EVALUATION OF PHENYLALANINE ISOTOPES USING AN ISOLATED ORGAN PERFUSION MODEL
Date
1999
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
Type
Degree Level
Masters
Abstract
The objective of the research was to evaluate the possibility of isotopic discrimination between two isotopomers of phenylalanine during the hydroxylation to tyrosine. Phenylalanine hydroxylation of L-[1-13C]phenylalanine and L-{ring- 2H5]phenylalanine were evaluated in three in vitro experiments, as well as an isolated liver perfusion model. The in vitro experiment involved an assay system using purified phenylalanine hydroxylase enzyme in a buffer medium. The isolated liver perfusion was set up to examine the first-pass hepatic metabolism of phenylalanine. Tyrosine production from the unlabeled phenylalanine as well as the two isotopomers was measured by spectrophotometric assay and high-performance liquid chromatography (HPLC). Gas chromatography-mass spectrometry (GC-MS) analysis was used to measure the enrichment of 13C-tyrosine and 2H4-tyrosine in a third in vitro experiment as well as labeled tyrosine and phenylalanine enrichments in the liver perfusion experiments. In a
kinetic assay, the rate of production of 2H4-tyrosine (from hydroxylation 2H5-
phenylalanine), was found to be significantly lower (p < 0.01) than the rate of production of unlabeled or 13C-tyrosine. When overall tyrosine production was measured by HPLC, the
2H4 -tyrosine production was found to be significantly lower than from both unlabeled and 13C-tyrosine (p < 0.01). 13C-Tyrosine was also found to be significantly lower than unlabeled tyrosine (p < 0.05). When measured by GC-MS, the enrichment of 13C- tyrosine was found to be significantly greater than the enrichment of 2H4-tyrosine (p < 0.05) indicating more production of tyrosine from 13C-phenylalanine than from 2H5- phenylalanine. In the liver perfusion experiments, mean phenylalanine enrichments were 15.57 ± 2.99 and 15.94 ± 3.16 for 13C-phenylalanine and 2H5-phenylalanine, respectively. Mean tyrosine enrichments were 12.18 ± 2.04 and 11.61 ± 1.94 for 13C- tyrosine and 2H4-tyrosine, respectively. There was no significant difference for either the phenylalanine (p > 0.05) or tyrosine (p > 0.05) enrichments. The lack of significant difference between enrichments of 13C-phenylalanine and 2H5-phenylalanine, or 13C- tyrosine and 2H4-tyrosine indicates that the discrimination at the enzymatic level is not
large enough to affect hepatic enrichment. This study suggests that, although discrimination may exist between L-[1-13C]phenylalanine and L-[ring-2H5]phenylalanine, the liver is not likely the site for such discrimination.
Description
Keywords
isotopic discrimination, phenylalanine, isolated liver perfusion model
Citation
Degree
Master of Science (M.Sc.)
Department
Pharmacy and Nutrition