Oral aspartame and plasma phenylalanine: pharmacokinetic difference between rodents and man,and relevance to CNS effects of phenylalanine

Summary The ingestion of aspartame, a phenylalanine-containing dipeptide, raises plasma phenylalanine levels. These increments are much greater in humans than rats, because the rat hydroxylates phenylalanine five times faster than man. Accordingly, dose comparisons of aspartame (or phenylalanine) between humans and rats have usually been corrected by a factor of five. Recently, a correction factor of sixty has been proposed (Wurtman and Maher, 1987); the rationale is based on a novel calculation of competitive phenylalanine transport into brain. An analysis of the logic behind this postulation reveals there to beno basis for accepting the higher dose conversion of 60 between rat and man.     

Acute effects of aspartame on systolic blood pressure in spontaneously hypertensive rats

Summary Exogenous tyrosine lowers blood pressure in spontaneously hypertensive rats (SHR). The artificial sweetener aspartame also elevates blood and brain tyrosine levels in rats by being hydrolyzed to phenylalanine, which is then rapidly hydroxylated to tyrosine in the liver. Hence we tested the ability of aspartame; its hydrolytic products phenylalanine, aspartic acid and methanol; and of tyrosine itself to lower blood pressure in SHR. For one week prior to experimentation rats were acclimated to the indirect blood pressure measurement technique; on the day of an experiment they received I.P. injections (mg/kg) of aspartame (12.5–200), tyrosine (25–200) or phenylalanine (100–200), or of aspartic acid or methanol in the doses theoretically contained within 200 mg/kg aspartame. Animals receiving 50, 100 or 200 mg/kg of aspartame exhibited maximum falls in blood pressure of 17.3, 24.2 and 19.3 mmHg, respectively. All changes were significant, as determined by ANOVA and the Newman-Keuls test (p<0.05). Tyrosine or phenylalanine also lowered blood pressure, but aspartic acid or methanol produced no significant effects. Co-administration of aspartame with valine, a large neutral amino acid that competes with phenylalanine or tyrosine for brain uptake, attenuated aspartame's hypotensive effect. These observations suggest that the neurochemical changes produced by aspartame lead to predicted tyrosine-induced changes in blood pressure.     

L-[U-14C] Tyrosine Metabolism of the Perfused Cat Brain with High Plasma Phenylalanine or without Plasma Tyrosine

Cat brain perfusion was carried out with standard artificial blood containing 2 mg/dl of L-tyrosine and also with standard blood not containing tyrosine, for 70 minutes in each group. In addition, another group was perfused with the tyrosine-containing artificial blood plus 50 mg/dl of L-phenylalanine. Each of these experiments was conducted to see how such blood would affect the tyrosine metabolism of the brain. The results are summarized briefly.

• 1 Tn the perfusion without addition of tyrosine, the tyrosine content of the brain decreased markedly. Furthcr, there was an outflow of the brain tyrosine into the venous blood. The free tyrosine of the brain is mainly supplied from the blood tyrosine.
• 2 In the high plasma phenylalanine perfusion, the transport of the blood tyrosine into the brain was inhibited competitively by the blood phenylalanine, but output of tyrosine from the brain to the blood was not disturbed.
• 3 Tn the high plasma phenylalanine perfusion, the incorporation of tyrosinc into the brain protein fraction from the brain acid soluble fraction was lower than that ofthe control group.
• 4 During the high plasma phenylalanine perfusion, there was observed a marked increase of phenylalanine, and a decrease of tyrosine, threonine, isoleucine and leucine in the brain.

     

Amino acids, monoamines and audiogenic seizures in genetically epilepsy-prone rats: effects of aspartame.

It has been suggested that aspartame facilitates seizures in man and animals because phenylalanine, one of its major metabolites, interferes with brain transport of neurotransmitter precursors and alters the synthesis of monoamine neurotransmitters such as norepinephrine, dopamine and/or serotonin. This facilitation is purportedly more likely in subjects predisposed to seizures. One test of this hypothesis would be to administer a wide range of aspartame doses to subjects whose seizure predisposition is dependent on abnormalities in monoaminergic function. Genetically epilepsy-prone rats (GEPRs) have a broadly based seizure predisposition that is based, in part, on widespread central nervous system noradrenergic and serotonergic deficits. Further reductions in the functional state of these neurotransmitters increases seizure severity in GEPRs. Thus, GEPRs appear ideally suited for testing the hypothesis that aspartame facilitates seizures by interfering with central nervous system monoamines. Oral administration of acute (50-2000 mg/kg) or sub-chronic (up to 863 mg/kg/day for 28 days) doses of aspartame did not alter seizure severity in either of two types of GEPRs. Not surprisingly, acute aspartame doses produced dramatic changes in plasma and brain amino acid concentrations. Hypothesized alterations in monoamine neurotransmitter systems were largely absent. Indeed, increases in norepinephrine concentration, rather than the hypothesized decreases, were the most evident alterations in these neurotransmitter systems. We conclude that aspartame does not facilitate seizures in GEPRs and that convincing evidence of seizure facilitation in any species is lacking.     

Availability of phenylalanine and tyrosine for brain norepinephrine synthesis in developmentally protein-malnourished rats

Ontogenetic changes in brain phenylalanine, tyrosine, and norepinephrine concentrations occurred in rats as sequelae of protein inadequacies incurred during prenatal and postnatal development. The malnourished pups, whose dams received a low protein diet (8% casein) starting 5 weeks prior to conception, showed significantly elevated brain phenylalanine and norepinephrine, but not tyrosine, at most ages examined compared with offspring from dams fed a normal diet (25% casein) throughout the same time interval. Although the brain tyrosine concentrations of the malnourished offspring were somewhat lower than the normal pups, these decreases were found only at some ages and were restricted to certain brain regions (mainly the telencephalon and the cerebellum). Also, the peripheral availabilities of phenylalanine and tyrosine in the protein-deprived rats during the study (birth to age 30 days) displayed alterations which corresponded to their brain concentrations of these amino acids (increases of the former and decreases of the latter versus the controls). Although the malnourished rats had lower plasma tyrosine concentrations than the normal pups, both groups of offspring consistently showed higher concentrations of this amino acid compared with their phenylalanine values throughout postnatal development. In the case of the deprived rats, the increased availability of the semiessential rather than the essential amino acid to their brains may explain, in part, why the amounts of tyrosine in their brain-stem regions were generally comparable to the normal animals. This, in turn, may indirectly account for the higher amine biosynthesis in the brains of the malnourished pups. Overall, the present data demonstrate that the nutritional status with respect to the amount of dietary protein during prepartum and postpartum development has an important role in determining the substrate availability of tyrosine and/or phenylalanine for brain norepinephrine metabolism.     

In vivo tyrosine hydroxylation rate in retina: effects of phenylalanine and tyrosine administration in rats pretreated with p-chlorophenylalanine

p-Chlorophenylalanine was administered to rats to inhibit hepatic phenylalanine hydroxylase activity. Two days later, phenylalanine injection was noted to produce substantial increases in serum phenylalanine levels, and relatively modest increments in serum tyrosine levels. Rats injected with p-chlorophenylalanine 2 days earlier showed a normal light-induced activation of retinal tyrosine hydroxylase activity in vivo, measured as dihydroxyphenylalanine accumulation following pharmacologic inhibition in vivo of aromatic L-amino acid decarboxylase activity. In addition, tyrosine injection into p-chlorophenylalanine-treated rats in the light produced anticipated increments in retinal tyrosine hydroxylation rate, showing the enzyme to be functionally normal. The acute administration of phenylalanine (62.5-500 mg/kg i.p.) to p-chlorophenylalanine-treated rats produced dose-related increments in retinal phenylalanine. In vivo tyrosine hydroxylation rate in retina was normal at all doses below 300 mg/kg. However, at the highest dose (500 mg/kg), when retinal phenylalanine levels were almost 5-times normal tyrosine hydroxylation rate consistently fell (to about half-normal values). These results demonstrate that very large elevations in tissue phenylalanine levels do not stimulate tyrosine hydroxylation in vivo, and that at extremely high levels phenylalanine inhibits tyrosine hydroxylation rate.     

Lack of effect of aspartame or of L-phenylalanine on photically induced myoclonus in the baboon, Papio papio

The effects of large doses of L-phenylalanine and of aspartame on seizure susceptibility and severity have been assessed in baboons Papio papio from Senegal which show photosensitive epileptic responses similar to primary generalised epilepsy in man. L-Phenylalanine, 50, 150 or 450 mg/kg, or aspartame, 300 or 1000 mg/kg, were administered orally. Peak plasma L-phenylalanine concentrations of approximately 2000 mumoles/l occurred 1-4 h after the highest dose of L-phenylalanine or aspartame. The plasma L-phenylalanine to large neutral amino acid ratio increased approximately 30-fold at this time. Compared with water administration there were no changes in epileptic responses 1-5 h after either treatment. In this primate model of epilepsy acute increases in plasma phenylalanine concentration are neither pro- nor anticonvulsant.     

Reduction of brain dopamine concentration with dietary tyrosine plus phenylalanine depletion: an [11C]raclopride PET study

OBJECTIVE: Extracellular dopamine concentrations were estimated through measurement of [(11)C]raclopride binding with positron emission tomography after dietary manipulation of the dopamine precursors tyrosine and phenylalanine. METHOD: Healthy male subjects were scanned on two occasions: once after receiving a balanced amino acid drink and once after receiving a drink mixture from which tyrosine and phenylalanine were omitted. RESULTS: Dietary tyrosine and phenylalanine depletion increased [(11)C]raclopride binding in the striatum by a mean of 6%. The change in [(11)C]raclopride binding correlated significantly with the fall in the ratio of tyrosine and phenylalanine to large neutral amino acids. CONCLUSIONS: This is the first demonstration of an effect of a dietary manipulation on brain dopamine release in humans. This result provides support for the further investigation of the role of dietary manipulations in the treatment of neuropsychiatric disorders.     

Aspartame and Seizure Susceptibility: Results of a Clinical Study in Reportedly Sensitive Individuals

Summary: The high intensity sweetener aspartame has been implicated anecdotally in seizure provocation. This possibility was investigated with a randomized, double-blind, placebo-controlled, cross-over study. After an extensive search, 18 individuals (16 adults and 2 children) who had seizures allegedly related to aspartame consumption were admitted to adult or pediatric epilepsy monitoring units where their EEG was monitored continuously for 5 days. Aspartame (50 mg/kg) or identically enpackaged placebo was administered in divided doses at 800, 1000, and 1200 h on study days 2 and 4. All meals were uniformly standardized on treatment days. No clin-ical seizures or other adverse experiences were observed after aspartame ingestion. Mean plasma phenylalanine (Phe) concentrations increased significantly after aspar-tame ingestion (83.6 pIM) as compared with placebo (52.3 μC M ).Results suggest that aspartame, in acute dosage of ε50 mg/kg, is no more likely than placebo to cause seizures in individuals who reported that their seizures were provoked by aspartame consumption.     

Association between amino acid alterations and hallucinations in alcoholic patients

Because available evidence suggests that alterations in the serotonergic as well as dopaminergic tones underlie hallucinatory activity, we decided to investigate whether serotonin and dopamine pathways are modified in alcoholics with a history of hallucinosis. Brain serotonin has been shown to depend on the plasma ratio of its precursor tryptophan over other amino acids competing with it for brain entry. Similarly, brain dopamine depends on the plasma ratio of its precursors phenylalanine and dopamine over their competitors. Amino acid abnormalities are common in alcoholics. For this reason, we assessed whether alcoholics who had experienced hallucinations have alterations in amino acids believed to be associated with neurotransmitter modifications. Patients with a history of hallucinations were found to have a tryptophan ratio significantly lower than that of patients without such a history, and a tyrosine + phenylalanine ratio significantly higher. These data suggest that amino acid abnormalities believed to result in decreased brain serotonin and in increased brain dopamine render certain individuals more vulnerable to hallucinatory experiences.     

Free amino acid pool in the brain of mice homozygous for the gene "dilute lethal"

The neurologic mutant "dilute lethal" (dl) mice, which reveal several neurologic and biochemical disturbances similar to human phenylketonuria, were used to investigate some aspects of amino acid disorder. We have studied the free amino pool in the brain of "dl" mice and of their control littermates as well as phenylalanine and tyrosine levels in brain and liver as a function of age and after phenylalamine overload. The tyrosine level decreased in brain and liver of affected mice whereas the phenylalanine/tyrosine ratio increased as a function of age. The significantly higher phenylalanine level and phenylalanine/tyrosine ratio in the liver of 20-day-old "dl" mice suggest a lower liver phenylalanine hydroxylase activity. After phenylalanine overload, the impairment of phenylalanine metabolism is predominant in the brain of "dl" mice, suggesting a disturbance in phenylalanine hydroxylation. A decrease in the level of several amino acids occurs in the brains of "dl" mice without or after phenylalanine overload; these facts might correspond to a disturbance in the transfer of amino acids to the brain and may lead to impairment in protein synthesis.     

Cerebral amino acid levels and uptake in rats after portocaval anastomosis: II. Regional studies in vivo.

Amino acid levels have been determined in plasma and in four cerebral regions of rats one month after portocaval shunt. Many plasma amino acids are significantly lowered (asparagine, glutamine, theonine, serine, alanine, valine, leucine, isoleucine, cystine, lysine), while others remain unchanged (taurine, glycine, proline, tryptophan, ornithine, histidine, arginine). Asparagine and glutamine levels are significantly higher than in normal rats, and a net increase of tyrosine (100%), phenylalanine (50%) and citrulline (50%) is evident. In the shunted rat brain the most prominent feature is a very large rise (up to fivefold) of tyrosine, phenylalanine, histidine, citrulline, tryptophan, and glutamine uniformly in the tested regions. Other neutral amino acids are slightly increased. Lysine and arginine are decreased in cerebellum and pons-medulla; taurine, in forebrain and cerebellum. Cerebral permeability to L-amino acids was studied in vivo. Neutral amino acid permeability is greatly increased, whereas basic amino acids show a net decrease in their rate of passage from blood to the brain. No changes are observed for GABA and glutamic acid. These data suggest an altered permeability of the cerebral capillary membranes, which seems to be selective for the different amino acid transport classes. Competitive inhibition experiments demonstrated that the increased brain permeability to neutral amino acids after portocaval shunt is due to an enhancement of the saturable transport. The sharp rise in the brain of some essential neutral amino acids (phenylalanine, tyrosine, trytophan, histidine), largely exceeding their changes in plasma, and the slight cerebral increase of other neutral amino acids despite their lowered level and the rise of competing amino acids in the plasma, is consistent with our observation of enhanced transport for the neutral class. In hepatic encephalopathy, correction of the altered plasma amino acid levels has been reported to improve the clinical status. If this result is connected to the concomitant correction of the brain amino acid levels, carefully selected competitive inhibition among various plasma amino acids could be a useful therapeutic tool in this pathologic condition. However, the increased activity of the neutral amino acid transport system adds a new factor to the problem, since it probably implies that the competing amino acids will accumulate to unphysiological levels in the brain.     

Suppressant effects of dexamethasone on the availability of plasma L-tryptophan and tyrosine in healthy controls and in depressed patients

Formation in the brain of serotonin from L-tryptophan (L-TRP) and noradrenaline from tyrosine are pathways related to the pathophysiology of major depression and to the regulation of the hypothalamic-pituitary-adrenal (HPA) axis. In the past, decrements in L-TRP availability and disorders in the HPA axis have repeatedly been observed in major depressed patients; both factors were shown to be inversely correlated. In order to investigate the relationships between glucocorticosteroid activity and the availability of L-TRP and tyrosine, the authors measured L-TRP, tyrosine, valine, leucine, isoleucine and phenylalanine in baseline conditions and after treatment with 1 mg dexamethasone in 16 healthy controls and in 50 depressed patients. The ratios between L-TRP and tyrosine and the sums of the amino acids known to compete with them during transport across the blood-brain barrier were computed as an index of (respectively) the serotonin and noradrenaline synthesis in the brain. We found significantly decreased plasma L-TRP and tyrosine levels after treatment with dexamethasone compared with basal levels. Accordingly, the plasma ratios between L-TRP and tyrosine and the sum of the competing amino acids were significantly reduced by dexamethasone administration. It was hypothesized that through these actions of dexamethasone on peripheral amino acids, the central noradrenaline and serotonin control over the HPA-axis could be altered.     

Plasma tyrosine in normal humans: Effects of oral tyrosine and protein-containing meals

Summary To test the effects of tyrosine ingestion and concurrent food consumption on plasma tyrosine levels and on the plasma tyrosine ratio, we measured plasma neutral amino acid levels in 11 subjects who consumed a diet containing 113 g protein and who also took 100 mg/kg/day of L-tyrosine (in three equally divided doses) before meals. Plasma tyrosine levels rose significantly (p<0.025) during the day when subjects consumed the diet alone; they increased markedly after tyrosine ingestion (p<0.005). Tyrosine administration did not affect plasma concentrations of the other neutral amino acids that compete with tyrosine for entry into the brain. Thus, the plasma tyrosine ratio increased from 0.13 to 0.21 (p<0.001) on the day fed subjects received the tyrosine. These observations indicate that tyrosine administration might increase brain tyrosine levels and perhaps accelerate catecholamine synthesis in humans with diseases in which catecholamine synthesis or release is deficient.     

Effects of maternal hyperphenylalaninemia on fetal brain development: A morphological study

We examined the effects of maternal hyperphenylalaninemia on body and brain growth, and the biochemical maturation of the fetal and neonatal rat brain. Elevated concentrations of plasma phenylalanine were induced in pregnant rats under two experimental conditions from the 14th through the 21st days of gestation. In the first treatment, pregnant rats were injected subcutaneously with alpha-methylphenylalanine (to inhibit maternal liver phenylalanine hydroxylase) at a dosage of 30 mg/100 g body weight plus phenylalanine supplementation (to increase maternal and fetal plasma phenylalanine) at a dosage of 60 mg/100 g body weight two times daily. In the second treatment, pregnant dams were injected with phenylalanine only at a dosage of 65 mg/100 g body weight three times daily. Treatment with alpha-methylphenylalanine/phenylalanine (mPhe/Phe) resulted in a 76% inhibition in the activity of maternal phenylalanine hydroxylase and a 25-fold increase in the mean daily concentration of phenylalanine in the maternal and fetal plasma. Phenylalanine treatment alone resulted in a 15-fold increase in plasma phenylalanine in the maternal and fetal animals. Significant reductions in body and brain weights in the fetal and neonatal rats were found in both treatment groups. Biochemical determinations indicated that the total DNA, RNA, and protein contents of the cerebra were reduced, with the reductions being greater in the mPhe/Phe- than the phenylalanine-treated rats. However, the retardation in body and brain growth of both treatment groups did not appear to be permanent because substantial recovery was noted in the rats after postnatal day 7. These results suggest that exposure of the fetus to high plasma concentrations of phenylalanine caused a delay in the biochemical maturation of the fetal rat brain.     

A new method for rapidly and simultaneously decreasing serotonin and catecholamine synthesis in humans

OBJECTIVE: The administration of amino acid (AA) mixtures that are selectively deficient either in tryptophan or phenylalanine plus tyrosine can decrease serotonin or catecholamine synthesis, respectively. In the present study, we assessed whether a mixture that was simultaneously deficient in tryptophan, phenylalanine and tyrosine could induce sufficient protein synthesis that plasma levels of all 3 monoamine precursors would decrease. DESIGN: Ten healthy volunteers (5 male, 5 female) were administered a tryptophan/phenylalanine/tyrosine-deficient AA mixture in an open-label study. Plasma concentrations of large neutral AAs were measured before and 5 hours after mixture ingestion. RESULTS: The tryptophan/phenylalanine/tyrosine-deficient mixture lowered plasma concentrations of the 3 AAs by 67;, 78% and 77%, respectively (p < or = 0.001); their ratio to other large neutral AAs was decreased more, namely, by 87%, 90% and 90% (p < or = 0.001). Mood lowering was seen on 3 subscales of the bipolar Profile of Mood States, that is, elated-depressed, composed-anxious and clearheaded-confused, as well as 2 visual analog scales, bored and irritated (p < or = 0.05). CONCLUSIONS: Acute tryptophan/phenylalanine/tyrosine depletion may be a suitable new method for rapidly decreasing serotonin and catecholamine transmission simultaneously.     

Decreased cerebrospinal fluid levels of neutral and basic amino acids in patients with Parkinson''s disease

We measured the CSF levels of 21, and the plasma levels of 26, amino acids in 31 patients with Parkinson's disease (PD) and in 45 matched controls. We used an ion-exchange chromatography method. When compared to controls, PD patients had lower CSF levels of taurine, alanine, valine, leucine, isoleucine, ethanolamine, citrulline, ornithine, lysine, histidine, arginine, and alpha-aminobutyric acid. PD patients not treated with levodopa or with dopamine agonists had higher CSF tyrosine and phenylalanine levels than those not treated with these drugs and also than controls. PD patients had higher plasma levels of phosphoserine, threonine, methionine, tyrosine, sarcosine and -aminoadipic acid, and lower plasma levels of valine, leucine, and tryptophan, than controls. The CSF/plasma ratio of many of these amino acids was significantly lower in PD patients than those of controls, suggesting that PD patients might have a dysfunction in the transport of neutral and basic amino acids across the blood–brain barrier.     

Behavioral, electroencephalographic, and biochemical changes in porta-cava shunted rats

Behavioral, electroencephalographic, and biochemical alterations have been studied in rats with porta-caval shunt, up to 45--50 days after the operation. No behavioral or electroencephalographic changes have been observed, while modifications of various amino acids both in plasma and brain have been found. Among all the amino acids considered of particular significance are the plasma and brain increases of tyrosine, phenylalanine, and tryptophan, since they are the precursors of biogenic amines. In fact, the increase of 5-HIAA found in regions of CNS which are richest in serotoninergic synapsis could indicate an increased turnover of 5-HT. On the other hand no significant alterations of turnover of catecholamines have been found.     

Metabolic changes in aromatic amino acids and monoamines in infantile autism and development of new treatment related to the finding

A new method for measurement of the turnover rate of aromatic amino acids and related compounds in vivo using stable isotopes was developed. Deuterium-and carbon 13-labeled phenylalanine and deuterium-labeled tryptophan were used as tracers. This method was applied for the analysis of amino acid and amine metabolism in infantile autism. Marked disturbances of uptake of deuterated phenylalanine and tryptophan from intestine into blood were found in a portion of autistic patients (group A). In another group of the patients a remarkable decrease of turnover of tyrosine in blood was found (group B). This phenomenon was confirmed by an experiment using carbon 13 labeled phenylalanine. These findings might suggest that supply of tyrosine and free tryptophan to the brain (in group A) or supply of tyrosine (group B) to the brain might be decreased. We postulated that in some of autistic patients there might exist decreases in synthesis of catecholamine or serotonin. Based on the hypothesis, we started a new treatment with L-DOPA and 5 HTP in small doses, and found significant effects in some patients. However, in some, the amino acids caused marked aggravation of the symptoms. Recently, Hayaishi and his colleagues reported that R-tetrahydrobiopterin (R-THBP) could enhance biosynthesis of catecholamine and serotonin in the brain. Therefore, we started a clinical trial concerning effects of R-THBP. In the beginning, 17 cases were treated and patients younger than 5 years old showed marked improvement. Then, a double blind trial with inactive placebo was performed.(ABSTRACT TRUNCATED AT 250 WORDS)     

A PET study following treatment with a pharmacological stressor,FG7142, in conscious rhesus monkeys

FG7142 is a benzodiazepine partial inverse agonist, which is known as a pharmacological stressor. Several reports demonstrated that FG7142 produced anxiety in humans, non-human primates, and rodents, and impaired working memory in non-human primates and rodents. In this study, we examined the effect of FG7142 on cerebral blood flow and glucose metabolism using positron emission tomography (PET) in conscious rhesus monkeys. Male rhesus monkeys were intramuscularly treated with FG7142 (0.2 or 1.0 mg/kg, n=5, respectively), and regional cerebral blood flow (rCBF) and regional cerebral metabolic rate of glucose (rCMRglc) were measured by PET 20 min and 40 min after treatment, respectively. During PET measurement, physiological parameters and plasma cortisol levels were monitored. FG7142 significantly decreased rCBF in the thalamus and rCMRglc in all brain regions examined in a dose-dependent manner without changes in physiological parameters. FG7142 also significantly increased plasma cortisol levels. The present study may provide an important insight into the understanding of the pathophysiology of anxiety and stress-related disorders in humans, and strongly suggesting that prevention of anxiety or stress is important when measuring conscious brain function.