Data on phenylalanine-to-tyrosine ratios in assessment of tetrahydrobiopterin (BH4)-responsiveness in patients with hyperphenylalaninemia

Blood phenylalanine-to-tyrosine (Phe/Tyr) ratio is an important indicator of metabolic control in phenylketonuria patients. We present the data that highlights the role of Phe/Tyr-ratio in the evaluation of tetrahydrobiopterin (BH4)-responsiveness in patients with hyperphenylalaninemia. Our data complements the results from the original research article by Tansek et al., 2012 [1]. We performed a BH4-loading test in 32 patients after four days of increased protein intake (2000 mg/kg body weight). Blood sampling was performed 96, 72, 48, 24, 16 h, and moments before oral administration of BH4 in a dose of 20 mg/kg body weight. Additional blood samples were collected 8 and 24 h after its administration. Phenylalanine (Phe) and Tyrosine (Tyr) levels were determined from dried blood spots by tandem mass spectrometry. Phe/Tyr-ratio reached a plateau after three days of increased dietary protein intake. Fifteen patients (47%) responded to BH4, defined as a decrease of Phe-of at least 30% after 24 h of BH4 administration. Phe/Tyr-ratios were significantly higher in non-responders compared to responders. In the responder group, Phe/Tyr-ratios decreased in average of 67% (p = 0.001) and 45% (p = 0.001) after 8 and 24 h of BH4 administration, respectively. Phe/Tyr-ratio decreased after 8 h of drug administration also in the non-responder group, but not 24 h after administration.


a b s t r a c t
Blood phenylalanine-to-tyrosine (Phe/Tyr) ratio is an important indicator of metabolic control in phenylketonuria patients. We present the data that highlights the role of Phe/Tyr-ratio in the evaluation of tetrahydrobiopterin (BH 4 )responsiveness in patients with hyperphenylalaninemia. Our data complements the results from the original research article by Tansek et al., 2012 [1] . We performed a BH 4 -loading test in 32 patients after four days of increased protein intake (20 0 0 mg/kg body weight). Blood sampling was performed 96, 72, 48, 24, 16 h, and moments before oral administration of BH 4 in a dose of 20 mg/kg body weight. Additional blood samples were collected 8 and 24 h after its administration. Phenylalanine (Phe) and Tyrosine (Tyr) levels were determined from dried blood spots by tandem mass spectrometry. Phe/Tyr-ratio reached a plateau after three days of increased dietary protein intake. Fifteen patients (47%) responded to BH 4 , defined as a decrease of Phe-of at least 30% after 24 h of BH 4 administration. Phe/Tyr-ratios were significantly higher in non-responders compared to responders. In the responder group, Phe/Tyr-ratios decreased in average of 67% ( p = 0.001) and 45% (p = 0.001) after 8 and 24 h of BH 4 administration, respectively. Phe/Tyr-ratio decreased after 8 h of drug administration also in the non-responder group, but not 24 h after administration. ©

Value of the Data
• The data supports the use of phenylalanine-to-tyrosine (Phe/Tyr) ratio in the assessment of tetrahydrobiopterin (BH 4 )-responsiveness. • The data could help physicians in predicting the response to BH 4 in patients with hyperphenylalaninemia. • The data could be used by field experts for the development of personalized treatments for patients with hyperphenylalaninemia. • Further studies of BH 4 -responsiveness could benefit from the addition of Phe/Tyr-ratio in metabolic monitoring. • Due to the rarity of hyperphenylalaninemia-causing errors of metabolism, additional data on treatment selection is beneficial.

Patients' characteristics
We included 32 patients (17 females) with a median age of 5.7 years. The youngest patients was 1 year old and the oldest 22 years. According to BH 4 -responsiveness criteria, 15 patients were responders (9 females) and 17 non-responders (8 females). Age difference between the two groups was not statistically significant ( p = 0.355), although metabolic phenotypes were differently distributed between the responder and non-responder groups. Nine patients with classic phenylketonuria (cPKU), 4 with mild phenylketonuria (mPKU) and 4 with mild hyperphenylalaninemia (MHP) were in the non-responder group, while in the responder group none had cPKU, 8 had mPKU and 7 MHP. No side effects were observed during the BH 4 -loading test. Detailed patients' characteristics are presented in Table 1 .

Tyrosine (Tyr) concentration and Phe/Tyr-ratio pre-administration of BH 4
We monitored blood Phe, Tyr-and Phe/Tyr-ratios in nine patients (5 females) at t -96 , t -72 , t -48 , t -24 and t 0 . After 3 days of increased dietary intake of protein, blood phenylalanine (Phe) concentrations reached a plateau. As observed with Phe, Phe/Tyr-ratio rose steadily for 3 days and also reached a plateau at a mean of 16.53 ( Fig. 1 ). Tyr-concentrations stayed relatively stable during the test. The average of mean Tyr-concentrations from different time points was 50.16 μmol/L (standard deviation = 1.29 μmol/L).

Phe/Tyr-ratios regarding to phenotype
We compared Phe/Tyr-ratios in patients with different metabolic phenotypes. The highest ratios were observed in cPKU patients compared to mPKU and MHP ( p < 0.001 for both comparisons). Phe/Tyr-ratios were comparable between mPKU and MHP whereas mPKU patients had slightly higher ratios ( Fig. 2 ). This difference was statistically significant only for Phe/Tyr-ratios at t −24 and t 0 ( p = 0.019 and p = 0.042, respectively). One day after receiving BH 4 , there was a significant fall of Phe/Tyr-ratio in both mPKU and MHP patients relative to baseline levels (t 0 ), however this finding was not observed in cPKU patients ( p = 0.012, p = 0.003 and p = 0.515, respectively). In the latter group, we can see an initial decrease of Phe/Tyr-ratios after 8 h of BH 4 administration which was followed by an increase to baseline concentrations 24 h after loading.

Phe/Tyr-ratio regarding to BH 4 -responsiveness
Phe/Tyr-ratios between BH 4 -responders and non-responders were significantly different throughout the observation period ( p < 0.001) and are shown in Fig. 3 . In the responder group, Phe/Tyr-ratio decreased in average of 67% ( p = 0.001) and 45% (p = 0.001) from baseline concentrations after 8 and 24 h of BH 4 administration, respectively. On the other hand, a decrease of Phe/Tyr-ratio from baseline was evident at 8 h after BH 4 -loading and not at 24 h in the nonresponder group as Phe/Tyr-ratio decreased in average 48% ( p < 0.001) and 6% ( p = 0.356) after 8 and 24 h from BH 4 -loading, respectively.
Dataset title: Phenylalanine-to-tyrosine ratio in assessment of tetrahydrobiopterin responsiveness in hyperphenylalaninemia Dataset description: Dataset includes the general patient information (gender, age) and their metabolic phenotype. Additionally, the data on Phe, Tyr-levels and Phe/Tyr-ratios at various time points is included.

Participants of the study and collection of samples
To assess the role of Phe/Tyr-ratio in BH 4 -reponsiveness assessment, we included (i) patients with at least one mutation that was BH 4 -responsive, (ii) patients with at least one mutation with unclear effect on BH 4 -responsiveness and (iii) patients that were tested for BH 4 -responsiveness before the results of genetic analyses. The data on mutations and genotypes related BH 4 - Fig. 3. Phe/Tyr-ratios and BH 4 -responsiveness. Average Phe/Tyr-ratios together with standard deviations are presented for two groups according to the response to BH 4 . Non-responders (blue) had higher Phe/Tyr-ratios throughout the observation period compared to responders (red).
Additionally, responders experienced a decrease of Phe/Tyr-ratios and had lower Phe/Tyr-ratios at 8 h and 24 h after BH 4 -administration, while in the non-responders group Phe/Tyr-ratios were lower only 8 h after drug administration. Abbreviation: Phe/Tyr-ratio -phenylalanine-to-tyrosine ratio.
responsiveness were obtained from BIOPKU database (available at: www.biopku.org/biopku ). Genetic characteristics of the included patients were previously described in the original research article by Tansek et al. [1] . Altogether, thirty-two patients (17 females) were selected for the study, were receiving dietary treatment for hyperphenylalaninemia and were regularly followed as outpatients.

Dietary protein intake and BH 4 -loading test
Included patients stopped taking their PKU nutritional supplements for five days during the study. At the same time, each patient received her/his own individual meal plan, prepared by a dietician based on her/his food choices. Protein intake was increased to 20 0 0 mg/kg body weight, corresponding to an approximate Phe-intake of 100 mg/kg body weight. Adequacy of dietary intake was controlled on site.
Four days after starting the study, each patient received 20 mg/kg body weight of BH 4 (Kuvan®, sapropterin dihydrochloride, Merck Sorono, Darmstadt, Germany) in one orallyadministered dose. For the determination of responsiveness, we used the reduction of blood Phe-after BH 4 administration as the main criteria, while having in mind that alternative definitions of responsiveness exist [3] . A "responder" was defined when the reduction of blood Phelevel was ≥ 30% after 24 h of BH 4 administration and a "non-responder" at smaller reductions [3 , 4] . In the first three days, blood samples were collected from only 9 patients. Day 4 (t 0 ) represents the day before the administration of BH 4 . Responsiveness to BH 4 was determined by the reduction from baseline of blood Phe, measured at t + 24 . Abbreviations: BH 4 -tetrahydrobiopterin, Phe -phenylalanine.

Blood collection and amino acids measurements
Blood collection was performed four days (-96 h; t -96 ), three days (-72 h; t -72 ), two days (-48 h; t -48 ), one day (-24 h; t -24 ), 16 h (t -16 ), and moments prior (t 0 ) to BH 4 administration, and 8 and 24 h (t + 8 ; t + 24 ) later ( Fig. 4 ). Measurements at t -96 , t -72 and t -48 were done only at 9 patients while all other measurements were done in all included patients. Following collection, blood was smeared on a special filter paper and dried at room temperature for approximately 3 h. Phe-and Tyr-concentrations were measured from dried blood spots by tandem mass spectrometry (Perkin Elmer PE 200 HPLC with AB Sciex 3200 QTrap).

Statistical analysis
IBM SPSS Statistics, version 26.0 (IBM, USA) was used for statistical analysis. Data was visualized in Microsoft Office 365 Excel (Microsoft Corporation, USA). The significance of Phe/Tyr-ratios change in responder and non-responder groups and in three metabolic phenotype groups was calculated with Wilcoxon matched-pairs signed-rank test. Mann-Whitney U test was used for comparison between responders and non-responders and between different phenotype groups. A p value of ≤ 0.05 was considered as statistically significant.

Ethics Statements
All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the 20 0 0 revision of Helsinki declaration. Written informed consent was obtained from the patients/patient's parents for inclusion into the study and for anonymized data publication and are available for review upon request. This article does not contain any studies with animal subjects performed by any of the authors.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: