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 pheny lalanine metabolites (phenylacetate (PAA), phenyllactate (PLA), and phenylp yruvate (PPA)) in body fluids, Here we describe a method to determine the c oncentrations of PAA, PPA, and PLA in the brain of normal and mutant orthol ogous mice, the latter being models of human PKU and non-PKU HPA. Stable is otope dilution techniques are employed with the use of [H-2(5)]-phenylaceti c acid and [2,3,3-H-2(3)]-3-phenyllactic acid as internal standards. Negati ve ion chemical ionization (NICI)-GC/MS analyses are performed on the penta fluorobenzyl ester derivatives formed in situ in brain homogenates. Unstabl e PPA in the homogenate is reduced by (NaBH4)-H-2 to stable PLA, which is l abeled with a single deuterium and discriminated from endogenous PLA in the mass spectrometer on that basis. The method demonstrates that these metabo lites 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. (C) 2000 Academic Press.