Normal vision and development in mice with low functional expression of Kir7.1 in heterozygosis for a blindness-producing mutation inactivating the channel.

K+ channel Kir7.1 expressed at the apical membrane of the retinal pigment epithelium (RPE) plays an essential role in retinal function. An isoleucine-to-threonine mutation at position 120 of the protein is responsible for blindness-causing vitreo-retinal dystrophy. We have studied the molecular mechanism of action of Kir7.1-I120T in vitro by heterologous expression and in vivo in CRISPR-generated knockin mice. Full-size Kir7.1-I120T reaches the plasma membrane but lacks any activity. Analysis of Kir7.1 and the I120T mutant in mixed transfection experiments, and that of tandem tetrameric constructs made by combining wild type (WT) and mutant protomers, leads us to conclude that they do not form heterotetramers in vitro. Homozygous I120T/I120T mice show cleft palate and tracheomalacia and do not survive beyond P0, whereas heterozygous WT/I120T develop normally. Membrane conductance of RPE cells isolated from WT/WT and heterozygous WT/I120T mice is dominated by Kir7.1 current. Using Rb+ as a charge carrier, we demonstrate that the Kir7.1 current of WT/I120T RPE cells corresponds to approximately 50% of that in cells from WT/WT animals, in direct proportion to WT gene dosage. This suggests a lack of compensatory effects or interference from the mutated allele product, an interpretation consistent with results obtained using WT/- hemizygous mouse. Electroretinography and behavioral tests also show normal vision in WT/I120T animals. The hypomorphic ion channel phenotype of heterozygous Kir7.1-I120T mutants is therefore compatible with normal development and retinal function. The lack of detrimental effect of this degree of functional deficit might explain the recessive nature of Kir7.1 mutations causing human eye disease.NEW & NOTEWORTHY Human retinal pigment epithelium K+ channel Kir7.1 is affected by generally recessive mutations leading to blindness. We investigate one such mutation, isoleucine-to-threonine at position 120, both in vitro and in vivo in knockin mice. The mutated channel is inactive and in heterozygosis gives a hypomorphic phenotype with normal retinal function. Mutant channels do not interfere with wild-type Kir7.1 channels which are expressed concomitantly without hindrance, providing an explanation for the recessive nature of the disease.

Branched chain amino acids catabolism as a source of new drug targets in pathogenic protists.

Leucine, isoleucine, and valine, collectively termed Branched Chain Amino Acids (BCAA), are hydrophobic amino acids (AAs) and are essential for most eukaryotes since in these organisms they cannot be biosynthesized and must be supplied by the diet. These AAs are structurally relevant for muscle cells and, of course, important for the protein synthesis process. The metabolism of BCAA and its participation in different biological processes in mammals have been relatively well described. However, for other organisms as pathogenic parasites, the literature is really scarce. Here we review the BCAA catabolism, compile evidence on their relevance for pathogenic eukaryotes with special emphasis on kinetoplastids and highlight unique aspects of this underrated pathway.

Prediction of semen analysis parameter improvement after varicocoelectomy using 1 H NMR-based metabonomics assays.

BACKGROUND: Varicocoele is the most common correctable cause of male infertility; however, predicting varicocoelectomy outcomes is difficult. "Omics" techniques have been increasingly used to develop new diagnostic and prognostics tools for several male infertility causes, and could be applied to study varicocoele. OBJECTIVES: The objective is to create metabolomics models capable of segregating men who improved semen analysis (SA) parameters or achieved natural pregnancy after microsurgical varicocoelectomy (MV) from those who did not, using hydrogen-1 nuclear magnetic resonance (1 H NMR) spectra of seminal plasma of pre-operative samples. MATERIAL AND METHODS: We recruited 29 infertile men with palpable varicocoele. 1 H NMR spectra of seminal plasma were obtained from pre-operative samples and used to create metabonomics models. Improvement was defined as an increase in the total motile progressive sperm count (TMC) of the post-operative SA when compared to the baseline, and pregnancy was assessed for 24 months after MV. RESULTS: Using linear discriminant analysis (LDA), we created a model that discriminated the men who improved SA from those who did not with accuracy of 93.1%. Another model segregated men who achieved natural pregnancy from men who did not. We identified seven metabolites that were important for group segregation: caprylate, isoleucine, N-acetyltyrosine, carnitine, N-acetylcarnitine, creatine, and threonine. DISCUSSION: We described the use of metabonomics model to predict with high accuracy the outcomes of MV in infertile men with varicocoele. The most important metabolites for group segregation are involved in energy metabolism and oxidative stress response, highlighting the pivotal role of these mechanisms in the pathophysiology of varicocoele. CONCLUSIONS: 1 H NMR spectroscopy of seminal plasma can be used in conjunction with multivariate statistical tools to create metabonomics models useful to segregate men with varicocoele based on the reproductive outcomes of MV. These models may help counseling infertile men with varicocoele regarding their prognosis after surgery.

Structural analysis of the woodland strawberry COI1-JAZ1 co-receptor for the plant hormone jasmonoyl-isoleucine.

The phytohormone jasmonoyl-isoleucine (JA-Ile) regulates fundamental plant processes. Fragaria vesca, the woodland strawberry, is a model plant for the Rosaceae family, in which the JA-Ile perception is poorly understood at the molecular level. JA-Ile promotes binding of JAZ repressor to COI1 protein in Arabidopsis to activate jasmonate (JA)-dependent responses. The aim of this work was to understand the molecular basis of the interaction between the F. vesca COI1 (FvCOI1) and JAZ1 (FvJAZ1) promoted by JA-Ile using a computational approach. Multiple sequence alignments and phylogenetic analyses of amino acid sequences were performed for FvCOI1, FvJAZ1 and their ortholog sequences. 3D structures for FvCOI1 and FvJAZ1 proteins were built by methods of homology modeling, using AtCOI1-JA-Ile-AtJAZ1 as template and then they were further refined and validated by molecular dynamics (MD) simulation. A molecular docking approach along with MDS analysis were used to gain insights into the interaction between a putative degron-like sequence present in FvJAZ1 with the FvCOI1-JA-Ile complex. FvCOI1 and FvJAZ1 showed high and moderate sequence identity, respectively, with the corresponding ortholog proteins from other plant species including apple, grape, tomato and Arabidopsis. Moreover, the FvJAZ1 has a variant C-terminal IPMQRK sequence instead of the canonical LPIARR degron sequence located in the Jas domain of AtJAZ1. The MD simulation results showed that the FvCOI1-JA-Ile-FvJAZ1 complex was stable, and the IPMQRK peptide of FvJAZ1 directly interacted with FvCOI1 and JA-Ile. The present research provides novel insight into the molecular interactions among key JA-signaling components in the model plant F. vesca, being few examples of characterized JA-Ile receptors at a structural level in plants.

Aspergillus fumigatus Intrinsic Fluconazole Resistance Is Due to the Naturally Occurring T301I Substitution in Cyp51Ap.

Aspergillus fumigatus intrinsic fluconazole resistance has been demonstrated to be linked to the CYP51A gene, although the precise molecular mechanism has not been elucidated yet. Comparisons between A. fumigatus Cyp51Ap and Candida albicans Erg11p sequences showed differences in amino acid residues already associated with fluconazole resistance in C. albicans The aim of this study was to analyze the role of the natural polymorphism I301 in Aspergillus fumigatus Cyp51Ap in the intrinsic fluconazole resistance phenotype of this pathogen. The I301 residue in A. fumigatus Cyp51Ap was replaced with a threonine (analogue to T315 at Candida albicans fluconazole-susceptible Erg11p) by changing one single nucleotide in the CYP51A gene. Also, a CYP51A knockout strain was obtained using the same parental strain. Both mutants' antifungal susceptibilities were tested. The I301T mutant exhibited a lower level of resistance to fluconazole (MIC, 20 µg/ml) than the parental strain (MIC, 640 µg/ml), while no changes in MIC were observed for other azole- and non-azole-based drugs. These data strongly implicate the A. fumigatus Cyp51Ap I301 residue in the intrinsic resistance to fluconazole.

Biochemical Characterization of Branched Chain Amino Acids Uptake in Trypanosoma cruzi.

Trypanosoma cruzi is the etiological agent of Chagas disease. During its life cycle, it alternates among vertebrate and invertebrate hosts. Metabolic flexibility is a main biochemical characteristic of this parasite, which is able to obtain energy by oxidizing a variety of nutrients that can be transported from the extracellular medium. Moreover, several of these metabolites, more specifically amino acids, have a variety of functions beyond being sources of energy. Branched chain amino acids (BCAA), beyond their role in ATP production, are involved in sterol biosynthesis; for example, leucine is involved as a negative regulator of the parasite differentiation process occurring in the insect midgut. BCAA are essential metabolites in most nonphotosynthetic eukaryotes, including trypanosomes. In view of this, the metabolism of BCAA in T. cruzi depends mainly on their transport into the cell. In this work, we kinetically characterized the BCAA transport in T. cruzi epimastigotes. Our data point to BCAA as being transported by a single saturable transport system able to recognize leucine, isoleucine and valine. In view of this, we used leucine to further characterize this system. The transport increased linearly with temperature from 10 to 45 °C, allowing the calculation of an activation energy of 51.30 kJ/mol. Leucine uptake was an active process depending on ATP production and a H(+) gradient, but not on a Na(+) or K(+) gradient at the cytoplasmic membrane level.

Production of serine protease inhibitors by mutagenesis and their effects on the mortality of Aedes aegypti L. larvae.

BACKGROUND: Dengue, transmitted primarily by the bites of infected Aedes aegypti L., is transmitted to millions of individuals each year in tropical and subtropical areas. Dengue control strategies are primarily based on controlling the vector, using insecticides, but the appearance of resistance poses new challenges. Recently, highly selective protease inhibitors by phage display were obtained for digestive enzymes of the 4th instar larvae (L4) midgut. These mutants were not confirmed as a larvicide due to the low yield of the expression of these inhibitors. In the present study, chimera molecules were constructed based on the mutations at positions P1-P4' selected previously. The T6, T23 and T149 mutants were mixed with another Kunitz inhibitor, domain 1 of the inhibitor boophilin (D1). METHODS: The chimeras T6/D1, T149/D1 and T23/D1 were expressed at high levels in P. pastoris yeast, purified by ionic exchange chromatography and their homogeneity was analyzed by SDS-PAGE. The chimera inhibitors were assayed against larval trypsin, chymotrypsin and elastase using specific chromogenic substrates. The inhibitors were assayed for their larvicide potential against L4. RESULTS: The chimeras exhibited strong inhibitory activities against the larval digestive enzymes in a dose-dependent manner. T6/D1, T149/D1 and T23/D1 exhibited strong larvicidal activity against L4 of Ae. aegypti with inhibitor concentrations in the µM range. A synergistic increase in mortality was observed when a mixture of the three chimeric inhibitors was tested. CONCLUSIONS: The strategy for constructing the chimeric inhibitors was successful. The chimeras showed strong larvicidal activity against Ae. aegypti. In the future, our findings can be used to design synthetic inhibitors for larvae digestive enzymes as an alternative method to control the dengue vector.

Supplemental leucine and isoleucine affect expression of cationic amino acid transporters and myosin, serum concentration of amino acids, and growth performance of pigs.

Leucine (Leu) participates in the activity of cationic amino acid (aa) transporters. Also, branched-chain aa [Leu, isoleucine (Ile), and valine (Val)] share intestinal transporters for absorption. We conducted an experiment with 16 young pigs (body weight of about 16 kg) to determine whether Leu and Ile affect expression of aa transporters b(0,+) and CAT-1 in the jejunum and expression of myosin in muscle, as well as serum concentration of essential aa, and growth performance in pigs. Dietary treatments were: wheat-based diets fortified with Lys, Thr, and Met; basal diet plus 0.50% Leu; basal diet plus 0.50% Ile, and basal diet plus 0.50% Leu and 0.50% Ile. After 28 days, the pigs were sacrificed to collect blood, jejunum, and semitendinosus and longissimus muscle samples. The effects of single and combined addition of Leu and Ile were analyzed. Leu alone or combined with Ile significantly decreased daily weight gain and reduced feed conversion. Leu and Ile, alone or in combination, significantly decreased expression of b(0,+) and significantly increased CAT-1. Ile alone or combined with Leu significantly decreased myosin expression in semitendinosus and significantly decreased it in longissimus muscle. Leu alone significantly decreased Lys, Ile and Thr serum concentrations; Ile significantly decreased Thr serum concentration; combined Leu and Ile significantly decreased Thr and significantly increased Val serum concentration. We conclude that dietary levels of Leu and Ile affect growth performance, expression of aa transporters and myosin, and aa serum concentrations in pigs.

Point mutations in the barley HvHAK1 potassium transporter lead to improved K+-nutrition and enhanced resistance to salt stress.

Members of group I KT-HAK-KUP transporters play an important role in K+ acquisition by plant roots, a process that is strongly affected by salt stress. A PCR-based random mutagenesis approach on HvHAK1 allowed identification of V366I and R591C substitutions, which confer enhanced K+-capture, and improved NaCl, LiCl and NH4Cl tolerance, to yeast cells. Improved K+-capture was linked to an enhanced Vmax. Results reveal an intrinsic protective effect of K+, and assign an important role to the 8th transmembrane domain, as well as the C-terminus, in determining the maximum capacity for the transport of K+ in KT-HAK-KUP transporters.

Enfermedad de orina a jarabe de arce / Maple syrup urine disease

Las enfermedades metabólicas pueden presentarse con síntomas, signos y laboratorios inespecíficos, que si no se consideran entre los diagnósticos diferenciales pueden retrasar el diagnóstico de estos pacientes, lo que lleva a un alto grado de secuelas neurológicas o muerte en etapas tempranas. La enfermedad de Orina a Jarabe de Arce es una enfermedad metabólica de baja incidencia caracterizada por la acumulación de niveles tóxicos de valina, isoleucina y principalmente leucina. Se presenta un paciente sin antecedentes que a los 11 días de vida comienza con mala actitud alimentaria, letargia y fontanela tensa. Descartadas las causas infectológicas se realizó un screening para enfermedades metabólicas. Se diagnosticó Leucinosis (Enfermedad de orina con olor a Jarabe de Arce) y se inició el tratamiento con restricción de leucina, valina e isoleucina en la dieta. A los pocos días del tratamiento el paciente mostró evidencias de mejoría clínica y en los parámetros de laboratorio.

Clinical signs, symptoms and lab tests of neonatal metabolic diseases may be unspecific and a high grade of suspicion is necessary to include them among the differential diagnosis avoiding a significant delay in recognizing this condition and consequent risk of neurologic handicap or early dead. Maple syrup urine disease is a congenital metabolic disorder with a low rate of prevalence and characterized by a toxic accumulation of the amino acids valine, isoleucine and mainly leucine. In this report we describe the history of a patient apparently healthy that on the 11th day after birth initiates symptoms like poor feeding, lethargy and tense fontanel. Excluded sepsis a work up for metabolic disease was performed, being diagnosed a leucinosis (Maple syrup urine disease). A dietary treatment with leucine, valine and isoleucine restriction was immediately initiated and a few days after the patient showed significant clinical and lab improvement. A short description and discussion of this disease is presented.

Induction of oxidative stress in rat brain by the metabolites accumulating in maple syrup urine disease.

Maple syrup urine disease (MSUD) is an inherited disorder caused by deficiency of branched-chain L-2-keto acid dehydrogenase complex activity. Affected patients present severe brain dysfunction manifested as convulsions, coma, psychomotor delay and mental retardation. However, the underlying mechanisms of these neurological findings are virtually unknown. In this study, we tested the in vitro effect of L-leucine, L-isoleucine and L-valine, the amino acids accumulating in MSUD, on the lipid peroxidation parameters chemiluminescence and thiobarbituric acid-reactive substances (TBA-RS), as well as on total radical-trapping antioxidant potential (TRAP) and total antioxidant reactivity (TAR) in cerebral cortex from 30-day-old rats. L-Leucine significantly increased chemiluminescence and TBA-RS measurements and markedly decreased TRAP and TAR values. L-Isoleucine increased chemiluminescence and decreased TRAP measurements, but TAR and TBA-RS levels were not altered by the amino acid. Finally, TRAP measurement was diminished by L-valine. The results indicate a stimulation of lipid peroxidation and a reduction of brain capacity to efficiently modulate the damage associated with an increased production of free radicals by the branched-chain amino acids (BCAAs) accumulated in MSUD. It is therefore tempting to speculate that oxidative stress may be implicated in the brain damage found in MSUD patients.

Inhibition of glutamate uptake into synaptic vesicles of rat brain by the metabolites accumulating in maple syrup urine disease.

Maple syrup urine disease is an inherited metabolic disorder characterized by tissue accumulation of branched-chain amino acids and their corresponding keto acids in the affected children. Although this disorder is predominantly characterized by neurological symptoms, only few studies were carried out to investigate its neuropathology. In this study we investigated the effect of the metabolites accumulating in maple syrup urine disease on the in vitro uptake of [3H]glutamate by synaptic vesicles of rat brain. Synaptic vesicle preparations from whole brain of male adult Wistar rats (200-250 g) were incubated with the branched-chain amino acids and their corresponding keto acids at final concentrations ranging from 0.25 to 10 mM for the determination of glutamate uptake. Glutamate uptake was significantly inhibited by L-leucine, L-isoleucine, L-2-ketoisocaproic acid and L-2-keto-3-methylvaleric acid by approximately 60%, whereas L-valine and L-2-ketoisovaleric acid showed no effect. We also verified that the metabolites probably act by competitive inhibition. Therefore, it is possible that extracellular glutamate levels may be increased in maple syrup urine disease and that excitotoxicity may be involved in the neuropathology of this disorder.

Deficiencia de la 2-metilacetoacetil-CoA tiolasa mitocondrial en Argentina / Mitochondrial 2-methylacetoacetyl-CoA thiolase deficiency in Argentina

A partir de la descripción de dos pares de hermanos, pertenecientes a familias no emparentadas, una argentina con antecedentes de consanguinidad y de ancestros irlandeses y la otra oriunda del Paraguay, en quienes se reconoció la deficiencia de la 2-metilacetoacetil-CoA tiolasa mitocondrial, comúnmente conocida como la deficiencia de la Beta-cetotiolasa (DCT; McKusick 203750; EC 2.3.1.9), intentamos delinear las consecuencias clínicas y bioquímicas de este defecto genético en el sexto paso del catabolismo de la isoleucina. La expresión fenotípica que presentaron los pacientes, correspondió a la forma clásica de DCT; el cuadro clínico uniforme se inició entre los 7 y 15 meses de edad y comprendió esencialmente, una asociación de uno o varios severos ataques cetoacidóticos e hiperglucemia constatada en dos de ellos. La demonstración por cromatografía en capa delgada de la tiglilglicina, dinitrofenilhidrazona de la butanona, aminoacidemia y aminoaciduria normales y un perfil único de ácidos orgánicos obtenido por cromatografía en fase gaseosa y espectrometría de masa (CG/EM) con excreción de grandes cantidades de los metabolitos característicos de la enfermedad, 2-metil-3-hidroxibutirato, 2-metil-acetato, tiglilglicina y 2-etilhidracrilato permitieron establecer el diagnóstico bioquímico de la DCT. El ensayo de la Beta-cetotiolasa en linfocitos y polimorfos nucleares de la única sobreviviente (VT), demostró una falta de activación por el ión K+ cuando se utilizó el acetoacetil-CoA como sustrato. Esta primera comunicación argentina sobre la DCT, permite inscribir tres aspectos ampliatorios respecto a las referencias previas: incorpora otros distintos orígenes étnicos de los pacientes, señala un análisis morfológico de material de autopsia sin cambios estructurales en cerebro, hígado y riñon y marca en la paciente VT, una disociación entre una clínica asintomática a partir de los 7 años y la persistente anormalidad bioquímica hasta la edad actual de 16 años. El conocimiento de la existencia de estas patologías en nuestro medio, aunado a la disponibilidad y acceso a la CG/EM de alta precisión y rapidez, permitirán reconocimientos precoces y mejores resultados terapéuticos. (AU)

Deficiencia de la 2-metilacetoacetil-CoA tiolasa mitocondrial en Argentina / Mitochondrial 2-methylacetoacetyl-CoA thiolase deficiency in Argentina

A partir de la descripción de dos pares de hermanos, pertenecientes a familias no emparentadas, una argentina con antecedentes de consanguinidad y de ancestros irlandeses y la otra oriunda del Paraguay, en quienes se reconoció la deficiencia de la 2-metilacetoacetil-CoA tiolasa mitocondrial, comúnmente conocida como la deficiencia de la Beta-cetotiolasa (DCT; McKusick 203750; EC 2.3.1.9), intentamos delinear las consecuencias clínicas y bioquímicas de este defecto genético en el sexto paso del catabolismo de la isoleucina. La expresión fenotípica que presentaron los pacientes, correspondió a la forma clásica de DCT; el cuadro clínico uniforme se inició entre los 7 y 15 meses de edad y comprendió esencialmente, una asociación de uno o varios severos ataques cetoacidóticos e hiperglucemia constatada en dos de ellos. La demonstración por cromatografía en capa delgada de la tiglilglicina, dinitrofenilhidrazona de la butanona, aminoacidemia y aminoaciduria normales y un perfil único de ácidos orgánicos obtenido por cromatografía en fase gaseosa y espectrometría de masa (CG/EM) con excreción de grandes cantidades de los metabolitos característicos de la enfermedad, 2-metil-3-hidroxibutirato, 2-metil-acetato, tiglilglicina y 2-etilhidracrilato permitieron establecer el diagnóstico bioquímico de la DCT. El ensayo de la Beta-cetotiolasa en linfocitos y polimorfos nucleares de la única sobreviviente (VT), demostró una falta de activación por el ión K+ cuando se utilizó el acetoacetil-CoA como sustrato. Esta primera comunicación argentina sobre la DCT, permite inscribir tres aspectos ampliatorios respecto a las referencias previas: incorpora otros distintos orígenes étnicos de los pacientes, señala un análisis morfológico de material de autopsia sin cambios estructurales en cerebro, hígado y riñon y marca en la paciente VT, una disociación entre una clínica asintomática a partir de los 7 años y la persistente anormalidad bioquímica hasta la edad actual de 16 años. El conocimiento de la existencia de estas patologías en nuestro medio, aunado a la disponibilidad y acceso a la CG/EM de alta precisión y rapidez, permitirán reconocimientos precoces y mejores resultados terapéuticos.

[Mitochondrial 2-methylacetoacetyl-CoA thiolase deficiency in Argentina]. / Deficiencia de la 2-metilacetoacetil-CoA tiolasa mitocondrial en Argentina.

From the description of two pairs of siblings belonging to unrelated families, one Argentine family with a history of consanguinity and Irish ancestry and the other family native of Paraguay, in whom mitochondrial 2-methylacetoacetyl-CoA thiolase deficiency, commonly known as beta-ketothiolase deficiency (beta-KTD, McKusick 203750; EC 2.3.1.9) was recognized. We tried to outline through this experience the clinical and biochemical consequences of this genetic defect in the 6th step of the isoleucine catabolism. The phenotyoic expression presented by the patients belonged to the classical form of beta-KTD. Seven to 15 months was the age at onset of the uniform clinical pattern this being essentially an association of one or several severe ketoacidotic episodes and hyperglycemia which was observed in two patients. The thin-layer chromatography of the tiglylglycine, and dinitrophenylhydrazone of the butanone were positive; aminoacidemia and aminoaciduria revealed normal levels. The organic acids having a unique profile obtained through gaschromatography and mass-spectrometry (GC/MS) showed excretion of large quantities of metabolites characteristic of the disease: 2-methyl-3-hydroxybutirate, 2-methylacetoacetic acid, tiglylglycine and 2-ethylhydracrilic acid which led us to establish the biochemical diagnosis of beta-KTD. The assay of the beta-ketothiolase in lymphocytes and polymorphonuclear leukocytes of the only surviving patient (VT) showed absence of activation by the K+ ion when the acetoacetyl-CoA was used as a substrate. This first Argentine report about beta-KTD leads us to mention three amplifying aspects with regards to previous literature: it adds other different ethnic ancestries of patients, points out a morphological analysis of autopsy material with unchanged structures in the brain, liver and kidneys and marks in the patient VT a dissociation between a symptom-free clinical pattern since age 7 and the persistent biochemical abnormality until the present age, 15 years. The knowledge of the existence of these diseases in our country together with the availability and access to GC/MS of high precision and speed, will allow early diagnosis and better therapeutic results.

Enfermedad de la orina olor a jarabe de arce / Maple syrup urine disease

La enfermedad de la orina olor a jarabe de arce, es una afección metabólica producida por deficiencia en la descarboxilación oxidativa de los quetoácidos provenientes de los aminoácidos ramificados valina, isoleucina y leucina. Los pacientes parecen normales hasta los 5 a 7 días de vida, cuando muestran rechazo a la alimentación, vómitos, olor a jarabe de arce en la orina y la piel y deterioro neurológico progresivo, que lleva rápidamente al coma. Se presentan tres niños con enfermedad de la orina olor a jarabe de arce clásica, leucina >2 000 µmol/1, que fueron diagnosticados a los 10, 20 y 21 días de vida, iniciándose inmediatamente el tratamiento en base a la dieta especial. el seguimiento está basado en evaluaciones clínicas, nutricionales y bioquímicas periódicas, para adecuar los requerimientos nutricionales a la evolución de la enfermedad. La evaluación psicométrica, escala de Bayley, mostró que el coeficiente de desarrollo mental estaba entre 50 y 82, relacionándose éste con la edad de diagnóstico. Actualmente los niños tienen entre 2 y 3 años de edad, se encuentran con dieta restringida en los 3 aminoácidos ramificados. Su control debe ser frecuente para evitar desbalances metabólicos y prevenir daños neurológicos ocasionados por éstos

A new syndrome with ethylmalonic aciduria and normal fatty acid oxidation in fibroblasts.

We describe four Italian male infants with a novel clinical phenotype characterized by orthostatic acrocyanosis, relapsing petechiae, chronic diarrhea, progressive pyramidal signs, mental retardation, and brain magnetic resonance imaging abnormalities. The first symptoms appeared after the termination of breast-feeding and introduction of formula feeding. Marked persistent 2-ethylmalonic aciduria was associated with abnormal excretion of C4-C5(n-butyryl-, isobutyryl-, isovaleryl-, and 2-methylbutyryl-)acylglycines and acylcarnitines and with intermittent lactic acidosis. Short- and branched-chain plasma acylcarnitine levels were also elevated. 2-Ethylmalonic aciduria is generally regarded as being indicative of a defect in fatty acid oxidation. Extensive studies of cultured fibroblasts failed to reveal such a defect. The observation of intermittent urinary excretion of 2-ethylhydracrylic acid pointed to involvement of the isoleucine R pathway in ethylmalonate biosynthesis. This hypothesis was tentatively corroborated by the biochemical responses to an oral isoleucine challenge in two patients. However, fibroblast studies showed normal oxidation rates of (14C)isoleucine (ul), indicating that this is not a defect of isoleucine oxidation expressed in skin fibroblasts. In one of two patients tested, cytochrome c oxidase activity was partially reduced (45%) in cultured fibroblasts. This unique clinical and biochemical phenotype identifies a new metabolic encephalopathy of yet undetermined cause.

Acetoacetyl CoA thiolase deficiency: a cause of severe ketoacidosis in infancy simulating salicylism.

A female child presented at one year of age with a febrile illness and loose stools, then developed severe ketoacidosis with vomiting; an apparent salicylate level of 11 mg/dl was measured. A sibling had died in similar circumstances nine years earlier. Investigation revealed that the child did not have salicylate intoxication, and that high levels of acetoacetate in blood and urine were giving readings indicative of the presence of salicylate on routine testing. Gas-liquid chromatographic analysis combined with mass spectrometry on urine samples revealed the presence of 2-methyl-acetoacetate, 2-methyl-3-hydroxybutyrate, and tiglyl glycine in appreciable amounts, indicating a defect in isoleucine catabolism located at the beta-ketothiolase step. The oxidation of 14C-isoleucine to CO2 in cultured fibroblasts confirmed that this pathway was defective. We present evidence that beta-ketothiolase deficiency is not simply a defect of isoleucine degradation; the deficient enzyme is the K+ dependent short-chain mitochondrial thiolase, which also plays a major catalytic role in ketone body and fatty acid oxidation.