Synthesis and tyrosinase inhibitory properties of some novel derivatives of kojic acid.

Tyrosinase is a multifunctional oxidase that is widely distributed in nature. It is a key enzyme in melanin biosynthesis and is involved in determining the color of mammalian skin and hair. In addition it is responsible for the undesirable enzymatic browning that occurs in plant-derived foods, limiting the shelf-life of fresh-cut products with the resultant economic loss. In recent years there has been considerable interest to study the inhibitory activity of tyrosinase and a number of inhibitory compounds derived from natural sources or partly/fully synthetic have been described. However, the current conventional methods to control tyrosinase action are inadequate. Considering the significant industrial and economic impact of the inhibitors of tyrosinase, this study was set to seek new potent inhibitors of this enzyme. A series of 3-hydroxypyridine-4-one derivatives were prepared in high yield and evaluated for their inhibitory activity on tyrosinase enzyme using dopachrome method. Our results show that all synthesized compounds have inhibitory effect on tyrosinase activity for the oxidation of L-DOPA. Among compounds studied those containing two free hydroxyl group (ie Va and V'a) were more potent than their analogues with one hydroxyl group (ie Vb and V'b). Also substitution of a methyl group on position N(1) of the hydroxypyridinone ring seems to confer more inhibitory potency.

Are antioxidants helpful for disease prevention?

Free radicals are produced continuously in the cells as part of normal cellular function, however excess production might play a role in pathophysiology of many disease conditions, including cancer, Alzheimer's disease, atherosclerosis and some of the drug-induced toxicity. Many basic research studies and observational epidemiologic studies in human suggest that antioxidants can prevent oxidative damage. However, this is still a controversial issue because the results of clinical trials have been inconsistent. This article provides a brief overview of some of the diseases which are associated with free radicals, then discusses the roles of some of dietary antioxidant supplements in disease prevention, with particular reference to the findings of latest clinical trials.

Methylmalonic acidaemia: examination of genotype and biochemical data in 32 patients belonging to mut, cblA or cblB complementation group.

Methylmalonic acidaemia (MMA) is a genetic disorder caused by defects in methylmalonyl-CoA mutase or in any of the different proteins involved in the synthesis of adenosylcobalamin. The aim of this work was to examine the biochemical and clinical phenotype of 32 MMA patients according to their genotype, and to study the mutant mRNA stability by real-time PCR analysis. Using cellular and biochemical methods, we classified our patient cohort as having the MMA forms mut (n = 19), cblA (n = 9) and cblB (n = 4). All the mut (0) and some of the cblB patients had the most severe clinical and biochemical manifestations, displaying non-inducible propionate incorporation in the presence of hydroxocobalamin (OHCbl) in vitro and high plasma odd-numbered long-chain fatty acid (OLCFA) concentrations under dietary therapy. In contrast, mut (-) and cblA patients exhibited a milder phenotype with propionate incorporation enhanced by OHCbl and normal OLCFA levels under dietary therapy. No missense mutations identified in the MUT gene, including mut (0) and mut (-) changes, affected mRNA stability. A new sequence variation (c.562G>C) in the MMAA gene was identified. Most of the cblA patients carried premature termination codons (PTC) in both alleles. Interestingly, the transcripts containing the PTC mutations were insensitive to nonsense-mediated decay (NMD).

[Determination of normal acylcarnitine levels in a healthy pediatric population as a diagnostic tool in inherited errors of mitochondrial fatty acid beta-oxidation]. / Determinación de valores normales de acilcarnitinas en una población infantil sana como herramienta diagnóstica de errores hereditarios de la beta-oxidación mitocondrial de los ácidos grasos.

INTRODUCTION: Acylcarnitine measurement in blood is a useful test for the diagnosis of inherited errors of mitochondrial fatty acid beta-oxidation. However, there are few data in the literature on the reference ranges of the various acylcarnitines and on whether these reference ranges are age- or sex-dependent. OBJECTIVES: To draw attention to inherited errors of mitochondrial fatty acid beta-oxidation and to establish reference acylcarnitine values in children. PATIENTS AND METHODS: A total of 309 blood samples from healthy children divided into four age groups (group A: <1 month; group B: 1-12 months; group C: 1-7 years; group D: 7-18 years) were obtained and analyzed using tandem mass spectrometry. RESULTS AND CONCLUSION: Reference acylcarnitine values in children are provided. No significant differences were found in relation to age or sex. Our results differ from those reported in the literature reviewed. Importantly, hydroxyacylcarnitines and glutaryl carnitine are absent when normal samples are processed. We review the literature on the main clinical and laboratory findings in mitochondrial fatty acid beta-oxidation deficiencies.

Determinación de valores normales de acilcarnitinas en una población infantil sana como herramienta diagnóstica de errores hereditarios de la Beta-oxidación mitocondrial de los ácidos grasos / Determination of normal acylcarnitine levels in a healthy pediatric population as a diagnostic tool in inherited errors of mitochondrial fatty acid Beta-oxidation

Introducción La determinación de acilcarnitinas en sangre es una prueba útil en el diagnóstico de los errores hereditarios de la β-oxidación mitocondrial de los ácidos grasos. Sin embargo, existen pocos datos en la literatura médica, relacionados con valores de referencia para acilcarnitinas y si esos valores dependen de la edad o el sexo. Objetivos Llamar la atención acerca de los errores innatos de la β-oxidación mitocondrial de los ácidos grasos y establecer valores de referencia para acilcarnitinas en niños. Pacientes y métodos Fueron tomadas muestras de sangre de 309 niños normales divididos en cuatro grupos de edad (grupo A, < 1 mes; grupo B, 1-12 meses; grupo C, 1-7 años; grupo D, 7-18 años) y fueron analizadas por espectrometría de masas en tándem. Resultados y discusión Se aportan valores de referencia para acilcarnitinas en niños. No se encontraron diferencias estadísticamente significativas relacionadas con la edad o el sexo. Nuestros resultados son diferentes cuando se comparan con los de la literatura médica encontrada. Es importante destacar la ausencia de hidroxiacilcarnitinas y glutarilcarnitina cuando se procesan muestras normales. Revisamos la bibliografía relacionada con los principales hallazgos clínicos y de laboratorio en las deficiencias de la β-oxidación mitocondrial de los ácidos grasos

Introduction Acylcarnitine measurement in blood is a useful test for the diagnosis of inherited errors of mitochondrial fatty acid β-oxidation. However, there are few data in the literature on the reference ranges of the various acylcarnitines and on whether these reference ranges are age- or sex-dependent. Objectives To draw attention to inherited errors of mitochondrial fatty acid β-oxidation and to establish reference acylcarnitine values in children. Patients and methods A total of 309 blood samples from healthy children divided into four age groups (group A: < 1 month; group B: 1-12 months; group C: 1-7 years; group D: 7-18 years) were obtained and analyzed using tandem mass spectrometry. Results and conclusion Reference acylcarnitine values in children are provided. No significant differences were found in relation to age or sex. Our results differ from those reported in the literature reviewed. Importantly, hydroxyacylcarnitines and glutaryl carnitine are absent when normal samples are processed. We review the literature on the main clinical and laboratory findings in mitochondrial fatty acid β-oxidation deficiencies

Biochemical, clinical and molecular findings in LCHAD and general mitochondrial trifunctional protein deficiency.

General mitochondrial trifunctional protein (TFP) deficiency leads to a wide clinical spectrum of disease ranging from severe neonatal/infantile cardiomyopathy and early death to mild chronic progressive sensorimotor poly-neuropathy with episodic rhabdomyolysis. Isolated long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency resulting from the common Glu510Gln mutation usually gives rise to a moderately severe phenotype with multiorgan involvement with high morbidity and mortality. However, isolated LCHAD deficiency can also be consistent with long-term survival in patients identified and treated from an early age. We present biochemical, clinical and mutation data in 9 patients spanning the full spectrum of disease. Fibroblast acylcarnitine profiling shows good correlation with clinical phenotype using the ratio C18(OH)/(C14(OH)+C12(OH)). This ratio shows a gradation of values, from high in four patients with severe neonatal disease (2.5+/-0.8), to low in two neuromyopathic patients (0.35, 0.2). Fibroblast fatty acid oxidation flux assays also show correlation with the patient phenotype, when expressed either as percentage residual activity with palmitate or as a ratio of percentage activity of myristate/oleate (M/O ratio). Fibroblasts from four patients with severe neonatal disease gave an M/O ratio of 4.0+/-0.6 compared to 1.97 and 1.62 in two neuromyopathic patients. Specific enzyme assay of LCHAD and long-chain 3-ketothiolase activity in patient cells shows lack of correlation with phenotype. These results show that measurements in intact cells, which allow all determinative and modifying cellular factors to be present, better reflect patient phenotype. Mutation analysis reveals a number of alpha- and beta-subunit mutations. Peripheral sensorimotor polyneuropathy, often as the initial major presenting feature but usually later accompanied by episodic rhabdomyolysis, is a manifestation of mild TFP protein deficiency. The mild clinical presentation and relative difficulty in diagnosis suggest that this form of TFP is probably underdiagnosed.

[Early diagnosis of neurometabolic diseases by tandem mass spectrometry. Acylcarnitine profile from cord blood]. / Diagnóstico temprano de enfermedades neurometabólicas por espectrometría de masas en tándem. Perfil de acilcarnitinas en la sangre del cordón umbilical.

INTRODUCTION: Tandem mass spectrometry (MS/MS) provides a multi-analyte technology for the detection of disorders characterised by the presence of abnormal concentrations of metabolites related to neurological deterioration. It has been recently recommended the use of this technique for early diagnosis of inherited metabolic diseases using cord blood. AIMS: To draw the attention to the inherited metabolic diseases detected by tandem mass spectrometry and to establish reference values for acylcarnitines in cord blood. PATIENTS AND METHODS: One hundred and thirty cord blood specimens from full-term and normal birth weight children (78 males and 52 females) were analysed by MS/MS. RESULTS AND CONCLUSION: Reference values for acylcarnitines from cord blood by MS/MS as a tool for the diagnosis of some neurometabolic diseases are provided. No statistical significant difference between sexes was found. We reviewed the literature related to the diagnosis of inherited metabolic diseases, with emphasis in fatty acid mitochondrial beta-oxidation using MS/MS.

Diagnóstico temprano de enfermedades neurometabólicas por espectrometría de masas en tándem. Perfil de acilcarnitinas en la sangre del cordón umbilical / Early diagnosis of neurometabolic diseases by tandem mass spectrometry. Acylcarnitine profile from cord blood

Introducción. La espectrometría de masas en tándem (MS/MS) ofrece una tecnología de análisis múltiple para la detección de trastornos caracterizados por la presencia de concentraciones anormales demetabolitos, asociada con deterioro neurológico. Recientemente, se ha recomendado el uso de esta técnica para el diagnóstico temprano de enfermedades metabólicas hereditarias, mediante el uso de la sangre del cordón umbilical. Objetivos. Llamar la atención acerca de los errores innatos del metabolismo detectados por MS/MS y establecer unos valores de referencia para acilcarnitinas en la sangre del cordón umbilical. Pacientes y métodos. Se analizaron 130 muestras de sangre del cordón umbilical de niños producto de embarazos a término y con pesos normales al nacer (78 niños y 52 niñas) mediante MS/MS. Resultados y conclusión. Se aportan valores de referencia para acilcarnitinas en la sangre del cordón umbilical por MS/MS, como herramienta para el diagnóstico temprano de algunas enfermedades neurometabólicas. No se encontraron diferencias estadísticamente significativas entre los sexos. Revisamos la bibliografía relacionada con el uso de MS/MS para el diagnóstico de errores innatos del metabolismo, y hacemos énfasis en trastornos de la b-oxidación mitocondrial de los ácidos grasos (AU)

Introduction. Tandem mass spectrometry (MS/MS) provides a multi-analyte technology for the detection of disorders characterised by the presence of abnormal concentrations of metabolites related to neurological deterioration. It has been recently recommended the use of this technique for early diagnosis of inherited metabolic diseases using cord blood. Aims. To draw the attention to the inherited metabolic diseases detected by tandem mass spectrometry and to establish reference values for acylcarnitines in cord blood. Patients and methods. One hundred and thirty cord blood specimens from full-term and normal birth weight children (78 males and 52 females) were analysed by MS/MS. Results and conclusion. Reference values for acylcarnitines from cord blood by MS/MS as a tool for the diagnosis of some neurometabolic diseases are provided. No statistical significant difference between sexes was found. We reviewed the literature related to the diagnosis of inherited metabolic diseases, with emphasis in fatty acid mitochondrial b-oxidation using MS/MS (AU)

Lipid-storage myopathy and respiratory insufficiency due to ETFQO mutations in a patient with late-onset multiple acyl-CoA dehydrogenation deficiency.

We report a patient with lipid-storage myopathy due to multiple acyl-CoA dehydrogenation deficiency (MADD). Molecular genetic analysis showed that she was compound heterozygous for mutations in the gene for electron transfer flavoprotein:ubiquinone oxidoreductase (ETFQO). Despite a good initial response to treatment, she developed respiratory insufficiency at age 14 years and has required long-term overnight ventilation. Thus, MADD is one of the few conditions that can cause a myopathy with weakness of the respiratory muscles out of proportion to the limb muscles.

Mutation and biochemical analysis in carnitine palmitoyltransferase type II (CPT II) deficiency.

Carnitine palmitoyltransferase type II (CPT II) deficiency has three basic phenotypes, late-onset muscular (mild), infantile/juvenile hepatic (intermediate) and severe neonatal. We have measured fatty acid oxidation and CPT II activity and performed mutation studies in 24 symptomatic patients representing the full clinical spectrum of disease. Severe and intermediate phenotypes show a clear correlation with biochemical indices and genetic analysis revealed causative mutations in most patients. Studies of mild phenotypes suggest a more complex interaction, with higher residual fatty acid oxidation, a wider range of CPT II activity (10-60%) but little evidence of genotype-phenotype correlation. Residual CPT II mutant protein from myopathic patients shows thermal instability at 41 degrees C. The common 'polymorphisms' V3681 and M647V are strikingly overrepresented in the myopathic patients, the implication being that they may significantly influence the manifestation of clinical disease and could therefore potentially be considered as a susceptibility variants. Among myopathic individuals, males comprised 88% of patients, suggesting increased susceptibility to clinical disease. A small number of symptomatic patients appear to have significant residual CPT II activity (42-60%) The synergistic interaction of partial deficiencies of CPT II, muscle adenosine monophosphate deaminase and possibly other enzymes of muscle energy metabolism in the aetiology of episodic myopathy deserves wider consideration.

Glutaryl-CoA dehydrogenase deficiency: region-specific analysis of organic acids and acylcarnitines in post mortem brain predicts vulnerability of the putamen.

The neurometabolic disorder glutaryl-CoA dehydrogenase (GCDH) deficiency is biochemically characterised by an accumulation of the marker metabolites 3-hydroxyglutaric acid, glutaric acid, and glutarylcarnitine. If untreated, the disease is complicated by acute encephalopathic crises, resulting in neurodegeneration of vulnerable brain regions, in particular the putamen. 3-hydroxyglutaric acid is considered the major neurotoxin in this disease. There are only preliminary data concerning glutaric acid concentrations in the brains of affected children and the distribution of 3-hydroxyglutaric acid and glutarylcarnitine has not been described. In the present study, we investigated post mortem the distribution of 3-hydroxyglutaric and glutaric acids as well as glutarylcarnitine in 14 different brain regions, internal organs, and body fluids (urine, plasma, cerebrospinal fluid) in a 14-year-old boy. 3-Hydroxyglutaric acid showed the highest concentration (62 nmol/g protein) in the putamen among all brain areas investigated. The glutarylcarnitine concentration was also highest in the putamen (7.1 nmol/g protein). We suggest that the regional-specific differences in the relative concentrations of 3-hydroxyglutaric acid contribute to the pattern of neuronal damage in this disease. These results provide an explanatory basis for the high vulnerability of the putamen in this disease, adding to the strong corticostriatal glutamatergic input into the putamen and the high excitotoxic susceptibility of neostriatal medium spiny neurons.

Carnitina libre y total en sangre de cordón umbilical / Free and total carnitine in umbilical cord blood

Se han encontrado concentraciones anormales de carnitina en niños asociadas con deficiencias primaria y secundaria de carnitina, con disminución de las concentraciones en sangre y tejidos, así como niveles elevados en algunos errores innatos del metabolismo. El presente estudio tiene como objetivo la obtención de valores de referencia para carnitina libre y total en sangre de cordón umbilical, como herramienta en el diagnóstico temprano de errores innatos del metabolismo. Se analizaron mediante espectrometría de masas en tándem (MS/MS) 130 muestras de sangre de cordón umbilical de nacimientos con embarazos a término y peso normal al nacer (78 niños y 52 niñas) de una población colombiana. No se encontró diferencia estadísticamente significativa dependiendo del sexo del recién nacido. Reportamos valores de referencia para carnitina libre y total en sangre de cordón umbilical de 18,5+/-3,4 micromol/L y 20,9 +/- micromol/L respectivamente.

Plasma free and total carnitine measured in children by tandem mass spectrometry.

Free and total carnitine quantification is important as a complementary test for the diagnosis of unusual metabolic diseases, including fatty acid degradation disorders. The present study reports a new method for the quantification of free and total carnitine in dried plasma specimens by isotope dilution electrospray tandem mass spectrometry with sample derivatization. Carnitine is determined by looking for the precursor of ions of m/z = 103 of N-butylester derivative, and the method is validated by comparison with radioenzymatic assay. We obtained an inter- and intra-day assay coefficient of variation of 4.3 and 2.3, respectively. Free and total carnitine was analyzed in 309 dried plasma spot samples from children ranging in age from newborn to 14 years using the new method, which was found to be suitable for calculating reference age-related values for free and total carnitine (less than one month: 19.3 +/- 2.4 and 23.5 +/- 2.9; one to twelve months: 28.8 +/- 10.2 and 35.9 +/- 11.4; one to seven years: 30.7 +/- 10.3 and 38.1 +/- 11.9; seven to 14 years: 33.7 +/- 11.6, and 43.1 +/- 13.8 micro M, respectively). No difference was found between males and females. A significant difference was observed between neonates and the other age groups. We compare our data with reference values in the literature, most of them obtained by radioenzymatic assay. However, this method is laborious and time consuming. The electrospray tandem mass spectrometry method presented here is a reliable, rapid and automated procedure for carnitine quantitation.

Plasma free and total carnitine measured in children by tandem mass spectrometry

Free and total carnitine quantification is important as a complementary test for the diagnosis of unusual metabolic diseases, including fatty acid degradation disorders. The present study reports a new method for the quantification of free and total carnitine in dried plasma specimens by isotope dilution electrospray tandem mass spectrometry with sample derivatization. Carnitine is determined by looking for the precursor of ions of m/z = 103 of N-butylester derivative, and the method is validated by comparison with radioenzymatic assay. We obtained an inter- and intra-day assay coefficient of variation of 4.3 and 2.3, respectively. Free and total carnitine was analyzed in 309 dried plasma spot samples from children ranging in age from newborn to 14 years using the new method, which was found to be suitable for calculating reference age-related values for free and total carnitine (less than one month: 19.3 ± 2.4 and 23.5 ± 2.9; one to twelve months: 28.8 ± 10.2 and 35.9 ± 11.4; one to seven years: 30.7 ± 10.3 and 38.1 ± 11.9; seven to 14 years: 33.7 ± 11.6, and 43.1 ± 13.8 æM, respectively). No difference was found between males and females. A significant difference was observed between neonates and the other age groups. We compare our data with reference values in the literature, most of them obtained by radioenzymatic assay. However, this method is laborious and time consuming. The electrospray tandem mass spectrometry method presented here is a reliable, rapid and automated procedure for carnitine quantitation

Neonatal screening for medium-chain acyl-CoA dehydrogenase deficiency.

Neonatal screening for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency has not yet been introduced in the UK, primarily because of uncertainty about the natural history of the disorder and concerns about the specificity of the screening test. To obtain data on these issues, we did a retrospective study in which we analysed the concentrations of acylcarnitines in stored neonatal blood spots, and reviewed patients with high octanoylcarnitine concentrations at age 7-9 years. The high morbidity and mortality associated with the disorder, and the specificity of acylcarnitine analysis seen in our study support the introduction of screening for MCAD deficiency.

Plasma coenzyme Q(10) in children and adolescents undergoing doxorubicin therapy.

The objective of this study was to test the hypothesis that doxorubicin treatment for cancer in childhood and adolescence causes a dose-related decrease in the concentration of plasma coenzyme Q(10). The concentration of plasma coenzyme Q(10) was measured before and after administration of doxorubicin in six patients, and before and after chemotherapy in six patients undergoing treatments that did not include doxorubicin. There was a significant increase in the concentration of plasma coenzyme Q(10) in post-treatment samples compared to pre-treatment samples in patients treated with doxorubicin (P=0.008; n=32), whereas there were no significant changes in plasma coenzyme Q(10) concentrations in patients treated with chemotherapy that did not include doxorubicin. (P=0.770; n=30). We hypothesise that the increase in plasma coenzyme Q(10) that was observed in patients undergoing doxorubicin treatment is due to release of coenzyme Q(10) from apoptotic or necrotic cardiac tissue. We conclude that the cardiotoxicity due to doxorubicin therapy does not involve acute myocardial depletion of coenzyme Q(10).

[Diagnostic error of mental retardation of neurometabolic origin confirmed by mass sequential spectrometry]. / Error diagnóstico de retraso mental de origen neurometabólico confirmado por espectrometría de masa secuencial.

INTRODUCTION: The metabolic screening test gives the first laboratory indication for neurometabolic alterations which can cause mental retardation. Some techniques such as thin layer chromatography, are still used in several countries to confirm the diagnosis of inborn errors of metabolism after a general screening test. PATIENTS AND METHODS: Two patients from a mentally retarded Colombian population were reported positive for the Nitrosonaphtol test, and remained positive to tyrosine metabolism alteration by thin layer chromatography, suggesting the correspondent management. In the present study we tried to confirm the last diagnosis, performing tandem mass spectrometry analysis of acylcarnitines and amino acids, on blood samples of all patients from the last study, which were found negative for any alteration. CONCLUSION: Is necessary to improve the diagnosis methods used in some countries in order to avoid mistakes that can change the life-style of the wrongly diagnosed patients.

The mitochondrial trifunctional protein: centre of a beta-oxidation metabolon?

The trifunctional enzyme comprises three consecutive steps in the mitochondrial beta-oxidation of long-chain acyl-CoA esters: 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase. Deficiencies in either 3-hydroxyacyl-CoA dehydrogenase activity, or all three activities, are important causes of human disease. The dehydrogenase and thiolase have a requirement for NAD+ and CoA respectively, whose levels are conserved within the mitochondrion and thus provide possible means for control and regulation of beta-oxidation. Using analysis of the intact CoA ester intermediates produced by the complex, we have examined the sensitivity of the complex to NAD+/NADH and acetyl-CoA. We consider the evidence for channelling within the trifunctional protein and propose a model for a beta-oxidation 'metabolon'.

Error diagnóstico de retraso mental de origen neurometabólico confirmado por espectrometría de masa secuencial / Diagnostic error of mental retardation of neurometabolic origin confirmed by mass sequential spectrometry

Introducción. El tamizaje metabólico general aporta los primeros indicios de laboratorio para alteraciones neurometabólicas que pueden causar retraso mental. Algunas técnicas, como la cromatografía en capa fina, todavía se utilizan en diferentes países para confirmar el diagnóstico de errores innatos del metabolismo después de dicho tamizaje. Pacientes y métodos. Dos pacientes de una población colombiana con retraso mental dieron positivo en la prueba de nitrosonaftol. Igual resultado se obtuvo al realizar posteriormente cromatografía en capa fina para verificar si existían alteraciones en el metabolismo de la tirosina, sugiriéndose el diagnóstico respectivo. En el presente estudio tratamos de confirmar el diagnóstico anterior mediante espectrometría de masa secuencial de acilcarnitinas y aminoácidos, en muestras de sangre de todos los pacientes estudiados en una investigación anterior. Dichos resultados fueron negativos pues no se observó alteración alguna. Conclusión. Es necesario mejorar los métodos diagnósticos empleados en algunos países para tratar de evitar errores que puedan cambiar el estilo de vida de los pacientes positivos falsos (AU)

Introduction. The metabolic screening test gives the first laboratory indication for neurometabolic alterations which can cause mental retardation. Some techniques such as thin layer chromatography, are still used in several countries to confirm the diagnosis of inborn errors of metabolism after a general screening test. Patients and methods. Two patients from a mentally retarded Colombian population were reported positive for the Nitrosonaphtol test, and remained positive to tyrosine metabolism alteration by thin layer chromatography, suggesting the correspondent management. In the present study we tried to confirm the last diagnosis, performing tandem mass spectrometry analysis of acylcarnitines and amino acids, on blood samples of all patients from the last study, which were found negative for any alteration. Conclusion. Is necessary to improve the diagnosis methods used in some countries in order to avoid mistakes that can change the life-style of the wrongly diagnosed patient (AU)