Determination of phenylalanine enantiomers in the plasma and urine of mammals and ᴅ-amino acid oxidase deficient rodents using two-dimensional high-performance liquid chromatography

Sui Wen Hsiao, Chiharu Ishii, Aogu Furusho, Chin Ling Hsieh, Yukiko Shimizu, Takeyuki Akita, Masashi Mita, Tadashi Okamura, Ryuichi Konno, Tomomi Ide, Ching Kuo Lee, Kenji Hamase

Research output: Contribution to journalArticlepeer-review

Abstract

A two-dimensional (2D) HPLC system focusing on the determination of phenylalanine (Phe) enantiomers in mammalian physiological fluids has been developed. ᴅ-Phe is indicated to have potential values as a disease biomarker and therapeutic molecule in several neuronal and metabolic disorders, thus the regulation of ᴅ-Phe in mammals is a matter of interest. However, the precise determination of amino acid enantiomers is difficult in complex biological samples, and the development of an analytical method with practically acceptable sensitivity, selectivity and throughput is expected. In the present study, a 2D-HPLC system equipped with a reversed-phase column in the 1st dimension and an enantioselective column in the 2nd dimension has been designed, following the fluorescence derivatization of the target amino acid enantiomers with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The analytical method was validated using both plasma and urine samples, and successfully applied to human, rat and mouse fluids. Trace levels of ᴅ-Phe were determined in the plasma, and the %ᴅ values were around 0.1% for all species. In the urine, relatively large amounts of ᴅ-Phe were observed, and the %ᴅ values for humans, rats and mice were 3.99, 1.76 and 5.25%, respectively. The relationships between the enzymatic activity of ᴅ-amino acid oxidase (DAO) and the amounts of intrinsic ᴅ-Phe have also been clarified, and high ᴅ-Phe amounts were observed (around 0.3% in the plasma and around 50% in the urine) in the DAO deficient rats and mice.

Original languageEnglish
Article number140540
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1869
Issue number1
DOIs
Publication statusPublished - Jan 2021

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biophysics
  • Biochemistry
  • Molecular Biology

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