The effect of casein glycomacropeptide versus free synthetic amino acids for early treatment of phenylketonuria in a mice model.

INTRODUCTION: Management of phenylketonuria (PKU) is mainly achieved through dietary control with limited intake of phenylalanine (Phe) from food, supplemented with low protein (LP) food and a mixture of free synthetic (FS) amino acids (AA) (FSAA). Casein glycomacropeptide (CGMP) is a natural peptide released in whey during cheese making by the action of the enzyme chymosin. Because CGMP in its pure form does not contain Phe, it is nutritionally suitable as a supplement in the diet for PKU when enriched with specific AAs. Lacprodan® CGMP-20 (= CGMP) used in this study contained only trace amounts of Phe due to minor presence of other proteins/peptides. OBJECTIVE: The aims were to address the following questions in a classical PKU mouse model: Study 1, off diet: Can pure CGMP or CGMP supplemented with Large Neutral Amino Acids (LNAA) as a supplement to normal diet significantly lower the content of Phe in the brain compared to a control group on normal diet, and does supplementation of selected LNAA results in significant lower brain Phe level?. Study 2, on diet: Does a combination of CGMP, essential (non-Phe) EAAs and LP diet, provide similar plasma and brain Phe levels, growth and behavioral skills as a formula which alone consist of FSAA, with a similar composition?. MATERIAL AND METHODS: 45 female mice homozygous for the Pahenu2 mutation were treated for 12 weeks in five different groups; G1(N-CGMP), fed on Normal (N) casein diet (75%) in combination with CGMP (25%); G2 (N-CGMP-LNAA), fed on Normal (N) casein diet (75%) in combination with CGMP (19,7%) and selected LNAA (5,3% Leu, Tyr and Trp); G3 (N), fed on normal casein diet (100%); G4 (CGMP-EAA-LP), fed on CGMP (70,4%) in combination with essential AA (19,6%) and LP diet; G5 (FSAA-LP), fed on FSAA (100%) and LP diet. The following parameters were measured during the treatment period: Plasma AA profiles including Phe and Tyr, growth, food and water intake and number of teeth cut. At the end of the treatment period, a body scan (fat and lean body mass) and a behavioral test (Barnes Maze) were performed. Finally, the brains were examined for content of Phe, Tyr, Trp, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and 5-hydroxyindole-acetic acid (5-HIAA), and the bone density and bone mineral content were determined by dual-energy x-ray absorptiometry. RESULTS: Study 1: Mice off diet supplemented with CGMP (G1 (N-CGMP)) or supplemented with CGMP in combination with LNAA (G2 (N-CGMP-LNAA)) had significantly lower Phe in plasma and in the brain compared to mice fed only casein (G3 (N)). Extra LNAA (Tyr, Trp and Leu) to CGMP did not have any significant impact on Phe levels in the plasma and brain, but an increase in serotonin was measured in the brain of G2 mice compared to G1. Study 2: PKU mice fed with mixture of CGMP and EAA as supplement to LP diet (G4 (CGMP-EAA-LP)) demonstrated lower plasma-Phe levels but similar brain- Phe levels and growth as mice fed on an almost identical combination of FSAA (G5 (FSAA-LP)). CONCLUSION: CGMP can be a relevant supplement for the treatment of PKU.

Tyrosine metabolism in health and disease: slow-release amino acids therapy improves tyrosine homeostasis in phenylketonuria.

OBJECTIVES: Phenylalanine (Phe) hydroxylase (PAH) deficiency leads to hyperphenylalaninemia (HPA) and tyrosine (Tyr) depletion. We investigated Tyr homeostasis in patients with PAH deficiency and the effect of a slow-release amino acids therapy in phenylketonuria (PKU). METHODS: We performed four complementary investigations: (1) Tyr concentrations were monitored in 114 patients (10.6 ± 11.9 years) with PKU on dietary treatment supplemented with traditional amino acid formulations (n=52, 1175 samples) or non-PKU HPA on a free diet (n=62, 430 samples); (2) Tyr metabolism in PKU was quantitatively evaluated in three patients by a simple Tyr oral loading test (100 mg/kg); (3) diurnal and (4) long-term Tyr concentrations were evaluated in 5 and 13 patients with PKU, respectively, who switched from traditional to slow-release amino acids therapy. RESULTS: 1) Tyr concentrations in the PKU population were subnormal and significantly lower than in non-PKU HPA (p<0.01); (2) the response to a Tyr loading test in PKU was normal, with basal Tyr concentrations reached within 12 h; (3) the diurnal metabolic profile in patients on slow-release amino acids therapy revealed higher morning fasting and nocturnal Tyr concentrations with respect to traditional therapy (p<0.01); (4) this picture was confirmed at follow-up, with normalization of morning fasting Tyr concentrations in patients on slow-release amino acids therapy (p<0.01) and unchanged Phe control (p=0.19). CONCLUSIONS: Slow-release amino acids therapy can improve Tyr homeostasis in PKU. If associated to optimized Phe control, such a metabolic goal may allow long-term clinical benefits in patients with PKU.

Long-term dietary intervention with low Phe and/or a specific nutrient combination improve certain aspects of brain functioning in phenylketonuria (PKU).

INTRODUCTION: In phenylketonuria (PKU), a gene mutation in the phenylalanine metabolic pathway causes accumulation of phenylalanine (Phe) in blood and brain. Although early introduction of a Phe-restricted diet can prevent severe symptoms from developing, patients who are diagnosed and treated early still experience deficits in cognitive functioning indicating shortcomings of current treatment. In the search for new and/or additional treatment strategies, a specific nutrient combination (SNC) was postulated to improve brain function in PKU. In this study, a long-term dietary intervention with a low-Phe diet, a specific combination of nutrients designed to improve brain function, or both concepts together was investigated in male and female BTBR PKU and WT mice. MATERIAL & METHODS: 48 homozygous wild-types (WT, +/+) and 96 PKU BTBRPah2 (-/-) male and female mice received dietary interventions from postnatal day 31 till 10 months of age and were distributed in the following six groups: high Phe diet (WT C-HP, PKU C-HP), high Phe plus specific nutrient combination (WT SNC-HP, PKU SNC-HP), PKU low-Phe diet (PKU C-LP), and PKU low-Phe diet plus specific nutrient combination (PKU SNC- LP). Memory and motor function were tested at time points 3, 6, and 9 months after treatment initiation in the open field (OF), novel object recognition test (NOR), spatial object recognition test (SOR), and the balance beam (BB). At the end of the experiments, brain neurotransmitter concentrations were determined. RESULTS: In the NOR, we found that PKU mice, despite being subjected to high Phe conditions, could master the task on all three time points when supplemented with SNC. Under low Phe conditions, PKU mice on control diet could master the NOR at all three time points, while PKU mice on the SNC supplemented diet could master the task at time points 6 and 9 months. SNC supplementation did not consistently influence the performance in the OF, SOR or BB in PKU mice. The low Phe diet was able to normalize concentrations of norepinephrine and serotonin; however, these neurotransmitters were not influenced by SNC supplementation. CONCLUSION: This study demonstrates that both a long-lasting low Phe diet, the diet enriched with SNC, as well as the combined diet was able to ameliorate some, but not all of these PKU-induced abnormalities. Specifically, this study is the first long-term intervention study in BTBR PKU mice that shows that SNC supplementation can specifically improve novel object recognition.

Sapropterina no tratamento da hiperfenilalaninemia com deficiência de BH4 / Sapropterin in the treatment of hyperphenylalaninemia with BH4 deficiency

Fenilcetonúria (FNC) é uma doença genética, autossômica recessiva, causada por mutações no gene localizado no cromossomo 12q22-q24, o qual codifica a enzima hepática fenilalanina-hidroxilase (FAH). Sua ausência ou deficiência impede a conversão hepática de fenilalanina (FAL), um dos aminoácidos essenciais e mais comuns do organismo, em tirosina, causando acúmulo de FAL no sangue e em outros tecidos. O aumento de fenilalanina no sangue em 98% dos casos é devido a mutações na codificação genética para a enzima fenilalanina-hidroxilase, enquanto 2% são devidos a defeitos no metabolismo da tetrahidrobiopterina (BH4), que é um cofator essencial para a atividade da fenilalanina-hidroxilase. A principal característica da doença não tratada é retardo mental, com piora durante a fase de desenvolvimento do cérebro e que se estabilizaria com a maturação completa deste órgão. O quociente de inteligência (QI) mede a extensão deste retardo e varia de leve a gravemente prejudicado. A HFA não tratada resulta em progressivo retardo mental, com QI < 50. A piora está relacionada aos níveis sanguíneos de FAL. Caso a doença seja diagnosticada logo após o nascimento e o paciente for mantido em dieta restrita em FAL, os sintomas podem ser prevenidos e a criança pode ter desenvolvimento e expectativa de vidas normais. Nesse sentido, o rastreamento no Brasil é realizado pelo teste do pezinho, cuja necessidade consta no Estatuto da Criança e do Adolescente, e está regulamentado pela portaria que estabeleceu o Programa Nacional de Triagem Neonatal para diagnóstico precoce de fenilcetonúria. No Protocolo Clínico e Diretrizes Terapêuticas do Ministério da Saúde para Fenilcetonúria foram incluídos os pacientes com níveis de FAL≥ 10mg/dl (600 µmol/l) em dieta normal 1,14 e todos os que apresentarem níveis de FAL entre 8 e 10 mg/dl persistentes (pelo menos em 3 dosagens consecutivas, semanais, em dieta normal). Dieta restrita em FAL é eficaz em reduzir os níveis sanguíneos de FAL e melhorar o QI e o prognóstico neuropsicológico dos pacientes com HFA. O tratamento deve ser iniciado tão cedo quanto possível, idealmente até o 10º dia de vida. O aleitamento materno deve ser encorajado e associado ao uso de fórmula isenta de FAL. Os níveis de FAL devem ser diminuídos rapidamente. Além da dieta, o tratamento clínico recomendado pelo PCDT do Ministério da Saúde para o controle metabólico dos pacientes é a utilização de fórmulas alimentares especiais. As fórmulas são medicamentos que devem conter as quantidades recomendadas de vitaminas e sais minerais adequadas à faixa etária do paciente. Sapropterina é uma forma sintética oral de BH4 (BH4 supplementation sapropterin dihydrochloride). Há relatos de casos de pessoas com FCN que apresentaram boa resposta após o uso de doses farmacológicas de BH4, com redução dos níveis de FAL. Todos tinham mutação no gene FAH. Pessoas com resposta ao BH4 são identificadas inicialmente por um teste com teste de tolerância a BH4. Resposta positiva é considerada como uma redução de 30% ou mais na concentração de FAL, 24 horas após a administração de BH4. A variação na intensidade da resposta é independente da gravidade da FCN, da dose de BH4 empregada no teste de tolerância, duração do teste e genótipo. Pessoas com mesmo genótipo mostram respostas diferentes. Há poucos resultados de uso de longa duração de BH4 que mostram que pode haver relaxamento da restrição dietética sem efeitos adversos. A maioria dos indivíduos dos estudos apresentava doença moderada ou leve. A Secretaria-Executiva da CONITEC realizou busca na literatura por artigos científicos, com o objetivo de localizar a melhor evidência científica disponível sobre o tema. A CONITEC em sua 14ª reunião ordinária realizada no dia 04 de abril de 2013, recomendou a não incorporação no SUS da sapropterina para o tratamento de hiperfenilalaninemia (HFA) com deficiência em tetrahidrobiopterina (BH4). Considerou-se que os estudos, a maioria de baixa qualidade metodológica, não conseguiram comprovar a superioridade do tratamento, principalmente no que diz respeito à ausência de dados específicos para o subgrupo com deficiência de BH4. Os membros da CONITEC presentes na 15ª reunião do plenário do dia 09/05/2013 deliberaram, por unanimidade, por não recomendar a sapropterina para o tratamento de hiperfenilalaninemia (HFA) com deficiência em tetrahidrobiopterina (BH4). A Portaria nº 34, de 6 de agosto de 2013 - Torna pública a decisão de não incorporar o medicamento sapropterina no tratamento da hiperfenilalaninemia com deficiência de BH4 no Sistema Único de Saúde (SUS).

Uncoupling of eNOS causes superoxide anion production and impairs NO signaling in the cerebral microvessels of hph-1 mice.

In this study, we used the GTP cyclohydrolase I-deficient mice, i.e., hyperphenylalaninemic (hph-1) mice, to test the hypothesis that the loss of tetrahydrobiopterin (BH(4)) in cerebral microvessels causes endothelial nitric oxide synthase (eNOS) uncoupling, resulting in increased superoxide anion production and inhibition of endothelial nitric oxide signaling. Both homozygous mutant (hph-1(-/-)) and heterozygous mutant (hph-1(+/-) mice) demonstrated reduction in GTP cyclohydrolase I activity and reduced bioavailability of BH(4). In the cerebral microvessels of hph-1(+/-) and hph-1(-/-) mice, increased superoxide anion production was inhibited by supplementation of BH(4) or NOS inhibitor- L- N(G) -nitro arginine-methyl ester, indicative of eNOS uncoupling. Expression of 3-nitrotyrosine was significantly increased, whereas NO production and cGMP levels were significantly reduced. Expressions of antioxidant enzymes namely copper and zinc superoxide dismutase, manganese superoxide dismutase, and catalase were not affected by uncoupling of eNOS. Reduced levels of BH(4), increased superoxide anion production, as well as inhibition of NO signaling were not different between the microvessels of male and female mice. The results of our study are the first to demonstrate that, regardless of gender, reduced BH(4) bioavailability causes eNOS uncoupling, increases superoxide anion production, inhibits eNOS/cGMP signaling, and imposes significant oxidative stress in the cerebral microvasculature.

Novel pharmacological chaperones that correct phenylketonuria in mice.

Phenylketonuria (PKU) is caused by inherited phenylalanine-hydroxylase (PAH) deficiency and, in many genotypes, it is associated with protein misfolding. The natural cofactor of PAH, tetrahydrobiopterin (BH(4)), can act as a pharmacological chaperone (PC) that rescues enzyme function. However, BH(4) shows limited efficacy in some PKU genotypes and its chemical synthesis is very costly. Taking an integrated drug discovery approach which has not been applied to this target before, we identified alternative PCs for the treatment of PKU. Shape-focused virtual screening of the National Cancer Institute's chemical library identified 84 candidate molecules with potential to bind to the active site of PAH. An in vitro evaluation of these yielded six compounds that restored the enzymatic activity of the unstable PAHV106A variant and increased its stability in cell-based assays against proteolytic degradation. During a 3-day treatment study, two compounds (benzylhydantoin and 6-amino-5-(benzylamino)-uracil) substantially improved the in vivo Phe oxidation and blood Phe concentrations of PKU mice (Pah(enu1)). Notably, benzylhydantoin was twice as effective as tetrahydrobiopterin. In conclusion, we identified two PCs with high in vivo efficacy that may be further developed into a more effective drug treatment of PKU.

Phenylalanine hydroxylase deficiency.

Phenylalanine hydroxylase deficiency is an autosomal recessive disorder that results in intolerance to the dietary intake of the essential amino acid phenylalanine. It occurs in approximately 1:15,000 individuals. Deficiency of this enzyme produces a spectrum of disorders including classic phenylketonuria, mild phenylketonuria, and mild hyperphenylalaninemia. Classic phenylketonuria is caused by a complete or near-complete deficiency of phenylalanine hydroxylase activity and without dietary restriction of phenylalanine most children will develop profound and irreversible intellectual disability. Mild phenylketonuria and mild hyperphenylalaninemia are associated with lower risk of impaired cognitive development in the absence of treatment. Phenylalanine hydroxylase deficiency can be diagnosed by newborn screening based on detection of the presence of hyperphenylalaninemia using the Guthrie microbial inhibition assay or other assays on a blood spot obtained from a heel prick. Since the introduction of newborn screening, the major neurologic consequences of hyperphenylalaninemia have been largely eradicated. Affected individuals can lead normal lives. However, recent data suggest that homeostasis is not fully restored with current therapy. Treated individuals have a higher incidence of neuropsychological problems. The mainstay of treatment for hyperphenylalaninemia involves a low-protein diet and use of a phenylalanine-free medical formula. This treatment must commence as soon as possible after birth and should continue for life. Regular monitoring of plasma phenylalanine and tyrosine concentrations is necessary. Targets of plasma phenylalanine of 120-360 µmol/L (2-6 mg/dL) in the first decade of life are essential for optimal outcome. Phenylalanine targets in adolescence and adulthood are less clear. A significant proportion of patients with phenylketonuria may benefit from adjuvant therapy with 6R-tetrahydrobiopterin stereoisomer. Special consideration must be given to adult women with hyperphenylalaninemia because of the teratogenic effects of phenylalanine. Women with phenylalanine hydroxylase deficiency considering pregnancy should follow special guidelines and assure adequate energy intake with the proper proportion of protein, fat, and carbohydrates to minimize risks to the developing fetus. Molecular genetic testing of the phenylalanine hydroxylase gene is available for genetic counseling purposes to determine carrier status of at-risk relatives and for prenatal testing.

Impact of neonatal protein metabolism and nutrition on screening for phenylketonuria.

OBJECTIVES: Early blood phenylalanine (Phe) elevation after birth enables screening for and anticipation of the diagnosis of phenylketonuria. The differential impact of factors involved in this phenomenon, however, has not been elucidated. To solve this question, phenotype, genotype, dietary Phe intake, timing of blood collection, and Phe metabolism were retrospectively analyzed in 21 phenylketonuria newborns and prospectively in 1. PATIENTS AND METHODS: Patients were assigned to 1 of 4 classes of phenylalanine hydroxylase (PAH) deficiency (severe, moderate, mild, and benign) on the basis of their Phe tolerance. Phe ingested, tolerated, and released from endogenous catabolism was assessed. RESULTS: From birth to screening test, the amount of Phe tolerated ranged from 704 to 1620 mg, according to the class of PAH deficiency. The amount of Phe ingested ranged only from 204 to 405 mg, whereas the endogenous Phe breakdown ranged from 812 to 1534 mg, resulting in a rate of Phe catabolism ranging from 262 to 341 mg/day, regardless of the class of PAH deficiency. CONCLUSIONS: The high rate of protein catabolism is the main determinant of neonatal hyperphenylalaninemia. It is sufficient to turn to positive the screening test in severe and moderate PAH deficiency. In mild and benign PAH deficiency, the outcome of screening procedures can be substantially altered by the concurrence of genetic and peristaltic factors. These results imply that the value of blood Phe at the screening test is not fully predictive of the phenylketonuria phenotype, and strengthen concerns regarding the reliability of early screening procedures.

Effects of tetrahydrobiopterin and phenylalanine on in vivo human phenylalanine hydroxylase by phenylalanine breath test.

BH(4) administration results in the reduction of blood phenylalanine level in patients with tetrahydrobiopterin (BH(4))-responsive phenylalanine hydroxylase (PAH) deficiency. The mechanism underlying BH(4) response remains unknown. Here, we studied the effects of BH(4) and phenylalanine on in vivo PAH activity of normal controls using the phenylalanine breath test (PBT) by converting l-[1-(13)C] phenylalanine to (13)CO(2). Phenylalanine oxidation rates were expressed as Delta(13)C ((13)CO(2)/(12+13)CO(2), per thousand) and cumulative recovery rates over 120min (CRR(120), %; total amount of (13)CO(2)/the administered dose of (13)C-phenylalanine). Under physiological conditions of blood phenylalanine, BH(4) administration reduced the Delta(13)C peak from 40.8 per thousand to 21.6 per thousand and CRR(120) from 16.9% to 10.2%. Under high blood phenylalanine conditions, administration of BH(4) increased the Delta(13)C peak from 30.7 per thousand to 46.0 per thousand, while the CRR(120) was similar between phenylalanine (19.9%) and phenylalanine+BH(4) (21.1%) groups. Corrected Delta(13)C and CRR(120) were calculated against serum phenylalanine levels to remove the effects of phenylalanine loading. After BH(4) administration, the corrected Delta(13)C peak increased from 82.7 per thousand to 112.6 per thousand, while the corrected CRR(120) was similar (47.6% and 45.6%). These results indicate that phenylalanine worked as a regulator of in vivo PAH by serving as both a substrate and an activator for the enzyme. Excessive dosages of BH(4) inhibited PAH under normal phenylalanine conditions and activated PAH under conditions of high phenylalanine. The regulation system is therefore designed to maintain phenylalanine levels in the human body. Appropriate BH(4) supplementation must be reviewed in patients with BH(4)-responsive PAH deficiency.

Evolución clínica de niños con niveles plasmáticos de fenilalanina entre 2.1 y 6.0 mg/dl en el período neonatal / Clinical evolution of children with phenylalanine plasma levels between 2.1 and 6.0 mg/dl during the neonatal period

Introducción: La Academia Americana de Pediatría (AAP) ha clasificado la Fenilquetonuria (PKU) e Hiperfenilalaninemia (HFA) según la tolerancia de la ingesta de fenilalanina (FA) en: PKU clásica: 20 mg FA/kg/día, PKU moderada: 21 y 25 mg FA/kg/día y PKU leve: 25 y 50 mg FA/kg/día, e HFA benigna con dieta normal, manteniendo un nivel plasmático de FA entre 2,0 y 10,0 mg/dl. Objetivo: Evaluar la evolución clínica de 67 niños con valores de FA plasmática entre 2.1 y 6.0 mg/dL en el período neonatal. Resultados: Del total, 29 niños tenía entre 0 y 2 años, 23 entre 2 y 4 años y 15 niños eran mayores de 4 años de edad. El estado nutricional de 45 niños era normal, 14 niños estaban con sobrepeso u obesidad, y 8 casos tenían riesgo nutricional. Se determinó que 4 niños tenían una ingesta menor de 20 mg FA/kg/día, dos niños entre 21 y 25 mg FA/kg/día, 15 casos entre los 26 a 50 mg FA/kg/día y 46 niños estaban con dieta normal. Conclusión: Los recién nacidos con niveles de FA entre 2.1 y 6.0 mg/dl durante el período neonatal, tienen una evolución clínica y nutricional diferente, que puede ir desde una PKU clásica a una HFA benigna, por lo cual se recomienda mantener un control frecuente de FA sanguínea y una vigilancia nutricional, con un mínimo de 2 años de seguimiento.

Introduction: The American Academy of Pediatric (AAP) has classified Phenylketonuria (PKU) and Hyperphenylalaninaemias (HPhe) according to tolerance of phenylalanine (Phe) intake in: Classic PKU (20 mg Phe/kg/day), moderate PKU (between 21 and 25 mg Phe/kg/day) and mild PKU (between 25 and 50 mg Phe/kg/day), and benign HPhe with normal diet, maintaining blood Phe levels between 2,0 and 10,0 mg/dL. Objective: To evaluate the clinical evolution of 67 children with blood Phe values between 2,1 and 6.0 mg/dl in the neonatal period. Results: Of the total, 29 children were aged between 0 and 2 years, 23 between 2 and 4 years and 15 children were older than 4 years of age. The nutritional state of 45 children was normal, 14 children were overweight or obese, and 8 were at nutritional risk. Four children had Phe intake below 20 mg/kg/day, two children between 21 and 25 mg/kg/day; 15 cases between 26 to 50 mg/kg/day and 46 children were on normal diet. Conclusion: Newborns with blood Phe levels between 2,1 and 6,0 mg/dl in the neonatal period, had a different clinical and nutritional evolution, which could go from the classic PKU to a benign HPhe. Thus, it is recommended to keep a frequent control of plasmatic Phe levels and nutritional monitoring for a minimum of 2 years of follow up.

Effects and clinical significance of tetrahydrobiopterin supplementation in phenylalanine hydroxylase-deficient hyperphenylalaninaemia.

In recent years several studies on tetrahydrobiopterin (BH4)-responsive phenylalanine hydroxylase (PAH) deficiency have been published. The molecular mechanisms of BH4 responsiveness are not conclusively understood, but there is evidence that BH4 responsiveness in hyperphenylalaninaemia (HPA) depends on the patient's genotype and residual PAH activity. As a BH4 preparation will soon obtain marketing approval as an alternative treatment for phenylketonuria (PKU), it is particularly important to evaluate this treatment and to define criteria to identify patients with a potential benefit from it. Most of the patients found to be BH4-responsive suffered from mild PKU or mild hyperphenylalaninaemia (MHP) and some of these would not be treated at all in many countries. Of patients with moderate and classic forms of PKU, only a few were classified as responders and the clinical significance of the effect size may be small.

Wild-type phenylalanine hydroxylase activity is enhanced by tetrahydrobiopterin supplementation in vivo: an implication for therapeutic basis of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency.

We previously proposed a novel disease entity, tetrahydrobiopterin (BH4)-responsive phenylalanine hydroxylase (PAH) deficiency, in which administration of BH4 reduced elevated levels of serum phenylalanine [J. Pediatr. 135 (1999) 375-378]. Subsequent reports indicate that the prevalence of BH4-responsive PAH deficiency is much higher than initially anticipated. Although growing attention surrounds treatment with BH4, little is known about the mechanism of BH4 responsiveness. An early report indicates that BH4 concentration in rat liver was 5 microM where Km for BH4 of rat PAH was estimated to be 25 microM in an oxidation experiment using a liver slice, suggesting relative insufficiency of BH4 in liver in vivo. In the present study, we developed a breath test for mice using [1-13C]phenylalanine in order to examine the BH4 responsiveness of normal PAH in vivo. The reliability of the test was verified using BTBR mice and its mutant strain lacking PAH activity, Pahenu2. BH4 supplementation significantly enhanced 13CO2 production in C57BL/6 mice when phenylalanine was pre-loaded. Furthermore, BH4 apparently activated PAH in just 5 min. These observations suggest that submaximal PAH activity occurs at the physiological concentrations of BH4 in vivo, and that PAH activity can be rapidly enhanced by supplementation with BH4. Thus, we propose a possible hypothesis that the responsiveness to BH4 in patients with PAH deficiency is due to the fact that suboptimal physiological concentrations of BH4 are normally present in hepatocytes and the enhancement of the residual activity may be associated with a wide range of mutations.

Dietoterapia en errores innatos del metabolismo / Nutritional treatment of inborn errors of metabolism

Dependiendo del defecto enzimático, algunos errores innatos del metabolismo (EIM) responden satisfactoriamente a la manipulación de la dieta. Según la alteración metabólica, se han identificado siete formas de tratamiento nutricional, las que permiten reestablecer el balance metabólico. Una de ellas es a través de la reducción del sustrato acumulado causado por la deficiencia primaria de una enzima o por la inhibición secundaria de una de ellas. Un ejemplo ampliamente descrito en la literatura es la fenilquetonuria en la que se ha demostrado que gracias al diagnóstico neonatal temprano, seguido de un tratamiento basado en una dieta restringida en fenilalanina revierten su acumulación previniendo con ello el da¤o neurológico que la enfermedad causa al no ser tratada temprana y adecuadamente. Otra forma de tratamiento es la suplementación de una sustancia nutritiva en déficit debido al defecto metabólico, como ocurre en los defectos del ciclo de la urea, en los cuales donde la arginina debe entregarse como fármaco. Algunos EIM con defecto parcial de la enzima tienen la posibilidad de estimular vías alternas para detoxificar o evitar la síntesis de sustancias nocivas a través drogas o megadosis de vitaminas, un ejemplo es la homocistinuria donde la betaína y piridoxina reducen la producción de homocisteína. Otros defectos enzimáticos abren vías metabólicas alternas, las que generan metabolitos tóxicos, el tratamiento en estos casos, consiste en proporcionar cofactores o megadosis de vitaminas para formar complejos no tóxicos que sean excretados por vía urinaria. Es importante se¤alar que el diagnóstico precoz y el seguimiento estricto a largo plazo, permite que un ni¤o con un EIM se desarrolle normalmente.

Long term effects of long chain polyunsaturated fats in hyperphenylalaninemic children.

Blood fatty acid status and visual function of 20 treated hyperphenylalaninemic (HPA) children, randomly allocated into two groups to receive supplementation of either long chain polyunsaturated fatty acids (LCPUFA), including docosahexaenoic acid (DHA), or placebo for 12 months, have been investigated three years after the end of the treatment. Although in the LCPUFA group blood DHA levels and P100 wave latency improved at the end of supplementation, they had returned to baseline after three years.

Effects of long-chain polyunsaturated fatty acid supplementation on fatty acid status and visual function in treated children with hyperphenylalaninemia.

BACKGROUND: Children with phenylalanine-hydroxylase deficiency (type-I hyperphenylalaninemia, HPA) follow a low-phenylalanine diet, severely restricted in animal foods and long-chain polyunsaturated fatty acids (LCPUFA). Consequently, they have a poor LCPUFA status, particularly for docosahexaenoic acid (DHA). DHA is relevant to visual and neural development. OBJECTIVE: To investigate the effects of a 12-month supplementation with LCPUFA in a double-blind, placebo-controlled trial in treated children with HPA. STUDY DESIGN: Twenty children with well-controlled HPA were randomly allocated to receive either a fat supplement (supplying 26% as fatty acids including DHA, 8%) or a placebo. The fatty acid composition of erythrocyte lipids and the visual evoked potentials were measured at baseline and after 12 months of supplementation. Reference data were obtained from healthy children of comparable age. RESULTS: At baseline children with HPA had a poorer DHA status and prolonged P100 wave latencies than the reference group. At the end of the trial the LCPUFA group showed a significant increase in DHA levels of erythrocyte lipids. In the LCPUFA group P100 wave latency decreased and was negatively associated with the DHA changes. CONCLUSIONS: A balanced dietary supplementation with LCPUFA in children with HPA is associated with an increase of the DHA pool and improved visual function.

Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency.

Serum phenylalanine concentrations decreased in 4 patients with hyperphenylalaninemia after loading with tetrahydrobiopterin. There were no abnormalities in urinary pteridine excretion or in dihydropteridine reductase activity. However, mutations were detected in the phenylalanine hydroxylase gene, suggesting a novel subtype of phenylalanine hydroxylase deficiency that may respond to treatment with cofactor supplementation.

Metabolic engineering as therapy for inborn errors of metabolism--development of mice with phenylalanine hydroxylase expression in muscle.

Treatment of many inherited liver enzyme deficiencies requires the removal of toxic intermediate metabolites from the blood of affected individuals. We propose that circulating toxins can be adequately cleared and disease phenotype influenced by enzyme expressed in tissues other than the liver. Phenylalanine hydroxylase (PAH) activity was constitutively expressed in skeletal and cardiac muscle of transgenic mice which carried the PAH cDNA under the transcriptional control of the mouse muscle creatine kinase promoter. Muscle PAH-expressing mice were bred to liver PAH-deficient, hyperphenylalaninemic mice to yield progeny that lack PAH activity in liver but express PAH in muscle. These mice exhibited hyperphenylalaninemia at baseline, but serum phenylalanine levels decreased significantly when the mice were supplemented with tetrahydrobiopterin (BH4), a required cofactor for PAH. This is the first demonstration that a liver-specific enzyme, when expressed in a heterologous tissue and supplied with necessary cofactors, can effectively clear toxic metabolites from the circulation of individuals with inherited enzyme deficiency. This result suggests that gene therapy targeted to heterologous tissues, such as muscle, will be effective in the treatment of selected inborn errors of metabolism.

Seguimiento de pacientes con hiperfenilalaninemia diagnosticados precozmente / Follow-up of patients with early diagnostic of hyperphenylalaninemia

Se presentan 17 pacientes con hiperfenilalaninemia diagnosticados en el período neonatal (20,3 ñ 12,6 días de vida), cuyas concentraciones iniciales de fenilalanina sérica eran de 16,5 a 46,2 mg por ciento. En todos el tratamiento dietético comenzó al hacer el diagnóstico y han sido seguidos en un policlínico especial del Instituto de Nutrición y Tecnología de los Alimentos de la Universidad de Chile. Se describen la dieta, los suplementos de minerales y vitaminas requeridos, que han permitido, durante el seguimiento, mantener concentraciones de fenilalanina en el suero entre 2 y 6 mg/dl, salvo transgresiones transitorias que han ocurrido en 2 casos. En 82,2 por ciento de los casos el crecimiento y el desarrollo han sido normales. Se subraya la importancia de tomar la primera muestra en tarjet de papel filtro a todos los recién nacidos antes de cumplir 10 días de vida, para hecer oportunamente el diagnóstico. Los programas se seguimiento integral facilitan el manejo y el desarrollo físico y mental normales de estos niños

Enzyme replacement therapy in ENU2 phenylketonuric mice using oral microencapsulated phenylalanine ammonia-lyase: a preliminary report.

The presence of an extensive enterorecirculation of amino acids between the intestine and the body allows the removal of systemic phenylalanine in PKU rats by oral microencapsulated phenylalanine ammonia lyase. The work presented in this article has the main goal of assessing the feasibility of yeast phenylalanine ammonia-lyase (PAL) loaded collodion microcapsules in reducing elevated plasma phenylalanine concentrations to standard levels in genetically mutated PKU mice, within a 30 day time frame. The distinguishing aspect from previous studies lies in the available animal model. Rather than artificial induction of elevated phenylalanine plasma levels, the mice representing the human phenylketonuric condition, are mutated strains which are deficient in the enzyme phenylalanine hydroxylase. The first in vivo study established a method for orally feeding microcapsules, both control and enzyme loaded, over 30 consecutive days, by mixing with soft, unripened cheese. Under this unique regime a decrease of 51.3% +/- 9.02% in phenylalanine plasma levels was observed after 23 days. Reduction in the phenylalanine plasma levels to within the desired maintenance range of 250-1000 umol/L was observed in 2 out of 4 PAL treated mice, with only 50% of the PAL dose used in previous rat studies by Chang's group. The second animal study confirmed the finding in the first in vivo study that there is no significant decrease in the plasma phenylalanine levels within the first seven days. This may be due to the severely deteriorated physical condition of the ENU mice used, the PAL enzyme preparations available or the fact that normal mice contain 10 times the amount of phenylalanine hydroxylase as compared with humans, thus requiring larger doses of PAL in order to be effective in a shorter time span.