Antiglycating potential of acesulfame potassium: an artificial sweetener

Abstract

Sweeteners have replaced the natural sugars in the food and beverage industry because of many reasons, such as hyperglycemia and cost. Saccharin, sucralose, aspartame and acesulfame-K are the most commonly used sweeteners. In the present study, the abovementioned artificial sweeteners were used to assess their glycating properties by established methods such as browning, fructosamine assay, determination of carbonyl content, protein aggregation, and measurement of fluorescence. Amadori and advanced glycation end products (AGEs) are formed as a result of the interaction between carbonyl groups of reducing sugars and amino groups of proteins and other macromolecules during glycation. The objective of this study was to investigate the influence of artificial sweeteners on the formation of AGEs and protein oxidation in an in vitro model of glucose-mediated protein glycation. The results indicated that the abovementioned artificial sweeteners do not enhance the process of glycation. On the other hand, acesulfame-K was found to have antiglycating potential as it caused decreased formation of Amadori products and AGEs. Further studies are essential in the characterization of Amadori products and AGEs produced as a result of interaction between sweeteners and proteins, which are interfered with by sweeteners. This study is significant in understanding the probable role of artificial sweeteners in the process of glycation and the subsequent effect on macromolecular alteration.

Full text

ARTICLE
Antiglycating potential of acesulfame potassium: an artificial
sweetener
Ahmad Ali,Tejashree Anil More,Amaritpal Kaur Hoonjan, and Subramanian Sivakami
Abstract: Sweeteners have replaced the natural sugars in the food and beverage industry because of many reasons, such as
hyperglycemia and cost. Saccharin, sucralose, aspartame and acesulfame-K are the most commonly used sweeteners. In the
present study, the abovementioned artificial sweeteners were used to assess their glycating properties by established methods
such as browning, fructosamine assay, determination of carbonyl content, protein aggregation, and measurement of fluores-
cence. Amadori and advanced glycation end products (AGEs) are formed as a result of the interaction between carbonyl groups
of reducing sugars and amino groups of proteins and other macromolecules during glycation. The objective of this study was to
investigate the influence of artificial sweeteners on the formation of AGEs and protein oxidation in an in vitro model of
glucose-mediated protein glycation. The results indicated that the abovementioned artificial sweeteners do not enhance the
process of glycation. On the other hand, acesulfame-K was found to have antiglycating potential as it caused decreased formation
of Amadori products and AGEs. Further studies are essential in the characterization of Amadori products and AGEs produced as
a result of interaction between sweeteners and proteins, which are interfered with by sweeteners. This study is significant in
understanding the probable role of artificial sweeteners in the process of glycation and the subsequent effect on macromolecular
alteration.
Key words: Amadori product, AGEs, artificial sweeteners, DNA damage, glycation, methyl glyoxal.
Résumé : Plusieurs facteurs tels l’hyperglycémie et le coût ont contribué au remplacement des sucres naturels par des édul-
corants dans l’industrie des aliments et des boissons. La saccharine, le sucralose, l’aspartame et l’acésulfame-potassium sont les
édulcorants les plus utilisés. Dans la présente étude, on utilise ces édulcorants artificiels afin de déterminer leurs propriétés de
glycation par des méthodes établies telles que le brunissement, le dosage de la fructosamine, la détermination du contenu en
carbonyle, l’agrégation des protéines et la mesure de la fluorescence. Les produits terminaux de glycation avancée (« AGE ») et
d’Amadori sont le résultat de l’interaction des groupes carbonyles de sucres réducteurs et des groupes aminés de protéines et
d’autres macromolécules durant la glycation. Le but de cette étude est d’examiner l’influence des édulcorants artificiels sur la
formation des AGE et l’oxydation des protéines dans un modèle in vitro de glycation de protéine induite par le glucose. D’après
les résultats, ces édulcorants artificiels n’amplifient pas le processus de glycation. D’autre part, l’acésulfame-potassium présente
un potentiel d’antiglycation, car il diminue la formation des produits AGE et d’Amadori. Il faut réaliser d’autres études sur la
caractérisation des produits AGE et d’Amadori qui sont le résultat de l’interaction des édulcorants et des protéines modifiées par
les édulcorants. Cette étude est signifiante pour la compréhension du rôle probable des édulcorants artificiels dans le processus
de glycation et leur effet subséquent dans la modification des macromolécules. [Traduit par la Rédaction]
Mots-clés : produit d’Amadori, AGE, édulcorants artificiels, dommage a
`l’ADN, glycation, méthylglyoxal.
Introduction
Nonenzymatic glycosylation, also known as glycation, is a spon-
taneous amino-carbonyl reaction between sugars and long-lived
proteins, nucleic acids, and lipids. It is also one of the post-
translational modification processes between the reducing sugars
and free amino groups of proteins. Glycation has been linked to a
number of diseases such as Alzheimer’s, diabetes mellitus, cata-
ract, dialysis-related amyloidosis, arthrosclerosis, Parkinson’s, as
well as physiological aging (Thorpe and Baynes 1996). Nonenzy-
matic reactions can occur both in vivo as well as in vitro and affect
the structure, function, or conformation of these biomolecules.
The process of glycation occurs continuously in the body even at
normal glucose levels and the damage caused by the advanced
glycation end products (AGEs) keep accumulating slowly over
time. The deleterious effects of these products are more signifi-
cantly observed when the blood glucose level increases above the
normal level in conditions like hyperglycemia.
The model system of methylglyoxal (MG) and amino acids
(Meeprom et al. 2015) has been used to study the late stages of
glycation between dicarbonyl intermediates formed during glyca-
tion and free amino groups of proteins. The formation of keto-
aldehydes during glycation is an important step that causes
protein cross-links to form and leads to the formation of radical
cation sites on the cross-linked proteins. The counter anions,
superoxide, and hydrogen peroxide generated from MG anion
can initiate free radical chain reactions, leading to damage of
biomacromolecules, such as DNA, protein, etc., in close prox-
imity to reaction sites (Yim et al. 1995).
Synthetic sugar substitutes, also known as artificial sweeteners,
amplify the sugar effect in taste, having less or no food energy.
Artificial sweeteners are universally used in the food, beverage,
Received 23 February 2017. Accepted 17 May 2017.
A. Ali, T.A. More, A.K. Hoonjan, and S. Sivakami. Department of Life Sciences, University of Mumbai, Vidyanagari, Santacruz (E), Mumbai 400098,
India.
Corresponding author: Ahmad Ali (email: ahmadali@mu.ac.in).
Copyright remains with the author(s) or their institution(s). Permission for reuse (free in most cases) can be obtained from RightsLink.
Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)
1
Appl. Physiol. Nutr. Metab. 00: 1–10 (0000) dx.doi.org/10.1139/apnm-2017-0119 Published at www.nrcresearchpress.com/apnm on 15 June 2017.
rich2/apn-apnm/apn-apnm/apn99914/apn1004d14z
xppws S⫽3 7/25/17 4:35 4/Color Fig: F1-F5,F7-F11,F13 Art: apnm-2017-0119 Input-1st dis

confectionary, and pharmaceutical industries (Chattopadhyay
et al. 2014). There are 2 generations of artificial sweeteners. The
first includes aspartame, saccharin, and cyclamate while the
second generation of products includes acesulfame-K, alitame,
neotame, and sucralose (Benton 2005). Acesulfame-K is the potas-
sium salt of the acesulfame ring molecule and saccharin is a
2-membered ring structure. Both of these sweeteners contain sul-
phur and nitrogen atoms and also a carbonyl group. Aspartame is
chemically the methyl ester of the standard amino acids L-aspartic
acid and L-phenylalanine; it contains 3 carbonyl groups, 1 hydroxyl
group, and 1 amino group each along with the benzene ring from
phenylalanine skeleton. Sucrose when chlorinated at 3 specific
hydroxyl groups gives sucralose as the product. In addition to the
original hydroxyl groups, there are chlorine atoms present as well
(Figs. 1A–1D). Based on scientific evidence and reports, including
several toxicological and clinical trial studies, 5 artificial sweeten-
ers (saccharin, aspartame, sucralose, neotame, and acesulfame-K)
are considered as safe for human use by the Food and Drug Ad-
ministration (FDA) in accordance with their acceptable daily in-
take (Lorenzo et al. 2015). On the other hand, breakdown products
of some of these sweeteners have been shown to have contentious
and metabolic health effects. Research is still proceeding to com-
pare the diabetogenic and mutagenic potency of most broadly
used artificial sweeteners (Shastry et al. 2012). There are very few
reports suggesting role of artificial sweeteners in the process of
glycation (Ali and Devrukhkar 2016).
The current study is based on the fact that the artificial sweet-
eners mentioned above have functional groups similar to sugars
that can react with amino groups of macromolecules, inducing
damage by glycation and glycoxidation. In the present study, ar-
tificial sweeteners were used to assess their glycating properties
by using established methods, such as browning, fructosamine,
and carbonyl assays; determination of protein aggregation; and
measurement of fluorescence of AGEs. Besides, the effect of
Fig. 1. Structures of the artificial sweeteners used in the present study. (A) Acesulfame-K (National Center for Biotechnology Information.
PubChem Compound Database; CID = 11074431, https://pubchem.ncbi.nlm.nih.gov/compound/11074431 (accessed 1 June 2017)). (B) Saccharin
(National Center for Biotechnology Information. PubChem Compound Database; CID = 5143, https://pubchem.ncbi.nlm.nih.gov/compound/
5143 (accessed 1 June 2017)). (C) Aspartame (National Center for Biotechnology Information. PubChem Compound Database; CID = 134601,
https://pubchem.ncbi.nlm.nih.gov/compound/134601 (accessed 1 June 2017)). (D) Sucralose (National Center for Biotechnology Information.
PubChem Compound Database; CID = 71485, https://pubchem.ncbi.nlm.nih.gov/compound/71485 (accessed 1 June 2017)). [Colour online.]
Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)
2 Appl. Physiol. Nutr. Metab. Vol. 00, 0000
Published by NRC Research Press
F1
rich2/apn-apnm/apn-apnm/apn99914/apn1004d14z
xppws S⫽3 7/25/17 4:35 4/Color Fig: F1-F5,F7-F11,F13 Art: apnm-2017-0119 Input-1st dis

acesulfame-K on glycation-induced damage to DNA by the MG–
lysine–ferric chloride and glucose–lysine–ferric chloride systems
was determined. Results obtained in the present work suggest the
antiglycating potential of acesulfame-K and probably insignifi-
cant roles of other sweeteners in the glycation process. This study
is significant in understanding the possible role of artificial sweet-
eners in the process of glycation and the subsequent effect on
macromolecular alteration.
Materials and methods
The following chemicals were purchased as follows: glucose from
MERCK chemicals; acrylamide, agarose, bovine serum albumin
(BSA), lysine, and MG from Sigma–Aldrich; aspartame, acesulfame-K,
saccharin, and sucralose were purchased from NeelChem Mumbai;
pBR322 from Thermofisher. All other chemicals used were of high-
analytical grade.
Methods
In vitro glycation of lysine with glucose and artificial sweeteners
An aqueous solution of lysine (100 mg/mL; pH adjusted to 7.4)
was incubated with glucose (100 mg/mL) and the artificial sweet-
eners sucralose, saccharin, acesulfame-K (100 mg/mL) at 37 °C for
5 days. Since aspartame has lower solubility in water, an aqueous
solution of lysine (14.2 mg/mL; pH adjusted to 7.4) was incubated
with glucose (14.2 mg/mL) and aspartame (14.2 mg/mL) at 55 °C for
5 days. All the incubations were carried out in 0.1 mol/L phosphate
buffer, pH 7.4, and contained 3 mmol/L sodium azide to prevent
bacterial contamination.
In vitro glycation of BSA with glucose and acesulfame-K
An aqueous solution of BSA (10 mg/mL) was incubated with
glucose (100 mg/mL) and acesulfame-K (100 mg/mL) in 0.1 mol/L
phosphate buffer (pH 7.4) at 37 °C for 14 days. Bacterial contami-
nation was prevented by addition of 3 mmol/L sodium azide.
Measurement of browning
Browning was measured at 420 nm using Shimadzu UV 1800
spectrophotometer (Rondeau et al. 2007).
Measurement of fructosamines
The concentration of fructosamine, an Amadori product, was
measured by Nitroblue tetrazolium (NBT) assay according to
Meeprom et al. (2013) with slight modifications. Ten microliters of
the glycated sample was incubated with 100 ␮L of 0.5 mmol/L NBT
in 0.1 mol/L carbonate buffer (pH 10.4) at 37 °C for 15 min. The
volume of the reaction mixture was made to 1 mL with D/W and
the absorbance was measured at 530 nm using a Shimadzu UV
1800 spectrophotometer.
Determination of carbonyl content
The carbonyl group in glycated sample was determined by the
conventional 2,4-dinitrophenylhydrazine (DNPH) method with
minor modifications (Meeprom et al. 2013). In this method, 400 ␮L
of 10 mmol/L DNPH in 2.5 mol/L hydrochloride (HCl) was added to
100 ␮L of glycated samples and incubated in dark for an hour.
Then, 500 ␮L of 20% (w/v) Trichloroacetic acid was added to the
tubes and kept on ice for 5 min for proteins to precipitate. Cen-
trifugation at 10 000 r/min for 10 min at 4 °C was carried out,
following which the protein pellet was washed with 500 ␮Lof
ethanol/ethyl acetate (1:1) mixture 3 times. Finally, the pellet was
resuspended in 250 ␮L of 6 mol/L guanidine HCl and volume made
to 1 mL with D/W for spectroscopic measurement at 370 nm using
a Shimadzu UV 1800 spectrophotometer.
Determination of protein aggregation
Amyloid cross ␤-structure, a common marker for protein aggre-
gation, was measured by using Congo red assay according to
Adisakwattana and colleagues (2014) with minor modifications.
Briefly, 50 ␮L of glycated BSA was incubated with 50 ␮Lof
100 ␮mol/L Congo red in 10% (v/v) ethanol/phosphate-buffered
saline for 20 min at 25 °C. The volume was raised to 1 mL with
D/W. Absorbance was measured at 530 nm using a Shimadzu
UV 1800 spectrophotometer.
Measurement of fluorescence
Formation of fluorescent AGEs was measured using Cary Eclipse
Fluorescence spectrophotometer (Varian). The fluorescent inten-
sity was measured at excitation and emission wavelengths of
370 nm and 440 nm, respectively (Sharma et al. 2002).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) of glycated proteins
Glycated protein samples were dialyzed extensively using
10 mmol/L phosphate buffer (pH 7.4) to remove unreacted glucose.
Dialyzed sample was mixed with equal proportion of 2× gel load-
ing dye, heated at 95 °C for 5 min. Twenty microliters of superna-
tant was loaded onto SDS-PAGE (10% resolving gel and 5% stacking
gel) and allowed to stack at 50 V, following which the run was
continued at 100 V. The gel was stained in Coomassie brilliant blue
R-250 staining solution for 45 min. Gel was de-stained until back-
ground was clear and observed under white light.
In vitro glycation of plasmid DNA in the presence of acesulfame-K
The role of sweetener (acesulfame-K) in the glycation-mediated
DNA strand breakage was performed according to a previous pub-
lication with minor modifications (Ali et al. 2014). pBR322 plasmid
DNA (0.5 ␮g) in 0.1 mol/L phosphate buffer (pH 7.4) was incubated
with lysine (20 mmol/L), glucose (250 mmol/L)/MG (20 mmol/L),
ferric chloride (100 ␮mol/L), sodium azide (250 mmol/L) in the
presence and absence of acesulfame-K (250 mmol/L), in a 10-␮L
reaction system, at 37 °C overnight. The reaction was stopped by
freezing the samples at −20 °C.
Agarose gel electrophoresis of glycated plasmid DNA sample
Ten microliters of samples were mixed with 3 ␮L 6× gel loading
dye and loaded on to 1% agarose gel. Electrophoresis carried out
initially at 90 V and once the samples left the well, voltage was
decreased to 85 V. Once the dye band reached two-thirds of gel
length, electrophoresis was terminated and gel was stained using
ethidium bromide solution (final concentration 5 ␮g/mL) for 20 min
in dark. Subsequently the gel was visualized under ultraviolet tran-
silluminator and bands analyzed with the help of control.
Statistical analysis
Statistical analysis was performed using Microsoft Excel 2007.
Results of each experiment were presented as means ± SE. Student’s
ttest (paired) was used to analyze the data for significance, where
results with pvalue ≤0.05 were accounted as statistically significant.
Results
In vitro glycation of lysine with glucose and artificial
sweeteners
Browning of lysine incubated with glucose and artificial
sweeteners
The intensity of browning of lysine incubated with glucose
and/or artificial sweeteners is presented in Fig. 2. Lysine was incu-
bated with glucose and saccharin, sucralose, and acesulfame-K at
37 °C for 5 days. Aspartame was incubated with lysine and glucose
mixture at a higher temperature (55 °C) for 5 days to compensate
for the lower concentration of aspartame used in the experimental
system because of its lower solubility in water. It was noted that
among the abovementioned artificial sweeteners, acesulfame-K
showed a significant reduction in the intensity of browning of
lysine and glucose mixture whereas others shows negligible
effect. The percentage inhibition of browning by acesulfame-K
is approximately 23.49%.
Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)
Ali et al. 3
Published by NRC Research Press
F2
rich2/apn-apnm/apn-apnm/apn99914/apn1004d14z
xppws S⫽3 7/25/17 4:35 4/Color Fig: F1-F5,F7-F11,F13 Art: apnm-2017-0119 Input-1st dis

Fig. 2. Browning of lysine incubated with glucose and artificial
sweeteners. Lysine (100 mg/mL) was incubated with glucose
(100 mg/mL); sucralose, saccharin, and acesulfame-K (100 mg/mL
each) were incubated with sodium azide (3 mmol/L) at 37 °C for
5 days. In case of aspartame (14.2 mg/mL), lysine and glucose
concentrations were changed (14.2 mg/mL), the temperature raised
to 55 °C, and they were incubated for 5 days. Absorbance was
measured at 420 nm post-incubation and Lys + GLU tube was used as
control (100%) for calculating the relative percentage of other tubes.
Results are expressed as means ± SE (n= 3). *, Statistical significance,
p< 0.05. ACE-K, acesulfame-K; ASP, aspartame; GLU, glucose;
Lys, lysine; SACC, saccharin; SUC, sucralose. [Colour online.]
Fig. 3. Fructosamines assay of glycated sample for determination of
Amadori products. Lysine (100 mg/mL) was incubated with glucose
(100 mg/mL); sucralose, saccharin, and acesulfame-K (100 mg/mL
each) were incubated with sodium azide (3 mmol/L) at 37 °C for
5 days. In case of aspartame (14.2 mg/mL), lysine and glucose
concentrations were changed (14.2 mg/mL), the temperature raised
to 55 °C, and they wereincubated for 5 days. Amount of early
Amadori product determined using Nitroblue tetrazolium assay,
with the resultant absorbance measured at 530 nm. Each value
expressed as relative percentage considering Lys + GLU as 100%.
Results are expressed as means ± SE (n= 3). *, Statistical significance,
p< 0.05. ACE-K, acesulfame-K; ASP, aspartame; GLU, glucose;
Lys, lysine; SACC, saccharin; SUC, sucralose. [Colour online.]
Fig. 4. Determination of carbonyl content of glycated lysine. Lysine
(100 mg/mL) was incubated with glucose (100 mg/mL); sucralose,
saccharin, and acesulfame-K (100 mg/mL each) were incubated with
sodium azide (3 mmol/L) at 37 °C for 5 days. In case of aspartame
(14.2 mg/mL), lysine and glucose concentrations were changed
(14.2 mg/mL), the temperature raised to 55 °C, and they were
incubated for 5 days. Amount of carbonyl content was estimated
using the 2,4-dinitrophenylhydrazine method and absorbance was
measured at 370 nm. Each value expressed as relative percentage
considering Lys + GLU as 100%. Results are expressed as means ± SE
(n= 3). *, Statistical significance, p< 0.05. ACE-K, acesulfame-K;
ASP, aspartame; GLU, glucose; Lys, lysine; SACC, saccharin;
SUC, sucralose. [Colour online.]
Fig. 5. Measurement of fluorescent AGEs of glycated lysine at
excitation and emission wavelengths of 370 and 440 nm, respectively.
Lysine (100 mg/mL) was incubated with glucose (100 mg/mL); sucralose,
saccharin, and acesulfame-K (100 mg/mL each) were incubated with
sodium azide (3 mmol/L) at 37 °C for 5 days. In case of aspartame
(14.2 mg/mL), lysine and glucose concentrations were changed
(14.2 mg/mL), the temperature raised to 55 °C, and they were incubated
for 5 days. Intensity of fluorescent AGEs was measured at the excitation
and emission wavelength of 370 and 440 nm, respectively. Each value
expressed as relative percentage considering Lys + GLU mixture as
100%. Results are expressed as means ± SE (n= 2). *, Statistical
significance, p< 0.05. ACE-K, acesulfame-K; AGEs, advanced
glycation end products; ASP, aspartame; GLU, glucose; Lys, lysine;
SACC, saccharin; SUC, sucralose. [Colour online.]
Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)
4 Appl. Physiol. Nutr. Metab. Vol. 00, 0000
Published by NRC Research Press
rich2/apn-apnm/apn-apnm/apn99914/apn1004d14z
xppws S⫽3 7/25/17 4:35 4/Color Fig: F1-F5,F7-F11,F13 Art: apnm-2017-0119 Input-1st dis

Measurement of fructosamines
To access the level of Amadori product formed during the gly-
cation process, fructosamine measurement was performed (Fig. 3).
The results indicated that the level of fructosamine was substantially
reduced in the glycated samples containing acesulfame-K (30.91%) as
well as saccharin (31.82%). In contrast to this, sucralose and aspar-
tame were found to show a negligible increase of 3.147% and 0.61%,
respectively.
Determination of carbonyl content
Protein oxidation of the glycated samples was measured by the
determination of carbonyl content (Fig. 4). It was observed that the
glycated samples containing saccharin and sucralose shows a marginal
decrease of 3.84% and 3.60%, respectively, whereas the one containing
acesulfame-K showed an extensive reduction of 34.26% in the carbonyl
content. On the other hand the glycated samples containing aspar-
tame shows a slight increase of 7.78% in the carbonyl content.
Formation of fluorescent AGEs
The amount of total AGEs was measured by spectrofluorimeter
with excitation and emission wavelengths of 370 and 440 nm, respec-
tively. It can be seen from the Figs. 5 and 6that acesulfame-K drasti-
cally reduced the amount of AGEs as compared with lysine and
glucose system.
Fig. 6. Determination of advanced glycation end products formation at excitation and emission wavelengths of 370 (A) nm and 440 nm (B),
respectively. Lysine (100 mg/mL) was incubated with glucose (100 mg/mL); sucralose, saccharin, and acesulfame-K (100 mg/mL each) were
incubated with sodium azide (3 mmol/L) at 37 °C for 5 days. In case of aspartame (14.2 mg/mL), lysine and glucose concentrations were
changed (14.2 mg/mL), the temperature raised to 55 °C, and they were incubated for 5 days. Excitation at 370 nm and emission spectrum from
400 nm to 520 nm was plotted. Graph of mean of 3 independent experiments was plotted. ACE-K, acesulfame-K; ASP, aspartame; GLU, glucose;
Lys, lysine; SACC, saccharin; SUC, sucralose.
A
B
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
400 420 440 460 480 500 520
Lys ONLY GLU ONLY Lys+GLU SUC ONLY
Lys+SUC Lys+GLU+SUC GLU+SUC SACC ONLY
Lys+SACC Lys+GLU+SACC GLU+SACC ACE-K ONLY
Lys+ACE-K Lys+GLU+ACE-K GLU+ACE-K
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
400 420 440 460 480 500 520
Lys ONLY GLU ONLY ASP ONLY Lys+GLU
Lys+ASP Lys+GLU+ASP GLU+ASP
Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)
Ali et al. 5
Published by NRC Research Press
F3
F4
F5-F6
rich2/apn-apnm/apn-apnm/apn99914/apn1004d14z
xppws S⫽3 7/25/17 4:35 4/Color Fig: F1-F5,F7-F11,F13 Art: apnm-2017-0119 Input-1st dis

In vitro glycation of BSA with glucose and acesulfame-K
Browning of BSA incubated with glucose and acesulfame-K
BSA (10.0 mg/mL) was incubated with glucose (100.0 mg/mL) and
acesulfame-K (100.0 mg/mL) at 37 °C for 5 days. The intensity of the
brown colour developed was measured at 420 nm. It is compre-
hended from the results that the intensity of browning of BSA +
glucose mixture decreases in the presence of acesulfame-K as
shown in Fig. 7. The intensity of browning of glucose-glycated BSA
decreases by 79.34% in the sample containing acesulfame-K.
Effect of acesulfame-K on the level of fructosamine of BSA
incubated with glucose
Amadori product, a fructosamine generated in the glycation
process, was measured by the fructosamine assay at 530 nm. The
effect of acesulfame-K on the level of fructosamine of BSA incu-
bated with glucose is presented in Fig. 8. The results indicated that
acesulfame-K markedly suppressed the formation of fructosamine. A
significant reduction of 66.29% in the level of fructosamine was ob-
served.
Effect of acesulfame-K on the level of protein oxidation of BSA
incubated with glucose
The protein oxidation mediated by the glycation process was
assessed by the determination of the carbonyl content. When BSA
was incubated with glucose, an increase in the level of carbonyl
content was noticed whereas acesulfame-K significantly reduced
the level of carbonyl content. At the end of the study, a decrease of
42.61% was observed in the solution of glycated BSA in the pres-
ence of acesulfame-K (Fig. 9).
Effect of acesulfame-K on the formation of amyloid cross-
␤
structure of BSA incubated with glucose
To access the level of protein aggregation called amyloid cross-␤
structure formed during the glycation of proteins, Congo red as-
say was performed. At the end of the experimental study, the
results demonstrated that the formation of amyloid cross-␤struc-
ture in the glycated BSA decreased by ⬃34.09% in the presence of
acesulfame-K (Fig. 10).
Effect of acesulfame-K on the formation of fluorescent AGEs of
BSA incubated with glucose
The formation of AGEs was observed by measuring the fluorescent
intensity in glycated BSA with glucose as shown in Figs. 11 and 12. The
Fig. 7. Browning of BSA incubated with glucose and acesulfame-K.
BSA (10.0 mg/mL) was incubated with glucose (100 mg/mL),
acesulfame-K (100 mg/mL), and sodium azide (3 mmol/L) at 37 °C for
14 days. Absorbance measured at 420 nm post-incubation. Results
are expressed as means ± SE (n= 3). *, Statistical significance,
p< 0.05. ACE-K, acesulfame-K; BSA, bovine serum albumin;
GLU, glucose. [Colour online.]
Fig. 8. Effect of acesulfame-K on the level of fructosamine of BSA
incubated with glucose. BSA (10.0 mg/mL) was incubated with
glucose (100 mg/mL), acesulfame-K (100 mg/mL), and sodium azide
(3 mmol/L) at 37 °C for 14 days. Amount of early Amadori product
was determined using Nitroblue tetrazolium assay, with the
resultant absorbance measured at 530 nm. Each value is expressed
as relative percentage considering glycated BSA as 100%. Results are
expressed as means ± SE (n= 3). *, Statistical significance, p< 0.05.
ACE-K, acesulfame-K; BSA, bovine serum albumin; GLU, glucose.
[Colour online.]
Fig. 9. Effect of acesulfame-K on the level of protein oxidation of
BSA incubated with glucose. BSA (10.0 mg/mL) was incubated with
glucose (100 mg/mL), acesulfame-K (100 mg/mL), and sodium azide
(3 mmol/L) at 37 °C for 14 days. Amount of carbonyl content was
estimated using the 2,4-dinitrophenylhydrazine method. And
absorbance measured at 370 nm. Each value is expressed as relative
percentage considering glycated BSA as 100%. Results are expressed
as means ± SE (n= 3). *, Statistical significance, p< 0.05. ACE-K,
acesulfame-K; BSA, bovine serum albumin; GLU, glucose. [Colour
online.]
Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)
6 Appl. Physiol. Nutr. Metab. Vol. 00, 0000
Published by NRC Research Press
F7
F8
F9
F10 F11-F12
rich2/apn-apnm/apn-apnm/apn99914/apn1004d14z
xppws S⫽3 7/25/17 4:35 4/Color Fig: F1-F5,F7-F11,F13 Art: apnm-2017-0119 Input-1st dis

results established that the addition of acesulfame-K into the solu-
tion greatly reduced the formation of fluorescent AGE throughout
the study period. Acesulfame-K decreased the formation of fluores-
cent AGEs by 50.71%.
SDS-PAGE of glycated proteins
When BSA was incubated with glucose at 37 °C for 14 days, a
characteristic aggregation was observed (Fig. 13; lane 2). On the
other hand, acesulfame-K decreased the aggregation caused by
glucose (Fig. 13; lane 4).
In vitro glycation of plasmid DNA
Effect of acesulfame-K on DNA damage caused by MG with lysine
and ferric chloride
The glycation system of MG with lysine and ferric chloride
caused damage by stand breakage (Fig. 14; lane 2). Sodium azide
inhibited the strand breakage (Fig. 14; lane 3). Also, acesulfame-K
inhibited the damage individually (Fig. 14; lane 4) as well as when
used along with sodium azide (Fig. 14; lane 5).
Effect of acesulfame-K on DNA damage caused by glucose with
lysine and ferric chloride
Glucose with lysine and ferric chloride, i.e., the glycation sys-
tem caused damage to plasmid DNA by stand breakage (Fig. 15;
lane 3). Sodium azide inhibited the strand breakage individually
(Fig. 15; lane 4) and also when used along with acesulfame-K
(Fig. 15; lane 6). Acesulfame-K caused no strand breakage (Fig. 15;
lane 2) and also inhibited DNA damage caused by the glycation
system (Fig. 15; lane 5).
Discussion
Advanced glycation end products have been implicated in a
number of pathophysiological complications, such as diabetes,
atherosclerosis, Alzheimer’s disease, and aging. Age-related dis-
eases exhibit increased levels of glycation and its end products,
further supporting the idea that sugars and their metabolites may
act as damaging molecules, especially when they accumulate in
the cells and tissues (Basta et al. 2004;Suji and Sivakami 2004).
Major pathways followed by AGEs to damage biomolecules in-
clude aggregation, precipitation, or through reactive oxygen spe-
cies formation. So far, many AGE structures have been identified
and their role in several health complications has been deduced.
Based on this information, synthetic molecules have been de-
signed so as to prevent the damage caused by glycation by inhib-
iting AGE formation or by preventing them from crosslinking
with the structural proteins. Alternatively, much attention is also
being paid to the utilization of natural substances, preferably
antioxidants, to prevention of glycation-mediated damage, posing
as potent candidates for therapeutics.
The change in food trends from natural sugars to artificially
synthesized sweet-tasting additives is rapid. Most commercially
manufactured edibles for human consumption have artificial
sweeteners being used for reasons ranging from weight control to
aid in dealing with diabetes, for instance, “diet beverages”. The
FDA has approved many of the chemically synthesized sweeteners
for human consumption. In a present study, the effect of artificial
sweeteners on the formation of glycation-mediated AGEs was an-
alyzed. Artificial sweeteners such as saccharin, sucralose, aspar-
tame, and acesulfame-K were assayed for their potential role in
glycation process. The results indicated that acesulfame-K re-
duced the level of Amadori products and carbonyl content in
lysine and glucose system of glycation. Subsequently, it also sig-
nificantly suppressed the formation of fluorescence AGEs in the
glycation mixture. Similar results were observed for acesulfame-K
in the BSA glycated samples as well. BSA alone resulted in the
generation of higher fluorescence than the glycated sample. Re-
cently it has been reported that proteins can show a blue-green
fluorescence phenomenon and this can be excited at the edge of
the long-wavelength ultraviolet range (⬃360–380 nm) and emits
in the deep-blue (⬃450 nm) (Sarkar et al. 2014). This fluorescence
exists in the protein prior to protein aggregations in monomeric
proteins, in disordered polypeptide chains, and in all single amino
Fig. 10. Effect of acesulfame-K on the formation of amyloid cross-␤
structure of BSA incubated with glucose. BSA (10.0 mg/mL) was
incubated with glucose (100 mg/mL), acesulfame-K (100 mg/mL), and
sodium azide (3 mmol/L) at 37 °C for 14 days. Amount of formation
of amyloid cross-␤structure was determined by Congo red assay and
absorbance was measured at 530 nm. Each value is expressed as
relative percentage considering glycated BSA as 100%. Results are
expressed as means ± SE (n= 3). *, Statistical significance, p< 0.05.
ACE-K, acesulfame-K; BSA, bovine serum albumin; GLU, glucose.
[Colour online.]
Fig. 11. Effect of acesulfame-K on the formation of fluorescent AGEs
of BSA incubated with glucose at emission wavelength of 440 nm.
BSA (10.0 mg/mL) was incubated with glucose (100 mg/mL),
acesulfame-K (100 mg/mL), and sodium azide (3 mmol/L) at 37 °C for
14 days. Intensity of fluorescent AGEs at the emission was measured
at wavelength of 440 nm. Each value is expressed as relative
percentage considering glycated BSA as 100%. Results are expressed
as means ± SE (n= 3). *, Statistical significance, p< 0.05. ACE-K,
acesulfame-K; AGEs, advanced glycation end products; BSA, bovine
serum albumin; GLU, glucose. [Colour online.]
Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)
Ali et al. 7
Published by NRC Research Press
F13
F14
F15
rich2/apn-apnm/apn-apnm/apn99914/apn1004d14z
xppws S⫽3 7/25/17 4:35 4/Color Fig: F1-F5,F7-F11,F13 Art: apnm-2017-0119 Input-1st dis

acids. Therefore, the higher fluorescence by BSA alone observed in
the present study may be because of the contribution of fluores-
cence and the abovementioned structural factors as well as self-
aggregation in the BSA. It can also be observed that glycated BSA
has shown more fluorescence than in the presence of acesulfame
potassium and glucose. It can be concluded that acesulfame po-
tassium interferes with the glycation process and prevents the
aggregation of the BSA.
When acesulfame-K was added to the BSA glycated samples, it
efficiently reduced the level of Amadori products and protein
carbonyl content in BSA. A significant reduction in the protein
aggregation was noticed in BSA glycated samples on the addition
of acesulfame-K as observed with the help of SDS-PAGE.
pBR 322 is a sensitive indicator of single strand breaks arising
owing to damage by glycation and free radicals (Levi and Werman
2001). The model system of MG/glucose, lysine, and ferric chloride
Fig. 12. Determination of advanced glycation end products formation spectrofluorimetrically at excitation at 370 nm and emission spectrum
from 380 nm to 520 nm. BSA (10.0 mg/mL) was incubated with glucose (100 mg/mL), acesulfame-K (100 mg/mL), and sodium azide (3 mmol/L) at
37 °C for 14 days. Emission spectrum from 360 nm to 520 nm was plotted in the presence of glucose and acesulfame potassium. Graph was
plotted for the mean value of 3 independent experiments. ACE-K, acesulfame-K; BSA, bovine serum albumin; GLU, glucose.
0
50
100
150
200
250
300
350
400
360 380 400 420 440 460 480 500 520
BSA ONLY GLU ONLY ACE-K ONLY BSA+GLU
BSA+ACE-K BSA+GLU+ACE-K GLU+ACE-K
Fig. 13. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
of glycated proteins. Bovine serum albumin (BSA) (10 mg/mL) was
incubated with glucose (100 mg/mL) and acesulfame-K (100 mg/mL)
in 0.1 mol/L phosphate buffer (pH 7.4) at 37 °C for 14 days and
dialyzed extensively with 10 mmol/L phosphate buffer (pH 7.4)
overnight. Dialyzed samples were analyzed on 10% resolving gel and
5% stacking gel. Lane 1, BSA; lane 2, BSA + glucose; lane 3, BSA +
acesulfame-K; lane 4, BSA + glucose + acesulfame-K. [Colour online.]
Fig. 14. Effect of acesulfame-K on DNA damage caused by
methylglyoxal (MG) with lysine and ferric chloride. pBR 322 DNA
(0.5 ␮g) was incubated with lysine (20 mmol/L), MG (20 mmol/L),
FeCl3 (100 ␮mol/L), sodium azide (250 mmol/L), and acesulfame-K
(250 mmol/L) in different combinations (mentioned in lane
description) at 37 °C overnight. Electrophoresed on 1% agarose gel
and stained using 5 ␮g/mL ethidium bromide. Lane 1, pBR 322 alone;
lane 2, pBR 322 + lysine (20 mmol/L) + MG (20 mmol/L) + FeCl3
(100 ␮mol/L); lane 3: lane 2 + sodium azide (250 mmol/L); lane 4:
lane 2 + acesulfame-K (250 mmol/L); lane 5, lane 2 + sodium azide
(250 mmol/L) + acesulfame-K (250 mmol/L).
Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)
8 Appl. Physiol. Nutr. Metab. Vol. 00, 0000
Published by NRC Research Press
rich2/apn-apnm/apn-apnm/apn99914/apn1004d14z
xppws S⫽3 7/25/17 4:35 4/Color Fig: F1-F5,F7-F11,F13 Art: apnm-2017-0119 Input-1st dis

has been used to study the glycation between carbonyl interme-
diates formed during glycation of MG/glucose and free amino
groups of proteins. It is proved that a substantial portion of hy-
drogen peroxide lethality involves damage to DNA by the oxidants
generated from the iron-mediated Fenton reaction (Henle and
Linn 1997). Thus, damage to DNA is mainly due to the generation
of free radicals during the late stage of glycation reactions. There
are several reports that suggest this mechanism of metal-induced
free radical mediated damage to DNA (Kang 2003;Suji and Sivakami
2007). Our biological system is home to many trace elements,
including iron, which may react with hydrogen peroxide to pro-
duce hydroxyl radical and then induce DNA strand breakage. It
has been shown earlier shown that MG, lysine, and ferric chloride
alone have no effect on the integrity of supercoiled DNA (Ali and
Sharma 2015). In our study also it was found that acesulfame-K
also does not cause damage to DNA by itself.
To check the effect of acesulfame-K on glycation-induced dam-
age to DNA, the plasmid DNA was incubated with MG + lysine +
ferric chloride in the presence and absence of acesulfame-K and/or
sodium azide. Surprisingly, it was seen that acesulfame-K pre-
vented the damage caused to DNA to a great extent. Also, in
collaboration with the sodium azide, acesulfame-K showed en-
hanced inhibitory action on preventing glycation-induced DNA
damage. Similar results were seen in the case of glucose + lysine +
ferric chloride glycation system. This suggests the antiglycating
potential of acesulfame-K. There are no reports in the literature
that suggest the free radical scavenging activity of acesulfame
potassium. However, 1 group showed that acesulfame potassium
does not have DNA-damaging property (Jeffrey and Williams 2000).
The antiglycating and prevention of DNA-imaging property of ace-
sulfame potassium is probably due to its effect on the inhibition
of early glycation products as can be seen in results obtained with
fructosamine assay, a method exclusively used for the measure-
ment of Amadori products. Therefore, it can be interpreted from
the results presented here that acesulfame potassium is not allow-
ing the generation of AGEs and AGE-mediated free radical gener-
ation.
Conclusion
Several approaches can be used to analyze the glycating/antig-
lycating potential of a molecule. These include the measurement
of Amadori products, carbonyl content, accumulation of AGEs,
and subsequent free radical formation. The purpose of this study
was to characterize the role of artificial sweeteners in the glyca-
tion process. It can be concluded from the results obtained in the
present study that most of the artificial sweeteners could not
induce glycation of proteins as none of them caused a significant
increase in the amount of Amadori product and AGEs. However,
acesulfame-K showed unexpected quality of reducing/interfering
with glycation, as evidenced by significant reduction in Amadori
product, carbonyl content, and the total AGEs. Further it was also
observed that acesulfame-K decreased the aggregation of glycated
BSA, a consequence of glycation process.
Free radicals are generated as a result of modifications of AGEs.
These free radicals can cause damage to macromolecules and one
of the best markers for this kind of analysis is double-stranded
DNA (pBR322). Glycation-induced free-radical mediated DNA dam-
age is analyzed by simple electrophoretic technique. Acesulfame-K
was found to reverse the glycation-induced DNA damage. All these
results indicate the antiglycating potential of acesulfame-K. In
most likelihood it is preventing the formation of early glycation
products (Amadori). Further characterization of this antiglycation
property of acesulfame and functional group involved is required
to deduce the exact mechanism. This is a preliminary work on the
roles of sweeteners in glycation.
Conflict of interest statement
We declare that we have no conflict of interest.
Acknowledgements
The study was funded by University of Mumbai (grant no. 179)
and University Grants Commission, New Delhi (grant no. F.20–
10(10)/2013(BSR)).
References
Adisakwattana, S., Thilavech, T., and Chusak, C. 2014. Mesona Chinensis Benth
extract prevents AGE formation and protein oxidation against fructose-
induced protein glycation in vitro. BMC Complement. Altern. Med. 14: 130.
doi:10.1186/1472-6882-14-130. PMID:24708679.
Ali, A., and Devrukhkar, J. 2016. In vitro study on glycation of plasma proteins
with artificial sweeteners. Acta Biol. (Szeged), 60(1): 65–70.
Ali, A., and Sharma, R. 2015. A comparative study on the role of lysine and BSA
in glycation-induced damage to DNA. Biosci. Bioeng. Commun. 1: 38–43.
Ali, A., Sharma, R., and Sivakami, S. 2014. Role of natural compounds in the
prevention of DNA and proteins damage by glycation. Bionano. Front. 7:
25–30.
Basta, G., Schmidt, A.M., and De Caterina, R. 2004. Advanced glycation end
products and vascular inflammation: implications for accelerated atheroscle-
rosis in diabetes. Cardiovasc. Res. 63: 582–592. doi:10.1016/j.cardiores.2004.
05.001. PMID:15306213.
Benton, D. 2005. Can sweeteners help control body weight and prevent obesity?
Nutr. Res. Rev. 18: 63–76. doi:10.1079/NRR200494. PMID:19079895.
Chattopadhyay, S., Raychaudhuri, U., and Chakraborty, R. 2014. Artificial
sweeteners – a review. J. Food Sci. Technol. 51(4): 611–621. doi:10.1007/s13197-
011-0571-1. PMID:24741154.
Henle, E.S., and Linn, S. 1997. Formation, prevention, and repair of DNA damage
by iron/hydrogen peroxide. J. Biol. Chem. 272: 19095–19098. doi:10.1074/jbc.
272.31.19095. PMID:9235895.
Jeffrey, A.M., and Williams, G.M. 2000. Lack of DNA-damaging activity of five
non-nutritive sweeteners in the rat hepatocyte/DNA repair assay. Food Chem.
Toxicol. 38: 335–338. doi:10.1016/S0278-6915(99)00163-5. PMID:10722887.
Fig. 15. Effect of acesulfame-K on DNA damage caused by glucose
with lysine and ferric chloride. pBR 322 DNA (0.5 ␮g) was incubated
with lysine (20 mmol/L), glucose (250 mmol/L), FeCl3 (100 ␮mol/L),
sodium azide (250 mmol/L), and acesulfame-K (250 mmol/L) in
different combinations (mentioned in lane description) at 37 °C
overnight. Electrophoresed on 1% agarose gel and stained using
5␮g/mL ethidium bromide. Lane 1, pBR 322 alone; lane 2, pBR 322 +
acesulfame-K (250 mmol/L); lane 3, pBR 322 + lysine (20 mmol/L) +
glucose (250 mmol/L) + FeCl3 (100 ␮mol/L); lane 4, lane 3 + sodium
azide (250 mmol/L); lane 5, lane 3 + acesulfame-K (250 mmol/L); lane 6,
lane 3 + sodium azide (250 mmol/L) + acesulfame-K (250 mmol/L)).
Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)
Ali et al. 9
Published by NRC Research Press
rich2/apn-apnm/apn-apnm/apn99914/apn1004d14z
xppws S⫽3 7/25/17 4:35 4/Color Fig: F1-F5,F7-F11,F13 Art: apnm-2017-0119 Input-1st dis

Kang, J.H. 2003. Oxidative damage of DNA induced by methylglyoxal in vitro.
Toxicol. Lett. 145(2): 181–187. doi:10.1016/S0378-4274(03)00305-9. PMID:14581171.
Levi, B., and Werman, M.J. 2001. Fructose triggers DNA modification and damage
in an Escherichia coli plasmid. J. Nutr. Biochem. 12(4): 235–241. doi:10.1016/
S0955-2863(00)00158-3. PMID:11287219.
Lorenzo, R.A., Pena, M.T., Fernández, P., González, P., and Carro, A.M. 2015.
Artificial sweeteners in beverages by ultra-performance liquid chromatography
with photodiode array and liquid chromatography tandem mass spectrometry.
Food Control, 47: 43–52. doi:10.1016/j.foodcont.2014.06.035.
Meeprom, A., Sompong, W., Chan, C.B., and Adisakwattana, S. 2013. Isoferulic
acid, a new anti-glycation agent, inhibits fructose- and glucose-mediated
protein glycation in vitro. Molecules, 18: 6439–6454. doi:10.3390/molecules
18066439. PMID:23722732.
Meeprom, A., Sompong, W., Suantawee, T., Thilavech, T., Chan, C.B., and
Adisakwattana, S. 2015. Isoferulic acid prevents methylglyoxal-induced protein
glycation and DNA damage by free radical scavenging activity. BMC Comple-
ment. Altern. Med. 15: 346. doi:10.1186/s12906-015-0874-2. PMID:26438049.
Rondeau, P., Armenta, S., Caillens, H., Chesne, H., and Bourdon, E. 2007. Assess-
ment of temperature effects on beta-aggregation of native and glycated
albumin by FTIR spectroscopy and PAGE: relations between structural changes
and antioxidant properties. Arch. Biochem. Biophys. 460(1):141–150. doi:10.
1016/j.abb.2007.01.014. PMID:17320036.
Sarkar, S., Sengupta, A., Hazra, P., and Mandal, P. 2014. Blue emission in pro-
teins. Available from https://arxiv.org/ftp/arxiv/papers/1404/1404.6859.pdf.
Sharma, S.D., Pandey, B.N., Mishra, K.P., and Sivakami, S. 2002. Amadori product
and age formation during nonenzymatic glycosylation of bovine serum
albumin in vitro. J. Biochem., Mol. Biol. Biophys. 6(4): 233–242. PMID:12186738.
Shastry, C.S., Yatheesh, C.K., and Aswathanarayana, B.J. 2012. Comparative eval-
uation of diabetogenic and mutagenic potential of artificial sweeteners –
aspartame, acesulfame-K and sucralose. Nitte Univ. J. Health Sci. 2(3): 80–85.
Suji, G., and Sivakami, S. 2004. Glucose, glycation and aging. Biogerontology, 5:
365–373. doi:10.1007/s10522-004-3189-0. PMID:15609100.
Suji, G., and Sivakami, S. 2007. DNA damage during glycation of lysine by meth-
ylglyoxal: assessment of vitamins in preventing damage. Amino Acids, 33:
615–621. doi:10.1007/s00726-007-0498-z. PMID:18004515.
Thorpe, S.R., and Baynes, J.W. 1996. Role of the Maillard reaction in diabetes
mellitus and diseases of aging. Drugs Aging, 9: 69–77. doi:10.2165/00002512-
199609020-00001. PMID:8820792.
Yim, H.S., Kang, S.O., Hah, Y.C., Chock, P.B., and Yim, M.B. 1995. Free radicals
generated during the glycation reaction of amino acids by methylglyoxal:
a model study of protein-cross-linked free radicals. J. Biol. Chem. 270(47):
28228–28233. doi:10.1074/jbc.270.47.28228. PMID:7499318.
Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)
10 Appl. Physiol. Nutr. Metab. Vol. 00, 0000
Published by NRC Research Press
rich2/apn-apnm/apn-apnm/apn99914/apn1004d14z
xppws S⫽3 7/25/17 4:35 4/Color Fig: F1-F5,F7-F11,F13 Art: apnm-2017-0119 Input-1st dis