Sustaining benefits of nutritional therapy in young adults with phenylketonuria - A 2 year prospective study.

INTRODUCTION: Phenylketonuria (PKU) is an inborn error of metabolism, which is caused by a deficiency in the enzyme phenylalanine hydroxylase (PAH). Life-long Phe-free diet impairs quality of life, especially in adolescents and young adults which take responsibility over their diet and therapy from their parents, but expect freedom in daily routine. METHODS AND
RESULTS: 105 patients with PKU were screened for eligibility for participating in this study. Data of 21 patients with genetic predictive value (GPV) ≤5 and age between 14 and 30 years were included in the analysis. Mean age of the study population was 22.6 ± 7.5 years, 8 patients (38%) were female. At baseline, structured counselling by a professional nutrionist was performed.Mean Phe-level at baseline was 926 ± 432 μmol/l, after six months Phe-levels were significantly reduced to 709 ± 314 μmol/l (p = .039), in total 4 additional patients (38% of the population) reached values within the therapeutic goal. After 12, 18 and 24 months, mean Phe-level elevated significantly to initial level (869 ± 427 μmol/l; p = .311). Mean daily intake of natural protein at baseline was 32.3 ± 24.3 g per day. There was a significant decrease after 6 months (26.9 ± 18.8 g/day; p = .049) and 12 months (25.9 ± 16.2 g/day; p = .30) compared to baseline. Values at 18 months (27.5 ± 9.2 g/day; p = .26) and 24 months (35.0 ± 22.3 g/day; p = .87) did not differ. Mean daily supplementation of Phe-free amino acids was 26.2 ± 19.2 g per day. In all follow-up examinations a significant increase compared to baseline values was calculated (42.4 ± 17.6 g/day after 6 moths (p = .028), 52.1 ± 29.9 g/day after 12 months (p ≤.01), 38.7 ± 20.3 g/day after 18 months (p < .01) and 39.3 ± 21.9 g/day after 24 months (p = .014)). At baseline, mean total protein intake (natural protein plus supplements) was 0.97 ± 0.42 g per kg body weight (g/kgBW). After 24 months the protein intake was within recommended levels. (1.23 ± 0.33 g/kgKB; p = .013). After 24 months, plasma Vitamin B12 increased to 424.8 ± 176.9 pg/ml (baseline 368.6 ± 205.6 pg/ml; p = .049) and Vitamin D increased to 30.4 ± 9.9 ng/ml (baseline 24.5 ± 10.1 ng/ml; p = .06).
CONCLUSION: Counselling by a professional nutrionist in young adults with PKU has clear short-term effects on plasma Phe-levels. Easy applicable therapeutic recommendations, as additional intake of amino acid supplement, are well tolerated and result in strict therapy adherence up to 24 months. Apart from that, the effects on Phe-levels seem only to sustain for about 6 months. More frequent nutritional counselling, i.e. at least two times per year, is recommended to preserve positive effects on Phe-levels. Lack of Vitamin B12 and Vitamin D still are common in PKU patients, but not necessarily need to be substituted. They can effectively be equalized by a well-balanced diet within 24 months.

Introduction

Phenylketonuria (PKU) is an inborn error of metabolism, which is caused by a deficiency in the enzyme phenylalanine hydroxylase (PAH), resulting in disturbances of phenylalanine (Phe) metabolism. [1] The elevated Phe concentrations in adult patients affect neurophysical functions, resulting in cognitive impairment and neuropsychiatric symptoms linked to amount of the elevation [2,3] as well as neurological symptoms [4]. The life-long treatment should result in blood Phe-levels of 120-360 μmol/l in childhood and < 600 μmol/l in adulthood and adolescence [5,6]. Our clinic follows European policy for blood phenylalanine target and recommends treatment for life. Life-long diet impairs quality of life, especially in adolescents and young adults who took responsibility over their diet and therapy from their parents, but expect freedom in daily routine. This is visible in loosen therapy adherence and therefore higher plasma Phe-levels in these patients. According to clinical experience, the treatment of PKU in young adults is often challenging and results in Phe levels far above the recommendations. Nutritional intervention is offered to every patient with blood Phe values >600 μmol/ (adults) or > 360 μmol/l (children). A nutritionist is present at the regular follow-up visits in our clinic at least once a year.

Treatment options are protein-restricted diet combined with Phe-free medical foods and amino acid mixtures (AAM) respectively the use of sapropterin dihydrochloride (Kuvan®, BioMarin Pharmaceutical Inc., Novato, California, USA) which is an effective treatment in patients with residual PAH activity [7,8]. Pegvaliase (Palynziq®, BioMarin Pharmaceutical Inc., Novato, California, USA) is a novel enzyme substitution therapy approved by the European Drug Administration (EDA) in May 2019 for the treatment of PKU in adults and children ≥16 years of age [9,10]. The selection of the right patient for a potential harmful treatment is essential for patient's contentedness and long-term therapy compliance and needs well-founded information for long-term effects of conservative treatment modalities. The individual Phe tolerance depends on residual phenylalanine hydroxylase activity [11] and varies between patients. The necessary AAM dosage also depends on the individual Phe tolerance, age and body weight. To account for metabolic imbalances and potential absorption deficiencies of amino acids from the AAM, it is generally suggested to provide additional 40% of the recommended [12,13] protein supply. The supplementation with Phe-free amino acids provide an important source of tyrosine and plays a key a role in suppressing blood phenylalanine concentrations. To avoid micronutrient deficiencies, as the patients' choice of natural food is extremely limited, the AAM contain significant amounts of vitamins, minerals and trace elements. Patients who follow a relaxed diet or lost therapy adherence take only a small amount or no AAM. Consequently, these patients might be at risk of insufficient micronutrient supply. Especially the supply with Vitamin B12 has been shown to be often far under recommendations [[14], [15], [16]]. Due to several, in particular cultural, differences in relationship and treatment modalities, a selective observation of different countries is necessary to improve individual patient care.

Main objective of this study was to investigate the long-term effects (24 months) of an intervention by professional nutritionists on plasma Phe-levels, daily protein intake, use of AAM and micronutrient supply in this unsteady and challenging subgroup of PKU patients.

Material and methods

105 patients (childrens and adults) with disturbances of phenylalanine metabolism are in regular care of the metabolic centre of Ulm. All patients were screened for eligibility for participating in this study. Exclusion criteria were the following: all-time maximum Phe-levels <600 μmol/l, age < 14 years or > 30 years, current or planned pregnancy, no availability of genetic information or genetic predictive value (GPV) >5. Common cut-off-values for GPV were used as follows: 0.0–2.7 for classic PKU, 2.8–6.6 for mild PKU and 6.7–10.0 for mild hyperphenylalaninemia. Among the study population 42 patients were classified as eligible for participation and were invited to a regular consultation in our clinic. All patients suffered from PKU and had dietary treatment in their whole childhood with Phe levels lower than 360 μmol/l in the first 10 years of their live (ECTPKU). 27 patients were willing to participate, 2 patients were excluded due to personal circumstances. Within the regular routine assessment, we collected data about daily Phe- and protein-intake (analysis of 3-day protocols) and clinical data. Retrospective data about protein intake were assessed out of clinical records. We performed routine clinical and neurological examination and collected blood samples for determination of plasma Phe-levels in our own lab (high-pressure liquid chromatography, Biochrom 30+, Harvard Bioscience Inc., Holliston, Massachusetts, USA) as well as determination of vitamins and micronutrients in plasma (plasma values of Vitamin C, Vitamin B12, Vitamin D, Vitamin E, plasma iron and zinc). The nutritional advice and recommendations are presented semi-structured to every single patient and try to follow the patients individual needs. Follow-Up assessments were arranged after 6, 12, 18 and 24 months for acquiring information about daily Phe- and protein-intake (analysis of 3-day protocols) and clinical data at every appointment, as well as determination of plasma Phe-levels. If there were any questions about the nutritional recommendations, those were answered by the same nutritionist who did the initial counselling. No changes to the initial recommendations were made during the follow-up period. Plasma levels of vitamins and micronutrients were re-assessed after 24 months. The study protocol is shown in Fig. 1.

Fig. 1

Study design.

All values are presented as the mean ± SD or as the absolute number. Differences between two groups were tested using the paired or unpaired t-test if normal distribution could be assumed. Otherwise, the Wilcoxon rank-sum test was performed. For categorical data, the chi-squared test or the Fisher's exact test was used. All differences were tested two-sided. Larger numbers of groups were tested using two-way analysis of variance. P-values of 0.05 are considered as indicating statistical significance. Data were analyzed using SPSS version 25.0 (SPSS Inc., Chicago, IL, USA). Written informed consent was obtained by patients or their legal representatives.

Results

A total of 105 patients was screened for participating in this study. Main exclusion criteria were the following: Age not within the inclusion range or treatment with Sapropterine (n = 58), not willing to participate (n = 15), no genetic information (n = 5) and personal circumstances avoiding regular follow-ups (n = 2). At least 25 patients were included in this study, of which 4 patients were lost to follow-up. In total, data of 21 patients were included in the analysis. Mean age of the study population was 22.6 ± 7.5 years, 8 patients (38%) were female. The mean time of nutritional counselling was 33 ± 11 min. Follow-up visits were performed after 6.5 ± 3.9 months (Follow-Up 1), 11.2 ± 3.3 months (Follow-up 2), 19.8 ± 7.4 months (Follow-Up 3) and 25.5 ± 5.7 months (Final Visit) after the baseline examination. The study protocol is shown in Fig. 1. Mean plasma Phe-level at baseline was 957 ± 389 μmol/l, only 4 patients (19%) reached the therapeutic goal (Phe <600 μmol/l). All patients were substituted with Phe-free amino acid mixture, no patient used glykomakropeptides (GMP). All baseline characteristics are presented in Table 1.

Table 1

Baseline characteristics.

	Study group (n = 21)
Female gender (n)	8 (38%)
Age (yrs)	22.6 ± 7.5
Mean GPV	0.9 ± 2.0 (0–5)
GPV ≤2.7 (classic) (n)	17 (80.1%)
Height	164 ± 18
Weight	65.5 ± 22.0
BMI	23.3 ± 4.9
Use of Kuvan®	0 (0%)
Plasma Phe (μmol/l)	957 ± 389
Phe within reference <600 μmol/l (n)	4 (19%)
Phe intake (mg/day)	1542 ± 1146
Natural protein intake (g/day)	32.3 ± 24.3
Use of Amino acids (n)	16 (76%)
Amino acid supplement (g/day)	26.2 ± 19.2

Changes in plasma Phe-levels and daily protein-intake

Retrospective data, according to plasma Phe-level (930 ± 414 μmol/l; p = .69), daily Phe-Intake (1530 ± 1139 g/day; p = .89), daily intake of natural protein (33.4 ± 26.1 g/day; p = .78) and supplemental intake of Phe-free amino acids (23 ± 15 g/day; p = .85) were not different to baseline values.

Mean Phe-level at baseline was 926 ± 432 μmol/l, only 4 patients (19%) had values within the therapeutic goal. Six months after the intervention, Phe-levels were significantly reduced to 709 ± 314 μmol/l (p = .039), in total 4 additional patients (38% of the population) reached values within the therapeutic goal. After 12 months, mean Phe-level elevated significantly to initial level (901 ± 398 μmol/l, p = .04 vs. Follow-up 1; p = .187 vs. baseline) and henceforth remained stable after 18 months (919 ± 306 μmol/l, p = .267) and 24 months 869 ± 427 μmol/l (p = .311) (Fig. 2A).

Fig. 2

Box plots Plasma Phe levels and daily protein intake.

A. Plasma Phe-levels (μmol/l) baseline vs. follow-up examinations. B. Daily Phe-intake (mg/day) baseline vs. follow-up examinations. C. Daily natural protein intake (g/day) baseline vs. follow-up examinations. D. Daily supplemental protein intake baseline vs. follow-up examinations. Significant differences are marked with stars as mentioned in the figure.

Mean daily Phe-Intake was calculated with 1542 ± 1146 mg per day at baseline. Six months after the intervention, the mean intake remained stable at 1435 ± 1051 mg/day (p = .91), followed by a decline to 1237 ± 730 mg/day after 12 months and 1316 ± 462 mg/day after 18 months. The difference to baseline was statistically significant for 12 months (p = .03), but not for 18 month's value (p = .11). At the final examination after 24 months daily Phe-Intake increased to 1725 ± 1097 mg/day back to initial values (p = .53) (Fig. 2B).

Mean daily intake of natural protein at baseline was 32.3 ± 24.3 g per day. There was a significant decrease after 6 months (26.9 ± 18.8 g/day; p = .049) and 12 months (25.9 ± 16.2 g/day; p = .30) compared to baseline. Values at 18 months (27.5 ± 9.2 g/day; p = .26) and 24 months (35.0 ± 22.3 g/day; p = .87) did not differ to baseline (Fig. 2C).

Mean daily supplementation of Phe-free amino acids at baseline was 26.2 ± 19.2 g per day. In all follow-up examinations a significant increase compared to baseline values was calculated. In detail 42.4 ± 17.6 g/day after 6 moths (p = .028), 52.1 ± 29.9 g/day after 12 months (p ≤.01), 38.7 ± 20.3 g/day after 18 months (p < .01) and 39.3 ± 21.9 g/day after 24 months (p = .014) (Fig. 2D).

At baseline, mean total protein intake (natural protein plus supplements) was 0.97 ± 0.42 g per kg body weight (g/kgBW). After 6 months, the total protein intake increased to 1.1 ± 0.33 g/kgBW (p = .041), which remained stable after 12 months (1.1 ± 0.34 g/kgBW; p = .024) and 18 months (1.0 ± 0.24 g/kgBW p = .044). Even after 24 months the protein intake was significantly higher compared to baseline (1.23 ± 0.33 g/kgKB; p = .013) (Fig. 3). All values are presented in Table 2.

Fig. 3

Box plots micronutrients and Vitamins.

Box plots of Vitamins and micronutrients baseline vs. final follow-up examination (24 months). Left box baseline, right box follow-up. Lower reference values are marked with red dotted line, respectively. Significant differences are marked with stars as mentioned in the figure.

Table 2

Overview of Phe-levels, vitamins and micronutrients in plasma as well as data out of 3-day protocols to all examination points.

	Retrospective	Baseline	Follow-Up 1 (6 months)	Follow-Up 2 (12 months)	Follow-Up 3 (18 months)	Follow-Up 4 (24 months)
Plasma Phe (μmol/l)	930 ± 414	926 ± 432	709 ± 314⁎, #	901 ± 398⁎	919 ± 306	869 ± 427
Phe within reference < 600 μmol/l (n)	4 (19%)	4 (19%)	8 (38%)⁎, #	5 (24%)⁎	7 (33%)	6 (29%)
Phe intake (mg/day)	1530 ± 1139	1542 ± 1146	1435 ± 1051	1237 ± 730#	1316 ± 462	1725 ± 1097
Natural protein intake (g/day)	33.4 ± 26.1	32.3 ± 24.3	26.9 ± 18.8⁎, #	25.9 ± 16.2#	27.5 ± 9.2	35.0 ± 22.3
Use of Amino acid mixture (AAM)(n)	16 (76%)	16 (76%)	21 (100%)	20 (95%)	19 (90%)	20 (95%)
Total supplemental protein intake (g/day)	23 ± 15	26.2 ± 19.2	42.4 ± 17.6⁎, #	52.1 ± 29.9#	38.7 ± 20.3#	39.3 ± 21.9#
Total protein intake (g/kgBW)	0.99 ± 0.38	0.97 ± 0.42	1.1 ± 0.33⁎, #	1.1 ± 0.34#	1.0 ± 0.24#	1.23 ± 0.33#
Vitamin A (μg/mL)	–	1.7 ± 0.4	–	–	–	1.8 ± 0.3
Vitamin B12 (pg/mL)	–	368.6 ± 205.6	–	–	–	424.8 ± 176.9#
Vitamin C (mg/L)	–	15.1 ± 5.3	–	–	–	12.4 ± 4.5
Vitamin D (ng/mL)	–	24.5 ± 10.1	–	–	–	30.4 ± 9.9#
Vitamin E (μmol/L)	–	25.8 ± 5.7	–	–	–	28.4 ± 9.9
Plasma iron (μmol/L)	–	19.1 ± 5.4	–	–	–	20.8 ± 7.7
Zinc (μmol/L)	–	10.9 ± 1.9	–	–	–	10.8 ± 1.9

AAM, amino acid mixture; BW, body weight; g, gram; kg, kilogram, Phe, phenylalanine.

p-Value <.05 compared to baseline.

p-Value <.05 compared to previous visit.

Vitamins and micronutrients

Concerning Vitamins and micronutrients, no significant changes were found after 24 months for Vitamin A (1.7 ± 0.4 vs. 1.8 ± 0.3 μg/mL; p = .70), Vitamin C (15.1 ± 5.3 vs. 12.4 ± 4.5 mg/l; p = .54), Vitamin E (25.8 ± 5.7 vs 28.4 ± 9.9 μmol/l; p = .18), plasma iron (19.1 ± 5.4 vs. 20.8 ± 7.7 μmol/l; p = .20) and zinc (10.9 ± 1.9 vs. 10.8 ± 1.9 μmol/l; p = .89).

At baseline examination 3 patients (14%) presented with lack of Vitamin B12 supply (<191 pg/mL) and 7 patients (33%) with lack of Vitamin D (<20 ng/mL). At the final examination after 24 months, plasma Vitamin B12 increased to 424.8 ± 176.9 pg/mL (baseline 368.6 ± 205.6 pg/ml; p = .049), all patients (100%) had values upon the lower reference levels. Plasma Vitamin D increased to 30.4 ± 9.9 ng/mL (baseline 24.5 ± 10.1 ng/mL; p = .06), only one patient (5%) had apparent lack of Vitamin D. All values are presented in Fig. 4and Table 2.

Fig. 4

Box plots total daily protein intake.

Total Daily Phe-intake (natural plus supplemental protein) (mg/day) baseline vs. follow-up examinations. Significant differences are marked with stars as mentioned in the figure.

Changes in body weight and BMI

Mean body weight did not significantly change during the follow-up period (65.5 ± 22.0 vs. 66.9 ± 19.4 kg; p = .49) as well as the body-mass index (BMI; 23.3 ± 4.9 vs. 23.4 ± 4.3 kg/m2; p = .15).

Discussion

According to well-known effects of strict diet in PKU, our study shows a reduction in Phe-levels and reduced amount of intake of natural protein within 6 months in adolescents and young adults with PKU. But in contrast to generally supposed ongoing effects of counselling by a professional nutrionist, we could clearly show the low half-life of specific changes in nutrition and health behavior after single interventions. After 12 months, plasma Phe-levels and protein intake are back to initial values and remain stable for the rest of the observation period. In our clinic blood phenylalanine is measured every 4 weeks. Thus it is supposed, that providing regular values to the patient more often does not additionally improve therapy adherence without professional counselling. One aspect for lose therapy adherance is certainly the management of PKU in Europe for the last decade, which permitted the loose permissive value of plasma Phe levels of 1200 μmol/l. Another aspect is the low burden of disease in most adult patients, most of which would not consider themselves as patients with an inborn error of metabolism. This is based on the low predictive power of blood phenylalanine on the clinical outcome from the second decade of life onwards. [17] There is clear evidence for improved quality of life in patients with relaxation of their Phe-restricted diet. [[18], [19], [20]] Conversely, establishing dietary limitations results in restrictions of daily life, which are not easy to accept in a cohort of patients with a desire to mobility and liberal lifestyle. Additionally, the long-term benefit of lower Phe-levels for the individual patient is not predictable. [17] Detailed cognitive testing and an assessment of quality of life in long-term follow-ups are needed to answer the question if life-long blood Phe control is able to prevent or improve cognitive impairment. Little harmful treatments as Sapropterine [8,21,22] are not applicable to the patients in this study.

In contrast to that, easy applicable therapeutic recommendations, as additional intake of amino acid supplement, are well tolerated and result in strict therapy adherence up to 24 months. Therefore, it was possible to reach the recommended intake of 1.2 g/kgBW of protein per day and keep it over the observational period. Additional intake of Phe-free amino acids despite of missing dietary adherence did not lead to an increase of body weight in the study population within two years.

Especially in this vulnerable cohort of patients, additionally treatment options as Pegvaliase are most welcome. Therefore, a detailed information should be given to every single patient concerning new therapeutic options and potential side effects. Further studies are necessary for finding reliable recommendations for a treatment, resulting in severe adverse events in 10% of the treated patients [23]. The available cognitive data for admission of the treatment [10,[23], [24], [25]] are based on depression and activity rating scalers, but neglect an persisting central executive impairment even in patients with well treated PKU and low blood Phe-levels [26] which needs to be analyzed in further studies.

Another clear result of this study is, that lack of Vitamin B12 and Vitamin D, despite of being well-described in literature for years, still are common in PKU patients. In contrast to other reports [27], the lack of vitamins not necessarily need to be substituted, but can effectively be equalized by a well-balanced diet within 24 months. For other tested vitamins and micronutrients, no relevant aberrations were found. If this is also effective for other micronutrients which are not included in this study, such as selen and folic acid [28] further studies are needed. PKU patients under regular diet with sufficient substitution are not able to increase their vitamin D levels. Therefore, patients under well-balanced diet still having a lack of vitamin D need additional substitution.

The positive effects of counselling by a professional nutrionist recreate disease consciousness, focuses on diet by preparing a nutrition protocol and activates recourses for therapy and diet-adherence. The short half-life of this positive effects should result in frequent repetitions of nutritional counselling and consideration of new therapeutic options, especially in young adults.

Conclusion

Counselling by a professional nutrionist in young adults with PKU has clear short-term effects on plasma Phe-levels and health behaviour. Though, we could clearly show a low half-life of specific changes in nutrition and health behavior after single interventions of about 6 months. Thus, we recommend more frequent nutritional counselling, i.e. at least two times per year, to preserve positive effects on Phe-levels.

Easy applicable therapeutic recommendations, as additional intake of amino acid supplement, are well tolerated and result in strict therapy adherence up to 24 months. Therefore, it was possible to reach the recommended intake of 1.2 g/kgBW of protein per day and keep it over the observational period. Additional intake of Phe-free amino acids despite of missing dietary adherence did not lead to an increase of body weight in the study population within two years. Lack of Vitamin B12 and Vitamin D still are common in PKU patients, but not necessarily need to be substituted. They can effectively be equalized by a well-balanced diet within 24 months. PKU patients under well-balanced diet still having a lack of vitamin D or other micronutrients need specific additional substitution.

Limitations

The small sample size impedes greater validity. Due to the rare condition of the disorder, randomized controlled trials are difficult to realize and result in reduced sample size. A long-term follow-up is needed to analyze the described effects after a longer period of time. The overall supply of micronutrients in total was not calculated, therefore specific information about the distribution of micronutrients between AAM and regular nutrition is missing. Another limitation is the lack of information about body composition. Thus, besides BMI, no information about positive or negative aspects of the intervention to health related aspects of body composition is available in this study. All information about nutrition are based on the information provided by the patient, therefore we need to accept a certain impreciseness in these information in lack of a more precise method for data acquisition.

Ethics approval and consent for publication

Positive ethical statement was obtained from the Ethics Committee (University of Ulm). All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all patients for being included in the study.

Availability of data and material

Original data is available upon personal request.

Funding

Research funding was provided by Dr. Schaer AG/Spa, Zona di Produzione Winkelau 9, 39014 Postal (BZ), Italy.

Authors' contributions

Johannes Krämer: first draft, concept of the manuscript, data interpretation, statistical analysis.

Declaration of Competing Interest

Johannes Krämer declares that he has no conflict of interest.