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: hic_moul@hotmail.com

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.

References

Al-Hilaly YK et al (2013) A central role for dityrosine

crosslinking of Amyloid-beta in Alzheimer’s disease. Acta

Neuropathol Commun 1:83. https://doi.org/10.1186/2051-

5960-1-83

Alam P, Siddiqi K, Chturvedi SK, Khan RH (2017) Protein

aggregation: from background to inhibition strategies. Int J

Biol Macromol 103:208–219. https://doi.org/10.1016/j.

ijbiomac.2017.05.048

Aloisi A, Barca A, Romano A, Guerrieri S, Storelli C, Rinaldi R,

Verri T (2013) Anti-aggregating effect of the naturally

occurring dipeptide carnosine on abeta1-42 fibril forma-

tion. PLoS ONE 8:e68159. https://doi.org/10.1371/journal.

pone.0068159

Attanasio F et al (2009) Protective effects of L- and D-carnosine

on alpha-crystallin amyloid fibril formation: implications

for cataract disease. Biochemistry 48:6522–6531. https://

doi.org/10.1021/bi900343n

Attanasio F et al (2013) Carnosine inhibits Abeta(42) aggrega-

tion by perturbing the H-bond network in and around the

central hydrophobic cluster. ChemBioChem 14:583–592.

https://doi.org/10.1002/cbic.201200704

Awasthi S, Saraswathi NT (2016) Sinigrin, a major glucosino-

late from cruciferous vegetables restrains non-enzymatic

glycation of albumin. Int J Biol Macromol 83:410–415.

https://doi.org/10.1016/j.ijbiomac.2015.11.019

Aydin AF, Kucukgergin C, Coban J, Dogan-Ekici I, Dogru-

Abbasoglu S, Uysal M, Kocak-Toker N (2018) Carnosine

prevents testicular oxidative stress and advanced glycation

end product formation in D-galactose-induced aged rats.

Andrologia. https://doi.org/10.1111/and.12939

Bellia F, Vecchio G, Rizzarelli E (2012) Carnosine derivatives:

new multifunctional drug-like molecules. Amino Acids

43:153–163. https://doi.org/10.1007/s00726-011-1178-6

Bermudez ML, Skelton MR, Genter MB (2018) Intranasal

carnosine attenuates transcriptomic alterations and

improves mitochondrial function in the Thy1-aSyn mouse

model of Parkinson’s disease. Mol Genet Metab

125:305–313. https://doi.org/10.1016/j.ymgme.2018.08.

002

Boldyrev AA, Aldini G, Derave W (2013) Physiology and

pathophysiology of carnosine. Physiol Rev 93:1803–1845.

https://doi.org/10.1152/physrev.00039.2012

Brazao V, Filipin Mdel V, Santello FH, Azevedo AP, Toldo MP,

de Morais FR, do Prado Jr. JC (2015) Immunomodulatory

properties and anti-apoptotic effects of zinc and melatonin

in an experimental model of chronic Chagas disease.

Immunobiology 220:626–633. https://doi.org/10.1016/j.

imbio.2014.11.018

Brender JR et al (2010) Role of zinc in human islet amyloid

polypeptide aggregation. J Am Chem Soc 132:8973–8983.

https://doi.org/10.1021/ja1007867

Cararo JH, Streck EL, Schuck PF, Ferreira Gda C (2015) Car-

nosine and related peptides: therapeutic potential in age-

related disorders. Aging Dis 6:369–379. https://doi.org/10.

14336/AD.2015.0616

Caruso G, Caraci F, Jolivet RB (2019) Pivotal role of carnosine

in the modulation of brain cells activity: multimodal

mechanism of action and therapeutic potential in neu-

rodegenerative disorders. Prog Neurobiol 175:35–53.

https://doi.org/10.1016/j.pneurobio.2018.12.004

Chilukuri H, Kulkarni MJ, Fernandes M (2018) Revisiting

amino acids and peptides as anti-glycation agents. Med-

ChemComm 9:614–624. https://doi.org/10.1039/

c7md00514h

Chinchansure AA, Korwar AM, Kulkarni MJ, Joshi SP (2015)

Recent development of plant products with anti-glycation

activity: a review. RSC Adv 5:31113–31138. https://doi.

org/10.1039/c4ra14211j

Chiti F, Dobson CM (2006) Protein misfolding, functional

amyloid, and human disease. Annu Rev Biochem. https://

doi.org/10.1146/annurev.biochem.75.101304.123901

Chmielewska K, Dzierzbicka K, Inkielewicz-Stepniak I, Przy-

bylowska M (2020) Therapeutic potential of carnosine and

its derivatives in the treatment of human diseases. Chem

Res Toxicol 33:1561–1578. https://doi.org/10.1021/acs.

chemrestox.0c00010

Chu A, Foster M, Samman S (2016) Zinc status and risk of

cardiovascular diseases and type 2 diabetes mellitus—a

systematic review of prospective cohort studies. Nutrients

8:707. https://doi.org/10.3390/nu8110707

Cuajungco MP, Faget KY (2003) Zinc takes the center stage: its

paradoxical role in Alzheimer’s disease Brain research.

Brain Res Rev 41:44–56. https://doi.org/10.1016/s0165-

0173(02)00219-9

DeGroot J et al (2004) Accumulation of advanced glycation end

products as a molecular mechanism for aging as a risk

factor in osteoarthritis. Arthritis Rheum 50:1207–1215.

https://doi.org/10.1002/art.20170

Erthal LC et al (2016) Regulation of the assembly and amyloid

aggregation of murine amylin by zinc. Biophys Chem

218:58–70. https://doi.org/10.1016/j.bpc.2016.09.008

Fournet M, Bonte F, Desmouliere A (2018) Glycation damage: a

possible hub for major. Pathophysiological disorders and

aging. Aging Dis 9:880–900. https://doi.org/10.14336/AD.

2017.1121

Frousios KK, Iconomidou VA, Karletidi CM, Hamodrakas SJ

(2009) Amyloidogenic determinants are usually not buried.

BMC Struct Biol 9:44. https://doi.org/10.1186/1472-6807-

9-44

Ghodsi R, Kheirouri S (2018) Carnosine and advanced glycation

end products: a systematic review. Amino Acids

50:1177–1186. https://doi.org/10.1007/s00726-018-2592-

9

Giulivi C, Traaseth NJ, Davies KJ (2003) Tyrosine oxidation

products: analysis and biological relevance. Amino Acids

25:227–232. https://doi.org/10.1007/s00726-003-0013-0

123

362 Biometals (2020) 33:353–364

Grzebyk E, Piwowar A (2016) Inhibitory actions of selected

natural substances on formation of advanced glycation

endproducts and advanced oxidation protein products.

BMC Complement Altern Med 16:381. https://doi.org/10.

1186/s12906-016-1353-0

Hamodrakas SJ (2011) Protein aggregation and amyloid fibril

formation prediction software from primary sequence:

towards controlling the formation of bacterial inclusion

bodies. FEBS J 278:2428–2435. https://doi.org/10.1111/j.

1742-4658.2011.08164.x

Handa O et al (2002) Inhibitory effect of polaprezinc on the

inflammatory response to Helicobacter pylori. Can J Gas-

troenterol 16:785–789. https://doi.org/10.1155/2002/

631070

Hara T, Takeda TA, Takagishi T, Fukue K, Kambe T, Fukada T

(2017) Physiological roles of zinc transporters: molecular

and genetic importance in zinc homeostasis. J Physiol Sci

67:283–301. https://doi.org/10.1007/s12576-017-0521-4

Heidari F et al (2020) Advanced glycation end-products and

advanced oxidation protein products levels are correlates

of duration of type 2 diabetes. Life Sci. https://doi.org/10.

1016/j.lfs.2020.118422

Hill TL, Blikslager AT (2012) Effect of a zinc L-carnosine

compound on acid-induced injury in canine gastric mucosa

ex vivo. Am J Vet Res 73:659–663. https://doi.org/10.

2460/ajvr.73.5.659

Hipkiss AR, Brownson C, Carrier MJ (2001) Carnosine, the

anti-ageing, anti-oxidant dipeptide, may react with protein

carbonyl groups. Mech Ageing Dev 122:1431–1445.

https://doi.org/10.1016/S0047-6374(01)00272-X

Hu JY et al (2017) Pathological concentration of zinc dramati-

cally accelerates abnormal aggregation of full-length

human Tau and thereby significantly increases Tau toxicity

in neuronal cells. Biochim Biophys Acta Mol Basis Dis

1863:414–427. https://doi.org/10.1016/j.bbadis.2016.11.

022

Kawahara M, Koyama H, Nagata T, Sadakane Y (2011) Zinc,

copper, and carnosine attenuate neurotoxicity of prion

fragment PrP106-126. Metallomics 3:726–734. https://doi.

org/10.1039/c1mt00015b

Kawahara M, Tanaka KI, Kato-Negishi M (2018) Zinc, carno-

sine, and neurodegenerative diseases. Nutrients. https://

doi.org/10.3390/nu10020147

Kazi TG, Afridi HI, Kazi N, Jamali MK, Arain MB, Jalbani N,

Kandhro GA (2008) Copper, chromium, manganese, iron,

nickel, and zinc levels in biological samples of diabetes

mellitus patients. Biol Trace Elem Res 122:1–18. https://

doi.org/10.1007/s12011-007-8062-y

Kheirouri S, Alizadeh M, Maleki V (2018) Zinc against

advanced glycation end products. Clin Exp Pharmacol

45:491–498. https://doi.org/10.1111/1440-1681.12904

Kim NH, Kang JH (2007) Protective effects of histidine

dipeptides on the modification of neurofilament-L by the

cytochrome c/hydrogen peroxide system. J Biochem Mol

Biol 40:125–129. https://doi.org/10.5483/bmbrep.2007.40.

1.125

Kim Y, Kim Y (2020) L-histidine and L-carnosine exert anti-

brain aging effects in D-galactose-induced aged neuronal

cells. Nutr Res Pract 14:188–202. https://doi.org/10.4162/

nrp.2020.14.3.188

Ko JK, Leung CC (2010) Ginger extract and polaprezinc exert

gastroprotective actions by anti-oxidant and growth factor

modulating effects in rats. J Gastroenterol Hepatol

25:1861–1868. https://doi.org/10.1111/j.1440-1746.2010.

06347.x

Krisko A, Radman M (2019) Protein damage, ageing and age-

related diseases. Open Biol 9:180249. https://doi.org/10.

1098/rsob.180249

Lee SR (2018) Critical role of zinc as either an antioxidant or a

prooxidant in cellular systems. Oxid Med Cell Longev

2018:9156285. https://doi.org/10.1155/2018/9156285

Lopez de la Paz M, Serrano L (2004) Sequence determinants of

amyloid fibril formation. Proc Natl Acad Sci USA

101:87–92. https://doi.org/10.1073/pnas.2634884100

Mahmood A, FitzGerald AJ, Marchbank T, Ntatsaki E, Murray

D, Ghosh S, Playford RJ (2007) Zinc carnosine, a health

food supplement that stabilises small bowel integrity and

stimulates gut repair processes. Gut 56:168–175. https://

doi.org/10.1136/gut.2006.099929

Matsukura T, Tanaka H (2000) Applicability of zinc complex of

L-carnosine for medical use. Biochem Biokhimiia

65:817–823

Matsuura N, Aradate T, Sasaki C, Kojima H, Ohara M, Hase-

gawa J, Ubukata M (2002) Screening system for the

Maillard reaction inhibitor from natural product extracts.

J Health Sci 48:520–526. https://doi.org/10.1248/jhs.48.

520

Meeprom A, Sompong W, Chan CB, Adisakwattana S (2013)

Isoferulic acid, a new anti-glycation agent, inhibits fruc-

tose- and glucose-mediated protein glycation in vitro.

Molecules 18:6439–6454. https://doi.org/10.3390/

molecules18066439

Mineo P, Vitalini D, La Mendola D, Rizzarelli E, Scamporrino

E, Vecchio G (2002) Electrospray mass spectrometric

studies of L-carnosine (beta-alanyl-L-histidine) complexes

with copper(II) or zinc ions in aqueous solution. Rapid

Commun Mass Spectrom 16:722–729. https://doi.org/10.

1002/rcm.633

Mo ZY, Zhu YZ, Zhu HL, Fan JB, Chen J, Liang Y (2009) Low

micromolar zinc accelerates the fibrillization of human tau

via bridging of Cys-291 and Cys-322. J Biol Chem

284:34648–34657. https://doi.org/10.1074/jbc.M109.

058883

Moulahoum H, Sanli S, Timur S, Zihnioglu F (2019) Potential

effect of carnosine encapsulated niosomes in bovine serum

albumin modifications. Int J Biol Macromol 137:583–591.

https://doi.org/10.1016/j.ijbiomac.2019.07.003

Mukherjee S et al (2017) Characterization and identification of

dityrosine cross-linked peptides using tandem mass spec-

trometry. Anal Chem 89:6136–6145. https://doi.org/10.

1021/acs.analchem.7b00941

Olechnowicz J, Tinkov A, Skalny A, Suliburska J (2018) Zinc

status is associated with inflammation, oxidative stress,

lipid, and glucose metabolism. J Physiol Sci 68:19–31.

https://doi.org/10.1007/s12576-017-0571-7

Portelius E, Brinkmalm G, Tran AJ, Zetterberg H, Westman-

Brinkmalm A, Blennow K (2009) Identification of novel

APP/Abeta isoforms in human cerebrospinal fluid. Neu-

rodegener Dis 6:87–94. https://doi.org/10.1159/

000203774

123

Biometals (2020) 33:353–364 363

Prasad AS (2014) Zinc is an antioxidant and anti-inflammatory

agent: its role in human health. Front Nutr. https://doi.org/

10.3389/fnut.2014.00014

Prokopieva VD, Bohan NA, Johnson P, Abe H, Boldyrev AA

(2000) Effects of carnosine and related compounds on the

stability and morphology of erythrocytes from alcoholics.

Alcohol Alcohol 35:44–48. https://doi.org/10.1093/alcalc/

35.1.44

Reeg S, Grune T (2015) Protein oxidation in aging: does it play a

role in aging progression? Antioxid Redox Signal

23:239–255. https://doi.org/10.1089/ars.2014.6062

Roberts BR, Ryan TM, Bush AI, Masters CL, Duce JA (2012)

The role of metallobiology and amyloid-beta peptides in

Alzheimer’s disease. J Neurochem 120(Suppl 1):149–166.

https://doi.org/10.1111/j.1471-4159.2011.07500.x

Sadowska-Bartosz I, Bartosz G (2015) Prevention of protein

glycation by natural compounds. Molecules

20:3309–3334. https://doi.org/10.3390/

molecules20023309

Sale C, Artioli GG, Gualano B, Saunders B, Hobson RM, Harris

RC (2013) Carnosine: from exercise performance to health.

Amino Acids 44:1477–1491. https://doi.org/10.1007/

s00726-013-1476-2

Shankar GM et al (2008) Amyloid-beta protein dimers isolated

directly from Alzheimer’s brains impair synaptic plasticity

and memory. Nat Med 14:837–842. https://doi.org/10.

1038/nm1782

Stewart AJ, Blindauer CA, Berezenko S, Sleep D, Sadler PJ

(2003) Interdomain zinc site on human albumin. Proc Natl

Acad Sci USA 100:3701–3706. https://doi.org/10.1073/

pnas.0436576100

Torreggiani A, Bonora S, Fini G (2000) Raman and IR spec-

troscopic investigation of zinc(II)-carnosine complexes.

Biopolymers 57:352–364. https://doi.org/10.1002/1097-

0282(2000)57:6\352:AID-BIP50[3.0.CO;2-Y

Tupe R et al (2015) Zinc inhibits glycation induced structural,

functional modifications in albumin and protects erythro-

cytes from glycated albumin toxicity. Int J Biol Macromol

79:601–610. https://doi.org/10.1016/j.ijbiomac.2015.05.

028

Tupe RS, Agte VV (2010) Role of zinc along with ascorbic acid

and folic acid during long-term in vitro albumin glycation.

Br J Nutr 103:370–377. https://doi.org/10.1017/

S0007114509991929

Tupe RS, Kemse NG, Khaire AA, Shaikh SA (2017) Attenua-

tion of glycation-induced multiple protein modifications by

Indian antidiabetic plant extracts. Pharm Biol 55:68–75.

https://doi.org/10.1080/13880209.2016.1228683

Witko-Sarsat V et al (1996) Advanced oxidation protein prod-

ucts as a novel marker of oxidative stress in uremia. Kidney

Int 49:1304–1313. https://doi.org/10.1038/ki.1996.186

Xiong M, Liu L, Liu Z, Gao H (2015) Inhibitory effect of zinc on

the advanced glycation end product-induced apoptosis of

mouse osteoblastic cells. Mol Med Rep 12:5286–5292.

https://doi.org/10.3892/mmr.2015.4088

Yamashita S, Sato M, Matsumoto T, Kadooka K, Hasegawa T,

Fujimura T, Katakura Y (2018) Mechanisms of carnosine-

induced activation of neuronal cells. Biosci Biotechnol

Biochem 82:683–688. https://doi.org/10.1080/09168451.

2017.1413325

Zhu K et al (2013) Antidiabetic and pancreas-protective effects

of zinc threoninate chelate in diabetic rats may be associ-

ated with its antioxidative stress ability. Biol Trace Elem

Res 153:291–298. https://doi.org/10.1007/s12011-013-

9675-y

Publisher’s Note Springer Nature remains neutral with

regard to jurisdictional claims in published maps and

institutional affiliations.

123

364 Biometals (2020) 33:353–364