This document summarizes a study that evaluated the toxic effects of aspartame on human erythrocyte membranes by measuring acetylcholinesterase (AChE) activity. Blood samples were taken from 40 healthy individuals and erythrocyte membranes were separated into a control group and a group incubated with aspartame. AChE levels were then measured spectrophotometrically. Results showed AChE levels were significantly lower in the aspartame group compared to the control, indicating that aspartame consumption decreases AChE activity in erythrocyte membranes. The study concludes that aspartame intake should be limited to prevent potential neurological side effects.

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Aspartame-induced Toxicity on Human Erythrocyte Membrane
Acetylcholine esterase Activity
Nevine El Kady, Usama Mohamed El-Barrany, Khaled Bayoumi, Mohamed Adly
Department of Forensic Medicine and Clinical Toxicology, Faculty of Medicine, Cairo University
Abstract
BACKGROUND: Aspartame is probably the most extensively studied food additive ever approved
by the FDA. Neurological disturbances have been implicated with aspartame (ASP) consumption and
the cholinergic system. The aim of this study was to evaluate the toxic effect of Aspartame on human
erythrocyte membranes through its effect on acetylcholinesterase (AChE) activity.
METHODS: A comparative in vitro study was performed, in which blood samples were collected
from 40 healthy individuals. Erythrocyte membranes were separated from blood of the different
samples, and then erythrocyte membranes from each sample were subdivided into 2 groups: group
(1) is considered as a control group and group (2) with incubation of erythrocyte membranes with
ASP. Acetyl cholinesterase was measured spectrophotometrically, and its levels were evaluated in
these groups.
RESULTS: Comparison between the acetyl cholinesterase level in the control and the ASP groups
showed statistically significant (p <0.05) lower level in the ASP group.
CONCLUSIONS: Aspartame consumption decreased the erythrocyte membrane acetyl
cholinesterase. Such information may in turn guide clinical practice on the use of Aspartame to avoid
long-term complications, and so, more studies should be done for more understanding of the side
effects of aspartame.
Keywords: Aspartame – Acetylcholine esterase – food additives – sweeteners - erythrocyte
membrane
Conflicts of interest: None declared.
INTRODUCTION
The original Food and Agriculture Organization (FAO)/World Health
Organization (WHO) definition of an additive was “non-nutritive substances added

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intentionally to food, generally in small quantities, to improve its appearance, flavor,
texture, or storage properties” [1 – 3].
For decades now, the food industry has continually created new chemicals to
manipulate, preserve, and transform our food. There are even foods products that are
made entirely from chemicals e.g. coffee creamers, sugar substitutes and some candies
[4].
Aspartame is a nutritive sweetening agent used in food and beverages. It
contains 4 kcal /g and is about 180–200 times as sweet as sucrose. The product was
discovered in 1965 and obtained final approval by the Food and Drug Administration
(FDA) as a food additive in 1981 and in carbonated beverages in 1983. Aspartame is
probably the most extensively studied food additive ever approved by the FDA
[5]used in more than 6000 different type of products [6].
The Joint Expert Committee on Food Additives (JECFA) and the European
Commission’s Scientific Committee on Food have determined this safe value: 40
mg/kg of body weight for aspartame, while FDA has set its ADI for aspartame at 50
mg/kg [6, 7].
Chronic exposure to aspartame was reported clinically to cause blurred
vision and brain tumors as well as eye problems, numbness, insomnia, nausea, slurred
speech, personality changes, loss of energy, hyperactivity, hearing problems,
neurological and behavioral disturbances [8,9].
Aspartame is also believed to cause negative effects on specific human
neurological functions including memory loss, seizures, headaches, confusion and
dizziness. Studies show that the sweetener might affect brain neurotransmitters and
receptors especially after long-term consumption. Acetylcholine esterase (AChE) is a

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key enzyme of the muscarinic cholinergic system, involved in learning and memory
that can be affected by the long term use of aspartame [10, 11].
It was concluded that long-term aspartame may be responsible for oxidative
stress and hepato-renal toxicity due to release of toxic metabolites, in which methanol
is considered to be one [9].
With reference to aspartame and other low-calorie sweeteners, most animal
studies have failed to show a carcinogenic activity. Only two experimental studies on
rats found an excess incidence of malignant neoplasms, especially lymphomas and
leukemia in females [12, 13]and hepatocellular and alveolar/bronchiolar carcinomas in
males only [14]. In 1981, an FDA statistician stated that the brain tumor data on
aspartame was so "worrisome" that he could not recommend its approval [15].
According to a research, aspartame leads to poorer diabetic control and
aggravation of diabetic complications [15].The Food and Drug Administration (FDA)
has reviewed more than 100 toxicologic and clinical studies that attest to its safety [3,
16].
Acetylcholine esterase is found at mainly neuromuscular junctions and
cholinergic brain synapses, where its activity serves to terminate synaptic transmission
[17, 18]. AChE is a key enzyme of the muscarinic cholinergic system, involved in
learning and memory [11, 19].
Acetylcholine esterase is found in many types of conducting tissue: nerve
and muscle, central and peripheral tissues, motor and sensory fibers, and cholinergic
and noncholinergic fibers. The activity of AChE is higher in motor neurons than in
sensory neurons. AChE is also found on the red blood cell membranes [20].That is

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why measurement of its activity in erythrocytes reflects disturbances in nervous
system [21].
Irreversible inhibitors of AChE may lead to muscular paralysis, convulsions,
bronchial constriction, and death by asphyxiation. Organophosphates (OP) are a class
of irreversible AChE inhibitors used in insecticides (e.g., malathion) and nerve gases
for chemical warfare[17, 18]. Carbamates are AChE inhibitors used for medical
purposes (e.g., physostigmine for the treatment of glaucoma) [19].
Reversible inhibitors occupy the esteratic site for short periods of time
(seconds to minutes) and are used to treat of a range of central nervous system
diseases. Tetrahydroaminoacridine and donepezil are FDA-approved to improve
cognitive function in Alzheimer's disease and pyridostigmine bromide is used to treat
myasthenia gravis [19].
Caffeic acid (3,4-dihydroxycinnamic acid) is a compound present in many
plants and in the diet as part of fruits, tea and wine andwas discovered to alter the
AChE activity, in vitro and in vivo and improve memory [17].
The determination of AChE activity is usually based on the quantification of
the decomposition product during such reaction. This includes photometric,
flourometric, manometrics, potentiometric, titrimetric, enzymatic methods,
radioisotopic assays, mass spectrometry and biosensors [22].
Thin layer chromatography (TLC) is one of the most wide spread analytical
methods used in the organic chemistry laboratory. A new, validated, sensitive and
cheap method for preliminary quantitative evaluation of acetylcholine esterase
inhibitory activity is high performance thin layer chromatography (HPTLC) [22].

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Aspartame is immediately absorbed from the lumen upon ingestion, and
metabolized to phenylalanine, aspartic acid and methanol. Studies concluded that low
concentrations of aspartame metabolites had no effect on the erythrocyte membrane
AChE enzyme activity, whereas high or toxic concentrations partially or remarkably
decreased the activity, respectively, with related neurological symptoms, including
learning and memory processes.Also almost the same degree of the enzyme inhibition
was observed in the erythrocyte membrane AChE activity of phenylketonuric (PKU)
patients “off diet” [10].
The aim of our study was to evaluate the toxic effect of Aspartame on
human erythrocyte membranes through its effect on acetylcholinesterase (AChE)
activity.
MATERIALS& METHODS:
This was an in vitro study, where blood samples were obtained from 40
healthy individuals above 12 years of age, including 14 females and 26 males.
Erythrocyte membranes will be separated from blood of the different samples, and
then erythrocyte membranes from each sample will be subdivided into 2 groups:
group (1): is considered as a control group in which erythrocyte membranes will be
left alone without incubation and without adding ASP and group (2): Incubation of
erythrocyte membranes with ASP.
Quantitative assessment of AChE levels was donespectrophotometrically
and its levels were evaluated in these two groups. Prepare AChE reaction mixture (50
μl), then add AChE standards or AChE test samples (50 μl), then Incubate at room

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temperature for 10 - 30 minutes. Monitor absorbance at 410 ± 5 nm and thaw all the
kit components to room temperature before starting the experiment [23].
RESULTS
Assessment of AChE levels spectrophotometrically in both groups showed
that the mean level of acetyl cholinesterase is lower in the ASP groupthan in the
control group(table 1) and Fig. 1.
Group Mean
(IU/L)
*N *Std.
Deviation
Minimum Maximum Median
(1) Control 4.5770 40 0.61320 3.80 5.80 4.4000
(2) *ASP 1.6135 40 0.51703 0.99 2.80 1.5000
*ASP: Aspartame, N: number, Std. Deviation:standard deviation.
Table (1): The mean, standard deviation, minimum, maximum and median of the results of the
acetyl cholinesterase results in the 2groups.
Figure (1): Mean acetylcholine esterase levels between the control and the ASP groups.
Comparison between the acetyl cholinesterase levels showed statistically
significant difference (p <0.05) between the acetyl cholinesterase level of the control
group and the ASP (table 2).
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Control ASP
Mean AChE (IU/L)

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Comparison between Control
and ASP Group
Mean
Difference
*Std. Error *P value 95% Confidence
Interval
2.96350(*) 0.20250 0.000
Upper
Bound
Upper
Bound
2.3196 3.6074
Table (2):Comparison between the acetyl cholinesterase level of the control and the ASP groups.
DISCUSSION
Aspartame was found to have numerous serious complications. There is very
little awareness regarding aspartame side effects because these are not widely reported
by the media and most people do not associate their symptoms with long-term use of
aspartame [15]. There are many accounts of situations in which ASP is believed to
have caused negative effects on specific human neurological functions. These include
memory loss, seizures, headaches, confusion and dizziness [11].
Numerous studies have implicated muscarinic cholinergic receptors and
memory. It has been suggested that nicotinic transmission may be important in delayed
response tasks, while the muscarinic system may be involved, in general working
memory processes. These studies lead us to hypothesize that it might exist an
alteration in brain muscarinic system after high or toxic doses of ASP consumption
and that the most available human tissue for AChE study was the human erythrocyte
membranes [24, 25].
In our study, comparison between the acetyl cholinesterase level of the
control group and the other groups showed statistically significant difference (p <0.05)
between the acetyl cholinesterase level of the control group and the ASP.
CONCLUSIONS
Aspartame consumption decreases the erythrocyte membrane
acetylcholinesterase levels. So, its intake is better to be avoided, as much as possible,

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to prevent their side effects and protect our health. Strict supervision of various food
additives’ companies and industries is necessary to prevent any fault and ensure their
safety. Further larger prospective studies should be done to fully understand the impact
of aspartame and guide clinical practice on its use to avoid long-term complications.
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