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Zinc enhances carnosine inhibitory effect against structuraland functional age-related protein alterations in an albuminglycoxidation model
Hichem Moulahoum . Faezeh Ghorbanizamani . Suna Timur .
Figen Zihnioglu
Received: 13 July 2020 / Accepted: 25 September 2020 / Published online: 30 September 2020
� Springer Nature B.V. 2020
Abstract Age-related complications including pro-
tein alterations seen in diabetes and Alzheimer’s
disease are a major issue due to their accumulation and
deleterious effects. This report aims to investigate the
effect of zinc supplementation on the anti-glycoxida-
tion activity of carnosine on the in vitro model of
albumin-based protein modification. Besides, the
therapeutic effect of this combination was tested
through the addition of the molecules in tandem (co-
treatment) or post initiation (post-treatment) of the
protein modification process. Glycation was induced
via the addition of glucose to which carnosine (5 mM)
alone or with various zinc concentrations (125, 250,
and 500 lM) were added either at 0 h or 24 h post-
glycation induction. On the other hand, protein
oxidation was induced using chloramine T (20 mM)
and treated in the same way with carnosine and zinc.
The different markers of glycation (advanced glyca-
tion end products (AGEs), dityrosine, and beta-sheet
formation (aggregation)) and oxidation (AOPP,
advanced oxidation protein products) were estimated
via fluorescence and colorimetric assays. Zinc addi-
tion induced a significant enhancement of carnosine
activity by reducing albumin modification that out-
performed aminoguanidine both in the co- and post-
treatment protocols. Zinc demonstrated a supplemen-
tary effect in combination with carnosine highlighting
its potential in the protection against age-related
protein modifications processes such as the ones
found in diabetes.
Keywords Zinc � Carnosine � Glycoxidation �Protein modification � Aggregation � Albumin
Introduction
Aging is a snowballing phenomenon resulting from
the accumulation of altered and dysfunctional proteins
that contribute to the predisposition to age-related
diseases (Krisko and Radman 2019). Glycation is a
central mechanism in aging and is associated with
diabetes mellitus. Its products contribute to long-term
hyperglycemia and the production and accumulation
of advanced glycation end-products (AGEs). This
alteration is a long process that may take weeks,
however, during this process, the interaction of the
aldehydes and ketones found in sugars with the
proteins’ amino groups (i.e. lysine and arginine)
results in the formation of intermediate products such
H. Moulahoum (&) � F. Ghorbanizamani �S. Timur � F. Zihnioglu
Biochemistry Department, Faculty of Sciences, Ege
University, 35100 Bornova, Izmir, Turkey
e-mail: [email protected]
S. Timur
Central Research Test and Analysis Laboratory
Application and Research Center, Ege University,
35100 Bornova, Izmir, Turkey
123
Biometals (2020) 33:353–364
https://doi.org/10.1007/s10534-020-00254-0(0123456789().,-volV)( 0123456789().,-volV)
as Schiff bases and Amadori products (Fournet et al.
2018; Heidari et al. 2020). AGEs attachment to
macromolecules is non-reversible even after the
correction of hyperglycemia and tends to accumulate
with time (DeGroot et al. 2004). Glycation is also
linked with an imbalance of the oxidative stress
system through the production of reactive oxygen
species and free radicals that induce protein oxidation
producing advanced oxidation protein products
(AOPP). These products (AGEs and AOPPs) are seen
as the intersection of several metabolic pathways
(Fournet et al. 2018).
Therapeutic strategies against age-related protein
alterations and their accumulation could be
approached from various directions among which
(i) inhibition of early or advanced glycation, (ii)
carbonyl quenching, (iii) concealing specific amino
acid residues, and (iv) blocking aggregation (Chilu-
kuri et al. 2018). There exist numerous molecules and
drugs that have an anti-glycation effect. However,
AGEs inhibitors are ingested daily through the con-
sumption of natural products, natural remedies,
nutraceuticals, and functional foods which attracted
the interest in these molecules for their therapeutic
potential against age-related diseases (Chilukuri et al.
2018; Chinchansure et al. 2015; Sadowska-Bartosz
and Bartosz 2015).
Proteins and peptides represent an important
reserve of biologically active molecules. Some bioac-
tive peptides are dormant in their parent proteins and
become active once released by proteolysis. Their
activities are dictated by their amino acid composition,
sequence, and length. As such, peptides could be seen
as a viable alternative to synthetic drugs due to the
advantages they propose including their good bio-
compatibility, wide range of action, low accumulation,
and non-toxicity (Chilukuri et al. 2018).
Carnosine (b-alanyl-l-histidine) is a naturally
occurring dipeptide found in the muscles, brains, and
hearts of many organisms. The main source comes
from dietary intake (meat and fish) but could be
synthesized endogenously by carnosine synthase
(Boldyrev et al. 2013; Sale et al. 2013). The potential
therapeutic benefits of carnosine were demonstrated
against diabetes, cancer, metabolic syndrome, catar-
acts, wound healing, and many other pathological
conditions (Chmielewska et al. 2020; Ghodsi and
Kheirouri 2018). Carnosine possesses various func-
tions such as ion chelating, acid–base buffering
activity, antitoxic activity, antioxidant, anti-inflam-
matory, immunity system enhancement, anti-aging,
anti-aggregation, neurotransmitter, and neuroprotec-
tion activities (Caruso et al. 2019; Ghodsi and
Kheirouri 2018). Hence, carnosine could be beneficial
in therapy against aging-related pathologies (Ber-
mudez et al. 2018; Caruso et al. 2019).
Carnosine has anti-glycating and anti-crosslinking
activities. It can inhibit AGE formation and/or assist
the unfolding of altered proteins and solubilize them
through hydrophobic regions disruption (Chilukuri
et al. 2018). Previously, we have reported the potential
effects of carnosine against protein glycation, aggre-
gation, and oxidation. The interaction sites were
shown to be different from the sites used by the
reference antiglycating molecule aminoguanidine
(Moulahoum et al. 2019). We speculated that despite
knowing the hotspot regions for the aggregation
process, each molecule interacts differently to reach
the same end result to prevent glycation and protein
modification. Based on the previous findings and
aiming to further explore the potential of carnosine,
we directed our focus on the effect of trace metal
supplementation on the anti-glycation activity of
carnosine.
Zinc is an essential element in the body that is
connected to many key mechanisms of cell growth and
development, immunity, structure/function of pro-
teins, and cell signaling (Hara et al. 2017). It is
required by more than 2500 macromolecules and 300
proteins and enzymes as part of their structures and
functions (Lee 2018). Zinc possesses many activities
and functions as an inflammatory status regulator and
an antioxidant either alone or by partaking in the
synthesis of other antioxidant enzymes (Olechnowicz
et al. 2018). Multiple evidence pointed to the impli-
cation of zinc levels in aging-related complications
and chronic diseases such as diabetes and Alzheimer’s
disease (Chu et al. 2016). Though the antioxidant
effect of zinc is well established and various reports
demonstrated its potential in inhibiting protein glyca-
tion (Kheirouri et al. 2018; Tupe and Agte 2010;
Xiong et al. 2015), there are contradictory information
on the beneficial or deleterious effects of zinc
supplementation in age-related diseases (Cuajungco
and Faget 2003). Carnosine is known for its metal
chelating activity and its zinc homeostasis mainte-
nance in the brain cells. Zinc carnosine (ZnC) has been
artificially prepared and the polymeric structure has
123
354 Biometals (2020) 33:353–364
been marketed as a food supplement. The combination
of zinc and carnosine would provide enhanced benefits
theoretically compared to each of them alone (Mah-
mood et al. 2007).
Despite the abundant literature on the anti-glyca-
tion effect of carnosine and zinc, there has been no
clear focus addressing their combinatory benefit over
age-related protein alteration. Therefore, the present
study aimed to explore the effects of carnosine in the
presence or absence of different zinc concentrations
(co- and post-treatment) over an in vitro albumin
protein modification model.
Materials and methods
Reagents
Bovine serum albumin (BSA), sodium azide, and
aminoguanidine (AG) were purchased from Sigma
Aldrich (St. Louis, MO, USA). Glucose and zinc
sulfate were procured from Merck (Germany). All
other reagents used in this work were of analytical
grade and unless mentioned otherwise, were obtained
from Sigma.
BSA protein glycation
The glycation procedure was performed following an
established method (Matsuura et al. 2002) with slight
modifications. Glycation was induced by mixing
bovine serum albumin (BSA, 10 mg/mL) with 0.5 M
glucose (Glc) in 3 mL phosphate buffer (0.1 M,
pH7.4, 0.02% sodium azide) and incubated at 60 �Cfor 48 h. Initially, two sets containing either BSA or
BSA ? Glc were tested for a time-dependent glyca-
tion assessment in order to determine the optimal time
for the post-treatment protocol to be initiated (1, 2, 3,
4, 5, 6, 18, 24, and 48 h). Treatments were either added
at 0 h or 24 h after the glycation initiation. Experi-
mental sets received carnosine (5 mM) alone or with
the addition of different zinc concentrations (125, 250,
and 500 lM) and were incubated according to the
scheme presented in Fig. 1. Negative control (BSA),
positive control (BSA ? Glc), and aminoguanidine, a
reference anti-glycation molecule (BSA ? Glc ?
AG), were prepared for each experimental set.
Additional sets with AG, Car, and different concen-
trations of zinc without BSA or glucose were put under
the same experimental conditions to determine any
changes of these molecules during the incubation
process. At the end of the protocol, samples were
dialyzed against phosphate buffer for 24 h to eliminate
unbound glucose. The samples were aliquoted and
stocked until glycation and aggregation markers
estimation. Experiments were performed in triplicates
for three independent experiments. Carnosine concen-
tration was based on the effective natural amounts
present in the brain and muscles which is between 2
and 10 mM (Prokopieva et al. 2000) and the 5 mM
dose was used according to the various reports
working with carnosine (Kim and Kim 2020; Moula-
houm et al. 2019; Yamashita et al. 2018). The zinc
doses were used as recommended by other authors
(Tupe et al. 2015).
AGEs and dityrosine estimation
AGEs estimation was performed using fluorimetry
which takes advantage of the fluorescence emitted by
the glycated proteins (Tupe and Agte 2010). AGEs
fluorescence intensities were measured using a regular
fluorescence spectrometer (Perkin Elmer Ltd., Eng-
land) where the excitation wavelength was kex = 370
nm, and the emission wavelength kem = 440 nm. In
addition, dityrosine (kex = 330 nm/kem = 415 nm)
was measured in the same conditions as AGEs. The
inhibitory effect of the different treatments was
estimated according to the following equation: Inhi-
bition (%) = 100% - [(Sample fluorescence/
C ? fluorescence) 9 100]. Sample fluorescence is
the value obtained by subtracting the fluorescence of
the treatment molecule alone from the fluorescence of
BSA ? Glc ? respective treatment molecule.
Cross b structure and high molecular weight
aggregate estimation
Cross beta aggregation in glycated albumin samples
was estimated using the Thioflavin T (ThT) fluori-
metric method (Tupe and Agte 2010) with modifica-
tions (Meeprom et al. 2013). Glycated samples (300
lL) were mixed with 3 mL ThT (64 lM) prepared in
phosphate buffer (0.1 M, pH7.4) and incubated for 1 h
(room temperature, in the dark). Samples were ana-
lyzed via fluorescence (kex = 435 nm and kem = 485
nm) and the inhibitory levels were determined
123
Biometals (2020) 33:353–364 355
according to the same formula used for AGE inhibi-
tion estimation.
BSA protein oxidation
The protein oxidation protocol was performed accord-
ing to the method described previously with slight
modification (Grzebyk and Piwowar 2016). Briefly,
BSA (10 mg/mL) was mixed with 20 mM chloramine
T (CT) in phosphate buffer (0.1 M, 0.02% sodium
azide, pH 7.4) for 2 h at 37 �C. Similar to the protocol
made for protein glycation, carnosine (5 mM) in the
absence or presence of three doses of zinc sulfate were
added (125, 250, 500 lM). Positive and negative
controls were prepared with BSA ? CT or BSA
alone, respectively. AG (5 mM) was used as a
reference molecule. The co-treatment set received
the molecules at 0 h while the post-treatment set
Fig. 1 Protocol design for the examination of zinc supplemen-
tation on the anti-protein glycation and anti-protein oxidation
effects of carnosine in vitro. (BSA) Bovine serum albumin;
(Glc) Glucose; (CT) Chloramine T; (AG) Aminoguanidine;
(Car) Carnosine; (Zn) zinc; (C-) BSA alone; (C ?) BSA with
either glucose or chloramine T
123
356 Biometals (2020) 33:353–364
received them at 1 h after the initiation of protein
oxidation. Once the incubation finished, samples were
dialyzed for 24 h against phosphate buffer and then
stored until further measurements. Experiments were
performed in triplicates for three independent
experiments.
AOPP estimation
Advanced oxidation protein products (AOPP) were
measured according to the methods described previ-
ously (Witko-Sarsat et al. 1996). In a 96-well
microplate, 70 lL of samples are deposited and mixed
with 100 lL of phosphate buffer. Glacial acetic acid
(20 lL), KI solution (10 lL, 1.16 M), and another 20
lL of glacial acetic acid were added sequentially to the
samples after which the plate was read at 340 nm
(UV). Results were calculated from a standard curve
using chloramine T (0–10 lM) prepared concomi-
tantly with the samples.
Statistical analysis
Results are presented as mean ± SD calculated from
triplicates of three independent experiments. The
statistical analysis was performed using a paired
two-tailed Student’s t-test for the time-dependent data
(Fig. 2). One-way analysis of variance (ANOVA)
with Tukey’s post hoc test was used for the protein
modification results (Figs. 3, 4) (Graphpad Prism 8.4,
CA, USA). Results were deemed significant when
p\ 0.05.
Result and discussion
Carnosine complexes with transition metals have been
garnering a lot of interest in research especially zinc
and copper due to their crucial roles in redox chemistry
and implication with physiological functions of var-
ious enzymes and peptides (Kawahara et al.
2011, 2018). Zinc carnosine complex (ZnC) is partic-
ularly studied due to the biological relevance of both
molecules separately and the significant pharmaco-
logical applications of the produced complex (Mat-
sukura and Tanaka 2000). Indeed, a zinc carnosine
compound has been synthesized and commercialized
under the name Polaprezinc or Z-103. It has been
shown to possess a protective effect against ulcers and
mucosal lesions as well as Helicobacter pylori-asso-
ciated gastritis (Handa et al. 2002; Hill and Blikslager
2012; Ko and Leung 2010). It has been shown that the
ZnC complex had a higher effect than the two
molecules separately. Furthermore, ZnC was shown
to form and perform at physiological pH especially
when the ligand/metal ratio is four (Mineo et al. 2002;
Torreggiani et al. 2000).
Earlier research reported the role of zinc or
carnosine alone in the prevention of protein glycation
and aggregation (Moulahoum et al. 2019; Tupe and
Agte 2010). However, it is not clear if the zinc
carnosine combination would produce enhanced
effects either to prevent the protein alteration process
(co-treatment) or reverse the deleterious modified
proteins (post-treatment). Therefore, the current study
explored the use of carnosine supplemented with
different zinc concentrations (125, 250, and 500 lM)
on the glycoxidation of proteins.
The first step was to validate the in vitro glycation
and oxidation models in a time-dependent manner to
allow the choice for the optimal time to explore the co-
and post-treatment application of the different mole-
cules. As such, BSA alone or in the presence of
glucose or chloramine T were incubated according to
the specific methods described before. AGEs and
AOPPs measurements were performed at defined time
intervals. Data showed a time-dependent increase of
AGEs during the 48 h of incubation with almost a 50%
increase between 24 h point and 48 h. Therefore, the
point of 24 h was chosen for the post-treatment study.
Similarly, AOPPs levels also increased linearly and
showed a large difference between 1 and 2 h which
resulted in choosing 1 h as the time point to start the
post-treatment protocol (Fig. 2).
Protein alterations are generally demonstrated
through the increase of AGEs and AOPPs due to the
presence of glucose and chloramine T, respectively.
Several studies have employed this experimental
model to explore many molecules both natural and
synthetic for their therapeutic potential in diabetes
(Grzebyk and Piwowar 2016; Tupe et al. 2017). The
glycation process brings various modifications to the
protein structures other than glycation, aggregation,
and oxidation. These changes could be estimated
through thiol groups, fructosamines, and protein
carbonyls that could be found both in vitro and
in vivo (Awasthi and Saraswathi 2016; Aydin et al.
2018; Grzebyk and Piwowar 2016; Tupe et al. 2017).
123
Biometals (2020) 33:353–364 357
We have reported previously the effect of carnosine
(2.5, 5, and 10 mM) and aminoguanidine (5 mM) on
reducing BSA glycation, aggregation, and oxidation
(Moulahoum et al. 2019). However, some unanswered
questions remained as to whether carnosine prevents
de novo protein alteration or correct the already
altered proteins. Furthermore, given the structure of
the dipeptide having a histidine and the metal chelat-
ing capacity of carnosine, we speculated that the
addition of a divalent transition metal would affect its
activity. As such, this work investigated the effect of
carnosine in addition to various concentrations of zinc
(125, 250, 500 lM) on different protein alteration
models where the molecules were either administered
at the start of the protocol or after the induction of
protein modification. Our data demonstrated that the
addition of zinc induced an enhancement of carnosine
activity in a dose-dependent manner on both experi-
mental protocols (co- and post-treatment) by reducing
AGEs, AOPPs, and aggregation levels. However, the
effect observed in the co-treatment protocol was
higher than the one observed in the post-treatment
Fig. 2 Time-dependent measurement of protein alterations for
a Advanced glycation end-products (AGEs) and b Advanced
oxidation protein products (AOPPs). BSA bovine serum
albumin, Glc glucose, CT Chloramine T. ns = non-significant,
*p\ 0.05, **p\ 0.01, and ***p\ 0.001 are the statistical
significance values for each time point between protein alone
(BSA) and modified proteins (BSA ? Glc or BSA ? CT).
n = 3 independent experiments with triplicates in each one
123
358 Biometals (2020) 33:353–364
model (Fig. 3a–b). These results suggest that zinc
carnosine combination exerts its inhibitory effect
against de novo altered proteins.
Zinc is an important nutrient that is linked with
various growth and development processes in living
organisms. It functions as a cofactor of many proteins
and enzymes and has been shown to have various
activities including antioxidant, anti-inflammatory,
and anti-apoptotic properties (Brazao et al. 2015;
Prasad 2014). Several studies indicated the therapeutic
benefits of zinc against different chronic diseases (e.g.
diabetes) where reduced zinc levels were observed
(Chu et al. 2016; Zhu et al. 2013). The metabolism of
zinc during diabetes has been shown to be negatively
affected and the low zinc level is speculated to be
caused by a loss of structure–function of proteins after
glycation (Kazi et al. 2008). Additionally, in vitro and
animal model reports have demonstrated that zinc
supplementation can decrease protein modifications
and glycation (Tupe et al. 2015; Xiong et al. 2015).
Other reports have demonstrated that zinc could be
a pro-aggregation molecule. Erthal et al. described the
regulatory effect of zinc on amylin’s assembly and
amyloid aggregation (Erthal et al. 2016). Their results
indicated an increased tendency for amylin oligomer-
ization in the presence of increased zinc amounts.
Furthermore, they showed that zinc’s effect does not
require the presence of histidine to induce aggregation.
Zinc was also shown to induce Tau protein aggrega-
tion in neuronal cells and results in apoptosis via its
interaction with two cysteine residues on the protein
(Hu et al. 2017; Mo et al. 2009).
Fig. 3 Protein alteration assessment through the estimation of
AGEs (a) and AOPPs (b) in a BSA protein modification model
treated with carnosine and zinc. BSA bovine serum albumin, Glcglucose, CT chloramine T, AG aminoguanidine, Car carnosine,
Zn zinc, C- BSA alone, C? BSA with either glucose or
chloramine T. ***p\ 0.001 vs. C-, ###p\ 0.001 vs. C? ,$p\ 0.05, $$p\ 0.01, and $$$p\ 0.001 vs. BSA ? Glc/CT ?
Car 5 mM. n = 3 of three independent experiments
123
Biometals (2020) 33:353–364 359
Research on human serum albumin has identified
five potential zinc sites on its structure which are
mainly positioned in the first and second domains.
They are mainly histidine, asparagine, and aspartate
(Stewart et al. 2003). Although the glycation hotspots
are generally associated with lysine and arginine (not
involved with zinc-binding regions), lysine amino
acids which are positioned close to zinc-binding
histidine and cysteine could be influenced by zinc
and inhibit their reaction with glucose (Tupe et al.
2015). A clear demonstration is seen in the co-
treatment set of the current study where protein
glycation was significantly lower than the post-
treatment.
Proteins are prone to form aggregates and even
though they differ in structure and sequence, they tend
to form cross-b structures similar to the ones observed
in amyloid fibrils (Alam et al. 2017; Chiti and Dobson
2006). Proteins possess various sequences (generally
hydrophobic and hidden) that influence the passage
from native to aggregates by forming b-sheets under
certain conditions (Frousios et al. 2009; Hamodrakas
2011; Lopez de la Paz and Serrano 2004). In vivo
protein glycosylation occurs at different sites contain-
ing arginine, lysine, and cysteine amino acids resulting
in the opening of the hidden hydrophobic regions.
With time, a transition from an alpha-helical form to a
beta-sheet structure takes place (amyloid formation).
In our study, the analysis of the beta-sheet formation
was performed by measuring the fluorimetric levels of
glycated proteins mixed with ThT. Data demonstrated
a high level of aggregates in the presence of glucose
Fig. 4 Cross-linking measurements demonstrated via beta-
sheet formation using ThT analysis (a) and dityrosine formation
(b) in a BSA protein modification model treated with carnosine
supplemented with zinc. BSA bovine serum albumin, Glcglucose, CT chloramine T, AG aminoguanidine, Car carnosine,
Zn zinc, C- BSA alone, C? BSA with either glucose or
chloramine T. ***p\ 0.001 vs. C-, ###p\ 0.001 vs. C ? ,$$p\ 0.01 and $$$p\ 0.001 vs. BSA ? Glc/CT ? Car 5 mM.
n = 3 of three independent experiments
123
360 Biometals (2020) 33:353–364
(C?). Treatment with AG or carnosine was able to
significantly prevent aggregation. Furthermore, the
supplementation of increasing zinc concentrations to
carnosine induced a higher anti-aggregative effect
(Fig. 4a). It has been shown that amyloid fibrils
formation in glycated a-crystallin is highly prevented
in the presence of carnosine (Attanasio et al. 2009). In
addition, carnosine was described as a suppressor of
beta-amyloid accumulation and fibril formation
(Aloisi et al. 2013; Attanasio et al. 2013). In a study
on patients with type II diabetes, islet cells containing
the highly amyloidogenic protein Human Islet Amy-
loid Polypeptides (hIAPP) treated with zinc demon-
strated that the treatment significantly reduced and
delayed amyloid fibrillogenesis (Brender et al. 2010).
Protein aggregation is a general term used to describe
cross-linked proteins. These aggregates may be cre-
ated in different forms and structures including
lysosomes, inclusion bodies, aggresomes, misfolding,
and plaques (Reeg and Grune 2015). Another marker
that is usually analyzed during protein alteration is the
formation of dityrosine cross-links that increases with
aging, protein oxidation (cellular stress, UV irradia-
tion, and pathological conditions). These dimers are
chemically stable compounds that get released after
oxidized proteins proteolysis (Giulivi et al. 2003). Our
analysis of the aggregation process demonstrated an
elevation of dityrosine levels which comply with the
literature. Treatment with AG, carnosine, and zinc
carnosine significantly decreased dityrosine levels.
Furthermore, the presence of zinc demonstrated a
dose-dependent effect that enhanced carnosine activ-
ity with higher concentrations (Fig. 4b). Beta-amy-
loids dimers isolated from AD brains have been shown
to possess a neurotoxic and disruptive effect (Shankar
et al. 2008). These dimers are stable and consistent due
to the presence of a dityrosine cross-link bond on the
amyloid precursor containing the beta-amyloid region
(Portelius et al. 2009; Roberts et al. 2012). It has been
shown in earlier studies that carnosine and its
homologs (homocarnosine and anserine) reduce the
cross-linking and aggregation via the interaction with
carbonyl and dityrosine groups. It is speculated that
this effect is mostly related to the antioxidant effect of
carnosine (Cararo et al. 2015; Hipkiss et al. 2001; Kim
and Kang 2007). Elevated amounts of zinc, iron, and
copper were found in amyloid plaques and have been
implicated in the pathogenesis of Alzheimer’s disease
(AD). Some reports suggested that the lack of
transition metals in AD and diabetes is probably
caused to their entrapment by beta-amyloids and ROS
generation via the implication of dityrosine residues.
As such, carnosine acts as a metal chelator and its
interaction with zinc could be seen as an interesting
approach due to its anti-aggregation and antioxidation
effect in addition to chelating the metals from the
amyloid plaques (Al-Hilaly et al. 2013; Mukherjee
et al. 2017).
The limitation of the current study is observed in
the need for clinical application to establish the
therapeutic potential of zinc carnosine. It is under-
standable that in vitro studies vary from what happens
in the living body due to the implication of various
factors and mechanisms such as the metabolic
response, absorption rate, and therapeutic doses. There
has been a concern about carnosine treatment due to
the degradation possibility executed by carnosinase in
the serum. As such, it is imperative to create novel
derivatives and treatment formulations that can over-
come the hydrolysis issue either by creating com-
plexes such as the one described in the current study or
developing encapsulation methods to protect the
peptide (Bellia et al. 2012; Moulahoum et al. 2019).
Conclusion
In this report, we demonstrated that the potential of
zinc supplementation is important in enhancing the
activity of carnosine against structural and functional
age-related protein alterations. Zinc has been shown to
form a complex in the presence of carnosine. Our data
showed that zinc supplementation induced a dose-
dependent enhancement of carnosine activity against
protein glycation, oxidation, and aggregation. Fur-
thermore, we were able to demonstrate that zinc
carnosine activity is more effective against de novo
formation of aggregates. It is speculated that the
effects observed are linked to the physical binding of
zinc onto the region of glycation sites or a comple-
mentary effect on carnosine by enhancing its anti-
glycation and antioxidant activities. These findings
suggest the important potential of zinc supplementa-
tion to carnosine and its prospective application in
age-related diseases such as AD and diabetes. Further
molecular, proteomic, and in vitro studies are neces-
sary for further approval of the current study.
123
Biometals (2020) 33:353–364 361
Acknowledgements This project received no funding from
neither private, public or non-profit organizations
Compliance with ethical standards
Conflict of interest The authors declare that they have no
conflict of interest.
Informed consent All authors read and approved the current
version for publication.
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