Quantitation of L-amino acids by substrate recycling between an aminotransferase and a dehydrogenase: application to the determination of L-phenylalanine in human blood.
A spectrophotometric recycling assay for the quantitation of L-phenylalanine (and phenylpyruvate) has previously been reported (Cooper et al., Anal. Biochem. 183, 210-214, 1989). The procedure involves the coupling of bacterial phenylalanine dehydrogenase with rat kidney cytosolic glutamine transaminase K. The latter enzyme possesses high affinity for phenylpyruvate. Recycling results in a > or = 50-fold increase in sensitivity over that of a conventional spectrophotometric "end point" analysis procedure. The spectrophotometric recycling procedure has now been adapted to the measurement of L-phenylalanine in microliter quantities of human blood. This procedure is 10 times more sensitive than provided by a commercial kit for the spectrophotometric measurement of L-phenylalanine in human blood. Moreover, the present results suggest that the recycling procedure adapted for fluorometry will be even more sensitive. By use of suitable dehydrogenases and amino acid aminotransferases it should be possible to quantitate amino acids (in addition to phenylalanine) in small quantities of human blood.
Nakamura
K
K
Sapporo Immunodiagnostic Laboratory, Japan.
Fujii
T
T
Kato
Y
Y
Asano
Y
Y
Cooper
A J
AJ
D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D001407:Bacillus / Q000201:enzymology; D003600:Cytosol; D006801:Humans; D007202:Indicators and Reagents; D007668:Kidney / Q000201:enzymology; D010649:Phenylalanine / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods; D013053:Spectrophotometry / Q000379:methods; D000637:Transaminases / Q000302:isolation & purification / Q000378:metabolism
D000596:Amino Acids; D007202:Indicators and Reagents; D010649:Phenylalanine; D000594:Amino Acid Oxidoreductases; C022342:phenylalanine oxidase; D000637:Transaminases; C020656:glutamine-pyruvate aminotransferase