Avances en el diagnóstico y tratamiento de la enfermedad de jarabe de arce, experiencia en Galicia / Advances in the diagnosis and treatment of maple syrup urine disease: experience in Galicia (Spain)

Introducción La enfermedad de la orina con olor a jarabe de arce (MSUD) es un raro trastorno de herencia autosómica recesiva en el que, debido a la deficiencia enzimática en el complejo deshidrogenasa de los α-cetoácidos de cadena ramificada, se produce acumulación de los aminoácidos leucina, isoleucina, valina y aloisoleucina y sus productos metabólicos en células y líquidos biológicos, lo que conduce a una disfunción cerebral grave. Pacientes y método Presentamos los tres casos de MSUD diagnosticados desde el año 2000 en Galicia, a partir de la puesta en marcha del Programa de Cribado Neonatal por espectrometría de masas en tándem (MS/MS) en muestra de sangre impregnada en papel. Se trata de dos formas clásicas y una intermedia de MSUD, una diagnosticada por el inicio clínico precoz y dos a través del cribado neonatal. Todos los casos fueron confirmados mediante estudio enzimático y genético. Se expone la situación clínica y bioquímica al diagnóstico y durante el seguimiento posterior. Los tres mantuvieron un adecuado control metabólico con concentraciones medias de leucina a lo largo del tiempo cercanos al rango normal (220, 177 y 252 μmol/l, respectivamente). Presentaron descompensaciones moderadas y sólo superaron de forma aislada, un día en dos de los pacientes, los 1.000 μmol/l de leucina. Los cocientes de desarrollo en los tres casos están en rango normal. Resultados A la vista de nuestros resultados, consideramos clave para mejorar el pronóstico de los pacientes con MSUD: la inclusión de esta enfermedad dentro de los programas de cribado neonatal con toma de muestra precoz, a los dos o tres días de vida; un tratamiento agresivo en la fase inicial y en las crisis por descompensación metabólica; un control evolutivo estricto con el fin de prevenir descompensaciones, y la monitorización de aminoácidos ramificados de forma cómoda para el paciente (sobre muestra de sangre en papel) manteniendo concentraciones de leucina inferiores a 300 μmol/l

Introduction Maple syrup urine disease (MSUD) is a rare autosomal recessive disorder caused by an inherited deficiency of branched chain α-ketoacid dehydrogenase activity. Accumulation of the amino acids leucine, isoleucine, valine and alloisoleucine and their metabolic products in cells and biological fluids results in severe brain dysfunction. Patients and methods We present three cases of MSUD diagnosed in Galicia since 2000, the year in which the Extended Newborn Screening Program by tandem mass spectrometry was started in this region. One of the patients was diagnosed on the basis of early clinical presentation and the others by neonatal screening. Enzymatic and molecular studies confirmed two classic cases of MSUD and an intermediate variant. We describe the clinical and biochemical details at confirmation of diagnosis and the long-term outcome of the three patients. Throughout follow-up, all the patients maintained adequate leucine levels, which were near the normal range (mean levels: 220, 177 and 252 μmol/L, respectively). Several moderate metabolic decompensations were observed but leucine levels only occasionally exceeded 1000 μmol/L (one day in two patients). IQ tests were performed in all patients and scores were within the normal range. In view of our results, we believe the following measures are essential to improve the prognosis of MSUD: inclusion of this disease in Expanded Neonatal Screening Programs with early samples (at 2-3 days of life); aggressive treatment in the initial phase and during acute decompensations; strict metabolic control to prevent crises, monitoring of branched-chain amino acids (dried blood spot sample), and maintenance of long term plasma leucine levels below 300 μmol/L

Effects of reduced free fatty acid availability on skeletal muscle PDH activation during aerobic exercise. Pyruvate dehydrogenase.

This study investigated the effect of reduced free fatty acid (FFA) availability on pyruvate dehydrogenase activation (PDHa) and carbohydrate metabolism during moderate aerobic exercise. Eight active male subjects cycled for 40 min at 55% Vo(2 peak) on two occasions. During one trial, subjects ingested 20 mg/kg body mass of the antilipolytic drug nicotinic acid (NA) during the hour before exercise to reduce FFA. Nothing was ingested in the control trial (CON). Blood and expired gas measurements were obtained throughout the trials, and muscle biopsy samples were obtained immediately before exercise and at 5, 20, and 40 min of exercise. Plasma FFA were lower in the NA trial (0.13 +/- 0.01 vs. 0.48 +/- 0.03 mM, P < 0.05), and the respiratory exchange ratio (RER) was increased with NA (0.93 +/- 0.01 vs. 0.89 +/- 0.01, P < 0.05), resulting in a 14.5 +/- 1.8% increase in carbohydrate oxidation compared with CON. PDHa increased rapidly in both trials at exercise onset but was approximately 15% higher (P < 0.05) throughout exercise in the NA trial (2.44 +/- 0.19 and 2.07 +/- 0.12 mmol x kg wet muscle(-1) x min(-1) for NA and CON at 40 min). Muscle glycogenolysis was 15.3 +/- 9.6% greater in the NA trial vs. the CON trial but did not reach statistical significance. Glucose 6-phosphate contents were elevated (P < 0.05) in the NA trial at 30 and 40 min of exercise, but pyruvate and lactate contents were unaffected. These data demonstrate that the reduction of exogenous FFA availability increased the activation of PDH and carbohydrate oxidation during moderate aerobic exercise in men. The increased activation of PDH was not explained by changes in muscle pyruvate or the ATP/ADP ratio but may be related to a decrease in the NADH/NAD(+) ratio or an epinephrine-induced increase in calcium concentration.

Diagnosis and treatment of maple syrup disease: a study of 36 patients.

OBJECTIVE: To evaluate an approach to the diagnosis and treatment of maple syrup disease (MSD). METHODS: Family histories and molecular testing for the Y393N mutation of the E1alpha subunit of the branched-chain alpha-ketoacid dehydrogenase allow us to identify infants who were at high risk for MSD. Amino acid concentrations were measured in blood specimens from these at-risk infants between 12 and 24 hours of age. An additional 18 infants with MSD were diagnosed between 4 and 16 days of age because of metabolic illness. A treatment protocol for MSD was designed to 1) inhibit endogenous protein catabolism, 2) sustain protein synthesis, 3) prevent deficiencies of essential amino acids, and 4) maintain normal serum osmolarity. Our protocol emphasizes the enhancement of protein anabolism and dietary correction of imbalances in plasma amino acids rather than removal of leucine by dialysis or hemofiltration. During acute illnesses, the rate of decrease of the plasma leucine level was monitored as an index of net protein synthesis. The treatment protocol for acute illnesses included the use of mannitol, furosemide, and hypertonic saline to maintain or reestablish normal serum sodium and extracellular osmolarity and thereby prevent or reverse life-threatening cerebral edema. Similar principles were followed for both sick and well outpatient management, especially during the first year, when careful matching of branched-chain amino acid intake with rapidly changing growth rates was necessary. Branched-chain ketoacid excretion was monitored frequently at home and branched-chain amino acid levels were measured within the time of a routine clinic visit, allowing immediate diagnosis and treatment of metabolic derangements. RESULTS: 1) Eighteen neonates with MSD were identified in the high-risk group (n = 39) between 12 and 24 hours of age using amino acid analysis of plasma or whole blood collected on filter paper. The molar ratio of leucine to alanine in plasma ranged from 1.3 to 12.4, compared with a control range of 0.12 to 0.53. None of the infants identified before 3 days of age and managed by our treatment protocol became ill during the neonatal period, and 16 of the 18 were managed without hospitalization. 2) Using our treatment protocol, 18 additional infants who were biochemically intoxicated at the time of diagnosis recovered rapidly. In all infants, plasma leucine levels decreased to <400 micromol/L between 2 to 4 days after diagnosis. Rates of decrease of the plasma leucine level using a combination of enteral and parenteral nutrition were consistently higher than those reported for dialysis or hemoperfusion. Prevention of acute isoleucine, valine, and other plasma amino acid deficiencies by appropriate supplements allowed a sustained decrease of plasma leucine levels to the therapeutic range of 100 to 300 micromol/L, at which point dietary leucine was introduced. 3) Follow-up of the 36 infants over >219 patient years showed that, although common infections frequently cause loss of metabolic control, the overall rate of hospitalization after the neonatal period was only 0.56 days per patient per year of follow-up, and developmental outcomes were uniformly good. Four patients developed life-threatening cerebral edema as a consequence of metabolic intoxication induced by infection, but all recovered. These 4 patients each showed evidence that acutely decreased serum sodium concentration and decreased serum osmolarity were associated with rapid progression of cerebral edema during their acute illnesses. CONCLUSIONS: Classical MSD can be managed to allow a benign neonatal course, normal growth and development, and low hospitalization rates. However, neurologic function may deteriorate rapidly at any age because of metabolic intoxication provoked by common infections and injuries. Effective management of the complex pathophysiology of this biochemical disorder requires integrated management of general medical care and nutrition, as well as control of several variables that influence endogenous protein anabolism and catabolism, plasma amino acid concentrations, and serum osmolarity.

Enzymatic method for branched chain alpha-ketoacid determination: application to rapid analysis of urine and plasma samples from maple syrup urine disease patients.

A new method for the determination of branched-chain alpha-ketoacid concentration using lactate dehydrogenase (E C 1.1.1.27) isozyme C4 (LDH C4) from mouse testes is proposed. The assay is performed on urine and plasma without previous treatment. Alpha-ketoglutarate and pyruvate are determined on the same sample using glutamate dehydrogenase (GDH, EC 1.4.1.2) and lactate dehydrogenase isozyme A4 (LDH5) respectively and subtracted from the total alpha-ketoacid concentration obtained with LDH C4. This value corresponds to the branched chain alpha-ketoacid. Results were linear within the concentration range 8 to 170 mumoles/L. Detection limit was 8 mumoles/L. Analytical recovery was higher than 91%. For microplate assays, recoveries were higher than 84% and the detection limit was 20 mumoles/L. Determinations performed with GDH, LDH A4 and LDH C4 allow differentiation of E3 deficiency from other clinical phenotypes of maple syrup urine disease. The method is simple and fast, and adaptation to microplates would allow screening of newborns.

[Erythrocyte ketone reductase activity, total plasma haloperidol and acute psychoses]. / Activité cétone réductase érythrocytaire, taux plasmatiques d'halopéridol et psychoses aiguës.

Haloperidol (HAL) is a widely used and clinically effective neuroleptic. Its metabolism differs in various animal species. In humans, reduced haloperidol (RHAL), a hydroxy metabolite of HAL, is produced by a cytosolic ketone reductase. Interconversion is known to occur whereby HAL is found in the plasma after administration of RHAL in vivo. Interconversion of HAL and RHAL has been observed in man. However, the capacity for reductive HAL is greater than its oxidation back from RHAL. RHAL, the resulting metabolite of HAL, is reported to be about 10-25% as active as HAL in an animal model. Large intersubject variation has been observed in the pharmacokinetics of HAL and RHAL. A wide variation in reductive drug-metabolizing has been observed in schizophrenic patients treated with HAL. Both high and low RHAL/HAL ratios or RHAL levels were reported to be linked to poor response in HAL-treated patients and might be correlated with the therapeutic window effect of HAL treatment. It is conceivable, therefore, that subjects with high reductive capacity relative to oxidative capacity may have less therapeutic response from the same dose of HAL than those with a low reductive capacity relative to oxidative capacity. This aim of this study was to investigate the HAL reduction among a sample of HAL-treated schizophrenic patients. Because ketone reductases are generally not tissue specific, we investigate the reductase activity in Red blood cells (as described by Inaba), before and during the treatment. Steady-state plasma drug levels during 2 weeks of treatment were quantified. We examined the relationships between fixed doses of HAL treatment, Red blood cells ketone reductase activity, plasma HAL and RHAL levels and the percentage of change of the Positive and Negative Syndrome Scale for Schizophrenia. The participants in this study were 15 inpatients consecutively being treated in the adult psychiatric wards of the University of Lille. All subjects met DSM III-R criteria for schizophrenia (paranoid form). Upon induction subjects were evaluated clinically by trained raters using the Positive and Negative Syndrome Scale for Schizophrenia (PANSS). Subjects were required to score 40 or higher on the general psychopathology subscale of the PANSS to continue participation. All subjects were drug free. Haloperidol was administered orally at three times daily dose. Patients were randomized to treatment at three orally fixed doses: 6 mg per day, 10 mg per day and 15 mg per day. Patients were treated for 2 periods of one week. At the end of each period, dosages could be modified according to the clinic evolution of the patient. PANSS was repeated by the same raters blinded to the haloperidol dosage, plasma concentration and Rbc haloperidol ketone reductase activity, at the beginning and at the end of each period. Blood samples were collected on the same day that clinical assessment were made. Multiple regression analysis (forward stepwise) revealed that Red blood cells reductase activity at D0 is an important variable predicting haloperidol plasma levels at week 2. Similarly Red blood cells reductase activity at D0 and D7 predicted Reduced haloperidol plasma concentrations at week 2. In this sample, no parameter was found to be consistency predicted the percentage change in the PANSS positive subscale from baseline, at week 2. Nevertheless, Red blood cells reductase activity at D0, Reduced haloperidol/haloperidol ratio at week 2, haloperidol plasma levels at week 2 and the dose of haloperidol at week 1 were important variables predicting the percentage change in the PANSS general subscale from baseline at week 2. These results suggest that the knowledge of reductase activity could predict the treatment response in acute schizophrenic patients.

Nonlinear relationship between circulating concentrations of reduced haloperidol and haloperidol: evaluation of possible mechanisms.

In patients taking haloperidol (HP), circulating concentrations of reduced haloperidol (RHP increase disproportionately to the dose or concentration of the parent drug. In the current study, we tested the hypothesis that the nonlinearity is due to preferential saturation of the reoxidation of RHP to HP, and two factors that could amplify the nonlinearity-concentration-dependent binding of RHP by plasma proteins, or by red blood cells. In 25 patients with schizophrenia who were taking HP, the unbound fraction of HP (0.085 +/- 0.016) and RHP (0.244 +/- 0.026) in plasma, and the blood:plasma ratio for each compound were independent of their concentration. Thus, saturable binding of RHP to plasma proteins or red blood cells can be excluded. HP reductase and RHP oxidase activity were measured in human liver cytosol and microsomal fractions, respectively. Because ketone reductase-catalysed formation of RHP is stereospecific, we examined each enantiomer of RHP separately. The Vmax for the oxidation of the S(-) and R(+) RHP enantiomers in four livers was 0.23 +/- 0.15 and 0.60 +/- 0.32 mumol/g protein per min (mean +/- SD), respectively. The Km was 110 +/- 40 and 70 +/- 10 microM, respectively. In contrast, HP reductase activity displayed greater capacity and was not saturable. The rate of production of RHP at a HP concentration of 122 microM (the limit of HP solubility) in the same livers was 2.6 +/- 0.7 mumol/g protein per min. Despite the observed nonlinearity between the enzymatic pathways in vitro, RHP concentrations in vivo are 2-3 orders of magnitude lower than the Km for oxidation of each enantiomer of RHP.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of haloperidol and reduced haloperidol in the plasma and blood of patients on depot haloperidol.

We developed a sensitive HPLC assay to measure haloperidol (HA) and its metabolite, reduced haloperidol (RH), in plasma and whole blood. The conditions under which HA might be converted to RH during collection and analysis of blood were examined. Provided the blood was kept at 0 degrees C, erythrocyte ketone reductase activity was insignificant. The solid phase extraction method did not generate RH. We studied ten patients taking 25-400 mg/month of HA decanoate and one patient for 4 weeks after the daily oral dose of 120 mg HA was ceased. In the patients on depot HA, the plasma and blood concentrations of HA were not significantly different (P greater than 0.1). For the first time, RH was detected in plasma patients on depot drug, but only in three cases. In contrast, RH was present in the blood of eight of these patients. The accumulation of RH in red blood cells was also evident in the patient on oral HA, in whom the mean ratio of RH concentrations in whole blood to plasma was 3.6 +/- 1.1. Plasma concentrations of HA correlated highly with total neuroleptic activity measured by a radioreceptor assay. Compared to plasma, analysis of concentrations of HA and RH in blood has the advantages of greater sensitivity, of using smaller volumes of blood and of avoiding the efflux of HA and RH during separation of plasma and red cells.

Assessment of effects of amino acids and branched chain keto acids on leucine oxidation in human lymphocytes.

Effects of amino acids and branched chain keto acids on leucine transamination and oxidation were assessed in peripheral human lymphocytes. Isoleucine (80-200 mumol/l) and valine (250-500 mumol/l) diminished transamination and oxidation of leucine up to 25%, glutamine (50-1000 mumol/l) up to 55%. alpha-Ketoisocaproic acid (KIC; 200 mumol/l) augmented the activity state of branched chain keto acid dehydrogenase by 40%. It is concluded that in peripheral human lymphocytes (1) isoleucine, valine and glutamine are physiological inhibitors of leucine catabolism, and (2) leucine can promote its own degradation via KIC.

Oxidation of leucine in human lymphocytes.

Appropriate conditions for analysis of leucine oxidation in peripheral human lymphocytes were determined. Lymphocytes, isolated by Ficoll centrifugation, were washed in phosphate-buffered saline without additions, with 5 mmol/l ADP or with 5 mmol/l ADP plus 25 mmol/l NaF for determination of transaminase activity, and total and basal activity of branched chain keto acid dehydrogenase (BCKA-D), respectively. Cells were incubated for 20-60 min with [1-14C]-leucine, or [1-14C]-alpha-ketoisocaproic acid (KIC) in the presence of various concentrations of the respective unlabelled substrates. Pyridoxal phosphate (0.1 mmol/l) augmented transaminase activity, coenzyme A, NAD and thiaminepyrophosphate (0.5 mmol/l each) enhanced BCKA-D activity. Apparent Km values for transamination and for BCKA-D were 40 and 17 microM, respectively. Total capacity for leucine transamination was about five times greater than for oxidation of KIC. The mean activity state of BCKA-D was 30%. Oxidation of KIC declined when 5.6 mmol/l glucose was added to the medium. It is concluded that: (1) BCKA-D is rate-limiting for leucine catabolism in peripheral human lymphocytes, (2) the BCKA-D complex is normally only partially active, and (3) flux of leucine through BCKA-D is inhibited by physiological glucose concentrations.

Kinetics of IMP:NAD+ oxidoreductase in serum of normal Iraqi individuals.

The general kinetic parameters of IMP:NAD+ oxidoreductase, were investigated at 37 degrees C in normal human serum. These included substrate - velocity relationship, Km (IMP), Km (NAD), as well as the effect of temperature and pH on the kinetics of the reaction. Optimum conditions of IMP, NAD+ oxidoreductase in serum of normal Iraqi individuals at 37 degrees C were investigated. The optimum IMP concentration was found to be 0.6166 mM and the optimum NAD+ concentration was 6.28 mM, while the optimum pH for the reaction was found to be 8.3. The enzyme shows low stability even when the sera were incubated at low temperature. The activity of the enzyme is proportional to the serum concentration and its optimal temperature for the reaction was 37 degrees C. The enzyme was found to be inhibited by XMP, GMP competitively with Ki values of 0.0724 mM and 0.1583 mM respectively. The inhibition by thyroid hormone (T4) was studied giving a Ki value of 2.25 X 10(-6) mM. The mechanism of enzyme action was found to follow ordered sequential mechanism.

Inosine 5'-phosphate dehydrogenase activity in normal and leukemic blood cells.

The enzyme inosinic acid dehydrogenase (EC 1.2.1 [14]) was measured and partially purified (10- to 15-fold) from normal and leukemic leukocytes. From the normal blood cells, the highest activities could be detected in lymphocytes and bone marrow cells. Dependent on the blast cell count, the leukemic IMP dehydrogenase had a higher mean specific activity than the enzymes of fractionated, immature bone marrow cells, or normal granulocytes. The partially purified enzymes from the various blood cells were apparently identical; they exhibited hyperbolic substrate saturation kinetics and were inhibited by a number of purine nucleotides. For the leukemic blast cell enzyme, the Km values for the substrates, IMP and NAD+, were 28 +/- 11; 227 +/- 98 microM, and 34 +/- 10; 240 +/- 67 microM for the partially purified enzyme from normal, immature bone marrow cells. The hypoxanthine-guanine and adenine phosphoribosyltransferase activities increased in the leukemic cells when compared with mature granulocytes, but nearly always showed similar activities when compared with fractionated bone marrow cells. Only one of the 30 investigated leukemic patients exhibited a marked decrease in hypoxanthine phosphoribosyltransferase activity of 0.5 nmol/mg/h. The phosphoribosyltransferase-specific activities of the leukemic cells are more variable than for the normal ones and no correlation of enzyme activities and blast cell count was apparent.

Serum xanthine dehydrogenase of carbon tetrachloride-induced hepatotoxicity in alloxan-diabetic rats.

Xanthine dehydrogenase activity was determined in blood serum of rats in which diabetes had been induced by alloxan administration. The results show that there is no statistical significance in the difference found for normal and diabetic rats. Alloxan produced an inhibition in the enzyme activity in animals in which a carbon tetrachloride hepatotoxicity had been induced.

The partial purification and properties of a human erythrocyte 4-nitroacetophenone reductase.

1. A soluble enzyme which catalyses the NADPH-dependent reduction of 4-nitroacetophenone to 4-nitrophenylmethylcarbinol has been partially purified from human erythrocytes. 2.inter-individual or intra-individual differences in the enzymic activity were small except for very low activity observed in one subject with glucose 6-phosphate dehydrogenase deficiency resulting in decreased levels of NADPH. 3. The enzyme was inactivated above 50 degrees or on storage at 4 degrees for longer than 24 h. The pH optimum was between 7-0-8-0. 4. the enzyme has been differentiated from NADPH-methaemoglobin reductase, NADPH-cytochrome c reductase, glutathione reductase, alpha,beta-unsaturated ketone reductase and aromatic alpha-keto acid reductase activities, but similarities exist between this enzyme and a rabbit kidney cortex aromatic aldehyde/ketone reductase.