Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received: September 30, 2015
Accepted : December 1, 2015
Published online : March 31, 2016

Original Article

Melatonin is not a carbonyl scavenger
Mohammad Moniruzzaman, Wakako Takabe, Yoshikazu Yonei
Anti-Aging Medical Research Center and Glycative Stress Research Center, Graduate School of Life and Medical Sciences,
Doshisha University, Kyoto, Japan.

Abstract

Objectives: In our previous study on the human skin accumulation of AGEs and lifestyle behaviors, as well as smoking andalcohol drinking, lack of sleep clearly enhanced AGE accumulation. We hypothesized that melatonin may participate in thisphenomenon. Here, we investigated the carbonyl scavenging capacity of melatonin in human serum albumin (HSA)/glucose
model in attempt to reveal an as yet undiscovered function of melatonin.
Methods: AGE-derived fluorescence intensity was measured by fluorescence spectroscopy at excitation 370 nm andemission 440 nm. Pentosidine, 3-deoxyglucosone (3-DG), glyoxal (GO), and methylglyoxal (MGO) were analyzed by highperformance liquid chromatography (HPLC). A system composed of glucose and HSA was employed as an in vitro modelin our study. Aminoguanidine was used as positive control.
Results: Melatonin neither inhibited fluorescent AGE formation in both HSA/glucose and collagen/glucose models
nor inhibited the generation of AGE intermediates such as 3-DG, GO, and MGO in HSA/glucose model. In contrast,aminoguanidine showed a dose-dependent inhibition of formation of these AGEs and carbonyl compounds.
Conclusion: The present study compelled us to conclude that melatonin has no direct influence on AGE generation. In
the phenomenon showing that a lack of sleep in the lifestyle behavior plays a role in promoting skin AGE accumulation, it islikely that a mechanism other than melatonin is involved.

KEY WORDS: melatonin, aminoguanidine, carbonyl compounds, human serum albumin

Introduction

resistant and ubiquitin-proteasome-resistant deposits, andconsequently affect specific protein functions. Therefore,
the formation of RCSs is considered the key event in theintermediate stage of protein glycation 4).
AGEs are not only markers but also principal causative
factors for the pathogenesis of diabetes complications,
including neuropathy, nephropathy, retinopathy, cataract 5, 6)
and other health disorders, such as atherosclerosis 7),
Alzheimer’s disease 7 - 9), and normal aging 10, 11). Thus, thediscovery of AGE inhibitors can offer a promising therapeutic
approach for the prevention of diabetes and other pathogeniccomplications. Both natural compounds and synthetic
compounds have been evaluated as inhibitors of AGEs
formation. The synthetic AGE inhibitors discovered to datecan be divided into three classes: (I) carbonyl-trapping agentsthat attenuate carbonyl stress; (II) metal ion chelators, which
suppress glycoxidation; and (III) cross-link breakers thatreverse AGE cross-links 12).
Aminoguanidine, a hydrazine-like molecule, is a
nucleophilic agent that traps reactive carbonyl intermediates

Melatonin is a somewhat mysterious substance. Inanimals, melatonin is secreted from the pineal gland during
the night 1). It acts as a hormone, functioning as a circadian
mediator for time information over the course of each day,and it is also able to eliminate free radicals (reactive oxygenspecies). Melatonin also exists in higher plants (edible plants),and is inadvertently obtained from daily meals 2, 3).
Glycation intermediates and the two reactive carbonylgroups, namely the reactive carbonyl species (RCSs), are ofgreat importance for the cytotoxicity of advanced glycation
endproducts (AGEs). The toxic AGE precursors, such as
3-deoxyglucosone (3-DG), glyoxal (GO), and methylglyoxal(MGO), are characteristic of individuals susceptible todiabetic complications. Although the chemical reactions
leading to AGE formation are nonenzymatic, the RCSlevels are ultimately controlled by enzymatic mechanismsinvolving glyoxalase, MG and 3-DG reductase. The RCSintermediates act as potent crosslinkers. Protein crosslinking
results in aggregates that form intracellular protease-

Contact Address: Professor Yoshikazu Yonei, MD, PhD
Anti-Aging Medical Research Center Graduate School of Life and Medical Sciences
Doshisha University 1-3 Tataramiyakodani Kyotanabe-shi, Kyoto, 610 -0394 Japan
Phone/Fax: +81-774 -65-6394 E-mail: yyonei@mail.doshisha.ac.jp
Co-authors: Moniruzzaman M, sumonbge2009@gmail.com ;Takabe W, wtakab e@mail.doshisha.ac.jp_ 1 _Glycative Stress Research 2016; 3 (1): 1-4(c) Society for Glycation Stress Research

Melatonin is not a Carbonyl Scavenger

such as 3-DG, GO, and MGO to form relatively nontoxicadducts. However, the drug was not ultimately approved forcommercial production due to side effects observed indiabetes patients in phase III clinical trials; such effects
may be related to the sequestration of pyridoxal, resulting invitamin B6 deficiency 13). Despite AG’s limitations, proof-ofconcept studies indicated that inhibition of AGE formation
by trapping reactive carbonyl species could be a reasonabletherapeutic approach for the treatment of diabetes
complications.
Free radicals are generated during the early stage ofglycation. Schiff bases are prone to oxidation, generating freeradicals and reactive carbonyl groups, and the glycated protein
can also catalyze the generation of free radicals. Therefore,
the generation of reactive carbonyl or dicarbonyl groups
is related to oxidative metabolism. At an early stage, antiglycation strategies involve scavenging of hydroxyl radicals
and superoxide radicals to alleviate oxidative stress, andreducing the generation of reactive carbonyl or dicarbonylgroups. Thus, these strategies inhibit glycation 4). Melatonin’sfunctions as an anti-oxidant include: I), direct free radicalscavenging, II), stimulation of anti-oxidative enzymes, andIII), augmenting the efficiency of other anti-oxidants 14).
Many anti-oxidants can protect against free radicals
generated during glycation 4).
In our previous results of the skin accumulation of AGEs,
which was detected as specific fluorescence, we examined
the relation to lifestyle behaviors, such as smoking andalcohol drinking, lack of sleep clearly, and promoted AGE
accumulation 15). We hypothesized that melatonin may
participate in this phenomenon. In this regard, because of
the direct free radical scavenging capacity of melatonin, andas part of revealing the undiscovered function of melatonin
we investigate whether it has reactive carbonyl scavenging
capacity or not.

10 μg/mL 3-DG, GO, and MGO standard solution wasprepared by mixing 0.4 mL of 50 μg/mL 3-DG, 0.4 mL of
50 μg/mL GO and 0.4 mL of 50 μg/mL MGO with 1.2 mL of
distilled water. Standard 1.0, 0.25, 0.05, 0.025, and 0 μg/mL
were prepared by serial dilution with distilled water from
10 μg/mL. 200 μL standard solutions, 100 μL of 200 mM
phosphate buffer and 30 μL of distilled water were added inmicrotube and mix properly. 200 μL of anti-glycation test
reagent with/without melatonin and aminoguanidine wereadded with 130 μL of distilled water in microtube and mix
properly. 170 μL of 6% perchloric acid was added separately
in both sample and standard solutions, and centrifuged at
12,000 rpm for 10 minutes. After centrifugation 400 μL of
supernatant was transferred into another microtube. Thiswas followed by the addition of 350 μL saturated sodium
bicarbonate, and finally by 50 μL of DAN as an internal
standard. After mixing, the reaction mixtures were allowed
to incubate at 4ºC for 24 hours. After incubation the reaction
mixtures were centrifuged at 15,000 rpm for 10 minutes, andthe supernatant was used for analysis by HPLC-UV. The
following analytical conditions were used: eluent 50 mM
phosphate : ACN = 89: 11, YMC-pack CN (YMC, Kyoto,Japan), S-5 μm, 150 × 4.6 mm ID, a flow rate of 1.0 mL/min, column temperature 40ºC, detection wavelength 268
nm, injection volume 20 μL. 3-DG, GO and MGO standard
curves were constructed by measuring peak areas of thestandard solutions. The effect of melatonin on 3-DG, GOand MGO formation was determined from the standard
curve. Two measurements (n = 2) were taken for eachsample.

Results

Melatonin did not demonstrate an anti-glycation effect in
the in vitro glucose HSA reaction model, i.e., an inhibitory
effect on the formation of fluorescent AGEs ( Fig. 1 ). At theconcentration of 1 mg/mL and 10 mg/mL of melatonin, %inhibitin of fluorescent AGE formation was 7.4% and 30.1%
respectively. On the contrary, aminoguanidine, as a positivecontrol, showed a dose-dependent inhibitory effect.
Carbonyl trapping activity of melatonin was examinedalso in the in vitro glucose HSA reaction model ( Fig. 2 ).
However, melatonin showed no activity, while aminoguanidine
dose-dependently reduced the formation of carbonylcompounds, 3-DG, GO and MGO.

Methods

Chemicals and instrument

Aminoguanidine, phosphoric acid, 40% glyoxal solution,
sodium bicarbonate (Na2HCO3), acetonitrile (ACN) andethanol were purchased from Wako Pure Chemical Industries,Ltd. (Osaka, Japan). Melatonin and MGO solution waspurchased from Sigma-Aldrich (St. Louis, MO, USA). 2, 3diaminonaphthalene (DAN) and 3-DG were purchased from(Dojindo Laboratories, Kumamoto, Japan). Analytical highperformance liquid chromatography (HPLC) was carried out
using a LC-20AT system equipped with SPD-20A UV/VIS
detector and LC-solution software (Shimadzu, Kyoto, Japan).

Discussion

Effects of melatonin on fluorescent AGE formation

Evaluation of the inhibitory effect of 3-DG, GOand MGO formation

The fluorescent AGE inhibition activity of melatoninhas been investigated in HSA/glucose model along withaminoguanidine as a reference compound. Fluorescent
AGEs inhibition activity of aminoguanidine increases dose
dependently. At a concentration of 10 mg/mL, aminoguanidine
inhibits fluorescent AGE formation by more than 95%. On
the other hand, no noticeable fluorescent AGE inhibition
activity has been observed with melatonin. Similar results
have also been found in the in vitro collagen I/glucose
reaction model (data not shown).

In order to evaluate the effect of melatonin on theformation of 3-DG, GO and MGO, 3 concentrations (10 mg/mL, 1 mg/mL and 0.1 mg/mL) were prepared by dissolving
in ethanol. The effect of aminoguanidine on the formationof 3-DG, GO and MGO was also analyzed as a referencecompound to compare the effect of melatonin. As advanced
glycation end product intermediates 3-DG, GO and MGO
were measured by HPLC (LC-20AT) equipped with a SPD20A UV/VIS detector and LC-solution software (Shimadzu).
_ 2 _Glycative Stress Research120100% Inhibition

8060
Melatonin

40

Aminoguanidine

200-200.1 mg/mL

1.0 mg/mL

10 mg/mLFig. 1. Effect of melatonin and aminoguanidine on fluorescent AGE formation.

Concentrations of melatonin and aminoguanidine were 0.1 mg/mL, 1 mg/mL and 10 mg/mL.
Results are expressed as means ± standard deviation, n = 3. AGE, advanced glycation endproduct.

100

Melatonin
Aminoguanidine100a) 3-DG

% inhibition% inhibition

60604020402000-20-200.1 mg/mL

1.0 mg/mL

10 mg/mL

0.1 mg/mL

Melatonin
Aminoguanidine

10 mg/mLa) 3-DG inhibition rate (%), b) GO inhibition rate (%),c) MGO inhibition rate (%). Concentrations ofmelatonin and aminoguanidine were 0.1 mg/mL, 1 mg/mL and 10 mg/mL (n = 2). 3-DG, 3-deoxyglucosone;GO, glyoxal ; MGO, methylglyoxal.

6040200-200.1 mg/mL

1.0 mg/mLFig. 2. Carbonyl trapping activity of melatonin.

b) GO

80% inhibitionc) MGO

8080100

Melatonin
Aminoguanidine

1.0 mg/mL10 mg/mL_ 3 _Melatonin is not a Carbonyl Scavenger
Melatonin has no direct action on AGE generation. In thephenomenon that lack of sleep in the lifestyle behavior plays
a role in promoting skin AGE accumulation, other mechanism
than melatonin may be involved.

Inhibitory effects on the formation of 3-DG, GOand MGO

The reactive carbonyl species (RCS) such as 3-DG, GO,and MGO act as potent crosslinkers. Formation of RCSs is
considered the central event in the intermediate phase ofprotein glycation 4). The inhibitory activity of aminoguanidineon the generation of 3-DG, GO, and MGO increases dosedependent manner. At a concentration of 10 mg/mL,aminoguanidine inhibits the reactive carbonyl species bymore than 90%. Historically, aminoguanidine, a hydrazinelike small molecule, was the first AGE inhibitor explored inclinical trials. It is a nucleophilic agent that traps reactive
carbonyl species such as 3-DG, GO, and MGO to form
relatively nontoxic adducts 16). No inhibitory activity of
melatonin against any reactive dicarbonyl has been foundat any concentration. Our findings suggest that there is nodirect role of melatonin in AGE formation.

Acknowledgements

This study was presented at “the 9th Meeting of theSociety for Glycative Stress Research” on September 5th,
2015, at Kyoto, Japan. This work was supported by theJapanese Council for Science, Technology and Innovation,SIP (Project ID 14533567 ), “Technologies for creating nextgeneration agriculture, forestry and fisheries” (fundingagency: Bio-oriented Technology Research Advancement
Institution, NARO), and JSPS KAKENHI Grant Number
26350917.

Conclusion

Conflict of Interest Statement

The present study revealed that melatonin neither
inhibit fluorescent formation nor inhibit the generation ofreactive carbonyl intermediates such as 3-DG, GO, and MGO.

The authors state that the performance of this study
entailed no issues representing a conflict of interest.

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