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 (BH(4)), 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 <5.3% and <6.2%, respectively, for the p.R158Q variant in lower tyrosine range. Recovery of tyrosine was 100%. Compared to the wild-type enzyme, the highest PAH activity at standard conditions (1 mmol/L L-Phe; 200 μmol/L BH(4)) was found for the mutant p.Y417C (76%), followed by p.E390G (54%), p.R261Q (43%), p.I65T (33%), p.E280A (15%), p.R158Q (5%), and p.R408W (2%). A relative high PAH activity was found in kidney (33% of the liver activity), but none in brain. CONCLUSIONS: This novel method is highly sensitive, specific, reproducible, and efficient, allowing the quantification of PAH activity in different cells or tissue extracts using minimum amounts of samples under standardized conditions. Copyright Â
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 (BH(4)), 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 PKUmice. 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 <5.3% and <6.2%, respectively, for the p.R158Q variant in lower tyrosine range. Recovery of tyrosine was 100%. Compared to the wild-type enzyme, the highest PAH activity at standard conditions (1 mmol/L L-Phe; 200 μmol/L BH(4)) was found for the mutant p.Y417C (76%), followed by p.E390G (54%), p.R261Q (43%), p.I65T (33%), p.E280A (15%), p.R158Q (5%), and p.R408W (2%). A relative high PAH activity was found in kidney (33% of the liver activity), but none in brain. CONCLUSIONS: This novel method is highly sensitive, specific, reproducible, and efficient, allowing the quantification of PAH activity in different cells or tissue extracts using minimum amounts of samples under standardized conditions. Copyright Â
Authors: Diane B Zastrow; Heather Baudet; Wei Shen; Amanda Thomas; Yue Si; Meredith A Weaver; Angela M Lager; Jixia Liu; Rachel Mangels; Selina S Dwight; Matt W Wright; Steven F Dobrowolski; Karen Eilbeck; Gregory M Enns; Annette Feigenbaum; Uta Lichter-Konecki; Elaine Lyon; Marzia Pasquali; Michael Watson; Nenad Blau; Robert D Steiner; William J Craigen; Rong Mao Journal: Hum Mutat Date: 2018-11 Impact factor: 4.878
Authors: Yair Anikster; Tobias B Haack; Thierry Vilboux; Ben Pode-Shakked; Beat Thöny; Nan Shen; Virginia Guarani; Thomas Meissner; Ertan Mayatepek; Friedrich K Trefz; Dina Marek-Yagel; Aurora Martinez; Edward L Huttlin; Joao A Paulo; Riccardo Berutti; Jean-François Benoist; Apolline Imbard; Imen Dorboz; Gali Heimer; Yuval Landau; Limor Ziv-Strasser; May Christine V Malicdan; Corinne Gemperle-Britschgi; Kirsten Cremer; Hartmut Engels; David Meili; Irene Keller; Rémy Bruggmann; Tim M Strom; Thomas Meitinger; James C Mullikin; Gerard Schwartz; Bruria Ben-Zeev; William A Gahl; J Wade Harper; Nenad Blau; Georg F Hoffmann; Holger Prokisch; Thomas Opladen; Manuel Schiff Journal: Am J Hum Genet Date: 2017-01-26 Impact factor: 11.025
Authors: Vikram K Raghu; Steven F Dobrowolski; Rakesh Sindhi; Kevin A Strauss; George V Mazariegos; Jerry Vockley; Kyle Soltys Journal: Mol Genet Metab Rep Date: 2022-04-21
Authors: Hiu Man Viecelli; Richard P Harbottle; Suet Ping Wong; Andrea Schlegel; Marinee K Chuah; Thierry VandenDriessche; Cary O Harding; Beat Thöny Journal: Hepatology Date: 2014-07-29 Impact factor: 17.425