Effects of oral aspartame on plasma phenylalanine in humans and experimental rodents

Summary All aspartame does given to humans cause greater elevations in plasma (and, presumably, brain) phenylalanine than in plasma tyrosine. In contrast, doses of aspartame usually used in experiments on rodents preferentially elevate tyrosine. Since phenylalanine can inhibit brain catecholamine synthesis while tyrosine is the antidote for this effect, we determined the aspartame dose that would be needed to elevate phenylalanine more than tyrosine in rodents, using published data. In general rodents need 60 times as much aspartame, on a mg/kg basis, as humans to obtain comparable elevations in phenylalanine with respect to tyrosine.     

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.     

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.     

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.     

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.     

Age-dependent utilization of phenylalanine for the synthesis of neuronal and glial proteins

1. Groups of 5-, 10-, 18- and 43-day old rats were injected intracerebrally with a single dose of [U14C] phenylalanine and its conversion to acid-soluble metabolites was determined in the cerebral cortex over a period of 24 h. Although the most rapid utilization of [14C] phenylalanine occurred initially in the oldest group, by 45 min the 18-day old cortex had the lowest levels of unchanged [14C] phenylalanine; by 24 h [14C] phenylalanine was, in turn, lowest in the 10-day old cortex. 2. The specific radioactivity of [14C] phenylalanine decreased rapidly at all ages for the first 2 h; it then continued to decrease in the two younger age groups while it remained virtually constant in the two older ones. 3. We also determined the incorporation of [14C] phenylalanine into, and the loss of radioactivity from, cortical, glial and neuronal proteins, isolated from animals injected on their 10th, 18th and 43rd day of life and followed over a period of two weeks. a. The proteins of the 10-day old cortex attained the highest specific radioactivity and were labeled at the highest rate. b. The glial proteins and particularly those of the 10-day old cortex were consistently more highly radioactive than the neuronal proteins; the rates of radioactivity decay from neuronal and glial proteins were parallel, however. c. The decay of the radioactivity followed a similar time course at all ages and for all proteins examined, exhibiting two phases, a rapid one extending from day 1 (day 3 in the 18-day old cortex) to day 7 and a slower phase extending through the entire second week.     

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.     

Chronic nicotine improves working and reference memory performance and reduces hippocampal NGF in aged female rats

The cholinergic system is involved in cognition and several forms of dementia, including Alzheimer's disease, and nicotine administration has been shown to improve cognitive performance in both humans and rodents. While experiments with humans have shown that nicotine improves the ability to handle an increasing working memory load, little work has been done in animal models evaluating nicotine effects on performance as working memory load increases. In this report, we demonstrate that in aged rats nicotine improved the ability to handle an increasing working memory load as well as enhanced performance on the reference memory component of the water radial arm maze task. The dose required to exert these effects (0.3mg/kg/day) was much lower than doses shown to be effective in young rats and appears to be a lower maintenance dose than is seen in light to moderate smokers. In addition, our study reports a nicotine-induced reduction in nerve growth factor (NGF) protein levels in the hippocampus of the aged rat. The effects of nicotine on hippocampal NGF levels are discussed as a potential mechanism of nicotine-induced improvements in working and reference memory.     

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.     

Oral administration of aspartame is not proconvulsant in rats

These experiments examined the potential for single or repeated doses of aspartame to exacerbate or facilitate the production of seizures in Fischer-344 rats. In adult animals, 1,000 mg/kg of aspartame given by gavage acutely or over a 14 day period had no significant effect on the rate of kindling induced by stimulation of the prepyriform cortex. A single dose of 1,000 mg/kg of aspartame had no effect on the number of animals developing tonic seizures after electroconvulsive shock, nor did aspartame affect the frequency or duration of seizure activity after pentylenetetrazol. In a second series of studies, young male and female rats were dosed with 1,000 mg/kg of aspartame on day 3-13 or 21-35 of age. Prior exposure to aspartame had no significant effect on the rate of kindling at 90 days of age. These experiments indicate that aspartame does not act a pro-convulsant in rats.     

Neuropsychological speed tests and blood phenylalanine levels in patients with phenylketonuria: A meta-analysis

Although pathogenesis of phenylketonuria is not completely understood, a low phenylalanine diet is effective to prevent severe neurological impairment, mental retardation and behavioural difficulties. Treatment recommendations heavily rely on neuropsychological research; however, single study results are ambiguous, what is reflected in substantial variation of US, British, German, Dutch and French recommendations for blood phenylalanine concentrations for adolescents and adults. We conducted a meta-analysis estimating the influence of age, phenylalanine level, and type of neuropsychological test on effect sizes (standardized differences between controls and patients) of computer-based speed measurements in phenylketonuric patients. The effect of blood phenylalanine level on effect size was more pronounced in children and adolescents than in adults, with choice reaction time being particularly sensitive for phenylalanine concentrations. Results corroborate all recommendations for children. With the exception of the US and Dutch recommendations, all recommendations for adolescents seem to be too liberal. The same effect size is predicted for adult phenylalanine concentrations between 750 and 1500 μmol/L not suggesting a preference for any of the published treatment recommendations for adulthood.     

Ultrastructural changes in rat optic nerve associated with hyperphenylalaninemia induced by para-chlorophenylalanine and phenylalanine.

The morphologic effects of hyperphenylalaninemia induced by treatment with para-chlorophenylalanine (PCP) plus phenylalanine on optic nerve were studied in developing F344 rats. PCP and phenylalanine were infected daily between days 5 and 20 days of life. At 20 days optic nerve of treated animals, as compared with saline-injected controls, showed enhanced neuroglial activity with broad astrocytic septae and debris-laden oligodendrocytes. In specimens obtained long after treatment with PCP and phenylalanine, continuing gliosis with evidence of focally abnormal myelination and axonal degeneration were observed. The results are consistent with a metabolic insult sustained in early development by astrocytes and oligodendrocytes, and are considered in relation to other work in experimental hyperphenylalaninemia and to human phenylketonuria.     

Effect of dietary phenylalanine on the plasma concentrations of phenylalanine, phenylethylamine and phenylacetic acid in healthy volunteers

1. Phenylethylamine has been proposed as a neuromodulator in several psychiatric and other brain disorders, and its concentration and that of its major metabolite, phenylacetic acid, in plasma may prove useful as state or trait markers in diagnosis, treatment or in the elucidation of biochemical mechanisms of these disorders. 2. The effect of dietary phenylalanine intake and changes in dietary phenylalanine intake on the plasma concentrations and changes in plasma concentrations, respectively, of phenylalanine, phenylethylamine and unconjugated and conjugated phenylacetic acid have been investigated. 3. Dietary phenylalanine affects the concentration of plasma phenylalanine on the following day, but has no effect on phenylethylamine or phenylacetic acid concentrations. Thus single measurements per subject of phenylethylamine or phenylacetic acid do not need to take dietary factors into account. 4. Changes in dietary phenylalanine (whether in absolute amount or in the proportion of phenylalanine in the diet) are significantly correlated with changes in unconjugated phenylacetic acid. Therefore, in longitudinal studies, dietary factors should be taken into account.     

Covalent coupling of rat liver phenylalanine hydroxylase

Optimal conditions have been determined for the coupling of rat liver phenylalanine hydroxylase (Phe H) to cyanogen bromide-Sepharose 4B. When 8 mg of ligand was reacted with 100 mg of matrix, 20 to 30 percent of the initial enzyme activity was covalently bound along with 90 percent of the protein. The coupled enzyme showed greater thermal stability from 40° to 60°, a broader base of optimal pH activity from 5.8 to 10.0, more resistance to proteolysis and less inhibition by various inhibitors. The uncoupled enzyme exhibited greater storage stability at 25° after 24 hr and at 0° after 18 days. Alteration of the microenvironment by introduction of sulfhydryl groups or of carriers having positive or negative charges had variable effects on the hydroxylase activity.     

A rapid procedure for the analysis of phenylalanine in brain tissue utilizing electron-capture gas chromatography

A rapid procedure has been developed for the analysis of phenylalanine in brain tissue. Perchloric acid extracts of brain tissue were made basic, and benzoyl chloride was added to derivatize the amine function. The aqueous layer was retained and made slightly acidic. To derivatize the carboxylic acid group, a solution of pentafluorobenzyl alcohol was added in the presence of the coupling agent dicyclohexylcarbodiimide and chloroform. After shaking for 15 min, the organic phase was retained and taken to dryness. The residue was taken up in toluene, washed, and an aliquot used for analysis on a gas chromatograph equipped with an automatic injector, a capillary column and an electron-capture detector. The procedure has been utilized for analysis of phenylalanine in brains of rats treated with vehicle or phenylalanine.     

Approaches to extrapolating animal toxicity data on organic solvents to public health

Synthesizing information about the acute neurotoxicity of organic solvents into predictive relationships between exposure and effect in humans is difficult because (1) data are usually derived from experimental animals whose sensitivity to the chemical relative to humans is unknown; (2) the specific endpoints measured in laboratory animals seldom translate into effects of concern in humans; and (3) the mode of action of the chemical is rarely understood. We sought to develop approaches to estimate the hazard and cost of exposure to organic solvents, focusing on the acute behavioral effects of toluene in rats and humans. Available published data include studies of shock avoidance behavior in rats and choice reaction time in humans. A meta-analysis of these data suggested that a 10% change in rat avoidance behavior occurs at a blood concentration of toluene 25 times higher than the concentration at which a 10% change in human choice reaction time occurs. In contrast, our in vitro studies of nicotinic acetylcholine receptors indicated that human and rat receptors do not differ in sensitivity to toluene. Analysis of other dose-response relationships for visual and cognitive functions in rats suggests that the apparent difference between rats and humans may be driven by the specific endpoints measured in the two species rather than by inherent differences in sensitivity to toluene. We also explored the hypothesis that dose-equivalence relationships may be used to compare the societal costs of two chemicals. For example, ethanol-induced changes in choice reaction time, for which societal costs are estimatable, may be used as a benchmark effect for estimating the monetary benefits of controlling exposure to organic solvents. This dose-equivalence method is applicable for solvents because this set of data fulfills three important assumptions about equivalence relationships based on a single effect: (1) a common dose metric (concentration of the chemical in the brain); (2) a common effect to provide a linking variable (choice reaction time); and (3) a common mode of action (interference with neuronal ion channel function).     

Phenylketonuria: brain phenylalanine concentrations relate inversely to cerebral protein synthesis

In phenylketonuria, elevated plasma phenylalanine concentrations may disturb blood-to-brain large neutral amino acid (LNAA) transport and cerebral protein synthesis (CPS). We investigated the associations between these processes, using data obtained by positron emission tomography with l-[1-¹¹C]-tyrosine (¹¹C-Tyr) as a tracer. Blood-to-brain transport of non-Phe LNAAs was modeled by the rate constant for ¹¹C-Tyr transport from arterial plasma to brain tissue (K₁), while CPS was modeled by the rate constant for ¹¹C-Tyr incorporation into cerebral protein (k₃). Brain phenylalanine concentrations were measured by magnetic resonance spectroscopy in three volumes of interest (VOIs): supraventricular brain tissue (VOI 1), ventricular brain tissue (VOI 2), and fluid-containing ventricular voxels (VOI 3). The associations between k₃ and each predictor variable were analyzed by multiple linear regression. The rate constant k₃ was inversely associated with brain phenylalanine concentrations in VOIs 2 and 3 (adjusted R²=0.826, F=19.936, P=0.021). Since brain phenylalanine concentrations in these VOIs highly correlated with each other, the specific associations of each predictor with k₃ could not be determined. The associations between k₃ and plasma phenylalanine concentration, K₁, and brain phenylalanine concentrations in VOI 1 were nonsignificant. In conclusion, our study shows an inverse association between k₃ and increased brain phenylalanine concentrations.     

A longitudinal study of somatosensory, brainstem auditory and peripheral sensory-motor conduction during vitamin E deficiency in the rat

A severe deficiency of vitamin E causes a characteristic neurological syndrome in man and experimental animals. In this study a number of electrophysiological modalities in vitamin E deficient and control rats have been investigated over a period of one year to define the time of onset and severity of the abnormalities associated with vitamin E deficiency in the rat. The mean velocities (n = 10) of the sensory evoked potentials were slower at all time points in the vitamin E deficient rats, with the central conduction velocities being more severely affected than the peripheral. Central conduction velocities, following both tibial and median nerve stimulation, were significantly delayed (P less than 0.005) after 8 months of deficiency. Differences in peripheral conduction following tibial stimulation became significantly delayed (P less than 0.005) after 11 months of deficiency. There were no significant differences in the brainstem auditory evoked potentials or peripheral sensory motor responses between the vitamin E deficient and control rats over the 1 year period. These results in the rat are essentially similar to those previously reported in vitamin E deficient man.     

Vitamin K-dependent blood clotting changes in female rats treated with oestrogens

Hexoestrol, diethylstilboestrol and ethinyloestradiol administered orally at a dose of 60 mg/kg/day to groups of five rats caused decreases in vitamin K-dependent blood clotting as measured by the Thrombotest reaction. These changes were self-limiting over a twenty-day study period and were not associated with external signs of bleeding. Pretreatment with testosterone and vitamin K1 did not significantly affect the clotting changes, but the antioestrogen clomiphene citrate exacerbated these, in several cases to lethal effect. Death was associated with haemorrhage. Hexoestrol-treated rats receiving clomiphene citrate in a sub-experiment did not show the increase in liver weight seen in animals given hexoestrol alone. In another sub-experiment, the level of clotting factor VII activity in the plasma of rats receiving hexoestrol at 60 mg/kg/day for four days was shown to be about half that of controls. There was probably no abnormal clotting inhibitor activity. It was deduced that the reduction in factor VII activity was more likely to be due to a decrease in synthesis as a result of liver dysfunction than changes in vitamin K availability. Clomiphene citrate may exacerbate the liver-dependent clotting disorders induced by oestrogens by preventing an adaptive increase in liver mass. It is concluded that the rat is a poor model species for investigating the blood clotting disorders seen in humans treated with oestrogens.     

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.