Dissertations / Theses on the topic 'Phenylalanine Phenylketonuria'

The presence of an extensive enterorecirculation of amino acids between the intestine and the body, allows for the removal of elevated systemic phenylalanine present in the phenylketonuric condition, by oral administration of microencapsulated phenylalanine ammonia-lyase(28). The work presented in this thesis, had the main goal of assessing the feasibility of phenylalanine ammonia-lyase (PAL) loaded collodion microcapsules, in reducing elevated plasma phenylalanine concentrations to standard levels in genetically mutated, ENU2 PKU mice, within a 30 day time frame. The distinguishing aspect from similar previous studies, originated with the available animal model. Rather than artificial induction of elevated phenylalanine plasma levels, the mice representing the human phenylketonuric condition, were mutated strains, deficient in the enzyme phenylalanine hydroxylase.
The first in vivo study established a method for orally feeding microcapsules, over 30 consecutive days, by mixing with soft, unripened cheese. The second animal study confirmed the finding in the first study that there is no significant decrease in the plasma phenylalanine levels within the first seven days of treatment. (Abstract shortened by UMI.)

Phenylketonuria (PKU) is an inherited metabolic disease that affects about one in 10,000 of the population worldwide. In the classical form of the condition, the hepatic enzyme phenylalanine hydroxlase is absent or much reduced. If untreated, severe or profound mental handicap customarily results due to the accumulation of dietary phenylalanine (phe) which is neurotoxic. The mechanism by which phe impairs growth in the immature nervous system is little understood, but myelin metabolism appears to be disturbed. Treatment is by reduction of phe in daily food intake. Treatment should ideally begin in the neonatal period if intellectual loss is to be avoided. However, the safe range of phe concentrations during treatment and the age at which treatment can be discontinued without further damage being inflicted are uncertain. The studies reported in this volume investigated neuropsychological outcomes of treatment control and cessation factors. In addition, the question of whether executive functions are especially vulnerable to elevated phe concentrations during treatment was addressed. Patient samples conformed to the practice adopted in the West of Scotland regional centre for the management of PKU of maintaining dietary treatment until age 10 or beyond. Almost exclusively, negative findings emerged. These suggested that, if control of phe intake conforms to current UK recommendations for the preschool and primary years, neither global nor specific intellectual deficit result. Furthermore, the data supported the view that cessation of treatment at 10 years of age does not have harmful consequences. These findings have direct implications for the formulation of clinical policy on the treatment of PKU, but it must be recognized that the history of the successful treatment of PKU and mass screening for the disease spans a mere three decades. Thus, treatment outcome research to date is based only on children and young adults. In future investigations, a life-span approach will be required before the issues raised in this thesis can be finally settled.

Maternal phenylketonuria is a disease process caused by the adverse effects of high maternal blood phenylalanine (PHE) on the fetus. Unless treated, maternal PKU results in teratogenic effects on the fetus that can lead to mental retardation, microcephaly, intrauterine growth retardation, congenital cardiovascular defects, low birth weight, spontaneous abortion and fetal death. Although PKU has been recognized as a major challenge for many years, surprisingly little is known about the pathophysiologic mechanism(s) of PHE toward the fetus. To more thoroughly investigate the pathogenesis of this heritable disease and to explore potential therapeutic actions, the genetic mouse model Pahenu2 was used. The overall goals of this project were to use the Pah enu2 mouse to examine the effect of maternal blood PHE level on: (1) The pregnancy outcome of maternal PKU offspring as measured by the incidence of spontaneous abortion and certain key measures of development at birth (i.e., head circumference, weight, and crown-rump length of offspring); and (2) The fetal nutritional status of maternal PKU offspring as assessed by the levels of PHE, tyrosine (TYR), and other essential amino acids (EAA) at birth. In this study, we clearly observed that elevated maternal blood PHE levels, whether they were caused by the maternal diet or maternal genotype, were responsible for the fetal abnormalities in maternal PKU. With regard to fetal developmental outcomes, significant reductions in birth weight, crown-rump length, and head circumference were seen in offspring gestated under high maternal blood PHE conditions. The incidence of fetal loss was significantly different between treatment and control groups. Reductions in the levels of alanine, glutamine, and glutamic acid were observed in fetal blood among offspring born to mutant mothers with high blood PHE levels. None of the branched chain amino acids were reduced in maternal PKU offspring. These findings strongly suggest that there are important maternal genotype and dietary components but no fetal genotype component to this maternal PKU model. Given that these maternal factors also appear to be the most important components of human maternal PKU, this model seems certain to provide a valid animal model to overcome the difficulties of human studies.

Phenylketonuria (PKU) and related forms of non-PKU hyperphenylalaninemias (HPA) result from deficiencies in phenylalanine hydroxylase (PAH), the hepatic enzyme that catalyses the conversion of phenylalanine (phe) to tyrosine (tyr). Patients are characterised by a metabolic phenotype comprising elevated levels of phe and some of its metabolites, notably phenyllactate (PLA), phenylacetate (PAA) and phenylpyruvate (PPA), in both tissue and body fluids. Treatment from birth with low-phe diet largely prevents the severe mental retardation that is its major consequence.
Mechanisms underlying the pathophysiology of PKU are still not fully understood; to this end, the availability of an orthologous animal model is relevant. A number of N-ethyl-N-nitrosourea (ENU) mutagenized mouse strains have become available. I report a new heteorallelic strain, developed by crossing female ENU1 (with mild non-PKU HPA) with a male ENU2/+ carrier of a 'severe' PKU-causing allele. I describe the new hybrid ENU1/2 strain and compare it with control (BTBR/Pas), ENU1, ENU2 and the heterozygous counterparts. The ENU1, ENU1/2 and ENU2 strains display mild, moderate and severe phenotypes, respectively, relative to the control and heterozygous counterparts.
I describe a novel method using negative ion chemical ionization gas chromatography/mass spectrometry (NICI-GC/MS) to measure the concentration of PLA, PAA and PPA in the brain of normal and mutant mice. Although elevated moderately in HPA and more so in PKU mice, concentrations of these metabolites are not sufficient to explain impaired brain function; however phe is present in brain at levels associated with harm.
Finally, I describe a new modality for treatment of HPA, compatible with better human compliance: it involves enzyme substitution with non-absorbable and protected phenylalanine ammonia lyase (PAL) in the intestinal lumen, to convert L-phenylalanine to the harmless metabolites (trans-cinnamic acid and trace ammonia). PAL, taken orally, substitutes for the deficient PAH enzyme and depletes body pools of excess phe. I describe an efficient recombinant approach to produce PAL enzyme. I also provide proofs of both pharmacologic and physiologic principles by testing PAL in the orthologous mutant mouse strains with HPA. The findings encourage further development of PAL for oral use as an ancillary treatment of human PKU.

It is widely appreciated by the medical community that subtle deficits in intellect, academic skills and executive functioning exist in early treated phenylketonuria (PKU). In this study, we described the relationship between intellectual outcome and concentration/variation in blood phenylalanine (Phe) during specific developmental periods (0-5 years, 6-10 years, >10 years). We also examined the association between mean number of blood Phe samples and maintenance of Phe within treatment range (120-360 ìmol/L) and within one standard deviation (SD) of index of dietary control, defined as the mean of 12-month median Phe. Retrospective data was collected from 55 patients receiving treatment at the University of Utah Metabolic Clinic. Index of dietary control (IDC) and SD blood Phe steadily increased and mean number of samples decreased during each developmental period. The correlation between IDC during 6-10 years of life and perceptual reasoning was -.370 (p = 0.006). Using multivariate linear regression, IDC during 0-5 years and 6-10 years were associated with a 0.5-point decrease and 0.3-point decrease in perceptual reasoning scores for every 100 ìmol/L increase in blood Phe, though associations were nonsignificant (p = 0.067; 0.082). SD of Phe was not associated with any measure of intelligence. The likelihood of IDC >360 ìmol/L in those 6-10 years was 32.3% lower for each additional blood Phe sample per year (p = 0.001). The present study suggests frequent blood Phe monitoring during ages 6-10 years may reduce blood Phe and prevent deficits in perceptual reasoning later in life.

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.

Phenylketonuria and hyperphenylalanemia are autosomal recessive inborn errors of phenylalanine metabolism that are caused by mutations in the phenylalanine hydroxylase gene. Due to the stringency of the present dietary therapy, alternative treatments are being studied. Phenylalanine ammonia-lyase (PAL) is one of the potential dietary supplements for these patients. PAL is a well-studied plant enzyme which breaks down phenylalanine into trans-cinnamic acid and ammonia (Camm and Towers, 1973). It is found in the cytoplasm of the plant cells and is naturally encapsulated by plant cell walls which may protect it against the acidic pH environment in the gastrointestinal tract. It presumably degrades ingested Phe that circulates in the intestinal lumen. In this study, red spring wheat seedlings (Triticum aestivum L.) found to contain high PAL activity naturally were investigated as a potential alternative oral therapy. Specifically, the objectives were (1) to evaluate different drying methods on generating concentrated and dried preparation of wheat seedlings containing high levels of PAL activity; (2) to examine the retention of PAL activity over three months of storage under various storage conditions; (3) to determine the stability of PAL activity in simulated human digestion condition to establish if further study of using plant source enzyme in vivo is warranted. Freeze-drying (FD) was found to have retained the most activity (>90 % recovery dry wt basis) compared to air-drying (AD) and vacuum-microwave drying (VMD) for both leaf and residual seed/root samples. Pre-freezing of leaf tissues at -18 °C before FD significantly retained the highest PAL activity compared to pre-freezing at -25 °C, -35 °C, and -80 °C (P<0.05). Over three months of storage, 60-80 % of PAL activity was recovered in leaf and —100 % was recovered in residual seed/root tissues after storage at -20 °C. After in vitro protein digestion, 36% and 42 % of PAL activity was recovered in fresh leaf and root tissues respectively; however, FD tissues were found to be susceptible to proteases and acidic environment and no activity was recovered after three hours of in vitro protein digestion. High performance liquid chromatography (HPLC) analysis of the residual Phe after in vitro protein digestion confirmed that fresh tissues had significantly higher conversion of Phe than that of FD tissues. Together, these results suggest that red spring wheat seedlings may have potential as a dietary supplement for phenylketonuric patients while further study to enhance PAL activity in plant preparations is required.

Phenylketonuria (PKU) is an inherited inborn error of phenylalanine (PHE) metabolism caused by deficiency of hepatic enzyme phenylalanine hydroxylase (PAH). Therefore, PHE accumulates in plasma leading to mental retardation and developmental delay. Kuvan® (Sapropterin dihydrochloride), a synthetic form of tetrahydrobiopterin (BH₄), has been shown to reduce plasma PHE levels in PKU, but not all patients respond to sapropterin treatment. The major mode of treatment remains nutritional management with dietary restriction of PHE and provision of sufficient protein. The dietary protein requirement in children with PKU remains unknown. Therefore the objectives of the current thesis were: 1) to identify sapropterin responsiveness in PKU children using a minimally invasive L-[1-¹³C] phenylalanine breath test (¹³C-PBT), and 2) to determine protein requirements in PKU children using the indicator amino acid oxidation (IAAO) technique. Experiment 1- Nine children with PKU (5-18y) underwent ¹³C-PBT tracer protocols twice, once before and once after 1-2 weeks of sapropterin therapy. ¹³CO₂ was measured using isotope ratio mass spectrometer (IRMS). The study protocol was tested in healthy children (n= 6) as proof of principle. Experiment 2- Four PKU children (5-18y) were recruited to participate in test protein intakes (ranging from deficiency to excess 0.2 – 3.2 g/kg/d) with the IAAO protocol using L-[1-¹³C] leucine, followed by collection of breath and urine samples over 8 hours. Results 1- ¹³CO₂ productions in all children with PKU pre-sapropterin treatment were low, except in one child (PKU04). Five children with PKU showed a significantly higher peak enrichment after sapropterin treatment at 20min. Three PKU children had no change in ¹³CO₂ production post sapropterin therapy. Results 2- The mean protein requirement, identified using 2-phase linear regression analysis was determined to be 1.85 g/kg/d. This result is significantly higher than the most recent PKU recommendations (2014) (1.14 – 1.33g/kg/d, based on 120-140% above current recommended dietary allowance RDA). These findings show that the ¹³C-PBT can be a minimally invasive method to examine in vivo PHE metabolism in PKU children responsive to sapropterin therapy. Also, current recommendations for optimal protein intake may be underestimated.
Land and Food Systems, Faculty of
Graduate

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.

The aim of this study was to evaluate the National Neonatal Screening Program in Sergipe State in Brazil Northeastern (PNTN/SE) for phenylketonuria (PKU). It was performed a cross-sectional study. Variables assessed were: phenylalanine blood concentrations at filter paper collected from the heel of 43.449 children (PKUneo); blood phenylalanine concentrations obtained by venipuncture in the children with abnormal PKUneo; children s age in the different program phases from January 2007 to June 2008; and the coverage in 2007. The suspected children were selected when PKUneo were above the cut-off level of 5 mg/dL. Furthermore, these children were classified by the venous concentration of phenylalanine in according to the literature, thereby obtaining the prevalence of hyperphenylalaninemy (HPA) and phenylketonuria from January 2007 to June 2008. The cases diagnosed before 2007 were not analysed. Finally, we verified the venous concentrations of phenylalanine at those children on dietetic treatment for the disease as much as the amount of phenylalanine present on their diet. The children s age at PKUneo collection was 107 days (MDP), the age when the assay was done was 2813 days and at the venous collection in the diagnosis confirmation was 5317 days. Twelve children were called based on the PKUneo cut-off. From these, the concentrations of phenylalanine collected by venipuncture were normal in five children, one child was classified as hyperphenylalaninemy and five as PKU with the prevalence of 1/43449 and 1/8690, respectively. The treatment for PKU began with 5112 days. The coverage of PNTN/SE/2007 was 78.93%, besides, 11% of the Sergipe´s children that have private health care. In conclusion, PNTN/SE presented satisfactory coverage, PKU and hyperphenylalaninemy prevalences compatible with the literature and adequate cut-off. On the other hand, the collection of PKUneo is late and the onset of treatment is delayed.
O objetivo deste trabalho foi avaliar o Programa Nacional de Triagem Neonatal no Estado de Sergipe no Nordeste do Brasil (PNTN/SE) para a fenilcetonúria (PKU). Foi realizado um estudo transversal. As variáveis estudadas foram: concentrações de fenilalanina no sangue coletado em papel-filtro do calcanhar de 43.449 crianças (PKUneo); concentrações de fenilalanina no sangue coletado por punção venosa realizada nas crianças convocadas após resultado de PKUneo alterado; idade das crianças nas diferentes fases do PNTN/SE, no período entre janeiro de 2007 a junho de 2008, e a cobertura do programa no ano de 2007. As crianças suspeitas foram selecionadas quando apresentavam concentrações de PKUneo acima do ponto de corte de 5 mg/dL. Além disso, classificamos estas crianças segundo as concentrações venosas de fenilalanina de acordo com a literatura, calculando, assim, a prevalência de PKU e da hiperfenilalaninemia (HPA) no período de janeiro de 2007 a junho de 2008, não utilizando os casos diagnosticados antes de janeiro de 2007 e depois de junho de 2008. Por fim, foram acompanhadas as concentrações venosas de fenilalanina das crianças classificadas como fenilcetonúricas e hiperfenilalaninêmicas em tratamento dietético, assim como a quantidade de fenilalanina ingerida na alimentação. A idade das crianças, na coleta do PKUneo, foi de 107 dias (MDP), na realização do ensaio foi de 2813 dias e na coleta para confirmação do diagnóstico foi de 5317 dias. Foram convocadas doze crianças após resultado de PKUneo alterado, das quais cinco tiveram concentrações venosas normais de fenilalanina, uma foi classificada como hiperfenilalaninêmica e cinco como fenilcetonúricas com prevalência de 1/43449 e 1/8690, respectivamente. A terapia nas cinco crianças com PKU foi iniciada com 5112 dias. A cobertura do PNTN/SE em 2007 foi de 79%, não sendo considerados nesse resultado os 11% da população coberta por planos privados de saúde. Deste modo, o PNTN/SE apresentou no período estudado cobertura satisfatória, prevalências de PKU e HPA compatíveis com àquelas encontradas na literatura e ponto de corte adequado. Em contrapartida, a coleta do PKUneo é tardia e o início do tratamento é demorado.

O presente trabalho teve como objetivo estimar a concentração de Phe em 22 amostras de sopas desidratadas instantâneas, por serem úteis na diversificação do cardápio de fenilcetonúricos. Foi analisada a concentração de glutamato monossódico (GMS) por ser uma provável fonte de N não protéico (NNP) que pode resultar em concentrações protéicas superestimadas. A concentração de proteína real estimada foi realizada após precipitação da proteína com TCA 10%, seguida da análise do N pelo método de Kjeldahl, o qual foi convertido para proteína por um fator de conversão (Fc) adequado. A legislação Brasileira estabelece um Fc de 5,75 para proteínas vegetais, 6,25 para proteínas da carne e misturas de proteínas e 6,38 para proteínas lácteas. A concentração de GMS foi determinada por método enzimático com eletrodo sensível a amônia. A concentração de proteína bruta (N totalxFc) variou entre 6,05 e 21,51%, tendo sido estes valores, na maioria das vezes, similares aos declarados no rótulo, indicando que os fabricantes utilizam o N totalxFc para expressar o conteúdo protéico. A concentração protéica real estimada foi baixa, variando entre 1,28 e 16,31%. A concentração de NNP teve uma variação de 0,33 a 1,27g/100g de amostra, representando de 11,10 a 81,33% do NT presente. A concentração de GMS variou entre 1,01 e 7,86g/100g de amostra, sendo que o N proveniente deste realçador de sabor contribuiu com 2,53 a 47,71% na quantidade total de N. A diferença entre a concentração de proteína bruta e real estimada se deve à presença de NNP, na forma de GMS. Com base nos valores protéicos reais estimados, foram calculados os teores de Phe que variaram entre 51,16 e 652,24mg de Phe/100g de amostra. Assim, recomenda-se que todos os alimentos adicionados de realçadores de sabor sejam analisados quanto à concentração de proteína real para que a Phe seja corretamente estimada.
The aim of this work was to estimate the concentration of Phe in 22 samples of commercially available dehydrated soups, as they are useful to add variety to the diet for phenilketonurics. The monosodium glutamate (MSG) contents had been analyzed as it is a likely source of non protein N (NPN) that might result in overestimated protein contents. The true protein content was accomplished after protein precipitation with 10% TCA and followed by N analysis according to the Kjeldahl method, which was converted to protein by a suitable conversion factor (Fc). The Brazilian legislation establishes a Fc of 5,75 for vegetables proteins, 6,25 for meat and blended proteins and 6,38 for milk proteins. The MSG concentration was determined by an enzymatic method employing an ammonia gas-sensitive electrode. The crude protein content (total NxFc) varied from 6,05 to 21,51% and were similar, in most cases, to those stated on the label, showing that manufacturers use total NxFc to express the protein content. Nevertheless, the true protein content was low, varying from 1,28 to 16,31%. The NPN concentration varied from 0,33 to 1,27g/100g of sample, which represents from 11,10 to 81,33% of the existing total N. The MSG concentration varied from 1,01 to 7,86g/100g of sample; the N arose from this flavor enhancer gives about 2,53 to 47,71% of the total quantity of N. The difference between the crude protein and true protein contents is due to the presence of MSG-like NPN. The Phe concentrations were calculated in accordance with the true protein values and varied from 51,16 to 652,24 mg/100g of sample. Thus, we recommend the analysis of all flavor-enhancer-added foods, in order to get reliable results for Phe estimation from the protein contents.

Thesis (Ph.D)--Chemistry and Biochemistry, Georgia Institute of Technology, 2007.
Committee Chair: Sheldon May; Committee Members: Nicholas Hud, Stanley Pollock, James Powers, and Gerald Pullman. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Microencapsulation of the enzyme phenylalanine ammonia-lyase (PAL) was developed for in vivo depletion of systemic phenylalanine (PHE) in Phenylketonuric (PKU) rats. Phenylalanine ammonia-lyase was successfully microencapsulated within artificial cells. The immobilized enzyme had an assayed activity of 20% $ pm$ 4% (Mean $ pm$ S.D.) of the free enzyme in solution. The immobilized enzyme maintained a higher enzyme activity at very low pH compared to the free enzyme in solution. The pH optimum of the free and immobilized enzyme was 8.5. This pH optimum corresponds to the average pH range of the small intestine.
PKU induced rats had a 10 to 20-fold increase in the amino acid phenylalanine in the systemic circulation. This amino acid elevation was comparable to the plasma phenylalanine increase in human PKU patients. Daily oral administration of artificial cells containing 5 units of the enzyme PAL, to PKU rats, lowered on an average the systemic phenylalanine level to 20% $ pm$ 8% (Mean $ pm$ S.D.) of the original levels in 7 days (P $<$ 0.001). After 7 days of this form of enzyme therapy, systemic blood phenylalanine levels of PKU treated rats were lowered to levels not significantly different from those of normal rats. Unlike control PKU rats, the treated group showed no signs of abnormal behavior or weight loss.
HPLC analysis of free amino acids in the plasma, cerebro-spinal fluid and brain of PKU rats was carried out in the following groups: (1) Normal rats, (2) PKU rats on a normal diet, (3) PKU rats on a high phenylalanine diet, (4) PKU rats on a phenylalanine free diet, and (5) PKU treated rats with PAL-loaded artificial cells. Amino acid compartmental distribution was different in all these groups.
The oral administration of PAL-loaded artificial cells decreased the hyperphenylalaninemia in all the compartments studied. It also corrected the imbalance of many amino acids, including tyrosine and tryptophan, in the CSF and the brain. This enzyme therapy was more effective in compartmental PHE level depletion, than a PHE-free diet.

Objective: The primary aim of this retrospective cohort study was to determine the association between the source of dietary protein intake and the sum of plasma concentration of large neutral amino acids (LNAA) in patients with Phenylketonuria (PKU). A secondary aim of the study was to examine the effect of dietary compliance on plasma concentration of LNAA. Methods: The analysis included combined participant data from two previous studies conducted at the Emory University School of Medicine. Subjects are males (n=34) and females (n=43) with PKU ages 4-50 years. A Student t-test was used to compare total combined plasma LNAA (excluding tryptophan and phenylalanine) by dietary compliance status (alpha=0.05). Correlation statistics were used to determine the association between the ratio of reported intact food protein to medical food protein on plasma levels of LNAA. Multiple regression analysis was used to examine the contribution of intact protein to medical food protein ratio and other variables to plasma LNAA. Results: The median ratio of intact protein to medical food protein reported was 0.354 (IQR: 0.188, 0.914). Median percent of PHE intake over the PHE intake recommendation was 31.64 (Interquartile range [IQR]; 7.44, 104.98). Plasma concentration of LNAA did not differ significantly between those with plasma PHE levels within the therapeutic range μmol/L (compliant; 611.7 μmol/L [n=19]) vs levels above the therapeutic range (non-compliant; 595.3 μmol/L [n=47]); p=0.613). There was an inverse marginal correlation between the ratio of intact protein to medical food protein and plasma concentration of LNAA for those who were compliant (r = -0.436, r = 0.1) although the association was not statistically significant (p=0.08). No correlation was found for patients who were non-compliant. Regression analysis revealed that plasma concentration of LNAA was not significantly affected by the ratio of intact protein to medical food protein ratio, age, or gender. Conclusions: Although not statistically significant, a negative trend was observed between plasma LNAA concentration and the intact protein to medical food protein ratio in patients compliant with the PHE prescription. This suggests that the ratio of intact dietary protein to protein coming from medical food, as reported by patient diet records, may promote increased plasma LNAA levels in the effective treatment of PKU. The majority of the sample (74%) were non-compliant with diet based on plasma PHE levels. Future studies are needed to determine the consequences of non-compliance by decreased intake of medical food protein or increased intake of intact protein on plasma LNAA concentration and downstream health effects.

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2020
Phenylketonuria (PKU; OMIM #261600) is an autosomal recessive disorder, caused mostly by missense mutation in the PAH gene, which encodes human phenylalanine hydroxylase (hPAH).Human PAH is a nonheme, homotetrameric, iron-containing enzyme, which catalyzes the conversion of LPhenylalanine (L-Phe) into L-Tyrosine, by para-hydroxylation of the aromatic side chain in the presence of the co-factor (6R)-L-erytro-5,6,7,8-tetrahydrobiopterin (BH4), non-hemic iron and molecular oxygen (O2). Presently, classical PKU patients must follow restricted diets in order to control L-Phe levels in the blood. Compliance is difficult and cognitive and social development impaired. PKU can be classified as a misfolding disease, since the majority of the PKU causing mutations result in misfolded hPAH variants which, in the cellular context, will form soluble aggregates targeted for degradation, leading to a loss-of-function phenotype. Therefore, pharmacological chaperone therapy imposes a viable alternative to PKU treatment. By stabilizing the variant protein, and avoiding its degradation, rescue of enzyme residual activity may be attained, improving patient’s quality of life by increasing dietary LPhe tolerance and alleviating some of the dietary restrictions imposed. Among the high number of PAH variants already identify, the p. G46S is considered an excellent model to the in vitro study of the hPAH aggregation process, as it presents a high tendency to selfassociate and form non-amyloid fibrils, when overexpressed in E.coli, and to be rapidly degraded in eukaryotic systems, representing the most severe form of Classical PKU phenotype. In the presence of maltose binding protein (MBP) added N-terminally to p.G46S, it is possible to obtain soluble metastable tetramers with a normal enzyme activity rate. Upon MBP tag cleavage with Factor Xa protease the hPAH p.G46S aggregates in a process efficiently followed in vitro. Our aim was to identify, among an in-house compound library, molecules able to modulate this behavior in vitro, and to characterize the underlying mechanism by studying the full-length (FL) and a truncated form of p.G46S representing the unstable N-terminal regulatory domain (N1-120) harboring the allosteric site. For this, the p.G46S forms were expressed in a prokaryotic system in fusion with the MBP tag and purified. The rate of self-association of the isolated FL tetramers and N1-120 dimers (variant and wild-type) were studied by real-time turbidimetry, upon cleavage of the MBP tag, in the presence and absence of studied molecules. Following in vitro studies, molecules with a positive impact on p.G46S aggregation were submitted to in cellullo studies to evaluate their influence on enzyme activity and stability. From the aggregation assays, five molecules had positive effects on the self-association pattern of the FL p.G46S, either by inhibiting aggregation or by delaying it. From those molecules, two (C4 and C14) also inhibited the wild-type and p.G46S RD1-120 aggregation, and one (C17) only impacted the aggregation of the wild-type RD1-120, suggesting that these molecules bind to the unstable N-terminal domain and particularly C4 and C14 will be interacting with helix a1 of the ACT domain, the main driver of p.G46S-RD1-120 instability while C17 is acting on a different region. In cellullo assays showed that C17 and C14 could improve enzyme activity when compared to p.G46S in the absence of compounds (control). Enzyme content in cell lysates, however, was at the same level as the p.G46S control, thus suggesting that these two compounds are promoting a stabilization of the enzymatic reaction, allowing it to present higher activity levels than it normally would have (activity chaperone), instead of acting as stabilizers of the protein helping it to avoid the cell protein control quality mechanisms (pharmacological chaperone). Although further studies were not carried out due to the restrictions and contingency plans applied by the Research institutions to respond to SARS-CoV-2_COVID-19 pandemics, here we present possible hit structures for the development of a new class of pharmacological/activity chaperones, specifically designed for the treatment of classical PKU.
A fenilcetonuria (PKU; OMIM #261600) e uma doenca autossomica recessiva, causada na sua maioria por mutacoes missense no gene PAH, que codifica a fenilalanina hidroxilase humana (hPAH). A hPAH e uma enzima homotetramerica, que catalisa a conversao da L-fenilalanina (L-Phe) em Ltirosina (L-Tyr), por para-hidroxilacao da cadeia lateral aromatica. Para que esta reacao ocorra, e necessaria a presenca do seu co-factor natural (6R)-L-erytro-5,6,7,8-tetrahidrobiopterina (BH4), de ferro nao-hemico e oxigenio molecular (O2). As mutacoes missense no gene PAH originam na sua maioria a expressao de variantes de hPAH conformacionalmente alteradas (misfolded), no contexto celular, tem tendencia a formar agregados soluveis que rapidamente se tornam alvos das vias celulares de degradação proteica, levando a um fenotipo patologico de perda de funcao. A PKU e entao considerada uma doença conformacional, na qual o equilibrio normal dos estados folded« unfolded e alterado, deslocando-se preferencialmente para o estado unfolded. Consequentemente, as proteinas resultantes misfolded são reconhecidas, levando a que a maquinaria celular de controlo de qualidade e degradacao proteica atue no sentido de acelerar a degradacao de proteinas misfolded devido a sua instabilidade e tendencia para agregacao. Nos doentes PKU, quando nao tratados o mais rapidamente possivel apos diagnostico, a função alterada da hPAH variante resulta na acumulacao de L-Phe para niveis neurotoxicos, causando alteracoes neurologicas, convulsoes, atraso no desenvolvimento, problemas comportamentais, etc. O tratamento disponivel assenta basicamente em dietas restritivas e suplementos alimentares desde o diagnostico (preferencialmente dentro dos primeiros dias de vida) a fim de controlar os niveis de L-Phe em circulacao no sangue. Atualmente, as guidelines internacionais indicam que a restricao dietetica deve ser mantida durante a vida e que os niveis de L-Phe circulante devem ser mantidos abaixo de 600 μM (considerando niveis normais de circulacao no sangue ≈120 μM). A aquiescencia para com esta terapeutica e dificil, principalmente durante a adolescencia e nos adultos, e o desenvolvimento cognitivo e social sao prejudicados. Ao longo dos anos tem-se vindo a estudar diferentes terapeuticas alternativas, de forma a que seja possivel controlar os niveis de L-Phe circulantes e seja possivel um alivio nas restricoes dieteticas. A terapeutica baseada na administracao de chaperones propoem-se como uma alternativa viavel, uma vez que a PKU e atualmente reconhecida como uma doenca conformacional. Ao estabilizar a enzima misfolded e a sua degradacao iminente, permite-se que a enzima esteja presente e mantenha a sua atividade residual. Um leve aumento da capacidade de processamento da L-Phe poderá entao levar a um aumento da qualidade de vida do doente, aliviando algumas das restricoes dietéticas impostas. Ate a data, foram descritas tres abordagens diferentes para a terapia por chaperones: modulacao de chaperones moleculares a fim de resgatar proteinas com folding incorreto (reguladores de proteostase); chaperones quimicos e chaperones farmacologicos com o objetivo de estabilizar a proteina alterada, evitando-se a degradacao pelos sistemas de controlo de qualidade proteica. Entre as varias variantes causadoras de PKU, a p.G46S e considerada como um excelente modelo para o estudo in vitro do processo de agregacao da hPAH, uma vez que apresenta uma elevada tendencia para se autoassociar e formar fibrilhas nao amiloides, quando sobre-expressa em E.coli, e para ser rapidamente degradada em sistemas eucariotas, representando um fenotipo classico de PKU. Assim, e na presença da proteina de ligacao a maltose (maltose binding protein - MBP) adicionada a N-terminal da cadeia polipeptidica da p.G46S, e possivel obter tetrameros soluveis, metastaveis e com uma taxa de atividade enzimatica normal. Apos a clivagem da MBP com a protease Factor Xa (FXa), e entao possivel seguir in vitro e de forma eficiente o processo de agregacao da proteina hPAH p.G46S. Tendo em consideracao as caracteristicas descritas da p.G46S, o objetivo do presente estudo e o de identificar, de entre uma biblioteca de compostos desenhados e sintetizados pelo Grupo de Quimica Bioorganica do Instituto de Investigacao do Medicamento (iMed.ULisboa), moleculas capazes de modular o comportamento auto-associativo in vitro, e caracterizar o mecanismo subjacente atraves do estudo da proteina inteira (Full lenght; FL) e de uma forma truncada da p.G46S representando o domínio regulador instavel N-terminal (RD1-120) da hPAH e que contem o local de ligacao alosterico ao substrato L-Phe . Para tal, as formas de p.G46S (FL e RD1-120) foram expressas num sistema procariota como proteinas de fusao com a MBP e posteriormente purificadas. A taxa de auto-associacao dos tetrâmeros FL isolados e dos dimeros RD1-120 foram estudados por turbidimetria em tempo real, apos a clivagem da MBP, na presenca (100 μM) e ausencia das moleculas estudadas. Apos estudos in vitro, as moléculas com efeito inibidor na agregacao da p.G46S foram sujeitas a estudos in celullo com a finalidade de avaliar a sua influencia na atividade e estabilidade enzimatica na celula. Desta forma, células embrionicas do rim humano (human embryonic kidney cells), HEK293T, foram transfetadas com vetores de expressao eucariotas capazes de expressar a proteina p.G46S. Como controlo do ensaio celulas HEK293T foram tambem transfetadas com vetor de expressao contendo o gene normal da hPAH (wild type; WT). Para monitorizacao da estabilidade e variacao na tendencia de agregacao da proteína nas celulas eucariotas, foram realizados ensaios de imunocitoquimica. Adicionalmente, avaliou-se de que forma a funcao biologica celular da p.G46S seria afetada pela presenca dos compostos atraves da medicao da atividade enzimatica em lisados celulares. Dos ensaios de agregacao observou-se que cinco moleculas (C4, C10, C12, C14 e C17) tiveram efeitos positivos sobre o padrao de auto-associacao da forma FL da p.G46S, quer inibindo a agregacao, quer atrasando-a. Destas moleculas, duas (C4 e C14) atrasaram tambem a agregacao das formas truncadas representantes do RD (p.G46S e WT), enquanto uma (C17) apenas diminuia a agregacao da forma WT RD1-120, sugerindo que a interacao destas moleculas com a enzima ocorrera no dominio regulador N-terminal e que em particular as moléculas C4 e C14 deverao interagir com a helice a1 do dominio ACT promotora da instabilidade proteica. Estas cinco moleculas foram submetidas a estudos de imunocitoquimica, que, infelizmente, foram pouco conclusivos, devido a sobre-expressao tanto da proteina p.G46S como da WT, que impediu a discriminacao de agregados proteicos na celula. Desta forma, nao foi possivel obter resultados conclusivos acerca da capacidade de os compostos modularem a agregacao da proteina in cellullo. Para a avaliacao da modulacao da atividade biologica da proteina na celula pelos compostos em estudo (50 μM), realizaram-se ensaios na presenca de 0,5% DMSO e de 50 μM de C6 como controlos negativos. Como esperado, em ambos os ensaios a atividade enzimatica foi reduzida (» 15%), quando comparada com a hPAH WT. Estes resultados encontram-se em concordancia com os dados obtidos por analise de Western blot, sendo que os niveis intracelulares da proteina alvo se encontravam tambem reduzidos, quando comparados com a WT. Dos compostos que melhoravam a agregacao de p.G46S in vitro, tanto o C14 como o C17 mostraram ser capazes de melhorar a atividade da proteina variante, quando comparado com o ensaio controlo (p.G46S/0,5% DMSO). No entanto, para corroborar os pressupostos assumidos nas experiencias in vitro, ensaios adicionais deverao ser realizados, uma vez que se verificaram diferencas significativas entre os ensaios de atividade independentes, efetuados nas células HEK293T. Ao contrario do que seria de esperar, observou-se nos immunoblots, tanto para C14 como para C17, uma quantidade de proteina p.G46S tambem reduzida em comparacao com a hPAH WT, sugerindo que estes compostos poderao estar a estabilizar o processo enzimatico de modo a que esta tenha maior capacidade para realizar a sua funcao biologica (chaperone de atividade), ao inves de estabilizar a conformacao proteica, evitando a sua degradacao pela maquinaria de controlo de qualidade e degradacao proteica celular (chaperone farmacologico). Embora nao tenha sido possivel efetuar ensaios adicionais devido as restricoes impostas pelo plano de contingencia de combate a pandemia SARS-CoV-2_COVID-19, o presente estudo serviu como base para a identificacao de estruturas hit para o desenvolvimento de uma nova classe de chaperones farmacologicos ou chaperones de atividade direcionados para tratamento da PKU classica.