Creatine Deficiency Syndromes

From ICNApedia - The Child Neurology Knowledge Environment

Creatine deficiency syndromes are a rare group of inborn errors of metabolism involving creatine synthesis l-arginine-glycine amidinotransferase deficiency (AGAT) and guanidinoacetate methyltransferase deficiency (GAMT) ) and creatine transport (creatine transporter deficiency).

Creatine metabolism

• Greek κreas = flesh
• first isolated from meat extract by Chevreul in 1835[1].
• Creatine and its phosphorylated form, phosphocreatine, play an essential role in energy storage and transmission in most tissues, predominantly in skeletal muscle and brain.

Biosynthesis and transport

• Creatine is either obtained by dietary intestinal absorption or by endogenous synthesis, primarily in kidney, pancreas, and liver.
• 95% of Creatine (α-methyl guanidine-acetic acid) is found in skeletal muscle[2] and most of the remaining 5% of the creatine pool is located in brain, liver, kidney, and testis.
• Two enzymes are involved in creatine biosynthesis.
  

  

  Creatine biosynthesis and transport
  • AGAT converts arginine and glycine into ornithine and guanidinoacetate.
  • Subsequently, GAMT catalyzesS-adenosyl-l-methionine-dependent methylation of guanidinoacetate to yield creatine and S-adenosyl-l-homocysteine. The liver is the principal organ where this methylation takes place
• The blood-brain barrier is poorly permeable to creatine and there is evidence of an autonomous creatine synthesis pathway in the CNS.
• Creatine is taken up from blood into creatine-requiring tissues actively by SLC6A8 , which spans the plasma membrane, against a large concentration gradient: plasma creatine = 50 μmol/L; intracellular (creatine + phosphocreatine)> 40 mmol/L
• Muscular creatine and phosphocreatine are nonenzymatically converted at an almost steady rate (2% of total creatine) to creatinine, which diffuses out of the cells and is eventually excreted into the urine[3]

Regulation of Creatine Metabolism

• The formation of guanidinoacetate is normally the rate-limiting step of creatine biosynthesis; consequently, the AGAT reaction is the most likely control step in the pathway
• Most important in this respect is the feedback repression of AGAT by creatine, the end product of the pathway, which most probably serves to conserve the dietary essential amino acids arginine and methionine.
• In folic acid deficiency, in which creatine biosynthesis is curtailed and the serum concentration of creatine is likely decreased, AGAT is overexpressed.
• An increase in the serum concentran of creatine, due either to an endogenous source or to dietary supplementation, results in concomitant decrease in the mRNA content, the enzyme level, and the enzymatic activity of AGAT, suggesting regulation of AGAT expression at a pretranslational level
• The expression of AGAT may be modulated by dietary and hormonal factors.
  • Thyroidectomy or hypophysectomy in rats decreases AGAT activity in the kidney. The original AGAT activity can be restored by injection of thyroxine or of growth hormone.
  • AGAT levels are also decreased in liver, pancreas, and kidney in conditions of dietary deficiency and disease (fasting, protein-free diets, vitamin E deficiency, or streptozotocin-induced diabetes)
• In mouse models GAMT expression in liver is higher in females than in males, suggesting that GAMT expression might be under the control of sex hormones . In contrast, however, removal of adrenal, pituitary, gonad, or thyroid and parathyroid glands and administration of large doses of insulin, estradiol, testosterone, cortisol, thyroxin, or growth hormone had only minor effects, if any, on GAMT activity in rat liver . There are some indications that GAMT activity in the liver may be influenced by dietary factors , but creatine does not interfere with the expression of GAMT or arginase in liver.
• In vitro Creatine, creatinine, and phosphocreatine do not seem to regulate allosteric activities of AGAT or GAMT.
• But AGAT is potently inhibited by ornithine, which may be pathologically implicated in, for instance, gyrate atrophy of the choroid and retina.
• All the enzymes involved in creatine metabolism (AGAT, GAMT, and creatine kinase) are sensitive to modification and inactivation by sulfhydryl reagents.
• Ammonia and AGAT,GAMT,SLC6A8
  • Ammonia exposure differentially alters regulation of AGAT, GAMT and SLC6A8, in terms of both gene expression and protein activity, in a cell-type-specific manner.
• Ammonia exposure decreases both creatine and its synthesis intermediate, guanidinoacetate, in brain cells, probably through the inhibition of AGAT enzymatic activity.
  • Oligodendrocytes, which might be affected by hyperammonemia, seem to be the major actors in the brain in terms of creatine synthesis trafficking and uptake. Ammonia exposure induces SLC6A8 in astrocytes and might increase the BBB permeability[4]

Physiologic role of creatine

• Creatine is essential in tissue energy metabolism, mainly in muscle and the nervous system.
  • Creatine is involved in adenosine triphosphate production through its involvement in the phosphocreatine energy system. This system can serve as a temporal and spatial energy buffer, as well as a pH buffer. As a spatial energy buffer, creatine and phosphocreatine are involved in the shuttling of adenosine triphosphate from the inner mitochondria membrane into the cytosol .
  • Creatine is also involved in regulating glycolysis. When tissue creatine is depleted activity of oxidative enzymes such as creatine kinase and citrate synthase are increased.
• Creatine supplementation increases muscle fiber diameter for both type 1 and 2 muscle fibers by 35 %
• Creatine, and in particular phosphocreatine, may stabilize membranes because of the zwitterion nature of phosphocreatine, with negatively charged phosphate and positively charged guanidine groups.
• In the CNS Creatine represents a neuromodulator, released in the brain in an action-potential-dependent manner. The creatine concentration in the brain may play a role in regulating appetite and body weight[5, 6]

Epidemiology

Creatine deficiency syndromes are rare and are due to inborn errors of creatine metabolism resulting in:

• GAMT deficiency, first described in 1994[7]
• AGAT deficiency, first described in 2001 [8], and
• SLC6A8 defect, first described in 2001[9].

• Worldwide, only 4 cases of AGAT and approximately 40 cases of GAMT have been reported to date[10, 11, 12].
• SLC6A8 deficiency prevalence seems to be slightly higher with up to 20 mutations being described[13, 14, 15]

Because the creatine deficiency syndromes have been only recently described, they remain unfamiliar to most pediatricians and neurologists and so are often unrecognized [66]. Furthermore, few laboratories offer the opportunity for diagnostic confirmation with biochemical and genetic tests. Thus, the frequency of creatine deficiency syndromes may be underestimated[17]

Pathophysiology

The mechanisms of brain damage in creatine deficiency syndromes are poorly understood.ATP and phosphocreatine are critical to energy production via oxidative phosphorylation and the creatine kinase-phosphocreatine system is assumed to play a critical role in the CNS energy metabolism.

Various guanidino compounds, including methylguanidine, argininic acid, N-acetylarginine, and homoarginine, has been shown to significantly inhibit brain Na+, K+-adenosine triphosphatase activity in animal studies. The Na+, K+-adenosine triphosphatase is necessary to maintain the ionic gradient for the neuronal excitability. This enzyme is present in high concentrations in the brain cellular membrane, consuming approximately 40-50% of the adenosine triphosphate generated in this tissue, and is highly responsive to changes in membrane fluidity .

• Neurologic dysfunction in GAMT deficiency has been attributed primarily to increased guanidinoacetate levels.
  • Guanidinoacetate may be epileptogenic[schulze] and may alter neurotransmission and decrease membrane fluidity.
  • Guanidinoacetate is an agonist of γ Amino butyric acid A (GABA A)receptor and thus affects GABA neurotransmission.
• However Guanidinoacetate levels are not increased in AGAT deficiency and SLC6A8 defects

• Elevated cellular creatine levels is reported to exert a direct antiapoptotic effect . In combination with the action of creatine kinase inside mitochondria, creatine prevents or delay mitochondrial permeability transition pore opening, an early event in apoptosis[18]
• Creatine has also been shown to exert an antioxidant effect via a mechanism that involves the scavenging of reactive oxygen species[19]

Clinical features

• Creatine deficiency syndromes are associated with severe neurologic phenotypes and lack any detectable creatine in the brain.
• Creatine levels correlate positively with recognition memory, and creatine supplements reduce mental fatigue [54] and [55] and protect against toxicity of glutamate and β amyloid in rat hippocampal neurons [56].
• Exposure to ammonium decreased creatine, phosphocreatine, adenosine diphosphate and creatine treatment protects axons from ammonium toxicity[20]

Creatine depletion in the brain was found to be associated with disruption of neuronal functions, such as loss of hippocampal mossy fiber connection[21], and with changes in mitochondrial structure, such as the intramitochondrial ubiquitous mitochondrial-creatine kinase-rich inclusion bodies[22] typical of several clinical pathologic conditions, including encephalomyopathies and mitochondrial myopathies.

Epilepsy, mental retardation, and autism remain the principal neurologic signs associated with creatine deficiency syndromes[23].

Abbreviations:AGAT = l-arginine-glycine amidinotransferaseb = BloodCr = CreatineCrn = Creatininedbs = Dry blood spotf = FibroblastsGAA = GuanidinoacetateGAMT = Guanidinoacetate methyltransferasel = LymphoblastsSLC6A8 = Creatine transporter Source:Nasrallah F, Feki M, Kaabachi N. Creatine and creatine deficiency syndromes: biochemical and clinical aspects. Pediatr Neurol 2010;42:163-171.

• GAMT deficiency
  • Nonspecific phenotype with varying age of onset.No clear genotype-phenotpe correlation has been established.
  • mental retardation( most common )
  • epilepsy
    • seizures usually start from age 10 months to 3 years
    • Seizure types include myoclonic, generalized tonic-clonic, partial complex seizures and drop attacks
    • Seizures are refractory to AED treatment in severe cases
  • developmental delay
  • muscular hypotonia & weakness
  • progressive extrapyramidal signs and symptoms
  • autistic or self-aggressive behavior
• AGAT deficiency
• Nonspecific phenotype
  • mild to moderate mental retardation
  • psychomotor delay during the first years of life with severe language delay later
  • epilepsy or movement disorders have not been reported[12].
• Normal Brain MRI but proton magnetic resonance spectroscopy examinations reveal brain creatine depletion in all cases[12]. No sufficient data to establish a genotype-phenotype correlation in this disease.
• SLC6A8 creatine transporter defect
  • By 2009 15 families have been diagnosed with a mutation in the SLC6A8 gene, but clinical descriptions have been published for only the first nine families[14]
  • mental retardation (mostly mild to moderate)
  • expressive speech and language delay
  • seizures (easily controlled)
  • autistic characteristics

Investigations and Diagnosis

Some GAMT and AGAT deficiencies are easily treatable and hence early diagnosis is important.

Analysis of creatine and its metabolites

• Urine guanidinoacetate, creatine concentration and calculation of creatine/creatinine and guanidinoacetate/creatinine ratios in urine
  • AGAT (low creatine with low guanidinoacetate excretion)
  • GAMT deficiencies (low creatine with high guanidinoacetate excretion)
  • SLC6A8 defects (high creatine/creatinine and guanidinoacetate/creatinine ratios in urine).
    

    

    Urine chromatography in creatine deficiency syndromes
• Guanidinoacetate and creatine can be measured in plasma, in cerebrospinal fluid, and in amniotic fluid for prenatal diagnosis.
• Age-related reference values for creatine and guanidinoacetate in plasma and urine have been reported[24, 25, 26]
• Creatine and guanidinoacetate concentrations can be measured using
  • high performance liquid chromatography[24, 27]
  • tandem mass spectrometry[4, 28]or gas chromatography
  • mass spectrometry[26, 29, 30].
• Creatinine concentrations in urine alone cannot be used for the diagnosis of creatine deficiency syndromes,
  • because normal concentrations can be found in affected patients[31]
  • Low urinary creatinine concentration and low 24-hour urine creatinine excretion are also observed in patients with vanishing muscle mass, representing an unspecific finding in several myopathies and muscular dystrophies<stromberger2003</cite>
  • Plasma creatine and guanidinoacetate should be measured to confirm altered results in urine and for the diagnosis of AGAT deficiency in patients with certain urea cycle defects who show very low guanidinoacetate concentration in urine, but not in plasma[30]

Enzymatic Study and Creatine Loading Test

• The enzyme activity of AGAT and GAMT is determined in cultured cells by the measure of formed product in the presence of a labeled substrate of the enzyme.
• Two different methods for AGAT activity measurement have been reported, both using cultured lymphoblasts as a source of the enzyme.
  • In the first method, cell homogenate supernatants are incubated with arginine and 14C-glycine[8]
  • The second method makes use of stable-isotope-labeled substrates (U-13C,15N) glycine and l-(guanido-15N2) arginine[32]. Formation of radiolabeled guanidinoacetate is measured by high performance liquid chromatography.
• GAMT assays in lymphoblasts and fibroblasts have been reported, using radiolabeled substrate (13C2-guanidinoacetate) and methyl donor (H3-S-adenosyl-l-methionine). The reaction product (13C2-2 H3-creatine) is analyzed by gas chromatography and mass spectrometry[33]
• The diagnosis of SLC6A8 can be confirmed by the creatine uptake test.

Creatine uptake studies: Fibroblasts are grown in medium supplemented with creatine. The cells are harvested and creatine content is measured by stable isotope dilution gas chromatography and mass spectrometry. In cells obtained from healthy controls, creatine uptake is observed after incubation with 25 μmol/L creatine. In cells from affected patients, no uptake at 25 μmol/L is observed; at 500 μmol/L, uptake is 25% of that found in controls.

When an inhibitor of SLC6A8, guanidinopropionate, is added to the incubation medium, uptake in controls decreases by 50% but uptake in the patients remains constant. This difference suggests that the creatine uptake observed in the patients at high creatine concentrations is not due to the action of SLC6A8, but may be passive diffusion or uptake by means of other transporters[32].

Mutation Studies[34]

• Mutation analysis has been performed by direct DNA sequence analysis of polymerase chain reaction products.
• AGAT deficiency was described only in three patients from one family. All three patients were homozygous for the single mutation p.W149X[12]
• In GAMT deficiency, prescreen by gradient gel electrophoresis denaturation method is applied, prior to DNA sequence analysis[35] The mutations causing GAMT deficiency are in contrast heterogeneous. In the literature, 29 patients affected with GAMT deficiency have been described of which 10 are of Portuguese origin and 17 of the 20 alleles contain the same mutation, c.59 G > C, pTrp20Ser [61], [86] and [87]. The c.327 G > A mutation, which occurred in 7 of 28 alleles, was investigated but there appears to be no hotspot in the spectrum of GAMT mutations.[34]
• A total of 20 pathogenic mutations in theSLC6A8 gene had been reported. Mutational analysis of the SLC6A8 gene by DNA sequence analysis resulted in the identification of nonsense mutations, single-amino-acid deletions, large deletions, missense mutations, and splice errors. As yet, no hotspot mutation has been found

Brain proton magnetic resonance spectroscopy

Therapeutic Strategies[34]

• GAMT deficiency
  • Patients with GAMT deficiency, especially if treated early, have shown favorable clinical response to oral supplementation of creatine monohydrate (350 mg/kg per day). A dietary restriction of arginine (15 mg/kg per day) in combination with ornithine supplementation (100 mg/kg per day) reduced the concentrations of guanidinoacetate
• AGAT deficiency
  • In patients with AGAT deficiency, guanidinoacetate concentration is low; early creatine substitution treatment alone (400 mg/kg per day) might effectively prevent neurologic sequelae].

The creatine deficiency caused by disorders of creatine synthesis (GAMT and AGAT deficiency) can be corrected by oral supplementation of creatine monohydrate.

• SLC6A8 deficiency (creatine transporter deficiency)
  • oral creatine substitution (340 mg/kg per day) does not result in an increase of brain creatine levels. Treatment results in no change in behavior or language, but a substantial increase in body weight. l-arginine improved neuropsychologic disorders in a child with SLC6A8 creatine transporter deficiency; however, Fons et al.[38]found no effectiveness of 9 months of l-arginine supplementation in four patients with the transporter deficiency.

References

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    This icnapedia page incorporates tables, figures and statements from this key review

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