BALVEDI, Mário Carlos Welin. L-arginine suplementation and muscle performance – Scientifc evidence. 73f. Technical Report. Professional Master´s in Exercise in Health Promotion. Research Center on Health Sciences. North University of Paraná, Londrina. 2015.
ABSTRACT
This technical report shows a detailed description of the phases of preparation of practical guide entitled: L-arginine Supplementation and Muscular Performance - Scientific Evidence. Additionally, we have shown the scientific manuscript submitted to the American Journal of Clinical Nutrition, which express the interface between research activities and practical content displayed on practical guide. The preparation of the Practical Guide was performed in 4 steps: (1) Selection of scientific papers, (2) Definition of the topic of the Practical Guide, (3) Final art of practical guide, and (4) Request the International Standard Book Number (ISBN). Based on the content of the selected papers, the following topics were discussed: (i) Purpose of guide, (ii) L-arginine: definitions and functions, (iii) Food sources of L-arginine, (iv) Mechanisms of actions of L-arginine on endothelial function during exercise, (v) Ergogenic effects of L-arginine - scientific evidence, (vi) Possible side effects and contraindications, (vii) There are optimal dose?, (viii) Our study, and (ix) Final considerations. Subsequently, a graphic designer was hired to conclude the processes of creation, layout and final art of practical guide. The material produced in this work will be submitted to editorial review, in order to request the ISBN number, and subsequent dissemination of the product. This practical guide was designed to meet the peculiar characteristics of the Professional Master in Physical Exercise in Health Promotion of this IES, in which the main scope is to present a technical product to help the professional work in the labor market. The compilation of information contained in each part of this technical report can substantially contribute to the understanding of the real benefits of L-arginine supplementation on endothelial and muscle health in different populations, thereby helping healthcare professionals in making decision regarding the use of this supplement in the practical and clinical context. Keywords: practical guide, L-arginine, physical exercise, muscle performance, muscular, supplement
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SUMÁRIO
1. INTRODUÇÃO ........................................................................................................ 7
2. REVISÃO DE LITERATURA – CONTEXTUALIZAÇÃO ........................................ 9
3. DESENVOLVIMENTO .......................................................................................... 13
4. CONCLUSÃO ....................................................................................................... 18
5. REFERÊNCIAS ..................................................................................................... 19
APÊNDICE A – Guia prático ................................................................................... 277
APÊNDICE B – Trabalho Apresentado em Evento Científico ............................... 4242
APÊNDICE C – Artigo Científico ............................................................................... 43
ANEXO A – Termo de consentimento Livre e Esclarecido ....................................... 73
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1. INTRODUÇÃO
O óxido nítrico [do termo inglês: Nitric Oxide (NO)] é uma importante
molécula endógena anti-aterogênica, envolvida em vários processos fisiológicos e
patológicos (Hou et al. 1999). A liberação de NO das células endoteliais promove
dilatação arterial para aumentar o fluxo sanguíneo (Wu e Meininger, 2002),
manutenção da elasticidade endotelial (Kinlay et al. 2001), inibição da adesão e
agregação de plaquetas nas paredes da artéria (Preli et al. 2002). No entanto, a
reduzida biodisponibilidade de NO têm sido associada a vários fatores de risco para
aterosclerose, incluindo hipercolesterolemia, hipertensão, diabetes tipo II, tabagismo
e envelhecimento - todas das quais são condições associadas com
comprometimento da função endotelial (Taddei et al. 2001; Barua et al. 2001;
Stühlinger et al. 2001;. Bitar et al. 2005; Hermann et al. 2006;. Schrage et al. 2007;
de Beer et al. 2013). Considerando que existe uma correlação positiva entre
biossíntese de NO e função endotelial (Barua et al. 2001), parece óbvio pensar que
intervenções com o objetivo de aumentar a biodisponibilidade de NO são
particularmente importantes para melhoria da saúde vascular e capacidade de
exercício em condições fisiológicas acometidas pela reduzida biossíntese de NO,
como o envelhecimento. Portanto, a manutenção de um sistema vascular saudável
apresenta significante relevância clínica para uma variedade de populações
associadas à disfunção endotelial.
O envelhecimento tem sido apontado como importante fator de risco
cardiovascular, e tem sido associado com progressivo comprometimento da função
endotelial (Bode-Böger et al. 2003; Schrage et al. 2007). Vários estudos em
humanos e animais têm demonstrado que a biodisponibilidade de NO e
vasodilatação dependente do endotélio (VDE) declinam progressivamente com a
idade (Taddei et al. 2000; Muller-Delp et al. 2002; Toma et al. 1985; Mayhan et al.
1990; Egashira et al. 1993; Schrage et al. 2007; Lesniewski et al. 2009), predispondo
a população idosa a maior risco de doença vascular aterosclerótica (Shimokawa,
1999). O envelhecimento também está associado com reduzida capacidade para
realizar exercícios (Holloszy and Kohrt, 1995) e isto pode ser atribuído, pelo menos
em parte, ao prejudicado fluxo sanguíneo e liberação de oxigênio para os músculos
ativos (Lawrenson et al. 2003; Poole et al. 2003; Proctor et al. 2003). Portanto,
estratégias de intervenções, visando melhorar a biodisponibilidade de NO, tais como
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o exercício físico regular (ex: o treinamento aeróbio) e/ou suplementos
vasodilatadores (ex: L-Arginina) poderiam ser potencialmente importantes para
melhorar a saúde vascular e função muscular durante o processo de
envelhecimento.
Enquanto não há dúvida de que o exercício físico regular promove
benefícios sobre a função endotelial e muscular (Warburton et al. 2006;. Golbidi e
Laher, 2013), um limitado número de estudos investigou os efeitos da
suplementação de L-arginina sobre função endotelial e desempenho muscular de
indivíduos idosos (Chauhan, 1996; Blum et al. 2000; Bode-Boger, 2003;. Wilson et
al. 2007), e resultados contraditórios têm sido relatados. Enquanto alguns autores
demonstraram melhora significativa na função endotelial (avaliada pela VDE) em
indivíduos idosos saudáveis (idade: 73,8 ± 2,7 anos) após suplementação de L-
arginina (16g/dia, durante 14 dias) (Bode-Boger et al. 2003), outros não observaram
diferenças significantes na vasodilatação em mulheres pós-menopáusicas
saudáveis, após um mês de suplementação de L-arginina (9 g/dia) (Blum et al.
2000), e até mesmo um efeito prejudicial da suplementação de L-arginina a longo
prazo (3g/dia, durante 6 meses) sobre a função endotelial (avaliada como
biodisponibilidade de NO e vasodilatação) em indivíduos idosos (idade 73 ± 9 anos)
com doença arterial periférica (DAP) (Wilson et al. 2007). Portanto, há ainda muito
para se aprender sobre os possíveis efeitos ergogênicos da suplementação de L-
arginina na função endotelial e desempenho muscular em diferentes populações,
sob condições saudáveis ou patológicas.
O presente estudo foi realizado para expandir as observações
supracitadas, com o objetivo de examinar os possíveis efeitos da suplementação de
L-arginina sobre a função endotelial e muscular em diferentes populações. O
entendimento dos possíveis efeitos benéficos da suplementação de L-arginina
poderá auxiliar profissionais da área da saúde na tomada de decisão em relação ao
uso deste suplemento no contexto prático e clínico.
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2. REVISÃO DE LITERATURA – CONTEXTUALIZAÇÃO
O processo fisiológico de envelhecimento é associado ao
progressivo declínio na função endotelial, que predispõe a população de idosos a
maior risco de trombose, doença vascular aterosclerótica, isquemia cardíaca e
doença vascular cerebral (Luscher et al. 1993; Wu e Thiagarajan, 1996; Shimokawa,
1999). O envelhecimento também está associado com a redução na capacidade de
realizar exercícios (Holloszy e Kohrt, 1995), e isto pode ser devido, em parte, ao
reduzido fluxo sanguíneo e liberação de oxigênio aos músculos exercitados
(Lawrenson et al. 2003; Poole et al. 2003; Proctor et al. 2003). Muller-Delp et al.
(2002) demonstraram que a vasodilatação dependente do endotélio é realmente
reduzida nas artérias e arteríolas do músculo ‘oxidativo’ sóleo em ratos velhos, mas
não em ratos novos, sugerindo assim uma diminuída distribuição do fluxo sanguíneo
periférico.
Teoricamente, a reduzida hiperemia muscular (aumento do volume
sanguíneo para o músculo exercitado) e consequente intolerância ao exercício,
observada em sujeitos idosos saudáveis (Proctor e Parker, 2006), pode ser
explicada pela diminuída biodisponibilidade de óxido nítrico [do inglês: Nitric Oxide
(NO)] – um potente mediador da vasodilatação dependente do endotélio (Taddei et
al. 2000; Schrage et al. 2007). De fato, estudos recentes confirmam que a
biodisponibilidade de NO e do aminoácido essencial L-Arginina (substrato para
síntese de NO) são reduzidos com o avanço da idade (Taddei et al. 2000, Delp et al.
2008; Sindler et al. 2011), e que esta redução está intimamente relacionada com o
aparecimento de fatores de risco cardiovasculares (Celermajer et al. 1994; Quyyumi
et al. 1995) e intolerância ao exercício físico (Proctor e Parker, 2006).
O importante papel vasodilatador da via do NO também tem sido
demonstrado em estudos envolvendo sujeitos jovens saudáveis (Schrage et al.
2004; Mortensen et al. 2007). Nestes estudos, os autores observaram que a inibição
local de NO [mediante a infusão intra-arterial de NG-monomethyl-L-arginine (L-
NMMA) ou NG-nitro-L-arginine methyl ester (L-NAME)] reduziu o fluxo sanguíneo
para os músculos ativos durante os exercícios de handgrip (Schrage et al. 2004) e
extensão de pernas (Mortensen et al. 2007), indicando que a biodisponibilidade de
NO é fundamental para o controle da vasodilatação endotelial durante o exercício
físico. Tal fato confirma a hipótese de que a menor disponibilidade de NO nas
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células endoteliais de sujeitos idosos pode, de fato, contribuir para o surgimento de
disfunções endoteliais (Lauer et al. 2001) e redução na capacidade de realizar
exercícios (Proctor e Parker, 2006).
O NO é um gás solúvel (molecular) que consiste na ligação co-
valente entre um átomo de nitrogênio e um átomo de oxigênio. A sua produção no
organismo humano ocorre quando o aminoácido L-arginina é convertido em L-
citrulina numa reação catalisada pela enzima óxido nítrico sintase (NOS) (Ckless et
al. 2007). A L-arginina é considerada um aminoácido semiessencial, porque o corpo
normalmente a produz em quantidades suficientes. No entanto, a suplementação de
L-arginina pode ser necessária em algumas condições especiais, como (1) a
desnutrição, (2) a produção de amônia em excesso, (3) as infecções, (4) as
perturbações da síntese de ureia, e/ou (5) a septicemia (Appleton, 2002).
Considerando que a L-arginina é o principal substrato endógeno para a síntese de
NO (Cynober et al. 1995), parece lógico pensar que a suplementação deste
aminoácido pode aumentar a produção de NO (Nichols et al. 1994) e,
consequentemente, a vasodilatação periférica (Álvares et al. 2011; Tang et al. 2011).
Como resultado, a suplementação de L-arginina pode aumentar a perfusão
sanguínea (fluxo sanguíneo aos tecidos corporais por meio de capilares) e
teoricamente promover maior liberação de nutrientes e oxigênio para os tecidos
ativos durante o exercício, aumentar a síntese proteica e facilitar a recuperação
muscular (Álvares et al. 2012).
A suplementação de L-arginina também pode melhorar a função
endotelial por vários mecanismos indiretos, tais como: (1) a diminuição plasmática
da concentração de endotelina-1, substância vasoconstritora presente em níveis
elevados em pacientes com disfunção endotelial (Boulanger e Luscher, 1990), (2) a
maior liberação de insulina, glucagon e hormônio do crescimento [do inglês: growth
hormone (GH)], que podem aumentar o fluxo sanguíneo e a vasodilatação (Böger et
al. 1996), e (3) a competição direta com a dimetilarginina assimétrica [do inglês:
asymmetric dimethylarginine (ADMA)] – um inibidor endógeno da NOS (Bode-Böger
et al. 2003). Sugere-se também que a L-arginina apresente propriedade
antioxidante, que consiste no sequestro de radicais superóxidos, reduzindo a
oxidação da lipoproteína de baixa densidade [do inglês: Low Density Lipoprotein
(LDL)] (Wascher et al. 1997). Em um estudo envolvendo suplementação de L-
arginina e alfa-tocoferol, em coelhos hipercolesterolêmicos, os autores observaram
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redução da formação de lesões e espessamento da camada íntima arterial,
aumentando o relaxamento dependente do endotélio e reduzindo a progressão das
placas ateroscleróticas (Böger et al. 1995). Adicionalmente, Wiel et al. (2000)
observaram que a L-arginina preveniu a disfunção endotelial e lesões nas artérias de
camundongos previamente tratados com lipopolissacarídeo (LPS). Os resultados
destes estudos e muito outros (Clarkson et al. 1996; Creager et al. 1992; Pieper et
al. 1996) sugerem que a suplementação da L-arginina pode ser uma estratégia não
farmacológica eficaz para tratamento de indivíduos com fatores de risco para
aterosclerose, tais como: hipercolesterolemia, hipertensão, diabetes mellitus,
insuficiência renal, hiper-homocisteinemia, cigarro e envelhecimento – condições
associadas com reduzida biossíntese de NO (Clarkson et al. 1996; Creager et al.
1992; Pieper et al. 1996). No entanto, as concentrações fisiológicas de L-arginina
(40-100 μmol·L–1) em sujeitos saudáveis, são suficientes para saturar a NOS
endotelial (saturação: 2–20 μmol·L–1) e produzir NO (Durante et al. 2007). Portanto,
teoricamente, a suplementação de L-arginina não deveria promover aumento na
atividade da enzima NOS e, por conseguinte, na produção de NO em sujeitos
saudáveis – esta condição foi denominada de “paradoxo da L-arginina” (Bogle et al.
1992; Bogle et al. 1996; Brunini et al. 2003; Bode-Böger et al. 2007).
Apesar dos efeitos benéficos da L-arginina não terem sido
confirmados em sujeitos jovens saudáveis (Álvares et al. 2012), o importante papel
vasodilatador deste aminoácido, mediado pela síntese de NO (Álvares et al. 2011;
Tang et al. 2011), pode ser de suma importância para promover maior fluxo
sanguíneo periférico, aumentar da tolerância ao exercício físico, preservar a aptidão
física e melhorar a qualidade de vida de sujeitos idosos. Neste contexto, Chen et al.
(2010) investigaram os efeitos de um suplemento contendo 5,2 g de L-arginina, em
uma mistura com L-citrulina, 500 mg de ácido ascórbico, 400 UI de vitamina E, 400
ug de ácido fólico, 300 mg de L-taurina, e 10 mg de ácido alfa-lipóico na forma de
pó, sobre desempenho máximo de ciclistas idosos (50-73 anos) do gênero
masculino. Os resultados deste estudo demonstraram significante aumento na
produção de potência (22.51 watts) referente ao limiar anaeróbio, no grupo L-
arginina comparado ao grupo placebo, indicando um potencial aumento da
capacidade aeróbia. Adicionalmente, Bode-Böger et al. (2003) demonstraram que a
suplementação de L-arginina pode atenuar os efeitos negativos do envelhecimento
sobre a função endotelial, presumidamente pela normalização da relação entre a
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arginina plasmática e concentrações de ADMA. Coletivamente, os resultados destes
estudos indicam que a suplementação de L-arginina pode ser uma estratégia
benéfica para reverter os efeitos negativos do envelhecimento sobre a função
endotelial e, consequentemente, melhorar a aptidão aeróbia de sujeitos idosos.
No entanto, até o momento, nenhum estudo foi conduzido para
avaliar se os efeitos benéficos da suplementação de L-arginina sobre a
vasodilatação dependente do endotélio podem refletir na melhora do desempenho
muscular em indivíduos idosos. Considerando que a biodisponibilidade de NO é
reduzida com o avanço da idade (Taddei et al. 2000, Delp et al. 2008; Sindler et al.
2011) e que a L-arginina é o principal substrato para a produção de NO (Cynober et
al. 1995), é provável que a suplementação de L-arginina possa favorecer o aumento
da produção de NO e, consequentemente, uma maior resposta vasodilatadora
durante o exercício físico em sujeitos idosos. Por conseguinte, os sujeitos idosos
poderiam aumentar o desempenho muscular, preservar a aptidão física e melhorar a
sua qualidade de vida. Portanto, o objetivo deste estudo foi investigar os possíveis
efeitos da suplementação aguda de L-arginina sobre a função endotelial e
desempenho muscular de mulheres idosas.
Com base no conhecimento teórico e prático produzido a partir deste
trabalho foram elaborados: 1) um guia prático (APÊNDICE A) com informações
científicas sobre o uso da suplementação de L-arginina para melhora do
desempenho muscular, 2) um resumo (APÊNDICE B) apresentado em congresso
científico internacional, e 3) um artigo científico (APÊNDICE C), no qual se
investigou os efeitos da suplementação de L-arginina sobre a função endotelial e
desempenho muscular de mulheres idosas. A compilação de informações contidas
em todas as partes deste relatório técnico poderá contribuir substancialmente para o
entendimento dos reais benefícios da suplementação de L-arginina sobre a saúde
endotelial e muscular de diferentes populações, auxiliando, assim, profissionais da
área da saúde na tomada de decisão em relação ao uso deste suplemento no
contexto prático e clínico.
A seguir apresentamos a descrição metodológica adotada para
elaboração do guia prático, de acordo com as características peculiares do curso de
Mestrado Profissional em Exercício Físico na Promoção da Saúde da presente IES,
cujo escopo principal é apresentar um produto técnico que auxilie na atuação
profissional direcionada ao mercado de trabalho.
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3. DESENVOLVIMENTO
O presente guia prático (APÊNDICE A) foi elaborado de acordo com
as seguintes etapas:
Etapa 1: Seleção de artigos científicos
Para seleção dos artigos científicos foram utilizados os seguintes
descritores da Medical Subject Headings (MeSH - MEDLINE), em língua inglesa:
arginine (arginina), nitric oxide (óxido nítrico) e vasodilation (vasodilatação), skeletal
muscle (músculo esquelético), hypertrophy (hipertrofia), muscle strength (força
muscular), athletic performance (desempenho atlético), na base de dados eletrônica
Pubmed (www.pubmed.com). Os termos foram combinados em 3 modos: [arginine +
vasodilation + nitric oxide], [arginine + skeletal muscle + hypertrophy] e [arginine +
muscle strength + athletic performance], utilizando-se o operador “AND” para
combinar os descritores utilizados. Dois pesquisadores efetuaram a busca na base
de dados selecionada, considerando o período de 1995 a 2014.
Foram identificados inicialmente 4.324 estudos, sendo 4.286 para a
combinação dos descritores arginine + vasodilation + nitric oxide, 25 para arginine +
skeletal muscle + hypertrophy, e 13 para arginine + muscle strength + athletic
performance (Figura 1). Os critérios para inclusão dos estudos foram: 1) artigos
originais, 2) estudos que avaliaram os efeitos a L-arginina sobre a função muscular
e/ou endotelial; 3) estudos realizados com sujeitos humanos, 4) estudos que
apresentaram resumo, 5) estudos publicados em jornais de língua inglesa, 6)
estudos com metodologia apropriada, e 7) estudos com análise estatística
adequada. Após a aplicação dos critérios, foram selecionados 5 artigos na busca
para combinação dos descritores arginine + vasodilation + nitric oxide, 2 para
arginine + skeletal muscle + hypertrophy, e 3 para arginine + muscle strength +
athletic performance, totalizando 10 estudos para elaboração do guia prático (Figura
1 e Tabela 1).
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Figura 1. Abordagem metodológica utilizada para seleção dos estudos.
Tabela 1. Artigos incluídos no estudo
Estudo Periódico Amostra Idade (anos)
Tipo de estudo
Yavuz et al. (2014) Biology of Sport 9 lutadores de elite do sexo
Masculino.
21-28 Transversal
Wax et al. (2013) International Journal of Exercise Science 19 homens jovens saudáveis. 18-21 Transversal
Alvares et al. (2012) Applied Physiology Nutrition and Metabolism 15 homens saudáveis, com
experiência prévia em TR. 20-30 Transversal
Wax et al. (2012) The Journal of Applied Research 19 mulheres jovens saudáveis,
treinadas e não treinadas
19-22 Transversal
Wax et al. (2012) Journal of the International Society of Sports
Nutrition
16 homens jovens saudáveis,
treinados e não treinados.
18-24 Transversal
Geer and Jones (2011) Journal of Strength and Conditioning
Research
12 homens jovens treinados. 19-26 Transversal
Angeli et al. (2007) Revista Brasileira de Medicina do Esporte 20 homens jovens saudáveis. 17-18 Transversal
Campbell et al. (2006) Nutrition 35 homens treinados. 30-50 Transversal
Santos et al. (2002) Isokinetics and Exercise Science 12 homens jovens saudáveis. 20-27 Transversal
Stevens et al. (2000) Medicine & Science in Sports & Exercises 13 homens jovens saudáveis. 19-23 Transversal
TR: treinamento resistido
16
Etapa 2: Definição dos tópicos do guia prático
Após a seleção dos artigos científicos, foram definidos os tópicos
para compor o conteúdo do guia prático. Os seguintes tópicos foram descritos:
(1) Nossa proposta
Este tópico apresenta uma breve descrição dos objetivos e proposta
do guia prático, bem como a finalidade de apresentação deste conteúdo como
requisito parcial para obtenção do título de Mestre no curso de Mestrado Profissional
em Exercício Físico na Promoção da Saúde, da Universidade Norte do Paraná
(UNOPAR).
(2) O aminoácido L-arginina
Este tópico apresenta uma breve descrição das características
bioquímicas do aminoácido L-arginina, bem como seu papel fundamental na síntese
de óxido nítrico (do termo em inglês: Ntitric Oxide [NO]).
(3) Fontes alimentares de L-arginina
Este tópico apresenta um quadro comparativo, com a descrição das
principais fontes alimentares que contém elevadas concentrações de L-arginina.
(4) Mecanismos de ação da L-arginina sobre a função endotelial
durante o exercício físico
Este tópico aborda os principais mecanismos de ação da L-arginina
sobre a produção de NO e função endotelial (por exemplo: vasodilatação) durante o
exercício físico.
(5) Efeitos ergogênicos da L-arginina - Evidências científicas
Este tópico apresenta um quadro comparativo dos resultados de
estudos que investigaram os possíveis efeitos da suplementação de L-arginina sobre
o desempenho muscular, em diferentes populações.
(6) Doses utilizadas
Este tópico apresenta um quadro comparativo dos resultados de
estudos que utilizaram diferentes doses de L-arginina, em diferentes populações.
17
(7) Possíveis efeitos colaterais e contraindicações
Este tópico aborda os possíveis efeitos colaterais e contraindicações
associados ao uso agudo e crônico de diferentes doses de L-arginina.
(8) Nosso estudo
Este tópico contém informações sobre os objetivos, metodologia,
resultados e conclusão do estudo científico desenvolvido em conjunto com o guia
prático.
(9) Considerações finais
Este tópico apresenta informações baseadas em evidências
científicas, sobre os possíveis efeitos benéficos da suplementação de L-arginina
para melhora do desempenho muscular.
Etapa 3: Arte final do guia prático
Após a descrição do conteúdo, um profissional de Design Gráfico foi
contratado para concluir os processos de criação, diagramação e arte final do guia
prático. Para tanto, foram utilizados os programas de edição de imagens
CorelDRAW (versão: 7.1) e Photoshop (versão: 7.1), considerando as dimensões do
guia de 21,0 x 29,7cm.
Etapa 4: Solicitação do International Standard Book Number
(ISBN)
O guia prático será posteriormente submetido à análise de mérito na
editora da Universidade Norte do Paraná (UNOPAR editora), visando à solicitação
do número ISBN, e subsequente divulgação do material.
18
4. CONCLUSÃO
Até o momento, não há suficientes evidências científicas para
recomendar o uso da suplementação de L-arginina como potencial adicional
ergogênico para aumentar o desempenho muscular em diferentes populações.
Futuros estudos são necessários para testar os possíveis efeitos agudos e crônicos
da suplementação de L-arginina, bem como os mecanismos primários de ação deste
suplemento sobre a função endotelial e muscular.
19
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27
APÊNDICE A – Guia prático
42
APÊNDICE B – Trabalho Apresentado em Evento Científico
EFEITOS DA SUPLEMENTAÇÃO AGUDA DE L-ARGININA SOBRE A FUNÇÃO ENDOTELIAL DE MULHERES IDOSAS Mário Carlos Welin Balvedi1, Douglas Kratki da Silva1, Guilherme Atsushi Muraoka1, Rafael Mendes Pereira1, Vanda Cristina Sanches1, Walquiria Batista de Andrade1 e Andreo Fernando Aguiar1. 1Centro de Ciências Biológicas e da Saúde, UNOPAR, Londrina, PR, Brasil. [email protected] Introdução: O processo fisiológico de envelhecimento é associado ao progressivo declínio na função endotelial, que pode ser explicado pela diminuída biodisponibilidade de óxido nítrico [do inglês: Nitric Oxide (NO)] e do aminoácido essencial L-Arginina (substrato para síntese de NO), que resulta na redução da hiperemia muscular. Objetivo: A proposta do presente estudo foi avaliar os possíveis efeitos da suplementação aguda de L-Arginina sobre a função endotelial (fluxo sanguíneo) em mulheres idosas. Metodologia: 20 mulheres idosas (idade: 65–80 anos) foram divididas em dois grupos (N = 10/grupo): Placebo (PLA – recebendo substância inerte) e L-arginina (ARG – recebendo L-arginina, Sigma Aldrich®). Os suplementos foram administrados na dose única de 8g (em idêntico formato e sabor), em uma solução contendo 400 ml de H2O. Ambos os grupos foram submetidos ao exame de ultrassonografia com Doppler (System FiVe; GE Medical Systems), para medida do fluxo sanguíneo da artéria femoral (2 a 3 cm proximal à bifurcação para os segmentos superficial e profundo da artéria), antes [10 min.] e após [80 min.] o momento de suplementação, de acordo com as propriedades farmacocinéticas da L-aginina. Os dados foram avaliados por meio de testes de ANOVA [grupo (2) x tempo (2)] para medidas repetidas. Foi adotado nível α de significância de P < 0,05. Resultados: Nenhuma diferença significante (P > 0,05) foi observada entre os grupos do momento pré para o momento pós-suplementação (Figura 1). Conclusão: A suplementação aguda de L-arginina não promove efeitos adicionais sobre a função endotelial (fluxo sanguíneo) em mulheres idosas. Balvedi MCW, Da Silva DK, Muraoka GA, Pereira RM, Sanches VC, Andrade WB, Aguia AF. Efeitos da suplementação aguda de L-arginina sobre a função endotelial de mulheres idosas. 37º Simpósio Internacional de Ciências do Esporte – Lições e sonhos na atividade física e no exercício (CELAFISCS). São Paulo, Brasil. 2014. p. 54-54.
43
APÊNDICE C – Artigo Científico
Acute L-Arginine supplementation fails to improve endothelial function
and muscle performance in older women
Artigo submetido para publicação no American Journal of Clinical Nutrition (Fator de
impacto: 6.9; Qualis A1)
ABSTRACT
Background: Age-related endothelial dysfunction (e.g., reduced vasodilatation) is associated
with reduced NO bioavailability and consequenty decline in exercise capacity. Given that L-
arginine (L-arg) is the only substrate for NO synthesis, it is expected that its supplementation
increases peripheral vasodilatation and, thus, improve muscle performance in older women.
Objective: This study examined the effects of L-arg supplementation on the peripheral
vasodilatation and muscle performance in older women. Design: In a double-blind,
randomized, placebo-controlled study, 20 old women were randomly assigned in a double-
blind fashion to either an L-arg (ARG, N = 10) or placebo (PLA, N = 10) group. Both groups
performed the Doppler ultrasound exam (to assess the femoral artery vasodilatation), and a
battery of muscle performance tests (to assess the isokinetic, isometric, and functional
strength), 80 minutes after L-arg supplementation (a single oral dose of 8g). Results: Femoral
artery blood flow (ARG: 443.9 ± 42.8 vs. PLA: 373.1 ± 40.8 ml∙min-1; P > 0.05) and area
(ARG: 0.45 ± 0.03 vs. PLA: 0.41 ± 0.02 cm2; P > 0.05) were similar between groups at basal
condition, and remained unchanged after supplementation. Following the bout of exercise,
blood flow increased ~160% above basal, with no significant (P > 0.05) difference between
the ARG and PLA groups. Additionally, there were no significant (P > 0.05) differences
between ARG and PLA groups for all strength variables (isokinetic, isometric and functional).
Conclusion: Acute L-arg supplementation provides no ergogenic effect on endothelial
function and muscle performance in older women.
44
INTRODUCTION
Nitric oxide (NO) is an important endogenous anti-atherogenic molecule involved in several
pathological and physiological processes (Hou et al. 1999). NO release from endothelial cells
promotes arterial dilatation to increase blood flow (Wu and Meininger, 2002), maintaining
endothelial elasticity (Kinlay et al. 2001), inhibition platelet adhesion and aggregation to
artery walls (Preli et al. 2002), and enhance aerobic capacity for exercise (Maxwell et al.
2005). Nonetheless, the reduced NO bioavailability has been associated with atherosclerosis
risk factors, including hypercholesterolemia, hypertension, type II diabetes,
hyperhomocysteinemia, smoking, and aging – all of which are conditions that are associated
with impairment of endothelial function (Taddei et al. 2001; Barua et al. 2001; Stühlinger et
al. 2001; Bitar et al. 2005; Hermann et al. 2006; Schrage et al. 2007; de Beer et al. 2013).
Aging is a well-documented cardiovascular risk factor, associated with progressive
impairment of endothelial function (Bode-Böger et al. 2003; Schrage et al. 2007). Several
human and animals studies have shown that NO bioavailability and endothelium-dependent
vasodilatation (EDV) progressively declines with age (Taddei et al. 2000; Muller-Delp et al.
2002; Toma et al. 1985; Mayhan et al. 1990; Egashira et al. 1993; Schrage et al. 2007;
Lesniewski et al. 2009). This condition predispose the elderly population to reduced blood
flow and oxygen delivery to contracting muscles (Lawrenson et al. 2003; Poole et al. 2003;
Proctor et al. 2003), reducing the exercise capacity (Holloszy & Kohrt, 1995). Therefore,
strategy of interventions improving NO bioavailability, such as regular physical exercise (e.g.,
aerobic training) and/or vasodilators agents (e.g., L-Arginine [L-arg]), would thus be expected
to enhanced vascular health and muscle function during aging process.
While there is no question that regular physical exercise improves endothelial and
muscle function (Warburton et al. 2006; Golbidi and Laher, 2013), a limited number of the
studies have investigated the effects of L-arg supplementation on the endothelial function in
45
older subjects (Chauhan 1996; Blum et al. 2000; Bode-Böger et al. 2003; Wilson et al. 2007),
and contradictory results have been reported. While some authors have reported a significant
improve in vascular endothelial function (assessed as endothelium-dependent vasodilatation
[EDV]) in healthy old subjects (age 73.8 ± 2.7 yr) after short-time L-arg supplementation (16
daily for 14 days) (Bode-Böger et al. 2003), others have reported no significant improve in
flow-mediated dilation (FMD) in healthy postmenopausal women following 1 month of L-arg
supplementation (9g/day) (Blum et al. 2000), and even a detrimental effect of long-term L-arg
supplementation (3g/day for 6 months) on endothelial function (assessed as NO
bioavailability and FMD) in older subjects (age 73 ± 9 yr) with peripheral arterial disease
(PAD) (Wilson et al. 2007). Therefore, there is still much to learn about the effects of L-arg
supplementation on endothelial and muscle function in elderly subjects.
The present study was designed to extend the above observations by examining the
effect of acute L-arg supplementation on endothelial function and muscle performance in
older women. Given that aging-related endothelial dysfunction (e.g. reduced vasodilatation) is
associated with reduced NO bioavailability, and that L-arg is the only substrate for NO
synthesis, we hypothesized that L-arg supplementation would increase peripheral
vasodilatation (e.g., femoral artery blood flow and area) and, thus, improve muscle
performance in older women.
46
METHODS
Experimental design
A 2 group, randomized, double-blind, placebo-controlled design with repeated measures was
performed to examine the effects of acute L-Arginine (ARG) supplementation on the
endothelial function and muscle performance in older women (Fig.1). Initially, all subjects
completed a 2-wk familiarization period (3 non-consecutive d per wk, for a total of 6
sessions) involving the muscular tests to minimize potential learning effects. Subsequently,
the subjects were matched according to age and body mass, and then randomly assigned in a
double-blind fashion to an L-Arginine (ARG, N = 10) or Placebo (PLA, N = 10) group. Both
the ARG and PLA groups completed a battery of muscular tests on 3 separate occasions (V1,
V2, and V3), with a 1-week interval between visits. During the first visit (V1) to the
laboratory, the participants were underwent a Doppler ultrasound examination to femoral
artery blood flow and area measurements (see blood flow measurement) at pre (-10 min) and
post (+ 80 min) supplementation, and immediately after the isokinetic strength test (see
isokinetic strength measurements). Moreover, the systemic arterial blood pressure, and heart
rate were registered before to collect blood samples at baseline (20 min. before
supplementation), after a 10-min period of quiet rest in the sitting position. On the second
visit (V2), the participants performed the maximal isometric strength test in order to assess
maximal strength in short-duration, high-intensity static activities (see isometric strength
measurements). Finally, on the third visit (V3), the participants completed a battery of lower-
body functional tests (Sit-stand, Tandem gait, e Timed Up and Go), in order to assess
submaximal functional capacity during daily life activities (see Lower-body functional tests).
The muscular tests (isokinetic, isometric, and functional) were performed on 3 separate
occasions (V1, V2, and V3, respectively) to be initiated at same time-point (80 min after
supplementation) and avoid the effects of prior fatigue on subsequent tests. To control any
47
influence of diet, each participant completed a 3-day dietary intake record (including 1
weekend day) prior to their first visit. Moreover, subjects were instructed to maintain their
habitual daily activities and diet throughout study, but to refrain from any strenuous activity
and avoid consuming foods rich in [NO2-] and [NO3-] for the 3-d period prior to each visit
(see dietary control).
Figure 1. Experimental design.
Subjects
Physically activity older women than 65 years were invited to attend a meeting aimed at
explaining the purpose and details of the study protocol. To qualify as participants, the
subjects were required to 1) be aged between 65 and 85 years, 2) not be smokers or alcoholic,
3) not be obese [body mass index (BMI) < 30 kg/m2], 4) not be hypertensive [blood pressure
< 140/90 mmHg (systolic/diastolic)], 5) have not ingested any supplement or medication that
could limit or improve the physical performance, 7) have a detailed description of their
lifestyle and daily food intake, and 8) have medical approval for the practice of physical
48
exercise. All subjects were free of any significant medical problems (i.e., prior stroke,
myocardial infarction, claudication, liver disease, and impaired renal function) and
cardiovascular risk factors (i.e., diabetes, low HDL, hypercholesterolaemia, and
hyperlipidemia). Twenty women [mean (SD): age 71.6 (5.9) yr, height 152.7 (5.6) cm, and
body mass 61.9 (8.6) kg] who met these criteria volunteered to participate in the study. The
physical characteristics of the ARG and PLA groups at baseline are presented in Table 1. All
subjects were informed of the procedures, risks, and benefits of the investigation and signed
an informed consent document approved by the Institutional Review Board of the University
(protocol no: 28443714.0.0000.0108). All procedures were performed according to the
principles outlined in the 1964 Declaration of Helsinki.
Table 1. Baseline hematology and physical characteristics of the L-
arginine (ARG) and placebo (PLA) groups
ARG (N = 10) PLA (N = 10)
Age (yr) 70.6 ± 2.2 72.5 ± 1.6
Body mass (kg) 62.9 ± 3.3 60.4 ± 2.1
Height (cm) 153.2 ± 2.3 152.2 ± 1.1
BMI (kg∙m2) 27.1 ± 1.0 26.1 ± 0.7
HR (beats/min) 76.5 ± 3.5 70.3 ± 2.8
Systolic BP (mm Hg) 121.5 ± 6.0 124.3 ± 5.5
Diastolic BP (mm Hg) 73.8.1 ± 1.7 75.8 ± 2.9
Total cholesterol (mg/dL) 189.3 ± 7.7 172.9 ± 7.1
HDL cholesterol (mg/dL) 50.3 ± 4.0 50.3 ± 4.8
LDL cholesterol (mg/dL) 120.1 ± 9.2 96.1 ± 7.5
Triglycerides (mg/dL) 146.9 ± 19.1 132.3 ± 19.7
Plasma insulin (µU/ml) 11.7 ± 2.2 12.9 ± 2.5
Femoral artery blood flow (ml∙min-1) 443.9 ± 42.8 373.1 ± 40.8
Femoral artery area (cm2) 0.45 ± 0.03 0.41 ± 0.02
Values are mean ± SEM. BMI, body mass index; HR, heart rate; BP,
blood pressure. There were no differences between the groups
49
Blood pressure and heart rate measurements
Systemic arterial blood pressure was registered with an automatic monitor (Omron MX3 Plus,
Bannockburn, USA), previously validated (Coleman et al. 2005). The subjects were placed in
a seated position and the measurement was performed in the left arm, according to the
recommendations of the V Brazilian Guidelines on Hypertension (V Brazilian Guidelines in
Arterial Hypertension, 2007) and American Heart Association (Pickering et al. 2005). The
heart rate was registered using a heart rate monitor (Polar RS800CX, Kempele, Finland).
Nutrient intake
To control any influence of diet, each participant was supervised by nutritionists to complete a
3-day dietary intake record (including 1 weekend day) prior to their first visit; standard
portions were used to assess the amount of food and drink consumed. The total energy intake
and macronutrient amounts were calculated using software for nutritional assessment
(Avanutri, version 3.1.4, Rio de Janeiro-RJ, Brazil). Participants were instructed to maintain
their habitual daily activities and diet throughout study, but to refrain from any strenuous
activity and avoid consuming foods rich in [NO2-] and [NO3-] for the 3-d period prior to each
visit. A list describing foods to be avoided was distributed to the subjects to simplify their
dietary choices for low NO2– and NO3– foods. This list was developed following the estimate
of NO2– and NO3– in food items, as proposed by Griesenbeck et al. (2009). The participants
were also instructed to report any adverse events from the supplements on their health status
during the laboratory visits. No adverse events were reported by the participants.
Supplementation
ARG group received orally 8 g of encapsulated L-arg (Sigma Aldrich®) dissolved in water
(400 mL), while the PLA group ingested an identical-looking and equivalent amount of the
placebo (as cornstarch), in a double-blind and randomized manner. The L-arg and placebo
50
were analyzed for purity and validated prior to the study. To ensure the double-blind design,
an individual who was not involved in the study was responsible for placing the supplements
into bags and labeling the capsules with the subjects’ names according to the randomization
list. We chose to provide 8 g of L-arginine because such a dose would be safe and well
tolerated when consumed orally (Böger, 2007) and has been reported to increase
vasodilatation in older subjects (Bode-Böger et al. 2003). The Doppler ultrasound (blood
flow) examination and physical tests were initiated 80 min after supplementation (Fig. 1),
based on the pharmacokinetics properties of L-arginine: it has been reported that the maximal
concentration of plasma L-arginine is reached ~90 min following the ingestion of 6 g of L-
arginine (Bode-Böger et al., 1998).
Blood flow measurement
On the first visit 1 (V1) to the laboratory, subjects were underwent a Doppler ultrasound
examination to femoral artery blood flow and area measurements (Fig. 2). Longitudinal
images of the femoral artery and blood velocity measurements were made as previously
described (26) using a 10-MHz linear array pulse Doppler ultrasound probe (Acuson Aspen,
Siemens, Malvern, Pennsylvania) placed on the skin surface 2–3 cm proximal to bifurcation
of the femoral artery into the superficial and profundus branch. The position was chosen to
minimize turbulence from the bifurcation and interference of blood flow to the inguinal region
(Rådegran, 1997). The probe insonation angle relative to the skin was 45 deg. Measurements
were taken in late diastole (Schrage, 2007). Femoral artery blood flow (Q) was calculated as
the product of cross-sectional area (CSA) and mean blood velocity (V) at the same location on
the vessel (Tang et al. 2011), according to the following equation:
51
Q = V x CSA.
Cross-sectional area was calculated by the following equation:
CSA = πr2,
where r = femoral artery radius as determined by quantitative analysis of the longitudinal
images obtained of the femoral artery. The equipment’s software automatically calculates the
artery blood flow and area following determination of vessel local by ultrasound’s operator.
To establish intra-rater reliability of blood flow measurement, the same experienced
ultrasound operator (a cardiology physician with more than 5 years experience in ultrasound
measurements) blinded in respect to treatments (ARG or PLA) performed all measurements.
Previous analysis revealed a strong and significant intra-rater reliability (ICC: > 0.94, P <
0.0001) for femoral artery diameter and blood flow velocity measurements (Billinger and
Kluding, 2009).
Figure 2. Doppler ultrasound image of femoral artery blood flow and area from a representative
subject from the L-arginine group (ARG, N = 10) at baseline (a,b), pre-exercise (c.d), and immediately
post-exercise (e,f). Note an increase in the blood flow from pre- to post-exercise, and no difference
from basal to- pre-exercise. Interestingly, area remained unchanged from basal to post-exercise.
52
Familiarization protocol
All subjects completed a 2-wk orientation program (3 non-consecutive d per wk, for a total of
6 sessions) before randomization (ARG or PLA), involving the muscular tests in an effort to
minimize potential learning effects and establish the reliability of the testing protocols. The
sessions consisted of repeated practicing of each protocol and involved teaching specific
exercise techniques for each test (isokinetic, isometric, and functional tests). Qualified
personnel supervised individually each participant during the familiarization period. Maximal
effort in each test was requested during the last 3 sessions to reduce any learning effects and
to make the data consistent. The intraclass correlation coefficients were ≥0.97 for each test,
indicating the elimination of the learning curve for the subjects. All familiarization sessions
and physical tests were performed at the same location, between 9 and 12 am.
Isokinetic strength test
On the first visit (V1) to the laboratory, the subjects performed 3 sets of 8 maximum
repetitions of unilateral (dominant leg) knee extension/flexion at 60o/s, with 1 min rest
between sets, using a Biodex System 3 Isokinetic Dynamometer (Biodex, Inc., Shirley, NY)
(Fig. 1). The range of motion at the knee joint was 90 to 10o of knee flexion (0° = full knee
extension). Prior to the isokinetic test, the subjects performed a specific warm-up that
consisted of 8 submaximal repetitions of knee extension/flexion at 60o/s, during which the
subjects were advised not to perform at maximal effort. Subjects were set up on the
dynamometer in a comfortable, upright, seated position. Straps with Velcro were used to
stabilize the thigh, pelvis, and trunk to prevent extraneous body movement. The axis of the
dynamometer was aligned with the axis of rotation of the right knee joint, according to the
body dimensions of each subject. Arms were placed across the chest with hands grasping the
straps. These settings adjustments were accurately identical in each subject. Qualified
personnel supervised each participant individually during every test. Subjects were given
53
verbal encouragement in an attempt to achieve maximal effort in each set, and they were
instructed to exhale during the contractions. The strength variables analyzed were peak torque
(Nm), mean torque (Nm), set total work (J), and average power. Calibration of the Biodex
dynamometer was performed according to the specifications of the manufacturer before each
test. The validity coefficient of this equipment is 0.99 (Drouin et al. 2004), and the test-retest
reliability for measuring peak torque during knee extension is high in older adults (0.89
[Symons et al. 2005]).
Isometric strength test
Maximal unilateral isometric force of the knee extensor muscles was recorded at 90° of knee
flexion with a dynamometer (Microfet, Hoggan Health Industries, Inc., Murray, UT), fixed in
a commercial knee extensor machine. Subjects were instructed to extend the dominant leg as
rapidly as possible upon command for four seconds. Four trials were conducted, with 1-min
rest between trials, and the greatest effort was recorded. Qualified personnel supervised each
participant individually during every test. Subjects were given verbal encouragement in an
attempt to achieve maximal effort, and they were instructed to exhale during the contractions.
Lower-body functional tests
Ability to performance daily life tasks is very importance for the older population since
functional capacity is a prerequisite for independent living. To examine the effects of oral L-
arg supplementation on lower extremity functional performance, the participants completed
the sit-stand (modified version from Gotshalk et al. 2002), Tandem gait (Gotshalk et al.
2002), and Timed Up and Go (Podsiadlo and Richardson, 1991) tests. Briefly, to test the
ability to rise from a chair without using arms, a straight-backed chair (40 cm high) was used.
From a seated position with arms folded across chest, the subject was instructed to stand up
and sit down as quickly as possible three times. The tandem gait test consisted of subjects
54
walking as fast as possible along a 6-m line with each foot in tandem position (heel of one
foot directly in front of and in contact with the toe of the other foot) (Gotshalk et al. 2002),
while being timed for speed. The Timed Up and Go test consisted of subjects stand up from
the chair, walking along a 3-m line to and around a cone, and returning to the chair as fast as
possible and sit down again. Each test was repeated 3 times, with 1-min rest between tests,
and the shortest time was recorded. A digital stopwatch was used for timing (to the neared
0.01 s), and each trial started on technician’s command. If the third test was the fastest, further
tests were conducted until a test slower than the previous one was performed.
Statistical analyses
Data are expressed as the means ± SEM. A two-way ANOVA with repeated measures on 2
factors (2 x 3; group x time) was used to identify differences in blood flow and area across
time and between ARG and PLA groups. A two-way ANOVA with repeated measures on 2
factors (2 x 3; group x set) was used to identify differences in strength variables between
ARG and PLA groups. When significant differences were confirmed with ANOVA, multiple
comparisons testing were performed using Bonferroni post hoc analysis to identify these
differences. Two-tailed unpaired Student t test was utilized to identify differences between
groups in isometric and functional strength data. The level of significance was set at P < 0.05.
Statistical analyses were performed using SPSS statistical analysis software (SPSS version
20.0; Chicago, IL, USA).
55
RESULTS
Participant characteristics
All participants (CR, N = 10; PL, N = 10) completed the study. The baseline characteristics of
the subjects are presented in Table 1. Both the CR and the PL groups had similar (P > 0.05)
baseline hematology and physical characteristics. In addition, no significant (P > 0.05)
differences in the daily dietary intakes were observed between the groups (data not shown).
Femoral artery blood flow
The representative Doppler image used to quantify femoral artery blood flow is shown in Fig.
3, and the corresponding data for each group is presented in Fig. 3. Rates of femoral artery
blood flow were similar between groups (mean ± SEM) at basal condition (ARG: 443.9 ±
42.8 vs. PLA: 373.1 ± 40.8 ml∙min-1; P > 0.05), and remained unchanged after
supplementation (pre-exercise, ARG: 402.5 ± 46.1 vs. PLA: 491.3 ± 45.8 ml∙min-1; P > 0.05).
Following the bout of exercise, femoral artery blood flow increased ~160% above basal, with
no significant (P > 0.05) difference between the ARG and PLA groups.
Figure 3. Femoral artery blood flow in L-arginine (ARG, N = 10) and placebo (PLA, N = 10) groups
at basal, pre-exercise, and immediately post-exercise. Values are means ± SEM. * P < 0.05 compared
to the basal and pre-exercise values for both ARG and PLA groups.
56
Femoral artery area
The representative Doppler image used to quantify femoral artery area is shown in Fig. 2, and
the corresponding data for each group is presented in Fig. 4. Femoral artery area were similar
between groups (mean ± SEM) at basal condition (ARG: 0.45 ± 0.03 vs. PLA: 0.41 ± 0.02
cm2; P > 0.05), and remained unchanged after supplementation (pre-exercise, ARG: 0.44 ±
0.01 vs. PLA: 0.42 ± 0.03 cm2; P > 0.05) and exercise (post-exercise, ARG: 0.45 ± 0.02 vs.
PLA: 0.42 ± 0.02 cm2; P > 0.05)
Figure 4. Femoral artery area in L-arginine (ARG, N = 10) and placebo (PLA, N = 10) groups at
basal, pre-exercise, and immediately post-exercise Values are means ± SEM. There were no
differences between the groups in each time point.
57
Isokinetic and isometric strength performance
The isokinetic and isometric strength performances for each group are presented in Table 3
and Fig. 5, respectively. There were no significant (P > 0.05) differences between ARG and
PLA groups over the 3 sets of the exercise for all isokinetic strength variables (mean torque,
peak torque, peak torque/BW, set total work, and average power), in both knee extensors and
flexors muscles. Additionally, there were no significant (P > 0.05) differences in isometric
strength between ARG and PLA groups.
Table 3. Isokinetic strength data of knee extensors and flexors muscles of the L-arginine
(ARG) and placebo (PLA) groups
ARG (N = 10) PLA (N = 10)
Variables Extension Flexion Extension Flexion
Mean torque (Nm)
First set 45.1 ± 4.3 30.4 ± 2.0 45.2 ± 3.6 29.0 ± 2.6
Second set 49.0 ± 5.2 32.7 ± 2.1 48.9 ± 3.8 29.5 ± 2.1
Third set 50.0 ± 5.2 33.6 ± 2.0 49.1 ± 3.5 30.6 ± 2.0
Peak torque (Nm)
First set 82.6 ± 6.9 47.6 ± 2.7 80.6 ± 5.4 40.7 ± 3.3
Second set 87.0 ± 8.1 49.9 ± 3.1 86.2 ± 5.0 44.1 ± 2.8
Third set 87.8 ± 8.9 51.4 ± 3.3 87.8 ± 4.8 48.2 ± 2.7
Peak torque/BW (%)
First set 131.1 ± 8.5 73.1 ± 2.4 134.8 ± 9.0 69.0 ± 6.6
Second set 136.6 ± 9.6 76.6 ± 3.1 143.4 ± 7.2 74.3 ± 5.6
Third set 144.1 ± 8.6 78.9 ± 3.3 146.5 ± 7.8 81.1 ± 5.4
Set total work (J)
First set 68.9 ± 5.2 45.9 ± 2.3 67.8 ± 6.5 36.4 ± 4.8
Second set 72.9 ± 5.8 47.1 ± 1.9 71.4 ± 6.2 40.9 ± 3.4
Third set 72.9 ± 2.4 48.9 ± 1.9 72.4 ± 5.8 44.9 ± 3.1
Average power (Watts)
First set 46.1 ± 4.3 28.9 ± 2.1 45.2 ± 3.7 23.0 ± 3.5
Second set 50.7 ± 5.6 30.9 ± 2.6 49.5 ± 3.8 26.4 ± 2.6
Third set 51.4 ± 5.8 32.7 ± 2.4 47.2 ± 2.7 27.2 ± 2.8
Values are mean ± SEM. There were no differences between the groups
58
Figure 5. Isometric peak torque for L-arginine (ARG, N = 10) and placebo (PLA, N = 10) groups.
Values are means ± SEM. There were no differences between the groups.
Lower body functional performance
The functional performance for each group is presented in Fig. 6. There were no significant
differences (mean ± SEM) in the Tandem gait (ARG: 16.8 ± 1.2 vs. 18.8 ± 1.3 sec, P > 0.05),
Sit Stand (ARG: 4.9 ± 0.1 vs. 5.1 ± 0.3 sec, P > 0.05), and Timed Up and Go (ARG: 7.2 ± 0.3
vs. 7.4 ± 0.4 sec, P > 0.05) tests between ARG and PLA groups.
Figure 6. Time required to complete the Tandem gait, sit stand and timed up and go tests for L-
arginine (ARG, N = 10) and placebo (PLA, N = 10) groups. Values are means ± SEM. There were no
differences between the groups in each test.
59
DISCUSSION
To our knowledge, this is the study first that has examined the effects of oral L-arg
supplementation on endothelium function and muscle performance in older women. Given
that aging-related endothelial dysfunction (e.g. reduced vasodilatation) is associated with
reduced availability of NO, and that L-arg is the only substrate for NO synthesis, we
hypothesized that L-arg supplementation would increase peripheral vasodilatation (e.g.,
femoral artery blood flow) and, thus, improve muscle performance in healthy older women.
The major and unexpected findings of this study were that L-arg supplementation does not
improve the lower body muscle performance (assessed as isokinetic, isometric, and functional
tests) and endothelial function (assess as femoral artery blood flow and area), during rest and
exercise conditions in older women.
In contrast to our hypothesis, L-arg supplementation did not promote any beneficial
effect on endothelial function (assess as femoral artery blood flow and dilatation) during rest
and exercise conditions. This result is contradictory to others previous studies that showed
improve in endothelial function after L-arg supplementation in older subjects (Chauhan et al.
1996; Bode-Böger et al. 2003). Considering that L-arg supplementation has been described
for increasing NO levels (Boger et al. 1998), it is unclear why the endothelial function
remained unchanged in ARG group in current study. There were three possibilities that may
explain the discrepancy with above-cited studies (by Chauhan et al. 1996; Bode-Boger et al.
2003). First, in contrast to our sample of normotensive older women (BP < 130/90 mmHg),
the older subjects cohort in studies from Chauhan et al. (1996) and Bode-Boger et al. (2003)
displayed BP up to 150/90 mmHg, indicating that the subjects could potentially to be
hypertensive (Aronow et al. 2011). Previous findings have shown that hypertensive subjects
have worse endothelial function (e.g., attenuated NO bioavailability) (Brunner et al. 2005;
Lüscher TF and Vanhoutte, 1986; Panza et al. 1990; Hermann et al. 2006) and may be more
60
responsive to L-arginine infusion, compared to normotensive subjects (Taddei et al. 1997). In
fact, a recent well-conducted meta-analysis showed that oral L-arginine supplementation is
more effective to improve endothelial function in subjects with impaired endothelial function
(e.g., lower FMD and NO bioavailability) (Bai et al. 2009). Thus, the lack of a significant
effect of L-arg supplementation on endothelial function in our subjects (physically active and
normotensive) may be due to preserved initial endothelial health. A possible mechanism that
could explain this condition is the preservation of NO bioavailability reported in physically
active older (Taddei et al. 2000) and well-trained young (Liu et al. 2009) subjects, but not in
sedentary elderly (Taddei et al. 2000) and hypertensive subjects (Forte et al 1997). Therefore,
L-arg supplementation may be necessary only for individuals with endothelial dysfunction.
Our results are consistent with this assertion.
Second, our examined the effects of L-arg supplementation on endothelial function of
the femoral artery, while the studies from Chauhan et al. (1996) and Bode-Böger et al. (2003)
investigated the brachial and coronary arteries, respectively. Previous studies have shown that
femoral artery exhibit blunted vasodilator responsiveness to endothelium-dependent
vasodilators agents compared to brachial artery (Newcomer et al. 2004), suggesting
fundamental differences between femoral and brachial arteries. Additionally, several animals
and human studies have reported differences in blood flow between forearm and leg for a
variety of stimulus (Laughlin et al. 1998; Imadojemu et al. 2001; Pawelczyk and Levine,
2002). These findings of heterogeneous vascular responses in both human and animals
suggest regional differences in endothelium function throughout vascular tree. Thus, it seems
plausible to think that L-arg supplementation may be unable to improve endothelial function
in blood vessels with blunted vasodilator responsiveness; this could explain the lack of effect
L-arg on femoral artery in our study and others (Tang et al. 2011), in contrast to that was
observed in brachial (Chauhan et al. 1996) and coronary (Bode-Böger et al. 2003) arteries.
61
These regional differences in function may be attributed to variation between blood vessels in
contribution of the endothelial factors (e.g., composition of neurotransmitters, receptor
expression and activation, structural proteins, and variation in the signal transduction
pathway) to the overall response (Hill et al. 2001).
Finally, the lack of effect of L-arg supplementation on endothelial function may be
attributed to compensatory mechanisms of loss NO bioavailability in early vascular aging
(Puzservoa et al. 2014). In study of Puzservoa et al. (2014) the authors reported that reduced
NO-dependent vasorelaxation in adult rats (age 22-week-old) was fully compensated by
accentuation of NO-independent mechanisms. This explanation has recently been suggested
in the study of Ballard et al. (2014) in which the authors showed no change in FMD after
blocking NO [following infusion of the inhibitor N(G)-monomethyl-L-arginine (L-NMMA)]
and vasodilator prostanoids (PN) (following infusion of the ketorolacin + L-NMMA) in
healthy older adults. The authors argue that compensatory vasodilator pathways [e.g.,
cytochrome P (CYP) 450 epoxygenases and endothelium-derived hyperpolarizing factor
(EDHF) may contribute to conduit artery FMD during aging. Therefore, it is likely that in our
healthy older women the vascular health (e.g., NO bioavailability and FMD) has been
maintained by NO-independent vasodilators mechanisms. This could explain, at least in part,
the lack of effects of L-arg supplementation on endothelial function. These protective
mechanisms has been confirmed in previous studies that failure to significantly alter FMD
with LNMMA (Pyke et al. 2010; Parker et al. 2011; Wray et al. 2013), indicating potential
involvement of additional vasodilators agents [e.g., vasodilators prostanoids (PN) (Duffy et al.
1999) and EDHF (Bellien et al. 2006; Bellien et al. 2008)]. However, there are several doubts
about the effects of vasodilators on endothelial function in different arteries, including the fact
that NO may not be obligatory to promote FMD (Pyke et al. 2010). Therefore, there is still
62
much to learn about the role of NO and L-arg supplementation on endothelial function,
especially in the elderly population.
The lack of effect of L-arg supplementation on endothelial function was associated
with no improvement in the muscle performance (e.g., strength and functional capacity) in
ARG group compared to PL group. This result is consistent with findings of previous studies
involving L-arg supplementation in young subjects (Álvares et al. 2012; Wax et al. 2012). For
example, Álvares et al. (2012) reported increase in muscle blood volume, but no additional
effect on muscle strength variables (peak torque, total work, and set total work), 80 min after
L-arg ingestion (single dose of 6 g). In addition, Wax et al. (2012) demonstrated that L-arg
supplementation [3000mg of L-arginine alpha-ketoglutarate (AAKG)] had no additional
effect on upper (barbell bench press) or lower body (leg press) 1RM strength and total load
volume (TLV) in either resistance trained or untrained men. However, others authors have
reported increases in anaerobic performance (Campbell et al. 2006; Elam et al. 1989) and
muscular endurance (Santos et al. 2001) after L-arg supplementation. The reasons for the
conflicting results are unclear but might be due to loading period (Wax et al. 2012), given that
the ergogenic effects of L-arg supplementation on muscle performance has been observed in
studies with chronic ingestion (2 – 8 weeks) (Santos et al. 2001; Elam et al. 1989; Campbell et
al. 2006), but not acute (Alvares et al. 2012; Greer and Jones, 2011; Wax et al. 2012; Wax et
al. 2013). Speculations suggest that the increased peripheral blood flow induced by chronic L-
arg supplementation is the underlying mechanism for improvement of muscle performance;
however, it seems strange to believe in the chronic effect of the L-arg supplementation since it
has no accumulative effect on the human body [terminal elimination half life of aprox. 75
min. (Bode Böger et al. 1998)]. In other words, the L-arg supplementation should improve
endothelial function and muscle performance in a single exercise bout to confirm your
probable ergogenic effect during long-term resistance training. However, our results did not
63
confirm this hypothesis in elderly women. Therefore, there is still doubt about the possible
ergogenic effects of L-arg supplementation to increase endothelial function and muscle
performance in older subjects.
A limitation of our study was that we have not investigated the possible cellular
mechanisms that could explain the lack of effects of L-arg supplementation in our elderly
women. It is possible that NO-independent vasodilators mechanisms (e.g., CYP, PN and
EDHF), hormonal changes (e.g., increased growth hormone and insulin), reduced oxidative
stress, and/or normalized plasma levels of L-arg and endogenous NOS inhibitors [e.g., levels
of asymmetric dimethylarginine (ADMA)] are associated with endothelial healthy and
consequenty lack of ergogenic effect of L-arg supplementation. We did not explore such a
mechanism in our study.
In conclusion, acute L-arg supplementation provides no ergogenic effect on
endothelial function and muscle performance in older women. Further research is required to
elucidate the real ergogenic benefits of chronic and acute L-arg supplementation, and
underlying mechanism(s) of action of this supplement on muscular and endothelial function,
before considering it as a potential ergogenic aid.
64
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73
ANEXO A - TERMO DE CONSENTIMENTO LIVRE E ESCLARECIDO
Titulo da pesquisa:
EFEITOS DA SUPLEMENTAÇÃO DE L-ARGININA SOBRE O DESEMPENHO MUSCULAR DE MULHERES IDOSAS
Prezado (a) Senhor (a): Gostaríamos de convidá-lo (a) a participar do projeto de pesquisa intitulado: Efeitos da suplementação de L-arginina sobre o desempenho muscular de mulheres idosas
O projeto será desenvolvido no Centro de Pesquisa em Ciências da Saúde (CPCS) da Universidade Norte do Paraná (UNOPAR), e terá duração de 4 semanas.
O objetivo da pesquisa será verificar os efeitos da suplementação de L-arginina sobre o desempenho muscular de mulheres idosas (acima de 65 anos).
O projeto envolverá as seguintes etapas:
(1) Apresentação do projeto aos participantes;
(2) Entrevista pessoal para seleção dos participantes;
(3) Três sessões de familiarização para os testes motores;
(4) Divisão dos participantes em dois grupos experimentais (L-arginina ou Placebo);
(5) Três momentos de avaliação, envolvendo testes motores, exame de ultrassonografia, coleta de sangue e aferição da pressão arterial. O 1º. dia de avaliação será realizado na Universidade Estadual de Londrina (UEL) e os outros dois na UNOPAR.
Não haverá qualquer risco para a integridade física, mental ou moral dos participantes dessa pesquisa. Todas as medidas de segurança serão adotadas para evitar potenciais riscos à integridade física dos participantes, durante a realização dos testes físicos. Normalmente os testes não causam dores ósseas e/ou musculares nos dias subsequentes. As informações serão utilizadas somente para os fins de pesquisa e serão tratadas com sigilo e confidencialidade, de modo a preservar a sua identidade. Gostaríamos de esclarecer que sua participação é totalmente voluntária, podendo recusar-se a participar, ou mesmo desistir a qualquer momento sem que isto acarrete qualquer ônus ou prejuízo à sua pessoa.
Londrina, ___ de ________ de 2014.
Andreo Fernando Aguiar Pesquisador Responsável
Eu, _________________________________________ tendo sido devidamente esclarecido
(a) sobre os procedimentos da pesquisa, concordo em participar voluntariamente da
pesquisa descrita acima.
Assinatura: ____________________________
Data: ___ / ____ / ______
