Monitoring of phenylketonuria: a colorimetric method for the determination of plasma phenylalanine using L-phenylalanine dehydrogenase.
A simple, rapid, accurate, and precise colorimetric assay for the determination of L-phenylalanine in plasma samples using L-phenylalanine dehydrogenase [L-phenylalanine:NAD+-oxidoreductase (deaminating)] from Rhodococcus sp. M 4 is described. The enzyme catalyzes the NAD-dependent oxidative deamination of L-phenylalanine. However, the equilibrium of reaction favors L-phenylalanine formation. By stoichiometric coupling of this reaction with diaphorase/iodonitro tetrazolium chloride (INT) the formed NADH converts INT to a formazan whereby the reaction is displaced in favor of phenylpyruvate. Using a kinetic approach the increase in absorbance at 492 nm shows linearity over more than 30 min. Deproteinized standard solutions of L-phenylalanine in the range from 30 to 1200 mumol/liter show a linearity between the dAformazan/30 min and the substrate concentration. In phenylketonuria (PKU) plasma samples no interferences caused by L-tyrosine or phenylpyruvic acid are seen. Applicability is demonstrated by comparative determination of plasma L-phenylalanine of treated PKU patients by the colorimetric method and automated amino acid analysis.
Wendel
U
U
Kinderklinik, Universität Düsseldorf, Federal Republic of Germany.
Hummel
W
W
Langenbeck
U
U
D000594:Amino Acid Oxidoreductases; D001322:Autoanalysis; D003641:Deamination; D005069:Evaluation Studies as Topic; D006801:Humans; D007700:Kinetics; D008991:Monitoring, Physiologic; D010649:Phenylalanine / Q000097:blood*; D010661:Phenylketonurias / Q000097:blood*; D012240:Rhodococcus / Q000201:enzymology; D013053:Spectrophotometry
D010649:Phenylalanine; D000594:Amino Acid Oxidoreductases; C022342:phenylalanine oxidase