Quantitative determination of aromatic amino acids and related compounds in rumen fluid by high-performance liquid chromatography

A rapid method for the quantitative determination of tyrosine (Tyr), phenylalanine (Phe), p-hydroxybenzoic acid (HBA), p-hydroxyphenylacetic acid (HPA), benzoic acid (BZA), p-hydroxyphenylpyruvic acid (HPY), phenylacetic acid (PAA), phenyllactic acid (PLA), tryptophan (Trp), indoleacetic acid (IAA), phenylpyruvic acid (PPY), phenylpropionic acid (PPA) and cinnamic acid (CNA) in goat rumen fluid was established by high-performance liquid chromatography (HPLC). The mobile phase used for isocratic elution was 50 mM sodium phosphate buffer (pH 6.5)–methanol (97:3, v/v). The flow-rate was 1.0 ml/min; column temperature 40°C and compounds were monitored at 215 nm with a UV absorbance detector after injection of 10 μl of filtered rumen fluid. Analysis was completed within 40 min. The minimum detectable limits of quantification (μM) of these compounds were Tyr, 2; Phe, 3; HBA, 1; HPA, 2; BZA, 2; HPY, 8; PAA, 3; PLA, 4; Trp, 2; IAA, 2; PPY, 15; PPA, 8 and CNA, 4. Detectable levels of Tyr, Phe, HPA, BZA, HPY, PAA, PLA, Trp and PPA were found in the deproteinized rumen fluid of goat fed a haycube and concentrate mixture. PAA was the predominant compound before and after feeding. The concentrations of HPA, BZA, PAA, PLA and PPA in the goat rumen fluid increased after feeding, while the concentration of Tyr decreased. Phe, HPY and Trp were minor components at all times. PPY, IAA and CNA were not detected and HBA was not completely resolved in the goat rumen fluid.     

Behavioral and Neurochemical Characterization of New Mouse Model of Hyperphenylalaninemia

Hyperphenylalaninemia (HPA) refers to all clinical conditions characterized by increased amounts of phenylalanine (PHE) in blood and other tissues. According to their blood PHE concentrations under a free diet, hyperphenylalaninemic patients are commonly classified into phenotypic subtypes: classical phenylketonuria (PKU) (PHE > 1200 µM/L), mild PKU (PHE 600-1200 µM/L) and persistent HPA (PHE 120-600 µM/L) (normal blood PHE < 120 µM/L). The current treatment for hyperphenylalaninemic patients is aimed to keep blood PHE levels within the safe range of 120-360 µM/L through a PHE-restricted diet, difficult to achieve. If untreated, classical PKU presents variable neurological and mental impairment. However, even mildly elevated blood PHE levels, due to a bad compliance to dietary treatment, produce cognitive deficits involving the prefrontal cortical areas, extremely sensible to PHE-induced disturbances. The development of animal models of different degrees of HPA is a useful tool for identifying the metabolic mechanisms underlying cognitive deficits induced by PHE. In this paper we analyzed the behavioral and biochemical phenotypes of different forms of HPA (control, mild-HPA, mild-PKU and classic-PKU), developed on the base of plasma PHE concentrations. Our results demonstrated that mice with different forms of HPA present different phenotypes, characterized by increasing severity of behavioral symptoms and brain aminergic deficits moving from mild HPA to classical PKU forms. In addition, our data identify preFrontal cortex and amygdala as the most affected brain areas and confirm the highest susceptibility of brain serotonin metabolism to mildly elevated blood PHE.     

Phenylalanine and its metabolites induce embryopathies in mouse embryos in culture

The aim of this study was to determine the teratogenicity of phenylalanine (Phe) and Phe metabolites in neurulating mouse embryos. Therefore, the system of whole embryo culture was employed and D9 (neurulating) mouse embryos were exposed to Phe, phenylethylamine (PEA), phenylpyruvic acid (PPA), phenylacetic acid (PAA), 2-OH phenylacetic acid (2-OH PAA), and phenyl-lactic acid (PLA) at concentrations ranging from 0.01 mM to 10 mM for 24 hours. After 24 hours, embryos were examined for morphological abnormalities and protein content by the Lowry method. Phe at 1 and 6 mM concentrations was not teratogenic; however, 10 mM inhibited cranial neural tube closure in 82% of the embryos. PEA was the most toxic factor and concentrations of 1 and 10 mM were embryo-lethal, whereas neural tube closure defects (NTDs) were observed in 67% of the embryos at 0.1 mM. 2-OH PAA was the second most toxic metabolite with concentrations of 1 and 10 mM producing NTDs in 10 and 100% of the embryos, respectively. PLA and PAA produced no NTDs at concentrations of 1 mM, 60% at 5 mM, and 100% at 10 mM. Finally, PPA produced approximately 50% NTDs at both 1 mM and 10 mM concentrations. PLA, PAA, 2-OH PAA, and PPA produced a significant reduction in embryonic protein, and PEA and 2-OH PAA reduced yolk sac protein values. PEA, 2-OH PAA, PPA, PAA, and PLA also produced craniofacial abnormalities, i.e., incomplete expansion of the forebrain, collapse of the optic vesicle, and hypoplasia of the mandible and/or the maxilla.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of Catalase and Superoxide Dismutase Activities on Oxidative Stress in the Brain of a Phenylketonuria Animal Model and the Effect of Lipoic Acid

Phenylketonuria (PKU) is an inherited metabolic disorder caused by deficiency of phenylalanine hydroxylase which leads to accumulation of phenylalanine and its metabolites in tissues of patients with severe neurological involvement. Recently, many studies in animal models or patients have reported the role of oxidative stress in PKU. In the present work we studied the effect of lipoic acid against oxidative stress in rat brain provoked by an animal model of hyperphenylalaninemia (HPA), induced by repetitive injections of phenylalanine and α-methylphenylalanine (a phenylalanine hydroxylase inhibitor) for 7 days, on some oxidative stress parameters. Lipoic acid prevented alterations on catalase (CAT) and superoxide dismutase (SOD), and the oxidative damage of lipids, proteins, and DNA observed in HPA rats. In addition, lipoic acid diminished reactive species generation compared to HPA group which was positively correlated to SOD/CAT ratio. We also observed that in vitro Phe inhibited CAT activity while phenyllactic and phenylacetic acids stimulated superoxide dismutase activity. These results demonstrate the efficacy of lipoic acid to prevent oxidative stress induced by HPA model in rats. The possible benefits of lipoic acid administration to PKU patients should be considered.     

Quantitative analysis of phenylalanine metabolites in urine to detect heterozygotes of phenylketonuria

Summary A method is described to detect carriers of phenylketonuria (PKU). 200 mg Phenylalanine per kilogram body weight are orally administered to 9 heterozygotes and 10 normal controls. Some phenylalanine metabolites in urine are quantitatively determined by gas chromatography. Both groups can be distinguished by the excreted amounts of mandelic acid (MA), phenylpyruvic acid (PPA), 2-hydroxyphenylacetic acid (2HOPAA) and phenylacetic acid (PAA).

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Zusammenfassung Ein Heterozygotentest für die Phenylketonurie (PKU) wird beschrieben. 200 mg Phenylalanin pro Kilogramm Körpergewicht werden 9 Heterozygoten und 10 Kontrollpersonen oral verabreicht. Einige Metaboliten des Phenylalanins werden nach bestimmten Zeiten im Urin gaschromatographisch quantitativ bestimmt. Gut unterscheidbar sind beide Versuchsgruppen mit Hilfe der Werte von Mandelsäure (MA), Phenylbrenztraubensäure (PPA), 2-Hydroxyphenylessigsäure (2HOPAA) und Phenylessigsäure (PAA).

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Supported by the Deutsche Forschungsgemeinschaft.     

Carcinogenic Effects in a Phenylketonuria Mouse Model

Phenylketonuria (PKU) is a metabolic disorder caused by impaired phenylalanine hydroxylase (PAH). This condition results in hyperphenylalaninemia and elevated levels of abnormal phenylalanine metabolites, among which is phenylacetic acid/phenylacetate (PA). In recent years, PA and its analogs were found to have anticancer activity against a variety of malignancies suggesting the possibility that PKU may offer protection against cancer through chronically elevated levels of PA. We tested this hypothesis in a genetic mouse model of PKU (PAH^enu2) which has a biochemical profile that closely resembles that of human PKU. Plasma levels of phenylalanine in homozygous (HMZ) PAH^enu2 mice were >12-fold those of heterozygous (HTZ) littermates while tyrosine levels were reduced. Phenylketones, including PA, were also markedly elevated to the range seen in the human disease. Mice were subjected to 7,12 dimethylbenz[a]anthracene (DMBA) carcinogenesis, a model which is sensitive to the anticancer effects of the PA derivative 4-chlorophenylacetate (4-CPA). Tumor induction by DMBA was not significantly different between the HTZ and HMZ mice, either in total tumor development or in the type of cancers that arose. HMZ mice were then treated with 4-CPA as positive controls for the anticancer effects of PA and to evaluate its possible effects on phenylalanine metabolism in PKU mice. 4-CPA had no effect on the plasma concentrations of phenylalanine, phenylketones, or tyrosine. Surprisingly, the HMZ mice treated with 4-CPA developed an unexplained neuromuscular syndrome which precluded its use in these animals as an anticancer agent. Together, these studies support the use of PAH^enu2 mice as a model for studying human PKU. Chronically elevated levels of PA in the PAH^enu2 mice were not protective against cancer.     

Inhibition of the Mitochondrial Respiratory Chain by Phenylalanine in Rat Cerebral Cortex

Phenylketonuria (PKU) is biochemically characterized by the accumulation of phenylalanine (Phe) and its metabolites in tissues of affected children. Neurological damage is the clinical hallmark of PKU, and Phe is considered the main neurotoxic metabolite in this disorder. However, the mechanisms of neurotoxicity are poorly known. The main objective of the present work was to measure the activities of the mitochondrial respiratory chain complexes (RCC) and succinate dehydrogenase (SDH) in brain cortex of Wistar rats subjected to chemically induced hyperphenylalaninemia (HPA). We also investigated the in vitro effect of Phe on SDH and RCC activities in the cerebral cortex of 22-day-old rats. HPA was induced by subcutaneous administration of 2.4 mol/g body weight -methylphenylalanine, a phenylalanine hydroxylase inhibitor, once a day, plus 5.2 M/g body weight phenylalanine, twice a day, from the 6th-21st postnatal day. The results showed a reduction of SDH and complex I + III activity in brain cortex of rats subjected to HPA. We also verified that Phe inhibited the in vitro activity of complexes I + III, possibly by competition with NADH. Considering the importance of SDH and RCC for the maintenance of energy supply to brain, our results suggest that energy deficit may contribute to the Phe neurotoxicity in PKU.     

DNA haplotype analyses of patients with hyperphenylalaninemia.

Linkage analysis of phenylketonurics has shown a strong association between the DNA haplotype at the phenylalanine hydroxylase (PAH) locus and phenylketonuria (PKU). Similarly, a genetic linkage between less severe forms of hyperphenylalaninemia (HPA) and the PAH locus has been suggested. In the present study we analyzed this linkage in more detail. Haplotypes at the PAH locus were determined for 19 individuals with moderately elevated plasma phenylalanine and normal urinary neopterin/biopterin ratios. Fourteen of these individuals had plasma phenylalanine levels of 4-10 mg/dl (mild HPA), and the other five had plasma phenylalanine levels of 10-19 mg/dl (atypical PKU). Thirteen of the 15 HPA families consisted of an affected child and at least one other sibling. Elevated plasma phenylalanine was seen to genetically segregate with specific PAH alleles in each family. Summation of the LOD scores for both categories of moderate plasma phenylalanine elevation gave a maximum value of 3.556 at theta = 0. At theta = 0 this gives a probability of linkage between the PAH locus and the locus for moderate phenylalanine elevations that is approximately 3,600:1. None of the alleles segregating with either mild HPA or atypical PKU were of haplotype 2 or 3, and 13/20 were of types 1 or 4. This is in agreement with the most deleterious mutations being on haplotypes 2 and 3 and with the less severe mutations being on haplotypes 1 and 4. chi 2 Analyses indicated no statistically significant correlation between HPA and a particular haplotype or restriction-enzyme site.     

Compound heterozygosity in nonphenylketonuria hyperphenylalanemia: the contribution of mutations for classical phenylketonuria

Hyperphenylalaninemia (HPA) results from defective hydroxylation of phenylalanine in the liver, in most cases because of defective phenylalanine hydroxylase. HPA is highly variable, ranging from moderate elevation of plasma phenylalanine with no clinical consequences to a severe disease, classical phenylketonuria (PKU). Non-PKU HPA was found in excess of PKU in Israel, while the opposite is true in Europe. To study the genetic basis of non-PKU HPA, we performed haplotype analysis at the phenylalanine hydroxylase locus in 27 families with non-PKU HPA. All individuals with this condition were compound heterozygotes. In six of these families, in which both PKU and non-PKU HPA were segregating, haplotype analysis showed that non-PKU HPA resulted from compound heterozygosity for a PKU mutation and a second mutation, with milder effect, which is probably expressed only when it interacts with the severe mutation. The involvement of PKU mutations in non-PKU HPA was further demonstrated in Jewish Yemenite families with non-PKU HPA, in which the individuals with this condition were carriers of the single PKU allele which exists in this community. In addition, two previously known PKU point mutations (R261Q and R408W) were found in individuals with non-PKU HPA. These mutations are associated, in our population, with the same haplotypes as those with which it is associated in Europe. Based on the above-mentioned genetic model for non-PKU HPA, successful prenatal diagnosis of this condition was performed in one family.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of calpastatin, minopontin, NIPSNAP1, rabaptin-5 and neuronatin in the phenylketonuria (PKU) mouse brain: possible role on cognitive defect seen in PKU

Phenylketonuria (PKU) is an inborn error of amino acid metabolism. Phenylalanine hydroxylase (PAH) deficiency results in accumulation of phenylalanine (Phe) in the brain and leads to pathophysiological abnormalities including cognitive defect, if Phe diet is not restricted. Neuronatin and 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) reportedly have role in memory. Therefore, gene expression was examined in the brain of mouse model for PKU. Microarray expression analysis revealed reduced expression of calpastatin, NIPSNAP 1, rabaptin-5 and minopontin genes and overexpression of neuronatin gene in the PKU mouse brain. Altered expression of these genes was further confirmed by one-step real time RT-PCR analysis. Western blot analysis of the mouse brain showed reduced levels of calpastatin and rabaptin-5 and higher amount of neuronatin in PKU compared to the wild type. These observations in the PKU mouse brain suggest that altered expression of these genes resulting in abnormal proteome. These changes in the PKU mouse brain are likely to contribute cognitive impairment seen in the PKU mouse, if documented also in patients with PKU.     

The proportion of tetrahydrobiopterin deficiency and PAH gene deficiency variants among cases with hyperphenyalaninemia in Western Iran

BACKGROUND:

Defects either in phenylalanine hydroxylase (PheOH) or in the production and recycling of its cofactor (tetrahydrobiopterin [BH4]) are the causes of primary hyperphenylalaninemia (HPA). The aim of our study was to investigate the current status of different variants of HPA Kurdish patients in Kermanshah province, Iran.

MATERIALS AND METHODS:

From 33 cases enrolled in our study, 32 were identified as HPA patients. Reassessing of pre-treatment phenylalanine concentrations and the analysis of urinary pterins was done by high-performance liquid chromatography method.

RESULTS:

A total of 30 patients showed PAH deficiency and two patients were diagnosed with BH4 deficiency (BH4/HPA ratio = 6.25%). Both of these two BH4-deficient patients were assigned to severe variant of dihydropteridine reductase (DHPR) deficiency. More than 75% of patients with PAH deficiency classified as classic phenylketonuria (PKU) according their levels of pre-treatment phenylalanine concentrations.

CONCLUSION:

Based on the performed study, we think that the frequency of milder forms of PKU is higher than those was estimated before and/or our findings here. Furthermore, the frequency of DHPR deficiency seems to be relatively high in our province. Since the clinical symptoms of DHPR deficiency are confusingly similar to that of classic PKU and its prognosis are much worse than classical PKU and cannot be solely treated with the PKU regime, our pilot study support that it is crucial to set up screening for BH4 deficiency, along with PAH deficiency, among all HPA patients diagnosed with HPA.     

Glycomacropeptide, a low-phenylalanine protein isolated from cheese whey, supports growth and attenuates metabolic stress in the murine model of phenylketonuria

Phenylketonuria (PKU) is caused by a mutation in the phenylalanine (phe) hydroxylase gene and requires a low-phe diet plus amino acid (AA) formula to prevent cognitive impairment. Glycomacropeptide (GMP) contains minimal phe and provides a palatable alternative to AA formula. Our objective was to compare growth, body composition, and energy balance in Pah(enu2) (PKU) and wild-type mice fed low-phe GMP, low-phe AA, or high-phe casein diets from 3-23 wk of age. The 2 × 2 × 3 design included main effects of genotype, sex, and diet. Fat and lean mass were assessed by dual-energy X-ray absorptiometry, and acute energy balance was assessed by indirect calorimetry. PKU mice showed growth and lean mass similar to wild-type littermates fed the GMP or AA diets; however, they exhibited a 3-15% increase in energy expenditure, as reflected in oxygen consumption, and a 3-30% increase in food intake. The GMP diet significantly reduced energy expenditure, food intake, and plasma phe concentration in PKU mice compared with the casein diet. The high-phe casein diet or the low-phe AA diet induced metabolic stress in PKU mice, as reflected in increased energy expenditure and intake of food and water, increased renal and spleen mass, and elevated plasma cytokine concentrations consistent with systemic inflammation. The low-phe GMP diet significantly attenuated these adverse effects. Moreover, total fat mass, %body fat, and the respiratory exchange ratio (CO(2) produced/O(2) consumed) were significantly lower in PKU mice fed GMP compared with AA diets. In summary, GMP provides a physiological source of low-phe dietary protein that promotes growth and attenuates the metabolic stress induced by a high-phe casein or low-phe AA diet in PKU mice.     

Large Neutral Amino Acid Supplementation Exerts Its Effect through Three Synergistic Mechanisms: Proof of Principle in Phenylketonuria Mice

Background

Phenylketonuria (PKU) was the first disorder in which severe neurocognitive dysfunction could be prevented by dietary treatment. However, despite this effect, neuropsychological outcome in PKU still remains suboptimal and the phenylalanine-restricted diet is very demanding. To improve neuropsychological outcome and relieve the dietary restrictions for PKU patients, supplementation of large neutral amino acids (LNAA) is suggested as alternative treatment strategy that might correct all brain biochemical disturbances caused by high blood phenylalanine, and thereby improve neurocognitive functioning.

Objective

As a proof-of-principle, this study aimed to investigate all hypothesized biochemical treatment objectives of LNAA supplementation (normalizing brain phenylalanine, non-phenylalanine LNAA, and monoaminergic neurotransmitter concentrations) in PKU mice.

Methods

C57Bl/6 Pah-enu2 (PKU) mice and wild-type mice received a LNAA supplemented diet, an isonitrogenic/isocaloric high-protein control diet, or normal chow. After six weeks of dietary treatment, blood and brain amino acid and monoaminergic neurotransmitter concentrations were assessed.

Results

In PKU mice, the investigated LNAA supplementation regimen significantly reduced blood and brain phenylalanine concentrations by 33% and 26%, respectively, compared to normal chow (p<0.01), while alleviating brain deficiencies of some but not all supplemented LNAA. Moreover, LNAA supplementation in PKU mice significantly increased brain serotonin and norepinephrine concentrations from 35% to 71% and from 57% to 86% of wild-type concentrations (p<0.01), respectively, but not brain dopamine concentrations (p = 0.307).

Conclusions

This study shows that LNAA supplementation without dietary phenylalanine restriction in PKU mice improves brain biochemistry through all three hypothesized biochemical mechanisms. Thereby, these data provide proof-of-concept for LNAA supplementation as a valuable alternative dietary treatment strategy in PKU. Based on these results, LNAA treatment should be further optimized for clinical application with regard to the composition and dose of the LNAA supplement, taking into account all three working mechanisms of LNAA treatment.     

Preliminary mutation analysis in the phenylanaline hydroxylase gene in Greek PKU and HPA patients

The presence of nine mutations in the phenylalanine hydroxlase (PAH) gene, previously described in phenylketonuria (PKU) patients of other Mediterranean and European populations, was assessed in 47 Greek PKU and 3 hyperphenylalaninaemia (HPA) patients. Of the nine mutations investigated, only five were detected, characterizing 31 % of the PKU alleles in our patients.     

High Levels of Orexin A in the Brain of the Mouse Model for Phenylketonuria: Possible Role of Orexin A in Hyperactivity Seen in Children with PKU

Phenylketonuria (PKU) is a metabolic disorder caused by phenylalanine hydroxylase deficiency leading to increased levels of phenylalanine in the brain. Hyperactivity is reportedly induced by a high level of orexin A, and therefore orexin A content was studied in the PKU mice. Hypothalamus and brain stem had higher levels of orexin A compared to cerebrum and cerebellum both in wild type and PKU mice brains as observed by radioimmunoassay method. Interestingly, all these regions of the brain in PKU mouse showed a higher level of orexin A compared to the wild type. Heart and plasma also had higher levels of orexin A in PKU compared to the wild type. Immunohistochemical analysis revealed an increased number of orexin A–stained cells in the brain and heart of PKU mouse compared to the wild type. This is the first report of increased level of orexin in the PKU mouse brain. Hyperactivity is commonly observed in children with PKU; thus these findings suggest that orexin A is a contributing factor for the hyperactivity.     

Molecular basis for nonphenylketonuria hyperphenylalaninemia.

Nonphenylketonuria hyperphenylalaninemia (non-PKU HPA) is defined as phenylalanine hydroxylase (PAH) deficiency with blood phenylalanine levels below 600 mumol/liter (i.e., within the therapeutic range) on a normal dietary intake. Haplotype analysis at the PAH locus was performed in 17 Danish families with non-PKU HPA, revealing compound heterozygosity in all individuals. By allele-specific oligonucleotide (ASO) probing for common PKU mutations we found 12 of 17 non-PKU HPA children with a PKU allele on one chromosome. To identify molecular lesions in the second allele, individual exons were amplified by polymerase chain reaction and screened for mutations by single-strand conformation polymorphism. Two new missense mutations were identified. Three children had inherited a G-to-A transition at codon 415 in exon 12 of the PAH gene, resulting in the substitution of asparagine for aspartate, whereas one child possessed an A-to-G transition at codon 306 in exon 9, causing the replacement of an isoleucine by a valine in the enzyme. It is further demonstrated that the identified mutations have less impact on the heterozygote's ability to hydroxylate phenylalanine to tyrosine compared to the parents carrying a PKU mutation. The combined effect on PAH activity explains the non-PKU HPA phenotype of the child. The present observations that PKU mutations in combination with other mutations result in the non-PKU HPA phenotype and that particular mutation-restriction fragment length polymorphism haplotype combinations are associated with this phenotype offer the possibility of distinguishing PKU patients from non-PKU individuals by means of molecular analysis of the hyperphenylalaninemic neonate and, consequently, of determining whether a newborn child requires dietary treatment.     

MAOB: a modifier gene in phenylketonuria?

Phenylketonuria (PKU), the most frequent inborn error of metabolism (1/15,000 live births), is an autosomal recessive condition caused by phenylalanine hydroxylase deficiency. Despite early and strict dietary control, some PKU children still exhibit behavioral and cognitive difficulties suggestive of a partly prenatal brain injury. The reported variability between the cognitive and clinical phenotypes within the same family raises the question of modifying genes in PKU. We suggest here that monoamine oxidase type B, MAOB, an enzyme degrading phenylethylamine, a very toxic metabolite of phenylalanine, could act as a modifying gene since a variant enzymatic activity of MAOB in PKU patients with similar phenylalanine levels would result in different phenylethylamine levels and different clinical outcomes. Finally the report of low MAOB, and consequently expectedly high phenylethylamine levels in neonates is consistent with a phenylethylamine-mediated brain injury possibly causing irreversible damages in PKU newborns prior to onset of the low protein diet.     

Oxidative Stress in Phenylketonuria: What is the Evidence?

Phenylketonuria (PKU) is an inborn error of amino acid metabolism caused by severe deficiency of phenylalanine hydroxylase activity, leading to the accumulation of phenylalanine and its metabolites in blood and tissues of affected patients. Phenylketonuric patients present as the major clinical feature mental retardation, whose pathomechanisms are poorly understood. In recent years, mounting evidence has emerged indicating that oxidative stress is possibly involved in the pathology of PKU. This article addresses some of the recent developments obtained from animal studies and from phenylketonuric patients indicating that oxidative stress may represent an important element in the pathophysiology of PKU. Several studies have shown that enzymatic and non-enzymatic antioxidant defenses are decreased in plasma and erythrocytes of PKU patients, which may be due to an increased free radical generation or secondary to the deprivation of micronutrients which are essential for these defenses. Indeed, markers of lipid, protein, and DNA oxidative damage have been reported in PKU patients, implying that reactive species production is increased in this disorder. A considerable set of data from in vitro and in vivo animal studies have shown that phenylalanine and/or its metabolites elicit reactive species in brain rodent. These findings point to a disruption of pro-oxidant/antioxidant balance in PKU. Considering that the brain is particularly vulnerable to oxidative attack, it is presumed that the administration of appropriate antioxidants as adjuvant agents, in addition to the usual treatment based on restricted diets or supplementation of tetrahydrobiopterin, may represent another step in the prevention of the neurological damage in PKU.     

Controlled Diet in Phenylketonuria and Hyperphenylalaninemia may Cause Serum Selenium Deficiency in Adult Patients: The Czech Experience

Phenylketonuria is an inherited disorder of metabolism of the amino acid phenylalanine caused by a deficit of the enzyme phenylalanine hydroxylase. It is treated with a low-protein diet containing a low content of phenylalanine to prevent mental affection of the patient. Because of the restricted intake of high-biologic-value protein, patients with phenylketonuria may have lower than normal serum concentrations of pre-albumin, selenium, zinc and iron. The objective of the present study was to assess the compliance of our phenylketonuric (PKU) and hyperphenylalaninemic (HPA) patients; to determine the concentration of serum pre-albumin, selenium, zinc and iron to discover the potential correlation between the amount of proteins in food and their metabolic control. We studied 174 patients of which 113 were children (age 1–18), 60 with PKU and 53 with HPA and 61 were adults (age 18–42), 51 with PKU and 10 with HPA. We did not prove a statistically significant difference in the concentration of serum pre-albumin, zinc and iron among the respective groups. We proved statistically significant difference in serum selenium concentrations of adult PKU and HPA patients (p?=?0.006; Mann–Whitney U test). These results suggest that controlled low-protein diet in phenylketonuria and hyperphenylalaninemia may cause serum selenium deficiency in adult patients.     

Co-administration of Creatine Plus Pyruvate Prevents the Effects of Phenylalanine Administration to Female Rats During Pregnancy and Lactation on Enzymes Activity of Energy Metabolism in Cerebral Cortex and Hippocampus of the Offspring

Phenylketonuria (PKU) is the most frequent inborn error of metabolism. It is caused by deficiency in the activity of phenylalanine hydroxylase, leading to accumulation of phenylalanine and its metabolites. Untreated maternal PKU or hyperphenylalaninemia may result in nonphenylketonuric offspring with low birth weight and neonatal sequelae, especially microcephaly and intellectual disability. The mechanisms underlying the neuropathology of brain injury in maternal PKU syndrome are poorly understood. In the present study, we evaluated the possible preventive effect of the co-administration of creatine plus pyruvate on the effects elicited by phenylalanine administration to female Wistar rats during pregnancy and lactation on some enzymes involved in the phosphoryltransfer network in the brain cortex and hippocampus of the offspring at 21 days of age. Phenylalanine administration provoked diminution of body, brain cortex an hippocampus weight and decrease of adenylate kinase, mitochondrial and cytosolic creatine kinase activities. Co-administration of creatine plus pyruvate was effective in the prevention of those alterations provoked by phenylalanine, suggesting that altered energy metabolism may be important in the pathophysiology of maternal PKU. If these alterations also occur in maternal PKU, it is possible that pyruvate and creatine supplementation to the phenylalanine-restricted diet might be beneficial to phenylketonuric mothers.