DOUGLAS KRATKI DA SILVA...3 DA SILVA, DOUGLAS KRATKI. Aminoácido L-citrulina e aptidão física:...
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0 Londrina - Paraná 2016 PROGRAMA DE PÓS-GRADUAÇÃO STRICTO SENSU MESTRADO EM EXERCÍCIO FÍSICO NA PROMOÇÃO DA SAÚDE DOUGLAS KRATKI DA SILVA AMINOÁCIDO L-CITRULINA E APTIDÃO FÍSICA: EVIDÊNCIAS CIENTÍFICAS
Transcript of DOUGLAS KRATKI DA SILVA...3 DA SILVA, DOUGLAS KRATKI. Aminoácido L-citrulina e aptidão física:...
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Londrina - Paraná 2016
PROGRAMA DE PÓS-GRADUAÇÃO STRICTO SENSU MESTRADO EM EXERCÍCIO FÍSICO NA PROMOÇÃO DA SAÚDE
DOUGLAS KRATKI DA SILVA
AMINOÁCIDO L-CITRULINA E APTIDÃO FÍSICA:
EVIDÊNCIAS CIENTÍFICAS
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DOUGLAS KRATKI DA SILVA
Cidade ano
AUTOR
Londrina - Paraná
2016
AMINOÁCIDO L-CITRULINA E APTIDÃO FÍSICA: EVIDÊNCIAS CIENTÍFICAS
Relatório Técnico apresentado à UNOPAR, como requisito parcial para a obtenção do título de Mestre Profissional em Exercício Físico na Promoção da Saúde. Orientador: Prof. Dr. Andreo Fernando Aguiar
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DOUGLAS KRATKI DA SILVA
AMINOÁCIDO L-CITRULINA E APTIDÃO FÍSICA: EVIDÊNCIAS CIENTÍFICAS
Relatório Técnico apresentado à UNOPAR, referente ao Curso de Mestrado
Profissional em Exercício Físico na Promoção da Saúde, como requisito parcial para
a obtenção do título de Mestre Profissional conferido pela Banca Examinadora:
_________________________________________ Prof. Dr. Andreo Fernando Aguiar
Universidade Norte do Paraná
_________________________________________ Prof. Dr. Rubens Alexandre da Silva Junior
Universidade Norte do Paraná
_________________________________________ Prof. Dr. Edilson Serpeloni Cyrino
(Membro Externo)
_________________________________________ Prof. Dr. Dartagnan Pinto Guedes
Coordenador do Curso
Londrina, 16 de dezembro de 2016.
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DA SILVA, DOUGLAS KRATKI. Aminoácido L-citrulina e aptidão física: evidências científicas. 64f. Mestrado Profissional em Exercício Físico na Promoção da Saúde. Centro de Pesquisa em Ciências da Saúde. Universidade Norte do Paraná, Londrina. 2016.
RESUMO
O presente material será apresentado em duas seções distintas, conforme
regimento do Curso de Mestrado Profissional em Exercício Físico na Promoção da
Saúde, da Universidade Norte do Paraná (UNOPAR). A primeira seção corresponde
à produção técnica (guia prático), intitulada: Aminoácido L-citrulina e aptidão física:
evidências científicas. O conteúdo deste guia prático está fracionado em 9 tópicos:
(1) Nossa proposta, (2) O aminoácido L-citrulina (definição, principal função,
biossíntese e metabolismo), (3) Fontes alimentares de L-citrulina, (4) Farmacologia
(absorção intestinal e níveis séricos), (5) Possíveis efeitos benéficos sobre a aptidão
física (evidências científicas), (6) Possíveis efeitos benéficos sobre a síntese prtéica,
(7) Possíveis efeitos colaterais (8) Considerações finais, e (9) Referências. Ao
término do conteúdo, um profissional de Design Gráfico foi contratado para concluir
o processo final de produção artística do guia (criação, diagramação e arte final).
Posteriormente, este material será submetido à análise editorial, visando à
solicitação do número ISBN, e subsequente divulgação do guia prático. A segunda
seção corresponde à produção científica, intitulada: Efeitos da suplementação de
citrulina malato sobre a recuperação muscular após exercício resistido em homens
jovens, que será submetida para publicação no periódico Medicine and Science in
Sports and Exercise. O compilado de informações contidas neste material foi
elaborado para atender as características peculiares do respectivo curso de Pós-
Graduação, cujo escopo principal é apresentar um produto técnico que possa
auxiliar Profissionais e Pesquisadores que atuam na área de Prescrição e
Orientação de Programas de Exercício Físico.
Palavras-chave: Exercício físico, Suplementação, Ergogênico, aptidão muscular,
aptidão aeróbica.
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DA SILVA, DOUGLAS KRATKI. Amino acid L-citrulline and physical fitness: scientific evidence. 64f. Professional Master´s in Exercise in Health Promotion. Research Center in Health Sciences. North University of Parana, Londrina. 2016.
ABSTRACT
The present material will be presented in two distinct sections, according to the
regiment of the Professional Masters Course in Physical Exercise in Healthy Health,
from the North University of Paraná (UNOPAR). The first section corresponds to the
technical production (practical guide) entitled: Amino acid L-citrulline and physical
fitness: scientific evidence. The content of this practical guide will be divided into 9
topics: (1) Our proposal, (2) The amino acid L-citrulline (definition, main function,
biosynthesis and metabolism), (3) Dietary sources of L-citrulline, (4) Pharmacology
(5) Potential beneficial effects on physical fitness (scientific evidence), (6) Potential
beneficial effects on protein synthesis, (7) Possible side effects (8) Final
considerations, and (9) References. A graphic design professional was hired to
complete the final artistic production process of the practice guide (creation, layout
and final art). Subsequently, this material will be submitted to editorial analysis,
seeking the ISBN number, and subsequent dissemination of the practical guide. The
second section corresponds to the scientific production, titled: Effects of citrulline
malate supplementation on muscle recovery after resistance exercise in young men,
which will be submitted for publication in the Medicine and Science in Sports and
Exercise. The compilation of information contained in this material was prepared to
meet the peculiar characteristics of the respective postgraduate course, whose main
scope is to present a technical product that can assist Professionals and
Researchers who work in the area of Prescription and Guidance of Physical Exercise
Programs.
Keywords: Exercise, Supplementation, Ergogenic, muscular fitness, aerobic fitness.
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SUMÁRIO 1. INTRODUÇÃO ....................................................................................................... 6
2. REVISÃO DE LITERATURA .................................................................................. 8
3. DESENVOLVIMENTO ...........................................................................................12
Etapa 1: Seleção de artigos científicos ................................................................. 12
Etapa 2: Descrição dos tópicos ............................................................................. 12
Etapa 3: Arte final ................................................................................................. 12
Etapa 4: Solicitação do International Standard Book Number (ISBN) ................... 12
4. REFERÊNCIAS .....................................................................................................13
APÊNDICE A – Guia prático......................................................................................17
APÊNDICE B – Trabalho Apresentado em Evento Científico ....................................38
APÊNDICE C – Artigo Científico ...............................................................................39
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1. INTRODUÇÃO
O óxido nítrico [do inglês Nitric Oxide (NO)] é uma importante
molécula endógena anti-aterogênica, envolvida em vários processos fisiológicos e
patológicos1. A libertação de NO das células endoteliais promove dilatação arterial
para aumentar o fluxo sanguíneo, manutenção da elasticidade endotelial, inibição da
adesão e agregação de plaquetas nas paredes da artéria2, e uma possível melhora
da capacidade aeróbia e/ou anaeróbica para realizar exercícios físicos3. O NO é um
gás solúvel (molecular) que consiste na ligação covalente entre um átomo de
nitrogênio e um átomo de oxigênio. A sua produção no organismo humano ocorre
quando o aminoácido L-arginina (L-arg) é convertido em L-citrulina (L-cit) numa
reação catalisada pela enzima óxido nítrico sintase (NOS)4.
O importante papel vasodilatador da via do NO tem sido
demonstrado em estudos envolvendo sujeitos jovens saudáveis5-6. 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 teste de
preensão manual5 e exercício de extensão de pernas6, indicando que a
biodisponibilidade de NO é fundamental para o controle da vasodilatação endotelial
durante o exercício físico. Estes resultados sugerem que a menor disponibilidade de
NO nas células endoteliais de sujeitos adultos jovens ou idosos pode contribuir para
o surgimento de disfunções endoteliais e redução na capacidade de realizar
exercícios físicos7. Por outro lado, o aumento da biodisponibilidade de NO em
condições normais e patológicas poderia ser favorável para aumentar a perfusão
sanguínea e consequentemente, melhorar a função muscular durante exercício.
Portanto, estratégias não farmacológicas com objetivo de aumentar
a biodisponibilidade de NO poderiam contribuir para aumentar a perfusão sanguínea
durante o repouso e exercício e, assim, melhorar a aptidão muscular em diferentes
condições relacionadas à saúde e doença. Neste contexto, o consumo de
suplementos vasodilatadores tem recebido considerável atenção nas duas ultimas
décadas, uma vez que seus possíveis efeitos ergogênicos poderiam contribuir para
o aprimoramento dos diferentes componentes da aptidão física relacionados à
saúde. Dentre os suplementos mais utilizados na atualidade, destaca-se a L-cit.
Tem sido descrito que a suplementação de L-cit pode promover
aumento da síntese de NO e acelerar a depuração de amônia (NH3 – Fator promotor
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de fadiga central e periférica) através do ciclo da ureia, resultando no aumento da
vasodilatação e eliminação de subprodutos no metabolismo muscular. Além disso, o
aumento da produção de NO induzido pela L-cit, parece ser benéfico para o
aumento da aptidão aeróbica e anaeróbica8-9, força muscular, perfusão sanguínea10-
12, e recuperação muscular13-14, contribuindo assim para o aprimoramento de
diversos componentes da aptidão física relacionados à saúde. Logo, a
suplementação de L-cit pode ser uma valiosa intervenção não farmacológica para
promoção da saúde e qualidade de vida em diferentes populações.
Todavia, grande parte dos Profissionais que atuam em diversos
contextos da promoção da saúde, ainda apresenta limitações práticas (por ex:
dificuldades com a leitura/interpretação de textos científicos, limitada compreensão
da língua inglesa, desconhecimento das bases de dados e acervos bibliotecários, e
desatualização científica) para acesso a informações científicas teóricas e práticas
que possam facilitar o entendimento dos possíveis efeitos ergogênicos da
suplementação de L-cit sobre os componentes da aptidão física relacionados à
saúde, bem como mecanismos de ação, as doses utilizadas, e seus possíveis
efeitos colaterais.
Assim, a proposta desta produção técnica será apresentar um
material didático (guia prático) de fácil acesso e compreensão, com informações
teórico-práticas relacionadas aos possíveis efeitos da suplementação de L-cit sobre
os componentes da aptidão física relacionados à saúde, com foco na aptidão
cardiorrespiratória, força e resistência muscular localizada (RML). Esperamos que
este material possa contribuir para a tomada de decisão em relação ao uso deste
suplemento em diversos contextos da promoção da saúde.
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2. REVISÃO DA LITERATURA
O American College of Sports Medicine define o termo ‘Aptidão
Física’ (AF) como “a habilidade para realizar tarefas diárias com vigor e agilidade,
sem fadiga excessiva, e com energia suficiente para usufruir das atividades de lazer
e atender as emergências imprevistas”. A AF tem sido considerada um constructo
multifatorial, que inclui essencialmente cinco componentes relacionados à saúde: (1)
a resistência cardiorrespiratória, (2) a composição corporal, (3) a resistência
muscular localizada (RML), a (4) força muscular e (5) a flexibilidade.
Os benefícios do exercício físico (EF) sobre os componentes da AF
relacionados à saúde, bem como as diretrizes para prescrição de EF em diferentes
condições de saúde e doença têm sido amplamente discutidos na literatura. No
entanto, estratégias complementares que possam “ampliar” os efeitos benéficos do
EF sobre os componentes da AF podem, potencialmente, contribuir para melhoria da
saúde e qualidade de vida de diferentes populações.
Neste sentido, o consumo de suplementos alimentares tem sido
recentemente indicado como uma importante intervenção não farmacológica capaz
de “ampliar” a capacidade do EF e, assim, contribuir para o aprimoramento dos
diversos componentes da AF relacionados à saúde. Dentre os suplementos mais
utilizados na atualidade destaca-se a L-cit, que pode ser descrita como a
combinação do aminoácido não essencial, a citrulina, e um sal ácido, o malato,
formando assim a citrulina malato (CM). A L-cit pode ser obtida pela ingestão de
frutas, como a melancia e melão, enquanto o malato é derivado das maças, sendo a
forma ionizada do ácido málico. Tanto a forma simples (L-cit), como a forma
combinada ao malato (CM), são comercializadas como suplementos
vasodilatadores, o que tem aumentado a sua procura em lojas de suplementos
alimentares. Tal fato destaca a importância de se entender os benefícios deste
suplemento no contexto prático e clínico para promoção da saúde.
Estudos recentes demonstram que o consumo de CM (6-8g/dia)
pode aumentar a aptidão aeróbia8 e anaeróbia9, bem como a resistência e força
muscular14-15, contribuindo assim, para o aprimoramento dos diversos componentes
da AF relacionados à saúde16. Logo, a suplementação de CM pode ser utilizada
como uma intervenção complementar ao EF para promoção da saúde e qualidade
de vida em diferentes populações, principalmente pelos seus efeitos benéficos sobre
a aptidão cardiorrespiratória e neuromuscular8,14,17.
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Os efeitos fisiológicos da suplementação de L-cit parecem estar
associados ao aumento da produção de NO10 e consequente melhora na resposta
vasodilatadora18. A L-cit pode ser convertida em L-arginina (principal substrato para
síntese de NO18), que subsequentemente será convertida em NO, numa reação
catalisada pela enzima NOS17-18. Tem sido descrito que a suplementação de L-cit
parece ser mais efetiva do que suplementação de L-arginina para aumentar a
produção de NO, uma vez que a L-cit não sofre efeitos de enzimas arginases
(quebram a L-arginina) no trato gastrointestinal e fígado. Assim, a suplementação de
L-cit tem sido mais indicada para aumentar as concentrações plasmáticas de L-
arginina, resultando no aumento da síntese de NO e consequente aumento da
vasodilatação e fluxo sanguíneo17.
A L-cit também esta envolvida no ciclo da ureia, juntamente com
ornitina e arginina. O ciclo da ureia, é um ciclo de reações bioquímicas que ocorrem
nos seres humanos para produzir ureia (NH2-CO-NH2) a partir do acido amoníaco
(NH3). A amônia é um acido tóxico, cuja alta concentração produzida durante
atividades aeróbicas e anaeróbicas, pode resultar em efeitos prejudicais para as
células musculares19, devido à redução na formação de glicogênio e consequente
fadiga. Estudos relatam que a L-cit pode acelerar a depuração de amônia, através
do ciclo da ureia, resultando na eliminação de subprodutos do metabolismo
muscular, e consequente melhora da função muscular8-9.
De fato, os efeitos benefícios da suplementação de L-cit sobre o
desempenho físico têm sido demonstrado em vários estudos envolvendo sujeitos
humanos e animais. Bailey et al.20 demonstraram aumento da resistência à fadiga e
quantidade total de trabalho durante teste de esforço em cicloergômetro em homens
adultos saudáveis suplementados com L-cit. Adicionalmente, Jourdan et al.13, em um
estudo crossover, encontraram aumento da taxa de síntese protéica em sujeitos
suplementados com L-cit + aminoácidos de cadeia ramificada (ACR), comparados a
suplementação de ACR (L-cit: 0.060 ± 0.006 vs. ACR: 0.049 ± 0.005, P = 0,03), após
três dias de baixa ingestão protéica.
Resultados similares foram observados em estudo com animais;
Takeda et al.21 demonstraram redução na produção de lactato, e recíproco aumento
o tempo até a exaustão, durante teste de esforço máximo em ratos suplementados
com L-cit. Adicionalmente, tem sido demonstrado que a suplementação de L-cit
pode preservar a função muscular e aumentar a síntese protéica em ratos privados
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de alimento22-23. Consistente com estes estudos, Moinard et al.24 demonstraram um
aumento de 9% na massa magra e 13% de redução na massa gorda em ratos
idosos saudáveis suplementados com L-cit + ACR, comparados ao grupo ACR. Os
autores concluíram que a suplementação de L-cit pode ser uma estratégia efetiva
para aumentar a massa muscular em populações com reduzida ingestão de
proteínas, e atenuar a perda de massa muscular durante o processo de
envelhecimento.
Todavia, resultados contraditórios foram observados no estudo de
Hickner et al.25 na qual investigaram os possíveis efeitos da suplementação de L-cit
sobre a resistência à fadiga em teste máximo de esteira em sujeitos adultos jovens
(18-34 anos). Os autores não encontraram diferença significante no tempo até
exaustão entre os grupos L-cit (3g) e placebo (9g), sugerindo que a administração
de L-cit não promove efeitos benéficos sobre a resistência à fadiga em teste
máximo. Adicionalmente, nenhum efeito preventivo foi observado sobre a função e
massa muscular de ratos suplementados com L-cit (0.81g/kg), após 14 dias de
imobilização do membro posterior (unilateral)26. Portanto, a utilização da
suplementação de L-cit como adicional ergogênico ainda parece ser prematura,
sugerindo que novos estudos sejam realizados para investigar os reais efeitos deste
suplemento em diversas populações e condições experimentais.
Não obstante, a combinação de L-cit e malato (formando a CM)
parece demonstrar melhores resultados do que a suplementação isolada de L-cit
para aumento da AF14-15,27. Glenn et al.15 observaram um aumento da resistência
muscular localizada (RML) e menor percepção subjetiva de esforço em mulheres
treinadas suplementadas com CM (8g CM + 8g dextrose), quando comparadas ao
grupo placebo (8g de dextrose), após seis séries de exercícios resistido (supino e
leg press) a 80% de 1RM até a falha concêntrica. Similarmente, Wax et al.28, em um
estudo crossover, demonstraram um aumento do número de repetições nos
exercícios de chin-ups (puxada na barra), reverse chin-ups (puxada invertida na
barra) e push-ups (flexão de braço), após três séries de exercício até a falha, em
indivíduos suplementados com CM. Os mesmos autores em outro estudo14,
demonstraram um aumento significante no número de repetições em sujeitos
suplementados com CM (8g), durante cinco séries de exercícios resistido (hack, leg
press, e cadeira extensora) a 60% de 1RM, até a falha muscular. Coletivamente, os
resultados destes estudos sugerem que a suplementação de CM pode ser uma
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estratégia eficaz para aumentar a RML em adultos jovens, contribuindo assim para
melhoria da saúde e qualidade de vida.
Interessantemente, doses menores do que 8g também parecem ser
efetivas para promover efeitos benéficos sobre a AF. López-Cabral et al.29 avaliaram
os efeitos da suplementação de CM sobre a concentração de lactato sanguíneo e
percepção subjetiva de esforço muscular em atletas de alto rendimento. Os autores
encontraram menor concentração de lactato e menor índice de percepção de fadiga
em atletas suplementados com 3 e 6 g de CM, comparados ao grupo placebo, após
13 dias de treinamento de pentatlo e patinação. O estudo concluiu que a
suplementação de CM pode acelerar a recuperação muscular e diminuir a fadiga em
atletas de alto rendimento, contribuindo para melhora do desempenho físico. Este
efeito benéfico também foi observado em jogadoras da categoria máster (51 ± 9
anos) de tênis, suplementadas com 8g de CM + 12g de dextrose, na qual
demonstraram um aumento da potência pico e a potência explosiva durante teste de
wingate, comparadas ao grupo placebo (12g de dextrose)30. Portanto, a
suplementação de CM parece ser uma estratégia benéfica para melhora da AF em
diferentes populações associadas à saúde e desempenho.
Logo, torna-se imprescindível “criar” estratégias teórico-práticas de
fácil acesso aos Profissionais da área de saúde, que possam contribuir para a
divulgação do conhecimento em relação aos possíveis efeitos benéficos da L-cit e
CM sobre os componentes da AF, bem como as doses utilizadas, os mecanismos de
ação, e os possíveis efeitos colaterais, e assim contribuir para a tomada de decisão
em relação ao uso deste suplemento em diversos contextos da promoção da saúde
e esporte.
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3. DESENVOLVIMENTO
O presente guia prático foi elaborado de acordo com as seguintes
etapas:
Etapa 1: Seleção de artigos científicos
Foram selecionados artigos científicos de diferentes bases de dados
utilizando-se os seguintes descritores em língua inglesa: citrulline malate (citrulina
malato), citrulline (citrulina), nitric oxide (óxido nítrico) e vasodilation (vasodilatação),
skeletal muscle (músculo esquelético), hypertrophy (hipertrofia), muscle strength
(força muscular), athletic performance (desempenho atlético), aerobic fitness
(aptidão aeróbica) and protein synthesis (síntese protéica). Dois pesquisadores
efetuaram a busca, considerando o período de 1995 a 2016.
Etapa 2: Descrição dos tópicos
Após a seleção dos artigos científicos, procedeu-se a descrição do
conteúdo do guia prático, constituindo dos seguintes tópicos: (1) Nossa proposta, (2)
O aminoácido l-citrulina (definição, principal função, biossíntese e metabolismo), (3)
Fontes alimentares de l-citrulina, (4) Farmacologia (absorção intestinal e níveis
séricos), (5) Possíveis efeitos benéficos sobre a aptidão física (evidências
científicas), (6) Possíveis efeitos benéficos sobre a síntese protéica, (7) Possíveis
efeitos colaterais (8) Considerações finais, e (9) Referências.
Etapa 3: Arte final
Após a descrição do conteúdo, um profissional de Design Gráfico foi
contratado para concluir os processos de diagramação e arte final do guia prático.
Para tanto, foi utilizado o programa de edição de imagens Photoshop (versão: 7.1),
considerando as dimensões do guia de 21,0 x 29,7cm (folha A4).
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.
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4. REFERÊNCIAS
1. Hou YC, Janczuk A, Wang PG. Current trends in the development of nitric oxide
donors. Current Pharmaceutical Design. 1999; 5:417-41.
2. Preli RB, Klein KP, Herrington DM. Vascular effects of dietary L-arginine
supplementation. Atherosclerosis. 2002; 162:1-15.
3. Maxwell AJ, Ho HV, Le CQ, Lin PS, Bernstein D, Cooke JP. L-arginine enhances
aerobic exercise capacity in association with augmented nitric oxide production.
Journal of Applied Physiology. 2001; 90:933-8.
4. Ckless K, van der Vliet A, Janssen-Heininger Y. Oxidative-nitrosative stress and
post-translational protein modifications: implications to lung structure-function
relations. Arginase modulates NFkappaB activity via a nitric oxide-dependent
mechanism. American Journal of Respiratory Cell and Molecular Biology. 2007;
36:645-53.
5. Schrage WG, Joyner MJ, Dinenno FA. Local inhibition of nitric oxide and
prostaglandins independently reduce forearm exercise hyperaemia in human.
The Journal of Physiology. 2004; 557:599-611.
6. Mortensen SP, Gonzalez-Alonso J, Damsgaard R, Saltin B, Hellsten Y. Inhibition
of nitric oxide and prostaglandins, but not endothelial-derived hyperpolarizing
factors, reduces blood flow and aerobic energy turnover in the exercising human
leg. The Journal of Physiology. 2007; 581:853-861.
7. Proctor DN, Koch DW, Newcomer SC, Le KU & Leuenberger UA. Impaired leg
vasodilation during dynamic exercise in healthy older women. Journal of Applied
Physiology. 2003; 95:1963-1970.
8. Bendahan D, Mattei JP, Ghattas B, Confort-Gouny S, Le Guern ME & Cozzone
PJ. Citrulline/malate promotes aerobic energy production in human exercising
muscle. British Journal of Sports Medicine. 2002; 36:282-289.
9. Pérez-Guisado J & Jakeman PM. Citrulline malate enhances athletic anaerobic
performance and relieves muscle soreness. The Journal of Strength and
Conditioning Research. 2010; 24:1215-1222.
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10. Ochiai M, Hayashi T, Morita M, Ina K, Maeda M, Watanabe F & Morishita K.
Short-term effects of L-citrulline supplementation on arterial stiffness in middle-
aged men. International Journal of Cardiology. 2012.
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14. Wax B, Kavazis AN, Weldon K & Sperlak J. Effects of supplemental citrulline
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15. Glenn MJ, Gray M, Wethington LN, Stone MS, Stewart Jr. RW & Moyen NE.
Acute citrulline malate supplementation improves upper-and lower-body
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17
APÊNDICE A – Guia prático
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22
23
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25
26
27
28
29
30
31
32
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35
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APÊNDICE B – Trabalho Apresentado em Evento Científico
EFEITOS DA SUPLEMENTAÇÃO DE CITRULINA MALATO SOBRE A RECUPERAÇÃO MUSCULAR APÓS EXERCÍCIO RESISTIDO EM HOMENS JOVENS INTRODUÇÃO: O aminoácido citrulina malato (CM) é formado pela combinação de L-citrulina e malato. Prévios estudos sugerem que a suplementação de CM pode aumentar a perfusão sanguínea durante exercício (mediante aumento da produção de óxido nítrico e consequente vasodilatação), e melhorar a taxa de síntese protéica muscular em diferentes condições fisiológicas. Por conseguinte, a suplementação de CM poderia melhorar a função muscular e acelerar o processo de recuperação pós-exercício. No entanto, esta hipótese ainda não foi testada durante a recuperação muscular após exercício resistido em sujeitos jovens não treinados. OBJETIVOS: Investigar os possíveis efeitos da suplementação isolada de CM sobre a recuperação muscular após uma única sessão de treinamento resistido (TR) em homens jovens. MÉTODOS: Foi empregado um desenho crossover e duplo cego, na qual 09 homens jovens (24 3,3 anos) não treinados foram suplementados com CM (6g/dia) e placebo (PLA, 6g/dia), em dois momentos análogos (M1 e M2), separados por um período washout de 1 semana. Em cada momento (M1 e M2), os participantes foram submetidos a 1 sessão de TR (3 séries de 10 repetições à 90% de 10RM) envolvendo dois exercícios para o músculo quadríceps (leg press e agachamento hack), e 3 subsequentes sessões (24, 48, e 72 horas após a sessão de TR) de teste de fadiga muscular (1 série até a fadiga voluntária no leg press, com carga para 10RM), a fim de analisar a recuperação muscular, mediante o número máximo de repetições até a falha. A suplementação de CM e PLA foi realizada 60 minutos antes das sessões de TR e testes de fadiga, considerando as propriedades farmacocinéticas da CM. A carga para 10RM foi determinada mediante a realização de duas sessões alternadas de teste para cada exercício (leg press e hack), antes do momento M1. Os dados foram avaliados por meio de testes de ANOVA para medidas repetidas, complementado pelo teste post-hoc de Bonferroni. RESULTADOS: Nenhum efeito do tempo ou interação (grupo x tempo) (P > 0,05) foi observado no número máximo de repetições até a falha, durante os testes de fadiga realizados nos momentos 24, 48 e 72h após a sessão de TR. CONCLUSÃO: A suplementação de CM não promove melhoria na função muscular durante o processo de recuperação após exercício resistido, indicando que a utilização deste suplemento para regeneração muscular ainda parece ser prematura. da Silva DK, Andrade WB, Jacinto JL, Estoche JM, Roveratti MC, Mario Balvedi CW, Oliveira DB, Sena BNS, da Silva Junior RA, Aguiar AF. Efeitos da suplementação de citrulina malato sobre a recuperação muscular após exercício resistido em homens jovens. VI Congresso Brasileiro de Metabolismo, Nutrição e Exercício (COMBRAMANE). Londrina, Brasil. 2016. p.26.
39
APÊNDICE C – Artigo Científico
Citrulline malate does not improve muscle recovery course after resistance
training in young men
Este artigo será submetido para publicação no Medicine and Science in Sports and Exercise
(Fator de impacto: 4.04; Qualis A1)
PURPOSE: The purpose of this study was to investigate the effects of citrulline malate
supplementation on muscle recovery course after a single session of conventional lower-body
resistance exercise in untrained young subjects. METHODS: Nine young men (24.0 ± 3.3 yr)
participated in a 2-period crossover, counterbalanced, double-blind study with repeated
measures. The subjects were supplemented with 6 g of citrulline malate (CIT) or placebo
(PLA) on 02 identical moments (M1 and M2), separated by a 7-day washout period. Subjects
served as their own control. The M1 and M2 moments consisted of a single session of
resistance training (0h), and 3 subsequent sessions of fatigue tests (at 24, 48 and 72h)
involving leg press and hack squat exercises. During fatigue tests were analyzed: number of
maximum repetitions, electromyographic signal (i.e., root mean square [RMS] and frequency
median [FM]), muscle soreness, and perceived exertion, and blood levels of creatine
phosphokinase (CPK), lactate, insulin, testosterone, and cortisol. RESULTS: There was no
main effect for time (P > 0.05) or group x time interaction (P > 0.05) in number of repetitions,
perceived exertion, RMS, FM, insulin, testosterone and cortisol and testosterone:cortisol ratio
during recovery course. Additionally, there was no group x time interaction (P > 0.05), but a
significant (P < 0.05) main effect for time was found for CPK and lactate. CONCLUSION:
Our data indicate that CIT supplementation (single 6 g dose at 60 minutes prior to training)
does not improve muscle recovery course after a single session of high-intensity resistance
training in untrained young men.
Key words: nutrition, supplementation, ergogenic, amino acid, muscle, fatigue
40
INTRODUCTION
Nutritional supplementation is one of the most popular non-pharmacological strategies used
by athletes and recreationally active adults to improve physical performance and muscle
recovery. Recently, nitric oxide (NO) related supplements have received special attention for
their possible ergogenic effects (Alvares et al. 2011; Bendahan et al. 2002; Bescos et al.
2012). In particular, the citrulline malate (CM) has been reported to augment aerobic energy
production (Bendahan et al. 2002), improve the elimination of ammonia (NH3) during course
of recovery from exhaustive exercise (Vanuxem et al. 1990; Sureda and Pons, 2012),
attenuating muscle soreness after high-intensity resistance exercise (Perez-Guisado and
Jakeman, 2010), and increasing performance during repeated bouts of high-intensity
resistance exercise (Perez-Guisado and Jakeman, 2010; Wax et al. 2015; Glenn et al. 2015).
CM is formed by combination of L-citrulline (L-cit) and malate (or malic acid - a salt
primarily found in apples). L-cit is a non-essential amino acid produced endogenously via two
key metabolic processes: 1) it is synthesized from glutamine in enterocytes of the intestinal
tract, where the enzyme ornithine transcarbamylase uses both ornithine and carbamoyl
phosphate to produce L-cit (Tonlinsom et al. 2011; Curis et al. 2007; Rougé et al. 2007), and
2) it is produced as a byproduct from the conversion of L-arginine (L-arg) to NO in a reaction
catalyzed by nitric oxide synthase (NOS) enzymes (Aguilo et al. 2000).
The possible ergogenic effects of CM have been attributed to three key mechanisms.
First, the L-cit produced by gut is released as such into the bloodstream, and approximately
83% of the circulating L-cit (L-cit bypasses hepatic and intestinal metabolism, remaining
unaffected by arginase enzymes action) is taken up by kidney (Windmueller et al. 1981; Van
de Poll et al. 2007a; Van de Poll et al. 2007b), where L-cit is converted to L-arg in cells of the
proximal tubules (Levillain et al. 1997). Given that L-arg is the main substrate for the NO
synthesis - an important modulator of blood flow (Bloomer et al. 2010), it has been suggested
that oral CM supplementation may indirectly increase the synthesis of NO (Perez-Guisado
41
and Jakeman, 2010) and, consequently, increasing blood flow for active muscles. As a result,
the CM supplementation could contribute to increase nutrient delivery for recruited muscles
and/or waste-product clearance (Little et al. 2008; Wilcock et al. 2006) such as plasma lactate
and ammonia, thereby improving muscle function (Briand et al. 1992).
Second, L-cit is an essential component participating in the urea cycle in the liver
(Curis et al. 2005), where L-arg produced from L-cit is catabolized by arginase into ornithine
and urea. Given that urea is the major vehicle to eliminate ammonia - a promoter of muscle
fatigue via anaerobic glycolysis and consequenty lactic acid production (Cutrufello et al.
2015), it has been suggested that L-cit supplementation may improve ammonia homeostasis
(Breuillard et al. 2015) and thus improving muscle function. Third, malate is an intermediate
of tricarboxylic acid (TCA) cycle, and its greater available after CM supplementation may
augment aerobic ATP production from the TCA cycle through anaplerotic reactions
(Bendahan et al. 2002), resulting in the decreasing muscle fatigue (Bendahan et al. 2002;
Perez-Guisado and Jakeman, 2010; Wax et al. 2015).
Based on these aforementioned mechanisms is postulate that CM supplementation
may increase muscle performance by several mechanisms, including reducing muscle
soreness (Perez-Guisado and Jakeman, 2010) and fatigue (Bendahan et al. 2002), improving
oxygen delivery for active muscle (Bendahan et al. 2002), increasing aerobic energy
production (Bendahan et al. 2002), and lowering lactate and ammonium production
(Breuillard et al. 2015). Despite an abundance of studies on ergogenic effect of CM, no study
to date has examined the effects of CM supplementation on time course (several time points)
of muscle recovery after lower-body resistance exercise in young subjects. To our knowledge,
only one previous study investigated the beneficial effects of CM supplementation (6g/day for
16 days) on muscle recovery (Bendahan et al. 2002). The authors used a model of finger
flexion exercise to induce muscle fatigue in sedentary young subjects, and found a 20%
increase in the rate of phosphocreatine (PCr) resynthesis during the recovery period.
42
However, there is a great limitation to this research because there was no placebo group or
blind condition in the design, and the training protocol used does not transcribe the exercises
and muscles involved in the conventional resistance training routines.
Therefore, the aim of this study was to investigate the effects of CM supplementation
on muscle recovery course after a single session of conventional lower-body resistance
exercise in untrained young subjects. Based on the physiological properties and beneficial
effects of CM on muscle fatigue and performance (Perez-Guisado and Jakeman, 2010; Wax et
al. 2015; Glenn et al. 2015), we hypothesized that CM supplementation would enhance
muscle recovery process after a single session of conventional resistance exercise in young
men.
43
METHODS
Subjects
Twelve healthy, recreationally active males were recruited from a university population, and 9
completed the study (1 withdrew because of illness, and 2 withdrew because of factors not
related to the study). Descriptive characteristics of the participants are presented in Table 1.
An a priori power analysis was conducted (G*Power v. 3.0.1) for an F test (repeated
measures, within factors for four time points). On the basis of a statistical power (1 – β) of
0.80, a moderately large effect size (0.5), and an overall level of significance of 0.05, least 8
subjects were required for this study. Eligibility criteria consisted of following: (1) not be
vegetarian or smoker, (2) not have ingested any nutritional supplement or anabolic steroids
for the 6 months prior to the start of study, (3) not have musculoskeletal and cardiorespiratory
disorders, (4) not have ingested any medication that could affect muscle recovery or the
ability to exercise intensely during the study, (5) not be involved in the practice of
systematized physical activity (more than 2 days per week), for the 6 months prior to the start
of study, and (6) not have medical restriction for the practice of exercise physical. 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: 1.748.002). All procedures were performed according to the principles outlined
in the 1964 Declaration of Helsinki.
Table 1. Participant characteristics (N = 9)
Age (y) 24.0 ± 3.3
Height (cm) 175.1 ± 4.8
Weight (kg) 77.4 ± 9.6
BMI (kg/m2) 25.3 ± 2.9
Glucose (mg/dL) 71.9 ± 19.0
Cholesterol (mg/dL 155.2 ± 31.0
Values are means ± SD.
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Experimental design
A 2-period crossover, counterbalanced, double-blind, placebo-controlled design with repeated
measures was performed to examine the effects of CM supplementation on time course of
muscle recovery after a single session of heavy-resistance training (RT) in young men (Fig.
1). For this purpose, all subjects were randomly supplemented with 1 of 2 treatments
[citrulline malate (CIT) or placebo (PLA)] on 02 identical moments (M1 and M2), separated
by a 7-day washout period. With this experimental approach, each subject was supplemented
with one of the treatments (M1) and then with the inverse treatment (M2), acting as their own
control. Before M1, all subjects completed 3 and 2 sessions of familiarization and 10-
repetition maximum (10RM) tests, respectively. During M1 and M2, all subjects underwent a
single session of RT (0h) and 3 subsequent sessions (at 24, 48 and 72h after RT session) of
fatigue tests (to assessed the time course of muscle recovery), performed at 60 minutes after
supplementation. In each session of fatigue test were assessed mechanical (i.e., number of
maximum repetitions), physiological (i.e., electromyographic signal), functional (i.e., muscle
soreness, and perceived exertion), metabolic (i.e., creatine phosphokinase [CPK], and lactate),
and anabolic (i.e., testosterone, cortisol, and insulin) indicators of muscle recovery. Moreover,
all participants completed a 3-day dietary intake record (including 1 weekend day) in order to
examine any influence of diet during study period (M1 and M2). All subjects had an adequate
intake of macronutrients during study period (Table 2).
We used a crossover design because of its advantages over a parallel group design.
First, crossover designs are not influenced by intersubject variability (e.g., genetic, food
intake, motivation, and life style) because the participant acts as its own control. Second,
crossover designs are statistically efficient and require fewer subjects than parallel designs.
Third, crossover designs allow carried out the experiment in a shorter time than a parallel
group study, thereby increasing participant’s adherence. Nevertheless, crossover studies
require a sufficiently long wash-out period between treatments to avoid potential carry-over
45
effects (influence from one treatment to another). Therefore, we used a 7-day washout period
between M1 and M2 moments to allow a sufficient period of muscle recovery, and ensure the
total elimination of CM of body. We ensured that the experimental approach used in the
present study provides an effective way to investigate the effects of CM supplementation on
muscle recovery in young subjects.
Figure 1. Experimental design
Table 2. 3-day dietary intake values between M1 and M2 moments
M1 M2 p
value
Carbohydrate (%) 61.1 ± 2.6 62.2 ± 1.7 0.40
Protein (%) 23.5 ± 2.4 23.8 ± 2.8 0.55
Fat (%) 15.4 ± 2.5 14.0 ± 2.8 0.38
Total kilocalories 2255.3 ± 1123.4 2196.1 ± 1092.4 0.59
Values are mean ± SD. Macronutrient amounts are expressed in
percentage of grams There were no differences between M1 and M2
moments.
46
Familiarization protocol
Before M1, all subjects completed 3 sessions of familiarization (three non-consecutive days)
with the leg press and hack squat exercises, in order to minimize any potential learning effects
and establish the reliability of the testing protocols (Fig. 1). The protocol consisted of 3 sets of
8–12 repetitions, with 2 and 3 min rests between the sets and exercises, respectively. Maximal
effort was requested in each exercise during the last two sessions in an attempt to achieve the
approximate load for 10 repetitions maximum. Qualified personnel individually supervised
each participant during the familiarization period. All session of familiarization were
performed at the same location, between 8 and 10 a.m.
Determination of 10 repetition maximum load
Before M1, 4 non-consecutive sessions (2 sessions for each exercise - leg press and hack
squat) of 10RM tests were carried out for each subject by determining the maximum load for
10 consecutive repetitions (Fig. 1). The 10RM test was preceded by a set of warm-up exercise
(~15 repetitions) for each exercise. After 2 min of rest, the 10RM attempts were performed
with a progressively increasing load (1-5 kg) for each attempt, and were separated by 4- to 5-
min rest intervals to allow adequate recovery. Only 3 attempts were allowed in each testing
session. Verbal encouragement was provided during all 10RM attempts. The exercises
execution technique was standardized and continuously monitored by the same experienced
rater in an attempt to assure the quality of the data, and found the 10RM within 3 attempts.
The intraclass correlation coefficients (ICC) test-retest were ≥0.93 for each 10RM test,
indicating the elimination of the learning curve for the subjects. All session of 10RM tests
were performed at the same location, between 8 and 10 a.m.
Resistance training
During M1 and M2, all subjects were submitted to a single session of RT (3 sets of 8-12
repetitions at 90% of 10RM, with 2 min rest between sets and exercises), involving the leg
press and hack squat exercises (bilateral), using commercial machines (Nakagym equipment,
47
São Paulo, Brazil). The velocity/cadence of muscle action was 30 repetitions per minute (1 s
concentric: 1 s eccentric), which was controlled with a metronome. This protocol was
designed to maximize the recruitment of quadriceps muscle, and the training stimulus was
similar to a session of conventional RT (3 sets of 8-12 repetitions at 70-75% of 1RM) for
novice individuals (ACSM, 2009). We chosen use the 10RM load (as opposed to 1RM)
because of its easy application in the practical context, and safety for novice practitioners.
Each training session began with general (moderate walking on treadmill for 10 min) and
specific (1 set of 12 repetitions with a self-selected load) warm-up exercises for quadriceps
muscle. Qualified personnel supervised each participant individually during every workout.
The session of RT were performed at the same location, between 8 and 10 a.m.
Supplementation protocol
During M1 and M2, the subjects ingested an identical looking and equivalent amount (6 g) of
CM (100% pure) or placebo dissolved in water (200 ml), in a counterbalanced, double-blind
and randomized manner. The CM and placebo were analyzed for purity and validated prior to
the study. We chosen to use a dose of 6g based on prior studies that showed an increase in
plasma citrulline concentration after intense exercise in cyclists, without any adverse effects
reported (Sureda et al. 2009; Sureda et al. 2010). The supplementation was performed 60
minutes before RT session and fatigue tests, based on prior studies that reported an ergogenic
effect of CM during multiple bouts of upper-body resistance training in young men (Perez-
Guisado and Jakeman, 2010; Wax et al. 2015). 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.
Nutrient intake
To control any influence of diet, each participant completed a 3-day dietary intake record
(including 1 weekend day) during the M1 and M2 moments. Standard portions were used to
assess the amount of daily food consumed, and then the values were converted to
48
macronutrient amounts using software for nutritional assessment (Avanutri, version 3.1.4, Rio
de Janeiro-RJ, Brazil). The participants were instructed to maintain their habitual daily diet,
and to refrain from any strenuous activity during experimental period. The water intake was
ad libitum. The participants were also instructed to report any adverse events from the
supplements on their health status during study period. No discomfort or adverse effect was
reported by subjects after CM ingestion.
Perceived exertion
Rating of perceived exertion (RPE) was measured immediately after muscular fatigue tests
(24, 48, and 72h after RT session) during M1 and M2 using the OMNI-RES scale (Robertson
et al. 2003). The subjects were instructed to report the RPE value indicating a number of the
OMNI-RES scale (0 “no effort” and 10 “maximal effort”) that best represented their overall
muscular effort (Robertson et al. 2003; Day et al. 2004), and the investigator used the same
question: “how hard do you feel your muscles are working” (Marcora, 2009). The score was
the value (0-10) reported in OMNI-RES scale.
Delayed-Onset Muscle Soreness
Delayed-onset muscle soreness (DOMS) was measured before muscular endurance tests (24,
48, and 72h after RT session) during M1 and M2 using a visual analog scale (VAS). The VAS
consists of a 10-cm line whose end points were labeled with “no pain” (left) and “unbearable
pain” (right). The subjects were instructed to palpate their quadriceps muscle and mark a
vertical line at a scale point that best represented their rating of momentary soreness. The
score was the distance (cm) from the left side of the scale to the point marked (Mattacolla et
al. 1997).
Muscular endurance tests and electromyographic (EMG) signal recordings
During M1 and M2, all subjects were submitted to a muscular fatigue test (1 set at 100% of
10RM until failure) in the leg press and hack squat exercises at 24, 48, and 72h after RT
session (Fig. 1), in order to examine the muscular endurance recovery. During the test, surface
49
EMG signals were recorded from the vastus lateralis (VL) muscle using a pre-amplified (gain:
1000) active bipolar surface electrode (Model EMG System Brazil Ltda, São José dos
Campos, São Paulo, Brazil) at a sampling rate of 2000 Hz. The subject’s skin was prepared by
removing the superficial dead skin and was sterilized with an alcohol swab. The electrode was
placed on a location near the center of the belly of the muscle according to the
recommendations of SENIAM (Surface EMG for Non-Invasive Assessment of Muscles) and
from our previous work (Aguiar et al. 2016), and the reference electrode was fixed at the right
styloid process. The EMG signals were filtered with a band-pass digital filter between 10 and
500 Hz to remove high frequency noise as well as low-frequency movement.
To determine the muscle activation and fatigue, two EMG parameters were computed:
(1) Root mean square (RMS) - the muscular activation from the EMG signals corresponding
to the second and before last contractions of the endurance test (e.g., to avoid the acceleration
and the deceleration portions of the concentric leg contractions during the extension phase of
movement). This parameter was computed by a moving RMS method executed on successive
250 ms (512 points) time-series windows (50% overlap) to obtain the RMS average values
during the entire leg press and hack squat exercises. (2) Median frequency (MF) – muscle
fatigue from the magnitude of the electromyographic spectral content evaluated by the MF
value of the power spectra (Short-fast Fourier transform, Hanning window processing) during
successive time windows (50% overlap) of 250 ms for the total time of the fatigue test.
Afterwards, least squares linear regression analysis was applied to the MF time series to
calculate the rate of decline in MF over time (MF/time slope as a muscle fatigue index), as
supported by previous studies (Larivière et al. 2002; Kienbacher et al. 2014; da Silva et al.
2015). All EMG signals and estimates (RMS and MF) were processed using software
MATLAB sub-routines (Version 8.0, Mathworks®, South Natick, MA, USA).
Blood collection and analysis
Blood samples were collected at pre- and post-training, and immediately after muscular
50
fatigue tests (24, 48, and 72h after RT session) during M1 and M2 for analyses of CPK,
lactate, insulin, glucose, cholesterol, testosterone, and cortisol concentrations. The blood
samples were coagulated and then centrifuged at 2,000 g for 15 min, and the serum frozen at -
80°C until analysis. All analyzes were performed in a laboratory equipped with automated
systems using commercial kits for the chemiluminescence (insulin, testosterone, and cortisol),
kinetic (CPK and lactate), and enzymatic (cholesterol, glucose) techniques.
Statistical analyses
Data are expressed as means ± SD. The normality and homogeneity for outcome measures
were tested using the Shapiro-Wilk’s and Levene’s tests, respectively. Independent variables
included the supplementation protocol (i.e., CIT vs. PLA) and time (i.e., 24, 48, and 72h).
Dependent variables included number of maximum repetitions, perceived exertion, muscle
soreness, EMG signals (RMS and FM), and blood analyses (i.e., CPK, lactate, insulin,
testosterone and lactate). Baseline characteristics and food intake (during M1 and M2)
between groups were compared using a paired t-test. Two way (group x time) ANOVA with
repeated measures was used to evaluate the data across time and between groups for all
independent variables. Violation of sphericity was adjusted by Greenhouse-Geisser. When
significant differences were confirmed with ANOVA, multiple comparisons testing were
performed using Bonferroni post hoc analysis to identify these differences. The significance
level was set at P ≤ 0.05. Statistical analyses were performed using SPSS statistical analysis
software (SPSS version 20.0; Chicago, IL, USA).
51
RESULTS
Total repetitions and perceived exertion in the fatigue tests during recovery. There was no
main effect for time (P > 0.05) or group x time interaction (P > 0.05) in total number of
repetitions and perceived exertion for the leg press (Fig. 2A) and squat (Fig. 2B) fatigue tests
during time course o recovery (24, 48, and 72 h after RT). Both CIT and PLA groups
achieved maximum effort (mean ± SD) in the leg press (total OMNI scale for the 3 times of
recovery, CIT: 10 ± 0 vs. PLA: 10 ± 0; P > 0.05) and squat (total OMNI scale for the 3 times
of recovery, CIT: 10 ± 0 vs. PLA: 10 ± 0; P > 0.05) fatigue tests, indicating that protocol was
reliable for muscle recovery analysis.
Figure 2. Number of maximum repetitions in the leg pres (A) and hack squat (B) fatigue test during
recovery period (at 24, 48, and 72h after resistance training session). Citrulline CIT, Placebo PLA.
Upper figure indicate the perceived exertion - OMNI scale (0-10) immediately after each exercise.
Data are means ± SD.
CPK and lactate blood levels after fatigue test during recovery. There was no significant
group x time interaction (P > 0.05), but a significant main effect for time (P < 0.05) indicated
an increase in serum CPK levels (Fig. 3A) from pre-training to 24h (CIT: 36% vs. PLA: 38%,
P > 0.05) and 48h (CIT: +26 vs. +31%, P > 0.05) of recovery in both CIT and PLA groups
52
(Fig. 3A). Similarly, there was no significant group x time interaction (P > 0.05) in plasma
lactate levels, but a significant main effect for time (P < 0.05) indicated a increase from pre-
training to post-training (CIT: +75 vs. PLA: +80%, P > 0.05), 24h (CIT: +60 vs. PLA: +72%,
P > 0.05), 48h (CIT: +64 vs. PLA: +76%, P > 0.05) and 72h (CIT: +69 vs. PLA: +72%, P >
0.05) of recovery in both CIT and PLA groups (Fig 3B).
Figure 3. Blood CPK (A) and lactate (B) levels at pre- and post-training session, and immediately
after fatigue tests (leg pres and hack squat exercises) during recovery period (at 24, 48, and 72h after
resistance training session). Citrulline CIT, Placebo PLA. * P < 0.05 compared to pretraining, # P <
0.05 compared to 24, 48 and 72h post-training. Data are means ± SD.
53
Delayed-Onset Muscle Soreness during recovery. There was no significant group x time
interaction (P > 0.05), but a significant main effect for time (P < 0.05) indicated a decrease in
perceived intensity of soreness (mean ± SD) of the quadriceps muscle from 24h (CIT: 4.2 ±
3.3 and PLA: 2.9 ± 1.8, P > 0.05) and 48h (CIT: 4.1 ± 3.8 and PLA: 3.2 ± 2.5, P > 0.05) to
72h (CIT: 2.3 ± 2.0 and PLA: 1.6 ± 2.0, P > 0.05) of recovery in both CIT and PLA groups.
Insulin, testosterone and cortisol blood levels after fatigue test during recovery. There was no
main effect for time (P > 0.05) or group x time interaction (P > 0.05) in blood insulin (Fig.
4A), testosterone (Fig. 4B) and cortisol (Fig. 4C) levels, and testosterone:cortisol ratio (Fig.
4D) after fatigue test during recovery period (24, 48, and 72 h after RT).
Figure 4. Blood insulin (A), testosterone (B), and cortisol (C) levels, and testosterone:cortisol ratio
(D) at pre- and post-training session, and immediately after fatigue tests (leg pres and hack squat
exercises) during recovery period (at 24, 48, and 72h after resistance training session). Citrulline CIT,
Placebo PLA. Data are means ± SD.
54
EMG signal in the fatigue tests during recovery. There was no significant main effect for time
(P > 0.05) or group x time interaction (P > 0.05) in RMS (Fig. 5A) and FM slope (Fig. 5B)
values for the fatigue tests during the recovery period (24, 48, and 72 h after RT). The total
RMS (mean ± SD, CIT: 322 ± 130 vs. PLA: 304 ± 100; P > 0.05) and FM slope (mean ± SD,
CIT: -0.090 ± 0,073 vs. PLA: -0.098 ± 0.051; P > 0.05) values (for the 3 times of recovery)
were similar between CIT and PLA groups during fatigue tests.
Figure 5. RMS (A) and FM (B) values during fatigue tests (leg pres and hack squat exercises) during
recovery period (at 24, 48, and 72h after resistance training session). Citrulline CIT, Placebo PLA.
Data are means ± SD.
55
DISCUSSION
To our knowledge, this is the first study to examine the effects of oral CM supplementation
on time course of muscle recovery after a single session of conventional lower-body RT in
untrained young subjects. Based on the physiological properties and beneficial effects of CM
on muscle recovery, fatigue and performance (Bendahan et al. 2002; Perez-Guisado and
Jakeman, 2010; Wax et al. 2015; Glenn et al. 2015), we hypothesized that CM
supplementation would enhance muscle recovery process after a single session of
conventional RT. For this, we used the main indicators of muscle function and regeneration
(e.g., mechanical, functional, metabolic, and anabolic) to demonstrate with accuracy the
possible effects of CM on muscle recovery after RT. Our results reject the hypothesis tested,
by showing by the first time that CM supplementation does not improve muscle recovery
process (indicate by number of maximum repetitions, RPE, DOMS, EMG signal, CPK,
lactate, and anabolic hormones) after conventional RT in previously untrained young subjects.
Surprisingly, no significance differences were found in the number of maximum
repetitions and perceived exertion (OMNI scale) in the fatigue tests during the course of
recovery between CIT and PLA conditions. In contrast to our results, previous performance
studies have shown that CM supplementation (8g) can reduced rating of perceived exertion
(Glenn et al. 2015) and increased performance (i.e., number of maximum repetitions) during
repeated bouts of high-intensity resistance exercise to failure (Perez-Guisado and Jakeman,
2010; Wax et al. 2015; Glenn et al. 2015). A direct comparison of our results with the
aforementioned studies may be complex, due to the differences between the studies aims
(e.g., recovery vs. performance), but a key point should be asked: If CM supplementation was
effective to improve muscle function (number of repetition performed) in normal, unfatigued
subjects (Perez-Guisado and Jakeman, 2010; Wax et al. 2015; Glenn et al. 2015), why our
results found no beneficial effect in fatigued subjects during the course of muscle recovery? A
possible explanation for this question may be the incapability of CM for attenuating muscle
56
damage during recovery period; the conventional RT session resulted in similar increase in
plasma lactate (~75% increase at posttraining) and CPK (>25% increase at 24h post training)
concentration between CIT and PLA conditions, which typically results in impairment of
muscle function. However, Wax et al. (2015) reported an increase in muscle performance
(i.e., number of repetitions performed) without any inhibitory effect of CM on lactate
production, indicating that beneficial effects of CM on muscle function are not attributed to
acid base balance. Therefore, it is likely that the lack of effects of CM on muscle function
(i.e., number of maximum repetition) observed in our study to be due to inability of CM for
attenuating RT-induced muscle damage (indicate by similar CPK levels between CIT and
PLA conditions), resulting in no beneficial effect on rating of perceived exertion (post-test)
and muscle soreness (pre-test) during recovery period. In fact, the muscle damage has been
considered a potential indicator of impaired of muscle function after intense exercise (Byrne
et al. 2004), and its negative impact could be superior to any beneficial effects of CIT on
muscle function. This could explain, at least partially, because the CIT supplementation
promoted positive effects on muscle performance (Perez-Guisado and Jakeman, 2010; Wax et
al. 2015; Glenn et al. 2015), perceived exertion (Glenn et al. 2015), and muscle soreness
(Perez-Guisado and Jakeman, 2010) in normal, unfatigued (without any exercise-induced
damage) conditions, but not in our study, where the muscle was exposed to a unfavorable
physiological conditions (e.g., high degree of muscle damage after intense exercise) during
recovery. Therefore, it seems inconsistent indicate the CM supplementation to improved
muscle regeneration and, thus, the muscle function during recovery course.
One possible explanation for lack of effects of CIT on muscle damage regeneration
may be its particular mechanisms of action. CIT has been shown to increase ATP production
and rate of phosphocreatine (PCr) resynthesis (Bendahan et al. 2002), improving blood flow,
and increasing ammonia clearance (Breuillard et al. 2015), but not improving rate of protein
synthesis, and expression of anabolic markers (i.e., insulin and mTOR) in human muscles
57
(Churchward-Venne et al. 2014). This lack of effect of CIT on muscular regeneration markers
could explain, at least partially, the inability of CIT for improving tissue and functional
properties during recovery course. This was supported by no differences in anabolism (i.e.,
insulin and testosterone concentrations, and testosterone/cortisol ratio) and regeneration (i.e.,
CPK concentration) markers between CIT and PLA conditions observed in our study. It is
likely that the inability of CIT for improving muscle regeneration after RT to be superior to its
small effect on metabolism and performance (Perez-Guisado and Jakeman, 2010; Wax et al.
2015; Glenn et al. 2015), thereby obscuring any beneficial effect of CIT on muscle function
(i.e., number of repetitions and EMG signals – RMS and FM) during recovery course.
Corroborating the lack of effects of CM on muscle function during recovery, our
results showed no difference in the electromyographic (EMG) indicators of muscle activation
(RMS) and fatigue (FM) between CIT and PLA conditions. To our knowledge, this the first
study to examine the effects of CM supplementation on physiological indicators of muscle
activation and fatigue during recovery period. Considering that a decline in MF and increase
in RMS are typically associated with muscle fatigue during isometric and dynamic
contractions (De Luca, 1993; Arab and Salavati, 2007; da Silva et al. 2008; Larivière et al.
2011; Adam and De Luca, 2003), it should be expected a inverse effect of CM
supplementation on these factors in the fatigue test during recovery period. However, the lack
of beneficial effects of CM supplementation on muscle function (e.g., number of repetitions
performed) was corroborate with no change in RSM and FM values, indicating that CM
supplementation may not be effective to improve the neuromuscular responses during
recovery course after RT. It is important to note that subjects achieved maximum effort in the
leg press (OMNI scale mean 10 ± 0) and hack squat (OMNI scale mean 10 ± 0) fatigue tests,
indicating that protocol was reliable for muscle recovery analysis. Therefore, the lack of
effects of CM on muscle recovery would be attributed physiological properties of CM, but not
to fatigue protocol used in this study. Reinforcing our findings, no difference was observed in
58
the perceived muscle soreness between CIT and PLA conditions during recovery period.
Although muscle soreness may be a poor indicator of exercise-induced muscle damage during
recovery (Nosaka et al. 2002), it usually reflects muscle fatigue. This supports the findings of
the present study that CM supplementation does not promote attenuating effects on muscle
fatigue (an indicator of function) during recovery course.
Naturally a few limitations from this study must be mentioned. First, we did not
analyze the plasma NO and cittruline concentration, but previous studies that used similar
doses (6 g) of CIT showed an increase in plasma citrulline concentration (Sureda et al. 2009;
Sureda et al. 2010). Second, we did not collect muscle biopsies for analysis of the possible
tissue markers (e.g. IGF-I, HGF, mTOR, and p70S6k) of regeneration; however, we analyzed
the major markers of muscle (i.e., CPK, insulin, testosterone, and cortisol) regeneration and
(i.e., number of repetitions performed, perceived exertion, muscle soreness, and EMG signs)
function. Finally, subjects’ meals were not provided and may not accurately reflect exact food
consumption; however, we did encourage subjects to duplicate intakes for 24 hr preceding
each session. Further studies are required to address these issues.
In conclusion, our data indicate that CM supplementation (single 6 g dose at 60 min.
prior to training) does not improve muscle recovery course after a single session of high-
intensity resistance training in previously untrained young men with adequate macronutrients
intake. Further studies are required to extrapolate these results for others populations (e.g.,
older and/or women) with different training status (e.g., recreational practitioners and/or
athletes).
59
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