Creatine in Skeletal Muscle Physiology

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Nonvitamin and Nonmineral Nutritional Supplements. https://doi.org/10.1016/B978-0-12-812491-8.00008-4
© 2019 Elsevier Inc. All rights reserved.
Creatine in Skeletal Muscle Physiology
Massimo Negro*, Ilaria Avanzato* and Giuseppe D’Antona*,**
*CRIAMS-Sport Medicine Centre Voghera, University of Pavia, Pavia, Italy, **Department of Public Health, Experimental and Forensic Medicine,
University of Pavia, Pavia, Italy
INTRODUCTION
In 1992 Roger Harris and his colleagues discovered that exogenous creatine (Cr) administration enhances muscle Cr and
phosphocreatine (PCr) content (Harris etal., 1992). Since then Cr has become the most popular dietary supplement in
the field of sport and exercise physiology. In particular, Cr was used for the first time in the Olympic Games in Barcelona
by successful sprinters and, subsequently, has been used to enhance the physical performance in healthy individuals and
athletes. At present, a huge body of evidence, from more than 25years of research in the field, corroborates the efficacy
of Cr supplementation to promote physiological function in many types of exercise of varying duration and intensity,
and to aid improvements in strength, skeletal muscle mass, and bone mineral density, in healthy individuals and those
with neuromuscular diseases (Bazzucchi etal., 2009; Bemben etal., 2010; Bosco etal., 1997; Candow etal., 2015;
Chilibeck etal., 2015; D’Antona etal., 2014; Devries and Phillips, 2014; Griffen etal., 2015; Grindstaff etal., 1997;
Gualano etal., 2011, 2014; Hespel etal., 2001; Martone etal., 2015; Metzl etal., 2001; Mihic etal., 2000; Pearlman and
Fielding, 2006; Phillips, 2015; Ramirez-Campillo etal., 2015; Volek etal., 2004; Wilkinson etal., 2016). Indeed, Cr has
also been recently recognized as playing a role as an antioxidant, antinflammatory, and immunomodulatory compound
(Riesberg etal., 2016), as well as having interesting physiological effects on thermoregulation and cognitive performance
(Twycross-Lewis etal., 2016). In this chapter we will focus on the effects of Cr supplementation in skeletal muscle
physiology with particular attention to the known effects in healthy athletes.
CREATINE BACKGROUND
The Biochemistry of Creatine
Creatine (N-aminoiminomethyl-N-methylglycine, Cr) is a guanidine compound, which is endogenously synthesized by the
kidneys, pancreas, and liver, through a process that involves three amino acids as group donors: (1) a methyl group from
methionine, (2) an acetic group and nitrogen atom from glycine, and (3) an amide group from arginine (Fig.2.7.1). After
its production, Cr is mainly stored by the cardiac and skeletal muscle and brain. To perform its physiological role, Cr is
transformed into PCr by Cr kinase. The phosphate group is provided by adenosine triphosphate (ATP), which is converted
into adenosine diphosphate (ADP). PCr is a high-energy reserve, available for the conversion of ADP to ATP. This process
is essential during very intensive physical activity and relative high-energy request. Cr kinase catalyzes the reversible
transfer of the N-phosphoryl group from phosphoryl Cr to ADP to regenerate ATP and restore Cr skeletal muscle content
(Wyss etal., 2000).
The distribution of Cr kinase isoforms at a subcellular level has led to differing points of view regarding the function
of the Cr system. Wallimann etal. (1992) gave a description of the role of the different isoforms of Cr kinase. Cr kinase
isoforms are typically found in the cell cytoplasm and, in particular, at sites with a great ATP demand, e.g., plasma
membranes, sarcoplasmic reticulum, and myofibrils (Wallimann et al., 1992). A mitochondrial Cr kinase is also found
across mitochondrial membranes and, in the presence of Cr, it guarantees the conversion to PCr when the ATP obtained
from oxidative phosphorylation is available. These and other observations have promoted the fundamental role of Cr and
PCr in an energy shuttle system (ESS) of high-energy phosphates between the mitochondrial sites of ATP production
and the cytosolic sites of ATP utilization (Wallimann etal., 1992). The degree of involvement of this ESS depends on
Chapter 2.7

60 PART | II Nonessential Nutrients
different physiological muscle fiber requirements. Thus, in fast-twitch muscle fibers, mainly anaerobic/glycolytic, the ATP
production based on the efficacy of the ESS function predominates, whereas in slow-twitch, oxidative, the ESS function is
less important. The Cr/PCr system has a number of additional functions (Wallimann etal., 1992). These include maintaining
the cellular ATP/ADP ratio and buffering the products of ATP hydrolysis, which protects the thermodynamic efficiency of
ATP splitting. ATP hydrolysis generates ADP, phosphate, and a hydrogen ion. When high-intensity exercise occurs, the ATP
must be hydrolyzed very rapidly and it is essential that both ADP and the hydrogen ion are buffered so that: (1) prevention
of a rise in [ADP] caused by the Cr/PCr system reduces the inhibition of ATPases; (2) local cellular hydrogen production,
due to high rates of ATP utilization, is buffered by the Cr/PCr system when the Cr kinase reaction leads to ATP resynthesis
(Brosnan and Brosnan, 2007). Fig.2.7.2 illustrates the aspects of the Cr kinase system described above.
The Metabolism of Creatine: Loss, Replacement, and Tissue Transport Regulation
Cr and PCr elimination is primarily based on their break down to creatinine and its excretion through the urine. The rate of
Cr loss in humans is about 1.7% of the total body pool per day (Wyss etal., 2000), considering that in a 70-kg man the total
body Cr content is about 120 g, with a turnover of about 2 g/day (D’Antona etal., 2014). Since more than 90% of Cr and
PCr is found in skeletal muscle, Cr losses (and creatinine excretion) vary in relation to gender and age. Creatinine excretion
changes during life in an almost linear manner, reaching a maximum in 18–29year olds, with mean rates of loss of about
24 mg/kg per 24 h, reducing to mean rates of about 13 mg/kg per 24 h in 70–79year olds. Women have mean rates about
20% lower than men (Cockcroft and Gault, 1976).
Generally food provides 50% of our Cr daily requirement (approximately 1 g/day), with the body synthesizing the other
50% endogenously. Exogenous dietary sources of Cr include meat and fish, with Cr concentrations ranging from 4 to 5
g/kg in meat and from 4 to 10 g/kg in fish (D’Antona etal., 2014). No dietary Cr is available for vegans, and vegetarians
have very few sources of Cr in foods. Both vegans and vegetarians essentially require de novo synthesis for all of their
Cr stores, but studies have shown that Cr synthesis is insufficient in these subjects, with a decrease of serum/muscle Cr
levels compared to omnivores (Delanghe etal., 1989; Lukaszuk etal., 2002). Cr synthesis rates change during ageing and
Arginine:Glycine amidinotransferase
Arginine + Glycine ® Ornithine + GAA
Guanidinoacetate methyltransferase
AdoMet + GAA ® Creatine + AdoHcy
H2N
H2N
H2N
NH
H
2NH2N
H3C
CH3
NH2
OH OH
OH OH
O
S
O
N
N
N
N
NH2
O
S
OH OH
O
NN
NN
+
NH3
+
NH2
+
NH2
+
NH3
+
O
−O
H
O
–O
+
++
+H2N
NH3
+
NH
NH
O
−O
H
O
–O
O
–O
H2N
NH2
+
NH
O
–O
+
+H3N
FIG.2.7.1 Pathway of creatine synthesis. GAA, Guanidinoacetate; AdoMet, S-adenosyl-l-methionine; AdoHcy, S-adenosyl-l-homocysteine. (From
Brosnan, J.T., Brosnan, M.E., 2007. Creatine: Endogenous Metabolite, Dietary, and Therapeutic Supplement. Annu. Rev. Nutr. 27, 241-261).

Creatine in Skeletal Muscle Physiology Chapter | 2.7 61
in individuals with a typical Western diet, aged between 20–39, 40–59, and over 60, the estimated rates of Cr synthesis
is about 7.7, 5.6, and 3.7 mmol/day, respectively. Women have mean rates of Cr synthesis of about 70%–80% of those
observed in men (Brosnan and Brosnan, 2007).
Typically, the tissues containing the most Cr (e.g., skeletal and cardiac muscle) are essentially unable to synthesize it.
A Cr transporter (CRT) mediates the uptake of Cr into different tissues such as the kidneys, cardiac muscle, brain, and
primarily skeletal muscle. Cr uptake into tissues occurs against a high concentration gradient: blood Cr concentrations
range from 50 to 100 μM while, for example, skeletal muscle Cr concentrations generally range from 5 to 10 mM. CRT is
a Na+-dependent neurotransmitter transporter (Gregor etal., 1995), enhanced by insulin. CRT activates Na+/K+-ATPase
and presumably increases the driving force for Cr uptake (Snow and Murphy, 2001). Cr transport may be regulated through
acute and chronic mechanisms. Acute regulation may be brought about by changes in the Cr concentration. Chronically, Cr
transport may be regulated by gene expression, translation, or posttranslational modification of the CRT. Furthermore, an
inverse relationship regulates Cr uptake and its intracellular concentration (Dodd etal., 1999). In fact, a high extracellular
Cr concentration initially causes an increase in transport, which is followed by a down-regulation of CRT expression and a
reduction in Cr transport (Loike etal., 1988).
CREATINE SUPPLEMENTATION: PROTOCOLS, PHARMACEUTICAL FORMS, AND THEIR
SAFETY
The most popular Cr intake protocol found in the literature includes a loading phase of 20 g/day for 5days (divided in four
daily doses), followed by a 2–5 g/day maintenance dose. Generally, skeletal muscle significantly increases its Cr content
over the first 2–3days of supplementation. During this time, the osmotic effects of Cr uptake are assumed to be responsible
for body water retention, leading to a reduction in urine output typically being observed (Ziegenfuss etal., 1998). Cr has
frequently been taken together with carbohydrates and this combination seems to increase its uptake into the skeletal
muscle, probably due to the effect of insulin. Different carbohydrate dosages were used in several trials, but the most
effective dosage to significantly improve Cr uptake in muscle was 100 g per 5 g of supplemented Cr (Green etal., 1996).
Exercise is another potent stimulus for Cr uptake by skeletal muscle. This finding was demonstrated for the first time
by Harris etal. (1992), in a typical one-leg study. Cr supplementation has been found to increase total muscle Cr (Cr
plus PCr) by about 25% and when coupled with exercise, by an average of 37%, without affecting muscle ATP levels
(Harris etal., 1992). Considerable interindividual variation exists in the degree of muscle loading after supplementation.
FIG.2.7.2 The creatine kinase/phosphocreatine system. ADP, Adenosine diphosphate; AT P, adenosine triphosphate; CK, creatine kinase; Cr, creatine;
PCr, phosphocreatine; CRT, Cr transporter; mtCK, mitochondrial CK isoforms; ANT, adenine nucleotide translocator; OP, oxidative phosphorylation;
G, glycolytic enzymes; CK-g, glycolytic CK; CK-c, cytosolic CK; CK-a, ATPases CK (transporters, pumps, enzymes). (Modified from Brosnan, J.T.,
Brosnan, M.E., 2007. Creatine: Endogenous Metabolite, Dietary, and Therapeutic Supplement. Annu. Rev. Nutr. 27, 241-261).

62 PART | II Nonessential Nutrients
Although the reason for this is far from understood, it is clear that presupplement muscle Cr concentration is critical
(Rawson etal., 2002). Indeed, an inverse relationship between the increase in muscle PCr following supplementation in the
young (between 20 and 32years of age) and their Cr presupplementation levels has been established (Fig.2.7.3).
Cr monohydrate (CM) is the form most frequently cited in scientific literature and most commonly found in dietary
supplement/food products (Jäger etal., 2011). Besides CM, other Cr forms (Table 2.7.1) were recently introduced into
the market, with claims that they exhibit a higher physical performance improvement, bioavailability, effectiveness, and/
or safety profile than CM (Andres etal., 2016). However, to support these marketing claims there is little to no evidence
available in the literature (Jäger etal., 2011).
At present, there are no clinically significant side effects reported following CM supplementation at recommended
doses, unlike weight gain—an attribute often required by many athletes and subjects affected by muscle diseases (Bender
etal., 2008; Dalbo etal., 2008; Kreider etal., 2003a; Schilling etal., 2001).
CREATINE SUPPLEMENTATION AND PHYSICAL PERFORMANCE
Cr supplementation and its effects on strength and muscular performance have been widely studied. The International
Society of Sports Nutrition’s (ISSN) position claims Cr to be the most effective food supplement available so far for
increasing high-intensity exercise and gaining lean muscle mass (Kreider etal., 2010). Typical studies indicate that Cr
supplementation during training can increase one-repetition maximum (1RM) strength and power. Recently, a metaanalysis
of 63 studies showed that Cr supplementation enhances 1RM leg press and 1RM squat by 3% and 8%, respectively (Lahners
etal., 2015). In several experimental conditions the potential of Cr on anaerobic performance parameters such as total
working capacity, peak power output, resistance capacity, total work performed, total strength expressed, and others, have
been extensively demonstrated (Wilborn, 2015).
Strength, power, and muscle-building athletes are typically users of Cr supplements, although evidence suggests some
benefits for endurance athletes could also be obtained. In particular, Cr supplementation has not been linked to functional
improvement in runners involved in distance races longer than 5000 m, but performance increases could be obtained over
shorter and/or intermittent training for distances under 2000 m (Earnest, 1997; Earnest etal., 1997). The Cr supplementation
effectiveness on endurance capacity can be attributed to several mechanisms: (1) PCr aids ATP resynthesis, in a decreasing
role in relation to duration and intensity of muscle work (Bangsbo etal., 1990); (2) Cr may help produce ATP aerobically,
considering the ESS function of Cr between the mitochondria and muscle fibers (Wallimann etal., 1992); and (3) muscle Cr
can support anaerobic glycolysis and this may lead to a reduced intramuscular lactate production (Earnest and Rasmussen,
2015). Furthermore, based on the osmotic effects of Cr uptake (Ziegenfuss etal., 1998), the use of Cr in endurance sports
65%
60%
55%
50%
45%
40%
35%
30%
25%
20%
15%
16 17 18
r = −0.76; p = 0.03
Initial muscle PCr (mmol/kg)
Increase in muscle PCr (%)
19 20 21 22 23
24
FIG.2.7.3 The increase in muscle phosphocreatine (PCr) after creatine supplementation is inversely related to presupplementation PCr levels.
(From Brosnan, J.T., Brosnan, M.E., 2007. Creatine: Endogenous Metabolite, Dietary, and Therapeutic Supplement. Annu. Rev. Nutr. 27, 241-261).

Creatine in Skeletal Muscle Physiology Chapter | 2.7 63
(alone or in combination with other compounds) has been speculated as a possible strategy for preserving hydration and
minimizing sweat loss (Beis etal., 2011; Easton etal., 2007; Francaux and Poortmans, 1999; Polyviou etal., 2012; Saab
etal., 2002; Watson etal., 2006). However, despite the fact that some effects of Cr supplementation on thermoregulatory
and cardiovascular responses have been reported, this does not seem to affect significantly muscle work capacity in hot
environments (Beis etal., 2011; Easton etal., 2007; Kilduff etal., 2004; Polyviou etal., 2012). Other studies have failed to
observe any effects of Cr on thermoregulation (Bennett etal., 2001; Branch etal., 2007; Oopik etal., 1998; Rosene etal.,
2015; Terjung etal., 2000).
Creatine Supplementation and Muscle Hypertrophy
In several strength and power/speed sports an increase in muscle mass is often required to improve performance by
athletes. Based on this statement, the hypertrophic effects of Cr supplementation have been investigated in different papers.
During a typical Cr loading period (first 5–7days), data indicate that individuals will experience approximately 0.6–2.0
kg gains in lean body mass (Earnest etal., 1995; Green etal., 1996; Kreider etal., 1998; Kreider, 2003). Furthermore, Cr
supplementation during chronic resistance exercise studies (approximately 6–8weeks) has shown to increase the lean body
mass by about 3 kg (Earnest etal., 1995; Kreider etal., 1996; Stout etal., 1999). Other studies indicate that 4–8weeks
of strength training coupled with Cr supplementation, in combination with other substances (e.g., glucose), can stimulate
greater gains in lean mass when compared to Cr supplementation alone (Kreider etal., 1998; Stout et al., 1997). These
results are typically observed in men, but similar gains in lean muscle mass have been also reported in women.
Initial studies investigating the role of Cr supplementation suggested that additional water retention in the muscle
primar ily contributes to the gain in mass. However, subsequent research suggested that Cr supplementation increases body mass
through greater muscle protein synthesis and therefore of muscle fiber hypertrophy. From this point of view the pioneering
TABLE2.7.1 Different Forms of Creatine and Their Creatine Content Percentage (Jäger etal., 2011)
Form of creatine Creatine content (%) Difference in CM (%)
Creatine anhydrous 100.0 +13.8
CM 87.9 0
Creatine ethyl ester 82.4 –6.3
Creatine malate (3:1) 74.7 –15.0
Creatine methyl ester HC1 72.2 –17.9
Creatine citrate (3:1) 66 –24.9
Creatine malate (2:1) 66 –24.9
Creatine pyruvate 60 –31.7
Creatine a-amino butyrate 56.2 –36.0
Creatine a-ketoglutarate 53.8 –38.8
Sodium creatine phosphate 51.4 –41.5
Creatine taurinate 51.4 –41.6
Creatine pyroglutamate 50.6 –42.4
Creatine ketoisocaproate 50.4 –42.7
Creatine orotate (3:1) 45.8 –47.9
Carnitine creatinate 44.9 –49.0
Creatine decanoate 43.4 –50.7
Creatine gluconate 40.2 –54.3
CM, Creatine monohydrate.

64 PART | II Nonessential Nutrients
contribution made by Volek etal. (1999) is very interesting, demonstrating that a Cr ingestion in resistance-trained males
increased significantly the muscle fiber cross-sectional area in each of the muscle fiber types observed: type I (35% vs.
11%), type IIA (36% vs. 15%), and type IIX (35% vs. 6%). Based on these findings, Willoughby and Rosene further
examined the effects of Cr supplementation on gene and myosin heavy-chain protein expression of contractile filaments
(Willoughby and Rosene, 2001, 2003). The conclusion by the authors indicated that increases in lean body mass are not
solely attributable to greater water retention in the muscle, but rather to regulation of protein synthesis through a different
gene expression of myogenic regulatory factors induced by Cr. These myogenic regulatory factors (e.g., MRF-4, Myf-5,
Myo-D, and myogenin) act to control gene expression by binding to DNA and subsequently promoting muscle-specific
gene transcription of fundamental muscular proteins such as myosin heavy chain, myosin light chain, α-actinin, troponin I,
and Cr kinase (Lowe etal., 1998). Furthermore, the influence of Cr supplementation on satellite cell (SC) function, based
on myonuclear domain theory, was explored by Olsen etal. (2006) and Safdar etal. (2008).
Olsen etal. (2006) demonstrated that Cr ingestion (load: 24 g/day, 6 g/serving, 4 servings/day, 7days; maintenance:
6 g/day, 1 serving/day, 15weeks) during a resistance exercise promotes the proliferation of SCs and stimulates the
myonuclei relative distribution in skeletal muscle. Safdar etal. (2008) referred that supplementation of Cr (load: 20 g/day,
10 g/ serving, 2 servings/day, 3days; maintenance: 5 g/day, 1 serving/day, 7days) induces proliferation and differentiation
of SCs thus activating genes of cytoskeletal remodeling.
Creatine Supplementation and Exercise-Induced Muscle Damage and Injuries
High-intensity muscle work leads to myofibrillar damage in athletes. Muscle recovery passes through structural reparation
and Cr supplementation has been demonstrated to regulate at least four important mechanisms involved in the regeneration
process (Kim J etal., 2015).
(1) The first mechanism concerns the inflammatory response induced by exercise-induced muscle damage. Santos etal.
(2004) showed that 20 g/day of Cr, administrated to 34 male runners for 5days before a competition, diminished
prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and lactate dehydrogenase (LDH) after a competition of
30 km. In agreement with these data, Bassit etal. (2008) described how 11 male triathletes who introduced 20 g/day
of Cr for 5days before a half-Ironman contest had reductions in postexercise levels of interferon-α (INF-α), TNF-α,
PGE2, and interleukin-1β (IL-1β). Deminice etal. (2013) referred that 0.3 g/kg of Cr ingested for 7days suppresses
the rise in TNF-α after a repeated bout of anaerobic running tests.
(2) The second possible Cr mechanism is to reduce oxidative stress (Lawler etal., 2002; Rahimi, 2011). One of the first
pieces of evidence for the antioxidant capacity of Cr was described by Lawler etal. (2002). Later a subsequent study
(Deminice and Jordao, 2012) indicated that Cr ingestion during the 28days before acute muscle activity diminishes
lipid hydroperoxides and thiobarbituric acid-reactive substances (TBARS) with an increase of total antioxidant
capacity and in particular glutathione (GSH) and glutathione disulfide (GSSG) ratio. In another clinical trial, for 7days
Rahimi (2011) administrated 20 g/day of Cr to individuals who went on to show decreased levels of 8-hydroxy-2-
deoxyguanosine (8-OHdG) and malonyldialdehyde (MDA) in the blood after a resistance exercise session. Contrary
to this, other studies reported that Cr supplementation does not decrease oxidative stress after exercise-induced muscle
damage (Deminice etal., 2013; Silva etal., 2013). Although the role of Cr on exercise-related oxidative stress is very
interesting, poor data are available at present and this mechanism needs to be investigated in more detail.
(3) The third mechanism of action of Cr possibly involves the regulation of muscle calcium trafficking. Muscle damage
may increase calcium concentrations in the cytosol due to an impaired sarcoplasmic reticulum function, leading to
further muscle damage (Beaton et al., 2002). Cr regulates the sarcoplasmic reticulum calcium pump function by
phosphorylating ADP to ATP and decreasing cytosolic calcium levels (Cooke etal., 2009; Korge etal., 1993). From
this point of view, Minajeva etal. (1996) proposed that an increase of muscle PCr, with Cr supplementation, promotes
ATP regeneration leading to an attenuation of calcium-related damage. However, this hypothesis needs investigation.
(4) The last mechanism has been associated with SC activation and proliferation (Olsen etal., 2006; Safdar etal., 2008).
SCs are known to play a key role during the regeneration process after muscle damage (Paulsen etal., 2012) and the
role of Cr supplementation has already been shown in the previous paragraph.
CONCLUSION AND FUTURE DIRECTION
At present, there is an important core reference that underlines how Cr supplementation is safe and can lead to significant
performance enhancement. The positive effect of Cr on muscle physiology is useful in several anaerobic/aerobic

Creatine in Skeletal Muscle Physiology Chapter | 2.7 65
exercise, resistance, and speed training programs, as well as intermittent exercise and muscle hypertrophy programs. Cr
supplementation may also prevent exercise-induced muscle damage, facilitating recovery after training sessions and/or
contests, and this can facilitate muscle sports-specific adaptation. However, a clear comprehension of how Cr can regulate
the complex muscle subcellular signal network during exercise recovery needs to be better established with other well-
designed studies. In addition, Cr promises to be effective on bone metabolism but available data on its effect on bone
accretion are still inconsistent (and beyond the topic of the chapter). In conclusion, further accurate study must be designed
with appropriate groups of subjects and longer duration treatments (>52weeks) to understand the unclear mechanisms by
which Cr supplementation can, in the long term, affect the physiology of the muscle.
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FURTHER READING
Crassous, B., Richard-Bulteau, H., Deldicque, L., Serrurier, B., Pasdeloup, M., Francaux, M., Bigard, X., Koulmann, N., 2009. Lack of effects of creatine
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Wyss, M., Kaddurah-Daouk, R., 2000. Creatine and creatinine metabolism. Physiol. Rev. 80 (3), 1107–1213.