Preliminary results of PBA-loaded nanoparticles development and the effect on oxidative stress and neuroinflammation in rats submitted to a chemically induced chronic model of MSUD.

Maple syrup urine disease (MSUD) is a genetic disorder that leads the accumulation of branched-chain amino acids (BCAA) leucine (Leu), isoleucine, valine and metabolites. The symptomatology includes psychomotor delay and mental retardation. MSUD therapy comprises a lifelong protein strict diet with low BCAA levels and is well established that high concentrations of Leu and/or its ketoacid are associated with neurological symptoms. Recently, it was demonstrated that the phenylbutyrate (PBA) have the ability to decrease BCAA concentrations. This work aimed the development of lipid-based nanoparticles loaded with PBA, capable of targeting to the central nervous system in order to verify its action mechanisms on oxidative stress and cell death in brain of rats subjected to a MSUD chronic model. PBA-loaded nanoparticles treatment was effective in significantly decreasing BCAA concentration in plasma and Leu in the cerebral cortex of MSUD animals. Furthermore, PBA modulate the activity of catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase enzymes, as well as preventing the oxidative damage to lipid membranes and proteins. PBA was also able to decrease the glial fibrillary acidic protein concentrations and partially decreased the reactive species production and caspase-3 activity in MSUD rats. Taken together, the data indicate that the PBA-loaded nanoparticles could be an efficient adjuvant in the MSUD therapy, protecting against oxidative brain damage and neuroinflammation.

DNA damage induced by alloisoleucine and other metabolites in maple syrup urine disease and protective effect of l-carnitine.

Maple syrup urine disease (MSUD) is an inherited deficiency of the branched-chain α-keto dehydrogenase complex, characterized by accumulation of the branched-chain amino acids (BCAAs) and their respective branched chain α-keto-acids (BCKAs), as well as by the presence of alloisoleucine (Allo). Studies have shown that oxidative stress is involved in the pathophysiology of MSUD. In this work, we investigated using the comet assay whether Allo, BCAAs and BCKAs could induce in vitro DNA damage, as well as the influence of l-Carnitine (L-Car) upon DNA damage. We also evaluated urinary 8-hydroxydeoguanosine (8-OHdG) levels, an oxidative DNA damage biomarker, in MSUD patients submitted to a restricted diet supplemented or not with L-Car. All tested concentrations of metabolites (separated or incubated together) induced in vitro DNA damage, and the co-treatment with L-Car reduced these effects. We found that Allo induced the higher DNA damage class and verified a potentiation of DNA damage induced by synergistic action between metabolites. In vivo, it was observed a significant increase in 8-OHdG levels, which was reversed by L-Car. We demonstrated for the first time that oxidative DNA damage is induced not only by BCAAs and BCKAs but also by Allo and we reinforce the protective effect of L-Car.

Investigation of inflammatory profile in MSUD patients: benefit of L-carnitine supplementation.

Maple Syrup Urine Disease (MSUD) is a metabolic disorder caused by a severe deficiency of the branched-chain α-keto acid dehydrogenase complex activity which leads to the accumulation of branched-chain amino acids (BCAA) leucine (Leu), isoleucine and valine and their respective α-keto-acids in body fluids. The main symptomatology presented by MSUD patients includes ketoacidosis, failure to thrive, poor feeding, apnea, ataxia, seizures, coma, psychomotor delay and mental retardation, but, the neurological pathophysiologic mechanisms are poorly understood. The treatment consists of a low protein diet and a semi-synthetic formula restricted in BCAA and supplemented with essential amino acids. It was verified that MSUD patients present L-carnitine (L-car) deficiency and this compound has demonstrated an antioxidant and anti-inflammatory role in metabolic diseases. Since there are no studies in the literature reporting the inflammatory profile of MSUD patients and the L-car role on the inflammatory response in this disorder, the present study evaluates the effect of L-car supplementation on plasma inflammatory cytokines interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interferon-gamma (INF-É£), and a correlation with malondialdehyde (MDA), as a marker of oxidative damage, and with free L-car plasma levels in treated MSUD patients. Significant increases of IL-1ß, IL-6, and INF-É£ were observed before the treatment with L-car. Moreover, there is a negative correlation between all cytokines tested and L-car concentrations and a positive correlation among the MDA content and IL-1ß and IL-6 values. Our data show that L-car supplementation can improve cellular defense against inflammation and oxidative stress in MSUD patients and may represent an additional therapeutic approach to the patients affected by this disease.

Nutrition management guideline for maple syrup urine disease: an evidence- and consensus-based approach.

In an effort to increase harmonization of care and enable outcome studies, the Genetic Metabolic Dietitians International (GMDI) and the Southeast Regional Newborn Screening and Genetics Collaborative (SERC) are partnering to develop nutrition management guidelines for inherited metabolic disorders (IMD) using a model combining both evidence- and consensus-based methodology. The first guideline to be completed is for maple syrup urine disease (MSUD). This report describes the methodology used in its development: formulation of five research questions; review, critical appraisal and abstraction of peer-reviewed studies and unpublished practice literature; and expert input through Delphi surveys and a nominal group process. This report includes the summary statements for each research question and the nutrition management recommendations they generated. Each recommendation is followed by a standardized rating based on the strength of the evidence and consensus used. The application of technology to build the infrastructure for this project allowed transparency during development of this guideline and will be a foundation for future guidelines. Online open access of the full, published guideline allows utilization by health care providers, researchers, and collaborators who advise, advocate and care for individuals with MSUD and their families. There will be future updates as warranted by developments in research and clinical practice.

Improved amino acid, bioenergetic metabolite and neurotransmitter profiles following human amnion epithelial cell transplant in intermediate maple syrup urine disease mice.

Orthotopic liver transplant (OLT) significantly improves patient outcomes in maple syrup urine disease (MSUD; OMIM: 248600), yet organ shortages point to the need for alternative therapies. Hepatocyte transplantation has shown both clinical and preclinical efficacy as an intervention for metabolic liver diseases, yet the availability of suitable livers for hepatocyte isolation is also limited. Conversely, human amnion epithelial cells (hAEC) may have utility as a hepatocyte substitute, and they share many of the characteristics of pluripotent embryonic stem cells while lacking their safety and ethical concerns. We reported that like hepatocytes, transplantation of hAEC significantly improved survival and lifespan, normalized body weight, and significantly improved branched-chain amino acid (BCAA) levels in sera and brain in a transgenic murine model of intermediate maple syrup urine disease (imsud). In the current report, we detail the neural and peripheral metabolic improvements associated with hAEC transplant in imsud mice, including amino acids associated with bioenergetics, the urea cycle, as well as the neurotransmitter systems for serotonin, dopamine, and gamma-aminobutyric acid (GABA). This stem cell therapy results in significant global correction of the metabolic profile that characterizes the disease, both in the periphery and the central nervous system, the target organ for toxicity in iMSUD. The significant correction of the disease phenotype, coupled with the theoretical benefits of hAEC, particularly their lack of immunogenicity and tumorigenicity, suggests that human amnion epithelial cells deserve serious consideration for clinical application to treat metabolic liver diseases.

Dihydrolipoamide dehydrogenase deficiency: a still overlooked cause of recurrent acute liver failure and Reye-like syndrome.

The causes of Reye-like syndrome are not completely understood. Dihydrolipoamide dehydrogenase (DLD or E3) deficiency is a rare metabolic disorder causing neurological or liver impairment. Specific changes in the levels of urinary and plasma metabolites are the hallmark of the classical form of the disease. Here, we report a consanguineous family of Algerian origin with DLD deficiency presenting without suggestive clinical laboratory and anatomopathological findings. Two children died at birth from hepatic failure and three currently adult siblings had recurrent episodes of hepatic cytolysis associated with liver failure or Reye-like syndrome from infancy. Biochemical investigation (lactate, pyruvate, aminoacids in plasma, organic acids in urine) was normal. Histologic examination of liver and muscle showed mild lipid inclusions that were only visible by electron microscopy. The diagnosis of DLD deficiency was possible only after genome-wide linkage analysis, confirmed by a homozygous mutation (p.G229C) in the DLD gene, previously reported in patients with the same geographic origin. DLD and pyruvate dehydrogenase activities were respectively reduced to 25% and 70% in skin fibroblasts of patients and were unresponsive to riboflavin supplementation. In conclusion, this observation clearly supports the view that DLD deficiency should be considered in patients with Reye-like syndrome or liver failure even in the absence of suggestive biochemical findings, with the p.G229C mutation screening as a valuable test in the Arab patients because of its high frequency. It also highlights the usefulness of genome-wide linkage analysis for decisive diagnosis advance in inherited metabolic disorders.

Erythrocyte glutathione peroxidase activity and plasma selenium concentration are reduced in maple syrup urine disease patients during treatment.

Maple syrup urine disease (MSUD) is an inherited disorder caused by a deficiency of the branched-chain alpha-keto acid dehydrogenase complex activity. In the present study we evaluated selenium levels in plasma from MSUD patients at diagnosis and under treatment and the activities of glutathione peroxidase, catalase and superoxide dismutase in erythrocytes from treated patients. We verified that MSUD patients present a significant selenium deficiency at diagnosis, which becomes more pronounced during treatment, as well as a decrease of erythrocyte glutathione peroxidase activity during treatment. In contrast, erythrocyte catalase and superoxide dismutase activities were not altered in these patients. Our present results suggest that the reduction of an important antioxidant enzyme activity may be partially involved in the pathomechanisms of this disorder and that plasma selenium levels must be corrected through dietary supplementation in MSUD patients.

Diagnosis of MSUD by newborn screening allows early intervention without extraneous detoxification.

Maple syrup urine disease (MSUD) is a genetic metabolic disorder resulting from the defective activity of branched-chain 2-ketoacid dehydrogenase complex. Due to the metabolic block, high concentrations of the branched-chain amino acids (BCAA) leucine, valine, isoleucine, and allo-isoleucine as well as their corresponding branched-chain 2-keto acids accumulate in patients on a BCAA-unrestricted diet or during episodes with increased protein catabolism. Early diagnosis and management are essential to prevent permanent brain damage. Newborn screening by tandem MS allows for detection of elevated BCAA concentrations in blood in patients with classical MSUD before they show severe encephalopathic symptoms. Here, we report that newborn screening by expanded tandem MS enables for reversing the intoxication in newborns with MSUD within 24-48 h without any need for extraneous detoxification and thus decreasing the risk of brain damage during a particularly vulnerable period.

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.

Significance of L-alloisoleucine in plasma for diagnosis of maple syrup urine disease.

BACKGROUND: The significance of plasma L-alloisoleucine, which is derived from L-isoleucine in vivo, for diagnosis of maple syrup urine disease (MSUD) was examined. METHODS: Branched-chain L-amino acids were measured by automatic amino acid analysis. RESULTS: Alloisoleucine reference values in plasma were established in healthy adults [1.9 +/- 0.6 micromol/L (mean +/- SD); n = 35], children 3-11 years (1.6 +/- 0.4 micromol/L; n = 17), and infants <3 years (1.3 +/- 0.5 micromol/L; n = 37). The effect of dietary isoleucine was assessed in oral loading tests. In controls receiving 38 micromol (n = 6; low dose) and 1527 micromol (n = 3; high dose) of L-isoleucine per kilogram of body weight, peak increases of plasma isoleucine were 78 +/- 24 and 1763 +/- 133 micromol/L, respectively; the peak increase of alloisoleucine, however, was negligible for low-dose (<0.3 micromol/L) and minor for high-dose (5. 5 +/- 2.1 micromol/L) load. In patients with diabetes mellitus, ketotic hypoglycemia, phenylketonuria, and obligate heterozygous parents of MSUD patients, alloisoleucine was not significantly different from healthy subjects. Therefore, a plasma concentration of 5 micromol/L was used as a cutoff value. In patients with classical MSUD (n = 7), alloisoleucine was beyond the cutoff value in 2451 of 2453 unselected samples. In patients with variant MSUD (n = 9), alloisoleucine was >5 micromol/L in all samples taken for establishment of diagnosis and in 94% of the samples taken for treatment control (n = 624). With the other branched-chain amino acids, the frequency of diagnostically significant increases was <45%. CONCLUSIONS: The present findings indicate that plasma L-alloisoleucine above the cutoff value of 5 micromol/L is the most specific and most sensitive diagnostic marker for all forms of MSUD.

Sugar nucleotide concentrations in red blood cells of patients on protein- and lactose-limited diets: effect of galactose supplementation.

Uridine diphosphate (UDP) galactose, a pivotal compound in the metabolism of galactose, is the obligate donor of galactose in the formation of complex glycoconjugates. The cellular UDPgalactose concentration has been thought to be maintained by the interconversion of UDPglucose and UDPgalactose by UDPgalactose-4-epimerase. However, recent findings of lower average red blood cell (RBC) UDPgalactose concentrations in galactose-1-phosphate uridyltransferase-deficient patients suggest that other factors play a role in determining its concentration. To test the hypothesis that the amount of galactose traversing the Leloir pathway contributes to the cellular UDPgalactose pool, we determined RBC UDPgalactose in patients with maple syrup urine disease (MSUD), phenylketonuria (PKU), and other metabolic diseases who were treated with a low-protein, and consequently, low-lactose diet. Six patients with MSUD were also supplemented with 19 g galactose/d and their UDPhexose concentrations were measured at intervals. We show that young patients with MSUD or PKU have decreased average RBC UDPgalactose concentrations when compared with similarly aged healthy subjects. Galactose supplementation of MSUD patients significantly increased their UDPgalactose concentrations in both RBCs and white blood cells (WBCs) from 29.5 +/- 1.5 to 42.3 +/- 5.8 nmol/g hemoglobin and from 69.0 +/- 7.5 to 193.0 +/- 49.0 nmol/g protein, respectively. Discontinuation of supplementation was associated with a return to basal values in RBCs and a reattainment of the pretreatment ratio of UDPglucose to UDPgalactose in WBCs. These observations demonstrate that dietary galactose is a factor in establishing the steady state concentrations of the uridine sugar nucleotides and imply that galactose metabolism modulates the achievement of an epimerase-mediated equilibrium.

Effect of selenium supplementation on the distribution of selenium among plasma proteins of a patient with maple syrup urine disease.

Selenium (Se) status was studied in a patient with classical maple syrup urine disease (MSUD) receiving Se supplement. The basal plasma Se concentration was 0.06 mumol/l increasing to 2.1 mumol/l after 40 days of supplementation. When the plasma Se distribution was analysed by gel filtration, a major peak was seen close to the high molecular weight proteins with a second peak in the albumin region. When the Se dose was decreased in a stepwise manner from 50 micrograms/day to 25 micrograms/day and then to 17 micrograms/day plasma Se decreased, but the proportion of plasma Se in the two protein peaks did not change. In a healthy girl not supplemented with Se, the proportion of plasma Se in the albumin region was somewhat lower. In the MSUD patient glutathione peroxidase activity was initially low, and increased ten-fold during Se supplementation. The study indicates that the Se requirement for plasma glutathione peroxidase activity was fulfilled at the lowest dose of Se used and that Se is incorporated into several plasma proteins after supplementation.

Selenium concentration and glutathione peroxidase activity in blood of New Zealand infants and children.

The blood selenium (Se) concentrations of New Zealand children were lower than those reported for children living in other countries. This low blood Se was primarily determined by the low dietary intake of the children which, in turn, reflects the low Se content of New Zealand soils. Blood Se also varied geographically, with age, and with differences in quantities and types of food eaten. Children with phenylketonuria and maple syrup urine disease on synthetic diets had low Se intakes and blood Se concentrations compared with children on normal diets, and blood Se was seen to decrease with the length of time on these diets. A strong correlation (r = 0.62, P less than 0.001) was found between the blood Se levels and glutathione peroxidase activities for 107 children. Glutathione peroxidase activities of the children were lower than activities observed in New Zealand adults, refelecting their lower blood Se concentrations.

Serum-selenium concentrations in patients with maple-syrup-urine disease and phenylketonuria under dieto-therapy.

Serum-selenium concentrations were measured in 2 dietetically treated patients with maple-syrup-urine disease, in 11 dietetically treated patients with phenylketonuria, in 37 healthy children of different ages and in 183 healthy adults. The estimations were performed by instrumental neutron activation analysis. The values of the dietetically treated patients were much lower than those of healthy children of the same age group. Within 8 to 12 weeks the serum-selenium concentrations decreased from normal values before therapy to very low values under dieto-therapy. During infancy the serum-selenium concentrations of healthy individuals show an increase to the adult range of values.