A microplate-based enzymatic assay for the simultaneous determination of phenylalanine and tyrosine in serum

Abstract

Background: Several methods have been reported for the quantitation of phenylalanine (Phe) or tyrosine (Tyr) in blood, but the simultaneous determination of Phe and Tyr usually requires sophisticated equipment. Here we describe a simple and reliable microplate assay for measurement of Phe and Tyr in serum of patients with phenylketonuria (PKU). Methods: Serum was deproteinised and incubated with phenylalanine ammonia-lyase at 37 °C for 2 h. Phe and Tyr were converted to trans-cinnamate and trans-coumarate, respectively, which were determined UV spectrophotometrically. Results: The assay demonstrated linearity up to 3200 μmol/l Phe and 640 μmol/l Tyr. The sensitivity of the assay was 15 μmol/l for Phe and 5 μmol/l for Tyr. Analytical recovery was 105% and 97% for Phe and Tyr, respectively. The between-run coefficient of variation (CV) varied with concentration but was 4.7–18.8% for Phe and 4.2–8.8% for Tyr. The within-run CV was 2.7% for Phe and 6.5% for Tyr. Results obtained with the assay correlated well with those of an ion exchange chromatographic method. Conclusions: This novel assay is sensitive, accurate, precise and suitable for routine monitoring of PKU patients.