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 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 degrees 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 micromol/l Phe and 640 micromol/l Tyr. The sensitivity of the assay was 15 micromol/l for Phe and 5 micromol/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.
Wibrand
Flemming
F
John F. Kennedy Institute, Gl. Landevej 7, 2600 Glostrup, Denmark. flw@kennedy.dk
D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002934:Cinnamates / Q000737:chemistry; D003374:Coumarins / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents; D016014:Linear Models; D010649:Phenylalanine / Q000097:blood*; D010650:Phenylalanine Ammonia-Lyase / Q000737:chemistry; D010661:Phenylketonurias / Q000097:blood* / Q000175:diagnosis; D012015:Reference Standards; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000097:blood*
D002934:Cinnamates; D003374:Coumarins; D007202:Indicators and Reagents; D014443:Tyrosine; D010649:Phenylalanine; D010650:Phenylalanine Ammonia-Lyase