ACTA ENDOCRINOLOGICA (BUC)

Phenylketonuria (PKU) is the most frequent inherited amino acid metabolic disorder, and it may also be treated by dietary means. The determination of phenylalanine (Phe) levels in the blood plasma is important not only in early diagnostic, but also in monitoring the treatment of PKU. Purpose. The aim of our work was to develop a simple, sensitive and highly accurate procedure to determine the plasma concentration of Phe. Procedure. The measurement of plasma Phe concentration involves two steps: a) separation of plasma (from the blood taken on heparin), isolation and preparation of a concentrated solution of amino acids (by ion-exchange column chromatography on Dowex-50X8 and evaporation of the eluate in vacuum at 40˚C), and b) determination of Phe concentration in the solution of amino acids by HPLC. This analysis was performed using a Dionex Ultimate 3000 instrument equipped with a Ultimate 3000 diode array detector (DAD). The values of Phe concentration in the plasma of several patients were calculated using a calibration curve made with standards of Phe (dilutions of a stock solution of 50 mg/ dL). The measurements in duplicate (plasma Phe) or a greater number of samples from the same concentration of standards of Phe showed extremely small sample to sample differences. Concentrations as low as 0.2 mg/dL could be determined. Conclusion. The whole procedure presented here is relatively simple, rather inexpensive, however very sensitive and highly accurate. Consequently, it is very adequate for confirming the diagnosis of PKU in patients with neonatal hyperphenylalaninemia, as well as for monitoring the plasma concentration of Phe in patients with PKU.

Introduction. The determination of phenylalanine (Phe) and tyrosine (Tyr) levels in blood plasma is very important not only in early diagnostic, but also in monitoring the treatment of phenylketonuria (PKU). Purpose. We present a simple, sensitive and accurate procedure to determine simultaneously the plasma concentrations of Phe and Tyr. Procedure. The measurement involves two steps: a) separation of plasma (from blood prelevated on heparin), isolation and preparation of a concentrated solution of amino acids (by ion-exchange column chromatography on Dowex- 50X8), and b) determination of Phe and Tyr concentrations in the solution of amino acids by HPLC (using a Dionex Ultimate 3000 instrument equipped with a diode array detector). The analytical column was a Thermo Scientific Acclaim 120, C18, 5 μm Analitic (4.6 x 250 mm), coupled with an Acclaim C18 guard column. The values of Phe and Tyr concentrations in plasma of several patients were calculated using a calibration curve made with standards of Phe (1834.4 μmol/L in deionized water) and Tyr (600 μmol/L in deionized water). Concentrations as low as 24 μmol/dL of Phe and 15 μmol/dL of Tyr could be determined. Conclusion. The whole procedure presented here is relatively simple, rather inexpensive, however very sensitive and accurate. Consequently, it is very adequate for confirming the diagnosis of PKU in patients with neonatal hyperphenylalaninemia, as well as for monitoring the plasma concentrations of Phe and Tyr in patients with PKU.