creatine

Let’s start with a lay introduction. Our muscles and brains are a major source of creatine. Contracting muscles and thinking brains require a lot of energy in the form of ATP. When the high energy third phosphate is removed from adenosine tri-phosphate energy is released to do work. Creatine is a much smaller which can serve as a reserve for this high energy phosphate.

The genes/proteins controlling creatine production

Three genes harboring activity affecting SNPs: GAMT, GATM and SLC6A8 genes.[1] The featured image was adapted from ref [2] These authors see the kidney as the primary site of AGAT/GATM that converts glycine and arginine to guanidinoacetate (GAA) and
ornithine. The kidney image was replaced with a GAMT tissue distribution form Protein Atlas. The Ellery review has the liver as the site of GAA is then methylated in the liver
via the activity of guanidino acetate methyl transerfase (GAMT). The liver cartoon was replaced by the GAMT image from Protein Atlas. Once synthesized creatine is released into the blood stream and taken up by most tissues via the creatine transporter (SLC6A8). The Protein Atlas images challenge many notions that creatine need to be produced in just a few locations.

GATM

Glycine amidino transferase, mitochondrial catalyzes the transfer of the amidino group of L-arginine onto the amino moiety of acceptor metabolites such as glycine, beta-alanine, gamma-aminobutyric acid (GABA) and taurine yielding the corresponding guanidine derivatives. Catalyzes the rate-limiting step of creatine biosynthesis, namely the transfer of the amidino group from L-arginine to glycine to generate guanidinoacetate, which is then methylated by GAMT to form creatine. Provides creatine as a source for ATP generation in tissues with high energy demands, in particular skeletal muscle, heart and brain. This enzyme is also called arginine glycine amino transferase. According to Science Direct GATM is located in the inner membrane space of the mitochondria.

Multiple reactions of GAMT/AGAT

A previous study from the Tsikas group found that Becker muscular dystrophy wasting could be reduced by

• Glucose lowering metformin seemed to act as a competitive inhibitor of AGAT. The 2023 study used 3 x 500mg metformin per day for six weeks.
• L-citrulline resulted in increased serum hArg and GAA concentrations, seemingly acting as an effector of AGAT activity. The 2023 study used three doses of 1.5g L-citrulline per day. [3]

The Tsikas study [3] used gas chromatography coupled with mass spectrometry to measure the small molecule substrates of AGAT/GAMT as well as those of citrullin to arginine conversion.

Why not just supplement with L-arginine?

These are some bullet points from the Tsikas discussion. [3]

• Supplementary Cit is preferred to supplementary Arg due to its favorable properties in the gastrointestinal tract including restricted metabolization.
• In adults, oral doses of several grams of Cit per day are required to increase circulating Arg concentrations.
• The Tsikas publication seemed to indicate that citrulline administration increases hArg and GAA formation most likely by increasing the bioavailability of Arg within AGAT-expressing cells.
• Add-on supplementation of metformin to Cit seems to “freeze” the activity of AGAT, while add-on supplementation of Cit to metformin seems to “re-activate” AGAT to produce hArg and GAA.

Ornithine, citrulline, arginine gymnastics [2]

This post is not going to even try o explain the biochemistry of these relationships explored by the Tsikas 2023 study. [3] Suffice it to say that supplementation with citrulline resulted in measurable changes in related amino acids as well as metabolic products of AGAT/GATM. [3] The one remarkable thing about the Tsikas publication is the mention of a reactive site thiol in AGAT/GATM.

Does this relate to autism?

An Italian cross-sectional study included 89 participants recruited at the Child Neurology and Psychiatry Unit, Department of Clinical and Experimental Medicine, University of Catania. Participants included 40 individuals with ASD, 22 with intellectual disability (ID) and 27 with typical development (TD) Citrulline and arginine were not significantly different The following is an adaptation of Table 3. ANOVA parmeters are not included. Only the followup Bonferroni corrections are shown for simplicity.

Amino acid	ASD (μmol/L)	ID (μmol/L)	TD (μmol/L)	ASD vs TD	ASD vs ID	ID vs TD
Orn	71.4 ± 17.3	78.2 ± 13.6	99.9 ± 61.2	p = 0.006 *	p = 1	p = 0.119
Phe	39.6 ± 7.8	40.5 ± 6.5	44.7 ± 10	p = 0.043 *	p = 1	p = 0.226
Tyr	47.9 ± 9.3	53.30 ± 11.6	58.7 ± 21.3	p = 0.010 *	p = 0.492	p = 0.581

Table 2 examined amino acid ratios. Citrulline/ arginine was not significantly different in ASD vs TD. Ratios of branched chain to aromatic amino acids tended to be higher in ASD vs TD. [4] Table 3 examined how ASD patient amino acids and ratios thereof compared to the reference range. There was a tendency for around 30% of the ASD patients to be above the reference range of combinations of branched chain to aromatic amino acids. [4] Table 4 examined the relationship between serum amino acid levels and intelligence and developmental quotients. There were no significant correlations. [4]

AGAT/GATM SNPs in ASD [2]

Most of the SNPs discovered in the Cameron study are in non coding regions…

GAMT

Guanidinoacetate N-methyltransferase converts guanidinoacetate to creatine, using S-adenosylmethionine as the methyl donor.

The folic acid and methionine cycles supply GAMT

This post will give a nod to folic acid pathways playing a putative role in autism. [6]

GAMT SNPs in autism

The vast majority of the polymorphisms are in non coding regions. Let’s take a total of ten minutes to look up the polymorphisms in coding regions.

Slc6A8

Sodium- and chloride-dependent creatine transporter 1 mediates the uptake of creatine. chloride(out) + creatine(out) + 2 Na⁺(out) = chloride(in) + creatine(in) + 2 Na⁺(in)

A recent review in Sports Medicine suggested that creatine supplementation could be beneficial for numerous neurodegenerative disorders. SLc6A8 expression is up regulated in the hippocampus, cerebral cortex, cerebellum, brain stem, and spinal cord. Expression is less in the white matter and basal ganglia. {7]

Slc6A8 polymorphisms in autism

Brain co expression summaries

Our cells are left with a choice of importing exogenous creatine or making their own.

mRNA by region from Protein Atlas

These mRNA expression data were obtained from ProteinAtlas.org. The PHANTOM database was one of many used for comparison. Note that these data do not differentiate between neurons, glia, vascular cells, and so on.

Cell by cell in mouse brains…

Braissant and Henry [8] took things to the cell type level in mouse brains in both wild types and those made deficient for GATM/AGAT, GAMT, and SLC6A8.

1. Some cells do not express GAMT. GATM, or Slc6A8.
2. Endogenous synthesis of Cr within CNS can be achieved between AGAT and GAMT expressing cells with paracrine exchange.
3. Individual cells may express both AGAT+GAMT.
4. A low proportion of brain cells only express SLC6A8.
5. i.e. Cr users-only)

Bulk Supplements

Perhaps neurotypicals might want to boost their brain power. How many of us might have SNPs in GAMT. GATM, and/or Slc6A8 that might not put us to the threshold of ASD but could befit from supplements. It seems that both our brain and muscles require creatine to be that storage of an extra high energy phosphate that is easily transferred to ADP.

• creatine monohydrate powder 100g for ~ $14
• L-citrulline powder, 100g for ~ $13
• L-arginine powder, 100 g ~ $13

One would think a healthy intake of folic acid, B6 and B12 would be in order.

References

1. Cameron JM, Levandovskiy V, Roberts W, Anagnostou E, Scherer S, Loh A, Schulze A. Variability of Creatine Metabolism Genes in Children with Autism Spectrum Disorder. Int J Mol Sci. 2017 Jul 31;18(8):1665. PMC free article
2. Ellery SJ, Dickinson H, McKenzie M, Walker DW. Dietary interventions designed to protect the perinatal brain from hypoxic-ischemic encephalopathy–Creatine prophylaxis and the need for multi-organ protection. Neurochem Int. 2016 PubMed
3. Tsikas D. Determination of equilibria constants of arginine:glycine amidinotransferase (AGAT)-catalyzed reactions using concentrations of circulating amino acids. Amino Acids. 2023 Feb;55(2):203-213. PMC free article
4. Randazzo M, Prato A, Messina M, Meli C, Casabona A, Rizzo R, Barone R. Neuroactive Amino Acid Profile in Autism Spectrum Disorder: Results from a Clinical Sample. Children (Basel). 2023 Feb 20;10(2):412. PMC free article
5. Hoxha B, Hoxha M, Domi E, Gervasoni J, Persichilli S, Malaj V, Zappacosta B. Folic Acid and Autism: A Systematic Review of the Current State of Knowledge. Cells. 2021 Aug 3;10(8):1976. PMC free article
6. Peral MJ, García-Delgado M, Calonge ML, Durán JM, De La Horra MC, Wallimann T, Speer O, Ilundáin A. Human, rat and chicken small intestinal Na+ – Cl- -creatine transporter: functional, molecular characterization and localization. J Physiol. 2002 Nov 15;545(1):133-44. PMC free article
7. Candow DG, Forbes SC, Ostojic SM, Prokopidis K, Stock MS, Harmon KK, Faulkner P. “Heads Up” for Creatine Supplementation and its Potential Applications for Brain Health and Function. Sports Med. 2023 Jun 27. doi: 10.1007/s40279-023-01870-9. Epub ahead of print. Erratum in: Sports Med. 2023 Jul 10; PubMed
8. Braissant O, Henry H. AGAT, GAMT and SLC6A8 distribution in the central nervous system, in relation to creatine deficiency syndromes: a review. J Inherit Metab Dis. 2008 Apr;31(2):230-9. free article
9. Bonilla DA, Kreider RB, Stout JR, Forero DA, Kerksick CM, Roberts MD, Rawson ES. Metabolic Basis of Creatine in Health and Disease: A Bioinformatics-Assisted Review. Nutrients. 2021 Apr 9;13(4):1238. PMC free article