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Citation: Hanusch, B.; Falkenstein, M.;
Volkenstein, S.; Dazert, S.; Lücke, T.;
Sinningen, K. No Impairment in Bone
Turnover or Executive Functions in
Well-Treated Preschoolers with
Phenylketonuria—A Pilot Study.
Nutrients 2024,16, 2072. https://
doi.org/10.3390/nu16132072
Academic Editor: Yajun Xu
Received: 21 May 2024
Revised: 20 June 2024
Accepted: 25 June 2024
Published: 28 June 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
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distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
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4.0/).
nutrients
Article
No Impairment in Bone Turnover or Executive Functions in
Well-Treated Preschoolers with Phenylketonuria—A Pilot Study
Beatrice Hanusch 1, Michael Falkenstein 2, Stefan Volkenstein 3, Stefan Dazert 4, Thomas Lücke 1
and Kathrin Sinningen 1, *
1Research Department of Child Nutrition, University Hospital of Pediatrics and Adolescent Medicine,
St. Josef-Hospital, Ruhr-University Bochum, 44791 Bochum, Germany
2ALA Institute, 44805 Bochum, Germany
3Department of Otorhinolaryngology, Head and Neck Surgery, Johannes Wesling Klinikum Minden,
Ruhr-University Bochum, 32429 Minden, Germany
4Department of Otorhinolaryngology, Head and Neck Surgery, St. Elisabeth-Hospital Bochum,
Ruhr-University Bochum, 44787 Bochum, Germany
*Correspondence: kathrin.sinningen@rub.de
Abstract: Patients with phenylketonuria (PKU) present signs of impaired executive functioning and
bone health in adolescence and adulthood, depending in part on the success of therapy in childhood.
Therefore, nine children with well-treated PKU (4–7 years old, 22.2% ♀, seven with a full set of data,
two included into partial analysis) and 18 age-, gender- and season-matched controls were analyzed
for differences in executive functioning and bone parameters in plasma. Plasma was analyzed
with commercially available kits. Cognitive performance in tonic alertness, visuo-spatial working
memory, inhibitory control and task switching was assessed by a task battery presented on a touch
screen. Regarding cognition, only the performance in incongruent conditions in inhibitory control
was significantly better in children with PKU than in controls. No further differences in cognitive
tests were detected. Furthermore, no significant difference in the bone turnover markers osteocalcin,
undercarboxylated osteocalcin and CTX were detected between children with PKU and controls,
while children with PKU had a significantly higher vitamin D concentration (69.44
±
12.83 nmol/L
vs. 41.87
±
15.99 nmol/L, p< 0.001) and trended towards lower parathyroid hormone concentrations
than controls (48.27
±
15.16 pg/mL vs. 70.61
±
30.53 pg/mL, p= 0.066). In this small group of
well-treated preschoolers with PKU, no impairments in cognitive performance and bone turnover
were observed, while vitamin D supplementation of amino acid supplements seems to be sufficient
to achieve good vitamin D status.
Keywords: preschool children; bone turnover; phenylketonuria; executive functioning; cognition;
calciotropic hormones
1. Introduction
Phenylketonuria (PKU; OMIM #261600) is a congenital defect of the Phenylalanine
(Phe) metabolism with an incidence of 19.3/100,000 newborns in 2020 in Germany [
1
,
2
].
Variants in the gene encoding phenylalanine hydroxylases lead to reduced activity of the
enzyme, subsequently causing the accumulation of Phe and its metabolites in the blood
and brain [
1
]. Classical symptoms of PKU include irreversible intellectual disability, motor
deficits and developmental problems [
1
]. Early diagnosis and start of treatment, which
entails the reduction of Phe intake from natural protein and the supplementation of amino
acids, can prevent the development of most symptoms [
1
,
3
]. As consumption of natural
protein needs to be reduced up until the point of the Phe blood concentrations meeting the
target range, overall protein and caloric restriction is not sufficient for normal growth [
3
].
Therefore, foods naturally higher in protein, such as baked goods and pastas, are available
as low-protein substitutes to ensure an adequate energy supply, while protein requirement
Nutrients 2024,16, 2072. https://doi.org/10.3390/nu16132072 https://www.mdpi.com/journal/nutrients
Nutrients 2024,16, 2072 2 of 16
is covered by Phe-free amino acid mixtures with added vitamins (such as vitamin D) and
minerals [
3
]. While a certain amount of Phe is required for healthy development, a higher
intake of Phe results in the elevation of blood Phe concentration and should be limited [
1
,
3
].
Adults, therefore, tend towards a vegan-like diet, to reduce the intake of natural Phe [
4
].
Tetrahydrobiopterin (BH
4
) is the co-factor of phenylalanine hydroxylases, as well as various
enzymes involved in neurotransmitter synthesis [
1
,
5
]. BH
4
is available as therapeutic agent
for patients responding to the treatment, which enables them to increase their intake of
natural protein by ≥100% and/or gain biochemical control [1].
The leading goal in the therapy of patients with PKU is the prevention of neurocog-
nitive deficits [
1
]. Despite the early diagnosis, children and adults with PKU achieve a
significantly lower IQ than controls, but within normal range [
6
–
8
]. Additionally, executive
functioning, e.g., working memory, sustained attention, problem solving and strategy was
found to be lower in adolescents and adults with PKU, but less so in children in most
but not all studies [
9
–
12
]. The effects of BH
4
usage and dietary control were also studied
in relationship with cognitive outcomes in patients with PKU, and lead to inconsistent
results [
6
,
9
–
12
]. A regression analysis of Phe levels early in life and attention in 57 children
with PKU between 7 and 14 years of age showed a significant influence of Phe levels
between ages 5 and 7 on attention control and reaction speed [
13
]. Additionally, lesions
sustained in the white matter of the brain were observed in patients with PKU of all ages
and seem to be negatively affected by Phe concentrations earlier in life [
7
,
8
,
14
,
15
]. The
ability to sustain attention, suppress impulsivity and renew and connect information at
preschool age is important for later academic achievements [
16
]. Accordingly, preschool
age might be especially interesting in the cognitive development of children with PKU. We
were especially interested in the attention and executive functioning (inhibition, working
memory and task switching) of preschoolers, as these are underlying mechanisms for
higher cognitive functions [
17
–
20
]. Executive functions correlate with general intelligence
but are not a measure for intelligence itself [
21
]. Inhibition can be tested by the Flanker
Task, in which an interfering item surrounding the goal item slows down the reaction
time [
17
]. Corsi Block Tapping Task can be used to measure the renewing and span of
the visuo-spatial subsystem [
22
]. Switching between tasks requires both inhibition and
working memory for accurate and speedy processing [17,19].
However, cognitive disturbances are not the only obstacle patients with PKU have to deal
with. For instance, a lower bone mineral density (BMD) was described in several studies in
children and adults with PKU [
23
–
27
], while some found reduced bone quality in some patients
with PKU [
28
–
32
]. A systematic review and meta-analysis of bone health in patients with PKU
found a frequency of BMD Z-Score between
−
1 and
−
2.5 in 28–45% of children, and 5–13%
showed BMD Z-Scores below
−
2.5 [
27
]. In European adults with PKU, mean BMD Z-Scores
were significantly lower than in the reference population, but a low BMD Z-Score (
≤−
2) only
occurred in 1.6–5.5% [
25
]. While low adherence to diet (and therefore a higher intake of Phe
than required for low Phe blood concentration) on the one hand is associated with reduced bone
quality and higher spontaneous osteoclastogenesis [
24
,
28
], dietary supplementation with amino
acids on the other hand might negatively affect bone mineralization [
23
,
33
–
35
]. Bone accrual in
childhood might influence bone health in older age. Even subclinical disturbances during early
childhood could have an impact on peak bone mass, with short-term effects being visible in
bone turnover markers rather than in changed BMD, which only depicts bone mineral status
but not the fluctuation in bone turnover [
36
–
38
]. On the other hand, single measurements of
bone turnover markers may be limited in their ability to reflect current bone status in children,
but an analysis of BMD is reliant upon radiation usage and therefore not without risk [
36
,
39
].
Bone turnover is the result of bone resorption and bone formation, which are tightly interwoven
with each another. To evaluate bone formation, certain markers from collagen synthesis and
osteoblast activity can be used, such as osteocalcin (OCN), alkaline phosphatases, or procollagen
1 N-Peptide [
40
]. Bone resorption can be measured by collagens released from bone matrix and
osteoclast activity markers, such as N- or C-terminal cross-linked telopeptide of type-1 collagen
(CTX) as well as Tartrate-resistant acid phosphatase type 5b activity [40].
Nutrients 2024,16, 2072 3 of 16
As described, the studied impairments in bone health and the cognition of patients
with PKU are manifold and complex. Since early intervention in cognitive development
and bone formation could have a positive effect on health in adulthood, the investigation
of corresponding parameters in young, well-treated children with PKU is of interest. We
hypothesized that even in young, well-treated children with PKU, the first signs of increased
bone turnover and deficits in executive functions compared to healthy age-matched controls
can be observed.
2. Materials and Methods
Nine children with PKU (4–7 years old) were recruited to participate during routine
check-ups and were characterized by daily Phe tolerance, which is matched to the child’s
weight and changes in blood Phe concentration, as well as blood/dried blood filter cards (DBS)
Phe concentrations and tyrosine concentrations in the months before, during and after study
participation (Figure 1A). Mean Phe and tyrosine concentrations were calculated for these
3 months from a mixture of plasma and DBS results, as DBS usually are collected regularly
by the parents. All participating patients were treated with protein restriction and amino
acid-supplements; therefore, no child with mild hyperphenylalaninemia was included [
1
].
Additionally, caregivers of children with PKU gave information on supplement use, other
than amino acid supplementation used in treatment. Eighteen age- and gender-matched
otherwise healthy preschoolers (4–6 years old) who underwent tonsillotomy or tonsillectomy
were recruited to participate as controls, during the same season as the children with PKU.
Exclusion criteria were a diagnosed learning disorder or metabolic disease other than PKU, and
little or no knowledge of the German language as assessed by clinic staff. In patients with PKU,
fasted blood was drawn in the morning on the same day as cognition was tested. For controls,
fasted blood was drawn in the morning of the operation. Two days after the operation, digital
cognitive testing was conducted at the hospital (Figure 1B). Written informed consent was given
by parents or legal guardians of all participants. All participating children gave spoken consent
prior to inclusion. The study was approved by the Ethics Committees of Ruhr-University
Bochum (No. 17-6311), in accordance with the Declaration of Helsinki.
Nutrients 2024, 16, x FOR PEER REVIEW 4 of 17
Figure 1. Study protocol for patients with phenylketonuria (A) and healthy controls (B). On the day
of study participation, children with phenylketonuria (PKU) gave fasted blood and took part in the
cognitive testing. One child did not give blood, while one child did not participate in cognitive tests.
Routine phenylalanine (Phe) and tyrosin blood measurements are carried out regularly multiple
times per month in pediatric patients with PKU. These values were collected after study participa-
tion for the timeframe of three months surrounding the study participation. In healthy controls,
fasted blood was drawn on the day of tonsillotomy or tonsillectomy. Two days after the operation,
controls participated in cognitive testing while still in the hospital.
2.1. Biomaterials
EDTA-blood was centrifuged at 3000 rpm at 4 °C for 10 min, and plasma was stored
at −80 °C until further analyses. The measurement of the plasma concentration of carbox-
ylated osteocalcin (OCN), undercarboxylated osteocalcin (uOCN), C-terminal telopeptide
of type 1 collagen (CTX), parathyroid hormone (PTH), and 25-hydroxy vitamin D (25-OH
D) were conducted via commercially available ELISA according to manufacturer’s in-
structions (OCN and uOCN: EIA kit, Takara, Saint-Germain-en-Laye, France; CTX: Serum
Crosslaps
®
, Immunodiagnostic Systems, Boldon Colliery, UK; PTH: Tecan, IBL Interna-
tional GmbH, Hamburg, Germany; 25-OH D: Immunodiagnostic Systems, Boldon Col-
liery, UK). The 25-OH D was further categorized into vitamin D sufficient (≥50 nmol/L),
insufficient (30–50 nmol/L), and deficient (<30 nmol/L), as defined by IOM [41]. Patients
with PKU underwent regular blood analysis of Phe and tyrosine. These data for the month
before, during and after enrolment in the study were extracted from the patients’ medical
records, and mean values were calculated. These were determined by the laboratory for
standard care, described as follows: from dried blood filter cards (DBS), discs of 3.0 mm
diameter were punched into Eppendorf reaction vials. A total of 20 µL wa ter (LCMS grade)
were added to each sample. From each plasma sample, 20 µL EDTA-plasma were trans-
ferred into Eppendorf vials. The extraction was carried out with 100 µL Methanol (LCMS
grade), containing isotope labeled amino acids (13C, 15N); Sigma-Aldrich, Germany. Af-
ter vertical shaking at 1000 rpm for 20 min at 20 to 25 °C, all vials were centrifuged at
16,000 RCF for 5 min. The samples were derivatized by 6-aminoquinolyl-N-hydroxy-
succinimidyl carbamate in acetonitrile (included in AccQ-Tag™ Ultra Derivatization Kit,
Waters, 65760 Eschborn, Germany) with borate buffer. All vials were incubated for 10 min
at ambient temperature. The chromatographic separation of partly isobaric compounds
was carried out on an ACQUITY UPLC
®
I-Class System with Cortecs™UPLC
®
, particle
Figure 1. Study protocol for patients with phenylketonuria (A) and healthy controls (B). On the day
of study participation, children with phenylketonuria (PKU) gave fasted blood and took part in the
cognitive testing. One child did not give blood, while one child did not participate in cognitive tests.
Routine phenylalanine (Phe) and tyrosin blood measurements are carried out regularly multiple
times per month in pediatric patients with PKU. These values were collected after study participation
for the timeframe of three months surrounding the study participation. In healthy controls, fasted
blood was drawn on the day of tonsillotomy or tonsillectomy. Two days after the operation, controls
participated in cognitive testing while still in the hospital.
Nutrients 2024,16, 2072 4 of 16
2.1. Biomaterials
EDTA-blood was centrifuged at 3000 rpm at 4
◦
C for 10 min, and plasma was stored
at
−
80
◦
C until further analyses. The measurement of the plasma concentration of carboxy-
lated osteocalcin (OCN), undercarboxylated osteocalcin (uOCN), C-terminal telopeptide of
type 1 collagen (CTX), parathyroid hormone (PTH), and 25-hydroxy vitamin D (25-OH D)
were conducted via commercially available ELISA according to manufacturer’s instructions
(OCN and uOCN: EIA kit, Takara, Saint-Germain-en-Laye, France; CTX: Serum Crosslaps
®
,
Immunodiagnostic Systems, Boldon Colliery, UK; PTH: Tecan, IBL International GmbH,
Hamburg, Germany; 25-OH D: Immunodiagnostic Systems, Boldon Colliery, UK). The
25-OH D was further categorized into vitamin D sufficient (
≥
50 nmol/L), insufficient
(30–50 nmol/L), and deficient (<30 nmol/L), as defined by IOM [
41
]. Patients with PKU
underwent regular blood analysis of Phe and tyrosine. These data for the month before,
during and after enrolment in the study were extracted from the patients’ medical records,
and mean values were calculated. These were determined by the laboratory for standard
care, described as follows: from dried blood filter cards (DBS), discs of 3.0 mm diameter
were punched into Eppendorf reaction vials. A total of 20
µ
L water (LCMS grade) were
added to each sample. From each plasma sample, 20
µ
L EDTA-plasma were transferred
into Eppendorf vials. The extraction was carried out with 100
µ
L Methanol (LCMS grade),
containing isotope labeled amino acids (13C, 15N); Sigma-Aldrich, Germany. After vertical
shaking at 1000 rpm for 20 min at 20 to 25
◦
C, all vials were centrifuged at 16,000 RCF
for 5 min. The samples were derivatized by 6-aminoquinolyl-N-hydroxysuccinimidyl
carbamate in acetonitrile (included in AccQ-Tag™ Ultra Derivatization Kit, Waters, 65760
Eschborn, Germany) with borate buffer. All vials were incubated for 10 min at ambient
temperature. The chromatographic separation of partly isobaric compounds was carried
out on an ACQUITY UPLC
®
I-Class System with Cortecs™UPLC
®
, particle size: 1.8
µ
m;
150
×
2.1 mm (Waters, 65760 Eschborn, Germany) using 0.1% formic acid in ULC water
and 0.1% formic acid in acetonitrile as mobile phase. After chromatographic separation,
detection was performed using a Xevo
®
TQS-micro (Waters, Germany) in ESI positive mode
quantification with MassLynx™ NT version 4.1 (Waters, Germany).
2.2. Cognitive Testing
All participants completed a computer-based cognitive test in a quiet room previously
developed in collaboration with the ALA Institute in Bochum, Germany. The tests are based
on established pen-and-paper as well as digital tests [
42
–
45
], which were first translated
into digital versions for adolescents [
46
–
48
] and were now adapted to match the abilities of
children between 4 and 7 years of age. Tests were presented on a touch-sensitive screen
(Hanns-G by Hannspree TH 225 HPB, 5928 PN Venlo, The Netherlands) and were practiced
once by the child with the help of study staff. During the actual measurement, each test
was explained to the child again by the staff using paper cards and cut-outs of the tests as
shown on the screen. All tests are depicted in Figure 1. The overall testing took 20–30 min,
with a short break between the practice round and the actual measurement.
2.3. Tonic Alertness
Tonic Alertness (Figure 2a) was measured by requesting the child to tap on the screen
as soon as a black fixation circle on a white screen changed into an illustrated colorful
rabbit. The response stimulus interval was 3300 ms (
±
20%) and maximal accepted reaction
time (RT) was set at 1500 ms. The test included 50 items and required the child to stay
alert for 3 min. The outcome variables were mean reaction time (RT, ms), the number of
missings (no reaction after 1500 ms), and the number of commission errors (reaction during
the presence of the fixation circle).
Nutrients 2024,16, 2072 5 of 16
Nutrients 2024, 16, x FOR PEER REVIEW 5 of 17
size: 1.8 µm; 150 × 2.1 mm (Waters, 65760 Eschborn, Germany) using 0.1% formic acid in
ULC water and 0.1% formic acid in acetonitrile as mobile phase. After chromatographic
separation, detection was performed using a Xevo
®
TQS-micro (Waters, Germany) in
ESI positive mode quantification with MassLynx™ NT version 4.1 (Waters, Germany).
2.2. Cognitive Testing
All participants completed a computer-based cognitive test in a quiet room previ-
ously developed in collaboration with the ALA Institute in Bochum, Germany. The tests
are based on established pen-and-paper as well as digital tests [42–45], which were first
translated into digital versions for adolescents [46–48] and were now adapted to match
the abilities of children between 4 and 7 years of age. Tests were presented on a touch-
sensitive screen (Hanns-G by Hannspree TH 225 HPB, 5928 PN Venlo, The Netherlands)
and were practiced once by the child with the help of study staff. During the actual meas-
urement, each test was explained to the child again by the staff using paper cards and cut-
outs of the tests as shown on the screen. All tests are depicted in Figure 1. The overall
testing took 20–30 min, with a short break between the practice round and the actual meas-
urement.
2.3. Tonic Alertness
Tonic Alertness (Figure 2a) was measured by requesting the child to tap on the screen
as soon as a black fixation circle on a white screen changed into an illustrated colorful
rabbit. The response stimulus interval was 3300 ms (±20%) and maximal accepted reaction
time (RT) was set at 1500 ms. The test included 50 items and required the child to stay
alert for 3 min. The outcome variables were mean reaction time (RT, ms), the number of
missings (no reaction after 1500 ms), and the number of commission errors (reaction dur-
ing the presence of the fixation circle).
Figure 2. Task battery for executive functioning in children 4–7 years of age. Tonic Alertness (a):
Children were instructed to tap the grey box as soon as the fixation dot vanished to display a colorful
rabbit. Corsi Block Tapping Task (b): tested working memory; children were instructed to observe
the 3
×
3 matrix on which the animals would appear. They were to remember the order in which
the animals appeared on the screen and then repeat this on the 3
×
3 matrix boxes by tapping on
them. Flanker Task (c): tested inhibitory control and consisted of congruent (-1-) and incongruent (-2-)
stimuli. Children had to tap the box on the side to which the middle fish swam. Switch Task (d): The
task consisted of three sections. (-1-) rabbits had to be tapped in ascending order, if clicked correctly a
grey imprint covered the tapped rabbits; (-2-) carrots needed to be tapped in ascending order; (-3-)
Carrots and rabbits had to be tapped alternately (switch) in ascending order (1 carrot–1 rabbit–2
carrots–2 rabbits. . .).
2.4. Corsi Block Tapping Task
The Corsi Block Tapping Task (Figure 2b) was used in a digital version and shortened
for usage on younger children. A square consisting out of 3
×
3 smaller blue squares was
presented on the screen. In these squares, small, illustrated animals appeared for 500 ms
with an inter-interval sequence of 1000 ms. Children were asked to remember the location
and the order in which these animals appeared. One to four animal-block sequences were
displayed and needed to be repeated in the same order. Twelve block sequences had to be
repeated with increasing length: 1-, 2-, 3-, and 4-animal-boxes, each three times. As younger
children tended to reproduce sequences in inverse order during the testing, additional
analysis for correctly tapped boxes, neglecting the correct order, was applied. Therefore,
outcome variables were the number of correctly remembered orders and paths, and the
number of correct boxes (leaving out the order).
2.5. Flanker Task
The Flanker Task (Figure 2c) examines the ability of subjects to suppress dominant
responses (i.e., inhibitory control) [
49
]. Orange fish were used as directional target stimuli
and appeared at the center of the screen within confounding variables (flankers vertically
arranged). Children were instructed to tap a square at the bottom of the screen indicating in
Nutrients 2024,16, 2072 6 of 16
which direction the fish in the middle was swimming (fish swimming right = tap square on
the right, fish swimming left = tap square on the left). In the congruent condition, the central
fish was flanked by fish swimming in the same direction. In the incongruent condition, the
central fish was flanked by fish swimming in the opposite direction. The top and bottom
fish were presented first for 100 ms, then the middle fish also appeared on the screen. All
three fish were visible to the children for 800 ms. Maximum reaction time was 2000 ms and
the response stimulus interval was set as 1000 ms (
±
20%). The outcome variables were the
inverse efficiency (IE) of the congruent and incongruent task (IE = reaction time of trial
×
(count of all items in trial/count of true reactions in trial)), as well as the difference in IEs
(Difference IE = IE incongruent
−
IE congruent). To avoid implausible results (e.g., due to
playing with computer buttons and ignoring the instruction), participants with error rates
≥50% in the task were excluded.
2.6. Switch Task
The Switch Task (Figure 2d) was developed as a digital version of the Trail Making
Task and was adapted to fit the abilities of the young cohort by using rabbits and carrots
instead of the usually used letters and numbers. The Switch Task consisted of three sections:
the first two sections were non-switch sections, in which one to six rabbits were presented
in an irregular order on the screen and should be tapped in an ascending order. The second
section presented one to six carrots in an irregular order and children should tap these
in an ascending order. The third section was the switching section, in which all rabbits
and all carrots were shown and children were asked to “feed the rabbits”: first tap one
carrot and then one rabbit; two carrots, two rabbits, and so on. In each section, fields that
were tapped correctly were overlaid by a fingerprint. Fields that were tapped incorrectly
were not overlaid by a fingerprint. In each section, the maximum time to finish the task
was 1.5 min. The outcome variables were the sum of total reaction time (RT) for rabbits
(items 2–6) and carrots (items 2–6), and switch costs, i.e., the processing time of the third
section minus the sum of the five items of the second section and the five items of the first
section (RT
2–6
Switch
−
(RT
2–6
rabbits + RT
2–6
carrots). Negative switch costs were regarded
as implausible and were excluded.
2.7. Statistical Analyses
The statistical software package IBM
®
SPSS
®
Statistics for Windows, version 29.0 (IBM
Corp., Armonk, NY, USA) was used for the statistical analyses. Descriptive data were
analyzed by the Chi-squared test or by Fisher’s exact test for groups smaller than five
observations. The QQ plots were used to test for normal distribution. Normally distributed
data were analyzed using parametric tests (Student’s t-test). Non-normally distributed data
were analyzed using non-parametric tests (Mann–Whitney U test). Values of p< 0.05 were
considered significant. Effect sizes for normally distributed data were calculated by using
Cohen’s d for groups larger than 20 and Hedges g for groups smaller than 20 observations;
Pearson’s r was used for non-normally distributed data. Normally distributed data are
presented as mean
±
standard deviation (SD), non-normally distributed data as median
(25–75th interquartile range). Pearson’s correlation was used to analyze the correlation
between normally distributed metric parameters.
3. Results
3.1. Characterization of Children with PKU and Controls
In total, 27 children participated in the study, and 9 patients with PKU were included,
2 of whom were missing either blood or the cognition test. The 18 controls were age-,
gender- and season-matched, 2 for each patient with PKU. Two controls were excluded in
blood analysis and another two controls were excluded in cognition analysis.
Nutrients 2024,16, 2072 7 of 16
3.2. Characterization of Dietary Control of Patients with PKU
All of the nine participating patients with PKU had mean Phe levels of 79.9–322.7
µ
mol/L
in the months before, during and after study participation. No child exceeded 360
µ
mol/L, and
two children with PKU had mean blood Phe concentrations that stayed below 120
µ
mol/L (Phe
in blood: patient 1: 79.9
µ
mol/L; patient 2: 101.7
µ
mol/L, Table 1). In parallel to clinical care
with food protocol and counselling whenever needed, six (66.7%) caregivers stated that they
calculated Phe ingestion daily, while two (22.2%) said they calculated most of the time, and
one caregiver stated that they calculated Phe consumption sometimes. All patients with PKU
used amino acid-supplements with tyrosine, and none were treated with BH
4
. Therefore, all
participants were affected by PKU and none were affected by mild hyperphenylalaninemia, as
defined in European Guidelines [
1
]. Furthermore, two children with PKU used supplements
with docosahexaenoic acid (DocOmega, Vitaflo, Steinbach, Germany), which also contains
Vitamin C, sodium, potassium, chloride, calcium and phosphate. No other supplements
were used.
Table 1. Characterization of patients with phenylketonuria (PKU) and controls (Co).
Parameter PKU Co p
n 9 18
Age (years) 5.4 ±1.2 5.1 ±0.8 0.464 a
Female n (%) 2 (22.2%) 4 (22.2%) 1.000
Winter/spring n (%) 2 (22.2%) 5 (27.8%) 1.000
Daily Phe tolerance (mg/d) 291.1 ±43.4 n.a. -
Mean Phe in blood * (µmol/L) 203.4 ±82.9 n.a. -
Mean Tyr in blood * (µmol/L) 80.0 ±34.2 n.a. -
Supplementation with AAS n (%) 9 (100%) n.a. -
* mean values of regular routine measurements from the patients record in the month of the study participation, prior
to and after inclusion,
a
Cohen’s d = 0.304, AAS—amino acid-supplements, n.a.—not applicable, Phe—phenylalanine,
Tyr—tyrosine.
3.3. Executive Functioning
Overall, 8 patients with PKU and 16 controls were included in the analysis of executive
functioning. No significant differences could be found in most subdomains of the executive
functioning between the patients with PKU and the controls (Table 2). Only the Flanker Task
patients with PKU had a significantly smaller IE in incongruent trials (p= 0.030; Table 2),
but no significant difference in IE in congruent trials or difference in IEs were detected.
Table 2. Executive functions in patients with phenylketonuria (PKU) and controls (Co).
Parameter of Executive Functioning PKU Co pEffect Size
Tonic Alertness ◦
Commission error (n) 6.00 ±5.60 8.27 ±7.44 0.461 −0.329 a
Missing (n) 2.5 [0.3–4.8] 5.0 [2.0–9.0] 0.213 −0.264 c
Average RT correct (ms) 701.2 [621.3–814.2] 738.8 [656.9–905.0] 0.506 −0.148 c
Corsi Block Tapping Task $
Correct order and path (n) 7.63 ±1.10 7.19 ±0.60 0.706 0.165 a
Correct boxes (n) 9.13 ±0.79 9.31 ±0.49 0.835 −0.092 a
Flanker Task ~
IE congruent (ms) 886.7 ±226.2 1029.9 ±161.5 0.139 −0.741 b
IE incongruent (ms) 1036.8 ±194.9 1239.9 ±157.9 0.030 −1.135 b
Difference IE (ms) 150.2 ±95.6 210.0 ±130.7 0.337 −0.472 b
Switch Task *,#
Sum RT lblast * (s) 17.8 [10.2–39.1] 17.5 [14.2–27.0] 1.000 0.000 c
Switch costs #(s) 29.6 [12.2–45.8] 21.2 [16.2–24.8] 0.606 −0.160 c
◦
n
PKU
= 8, n
Co
= 15.
$
n
PKU
= 8, n
Co
= 16.
~
n
PKU
= 6, n
Co
= 11. * n
PKU
= 6, n
Co
= 11.
#
n
PKU
= 5, n
Co
= 9.
a
Cohen’s d,
b
Hedges g,
c
Pearsons r. IE—inverse efficiency; RT—reaction time; significant results are marked
in bold.
Nutrients 2024,16, 2072 8 of 16
3.4. Bone Health
Overall, 8 patients with PKU and 16 controls were included into plasma analysis. No signif-
icant differences in CTX (PKU: 1.84
±
0.24 ng/mL, Co: 1.69
±
0.58 ng/mL, p= 0.502,
d = 0.295
),
OCN (PKU: 13.5 [12.0–17.1] ng/mL, Co: 14.4 [12.9–17.3] ng/mL, p= 0.714,
r = −0.087
), uOCN
(PKU: 21.5 [10.6–31.6] ng/mL, Co: 31.4 [24.0–33.8] ng/mL, p= 0.235, r =
−
0.272) and the ratio of
uOCN/OCN (PKU: 1.33 [0.77–1.95], Co: 1.76 [1.18–2.14], p= 0.416, r =
−
0.201) were detected
between groups (Figure 3). PTH was lower in children with PKU (5.1
±
1.6 pmol/L) than in con-
trols (7.5
±
3.2 pmol/L), but this was not significant (p= 0.066, d =
−
0.839, Figure 3). Children
with PKU had a significantly higher 25-OH D plasma concentration (
69.44 ±12.83 nmol/L
)
than controls (41.87
±
15.99 nmol/L, p< 0.001, d = 1.831, Figure 3). Applying cut-offs defined
by IOM [
41
], 25-OH D blood concentration was further categorized into vitamin D sufficient
(
≥
50 nmol/L), insufficient (30–50 nmol/L), and deficient (<30 nmol/L). While no participant
with PKU was vitamin D insufficient or deficient, eight (50%) participants in the control group
were insufficient and three (18.8%) 25-OH D deficient. Therefore, patients with PKU were
significantly more often sufficient in vitamin D than controls (p= 0.007). The correlation between
PTH and 25-OH D was not significant (r = −0.282, p= 0.182).
Nutrients 2024, 16, x FOR PEER REVIEW 9 of 17
Figure 3. Parameters in plasma relevant for bone health in children with phenylketonuria (PKU)
and controls (Co). (a) c-terminal telopeptides of type 1 collagen* (CTX; n
PKU
=
8; n
Co
=
16); (b) para-
thyroid hormone* (PTH; n
PKU
=
8; n
Co
=
16); (c) 25-hydroxy vitamin D* (25-OH D; n
PKU
=
8; n
Co
=
16); (d) undercarboxylated osteocalcin° (uOCN; n
PKU
=
6; n
Co
=
12); (e) carboxylated osteocalcin°
(OCN; n
PKU
=
8; n
Co
=
14); (f) ratio of undercarboxylated osteocalcin to carboxylated osteocalcin°
(n
PKU
=
6; n
Co
=
13). * t-test; ° Mann–Whitney U test. Lines: mean ± standard deviation.
4. Discussion
Older children and adults with PKU showed bone health and executive performance
deficits in previous studies [6–14,23–35]. Therefore, an analysis of bone turnover and ex-
ecutive functioning in preschool children with PKU and matched controls was conducted.
Neither bone resorption and formation markers, nor most parameters of executive func-
tioning, differed between young children with well-treated PKU and age- and gender-
matched controls. Most interestingly, we did find significantly higher vitamin D levels in
children with PKU than in controls, even though they were recruited during the same
season.
None of the children with PKU exceeded the recommended blood Phe concentration
of 360 µmol/L in the months before, during and after participation in the study, and two
participants had mean Phe concentrations slightly below 120 µmol/L [1]. As the American
College of Medical Genetics and Genomics stated, there is no evidence for the recommen-
dation of blood Phe concentrations of 60–120 µmol/L, but low Phe levels are safe if ade-
quately monitored to avoid prolonged phases of blood Phe concentrations below 30
µmol/L [3]. Therefore, all participants with PKU had good compliance and all consumed
amino acid supplements.
The prevention of neurocognitive deficits in patients with PKU is the leading treat-
ment goal in the care of the metabolic disease [1]. We observed no difference in perfor-
mance in the Corsi Block Tapping Task, Tonic Alertness Task and Switch Task in well-
treated preschool children with PKU and healthy controls. As Townsend and Ashby sug-
gested in 1978, and Bruyer and Brysnaert discussed in 2011, speed and accuracy in some
cognitive testing can influence each other in a speed–accuracy trade-off [50]. Therefore,
we used the inverse efficiency (IE) as discussed to evaluate Flanker Task performance [50].
The IE penalizes faster RT if this comes with a higher number of false reactions. Therefore,
higher IE indicates poorer performance in the Flanker Task [51]. We observed significantly
beer performance in processing the incongruent stimuli in children with PKU vs.
Figure 3. Parameters in plasma relevant for bone health in children with phenylketonuria (PKU) and
controls (Co). (a) c-terminal telopeptides of type 1 collagen * (CTX; n
PKU
= 8; n
Co
= 16); (b) parathy-
roid hormone * (PTH; n
PKU
= 8; n
Co
= 16); (c) 25-hydroxy vitamin D * (25-OH D; n
PKU
= 8;
nCo = 16
);
(d) undercarboxylated osteocalcin
◦
(uOCN; n
PKU
= 6; n
Co
= 12); (e) carboxylated osteocalcin
◦
(OCN; n
PKU
= 8; n
Co
= 14); (f) ratio of undercarboxylated osteocalcin to carboxylated osteocalcin
◦
(nPKU = 6; nCo = 13). * t-test; ◦Mann–Whitney U test. Lines: mean ±standard deviation.
4. Discussion
Older children and adults with PKU showed bone health and executive performance
deficits in previous studies [
6
–
14
,
23
–
35
]. Therefore, an analysis of bone turnover and exec-
utive functioning in preschool children with PKU and matched controls was conducted.
Neither bone resorption and formation markers, nor most parameters of executive function-
ing, differed between young children with well-treated PKU and age- and gender-matched
controls. Most interestingly, we did find significantly higher vitamin D levels in children
with PKU than in controls, even though they were recruited during the same season.
None of the children with PKU exceeded the recommended blood Phe concentration
of 360
µ
mol/L in the months before, during and after participation in the study, and
two participants had mean Phe concentrations slightly below 120
µ
mol/L [
1
]. As the
American College of Medical Genetics and Genomics stated, there is no evidence for the
Nutrients 2024,16, 2072 9 of 16
recommendation of blood Phe concentrations of 60–120
µ
mol/L, but low Phe levels are
safe if adequately monitored to avoid prolonged phases of blood Phe concentrations below
30
µ
mol/L [
3
]. Therefore, all participants with PKU had good compliance and all consumed
amino acid supplements.
The prevention of neurocognitive deficits in patients with PKU is the leading treatment
goal in the care of the metabolic disease [
1
]. We observed no difference in performance in the
Corsi Block Tapping Task, Tonic Alertness Task and Switch Task in well-treated preschool
children with PKU and healthy controls. As Townsend and Ashby suggested in 1978, and
Bruyer and Brysnaert discussed in 2011, speed and accuracy in some cognitive testing can
influence each other in a speed–accuracy trade-off [
50
]. Therefore, we used the inverse
efficiency (IE) as discussed to evaluate Flanker Task performance [
50
]. The IE penalizes
faster RT if this comes with a higher number of false reactions. Therefore, higher IE indicates
poorer performance in the Flanker Task [
51
]. We observed significantly better performance
in processing the incongruent stimuli in children with PKU vs. controls. Matching our
results, Paermentier et al. observed no significant difference in the spatial working memory
of pre-school children with hyperphenylalaninemia and controls, but better performance
in inhibitory control in pre-school children with hyperphenylalaninemia. As they included
children with moderate hyperphenylalaninemia and phenylketonuria, they were able to
compare these two groups as well, and found lower performance in inhibitory control
and spatial working memory in children with PKU compared to children with moderate
hyperphenylalaninemia (none treated with protein restriction) [
52
]. As all participants
in our study had PKU, we were not able to compare participants with classical PKU
and hyperphenylalaninemia in our cohort. In the Netherlands, children with PKU were
recruited to participate in a study analyzing executive functioning and its associations to
Phe blood concentrations in 1997–1998 [
13
,
53
–
55
]. Comparable to our results, children with
Phe blood levels below 360
µ
mol/L did not differ from healthy controls in inhibition and
attention flexibility [
53
,
54
], whereas in the baseline speed task (comparable to the attention
testing we used), all patients with PKU had longer RTs than age-matched controls [
13
]. In
2012–2015, all patients were re-invited to participate. In this adult group of early-treated
patients, a significant correlation of Phe levels during early childhood (0–12 years), as well
as during adolescents (13–17 years), and executive functioning was observed. For inhibitory
control (Flanker Task), a greater increase in blood Phe levels from childhood to adulthood
correlated with poorer performance in the task, while cognitive flexibility was correlated
with Phe levels in early childhood [
55
]. On the contrary, another study revealed a trend
towards higher error rates in NoGo trials of the Go-NoGo test (reduced inhibitory control)
in young adult female patients with PKU compared to healthy age-matched controls. These
results did not correlate with the average Phe blood concentration during the first six years
of the lives of these patients, but came with a relative increase in brain activity in the right
middle frontal gyrus [
56
]. Another study showed that 29 out of 30 patients with PKU had
lesions, especially in the parietal and occipital lobes [
57
]. Additionally, working memory,
cognitive flexibility, sustained attention and processing speed were measured in these
patients with PKU and compared to controls, describing significantly lower performance
in the patients. Neither historical nor current metabolic control in those patients was
associated with performance in the cognitive testing and the white matter lesions, after
correcting for multiple comparisons [
57
]. In children with PKU, Hood et al. observed
correlations of mean diffusivity of two white matter brain regions with lifetime exposure
to Phe and with performance in executive functioning testing [
58
]. Similarly, 86 children
(8–17 years old) showed significant improvement in parent-reported executive functioning
after blood Phe concentration was reduced due to treatment with BH
4
[
59
]. We observed
no difference in the performance of visuo-spatial working memory, task switching or tonic
alertness, but better performance in the inhibition of incongruent stimuli in children with
PKU compared to healthy controls. Since a significant difference was neither observed in
processing the congruent stimuli nor in the difference of IE, further analysis of inhibitory
control in young children with PKU is needed. As children included into the present study
Nutrients 2024,16, 2072 10 of 16
were well treated, with no patient exceeding the upper recommendation of Phe level for the
age group, it might be possible for executive functioning to still be unaffected in these young
children, which in turn does not exclude the possibility for the occurrence of damages in
the brain and performance issues later in life. Because the children included into our pilot
study had lower blood Phe concentrations than those in the study of Paermentier et al.,
their performance in the inhibition task seems comparable to the children with moderate
hyperphenylalaninemia [52].
In the IDEFICS study, children between 3 and 15 years of age from eight European
countries had a mean 25-OH D serum concentration of 45.2 (
±
16.7) nmol/L, with 37%
of children being classified as vitamin D sufficient and significant differences between
countries [
60
]. Controls in our sample reached vitamin D sufficiency in 31.3% of cases,
consistent with the IDEFICS observations. The 560 German children included in the
IDEFICS study had a mean 25-OH D serum concentration of 36.1 (
±
15.1) nmol/L [
60
]. In
2007, data from the German KiGGS cohort study on 25-OH D in the serum of 10,115 children
living in Germany were published. In the group of 3–6 year olds, median serum 25-OH
D was 44.1 nmol/L (p5–p95: 15.0–95.8 nmol/L) [
61
], which corresponds to the controls
in our study. On the contrary, median serum 25-OH D in 1–2 year olds in KiGGS was the
highest of all age groups at 61.9 nmol/L (p5–p95: 19.4–115.0 nmol/L) [
61
], matching the
25-OH D concentrations of patients with PKU in our pilot study. It is recommended for
infants in Germany to be given vitamin D supplementation until their second summer of
life (e.g., up until about 1.5 years of age) [
62
]. Kunz et al. found clear differences in the
vitamin D status in German children according to season [
63
]. In 967 participants between
0 and 17 years of age, vitamin D sufficiency in 2013–2014 was observed to be 56%/39.7%
during summer/autumn, while only 29.2% and 28.1% reached sufficiency in winter and
spring [
63
]. Overall, children in our pilot study were mostly recruited during summer
and autumn, when UV exposure and time spent outside tend to be higher. Therefore, the
results presented here fit into the current literature for healthy children living in Germany.
Accordingly, the good vitamin D status of the children recruited with PKU is interesting.
As vitamin D is either taken in by food sources or dietary supplements, or is produced
by the skin as it is exposed to ultraviolet (UV) radiation [
64
], we matched patients and
controls not only for age and gender, but also for recruitment season. Thus, differences in
sun exposure are less likely and differences in vitamin D from foods or supplements might
account for the higher vitamin D concentrations in patients. The recommended vitamin D
supplementation in infants explains the higher 25-OH D concentrations in younger children,
as described in KiGGS [
61
] and by Geserick et al. [
65
]. All patients with PKU consumed
amino acid supplements containing 2.5–5
µ
g vitamin D/portion (xPHE energy, xPHE enjoy,
and xPHE hello, metaX, Friedberg, Germany; PKU gel and PKU cooler, Vitaflo, Steinbach,
Germany; Glytactin, Cambrook, Dali, Nicosia, Cyprus). The parents or guardians stated
that the included children with PKU consumed about 2.5 to 3 portions of amino acid
supplements and no further Vitamin D supplement each day, resulting in a vitamin D
intake of 6.25–9
µ
g per day from these amino acid supplements. At the same time, healthy
children between 6 and 8 years of age in Germany consume on average 1.8
µ
g of vitamin
D from food [
66
]. Therefore, the higher vitamin D plasma concentration in PKU patients
probably results from intake of amino acid supplements. In their study on bone mineral
density in patients (aged 8–20 years) Geiger et al. also did not find a patient with PKU with
vitamin D deficiency [
30
]. In contrast, Demirads et al. found that of 60 patients with PKU
(aged 1–39 years), 12% were not vitamin D sufficient, with 4 using Phe-free amino acid
mixtures (1 without added vitamin D) and 3 who did not adhere to a restrictive diet [32].
Next to a significantly higher 25-OH D plasma concentration in children with PKU,
we found lower PTH plasma concentrations in these children compared to controls, which
did not reach significance. Several studies described a negative non-linear relationship
between 25-OH D and PTH in children, with a plateau at about 75 nmol/L 25-OH D [
67
–
69
].
Therefore, the trend for lower PTH levels in our patients with PKU can be a result of higher
25-OH D levels, but the lack of significance might be a result of the small group of analyzed
Nutrients 2024,16, 2072 11 of 16
children. Additionally, the lack of a significant correlation between PTH and 25-OH D
could be a result of the non-linearity of the relationship between the two hormones, with
a flattening of the curve before the plateau [
67
]. As no data on PTH from the IDEFICS
observations or the KiGGS [
70
] on PTH in children exist, comparing controls in our study to
healthy children living in Germany was not feasible. Sahin et al. [
67
] and Vissing Landgrebe
et al. [
71
] measured PTH in children. Compared to both studies [
67
,
71
], PTH in our controls
was higher than expected, but 25-OHD was lower. The regression analysis conducted by
Vissing Landgrebe et al. resulted in an increase of 1 pmol/L of PTH for each 4.6 nmol/L
decrease of vitamin D in serum [
71
]. As the controls in our study were younger and had
vitamin D concentrations of 21 nmol/L lower than the participants in Vissing Landgrebe
et al.’s study, higher PTH concentration might result from these differences [
71
]. Higher
25-OHD concentrations were also observed in Sahin et al.’s study, with children of the
same age as in our study [
67
]. As for patients with PKU, PTH concentration in plasma
was also slightly higher than in the studies referred to, but still within, their standard
deviation [67,71].
Bone formation and resorption are continuous processes that do not only occur during
growth or bone mass accrual. In 395 7-year-old children from Portugal, no correlation
of total body bone mineral content and bone formation and resorption parameters was
observed after age, body size, and season were considered. Single measurements of these
markers may be limited in their ability to reflect current bone status in children [
39
]. As
a marker of bone resorption, CTX was analyzed in this pilot study. In children, CTX
rises from birth to puberty, with a median CTX concentration of about 1.5 ng/mL at
6 years and a p95 of about 2.5 ng/mL [
65
]. According to these reference values, the bone
resorption in children included into our pilot study was within the normal range. For
OCN, a bone formation marker, Geserick et al. observed high values during the first year
of life [
65
], which subsequently fell to a nadir between 3–4 years of age and then rose again.
Previously, Johansen et al., van Summeren et al. and Popko et al. found uOCN and OCN
serum concentrations comparable with those we found in plasma [
72
–
74
], whereas Tubic
et al. found uOCN at a mean concentration of 7 ng/mL and OCN at a concentration of
75.6 ng/mL, and Geserick et al. described median OCN at 3–4 years of age of 74.5 ng/mL
in boys and 78.1 ng/mL for girls [
65
,
75
]. While van Summeren et al. and Popko et al. used
the same ELISA assays we used here [
73
,
74
], Johansen et al. used radio-immunoassay [
72
],
Tubic et al. and Geserick et al. used an electrochemiluminescence immunoassay on
Cobas instruments, which explains the contradicting results [
65
,
75
]. To our knowledge,
this is the first study that analyzed uOCN in well-treated young children with PKU,
which was described to have hormonal effects in mice acting on glucose sensitivity and
neurotransmitter production [76].
A limiting factor of this study was the small number of patients included due to
its design as a pilot study. In addition, only eight patients with PKU were able to give
blood, and one child with PKU was not able to participate in cognitive testing. As the
drawing of blood in healthy children is problematic, children undergoing a small surgical
procedure were used as controls. To secure no influence of sedation on the test results of
cognitive battery, children were tested two days after the surgery. It cannot be excluded
that pain or malaise could have influenced the performance of controls. Furthermore, the
small amount of blood collected did not allow for a thorough analysis of bone-related
parameters. Children with PKU did not only give blood for study purposes, but also for
routine check-up, to minimize the times children had to be punctured. Moreover, the age
range of 4 to 7 years is a timeframe during childhood when growth spurts occur [
77
] and
cognition is developing rapidly [
78
]. Because growth spurts are difficult to predict [
77
],
we cannot rule out that the children included in this analysis were undergoing a spurt at
the time of blood collection, which in turn might have influenced the parameters of bone
formation and resorption. Parameters such as height or weight would have been helpful
in interpreting the results, but were regrettably not collected. As cognitive functions are
rapidly developing during early childhood [
78
], the tests might have been too difficult for
Nutrients 2024,16, 2072 12 of 16
younger children and/or too easy for older children. We tried to eradicate this by matching
patients with controls of the same age, but since calendar age does not necessarily reflect
brain maturity [
78
], results have to be interpreted with caution. Additionally, although
based on well-established pen-and-paper tests, the battery used for cognitive testing was
not validated; however, this is in part compensated by the direct comparison of patients
with controls. Vitamin D supplementation in controls was not documented. As 25-OHD
concentration in these children was comparable to children in the German KiGGS cohort
study [
61
], it can be expected that the children did not take any vitamin D supplementation.
Additionally, the usual diet in children with PKU and early-life management was not
analyzed, but could have influenced data on cognition and bone formation and resorption.
This data should be included in future studies. As this study was a pilot study, we are
interested in further investigating bone health, cognition and the relationship between these
parameters with habitual diet, physical activity and Phe concentration in older individuals.
In particular, we would like to include older individuals to study a broader spectrum of
parameters, as this allows for larger volumes of blood to be obtained and reduces the
impact of developmental spurts.
5. Conclusions
In conclusion, we did not find an early indication for preventable comorbidities in
well-treated young children with PKU compared to healthy age-, gender- and season-
matched controls. No child with PKU included was vitamin D insufficient or deficient, with
no effect on bone formation or resorption markers. Additionally, no difference in executive
functioning was observed in the children with PKU compared to the controls. Nevertheless,
many studies describe impairments in bone turnover and cognitive performance in older
children with PKU. Continuous treatment and regular check-ups should be performed to
prevent the development of these impairments and to ensure early intervention.
Author Contributions: Conceptualization, K.S.; methodology, M.F.; formal analysis, B.H.; investi-
gation, K.S. and B.H.; resources, S.V., S.D. and T.L.; data curation, K.S. and B.H.; writing—original
draft preparation, B.H.; writing—review and editing, T.L., K.S., S.D., S.V. and M.F.; visualization,
B.H.; supervision, K.S. and T.L.; project administration, K.S. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The study was conducted in accordance with the Declaration
of Helsinki, and approved by the Ethics Committees of Ruhr-University Bochum (No. 17-6311).
Informed Consent Statement: Written informed consent was given by parents or legal guardians of
all participants.
Data Availability Statement: The datasets used and analyzed during the current study are available
from the corresponding author on reasonable request.
Acknowledgments: We thank Ludger Blanke for his great work on adapting the preexisting computer-
based cognitive testing for touch-screen use by younger children. We also want to extend our gratitude
to Markus Mallek for adding the standard procedure of Phe and tyrosine measurements. Furthermore,
we thank all participating patients with PKU, healthy controls and families, as well as the recruiting
doctors at the Department of Otorhinolaryngology-Head and Neck Surgery and the University
Hospital of Pediatrics and Adolescent Medicine, Ruhr-University Bochum. Figure 1was drawn
in part using images from Servier Medical Art. Servier Medical Art by Servier is licensed under a
Creative Commons Attribution 4.0 Unported License (https://creativecommons.org/licenses/by/4.
0/, accessed on 18 June 2024).
Conflicts of Interest: The authors declare no conflicts of interest.
Nutrients 2024,16, 2072 13 of 16
Abbreviations
25-OH D 25-hydroxy vitamin D
BH4Tetrahydrobiopterin
BMD bone mineral density
CTX C-terminal telopeptide of type 1 collagen
DBS dried blood filter cards
IE inverse efficiency
OCN carboxylated osteocalcin
Phe phenylalanine
PKU phenylketonuria
PTH parathyroid hormone
RT reaction time
SD standard deviation
uOCN undercarboxylated osteocalcin
UV ultraviolet
WHO World Health Organization
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