Large Neutral Amino Acids (LNAAs) Supplementation Improves Neuropsychological Performances in Adult Patients with Phenylketonuria.

Phenylketonuria is an inborn error of phenylalanine (Phe) metabolism diagnosed by newborn screening and treated early with diet. Although diet prevents intellectual disability, patients often show impairment of executive functions, working memory, sustained attention, and cognitive flexibility. Large neutral amino acids (LNAAs) have been proposed as a dietary supplement for PKU adults. Few studies show that LNAAs may help in improving metabolic control as well as cognitive functions. In this study, 10 adult PKU patients with poor metabolic control were treated for 12 months with LNAAs (MovisCom, 0.8–1 g/kg/day) and underwent Phe and Tyrosine (Tyr) monitoring monthly. Neuropsychological assessment was performed at T0, T+3, and T+12 months by using the American Psychological General Well-Being Index, the Wisconsin Card Sorting Test, the Test of Attentional Performance, and the 9-Hole Peg Test. No change in plasma Phe levels was observed during LNAAs supplementation, while Tyr levels significantly improved during LNAAs supplementation (p = 0.03). Psychometric tests showed an improvement of distress and well-being rates, of executive functions, attention, and vigilance, whereas no difference was noted regarding hand dexterity. This study adds evidence of the advantage of LNAAs supplementation in improving cognitive functions and well-being in patients with PKU with poor metabolic control.


Introduction
Phenylketonuria (PKU; MIM 261600) is an autosomal recessive disorder caused by the deficiency of phenylalanine hydroxylase (PAH, EC 1.14.16.1), the hepatic enzyme that converts phenylalanine (Phe) to tyrosine (Tyr), using tetrahydrobiopterin (BH4) as coenzyme. More than 500 mutations of the PAH gene have been described so far (http://www.pahdb.mcgill.ca; http://www.biopku.org). PKU is classified into classic PKU (cPKU), moderate PKU (moPKU), and mild PKU (mPKU) according to plasma Phe levels at diagnosis and tolerance, defined as the highest Phe intake able to keep blood Phe levels within the safe range [1]. Hyperphenylalaninemia is also divided in mild hyperphenylalaninemia (HPA)-gray zone, requiring dietary protein restriction, and mild hyperphenylalaninemia where no treatment is required as Phe levels are below 360 µmol/L [1]. If untreated, PKU leads to neurological abnormalities, while early treatment with a Phe-restricted diet prevents brain damage and results in dosed 2-h post prandial Phe and Tyr levels in 12 PKU patients supplemented with 0.5 g/kg/day LNAAs for 4 weeks. The study confirmed a 58% reduction of Phe from baseline and a significant raise of Tyr levels. Finally, a significant increase of Tyr levels was found after a 12-month period of LNAAs supplementation in 12 adult PKU patients [26].
Taken together, literature data published so far on the effect of LNAAs supplementation in PKU are still scarce. Studies differ in LNAAs dose, therapy duration, and endpoints. However, the possible role of LNAAs in improving neurotransmitter derangement in PKU and, subsequently, neuropsychological performances deserve further attention and additional evidence. In the present study, we aimed at evaluating the effect of a long-term (12 months) high dose LNAAs supplementation (0.8-1 g/kg/day) on plasma Phe and Tyr levels, on executive function, vigilance, attention, and on the perceived patients' quality of life.

Patients
Ten adult patients (6 F/4 M, mean age 23.6 ± 4.5 years; range 18-32 years) with PAH deficiency were recruited at the Department of Maternal and Child Health, Federico II University Hospital, after Ethical Committee approval and signed informed consent (Ethical Committee Approval n. 30/15). The study was carried out in accordance with the Declaration of Helsinki and ICH/GCP. Inclusion criteria were age ≥18 years, early dietary intervention after neonatal diagnosis, PKU phenotype requiring dietary treatment and amino acid supplementation, complete PAH genotyping, normal intelligence (IQ > 80), poor metabolic control during the last 12 months, adherence to study protocol after informed consent signature.
Exclusion criteria were age <18 years, subjects with primary BH4 deficiency or with HPA not requiring dietary restriction, incomplete PAH genotyping, concomitant chronic diseases, patients not able to adhere to study procedure.

Supplementation with LNAAs
Patients were supplemented for 12 months with LNAAs (PheLNAA, MovisCom) at the dosage of 0.8-1 g/kg/day divided in 3 daily doses at main meals, with complete replacement of the previous non-LNAA formula. The nutritional facts of the LNAAs formula used in this study is reported in Table 2. Patients were asked to continue their diet without changing food habits, whereas the previous amino acid and vitamin supplements were withdrawn and replaced with PheLNAA.
Each patient underwent Phe, Tyr, Phe/Tyr ratio monitoring monthly. A 3-days meal diary was filled-in before each blood withdrawal.

Plasma Phe and Tyr Determinations
Median plasma Phe (µmol/L), Tyr (µmol/L), and Phe/Tyr ratio were calculated from plasma Phe and Tyr values obtained during the 12 months preceding the inclusion into the study and compared with median plasma Phe and Tyr dosed during the study.
Phe, Tyr, and Phe/tyr ratio were dosed by HPLC (Agilent Technologies 1200 Series LC System).

Neuropsychological Assessment
Neuropsychological assessment was performed at T0, T+3, and T+12 months by using the American Psychological General Well-Being Index (PGWBI) for the evaluation of the perceived level of quality of life [29,30], the Wisconsin Card Sorting Test (WCST) for the evaluation of executive functions [31], the Test of Attentional Performance (TAP test) for the evaluation of alertness and concentration [32], and the 9-Hole Peg Test (HPG test) for the evaluation of digital dexterity [33].

Psychological General Well-Being Index (PGWBI)
The Psychological General Well-Being Index (PGWBI) is a self-report questionnaire of 22 items organized in six subscales: Anxiety, Depression, Well-being, Self-control, Health, and Vitality. Responses for each item are evaluated on a six-point Likert scale ranging from 0 to 5. Higher scores indicate better well-being. The subscales sum provides a global index score for subjective well-being (range 0-110). Considering "distress" as the reverse of well-being, a global score <60 suggests a "Severe Distress"; from 60 to 72 a "Moderate Distress"; and >72 "No Distress" category [30].

Wisconsin Card Sorting Test (WCST)
The Wisconsin Card Sorting Test (WCST) is a neuropsychological tool to test abstract reasoning and the ability to change cognitive strategies when environmental circumstances change. The test evaluates executive functions and flexibility. The WCST explores the following frontal lobe functions: strategic planning, organized searching, utilizing environmental feedback to shift cognitive sets, directing behavior to achieve a goal, and modulating impulsive responding. The test is used with subjects aged from 6.5 years to 89 years. In the present study, the following test variables were evaluated: (1) perseverative errors, reflecting problems in switching from one rule to another; (2) non-perseverative errors, associated with absent-mindedness of attention; (3) total of all errors, which explores functional status of cognitive flexibility and attention; (4) total amount of categories completed, which tests cognitive processing success; (5) total amount of cards to complete the first category, which reflects the efficiency/speed of identifying the first classification algorithm.

Test of Attentional Performance (TAP Test)
The Test of Attentional Performance (TAP test) is a computer-based test exploring eight attention domains such as spatial attention, sustained attention, vigilance, and working memory. The test quantifies the speed (mean, median and standard deviation, msec) and the accuracy of the answers (errors, omissions). The computer-based test to assess vigilance gives mean, median and SD in ms for the first section, the second section, and the total time to complete the tasks.

9-Hole Peg Test (HPG)
The 9HPT is a brief, standardized, quantitative test of upper extremity function. It explores fine motor function and eye-hand coordination. Participants performed two consecutive rounds both with the dominant and non-dominant hand. The score for the HPT was an average of the 2 rounds for each hand.

Statistical Analysis
Normality of continuous data was assessed by the Levene test. The Student's t-test was used to compare means and the Mann-Whitney U-test to compare medians. Pearson's correlation coefficient (r) was used to measure the strength of the association between two variables. A two-tailed p-value <0.05 was assumed as statistical significant. Statistical analysis was performed by SPSS 17.0 software.

Phenotypic Classification
Ten adult PKU patients (6 females/4 males) completed the study: 7 patients were classified as cPKU, 1 patient as moPKU and 2 patients as mPKU. In patient #2, the neonatal pre-treatment Phe level (1200 µmol/L) was discordant with the observed historical tolerance (450 mg Phe/day) and was classified as mPKU according to tolerance, as detailed in the Methods section. All patients received complete genotyping (Table 1).

Phe, Tyr, and Phe/Tyr Ratio
All patients received a daily LNAAs dose of 0.8-1 g/kg/day divided in three administrations at main meals. No significant change in plasma Phe levels was observed during the LNAAs supplementation except for patient #4 who had a significant raise of Phe levels due to worsening of dietary compliance.
Hence, in adult patients with poor compliance to dietary treatment, we could not demonstrate a reduction of plasma Phe and an improvement of the metabolic control under LNAAs supplementation.
Plasma Tyr levels significantly raised in 9/10 patients and the Phe/Tyr ratio significantly declined in 5/10 patients (Table 3). Considering the PKU patients as a group, Tyr levels significantly improved during LNAAs supplementation (p = 0.03). Biochemical data were collected during the 12 months before the enrollment into the study and during the following 12 months of LNAAs supplementation. Phe and Tyr levels are expressed in µmol/L. Data are reported as medians (10 • -90 • centile). * p < 0.05; ** p < 0.01. Columns 8 and 9 report, for each patient, plasma Phe and Tyr levels at T0, T3, and T12 when patients performed the psychometric evaluation.

Psychological General Well-Being Index (PGWBI)
All patients completed the PGWBI health related quality of life test (Figure 1). The descriptive analysis showed that 3/10 patients presented at T0 with severe distress (total score 56, 46, and 52, respectively), 1/10 patients with a borderline-mild distress (total score 72), and 6/10 patients with positive well-being. After 3 months of LNAAs supplementation (T3), all patients showed an improvement of their general well-being, confirmed at the end of the study, despite the difference was not statistically significant (mean score ± SD: T0 78 ± 21; T3 87 ± 14; T12 88.4 ± 14). The improvement was more evident in those patients showing severe distress scores at T0: patient #1 improved his distress from severe to mild-moderate (Total score: T0 56, T3 69, T12 70) with better scores in the anxiety, self-control, general health, and vitality subscales; patient #3 improved from a severe distress to a positive well-being condition (Total score: T0 46, T3 73, T12 75) with relevant improvement in the depression, positive well-being, self-control, and vitality subscales; patient #8 improved from a severe to a mild distress condition (Total score: T0 52, T3 70, T12 71) with better rating in the anxiety, depression, positive well-being, and self-control subscales. Patient #5, presenting at T0 with a borderline-mild distress score (T0: 72), improved his perceived general well-being to a normal score (T3: 77, T12: 78) mainly due to better anxiety and vitality scores.

Wisconsin Card Sorting Test (WCST)
PKU patients were able to complete all six card classification categories (6/6) as proposed by the WCST (n. of categories completed) at T0, T3, and T12, except patient #2 that was able to complete 5 out of 6 categories at T0 and 6 out of 6 categories after 3 and 12 months of LNAAs supplementation (T3, T12). In order to accomplish the test, at T3 and T12, PKU patients needed less cards compared to T0 (83.5 and 82.8 vs. 93.6 out of 128 available cards). The total number of errors and the percent of errors significantly decreased during the LNAAs supplementation along with a significant improvement of the performance centile (p < 0.05). A tendency toward significancy was observed in the areas of perseverative errors (p = 0.06) and the % of perseverative errors (p = 0.08), reflecting a more flexible and adaptive behavior regarding the task. The significant reduction of non-perseverative errors (p = 0.03) and the % of non-perseverative errors (p = 0.001) reflected a reduction of the random responses during the task. Finally, the % of conceptual level responses significantly increased (p = 0.03), indicating that the reduction of errors was obtained for a raise of intentional right responses excluding random "rights" (Table 4).

Test of Attentional Performance (TAP test)
Vigilance and sustained attention were explored by the TAP test. Vigilance improved in all patients. The time to complete the total tasks, expressed in ms, significantly improved after 3 and 12 months of LNAAs supplementation (Mean ± SD: T0 706 ± 82 ms, T3 614 ± 64 ms; T12 609 ± 60 ms, p < 0.05). The total time (ms) to complete the tasks proposed to assess sustained attention improved after 3 and 12 months of LNAAs supplementation (Mean ± SD: T0 727 ± 147 ms, T3 597 ± 106 ms, T12 589 ± 98 ms; p < 0.01). In addition, the number of errors throughout the test significantly decreased as well as the number of omissions (Table 5).

9-Hole Peg Test (HPG)
The fine motor function and eye-hand coordination were explored by the 9-Hole Peg test for hand dexterity. Each patient performed two consecutive rounds both with the dominant and non-dominant hand and the performance was expressed in seconds. No significant difference in hand dexterity was observed during LNAAs supplementation in the analyzed group (mean ± SD T0 23 ± 3, T3 21 ± 3.6, T12 21.4 ± 2 s, p > 0.05). Only patient #1 and #5 improved their speed in the HPG test: patient #1 improved the speed of 3 s at T3 and of 4 s at T12 compared to 20 s at T0; patient #5 improved the speed of 8 s at both T3 and T12 compared to 24 s at T0.

Discussion
PKU is a rare metabolic disorder affecting 1:10,000 live births. After the success of the newborn screening programs and the efficacy of early low-Phe dietary intervention in averting the risk of intellectual disability in children, the attention of the scientific community is increasingly shifting from childhood to adulthood. Indeed, adults living with PKU experience deep psychological discomfort and neurological symptoms such as attention deficit, irritability, anxiety, headaches, and worst executive functions compared to age-matched controls. In childhood as in adulthood, the follow-up programs have emphasized plasma Phe levels as the most important outcome measure, although this now appears reductive. Therefore, great efforts have been made to identify novel biomarkers besides Phe to monitor patients' health, as well as neuropsychological measures susceptible of improvement. In addition, alternative pharmacological therapies have been developed such as Sapropterin and Pegvaliase (Biomarin).
The present study explored the effects of a medical formula enriched with LNAAs in 10 adult patients with PKU with a story of poor compliance to diet in the year before enrolment.
LNAAs formulas have been developed for PKU patients with poor compliance to diet. In this study, we choose to monitor plasma Phe and Tyr levels over a long time-period (12 months) and patients were asked to keep their dietary habits unchanged during the trial in order to obtain Phe and Tyr measures as realistic as possible. Under these conditions, we choose to supplement with the highest dose of LNAAs used so far in clinical trials (0.8-1 g/kg/day) [21,26] with the hope to achieve the best competition between dietary Phe and LNAAs at the level of the intestinal LAT transporter. Unfortunately, the present study failed to show a reduction of plasma Phe levels over the long period. This observation is consistent with previous studies [23,24] despite those studies using lower doses of LNAAs preparations (0.25 and 0.5 g/kg/day, respectively). Conversely, we could not replicate the data on plasma Phe from other studies [21,22,25] even if the LNAAs dose was as high as the dose used in the first report. In our opinion, this could be a consequence of the different study time-length. In previous studies, supplementation with LNAAs spanned from 1 to 4 weeks, a short period in which even patients poorly compliant to diet could better control their dietary Phe intake while knowing to be enrolled in a trial. Indeed, in the present study, the first plasma Phe value after the first month of Phe supplementation was lower by 23% to 50% from the baseline in 4 out of 10 patients. As a matter of fact, during the first weeks, those patients spontaneously reduced their dietary Phe amounts, as outlined by meals diaries. However, in all patients, Phe levels gradually raised during the following months as they went back to their previous dietary habits. This observation is in agreement with the data by Burlina and colleagues [26] which show a reduction of plasma Phe after the first 6-months of LNAAs supplementation but not after 12 months.
LNAAs formulas, compared to other amino acids mixtures, contain a high amount of Tyr and tryptophan per 100 g. During the study, we could observe a significant improvement of blood Tyr levels. Due to PAH deficiency, in PKU, Tyr becomes an essential amino acid. Tyr is the precursor of dopamine and norepinephrine, putatively involved in motor function, mood, arousal, attention, and anxiety [16]. In addition, the accumulation of Phe competes with the available Tyr and tryptophan (a precursor of serotonin) to cross the BBB. Therefore, the main effect of this disorder on the nervous system consists in a deficiency of dopamine and serotonin neurotransmitters.
In this study, the perceived level of quality of life, executive functions, vigilance, and attention span were evaluated by the Psychological General Well-Being Index (PGWBI), the Wisconsin Card Sorting Test (WCST), the TAP test, and the HPG test. The results showed an improvement of distress and well-being rates, of executive functions, sustained attention, and vigilance, whereas no difference was noted regarding hand dexterity. The improvement was observed after the first 3 months of LNAAs supplementation and was maintained at the end of the study. As Phe levels remained well above the desired levels in all patients, we can speculate that the difference in psychological performances may be due to the rise of LNAAs in the brain, including Tyr.
The present study has some limitations, including the low sample size and the lack of placebo. We also do not have data on the levels of neurotransmitter metabolites and we could not correlate the levels of plasma Phe and Tyr with the levels in the cerebrospinal fluid (CSF) nor the improvement of the psychological performance with the variation of neurotransmitters. We and others [26] suggest that the improvement of blood Tyr may be beneficial to attenuate the neurotransmitter imbalance in PKU, however we cannot exclude the opposite hypothesis, i.e., that a high dose of LNAAs may have a detrimental effect on Tyr transport across the BBB. Despite those limitations, these results add further evidence of the advantage of LNAAs supplementation in improving cognition and well-being in patients with PKU and poor metabolic control. In future studies, it would be interesting to correlate plasma amino acids and neurotransmitter metabolites with clinical data and to assess whether the cessation of LNAAs treatment would cause a decline of psychometric scores after initial improvements.

Conflicts of Interest:
The authors declare no conflict of interest.