Simultaneous determination of phenylalanine, leucine and galactose in a dried-blood disc by semi-micro FIA using an enzyme-immobilized column and its application to neonatal mass-screening for inborn errors of metabolism

Abstract

We have developed a simultaneous determination of phenylalanine (Phe), leucine (Leu) and galactose (Gal) by a semi-micro flow-injection analysis system using an enzyme-immobilized column. This method was applied to preliminary neonatal mass-screening for inborn errors of metabolism. Enzyme-immobilized gel was prepared with Phe, Leu or Gal dehydrogenase using a TSK-gel Tresyl 5PW (Tosoh Co., Tokyo), which was easily linked to the amino group of protein and packed into a column (1.5 i.d. × 100 mm). The enzyme-immobilized columns were connected to high-pressure 6-way valves. In the proposed method, the detection limits (S/N = 3) of Phe, Leu and Gal were 1.6 × 10-13 3.4 × 10-13 and 1.6 × 10-13 mol/assay, respectively. The measurable ranges for Phe, Leu and Gal were 0.3-19.6, 0.8-18.4 and 0.4-18.2 mg/dl on the standard dried-blood disc (3 mm i.d.) on filter paper, respectively. The accuracy of an intra-assay with each point of the standard disc (n = 5) was less than 2.4%. The mean recoveries (n = 11) of Phe, Leu and Gal from a dried-blood disc were 77.4, 78.5 and 97.4%, respectively. Further, we measured the Phe, Leu and Gal concentrations of neonatal blood-disc samples by the proposed method. The levels (mean ± SD, n = 30) were 1.5 ± 2.1 (Phe), 2.4 ± 1.4 (Leu) and 2.6 ± 2.1 (Gal) mg/dl, respectively. The proposed method could quantitatively measure amino acids and galactose in one dried-blood disc on filter paper for 17 min.