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

We have developed a simultaneous determination of phenylalanine (Phe), leuc ine (Leu) and galactose (Gal) by a semi-micro flow-injection analysis syste m using an enzyme-immobilized column. This method was applied to preliminar y neonatal mass-screening for inborn errors of metabolism. Enzyme-immobiliz ed gel was prepared with Phe, Leu or Gal dehydrogenase using a TSK-gel Tres yl 5PW (Tosoh Co., Tokyo), which was easily linked to the amino group of pr otein and packed into a column(1.5 i.d. x 100 mm). The enzyme-immobilized c olumns 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 x 10(-13), 3. 4 x 10(-13) and 1.6 x 10(-13) mol/assay, respectively. The measurable range s for Phe, Leu and Gal were 0.3-19.6, 0.8-18.4 and 0.4-18.2 mg/dl on the st andard dried-blood disc (3 mm i.d.) on filter paper, respectively. The accu racy of an intra-assay with each point of the standard disc (n = 5) was les s than 2.4%. The mean recoveries (n = II) of Phe, Leu and Gal from a dried- blood disc were 77.4, 78.5 and 97.4%,.respectively. Further, we measured th e Phe, Leu and Gal concentrations of neonatal blood-disc samples by the pro posed 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 metho d could quantitatively measure amino acids and galactose in one dried-blood disc on filter paper for 17 min.