1. INTRODUCTION:

Aspartame is a dipeptide derivative (L-aspartyl L-phenylalanine methyl ester) figure (1)¹. It is about180- 200 times sweeter than sucrose, it has a clean sugar-like taste, with no undesirable metallic or bitter taste. It is far cheaper than sugar and is an attractive option for manufacturers², it can be found in more than 6000 products, for example soft drinks, candies, tabletop sweeteners, and some pharmaceutical preparations such as vitamins and sugar-free cough drops³.

Figure(1): The chemical structure of aspartame⁴

After oral ingestion, aspartame is absorbed from the intestinal lumen and metabolized by gut esterase and peptidases into phenylalanine (50%) -the precursor for two neurotransmitters of the catecholamine family, aspartic acid (40%) -an excitatory amino acid, and methanol (10%)-which is oxidized to cytotoxic formaldehyde and formic acid⁵. The safety of aspartame and its metabolites has been discussed frequently⁶. Blood concentrations of aspartame metabolites increase after consumption⁷. An important consideration is that Aspartame metabolites are also found naturally in foods. For example, tomato juice has 6 times more methanol than an equivalent volume of an aspartame-sweetened beverage but the difference is that naturally occurring phenylalanine, aspartic acid, and methanol are released in the blood stream at different rates because they excite as complex compounds and need time to be metabolized⁷. While in aspartame-sweetened products, the metabolites are released rapidly and in greater concentrations in blood stream and that could be the reason for aspartame toxic effects⁸.

The effect of consumption of aspartame on the plasma levels of component amino acids was extensively investigated⁹ due to several potential adverse effects of very high levels of aspartic acid and phenylalanine especially neurotoxicity observed in patients with the disease phenylketonuria (PKU) and hyperphenylalaninemia (hyperPhe), which are autosomal recessive inborn errors of amino acid metabolism resulting from absent or decreased L-phenylalanine hydroxylase (PAH) in the liver¹⁰, resulting in high plasma phenylalanine; and potential behavioral effects due to competitive inhibition of brain uptake of tryptophan, a precursor of serotonin¹¹

Also methanol is being increasingly recognized as a substance that damages the liver cells¹² where it is oxidized to formaldehyde and later to formate¹³ . These processes are accompanied by elevation of NADH level and the formation of superoxide anion, which may be involved in lipid peroxidation¹⁴ .

After aspartame consumption, there are many enzymes that are found in the serum that did not originate from the extracellular fluid¹⁵it’s thought that aspartame causes tissue damage so some of these enzymes find their way into the serum through leakage arising from altered membrane in the tissues¹⁶.

The European Food Safety Authority set the ADI (Acceptable Daily Intake)of aspartame for humans at 40mg/kg of body weight¹⁷. The ADI of aspartame is also species dependent. It is accepted that rats and mice may have an aspartame dosage of 250mg/kg/of body weight ¹⁸, the reason of this wide difference is that mice and rats has larger amounts of the tetrahydrofolate that aids in the metabolism of methanol¹⁹.

Because of the arising usage of aspartame in our daily life we conducted this research, in which The status of the lipid profile as well as altered levels of blood parameters such as blood glucose, marker enzymes ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), Γgt (Gamma-glutamyl transpeptidase), and uric acid are being measured on Balb-c mice given the ADI dose of Aspartame (250mg/kg/bd) by oral gavage to study the safety of Aspartame.

2. MATERIALS AND METHODS:

2.1. Chemicals:

Pure aspartame powder was purchased from AVCENNA, LABS for pharmaceutical industries (Damascus, Syria), and all other chemical used were of analytical grade obtained from faculty of pharmacy Tishreen University, Syria.

2.2. Experimental design:

6-week-old Balb-c mice with an average body weight of 23 ±2.4g were purchased (n=16) from the animal house of the Research Center, Damascus, Syria. The mice were acclimatized for 7 days before the onset of the experiment. Animals were maintained at 24°C, good ventilation, and 12 h light/dark cycle. The mice were fed a standard diet, and water ad libitum. The mice were divided into two groups (8 mice per group) one is a control group and the other is the experimental group.

The experimental group received the ADI of aspartame for mice (250mg/Kg BW) which was calculated according to human ADI²⁰ and corrected for mice according to Fernstrom²¹.

Mice in the treated group received the sweetener dissolved in water by oral gavage daily for 15days, 30 days, while mice in the control group received physiological solution by oral gavage also to mimic the psychological effects in all tested groups.

2.3. Sample collection:

Blood samples were taken in two phases, the first one is after 15 days, half the experimental group were fastened for 24 hours then anesthetized using Chloroform. Blood samples were collected from the heart and then putten in test tubes with heparin as an anti-coagulated factor and then we conducted the biochemical estimation.

2.4. Biochemical determinations:

The following tests were done under these principle:

Fasting blood glucose²², Total Cholesterol, Triglyceride²³, LDL (low-density lipoprotein), HDL (high-density lipoprotein⁾²⁴, ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), Γgt (Gamma-glutamyl transpeptidase)²⁵, Uric Acid²⁶.

2.5. Statistical Analysis:

Data are expressed as mean±standard deviation (SD). All data were analyzed with the SPSS. Statistical significance between the different groups was determined by One way-analysis of variance (ANOVA). When the groups showed significant difference then Tukey’s multiple comparison tests was followed and the significance level was fixed at P<0.05.

3. RESULT AND DISCUSSION:

3.1. Effect of aspartame on blood glucose levels:

after oral administration of aspartame by oral gave the levels of fasting blood gloucose were measured, the data are presented in Figure (2).

Figure (2): Effect of Aspartame on blood glucose levels after (15d, 30d) oral administration in Bala-C mice

The study showed that there was no significant changes in the level of blood glucose after 15 days of administration. However, after 30 days the difference was significant. and there was a change in the weight of this mice due to changes of dietary habits²⁷ These result is similar to a study conducted on rats by Cowan et al, They showed that giving Aspartame (5–7mg/kg/d) in drinking water for 8 week elevated fasting glucose levels²⁸.

Kate S Collison and her Colleagues studied the effect of Aspartame alone (50mg/Kg bw/day) and with combination of Monosodium Glutamate (120mg/Kg bw/day) on C57BL/6J mice. They found that there was significant increase in fasting blood glucose together with reduced insulin sensitivity during an Insulin Tolerance Test (ITT⁾²⁹.

A study was done by Palmnäs et al showed that Low-dose aspartame (5–7mg/kg/d) consumed in drinking water over an 8-week period resulted in elevated fasting glucose levels and impaired insulin tolerance in diet-induced obese rats²⁸ .

3.2. Effect of aspartame on marker enzymes:

The data are presented as bar diagram Figure (3) with mean±SD. The marker enzymes (AST, ALT, ALP and γGT) levels in the blood of Balb-c mice treated with aspartame for 15 d, 30d were consequently increased in serum when compared to control.

Figure (3): Effect of Aspartame on marker enzymes after (15d,30d) oral administration in Bala-C mice

The consequent increase in level of these enzymes ALP, AST, ALT and γGT in serum confirms that damage has been inflicted on the hepatic membrance³⁰.

That’s similar to a study made by Mohames El-Sayed and his colleagues who also studied the effect of aspartame and aspirin on the liver. The study was conducted on two groups of rats: one group was given aspartame within the limits of ADI i.e. (250mg/kg BW) and the other group was given 4 times the daily allowable limit of aspartame (1000mg/kg BW), they were given aspartame daily by dissolving it with little water and orally administered it for 8 weeks. The study found a noticeable rise in liver enzymes ALT and ALP, and there was no noticeable difference for ALP levels between the two groups while there was a noticeable increase in ALT with increasing dose Given aspartame³¹. Aspartame-induced liver inflammation and necrosis is associated with GSH depletion³² and a decrease in glutathione peroxidase and glutathione reductase activities³³.

3.3. Effect of aspartame on lipid profile and Uric Acid:

The data are presented in Table (4) with mean±SD. The cholesterol, TG, LDL levels showed a marked increase in aspartame treated groups and this increase was more marked in 30 d aspartame exposed mice.

There was no significant changes observed in uric acid³⁴ and HDL levels

Table (4): effect of aspartame on the lipid profile after(15d,30d) oral administration in Balb-c mice

As we can see in our study, aspartame administration also increased the lipid profile (cholesterol, TG, LDL).³⁵

A study conducted in Brazeil found that animals exposed to aspartame during the prenatal period presented a higher consumption of sweet foods during adulthood and a greater susceptibility to alterations in metabolic parameters, such as increased glucose, LDL and triglycerides. These effects were observed in both males and females³⁶.

This increase could be associated with the up-regulation of leptin³⁷ and down-regulation of adiponectin and peroxisome proliferator activated receptor-γ (PPAR-γ) expression³⁸.

4. CONCLUSION:

The present results give further data to support the idea that aspartame alters biochemical indices and lipid profile in mice after consumption and it is a time-dependent change. This investigation clearly shows that Aspartame might not be safe, and it has several toxicities and these effects could result from its metabolites. Since aspartame consumption is on the rise, the safety of this sweetener should be revisited, that is why more studies should be done on both animals and humans to be surer of its safety.

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Received on 13.04.2020 Modified on 15.06.2020

Research J. Pharm. and Tech. 2021; 14(5):2387-2390.

DOI: 10.52711/0974-360X.2021.00421