Phenylalanine determination in fish serum: adaptation of a mammalian method to fish.

Biochemical methods originally designed for mammalian tissues are not, in all instances, directly adaptable to tissues from other species, such as fish. Consideration must be given to the biochemical profile of the research animal to which an analytical method is to be applied. In particular, when adapting a method for an amino acid such as phenylalanine from mammalian serum to fish serum, one must be aware of the relative concentration of the amino acid and also the total amino acid concentration in each species. Chance (1) reported that the total amino acid concentration of salmon plasma was two to three greater than that of swine. The level of each essential amino acid was three to six times higher in salmon than in pigs. We herein report a fluorimetric assay adapted from a mammalian method for the quantitat,ive determination of phcnylalaninc in fish serum. The fluorimetric method is based on the interaction of phcnylalanine with ninhydrin and the peptide leucyl-alanine. The greater concentration of amino acids in fish serum necessitates adding more of the ninhydrin-peptide reagent to the reaction to obtain quantitative results. McCaman and Robins (2) reported a quantitative fluorimetric method for phenylalanine in mammalian serum. The reproducibility [(SF/X) X 1001 was one to t.hree percent, and the accuracy (recovery from spiked sample) was 97%. This fluorimetric assay was based on the observation of Lowe et al. (3) that the fluorescence resulting from the reaction of phcnylalanine and ninhydrin was increased by the addit’ion of the peptide glycyl-m-phenylalanine. However, this fluorrsccnt reaction was not specific for phenylalanine, whereas McCaman and Robins found that using the peptide L-leucyl-L-alanine gave a specific, sensitive reaction for phenplalanine. This procedure involved adding 0.3 M succinate buffer (pH 5.8)) 30 nlM ninhydrin, and 5 mM L-leucyl-L-alanine in a volume ratio of 10:4:2, respectively, to deproteinized serum filtrate. After incubation of this mixture for 2 hr at, 6O”C, a copper solution was added and the resulting fluorescence measured at 392 nm excitation and 492 nm fluorescence. A modification of this method was outlined in Sigma Tentative Technical Bulletin No. 60-F1 (4) in which 0.6 M succinate buffer (pH