Effect of experimental hyperphenylalaninemia induced by dietary phenylalanine plus α-methylphenylalanine administration on amino acid concentration in neonatal chick brain,plasma, and liver

Supplementation of 5% phenylalanine plus 0.4% -methylphenylalanine to the standard diet or 1% phenylalanine plus 0.08% -methylphenylalanine to the drinking water produced phenylketonuria-like conditions in 5-day-old chicks. An increase of 10 to 15-fold in the phenylalanine content was observed in plasma or brain of animals after 9 days of both types of treatment. A smaller but significant increase was also observed in liver. However, practically no changes were found in the levels of tyrosine in the same conditions. Thus, the high values of plasma and brain phenylalanine/tyrosine ratio obtained by these treatments were mainly due to an increase in the phenylalanine levels, without increasing those of tyrosine. Chronic hyperphenylalaninemia induced a nonsignificant decrease in the most of amino acid contents in brain, especially after 9 days of treatment, although the levels of glycine and serine were significantly increased. A similar decrease was found in the plasma and liver concentration of various amino acids, although the variations observed in the liver were smaller than those found in plasma and brain.     

Inhibition of brain and liver 3-hydroxy-3-methylglutaryl-CoA reductase and mevalonate-5-pyrophosphate decarboxylase in experimental hyperphenylalaninemia

Experimental hyperphenylalaninemia has been induced in 5-day-old chicks by dietary treatments with phenylalanine and -methylphenylalanine. An increase of nearly 8-fold in plasma Phe/Tyr ratio was found after 4 days of supplementation the standard diet with 5% phenylalanine plus 0.4% -methylphenylalanine. The increase in this ratio was about 13-fold after 9 days of the same treatment. Similar results were observed in brain and liver, although the increases were smaller than those found in plasma. Total body, brain and liver weight decreased after 9 days of treatment. Phenylalanine plus -methylphenylalanine administration to 5-day-old chicks produced a significant decrease in the 3-hydroxy-3-methylglutary-CoA reductase and mevalonate-5-pyrophosphate decarboxylase specific activities from both brain and liver. These results demonstrated for the first time that experimental hyperphenylalaninemia inhibited different enzyme activites directly implicated in the regulation of cholesterogenesis. Therefore, a reduced cholesterol synthesis in brain may evidenciate the theory of an impaired myelination leading to mental retardation in phenylketonuria patients.     

Cerebral glycine content and phosphoserine phosphatase activity in hyperaminoacidemias

Chronic hyperphenylalaninemia maintained with the aid of a suppressor of phenylalamine hydroxylase, -methylphenylalanine, increases the glycine concentration and the phosphoserine phosphatase activity of the developing rat brain but not that of liver or kidney. Similar increases occur after daily injections with large doses of phenylalanine alone, while tyrosine, isoleucine, alanine, proline, and threonine, were without effect. Treatment with methionine, which increases the phosphoserine phosphatase activity of the brain and lowered that of liver and kidney, left the cerebral glycine level unchanged. When varying the degrees of gestational or early postnatal hyperphenylalaninemia, a significant linear correlation was found between the developing brains' phosphoserine phosphatase and glycine concentration. Observations on the uptake of injected glycine and its decline further indicate that coordinated rises in the brain's phosphoserine phosphatase and glycine content associated with experimental hyperphenylalaninemia denote a direct impact of phenylalanine on the intracellular pathway of glycine synthesis in immature animals.     

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.     

Characterization of experimental phenylketonuria augmentation of hyperphenylalaninemia with α-methylphenylalanine and p-chlorophenylalanine

Phenylalanine in conjuction with p-chlorophenylalanine or α-methylphenylalanine was administered to suckling rats to induce hyperphenylalaninemia reminiscent of untreated phenylketonuria, and developmental parameters were monitored. The experimental model utilizing p-chlorophenylalanine was found to be unsatisfactory, in that the drug had general deleterous effects on growth, numerous side effects including increased mortality, and affected brain levels of biogenic monoamine neurotransmitters. The model utilizing α-methylphenylalalanine was relatively free from nonspecific effects and thus, changes observed in the animals were attributable to expereimental phenylketonuria. The latter animals had slightly decreased body and brain weights, and exhibited grossly elevated serum phenylalanine and urinary excretion of phenylketone metabolites. Hyperphenylalaninemia produced greatly disrupted brain amino acids at 10 days of age (prior to the formalization of the blood-brain barrier and specific transport systems) which was limited by 30 days of age to changes in glycine, γ-aminobutyric acid and the aliphatic and aromatic amino acids which compete for uptake in t he brain by a common carrier. These animals also exhibited a myelin deficit and changes in proteins from isolated nerve cell preparations. Mature animals which had daily treatment up to 60 days of age results obtained with animal models and the clinical findings in untreated phenylketonuric patients.     

Effect of α-Methylphenylalanine and Phenylalanine on Brain Polyribosomes and Protein Synthesis

Abstract: A chronic hyperphenylalanemia was effectively produced in developing mice by daily administrations of phenylalanine (2 mg/g body wt) and a phenylalanine hydroxylase inhibitor α-methyl-D, L-phenylalanine (0.43 mg/g body wt). The presence of α-methylphenylalanine in newborn mice inhibited 65–70% of hepatic phenylalanine hydroxylase activity within 12 h. Since this maximum inhibition persisted for 24 h or longer, decreased enzyme activity was maintained by daily administrations. Whereas concentrations of phenylalanine increased approximately 40-fold in both plasma and brain following injection of α-methylphenylalanine and phenylalanine, plasma levels of tyrosine were not altered significantly. Concomitant with changes in phenylalanine concentrations we observed the brain polyribosomes' disaggregation, which reached a maximum 3 h after injection and persisted as long as 18 h. Polyribosomes did not become refractory to as many as 10 daily injections of α-methylphenylalanine and phenylalanine. In addition to polyribosome disaggregation, chronic hyperphenylalanemia reduced the rates of polypeptide chain elongation on polyribosomes isolated from brain homogenates.     

Effects of α-methylphenylalanine plus phenylalanine treatment during development on myelin in rat brain

The effects of hyperphenylalaninemia induced by treatment with -methylphenylalanine (MPA) plus phenylalanine (PHE) on body and brain weight, on myelin and synaptosome formation, and on the lipids and fatty acids of myelin were studied in rats. The administration of MPA (2.4 mol/g body wt) plus PHE (2.6 mol/g body wt) for 25 and 35 days beginning on the fifth postnatal day did not affect brain development. On doubling the dosage of PHE, body and brain weights and myelin yields were significantly lowered. The lipid composition of myelin from the brains of treated animals was largely unaffected; however, the concentration of sulfatides was significantly reduced. Unsaturated fatty acid levels in myelin from hyperphenylalaninemic rat brains were reduced while long-chain fatty acids were unaffected. We conclude that as in hyperphenylalaninemia induced by other methods, MPA+PHE treatment impairs body and brain growth, reduces myelin formation, and causes inhibition of fatty acid desaturation in the brain.     

Chronic Hyperphenylalaninemia Produces Cerebral Hyperglycinemia in Immature Rats

The amino acid content of three tissues was measured in 10-day-old rats made hyperphenylalaninemic from age 3 to 10 days by daily injection of phenylalanine plus alpha-methylphenylalanine to inhibit phenylalanine hydroxylase (PAH). At 12 h after the last injection, the concentrations of alanine, valine, methionine, isoleucine, and leucine in the cerebral hemispheres were depressed by 25-50%, whereas that of glycine was elevated 2.3-fold. In the spinal cord, the levels of phosphoserine, methionine, and leucine were decreased by 40-50%, and those of serine and threonine increased by 50%. Tyrosine and phenylalanine concentrations were high in all tissues, 2-3 and 15-30 times normal, respectively; of the amino acids investigated, they were the only ones changed in the liver. Cerebral hyperglycinemia was also produced by chronic treatment with phenylalanine plus p-chlorophenylalanine to inhibit PAH, but not by acute (12 h) hyperphenylalaninemia. An increase in cerebral phosphoserine phosphatase activity was greater in rats treated with phenylalanine plus PAH inhibitor than with inhibitor alone. The content of brain glycine normally declines with age from birth to 15 days; this decrease was prevented by chronic hyperphenylalaninemia. Attempts to reduce the cerebral glycine content of the hyperphenylalaninemic rats were unsuccessful. However, one of the therapeutic protocols, methionine loading, may be useful because it increased the methionine and decreased the phenylalanine contents in the brain.     

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 (-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.     

Developmental changes of cerebral phenylalanine uptake from severely elevated blood levels

Brain phenylalanine concentrations at plasma levels raised to that in phenylketonuric subjects were studied in rats from fetal through postnatal life. Suppression of the hepatic phenylalanine hydroxylase with methylphenylalanine, and injections of age-adjusted doses of phenylalanine on the next day, assured the persistence of the same elevation of plasma levels for at least four hours prior to assay. The net phenylalanine uptake determined under these conditions underwent several-fold decreases between the fourth day and the end of the suckling period, and by about the age of 30 days it was as low as in adulthood. The development of transport properties studied here could contribute to the change with age in the vulnerability of the brain to the same degree of hyperphenylalaninemia and, since the cerebral phenylalanine uptake may decrease to non-damaging levels during childhood, it is pertinent to defining the age at which the rigorous diet of phenylketonurics might be safely relaxed.Dedicated to Henry McIlwain.     

Inhibition of phenylalanine hydroxylase activity by alpha-methyl tyrosine, a potent inhibitor of tyrosine hydroxylase.

Inhibitors of phenylalanine hydroxylase and tyrosine hydroxylase were used in the assay of phenylalanine hydroxylase in liver and kidney of rats and mice. Parachlorophenylalanine (PCPA), methyl tyrosine methyl ester and dimethyl tyrosine methyl ester showed 5–15% inhibition while α-methyl tyrosine seemed to inhibit phenylalanine hydroxylase to the extent of 95–98% at concentrations of 5 × 10 ⁻⁵M –1 × 10 ⁻⁴M. After a phenylketonuric diet (0.12% PCPA + 3% excess phenylalanine), the liver showed 60% phenylalanine hydroxylase activity and kidney 82% that present in pair-fed normals. Hepatic activity was normal after 8 days refeeding normal diet whereas kidney showed 63% of normal activity. The PCPA-fed animals showed 34% in liver and 38% in kidney as compared to normals; in both cases normal activity was noticed after refeeding. The phenylalanine-fed animals showed activity similar to that seen in phenylketonuric animals. The temporary inducement of phenylketonuria in these animals may be due to a slight change in conformation of the phenylalanine hydroxylase molecule; once the normal diet is resumed, the enzyme reverts back to its active form. This paper also suggests that α-methyl tyrosine when fed in conjunction with the phenylketonuric diet may suppress phenylalanine hydroxylase activity completely in the experimental animals thus yielding normal tyrosine levels as seen in human phenylketonurics.     

Acute effects of aspartame on large neutral amino acids and monoamines in rat brain

The dipeptide aspartame (APM; aspartylphenylalanine methylester), an artificial sweetener, was studied $\text{in vivo}$ for its ability to influence brain levels of the large neutral amino acids and the rates of hydroxylation of the aromatic amino acids. The administration by gavage of APM (200 mg/kg) caused large increments in blood and brain levels of phenylalanine and tyrosine by 60 minutes. Brain tryptophan level was occasionally reduced significantly, but the brain levels of the branched-chain amino acids were always unaffected. Smaller doses (50, 100 mg/kg) also raised blood and brain tyrosine and phenylalanine, but did not reduce brain tryptophan levels. At the highest dose (200 mg/kg), APM gavage caused an insignificant increase in dopa accumulation (after NSD-1015), and a modest reduction in 5-hydroxytryptophan accumulation. No changes in the brain levels of serotonin, 5-hydroxyindoleacetic acid, dopamine, dihydroxyphenylacetic acid, homovanillic acid, or norepinephrine were produced by APM administration (200 mg/kg). These results thus indicate that APM, even when administered in amounts that cause large increments in brain tyrosine and phenylalanine, produce minimal effects on the rates of formation of monoamine transmitters.     

Lipid composition of brain myelin from normal and hyperphenylalaninemic chick embryos

The influence of hyperphenylalaninemia on the lipid composition of brain myelin has been investigated in 19-day-old chick embryos. CNP-ase activity was used as myelin marker enzyme for myelin isolation. CNP-ase activity was significantly lower in hyperphenylalaninemic myelin when compared with control. No significant differences were observed after experimental treatment in the total lipid content of myelin as well as in the proportion of cholesterol:phospholipid:galactolipid. Nevertheless, a clear increase in the percentage of esterified cholesterol was found. No appreciable alterations were observed in the phospholipid composition of brain myelin from both control and hyperphenylalaninemic chick embryos. However, the ratio of unsaturated to saturated fatty acids in serine plasmalogen and sphingomyelin was considerably increased by this treatment. This ratio in choline and ethanolamine phosphatides from treated embryos did not differ from that of controls.     

Decrease of homovanillic, dihydroxyphenylacetic acid and cyclic-adenosine-3',5'-monophosphate content in the rat caudate nucleus induced by the acute administration of an aminoacid mixture lacking tyrosine and phenylalanine.

The oral administration of an aminoacid mixture lacking tyrosine and phenylalanine induces, in rats, a profound depletion of tyrosine in serum and in brain. Brain tyrosine is maximally depleted by 73%, 2 h after treatment, when there is a concomitant decrease in the levels of HVA (by 50%), DOPAC (by 30%) and c-AMP (by 28%) in the basal ganglia. However, 4 to 8 h after treatment, when brain tyrosine is still depleted by 47 and 28%, respectively, DA metabolites and c-AMP levels have returned to normal. Our findings indicate that striatal tyrosine hydroxylase is fully saturated in v i m by the concentrations of tyrosine normally present in the basal ganglia. The results also suggest indirectly that decreased DA turnover results in decreased nerve activity, as judged by the decreased cyclic AMP levels.     

Effect of experimental hyperphenylalaninemia on biogenic amine synthesis at later stages of brain development

The effects of experimental hyperphenylalaninemia on catecholamine and serotonin synthesis in brain at a later stage of brain development were investigated. A group of 35-day-old rats treated with normal chow supplemented with 5% Phe + 0.4% alpha-methylphenylalanine, alpha MP, for the previous 10 days showed decreases in dopa, norepinephrine, and epinephrine versus controls. A group treated with a normal diet supplemented with 0.4% alpha MP showed similar decreases and these differences could be attributed to the presence of the phenylalanine hydroxylase and tyrosine hydroxylase inhibitor, alpha MP, rather than the hyperphenylalaninemia condition. No differences in dopamine were observed. Serotonin and 5-hydroxyindoleacetic acid (5HIAA) were decreased 50% in the HyPhe condition and were unaffected in the presence of alpha MP alone, indicating that the decreases in serotonin and 5HIAA were due to the increases in phenylalanine rather than the presence of the inhibitor. These abnormalities in serotonin metabolism at later stages of brain development may be relevant to early discontinuation of dietary therapy in the PKU patient and implies a role in tryptophan supplementation to increase intracerebral serotonin values.     

Polyribosome disaggregation and cell-free protein synthesis in preparations from cerebral cortex of hyperphenylalaninemic rats

Abstract— Seven-day-old rats were injected intraperitoneally with l -phenylalanine (1 g/kg) and the time course of brain polyribosome disaggregation and changes in brain levels of phenylalanine, tryptophan and tyrosine were determined. Disaggregation of brain polyribosomes preceded the increase in levels of phenylalanine in brain, and followed the same time course as depletion of tryptophan from brain. The effects of several metabolites of phenylalanine (which are formed in phenylketonuria) on protein synthesis in vitro was determined for brain and liver systems. None of the compounds tested was inhibitory at concentrations below 10 mM and in all cases hepatic protein synthesis was more sensitive to inhibition than was the corresponding system from brain. Ribosomal dimers, formed in brain after injection of phenylalanine, were incapable of supporting high levels of protein synthesis in vitro, a finding that suggested that the inhibition of protein synthesis in vitro in cell-free systems of brain tissue after injection of phenylalanine into young rats was mediated by disaggregation of brain polyribosomes associated with tryptophan deficiency in brain.     

Modulation of cerebral catecholamine concentrations during hyperphenylalaninaemia.

Hyperphenylalaninaemia induced by daily injections of alpha-methylphenylalanine plus phenylalanine caused 20-40% decreases in cerebral dopamine (3,4-dihydroxyphenethylamine) and noradrenaline in 7- and 11-day-old rats. alpha-Methylphenylalanine alone as well as phenylalanine alone caused cerebral dopamine depletion. However, the effects were not additive, in that the depletion caused by alpha-methylphenylalanine was greater, not less, than that after treatment with both it and phenylalanine. Increased concentrations of tyrosine in the brain, owing to administered or endogenously formed tyrosine, could overcome the effect of excess phenylalanine on cerebral dopamine content. The fact that the inhibition of tyrosine hydroxylase by phenylalanine (or alpha-methylphenylalanine) in vitro was overcome by tyrosine concentrations similar to those effective in vivo further implicates the tyrosine hydroxylase inhibition as the mechanism underlying the dopamine depletion in hyperphenylalaninaemia. These results provide a theoretical basis for elevation, by tyrosine supplementation, of the cerebral phenylalanine/tyrosine ratio as a possible treatment modality for phenylketonuria.     

Liver phenylalanine hydroxylase activity in relation to blood concentrations of tyrosine and phenylalanine in the rat

The plasma concentration of phenylalanine and tyrosine decreases in normal rats during the first few postnatal days; subsequently, the concentration of phenylalanine remains more or less constant, whereas that of tyrosine exhibits a high peak on day 13. The basal concentrations of the two amino acids were not altered by injections of thyroxine or cortisol, except in 13-day-old rats, when an injection of cortisol decreased the concentration of tyrosine. In young rats (13-15 days old), treatment with cortisol increased the activity of phenylalanine hydroxylase in the liver (measured in vitro) and accelerated the metabolism of administered phenylalanine: the rate constant of the disappearance of phenylalanine from plasma and the initial increase in tyrosine in plasma correlated quantitatively with the activity of phenylalanine hydroxylase in the liver. In adult rats, the inhibition of this enzyme (attested by assay in vitro) by p-chlorophenylalanine resulted in a proportionate decrease in tyrosine formation from an injection of phenylalanine. However, the quantitative relationship between liver phenylalanine hydroxylase activity and phenylalanine metabolism within the group of young rats was different from that observed among adult rats.     

Effects of Dietary Amino Acids on Brain Amino Acids and Transmitter Amines in Rats with a Portacaval Shunt

The etiologic relationship between disturbances in metabolism of amino acids and amines and hepatic coma was investigated by examining the effects of diets containing various mixtures of amino acids on brain amine metabolism in rats with a portacaval shunt, using a method for simultaneous analysis of amino acids and amines. Rats with a portacaval shunt were fed on four different amino acid compositions with increased amounts of various amino acids suspected to be etiologically related to hepatic coma, such as methionine, phenylalanine, tyrosine, and tryptophan. The animals were killed 4 weeks after operation. During the experimental period, these animals did not become comatose, but exhibited various behavioral abnormalities. Marked increase in the plasma and brain levels of the augmented amino acids, especially methionine and tyrosine, were observed in rats with a portacaval shunt. Brain noradrenaline, dopamine, and serotonin levels were significantly decreased when the brain tyrosine level was increased. These results indicate that in rats with a portacaval shunt the dietary levels of amino acids greatly influence the brain levels of both amino acids and transmitter amines.     

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.