Quantification of phenylalanine hydroxylase activity by isotope-dilution liquid chromatography–electrospray ionization tandem mass spectrometry

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Abstract

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 (r2  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 BH4) 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.

Highlights

► Activity of mutant PAH may predict the response to tetrahydrobiopterin. ► LC–ESI-MSMS PAH assay for the quantification of Phe and Tyr was developed. ► This novel method allows the quantification of PAH activity in cells or tissue extracts.

Introduction

Deficiency of phenylalanine hydroxylase (PAH, EC 1.14.16.1) is causing phenylketonuria (PKU, OMIM 261600), an autosomal recessively inherited disease presenting with elevated blood phenylalanine (Phe) levels [1], [2]. The phenotypic severity of PKU is characterized by the type of mutation, and thus by residual PAH enzyme activity. The fully functional homotetrameric PAH catalyzes hydroxylation of Phe to tyrosine (Tyr) in the presence of cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and molecular oxygen [3], [4]. According to the Locus Knowledgebase (PAHdb, www.pahdb.mcgill.ca), about 60% of mutations in the PAH gene are missense mutations, which may lead to a misfolding of the protein [5], [6], disturbing the complex enzyme regulation and changes in kinetics, due to altered affinities for the Phe substrate and the BH4 cofactor.

The incidence of PKU is about 1 in 10,000 newborns in Caucasian populations [2]. For most patients, therapy consists in a life-long dietary restriction of Phe to prevent neurological impairment. Recently, it has been reported that a subgroup of PKU patients (mild to moderate phenotype) can benefit from a pharmacological therapy with BH4 (sapropterin dihydrochloride; Kuvan®) [7], [8]. Newborn screening program for PKU, initially based on the Guthrie test [9], has been established for the early detection of PKU patients. Today, electrospray ionization tandem mass-spectrometry (ESI-MSMS or TMS) is the method of choice for fast screening and monitoring of Phe and Tyr levels in dried blood spots (DBS) [10].

Of the over 550 disease-causing mutations listed in the PAHdb [11], 88 were expressed in different in vitro cell systems to estimate the residual PAH activity. Expression systems like Escherichia coli, eukaryotic cell lines, or cell-free systems were most commonly used systems [12], [13], [14]. In addition to cell systems, PAH activity was studied in rat liver biopsy samples [15]. Expression of recombinant PAH in bacteria was applied for characterization of physical and chemical properties of the enzyme [16].

Previous methods for PAH activity measurement were based on the determination of 14C-labeled Tyr produced [3] or release of 3H [17]. Other methods are based on detection of Tyr by fluorescence coupled to HPLC [18], colorimetric assays [19], or fluorescence monitoring [20]. Recently described method by Gersting et al. [20] was developed for characterization of purified mutant PAH proteins at different Phe and BH4 concentrations.

In our novel assay, we applied liquid chromatography (LC) with ESI-MSMS for the quantification of Tyr produced from Phe. Prior to analysis, the amino acids are derivatized to propyl chloroformate derivatives, using the commercially available Phenomenex EZ:faast™ kit. Our method allows for short analysis times and lower limit of detection and is optimized for determination of PAH enzyme activity of recombinantly expressed mutant proteins in COS-1 and other cell lines, as well as in mice liver, kidney, and brain. Thus, it allows comparison between different mutant proteins at standard conditions.

Section snippets

Materials

The Phenomenex EZ:faast™ kit for LC with ESI-MSMS amino acid analysis was purchased from Phenomenex (Torrance, CA, USA). L-phenylalanine-d5 and L-tyrosine-d4 standard reagents were purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA) whereas L-phenylalanine-d8 was obtained from C/D/N Isotopes Inc. (Pointe Claire, Quebec, Canada). L-Phenylalanine and L-Tyrosine, as well as the DMEM cell culture medium were purchased from Sigma Aldrich (St. Louis, MO, USA). RPMI 1640 medium was

Linearity and limit of detection

Fig. 1 depicts linear regression of calibration standards for Phe and Tyr. Both calibration curves were linear with correlation coefficients of r2  0.99.

The LOD was 105 nmol/L for Phe and 398 nmol/L for Tyr and LOQ 147 nmol/L for Phe and 574 nmol/L for Tyr.

Imprecision and recovery

Inter- and intra-assay analyses were performed with the wild-type enzyme, with medium activity (p.R261Q), and low activity (p.R158Q) PAH variants, transfected into COS-1 cells. Six samples were each prepared either on the same day (intra-assay) or

Discussion

Molecular mechanisms of PKU and other hyperphenylalaninemias were established over several decades through investigations of mutations within the PAH gene [30]. Based on genotype findings and description of BH4-responsive forms of PKU [31], functional assays of mutation effects in vitro have proven to be very fruitful for the characterization of PAH mutations, building a bridge between patient and pure protein. Although, these data sets tend to overestimate PAH activities in vivo. Residual in

Acknowledgments

This work was supported by the Swiss National Science Foundation grant no. 31003A-119982 (to NB and BT). We would like to thank Dr. L. Ruiz Desviat for providing us with the PAH expression plasmid and Dr. H. M Viecelli for the mouse tissue samples and Dr. R. Dummer for keratinocytes. In addition, we thank the mass spectrometry group of the Children's Hospital in Zurich for expert technical assistance.

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