A fenilcetonúria (PKU), uma das alterações do metabolismo detectadas por meio da triagem neonatal (TN), pode acarretar alterações no desenvolvimento global do indivíduo. O objetivo deste estudo foi caracterizar as habilidades comunicativas, enfocando as habilidades do desenvolvimento nas áreas de linguagem, pessoal-social, motora fina-adaptativa e motora grossa de crianças com fenilcetonúria, diagnosticadas e tratadas precocemente e correlacionar estas habilidades com os níveis de fenilalanina plasmática. Foram avaliadas 20 crianças, sendo 10 crianças (5 do sexo feminino e 5 do masculino) com PKU detectada pela TN e que realizavam tratamento e acompanhamento no Laboratório do Teste do Pezinho, na Associação de Pais e Amigos dos Excepcionais (APAE)-Bauru e 10 crianças do grupo controle. As crianças pertenciam à faixa etária de 3 a 6 anos e 11 meses e foram pareadas quanto a sexo, idade, escolaridade e situação socioeconômica. A avaliação foi feita por meio do Teste de Screening de Desenvolvimento de Denver II (TSDD-II), Escala de Desenvolvimento Comportamental de Gesell e Amatruda (EDCGA), Teste de Vocabulário por Imagens Peabody (TVIP) e Escala de Avaliação do Desenvolvimento da Linguagem (ADL). Os resultados indicaram que as crianças apresentaram maiores comprometimentos nas áreas pessoal-social, linguagem e motora fina adaptativa. Não foram encontradas diferenças significantes entre os grupos na área motora grossa. As crianças com PKU, mesmo fazendo o acompanhamento e o tratamento indicado, não conseguiram manter, durante toda sua vida, índices de Phe nos limites de normalidade estabelecidos, o que pode ter contribuído para as dificuldades apresentadas nas habilidades avaliadas. / Phenylketonuria (PKU), one of the metabolic disorders detected by the newborn screening (NS), can cause alterations in ones global development The aim of this study is to characterize the profile of the abilities of communication focussing upon the developmental abilities in the areas of language, personal-social, fine motor-adaptive and gross motor in children with early diagnosis and treatment and correlate these abilities with the phenylalanine level serum. 20 children were evaluated, of which 10 (5 female and 5 male) with early classic phenylketonuria detected by Neonatal Screening and who were carrying through treatment and accompaniment in the Laboratory Teste do Pezinho at APAE in BAURU SP, and 10 being the control group. The children belonged to the etária band between 3 and 6 years and eleven months and were equalized regarding sex, socio-economic condition, and school instruction. The evaluation was made through the Screening Test Development Denver II (TSDD-II), Gesell and Amatruda Developmental Scales (EDCGA), Peabody Picture Vocabulary Test (TVIP) and Language Developmental Evaluation Scale (ADL). This study shows that even children with early diagnosis and treatment showed alteration in the abilities of personal-social, language and fine motor-adaptative. Dont showed differences in both groups in the gross motor area. The children with phenylketonuria, although carrying through treatment and accompaniment, couldnt keep the levels of phenylalanine within the stablished limits of normality during their lives, which may have contributed to the presented difficulties in the evaluated abilities.

Absence of functional phenylalanine hydroxylase results in phenylketonuria (PKU). Viable treatments remain few, expensive and secondary conditions such as osteopenia occur in most PKU patients. Objective 1: Given the recently described roles of gut microbes to aid host digestion, an orally administered genetically engineered probiotic as the delivery vehicle for enzyme replacement therapy was created. The engineered probiotic, pHENOMMenal, produced phenylalanine ammonia lyase with significant production of trans-cinnamate (phenylalanine cleavage product) in vitro and resulted in a reduction of 515 μM in blood phenylalanine when fed to PKU animals for 14 days (from 2307µM ± 264µM to 1792µM ± 261µM, n = 6, P < 0.05). The control probiotic produced no change in blood phenylalanine. Thus, pHENOMMenal treatment in PKU mice demonstrated engineered microbes could compensate for a metabolic deficiency of the host. Objective 2: Evaluate the PAHenu2 mouse model of PKU for a genetic discrepancy causing ocular enlargement and delayed development observed only after the PAHenu2 mutation was crossed to the C57BL/6J mouse. When compared to healthy littermates, ELISA indicated a consistent but insignificant decrease in plasma IGF-1 and an increase in ocular IGF-1 in PKU animals. SNP screening demonstrated a differential inheritance of IGF-1 alleles in healthy and PKU animals based on PAH allele inheritance. Ocular and developmental phenotypes in the PAHenu2 colony match those described in previous IGF-1 studies. Understanding the IGF-1 inheritance discrepancy will enable better osteopenia research using PAHenu2 mice and allow breeding of a healthier mouse colony for continued research. Collectively the results from this work describe a new therapeutic approach for treatment of PKU as well as a better understanding of the PAHenu2 mouse model to study this disease.

A fenilcetonúria (PKU) é uma doença metabólica causada pela deficiência da enzima fenilalanina hidroxilase, levando ao acúmulo de fenilalanina. As principais características clínicas dos pacientes com PKU não tratados são o comprometimento neuropsicológico e o retardo no desenvolvimento. O estresse oxidativo tem sido detectado em muitos erros inatos do metabolismo, incluindo PKU. A silibinina é um flavonoide proveniente da planta cardo de leite (Silybum marianum) que apresenta propriedades antioxidantes e que, após administração, é amplamente distribuída pelos tecidos. Neste trabalho, nós investigamos os efeitos da silibinina in vivo e in vitro contra o estresse oxidativo causado por elevados níveis de fenilalanina. Ratos machos e fêmeas, com 12 dias de vida no início dos experimentos, receberam injeções subcutâneas de α- metilfenilalanina e fenilalanina para realizar o modelo agudo de hiperfenilalaninemia, e o tratamento com silibinina consistiu em injeções intraperitoniais da substância na dose de 20 mg/kg. Os animais foram mortos no 14° dia de vida. Para realizar os experimentos in vitro, homogeneizados de córtex cerebral de ratos de 14 dias de vida foram incubados com fenilalanina e silibinina. In vitro e in vivo, a silibinina foi capaz de prevenir a inibição provocada pela fenilalanina nas atividades das enzimas catalase, glutationa peroxidase e glicose-6-fosfato desidrogenase. Não foram verificadas diferenças entre os grupos nas atividades da superóxido dismutase e da glutationa redutase. Além disso, a silibinina preveniu as alterações provocadas pela fenilalanina no conteúdo de carbonilas proteicas, nas substâncias reativas ao ácido tiobarbitúrico e na produção de espécies reativas. A silibinina preveniu o dano oxidativo induzido pela fenilalanina e pode ser uma potencial terapia complementar para o tratamento da PKU. / Phenylketonuria (PKU) is a metabolic disorder caused by a deficiency of phenylalanine hydroxylase, leading to accumulation of phenylalanine. The main clinical features of non-treated PKU patients are neuropsychological impairment and developmental retardation. Oxidative stress has been related to many inborn errors of metabolism including PKU. Silibinin is a flavonoid derived from the herb milk thistle (Silybum marianum) which presents antioxidant properties and is widely distributed into tissues after administration. In this study, we investigated the in vivo and in vitro effects of silibinin against oxidative stress caused by high levels of phenylalanine. Male and female rats, 12 days old at the beginning of experiments, received subcutaneous injections of α- methylphenylalanine and phenylalanine to produce hyperphenylalaninemia, and intraperitoneal injections of 20 mg/kg silibinin. The animals were killed on the 14th day of life. To perform in vitro experiments, cerebral cortex homogenates of 14 days old rats were incubated with phenylalanine and silibinin. In vivo and in vitro, silibinin was able to prevent the inhibition provoked by phenylalanine on the activities of catalase, glutathione peroxidase and glucose-6-phosphate dehydrogenase. No differences were found among the groups in the activities of superoxide dismutase and glutathione reductase. Moreover, silibinin prevented the alterations provoked by phenylalanine on protein carbonyl content, thiobarbituric acid-reactive substances and production of reactive species. Silibinin prevented oxidative damage induced by phenylalanine and may be a potential adjunctive therapy to PKU treatment.

El temblor en la fenilcetonuria es un trastorno del movimiento muy prevalente entre pacientes de diagnóstico precoz y tardío. Se trata de un temblor rápido y muy regular, localizado fundamentalmente en extremidades superiores, cuya amplitud aumenta con el movimiento sobre todo en los pacientes de diagnóstico tardío, en quienes puede causar una discapacidad para las tareas cotidianas. Los datos neurofisiológicos sugieren que el temblor está causado por una disfunción de circuitos neuronales a nivel del sistema nervioso central, pudiendo ser considerado como un indicador de daño cerebral. El temblor en la PKU se asocia a un descenso en las concentraciones plasmáticas de CoQ10 en los pacientes con un adecuado control metabólico, así como a una edad tardía de diagnóstico de la enfermedad. Debido a la asociación descrita entre el déficit de CoQ10 y el aumento del estrés oxidativo en la PKU, nuestros resultados sugieren que el déficit de coenzima Q10 podría actuar induciendo un daño oxidativo a nivel de los circuitos implicados en la fisiopatología del temblor en estos enfermos. Los pacientes fenilcetonúricos en tratamiento dietético presentan alteraciones significativas en el volumen de la sustancia blanca y sustancia gris, cuya aparición está relacionada con la duración y el adecuado cumplimiento del tratamiento dietético. Existe una distribución específica de esta pérdida de volumen a nivel de las áreas motoras corticales, ganglios basales y sustancia blanca periventricular, cuyo significado clínico deberá ser ampliado en futuros trabajos. En cuanto a las nuevas opciones terapéuticas en la PKU, la prueba de sobrecarga combinada con Phe/BH4 es una herramienta muy útil para la selección de los pacientes candidatos a iniciar un tratamiento con BH4. Considerando lo difícil que resulta predecir la respuesta a la BH4 a partir del genotipo, recomendamos realizar la prueba de sobrecarga a todos los pacientes PKU, exceptuando solo aquellos pacientes con mutaciones nulas en ambos alelos. Finalmente, consideramos que el tratamiento con BH4 es seguro y eficaz en un grupo seleccionado de pacientes PKU con fenotipos leves y moderados. La utilización de dosis bajas de BH4 permite liberalizar la dieta y retirar la fórmula especial, garantizando un desarrollo nutricional y cognitivo adecuados sin que se hayan observado efectos indeseados. En conclusión, pese a un adecuado control metabólico, los pacientes fenilcetonúricos en tratamiento dietético presentan signos clínicos de disfunción neurológica así como anomalías estructurales en el parénquima cerebral. En su origen están implicados múltiples factores; en primer lugar relacionados con el inicio, la duración y el adecuado cumplimiento dietético, pero también alteraciones metabólicas secundarias como el déficit de CoQ10 y el aumento del estrés oxidativo. El tratamiento con tetrahidrobiopterina representa una alternativa terapéutica a la dieta restrictiva en Phe en un grupo seleccionado de pacientes con fenotipos leves y moderados, en quienes permite optimizar el control metabólico y garantizar un adecuado desarrollo cognitivo y nutricional. / BRAIN INJURY IN PHENYLKETONURIA: PATHOPHYSIOLOGY AND NOVEL THERAPEUTIC APPROACH” Tremor is a common sign in phenylketonuric patients under dietary treatment. It is more frequent and severe among late-treated PKU patients. The cause of tremor is unknown, but neurophysiological data suggest that it is more dependen ton abnormalities in central nervous system neural networks than on peripheral circuits involving mechanical oscillations. If such is the case, tremor may be an index of cerebral damage in PKU patients. Tremor is associated with low concentrations of CoQ10 in patients under dietary treatment, potentially leading to increased oxidative stress damage in central neural networks. Treated patients may show significant gray and white matter volume changes related to the duration and strict observation of dietary treatment. Voxel-based maps revealed a significant gray and white matter reduction in motor and premotor cortex, thalamus and periventricular white matter. Further studies are needed to investigate whether the presence of neurologic symptoms may be explained by specific anatomic alterations. Regarding new therapeutic options in PKU, we found that Phe/BH4 loeading should be recommended for al PKU patients before starting BH4 therapy considering the difficulties in predicting a good response on the basis of patients genotype. Finally, our data confirm that BH4 is a safe and effective therapy in a selected group of mild and moderate PKU patients. Low doses of BH4 in monoteraphy allow for withdrawal of the special formula and guarantee a good clinical and nutritional outcome with no adverse side effects in PKU patients.

Background Tetrahydrobiopterin (BH4)-sensitive phenylketonuria (PKU) can be treated with sapropterin dihydrochloride. We studied metabolic control and health-related quality of life (HRQoL) in PKU patients treated with BH4. Subjects and methods Based on the review of neonatal BH4 test results and mutation analysis in 41 PKU patients, 19 were identified as potentially BH4-sensitive (9 females, 10 males, age 4–18 years). We analyzed phenylalanine (phe) concentrations in dried blood samples, nutrition protocols, and HRQoL questionnaires (KINDL®) beginning from 1 year before, during the first 42 days, and after 3 months of BH4 therapy. Results Eight BH4-sensitive patients increased their phe tolerance (629±476 vs. 2131±1084 mg, p00.006) while maintaining good metabolic control (phe concentration in dried blood 283±145 vs. 304±136 μM, p01.0). Six of them were able to stop dietary protein restriction entirely. BH4- sensitive patients had average HRQoL scores that were comparable to age-matched healthy children. There was no improvement in HRQoL scores after replacing classic dietary treatment with BH4 supply, although personal reports given by the patients and their parents suggest that available questionnaires are inappropriate to detect aspects relevant to inborn metabolic disorders. Discussion BH4 can allow PKU patients to increase their phe consumption significantly or even stop dietary protein restrictions. Unexpectedly, this does not improve HRQoL as assessed with KINDL®, partly due to high scores even before BH4 therapy. Specific questionnaires should be developed for inborn metabolic disorders.

Phenylketonuria (PKU) is one of the most common amino acids metabolisms disorders. Even though such disorders are rather scarce, it was the first case of all metabolism disorders. Phenylketonuria was first described by A. Fioling in 1934. PKU is an autosomic recessive hereditary metabolism disease that is followed by disorders in amino acids metabolism, which disorders the development of the nervous system. The earlier the treatment begins, the better prognosis of the disease is. If the baby is not being treated, his health gets worse already during the first 6 months of his life. During the first months he often does not eat well, sucks wanly, vomits. After some time after birth there appear the jumps. The baby is lacking vitality, sleepy or too much irritable. In many cases the muscular tonicity might be varied (either higher or lower). A specific smell or color of the body or urine of babies who have phenylketonuria may be common. Over time, growth, physical and mental development of the baby is out of order. Children, who have phenylketonuria, have emotional and action problems Moreover, just after birth phenylketonuria causes serious and incurable mental disorders The intelligence quotient of the majority of patients is lower than 60 points. There is a general babies’ check in order to diagnose phenylketonuria on time. The researches are being held in the Medical Genetics Centre of Vilnius Santariskes Clinic since 1975. The average of babies who have phenylketonuria is... [to full text]

Denaturing gradient gel electrophoresis (DGGE) and sequencing of the PAH locus has found 38 different mutations on 141 chromosomes in the PKU patients resident in Quebec; mutation analysis is now 92.5% complete. Two novel disease producing alleles (K421, R157N) and one silent allele (IVS6 nt-55) were discovered in this project; these mutations remain unique to the Quebec population. Three novel mutation-(haplotype) combinations were also found (S67P (H1), G218V (H2), V245A (H7)); they are not at hypermutable sites and are therefore compatible with a single homologous recombination event between two different haplotypes. Whereas mutation types (missense 64%, nonsense 6%, splice 9%, frameshifts 6%, silent 15%), resemble those in world populations, the Quebec allele profile differs from that of any European population, reflecting range expansion, founder effects, genetic drift and assimilation. Furthermore, when analyzed by geographic region a stratification of PAH alleles is apparent, reflecting the different demographic histories of Western and Eastern Quebec and Montreal.

Introdução: Os Erros Inatos do Metabolismo ocorrem devido a um defeito enzimático que determina um bloqueio em uma via metabólica, sendo a maioria herdada de forma autossômica recessiva. A fenilcetonúria (PKU) é um erro inato do metabolismo associado à atividade deficiente da enzima hepática fenilalanina hidroxilase, a qual converte a fenilalanina em tirosina. Em conseqüência, os pacientes apresentam níveis séricos elevados de fenilalanina. Quando não diagnosticada e tratada precocemente a PKU causa danos neurológicos irreversíveis. O tratamento consiste em uma dieta restrita em fenilalanina, complementada com uma fórmula metabólica. Semelhante a outras doenças crônicas, o sucesso do tratamento depende dos pacientes e suas famílias. O Ambulatório de Tratamento de Distúrbios Metabólicos do Serviço de Genética Médica do Hospital de Clínicas de Porto Alegre iniciou suas atividades em 1991 e atualmente tem 65 pacientes em acompanhamento, sendo um serviço de referência no tratamento da fenilcetonúria. Estima-se que a adesão dos pacientes seja insatisfatória. Objetivo: O presente trabalho tem por objetivo identificar os fatores associados à adesão ao tratamento dos pacientes acompanhados no Serviço de Genética Médica do Hospital de Clínicas de Porto Alegre. Métodos: Estudo transversal de base ambulatorial que incluiu 56 pacientes com diagnóstico de fenilcetonúria clássica ou atípica. Os pacientes foram classificados em aderentes e não aderentes de acordo com a mediana de phe dos últimos 12 meses de tratamento. Os dados foram coletados a partir de revisão de prontuário e entrevista com pacientes e familiares. Três questionários foram aplicados aos pacientes ou familiares e continham questões sobre condições socioeconômicas, escolaridade, variabilidade intra-familial, conhecimento sobre a doença, percepção da dieta e principais problemas associados ao tratamento da fenilcetonúria. Resultados: A mediana de idade dos pacientes foi de 12 anos. Dezoito pacientes (32,1%) pacientes foram classificados como aderentes, sendo que 11 deles apresentavam idade superior a 13 anos. Fatores como convívio com os familiares e nível de escolaridade da mãe influenciaram na adesão dos pacientes ao tratamento. Outros fatores analisados como classe socioeconômica e conhecimento sobre a doença não foram associados à adesão ao tratamento. Conclusão: Os pacientes possuem uma adesão insatisfatória ao tratamento. Embora diversos fatores possam estar associados à mesma, outros parecem não ter influência direta sobre a adesão. A adesão ao tratamento é um assunto complexo, e que necessita do entendimento e conhecimento por parte dos pacientes e de seus familiares em relação ao tratamento e a doença, e principalmente suporte e confiança na equipe médica. As dificuldades associadas ao tratamento da PKU devem ser trabalhadas em conjunto a fim de serem encontradas para cada caso, as intervenções mais efetiva. / Background: The Inborn Errors of Metabolism occurs, mostly inherited as an autosomal recessive trait. Phenylketonuria (PKU) is an inborn error of metabolism associated with deficient activity of the hepatic enzyme phenylalanine hydroxylase, which converts phenylalanine to tyrosine. As a result, patients have high serum levels of phenylalanine . When not diagnosed and treated promptly cause irreversible neurological damage. The treatment involves a diet restricted in phenylalanine, complemented with a metabolic formula . Similar to other chronic treatment success depends on patients and their families. The Ambulatory Treatment of Metabolic Disorders, Medical Genetics Service, Hospital de Clinicas de Porto Alegre starting its activities in 1991 and currently has 65 patients monitored, and a referral service for the treatment of PKU. It is estimated that compliance of patients is unsatisfactory. Objective: This study aims to identify factors associated with treatment adherence of patients followed in the Department of Medical Genetics, Hospital de Clinicas de Porto Alegre. Methods: Cross-sectional study that included 56 ambulatory patients with classical or atypical phenylketonuria. Patients were divided into compliant and noncompliant according to the median of phenylalanine in the last 12 months of treatment. Data were collected from chart review and interviews with patients and families. Three questionnaires were administered to patients or relatives and contained questions on socioeconomic status, educational level, family status, housing conditions and intra-familial variability, knowledge about the disease, perception of diet and major problems related to treatment of phenylketonuria. Results: The median age of the 56 patients was 12 years. Eighteen patients (32.1%) patients were classified as compliant, with 11 of them were older than 13 years. Factors such as contact with family members and mother's education level influenced the patients' adherence to treatment. Other factors such as socioeconomic status and knowledge about the disease were not associated with adherence to treatment. Conclusions: Patients have a poor adherence to treatment. Although several factors may be associated with it, others seem to have no direct influence on adherence. Treatment adherence is a complex issue and requires the understanding and knowledge of patients and their families regarding treatment and disease, and mainly support and confidence in the medical team. The difficulties associated with the treatment of PKU should be worked together in order to be found for each case, the most effective interventions.

Introdução: Estudos recentes, utilizando vários protocolos, têm demonstrado que pacientes com Hiperfenilalaninemia por deficiência de fenilalanina hidroxilase (HPA-PAH) podem apresentar redução das concentrações plasmáticas de fenilalanina (Phe) mediante a administração de tetrahidrobiopterina (BH4). Objetivo: Determinar, em uma amostra de pacientes brasileiros com HPA-PAH, a responsividade à administração de dose única de BH4 por meio de um protocolo incluindo o teste de sobrecarga simples de Phe e o teste combinado de sobrecarga de Phe+BH4. Métodos: Foram incluídos no estudo pacientes com idade ≥ 4 anos, em tratamento dietético e que possuíam mediana de Phe plasmática inferior a 10mg/dL no ano anterior à inclusão. Foram realizados teste de sobrecarga simples de Phe, utilizando 100mg/kg de L-Phe (Teste 1) e teste combinado de Phe+BH4, utilizando 100mg/kg de L-Phe e 20mg/kg de BH4 (Teste 2), com intervalo de uma semana entre ambos. A ingestão do BH4 ocorreu após três horas da ingestão da Phe. Foram realizadas coletas de sangue no ponto basal e após três, 11 e 27h da ingestão de Phe (T0, T1, T2 e T3 dos testes 1 e 2, respectivamente). Para ser considerado responsivo, o paciente deveria apresentar evidência de redução dos níveis de Phe associada à administração do BH4 de acordo com pelo menos um dos seguintes critérios: critério A – análise das diferenças, em percentual, dos valores de Phe nos pontos T1 e T2 dos Testes 1 e 2; critério B – análise das diferenças, em percentual, dos valores de Phe nos pontos T1 e T3 dos Testes 1 e 2 e critério C – análise da diferença, em percentual, das áreas abaixo da curva de Phe entre os Testes 1 e 2. A classificação de responsividade foi também comparada com quatro critérios adicionais: critério D – análise da diferença, em percentual, dos valores de Phe nos pontos T1 e T2 do Teste 2; critério E – análise da diferença, em percentual, dos valores de Phe nos pontos T1 e T3 do Teste 2; cinco pacientes participaram de estudo anterior do grupo e foram também classificados através do critério F – análise da diferença, em percentual, dos valores de Phe após 8h da sobrecarga simples com BH4 e do critério G – análise da diferença, em percentual, dos valores de Phe após 24h da sobrecarga simples com BH4. Para todos os critérios foi utilizado como ponte de corte redução ≥ 30%. Resultados: Dezoito pacientes, com mediana de idade de 12 anos, participaram do estudo. Dez pacientes apresentavam a forma Leve de HPA-PAH e oito a forma Clássica. Seis pacientes foram considerados responsivos de acordo com os critérios adotados (Clássica: 2; Leve: 4). Houve concordância de responsividade entre os critérios A e B em relação ao C (Índice Kappa=0,557; p=0,017). Dos pacientes com genótipo disponível (n=16), seis possuíam dados de responsividade ao BH4 descritos na literatura, que foram concordantes com os encontrados no presente estudo. Conclusão: Dados relativos à responsividade, tipo de HPA-PAH e genotipagem estão de acordo com descrito na literatura. Haja vista a diferença de responsividade dos pacientes ao BH4 conforme o critério de classificação utilizado, salienta-se a importância de uma definição cautelosa de responsividade e que não seja baseada em um único critério. A comparação entre a sobrecarga simples de Phe e combinada de Phe+BH4 parece ser um critério adequado para avaliar responsividade ao BH4 em pacientes com HPA-PAH que apresentam bom controle metabólico quando em tratamento dietético. / Introduction: Recent studies using different protocols showed that patients with hyperphenylalaninemia due to phenylalanine hydroxylase deficiency (HPA-PAH) may have a reduction in phenilalanine (Phe) plasma concentrations after tetrahydrobiopterin (BH4) administration. Objective: To determine responsiveness to the administration of a single dose of BH4, in a sample of Brazilian patients with HPA-PAH using a protocol that includes the simple Phe loading test and the combined Phe+BH4 loading test. Methods: Patients included in the study were ≥ 4 years of age; their median Phe plasma concentration was ≤ 10mg/dL, and all underwent dietary treatment in the 12 months before inclusion in the study. Participants received a simple Phe loading test using 100mg/kg L-Phe (Test 1) and a combined Phe+BH4 loading test using 100mg/kg L-Phe and 20mg/kg /BH4 (Test 2) at a one-week interval. BH4 was ingested three hours after Phe ingestion. Blood samples were collected at baseline and three, 11 and 27h after Phe ingestion (time points T0, T1, T2 and T3 in Tests 1 and 2). To be classified as responsive, there should be evidence that the patient had a reduction in Phe levels associated with BH4 administration according to at least one of the criteria used: criterion A – analysis of percentage differences of the Phe values at time points T1 and T2 for Tests 1 and 2; criterion B – analysis of percentage differences of Phe values at time points T1 and T3 for Tests 1 and 2; and criterion C – analysis of percentage differences of the areas under the Phe curve for Tests 1 and 2. Responsiveness classifications were also compared according to four additional criteria: criterion D – analysis of percentage differences of the Phe values at time points T1 and T2 for Test 2; criterion E – analysis of percentage differences of Phe values at time points T1 and T3 for Test 2; criterion F – analysis of percentage difference of Phe values 8h after simple BH4 loading used for five patients that participated in a previous study conducted by the same authors; and criterion G – analysis of percentage difference of Phe values 24 h after simple BH4 loading, also used for the patients in the same previous study. The cut-off point for all criteria was a reduction of ≥ 30%. Results: Eighteen patients (median age = 12 years) were included in the study. Ten patients had mild HPA-PAH and eight, classical HPA-PAH. Six patients were responsive according to the criteria used (Classical: 2; Mild: 4). Responsiveness was concordant for criteria A and B when compared with criterion C (kappa=0.557; p=0.017). Of the patients whose genotype was available (n=16), six had data about BH4 responsiveness described in the literature, and these data were in agreement with our findings. Conclusion: Data about responsiveness, HPA-PAH type and genotype were in agreement with those described in the literature. The difference in BH4 responsiveness of patients according to classification criterion emphasizes the importance of a careful definition of responsiveness not based on a single criterion. The comparison of simple Phe loading and combined Phe+BH4 loading seems to be an adequate criterion to evaluate responsiveness to BH4 in patients with HPA-PAH and good metabolic control when following a dietary treatment.

Introdução: A Hiperfenilalaninemia por deficiência de fenilalanina hidroxilase (HPAPAH) é um erro inato do metabolismo no qual ocorre aumento dos níveis séricos de fenilalanina (Phe). Estudos recentes, realizados em várias populações, demonstraram que pacientes com HPA-PAH podem apresentar redução das concentrações plasmáticas de Phe mediante a administração oral de tetrahidrobiopterina (BH4). Objetivo: Identificar em uma amostra de pacientes brasileiros com HPA-PAH aqueles que são responsivos à administração de BH4 por via oral. Métodos: Para um paciente ser incluído no estudo, era necessário ter diagnóstico de HPA-PAH e idade igual ou superior a 7 anos, estar em tratamento dietético e apresentar nível de Phe igual ou superior a 6 mg/dL em todas as medidas realizadas no ano anterior à inclusão no estudo. No dia anterior à sobrecarga de BH4 (Dia 1), os pacientes foram submetidos a três coletas de sangue para mensuração dos níveis de Phe. No Dia 2, os pacientes receberam dose única de 20mg/Kg de BH4. As coletas de sangue foram, então, realizadas nos pontos de hora: 0, 4 e 8h (Dia 2) e 24h (Dias 3) após a ingestão do medicamento. Os níveis de Phe foram determinados através da espectrometria de massa in tandem. Foram utilizados dois critérios para definir a presença de responsividade ao BH4: Critério 1: redução 30% de Phe após 8h da administração do medicamento; Critério 2: redução 30% de Phe após 24h da administração do medicamento. Resultados: Dezoito pacientes foram incluídos no estudo, com mediana de idade de 14 anos, sendo 66,7% do sexo masculino. Onze apresentavam a forma clássica da doença e três a forma atípica. Três (forma clássica: 1, forma atípica: 2) e cinco (forma clássica: 2, forma atípica: 2 e forma não-definida: 1) pacientes foram considerados responsivos ao BH4 conforme critérios 1 e 2, respectivamente. Os níveis de Phe plasmáticos do dia anterior ao teste de sobrecarga não demonstraram variação nos pontos de hora (p=0,523). Entretanto, quando comparamos os níveis de Phe nos pontos de hora do dia pré e pós BH4, encontrou-se variação significativa entre eles (p=0,006). A análise da associação genótipo-fenótipo, para os pacientes com dados disponíveis (n=6) mostrou que a mesma é multifatorial. Conclusão: Nossos achados estão de acordo com a literatura, e indicaram que um número considerável de pacientes brasileiros com HPA-PAH poderá ser beneficiado com a administração oral de BH4. / Introduction: Hyperphenylalaninemia by phenylalanine hydroxylase deficiency (HPAPAH) is an inborn error of metabolism in which increased serum levels of phenylalanine (Phe) occur. Recent studies on several populations showed that patients with HPA-PAH can have their serum levels reduced when receiving oral tetrahydrobiopterin (BH4). Objective: to identify in a sample of Brazilian HPA-PAH the patients who are responsive to the oral administration of BH4. Methods: the following inclusion criteria were used: diagnosis of HPA-PAH, age 7 years, on dietary treatment and Phe levels 6 mg/dL in all tests performed one year prior to the inclusion in this study. On the day before the BH4 challenge (Day 1) 3 blood samples were obtained to measure Phe levels. Blood samples were also obtained at time points 0, 4, 8 hours (Day 2) and 24 h (Day 3) after the intake of the medication. Phe levels were determined by tandem mass spectrometry. Criteria used to define responsiveness to BH4 were: Criterion 1: Phe reduction 30% 8 hours after BH4 administration; Criterion 2: Phe reduction 30% 24 hours after BH4 administration. Results: a total of 18 patients with a mean age of 14 years were included in this study; of those, 66.7% were male. Eleven presented the classical form of the disease and 3, the atypical form. Three patients (classical form: 1, atypical form: 2) and 5 (classical form: 2; atypical form: 2; undefined form: 1) were considered responsive to BH4 according to criteria 1 and 2, respectively. Phe serum levels on the Day 1 did not show any change on the established time point schedule (p=0.523). However, when comparing levels of Phe between Days 1 and 2, significant variation was found (p=0.006). The phenotype – genotype association analysis of patients with available data (n=6) showed that the association is multifactorial. Conclusion: In accordance with the literature, our findings show that many Brazilian patients with HPA-PAH can benefit from the oral administration of BH4.