Phenylketonuria (PKU)

Phenylalanine, extracted from neonatal samples, is combined with the enzymatic reagent, phenylalanine dehydrogenase. In the presence of NAD+, the enzyme catalyzes the oxidative deamination of phenylalanine to phenylpyruvate and generates NADH. NADH acts as a co-factor for the reduction of resazurin to resorufin by the enzyme diaphorase. The intensity of the resorufin fluorescent signal is directly proportional to the concentration of Phenylalanine present in the sample.

Phenylketonuria (PKU) is a disorder of amino acid metabolism caused by a deficiency of a liver enzyme, Phenylalanine Hydroxylase (PAH), which catalyzes the transformation of phenylalanine (Phe) into tyrosine. This led to an accumulation of large amounts of Phe in the blood. The activity of PAH is conditioned by the presence of a cofactor, tetrahydrobiopterin. 

If untreated, symptoms begin in the first few months of life and may range from very severe to mild. Clinical manifestations include irreversible intellectual disability, growth retardation, microcephaly, seizures, tremor, vomiting, and typical odor. These patients often have light skin and hair, resulting from tyrosine deficiency.

In addition, a more severe form of hyperphenylalaninemia exists, malignant PKU. This abnormality is not caused directly by PAH deficiency, but by a synthesis defect of its cofactor, tetrahydrobiopterin. With approximately 1-2% of cases of phenylketonuria, malignant PKU is characterized by both hyperphenylalaninemia and neurotransmitter deficiency (serotonin and dopamine). Clinical signs include psychomotor retardation, microcephaly, myoclonic epilepsy, hyperthermia, swallowing difficulties, and hypersalivation.

The diagnosis of PKU is based on measuring the increase of phenylalanine levels in blood. Three forms are mostly distinguished, depending on the level of PAH activity impairment and the resulting plasma phenylalanine level: Conventional PKU, Moderate PKU and hyperphenylalaninemia.