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Karnataka and Tamil Nadu have been clashing over the fair distribution of water for over a hundred years. In recent years, this conflict has translated into heated conversations in social media platforms.

3-Methylglutaconic aciduria (3-MGA-uria) syndromes comprise a heterogeneous group of diseases associated with mitochondrial membrane defects. Whole-exome sequencing identified compound heterozygous mutations in TIMM50 (c.[341 G>A];[805 G>A]) in a boy with West syndrome, optic atrophy, neutropenia, cardiomyopathy, Leigh syndrome, and persistent 3-MGA-uria. A comprehensive analysis of the mitochondrial function was performed in fibroblasts of the patient to elucidate the molecular basis of the disease. TIMM50 protein was severely reduced in the patient fibroblasts, regardless of the normal mRNA levels, suggesting that the mutated residues might be important for TIMM50 protein stability. Severe morphological defects and ultrastructural abnormalities with aberrant mitochondrial cristae organization in muscle and fibroblasts were found. The levels of fully assembled OXPHOS complexes and supercomplexes were strongly reduced in fibroblasts from this patient. High-resolution respirometry demonstrated a significant reduction of the maximum respiratory capacity. A TIMM50-deficient HEK293T cell line that we generated using CRISPR/Cas9 mimicked the respiratory defect observed in the patient fibroblasts; notably, this defect was rescued by transfection with a plasmid encoding the TIMM50 wild-type protein. In summary, we demonstrated that TIMM50 deficiency causes a severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology, such as the maintenance of proper mitochondrial morphology, OXPHOS assembly, and mitochondrial respiratory capacity.

Miren Gutierrez. Cham, Switzerland: Palgrave Macmillan, 2018. xiii and 176 pp., figures, tables, glossary, bibliography, index. $54.99 cloth (ISBN 978-3-319-78318-5); $39.99 electronic (ISBN 978-3-...

Thiamine is a key cofactor for energy metabolism in brain tissue. There are four major genetic defects (SLC19A2, SLC19A3, SLC25A19 and TPK1) involved in the metabolism and transport of thiamine through cellular and mitochondrial membranes. Neurological involvement predominates in three of them (SLC19A3, SCL25A19 and TPK1), whereas patients with SLC19A2 mutations mainly present extra-neurological features (e.g. diabetes mellitus, megaloblastic anaemia and sensori-neural hearing loss). These genetic defects may be amenable to therapeutic intervention with vitamins supplementation and hence, constitutes a main area of research.We conducted a literature review of all reported cases with these genetic defects, and focused our paper on treatment efficacy and safety, adverse effects, dosing and treatment monitoring. Expert commentary: Doses of thiamine vary according to the genetic defect: for SLC19A2, the usual dose is 25-200 mg/day (1-4 mg/kg per day), for SLC19A3, 10-40 mg/kg per day, and for TPK1, 30 mg/kg per day. Thiamine supplementation in SLC19A3-mutated patients restores CSF and intracellular thiamine levels, resulting in successful clinical benefits. In conclusion, evidence collected so far suggests that the administration of thiamine improves outcome in SLC19A-2, SLC19A3- and TPK1-mutated patients, so most efforts should be aimed at early diagnosis of these disorders.

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.

Lysosomal acid lipase (LAL) is a lysosomal key enzyme involved in the intracellular hydrolysis of cholesteryl esters and triglycerides. Patients with very low residual LAL activity present with the infantile severe form Wolman disease (WD), while patients with some residual activity develop the less severe disorder known as Cholesteryl ester storage disorder (CESD). We present the clinical, biochemical, and molecular findings of 23 Spanish patients (22 families) with LAL deficiency. We identified eight different mutations, four of them not previously reported. The novel c.966+2T>G mutation accounted for 75% of the Wolman disease alleles, and the frequent CESD associated c.894G>A mutation accounted for 55% of the CESD alleles in our cohort. Haplotype analysis showed that both mutations co-segregated with a unique haplotype suggesting a common ancestor. Our study contributes to the LAL deficiency acknowledgement with novel mutations and with high frequencies of some unknown mutations for WD.

CoQ deficiency study group: et al., 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., This work was supported by grants from the Instituto de Salud Carlos III (FIS: PI12/01683, PI14/00005, PI14/00028 and PI14/01962), the Explora Ciencia Program (SAF2013-50139-EXP), the Departamento de Ciencia, Tecnología, from Universidad del Gobierno de Aragón (Grupos Consolidados B33), the Fondo Europeo de Desarrollo Regional (FEDER Funding Program) from the European Union. The CIBERER is an initiative of the ISCIII. UCLH/UCL received a proportion of funding from the Department of Health sNIHR Biomedical Research Centers funding scheme.

Resumen Fundamento y objetivo Los sindromes de deficiencia cerebral de creatina (Cr) constituyen un grupo de enfermedades neurometabolicas caracterizadas por deficiencia o ausencia de Cr en el cerebro. Cursan con retraso del desarrollo/mental y trastornos del lenguaje, y puede asociarse a epilepsia o a trastornos del movimiento. Se conocen 3 defectos: 2 de la sintesis —deficiencia de guanidinoacetato metiltransferasa (GAMT) y argininaglicina amidinotransferasa (AGAT)— y uno del transporte (CRTR). En este trabajo presentamos los 3 primeros pacientes espanoles con deficiencia de GAMT y comparamos su fenotipo clinico y respuesta al tratamiento con otros casos publicados. Pacientes y metodo Los pacientes presentan retraso mental, epilepsia y conducta autista. La paciente 1 asocia corea grave. El diagnostico se realizo mediante estudios bioquimicos para cuantificar metabolitos especificos y actividad enzimatica y geneticos del gen GAMT. Resultados En orina y plasma se detecto aumento de guanidinoacetato. La resonancia magnetica con espectroscopia revelo reduccion marcada de Cr cerebral. Los estudios enzimaticos mostraron disminucion de la actividad GAMT en fibroblastos y el estudio molecular revelo mutaciones en el gen GAMT. Tras el diagnostico, se inicio tratamiento con suplemento de Cr, y se asocio en los pacientes 2 y 3 una dieta restringida en arginina y suplemento de ornitina, con mejoria parcial. Conclusiones Los pacientes con deficiencia de GAMT presentan un fenotipo inespecifico pero relativamente constante. Deben buscarse los sindromes de deficiencia cerebral de Cr en pacientes con retraso mental/psicomotor de etiologia desconocida, especialmente si se acompanan de trastornos del movimiento y epilepsia. Es importante el diagnostico precoz en casos tratables como la deficiencia de GAMT.

Creatine transporter deficiency is a recently identified X-linked inborn error of metabolism. The natural course of the disease is not well delineated since clinical data from adult patients have scarcely been reported. A progressive course of the disease has been noted in a few described cases. We report the first two Spanish adult patients with creatine transporter deficiency and compare their clinical phenotype and the evolution of the disease with those of other published cases. The two brothers were identified in a study of a cohort of 610 mentally handicapped male patients. The disease was detected by biochemical studies and confirmed by DNA studies. The most significant clinical features were mental retardation, epilepsy and autistic behaviour, and these symptoms did not worsen, in contrast to other reports. They did not present gastrointestinal problems or movement disorders. Creatine transporter deficiency could be an underdiagnosed metabolic disorder and should be considered in adult patients with mental retardation. Clinical presentation of this disorder showed marked differences among adult patients and the course of the disease was static in our cases. Detection of additional adult patients might allow better understanding of the phenotypic outcome at a later age.

Mutations in NFU1 were recently identified in patients with fatal encephalopathy. NFU1 is an iron-sulfur cluster protein necessary for the activity of the mitochondrial respiratory chain complexes I-II and the synthesis of lipoic acid. We report two NFU1 compound heterozygous individuals with normal complex I and lipoic acid-dependent enzymatic activities and low, but detectable, levels of lipoylated proteins. We demonstrated a leaky splicing regulation due to a splice site mutation (c.545+5G>A) that produces small amounts of wild type NFU1 mRNA that might result in enough protein to partially lipoylate and restore the activity of lipoic acid-dependent enzymes and the assembly and activity of complex I. These results allowed us to gain insights into the molecular basis underlying this disease and should be considered for the diagnosis of NFU1 patients.

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.

Cofactor disorders of mitochondrial energy metabolism are a heterogeneous group of diseases with a wide variety of clinical symptoms, particular metabolic profiles and variable enzymatic defects. Mutations in NFU1, BOLA3, LIAS and IBA57 have been identified in patients with deficient lipoic acid-dependent enzymatic activities and defects in the assembly and activity of the mitochondrial respiratory chain complexes. Here, we report a patient with an early onset fatal lactic acidosis presenting a biochemical phenotype compatible with a combined defect of pyruvate dehydrogenase (PDHC) and 2-ketoglutarate dehydrogenase (2-KGDH) activities, which suggested a deficiency in lipoic acid metabolism. Immunostaining analysis showed that lipoylated E2-PDH and E2-KGDH were extremely reduced in this patient. However, the absence of glycine elevation, the normal activity of the glycine cleavage system and the normal lipoylation of the H protein suggested a defect of lipoic acid transfer to particular proteins rather than a general impairment of lipoic acid biosynthesis as the potential cause of the disease. By analogy with yeast metabolism, we postulated LIPT1 as the altered candidate gene causing the disease. Sequence analysis of the human LIPT1 identified two heterozygous missense mutations (c.212C>T and c.292C>G), segregating in different alleles. Functional complementation experiments in patient's fibroblasts demonstrated that these mutations are disease-causing and that LIPT1 protein is required for lipoylation and activation of 2-ketoacid dehydrogenases in humans. These findings expand the spectrum of genetic defects associated with lipoic acid metabolism and provide the first evidence of a lipoic acid transfer defect in humans.

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.

Item does not contain fulltext BACKGROUND: Creatine transporter deficiency is a monogenic cause of X-linked intellectual disability. Since its first description in 2001 several case reports have been published but an overview of phenotype, genotype and phenotype-genotype correlation has been lacking. METHODS: We performed a retrospective study of clinical, biochemical and molecular genetic data of 101 males with X-linked creatine transporter deficiency from 85 families with a pathogenic mutation in the creatine transporter gene (SLC6A8). RESULTS AND CONCLUSIONS: Most patients developed moderate to severe intellectual disability; mild intellectual disability was rare in adult patients. Speech language development was especially delayed but almost a third of the patients were able to speak in sentences. Besides behavioural problems and seizures, mild to moderate motor dysfunction, including extrapyramidal movement abnormalities, and gastrointestinal problems were frequent clinical features. Urinary creatine to creatinine ratio proved to be a reliable screening method besides MR spectroscopy, molecular genetic testing and creatine uptake studies, allowing definition of diagnostic guidelines. A third of patients had a de novo mutation in the SLC6A8 gene. Mothers with an affected son with a de novo mutation should be counselled about a recurrence risk in further pregnancies due to the possibility of low level somatic or germline mosaicism. Missense mutations with residual activity might be associated with a milder phenotype and large deletions extending beyond the 3' end of the SLC6A8 gene with a more severe phenotype. Evaluation of the biochemical phenotype revealed unexpected high creatine levels in cerebrospinal fluid suggesting that the brain is able to synthesise creatine and that the cerebral creatine deficiency is caused by a defect in the reuptake of creatine within the neurones.

Recent investigations have suggested creatine (Cr) as an additional biomarker of mitochondrial diseases. With the aim of corroborating previous findings, we have studied plasma Cr in a cohort of 33 patients with different mitochondrial diseases. Cr was clearly increased in 9 out of 33 patients. Therefore, positive patients represent only 28% of the total number, suggesting that Cr is not a sensitive biomarker of mitochondrial diseases although it does present an acceptable specificity (83%). High plasma Cr, together with other biomarkers, might be useful to reinforce the diagnosis of mitochondrial diseases.

Primary coenzyme Q₁₀ (CoQ₁₀) deficiencies are associated with mutations in genes encoding enzymes important for its biosynthesis and patients are responsive to CoQ₁₀ supplementation. Early treatment allows better prognosis of the disease and therefore, early diagnosis is desirable. The complex phenotype and genotype and the frequent secondary CoQ₁₀ deficiencies make it difficult to achieve a definitive diagnosis by direct quantification of CoQ₁₀. We developed a non-radioactive methodology for the quantification of CoQ₁₀ biosynthesis in fibroblasts that allows the identification of primary deficiencies. Fibroblasts were incubated 72 h with 28 μmol/L (2)H₃-mevalonate or 1.65 mmol/L (13)C₆-p-hydroxybenzoate. The newly synthesized (2)H₃- and (13)C₆- labelled CoQ₁₀ were analysed by high performance liquid chromatography-tandem mass spectrometry. The mean and the reference range for (13)C₆-CoQ₁₀ and (2)H₃-CoQ₁₀ biosynthesis were 0.97 (0.83-1.1) and 0.13 (0.09-0.17) nmol/Unit of citrate synthase, respectively. We validated the methodology through the study of one patient with COQ2 mutations and six patients with CoQ₁₀ deficiency secondary to other inborn errors of metabolism. Afterwards we investigated 16 patients' fibroblasts and nine showed decreased CoQ₁₀ biosynthesis. Therefore, the next step is to study the COQ genes in order to reach a definitive diagnosis in these nine patients. In the patients with normal rates the deficiency is probably secondary. In conclusion, we have developed a non-invasive non-radioactive method suitable for the detection of defects in CoQ₁₀ biosynthesis, which offers a good tool for the stratification of patients with these treatable mitochondrial diseases.

Background Cystinosis is an autosomal recessive genetic disorder due to mutations in CTNS gene, which causes a defect of cystinosin, impairing the transport of free cystine out of lysosomes and causing irreversible damage to various organs, particularly the kidney. The diagnosis of Cystinosis is carried out by measuring the cystine content in leucocytes. Accurate quantification of cystine is of capital importance not only for the diagnosis, but also for monitoring the effectiveness of cysteamine treatment. Here we describe an improvement to measure cystine in granulocytes using high-pressure-liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS). Method Granulocytes were isolated from heparinized blood by isopycnic centrifugation. After lysis and deproteinization, the concentration of di-butylated cystine was measured by HPLC-ESI-MS/MS, using deuterium labelled, d6-cystine, as internal standard. Results The assay was linear to at least 50 μmol/L with a good precision. Within-day and between-day coefficients of variation were about 6%. The recovery was higher than 98%. Control values were clearly distinguishable from pathological levels, even if patients were under treatment. A good correlation was observed with cystine binding protein (CBP) assay, one of the most sensitive and specific methods. Conclusion This method results in good analytical performance, and is useful for diagnosis and follow up of Cystinosis. This method offers several advantages over the CBP assay: it is less expensive, easier, quicker and it does not require radioactivity. In addition, when comparing with the HPLC-ESI-MS/MS method previously described by Chabli et al. 2006, our assay exhibits more sensitivity and is faster.

This work examined nine patients with creatine deficiency syndrome (CDS): six with a creatine transport (CRTR) defect and three with a GAMT defect. Eleven nucleotide variations were detected: six in SLC6A8 and five in GAMT. These changes were analyzed at the mRNA level and specific alleles (most of which bore premature stop codons) were selected as nulls because they provoked nonsense-mediated decay activation. The impact of these CDS mutations on metabolic stress (ROS production, p38MAPK activation, aberrant proliferation and apoptosis) was analyzed in patient fibroblast cultures. Oxidative stress contributed toward the severe form of CDS, with increases seen in the intracellular ROS content and the percentage of apoptotic cells. An altered cell cycle was also seen in a number of CRTR and GAMT fibroblast cell lines (mostly those carrying null alleles). p38MAPK activation only correlated with oxidative stress in the CRTR cells. Based on intracellular creatine levels, the contribution of energy depletion toward metabolic stress was demonstrable only in selected CRTR cells. Together, these findings suggest that the apoptotic response to genotoxic damage in the present CDS cells may have been triggered by different cell signaling pathways. They also suggest that reducing oxidative stress could be helpful in treating CDS. Hum Mutat 32:1-10, 2011. © 2011 Wiley-Liss, Inc.

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.

Brain creatine (Cr) deficiencies are a group of inborn errors of metabolism that are characterized by an absence or severe reduction of brain Cr. Clinically, these patients can display psychomotor/mental retardation and language disorders, commonly associated with epilepsy or movement disorders. Three metabolic defects are known: two affect synthesis - guanidinoacetate metiltransferase (GAMT) and glycine amidinotransferase (AGAT) deficiencies- and one affect the Cr transporter (CRTR). We present the first three Spanish patients with GAMT deficiency, and we compare their clinical phenotype and treatment response with other published cases.The three patients presented mental retardation, epilepsy and autistic behaviour. Patient 1 also had severe chorea. Diagnosis was done by biochemical and genetic procedures (guanidinoacetate quantification, determination of GAMT activity and mutation analysis in the GAMT gene).An increase of guanidinoacetate was detected in urine and plasma. Brain magnetic resonance spectroscopy revealed low Cr levels. Enzymatic studies revealed a decreased GAMT activity in fibroblasts. Molecular analysis detected pathogenic mutations in the GAMT gene. After the deficiency was confirmed, the patients started treatment with Cr. In addition, patient 2 and 3 received an arginine-restricted diet and ornithine supplements. All them showed a partial improvement.Patients with GAMT deficiency have an unspecific but relatively constant clinical presentation. Brain Cr deficiency should be considered in patients with mental retardation of unknown aetiology, specially in those with movement disorders or epilepsy. Early diagnosis is important in cases with known treatment such as GAMT deficiency.

Objectives To report the prevalence of creatine transporter deficiency in males with mental retardation and to study whether a protein-rich food intake might be a potential diagnostic pitfall. Design and methods We determined creatine/creatinine ratio in urine samples from 1600 unrelated male patients with mental retardation and/or autism. Urine creatine was analyzed by HPLC-MS/MS. Results Thirty-three of 1600 cases showed increased urine creatine/creatinine ratio. Four out of these thirty-three cases were definitively diagnosed with creatine transporter deficiency, while the other 29 were false positive results. Significantly higher values were observed for urine Cr/Crn ratio in healthy volunteers after a meal based on beef or oily fish as compared to eggs, pasta or salad (Wilcoxon test: p Conclusions False positive results may be observed in biochemical screening for creatine transporter deficiency, and they may be due to intake of meals rich in creatine prior to urine samples analysis.

The increasing number of patients with creatine deficiency syndromes (CDS) stresses the need to develop screening procedures for the identification these inherited disorders. Guanidinoacetate (GAA) and creatine (Cr) are reliable biochemical markers of CDS and several analytical methods to measure both metabolites have been developed. High-pressure liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) is quick and sensitive but, unlike HPLC and gas chromatography–mass spectrometry (GC/MS), it is unavailable in most laboratories. Thus, we decided to evaluate comparatively HPLC–MS/MS, GC/MS and HPLC methods, as well as to establish reference values in a healthy paediatric population. According to our results, these three methods may be suitable for analysing GAA in urine. Furthermore, Passing-Bablock plots showed good agreement among all three. However, when comparing the Cr/Crn ratio, our results revealed that while HPLC–MS/MS data were in agreement with those of GC/MS, a constant and proportional error was observed when compared with those of HPLC. Consequently, the Cr/Crn ratio obtained by the last method should be evaluated with caution. Our reference values for GAA and Cr/Crn ratio in urine negatively correlate with age. Concerning GAA and Cr measurements in plasma, it is interesting to note that in contrast to what was occurring in urine, GAA concentration increased significantly with age, while we did not find any significant difference for Cr values within the same age group.

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.

The purpose of this study was to investigate and to compare the frequency and possible aetiology of subclinical hypothyroidism (SH) in healthy and sick children developing neurological disorder changes. One hundred and eighty-seven male and female children between 1 month and 4 years old, 64 with and 123 without neurological disorders, were studied in the state of Mérida, Venezuela. Serum levels of thyrotropin (TSH), free thyroxine (FT4) and urinary iodine were measured by immunofluorescence and by the Sandell-Koltoff's method. Children were diagnosed as having SH if they had high levels of TSH and normal levels of FT4. Antithyroglobulin and antiperoxidase antibodies were measured in children with SH. To establish the frequency of SH, the TSH reference levels on the commercial kit (3.8 uU/mL) and the TSH reference levels of our group of healthy children were used. The latter was calculated (X + 2SD) from the values of TSH in the 123 healthy children (4.98 uU/mL). The frequency of SH in all children was of 15% when the TSH level from the commercial kit was used and of 6.4% when it was of our group of children (p0.001). The frequency of SH value was of 17.2% in children with neurological disorders and of 13.8% in healthy children when the commercial kit's TSH level was used. The frequency of SH in children with neurological disorders was of 7.8% and of 5.7% in healthy children, according to our TSH reference level. This difference was not statistically significant. No significant differences were found in the urinary iodine levels or in the presence of thyroid autoantibodies among the two groups of children with or without SH. There was no association between iodine urinary levels, presence of SH and neurological disorders. We conclude that: 1. In order to make suitable SH level diagnosis in children, we must establish our own levels of reference for TSH. 2. The frequency of SH in children from Mérida state is high, being slightly higher in children with neurological disorders. 3. The aetiology of SH is not due to iodine deficiency or immunological factors.

The incidence of hypothyroidism is higher among children with Down syndrome than among children in the general population. The frequency of hypothyroidism is higher in the areas of endemic goiter than in other areas. The aim of this paper was to study the concentrations of TSH and FT4 in children with Down syndrome residents of Mérida, a region of Venezuelan Andes. At the Centro de Estudio y Prevención del Retardo Mental y Alteraciones en el Desarrollo (CEPREMAD), the thyroid function was studied in 48 children (1 month to 6 years old), who had Down syndrome, and in 123 healthy children of similar ages. All the children were referred to the Center for thyroid function evaluation. Two (4.2%) of the 48 children with Down syndrome had congenital hypothyroidism and 22 (45.8%) had subclinical hypothyroidism (high concentration of thyrotropin-TSH). Among the control children, only 14% had elevated levels of TSH. There were no differences in relation to the gender. In conclusion in children with Down syndrome, the frequency of high concentrations of TSH was three times higher than the frequency among the healthy children. The frequency of hypothyroidism was similar to that found in areas without endemic goiter.