Genetics is concerned with inheritance, genomics with the study of genomes. Bioinformatics provides the tools to study the interface between the two. If a particular locus in the human genome could have 100 discrete alleles, then the genome (comprising an estimated 80,000 genes), could harbor 8 million different alleles. To record information about each of these alleles in a meaningful and systematic fashion is a task for the Mutation Database domain of bioinformatics. The HUGO Mutation Database Initiative is an international effort to capture, record and distribute information about variation in genomes. This initiative comprises a growing number of Locus-Specific Mutation databases, and a few large Federated Genomic databases [Cotton et al., 1998]. / Here I present work on a well recognized prototypical Locus-Specific database: PAHdb. PAHdb is a relatively large curated relational database. / This graduate project has had two major aims: to improve PAHdb , by careful analysis of version 1.0 and revision of its design, resulting in PAHdb version 2.0; to document the redesign process and share the experience by the conception of guidelines for content and structure of mutation databases in general. (Abstract shortened by UMI.)

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.

Dietary intake data of 15 treated patients with phenylketonuria (PKU) (7-17 years) and six control siblings (6-14 years) were used to evaluate the effectiveness of medical foods to balance energy, protein, vitamin B-6, and individual amino acids from natural foods and to evaluate vitamin B-6 status. Four-day diet records were computer-analyzed and the contribution of medical foods and natural foods to the total diets of the patients was determined. A fasting blood sample and two 24-hour urine collections were obtained from each subject. In eight patients consuming a strict diet, natural foods provided 0.9 g protein and 39 mg phenylalanine (phe)/100 calories, a significant reduction from control intakes of 3.3 g protein and 153 mg phe/100 calories. However, plasma phe levels were above the acceptable treatment range. Medical foods were consumed in less than the recommended quantities to meet approximately 120% of the Recommended Dietary Allowances (RDAs) for protein. These foods contributed 73% of the total protein RDAs but only met 22% of the energy RDAs of patients consuming the strict diet. Taste qualities of certain L-amino acids (L-AAs; Lmethionine, L-glutamic and L-aspartic acids), which constitute the protein in elemental medical foods (EMFs) for school-aged patients, make acceptance of these products difficult. Intakes of each of these L-AAs were above nutritional standards and suggested that their levels could be safely lowered in EMFs. Natural foods and medical foods provided 0.057 and 0.046 mg vitamin B-6/g protein, respectively, above the 0.020 RDA standard and the mean control intake of 0.018 mg vitamin B-6/g protein. The mean plasma pyridoxal 5'- phosphate (PLP) concentration for the patients with PKU was over twice that of the mean control concentration and above literature values. However, more than half of the patients excreted less than 30% of their vitamin B-6 as 4-pyridoxic acid; values below the criterion suggested for inadequate status. This work provides data for a better understanding of vitamin B-6 metabolism and status in PKU and supports the design and testing of a new EMF to balance energy, protein, and amino acids from natural foods and which may improve dietary adherence. / Graduation date: 1991

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.

Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliography.

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. / Land and Food Systems, Faculty of / Graduate

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.

No description available.

Metabolomics is the systematic analysis of low-molecular weight compounds (metabolites) within biological systems that represent molecular endpoints of gene expression and environmental exposures. A major goal of metabolomics is achieving better understanding of the pathophysiology of complex disease processes while elucidating mechanisms of action of nutrients, toxins, and/or drugs. Multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS) is a high-throughput microseparation platform that is ideal for the analysis of polar/ionic metabolites from volume-restricted biological samples. This thesis includes two major metabolomics projects using MSI-CE-MS that are aimed at contributing new advances in public health and chronic disease prevention. Chapter II presents an analysis of the metabolome from patients with phenylketonuria (PKU) — a genetic disease affecting phenylalanine (Phe) metabolism that requires lifelong dietary restriction to prevent irreversible intellectual disabilities. A targeted and nontargeted metabolomics approach using matching urine and plasma samples was conducted to confirm known markers of PKU and identify new markers associated with dietary adherence and disease progression. Along with increased excretion of Phe catabolites in urine, high plasma Phe was associated with decreased excretion of acylcarnitines and greater excretion of histidine catabolites, suggesting impaired fatty acid oxidation and micronutrient deficiencies, respectively. Overall, this may provide a strategy to objectively monitor dietary adherence beyond standard dietary records or patient recall. Chapter III investigates the impact of electronic cigarette smoke exposure on the placental metabolome as a model cell line of fetal development. Evidence of altered amino acid metabolism, in addition to changes in acylcarnitines and metabolites associated with cellular proliferation, were observed in more susceptible first trimester placental cells and were attributed to flavouring agents irrespective of nicotine dosage. This work supports the hypothesis that flavoured e-cigarette formulations pose a significant health risk in comparison to unflavoured formulations and supports the need for further risk assessment and careful regulation of these products to prevent deleterious birth outcomes in pregnant mothers. / Thesis / Master of Science (MSc)

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