Quantification of phenylalanine hydroxylase activity by isotope-dilution liquid chromatography–electrospray ionization tandem mass spectrometry
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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2021, Organic and Biomolecular ChemistryL-phenylalanine-imprinted polydopamine-coated CdS/CdSe n-n type II heterojunction as an ultrasensitive photoelectrochemical biosensor for the PKU monitoring
2020, Biosensors and BioelectronicsCitation Excerpt :Since these methods usually require a long time for sample processing, modern system based on developed electrochemical analysis, with the advantages of rapid response, high sensitivity, and simple handling can be used for the development of next-generation clinical diagnostic technology (Moreira et al., 2018; Xu et al., 2019b). Because PKU is a disease caused by the deficiency of the metabolic phenylalanine hydroxylase (PAH) enzyme, PAH activity measurements has been proposed as a method of PKU diagnosis (Heintz et al., 2012), while complicated handling procedures and the requirement for special care with enzymatic activity make these methods less robust and difficult for continuous in situ analysis (Charkhabi et al., 2018; Sassolas et al., 2012; Wick et al., 2019). Accordingly, PKU treatment demands urgent innovative interventions, developing a method based on direct detection of L-Phe as the most effective PKU marker without the employment of bio-recognizers, which is the most appropriate pathway to overcome the above-mentioned limitations and thus suitably μM analysis of L-Phe(Cheung et al., 2019; Idili et al., 2019; Zhou et al., 2018).
In vitro residual activities in 20 variants of phenylalanine hydroxylase and genotype-phenotype correlation in phenylketonuria patients
2019, GeneCitation Excerpt :Different methods to detect the in vitro PAH activity were used in previous studies, such as the colorimetric assay (Guroff et al., 1967), thin layer chromatography (TLC) based on the generation of 14C-labeled Tyr transformed from 14C-labeled Phe (Gamez et al., 2000), HPLC coupled to fluorescence detection (Bailey and Ayling, 1980) and continuous real-time PAH activity assay based on automated fluorescence (Gersting et al., 2010). In 2012, a new method of LC-ESI-MS/MS for quantification of L-Phe and L-Tyr at standardized BH4 and Phe concentrations was estimated with high accuracy and sensitivity, the limit of detection (LOD) was 0.1 μmol/L for Phe and Tyr (Heintz et al., 2012). In our study, we have applied this method to detect the in vitro residual activities of 20 PAH variants.
Relationship between genotype, phenylalanine hydroxylase expression and in vitro activity and metabolic phenotype in phenylketonuria
2018, Molecular Genetics and MetabolismCitation Excerpt :As mentioned previously, not only different cell systems but also different PAH assays have been used to measure enzyme activity. Different methods, such as thin-layer chromatography (TLC) with 14C-Phe to 14C-Tyr conversion [27], HPLC with fluorometric detection [29], or sophisticated mass-spectrometry methods involving the use of stable isotopes [18], have been employed to measure PAH activity at different temperatures, after different reaction times and using different substrates (BH4 or 6-methyl-tetrahydropterin; 6MTHP). It is difficult to believe that these strategies produce exactly same results, but there is concordance for variants with a very low activity (see above).
Three novel variants (p.Glu178Lys, p.Val245Met, p.Ser250Phe) of the phenylalanine hydroxylase (PAH) gene impair protein expression and function in vitro
2018, GeneCitation Excerpt :These results indicate that the mutant PAH proteins are likely degraded by proteases soon after synthesis because of aberrant folding or misassembly. PAH activity in human kidney was thought to be undetectable, but in kidney of wild type mice, PAH activity was around 30% of liver (Heintz et al., 2012). HEK293T cell was supposed to express low level of or no PAH since this cell line is derived from human embryo kidney.