Lower n-3 long-chain polyunsaturated fatty acid values in patients with
phenylketonuria: a systematic review and meta-analysis
Szimonetta Lohner1, Katalin Fekete2, Tamás Decsi1
1 Department of Pediatrics, University of Pécs, Hungary
2 Department of Biochemistry and Medical Chemistry, University of Pécs, Hungary
Department of Paediatrics, University of Pécs
József A. u. 7., H-7623 Pécs, Hungary
Tel. + 36 72 535 900
Fax: + 36 72 535 971
LCPUFA; long-chain polyunsaturated fatty acid
PUFA; polyunsaturated fatty acid
LA; linoleic acid
ALA; alpha-linolenic acid
EPA; eicosapentaenoic acid
DHA; docosahexaenoic acid
AA; arachidonic acid
RCT; randomized controlled trial
MD; mean difference
The mainstream of phenylketonuria (PKU) management is lifelong restriction of protein
intake; however, this dietary restriction may be accompanied by insufficient dietary intake of
long-chain polyunsaturated fatty acids (LCPUFA). The objective of this review was to assess
whether significant depletion of LCPUFA can be detected in PKU patients on low-protein
diet and whether LCPUFA supplementation is an effective way to increase the availability of
LCPUFA in PKU patients. The method included structured search strategy on Ovid
MEDLINE, Scopus, LILACS and the Cochrane Library CENTRAL databases, with formal
inclusion/exclusion criteria, data extraction procedure and meta-analysis. We evaluated nine
case-control studies and six randomized controlled trials, dated from the inception of the
databases to 2012. The meta-analysis of the case-control studies showed significantly lower
values of both eicosapentaenoic acid and docosahexaenoic acid (DHA) in all biomarkers
investigated and that of arachidonic acid in total plasma lipids in PKU patients as compared
to healthy controls. There were sufficient data to demonstrate that dietary DHA
supplementation of patients with PKU significantly increases the contribution of DHA to
total plasma lipids.
In summary, suboptimal LCPUFA status, especially that of n-3 LCPUFA can be detected in
PKU patients. Supplementing DHA to the diet of PKU patients may improve their LCPUFA
status; however, further research is needed to determine the optimal supplementation dosage
and to establish beneficial functional outcomes.
Keywords (MeSH): Phenylketonurias; Fatty Acids, Unsaturated; Docosahexaenoic Acid;
Phenylketonuria (PKU) is a hereditary disease characterized by deficiency of the liver
enzyme phenylalanine (Phe) hydroxylase, which plays an essential role in the breakdown of
the amino acid Phe to tyrosine. In the classic form of PKU, a deficiency of the enzyme leads
to blood phenylalanine concentrations higher than 1200 μmol/L. In the condition called
hyperphenylalaninaemia the activity of phenylalanine hydroxylase allows some conversion of
Phe, but the blood Phe concentrations persistently remain above 400 μmol/L. The diagnosis
of PKU can be established within a few days after birth, because Phe concentrations become
elevated rapidly after starting a normal dietary protein intake. A special diet low in Phe is the
way generally applied to avoid the neurotoxicity caused by high concentrations of Phe in the
blood. Untreated phenylketonuria is associated with progressive intellectual impairment
accompanied by psychiatric disorders including severe behavioral disturbances (psychotic,
autistic and aggressive disorders) . Dietary restrictions have to be started immediately after
suspecting the diagnosis in the newborn and they should be maintained until the exact
diagnosis set up. Phe concentrations should be kept between 120 and 360 μmol/L ;
however, the strictness of blood Phe control can be relaxed somewhat as the child gets older.
This type of strict metabolic control usually provides not only lower amounts of vitamins and
minerals, but also lower amounts of saturated and polyunsaturated fatty acids (PUFA). In
infancy and childhood, long-chain PUFA (LCPUFA) are important for normal
neurodevelopment [3,4], a deficiency may contribute to disturbances in the function of the
central nervous system, which are detectable as early as in preschool and school-age children
with PKU . LCPUFA intake may be especially deficient after the age of two years,
because, due to their high protein content, .nutrients rich in LCPUFA (like fatty fish, eggs or
offals) are excluded from the diet of children with PKU.
Narrative overviews summarizing the nutritional challenges in PKU and other inborn errors
of metabolism [6,7] and a systematic review assessing the effect of LCPUFA
supplementation on functional outcome and cognitive function in children  are also
available. However, there is no systematic review on PUFA status in PKU or on the
effectiveness of supplementation with LCPUFA. The aim of this systematic literature review
was to collect all available data on both aspects, i.e. on the LCPUFA status of patients with
PKU and on the effects of LCPUFA supplementation on different biomarkers in PKU. The
data collection in the present review is based on the methodology developed by Hooper et al
2. Approaches for determining the LCPUFA status in PKU patients
2.1. Inclusion criteria
To be included into the review, a study was required to meet the following characteristics: 1.
be a study on patients of any age with PKU on low-protein diet and 2. be a case-control study
reporting n–3 and/or n–6 LCPUFA status in patients and in healthy controls; or randomized
controlled trial (RCT) investigating the effect of LCPUFA supplementation on LCPUFA
status of patients with PKU.
Ovid MEDLINE (www.ovid.com), Scopus (www.scopus.elsevier.com), LILACS
(www.bireme.br) and the Cochrane Library CENTRAL database
(www.thecochranelibrary.org) were searched from inception to February 2011. The search
was repeated in a reduced form in May 2012. Text terms with appropriate truncation and
relevant indexing terms were used to identify articles. The search was in the form [n–3
LCPUFA terms] or [n–6 LCPUFA terms] and [PKU terms]. The Ovid MEDLINE search
strategy is shown in Supplemental Table 1. The search of the three other databases was based
also on this strategy. The electronic search was supplemented with articles in the reference
lists of the relevant studies and review articles. We did not apply any language restriction.
Data extraction and analysis
Two reviewers independently searched databases, selected studies to be included in the
review and extracted data. If the two reviewers disagreed, the study was discussed in detail
until they reached a consensus. Data for each study included were exported into a Microsoft
Office Excel 2007 database file. To provide a standardized format, units of measurement
were recalculated to percentage contribution of LCPUFA to total fatty acid composition of
the relevant biomarker (% weight/weight) from original data in the publication. When it was
not possible to convert data, we contacted the authors for details.
Statistical analyses were performed using the Review Manager 5.1 Software (The Cochrane
Collaboration, Oxford, United Kingdom), with the random-effects model. The confidence
interval (CI) was established at 95%. P values of less than 0.05 were considered to indicate
statistical significance. Statistical heterogeneity was assessed using the I2 statistics (I2 of 50%
or more indicating the presence of significant heterogeneity).
3. Reported findings
The flow diagram of the literature search for this review is shown in Figure 1. We included
nine case-control studies and six RCTs in this systematic review, with a total of 713 and 273
Case-control studies investigating fatty acid compositional differences
between PKU patients and healthy controls
In the case-control studies included, fatty acid composition of the following 5 biomarkers was
reported: total plasma lipids, plasma phospholipids, plasma cholesteryl esters, total
erythrocyte membrane lipids and erythrocyte phospholipids. Basic characteristics of the
studies included are presented in Table 1. In three papers the diagnosis of participants was
classic PKU [10,11,12], two studies investigated also patients with moderate or mild PKU
subtypes (Phe levels lower than 1200 or 600 μmol/L) [13,14], whereas in four papers no
further subclassification of PKU was described [15,16,17,18]. All patients followed amino
acid-modified diets. Bad metabolic compliance of participants (Phe values higher than 600
μmol/L, in spite of dietary intervention) was reported in two of the studies included [17,18].
In this review we focused on linoleic acid (LA) and arachidonic acid (AA) from the n-6 series
and alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA) from the n-3 series. We conducted meta-analysis in cases of fatty acids and
biomarkers used in at least three independent studies.
3.1.1. Fatty acid composition of total plasma lipids
Seven papers reported fatty acid composition of plasma total lipids. Analysis showed
significantly lower contribution of the n-6 LCPUFA, AA (MD: -0.75; 95% CI: -1.31, -0.18;
12 studies; 596 participants; I2 = 68%) and the n-3 LCFUFA, EPA (MD: -0.20; 95% CI: -
0.27, -0.13; 11 studies; 505 participants; I2 = 77%) and DHA (for data see Figure 2) to plasma
total lipids of patients with PKU compared to healthy controls, while in the values of the
essential precursors, LA (MD: 1.27; 95% CI: -0.23, 2.76; 12 studies; 596 participants; I2 =
65%) and ALA (MD: 0.05; 95% CI: 0.00, 0.10; 11 studies; 569 participants; I2 = 73%) there
was no significant difference between the two groups.
3.1.2. Fatty acid composition of plasma phospholipids
Four papers reported fatty acid composition of plasma phospholipids. Primary analysis
showed significantly lower EPA (MD: -0.41; 95% CI: -0.68, -0.14; 6 studies; 268
participants; I2 = 94%) and DHA (Figure 3) values in patients with PKU than in controls. No
significant differences were found between the two groups with respect to AA (MD: 0.00;
95% CI: -0.80, 0.81; 6 studies; 268 participants; I2 = 30%), LA (MD: 2.62; 95% CI: -0.46,
5.71; 6 studies; 268 participants; I2 = 88%) or ALA values (MD: -0.07; 95% CI: -0.17, 0.03;
5 studies; 241 participants; I2 = 97%).
3.1.3. Fatty acid composition of erythrocyte membrane total lipids
Comparison of erythrocyte membrane total lipid fatty acid composition in patients with PKU
and healthy controls was present in 8 studies. The primary analysis showed significantly
lower EPA (MD: -0.08; 95% CI: -0.16, -0.01; 7 studies; 260 participants; I2 = 73%) and DHA
(Figure 4) values in patients as compared to controls; however, there was no significant
difference in AA values (MD: -0.32; 95% CI: -1.07, 0.43; 8 studies; 351 participants; I2 =
71%). There was no difference in LA values between the two groups (MD: 0.53; 95% CI: -
0.18, 1.23; 8 studies; 351 participants; I2 = 52%), whereas ALA values were significantly
higher in patients than in controls (MD: 0.03; 95% CI: 0.01, 0.05; 7 studies; 324 participants;
I2 = 43%).
3.1.4. Fatty acid composition of erythrocyte phospholipids
Data relating to the erythrocyte phospholipid fraction were sufficient only for a meta-analysis
of DHA; values were significantly lower in PKU patients than in controls (Figure 5).
3.1.5. Fatty acid composition of plasma cholesteryl esters
There were insufficient data to conduct a meta-analysis comparing fatty acid composition of
plasma cholesteryl esters in subjects with PKU and controls.
Effects of LCPUFA supplementation on DHA concentrations in PKU
We included six RCTs with parallel design. In two studies participants were newborns
[19,20], in three studies infants and children between 1 and 18 years [21,22,23] were
included, while one study included both children and adults . Five studies were carried
out in Europe, one in the USA .
In this report we primarily focus on the effect of LCPUFA supplementation on DHA status.
We found eight biomarkers used to characterize changes in DHA values. From these, only
total plasma DHA was used in at least three independent studies; nevertheless, the effect of
LCPUFA supplementation on every biomarker of DHA status is shown in Figure 6. Agostoni
et al  applied supplementation with 2.5–4 g fish oil (18 g EPA, 4 g DPA and 12 g
DHA/100 g fatty acid) daily for 6 months; Agostoni et al  used 1 capsule (37 mg AA,
27.5 mg EPA, 20 mg DPA and 40 mg DHA/0.5 g capsule) per 4 kg body weight for 1 year;
Yi et al  used microalgae oil capsules (10 mg/kg/day DHA) for 4.5 months of
supplementation; Koletzko et al  used a supplemented formula (0.46 g AA and 0.27 g
DHA/100 g fatty acids) for 1 year; Agostoni et al  used a supplemented formula with
slightly different composition (0.7 g AA and 0.3 g DHA/100 g fatty acids) for 1 year while
Cleary et al  gave essential fatty acid supplemented protein substitute (17.2 g LA and 4.5
g ALA/100 g fatty acid) for 20 weeks. Although different dosages and forms of n-3 LCPUFA
supplementation were used in the different studies, these were all effective in significantly
increasing DHA values of different biomarkers (Figure 6).
4. Potential causes of inadequate LCPUFA supply in PKU patients and efficacy of
The mainstream of phenylketonuria management is the restriction of dietary intake of
phenylalanine, immediately, after confirmation of the diagnosis. This means that patients
with phenylketonuria have to consume a phenylalanine free formula and avoid foods rich in
protein (e.g. meat, fish, egg, cheeses, normal bread and seeds), moreover, they have to avoid
foods and drinks containing aspartame, flour, soya, beer or cream liqueurs. Low protein
natural foods (potatoes, some vegetables and most cereals) can be consumed in restricted
amounts . This strict diet may lead to the insufficient intake of some essential nutrients.
The potentially inadequate dietary LCPUFA supply of patients suffering from PKU on Phe-
restricted diet has gained attention recently. There are two main sources of LCPUFA in the
human organism: dietary intake and endogenous synthesis from their essential metabolites,
LA and ALA (Figure 7). However, the endogenous synthesis of AA and DHA is limited ,
therefore, the lack of preformed LCPUFA in the diet may lead to deficiency. Moreover, in
PKU a possible inhibitory effect of Phe metabolites on endogenous DHA synthesis is
The connection between elevated plasma Phe levels and neurological disorders seen in
patients with PKU is well-established . The optimum outcome is mainly dependent on
metabolic control with diet and this control varies during the patient’s life. However,
functional deficits may also be present in PKU patients treated early and well. Patients with
PKU have lower intelligence quotients as compared to healthy controls . Subtle
abnormalities in phenylketonuria include the impairment of executive abilities (such as
planning, problem solving, information processing and sustained attention . Besides,
children with phenylketonuria have behavioral abnormalities, motor dysfunction and
impaired memory .
LCPUFA are shown to have important role in cognitive development, in maturation of visual
acuity and development of motor functions in full-term and preterm infants . On the basis
of these considerations it has been hypothesized that LCPUFA deficiency, especially
inadequate DHA supply, might contribute also to the neurological abnormalities observed in
patients with PKU .
In this review we included 9 case-control studies (divided into 16 arms) and six RCTs
(divided into 13 arms) in order to assess whether significant deficiency of LCPUFA can be
demonstrated in patients with PKU. We also investigated whether LCPUFA status of PKU
patients can be improved by supplementing their diet with LCPUFA. We found that in PKU
patients on low-protein diet blood levels of the two principal n-3 LCPUFA, EPA and DHA,
were consequently and significantly reduced in different blood biomarkers; whereas the
values of the principal n-6 LCPUFA, AA were significantly reduced only in plasma total
lipids. These data indicate that supplementation with oils containing DHA may be an
effective way for improving the n-3 LCPUFA status of patients with PKU. Special dietary
products for infants with PKU are usually enriched with n-3 and n-6 LCPUFA, however,
supplementation should be applied routinely also after infancy. This may be achieved
relatively easily since large numbers of various capsules containing fish oil are available.
A limitation of the present review was that in the different studies different dosages of
LCPUFA were used for supplementation, and therefore subgroup analysis according to
supplement dosage was not possible. A further limitation was the lack of description of
compliance and blood Phe levels of the patients with PKU on low-protein diet; included
therefore, subgroup analysis evaluating the potential influence of different blood Phe levels
on PUFA metabolism was not possible. We assume that insufficient LCPUFA intakes
together with metabolic impairment of LCPUFA synthesis may lead to decreased LCPUFA
levels seen in patients with PKU.
Summary and future studies
The data systematically reviewed here indicate that in patients with PKU n-3 LCPUFA
supply is insufficient. However, data from RCTs suggest that DHA status in patients with
PKU may be reflectively improved by dietary supplementation. Further studies are needed to
investigate whether LCPUFA deficiency is present in all age groups, especially in
adolescents and adults who usually follow more relaxed diets. Moreover, there is only limited
evidence about the optimal LCPUFA supplementation dosage in the different age groups.
This study was financially supported by the Hungarian National Research Fund (OTKA
K104720). The authors confirm that there are no financial or other relationships, which might
lead to a conflict of interest in the publication of this work.
 Bone A, Kuehl AK, Angelino AF. A neuropsychiatric perspective of phenylketonuria I:
overview of phenylketonuria and its neuropsychiatric sequelae. Psychosomatics 2012;
 Demirkol M, Giżewska M, Giovannini M, Walter J. Follow up of phenylketonuria
patients. Mol Genet Metab 2011;104:S31-39.
 Decsi T, Koletzko B. Role of long-chain polyunsaturated fatty acids in early human
neurodevelopment. Nutr Neurosci 2000;3:293-306.
 Campoy C, Escolano-Margarit MV, Anjos T, Szajewska H, Uauy R. Omega 3 fatty acids
on child growth, visual acuity and neurodevelopment. Br J Nutr 2012;107:S85-S106.
 Koletzko B, Beblo S, Demmelmair H, Hanebutt FL. Omega-3 LC-PUFA supply and
neurological outcomes in children with phenylketonuria (PKU). J Pediatr Gastroenterol Nutr
 Fekete K, Decsi T. Long-chain polyunsaturated fatty acids in inborn errors of metabolism.
 Giovannini M, Verduci E, Salvatici E, Paci S, Riva E. Phenylketonuria: nutritional
advances and challenges. Nutr Metab (Lond) 2012;9:7.
 Agostoni C, Braegger C, Decsi T et al. Supplementation of N-3 LCPUFA to the diet of
children older than 2 years: a commentary by the ESPGHAN Committee on Nutrition. J
Pediatr Gastroenterol Nutr 2011;53:2-10.
 Hooper L, Ashton K, Harvey LJ, Decsi T, Fairweather-Tait SJ. Assessing potential
biomarkers of micronutrient status by using a systematic review methodology: methods. Am J
Clin Nutr 2009;89(suppl):1953S–9S.
 Acosta PB, Yannicelli S, Singh R et al. Intake and blood levels of fatty acids in treated
patients with phenylketonuria. J Pediatr Gastroenterol Nutr 2001;33:253-9.
 Sanjurjo P, Perteagudo L, Rodríguez Soriano J, Vilaseca A, Campistol J. Polyunsaturated Download full-text
fatty acid status in patients with phenylketonuria. J Inherit Metab Dis 1994;17:704-9.
 van Gool CJ, van Houwelingen AC, Hornstra G. The essential fatty acid status in
phenylketonuria patients under treatment. J Nutr Biochem 2000;11:543-7.
 Lage S, Bueno M, Andrade F et al. Fatty acid profile in patients with phenylketonuria
and its relationship with bone mineral density. J Inherit Metab Dis 2010; [Epub ahead of
 Vilaseca MA, Lambruschini N, Gómez-López L et al. Long-chain polyunsaturated fatty
acid status in phenylketonuric patients treated with tetrahydrobiopterin. Clin Biochem
 Galli C, Agostoni C, Mosconi C, Riva E, Salari PC, Giovannini M. Reduced plasma C-
20 and C-22 polyunsaturated fatty acids in children with phenylketonuria during dietary
intervention. J Pediatr 1991;119:562-7.
 Giovannini M, Verduci E, Radaelli G et al. Long-chain polyunsaturated fatty acids
profile in plasma phospholipids of hyperphenylalaninemic children on unrestricted diet.
Prostaglandins Leukot Essent Fatty Acids 2011;84:39-42.
 Moseley K, Koch R, Moser AB. Lipid status and long-chain polyunsaturated fatty acid
concentrations in adults and adolescents with phenylketonuria on phenylalanine-restricted
diet. J Inherit Metab Dis 2002;25:56-64.
 Yi SH, Kable JA, Evatt ML, Singh RH. A cross-sectional study of docosahexaenoic acid
status and cognitive outcomes in females of reproductive age with phenylketonuria. J Inherit
Metab Dis 2011;34:455-63.
 Agostoni C, Harvie A, McCulloch DL et al. A randomized trial of long-chain
polyunsaturated fatty acid supplementation in infants with phenylketonuria. Dev Med Child