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.

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.

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: 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.

No description available.

Cardiovascular disease is the leading cause of mortality in the developed world with up to fifty percent of cases being due to sudden cardiac death. Changes in sympatho-vagal balance underpin many cardiovascular conditions including heart failure and myocardial infarction. Neuraxial modulation of the autonomic nervous system is an emerging therapy to prevent ventricular arrhythmias, the main cause of sudden cardiac death. <b>Chapter One</b> reviews our current understanding of how the cardiac autonomic nervous system influences ventricular arrhythmogenesis. A particular focus was on the controversial role of cholinergic receptors and nitric oxide (NO) in parasympathetic protection from ventricular arrhythmias. Tetrahydrobiopterin (BH₄), a critical cofactor for both tyrosine hydroxylase and NO synthases, and the co-transmitter neuropeptide-Y (NPY) may also influence sympathetic triggering of ventricular arrhythmias. This leads to the specific aims of the thesis which were to determine the mechanisms of the cholinergic antifibrillatory effect, investigate the role of cotransmission in arrhythmogenesis and, the mechanistic role of BH4 in autonomic cardiovascular control. <b>Chapter Two</b> detailed the experimental approach taken to investigate the hypotheses. A novel Langendorff heart preparation was developed with intact autonomic nerves to investigate how the stable analogue of acetylcholine, carbamylcholine (CCh) raises ventricular fibrillation threshold (VFT) and whether exogenous or endogenously released NPY lowers VFT. These actions are further investigated using optical mapping, dye free imaging of ventricular cell monolayers, immunohistochemistry, ELISA assays and measurements of NO metabolite production. To investigate the role of BH4 in the sympathetic control of the heart, an IRES-cre recombinase strategy was used to produce genomic deletion of GCH1 (the gene encoding BH4) in sympathetic neurons. Biopterins and plasma catecholamines were measured using HPLC, and blood pressure and heart rate via tail cuff plethysmography. <b>Chapter 3</b> showed that CCh increased VFT, prolonged action potential duration and flattened the electrical restitution curve. This effect required stimulation of both muscarinic and nicotinic receptors and the generation of nNOS derived NO utilising a cGMP dependent pathway. These observations are in keeping with established evidence demonstrating the obligatory role of the muscarinic receptor and indicate that the role of NO is likely to be via modulation of cholinergic neurotransmission. <b>Chapter 4</b> studied the role of the sympathetic co-transmitter NPY. NPY has been shown to increase ventricular myocyte calcium dynamics. Plasma levels are also increased post myocardial infarction and during heart failure, and correlate with outcomes. Perfusion of NPY decreased VFT via a Y1 receptor dependent mechanism and increased arrhythmic activity in myocyte monolayers. Direct sympathetic stimulation resulted in NPY release and remained pro-arrhythmic despite &beta;-blockade, an effect that could be abolished by combined &beta;-Y₁ receptor blockade. These observations indicated that NPY may be a novel, pro-arrhythmic trigger amenable to therapeutic pharmacological modulation. <b>Chapter 5</b> details the generation and phenotyping of two tissue specific Gch1 knockout mouse models. Whilst one model failed to produce significant lowering of BH₄ in sympatho-adrenal tissue, the other did result in a marked neuro-motor phenotype. A biochemical rescue or alternative genomic modification approach would be required to study the cardiovascular phenotype of sympathetic Gch1 deletion in more detail. <b>Chapter 6</b> is a concluding discussion summarising the main findings of the thesis, placing them in a clinical context and discussing avenues for further research.

La tétrahydrobioptérine (BH4) est le cofacteur requis pour l’activité des enzymes responsables de la synthèse de la dopamine, la sérotonine et l’oxyde nitrique. Par conséquent, la BH4 est nécessaire au bon déroulement de nombreux processus physiologiques centraux et périphériques dont la neurotransmission, la réponse inflammatoire, la régulation du stress oxydatif, la fonction vasculaire et endothéliale et le métabolisme. Or, de par sa structure chimique, la BH4 est une molécule facilement oxydable et dégradable, et des diminutions des niveaux centraux de BH4 ont été observées dans de nombreuses maladies neuropsychiatriques à composante inflammatoire dont la maladie d’Alzheimer et la dépression majeure. Le déficit de BH4 apparait donc comme un mécanisme pouvant participer à l’étiologie de ces pathologies et en aggraver les symptômes. Malgré ces observations, les effets d’une administration de BH4 sur la neurophysiologie et les comportements en s’y rapportant restent largement inexplorés. L’objectif de cette thèse a donc été de caractériser les effets d’une administration périphérique de BH4 sur la fonction cérébrale et le comportement chez la souris. Nous nous sommes intéressés aux effets de BH4 sur le système dopaminergique mésolimbique et la motivation en condition physiologique puis en situation d’inflammation aigue. Dans un second temps, nous avons exploré le potentiel thérapeutique de la BH4 dans le traitement de la maladie d’Alzheimer. Nous avons tout d’abord démontré que la BH4 traversait la barrière hémato-encéphalique et qu'une injection périphérique permettait d’augmenter les niveaux centraux de BH4. Nos travaux ont montré qu’en condition physiologique, l’administration de BH4 potentialise la libération de dopamine dans le nucleus accumbens et les comportements motivés. En condition de neuroinflammation aiguë induite par le LPS, l’administration de BH4 permet d’atténuer la neuroinflammation. Ces données suggèrent un potentiel bénéfique de la BH4 sur les troubles motivationnels induits par l’inflammation. Dans notre deuxième étude, nous avons démontré que l’administration chronique de BH4 permet de corriger les déficits mnésiques observés dans le modèle murin triple transgénique de la maladie d’Alzheimer. La supplémentation en BH4 induit également une diminution de la neuroinflammation ainsi qu’une amélioration de la tolérance au glucose. Cependant, ces améliorations mnésiques, métaboliques et inflammatoires ne s’accompagnent pas d’une diminution des pathologies amyloïde et tau.Ainsi, l’ensemble de ces travaux a permis une meilleure caractérisation des effets neurobiologiques et comportementaux de la BH4 et renforce son potentiel thérapeutique. / Tetrahydrobiopterin (BH4) is the required cofactor for the activity of the enzymes involved in the synthesis of dopamine, serotonin and nitric oxide. BH4 is therefore necessary for many central and peripheral physiological processes including neurotransmission, inflammatory response, oxidative stress regulation, vascular and endothelial function, and metabolism. However, BH4 can be easily oxidized and degraded and decreased BH4 brain levels has been observed in many neuropsychiatric diseases including Alzheimer's disease and major depression. Consequently, the decrease in BH4 levels in these pathologies could contribute to the onset and aggravation of symptoms. Despite these observations, the effects of BH4 administration on brain function and related behavior remain largely unexplored. The aim of this thesis was to characterize the effects of peripheral BH4 administration on brain function and behavior in mice. We investigated the effects of BH4 on the mesolimbic dopaminergic system and motivation in physiological condition and during acute inflammation. Then, we explored the therapeutic potential of BH4 in the treatment of Alzheimer's disease. We first demonstrated that BH4 crossed the blood-brain barrier and that a peripheral injection of BH4 increased its cerebral levels. Our results also showed that under physiological condition, BH4 administration potentiates dopamine release into the nucleus accumbens and motivated behaviors. In condition of acute LPS-induced neuroinflammation, BH4 administration helps to reduce neuroinflammation. Therefore, BH4 may have beneficial effects on dopaminergic and motivational disturbances induced by inflammation. In our second study, we demonstrated that chronic administration of BH4 reversed memory deficits observed in the transgenic triple murine model of Alzheimer's disease. We also observed a decrease in neuroinflammation and an improvement in glucose tolerance. However, these memory, metabolic and inflammatory improvements are not accompanied by a decrease in amyloid and tau pathologies. This work had contributed to a better characterization of the neurobiological and behavioral effects of the BH4 and reinforces its therapeutic potential.

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.