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Short-chain enoyl-CoA hydratase (ECHS1) is a mitochondrial beta-oxidation enzyme involved in the metabolism of acyl-CoA fatty acid esters, as well as in valine metabolism. ECHS1 deficiency has multiple manifestations, including Leigh syndrome early at birth or in childhood with poor prognosis, to cutis laxa, exercise-induced dystonia and congenital lactic acidosis.Here we describe the case of a newborn with mutations in ECHS1 that caught our attention after the incidental finding of 3-hydroxy-butyryl/3-hydroxy-isobutyryl/malonylcarnitine (C4OH/C3DC) and tiglylcarnitine (C5:1) on blood spot in the newborn screening (NBS) program. Diagnosis was suspected based on the analysis of organic acids on dried urine spot. A moderate increase of 2-methyl-2,3-dihydroxybutyric acid, was detected, which is a known marker of this disease. Exome analysis showed c.404A>G (p.Asn135Ser) mutation in homozygosis in the ECHS1 gene. The child was therefore admitted to the hospital. Initial examination showed little response to auditory stimuli and mild hypertonia of the extremities. Clinical deterioration was evident at 4 months of age, including neurological and cardiac involvement, and the patient died at 5 months of age. This case illustrates how an incidental detection in the NBS Program can lead to the diagnosis ECHS1 deficiency. Although it is a severe disease, with no treatment available, early detection would allow adequate genetic counseling avoiding the odyssey that suffered most of these families. Keywords: ECHS1 deficiency, Mutations in ECHS1, Newborn screening, 2-methyl-2,3-dihydroxybutyric acid, 3-hydroxy-butyrylcarnitine/3-hydoxy-isobutyrylcarnitine, Tiglylcarnitine

Objectives: Early diagnosis of inborn errors of metabolism (IEM) is crucial to ensure early detection of conditions which are treatable. This study reports on targeted metabolomic procedures for the diagnosis of IEM of amino acids, acylcarnitines, creatine/guanidinoacetate, purines/pyrimidines and oligosaccharides, and describes its validation through external quality assessment schemes (EQA)., Methods: Analysis was performed on a Waters ACQUITY UPLC H-class system coupled to a Waters Xevo triple-quadrupole (TQD) mass spectrometer, operating in both positive and negative electrospray ionization mode. Chromatographic separation was performed on a CORTECS C18 column (2.1 × 150, 1.6 µm). Data were collected by multiple reaction monitoring., Results: The internal and EQA results were generally adequate, with a few exceptions. We calculated the relative measurement error (RME) and only a few metabolites displayed a RME higher than 30 % (asparagine and some acylcarnitine species). For oligosaccharides, semi-quantitative analysis of an educational panel clearly identified the 8 different diseases included., Conclusions: Overall, we have validated our analytical system through an external quality control assessment. This validation will contribute to harmonization between laboratories, thus improving identification and management of patients with IEM., (© 2024 Walter de Gruyter GmbH, Berlin/Boston.)

ADCK2 haploinsufficiency-mediated mitochondrial coenzyme Q deficiency in skeletal muscle causes mitochondrial myopathy associated with defects in beta-oxidation of fatty acids, aged-matched metabolic reprogramming, and defective physical performance. Calorie restriction has proven to increase lifespan and delay the onset of chronic diseases associated to aging. To study the possible treatment by food deprivation, heterozygous Adck2 knockout mice were fed under 40% calorie restriction (CR) and the phenotype was followed for 7 months. The overall glucose and fatty acids metabolism in muscle was restored in mutant mice to WT levels after CR. CR modulated the skeletal muscle metabolic profile of mutant mice, partially rescuing the profile of WT animals. The analysis of mitochondria isolated from skeletal muscle demonstrated that CR increased both CoQ levels and oxygen consumption rate (OCR) based on both glucose and fatty acids substrates, along with mitochondrial mass. The elevated aerobic metabolism fits with an increase of type IIa fibers, and a reduction of type IIx in mutant muscles, reaching WT levels. To further explore the effect of CR over muscle stem cells, satellite cells were isolated and induced to differentiate in culture media containing serum from animals in either ad libitum or CR diets for 72 h. Mutant cells showed slower differentiation alongside with decreased oxygen consumption. In vitro differentiation of mutant cells was increased under CR serum reaching levels of WT isolated cells, recovering respiration measured by OCR and partially beta-oxidation of fatty acids. The overall increase of skeletal muscle bioenergetics following CR intervention is paralleled with a physical activity improvement, with some increases in two and four limbs strength tests, and weights strength test. Running wheel activity was also partially improved in mutant mice under CR. These results demonstrate that CR intervention, which has been shown to improve age-associated physical and metabolic decline in WT mice, also recovers the defective aerobic metabolism and differentiation of skeletal muscle in mice caused by ADCK2 haploinsufficiency., Competing Interests: IN-E is employed by Atsena Therapeutics, Durham, NC, United States. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hernández-Camacho, Fernández-Ayala, Vicente-García, Navas-Enamorado, López-Lluch, Oliva, Artuch, Garcia-Villoria, Ribes, de Cabo, Carvajal and Navas.)

Pathogenic mutations in NDUFAF4 have been reported in very few cases. Here we present new data to further delineate the phenotypic spectrum of NDUFAF4 deficiency. We describe two siblings presenting with facial dysmorphia and lactic acidosis in the neonatal period. Later on, they developed fatal early encephalopathy with apneic episodes, irritability, central hypoventilation, liver involvement and hyperammonemia. Abnormality of the cerebral white matter was demonstrated in one case, and cardiomyopathy in the other. Urine organic acid profile showed an increased excretion of lactate, Krebs cycle metabolites and 3-methylglutaconate. Whole-exome sequencing identified a novel homozygous nonsense mutation in NDUFAF4 (c.478G > T; p.Glu160Ter), encoding a mitochondrial complex I assembly factor. The disruptive effect of the mutation was corroborated by the absence of NDUFAF4 expression in patient fibroblasts. OXPHOS assembly studies demonstrated almost undetectable levels of fully assembled complex I and complex I-containing supercomplexes and an abnormal accumulation of SCIII 2 IV 1 supercomplexes. Morphologically, fibroblasts showed rounder mitochondria and a diminished degree of branching of the mitochondrial network. Cellular respiratory capacity in fibroblasts was also markedly reduced. In sum, we provide insights into the physiopathological mechanisms underlying NDUFAF4 deficiency and expand the knowledge about the clinical and biochemical spectrum of this disorder., (Copyright © 2020 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

We evaluated the coenzyme Q₁₀ (CoQ) levels in patients who were diagnosed with mitochondrial oxidative phosphorylation (OXPHOS) and non-OXPHOS disorders (n=72). Data from the 72 cases in this study revealed that 44.4% of patients showed low CoQ concentrations in either their skeletal muscle or skin fibroblasts. Our findings suggest that secondary CoQ deficiency is a common finding in OXPHOS and non-OXPHOS disorders. We hypothesize that cases of CoQ deficiency associated with OXPHOS defects could be an adaptive mechanism to maintain a balanced OXPHOS, although the mechanisms explaining these deficiencies and the pathophysiological role of secondary CoQ deficiency deserves further investigation., (Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

The analysis of acylcarnitines (AC) in plasma/serum is established as a useful test for the biochemical diagnosis and the monitoring of treatment of organic acidurias and fatty acid oxidation defects. External quality assurance (EQA) for qualitative and quantitative AC is offered by ERNDIM and CDC in dried blood spots but not in plasma/serum samples. A pilot interlaboratory comparison between 14 European laboratories was performed over 3 years using serum/plasma samples from patients with an established diagnosis of an organic aciduria or fatty acid oxidation defect. Twenty-three different samples with a short clinical description were circulated. Participants were asked to specify the method used to analyze diagnostic AC, to give quantitative data for diagnostic AC with the corresponding reference values, possible diagnosis, and advice for further investigations.Although the reference and pathological concentrations of AC varied among laboratories, elevated marker AC for propionic acidemia, isovaleric acidemia, medium-chain acyl-CoA dehydrogenase, very long-chain acyl-CoA dehydrogenase, and multiple acyl-CoA dehydrogenase deficiencies were correctly identified by all participants allowing the diagnosis of these diseases. Conversely, the increased concentrations of dicarboxylic AC were not always identified, and therefore the correct diagnosis was not reach by some participants, as exemplified in cases of malonic aciduria and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency. Misinterpretation occurred in those laboratories that used multiple-reaction monitoring acquisition mode, did not derivatize, or did not separate isomers. However, some of these laboratories suggested further analyses to clarify the diagnosis.This pilot experience highlights the importance of an EQA scheme for AC in plasma.

Thiamine transporter-2 deficiency is caused by mutations in the SLC19A3 gene. As opposed to other causes of Leigh syndrome, early administration of thiamine and biotin has a dramatic and immediate clinical effect. New biochemical markers are needed to aid in early diagnosis and timely therapeutic intervention. Thiamine derivatives were analysed by high performance liquid chromatography in 106 whole blood and 38 cerebrospinal fluid samples from paediatric controls, 16 cerebrospinal fluid samples from patients with Leigh syndrome, six of whom harboured mutations in the SLC19A3 gene, and 49 patients with other neurological disorders. Free-thiamine was remarkably reduced in the cerebrospinal fluid of five SLC19A3 patients before treatment. In contrast, free-thiamine was slightly decreased in 15.2% of patients with other neurological conditions, and above the reference range in one SLC19A3 patient on thiamine supplementation. We also observed a severe deficiency of free-thiamine and low levels of thiamine diphosphate in fibroblasts from SLC19A3 patients. Surprisingly, pyruvate dehydrogenase activity and mitochondrial substrate oxidation rates were within the control range. Thiamine derivatives normalized after the addition of thiamine to the culture medium. In conclusion, we found a profound deficiency of free-thiamine in the CSF and fibroblasts of patients with thiamine transporter-2 deficiency. Thiamine supplementation led to clinical improvement in patients early treated and restored thiamine values in fibroblasts and cerebrospinal fluid., (© The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Aminoglycoside antibiotics, such as gentamicin, may induce premature termination codon (PTC) readthrough and elude the nonsense-mediated mRNA decay mechanism. Because PTCs are frequently involved in lysosomal diseases, readthrough compounds may be useful as potential therapeutic agents. The aim of our study was to identify patients responsive to gentamicin treatment in order to be used as positive controls to further screen for other PTC readthrough compounds. With this aim, fibroblasts from 11 patients affected by 6 different lysosomal diseases carrying PTCs were treated with gentamicin. Treatment response was evaluated by measuring enzymatic activity, abnormal metabolite accumulation, mRNA expression, protein localization, and cell viability. The potential effect of readthrough was also analyzed by in silico predictions. Results showed that fibroblasts from 5/11 patients exhibited an up to 3-fold increase of enzymatic activity after gentamicin treatment. Accordingly, cell lines tested showed enhanced well-localized protein and/or increased mRNA expression levels and/or reduced metabolite accumulation. Interestingly, these cell lines also showed increased enzymatic activity after PTC124 treatment, which is a PTC readthrough-promoting compound. In conclusion, our results provide a proof-of-concept that PTCs can be effectively suppressed by readthrough drugs, with different efficiencies depending on the genetic context. The screening of new compounds with readthrough activity is a strategy that can be used to develop efficient therapies for diseases caused by PTC mutations.

Glutaric acidemia type I (GA-I) is a treatable autosomal recessive disorder of lysine, hydroxylysine, and tryptophan metabolism caused by glutaryl-CoA dehydrogenase (GCDH) deficiency. Presentation and progression of disease are variable ranging from asymptomatic carrier state to catastrophic encephalopathy. GA-I usually presents before age 18 months, usually triggered by childhood infection, with mild or severe acute encephalopathy, striatal degeneration, and movement disorder, most often acute dystonia. At a presymptomatic stage diagnosis is suggested clinically by macrocephaly, radiologically by widened Sylvian fissures and biochemically by the presence of excess 3-hydroxyglutaric acid and glutaric acid in urine. Treatment consists of lysine-restricted diet and carnitine supplementation, specific diet restrictions, as well as symptomatic and anticatabolic treatment of intercurrent illness. Presymptomatic diagnosis and treatment are essential to prognosis. We report the case of 16-year-old macrocephalic female with late-onset GA-I and unusual paucisymptomatic presentation with fainting after exercise and widespread white matter signal changes at MRI. She was compound heterozygote for a novel mutation (IVS10-2A>G) affecting splicing at GCDH and a common missense mutation (c. 1240C>T; p.Arg402Trp, R402W). Interestingly, the site of the novel mutation is the nucleotide position of a common mutation found almost exclusively in patients of Chinese/Taiwanese origin (IVS10-2A>C).

Coenzyme Q10 (CoQ10) plays a key role in the exchange of electrons in lysosomal membrane, which contributes to protons' translocation into the lumen and to the acidification of intra-lysosomal medium, which is essential for the proteolytic function of hydrolases responsible -when deficient- of a wide range of inherited lysosomal diseases such as Sanfilippo syndromes. Our aim was to evaluate whether treatment with CoQ10 or with an antioxidant cocktail (α-tocopherol, N-acetylcysteine and α-lipoic acid) were able to ameliorate the biochemical phenotype in cultured fibroblasts of Sanfilippo patients. Basal CoQ10 was analyzed in fibroblasts and Sanfilippo A patients showed decreased basal levels. However, no dysfunction in the CoQ10 biosynthesis pathways was found, revealing for the first time a secondary CoQ10 deficiency in Sanfilippo A fibroblasts. Cultured fibroblasts from five patients affected by Sanfilippo A and B diseases were treated with CoQ10 and an antioxidant cocktail. Upon CoQ10 treatment, none of the Sanfilippo A fibroblasts increased their residual enzymatic activity, but the two Sanfilippo B cell lines showed a statistically significant increase of their residual activity. The antioxidant treatment had no effect on the residual activity in all tested cell lines. Moreover, one Sanfilippo A and two Sanfilippo B fibroblasts showed a statistically significant reduction of glycosaminoglycans accumulation both, after 50 μmol/L CoQ10 and antioxidant treatment. Fibroblasts responsive to treatment enhanced their exocytosis levels. Our results are encouraging as some cellular alterations observed in Sanfilippo syndrome can be partially restored by CoQ10 or other antioxidant treatment in some patients.

3-Methylglutaconic aciduria (3-MGA-uria) is a heterogeneous group of syndromes characterized by an increased excretion of 3-methylglutaconic and 3-methylglutaric acids. Five types of 3-MGA-uria (I to V) with different clinical presentations have been described. Causative mutations in TAZ, OPA3, DNAJC19, ATP12, ATP5E, and TMEM70 have been identified. After excluding the known genetic causes of 3-MGA-uria we used exome sequencing to investigate a patient with Leigh syndrome and 3-MGA-uria. We identified a homozygous variant in SERAC1 (c.202C>T; p.Arg68*), that generates a premature stop codon at position 68 of SERAC1 protein. Western blot analysis in patient's fibroblasts showed a complete absence of SERAC1 that was consistent with the prediction of a truncated protein and supports the pathogenic role of the mutation. During the course of this project a parallel study identified mutations in SERAC1 as the genetic cause of the disease in 15 patients with MEGDEL syndrome, which was compatible with the clinical and biochemical phenotypes of the patient described here. In addition, our patient developed microcephaly and optic atrophy, two features not previously reported in MEGDEL syndrome. We highlight the usefulness of exome sequencing to reveal the genetic bases of human rare diseases even if only one affected individual is available., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: CblC deficiency produces a combination of methylmalonic aciduria (MMA) and homocystinuria (HCU), and is the most common error of cobalamin metabolism. Patients present a wide spectrum of symptoms, ranging from early severe multisystemic forms, to milder late-onset phenotypes. Cognitive and visual impairment are nearly constant. Hydroxocobalamin (OHCbl), betaine, folinic acid, levocarnitine and eventually dietary protein restriction are the main therapeutic approaches. Although early introduction of OHCbl is crucial, no standardized protocols regarding dose adaptation exist. No reports on long-term outcomes after high doses of this vitamin have been published., Methods: In this study five patients with CblC deficiency (early severe forms) were treated with high doses of OHCbl for 18 to 30months. Clinical examinations, neurological assessment, and biochemical studies (plasma total homocysteine (tHcy), amino acids, hydroxocobalamin, and methylmalonic acid in urine) were periodically performed., Results: Variable clinical and biochemical outcomes were observed in patients treated with high doses of OHCbl. The best biochemical response was observed in those children with the worse metabolic control. By contrast, those patients with a concentration of tHcy around 50μmol/l or less showed only minor changes. Clinically, a considerable improvement was observed in those patients with severe problems in communication, expressive language and behavior., Conclusions: According to our study, high OHCbl doses in CblC deficiency could have a greater benefit in those children with a prior history of suboptimal metabolic control, and also in those with severe neurological phenotypes. More specifically, we observed improvements in communication skills and behavior. These results should encourage further prospective trials to determine the optimal OHCbl regimen and to generate protocols and guidelines in this rare disorder., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Mitochondrial DNA depletion syndrome (MDS) is a clinically heterogeneous group of mitochondrial disorders characterised by a quantitative reduction of the mitochondrial DNA copy number. Three main clinical forms of MDS: myopathic, encephalomyopathic and hepatocerebral have been defined, although patients may present with other MDS associated clinical symptoms and signs that cover a wide spectrum of onset age and disease. We studied 52 paediatric individuals suspected to have MDS. These patients have been divided into three different groups, and the appropriate MDS genes have been screened according to their clinical and biochemical phenotypes. Mutational study of DGUOK, MPV17, SUCLA2, SUCLG1 and POLG allowed us to identify 3 novel mutations (c.1048G>A and c.1049G>T in SUCLA2 and c.531+4A>T in SUCLG1) and 7 already known mutations in 10 patients (8 families). Seventeen patients presented with mtDNA depletion in liver or muscle, but the cause of mtDNA depletion still remains unknown in 8 of them. When possible, we quantified mtDNA/nDNA and CS activity in the same tissue sample, providing an additional tool for the study of MDS. The ratio (mtDNA/nDNA)/CS has shed some light in the discrepant results between the mtDNA copy number and the enzymatic respiratory chain activities of some cases., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Mevalonic aciduria (MA) represents the severest form of mevalonate kinase deficiency due to recessively inherited, loss-of-function MVK mutations. MA is an early-onset disorder characterized by a marked failure to thrive, diverse neurologic symptoms, dysmorphic features, and recurrent febrile episodes. However, significant clinical differences have been reported in the few cases published to date. Here we describe 2 unrelated Spanish patients with MA, emphasizing the clinical heterogeneity observed. One patient presented with the severe classic MA phenotype due to the homozygous p.Ile-268-Thr MVK genotype, with a poor response to conventional treatments. However, the anti-interleukin 1 agent anakinra in this patient resulted in improvement in many clinical and laboratory parameters. The second patient presented with an atypical milder phenotype because of an older age at disease onset, mild neurologic symptoms, absence of febrile episodes and dysmorphic features, and moderate-to-good response to conventional treatments. The novel p.Arg-241-Cys MVK mutation, associated with the already known p.Ser-135-Leu mutation, detected in this patient expands the genetic diversity of mevalonate kinase deficiency. This atypical presentation of MA suggests that it should be included in the differential diagnosis of unclassified patients with psychomotor retardation, failure to thrive or ataxia, even in the absence of febrile episodes.

We report on ten individuals with a fatal infantile encephalopathy and/or pulmonary hypertension, leading to death before the age of 15 months. Hyperglycinemia and lactic acidosis were common findings. Glycine cleavage system and pyruvate dehydrogenase complex (PDHC) activities were low. Homozygosity mapping revealed a perfectly overlapping homozygous region of 1.24 Mb corresponding to chromosome 2 and led to the identification of a homozygous missense mutation (c.622G > T) in NFU1, which encodes a conserved protein suggested to participate in Fe-S cluster biogenesis. Nine individuals were homozygous for this mutation, whereas one was compound heterozygous for this and a splice-site (c.545 + 5G > A) mutation. The biochemical phenotype suggested an impaired activity of the Fe-S enzyme lipoic acid synthase (LAS). Direct measurement of protein-bound lipoic acid in individual tissues indeed showed marked decreases. Upon depletion of NFU1 by RNA interference in human cell culture, LAS and, in turn, PDHC activities were largely diminished. In addition, the amount of succinate dehydrogenase, but no other Fe-S proteins, was decreased. In contrast, depletion of the general Fe-S scaffold protein ISCU severely affected assembly of all tested Fe-S proteins, suggesting that NFU1 performs a specific function in mitochondrial Fe-S cluster maturation. Similar biochemical effects were observed in Saccharomyces cerevisiae upon deletion of NFU1, resulting in lower lipoylation and SDH activity. Importantly, yeast Nfu1 protein carrying the individuals' missense mutation was functionally impaired. We conclude that NFU1 functions as a late-acting maturation factor for a subset of mitochondrial Fe-S proteins., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Creatine transporter (CRTR) deficiency is one of the most frequent causes of X-linked mental retardation. The lack of an effective treatment for this disease, in contrast to creatine (Cr) biosynthesis disorders that respond to Cr monohydrate (CM), led us to analyze the efficacy of a lipophilic molecule derived from Cr, creatine ethyl ester (CEE), in fibroblasts and patients with CRTR deficiency. CM and CEE uptake studies were performed in six controls and four fibroblast cell lines from patients. We found a significant increase in Cr uptake after 72 h of incubation with CEE (500 micromol/L) in patients and control fibroblasts compared to incubation with CM. Subsequently, we assayed the clinical effect of CEE administration in four patients with CRTR deficiency. After 1 year of treatment, a lack of significant improvement in neuropsychological assessment or changes in Cr level in brain (1)H MRS was observed, and CEE was discontinued. In conclusion, this 12-month trial with CEE did not increase the brain concentration of Cr. Our in vitro data lend support to the idea of a certain passive transport of CEE in both pathological and control cells, although more lipophilic molecules or other cell systems that mimic the BBB should be used for a better approach to the in vivo system., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC) is the most frequent genetic disorder of vitamin B(12) metabolism. The aim of this work was to identify the mutational spectrum in a cohort of cblC-affected patients and the analysis of the cellular oxidative stress and apoptosis processes, in the presence or absence of vitamin B(12). The mutational spectrum includes nine previously described mutations: c.3G>A (p.M1L), c.217C>T (p.R73X), c.271dupA (p.R91KfsX14), c.331C>T (p.R111X), c.394C>T (p.R132X), c.457C>T (p.R153X), c.481C>T (p.R161X), c.565C>A (p.R189S), and c.615C>G (p.Y205X), and two novel changes, c.90G>A (p.W30X) and c.81+2T>G (IVS1+2T>G). The most frequent change was the known c.271dupA mutation, which accounts for 85% of the mutant alleles characterized in this cohort of patients. Owing to its high frequency, a real-time PCR and subsequent high-resolution melting (HRM) analysis for this mutation has been established for diagnostic purposes. All cell lines studied presented a significant increase of intracellular reactive oxygen species (ROS) content, and also a high rate of apoptosis, suggesting that elevated ROS levels might induce apoptosis in cblC patients. In addition, ROS levels decreased in hydroxocobalamin-incubated cells, indicating that cobalamin might either directly or indirectly act as a scavenger of ROS. ROS production might be considered as a phenotypic modifier in cblC patients, and cobalamin supplementation or additional antioxidant drugs might suppress apoptosis and prevent cellular damage in these patients.

We established an analytical methodology for guanidinoacetate and creatine determination by gas chromatography-mass spectrometry with the use of stable isotopes as internal standards. The method showed good precision and high sensitivity, and it requires minimal sample handling. We determined the reference values in urine and plasma. In urine both guanidinoacetate concentration and creatine/creatinine ratio decrease as age increases, but no significant differences were found in plasma. In addition, 15 patients with urea cycle defects were analysed and showed low guanidinoacetate concentrations when compared with age-matched controls. We concluded that guanidinoacetate concentration is a parameter to be considered in the follow-up of patients with urea cycle defects, and arginine should be supplemented in sufficient amounts, as the brain seems to be impermeable to creatine influx, but not to its precursor, arginine, which is needed for creatine, protein, and NO synthesis.