Accepted Manuscript

Creatine biosynthesis and transport in health and disease

Marie Joncquel-Chevalier Curt, Pia-Manuela Voicu, Monique Fontaine, Anne-

Frédérique Dessein, Nicole Porchet, Karine Mention-Mulliez, Dries Dobbelaere,
Gustavo Soto-Ares, David Cheillan, Joseph Vamecq

PII: S0300-9084(15)00342-9
DOI: 10.1016/j.biochi.2015.10.022
Reference: BIOCHI 4862

To appear in: Biochimie

Received Date: 2 July 2015

Accepted Date: 27 October 2015

Please cite this article as: M. Joncquel-Chevalier Curt, P.-M. Voicu, M. Fontaine, A.-F. Dessein,
N. Porchet, K. Mention-Mulliez, D. Dobbelaere, G. Soto-Ares, D. Cheillan, J. Vamecq, Creatine
biosynthesis and transport in health and disease, Biochimie (2015), doi: 10.1016/j.biochi.2015.10.022.

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REVIEW

Creatine biosynthesis and transport in health and disease

Marie Joncquel-Chevalier Curta,b, Pia-Manuela Voicuc, Monique Fontainea,b, Anne-Frédérique

Desseina, Nicole Porcheta,b, Karine Mention-Mulliezb,d, Dries Dobbelaereb.d, Gustavo Soto-
Arese, David Cheillanf,g and Joseph Vamecqa,b,h,*

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a
Biochemistry and Molecular Biology, Hormonology-Metabolism-Nutrition & Oncology (HMNO),

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Center of Biology & Pathology (CBP) Pierre-Marie Degand, CHRU Lille, 59037 Lille, France, b RADEME
Research Team for Rare Metabolic and Developmental Diseases, EA 7364, Université Lille 2, Lille,
France, c Clinical Chemistry Laboratory, Dr Schaffner Hospital of Lens, 62307 Lens, France, d Medical

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Reference Center for Inherited Metabolic Diseases, Jeanne de Flandres Hospital, CHRU Lille, 59037
Lille, France, e Department of Neuroradiology, Roger Salengro Hospital, CHRU Lille, 59037 Lille,
France, f Hospices Civils de Lyon, Service Maladies Héréditaires du Métabolisme et Dépistage
Néonatal, Groupement Hospitalier Est, 69677 Bron, France, g Lyon University, INSERM U1060,

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CarMeN Laboratory, University Lyon-1, INSA-Lyon, F-69600 Oullins, France, h Inserm, Lille, France.
* Corresponding author. Tel.: +33 676 06 10 85. E-mail address: joseph.vamecq@inserm.fr
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ABSTRACT
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Creatine is physiologically provided equally by diet and by endogenous synthesis from arginine and
glycine with successive involvements of arginine glycine amidinotransferase [AGAT] and
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guanidinoacetate methyl transferase [GAMT]. A specific plasma membrane transporter, creatine

transporter [CRTR] (SLC6A8), further enables cells to incorporate creatine and through uptake of its
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precursor, guanidinoacetate, also directly contributes to creatine biosynthesis. Breakthrough in the

role of creatine has arisen from studies on creatine deficiency disorders. Primary creatine disorders
are inherited as autosomal recessive (mutations affecting GATM [for glycine-amidinotransferase,
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mitochondrial]) and GAMT genes) or X-linked (SLC6A8 gene) traits. They have highlighted the role of
creatine in brain functions altered in patients (global developmental delay, intellectual disability,
behavioral disorders). Creatine modulates GABAergic and glutamatergic cerebral pathways,
presynaptic CRTR (SLC6A8) ensuring re-uptake of synaptic creatine. Secondary creatine disorders,
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addressing other genes, have stressed the extraordinary imbrication of creatine metabolism with
many other cellular pathways. This high dependence on multiple pathways supports creatine as a
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cellular sensor, to cell methylation and energy status. Creatine biosynthesis consumes 40% of methyl
groups produced as S-adenosylmethionine, and creatine uptake is controlled by AMP activated
protein kinase, a ubiquitous sensor of energy depletion. Today, creatine is considered as a potential
sensor of cell methylation and energy status, a neurotransmitter influencing key (GABAergic and
glutamatergic) CNS neurotransmission, therapeutic agent with anaplerotic properties (towards
creatine kinases [creatine-creatine phosphate cycle] and creatine neurotransmission), energetic and
antioxidant compound (benefits in degenerative diseases through protection against energy
depletion and oxidant species) with osmolyte behaviour (retention of water by muscle). This review
encompasses all these aspects by providing an illustrated metabolic account for brain and body
creatine in health and disease, an algorithm to diagnose metabolic and gene bases of primary and
secondary creatine deficiencies, and a metabolic exploration by 1H-MRS asssessment of cerebral
creatine levels and response to therapeutic measures.
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1. Introduction
Creatine (from the Greek word « kreas » meaning meat) was first described by the French chemist
Eugène Chevreul in the 1830ies [1]. Bodybuilding effects of creatine have been widely publicized
towards the large public and they are accounted for by intracellular water retention rather than by
significant increase in muscle mass due to osmolyte properties of creatine [2-4]. Creatine is also long
known for its stimulatory effects on muscle function and energetics, and it contributes to a transient
intracellular storage of metabolic energy by the phosphate high energy bond exchange operated

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with adenosine triphosphate (ATP) and catalyzed by creatine kinases (CK) [5-8]. Phosphocreatine also
referred to as creatine phosphate represents the phosphorylated form of creatine (Fig. 1) and is a
convenient form of energy storage in tissues with high energetic needs such as muscle and brain [5-
8]. The creatine-phosphocreatine reversible conversion (creatine cycle) is polarized in the cell

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between mitochondria and extramitochondrial sites. Body stores in creatine, the substrate for
creatine kinases, are maintained physiologically in an equal manner by nutritional supply and
endogenous biosynthesis [9]. Biosynthesis of creatine involves two enzymes, L-arginine : glycine

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amidinotransferase [AGAT] (EC 2.1.4.1), and N-guanidinoacetate methyltransferase [GAMT] (EC
2.1.1.2), along with a membrane carrier, SLC6A8 (Fig. 2). Table 1 provides a survey of genes and
related proteins involved in creatine biosynthesis, transport, and creatine-phosphocreatine cycle,
along with main animal models and diseases, the information contained in this table being issued

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from works repeatedly cited throughout this review but also from works addressing more specific
issues [10-29]. The present review is dedicated to the biology of creatine biosynthesis, transport and
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related deficiencies completed by a short subsection on CK appearing thereafter.

2. Creatine/phosphocreatine cycle
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It should be kept in mind that the creatine/phosphocreatine cycle is expected to be impaired in

disorders of creatine biosynthesis and transport through a reduced supply in creatine. This reduced
substrate availability for CK may be, however, corrected by dietary supplementation in creatine and
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by other appropriate strategies to an extent depending on the underlying defect. Regarding the
working-up of CK cycle, in mitochondria, consumption of ATP and formation of phosphocreatine by
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mitochondrial CK (MitCK) (sarcomeric MitCK or sMitCK in striated muscle and ubiquitous MitCK or
uMitCK in most other tissues) maintains an ADP/ATP ratio favorable for the activity of ATP synthase
to proceed. At ATP requiring sites (for instance, muscle contraction, plasma membrane Na+/K+
ATPase), cytosolic CK (MM-CK, BB-CK or MB-CK isoenzymes) restitutes ATP and creatine, the
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resulting rise in the concentration of unphosphorylated creatine acting back to stimulate

consumption of mitochondrial ATP synthase-driven formation of ATP and hence oxidative
phosphorylations through a favorable ADP/ATP ratio. Regarding creatine kinases, in contrast to what
is observed for enzymes involved in creatine biosynthesis and transport, little patients with gene
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deficiency have been reported [30-31], essentially because CK exist under various isoenzyme forms
which may partially compensate for a deficiency of one of the isoforms [32-34]. Only deficiency of
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the muscle and not brain isoform of CK has been described to date, with a clinical picture of
myocardial infarction associated with little or no rise in blood CK, and a drop in skeletal muscle CK
content [30, 31, 35-37]. This muscle CK deficiency is caused by a mutant protein in which the
interaction between E79 and K138, a saline bond between a glutamic acid and a lysine, is disrupted
[38]. Close linkage of myotonic dystrophy to a polymorphism of CKM gene was also reported [39]
without, however, validation of involvement of the latter gene in this disease [40]. On the other
hand, animal models for a deficiency (known-out mice) of each of the CK isoforms have been
extensively described as well as combined CK deficiencies [16-18, 23-25]. As documented in this
review for deficiencies of proteins in creatine biosynthesis and transport, deficiencies secondary to
other genetic disorders have been also described for CK, for instance in 3-oxothiolase deficiency and
2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiencies in which 2-methyl,3-hydroxybutyrate
may act by inhibiting total and mitochondrial CK activities [41]. In contrast to the low number of
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reports describing CK deficiencies in the humans, CK abnormalities have been described in a great
many pathophysiological states and patients. This results from the involvement of CK in many cellular
processes and its easy leakage from dying cells to body fluids. Increased serum levels of MM-CK, BB-
CK and MB-CK isoforms are for instance observed during the course of skeletal muscle (myolysis,
rhabdomyolysis), brain (infarction) and heart (infarction) cell necrosis, respectively. CKB is actually a
marker of several disease states including cancer [42], cerebral disorder [43] and renal diseases [44].
CK in health and disease actually encompass a series of topics developed elsewhere in general
reviews [6, 45, 46] and including energetic aspects of neurodegenerative disorders [47].

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3. Creatine biosynthesis and transport, and creatine-phosphocreatine cycle

3.1. Metabolism of creatine

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The bulk of body creatine is present in skeletal muscle in which creatine and phosphocreatine
concentrations may reach 30 mM. Lower but substantial concentrations are also recovered in brain
(10 mM) [7]. Body needs in creatine is permanent to allow muscle mass development and to

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counteract physiological removal in the urine under the form of creatinine. Body stores in creatine
are maintained by two ways: nutritional supply and endogenous synthesis. Each of these routes
equally contributes to physiological body creatine stores [9].
The endogenous synthesis of creatine requires two amino acids : glycine and arginine and

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two enzymes, L-arginine : glycine amidinotransferase (AGAT]) (EC 2.1.4.1), and N-guanidinoacetate
methyltransferase (GAMT) (EC 2.1.1.2), along with a membrane carrier, SLC6A8 (Fig. 2).
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The first step of creatine biosynthesis is catalyzed by AGAT, and occurs mainly in kidney, mainly in
the mitochondria intermembrane space and to a lesser extent in cytoplasm [48, 49]. The two
mitochondrial and cytosolic isoforms of AGAT are encoded by the GATM gene and produced by
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alternative splicing. The mitochondrial AGAT isoform precursor contains 423 amino acids and is
cleaved by 37 aminoacids, giving rise to the mitochondria mature protein with 386 amino acids; the
cytoplasmic mature form is immediately produced as a 391 amino acids-containing protein [14, 15].
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AGAT operates a transfer of an amidino residue from arginine to glycine with, as a result, formation
of L-ornithine and guanidinoacetate (GAA).
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In a second step catalyzed by GAMT, a methyl group from S-adenosyl methionine (SAM), a methyl
donor, is transferred to GAA. This step occurs essentially in liver and produces creatine and S-
adenosyl homocysteine (SAH). Interestingly, this biosynthesis step is physiologically known to
consume 40% of methyl groups produced by cells as S-adenosylmethionine [50].
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After endogenous synthesis or in turn nutritional supply [9, 51], creatine may be released in
blood stream. Circulating creatine may enter cells via a specific plasma membrane Na+/Cl-
transporter, creatine transporter [CRTR], and may be transiently phosphorylated to phosphocreatine
by creatine kinases [7]. The latter enzymes catalyze a reversible reaction. So when energetic needs
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are high (muscular exercice), phosphocreatine can restitute the high energy phosphate bond to ADP
(adenosine diphosphate) to regenerate ATP which then becomes directly available for ATP-driven
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events.
Creatine and phosphocreatine may be subject to a non enzymatic dehydration and
cyclisation to form creatinine. About 1.7 % of body pool of creatine/phosphocreatine are daily
converted to creatinine [9]. This end product freely diffuses out the cell to be removed ultimately in
the urine. Urinary excretion of creatinine physiologically depends on the muscular mass and is
routinely used as a marker of the renal function, the alteration of which results in excessive retention
of creatinine in blood.

3.2. Regulation
Endogenous synthesis of creatine is modulated by several factors. A major regulation is
operated at the level of AGAT, the creatine biosynthesis-initiating and rate-limiting step (Fig. 2).
AGAT is subject to a negative feedback exerted by high levels of intermediate and product, ornithine
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and creatine, respectively. On the opposite, a low nutritional supply in creatine is associated with a
sustained increase in AGAT activity contributing to creatine homeostasis [7].
Creatine transporter (SLC6A8) is regulated by extracellular creatine levels in a time-
dependent manner, negative control by excess creatine occurring more rapidly than positive
regulation by creatine deficiency. SLC6A8 is also sensitive to inhibition by GAA [7].
Recently, it has been unexpectedly found that female and male populations might be
discriminated as regards to urine but not blood values including creatine on creatinine ratio and
guanidinoacetate on creatinine ratio values. These gender differences previously observed in healthy

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populations were proposed to result from differential effects of testosterone and estrogen in
adolescents and adults, and by estrogen effects in prepubertal age on SLC6A8 function [52]. Other
hormonal effects affecting creatine biosynthesis include stimulatory effects of growth hormone and
thyroxine on AGAT [53]. On the other hand, an important finding lies in the regulation of SLC6A8 by

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the AMP-activated protein kinase [54]. This protein kinase is a sensor of cellular energetic status and
is known to couple substrate transport to capacity of cells to yield energy. The AMP-activated protein
kinase is activated in conditions of energy depletion. It was shown by Li et al. [54] to inhibit SLC6A8

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via the mTOR pathway. The biological significance of this observation is that when cell is in energy
depletion, creatine which buffers ATP levels is not longer necessary (because of the lack of ATP) and
as a result its cellular uptake by SLC6A8 is consequently reduced. These regulatory aspects
completing intra-metabolic regulations given in Fig. 2 are illustrated in Fig. 3.

3.3. The particular case of brain

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Though representing 2% of total body weight, brain actually utilizes 20% of the whole body
energy production. Indeed, high costs in ATP are dedicated to transmembrane ionic gradients and
neurotransmission. Taking into account that energetic stores are low in cerebral cells, energetic
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substrates are supplied continuously by circulation, explaining why brain is highly dependent on
vascular integrity and supply. Creatine might help brain intracellular energy routing thanks to the
creatine-phosphocreatine cycle. In this respect, phosphocreatine and ATP are energetic compounds
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directly available for brain functions [55]. Importantly, CNS cells possess proteins enabling creatine
synthesis and transport. However, as shown in Fig. 4, CNS pathways physiologically ensuring creatine
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content of mature brain require intercellular cooperation to meet creatine formation or import since
only 12% of CNS cells possess the entire biosynthetic pathway (AGAT plus GAMT) [56, 57].
Approximately 43% of cells contain only one of the two biosynthetic enzymes, the remaining 45%
CNS cells being devoid of both AGAT and GAMT. The fact that AGAT and GAMT may be distributed in
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different cells explains that GAA produced in a cell needs transport to another to complete synthesis
into creatine. This transport is performed by SLC6A8 which may act not only as a creatine but also as
a GAA transporter. In mature brain, blood brain barrier (BBB) is little or no permeable to
physiological concentrations of creatine as a result of a low expression of SLC6A8 at this level notably
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in astrocytes. In immature/developing brain (during the first years of life), SLC6A8 is in contrast
highly expressed in brain structures contributing to BBB, explaining why immature brain substantially
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covers its needs in creatine from circulating creatine rather than exclusively from local synthesis.
Creatine has also been attributed a modulatory role in neurotransmission, acting on GABAA receptor
chloride channel complex for which it represents a competitive antagonist [58, 59] and on the NMDA
receptor [60]. This putative role of creatine as a brain neurotransmitter along with its attributed
neuromodulatory effects are illustrated in Fig. 5.

3.4. The role of creatine as an antioxidant

Creatine exhibits antioxidant activity through either direct interaction with oxidant species or
metabolic action conferring antioxidant protection (for a recent consideration, see [61]). Oxidant
species scavenged by creatine include superoxide anion radical (O2°-), peroxinitrite (ONOO-) and 2,2’-
azino-bis(3-ethylbenzothiazolamine-6-sulfonate stable radical cation (ABTS+) [62]. The effective
antioxidant activity of creatine, at least in acellular systems, has been reported to be lower than that
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of reduced glutathione, the two antioxidant potentials being additive [62]. In muscle, because of its
wide dispersion in the sarcoplasm, creatine is readily available to free radicals generated during
physical exercice [62].
Creatine is a substrate for creatine kinases, including mitochondrial creatine kinase(s). As
mentioned above, creatine availability to mitochondrial kinases allows to maintaining an ADP/ATP
ratio favorable for mitochondrial respiratory chain to proceed at substantial rates. By preventing
stasis in the electron transfer chain, mitochondrial creatine kinases reduce the local generation of
oxidant species (such as superoxide anion radicals) through a mitochondrial CK-dependent ADP re-

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cycling activity as highlighted experimentally by Meyer et al. [63] during exposure to high glucose or
mimicking conditions. Reactive oxygen species generated by high glucose supply is sensitive to
creatine [64]. In these experiments, creatine not only acts as a substrate but also helps to preserving
the mitochondrial CK [63,64]. Creatine like physical exercise may reduce oxidative stress in murine

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tissues and body fluids [65]. As a general rule, creatine exhibits protections in many experimental
models in which oxidative stress takes place and which include exposures to mitochondrial or nuclear
DNA mutagenesis [66,67], nitropropionate [68], UV [66,69], (hydrogen or/and t-butyl)

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hydroperoxides [67,70,71], peroxinitrites [70], respiratory chain inhibitor rotenone [71],
proconvulsant drug pentylenetetrazole [72], RNA-damaging conditions[73], guanidinoacetate [74],
acute exercice [75], ischemia/reperfusion injury [76], and sedentary lifestyle [77]. Part of
improvements mediated by creatine in some of these models may, however, result from other

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properties; for instance, neuromodulation of GABAergic and glutamergic pathways mentioned below
in this review may also, theoretically, protect against pentylenetetrazole-induced seizures. In
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combination with pyruvate, creatine has been shown to be effective in reducing oxidative stress
caused by hypertryptophanemia in rats [78]. Part of the antioxidant protection conveyed by creatine
may result from either promoting a rise or preventing a drop in cellular antioxidant defenses
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[6,72,76,79], though in a short-term study the antioxidant activity of creatine was not associated
with changes in antioxidant enzymes [80]. Moreover, some studies indicate that creatine, secondarily
to its ability to increase muscle performance, may promote oxidative stress and lower antioxidant
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status of healthy athletes [81] or in turn may be without effect of oxidative stress as in the case of
spontaneously hypertensive rats [82].
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3.5. Animal models of primary creatine disorders

3.5.1. Animal models for AGAT deficiency

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Gatm-/- mice have been developed and studied. At the muscular level, magnetic resonance spectra
of muscle in these AGAT-deficient mice compared with those in the wild type mice indicate a near-
complete absence of creatine and phosphocreatine along with increased inorganic phosphate/β-ATP
ratio and a halving of ATP levels [83]. OXPHOS enzyme activities pondered by the citrate synthase
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content are decreased for complexes I, III and IV and, like the overall mitochondrial content, are
increased for complexes II and V (ATP synthase) [83]. Skeletal muscle is morphologically atrophic,
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mice having a reduced grip strength. All these skeletal muscle abnormalities may be reversed by
creatine intake [83]. At a more general level, AGAT-deficient mice display creatine deficiency and
exhibit multiple anomalies including sterility. The animals are lean and short; body mass index,
locomotor activity and blood leptin are low. Scoliosis attests for chronic hypotonia. Biochemical
findings indicate a drop in nucleoside triphosphate levels and acetyl-CoA carboxylase activity in
several tissues including muscle, adipose tissue and liver. There is also activation of the AMP-
activated protein kinase (AMPK). Interestingly, the Gatm-/- mice present with an improved glucose
tolerance and a protection towards the high fat diet-induced metabolic syndrome. The phenotype of
these animals is well responsive to diet creatine supplementation [84]. Importantly, leptin appears to
be essential in AMPK activation and resistance to metabolic syndrome exhibited by AGAT-deficient
mice [85].
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3.5.2. Animal models for GAMT deficiency

A knockout mouse model for GAMT deficiency, Gamt -/- mice, generated by gene targeting in
embryonic stem cells, showed increased levels of GAA and reduced creatine and creatinine levels in
brain, serum, and urine like it is seen in human GAMT deficiency [20] The GAMT-deficient animals
were more subject to neonatal mortality, muscular hypotonia, male sterility, and reduction in body
weight caused by reduced body fat mass, not depending on leptin. This marked reduction in body
weight developed throughout the life and was the most obvious symptom of GAMT deficiency in
mice. High and low levels of phosphoGAA and creatine phosphate, respectively, were detected by 31P

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MRS study of heart, skeletal muscle and brain in deficient mice [20]. Interestingly in hindleg muscle,
phosphoGAA in deficient mice was at concentrations found for phosphocreatine in wide type mice.
This has led to the proposal that in GAMT-deficient mice, phosphoGAA might substitute to
phosphocreatine as a donor of phosphate high energy bond. Administration of creatine to the GAMT-

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deficient mice progressively reversed the pathological MRS signals into those found in the
littermates. The putative GAA/PhosphoGAA cycle taking place in deficient animasl was less efficient
than the creatine/phosphocreatine cycle and limited by the lower affinity of CK for phosphoGAA (vs

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phosphocreatine), however partially compensing for the lack of phosphocreatine in skeletal muscle
of mice lacking guanidinoacetate methyltransferase [86]. Under high-intensity electrical stimulation,
deficient muscle develops lower force and impaired performance [87], and in a similar manner,
though cardiac function is not affected in basal working conditions, it becomes markedly abnormal

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under inotropic stimulation or ischemia/reperfusion [88]. Normal cardiac function in basal conditions
might result from adaptive compensations in the GAMT-deficient animals [89], though this view
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along with the proposal for negative impact of creatine deficiency in high workload conditions for
skeletal muscle and heart have been recently questioned [90] along with changes in mitochondrial
organization and compartmentation of high-energy phosphates in hearts from GAMT-deficient
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animals [91]. Regarding the brain depletion in creatine, restoration of brain levels occurs in GAMT-
deficient mice through supplementation in creatine like it is observed for this disorder in the humans
[92]. Finally, though GAMT deficient animals exhibit biochemical phenotyping (tissue and body fluids
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metabolite profiling) well paralleling the biochemical phenotype of human patients, the deficient
animals develop only a mild cognitive deficient contrasting with severity seen in the humans [93].
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3.5.3. Animal models for CRTR deficiency

Mice with a ubiquitous knockout of CRTR gene (exons 2-4 of SLC6A8 flanked by loxP sites) were
shown to exhibit learning and memory deficits seen in the affected human patients. In affected male
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mice (SLC6A8 -/y); creatine was absent in brain and significantly lowered in heart and testes. These
mice had increased hippocampal and prefrontal concentrations in serotonin and 5-hydroxyindole
acetic acid [21]. The female carrier mice SLC6A8 (+/-) had reduced levels of creatine and SLC6A8
transcripts in brain. They showed hyperactivity and moderate cognitive deficits like some human
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carriers. [13]. The intriguing question to know how creatine biosynthesis and homeostasis might
adapt in the whole body SLC6A8 knockout mice (SLC6A8 (-/y)) has been explored in skeletal muscle,
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showing a 3-fold increase in AGAT without, however, changes in GAMT gene and protein expressions
and as an overall result, a 1.5-fold stimulated creatine biosynthesis. This up regulation of creatine
biosynthesis was not sufficient to restore creatine levels seen in the wild type muscle. [19], muscle
lack in creatine and severely reduced serum creatine observed in the animal model [21] contrasting
with higher circulating and muscle creatine levels in human patients with CRTR deficiency. A brain-
specific SLC6A8 knockout (male) mouse model, with a phenotype similar to that seen in human
patients, has been also developed. This animal model, serving for evaluation of potential therapies
for CRTR deficient patients, was successfully treated with cyclocreatine which induced a rise in brain
cyclocreatine and cyclocreatine phosphate and improved cognition, spatial learning and memory
[94]. Recently, another ubiquitous knockout mouse (deletion of 5-7 exons in the SLC6A8 gene),
shown to lack creatine in brain and to exhibit cognitive defects seen in the humans, has been
proposed as an alternative animal model for preclinical studies of the human CRTR deficiency [10].
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Finally, overexpression of the CRTR activity may also be pathogenic. Heart with overexpressed CRTR
in a transgenic mouse model has increased levels of creatine associated with enhanced free ADP
levels and left ventricular hypertrophy and dysfunction [95, 96]. Regulating intracellular creatine
stores through down-regulation of creatine uptake might prevent excessive creatine and resulting
depletion of ATP stores [95, 97].

4. Primary creatine deficiency disorders

These disorders are directly caused by mutations in either GATM, GAMT or SLC6A8 genes.

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4.1. General clinical and diagnostic aspects
Clinical expression of these disorders is predominated by neurological impairments with as a
common feature intellectual disability and language delay, stressing a unique specific role of creatine

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in brain. Accordingly, a severe drop in brain creatine is recovered in these patients [98].
The exact frequency of these disorders is still overlooked and remains to be clarified. In this
respect, the prevalence of creatine deficiency syndromes has been estimated to be 2,7 % in a cohort

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of children with psychomotor delay of unknown origin. This prevalence increases to 4.4% when
focusing on male patients [99].
The diagnosis of creatine deficiency syndromes proceeds in several steps : i) biological
determination in blood and urine of creatine and metabolites and/or cerebral proton magnetic

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resonance spectrometry (1H-MRS), ii) molecular studies of the gene suspected, iii) functional cellular
assessment of creatine enzyme and transporter activities. Strategically, 1H-MRS should have a more
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privilegiate place in the diagnosis, follow-up and prognosis of patients with a creatine deficiency
syndrome ; however, it is cost expensive with data interpretation requiring a specialized expertise
and in practice is only occasionally performed. In this context, biological measurements still remain
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the first line of exploration for the diagnosis of creatine deficiency syndromes.

4.2. Molecular basis for pathophysiological drop of brain creatine

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The drop in brain creatine levels is a common feature to creatine deficiency disorders. In
these conditions, the prominence of brain clinical signs argues for a major role of creatine in brain
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function. Though mechanisms for collapse in brain creatine in either GATM (AGAT) or GAMT
deficiency has been long understood as the result of a severe impairment of one of the two creatine
biosynthesis steps, mechanisms involved in creatine transporter deficiency may be understood on
lights of the events depicted in Fig. 4 and mentioned above in the text. The deficient creatine
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transporter function impairs not only the supply of brain cells with creatine from circulating
(immature brain) or local (mature brain) origins but also directly affects creatine biosynthesis by
cancelling the bridging of the formation of an intermediate (GAA) in one cerebral cell to its utilization
(towards creatine synthesis) in another cerebral cell. Of interest in the scope of these creatine
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deficiency disorders is the taurine transporter which has been shown to be active on GAA and
catalyzing its efflux from brain to blood, a feature having GAA detoxifying properties, notably in
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GAMT deficiency [100]. Finally, it is important that creatine deficient brain is not only affected by the
loss of the related energetic shuttle but also from other properties conveyed by creatine. In this
respect, creatine has been reported to be endowed with antioxidant and antiapoptotic protective
effects against for instance neurodegenerative disorders and cerebral cell ammoniac toxicity [95,
101].

4.3. AGAT deficiency

AGAT deficiency was first reported in 2001 in two sisters (4 and 6 years-old), both with
intellectual disability, major language and walk difficulties, and one with febrile seizures at 18
months [102]. AGAT deficiency was later found in a brother diagnosed at birth [103]. Familial inquiry
revealed AGAT deficiency in a cousin with psychomotor delay, absence of language acquisition at 2
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years, behavioral disturbances, moderate generalized hypotonia, hand stereotypes and discrete
microcephalia [104].
AGAT deficiency is inherited as an autosomal recessive trait. GATM gene is located on
chromosomal 15q21.1 and contains 9 exons [105] and is essentially expressed in kidney and to a
lesser extent in pancreas, liver and CNS. As mentioned above, the protein catalyzes the initial step in
creatine biosynthesis, producing GAA and ornithine. A deficient activity then results in a marked drop
in plasma and urinary GAA and a decrease in creatine [106].
The diagnosis of AGAT deficiency is suspected when plasma and urinary GAA levels are low.

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Decreased creatine alone is not a reliable marker of the enzyme deficiency. Measurements of AGAT
activity may be done on lymphoblasts or lymphocytes [102, 107]. The identification of GATM
mutations validates the diagnosis. Five private homozygous mutations of GATM gene have been
reported in five distinct families. They include c.446G>A [p.(Trp149X)] [102-104], c.484+1G>T

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[p.Ala97ValfsX11] [108, 109], c.1111dup [p.(Met371AsnfsX6] [110], c.505C>T [p.(Arg169X)] and
c.608A>C [p.(Tyr203Ser)] [101] mutations. 1H-MRS reveals a complete creatine deficiency in brain.
Treatment of AGAT deficiency consists of creatine supplementation (300 à 400 mg/kg/d) to

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restore cerebral creatine levels. In two creatine-supplemented patients (sisters with AGAT deficiency
mentioned above), behavioral disturbances were partially corrected, and motor along with visual
performances were improved [104]. Moderate intellectual handicap, however, persisted after
several years of treatment. In the cousin of the two sisters, creatine supplementation was started at

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the age of 2 years [104]. After a follow-up of 3 years, the patient still exhibited a moderate
psychomotor delay and language difficulties. His social interaction and sustained attention activity
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were improved by the creatine administration [104]. A fourth patient with AGAT deficiency
diagnosed at birth had the opportunity to be supplemented by creatine since the age of 4 months.
Interestingly enough, his psychomotor development was found to be normal at 18 months of age
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[103]. This major improvement might lie in the high concentration of SLC6A8 in immature brain over
the earliest years of life. In two new sisters with AGAT deficiency, 13 months of oral creatine
supplementation highly improved muscle strength and partially resolves language delay and
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intellectual disability [111]. The longest term study on treatment of AGAT patient who has normal
neurodevelopment at the age of 9 years is encouraging and suggests that early diagnosis followed by
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immediate treatment may positively impact developmental outcome [109]. As a whole, AGAT
deficiency responds to creatine supplementation better than GAMT deficiency. The reason is that
GAMT in contrast to AGAT deficiency exhibits a high plasma level of GAA which is highly toxic for
brain and is not completely resolved by creatine supplementation (see below). As a corollary,
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creatine supplementation better prevent neurological sequels in AGAT than GAMT deficiency. Earlier
diagnosis and therapeutic measures promote efficacy of creatine supplementation.

4.4. GAMT deficiency

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GAMT deficiency was the first recognized inborn error of creatine metabolism [112]. The
inaugural case report was a 22 months-old child with a severe developmental delay associated with a
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progressive extrapyramidal syndrome. Nowadays, about 110 GAMT-deficient patients with at least
25 distinct mutations have been inventoried in the world [113-115].
Like AGAT, GAMT deficiency is inherited as an autosomal recessive trait. GAMT gene is
located on the short arm of chromosome 19 at the p13.3 locus and has 6 exons [116]; it is expressed
essentially in liver and to a lesser extent in kidney, pancreas and CNS. As mentioned above, the gene
product catalyzes the methylation of GAA, using SAM as a methyl donor, and generating SAH and
creatine. Deficient enzyme activity results in a substantial lack of creatine biosynthesis and in
accumulation of GAA, a pro-convulsing metabolite highly toxic for CNS [117].
Clinical presentation of patients with GAMT deficiency is relatively heterogeneous. The onset
of clinical signs is variable and usually occurs between early childhood (3-6 months) and 3 years
[116]. Very often, the global development is delayed and major language difficulties take place along
with intellectual disability and behavioral disturbances [118, 119]. However, in some cases,
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neurological impairment is mild and normal neurodevelopmental outcomes were even reported
notably in patients diagnosed during the newborn period in a context of familial antecedents for
GAMT deficiency ([120] and references therein). Affected children may develop hypotonia and/or
dyskinesia, and sometimes pharmaco-resistant epilepsy [98, 119, 121,122]. Behavioral disturbances
(autistic signs with auto-mutilating syndrome and hyperkinetic signs) may be observed later [118,
122]. Interestingly, GAMT deficient patients are not affected by neuropathy, auditive and visual
impairments.
Diagnosis of GAMT deficiency is evoked when a major increase of GAA is observed in body

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fluids (urine, plasma, CSF). As a hallmark of GAMT deficiency, GAA levels are increased and creatine is
reduced in body fluids. Lowering of creatine levels is more drastic in plasma than in the urine [106].
Biochemical assay of GAMT points out a drop in the enzyme activity. First performed on liver
biopsies, direct measurements of GAMT activity are now available on lymphoblasts and cultured

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fibroblasts [123-125].
The search for gene mutations completes the diagnosis exploration of GAMT-deficient patients [113-
115, 126], and until now, 45-50 mutations spanning throughout the entire gene have been reported

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[120, 127]). Two mutations predominate and are recovered in the half of GAMT-deficient patients : a
missense c.59G>C mutation in which a tryptophane is replaced by a serine at the position 20 of the
mature protein (p.Trp20Ser) and a c.327G>A mutation which induces an abnormal splicing [126]. In a

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French cohort of patients with GAMT deficiency, new mutations were c.289C > T, c.391 + 15G > T,
c.577C > T, c.299_311 dup13 and c.506G>A [122]. Recently, 19 missense variants in GAMT were the
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focus of the work of Mercimek-Mahmutoglu et al. [120].
An effective diagnostic tool, also of interest for the other two primary creatine deficiency
disorders, lies in the 1H-MRS exploration which visualizes a complete or near complete creatine
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deficiency in brain. This exploration may serve also to monitor replenishing of cerebral creatine
stores under oral supplementation (see below). The electroencephalogram (EEG) in patients may be
abnormal with multifocal or bilateral peak/short wave discharges. The magnetic resonance image
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(MRI) may be normal or in turn may show, in T2 resolution, hypersignals of globus pallidus and peri-
aqueductal area [121, 128]. GAMT deficiency may impact negatively respiratory chain, and this
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secondary respiratory chain defect may lead to an erroneous diagnosis of mitochondrial

encephalopathy inasmuch clinical and radiological aspects of GAMT deficiency and respiratory chain
disorders may overlap [128, 129].
Therapy of GAMT deficiency has a double metabolic goal: restoring normal brain stores in
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creatine and preventing excess GAA formation through inhibition of AGAT activity by creatine (see
the corresponding negative feed-back illustrated on Fig. 2).
Oral creatine supplementation at a dose of 0.35 to 2 g/kg/day has averred to be safe (free of side
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effects). Reported therapeutic effects include improvement of hypotonia (acquirement of the control
of head standing up, of settled position and walking on all fours), resolution of dyskinesia, epilepsy
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and EEG abnormalities, improvement of attention and behavior, and disappearance of MRI signs
relative to globus pallidus [130]. A clinical trial with restricted supply in arginine (15 mg/kg/d which
corresponds to 0.4 g/kg/d of natural proteins) and ornithine supplementation (100 mg/kg/d) along
with a daily supply of 1.1 g/kg creatine over a period of 14 months induced a substantial decrease of
plasma and urinary GAA, a major improvement of the clinical status along with a complete resolution
of seizures and, at the EEG examination, of epileptogenetic activity [117]. Clinical trials of GAMT
deficiency addressing supplementation in the deficient product (creatine monohydrate) and
inhibition of the intermediate guanidinoacetate formation (restricted nutritional supply in arginine,
supplementation in L-ornithine and administration of sodium benzoate as glycine-scavenging
therapy) were recently reviewed [119]. Fig. 6 illustrates recovery of the brain creatine peak seen on
the 1H-MRS spectra in a patient given creatine supplementation.
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4.5. CRTR deficiency

Since its first report in 2001 [131], CRTR deficiency has been meanwhile described and
diagnosed in more than 150 patients and 60 families [132, 133]. It is an X-linked disorder with the
SLC6A8 gene located on Xq28 and contains 13 exons [134]. This gene is ubiquitously expressed in
tissues with high expressions in skeletal muscle, kidney and heart and low expressions in adult brain
and other tissues [134]. CRTR deficiency impairs the cellular uptake of creatine. Though this
pathology has been sometimes overlooked, SLC6A8 gene mutation may account for 1% of male
patients with intellectual disability [135]. In a study highlighting CRTR deficiency as a cause of

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intellectual disability as important as fragile X syndrome, a prevalence of 2.1 % was observed [136].
In our hands, a prevalence of no less than 0.16% for CRTR deficiency was determined on a large
cohort of more than 6,000 neurological patients [122].
Clinical signs in CRTR deficiency are usually predominated by intellectual disability and severe

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motor and language impairments associated with epilepsy or autistic behavior [122, 137]. Anselm et
al. [138] reported two SLC6A8 -deficient boys aged of 12 and 30 months with a severe clinical
phenotype associating intellectual disability, a lack of language acquisition, hypotonia, myopathy and

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extra-pyramidal signs. Fifty percents of CRTR deficiency carrier woman may be symptomatic with
language difficulties [137]. Nevertheless, clinical presentations similar to those in affected male
patients may characterize female carriers [139]. As for other X-linked syndromes, this phenotypic
heterogeneity may result from random inactivation of the X-chromosome in woman.

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MRI may be normal or may point out non specific brain abnormalities including cerebral or
cerebellar atrophy. As in other creatine deficiency syndromes, 1H-MRS in SLC6A8 -deficient brain is
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characterized by a severe drop in creatine peak intensity [121].
Biological diagnosis of CRTR deficiency is based on an enhanced urinary creatine to creatinine
ratio. The determination of this ratio has an important diagnostic value since creatine values not
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corrected by creatinine may be normal in the urine from SLC6A8-deficient patients. Plasma creatine
levels may be either increased or normal. Urinary and plasma GAA levels are in normal range values.
Biochemical assessment of CRTR is measured by cell uptake of creatine on cultured fibroblasts, using
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various concentrations of creatine along with or without guanidinopropionate, a specific inhibitor of

CRTR [131]. Biochemical investigations are confirmed by molecular genetic studies of SLC6A8.
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In contrast to other creatine deficiency syndromes, oral supplementation in creatine failed to

restore brain creatine contents and to resolve patient signs in initial studies [140]. Supplementation
of four patients with L-arginine, a precursor of creatine, over a period of 9 months was also
unsuccessful [141], and general inefficacy of nutritional measures in improving brain was further
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stated by other studies [137, 142, 143]. On the other hand, after encouraging results on cultured
fibroblasts, administration over 1 year of a lipophilic analogue of creatine to four SLC6A8 deficient
patients did not induce significant clinical benefits and changes in brain levels of creatine [144]. In
two other studies, designed treatments failed to improve brain creatine content as assessed by 1H-
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MRS [145,146]. In the first of these studies, a 4-6 years treatment combining creatine monohydrate,
L-arginine, and glycine did not aver to offer persisting clinical benefit to 9 CRTR-deficient boys [145].
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The other study using creatine supplementation alone or with its precursors L-glycine and L-arginine
was performed on 4 male and 2 female patients with no improvement in cognitive tasks and
psychiatric behaviours while some clinical benefit was observed in muscular manifestations [146].
Meanwhile, some hope emerges from discovery of creatine pro-drugs by-passing SCL6A8 to
enter brain and other cells like cyclocreatine shown to improve cognition in SLC6A8 KO mice [94].
Moreover, recent clinical studies also question the inefficacy of supplementations and even report
beneficial effects with creatine and/or arginine supplementations in patients with CRTR deficiency
[122]. In a heterozygous female patient with intractable epilepsy, treatment with creatine combined
with arginine and glycine completely resolved seizures [147]. Creatine supplementation was also
described to improve neurological, language and behavioral status and was associated with a rise in
the brain creatine peak as demonstrated by MRS in a child with CRTR deficiency [148]. L-arginine-
based therapy positively impacted daily living skills, lowered the frequency of epileptic episodes and
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induced a mild increase in brain creatine and phosphocreatine MRS signals in SLC6A8 deficient
patients [149].
Regarding brain cell penetration of creatine, there are other aspects in addition to those
presented above in the subsection titled “3.3. The particular case of brain” that warrants
consideration. CRTR deficiency is a X-linked disorder and therefore in female patients X-inactivation
induces a double cell population, one in which the active X shares the mutation and the other in
which the active X does not. Sometimes, the X-inactivation pattern may be skewed due to altered
viability of cells expressing the muted X [150]. In any case the CNS cells from a female patient may

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have a reduction in the total intra-organ (mature brain) and blood-organ (immature brain) capacity
to transfer/transport creatine. As a result, brain creatine levels (assessed by 1H-MRS) in a female
SLC6A8-deficient patient may be reduced in comparison to normal brain. Due to the parallel
existence of cells expressing a normal SLC6A8, a therapy with creatine especially at doses inducing

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blood concentrations higher than physiological ones is expected to have a beneficial effect on brain
creatine content and hence on neurological signs (higher the dose, higher the expected benefit). Due
to the fact also that the X-inactivation pattern may be skewed, CNS cells expressing the non-muted X

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may be prominent and associated with normal brain creatine levels at the 1H-MRS examination, a
feature which may lead to a misdiagnosis of these female patients (see Van de Kamp et al [95] for
additional consideration). At pharmacological doses of creatine, part of recovery in brain and by-pass
of BBB is not absolutely excluded in SLC6A8-deficient male or female patients. As in some other

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neuropathological disorders, one may not absolutely rule out some disruption in BBB due to the
pathogenesis process or as evoked above due to deaths of cells with the active muted X. In addition,
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some limited regions of brain involved in hormonal secretions and hence potentially influencing
growth are known to be physiologically devoid of BBB [151]. Therefore administration of creatine
might at least correct their creatine contents, the extent to which these regions may be affected in
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CRTR deficiency remaining, however, to be clarified.

4.6. Prenatal diagnosis of creatine deficiency disorders

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Molecular biology techniques are suitable for a prenatal diagnosis in gestation at risks. GAMT
and GATM (AGAT) gene studies are carried out on DNA isolated from fetal cells after amniocentesis
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or chorionic biopsy performed between 15 and 18 weeks or between 10 and 12 weeks of

amenorrhea, respectively. The gene chosen for prenatal investigations is the one responsible for
creatine deficiency in the fetus family. Prenatal diagnosis in gestational women carriers of CRTR
deficiency is also available and requires first to determine fetus sex on caryotype of fetal cells from
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either amniocentesis or chorionic biopsy. When fetus is a boy, SLC6A8 gene is studied on the fetal
DNA [133]. When fetus is a girl, the pre-existence of an index case in the family may also represent a
medical indication for prenatal diagnosis.
Among biochemical methods, a LC/MS-MS procedure [152] was adapted for measurements
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of creatine and GAA on amniotic fluid in GAMT deficiency [153]. Amniotic fluid samples from
pregnant women (15-20 weeks of amenorrhea) with a fetal risk of trisomy 21 served to generate
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reference GAA and creatine values. These values were successfully used for prenatal diagnosis of
GAMT deficiency [153].

5. Secondary disorders of creatine metabolism

Abnormal levels of creatine and/or GAA may be observed out the scope of a primary creatine
deficiency syndrome. In fact, creatine biosynthesis is interconnected with various metabolic
pathways including ornithine metabolism, urea and methylation cycles. Impairment of these
pathways may secondarily impact creatine homeostasis as highlighted in Fig. 7 and legend, and
developed thereafter.
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5.1. Ornithine delta-aminotransferase (OAT) deficiency and Hyperornithinemia-Hyperammonemia-

Homocitrullinuria (Triple H, SLC25A15 deficiency)

5.1.1. OAT deficiency

Steady-state concentrations of ornithine considered as a substrate initiating urea cycle are regulated
by mitochondrial ornithine delta-aminotransferase (OAT) (EC 2.6.1.13) (Fig. 7). OAT deficiency is an
autosomal recessive disorder biologically characterized by a huge increase in plasma ornithine.
Clinical signs of this hyperornithinemia are gyral atrophy of choroid and retina with myopia and, in

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childhood, disturbed nocturnal vision which progressively evolves to blindness at the adult age.
Interestingly, most patients with OAT deficiency present normal IQ [154] while some patients
develop intellectual disability and proximal myopathy, signs which along visual impairment are not
fully understood and might be accounted for by the secondary defect in creatine reported in OAT

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deficiency [155]. The metabolic reason why creatine may be reduced in OAT deficiency lies in the
AGAT step in creatine biosynthesis which converts arginine and glycine to ornithine and GAA. Excess
ornithine in OAT deficiency (Fig. 7) lowers AGAT activity via the product inhibition mechanism

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highlighted in Fig. 2, and hence, reduces secondarily GAA and creatine biosynthesis and levels [155].
Neuropsychological status and creatine metabolism has been studied conjointly in 7 patients with
OAT deficiency at an age range of 11 to 27 years. Neurocognitive disturbances were observed in 5 of
these patients. Creatine metabolism markers (GAA and creatine) were decreased in plasma and urine

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in all patients as well as a severe deficit in brain creatine content was shown by 1H-MRS imagery.
These results argued in OAT deficient patients for the pathogenesis link between alterations of CNS
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and a secondary creatine deficiency [156].

5.1.2. SLC25A15 deficiency

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Ornithine is a product of the arginase reaction and is formed by cleavage of arginine to ammonia and
ornithine in the cytoplasm. To re-initiate a new urea cycle ornithine needs to enter mitochondria for
being available for the ornithine transcarbamylase step and to condense with carbamyl-phosphate to
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form citrulline. The entry and hence consumption of ornithine in the mitochondrial matrix, the site
for the OTC reaction, depends on a mitochondrial inner membrane ornithine transporter referred to
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as SLC25A15. In SLC25A15 deficiency, ornithine is no longer consumed by the OTC and the urea cycle,
and thus it accumulates as a result of its disrupted degradation. Ammonia also increases in this
disorder as a result of a deficient urea cycle which is no longer alimented by ornithine, and finally
homocitrulline also increases due to the fact that carbamyl phosphate which is no longer consumed
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by OTC by lack of locally available ornithine. In fact, carbamylphosphate is directed in an alternative

pathway: the carbamoylation of lysine by lysine transcarbamylase responsible for the synthesis of
homocitrulline. This combined pathological rise in ornithine, ammonia and homocitrulline explains
the name of triple H (Hyperornithinemia-Hyperammonemia-Homocitrullinuria) given to SLC25A15
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deficiency [157, 158]. In this disorder, a low creatine urinary excretion attributed to its reduced
formation is observed [159] with creatine metabolism proposed to be impacted according to
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mechanisms similar to those mentioned above for OAT deficiency, namely inhibition by excess
ornithine of AGAT to which adds its inhibition by ammonia. SLC25A15 deficiency is usually associated
with the development of vomiting, hepatomegaly, failure to thrive and neurological signs which may
include cognitive impairment, behavior disturbances, seizures, consciousness changes and pyramidal
signs. Though exceptionally absent before adult age [160], neurological signs are found in all patients
in long-term studies which also document severe outcome of neurological impairments [161].
Factors contributing to brain toxicity include altered mitochondrial bioenergetics and oxidative stress
mainly caused by homocitrulline and to a lesser extent by ornithine [162]. The deficit in brain
creatine and ammonia toxicity have also to be taken into account, intellectual disability and
behavioral disturbances recalling signs seen in primary creatine deficiency disorders.

5.2. Primary urea cycle defects : ASS, ASL and OTC deficiencies
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5.2.1. Urea cycle disorders in general

Secondary alterations of the creatine biosynthesis pathway have been also reported in patients
suffering from urea cycle disorders. Defects of the urea cycle which reduce the production of
arginine (Figs 7 and 8) include primary deficiencies of ornithine transcarbamylase (OTC) (EC 2.1.3.3),
arginosuccinate synthetase (ASS) (EC 6.3.4.5) and arginosuccinate lyase (ASL) (EC 4.3.2.1).
Arginine plays a major role in CNS acting as a substrate for protein synthesis and nitric oxide
formation but also as a donor of guanidino groups for creatine biosynthesis [163]. This explains why

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insufficiency in arginine production, like in some urea cycle defects, may cause a secondary creatine
deficiency disorder. This is in practice corroborated by laboratory and brain 1H-MRS data. Arias et al.
[163] found in children with defective urea cycle (8 OTC, 3 ASS and 3 ASL primarily deficient patients)
a drop of plasma GAA levels among with a collapse of cerebral creatine on 1H-MRS examination.

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Secondary creatine deficiency in defective urea cycle remains sometimes debated. Whereas
Roze et al. [164] found a decrease of creatine and GAA in ASL deficient patient and similar results
were reported by Choi et al. [165] in a case of ASS deficiency; the latter authors did not find a

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reduction of brain creatine in a 6 years-old child with OTC deficiency [165]. This apparent discrepancy
of urea cycle defects to decrease brain creatine might reflect the heterogeneity of this class of
disorders or in turn difficulties in quantifying metabolite levels by cerebral 1H-MRS. Hyperamonemia
taking place in these urea cycle defects also impairs creatine biosynthesis. Recent studies on cultured

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rat cerebral cells highlighted the inhibition of AGAT by ammonium (Fig. 7) as a mechanism for the
drop in cerebral creatine in patients [166].
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5.2.2. The particular case of arginase deficiency
In contrast to other disturbances of the urea cycle, arginase deficiency (EC 3.5.3.1) enhances plasma
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levels of arginine. Increased availability of arginine for AGAT stimulates biosynthesis of GAA and
creatine. Their accumulation mimics, part of biological/metabolic abnormalities seen in GAMT and
SLC6A8 deficiencies. This stimulation of creatine biosynthesis in arginase deficiency is problematic
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because rise in GAA levels induces brain damage like in GAMT deficiency in which severity of CNS
impairment is correlated to excess GAA [167]. In contrast to CRTR deficiency, increases seen in
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plasma and urine levels are not associated with reduced brain contents in creatine.

5.3. Primary defects of homocysteine/methionine methylation cycle

Creatine metabolism also tightly interacts with the homocysteine/methionine methylation
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cycle (Fig. 8). Indeed, 40% of methyl groups provided by this cycle are consumed during creatine
synthesis from GAA acting as a methyl group acceptor [50]. The GAA-methyl group supplying route
(i.e. homocysteine/methionine methylation cycle) is branched on folate and cobalamine metabolic
pathways (Fig. 8), explaining why their alterations may also ultimately impact creatine metabolism.
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Bodamer et al. [168] have explored 5 patients with methylmalonic aciduria combined to
homocystinuria caused by cobalamine C (Cbl C) deficiency. This pathology induces a defective
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remethylation of homocysteine and hence a depletion in methionine and excess homocysteine.

Clinically, patients present neurological signs: a delay in psychomotor development, hypotonia,
epilepsy and nystagmus. This disease disrupts creatine metabolism as assessed by increased plasma
GAA concentrations in all patients (14,9 ± 4,8 μmol/L versus 1,4 et 5,4 μmol/L for control values).
Importantly, plasma creatine levels (43,8 ± 20,7 μmol/L) remain in normal range values (14 to 103
μmol/L). The increase of GAA in cobalamine C deficiency is explained by a partial inhibition of GAMT
by increased tissue concentrations of SAH. Cerebral creatine is normal.
Baric et al. [169] reported alterations of creatine metabolism in a child with a deficiency of S-
adenosyl homocysteine hydrolase (SAHH) (EC 3.3.1.1) which normally hydrolyzes SAH in
homocysteine and adénosine. Clinically, the child had a psychomotor delay and hypotonia. Cerebral
MRI showed white matter atrophy along with myelinating abnormalities and biological
measurements, increases of SAH and GAA by 150-fold and 2.5 to 7-fold, respectively. Increased levels
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of GAA along with a lowered creatine concentration are observed in CSF. As a whole, this observation
supports that SAH inhibits GAMT and hence causes accumulation of GAA. In view of its neurological
toxicity, GAA accumulation might be incriminated in neurological impairment seen in this disorder.
The impact on this neurological outcome of supplementing creatine to inhibit AGAT (see Fig. 2) and
limit GAA formation (in the same time as supplying creatine) is awaited.

5.4. Miscellaneous
Increased levels of GAA have been reported in succinate semialdehyde dehydrogenase

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(SSADH) (EC 1.2.1.24) deficiency [170]. This rare disorder which is inherited as an autosomal
recessive trait affects the metabolism of neurotransmitter GABA (gamma-aminobutyric acid) in a way
depicted on Fig. 9. Patients have delay in psychomotor development, hypotonia, ataxia and epilepsy.
SSADH deficiency increases brain GABA levels by disrupting GABA catabolism. Alternative

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metabolism of GABA takes place and causes increased AGAT-driven formation of gamma-
guanidinobutyrate. Acting as a guanidino group donor, the latter may affect GAMT by competition
with the natural substrate, GAA. GAA being less metabolized by GAMT may then accumulate in

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SSADH deficiency, being responsible for a part of the brain toxicity (Fig. 9).
Several drugs may interfere with urea cycle and may cause hyperammonemia which as
mentioned above inhibits AGAT and hence creatine biosynthesis. Inhibition of brain catabolism and
increased brain GABA levels may be also induced pharmacologically by several antiepileptic drugs.

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When drug exposure occurs in the perinatal period, excess GABA, neuroprotective on mature brain,
exhibits toxicity towards immature brain directly or through interference with creatine metabolism
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according to mechanisms similar to those encountered in SSADH deficiency [171] and depicted on
Fig. 9. Note that drugs such as valproate may combine hyperammonemia and brain GABA increase-
triggered mechanisms. Importantly, these interferences of valproate with creatine metabolism may
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induce false positive cases on the clinical chemistry screening.

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6. Diagnostic strategy and decisional tree

Intellectual disability in children and adolescents represents a major concern of public health.
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Facing intellectual disability of unknown origin, the search for a creatine deficiency syndrome may be
indicated especially when, besides a familial intellectual disability history or a severe language
acquisition deficit, co-exist an extra-pyramidal syndrome, behavioral disturbances, and/or the
absence of dysmorphic features [121] (see Fig. 10). Suspicion of a creatine deficiency syndrome is
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first explored by the assay of urinary and plasma GAA and creatine. Though in the plasma, these
metabolites are directly quantitated by their respective concentrations; in the urine, the metabolites
concentrations are pondered by those of creatinine. In the urine, creatine on creatinine ratio and
GAA on creatinine ratio are calculated in determining reference ratio data and in detecting
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pathological values. Abnormal GAA or creatine plasma concentrations and urinary ratios should
prompt a second metabolite measurement for diagnosis confirmation and may be completed by
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brain 1H-MRS imagery to visualize the putative drop in cerebral creatine/phosphocreatine peak
intensity. Usually genetic studies are first line studies to confirm the diagnosis. If necessity, enzyme
level or creatine uptake is measured to confirm biochemical diagnosis of deficient AGAT, GAMT or
CRTR functions. The determination of the protein activity may be performed prior to or after the
gene study. In the latter case, the functional test validates or invalidates the disease-causative nature
of the gene mutations, especially when a new mutation arises. Enzyme activities are carried out on
lymphoblasts or cultured fibroblasts. Gene studies are of prime importance in providing the
molecular basis of the creatine deficiency disorder. These studies confirm the diagnosis and are
suitable for genetic counseling and prenatal diagnosis [121]. Fig. 10 highlights the diagnostic
explorative rationale in creatine deficiency syndromes through a decisional tree partially adapted
from previous work [121]. Fig. 10 provides the reader with a general strategy to diagnose and
explore patients with a creatine deficiency, and to some extent provides immediately the reader with
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a pragmatic synopsis of major clinical, paraclinical, laboratory, enzyme and genetic features of
patients with defective creatine biology.

7. Treatment and innovative perspective for primary creatine defects

Cure of the CRTR deficient patients still remains a real challenge. As mentioned above, primary
creatine defects may be subject to efficient supplementations or dietary measures in which creatine
occupies a central place restoring brain creatine levels in deficient creatine biosynthesis but not
satisfactorily in CRTR deficiency though a positive impact becomes to be described in patients with

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deficient CRTR (see above). In these patients, creatine may be supplemented with its precursors
(arginine and glycine) since enzymes of creatine biosynthesis are not affected.
The usual creatine form supplemented to patients, and also in healthy subjects, is creatine
monohydrate. It requires a transit via the CRTR to recovering creatine within cells. Though small

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differences exist in kinetics, creatine pyruvate, tri-creatine citrate and creatine monohydrate (three
CRTR-dependent enhancers of intracellular creatine concentrations) were found to have similar
capacities to augment plasma creatine levels in healthy subjects, creatine pyruvate inducing the

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higher increase in blood creatine [172]. Phosphocreatine and phosphocreatine-magnesium complex
have been also reported to be a useful creatine-supplementing compound, being used in some
studies as a referent compound form which when given externally to cells may increase intracellular
creatine and protect brain [173]. Extracellular phosphocreatine has been also recently described to

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be a product of extracellular CK in a tumoral context, and to enter cells via CRTR in order to
regenerate intracellular creatine. [174]. Several other analogs of creatine have been also designed
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and evaluated. Cyclocreatine is a cyclic analog of creatine which enters cells mainly via the CRTR but
also in part independently of CRTR. It is not a prodrug of creatine since it does not release creatine
moieties once in the cells [175]. Nevertheless, cyclocreatine may substitute to creatine in the
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reactions catalyzed by creatine kinase, generating cyclocreatine phosphate. Even if it may improve
cognition in CRTR deficient brain [95], cyclocreatine remains a poor substrate for creatine kinases,
and for this reason, is not longer considered as a future lead compound for replacing or restoring
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brain creatine levels in CRTR deficiency. In turn, different creatine amides and esters (in the form of
acetate or fumarate salt) were designed as pro-drugs of creatine. Creatine amides evaluated for
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neuroprotective properties include amides formed by condensation of the carboxyl group of creatine
with the amine exhibited by an α (phenylalanine, tyrosine, glycine) or a γ (GABA) amino acid which
further harbors an amide or an ester function in replacement of the amino acid carboxylic group
[176]. These creatine amides were supported to act both as prodrugs of creatine and as substrates
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for CK, with creatylglycine ethyl ester having the best pharmacological profile of the studied series
[176]. Creatylglycine ethyl ester (fumarate salt) (referred to as creamide) has been shown to protect
against focal cerebral ischemia/reperfusion in rats to an extent comparing favorably with
phosphocreatine, as a referent compound [173]. Creamide was shown to maintaining cell energy
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(ATP levels) status, reducing lactate production and lowering oxidative stress, acting as a substrate
for CK and a partial agonist of the NMDA receptor/calcium channel complex [177]. Creamide thus
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presents with promising potential in protecting brain. Evaluating its efficacy in CRTR deficient
conditions is still awaited.
Creatine ethyl ester, the hydrolysis of which yields creatine, initially appearing as a good drug
candidate finally averred to be a prodrug more of creatinine than creatine as a result of rapid non-
enzymatic cyclisation preceding the hydrolysis step [178]. In people practicing bodybuilding and
taking this compound as a supplement, this may cause a huge rise in serum creatinine, inducing a
diagnostic pitfall with renal deficiency [179,180]. In contrast to creatine monohydrate, substantial
amounts of creatine ethyl ester are converted to creatinine in gastro-intestinal tract [181]. Though
creatine ethyl ester only moderately increases cell creatine content [182], it may improve brain
function [183], a benefit which might be linked to the ability of the creatine ester to be a substrate
for creatine kinase [184]. Because, it does not increase cell creatine content in CRTR deficient
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conditions, it is no longer considered as a privilegiate approach to treat patients with CRTR deficiency
[182] as more as chronic treatment of these patients fails to increase brain creatine levels [144].
A recent breakthrough with innovative perspective in creatine prodrugs lies in new fatty esters of
creatine designed to improve permeability through cell membranes in a way independent of the
CRTR and evaluated in an experimental cell-based model of blood brain barrier [185]. The galenic
formulation of the lead compound, dodecylcreatine ester, has been recently optimized through
encapsulation in lipid nanocapsules, highlighting the suitability of this strategy for treating CRTR-
deficient conditions, and making this compound a lead for future therapeutic management of

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deficient creatine levels in brain [186]. The last but not least, S-adenosyl methionine, the methyl
donor involved in creatine biosynthesis, has been recently evaluated as an adjunct of supplemented
creatine and precursors (arginine and glycine) in patients with CRTR deficiency [187]. The compound
aimed at stimulating creatine formation in brain fails to enhance cerebral creatine levels while, at

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non neurotoxic doses (here, neurotoxicity referring to sleep and behavioral disturbances), it may
impact positively scores for speech, language and communications skills, improving decision-making
and social skills in everyday life along with muscle mass gain [187].

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8. Conclusions

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7.1. Creatine is a guanidino compound which in the humans is physiologically provided equally by the
diet and endogenous synthesis from arginine and glycine. This biosynthesis is catalyzed by two
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proteins, AGAT and GAMT. A plasma membrane transporter, SLC6A8, further supplies creatine to
cells which do not produce it, and intracellular creatine may enter the creatine-phosphocreatine
cycle to stimulate mitochondrial oxidative phosphorylations in a way coupled to the transfer of
M

energy at various cellular sites through high energy phosphate bond exchanges catalyzed by creatine
kinases.
D

7.2. Though widely publicized, the snapshot of creatine as being solely a muscle energetic and body-
building up compound is now somewhat obsolete. Compelling evidence for a role of creatine in brain
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physiology has been given by the study of primary disorders of creatine biosynthesis and transport.
Brain physiologically secures its needs in creatine through uptake from the systemic circulation in
immature young brain (before 2 years) and, in mature CNS, through local biosynthesis as a result of
high and low SLC6A8 levels at immature and mature blood brain barrier, respectively. Cerebral
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creatine biosynthesis enzymes and SLC6A8 are distributed unequally in the various nervous cell
populations, transition of metabolites from a cell to another being often required to aliment the
cellular pool of creatine.
C

7.3. In brain, SLC6A8 acting as a plasma membrane creatine transporter takes immediately part to
local creatine biosynthesis as highlighted by deficiency of its function which induces a collapse in
AC

brain creatine content to the same extent as deficiencies of the biosynthesis enzymes do. SLC6A8
may be present in the presynaptic aspect of neurons to promote creatine re-uptake, a feature
consistent with its homology with other neurotransmitter transporters. In this respect, creatine is a
neurotransmitter which may be released in the synapse, may be re-uptaken by presynaptic SLC6A8,
and may either depress post-synaptic GABAergic neurotransmission or stimulate post-synaptic
glutamatergic pathways.

7.4. The growing-up number of secondary creatine disorders, i.e. creatine drop in diseases caused by
mutations of genes not coding for creatine biosynthesis and transport proteins, further highlight that
biosynthesis and transport of creatine are sensitive to a multitude of metabolic pathways. This high
dependence of creatine biosynthesis and transport on multiple pathways might support creatine as a
cellular sensor responding to cell methylation capacity and to cell energy content. In these respects,
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creatine biosynthesis is physiologically known to consume 40% of methyl groups produced by cells as
S-adenosylmethione, and cell incorporation of creatine (creatine transporter) is under control by the
AMP activated protein kinase, a ubiquitous cell sensor of energy depletion.

7.5. Creatine is also endowed with antioxidant properties which add to creatine-driven improvement
of cell energetic status to offer unique therapeutic protective potentialities notably towards human
degenerative diseases.

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7.6. Taken as a whole, our current knowledge on the biology of creatine in health and disease
therefore features this guanidino compound in a myriad of roles as a potential sensor of cell
methylation and energy status, as a neurotransmitter capable of modulating key (GABAergic and
glutamatergic) CNS neurotransmission pathways, as a therapeutic agent endowed with anaplerotic

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properties (towards creatine kinases [creatine-creatine phosphate cycle] and creatine-driven
neurotransmission) , energetic and antioxidant actions (benefits in degenerative diseases through
protection against energy depletion and oxidant species, respectively) along with osmolyte

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behaviour (retention of water by muscle and body-building properties).

Acknowledgements

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This work was supported by grant from the French Ministère de la Santé for a PHRC (Programme
Hospitalier de Recherche Clinique) dedicated to a study titled “Creatine and Guanidinoacetate : Pilot
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evaluation of the relative importance of abnormalities of creatine metabolism in the intellectual
disability in children”..
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D
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C EP
AC
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Table 1 –Proteins and related genes involved in creatine and phosphocreatine metabolic pathways
CREATINE BIOSYNTHESIS AND TRANSPORT* CREATINE PHOSPHOCREATINE CYCLE**
CREATINE BIOSYNTHESIS CREATINE TRANSPORT HIGH ENERGY PHOPHATE BOND EXCHANGE
FIRST STEP SECOND STEP (cell uptake) MITOCHONDRIAL EXTRA-MITOCHONDRIAL (CYTOPLASMIC)

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PROTEIN
- Complete name L-arginine : glycine Guanidinoacetate Solute carrier family 6 member 8 sarcomeric ubiquitous cytoplasmic muscle cytoplasmic brain
amidinotransferase methyltransferase (alternative names : Na+/Cl-- mitochondrial mitochondrial creatine kinase creatine kinase
dependent creatine transporter) creatine kinase creatine kinase
- Abbreviated name AGAT GAMT SLC6A8 sMitCK uMitCK MM-CK, CK-M, CKM, BB-CK, CK-B, CKB,

SC
(alternative abbreviated names : UMTCK CKMM CKBB
CT1, CRTR, CRT)
- EC number EC 2.1.4.1 EC 2.1.1.2 McKusick 300036 EC 2.7.3.2 EC 2.7.3.2 EC 2.7.3.2 EC 2.7.3.2
- Structure monomeric protein of 386 monomeric protein of 236 aas monomeric proteins of 635 aas Dimeric and Dimerci and Dimeric (MM) Dimeric (BB)
aas and 391 aas 26-31 KDa with 12 transmembrane domains octameric octameric (heterodimeric : MB) (heterodimeric MB)

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- Subcellular Mitochondria Cytoplasm, nuclear? Sarcolemnal and plasma Mitochondrial intermembrane and cristae spaces cytoplasm cytoplasm
localization intermembrane space (386 membranes (dimeric form) along with contact sites (octameric
aas) and cytoplasm (391 aas) form) with mitochondrial outer and inner
membranes
- Main tissue kidney liver Muscle, ubiquitous striated muscle most cell types (brain, Muscle (MM) Brain (BB)

AN
expressions kidney, sperm, etc.)

GENE
- Complete name glycine amidinotransferase, Guanidinoacetate Solute carrier family 6 member 8 Creatine kinase, Creatine kinase, Creatione kinase, Creatione kinase,
mitochondrial methyltransferase sarcomeric mitochondrial 1 muscle type brain type
mitochondrial

M
- Abbreviated name HGNC Approved Gene
Symbol : GATM
Alternative : AGAT
- Structure 17 kb long and contains 9
exons.
HGNC Approved Gene Symbol HGNC Approved Gene Symbol: HGNC Approved Gene HGNC Approved Gene HGNC Approved Gene HGNC Approved Gene
: GAMT SLC6A8 Symbol: CKMT2 Symbol: CKMT1B Symbol: CKM Symbol: CKB
5 kb long and contains 6 exons 8.4 Kb long and contains 13 exons 37 kb long and 5.5 kb long and 22 Kb long and 22 Kb long and
contains 11 exons contains 9 exons contains 8 exons contains 8 exons

D
- Chromosome 15q21.1 19p13.3 Xq28 5q14.1 15q15.3 19q13.32 14q32.32
location
- Gene with a similar CT2 (SLC6A10) expressed in sperm CKTM1A (telomeric to
sequence or coding a (REF 6), brain the CKMT1B gene on

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similar protein 16p11.2 chromosome 15q15)
function 15q15.3

DEFICIENCY
- Inborn error of L-arginine : glycine Guanidinoacetate (X-linked) creatine transporter
metabolism (with amidinotransferase methyltransferase deficiency, deficiency, SLC6A8 deficiency, CRTR Only patients with deficiency of the CKM gene have been described to-date
usual diseasenames) deficiency, AGAT deficiency, GAMT deficiency, cerebral deficiency, Cerebral creatine
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cerebral creatine deficiency creatine deficiency syndrome- deficiency syndrome 1 (CCDS1)
syndrome-3 (CCDS3) 2 (CCDS2)
- OMIM number 612718 612736 300352 123295 123290 123310 123280
(613415 for CKTM1A)

KO mice GATM-/- mouse GAMT-/- SLC6A8 -/y ScCKmit-/- mice UbCKmit-/- mice M-CK-/- mice B-CK-/- mice
M-CK/ScCKmit --/-- B-CK/UbCKmit--/-- M-CK/ScCKmit --/-- B-CK/UbCKmit--/--
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mice mice mice

* and **: for details and illustration of enzyme reactions, see Figs 1 and 2. For references, see the text.
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Legends to Figures

Fig. 1. Creatine, phosphocreatine and creatinine : chemical structures, enzymatic (A) and non
enzymatic (B) conversions. Abbreviations are CK, creatine kinase(s); ADP, adenosine diphosphate;
ATP, adenosine triphosphate. Phosphocreatine is also referred to as creatine phosphate or
phosphoryl-creatine.

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Fig. 2. Creatine metabolism : biosynthesis, transport and intra-regulatory interactions. Kidney is the
major site for the expression of AGAT which converts arginine and glycine to guanidinoacetate (and
L-ornithine). For pursuing creatine biosynthesis, guanidinoacetate is methylated essentially in liver by
GAMT using S-adenosylmethionine as a methyl donor. Creatine from de novo biosynthesis or from

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diet source transits by blood and is up-taken by tissues or cells expressing Na+-dependent SLC6A8, a
plasmic membrane transporter responsible for intracellular incorporation of creatine. Cellular
creatine may be phosphorylated to creatine phosphate by creatine kinase(s) in muscle and many

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other cell types including CNS cells. Creatinine, the formation of which is correlated with muscle
mass, is an end-product of metabolism which is removed in the urine, stagnation in blood being
observed when renal function is altered. Dashed red lines/arrows/symbols emphasize the major

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regulations taking place in creatine metabolism. Abbreviations are: AGAT, L-arginine:glycine
amidinotransferase; GAMT, N-guanidinoacetate methyltransferase; SLC6A8, creatine transporter.
AN
Fig. 3. Hormonal and emerging regulatory aspects of creatine metabolism. Mechanisms conveyed by
sex hormons are in part currently hypothetic and might explain some unexpected gender differences
recently observed in the metabolic patterns observed in the urinary excretions of creatine and
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guanidinoacetate (expressed vs urinary creatinine) in a series of more than 6,000 subjects [52].
Abbreviations are as in Fig. 2. SGK-1: serum- and glucocorticoid-inducible kinase 1. Other comments
are in the text.
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Fig. 4. CNS pathways physiologically ensuring creatine content of mature brain. CNS cells including
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neurons, microglial cells and astrocytes contribute to intracerebral creatine biosynthesis through cell
populations catalyzing only the first AGAT step (A) (22% cells) or the second GAMT step (B) (21%
cells) or both steps (C) (12% cells) or none of these steps (44% cells) (these estimations are from the
work of Braissant [56]). Mature brain cell creatine import rests on local production instead of on
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blood supply, because astrocytes which contribute to BBB are physiologically lacking the creatine
transporter (SLC6A8). Due to the presence of the transporter in endothelial cells which also
contribute to BBB (not shown), pharmacological supplementation in creatine may increase
C

substantially intracerebral contents at least when the transporter is not deficient in cerebral vascular
endothelial cells. Other considerations and the situation of immature brain are developed in the text.
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Fig. 5. Creatine as a neurotransmitter modulating brain neurotransmission. A complete equipment of

the afferent neuron with creatine biosynthesis proteins and substrates, and with synaptic and extra-
synaptic creatine transporter SLC6A8 has been illustrated. In practice and as indicated in Fig. 4, a
neuron will contain none, part or totality of these proteins. AGAT, arginine glycine
amidinotransferase; GAMT, guanidinoacetate methyltransferase

Fig. 6. Brain 1H- magnetic resonance spectroscopy in a case of GAMT deficiency prior to (A) and after
(B) a 12 months-period of creatine supplementation. MRS monovoxel (Short TE 35 ms) examinations
of cerebral gray nuclei were performed in a patient with GAMT deficiency in the absence (A) and the
presence (B) of creatine supplementation. Though in the former conditions, the examination shows
little or no creatine peak signal, in the supplemented state, a substantial rise in creatine is induced,
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normalizing the brain content in creatine. Labels are : mIno, myo-inositol ; Cho, choline ; Cr :
creatine/phosphocreatine, NAA :N-acetyl aspartate), lac, lactate.

Fig. 7. Metabolic and regulatory interconnections of creatine, ornithine, urea and methylation-based
pathways. Abnormalities in ornithine metabolism (ornithine oxoglutarate transaminase deficiency)*
and transport (SLC25A15 deficiency)*’ may lead to product inhibition of AGAT and hence functional
AGAT deficiency under enhanced levels of ornithine. In these disorders, inhibition by ammonia
resulting from a functional drop in urea cycle activity may also contribute to AGAT depression.

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Urea cycle disorders may also impact AGAT activity either via an increase of the enzyme product
ornithine, via a reduced supply in the enzyme substrate arginine** or via direct ammonia-driven
enzyme toxicity***.
**** Arginase deficiency does not lead to deficient but to stimulated creatine formation through

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increase in arginine availability for creatine synthesis. In these conditions, guanidinoacetate
formation is also enhanced and hence, like in GAMT deficiency, brain toxicity of this metabolite
contributes to neurological signs.

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***** Methylation cycle disorders may impair the GAMT step by reducing formation of the methyl
donor S-adenosylmethionine or its recycling needed for sustained methylation of guanidinoacetate
to creatine. Methylation cycle disorders may induce functional deficiency of GAMT activity, and
imbrications between creatine and methyl donor biosyntheses are further detailed in Fig. 8.

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Note the dual subcellular localization of AGAT : cytoplasm and mitochondrial intermembrane space
[14, 15, 48]. Abbreviations are CPS, carbamoylphosphate synthase; OAT, ornithine oxoglutarate
AN
transaminase; OTC, ornithine transcarbamylase; ASS, arginosuccinate synthase; ASL, arginosuccinate
lyase; ARGase, arginase; AGAT, arginine glycine amidinotransferase; GAMT, guanidinoacetate
methyltransferase.
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Fig. 8. Creatine, methylation cycle and other co-working homocysteine pathways. Vitamin
requirements of steps involved in homocysteine (HCys) metabolism are indicated between brackets.
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Known impairments of this metabolism causing creatine deficiency are presented in the text.
Abbreviations are HCys, homocysteine, Cys, cysteine; AGAT, arginine glycine amidinotransferase;
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GAMT, guanidinoacetate methyltransferase; SAM, S-adenosylmethionine; SAH, S-

adenosylhomocysteine, THF, tetrahydrofolate, MTHFR, 5.10-methylene-tetrahydrofolate reductase.

Fig. 9. Interaction between abnormal GABA metabolism and creatine biosynthesis in succinate
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semialdehyde dehydrogenase deficiency (4-hydroxybutyric aciduria). Abbreviations are SSA,

succinate semialdehyde; SSADH, succinate semialdehyde dehydrogenase; SSAR, succinate
semialdehyde reductase (or 4-hydroxybutyrate dehydrogenase); KC, Krebs’cycle; GAD, glutamate
decarboxylase; GABAT, GABA transaminase; AGAT, arginine glycine amidinotransferase; GAMT,
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guanidinoacetate methyltransferase; SLC6A8, plasma membrane creatine transporter. Adapted from

Vamecq et al. [171].
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Fig. 10. Diagnostic exploration tree for identifying the molecular basis of creatine disorders.
Neurological impairment is a major clinical appeal sign. Its diagnosis value is strengthened by onset in
the first years of life, unsolved underlying disease and characterization by intellectual disability,
speech delay, motor disturbances, hypotonia, epilepsy and/or autistic behavior with a special
emphasis on unsolved delays in language and/or motor acquisitions. These clinical appeal events are
recommended to prompt an exploration of creatine metabolism and transport, and importantly, as
soon as diagnosis exploration is committed, it should (i) either validate a primary, or in turn, a
secondary creatine deficiency disorder or (ii) invalidate this diagnosis issue if normalization of initial
abnormalities indicates a non-specific underlying cause. A brain defect in creatine content shown by
1
H-MRS must trigger gene and/or enzyme studies. In the case of a new gene anomaly, protein
function is evaluated to determine whether or not mutation is disease causing or not. Abbreviations
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are : OAT-D, Ornithine delta-aminotransferase deficiency; HHH, Hyperornithinemia-

Hyperammonemia-Homocitrullinuria (Triple H, SLC25A15 deficiency), OTC-D, ornithine
transcarbamylase deficiency, ASS-D, arginosuccinate synthetase deficiency; ASL-D, arginosuccinate
lyase deficiency; Cbl C-D, cobalamine C deficiency; SAHH-D, S-adenosyl homocysteine hydrolase
deficiency; SSADH-D, succinate semialdehyde dehydrogenase deficiency.

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35

A
HIGH ENERGY
PHOSPHATE

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ADP O BOND EXCHANGE

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II
NH2 HO-P-OH
ATP I

SC
HN=C NH
N-CH2-COOH HN=C phosphocreatine

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H3C
creatine N-CH2-COOH

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H 3C

M
II O

D
HN C
DEGRADATION WITH

TE C CH2 B
LOSS OF THE
HIGH ENERGY
EP
HN
N BOND EXCHANGE
I
creatinine
C

CH3 PROPERTIES
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(Fig. 1)
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L-Arginine Glycine some cells

- - (GATM + GAMT)

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AGAT
L-Ornithine Guanidinoacetate

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kidney (GATM) S-adenosyl-methionine
diet GAMT

SC
intake S-adenosyl -homocysteine
urine

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AN
Creatine
Creatine Creatine
blood Creatinine

M
+/-
plasma
SLC6A8 SLC6A8 membranes

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muscle
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Creatine
Guanidinoacetate Creatine Creatine kinases Creatine -P
GAMT Non enzymatic
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S-adenosyl-methionine conversion
Non enzymatic
S-adenosyl- conversion Creatinine
C

liver (GAMT) homocysteine

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(Fig. 2)
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growth hormone
testosterone thyroid hormones
signaling

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NH2 H2N-CH2 -COOH + = stimulation
HN=C glycine
+ or up-regualtion

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NH-CH2-CH2-CH2-CH-COOH

arginine NH2 +
AGAT
+ - = inhibition or

SC
NH2 down-regulation
H2N-CH2-CH2-CH2-CH-COOH HN=C S-adenosylmethionine
ornithine NH2 NH-CH2-COOH

guanidinoacetate S-adenosylhomocysteine
phosphocreatine

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NH2
GAMT HN=C creatine

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N-CH2-COOH
H3C
creatinine

ESTROGEN

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INCREASED RECOVERY
OF CREATINE AND guanidinoacetate SIGNALING
GUANIDINOACETATE creatine
IN THE URINE FROM creatinine

D
FEMALE SUBJECTS

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-
SLC6A8 SLC6A8
SLC6A8 = creatine transporter + ACTIVATION OF
AMP-ACTIVATED
CELL ENERGY
DEPLETION
CELL
PROTEIN KINASE (ATP)

SGK-1
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NH2 NH2 ATP ADP
HN=C HN=C

guanidinoacetate NH-CH2-COOH GAMT

H3C
N-CH2-COOH

creatine
phosphocreatine BLOOD
C
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creatinine
S-adenosylmethionine S-adenosylhomocysteine URINE

(Fig. 3)
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EXTRA-CNS DIETARY CREATINE

CELLS/TISSUES
BLOOD

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de novo CREATINE
CREATINE BIOSYNTHESIS
CREATINE
SLC6A8

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CNS
INCOMPLETE CREATINE X

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BIOSYNTHESIS PATHWAY
NO CREATINE TRANSPORT

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ARGININE ORNITHINE

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GLYCINE GUANIDINOACETATE
AGAT

AN
A

M
X
SLC6A8

NO CONTRIBUTION TO
D

D
CREATINE SYNTHESIS
SAM SAH
CREATINE CREATINE

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GUANIDINOACETATE
GAMT

B INCOMPLETE CREATINE
BIOSYNTHESIS PATHWAY
SLC6A8
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ARGININE ORNITHINE SAM SAH
GLYCINE GUANIDINOACETATE CREATINE
C

C
AGAT GAMT
AC

COMPLETE CREATINE
SLC6A8 BIOSYNTHESIS PATHWAY

(Fig. 4)
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CREATINE
BIOSYNTHESIS
PROTEINS

AGAT

GAMT
EXTRASYNAPTIC SLC6A8 EXTRASYNAPTIC SLC6A8

PT
(transport of creatine) (transport of guanidinoacetate)

RI
: arginine
: glycine
: guanidinoacetate

SC
: creatine

U
AFFERENT NEURON
AN
M

SYNAPTIC SLC6A8 SYNAPTIC SLC6A8

(presynaptic re-uptake (presynaptic re-uptake
{ ED

of creatine) of creatine)

- +
SYNAPTIC CLEFT
T

GABAA NMDA
receptor receptor
chloride calcium
channel channel
EP

++
Ca
{

-
Cl stimulation of
C

glutamatergic transmission EFFERENT NEURON

competitive antagonism of
GABAergic transmission
AC

(Fig 5)
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ACCEPTED MANUSCRIPT

NAA

PT
Cho
m-Ino

RI
Lac

SC
Lipids

U
AN
4 3 2 1 0
M

NAA

B
D
TE
Creatine

Cho
EP

m-Ino
C

Lac Lipids
AC

4 3 2 1 0
(Fig. 6)
 ACCEPTED MANUSCRIPT

41

fumarate
NH2
ASL HN=C
NH-CH2-CH2-CH2-CH-COOH
CYTOSOL

PT
H2N-CH2 -COOH
arginine
arginosuccinate ** NH2
NH2

ARGase AGAT
glycine

RI
O=C
NH2 ****
urea guanidinoacetate
ASS
UREA H2N-CH2-CH2-CH2-CH-COOH HN=C
NH2 S-adenosyl-
methionine
*****

SC
aspartate CYCLE ornithine NH2
PRODUCT NH-CH2-COOH

INHIBITION S-adenosylhomocysteine
citrulline GAMT
OF AGAT NH2
creatine
HN=C

U
AGAT N-CH2 -COOH
SLC25A15 H3 C

AN
OAT

O=C
NH2 *’ ornithine
H2 N-CH2-CH2 -CH2-CH-COOH
* H2 C
CH2 -CH-COOH

CH=N
HN-CH2-CH2 -CH2 -CH-COOH

M
NH2
∆1-pyrrolidine-5-
citrulline NH 2
carboxylate
mitochondrial OTC O
2-oxoglutarate

D
inner membrane II
HO-P-OH
I proline

TE
carbamoylphosphate O
O=C
NH2

MITOCHONDRIA glutamate
EP
CPS

*** NH3 CO2

C

mitochondrial mitochondrial
outer membrane intermembrane space
AC

(Fig. 7)
 ACCEPTED MANUSCRIPT

42

arginine glycine
Proteins

PT
methionine
AGAT

RI
adenosyltransferase
Serine-hydroxy Serine ornithine
methyltransfrase METHIONINE

SC
THF SAM
Glycine guanidinoacetate
DMG
5,10 GAMT

U
methylene Methionine METHYLATION CREATINE

AN
THF Synthase
CYCLE SAH creatine BIOSYNTHESIS
FOLATE (B12) Bétaïne
MTHFR CYCLE

M
(B2) SAH
5-methyl THF HCys hydrolase

D
Cystathionine

TE
Beta-Synthase Serine
(B6)
HCys TRANS-
EP
Cystathionine
SULFURATION
PATHWAY
C

α-ketobutyrate
AC

Cys SO42-

(Fig. 8)
 ACCEPTED MANUSCRIPT

43

2-oxo-glutarate
HOOC-CH-CH2 -CH2 -COOH

NH2
KC

PT
HOOC-CH2 -CH2 -COOH SSADH
succinic acid SSADH
DEFICIENCY

RI
O=CH-CH2 -CH2 -COOH
glutamate succinic semialdehyde
HOOC-CH-CH2 -CH2 -COOH

NH2
SSAR

SC
H2N-CH2 -CH2 -CH2 -COOH
HO-CH2 -CH2 -CH2 -COOH
GAD GABA GABA T
4-hydroxybutyrate

GABA METABOLISM

U
CREATINE BIOSYNTHESIS INCREASED BRAIN GABA

AN
NH2 H2N-CH2 -COOH H2N-CH2 -CH2 -CH2 -COOHAS AN AMIDINO
HN=C glycine GABA arginine ACCEPTOR
NH-CH2-CH2-CH2-CH-COOH AGAT AGAT METABOLITE
arginine ornithine

M
NH2
NH2 NH2
HN=C HN=C
H2N-CH2-CH2-CH2-CH-COOH
NH-CH2-COOH NH-CH2-CH2-CH2-COOH
ornithine γ-guanidinobutyrate

D
NH2 guanidinoacetate
S-adenosylmethionine BRAIN

TE
TOXICITY
GAMT
SLC6A8
S-adenosylhomocysteine
EP
NH2 NH2
HN=C creatine HN=C creatine
N-CH2-COOH N-CH2-COOH
H 3C H3C
C

(Fig. 9)
AC
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ACCEPTED MANUSCRIPT

Patient (boy / girl) with intellectual disability +/- speech delay

+/- Epilepsy +/- autistic behavior +/- familial history

Absence/Low Normal
creatine creatine
Urinary Creatine (CT)/Creatinine ratio and peak peak
Guanidinoacetate (GAA)/Creatinine ratio Brain 1H-MRS
Analysis of creatine peak

Abnormal Normal Does a clinical

result result suspicion persist Exclusion of a
in a female patient creatine deficiency

PT
Second sample with normal
syndrome
CT/Creatinine + GAA/Creatinine ratios on Normalisation
urine and CT + GAA on plasma metabolite levels? no
Confirmation of an
abnormal result yes

RI
URINE URINE URINE
GAA/Creatinine ratio : ↓ GAA/Creatinine ratio : ↑ GAA/Creatinine ratio : N
CT/Creatinine ratio : ↓ ou N CT/Creatinine ratio : ↓ ou N CT/Creatinine ratio : ↑
PLASMA

SC
PLASMA PLASMA
GAA : ↓ CT : ↓ ou N CT : ↑
GAA : ↑ GAA : N

(Arginase/Cbl C-D/ (Non specific elevation of CT :

U
(OAT-D / ASL-D/ASS-D/OTC-D, HHH) SAHH-D/SSADH-D) muscular lysis, non fasting sample)
or or or
AN
Suspicion for Suspicion for Suspicion for
AGAT Deficiency GAMT Deficiency Creatine transporter
Deficiency

GATM GAMT
M

gene studies gene studies SLC6A8 normal

gene studies gene structure
AGAT activity GAMT activity functional studies and
measurements measurements of creatine transport protein function
D

abnormal GATM, GAMT or SLC6A8 gene structure

TE

(if molecular genetic test results are inconclusive, protein activities are measured)
DIAGNOSIS CONFIRMATION OF A PRIMARY
CREATINE DEFICIENCY DISORDER

(Fig. 10)
C EP
AC
 ACCEPTED MANUSCRIPT
HIGHLIGHTS

Primary creatine disorders highlight a role of creatine in brain physiology

SLC6A8 takes part in creatine transport and re-uptake but also in biosynthesis

Secondary creatine defects stress dependence of creatine metabolism on many pathways

The high dependence on multiple metabolic pathways supports creatine as a cell sensor

PT
Creatine is a potential sensor of cell methylation and energy status

RI
U SC
AN
M
D
TE
C EP
AC