Elevated Phenylalanine Levels Research Articles

Evidence that L-carnitine and selenium supplementation reduces oxidative stress in phenylketonuric patients.

It is well established that the involvement of reactive species in the pathophysiology of several neurological diseases, including phenylketonuria (PKU), a metabolic genetic disorder biochemically characterized by elevated levels of phenylalanine (Phe). In previous studies, we verified that PKU patients (treated with a protein-restricted diet supplemented with a special formula not containing L-carnitine and selenium) presented high lipid and protein oxidative damage as well as a reduction of antioxidants when compared to the healthy individuals. Our goal in the present study was to evaluate the effect of Phe-restricted diet supplemented with L-carnitine and selenium, two well-known antioxidant compounds, on oxidative damage in PKU patients. We investigated various oxidative stress parameters in blood of 18 treated PKU patients before and after 6 months of supplementation with a special formula containing L-carnitine and selenium. It was verified that treatment with L-carnitine and selenium was capable of reverting the lipid peroxidation, measured by thiobarbituric acid-reactive species, and the protein oxidative damage, measured by sulfhydryl oxidation, to the levels of controls. Additionally, the reduced activity of glutathione peroxidase was normalized by the antioxidant supplementation. It was also verified a significant inverse correlation between lipid peroxidation and L-carnitine blood levels as well as a significant positive correlation between glutathione peroxidase activity and blood selenium concentration. In conclusion, our results suggest that supplementation of L-carnitine and selenium is important for PKU patients since it could help to correct the oxidative stress process which possibly contributes, at least in part, to the neurological symptoms found in phenylketonuric patients.

Newborn screening for T-cell deficiency

Newborn screening for T-cell deficiency is ongoing in two states, and the published results of 1 year of screening in Wisconsin are favorable. In this review, the history, methodology, results, challenges, and future direction of screening are discussed. As a concept, newborn blood screening (NBS) has evolved from the detection of elevated levels of phenylalanine on filter paper to the application of DNA-based technologies to identify T-cell lymphopenia in infants with severe combined immunodeficiency (SCID). After diagnosis by NBS, one infant with SCID received a hematopoietic stem cell transplant (HSCT), and several others are currently undergoing evaluation for HSCT. The utility of NBS for T-cell deficiency is dependent on the NBS program to define a rigorous screening protocol, ensure seamless follow-up of all identified infants, and demand a renewed emphasis on the training of future clinical immunologists to care for these patients. DNA-based NBS assays are currently being evaluated to identify infants with other serious diseases. Newborn screening for T-cell deficiency has ushered in a new era in the detection of infants with primary immunodeficiencies.

Pleiotropic physiological consequences of feedback-insensitive phenylalanine biosynthesis in Arabidopsis thaliana

A large proportion of plant carbon flow passes through the shikimate pathway to phenylalanine, which serves as a precursor for numerous secondary metabolites. To identify new regulatory mechanisms affecting phenylalanine metabolism, we isolated Arabidopsis thaliana mutants that are resistant to the phytotoxic amino acid m-tyrosine, a structural analog of phenylalanine. Map-based cloning identified adt2-1D, a dominant point mutation causing a predicted serine to alanine change in the regulatory domain of ADT2 (arogenate dehydratase 2). Relaxed feedback inhibition and increased expression of the mutant enzyme caused up to 160-fold higher accumulation of free phenylalanine in rosette leaves, as well as altered accumulation of several other primary and secondary metabolites. In particular, abundance of 2-phenylethylglucosinolate, which is normally almost undetectable in leaves of the A. thaliana Columbia-0 accession, is increased more than 30-fold. Other observed phenotypes of the adt2-1D mutant include abnormal leaf development, resistance to 5-methyltryptophan, reduced growth of the generalist lepidopteran herbivore Trichoplusia ni (cabbage looper) and increased salt tolerance.

Screening for Phenylketonuria

Newborn screening for phenylketonuria (PKU) started with Robert Guthrie (1916–1995) who developed the bacterial inhibition test for the semiquantitative analysis of phenylalanine, which was the first test suitable for high throughput analysis. In addition, he introduced the ‘Guthrie filter card’ as a transport medium for dried blood which is still used today. Realizing the potential of his approach for early diagnosis and the fact that a low-phenylalanine diet prevented the neurological sequelae of untreated PKU, Robert Guthrie became the first and utmost advocate of newborn screening for PKU. Following the first PKU newborn screening program in Buffalo, N.Y., USA, in the 1960s, additional newborn screening programs were initiated around the world in the 1960s and 1970s. Newborn screening has since been recognized as an important public health measure, and most countries have ongoing newborn screening programs for PKU and other inborn errors of metabolism. Since the first programs, it has been recognized that early diagnosis of PKU and subsequent initiation of a low phenylalanine diet results in normal neurological outcomes – in contrast to the severe mental retardation in untreated PKU. Today’s newborn screening laboratories use photometric assays or tandem mass spectrometry for analysis of phenylalanine rather than the bacterial inhibition test. This has led to an increased number of cases with hyperphenylalaninemia that often do not require dietary treatment. Any elevated phenylalanine level in a neonate needs to be followed by a second specimen for repeat analysis of phenylalanine and tyrosine. Confirmation of elevated phenylalanine levels with low to normal tyrosine levels requires analysis of urine pterines and dihydropterine reductase activity in red cells to rule out an inborn error of biopterin metabolism. In any neonate whose blood phenylalanine levels exceed 6 mg/dl, a tetrahydrobiopterin loading test should be performed and dietary therapy should be initiated. The level at which dietary therapy is started may be different between the USA, UK and continental Europe.

Is leucine induced p70S6 kinase phosphorylation following resistance exercise dependent on elevated phenylalanine levels in human skeletal muscle?

The purpose of this study was to investigate the specific role of leucine in the stimulation of the mammalian target of rapamycin signalling pathway. Six male subjects performed four heavy resistance exercise sessions, each separated by approximately one week. Subjects were randomly supplemented with one of four drinks: placebo (flavored water), leucine or essential amino acids (EAA) with and without leucine. Immediately following each exercise session, four subjects were infused with a flooding dose of L‐[2H5] phenylalanine (Inf) while two subjects served as controls (Ctrl). Muscle biopsies were taken before and one hour after exercise. In the Ctrl group, resistance exercise resulted in a substantial increase (45‐fold) in p70S6 kinase phosphorylation when all EAA were ingested, whereas ingestion of leucine alone had no greater effect than that of placebo. In the Inf group, however, ingestion of leucine alone and EAA increased p70S6k phosphorylation to a similar extent (35‐fold). The divergent signalling response in the two groups suggests that leucine alone is insufficient to increase p70S6k phosphorylation. Indeed, in the Inf group, there was a strong correlation (r=0.91) between p70S6kphosphorylation and the product of muscle leucine and phenylalanine levels. These results suggest that the stimulatory effect of leucine on p70S6kphosphorylation is dependent on elevated muscle phenylalanine levels. Supported by the Swedish National Centre for Research in Sports

Effects of phenylalanine on the survival and neurite outgrowth of rat cortical neurons in primary cultures: possible involvement of brain-derived neurotrophic factor

Phenylketonuria (PKU) is characterized by elevated levels of phenylalanine (Phe) in plasma and cerebrospinal fluid of PKU patients, leading to mental retardation. The developmental delay in the cerebral cortex is one of the characteristic pathologic changes in untreated phenylketonuria patients. This is thought to be due to the toxic effects of Phe and/or its metabolites; however, the underlying mechanisms are as yet unknown. In this study, using a model system in which cultured cortical neurons were induced with Phe, we observed that Phe inhibited the longest neurite outgrowth and induced the neuronal death. We further demonstrated that the expression of BDNF mRNA and protein was significantly decreased by Phe, together with a decrease in extracellular signal-regulated kinase (ERK) and Akt phosphorylation activity. There was no change in expression of TrkB mRNA and protein. Considering the important role of BDNF in normal brain development and function, these L: -Phe-induced changes in BDNF in PKU brain may be a critical element of the neurological symptoms of PKU.

Psychosocial issues and outcomes in maternal PKU

Elevated phenylalanine (Phe) levels in pregnant women with PKU are teratogenic. Fetal damage due to elevated maternal Phe levels during pregnancy is known as maternal phenylketonuria (MPKU). The risk of birth defects in MPKU, including global developmental delays, microcephaly, congenital heart disease, and low birth weight, can be dramatically reduced by controlling Phe levels during pregnancy (metabolic control). Phe levels should be maintained in the range of 120–360 μmol/L, ideally starting before pregnancy begins (i.e., when planning a pregnancy). If control is not achieved before pregnancy (e.g., if the pregnancy was unplanned), good outcomes are still possible if metabolic control is established by 8 weeks of pregnancy. Unfortunately, metabolic control before and during pregnancy can be poor. As well, many mothers stop treatment after pregnancy, which can decrease the mother’s ability to focus on her child and increase her risk of behavioral and psychological problems. This can have a negative effect on the home environment. Many factors affect adherence to the strict diet used to control Phe levels, including poor access to medical care, lack of reimbursement for medical foods (in some regions, such as parts of the United States), practical difficulties with implementing the diet, financial constraints, demographics, and psychosocial issues. A comprehensive treatment approach that begins prior to pregnancy and continues after the infant is born may help to improve the management of MPKU. This approach should include education of girls about MPKU at an early age, interventions to prevent unplanned pregnancies, psychosocial support, improved treatment access and reimbursement for medical foods, and treatment guidelines. Treatments such as sapropterin may also have a role in improving metabolic control during pregnancy.

Coenzyme Q 10 in phenylketonuria and mevalonic aciduria

Mevalonic aciduria (MVA) and phenylketonuria (PKU) are inborn errors of metabolism caused by deficiencies in the enzymes mevalonate kinase and phenylalanine 4-hydroxylase, respectively. Despite numerous studies the factors responsible for the pathogenicity of these disorders remain to be fully characterised. In common with MVA, a deficit in coenzyme Q 10 (CoQ 10) concentration has been implicated in the pathophysiology of PKU. In MVA the decrease in CoQ 10 concentration may be attributed to a deficiency in mevalonate kinase, an enzyme common to both CoQ 10 and cholesterol synthesis. However, although dietary sources of cholesterol cannot be excluded, the low/normal cholesterol levels in MVA patients suggests that some other factor may also be contributing to the decrease in CoQ 10.The main factor associated with the low CoQ 10 level of PKU patients is purported to be the elevated phenylalanine level. Phenylalanine has been shown to inhibit the activities of both 3-hydroxy-3-methylglutaryl-CoA reductase and mevalonate-5-pyrophosphate decarboxylase, enzymes common to both cholesterol and CoQ 10 biosynthesis. Although evidence of a lowered plasma/serum CoQ 10 level has been reported in MVA and PKU, few studies have assessed the intracellular CoQ 10 concentration of patients. Plasma/serum CoQ 10 is influenced by dietary intake as well as its lipoprotein content and therefore may be limited as a means of assessing intracellular CoQ 10 concentration. Whether the pathogenesis of MVA and PKU are related to a loss of CoQ 10 has yet to be established and further studies are required to assess the intracellular CoQ 10 concentration of patients before this relationship can be confirmed or refuted.

Sapropterin [Phenoptin]* is beneficial for children with phenylketonuria and elevated phenylalanine levels,

Sapropterin [Phenoptin]* is beneficial for children with phenylketonuria and elevated phenylalanine levels,

Elevated phenylalanine levels interfere with neurite outgrowth stimulated by the neuronal cell adhesion molecule L1 in vitro

Elevated levels of phenylalanine (Phe) as observed in patients with phenylketonuria interfere with proper neuronal development, leading to severe psychomotor deficits and mental retardation. We have analyzed the effects of Phe on neurite outgrowth in vitro. When expressed in fibroblasts, the neuronal cell adhesion molecules L1 and plexin B3 strongly increase the length of neurites emanating from cerebellar neurons in co-culture experiments. Elevated Phe blocks L1-mediated, but not plexin B3-mediated outgrowth, whereas tyrosine is ineffective. Elevated Phe also interferes with aggregation of fibroblasts overexpressing L1, suggesting that the pathological effect of elevated Phe occurs by interfering with L1-mediated cell adhesion.

Phenylalanine ammonia-lyase modified with polyethylene glycol: Potential therapeutic agent for phenylketonuria

Phenylketonuria (PKU) is an autosomal recessive genetic disease caused by the defects in the phenylalanine hydroxylase (PAH) gene. Individuals homozygous for defective PAH alleles show elevated levels of systemic phenylalanine and should be under strict dietary control to reduce the risk of neuronal damage associated with high levels of plasma phenylalanine. Researchers predict that plant phenylalanine ammonia-lyase (PAL), which converts phenylalanine to nontoxic t-cinnamic acid, will be an effective therapeutic enzyme for the treatment of PKU. The problems of this potential enzyme therapy have been the low stability in the circulation and the antigenicity of the plant enzyme. Recombinant PAL originated from parsley (Petroselinum crispum) chemically conjugated with activated PEG2 [2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine] showed greatly enhanced stability in the circulation and was effective in reducing the plasma concentration of phenylalanine in the circulation of mice. PEG-PAL conjugate will be an effective therapeutic enzyme for the treatment of PKU.

423. Increased Incidence of Hepatocellular Change and Neoplastic Disease in AAV-WPRE Treated Pahenu2 Mice

Phenylketonuria (PKU) is among the most common human birth defects occurring in approximately 1 in 16,000 births in the United States. The disease is caused by a single gene defect in the enzyme phenylalanine hydroxylase (PAH) which results in elevated serum phenylalanine (Phe) levels. Our laboratory is involved in the investigation of AAV applications for the treatment of this disease in a mouse model for PKU.

Wild-type phenylalanine hydroxylase activity is enhanced by tetrahydrobiopterin supplementation in vivo: an implication for therapeutic basis of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency

We previously proposed a novel disease entity, tetrahydrobiopterin (BH 4)-responsive phenylalanine hydroxylase (PAH) deficiency, in which administration of BH 4 reduced elevated levels of serum phenylalanine [J. Pediatr. 135 (1999) 375–378]. Subsequent reports indicate that the prevalence of BH 4-responsive PAH deficiency is much higher than initially anticipated. Although growing attention surrounds treatment with BH 4, little is known about the mechanism of BH 4 responsiveness. An early report indicates that BH 4 concentration in rat liver was 5 μM where K m for BH 4 of rat PAH was estimated to be 25 μM in an oxidation experiment using a liver slice, suggesting relative insufficiency of BH 4 in liver in vivo. In the present study, we developed a breath test for mice using [1- 13C]phenylalanine in order to examine the BH 4 responsiveness of normal PAH in vivo. The reliability of the test was verified using BTBR mice and its mutant strain lacking PAH activity, Pah enu2. BH 4 supplementation significantly enhanced 13CO 2 production in C57BL/6 mice when phenylalanine was pre-loaded. Furthermore, BH 4 apparently activated PAH in just 5 min. These observations suggest that submaximal PAH activity occurs at the physiological concentrations of BH 4 in vivo, and that PAH activity can be rapidly enhanced by supplementation with BH 4. Thus, we propose a possible hypothesis that the responsiveness to BH 4 in patients with PAH deficiency is due to the fact that suboptimal physiological concentrations of BH 4 are normally present in hepatocytes and the enhancement of the residual activity may be associated with a wide range of mutations.

874. Hepatocellular Morphology in the Pahenu2 Mouse Model of PKU Is Altered Following Portal Vein Administration of rAAV-mPAH-WPRE Vectors

Phenylketonuria (PKU) is among the most common human birth defects. The disease is caused by a defect in the enzyme phenylalanine hydroxylase (PAH) resulting in elevated serum phenylalanine (Phe) levels, leading to severe mental retardation. The current treatment for PKU is a restrictive diet. We developed a recombinant AAV vector carrying the mouse PAH gene (rAAV-mPAH) and delivered it through portal vein injection. Male Pahenu2 mice responded at vector doses of 6 × 1010 I.U. or above, lowering serum Phe to therapeutic levels. Female mice were unresponsive to similar doses. A dominant negative interaction between normal subunits of the PAH tetramer made from the vector delivered gene and endogenous PAH missense subunits is a possible explanation. To overcome a dominant negative effect, a Woodchuck Hepatitis Virus post-transcriptional regulatory element was added to the rAAV-mPAH vector (rAAV-mPAH-WPRE). Portal vein delivery to male Pahenu2 mice resulted in reduction of serum Phe levels at about 10% of previous doses.

Developmental timing of exposure to elevated levels of phenylalanine is associated with ADHD symptom expression.

JOURNAL ARTICLE REVIEWS: PHENYLALANINE TOXICITY: NEURODEVELOPMENTAL IMPACTS OF PRE- AND POSTNATAL EXPOSURE

Developmental timing of exposure to elevated levels of phenylalanine is associated with ADHD symptom expression.

This study addresses attention deficit hyperactivity disorder (ADHD), with a focus on how the timing of a known biological insult affects ADHD symptom expression. The sample consists of children exposed to elevated levels of phenylalanine, either postnatally as in Phenylketonuria (PKU; n = 46) or prenatally as in Maternal PKU (MPKU; n = 15). Non-hyperphenylalaninemic siblings of children with PKU (n = 18) serve as controls. Results indicate that elevated levels of phenylalanine are associated with ADHD symptoms. The manifestations of the symptom expression are dependent on exposure timing: prenatal exposure is associated with a higher likelihood of expressing hyperactive/impulsive symptoms and postnatal exposure is associated with a higher likelihood of expressing inattentive symptoms. This toxicity is dose-dependent and higher levels of phenylalanine appear more detrimental.

Timing is everything: executive functions in children exposed to elevated levels of phenylalanine.

This study addresses how the timing of a known biological insult affects the developmental progression of executive functions. The sample consisted of children exposed to elevated levels of phenylalanine, either postnatally, as in phenylketonuria (PKU; n = 46), or prenatally, as in maternal PKU (n = 15). Nonhyperphenylanemic siblings of children with PKU (n = 18) served as controls. Results indicated that elevated levels of phenylalanine are toxic to the neurological systems that manage executive functions and cognitive tempo. This toxicity is dose dependent, with higher levels of phenylalanine being more detrimental. Executive function difficulties noted in PKU are consistent with attention deficit hyperactivity disorder (ADHD)-inattentive type, whereas maternal PKU offspring had executive function difficulties consistent with ADHD-combined type.

Characterization of the Mouse Phenylalanine Hydroxylase Mutation Pahenu3

Phenylketonuria (PKU) is an inborn error of metabolism that is inherited in an autosomal recessive manner. It arises from a deficiency of phenylalanine hydroxylase, which is responsible for converting phenylalanine to tyrosine and thereby hastening its catabolism. To produce mouse models for the study of PKU, male mice were mutagenized with ethylnitrosourea and their progeny were screened for the elevated phenylalanine levels characteristic of phenylalanine hydroxylase deficiency. Of three mutant alleles recovered, two (Pahenu1 and Pahenu2) were characterized previously and shown to be missense mutations. Sequencing of phenylalanine hydroxylase cDNA from the third mutant allele, Pahenu3, revealed that two differently sized transcripts were being produced. These transcripts contained either a 5-nucleotide insertion or a 5-nucleotide deletion and both of these modifications occurred at the same location, the exon 11–exon 12 junction. Sequencing of the exon 11–intron 11 boundary revealed a T → G transversion in the invariant GT dinucleotide of the wild-type 5′ splice donor site. The analogous human Pah mutation would be called c.1199 + 2T > G. Sequence analysis also revealed two cryptic splice donor sites, upstream and downstream of the wild-type splice site, that appear to be used when the wild type is ablated and to thereby yield the observed differently sized transcripts. The 5-nucleotide insertion and the 5-nucleotide deletion are both predicted to cause frame shifting in exon 12 and exon 13, leading to premature termination.

Is there a relationship between 3-hydroxy-3-methylglutaryl coenzyme a reductase activity and forebrain pathology in the PKU mouse?

Previous reports have suggested that elevated levels of phenylalanine inhibit cholesterol synthesis. The goals of this study were to investigate if perturbations in cholesterol synthesis exist in the PAH(enu2) genetic mouse model for phenylketonuria (PKU), and if so, initiate studies determining if they might underlie the white matter pathology that exists in PKU forebrain. Gross sections and electron microscopy showed that select tracts were hypomyelinated in adult PKU mouse forebrain but not hindbrain. The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the rate controlling enzyme in the cholesterol biosynthetic pathway, was examined in isolated microsomes from forebrain, hindbrain, and liver to assess if perturbations in cholesterol biosynthesis were occurring. HMGR activity was normal in unaffected PKU hindbrain and was increased 2-4-fold in PKU liver compared to control. HMGR activity in the forebrain, however, was decreased by 30%. Because normal numbers of MBP-expressing glia (oligodendrocytes) were present, but the number of glia expressing HMGR was reduced by 40% in the hypomyelinated tracts, the decreased HMGR activity seemed to result from a down-regulation of HMGR expression in affected oligodendrocytes. Exposure of an oligodendrocyte-like glioma cell line to physiologically relevant elevated levels of Phe resulted in a 30% decrease in cholesterol synthesis, a 28% decrease in microsomal HMGR activity, and a 28% decrease in HMGR protein levels. Measurement of HMGR activity after addition of exogenous Phe to control brain microsomes revealed that Phe is a noncompetitive inhibitor of HMGR; physiologically relevant elevated levels of exogenous Phe inhibited HMGR activity by 30%. Taken together, these data suggest that HMGR is moderately inhibited in the PKU mouse. Unlike other cell types in the body, a subset of oligodendrocytes in the forebrain seems to be unable to overcome this inhibition. We speculate that this may be the cause of the observed pathology in PKU brain.

The response of sea bream following abrupt hyposmotic exposure

The response of a marine teleost, silver sea bream Sparus sarba, to abrupt hyposmotic exposure was investigated over a 120‐h period following direct exposure from sea water (SW, 33‰) to a hyposmotic environment of 6‰. Aspects of serum chemistry, stored metabolites and gill morphology were used to gain further insight into the biochemical, physiological and morphological alterations that take place following low salinity exposure of a marine fish. Rapid (<1 h) reductions in serum [Cl−] occurred while serum [Na+] exhibited only transient perturbations during initial exposure. Serum total [Ca] declined 24 h after exposure and returned to pre‐exposure levels by 120 h. Despite a tendency for muscle moisture to increase during the early stages of low salinity exposure to significant change could be detected. The response of the branchial chloride cell (CC) was rapid, with apical and fractional exposure area increasing after 6 h. The number of CCs exposed at the branchial surface reduced after 6 h but subsequently increased to elevated levels. Serum cortisol levels had increased three fold 1 h after hyposmotic exposure and stabilised at pre‐exposure levels within 12–24 h. Serum triiodothyronine (T3) levels exhibited a biphasic response, significantly elevating and decreasing after 3 and 6 h respectively. Significant post‐hyposmotic exposure elevations in serum glucose and protein occurred after 1 h, peaking at 3 h and returning to lower levels after 6 h. Total free ninhydrin reactive substances were significantly elevated 3 h post‐hyposmotic exposure, a phenomenon attributable to elevated levels of ammonia, alanine, arginine, glycine, isoleucine, lysine, methionine, phenylalanine, serine, taurine, threonine and valine. Of these substances, glycine, lysine, serine and taurine remained elevated for up to 12 h or longer. Serum urea levels elevated 1 h after exposure to 6%‰ and returned to SW levels 3 h post‐exposure. The relevance of these results is discussed within the context of current knowledge on the effects of hyposomotic adaptation on marine fish.