Quantification of phenylalanine hydroxylase activity by isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry
Quantification of phenylalanine hydroxylase activity by isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry
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DOI：
10.1016/j.ymgme.2011.12.025
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发表时间：
2012-04-01
影响因子：
3.8
通讯作者：
Blau, Nenad
中科院分区：
文献类型：
--
作者：
Heintz, Caroline;Troxler, Heinz;Blau, Nenad
Background: Residual phenylalanine hydroxylase (PAH) activity is the key determinant for the phenotype severity in phenylketonuria (PKU) patients and correlates with the patient's genotype. Activity of in vitro expressed mutant PAH may predict the patient's phenotype and response to tetrahydrobiopterin (BH4), the cofactor of PAH.Methods: A robust LC-ESI-MSMS PAH assay for the quantification of phenylalanine and tyrosine was developed. We measured PAH activity a) of the PAH mutations p.Y417C, p.I65T, p.R261Q, p.E280A, p.R158Q p.R408W, and p.E390G expressed in eukaryotic COS-1 cells; b) in different cell lines (e.g. Huh-7, Hep3B); and c) in liver, brain, and kidney tissue from wild-type and PKU mice.Results: The PAH assay was linear for phenylalanine and tyrosine (r(2)>= 0.99), with a detection limit of 105 nmol/L for Phe and 398 nmol/L for Tyr. Intra-assay and inter-assay coefficients of variation were
