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.

Variations in genotype-phenotype correlations in phenylketonuria patients.

Phenylalanine hydroxylase deficiency is a trait inherited in an autosomal recessive pattern; the associated phenotype varies considerably. This variation is mainly due to the considerable allelic heterogeneity in the phenylalanine hydroxylase enzyme locus. We examined the genotype-phenotype correlation in 54 phenylketonuria (PKU) patients from Minas Gerais, Brazil. Two systems were used. The first was a phenotype prediction system based on arbitrary values (AV) attributed to each mutation and the second was a correlation analysis. An AV was assigned to each mutation: AV = 1 for classical PKU mutation; AV = 2 for moderate PKU mutation; AV = 4 for mild PKU mutation, and AV = 8 for non-PKU hyperphenylalaninemia mutation. The observed phenotype for AV analysis was the clinical diagnosis established by the overloading phenylalanine test. Among the 51 PKU patients that we analyzed based on this trait, in 51% the predicted phenotype did not match the observed phenotype; the highest degree of concordance was found in patients with null/null genotypes. The genotype was observed to be a good predictor of the clinical course of the patients and significant correlations were found between phenylalanine values at first interview and predicted residual activity, genotype and arbitrary value sum.

Molecular characterization of phenylketonuria in South Brazil.

Phenylketonuria (PKU) is an autosomal recessive disorder due to phenylalanine hydroxylase (PAH) deficiency. The PAH gene, located at 12q22-q24.1, includes about 90kb and contains 13 exons. To date, more than 420 different alterations have been identified in the PAH gene. To determine the nature and frequency of PAH mutations in PKU patients from South Brazil, mutation analysis was performed on genomic DNA from 23 unrelated PKU patients. The 13 exons and flanking regions of the PAH gene were amplified by PCR and the amplicons were analyzed by single strand conformation polymorphism (SSCP). Amplicons that showed abnormal migration patterns were analyzed by restriction endonuclease digestion and/or sequencing. Twenty-two previously reported mutations were identified including R261X, R408W, IVS2nt5g-->c, R261Q, and V388M. Polymorphisms were observed in 48.8% of the PKU patients, the most frequent being IVS2nt19t-->c, V245V, and IVS12nt-35c-->t. In addition, two novel sequence variants were identified: 1378g-->t in the 3(')-untranslated region in exon 13 which may be disease-causing and an intron 12 polymorphism, IVS12nt-15t-->c. The mutation spectrum in the patients from Southern Brazil differed from that observed in patients from other Latin American countries and further defined the molecular heterogeneity of this disease.

Hyperphenylalaninemia reduces creatine kinase activity in the cerebral cortex of rats.

Phenylketonuria (PKU) is a metabolic disorder accumulating phenylalanine (Phe) and its metabolites in plasma and tissues of the patients. Considering that phenylalanine is the main neurotoxic metabolite, and brain energy homeostasis seems to be affected in phenylketonuria, our main objective was to investigate the effect of acute and chronic hyperphenylalaninemia (HPA) on creatine kinase (CK) activity in brain cortex of Wistar rats. Hyperphenylalaninemia was induced by subcutaneous administration of 5.2 micromol phenylalanine + 2.4 micromol alpha-methylphenylalanine (phenylalanine hydroxylase (PAH) inhibitor)/g of body weight. We also investigated the in vitro effect of phenylalanine and/or alpha-methylphenylalanine on creatine kinase activity in the brain cortex of non-treated rats. The results showed that phenylalanine significantly inhibited creatine kinase activity in vitro and reduced the enzyme activity in vivo. Considering the importance of creatine kinase for the maintenance of energy homeostasis in brain, if this enzyme inhibition also occurs in phenylketonuric patients, it is possible that creatine kinase inhibition may be one of the mechanisms by which phenylalanine is neurotoxic in phenylketonuria.

Phenylalanine and phenylpyruvate inhibit ATP diphosphohydrolase from rat brain cortex.

The main objective of the present study was to characterize the inhibition by phenylalanine and phenylpyruvate of ATP diphosphohydrolase activity in synaptosomes from the brain cortex of rats. This enzyme participates together with a 5'-nucleotidase in adenosine formation from the neurotransmitter, ATP, in the synaptic cleft. The inhibition of ATP diphosphohydrolase was competitive for nucleotide hydrolysis but 5'-nucleotidase was not affected by these metabolites. Furthermore, the two substances inhibited enzyme activity by acting at the same binding site. If the enzyme inhibition observed in vitro also occurs in the brain of PKU patients, it may promote an increase in ATP levels in the synaptic cleft. In this case, the neurotoxicity of ATP could possibly be one of the mechanisms leading to the characteristic brain damage of phenylketonuria.

Molecular basis of phenylketonuria in Cuba.

In this study we report the mutation analysis performed in Cuban PKU patients using DGGE and direct sequencing. Sixteen different mutations have been detected, which account for 91% of the total mutant alleles. Haplotype analysis and genealogical data support the European (mainly Spanish) origin of the mutations. Two mutations were found at unexpectedly high frequencies, E280K and R261Q, possibly due to consanguinity and genetic drift, among other factors. Hum Mutat 18:252, 2001.

Reduced Na(+), K(+)-ATPase activity in erythrocyte membranes from patients with phenylketonuria.

Na(+), K(+)-ATPase activity was determined in erythrocyte membranes from 12 phenylketonuric patients of both sexes, aged 8.8 +/- 5.0 y, with plasma phenylalanine levels of 0.64 +/- 0.31 mM. The in vitro effects of phenylalanine and alanine on the enzyme activity in erythrocyte membranes from healthy individuals were also investigated. We observed that Na(+), K(+)-ATPase activity was decreased by 31% in erythrocytes from phenylketonuric patients compared with normal age-matched individuals (p < 0.01). We also observed a significant negative correlation between erythrocyte Na(+), K(+)-ATPase activity and plasma phenylalanine levels (r = -0.65; p < 0.05). All PKU patients with plasma phenylalanine levels higher than 0.3 mM had erythrocyte Na(+), K(+)-ATPase activity below the normal range. Phenylalanine inhibited in vitro erythrocyte Na(+), K(+)-ATPase activity by 22 to 34%, whereas alanine had no effect on this activity. However, when combined with phenylalanine, alanine prevented Na(+) K(+)-ATPase inhibition. Considering that reduction of Na(+), K(+)-ATPase activity occurs in various neurodegenerative disorders leading to neuronal loss, our previous observations showing a significant reduction of Na(+), K(+)-ATPase activity in brain cortex of rats subjected to experimental phenylketonuria and the present results, it is proposed that determination of Na(+), K(+)-ATPase activity in erythrocytes may be a useful peripheral marker for the neurotoxic effect of phenylalanine in phenylketonuria.

Mutations of the phenylalanine hydroxylase (PAH) gene in Brazilian patients with phenylketonuria.

In the present study, 115 Brazilian families with phenylketonuria (PKU), mainly from the Southeast of the country, were studied using three laboratory methods (DGGE, SSCP, and sequencing). All 13 exons of the PAH gene were analyzed, including the splicing sites and the promoter region. We identified 50 distinct mutations and characterized 91% of the mutant alleles. The five most prevalent mutations of the 50 mutations identified (50% of the PKU alleles) were IVS10nt-11G-->A (17.4%), followed by R261Q (12.2%), V388M (9.1%), R252W (6.5%), and R270K (4.8%). The other mutations were rare. The mutation spectrum included 10 novel mutations (IVS5nt-54A-->G, IVS6nt17G-->T, E205A, F240S, K274E, I318T, L321L, C357G, IVS11nt17G-->A, and S411X). To characterize the origin and distribution of the PAH alleles we determined the association between the detected mutations and the PCR/RFLP haplotypes and VNTR alleles located on the PAH gene. For those patients whose mutant alleles were detected, we calculated the correlation with pretreatment phenylalanine levels, thus establishing a genotype/phenotype correlation. The present results confirm the marked heterogeneity observed at the PAH locus and contribute to the understanding of the distribution and frequency of PKU mutations in the Brazilian population.

Ten novel mutations in the phenylalanine hydroxylase gene (PAH) observed in Brazilian patients with phenylketonuria.

In the present study we report on the identification of ten novel mutations in the phenylalanine hydroxylase (PAH) gene of Brazilian patients with phenylketonuria (PKU): IVS5-54A>G, IVS6+17G>T, E205A, F240S, K274E, I318T, L321L, C357G, IVS11+17G>A and S411X. These mutations were detected during the characterization of the PAH genotypes of 115 patients with PKU from the southeast region of Brazil. The results obtained confirm the high heterogeneity of the PAH gene and provide information about the distribution of PKU mutations in the Brazilian population.

Effect of phenylalanine and p-chlorophenylalanine on Na+, K+-ATPase activity in the synaptic plasma membrane from the cerebral cortex of rats.

Na+, K+-ATPase activity was measured in synaptic plasma membrane from cerebral cortex of Wistar rats subjected to experimental phenylketonuria, i.e., chemical hyperphenylalaninemia induced by subcutaneous administration of 5.2 micromol phenylalanine / g body weight (twice a day) plus 0.9 micromol p-chlorophenylalanine / g body weight (once a day). The treatment was performed from the 6th to the 14th postpartum day and rats were killed 12 h after the last injection. Synaptic plasma membrane from cerebral cortex was prepared by a discontinuous density sucrose gradient for Na+, K+-ATPase activity determination. The results showed that the enzyme activity was decreased by 30% in animals subjected to experimental phenylketonuria when compared to control. The in vitro effects of the drugs on Na+, K+-ATPase activity were also investigated. Phenylalanine and p-chlorophenylalanine inhibited the enzyme activity and this inhibition was reversed by alanine. In addition, competition between phenylalanine and p-chlorophenylalanine for binding to the enzyme was observed, suggesting a common binding site for these substances. Our results suggest that reduction of Na+, K+-ATPase activity may be one of the mechanisms related to the brain dysfunction observed in human PKU.

Platelet Na+, K+-ATPase activity as a possible peripheral marker for the neurotoxic effects of phenylalanine in phenylketonuria.

The in vitro effects of phenylalanine or alanine alone or combined on Na+, K+-ATPase activity in membranes from human platelets were investigated. The enzyme activity was assayed in membranes prepared from platelet-rich plasma of healthy donors. Phenylalanine or alanine were added to the assay to final concentrations of 0.3 to 1.2 mM, similar to those found in plasma of phenylketonuric patients. Phenylalanine inhibited Na+, K+-ATPase activity by 20-50% [F(4,25)=11.47 ; p<0.001]. Alanine had no effect on Na+, K+-ATPase activity but when combined with phenylalanine prevented the enzyme inhibition. These results, allied to others previously reported on brain Na+, K+-ATPase activity, may reflect a general inhibitory effect of phenylalanine on this important enzyme activity. Therefore, it is possible that measurement of Na+, K+-ATPase activity in platelets from PKU patients may be a useful peripheral marker for the neurotoxic effects of phenylalanine.

Alanine prevents the decrease of Na+,K+-ATPase activity in experimental phenylketonuria.

Our objective was to investigate the effect of alanine administration on Na+,K+-ATPase activity in cerebral cortex of rats subjected to chemically-induced phenylketonuria. Wistar rats were treated from the 6th to the 28th day of life with subcutaneous injections of either 2.6 micromol alanine or 5.2 micromol phenylalanine plus 2.6 micromol alpha-methylphenylalanine per g body weight or phenylalanine plus alpha-methylphenylalanine plus alanine in the same doses or equivalent volumes of 0.15 M saline. The animals were killed on the 29th or 60th day of life. Synaptic plasma membrane from cerebral cortex was prepared for Na+,K+-ATPase activity determination. The results showed that alanine injection prevents the decrease of Na+,K+-ATPase activity in animals subjected to experimental phenylketonuria. Therefore, in case the same effects are achieved with ingested alanine, it is possible that alanine supplementation may be an important dietary adjuvant for phenylketonuric patients.

Plasma zinc, copper, and erythrocyte superoxide dismutase in children with phenylketonuria.

Children with phenylketonuria (PKU) are treated with semisynthetic diets restricted in phenylalanine (PHE). The formulae must supply those trace elements and vitamins that are usually supplied by whole protein foods. We studied the effects of phenylalaline restricted diets in 42 children with PKU (P) and 31 normal (N) children, aged 1-12 y, divided into two groups (below and above 7 y). Plasma zinc and copper were analyzed by means of atomic spectrophotometry, and superoxide dismutase (CuZnSOD) activity was measured in erythrocytes, through NBT inhibition and its profile, as determined by isoelectric focalization. Plasma zinc of PKU children > or = 7 years old was significantly lower than that in the control group (17 mumol/L versus 20 mumol/L) but still within the normal range; in children < 7 years no substantial differences were found between the two groups. Plasma copper was not statistically different between PKU and normal children. Qualitative activity of CuZnSOD presented the same electrophoretic profile in both normal and PKU. Quantitative activity was not different in both P (1210 U/g Hb < 7 versus 1328 U/g hemoglobin (Hb) > or = 7) and N (1675 U/g Hb < 7 versus 1367 U/g Hb > or = 7). We concluded that children with PKU presented normal mean levels of zinc and copper, with preserved function, measured by enzyme activity.

New approaches to the treatment of phenylketonuria.

Phenylketonuria (PKU) is the most common of all aminoacidopathies and is caused by autosomal recessive deficiency of the hepatic phenylalanine hydroxylase system. The diagnosis of PKU should be multifactorial and based on a protein overload test that reveals increased plasma phenylalanine levels during the ingestion of a normal diet, a phenylalanine tolerance test, and in vitro and in vivo activity of the liver enzyme. An individualized diagnosis that characterizes the severity of the disease in each patient provides objective and effective criteria for the dietary treatment of each particular case.

Molecular basis of phenylketonuria in Venezuela: presence of two novel null mutations.

This report describes the mutational spectrum and linked haplotypes of the phenylalanine hydroxylase gene in Venezuela. In this study, we have detected European mutations such as IVS10nt-11, R243Q, and R408W on the same haplotype background (6.7, 1.8, and 2.3, respectively) as in Europe. In this sample, we have found two novel mutations: S349L detected in two homozygous siblings on the background of haplotype 6.7, and a small deletion, P314fsdelC, that results in a frameshift and a premature stop codon detected on the background of haplotype 4.3. The definite demonstration that mutation S349L results in a nonfunctional protein was shown by expression analysis in prokaryotic and eukaryotic systems. This mutation results in an unstable phenylalanine hydroxylase (PAH) protein completely devoid of enzymatic activity well correlated with the severe form of the disease exhibited by the homozygous patients.

ATP diphosphohydrolase activity in synaptosomes from cerebral cortex of rats subjected to chemically induced phenylketonuria.

ATP diphosphohydrolase (apyrase) (EC 3.6.1.5) activity was measured in synaptosomes from cerebral cortex of Wistar rats of both sexes subjected to experimental phenylketonuria, i.e., chemical hyperphenylalaninemia induced by subcutaneous administration of 5.2 mumol phenylalanine/g body weight (twice a day) plus 0.9 mumol p-chlorophenylalanine/g body weight (once a day). ATP diphosphohydrolase specific activity (nmol Pi min-1 mg protein-1) of synaptosomes was significantly decreased compared to controls for both ATP (from 147.6 to 129.9) and ADP (from 70.2 to 63.1) hydrolysis one hour after single administration of the drugs to 35-day old rats. Chronic treatment was performed from the 6th to the 28th postpartum day. The enzyme specific activity of synaptosomes was measured one week after the last administration of the drugs and was significantly reduced compared to controls for both ATP (from 164.1 to 150.2) and ADP (from 76.3 to 62.1) hydrolysis. The in vitro effects of the drugs on the synaptosome enzyme specific activity were also investigated. Phenylalanine alone or associated with p-chlorophenylalanine significantly reduced enzyme specific activity for both ATP (from 150.2 to 136.0) and ADP (from 70.5 to 59.3) nucleotides as substrates. Since ADP diphosphohydrolase seems to play an important role in neurotransmission, these findings may be related to the neurological dysfunction characteristic of human phenylketonuria.

Molecular analysis of the phenylalanine hydroxylase gene in Mexican phenylketonuric patients.

The molecular analysis of the human phenylalanine hydroxylase (PAH) gene in Mexican phenylketonuric (PKU) patients is described. We analyzed the restriction fragment length polymorphism (RFLP) haplotypes of five probands and ten non-affected relatives, belonging to four unrelated PKU families. Twenty-nine alleles were typified, corresponding to 12 different haplotypes. Eight RFLP haplotypes corresponded to those described in other populations, while the remaining were unreported haplotypes, appearing both on normal and PKU chromosomes. Using the polymerase chain reaction (PCR) and the allele-specific oligonucleotide assay (ASO), we also screened for the IVS10 mutation, one of the most common PAH gene mutations in Mediterranean countries. Forty-two percent of the PKU chromosomes analyzed bore the IVS10 mutation, although it was present in the heterozygous state in all cases. Our data show an important genetic heterogeneity at the PAH locus in the Mexican population, and report the genetic influence of the Spanish immigration to the American continent.

Effect of phenylalanine and its metabolites on ATP diphosphohydrolase activity in synaptosomes from rat cerebral cortex.

The in vitro effects of phenylalanine and some of its metabolites on ATP diphosphohydrolase (apyrase, EC 3.6.1.5) activity in synaptosomes from rat cerebral cortex were investigated. The enzyme activity in synaptosomes from rats subjected to experimental hyperphenylalaninemia (alpha-methylphenylalanine plus phenylalanine) was also studied. In the in vitro studies, a biphasic effect of phenylalanine on both enzyme substrates (ATP and ADP) was observed, with maximal inhibition at 2.0 mM and maximal activation at 5.0 mM. Inhibition of the enzyme activity was not due to calcium chelation. Moreover, phenylpyruvate, when compared with phenylalanine showed opposite effects on the enzyme activity, suggesting that phenylalanine and phenylpyruvate bind to two different sites on the enzyme. The other tested phenylalanine metabolites phenyllactate, phenylacetate and phenylethylamine) had no effect on ATP diphosphohydrolase activity. In addition, we found that ATP diphosphohydrolase activity in synaptosomes from cerebral cortex of rats with chemically induced hyperphenylalaninemia was significantly enhanced by acute or chronic treatment. Since it is conceivable that ATPase-ADPase activities play an important role in neurotransmitter (ATP) metabolism, it is tempting to speculate that our results on the deleterious effects of phenylalanine and phenylpyruvate on ATP diphosphohydrolase activity may be related to the neurological dysfunction characteristics of naturally and chemically induced hyperphenylalaninemia.

Phenylalanine hydroxylase expression in liver of a fetus with phenylketonuria.

The expression and activity of phenylalanine hydroxylase was studied in the liver of a fetus aborted after prenatal diagnosis of phenylketonuria. No phenylalanine hydroxylase enzymatic activity or immunoreactive protein was detectable in the PKU liver specimen, though both enzymatic activity and immunoreactive protein were detectable in control specimens of similar gestational age. Phenylalanine hydroxylase messenger RNA of normal size was present in the PKU fetal liver at normal abundance. These results confirm the genetic diagnosis of PKU in this fetus and indicate that the mutations in this fetus affect translation or stability of the phenylalanine hydroxylase protein.