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

Background: Several methods have been reported for the quantitation of pbenylalanine (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 degreesC for 2 h. Phe and Tyr were converted to transcinnamate and trans-coumarate, respectively, which were determined UV spectrophotometrically. Results: The assay demonstrated linearity up to 3200 mumol/l Phe and 640 mumol/l Tyr. The sensitivity of the assay was 15 mumol/l for Phe and 5 mumol/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. (C) 2004 Elsevier B.V. All rights reserved.