Nutrition 32 (2016) 723–724
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Nutrition
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Research Letter
Guanidinoacetic acid
increases skeletal muscle
creatine stores
in healthy men
Guanidinoacetic acid (GAA; also known as glycocyamine,
betacyamine, or guanidinoacetate) is an experimental dietary
additive that enhances serum creatine bioavailability and affects
blood-derived metabolic markers of methylation in humans [1].
Because creatine is an important compound in cellular bioener-
getics [2], consumption of GAA has been recognized as an effec-
tive investigational intervention to facilitate creatine-mediated
energy provision in health and disease [3]. At the same time,
the methylation of GAA to form creatine consumes w40% of
available methyl groups, potentially depleting the sources of
methyl groups in the body, including methionine, folate, betaine,
and choline [4]. However, to our knowledge, no human study so
far has evaluated the effects of GAA consumption on creatine
bioavailability and/or use of methyl groups in tissues with
high-energy requirements, such as skeletal muscle or brain. In
this pilot study, we evaluated the effects of 4-wk GAA supple-
mentation on total creatine and choline levels in human skeletal
muscle of healthy men.
Four healthy young and physically active men (age 25
3 y,
body mass index 24.3
1.6 kg/m2, physical activity 15
2
h/wk) were assigned to receive 3 g/d of oral GAA for 4 wk in
this open-label, repeated-measure pilot study. Sample size
(N ¼ 4) was in accordance with the power analysis (effect
size ¼ 0.9, a error probability ¼ 0.05, power ¼ 0.95) for the pri-
mary outcome measure. Our primary outcome measure was the
change in total creatine levels in the right vastus medialis muscle
assessed at baseline and at 4 wk. Secondary outcome measures
were total choline levels, intramyocellular and extramyocellular
triacylglycerol contents, and muscle water. In vivo metabolite
concentrations were determined by 1.5 T magnetic resonance
spectroscopy using a four-element body matrix receiver coil
(Siemens Avanto Tim, Erlangen, Germany) and calculated using
the muscle water signal as an internal intensity reference [5].
Participants were obliged to maintain their usual diets (apart
from the intervention) and physical activity levels during the
study. All participants gave written informed consent before
This study was supported by the Serbian Ministry of Education, Science and
Technological Development (Grant No. 175037), and the Faculty of Sport and
Physical Education, University of Novi Sad (2015 Annual Award). The authors
have no conflicts of interest to declare.
0899-9007/Ó 2016 Elsevier Inc. All rights reserved.
study inclusion, and the study was approved by the local institu-
tional review board in accordance with the Declaration of
Helsinki.
The results demonstrated that supplemental GAA signifi-
cantly augmented total creatine levels by 15.9% (95% confidence
intervals, 12.7% to 44.5%; P ¼ 0.03) when averaged across par-
ticipants (Table 1). Furthermore, a trend has been found for
increased choline concentration in the vastus medialis muscle
(6.1% corresponding to 0.8 mM, P ¼ 0.12) from before to after
GAA administration. Myocellylar triacylglycerols and muscle
water were not affected by the intervention (P > 0.05).
In this study, supplemental GAA evidently functioned as a
source of creatine, leading to an increase in the body’s creatine
stores in the muscle. It appears that GAA is absorbed from the
intestinal tract, enters the circulation, and is methylated to yield
creatine. The relative increase in muscle creatine following GAA
intake in this study is higher compared with similar studies using
creatine loading [6], suggesting that GAA supplementation might
be more effective than creatine supplementation at increasing
creatine levels in the skeletal muscle. The mechanism leading
to higher levels of muscle creatine after the intervention could
be a result of transport of newly synthesized hepatic creatine,
endogenous synthesis inside the skeletal muscle, or a combina-
tion of both [7]. A biotransformation trial using isotopically
labeled GAA to monitor possible intramuscular methylation of
dietary GAA to creatine would have improved the significance
of this study, together with a direct comparison with creatine
supplementation. Large-scale pharmacokinetic studies recruit-
ing more participants during longer intervention periods, with
dose-escalation trials are warranted to more fully explore tissue
distribution and utilization of GAA after ingestion.
These findings are accompanied by a tendency for an
augmented total choline pool in the muscle after the interven-
tion, suggesting an increased uptake of methyl donors by target
tissue for enhanced intramuscular synthesis of creatine from
exogenous GAA or related reactions. It is well known that
S-adenosyl-L-methionine (SAMe) acts as the primary methyl
donor for the methylation of GAA to creatine [8]. Unfortunately,
we were unable to evaluate muscle SAMe levels for this study.
However, elevated choline levels perhaps indicate amplified
remethylation of tissue homocysteine, an intermediate product
that accumulates during creatine synthesis from GAA. Future
studies should evaluate the metabolic behavior of other relevant
methyl donors (e.g., SAMe, folates, dimethylglycine) during GAA
loading in both circulation and target tissues. Additionally, GAA
seems to be neurotoxic when accumulates in the brain of exper-
imental animals [9], and patients with inherited disorders of
creatine metabolism [10]. The relative risk for neurotoxicity by
increasing dietary GAA intake is currently unknown and needs
exploration, yet previous studies reported no major side effects
when GAA was orally administered in dosages 3 g/d for 4 wk
724 Research Letter / Nutrition 32 (2016) 723–724

Table 1 [5] Wang X, Salibi N, Fayad LM, Barker PB. Proton magnetic resonance spec-
Changes in skeletal muscle metabolites during the study (N ¼ 4) troscopy of skeletal muscle: a comparison of two quantitation techniques.
J Magn Reson 2014;243:81–4.
Variable Baseline At follow-up P-value* [6] Greenhaff PL, Bodin K, Soderlund K, Hultman E. Effect of oral creatine
Creatine (mmol/L VOI) 25.5
4.9 28.9
2.3 0.03 supplementation on skeletal muscle phosphocreatine resynthesis. Am J
Choline (mmol/L VOI) 11.5
6.0 12.2
1.1 0.12 Physiol 1994;266:E725–30.
[7] McBreairty LE, Robinson JL, Furlong KR, Brunton JA, Bertolo RF. Guanidi-
Water (ppm) 448
93 402
116 0.65
noacetate is more effective than creatine at enhancing tissue creatine
Intramyocellular TGs (ppm) 19.1
7.8 20.0
3.1 0.44
stores while consequently limiting methionine availability in Yucatan
Extramyocellular TGs (ppm) 15.5
2.7 41.2
13.1 0.24
miniature pigs. PLoS One 2015;10:e0131563.
TG, triacylglycerol; VOI, volume of interest [8] Brosnan JT, da Silva R, Brosnan ME. Amino acids and the regulation of
Values are mean
SD methyl balance in humans. Curr Opin Clin Nutr Metab Care 2007;10:52–7.
*
P-value from two-sided t test (baseline vs. postadministration). [9] Zugno AI, Pereira LO, Mattos C, Scherer EB, Netto CA, Wyse AT. Guanidinoa-
cetate administration increases acetylcholinesterase activity in striatum of
rats and impairs retention of an inhibitory avoidance task. Metab Brain Dis
2008;23:189–98.
[10] Schulze A. Creatine deficiency syndromes. Mol Cell Biochem 2003;244:
in healthy men and women [1,11]. No neurologic adverse events 143–50.
were reported during the present study. [11] Ostojic SM, Niess B, Stojanovic M, Obrenovic M. Creatine metabolism and
In conclusion, supplemental GAA could be used as an experi- safety profiles after six-week oral guanidinoacetic acid administration in
healthy humans. Int J Med Sci 2013;10:141–7.
mental agent to positively affect creatine pool in the skeletal
muscle of healthy men. This might be relevant for restoring
Sergej M. Ostojic, M.D., Ph.D.
cellular bioenergetics in both health and disease, including
Biomedical Sciences Department
high-intensity exercise that depletes creatine stores, or disorders
Faculty of Sport and Physical Education
characterized by low muscle creatine, such as neurodegenerative
University of Novi Sad
diseases, mitochondrial myopathies, cachexia, or chronic fatigue
Serbia
syndrome. A key strength of the present study was the use of
the gold standard magnetic resonance spectroscopy method to University of Belgrade School of Medicine
noninvasively evaluate muscle metabolites in vivo. A significant Belgrade
limitation was the limited size of the data set. Serbia
Patrik Drid, Ph.D.
Biomedical Sciences Department
References Faculty of Sport and Physical Education
University of Novi Sad
[1] Ostojic SM, Stojanovic MD, Drid P, Hoffman JR. Dose-response effects of Serbia
oral guanidinoacetic acid on serum creatine, homocysteine and B vitamins
levels. Eur J Nutr 2014;53:1637–43. Jelena Ostojic, Ph.D.
[2] Wallimann T, Tokarska-Schlattner M, Schlattner U. The creatine kinase
system and pleiotropic effects of creatine. Amino Acids 2011;40:1271–96.
Clinical Center of Vojvodina
[3] Ostojic SM. Advanced physiological roles of guanidinoacetic acid. Eur J Nutr Novi Sad
2015;54:1211–5. Serbia
[4] Obeid R. The metabolic burden of methyl donor deficiency with focus on
the betaine homocysteine methyltransferase pathway. Nutrients 2013;5:
3481–95. http://dx.doi.org/10.1016/j.nut.2015.11.006