Status of nutrients important in brain function in phenylketonuria: a systematic review and meta-analysis

Phenylketonuria (OMIM 261600) is a rare genetic disorder characterized by impaired conversion of phenylalanine (phe) to tyrosine (tyr) due to deficient activity of the hepatic enzyme L-phenylalanine-4- hydroxylase [1]. If untreated, the resulting high blood phe concentrations cross the blood brain barrier causing detrimental effects on brain development and function [2]. For the majority of patients with PKU, treatment is a phe-restricted diet that aims to prevent excessive accumulation of phe while also meeting requirements for growth and development [3]. Because of the severe restriction of many protein-containing foods, the diet requires supplementation with a phe-free protein substitute (PS) and a wide range of other nutrients including omega-3 fatty acids, vitamins, minerals and trace elements. However, nutrient intakes [4‐6] and status [7‐9] may be affected by factors such as poor dietary compliance, bioavailability of different nutrient sources, different management approaches in metabolic centres [2] and historical differences in the composition of the PS e.g. changes in selenium fortification practices [2, 4, 10‐12]. Despite early and continuous low phe dietary management, PKU patients have been shown to have suboptimal neuropsychological function compared to healthy controls, in particular lower IQ, slower information processing [13, 14] and suboptimal executive functioning [15‐17].

Tyr deficiency has been suggested as one reason for the decreased neuropsychological performance and cognitive dysfunction in PKU [18]. It has been suggested that tyr supplementation could have an important role in the treatment of PKU [19], however blood tyr concentrations do not correlate with cognitive outcomes in PKU, tyr supplementation alone does not prevent severe mental retardation [20] and 24 h tyr plasma levels show high variability. Reduced serotonin synthesis may be the result of reduced tryptophan (trp) brain concentrations caused by reduced blood-brain barrier transport of trp at elevated plasma phe concentrations [21, 22]. Different hypotheses focusing on the pathogenesis of PKU and disturbed amino acids transport from blood to brain on cerebral neurotransmitter and protein synthesis have been proposed, a detailed examination of this evidence is beyond the scope of this review.

Under normal physiological conditions, many other nutrients have an important role in brain development and function affecting multiple processes regulating neurotransmitter pathways, synaptic transmission, membrane fluidity and signal-transduction pathways [23]. Table 1 provides a summary of the key role of nutrients involved in brain development and functioning such as structural components of membranes, antioxidants, neurotransmitter precursors and co-factors [23‐29]. For many of these nutrients, food sources are restricted in PKU diets (Table 1) and reduced levels of biomarkers for some of these nutrients have been observed in PKU [5, 30‐36]. Poor status of some antioxidant nutrients have been associated with impaired cerebral function [30] and neuropsychological disturbances in PKU [37]. Improvements in markers of cognition (visual function and fine motor skills) have been found following supplementation with docosahexaenoic acid (DHA) in children with PKU [31, 38]. In addition to raised phe, sub-optimal levels of some of these nutrients could have an impact on cognition in PKU patients. Given this background, this is the first systematic review to investigate the status of multiple nutrients involved in brain function. Although the review will not investigate the functional outcomes of any altered levels of these nutrients found, there is a rationale that in PKU optimal levels of these nutrients, are key to achieve an optimal cognitive potential. Specifically, we have systematically reviewed, and performed meta-analysis where possible, the nutrient status of DHA, eicosapentanoic acid (EPA), phospholipids, selenium, vitamins B₆, B₁₂, E, C, A, D, folic acid, choline, uridine, calcium, magnesium, zinc, iron, iodine and cholesterol that are important for optimal brain function. This will indicate whether differences in nutrient status exist between individuals with PKU and healthy, non-PKU controls.

Sixty-five publications were identified that provided outcomes for one or more of the nutrients reviewed, and the characteristics of these studies and the PKU populations included are summarized in Table 2.

Table 3 summarises the studies included in the systematic review and the differences in the outcomes in nutrient status markers between PKU patients and healthy controls in the individual studies. Where ≥4 studies were retrieved for a nutrient, meta-analyses of the data were performed and the overall effect estimate is presented (SMD with confidence intervals [CI]), illustrated in forest plots), although it should be noted that there was significant heterogeneity within the datasets for all nutrients. When the heterogeneity is high (I² > 95%) and there is no statistically significant difference between the groups the overall conclusion is estimated and presented in the forest plots. Otherwise when heterogeneity is high for a given nutrient, the overall effect is not reported as it would not correctly represent the between group difference. Where < 4 studies were retrieved, the forest plots have been included to provide a graphical display of the data from individual studies which shows to which extent study results overlap without presenting the overall estimate.

To the best of our knowledge, this is the first systematic review and meta-analysis of published studies investigating differences between PKU patients and healthy controls in the status of multiple nutrients involved in brain function. The nutrients investigated affect multiple structural and metabolic pathways within the brain and the potential implications of the altered nutrient status found or indeed the lack of data for some of these nutrients is discussed.

DHA, EPA, cholesterol, PL and choline are key structural components of neuronal membranes. Lower status of DHA, EPA and cholesterol levels are suggested by the meta-analyses for PKU patients compared to healthy controls. In healthy individuals, there is evidence of limited endogenous synthesis of DHA and EPA from the parent n-3 essential fatty acid, α linolenic acid [48‐50] and in PKU patients the by-products of excessive phe may further inhibit synthesis of DHA [51]. Our findings of poorer DHA and EPA status concur with a previous meta-analysis carried out by Lohner et al. [36], who found suboptimal n-3 LC-PUFA status in PKU patients. As there have been many studies that have reported poor fatty acids status of PKU patients over the last 20 yrs, dietary advice has been modified and some PS have since been fortified with DHA, EPA and the parent essential fatty acids. Interestingly, some recent studies [52‐54], where a few or all the patients were taking a fortified PS (essential or long chain n-3 fatty acids) or a modified fat diet, found no difference in DHA and EPA status in PKU patients compared to healthy controls.

Cholesterol is also a key component of neuronal membranes with 24S-hydroxycholesterol (24 s-OHC) [25] a specific metabolite of brain cholesterol metabolism measurable in blood. Decreased levels of plasma 24 s-OHC in PKU individuals have been reported which may indicate reduced cholesterol synthesis in the brain and/or disturbed conversion from cholesterol to 24S-OHC [55]. Some animal studies have reported that cholesterol synthesis maybe suppressed in PKU with reduced activity of two key cholesterol synthesis enzymes [55]. Furthermore, Colome et al., 2003 [44] reported a relationship between high plasma phenylalanine levels and an inhibition of cholesterogenesis, although the low fat and saturates intakes typical in PKU diets may also result in lower serum cholesterol levels. The finding of significantly lower total plasma cholesterol levels in PKU patients in this meta-analysis may suggest altered brain levels and given the importance of this nutrient for brain function, further studies directly measuring 24 s-OHC in patients with PKU should be considered.

A phe-dependent down regulation of the hydroxymethylglutaryl (HMG)-CoA reductase and the mevalonate-5-pyrophosphate decarboxylase activities, may potentially reduce the synthesis of both cholesterol and coenzyme Q10 (CoQ10) through their common mevalonate pathway [56‐58]. CoQ10, a lipid component acting as an electron carrier in the mitochondrial respiratory chain, has been negatively correlated with phe levels in serum of PKU patients [59‐61]. However, it is suggested that that HPA has less profound effect upon CoQ10 synthesis than cholesterol synthesis [57, 62] in PKU low plasma levels of CoQ10 may be caused by increased oxidative stress [45].

Only two studies reporting on PL status were identified; Galli et al. reported no significant difference in 15 children with PKU compared to controls and Pietz et al., using ³¹P-MR spectroscopy [34] reported significantly lower baseline brain membrane bound PL levels, but no differences in mobile PL or catabolic metabolites in 11 PKU patients.

Antioxidant nutrients such as zinc, selenium and vitamins A, C and E play a key role in detoxifying reactive intermediates in the brain. Where intakes of antioxidant nutrients are poor, such as in PKU, antioxidant defenses may be compromised, contributing to the highly oxidative environment observed in PKU. Increased markers of oxidative stress have been observed in PKU populations; for example, Colome et al. [44] found plasma lipid peroxidation to be increased in PKU and Artuch et al. [45] observed that ubiquinone-10 concentrations were significantly lower in PKU patients compared with healthy controls. High levels of circulating phe are known to exacerbate excessive production of reactive oxygen species [63]. It is suggested that systemic oxidative stress causes increased lipid peroxidation and altered plasmatic fatty acid profiles [64]. Also associated with a high number of double bonds, DHA/EPA are particularly susceptible to peroxidative breakdown, offering an additional explanation for the lower levels of these fatty acids as found in the current meta-analysis.

For the antioxidant nutrients, we found no apparent difference in status for vitamin E and zinc, too few studies identified to carry out meta-analyses for vitamins A and C, or the degree of heterogeneity was too high to draw a definitive conclusion for selenium. Selenium is a nutrient of particular concern in PKU given that dietary sources are very limited, and intakes are typically very poor when compliance with the PS prescription is inadequate. The selenium content of vegetation varies depending on the concentrations in soil, with Europe and China typically having lower concentrations than North America [65]. Selenium supplementation of PKU diets was introduced in the mid-1980s [6] when selenium essentiality became more widely accepted. Although there may be a suggestion from the limited cluster analysis in this review and evidence from intervention studies [66, 67] that selenium supplementation is effective in improving status, it is also observed that 14 of the 16 studies in this review had confidence intervals on the left side of the plot, suggesting lower levels in PKU patients compared to controls. Therefore, although improvement in selenium status is observed in clinical practice with supplementation, the findings from this meta-analysis are inconclusive because of the high heterogeneity within the data. This may be due to several factors including: variable selenium intakes depending on whether the PS was fortified/not over the timeframe of the literature review; variation in patient compliance with PS over a wide age range and bioavailability of different sources of selenium [68] and/or low concentrations of selenium in grains and vegetables (many studies identified were in European populations). Furthermore, both the PKU and the healthy control groups in the studies were found to have selenium status that was low or close to lower European population norms in some studies [44, 45, 67, 69‐74] Therefore, at this time further research is warranted investigating selenium status in PKU and the effects of supplementation including measurement of plasma selenoprotein P (SEPP1) as a more informative marker of status.

Adherence to the phe-restricted diet is necessary to achieve plasma phe control including compliance with PS as the predominant source of many nutrients in the PKU diet, such as vitamin B₁₂, calcium, vitamin D, zinc and iron and to ensure adequate micronutrient status. However, patients who are non- or poorly- compliant with their PS prescription are at greatest risk of suboptimal intakes of these nutrients. For example, there have been several case studies reporting poor vitamin B₁₂ status in patients as a consequence of a relaxed PKU diet [7, 75] and low intakes of calcium have been reported in patients with PKU [76‐78] particularly if compliance is poor in older children/adults. The meta-analysis found no difference in plasma calcium levels in PKUs compared to healthy controls; however, plasma calcium is not a suitable biomarker of status [79]. So no conclusion about the status of calcium related to brain function in PKU can be drawn from this finding. We did not find a between group difference in vitamin D status between PKUs and healthy controls, however, minimal cutaneous synthesis is now assumed and recommended dietary intakes for vitamin D have increased globally in recent years [80, 81]. There is also emerging knowledge of the role of vitamin D as a ‘neuro-steroid’ and regulator of brain serotonin synthesis [82, 83]. Given this increasing knowledge on vitamin D requirements, dietary intakes of vitamin D by those with PKU should be monitored to ensure recommended intakes are achieved.

Intakes of folic acid are much less likely to be compromised given that good food sources, such as fruit and vegetables, are allowed in controlled or unrestricted quantities in the diet and that PS are routinely supplemented. In the meta-analysis for folic acid the excessively high heterogeneity across the studies undermined its reliability to draw a conclusion on between group differences in status. Nonetheless 6 of the 7 studies [84‐89] had confidence intervals on the right side of the plot (Fig. 6), suggesting positive differences between PKU patients and healthy controls. Intakes of folic acid by patients who are compliant with their PS prescription may be higher than desired when combined with intakes from normal foods that are also good sources of folic acid, and indeed high serum folate levels have been observed in PKU [90‐92]. Vitamin B₁₂ deficiency may be masked where circulating folate levels are high [93] and Walter et al. [9] reported that functional vitamin B₁₂ deficiency can occur in the presence of normal B₁₂ concentrations in PKU. Although we observed no apparent difference in vitamin B₁₂ status in PKU patients compared to healthy controls suggesting that supplementation is effective in maintaining nutrient status comparable to controls, the physiological effect of potentially high folate intakes and levels needs consideration.

The systematic review found limited or no publications matching our entry criteria on nutritional status of some nutrients (vitamins A, C, B₆, choline, uridine, iodine and PL). Although the status of some nutrients such as vitamin C is unlikely to be compromised in PKU, others such as choline and iodine warrant consideration. In healthy individuals, de-novo synthesis of choline is not sufficient to meet requirements and recommended or adequate intakes (AI) have been established [94, 95], however, sub-optimal intakes of choline are reported in the general population compared to the recommended AI [96, 97]. The main choline-rich food groups are restricted in PKU, and many PS are supplemented with choline. Dezortova et al. reported significantly lower brain choline concentrations (p < 0.05) in 15 adult PKU patients on a moderate/strict diet compared to controls [33]. Whereas, Sijens [98] reported no significant difference in brain choline levels between 10 adult PKU patients (including both compliant and non-compliant) and those of healthy controls. No detail on dietary intake of choline was provided in these studies. Because of the wide-ranging role of choline in brain function, if present, inadequate choline status in PKU may have an impact on neurological outcomes and so dietary intakes should be monitored and studies investigating choline status should be considered. This systematic review revealed no publications matching our inclusion criteria comparing iodine status of PKU patients with healthy controls. However, in PKU, an altered free thyroid hormone concentrations in PKU, in association with an impaired activity of thyroxine-5-deiodinase enzyme is described [35, 99],

Given that intakes of iodine from normal foods in PKU diets are likely to be very low and that iodine has an important role in thyroid function and brain development [100], the possibility of insufficiency among poorly compliant patients in particular should be considered.