What we know that could influence future treatment of phenylketonuria.

Phenylketonuria (PKU), a Mendelian autosomal recessive phenotype (OMIM 261600), is an inborn error of metabolism that can result in impaired postnatal cognitive development. The phenotypic outcome is multifactorial in origin, based both in nature, the mutations in the gene encoding the L-phenylalanine hydroxylase enzyme, and nurture, the nutritional experience introducing L-phenylalanine into the diet. The PKU story contains many messages including a framework to appreciate the complexity of this disease where phenotype reflects both locus-specific and genomic components. This knowledge is now being applied in the development of patient-specific therapies.

Measurement of phenyllactate, phenylacetate, and phenylpyruvate by negative ion chemical ionization-gas chromatography/mass spectrometry in brain of mouse genetic models of phenylketonuria and non-phenylketonuria hyperphenylalaninemia.

Phenylketonuria (PKU) (OMIM 261600) is the first Mendelian disease to have an identified chemical cause of impaired cognitive development. The disease is accompanied by hyperphenylalaninemia (HPA) and elevated levels of phenylalanine metabolites (phenylacetate (PAA), phenyllactate (PLA), and phenylpyruvate (PPA)) in body fluids. Here we describe a method to determine the concentrations of PAA, PPA, and PLA in the brain of normal and mutant orthologous mice, the latter being models of human PKU and non-PKU HPA. Stable isotope dilution techniques are employed with the use of [(2)H(5)]-phenylacetic acid and [2,3, 3-(2)H(3)]-3-phenyllactic acid as internal standards. Negative ion chemical ionization (NICI)-GC/MS analyses are performed on the pentafluorobenzyl ester derivatives formed in situ in brain homogenates. Unstable PPA in the homogenate is reduced by NaB(2)H(4) to stable PLA, which is labeled with a single deuterium and discriminated from endogenous PLA in the mass spectrometer on that basis. The method demonstrates that these metabolites are easily measured in normal mouse brain and are elevated moderately in HPA mice and greatly in PKU mice. However, their concentrations are not sufficient in PKU to be "toxic"; phenylalanine itself remains the chemical candidate causing impaired cognitive development.

A heteroallelic mutant mouse model: A new orthologue for human hyperphenylalaninemia.

Hyperphenylalaninemias (HPA) are Mendelian disorders resulting from deficiencies in the conversion of phenylalanine to tyrosine. The vast majority are explained by a primary deficiency of phenylalanine hydroxylase (PAH) activity. The majority of untreated patients experience irreversible impairment of cognitive development. Although it is one of the best known hereditary metabolic disorders, mechanisms underlying the pathophysiology of the disease are still not fully understood; to this end, the availability of an orthologous animal model is relevant. Various mutant hyperphenylalaninemic mouse models with an HPA phenotype, generated by N-ethyl-N'-nitrosourea (ENU) mutagenesis at the Pah locus, have become available. Here we report a new hybrid strain, ENU1/2, with primary enzyme deficiency, produced by cross breeding. The ENU1, ENU1/2, and ENU2 strains display mild, moderate, and severe phenotypes, respectively, relative to the control strain (BTBR/Pas). The Pah enzyme activities of the various models correlate inversely with the corresponding phenylalanine levels in plasma and brain and the delay in plasma clearance response following a phenylalanine challenge. The maternal HPA effect on the fetus correlates directly with the degree of hyperphenylalaninemia, but only the ENU2 strain has impaired learning.

A different approach to treatment of phenylketonuria: phenylalanine degradation with recombinant phenylalanine ammonia lyase.

Phenylketonuria (PKU), with its associated hyperphenylalaninemia (HPA) and mental retardation, is a classic genetic disease and the first to have an identified chemical cause of impaired cognitive development. Treatment from birth with a low phenylalanine diet largely prevents the deviant cognitive phenotype by ameliorating HPA and is recognized as one of the first effective treatments of a genetic disease. However, compliance with dietary treatment is difficult and when it is for life, as now recommended by an internationally used set of guidelines, is probably unrealistic. Herein we describe experiments on a mouse model using another modality for treatment of PKU compatible with better compliance using ancillary phenylalanine ammonia lyase (PAL, EC 4.3.1.5) to degrade phenylalanine, the harmful nutrient in PKU; in this treatment, PAL acts as a substitute for the enzyme phenylalanine monooxygenase (EC 1.14.16.1), which is deficient in PKU. PAL, a robust enzyme without need for a cofactor, converts phenylalanine to trans-cinnamic acid, a harmless metabolite. We describe (i) an efficient recombinant approach to produce PAL enzyme, (ii) testing of PAL in orthologous N-ethyl-N'-nitrosourea (ENU) mutant mouse strains with HPA, and (iii) proofs of principle (PAL reduces HPA)-both pharmacologic (with a clear dose-response effect vs. HPA after PAL injection) and physiologic (protected enteral PAL is significantly effective vs. HPA). These findings open another way to facilitate treatment of this classic genetic disease.