Phenylalanine Exposure Research Articles

Transient brain structure changes after high phenylalanine exposure in adults with phenylketonuria.

Phenylketonuria is a rare metabolic disease resulting from a deficiency of the enzyme phenylalanine hydroxylase. Recent cross-sectional evidence suggests that early-treated adults with phenylketonuria exhibit alterations in cortical grey matter compared to healthy peers. However, the effects of high phenylalanine exposure on brain structure in adulthood need to be further elucidated. In this double-blind, randomised, placebo-controlled crossover trial, we investigated the impact of a four-week high phenylalanine exposure on the brain structure and its relationship to cognitive performance and metabolic parameters in early-treated adults with phenylketonuria. Twenty-eight adult patients with early-treated classical phenylketonuria (19-48 years) underwent magnetic resonance imaging before and after the four-week phenylalanine and placebo interventions (four timepoints). Structural T1-weighted images were preprocessed and evaluated using DL+DiReCT, a deep-learning-based tool for brain morphometric analysis. Cortical thickness, white matter volume, and ventricular volume were compared between the phenylalanine and placebo periods. Brain phenylalanine levels were measured using 1H spectroscopy. Blood levels of phenylalanine, tyrosine, and tryptophan were assessed at each of the four timepoints, along with performance in executive functions and attention. Blood phenylalanine levels were significantly higher after the phenylalanine period (1441µmol/L) than after the placebo period (873µmol/L, P<0.001). Morphometric analyses revealed a statistically significant decrease in cortical thickness in 17 out of 60 brain regions after the phenylalanine period compared to placebo. The largest decreases were observed in the right pars orbitalis (point estimate=-0.095mm, P<0.001) and the left lingual gyrus (point estimate=-0.070mm, P<0.001). Bilateral white matter and ventricular volumes were significantly increased after the phenylalanine period. However, the structural alterations in the Phe-placebo group returned to baseline measures following the washout and placebo period. Additionally, elevated blood and brain phenylalanine levels were related to increased bilateral white matter volume (rs=0.43 to 0.51, P≤0.036) and decreased cortical thickness (rs=-0.62 to -0.39, not surviving FDR correction) after the phenylalanine and placebo periods. Moreover, decreased cortical thickness was correlated with worse cognitive performance after both periods (rs=-0.54 to -0.40, not surviving FDR correction). These findings provide evidence that a four-week high phenylalanine exposure in adults with phenylketonuria results in transient reductions of the cortical grey matter and increases in white matter volume. Further research is needed to determine the potential long-term impact of high phenylalanine levels on brain structure and function in adults with phenylketonuria.

Neurotoxicity of phenylalanine on human iPSC-derived cerebral organoids.

Phenylketonuria (PKU) is a common genetic metabolic disorder that causes phenylalanine accumulation in the blood. The most serious symptoms are related to the brain, as intellectual disability, seizure, and microcephaly are commonly found in poorly treated PKU patients and the babies of maternal PKU. However, the mechanism of hyperphenylalaninemia on human neurodevelopment is still unclear. Here we utilized human induced pluripotent stem cell (iPSC)-derived cerebral organoids to investigate the neurotoxicity of hyperphenylalaninemia. Cerebral organoids at days 40 or 100 were treated with different concentrations of phenylalanine for 5 days. After phenylalanine treatments, the cerebral organoids displayed alterations in organoid size, induction of apoptosis, and depletion of neural progenitor cells. However, phenylalanine did not have an impact on neurons and glia, including astrocytes, immature oligodendrocytes, and mature oligodendrocytes. Remarkably, a reduction in the thickness of the cortical rosettes and a decrease in myelination at the intermediate zone were inspected with the elevated phenylalanine concentrations. RNA-seq of phenylalanine-treated organoids revealed that gene sets related to apoptosis, p53 signaling pathway, and TNF signaling pathway via NF-kB were enriched in upregulated genes, while those related to cell cycle and amino acid metabolism were enriched in downregulated genes. In addition, there were several microcephaly disease genes, such as ASPM, LMNB1, and CENPE, ranked at the top of the downregulated genes. These findings indicate that phenylalanine exposure may contribute to microcephaly, abnormal cortical expansion, and myelination lesions in the developing human brain.

Risk Assessment of "Other Substances" – L-phenylalanine and DL-phenylalanine

The Norwegian Scientific Committee for Food Safety (Vitenskapskomiteen for mattrygghet, VKM) has, at the request of the Norwegian Food Safety Authority (Mattilsynet; NFSA), assessed the risk of "other substances" in food supplements and energy drinks sold in Norway. VKM has assessed the risk of doses given by NFSA. These risk assessments will provide NFSA with the scientific basis while regulating the addition of "other substances" to food supplements.&#x0D; "Other substances" are described in the food supplement directive 2002/46/EC as substances other than vitamins or minerals that have a nutritional and/or physiological effect. It is added mainly to food supplements, but also to energy drinks and other foods. VKM has not in this series of risk assessments of "other substances" evaluated any claimed beneficial effects from these substances, only possible adverse effects.&#x0D; The present report is a risk assessment of L-phenylalanine and DL-phenylalanine and is based on previous risk assessments.&#x0D; According to information from the Norwegian Food Safety Authority, L- and DL-phenylalanine are ingredients in food supplements sold in Norway. NFSA has requested a risk assessment of the following doses of L-phenylalanine and DL-phenylalanine in food supplements: L-phenylalanine 100, 250, 500, 750 and 1000 mg/day and DL-phenylalanine 50 and 75 mg/day.&#x0D; L-phenylalanine is an essential amino acid which means it has to be obtained from the diet. Amino acids are building blocks for proteins and present in protein rich food such as milk, meat, fish, eggs and cheese.&#x0D; No data on adverse health effects after chronic ingestion of supplemental phenylalanine in apparently healthy subjects are available, thus no tolerable upper intake level (UL) can be established. Patients with phenylketonuria (PKU), a genetic disorder that impairs phenylalanine hydroxylase (PAH), an enzyme involved in the metabolism of phenylalanine, must keep plasma levels of phenylalanine low in order to maintain normal growth and brain development. In Norway, all newborns are routinely screened for PKU three days after birth.&#x0D; The mean dietary intake of phenylalanine in the EU population range from 0.4-4.1 g/day corresponding to 79.0 mg/kg bw per day for adolescents (10-17 years) and 58.7 mg/kg bw per day for adults, respectively (EFSA, 2013). The sweetener aspartame contains phenylalanine. Taking the molecular weight of phenylalanine into account, the proportion of to phenylalanine exposure from aspartame is 56%. The ADI of 40 mg aspartame/day/kg bw (providing 22.4 mg phenylalanine/day/kg bw) JECFA (1981) was re-evaluated and maintained in 2013, based on the notion that elevated plasma levels of phenylalanine in pregnant women leads to developmental toxicity in their children (EFSA, 2013).&#x0D; The literature search did not provide novel information on adverse health effects related to intake of L-phenylalanine and no information related to DL-phenylalanine.&#x0D; VKM concludes that:&#x0D; In adults (≥ 18 years), the specified doses 100, 250, 500, 750 and 1000 mg/day L-phenylalanine in food supplements are considered unlikely to cause adverse health effects.&#x0D; &#x0D; In adolescents (14 to &lt; 18 years), the specified doses 100, 250, 500, 750 and 1000 mg/day L-phenylalanine in food supplements are considered unlikely to cause adverse health effects.&#x0D; In children (10 to &lt; 14 years), the specified doses 100, 250, 500, 750 and 1000 mg/day L-phenylalanine in food supplements are considered unlikely to cause adverse health effects. Although the highest dose provides 23 mg/kg bw per day which slightly exceeds 22.4 mg/kg bw per day, it is considered unlikely to cause adverse health effects in healthy children 10 to &lt; 14 years.&#x0D; None of the above conclusions are applicable for patients with phenylketonuria (PKU).&#x0D; No conclusion can be made regarding DL-phenylalanine.&#x0D; &#x0D; Children below 10 years were not included in the terms of reference.

The Effect of Phenylalanine Exposure on Cell Migration

Phenylketonuria (PKU) is an inborn error of metabolism that results when the amino acid phenylalanine (Phe) cannot be catabolized to tyrosine due to a mutation in phenylalanine hydroxylase enzyme. The inability to regulate Phe levels in individuals with PKU leads to mental retardation, seizures and other symptoms that can be avoided with early detection and a Phe‐restricted diet. When PKU mothers do not adhere to a Phe‐restricted diet during pregnancy, Phe can become teratogenic to the fetus and result in a condition known as Maternal PKU. Maternal PKU is characterized by multiple congenital defects, including cardiovascular malformations (CVM) such as coarctation of the aorta, tetralogy of Fallot, ventricular septal defects and hypoplastic left heart syndrome. However, the mechanisms responsible for Phe‐induced CVMs are poorly understood. Previous studies have shown that cardiac neural crest cells are important in formation of the outflow tract (OFT) and aortic arch arteries (AAA). Cell migration of the neural crest cells is a central process in the development of the heart.Study ObjectiveSince congenital CVM of the OFT and AAA are often observed in maternal PKU, in this study we aimed to determine if exposure to high Phe levels perturbs cell migration and proliferation.MethodsWe conducted migration assays on human fibroblasts cells and chick neural crest cells tubes to determine if migration was affected by Phe. Migration was assessed by classic scratch wound assay and by Silicon elastomeric masks migration study. Additionally, in the human fibroblast cells we stained for markers of proliferation to determine if it was affected by the Phe exposure. The chick neural tubes were dissected and plated on a 3‐D collagen matrix. Migration of neural crest cells was observed over 12–24 hours and the area of migration was quantified.ResultsPhe doses tested were 0.1mM, 1mM, and 10mM. We observed migration in human fibroblast was decreased in the higher doses (1mM and 10mM) but was increased in the 0.1mM dose in both methods observed. Proliferation was decreased in the cells exposed to 0.1mM Phe but was not changed in the higher doses (1mM and 10mM). Chick neural tubes were exposed to 1.5mM Phe and we observed increase in the area of migration.ConclusionPhenylalanine has a dose‐dependent effect on cell migration in vitro. Additionally, it has an effect on proliferation rates. While these results are preliminary they provide a foundation for more mechanistic studies to understand the effects of MPKU on development.Support or Funding Information2015 Fulbright Visiting Scholar Program for Iraq, UCO CMS CURE STEM program, and UCO office of Research and Grants

Chimeric human mitochondrial PheRS exhibits editing activity to discriminate nonprotein amino acids.

Mitochondria are considered as the primary source of reactive oxygen species (ROS) in nearly all eukaryotic cells during respiration. The harmful effects of these compounds range from direct neurotoxicity to incorporation into proteins producing aberrant molecules with multiple physiological problems. Phenylalanine exposure to ROS produces multiple oxidized isomers: tyrosine, Levodopa, ortho-Tyr, meta-Tyr (m-Tyr), and so on. Cytosolic phenylalanyl-tRNA synthetase (PheRS) exerts control over the translation accuracy, hydrolyzing misacylated products, while monomeric mitochondrial PheRS lacks the editing activity. Recently we showed that "teamwork" of cytosolic and mitochondrial PheRSs cannot prevent incorporation of m-Tyr and l-Dopa into proteins. Here, we present human mitochondrial chimeric PheRS with implanted editing module taken from EcPheRS. The monomeric mitochondrial chimera possesses editing activity, while in bacterial and cytosolic PheRSs this type of activity was detected for the (αβ)2 architecture only. The fusion protein catalyzes aminoacylation of tRNA(Phe) with cognate phenylalanine and effectively hydrolyzes the noncognate aminoacyl-tRNAs: Tyr-tRNA(Phe) and m-Tyr-tRNA(Phe) .

Altered DNA methylation in PAH deficient phenylketonuria.

While phenylalanine (PHE) is the toxic insult in phenylketonuria (PKU), mechanisms underlying PHE toxicity remain ill-defined. Altered DNA methylation in response to toxic exposures is well-recognized. DNA methylation patterns were assessed in blood and brain from PKU patients to determine if PHE toxicity impacts methylation. Methylome assessment, utilizing methylated DNA immunoprecipitation and paired-end sequencing, was performed in DNA obtained from brain tissue of classical PKU patients, leukocytes from poorly controlled PKU patients, leukocytes from well controlled PKU patients, and appropriate control tissues. In PKU brain tissue, expression analysis determined the impact of methylation on gene function. Differential methylation was observed in brain tissue of PKU patients and expression studies identified downstream impact on gene expression. Altered patterns of methylation were observed in leukocytes of well controlled and poorly controlled patients with more extensive methylation in patients with high PHE exposure. Differential methylation of noncoding RNA genes was extensive in patients with high PHE exposure but minimal in well controlled patients. Methylome repatterning leading to altered gene expression was present in brain tissue of PKU patients, suggesting a role in neuropathology. Aberrant methylation is observed in leukocytes of PKU patients and is influenced by PHE exposure. DNA methylation may provide a biomarker relating to historic PHE exposure.

Executive functioning of 4 children with hyperphenylalaninemia from childhood to adolescence.

Hyperphenylalaninemia is a variant of phenylketonuria, and debate remains as to what, if any, active management of this condition is required to preserve cognitive function and psychological well-being. This study is the first to examine longitudinally the executive function (EF) in adolescents with hyperphenylalaninemia. Two sibling pairs with mild hyperphenylalaninemia underwent neuropsychological examination in early childhood and again in adolescence using EF tests that were highly sensitive to phenylalanine exposure. By early adolescence, none of the 4 children demonstrated EF impairment. The children demonstrated a typical developmental trajectory of EF from childhood to adolescence, given phenylalanine exposure consistent with their condition.

Methylome repatterning in a mouse model of Maternal PKU Syndrome

Maternal PKU Syndrome (MPKU) is an embryopathy resulting from in utero phenylalanine (PHE) toxicity secondary to maternal phenylalanine hydroxylase deficient phenylketonuria (PKU). Clinical phenotypes in MPKU include mental retardation, microcephaly, in utero growth restriction, and congenital heart defects. Numerous in utero toxic exposures alter DNA methylation in the fetus. The PAHenu2 mouse is a model of classical PKU while offspring born of hyperphenylalaninemic dams model MPKU. We investigated offspring of PAHenu2 dams to determine if altered patterns of DNA methylation occurred in response to in utero PHE exposure. As neurologic deficit is the most prominent MPKU phenotype, methylome patterns were assessed in brain tissue using methylated DNA immunoprecipitation and paired-end sequencing. Brain tissues were assessed in E18.5–19 fetuses of PHE unrestricted PAHenu2 dams, PHE restricted PAHenu2 dams, and heterozygouswt/enu2 control dams. Extensive methylome repatterning was observed in offspring of hyperphenylalaninemic dams while the offspring of PHE restricted dams displayed attenuated methylome repatterning. Methylation within coding regions was dominated by noncoding RNA genes. Differential methylation of promoters targeted protein coding genes. To assess the impact of methylome repatterning on gene expression, brain tissue in experimental and control animals were queried with microarrays assessing expression of microRNAs and protein coding genes. Altered expression of methylome-modified microRNAs and protein coding genes was extensive in offspring of hyperphenylalaninemic dams while minimal changes were observed in offspring of PHE restricted dams. Several genes displaying significantly reduced expression have roles in neurological function or genetic disease with neurological phenotypes. These data indicate in utero PHE toxicity alters DNA methylation in the brain which has downstream impact upon gene expression. Altered gene expression may contribute to pathophysiology of neurologic presentation in MPKU.

Depression, anxiety, and stress in young adults with phenylketonuria: associations with biochemistry.

Increasing concurrent phenylalanine to "off-diet" levels in adults with phenylketonuria (PKU) has been shown to lead to mood disturbances. However, the impact of controlled phenylalanine exposure across the developmental life span and mood stability remains questionable. The aim of this study was to investigate correlations between lifetime and concurrent phenylalanine (Phe) and tyrosine (Tyr) with levels of depression, anxiety, and stress in young adults maintaining a continuously treated diet for PKU. Eight young adults (6 females, 2 males) aged 15 to 25 years (mean = 19.37, SD = 3.62) recruited through the Royal Children's Hospital Brisbane, with early and continuously treated PKU completed the Depression Anxiety Stress Scale, which was correlated with lifetime and concurrent measures of Phe, Tyr, and Phe-Tyr ratios. There was a strong significant correlation between lifetime Phe and levels of anxiety and stress (p < .01). Greater Phe-to-Tyr blood ratios were indicative of increased depression and anxiety levels (p < .01). An interaction between biochemical regulation and depression, anxiety, and stress levels was present in young adults with continuously treated PKU who were actively attempting to maintain dietary control. Strong associations were present with lifetime levels, suggesting a developmental impact of PKU-related biochemical exposure and the emergence of mood disturbances.

Convergent patterns of association between phenylalanine hydroxylase variants and schizophrenia in four independent samples

Recessive mutations in the phenylalanine hydroxylase (PAH) gene predispose to phenylketonuria (PKU) in conjunction with dietary exposure to phenylalanine. Previous studies have suggested PAH variations could confer risk for schizophrenia, but comprehensive follow-up has not been reported. We analyzed 15 common PAH "tag" SNPs and three exonic variations that are rare in Caucasians but common in African-Americans among four independent samples (total n = 5,414). The samples included two US Caucasian cohorts (260 trios, 230 independent cases, 474 controls), Bulgarian families (659 trios), and an African-American sample (464 families, 401 controls). Analyses of both US Caucasian samples revealed associations with five SNPs; most notably the common allele (G) of rs1522305 from case-control analyses (z = 2.99, P = 0.006). This SNP was independently replicated in the Bulgarian cohort (z = 2.39, P = 0.015). A non-significant trend was also observed among African-American families (z = 1.39, P = 0.165), and combined analyses of all four samples were significant (rs1522305: chi(2) = 23.28, 8 d.f., P = 0.003). Results for rs1522305 met our a priori criteria for statistical significance, namely an association that was robust to multiple testing correction in one sample, a replicated risk allele in multiple samples, and combined analyses that were nominally significant. Case-control results in African-Americans detected an association with L321L (P = 0.047, OR = 1.46). Our analyses suggest several associations at PAH, with consistent evidence for rs1522305. Further analyses, including additional variations and environmental influences such as phenylalanine exposure are warranted.

Relation of Prenatal Phenylalanine Exposure to Infant and Childhood Cognitive Outcomes: Results From the International Maternal PKU Collaborative Study

The primary aims of this study were to model the form of the relation between prenatal exposure to phenylalanine (Phe) and measures of offspring intellectual development and to estimate the developmental relations of maternal demographic, pregnancy-related, and perinatal variables on offspring intelligence during infancy and childhood. The participants were the 413 children and their mothers from the International Maternal PKU Collaborative Study. Results supported a nonlinear relation between prenatal Phe exposure and offspring cognitive outcomes, with damage to the developing fetus if average Phe levels are above approximately 360 micromol/L. Moreover, prenatal Phe exposure had a strong effect on offspring outcomes at 1 year of age and was the only one of the background, pregnancy-related, or perinatal variables to influence directly offspring outcomes at 2, 4, and 7 years of age. The present study was able to document the importance of prenatal exposure to Phe for predicting offspring cognitive outcomes in the presence of other predictors of these outcomes.

The National Institute of Child Health and Human Development and Phenylketonuria

The National Institute of Child Health and Human Development (NICHD) was established shortly after the Guthrie test for screening newborn infants for phenylketonuria (PKU) was introduced. The NICHD supported the study demonstrating the long-term efficacy of screening and a low-phenylalanine diet in preventing mental retardation. With the identification of the adverse impact on fetal development of high intrauterine phenylalanine exposure from a mother with PKU, the NICHD organized and supported the study reported here, demonstrating the protective effect of phenylalanine restriction of the mother's diet during pregnancy. The study provides clear guidance for the management of pregnancy in women with PKU.

Diet, genetic susceptibility and carcinogenesis

At least six types of gene-environment interactions (GEI) have been proposed (Kouhry and Wagener, 1993). In the first type, neither the environmental exposure (EE) nor the genetic risk factor (GRF) have any effect by themselves, but interaction between them causes disease. This is the case of phenylalanine exposure and the phenylketonuria genotype. Type 2 is a situation in which the GRF has no effect on disease in the absence of exposure, but exacerbates the effects of the latter. This is the most important type of GEI in relation to metabolic susceptibility genes and human carcinogenesis. The third type is the converse of the second (EE is ineffective per se, but enhances the effect of GRF). Type 4 occurs when both FE and GRF increase the risk for disease, but the combination is interactive or synergistic: an example is the interaction between Xeroderma Pigmentosum and LWV radiation. Types 5 and 6, according to the classification proposed by Kouhry, refer to cases in which the GRF is protective. The model of GEI that is emerging as the most important in chemical carcinogenesis refers to metabolic susceptibility genes. The general population can be divided into subgroups depending on their susceptibility to the action of carcinogens, based on their ability to metabolize such compounds to electrophilic, reactive metabolites (which form adducts with DNA), or, respectively, electrophobic metabolites that are excreted. The present contribution is a short review of the relevant literature, with particular emphasis on some polymorphisms involved in dietary exposures. In addition, the practical implications of genetic testing in this field are discussed.

Protein Intake and Plasma Phenylalanine Concentrations in Maternal Phenylketonuria

LEARNING OUTCOME: To define how protein intake is related to plasma phenylalanine concentrations and birth outcome in maternal phenylketonuria Women with phenylketonuria (PKU) who do not follow a low phenylalanine (PHE) diet are at high risk of giving birth to offspring with problems such as microcephaly, mental retardation, congenital heart disease and low birth weight. The Maternal PKU Collaborative Study (MPKUCS) examined the efficacy of a low PHE diet in preventing such problems. The diet is prescribed to meet the Recommended Dietary Allowances for pregnancy and is based on medical foods which provide a PHE-free source of protein. The subjects were women with PKU who had plasma PHE concentrations greater than 600 μmol/L at enrollment and who were treated with a low PHE diet. Weekly blood PHE concentrations were determined and the average PHE exposure was calculated. Monthly diet records on 249 women were collected and analyzed for 19 nutrients. Nutrient intake was correlated with average PHE exposure and birth outcome using Spearman correlation coefficients. The best predictor of offspring birth length, weight and head circumference was the number of weeks gestation after which maternal plasma PHE concentration was consistently below 600 μmol/L. Protein intake was negatively correlated with average PHE exposure during pregnancy (p=0.0001). Women with the highest protein intakes had the lowest plasma PHE concentrations and the best birth outcomes. When managing nutritional therapy of pregnant women with PKU, adequate protein intake should be emphasized. Since medical foods are the primary source of protein in the maternal PKU diet, every effort should be made to ensure adequate medical food intake.