Plasma Guanidinoacetate Research Articles

Response to therapy of creatine transporter deficiency caused by a hypomorphic variant in SLC6A8

Cerebral creatine deficiency syndromes (CCDS) are rare inherited metabolic disorders caused by defective biosynthesis or transport of creatine. These conditions are characterized by reduced accumulation of creatine in the brain, mild to severe intellectual disability, global developmental delay, and speech-language disorders. The amount of brain creatine reduction needed to cause symptoms is not known. Here we report a new patient with creatine transporter deficiency (CTD) who presented at 15 months of age with seizures and global delays with no speech at 3 years of age. Brain MRI was normal, but brain MRS indicated creatine levels reduced to about 20 % of normal. He had normal levels of creatine and guanidinoacetate in plasma, but increased creatine/creatinine ratio in urine. DNA sequencing identified a hemizygous c.832C > T (p.Arg278Cys) variant in the creatine transporter gene SLC6A8. Fibroblasts from this patient had about 25 % of normal creatine transport activity, a value much higher than that measured in patients whose variants introduced premature stop codons in SLC6A8. The child was started on supplements of creatine, glycine, and arginine. His speech improved dramatically, and he had no more seizures, even during episodes of fever. Despite the clinical improvement, a repeat MRS demonstrated similar levels of brain creatine. This study suggests that a reduction in creatine transporter activity to 25 % or less is sufficient to cause symptoms of brain creatine deficiency and that functionally milder forms of CTD might respond to supplements aimed at replenishing brain creatine.

Reduced guanidinoacetate in plasma of patients with autosomal dominant Fanconi syndrome due to heterozygous P341L GATM variant and study of organoids towards treatment.

Autosomal dominant Fanconi syndrome due to a GATM variant (GATM-FS), causes accumulation of misfolded arginine-glycine amidinotransferase (AGAT) in proximal renal tubules leading to cellular injury. GATM-FS presents during childhood and progresses to end-stage kidney disease (ESKD) in adults. We study creatine metabolism in two individuals of unrelated families with a known GATM variant and the effect of creatine supplementation in kidney organoids. Plasma and urine metabolites were measured by mass spectrometry. Brain creatine was assessed by magnetic resonance spectroscopy (MRS). Guanidinoacetate (GAA) synthesis by the AGAT mutant was measured in patient-derived immortalized lymphocytes using stable isotopes of arginine and glycine. The effect of creatine on GATM expression was assessed in human kidney cells and organoids. Several family members from two unrelated families were diagnosed with Fanconi syndrome and had the c.1022C>T (p. P341L) variant in GATM. Two affected individuals in both families had moderately reduced plasma GAA levels. In comparison to wild-type cells, GAA synthesis by patient-derived GATM P341L+/- lymphoblastoid cell lines (LCL) was reduced, but not absent as in GATM cells from a patient with creatine deficiency syndrome. In vitro studies on human kidney organoids revealed reduced AGAT expression after treatment with creatine. Finally, we showed in one patient that creatine supplementation (5 g daily) substantially increased plasma creatine levels. We report low plasma and urine GAA in patients with autosomal dominant GATM-FS and show that creatine downregulates AGAT in human kidney cells.

Creatine homeostasis and the kidney: comparison between kidney transplant recipients and healthy controls

Creatine is a natural nitrogenous organic acid that is integral to energy metabolism and crucial for proper cell functioning. The kidneys are involved in the first step of creatine production. With kidney transplantation being the gold-standard treatment for end-stage kidney disease, kidney transplant recipients (KTR) may be at risk of impaired creatine synthesis. We aimed to compare creatine homeostasis between KTR and controls. Plasma and urine concentrations of arginine, glycine, guanidinoacetate, creatine and creatinine were measured in 553 KTR and 168 healthy controls. Creatine intake was assessed using food frequency questionnaires. Iothalamate-measured GFR data were available in subsets of 157 KTR and 167 controls. KTR and controls had comparable body weight, height and creatine intake (all P > 0.05). However, the total creatine pool was 14% lower in KTR as compared to controls (651 ± 178 vs. 753 ± 239 mmol, P < 0.001). The endogenous creatine synthesis rate was 22% lower in KTR as compared to controls (7.8 ± 3.0 vs. 10.0 ± 4.1 mmol per day, P < 0.001). Despite lower GFR, the plasma guanidinoacetate and creatine concentrations were 21% and 41% lower in KTR as compared to controls (both P < 0.001). Urinary excretion of guanidinoacetate and creatine were 66% and 59% lower in KTR as compared to controls (both P < 0.001). In KTR, but not in controls, a higher measured GFR was associated with a higher endogenous creatine synthesis rate (std. beta: 0.21, 95% CI: 0.08; 0.33; P = 0.002), as well as a higher total creatine pool (std. beta: 0.22, 95% CI: 0.11; 0.33; P < 0.001). These associations were fully mediated (93% and 95%; P < 0.001) by urinary guanidinoacetate excretion which is consistent with production of the creatine precursor guanidinoacetate as rate-limiting factor. Our findings highlight that KTR have a disturbed creatine homeostasis as compared to controls. Given the direct relationship of measured GFR with endogenous creatine synthesis rate and the total creatine pool, creatine supplementation might be beneficial in KTR with low kidney function.Trial registration ID: NCT02811835.Trial registration URL: https://clinicaltrials.gov/ct2/show/NCT02811835.

ClinGen variant curation expert panel recommendations for classification of variants in GAMT, GATM and SLC6A8 for cerebral creatine deficiency syndromes

Cerebral creatine deficiency syndromes (CCDS) are inherited metabolic phenotypes of creatine synthesis and transport. There are two enzyme deficiencies, guanidinoacetate methyltransferase (GAMT), encoded by GAMT and arginine-glycine amidinotransferase (AGAT), encoded by GATM, which are involved in the synthesis of creatine. After synthesis, creatine is taken up by a sodium-dependent membrane bound creatine transporter (CRTR), encoded by SLC6A8, into all organs. Creatine uptake is very important especially in high energy demanding organs such as the brain, and muscle. To classify the pathogenicity of variants in GAMT, GATM, and SLC6A8, we developed the CCDS Variant Curation Expert Panel (VCEP) in 2018, supported by The Clinical Genome Resource (ClinGen), a National Institutes of Health (NIH)-funded resource. We developed disease-specific variant classification guidelines for GAMT-, GATM-, and SLC6A8-related CCDS, adapted from the American College of Medical Genetics/Association of Molecular Pathology (ACMG/AMP) variant interpretation guidelines. We applied specific variant classification guidelines to 30 pilot variants in each of the three genes that have variants associated with CCDS. Our CCDS VCEP was approved by the ClinGen Sequence Variant Interpretation Working Group (SVI WG) and Clinical Domain Oversight Committee in July 2022. We curated 181 variants including 72 variants in GAMT, 45 variants in GATM, and 64 variants in SLC6A8 and submitted these classifications to ClinVar, a public variant database supported by the National Center for Biotechnology Information. Missense variants were the most common variant type in all three genes. We submitted 32 new variants and reclassified 34 variants with conflicting interpretations. We report specific phenotype (PP4) using a points system based on the urine and plasma guanidinoacetate and creatine levels, brain magnetic resonance spectroscopy (MRS) creatine level, and enzyme activity or creatine uptake in fibroblasts ranging from PP4, PP4_Moderate and PP4_Strong. Our CCDS VCEP is one of the first panels applying disease specific variant classification algorithms for an X-linked disease. The availability of these guidelines and classifications can guide molecular genetics and genomic laboratories and health care providers to assess the molecular diagnosis of individuals with a CCDS phenotype.

Guanidinoacetate Methyltransferase Deficiency, a Treatable Neurodevelopmental Disorder

AbstractGuanidinoacetate methyltransferase (GAMT) deficiency is an autosomal recessive inborn error of creatine synthesis that results in intellectual disability, epilepsy, expressive language delay, and dystonia. We report data from two siblings with an uncommon GAMT deficiency phenotype and their clinical, biochemical, imaging, and treatment findings. The older sibling had intellectual disability, epilepsy, and generalized dystonia. The younger sibling had intellectual disability and generalized dystonia. After treatment with creatine, verbal fluency improved, as well as dystonia and aggression. This study confirms that in patients with unexplained intellectual disability, epilepsy, and/or movement disorders, GAMT deficiency should be considered. GAMT-associated cerebral creatine deficiency syndrome is a potentially treatable condition and can be identified by elevated levels of guanidinoacetate in plasma or urine or by a significantly decreased creatine peak on magnetic resonance spectroscopy.

Laboratory Diagnosis of Cerebral Creatine Deficiency Syndromes by Determining Creatine and Guanidinoacetate in Plasma and Urine.

Cerebral creatine deficiency syndromes are caused by the dysfunctional creatine biosynthesis or transport and comprise three hereditary neurodevelopmental defects including arginine-glycine amidinotransferase (AGAT), guanidinoacetate methyltransferase (GAMT), and creatine transporter deficiencies. All conditions are characterized by seizures, intellectual disability, and behavioral abnormalities. Laboratory diagnosis of these disorders relies on the determination of creatine and guanidinoacetate concentrations in both plasma and urine. Here we describe a rapid quantitative UPLC/MS/MS method for the simultaneous determination of these analytes using a normal-phase HILIC column after analyte derivatization. The approach is suitable for neonatal screening follow-ups and monitoring of the treatment for creatine deficiency syndromes.

Creatine metabolism in patients with urea cycle disorders

The urea cycle generates arginine that is one of the major precursors for creatine biosynthesis. Here we evaluate levels of creatine and guanidinoacetate (the precursor in the synthesis of creatine) in plasma samples (ns = 207) of patients (np = 73) with different types of urea cycle disorders (ornithine transcarbamylase deficiency (ns = 22; np = 7), citrullinemia type 1 (ns = 60; np = 22), argininosuccinic aciduria (ns = 81; np = 31), arginase deficiency (ns = 44; np = 13)). The concentration of plasma guanidinoacetate positively correlated (p < 0.001, R2 = 0.64) with levels of arginine, but not with glycine in all patients with urea cycle defects, rising to levels above normal in most samples (34 out of 44) of patients with arginase deficiency. In contrast to patients with guanidinoacetate methyltransferase deficiency (a disorder of creatine synthesis characterized by elevated guanidinoacetate concentrations), creatine levels were normal (32 out of 44) or above normal (12 out of 44) in samples from patients with arginase deficiency. Creatine levels correlated significantly, but poorly (p < 0.01, R2 = 0.1) with guanidinoacetate levels and, despite being overall in the normal range in patients with all other urea cycle disorders, were occasionally below normal in some patients with argininosuccinic acid synthase and lyase deficiency. Creatine levels positively correlated with levels of methionine (p < 0.001, R2 = 0.16), the donor of the methyl group for creatine synthesis. The direct correlation of arginine levels with guanidinoacetate in patients with urea cycle disorders explains the increased concentration of guanidino compounds in arginase deficiency. Low creatine levels in some patients with other urea cycle defects might be explained by low protein intake (creatine is naturally present in meat) and relative or absolute intracellular arginine deficiency.

Solute carrier SLC16A12 is critical for creatine and guanidinoacetate handling in the kidney.

A heterozygous mutation (c.643C.A; p.Q215X) in the creatine transporter SLC16A12 has been proposed to cause a syndrome with juvenile cataracts, microcornea, and glucosuria in humans. To further explore the role of SLC16A12 in renal physiology and decipher the mechanism underlying the phenotype of humans with the SLC16A12 mutation, we studied Slc16a12 knockout (KO) rats. Slc16a12 KO rats had lower plasma levels and increased absolute and fractional urinary excretion of creatine and its precursor guanidinoacetate (GAA). Slc16a12 KO rats displayed lower plasma and urinary creatinine levels, but the glomerular filtration rate was normal. The phenotype of heterozygous rats was indistinguishable from wild-type (WT) rats. Renal artery to vein (RAV) concentration differences in WT rats were negative for GAA and positive for creatinine. However, RAV differences for GAA were similar in Slc16a12 KO rats, indicating incomplete compensation of urinary GAA losses by renal GAA synthesis. Together, our results reveal that Slc16a12 in the basolateral membrane of the proximal tubule is critical for the reabsorption of creatine and GAA. Our data suggest a dominant-negative mechanism underlying the phenotype of humans affected by the heterozygous SLC16A12 mutation. Furthermore, in the absence of Slc16a12, urinary losses of GAA are not adequately compensated by increased tubular synthesis, likely caused by feedback inhibition of the rate-limiting enzyme l-arginine:glycine amidinotransferase by creatine in proximal tubular cells.NEW & NOTEWORTHY SLC16A12 is a recently identified creatine transporter of unknown physiological function. A heterozygous mutation in the human SLC16A12 gene causes juvenile cataracts and reduced plasma guanidinoacetate (GAA) levels with an increased fractional urinary excretion of GAA. Our study with transgenic SLC16A12-deficient rats reveals that SLC16A12 is critical for tubular reabsorption of creatine and GAA in the kidney. Our data furthermore indicate a dominant-negative mechanism underlying the phenotype of humans affected by the heterozygous SLC16A12 mutation.

Low-Dose Creatine Supplementation Lowers Plasma Guanidinoacetate, but Not Plasma Homocysteine, in a Double-Blind, Randomized, Placebo-Controlled Trial1–3

Background: Creatine synthesis from guanidinoacetate consumes ∼50% of s-adenosylmethionine (SAM)–derived methyl groups, accounting for an equivalent proportion of s-adenosylhomocysteine (SAH) and total homocysteine (tHcys) synthesis. Dietary creatine inhibits the synthesis of guanidinoacetate, thereby lowering plasma tHcys in rats.Objective: We tested the hypotheses that creatine supplementation lowers plasma guanidinoacetate, increases blood SAM, lowers blood SAH, and lowers plasma tHcys.Methods: Bangladeshi adults were randomly assigned to receive 1 of 4 treatments for 12 wk: placebo (n = 101), 3 g/d creatine (Cr; n = 101), 400 μg/d folic acid (FA; n = 153), or 3 g/d creatine plus 400 μg/d folic acid (Cr+FA; n = 103). The outcomes of plasma guanidinoacetate and tHcys, as well as whole blood SAM and SAH, were analyzed at baseline and week 12 by HPLC. Treatment effects of creatine supplementation were examined with the use of the group comparisons of Cr vs. placebo and Cr+FA vs. FA.Results: Plasma guanidinoacetate declined by 10.6% (95% CI: 4.9, 15.9) in the Cr group while increasing nonsignificantly in the placebo group (3.7%; 95% CI: −0.8, 8.5) (Pgroup difference = 0.0002). Similarly, plasma guanidinoacetate declined by 9.0% (95% CI: 3.4, 14.2) in the Cr+FA group while increasing in the FA group (7.0%; 95% CI: 2.0, 12.2) (Pgroup difference < 0.0001). Plasma tHcys declined by 23.4% (95% CI: 19.5, 27.1) and 21.0% (95% CI: 16.4, 25.2) in the FA and Cr+FA groups, respectively (Pgroup difference = 0.41), with no significant changes in the placebo or Cr groups (Pgroup difference = 0.35). A decrease in guanidinoacetate over time was associated with a decrease in tHcys over time in the Cr+FA group (β = 0.30; 95% CI: 0.17, 0.43; P < 0.0001).Conclusions: Our findings indicate that whereas creatine supplementation downregulates endogenous creatine synthesis, this may not on average lower plasma tHcys in humans. However, tHcys did decrease in those participants who experienced a decline in plasma guanidinoacetate while receiving creatine plus folic acid supplementation. This trial was registered at clinicaltrials.gov as NCT01050556.

Guanidinoacetate methyltransferase (GAMT) deficiency: Outcomes in 48 individuals and recommendations for diagnosis, treatment and monitoring

We collected data on 48 patients from 38 families with guanidinoacetate methyltransferase (GAMT) deficiency. Global developmental delay/intellectual disability (DD/ID) with speech/language delay and behavioral problems as the most affected domains was present in 44 participants, with additional epilepsy present in 35 and movement disorder in 13. Treatment regimens included various combinations/dosages of creatine-monohydrate, l-ornithine, sodium benzoate and protein/arginine restricted diets. The median age at treatment initiation was 25.5 and 39months in patients with mild and moderate DD/ID, respectively, and 11years in patients with severe DD/ID. Increase of cerebral creatine and decrease of plasma/CSF guanidinoacetate levels were achieved by supplementation with creatine-monohydrate combined with high dosages of l-ornithine and/or an arginine-restricted diet (250mg/kg/d l-arginine). Therapy was associated with improvement or stabilization of symptoms in all of the symptomatic cases. The 4 patients treated younger than 9months had normal or almost normal developmental outcomes. One with inconsistent compliance had a borderline IQ at age 8.6years. An observational GAMT database will be essential to identify the best treatment to reduce plasma guanidinoacetate levels and improve long-term outcomes.

Evidence-Based Treatment of Guanidinoacetate Methyltransferase (GAMT) Deficiency

BackgroundGuanidinoacetate methyltransferase (GAMT) deficiency causes cerebral creatine deficiency. Patients can have autistic behavior, seizures, intellectual disability, and severe speech delay. The goal of therapy is to increase creatine while reducing potentially neurotoxic guanidinoacetate concentrations. Here we evaluate how different therapies affect plasma guanidinoacetate levels in patients with GAMT deficiency. MethodsRetrospective analysis of data from five new patients with GAMT deficiency (four with delays and seizures, one diagnosed at birth). ResultsThe four symptomatic patients had decreased brain creatine by magnetic resonance spectroscopy and three also had abnormal globi pallidi by MRI. GAMT sequencing identified four previously reported mutations and one novel missense mutation (c.233T>A/p.V78E). Treatment with creatine (250–1000mg/kg/day), ornithine (100–800mg/kg/day), and sodium benzoate (50–135mg/kg/day) supplements along with dietary protein restriction (0.8–1.5g/kg/day) improved seizures and development with all patients becoming verbal. The patient treated at birth remains developmentally normal. Reduction in glycine and increase in ornithine levels significantly decreased plasma guanidinoacetate, with glycine levels being the best predictor of guanidinoacetate levels. In contrast, arginine levels were not significantly correlated with plasma guanidinoacetate. ConclusionsOur results show that supplements of creatine, sodium benzoate (to reduce glycine) and ornithine reduce guanidinoacetate levels in patients with GAMT deficiency (dietary therapy was not evaluated in our study). Normal development with early therapy renders GAMT deficiency an ideal candidate for inclusion in newborn screening panels.

Biochemical, molecular, and clinical diagnoses of patients with cerebral creatine deficiency syndromes

Cerebral creatine deficiency syndromes (CCDS) are a group of inborn errors of creatine metabolism that involve AGAT and GAMT for creatine biosynthesis disorders and SLC6A8 for creatine transporter (CT1) deficiency. Deficiencies in the three enzymes can be distinguished by intermediate metabolite levels, and a definitive diagnosis relies on the presence of deleterious mutations in the causative genes.Mutations and unclassified variants were identified in 41 unrelated patients, and 22 of these mutations were novel. Correlation of sequencing and biochemical data reveals that using plasma guanidinoacetate (GAA) as a biomarker has 100% specificity for both AGAT and GAMT deficiencies, but AGAT deficiency has decreased sensitivity in this assay. Furthermore, the urine creatine:creatinine ratio is an effective screening test with 100% specificity in males suspected of having creatine transporter deficiency. This test has a high false-positive rate due to dietary factors or dilute urine samples and lacks sensitivity in females.We conclude that biochemical screening for plasma GAA and measuring of the urine creatine:creatinine ratio should be performed for suspected CCDS patients prior to sequencing. Also, based on the results of this study, we feel that sequencing should only be considered if a patient has abnormal biochemical results on repeat testing.

Creatine Supplementation Prevents the Accumulation of Fat in the Livers of Rats Fed a High-Fat Diet ,

The aim of the present study was to examine the effects of creatine supplementation on liver fat accumulation induced by a high-fat diet in rats. Rats were fed 1 of 3 different diets for 3 wk: a control liquid diet (C), a high-fat liquid diet (HF), or a high-fat liquid diet supplemented with creatine (HFC). The C and HF diets contained, respectively, 35 and 71% of energy derived from fat. Creatine supplementation involved the addition of 1% (wt:v) of creatine monohydrate to the liquid diet. The HF diet increased total liver fat concentration, liver TG, and liver TBARS and decreased the hepaticS-adenosylmethionine (SAM) concentration. Creatine supplementation normalized all of these perturbations. Creatine supplementation significantly decreased the renal activity of L-arginine:glycine amidinotransferase and plasma guanidinoacetate and prevented the decrease in hepatic SAM concentration in rats fed the HF diet. However, there was no change in either the phosphatidylcholine:phosphatidylethanolamine (PE) ratio or PEN-methyltransferase activity. The HF diet decreased mRNA for PPARa as well as 2 of its targets, carnitine palmitoyltransferase and long-chain acylCoA dehydrogenase. Creatine supplementation normalized these mRNA levels. In conclusion, creatine supplementation prevented the fatty liver induced by feeding rats a HF diet, probably by normalization of the expression of key genes of aβ-oxidation.

Simultaneous determination of creatine and guanidinoacetate in plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS)

Background Guanidinoacetate (GAA) and creatine are reliable biochemical markers for primary and secondary creatine defects. We describe a method by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) for simultaneous determination of plasma GAA and creatine. We analyzed 283 healthy subjects from 0 to 63 years old to obtain age-related control values. Methods Plasma samples were extracted with acetonitrile containing 13C2-GAA and d3-creatine. Samples were analyzed by LC–MS/MS in positive ionisation mode, after derivatization to butyl-esters. Optimal chromatographic separation was achieved using a column Supelcosil™ LC-4.6 mm with isocratic elution in 5 min. Results Run time was 5 min. Standard curves were linear from 0.05 to 200 μmol/L for creatine and from 0.02 to 40 μmol/L for GAA. Limit of detection (LOD) and limit of quantitation (LOQ) were respectively 0.005 and 0.05 μmol/L for creatine; LOD and LOQ were 0.002 and 0.02 μmol/L respectively for GAA. Intra and inter-assay CVs for creatine and GAA were <8%. Recovery experiments adding 50 and 100 μmol/L creatine and 10 and 20 μmol/L GAA were 102.1% and 101.2%, for creatine; 102.95% and 96.45% for GAA. The method was applied to 283 plasma controls from healthy subjects to obtain control values in three specific age ranges: 0–12, 13–20, >20 years old. Conclusion A rapid and high sensitive LC–MS/MS method was developed and validated for determination of creatine and GAA in plasma and it could also be applied to other biological materials, such as CFS and urines. This method is useful for diagnoses of primary and also for secondary creatine defects that may occur in inherited metabolic diseases in which precursors of creatine biosynthesis are involved.

Methylation cycle, arginine-creatine pathway and asymmetric dimethylarginine in paediatric renal transplant

Hyperhomocysteinaemia represents an important cause of morbidity in recipients of renal transplants, but few investigations have been carried out to evaluate the status of the methylation cycle and its relation with levels of new cardiovascular biomarkers, such as asymmetric dimethylarginine (ADMA). Twenty-six children and adolescents aged 7-18years (17 male, 9 female) with stable renal transplants were recruited for the study. None had received treatment with folate, vitamin B(12) or statins. Levels of ADMA in plasma and of components of the methylation cycle and arginine (Arg)-creatine pathway in plasma and urine were analysed by specific analytical methods. Results were compared to those obtained by us with identical methods in healthy children of similar age. Concentrations of homocysteine (Hcys), S-adenosylhomocysteine (SAH) and ADMA were significantly higher, while S-adenosylmethionine (SAM)/SAH and Arg/ADMA ratios were significantly lower than controls. Arg/ADMA ratio correlated with plasma guanidinoacetate. The components of the methylation cycle, Hcys and SAH correlated with renal function. Children with renal transplant showed low methylation power (SAM/SAH) mainly due to increased levels of SAH which acts as a cardiovascular biomarker. Elevated values of ADMA and low Arg/ADMA coefficients also represent a novel finding because it inhibits nitric oxide synthesis contributing to endothelial dysfunction and cardiovascular risk in such patients.

Secondary creatine deficiency in ornithine delta-aminotransferase deficiency

Aims Ornithine delta-aminotransferase (OAT) deficiency causes gyrate atrophy (GA) of the retina, as a consequence of high plasma ornithine concentrations. Because creatine synthesis requires the conversion of arginine and glycine into ornithine and guanidinoacetate, high ornithine concentration inhibits this reaction thus causing secondary creatine deficiency. The aim of this study was to evaluate the neuropsychological features and creatine metabolism in patients with GA. Methods The study involved 7 GA patients, aged from 11 to 27 years who underwent neuropsychological evaluation and cerebral proton magnetic resonance spectroscopy (MRS). Results Neurocognitive impairment was found in 5/7 patients, including mental retardation (3/7), school failure (1/7), major visuospatial dyspraxia (1/7), aggressive behavior (3/7) and epilepsy (2/7). Two patients had normal neuropsychological evaluation. Cerebral proton magnetic resonance spectroscopy revealed a profound creatine deficiency in all patients. MRS data were confirmed by decreased levels of creatine and/or guanidinoacetate in plasma and urine in all patients. Conclusions In our group of patients with GA, we found a high prevalence of neurological impairment, not reported so far, and possibly related to secondary creatine deficiency and hyperornithinemia. We propose to treat mentally retarded GA patients with high doses of creatine, as it may normalize brain creatine levels and help to reduce ornithine levels.

Reduced brain choline in homocystinuria due to remethylation defects

To investigate whether secondary impairment of the transmethylation pathway is a mechanism underlying the neurologic involvement in homocystinuria due to remethylation defects. Twelve patients with neurologic disease due to remethylation defects were examined by brain magnetic resonance spectroscopic imaging ((1)H MRSI). Brain N-acetylaspartate, choline-containing compounds (Cho), and creatine (Cr) were quantified and compared to with controls. Metabolites of remethylation cycle and creatine biosynthesis pathway were measured in plasma and urine. MRSI revealed isolated Cho deficiency in all regions examined (mean concentration units +/- SD, patients vs controls): frontal white matter (0.051 +/- 0.010 vs 0.064 +/- 0.010; p = 0.001), lenticular nucleus (0.056 +/- 0.011 vs 0.069 +/- 0.009; p < 0.001), and thalamus (0.063 +/- 0.010 vs 0.071 +/- 0.007; p = 0.006). In contrast to controls, the Cho/Cr ratio decreased with age in patients in the three brain regions examined. Low creatine urinary excretion (p < 0.005), normal urine and plasma guanidinoacetate, and a paradoxical increase in plasma S-adenosylmethionine (p < 0.005) concentrations were observed. Patients with homocystinuria due to remethylation defects have an isolated brain choline deficiency, probably secondary to depletion of labile methyl groups produced by the transmethylation pathway. Although biochemical studies suggest mild peripheral creatine deficiency, brain creatine is in the reference range, indicating a possible compartmentation phenomenon. Paradoxical increase of S-adenosylmethionine suggests that secondary inhibition of methylases contributes to the transmethylation defect in these conditions.

Creatine metabolism in combined methylmalonic aciduria and homocystinuria

Methylation is an important aspect of many fundamental biological processes including creatine biosynthesis. We studied five patients with an inborn error of cobalamin metabolism to characterize the relation between homocysteine and creatine metabolism. Plasma guanidinoacetate concentrations were increased, 14.9 +/- 4.8 micromol/L (p < 0.0001), whereas plasma creatine concentrations were in the low reference range, 43.8 +/- 20.7 micromol/L (p = not significant). Individuals with combined methylmalonic aciduria and homocystinuria have a functional impairment of the creatine synthetic pathway probably secondary to a relative depletion of labile methyl groups. The neurotoxic effects of guanidinoacetate may be partly responsible for the observed neurological phenotype.

Plasma guanidino compounds are altered by oral creatine supplementation in healthy humans

Although creatine is one of the most widely used nutritional supplements for athletes as well as for patients with neuromuscular disorders, the effects of oral creatine supplementation on endogenous creatine synthesis in humans remains largely unexplored. The aim of the present study was to investigate the metabolic consequences of a frequently used, long-term creatine ingestion protocol on the circulating creatine synthesis precursor molecules, guanidinoacetate and arginine, and their related guanidino compounds. For this purpose, 16 healthy young volunteers were randomly divided to ingest in a double-blind fashion either creatine monohydrate or placebo (maltodextrine) at a dosage of 20 g/day for the first week (loading phase) and 5 g/day for 19 subsequent wk (maintenance phase). Fasting plasma samples were taken at baseline and at 1, 10, and 20 wk of supplementation, and guanidino compounds were determined. Plasma guanidinoacetate levels were reduced by 50% after creatine loading and remained approximately 30% reduced throughout the maintenance phase. Several circulating guanidino compound levels were significantly altered after creatine loading but not during the maintenance phase: homoarginine (+35%), alpha-keto-delta-guanidinovaleric acid (+45%), and argininic acid (+75%) were increased, whereas guanidinosuccinate was reduced (-25%). The decrease in circulating guanidinoacetate levels suggests that exogenous supply of creatine chronically inhibits endogenous synthesis at the transamidinase step in humans, supporting earlier animal studies showing a powerful repressive effect of creatine on l-arginine:glycine amidinotransferase. Furthermore, these data suggest that this leads to enhanced utilization of arginine as a substrate for secondary pathways.

Enzyme assay for diagnosis of guanidinoacetate methyltransferase deficiency.

Creatine in the human body is derived from two sources: the diet and endogenous synthesis. Biosynthesis involves two enzymes: l-arginine:glycine amidinotransferase (EC 2.1.4.1) and guanidinoacetate N -methyltransferase (GAMT; EC 2.1.1.2). Defects in both of these enzymes lead to inborn errors of metabolism (1)(2). Deficiency of l-arginine:glycine amidinotransferase has been reported very recently in two patients by Item et al. (3) and Bianchi et al. (4). GAMT deficiency was first described in 1994 (1)(5). The clinical presentation of the disease varies widely (2)(6). Some patients present with intractable epilepsy, extrapyramidal movement disorder, and severe retardation, whereas other patients have much milder symptoms and show only mild retardation and autistic behavior. The aspecific clinical picture of GAMT deficiency and the relatively good treatment options makes a good diagnostic strategy important. Magnetic resonance spectroscopy of the brains of affected patients shows absence of the creatine/creatine phosphate peak. However, magnetic resonance spectroscopy may not be performed on all patients with mild symptoms. Screening for metabolic disorders may also aid in the diagnosis because generalized increases in amino acids, organic acids, and other metabolites can be found when their concentrations are calculated relative to the creatinine concentration (2). The relatively low creatinine excretion in GAMT patients, which is attributable to the creatine deficiency, gives falsely increased values for a range of metabolites when they are calculated relative to creatinine. When GAMT deficiency is suspected, analysis of guanidinoacetate in plasma should be performed (7)(8). Confirmation of the defect can be obtained by enzymatic and DNA analysis (9)(10). Ilas et al. (11) described an assay for the determination of GAMT in fibroblasts and lymphoblasts. This method uses 14C-labeled guanidinoacetate as substrate. Product formation ([14C]creatine) is determined by HPLC with detection …