Conversion Of Phenylalanine Research Articles

Phenylalanine and Tyrosine Metabolism in Chronic Kidney Failure

In chronic kidney failure, there is impairment in the conversion of phenylalanine to tyrosine. As a result, tyrosine and the tyrosine/phenylalanine ratio are reduced in plasma and many tissues, and phenylalanine concentrations tend to be normal or slightly increased. Although animal studies indicate that the kidney is not a major contributor to the conversion of phenylalanine to tyrosine, human studies conducted in the postabsorptive state suggest that the kidney plays a major role in the uptake of phenylalanine and its hydroxylation and release as tyrosine. The human splanchnic bed in the postabsorptive state also displays net uptake of both phenylalanine and tyrosine and hydroxylation of substantial amounts of phenylalanine to form tyrosine. In chronic renal failure (CRF) patients, splanchnic uptake of tyrosine appears to be reduced in the postabsorptive state. After an amino acid meal, there is net release of phenylalanine from the splanchnic bed in normal subjects and to an even greater degree in CRF patients; tyrosine is released postprandially in both normal subjects and CRF patients. In the postabsorptive state, tyrosine release from the kidney is largely derived from the hydroxylation of phenylalanine. In CRF, the release of tyrosine from the kidney is reduced and this reduction may be marked with advanced CRF. These observations, as well as isotope studies indicating normal phenylalanine flux, reduced tyrosine flux and impaired conversion of phenylalanine to tyrosine in CRF patients, raise the possibility that tyrosine may be an essential amino acid in this condition. Further research will be necessary to answer this question. Oxidative stress, which often increases in CRF patients, may lead to increased formation of chlorotyrosine and nitrotyrosine in plasma proteins and of nitrotyrosine in the brain. Increased nitrotyrosine is also found in kidneys of patients with diabetic nephropathy or allograft nephropathy. Increased serum concentrations of oxidation products of phenylalanine have also been observed in patients with CRF. Impaired urinary excretion also may lead to accumulation of metabolic products of both phenylalanine and tyrosine in CRF. It is not known whether the elevated protein chlorotyrosine or nitrotyrosine or increased oxidative products of phenylalanine cause adverse metabolic or toxic effects in patients with CRF.

Conversion of Phenylalanine into Styrene by 2,4-Decadienal in Model Systems

2,4-Decadienal was heated under an inert atmosphere and in the presence of phenylalanine to investigate whether this secondary lipid oxidation product is a final product of lipid oxidation or it reacts with the amino acid. The results obtained showed that, in the presence of the alkadienal, the amino acid was degraded to styrene. This reaction was favored in dry systems at pH approximately 6 and in the absence of oxygen. If oxygen was present, the alkadienal was oxidized and the Strecker degradation of the amino acid was produced. The activation energy for the formation of styrene from phenylalanine was 150.4 kJ/mol. The reaction mechanism is suggested to be produced either by an electronic rearrangement of the imine produced between the aldehyde and the amino acid with the formation of styrene, 2-pentylpyridine, carbon dioxide, and hydrogen, or by Michael addition of the amino compound to the alkadienal followed by beta-elimination to produce the same compounds. Both reaction schemes were supported on the results obtained by studying both the degradation of phenylethylamine and phenylalanine methyl ester produced by 2,4-decadienal, and the formation of ethylbenzene in decadienal/phenylalanine reaction mixtures heated in the presence of platinum oxide. All these results suggest that, analogously to carbohydrates, certain lipid oxidation products may degrade appropriate amino acids to their corresponding vinylogous derivatives.

Substrate–protein interaction energy in the enzyme phenylalanine hydroxylase: DFT and ab initio results

The enzyme phenylalanine hydroxylase catalyzes the conversion of phenylalanine to tyrosine, which is in turn converted to a series of neurologically important compounds. These compounds can then inhibit the initial metabolism of phenylalanine by docking in the phenylalanine hydroxylase active site. We use second order Moller–Plesset theory (MP2) and several Density Functional Theory methods to estimate the contribution to the total substrate/protein interaction energy from the electrostatic and dispersion interactions between the substrate and Phe254 in the enzyme. We then study several mutations at the residue 254 site. Our results for one mutation, F254I, agree with an observed loss of function of phenylalanine hydroxylase. We predict several additional mutations that may also cause loss of function in the enzyme.

PKU is a reversible neurodegenerative process within the nigrostriatum that begins as early as 4 weeks of age in Pahenu2 mice

Phenylketonuria (PKU) is a common genetic disorder in humans that arises from deficient activity of phenylalanine hydroxylase (PAH), which catalyzes the conversion of phenylalanine to tyrosine. There is a resultant hyperphenylalanemia with subsequent impairment in cognitive abilities, executive functions and motor coordination. The neuropathogenesis of the disease has not been completely elucidated, however, oxidative stress is considered to be a key feature of the disease process. Hyperphenylalanemia also adversely affects monoaminergic metabolism in the brain. For this reason we chose to evaluate the nigrostriatum of Pah enu2 mice, to determine if alterations of monoamine metabolism resulted in morphologic nigrostriatal pathology. Furthermore, we believe that recent developments in adeno-associated virus (AAV)-based vectors have greatly increased the potential for long-term gene therapy and may be a viable alternative to dietary treatment for this metabolic disorder. In this study we identified neurodegenerative changes with regenerative responses in the nigrostriatum of Pah enu2 mice that are consistent with oxidative injury and occurred as early as 4 weeks of age. These neuropathologic changes were reversed following portal vein delivery of a recombinant adeno-associated virus-mouse phenylalanine hydroxylase-woodchuck hepatitis virus post-transcriptional response element (rAAV-mPAH-WPRE) vector to Pah enu2 mice and corresponded to rapid reduction of serum Phe levels.

Tomato aromatic amino acid decarboxylases participate in synthesis of the flavor volatiles 2-phenylethanol and 2-phenylacetaldehyde.

An important phenylalanine-derived volatile compound produced by plants is 2-phenylethanol. It is a major contributor to flavor in many foods, including fresh fruits, such as tomato, and an insect-attracting scent in roses and many other flowers. Despite the centrality of 2-phenylethanol to flavor and fragrance, the plant genes responsible for its synthesis have not been identified. Here, we describe a biosynthetic pathway for 2-phenylethanol and other phenylalanine-derived volatiles in tomato fruits and a small family of decarboxylases (LeAADC1A, LeAADC1B, and LeAADC2) that can mediate that pathway's first step. These enzymes each catalyze conversion of phenylalanine to phenethylamine and tyrosine to tyramine. Although tyrosine is the preferred substrate in vitro, phenylalanine levels in tomato fruits far exceed those of tyrosine, indicating that phenylalanine is a physiological substrate. Consistent with this view, overexpression of either LeAADC1A or LeAADC2 in transgenic tomato plants resulted in fruits with up to 10-fold increased emissions of the products of the pathway, including 2-phenylacetaldehyde, 2-phenylethanol, and 1-nitro-2-phenylethane. Further, antisense reduction of LeAADC2 significantly reduced emissions of these volatiles. Besides establishing a biosynthetic route, these results show that it is possible to change phenylalanine-based flavor and aroma volatiles in plants by manipulating expression of a single gene.

Renal amino acid, fat and glucose metabolism in type 1 diabetic and non-diabetic humans: effects of acute insulin withdrawal

The aim of this study was to test the hypothesis that type 1 diabetes alters renal amino acid, glucose and fatty acid metabolism. We studied five C-peptide-negative, type 1 diabetic subjects during insulin replacement (glucose 5.6 mmol/l) and insulin deprivation (glucose 15.5 mmol/l) and compared them with six non-diabetic subjects. Leucine, phenylalanine, tyrosine, glucose and palmitate tracers were infused after an overnight fast and samples were obtained from the renal vein, femoral vein and femoral artery. Insulin deprivation significantly increased whole-body fluxes (20-25%) of phenylalanine, tyrosine and leucine, and leucine oxidation (50%). Kidney contributed 5-10% to the whole-body leucine and phenylalanine flux. A net uptake of phenylalanine, conversion of phenylalanine to tyrosine (5 micromol/min) and net release of tyrosine (approximately 5 micromol/min) occurred across the kidney. Whole-body (three-fold) and leg (two-fold) leucine transamination increased but amino acid metabolism in the kidney did not alter with diabetes or insulin deprivation. Insulin deprivation doubled endogenous glucose production, renal glucose production was unaltered by insulin deprivation and diabetes (ranging between 100 and 140 micromol/min). Renal palmitate exchange was unaltered by insulin deprivation. In conclusion, kidney post-absorptively accounts for 5-10% of whole-body protein turnover, 15-20% of leucine transamination and 10-15% of endogenous glucose production, and actively converts phenylalanine to tyrosine. During insulin deprivation, leg becomes a major site for leucine transamination but insulin deprivation does not affect renal phenylalanine, leucine, palmitate or glucose metabolism. Despite its key metabolic role, insulin deprivation in type 1 diabetic patients does not alter many of these metabolic functions.

411. The Metabolic Basis of Sexual Dimorphism in PKU Mice after PAH Gene Treatment

Phenylketonuria (PKU) is a metabolic disorder secondary to a hepatic deficiency of phenylalanine hydroxylase (PAH) that predisposes affected individuals to the development of severe mental retardation. PAH is the enzyme that catalyzes the conversion of phenylalanine to tyrosine, and its deficiency leads to hyperphenylalaninemia as well as hypopigmentation in patients. We have previously reported a transgene delivery system based on phiBT1 bacteriophage integrase that result in targeted insertion of transgenes into the mammalian genome, and the use of it to transfer a CAG-driven mouse PAH cDNA expression cassett in the hepatocytes of PKU mice.

Neonatal Screening by Tandem Mass Spectrometry

After completing this article, readers should be able to: 1. List the major categories of diseases detectable by expanded newborn screening. 2. Give examples of the benefits and limitations of screening by tandem mass spectrometry. 3. Describe the appropriate follow-up for an abnormal screening result. The screening of newborns for inherited metabolic disorders is a well-established public health activity, first implemented in the early 1960s for the presymptomatic identification of patients who have phenylketonuria (PKU). Since then, significant technological advances, most importantly the development of tandem mass spectrometry (MS/MS), have enabled the detection of an increasing number of metabolic disorders in newborn blood. This article reviews the basic technology of MS/MS and its application to newborn screening, with the aim of highlighting the benefits and pointing out some limitations of this powerful technology. Early screening for PKU used the bacterial inhibition assay of Guthrie, in which inhibition of bacterial growth on an agar medium, introduced by the addition of a phenylalanine analog, was overcome by the presence of dried blood discs containing high levels of phenylalanine (such as from a PKU patient). This approach later was adapted for the detection of leucine in patients who have maple syrup urine disease (MSUD), methionine in patients who have classic homocystinuria, and tyrosine in patients who have tyrosinemia. Over the years, other techniques have been implemented to screen for additional disorders, including congenital hypothyroidism, biotinidase deficiency, galactosemia, hemoglobinopathies, and congenital adrenal hyperplasia. The classic criteria used to determine whether a disorder is suitable for screening include: 1) the disorder is clinically and biochemically well defined, 2) it is associated with significant morbidity or mortality, 3) an effective treatment is available, and 4) there is a simple and safe screening test. (1) By the early 1990s, all states offered screening for at least PKU and congenital hypothyroidism, …

1097. Complete and Persistent Phenotypic Correction of Phenylketonuria in Mice by Site-Specific Genome Integration of Phenylalanine Hydorxylase cDNA

Top of pageAbstract Phenylketonuria (PKU) is a metabolic disorder that predisposes affected children to severe and irreversible mental retardation, and is secondary to a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH), that catalyses the conversion of phenylalanine to tyrosine. In an attempt to correct this metabolic deficiency, we explored the potential of using a bacteriophage integrase system to achieve site-specific genome integration of murine PAH cDNA in the livers of Phenylketonuric mice. The integrase from Streptomyces phage |[Phi]|BT1 mediates efficient recombination between the phage attachment site attP and the bacterial attachment site attB, results in the integration of phage DNA into the bacterial genome in a site-specific manner. Here we showed that this phage enzyme functioned efficiently both in Escherichia coli and mouse cells in vitro. Several naturally occurring |[ldquo]|pseudo|[rdquo]| attP sites in the mouse genome, which contain partial sequence identity to the wild type attP site and are recognizable by the phage integrase, have been identified and molecularly characterized. In the presence of the phage integrase, naked plasmid DNA containing a wild-type attB site can be integrated specifically into these pseudo attP site permanently both in culture mouse cells and in the livers of mice. We further demonstrated that the addition of nuclear localization signal sequences to the C-terminus of the phage integrase enzyme enhanced the efficiency for transgene integration into the mouse genome. Using this phage integration system we delivered mouse PAH cDNA to the livers of PKU mice by hydrodynamic injection of plasmid DNA and showed that serum phenylalanine levels in the treated mice decreased significantly and persistently. After 3 applications, serum phenylalanine levels in all treated mice were reduced to the normal range and remained stable for more than 6 weeks. Furthermore, their fur color also changed from gray to black, indicating the reconstitution of melatonin biosynthesis as a result of available tyrosine derived from phenylalanine hydroxylation. Thus the bacterio-phage integrase represents an effective site-specific genome integration system in mammalian cells and can be of great value in plasmid DNA-mediated gene therapy of a multitude of genetic disorders.

Effect of temperature, pH, and metals on the stability and activity of phenylalanine hydroxylase from Chromobacterium violaceum

Phenylalanine hydroxylase (PAH) is a non-heme iron dioxygenase catalyzing the conversion of phenylalanine to tyrosine and is present in both prokaryotic and eukaryotic organisms. A relatively simple PAH is expressed by Chromobacterium violaceum, a gram-negative bacterium found in tropical and subtropical regions. The effects of temperature, pH and metals on the stability and catalytic activity of Chromobacterium violaceum PAH were determined by steady-state kinetics, circular dichroism (CD) and differential scanning calorimetry (DSC). The k cat and K M for phenylalanine were determined between 7 and 40 °C. The K M remained constant between 20 and 40 °C but rapidly increased below 20 °C. The half-life of the enzyme at 47 °C is 66 ± 4 min in the presence of Fe(II) and 8 ± 1 min in the presence of EDTA. The melting temperature of the protein determined by CD and DSC is 53 ± 2 °C in the presence of EDTA and 63 ± 2 °C in the presence of Fe(II). Co(II) stabilizes the enzyme ( T m = 63 ± 2 °C) and inhibits the catalytic activity by displacing iron from the active site. The optimum pH for catalytic activity and stability is 7.4. In conclusion, PAH is adapted for optimal phenylalanine binding at temperatures above 20 °C and Fe(II) enhances the resistance of the enzyme to thermal denaturation.

Speed of decision-making and set-switching: Subtle executive deficits in children with treated phenylketonuria

Phenylketonuria (PKU) is an inborn error of protein metabolism affecting about 1 in 10,000 children in which conversion of dietary phenylalanine into tyrosine is impaired. Unmetabolised phenylalanine accumulates in body tissue and is neurotoxic to the developing organism. The untreated child is almost invariably brain damaged and severely learning disabled. These gross neuropathic effects can be prevented by early implementation of a phenylalanine-restricted diet. Nonetheless, treated children may suffer subtle neuropsychological deficits in the form of mild executive dysfunction, possibly because of depletion of the neurotransmitter dopamine in prefrontal cortex. Dopamine is a downstream metabolite in the phenylalanine pathway but, because of the phenylalanine-tyrosine block, may be in short supply in treated PKU. We investigated the dopamine/executive hypothesis by comparing the performance of 22 early and continuously treated PKU children with controls, matched for non-executive ability, on the test of everyday attention for children. We found the PKU children did not show deranged selective or sustained attention, but were significantly slow on set-switching (creature counting), stop-go (walk/don’t walk), stroop-like (opposite worlds) and target-detection (sky search) tasks. These findings add weight to a growing body of evidence suggesting that rapid decision-making is subtly impaired in PKU, possibly due to prefrontal neurotransmitter insufficiency, and that speeded strategic and choice-reaction tests are particularly sensitive tools for investigating executive dysfunction. How these findings translate into daily domestic and scholastic functioning has yet to be established.

Amino acid and protein metabolism in chronic renal failure

Recent studies in humans have demonstrated the importance of the kidney in amino acid metabolism. In particular, it has been shown that conversion of phenylalanine to tyrosine occurs in the kidney and at a rate similar to that of the liver. Furthermore, unlike the liver, the kidney releases more tyrosine than it produces. In this review, the importance of this metabolic role of the kidney is discussed in context of chronic renal failure. The effect of renal failure on skeletal muscle protein metabolism is also discussed.

Immunolocalization of phenylalanine ammonia-lyase and cinnamate-4-hydroxylase in differentiating xylem of poplar

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) and cinnamate-4-hydroxylase (C4H; EC 1.14.13.11) are pivotal enzymes involved in lignification. We synthesized peptides as the epitopes according to the amino acid sequences of these enzymes, coupled them with hemocyanin, and injected them into mice. The antiserums against peptides of PAL and C4H specifically detected PAL and C4H in the crude enzymes extracted from differentiating xylem of poplar, respectively. PAL and C4H were localized in differentiating xylem of poplar. PAL labeling was mainly localized in the cytosol, and somewhat localized on the rough-endoplasmic reticulum (r-ER) and the Golgi apparatus. In contrast, C4H was mainly observed on r-ER and the Golgi apparatus. These findings suggest that conversion of phenylalanine to cinnamic acid occurs in the cytosol and the following reaction occurs near the membrane of r-ER and the Golgi apparatus. The possibility of coordinated localization of PAL and C4H is discussed. To cite this article: T. Sato et al., C. R. Biologies 327 (2004).

Morning preprandial plasma ghrelin and catecholamine concentrations in patients with phenylketonuria and normal controls: evidence for catecholamine-mediated ghrelin regulation.

Patients with phenylketonuria (PKU) have a diet-controlled deficiency in the conversion of phenylalanine (Phe) to tyrosine (Tyr), leading to decreased production of noradrenaline, adrenaline, and dopamine. Poor diet control results in high plasma Phe and low plasma Tyr and catecholamine concentrations. Ghrelin, a recently described gastrointestinal hormone that is elevated in the fasting state and low in the fed state, is considered a major appetite-stimulating hormone, possibly involved in the generation of obesity and insulin resistance. We evaluated morning preprandial plasma ghrelin levels in 14 diet-controlled and 15 poorly controlled PKU patients and 20 age- and body mass index (BMI)-matched healthy children (controls) and correlated its concentrations with those of Phe and catecholamines as well as with their BMI and 24-h nutrient intake. Plasma ghrelin levels were measured by RIA, plasma catecholamine concentrations were determined by HPLC with electrochemical detection, and Phe and Tyr levels were measured in an amino acid analyzer. The ghrelin concentration (744 +/- 25 ng/liter) in diet-controlled patients did not differ from that in controls (802 +/- 26 ng/liter; P > 0.05). On the contrary, the ghrelin concentration was significantly reduced in poorly controlled patients (353 +/- 23 ng/liter; P < 0.0001). Ghrelin correlated negatively with Phe in all three groups, whereas it correlated positively with catecholamine levels and energy intake and negatively with BMI only in diet-controlled patients and controls. We conclude that ghrelin secretion may receive positive direct or indirect input from catecholamines. The absence of a correlation between ghrelin and catecholamines, energy intake, or BMI in PKU patients on an inadequate diet may be due to dysregulation of their neuroendocrine system and might be affected by high Phe levels in the stomach and/or central nervous system.

877. Evidence for Dominant-Negative Interference in the Pahenu2 Mouse Model of PKU

Top of pageAbstract Phenylketonuria (PKU) is a metabolic disease caused by the deficiency of the enzyme phenylalanine hydroxylase (PAH). PAH is the obligate enzyme in the conversion of phenylalanine (phe) to tyrosine (tyr). Even though a successful treatment modality has been in place since the early 1970s, its success has led to an increase in maternal PKU syndrome. The syndrome results in a serious increase in risks of birth defects to children born of PKU women, especially when the mothers' blood phe levels do not reach a normal range before or very early in pregnancy. A mouse model for PKU was created in 1993 by ENU mutagenesis of male germ cells. The induced missense mutation, F263S, is located in exon 7 of the gene and renders the enzyme inactive. The mouse has “classic” PKU with elevated blood phe levels, cognitive deficiencies, and it exhibits maternal PKU syndrome. We have successfully treated these mice using liver-directed gene therapy with AAV2 containing the mouse PAH gene driven by a Chicken-β-actin hybrid promoter. However unusually high vector doses were needed to achieve normal blood phe levels (see abstract by Laipis et. al.). Message levels were measured in the mice by Northern blot: while PAH is very abundant in the liver, no differences were detected between wild type, heterozygote and null mice. At the protein level, wild type mice have two times as much protein as heterozygote and null mice. When considering that the PAH enzyme is a tetramer, or a dimer of dimers, it is possible to explain the heterozygote protein levels by assuming simple unstability of the missense protein. However, null mice show significant protein levels, about equivalent to heterozygotes, thus the F263S protein cannot be completely unstable. We hypothesize that the missense protein could tetramerize with the introduced protein during gene therapy in a dominant-negative fashion, reducing the total potential activity and thus the effectiveness of the treatment. To investigate this hypothesis, we performed calcium phosphate transfections on HEK-293 cells using plasmid DNA under the CBA promoter containing either the normal mPAH or mPAH-F263S gene. When mixing a one to one ratio of mPAH to mPAH-F263S, the PAH specific activity in the cell lysate drops to one half that observed with only mPAH. The reduction in PAH activity strongly suggests that the monomers can interact. A ribozyme was previously designed to cleave the mouse PAH mRNA. The ribozyme was tested in vitro and then cloned into an AAV vector. Simultaneously a ribozyme-resistant form of mPAH was cloned with strategically placed silent mutations (named mPAH-HdRz). In cell transfection experiments, the one to one combination of mPAH-HdRz and mPAH-F263S also drops to approximately one half the activity seen in mPAH-HdRz alone. When the ribozyme is added to the same combination, PAH activity is restored to near normal levels. Since the ribozyme has been shown not to cleave the mPAH-HdRz mRNA, we conclude that the combination of mPAH and mPAH-F263S proteins cause the observed reductions in total PAH activity. Dominant-negative interference is thus the most likely cause of the high vector doses needed to cure the Pahenu2 mice. Further work is underway to confirm the hypothesis in vivo.

White Matter Lesions in Phenylketonuria: Evaluation with Magnetic Resonance Imaging and Magnetic Resonance Spectroscopy

Phenylketonuria (PKU) is a congenital metabolic autosomic recessive disease, caused by a deficit in the liver of phenyl-alanine hydroxylase, the enzyme responsible for conversion of phenyl-alanine (PHE) into tyrosine. Reduction of this enzymatic activity is responsible for increased phenyl-alanine in blood and tissues and, above all, in brain. Accumulation of PHE causes neural damage which produces a typical clinical picture with mental retardation, psychiatric symptoms and epilepsy. It is now possible to diagnose this disease early (with neonatal screening), before irreversible clinical symptoms reflecting central nervous system injury appear. Early diagnosis allows timely onset of therapy (the only possible) consisting of a special diet with reduced intake of PHE (integrated with a mix of aminoacids) whose objective is to keep levels of PHE low in the blood (3–6 mg/dl). Magnetic Resonance Imaging (MRI) is the elective diagnostic tool to evaluate in vivo the involvement of the brain in PKU. Previous MRI morphological studies in patients with PKU have reported various focal symmetrical lesions in periventricular white matter (especially parieto-occipital) of patients with PKU with PHE blood values higher than 10 mg/dl. These lesions, whose importance is not yet clear, seem to represent a reversible structural alteration of myelin, since they regress if blood PHE decreases. Proton magnetic resonance spectroscopy (1H-MRS) can measure in vivo brain metabolites which could help determine the nature of white matter lesions. In particular, changes in NAA (a marker of neuronal integrity) or mI (a potential astrocytic marker) could point to possible neurochemical dysfunction, whereas Cho levels may parallel the degree of the tissue myelination. The purpose of the present study was to evaluate morphologically and biochemically the regional specificity of white matter lesions with structural MRI and with 1H-MRSI. The study included 12 patients with PKU ten to 42 years of age. All patients underwent structural MRI scans while eight of them were also studied with 1H-MRSI. Structural MRI lesions in white matter were analyzed both qualitatively (signal intensity) and quantitatively (location and extension). 1H-MRSI metabolites were measured as the ratio of the area under each peak: NAA/Cr, NAA/Cho, Cho/Cr. Analysis of location and extension of the lesion on structural MRI data showed limited involvement of parieto-occipital white matter in three cases (with isointense or vaguely hypointense lesions in T1, and moderately hyperintense lesions in T2); medium involvement in six cases (with fairly hypointense or isointense lesions in T1, fairly or moderately hyperintense lesions in T2); serious involvement in three cases (with isointense or fairly hypointense lesions in T1, and fairly hyperintense lesions in T2). As for 1H-MRSI data, ANOVA showed a significant reduction of NAA/Cho and increase in Cho/Cr in white matter lesions, but no change in NAA/Cr. No correlation was found between clinical parameters and morphological or spectroscopic data. In conclusion, our morphological MRI data confirmed the presence of multiple signal alterations, focal and symmetrical, in deep periventricular white matter (especially posterior), with occasional involvement of subcortical white matter. However, these lesions do not seem to be strongly predictive of clinical outcome. 1H-MRSI data suggest increased Cho levels in white matter lesions. Since Cho is thought to reflect membrane turnover, these data may support the demyelinating nature of lesions, consistent with earlier post mortem studies.

Simultaneous measurement of phenylalanine and tyrosine in phenylketonuric plasma and dried blood by high-performance liquid chromatography

Phenylketonuria (PKU) is a disorder characterized by an interruption in the conversion of phenylalanine to tyrosine, a reaction catalyzed by phenylalanine hydroxylase (PAH). Animal models of PKU used in this study were induced by daily subcutaneous injections of pups with α-methylphenylalanine plus phenylalanine in utero and postnatally from day 4 to day 14. Dry blood and plasma were utilized to measure phenylalanine concentration in PKU rats. The results indicated that the concentration of phenylalanine is higher and more stable in plasma than dry blood. Precolumn derivatization of dried blood and plasma free amino acids were conducted with phenylisothiocyanate (PITC). The phenylthiocarbamyl (PTC) derivatives were separated on a reversed-phase C-18 column (15 cm×4.6 mm). A gradient high-performance liquid chromatography method with two eluents, 0.1 M sodium acetate buffer and 100% acetonitrile was developed to facilitate the separation of nine amino acids within 11 min. Tyrosine and phenylalanine eluted the column at 5.4 and 9.4 min, respectively. This method provides a quick and reliable technique for neonatal screening.

Hepatic tyrosine aminotransferase activity is affected by chronic heat stress and dietary tyrosine in broiler chickens

1. Two experiments were conducted to investigate the impact of high temperature and dietary tyrosine (Tyr) content on performance and activity of hepatic tyrosine aminotransferase (EC 2.6.1.5.), an enzyme that catalyses the first step in the metabolic degradation of Tyr in broiler chickens. 2. Two-week-old birds were allocated to one of three temperature treatments: 24 ° C (control), 36 ° C (heat stress, HS) and 24 ° C pair-fed (24PF) for 2 weeks and fed on diets containing 100% (Experiment 1) and 50, 100 and 200% (Experiment 2) of the NRC requirement for Tyr. 3. In Experiment 1, exposure of chickens to 36 ° C for 2 weeks caused significant increase in hepatic tyrosine aminotransferase activity but no significant change in activity of hepatic phenylalanine 4-hydroxylase (EC 1.14.16.1) (an enzyme that catalyses conversion of phenylalanine to Tyr) compared with the 24PF birds. No significant changes attributable to heat stress were detected in hepatic glutamic-oxaloacetic transaminase (EC 2.6.1.1) activity. 4. In Experiment 2, heat stress caused reductions in weight gain and feed intake in chickens on all diets, compared with their control counterparts. Hepatic tyrosine aminotransferase activity was increased by heat stress compared with their 24PF counterparts in chickens fed on the 100 and 200% Tyr diets, while in chickens fed the 50% Tyr diet, it was reduced by heat stress. 5. From these results, it is suggested that hepatic tyrosine aminotransferase activity is affected by heat stress and dietary Tyr content and the increased tyrosine aminotransferase activity with, in part, relatively low phenylalanine hydroxylase activity in hepatic tissues may be involved in the Tyr metabolism characteristic of heat-stressed chickens.

Oxidative stress induced by phenylketonuria in the rat: Prevention by melatonin, vitamin E, and vitamin C.

Phenylketonuria (PKU) is an autosomal recessive disorder caused by a deficiency of the phenylalanine hydroxylation system and is characterized by a block in the conversion of phenylalanine (PHE) to tyrosine. We examined the effects of maternal hyperphenylalaninemia on the morphological and biochemical development of pup rat brain and cerebellum. In our model of PKU we evaluated a number of markers of oxidative stress such as Ehrlich adducts formation, lipid peroxidation, as well as the levels of reduced and oxidized glutathione, and the activities of the enzymes glutathione peroxidase and glutathione reductase. We also studied the expression of heme-oxigenase-1 and mitogen-activated protein kinase 1/2 (MAPK 1/2) as additional markers of oxidative stress. We demonstrate that PKU strongly increased most of the oxidative stress markers studied and induced significant morphological damage. We also showed that daily administration of melatonin (20 mg/kg BW), vitamin E (30 mg/kg BW), and vitamin C (30 mg/kg BW) until delivery prevented the oxidative biomolecular damage in the rat brain and cerebellum. Although no significant differences were observed among the antioxidants studied, it should be noted that the doses of melatonin were less than those for vitamins E and C. We conclude that PKU induces a clear state of oxidative stress that is somehow involved in the brain and body damage occurring in this inborn error. Moreover, melatonin and other antioxidants are capable of preventing completely the damage induced by PKU.

Identification and characterization of a novel liver-specific enhancer of the human phenylalanine hydroxylase gene.

The liver-specific phenylalanine hydroxylase catalyzes the conversion of phenylalanine to tyrosine. Genetic defects in the gene result in the autosomal recessive disorder phenylketonuria. We have identified a phenylalanine hydroxylase mutation, designated as -4173_-407del, in a hyperphenylalaninemic patient with a 3.7-kb deletion in the 5'-flanking region of the phenylalanine hydroxylase gene. Characterization of the deleted sequence has led to the identification of a novel liver-specific DNase I hypersensitive site located 3.3 kb upstream of the RNA initiation site of the phenylalanine hydroxylase gene. We show that this site comprises a liver-specific enhancer with cAMP responsiveness. We further show by mutational analysis that the enhancer carries a major hepatocyte nuclear factor-1-binding site important for the enhancer function but not for cAMP responsiveness. In transient transfection assays with a reporter gene, we demonstrate that a phenylalanine hydroxylase plasmid construct carrying the -4173_-407del mutation is severely impaired in phenylalanine hydroxylase transcriptional activity. Our data indicate that the 3.7-kb deletion uncovered in the genomic DNA of the phenylketonuria proband is linked to the observed phenylketonuria phenotype as a result of a deletion of the newly identified liver-specific enhancer. Our systematic approach to the analysis and subsequent discovery of the novel deletion mutation may be applicable to the search for other mutations in the 5' regulatory region of phenylalanine hydroxylase and other genes.