Omega-3 LC-PUFA Supply and Neurological Outcomes in Children With Phenylketonuria (PKU)

Journal of Pediatric Gastroenterology and Nutrition 48:S2 S7 # 2009 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition Omega-3 LC-PUFA Supply and Neurological Outcomes in Children With Phenylketonuria (PKU) Berthold Koletzko, Skadi Beblo, Hans Demmelmair, and Fabienne L. Hanebutt Division of Metabolic Diseases and Nutritional Medicine, Dr von Hauner Children s Hospital, University of Munich Medical Centre, Munich, Germany ABSTRACT Children with phenylketonuria (PKU) follow a diet with very low intakes of natural protein, which is devoid of food sources of the omega-3 docosahexaenoic acid (DHA). A resulting DHA depletion has been demonstrated in PKU children and may account for detectable subtle neurological deficits that are not explained by variation in plasma phenylalanine concentrations. We supplemented 36 children with PKU ages 1 to 11 years for 3 months with encapsulated fish oil providing a daily dose of 15 mg DHA/kg body weight. DHA supplementation resulted in significantly faster visual evoked potential latencies, indicating more rapid central nervous system information processing. In addition, DHA significantly improved outcomes in a test of motor function and coordination. No changes over time were seen in age-matched healthy controls. Because the PKU children had a good supply of the omega-3 precursor alphalinolenic acid, these observations lead us to conclude that endogenous conversion of alpha-linolenic acid is not sufficient to provide adequate amounts of DHA that support optimal function, and hence DHA appears to be a conditional essential substrate for children with PKU. Because early treated PKU children are healthy, with normal fatty acid turnover, these data may indicate a need to supply some DHA to children in general. Further studies are ongoing aiming at establishing quantitative DHA requirements in children. JPGN 48:S2 S7, 2009. Key Words: Arachidonic acid Docosahexaenoic acid Phenylketonuria Rostock Oseretzky test Visual evoked potential latencies. # 2009 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition Long-chain polyunsaturated fatty acids (LC-PUFA) are indispensible components of structural lipids in all tissues. Docosahexaenoic acid (DHA, C22:6n-3) and arachidonic acid (AA, C20:4n-6) are the quantitatively most important LC-PUFA of the n-3 and n-6 series, respectively. These 2 acids and the other LC-PUFA are derived from the essential fatty acids alpha-linolenic acid (C18:3n-3) and linoleic acid (C18:2n-6), respectively (1). Delta-6 desaturation of n-3 and n-6 precursor essential fatty acids converts alpha-linolenic acid to C18:4n-3 and linoleic acid to C18:3n-6, and these intermediates are elongated to C20 products and further desaturated via delta-5-desaturase, which yields eicosapentaenoic acid (EPA, C20:5n-3) and AA (C20:4n-6) (Fig. 1). Although AA is the major conversion product on the n-6 side, EPA serves as an intermediate of which a significant portion is further elongated to C22:5n-3 and to C24:5n-3, which in turn is converted by delta-6 desaturation to C24:6n-3. This C24 intermediate does not accumulate but undergoes partial peroxisomal Correspondence to Professor Berthold Koletzko, Dr von Hauner Children s Hospital, University of Munich Medical Centre, Lindwurmstr. 4, 80337 München, Germany (e-mail: office.koletzko@med. uni-muenchen.de). The authors report no conflicts of interest. S2 beta-oxidation, which leads to the biologically important product DHA (2). Activities of the desaturating enzyme have been demonstrated in humans, including neonates (3,4), but the rate of endogenous essential fatty acid conversion is low and dose not suffice to establish similar LC-PUFA levels in subjects devoid of exogenous LC- PUFA supply as compared with subjects with a habitual intake of a LC-PUFA containing diet (5,6). Studies in infants found both AA and DHA reduced without a dietary supply of preformed LC-PUFA (7,8), whereas in adults usually only DHA levels are found markedly dependent on dietary LC-PUFA intake, presumably because of the lesser endogenous DHA synthesis in the more complex synthesis pathway as compared with n-6 AA (6). The different relative contribution of endogenous conversion to the blood levels of AA and DHA is also demonstrated by the differential effects of single nucleotide polymorphisms of the enzymes delta-6 desaturase (fatty acid desaturase 2) and delta-1 desaturase (fatty acid desaturase 2) that we first described in humans (9). Reconstructed haplotypes with rare polymorphisms of these fatty acid desaturases explained almost 30% of the variation of AA in plasma phospholipids of adults consuming self-selected diets, but only about 1% of the variation of DHA (9). These data lead us to conclude that endogenous synthesis contributes very

OMEGA-3 LC-PUFA SUPPLY AND NEUROLOGICAL OUTCOMES S3 Linoleic acid Omega-6 Omega-3 C18:2 C18:3 D6-Desaturation alpha-linolenic acid C18:3 C18:4 Elongation Eicosanoids C20:3 C20:4 D5-Desaturation Arachidonic acid (AA) Eicosanoids C20:4 C20:5 Elongation Eicosanoids C24:4 C22:4 C22:5 C24:5 D6 D6 C24:5 C22:5 C22:6 C24:6 Oxidation Docosahexaenoic acid (DHA) Docosanoids FIG. 1. Major metabolic pathways for the conversion of the essential fatty acids linoleic acid (omega-6) and a-linolenic acid (omega-3) to long-chain polyunsaturated fatty acids (LC-PUFA). little to plasma DHA levels, but they are primarily predicted by dietary preformed DHA intake, for example, from fatty fish. Indeed, the importance of preformed intakes was clearly demonstrated in numerous studies showing marked effects of the intake of n-3 LC-PUFA with foods or supplements on DHA blood levels. (6,10,11). Phospholipids in human plasma and cell membranes typically contain LC-PUFA in the range of 10% to 15% of all fatty acids. The amount and composition of LC- PUFA in cell membranes has been shown to modulate a variety of membrane functions, such as membrane fluidity, the activity of membrane bound enzymes, receptors and ion channels, trans-membrane transport, and humoral and electrical signal transduction. In particular, the availability of DHA during prenatal and postnatal development has been shown to be related to central nervous tissue function and neural development in both animal and human studies (12). In addition, LC-PUFA serve as precursors for the formation of bioactive eicosanoids such as prostaglandins, thromboxanes, leukotrienes, and docosanoids such as resolvins, docosatrienes, and neuroprotectins (13). Eicosanoids modulate various tissue functions such as thrombocyte aggregation, postnatal closure of the ductus arteriosus, inflammatory reactions and postnatal development of immune phenotypes. Resolvins and protectins act as anti-inflammatory and proresolution mediators (13). Numerous immunological markers, such as lymphocyte proliferation, natural killer cell activity and cytokine profiles, are strongly influenced by LC-PUFA (14). Lipoxins, resolvins, and protectins derived from LC-PUFA can protect organs from collateral damage during inflammation, stimulating the clearance of inflammatory debris and promoting mucosal antimicrobial defence (15). A dietary supply of preformed DHA has been recommended for pregnant and lactating women, and for infants, because n-3 LC-PUFA supply in pregnancy was shown to reduce early preterm birth, and DHA status during prenatal and postnatal development has been linked to benefits for child outcomes in several studies (16). However, little is known on whether a dietary DHA supply is beneficial for neural function beyond the phase of rapid brain growth during the first and second year of life, and extrapolation of nutrient intake values from other age groups appears inappropriate (17). We aimed at investigating this question in children who follow a diet with a very low dietary intake of preformed DHA. Food sources of DHA are animal-derived foods such as fatty fish, and to a lesser extent also eggs, meat, and milk, whereas usual plant-derived foods do not provide appreciable amounts of DHA. The average dietary intake of preformed AA and DHA in European adults consuming omnivorous diets is estimated at about 200 mg/day of each AA and DHA (18), whereas very little DHA intakes are found in subjects with a low fish intake and in strict vegetarians and vegans (19). No appreciable DHA intake from foods is also found in children with inborn errors of amino acid metabolism who must strictly follow a protein restricted diet, such as children with phenylketonuria (PKU). PHENYLKETONURIA Phenylketonuria is the most common autosomal recessive inborn error of amino acid metabolism in Europe, with an incidence rate of approximately 1:10,000 neonates (20). PKU results from a deficient activity of the hepatic enzyme phenylalanine hydroxylase (PAH). The PAH gene has been mapped to chromosome 12q24.1, and several hundred mutations have been found in PKU (21,22). Defects in PAH impair the oxidation of the indispensible amino acid phenylalanine to tyrosine. Thus tyrosine, an important precursor of melanin and neurotransmitters, such as dopamine, becomes an essential amino acid. Untreated subjects with PKU show markedly elevated blood and urine concentrations of phenylalanine and phenylalaninemetabolites, associated with severe mental retardation, microcephaly, epilepsy, and reduced skin and hair pigmentation (23). Early dietary treatment with strict limitation of dietary intake of natural protein, combined with the supplementation of amino acid mixtures devoid of phenylalanine, leads to near normal plasma amino acids levels, normal growth and development, and prevents the occurrence of all above mentioned symptoms. Thus, neonatal screening programs have been introduced worldwide to allow for early detection and dietary treatment of individuals with PKU, which prevent mental retardation and enable normal development of affected children.

S4 KOLETZKO ET AL. To achieve this goal, however, children with PKU must follow a strictly controlled protein-restricted diet. School age children with classical PKU typically tolerate only 200 to 400 mg phenylalanine/day equivalent to only 4 to 8 g natural protein/day. Therefore, added phenylalaninefree synthetic amino acid mixtures must supply approximately 75% of the total protein requirements (24). In practice, the PKU diet is similar to a vegan diet in that it is devoid of all animal-based foods and also of some plantbased foods with significant protein contents, such as cereals, bread, pasta, and protein rich vegetables, which must also be avoided and be replaced in part by lowprotein dietetic products. Therefore, children with PKU do not have any appreciable food supply of DHA, except during infancy when they may receive some breast milk or infant formula preparations supplying DHA (25). PKU AND LC-PUFA: WHAT IS THE CONNECTION? The basically absent dietary DHA supply in PKU children is reflected by markedly reduced DHA levels in plasma and erythrocyte phospholipids (26 28). In addition to absent dietary supply, a possible inhibitory effect of phenylalanine metabolites, especially of phenylpyruvate and phenyllactate, on endogenous DHA synthesis has been discussed but there is no firm evidence for this hypothesis (29). In children affected by PKU, blood levels of DHA are reduced to a greater extent than those of AA, relative to values found in healthy omnivorous children (25,28 31). We hypothesized that this lasting restriction in DHA supply might induce adverse effects on neural function in PKU children. Although severe neurological damage is completely prevented in PKU affected individuals by adequate dietary therapy, subtle neurological deficits persist and are detectable. For example, the IQ of well and early treated PKU children participating in the German Collaborative Study was 3 6 points lower than that of their healthy siblings (32). Of importance, the authors noted that after early and strict treatment during the preschool years, variation of phenylalanine levels in the range observed in the study participants did not influence IQ development at school age. Thus, other factors than the degree of phenylalanine control appear to influence cognitive outcomes. In another study, early and continuously treated PKU patients of ages 8 to 20 years were compared with controls, matched individually for age, sex, and educational level of both parents, with respect to behaviour and school achievements (33). PKU patients, as a group, demonstrated more problems in task-oriented behaviour and average academic performance than did matched controls. Although the teacher rating on average academic performance of the PKU patients was associated with recent level of phenylalanine control, other behavioural outcomes were not. DHA SUPPLY AND NEURAL FUNCTION IN PKU CHILDREN? Because the subtle functional deficits in early and well-treated patients with PKU cannot be fully explained by variations in plasma phenylalanine levels, it seems possible that metabolic imbalances induced by the strict diet, such as an absent DHA supply, might contribute to neurological abnormalities. Current dietetic products for PKU infants without phenylalanine are usually enriched with both DHA and AA and normalize blood LC-PUFA levels as compared to levels typically found in fully breastfed infants (25,34 37), but LC-PUFA supplementation is not routinely provided in PKU after infancy. The effects of LC-PUFA supplementation in children have been assessed in a double-blind, placebo-controlled clinical trial in 20 patients with classical PKU assigned to receive LC-PUFA or placebo (olive oil) supplements for 12 months (38). The active capsules supplied 26% fatty acids as LC-PUFA and contained equivalent amounts of n-3 and n-6 fatty acids, since the authors proposed that it is important to supply both n-3 and n-6 fatty acids to avoid biochemical imbalances (34). Of the 20 enrolled children, 18 completed the study. Visual evoked potentials (VEP) were measured. P100 wave latency times before intervention were significantly prolonged in children with PKU compared with a healthy reference group, which is in agreement with previous observations (39,40). After the intervention period, P100 latencies were significantly improved in the group receiving LC-PUFA. We evaluated the effects of supplementing PKU children ages 1 year to 11 years for 3 months with encapsulated fish oil providing EPA (C20:5n-3) and DHA (C22:6n-3) but no major amounts of n-6 LC-PUFA. Out of 65 PKU children visiting our outpatient clinic during the enrollment period, 52 fulfilled the set inclusion criteria including good metabolic control with all measured phenylalanine values being <360 mmol/l over the previous 6 months (41). Of these, 38 children and their caregivers agreed to participate in the study, and36childrenages6.3 0.6 years (mean SD) completed the protocol. At baseline, a clinical examination, routine blood tests (including phenylalanine concentration), and VEP were performed. The PKU children were supplied with fish oil capsules (Ameu; Omega- Pharma, Berlin, Germany) providing 15 mg DHA/kg of body weight daily. Each capsule contained 500 mg of fish oil (35% of omega-3 fatty acids: 18% of eicosapentaenoic, 12% DHA). The capsule coating (gelatin) contained 3 mg of phenylalanine. Otherwise, dietary treatment remained unchanged. After 90 days, the clinical, laboratory, and VEP examinations were repeated. Plasma phenylalanine did not change in the PKU children from baseline (266 14 mmol/l) to the end of intervention (271 21 mmol/l (not significant). Results

OMEGA-3 LC-PUFA SUPPLY AND NEUROLOGICAL OUTCOMES S5 135 5' Fovea 125 P <0.0001 10' 117.5 135 5' Fovea 125 10' 117.5 n.s. 15' PKU Contr. PKU Contr. P <0.01 30' Total retina 15' Baseline 3 mon. Baseline 3 mon. 30' Total retina n.s. 105 PKU Contr. PKU Contr. FIG. 2. Latencies of visual evoked potentials in children with phenylketonuria and healthy, age-matched control subjects at baseline (values are P100 latencies, expressed as milliseconds after stimulation with 4 different pattern sizes; 2 sample t test). were compared with 30 healthy control children ages 6.6 0.5 years (not significant). At baseline, the children with PKU had significantly longer VEP latencies than healthy aged matched controls, when tested with pattern sizes for stimulation of the fovea (5 0 ), the entire retina (30 0 ) and two intermediate sizes (10 and 15 0 ) (Fig. 2). These data indicate that PKU children had a slower speed of information processing from the retina via a monosynaptic pathway to the visual cortex. Supplementation with fish oil capsules over 3 months resulted in a marked increase of EPA and DHA levels in blood lipids, but no significant change of AA levels (41). Fish oil supplementation also led to shorter P100-latencies in PKU patients, with significant differences in the pattern size 5 0 and 15 0 for foveal stimulation (Fig. 3). These data indicate an enhanced speed of information processing in children with PKU after 3 months of n-3 LC-PUFA supply. In contrast, there was no change of VEP latencies in the healthy controls over 3 months. We also assessed body coordination and fine motor skills in patients and controls using the motometric Rostock-Oseretzky Scale (ROS) by Kurth (42). The ROS test evaluates different specific motor functions, including static and dynamic balance, fine motor ability, and motor-rhythmic coordination. At baseline, PKU children under good metabolic control showed a significantly poorer motor skill performance in the ROS test than healthy controls (Fig. 4). After 3 months of fish oil supplementation ROS scores of PKU patients had significantly improved and came much closer to healthy controls (Fig. 4). The score of PKU children improved in the subtests for fine motor skills, especially coin sorting (P ¼ 0.046) and dynamic balance (P ¼ 0.004). Because the age-matched control children showed no change in ROS performance over the same time interval (Fig. 4), 105 Baseline 3 mon. Baseline 3 mon. Fig. 3. Effect of fish oil supplementation for 3 months on visual evoked potential latencies and plasma phenylalanine concentrations in children with phenylketonuria (t test paired, 2 samples for means; n.s., not significant). the observed improvements in the PKU group cannot be explained by an age-dependent developmental progress or by a training effect. We conclude from these observations that a supply of preformed n-3 LC-PUFA is required to achieve normal neural function in children with PKU. Although these children, whose diet was continuously monitored by experienced pediatric dieticians, had a good dietary supply of the n-3 precursor fatty acid ALA, this did not suffice to achieve a similar DHA status as in healthy omnivorous children. Thus, the average conversion of ALA is not sufficient to maintain a DHA status that supports optimal functional outcomes. Therefore, DHA must be considered a conditional essential substrate that should be supplied to children with PKU. Because children with early and adequately treated PKU are generally healthy and have a normal energy metabolism, fatty acid absorption, distribution, metabolism, and excretion, FIG. 4. Motor development index of the Rostock-Oseretzky scale in children with phenylketonuria and in healthy, age-matched controls at baseline and after 3 months show an improvement in patients following fish oil supplementation, but no change in controls. n.s., Not significant.

S6 KOLETZKO ET AL. these findings in PKU children and habitually low DHA may have implications for healthy children without an inborn error of amino acid metabolism. It appears likely that a preformed DHA supply is generally necessary for optimal functional outcomes in populations of children even beyond infancy. This question obviously has major implications for deriving nutrient intake values for children, for food policy and for practical food choices. Therefore, further studies are needed to corroborate these findings. In addition to a required confirmation of the observed DHA effects in a further study, quantitative needs should be established. The daily dose of 15 mg DHA/kg that we used in our studies may well be higher than the dose needed for optimal outcomes. With financial support provided by the 7th Framework Research Programme of the European Commission, we are currently embarking on a multicentre trial in school age children with PKU. These children will be randomised to receive different dosages of a DHA supplement and will be tested for functional outcomes both before and after supplementation. If successful, this ongoing randomized clinical trial may achieve to establish quantitative DHA requirements in children and thus contribute to a better evidence base for establishing nutrient intake values. Acknowledgments: The preparation of this review has been carried out partially with financial support from the Commission of the European Communities, within the FP 6 Priority 5 Food quality and safety (Early nutrition programming long term follow up of efficacy and safety trials and integrated epidemiological, genetic, animal, consumer and economic research, EARNEST, Food-CT-2005-007036 and Harmonising micronutrient recommendations across Europe with special focus on vulnerable groups and consumer understanding, EURRECA, FP6-036196-2), and the FP 7, Theme 2: Food, Agriculture and Fisheries, and Biotechnologies (The Effect of Diet on the Mental Performance of Children, NUTRIMENTHE, 212652). This manuscript does not necessarily reflect the views of the Commission and in no way anticipates its future policy in this area. 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