Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : January 7, 2023
Accepted : January 16, 2023
Published online : March 31, 2023
doi:10.24659/gsr.10.1_16

Original article

Mass spectrometric and immunological evaluation of AGEs during incubationof ribose with gelatin
Nana Katsuta 1), Mime Nagai 2), Ryoji Nagai 1, 2)
1) Laboratory of Food and Regulation Biology, Graduate School of Bioscience, Tokai University
2) Laboratory of Food and Regulation Biology, Department of Food and Life Sciences,
School of Agriculture, Tokai University, Kumamoto, Japan

Abstract

Ribose produces advanced glycation end-products (AGEs) more rapidly than glucose. It has also been reported that N ε(carboxymethyl)arginine (CMA) is formed in collagen, which is the most abundant protein in organisms. In this study,
AGEs produced by the reaction of ribose with gelatin, a soluble collagen, were evaluated by immunochemical methods
using anti-AGEs antibodies, mass spectrometry, and fluorescence intensity measurements. Measurement by enzymelinked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed
that N ε-(carboxymethyl)lysine (CML), N δ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine (MG-H1) and CMA weresignificantly increased in the ribose-gelatin sample compared to the gelatin alone sample after a 7-day incubation at 37 °C.
The fluorescence intensity at excitation and emission of 370 nm and 440 nm, respectively, was also significantly increased,
whereas the AGE structure detected was unknown. In addition, the pericarp hot water extract of Trapa bispinosa (TBE) and/or lutein were added to this reaction system to evaluate the inhibitory effect of AGE formation. As a result, inhibition of CML,
MG-H1, and CMA formation was observed at ≥ 5 μg/mL of TBE in ELISA and LC-MS/MS. However, no similar inhibitory
effect was detected in fluorescence measurements, suggesting that TBE does not inhibit the formation of fluorescent AGEs.
Since the inhibition of AGE formation by lutein was not confirmed, it may be a difficult compound to evaluate by in vitro
studies. This study showed that ELISA and LC-MS/MS measurement can detect three types of AGEs formation in the ribosegelatin system and that they are useful for screening compounds, e.g., TBE, that inhibit AGE formation.

KEY WORDS: advanced glycation end-products (AGEs), fluorescence, ELISA, LC-MS/MS, Trapa bispinosa Roxb.

Introduction

According to a 2020 report by the World HealthOrganization (WHO), non-communicable diseases (NCDs),
including cardiovascular disease, cancer, and diabetes,
account for approximately 70 % of global deaths 1). Thesediseases are also referred to as age-related diseases because
their onset and progression are associated with aging.
Advanced glycation end-products (AGEs) are thought tocontribute to the onset and progression of aging-relateddiseases, and it has been reported that AGEs increase andaccumulate in the body with aging, diabetic complications,atherosclerosis, and kidney disease 2-6). AGEs are also involvedin the inactivation of enzymes and changes in protein
conformation 7). Therefore, it is thought that suppressing theformation of AGEs in the body can prevent the onset andprogression of age-related diseases, and searches for naturalcompounds to inhibit AGE formation are being conductedworldwide.
To find compounds that inhibit AGEs formation, it isimportant to establish a model system in which AGEs are
rapidly produced and an assay method that can accurately
evaluate AGEs content. A mixture of reducing sugars or
carbonyl compounds and proteins is incubated, and thelevel of AGEs is usually measured by the enzyme-linked
immunosorbent assay (ELISA). In the conventional methods,
glucose, which has the highest physiological concentration
among reducing sugars, and albumin have been used.
However, glucose requires about 8 weeks or more to produce
AGEs when incubated at 37 °C, and albumin is difficult
to produce N ε-(carboxymethyl)arginine (CMA), which is
produced in large amounts in collagen glycation 8-10).

Address correspondence to: Ryoji Nagai, Ph.D.
Laboratory of Food and Regulation Biology, Department of Food and Life Sciences,
School of Agriculture, Tokai University
Toroku 9-1-1, Higashi-ku, Kumamoto City, Kumamoto 862-8652, Japan.
TEL &FAX: +81-96-386 -2692 e-mail: nagai-883@umin.ac.jp
Co-authors; Katsuta N, katsuta7355@gmail.com;
Nagai M, nm406537@tsc.u-tokai.ac.jp_ 16 _

Glycative Stress Research 2023; 10 (1): 16 -26(c) Society for Glycative Stress ResearchGlycative Stress Research

In addition, AGEs have been measured so far byimmunochemical methods using anti-AGEs antibodies
and fluorescence properties, but in many cases the epitope
structure of the antibodies and the AGEs structure detectedby fluorescence are unknown 7). Therefore, it may not be
possible to discover the inhibitors for AGEs formation that
actually show efficacy in humans. In addition, the serumis occasionally heated at 100 °C for 15 minutes beforehandin measuring blood AGEs, and it has been pointed out thatN ε-(carboxymethyl)lysine (CML) 11) and pentosidine 12) areartificially generated by this pretreatment. Trapa bispinosaRoxb. is an annual aquatic grass cultivated as an edible
and medicinal plant, and has been reported to possess
various functions, i.e., antibacterial activity, postprandial
hyperglycemia inhibition 13-15). In vitro and in vivo studies
have also revealed that it has anti-glycation properties, and
it is being studied especially from the viewpoint of cataract
prevention 16, 17). Lutein is a type of carotenoid, rich in green
and yellow vegetables, and is recognized for its antioxidant
capacity 18, 19). In humans, lutein is present in the lens andretina, and higher serum concentrations have been suggested
to decrease the risk of developing ocular diseases such as
age-related macular degeneration 20).
In this study, we established a system in which several
AGEs are produced in a short incubation period of 7 days
using the ribose-gelatin, and used fluorescence measurements,
indirect ELISA, and liquid chromatography-tandem mass
spectrometry (LC-MS/MS) to compare fluorescent intensity
and AGE contents such as CML, N δ-(5-hydro-5-methyl4 -imidazolone-2-yl)- ornithine ( MG -H1), and CMA.
Furthermore, this reaction system was used to evaluate theinhibition of AGE formation by the pericarp of T. bispinosahot water extract (TBE) and lutein.

Methods

Sample preparation

The pericarp hot water extract of T. bispinosa (TBE)(Hayashikane Sangyo Co., Ltd., Yamaguchi, Japan) andlutein 20 % suspension (Koyo Mercantile Co., Ltd., Tokyo,
Japan) were provided by Santen Pharmaceutical Co., Ltd.
(Osaka, Japan). Lutein 20 % suspension is a sample ofsafflower oil containing 20.0 % marigold pigment; therefore,
it was regarded that 1 mL of lutein 20 % suspension contains
200 mg of lutein for the experiment. The concentrationsof TBE and lutein added in this study were determinedbased on the data of 10 :1 ratio of TBE and lutein in Sante
Wellvision (Santen Pharmaceutical Co., Ltd.), a dietary
supplement developed with a focus on aging of the eyes andother parts of the body, and previous studies 21, 22).
The samples were prepared with 60 mM D (-)-ribose(Wako Pure Chemical, Osaka, Japan) and 4 mg/mL gelatinfrom porcine skin, Type A (Sigma-Aldrich, Darmstadt,
Germany) in 200 mM phosphate buffer ( NaPB, pH 7. 2)
and sterile filtered through a 0.45 μm filter ( DISMIC
25CS045AS, Advantec Toyo Kaisha, Ltd., Tokyo, Japan).
TBE and lutein were dissolved in dimethyl Sulfoxide(DMSO, Fujifilm Wako Pure Chemical Corporation, Osaka,
Japan). Then, only TBE was filtered through a 0.45 μm
filter (NP-44513-AEF, Tomsic Ltd., Tokyo, Japan), and theribose and gelatin solutions were mixed in equal volumes
in the presence of TBE (final concentrations 0, 1, 5, 10, 50)
and lutein (final concentrations 0, 0.1, 1, 5, 10), to make a
mixture solution with a final concentration of 30 mM ribose
and 2 mg/mL gelatin. A sample of 200 mM NaPB mixed
with an equal volume of 4 mg/mL gelatin solution was used
as a negative control for AGE formation. After incubation at
37 °C for 7 days, each sample was placed in a cellulose tubing(24/32, EIDIA Co., Ltd., Tokyo, Japan) and dialyzed in 2
L of water at 4 °C for 12 hours. Protein quantification was
performed by the bicinchoninic acid (BCA) method.

Fluorescence measurement

Each sample was diluted to 100 μg/mL in phosphatebuffered saline (PBS) and added to 96-well plates (96well, black, flat bottom, Tecan Trading AG, Männedorf,
Switzerland) at 200 μL. Fluorescence intensity was measured
using a Microplate Reader (Tecan infinite M PLEX, Tecan
Japan Co., Ltd., Kanagawa, Japan) at 370 nm excitation and
440 nm emission.

Indirect ELISA

The assay was conducted based on the previous studies. Briefly, each sample was diluted in PBS to a final
concentration of 0.2 or 1 μg/mL. It was added to each well ofa 96-well plate (Clear Flat-Bottom Immuno Nonsterile 96Well Plates, Thermo Fisher Scientific, Waltham, MA, USA)in 100 μL and coated by incubation at 25 °C for 2 hours. The
wells were then washed three times with PBS containing
0.05% Tween 20 (washing buffer). 200 μL of PBS containing
0.5 % gelatin was added to each well and incubated at 25°C
for 1 hour for blocking. After washing three times withwashing buffer, 100 μL of primary antibodies, monoclonalanti-CML antibody (0.5 μg/mL) 24), monoclonal anti-MG-H1
antibody (0.5 μg/mL) 25) and monoclonal anti-CMA antibody(1 μg/mL) 10), was added and incubated at 25 °C for 1 hour.
The wells were washed three times and incubated witha secondary antibody, at 5,000-fold diluted horseradish
peroxidase (HRP)-conjugated goat anti-mouse IgG ( H + L)
antibody (Kirkegaard & Perry Laboratories, Inc., Gaithersburg,
MD, USA), for 1 hour at 25 °C. After washing the wells
three times, 100 μL of a development solution consistingof 10 mL of citrate-phosphate buffer (pH 5.0), one OPD
Tablet (o-Phenylenediamine. 2HCl 5mg/ Tablet), and 5.9 mMhydrogen peroxide was added to each well and allowed toreact. The reaction was terminated with 100 μL of 1.0 M
sulfuric acid, and absorbance at 492 nm was measured using
a Microplate Reader.
9, 23)

Pretreatment of samples for AGE measurement by
LC-MS/MS

Pretreatment for measurement was based on previous
studies 9, 22, 26). Briefly, 50 μL of sample (25 μg protein) was
reduced by adding an equal volume of 100 mM sodium
borate buffer (pH 9.1) and one-tenth volume (5 μL) of 2 M
sodium tetrahydroborate (Fujifilm Wako Pure Chemical)
containing 0.1 M sodium hydroxide (NaOH, Fujifilm Wako
Pure Chemical), and allowed to stand at 25 °C for 4 hours.
Then, 10 μL of 1 μM [ 2 H 2 ] CML and [ 2 H 3 ] MG-H1_ 17 _

Mass spectrometric and immunological evaluation of AGEs during incubation of ribose with gelatin(PolyPeptide Laboratories France SAS, Strasbourg, France),
10 μL of 10 μM [13 C 6 ] CMA, 5 μL of 1 mM [13 C 6 ] lysine
and [ 13 C 6 ] arginine (Cambridge Isotope Laboratories,
Tewksbury, MA, USA), and 1 mL of 6 M HCl were added
to each sample and hydrolyzed by incubation at 100 °C for
18 hours. After drying, the sample was resuspended in 1 mL
water, passed over a Strata-X-C column (Phenomenex,
Torrance, CA, USA), washed with 3 mL of 2 % (v/v) formic
acid solution, and eluted with 2 mL of 7 % (v/v) ammonia
solution. Then, it was dried, resuspended in 1 mL of asolution containing 20 % (v/v) acetonitrile and 0.1% (v/v)
formic acid, and filtered through a 0.45 μm filter (Millex-LH
filter, Merck KGaA, Darmstadt, Germany).

Measurement of AGEs levels by LC-MS/MS

Measurements by LC -MS/MS were performed as
described previously 27). The precursor ions of CMA, [13 C 6 ]CMA, arginine, and [13 C 6 ] arginine were m/z 233, 239, 175,
and 181, respectively, the fragment ions were m/z 116, 121, 70,
and 74, respectively, the collision energy were 16, 16, 23, and23 V, respectively, detected in positive ion mode. Otherwise,
the measurement conditions were similar to previous studies 26).
CML values were normalized to lysine, MG-H1 and CMA
values were normalized to arginine values. Fig. 3-a, b, cshow the structural formula of the measured AGEs and thetype and site of stable isotope labeling, m/z of precursor ionsand fragment ions of AGEs and their internal standards.

Statistical analysis

The web application PlotsOfData and Microsoft Excel
2019 were used to draw the measured data 28). All data areexpressed as dots, and means are indicated by horizontal
lines. Statistical tests were performed using Dunnett's test,with p-values < 0.05 considered significant. All statisticalanalyses were performed using EZR (Saitama Medical
Center, Jichi Medical University, Saitama, Japan), which
is a graphical user interface for R (The R Foundation for
Statistical Computing, Vienna, Austria) 29).

Results

Evaluation of inhibition of AGE formation byfluorescence measurement

The fluorescence intensity of the ribose-gelatin sample,
a positive control for AGE formation, was measured at an
excitation wavelength (370 nm) and an emission wavelength(440 nm) and was significantly higher than that of the negative
control incubated with gelatin alone (A vs B: p < 0.001,
Fig. 1 ). The addition of lutein 1 μg/mL alone subsequently
resulted in significantly higher fluorescence intensity comparedto the positive control (B vs C: p = 0.001). Whereas, it wassignificantly lower in the TBE 5 μg/mL/lutein 0.1 μg/mL and
TBE 50 μg/mL/lutein 1 μg/mL groups ( B vs G: p = 0.029, B
vs M: p < 0.001).

Evaluation of inhibition of AGE formation byindirect ELISAELISA assay showed a significant increase in CML

and MG-H1 and CMA all in the ribose-gelatin mixture (Avs B: p < 0.001, Fig. 2-a, b, c). In contrast, CML, MG-H1,
and CMA were significantly decreased in the TBE 5 μg/mL/
lutein 0.1 μg/mL group; TBE 10 μg/mL/lutein 0, 0.1, 1, 5, 10
μg/mL group; and TBE 50 μg/mL/lutein 1 μg/mL group (Bvs G, H, I, J, K, L, M: p < 0.001, Fig. 2-a, b, c, respectively).
In the TBE 1 μg/mL alone and TBE 1 μg/mL/lutein 0.1
μg/mL groups, only the CMA formation was significantly
inhibited (B vs D: p = 0.027, B vs E: p < 0.001, Fig. 2-c).

Evaluation of AGE formation inhibition
by LC-MS/MS

LC-MS/MS clearly detected the peaks of CML, MGH1, and CMA (Fig. 3-d, e, f ). The CML, MG-H1, and CMA
levels were significantly increased in the ribose-gelatin
mixture compared to the gelatin alone sample (A), a negative
control for AGE formation (A vs B: p < 0.001, Fig. 4-a, b, c).
Significant increases in MG-H1 and CMA were detected
when lutein 1 μg/mL was added alone compared to thepositive control (B vs C: p < 0.001, respectively, Fig. 4-b, c).
In contrast, CML, MG-H1, and CMA were significantly
decreased in the groups with TBE 5 μg/mL/lutein 0.1 μg/mL,
TBE 10 μg/mL/lutein 0, 0.1, 1, 5, 10 μg/mL, and TBE 50
μg/mL/lutein 1 μg/mL (B vs G, H, I, J, K, L, M: p < 0.001,
respectively, Fig. 4-a, b, c). A significant inhibition of CML
and CMA formation was detected in the groups with TBE
1 μg/mL/lutein 0, 0.1, 1 μg/mL (B vs D, E, F: p < 0.001,
respectively, Fig. 4-a, c).

Discussion
Using a ribose-gelatin system in which AGEs areproduced after one week of incubation, we compared theincrease in CML, an oxidation-dependent AGE; MGH1, which is produced via methylglyoxal from abnormalcarbohydrate metabolism; and CMA, which is produced
mostly from collagen, using ELISA and mass spectrometry
10, 30, 31). Subsequently, changes in fluorescence intensity at
excitation and emission of 370 nm and 44 0 nm, which areoften used as a conventional way to evaluate AGEs 32). ELISA
and LC-MS/MS measurements showed that CM L, MGH1, and CMA levels increased significantly after one weekof reaction. Fluorescence intensity concurrently increased
significantly, suggesting that progression of the Maillard
reaction was detectable in three ways.
Using this reaction system, we evaluated the abilityof TBE, which has already been reported to inhibit AGE
formation, and lutein, which is known to have antioxidant
properties, to inhibit AGE formation. As a result, significant
inhibition of CML, MG-H1, and CMA formation wasobserved at TBE levels of 5 μg/mL or higher by ELISA
and LC-MS/MS measurements. It has been reported that
TBE inhibits ribose-derived MG-H1 generation but notmethylglyoxal-derived MG-H1 22). Other reports indicatethat polyphenols including gallic acid, ellagic acid, andeugeniin are present in TBE, and that ellagic acid inhibits
CMA formation via antioxidant rather than carbonyl trap
17, 33). Therefore, it is thought that TBE inhibits the pathway
by which CML, MG-H1, and CMA are produced from the_ 18 _Glycative Stress Research

adduct of ribose and protein. Note that even at 1 μg/mL of
TBE, significant inhibition of CML and CMA was detectedby ELISA and LC-MS/MS, and the difference from thepositive control was slight.
Although the Dunnett's test does not allow thecomparison of groups with different concentration of lutein,
as far as the measurement results of the TBE 1 μg/mL/lutein
0, 0.1, 1 μg/mL group (D, E, F) or the TBE 10 μg/mL/lutein
0, 0.1, 1, 5, 10 μg/mL group (H, I, J, K, L) were compared( Fig. 2, 4 ), the inhibitory effect of lutein on AGE formation
was almost negligible. In a previous report in which TBE
and lutein were administered to diabetic rats, lutein did not
suppress AGEs during short-term administration, while did
show a suppressive effect during long-term administration 17).
In a similar case, we previously reported that administrationof citric acid inhibited cataracts in diabetic rats, even though
citric acid does not inhibit AGE formation in vitro 34). Lutein,a lipophilic carotenoid, is thought to exhibit antioxidative
effects in vivo by translocation to the retina and lens.
Therefore, it is likely that evaluation of the inhibitory effectof lutein on AGE formation in vitro is difficult.
Since almost the same inhibitory effect was detected byboth ELISA and LC-MS/MS, it is considered that ELISA issufficient for the first screening of candidate compounds for
AGE inhibitors. In contrast to this result, the fluorescencemeasurement showed a significant decrease in intensity
under the conditions of TBE 5 μg/mL/lutein 0.1 μg/mL
or TBE 50 μg/mL/lutein 1 μg/mL. Fluorescent AGEs suchas pentosidine and crossline are reported 35, 36). Since theformation pathways of each AGE structure are diverse, theinhibitory effect of TBE was also different, which may be
the reason for the discrepancy between the fluorescencemeasurement versus ELISA and LC-MS/MS results. No
significant decrease in fluorescence intensity was observedin the group with 10 μg/mL of TBE. It is thought thatwhen sugar and protein are mixed and reacted, fluorescent
substances other than AGEs are also generated, and the
addition of lutein, which has conjugated double bonds andis fluorescent, affected the fluorescence intensity, resulting
in inconsistent results. Aminoguanidine, a known inhibitorof AGEs formation, inhibits the formation of fluorescent
AGEs in vitro 37), whereas its inhibitory effect on CML and
CMA formation was less effective in vitro 9). Therefore, themechanism of inhibition of AGEs formation by TBE may bedifferent from that of aminoguanidine.

Fig. 1. Measurement of fluorescence intensity.

The horizontal axis shows the alphabet identifying each group of samples (groups from A to M, respectively n = 4) and the final
concentrations of TBE and lutein. The fluorescence intensity of each reaction solution diluted to 100 μg/mL and measured at an
excitation of 370 nm and emission of 440 nm is shown. Negative control for AGE formation (group A) is gelatin alone, while positive
control (group B) and TBE-lutein added group (groups from C to M) are ribose-gelatin mixtures. Data are shown as dots, with means
indicated by horizontal lines. Means of the positive control for AGE formation (group B) and the other groups were compared byDunnett's test, and significant differences are shown in the graph. AGE, advanced glycation end-product; TBE, Trapa bispinosa extract.

_ 19 _

Mass spectrometric and immunological evaluation of AGEs during incubation of ribose with gelatin_ 20 _Glycative Stress Research

Fig. 2. Measurement of AGEs by indirect ELISA.

The horizontal axis shows the alphabet identifying each group of samples (groups from A to M, respectively n = 4) and the final
concentrations of TBE and lutein. The vertical axis shows the absorbance at 492 nm. The negative control for AGE formation (group A) is
gelatin only, and the positive control (group B) and the TBE and lutein supplemented groups (groups C-M) are ribose-gelatin mixture. The
development time was ( a ) 3 minutes for CML, ( b) 5 minutes for MG-H1, and (c ) 7 minutes for CMA. Data are shown as dots, with means
indicated by horizontal lines. Means of the positive control for AGE formation (group B) and the other groups were compared by Dunnett's
test, and significant differences are shown in the graph. AGEs, advanced glycation end-products; CML, N ε-(carboxymethyl)lysine; MG-H1,
N δ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine; CMA, N ε-(carboxymethyl)arginine; TBE, Trapa bispinosa extract.

_ 21 _

Mass spectrometric and immunological evaluation of AGEs during incubation of ribose with gelatin(a) CMLHO(b) MG-H1
HO

m/z 114 (ISTD: m/z 117 )O†(c) CMA

†

NH
O

H

NH

N
NH

HN

NH

O

H 2N

NH 2

OH

m/z 130
m/z 205 (ISTD: m/z 207)

O

O

*

NH

*

*

*

O

*

* NH 2
OH

OH

m/z 116 (ISTD: m/z 121)m/z 229 (ISTD: m/z 232)m/z 233 (ISTD: m/z 239)
Precursor ion
Fragment ion

Fig. 3. Structural formulas and chromatograms of measured AGEs.

Structural formula of ( a ) CML, ( b) MG-H1, and (c ) CMA. Deuterium and carbon-13 in the internal standards are marked by adagger (†) and asterisk ( *), respectively. Precursor ions are shown as solid lines and fragment ions as dashed lines. In each sample,
fragment ion peaks of (d ) CML, (e ) MG-H1, and ( f ) CMA were detected. Typical fragment ion peaks of CML, MG-H1, and CMA
detected in the ribose-gelatin mixture sample, which is the positive control for AGEs formation (group B), are shown in (i), eachinternal standard is shown in (ii), respectively. AGEs, advanced glycation end-products; CML, N ε-(carboxymethyl)lysine; MG-H1,
N δ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine; CMA, N ε-(carboxymethyl)arginine; ISTD, internal standard.

_ 22 _Glycative Stress Research_ 23 _

Mass spectrometric and immunological evaluation of AGEs during incubation of ribose with gelatin

Fig. 4. Measurement of AGEs by LC-MS/MS.

The horizontal axis shows the alphabet identifying each group of samples (groups from A to M, respectively n = 4) and the final
concentrations of TBE and lutein. ( a ) CML levels normalized to lysine, ( b) MG-H1 levels normalized to arginine levels, and (c ) CMA levels
normalized to arginine levels in the samples are shown on the vertical axis. Negative control for AGE formation (group A) is gelatin alone,while positive control (group B) and TBE-lutein added group (groups from C to M) are ribose-gelatin mixtures. Data are shown as dots,with means indicated by horizontal lines. Means of the positive control for AGE formation (group B) and the other groups were compared byDunnett's test, and significant differences are shown in the graph. AGEs, advanced glycation end-products; CML, N ε-(carboxymethyl)lysine;
MG-H1, N δ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine; CMA, N ε-(carboxymethyl)arginine; TBE, Trapa bispinosa extract.

Conclusion

Funding support

This study showed that by using a ribose-gelatin system
instead of glucose or BSA, MG-H1, rich in living organisms,
was produced in as short a period as 7 days, in addition to
CML and CMA, which have been reported previously 9, 38).
Furthermore, the possibility of using ELISA to search forcompounds inhibiting AGE formation, such as TBE, wassupported by the quantification of AGEs using mass
spectrometry.

This work was supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports,
Science and Technology (JSPS KAKENHI # 20K05895).

Acknowledgement
The publication of this study was supported by the
Isyoku-Dogen Research Foundation.

Conflict of interest declaration
None for this study.

_ 24 _Glycative Stress Research

References
1) World Health Organization, author. Noncommunicable

Diseases Progress Monitor 2020. World Health Organization;[cited 2023 january 7]. 2020. Available from:https://www.who.int/publications/i/item/9789240000490
2) Ahmed MU, Brinkmann Frye E, Degenhardt TP, et al.
N-epsilon-(carboxyethyl)lysine, a product of the chemical
modification of proteins by methylglyoxal, increases withage in human lens proteins. Biochem J. 1997; 324: 565570.
3) Verzijl N, DeGroot J, Thorpe SR, et al. Effect of collagenturnover on the accumulation of advanced glycation end
products. J Biol Chem. 2000; 275: 39027-39031.
4) Singh VP, Bali A, Singh N, et al. Advanced glycation end
products and diabetic complications. Korean J Physiol
Pharmacol. 2014; 18: 1-14.
5) Nagai R, Hayashi CM, Xia L, et al. Identification inhuman atherosclerotic lesions of GA-pyridine, a novel
structure derived from glycolaldehyde-modified proteins.
J Biol Chem. 2002; 277: 48905-48912.
6) Tanji N, Markowitz GS, Fu C, et al. Expression of advanced
glycation end products and their cellular receptor RAGE
in diabetic nephropathy and nondiabetic renal disease. J Am
Soc Nephrol. 2000; 11: 1656-1666.
7) Nagai R, Shirakawa J, Fujiwara Y, et al. Detection of
AGEs as markers for carbohydrate metabolism and protein
denaturation. J Clin Biochem Nutr. 2014; 55: 1-6.
8) Nagai R, Matsumoto K, Ling X, et al. Glycolaldehyde, areactive intermediate for advanced glycation end products,
plays an important role in the generation of an active
ligand for the macrophage scavenger receptor. Diabetes.
2000; 49: 1714 -1723.
9) Sugawa H, Ohno R, Shirakawa J, et al. Eucommia ulmoides
extracts prevent the formation of advanced glycation end
products. Food Funct. 2016; 7: 2566-2573.
10) Kinoshita S, Mera K, Ichikawa H, et al. N ε-(Carboxymethyl)
arginine is one of the dominant advanced glycation end
products in glycated collagens and mouse tissues. Oxid
Med Cell Longev. 2019; 2019: 9073451.
11) Miki Hayashi C, Nagai R, Miyazaki K, et al. Conversionof Amadori products of the Maillard reaction toN(epsilon)-(carboxymethyl) lysine by short-term heating:possible detection of artifacts by immunohistochemistry.
Lab Invest. 2002; 82: 795-808.
12) Nakano M, Kubota M, Owada S, et al. The pentosidine
concentration in human blood specimens is affected byheating. Amino Acids. 2013; 44: 1451-1456.
13) Adkar P, Dongare A, Ambavade S, et al. Trapa bispinosaRoxb.: A Review on Nutritional and Pharmacological
Aspects. Adv Pharmacol Sci. 2014; 2014: 959830.
14) Rahman MM, Wahed MII, Biswas MHU, et al. In vitro
antibacterial activity of the compounds of Trapa bispinosaRoxb. Journal of Medical Sciences. 2001; 1: 214-216.
15) Takeshita S, Ishioka Y, Yagi M, et al. The effects of water
chestnut (Trapa bispinosa Roxb.) on the inhibition of
glycometabolism and the improvement in postprandial
blood glucose levels in humans. Glycative Stress Res.
2016; 3: 124 -132.
16) Uemura T, Takeshita S, Yamada M, et al. The effectivenessof the peel extract of water chestnut (Trapa bispinosaRoxb.) in an α-crystallin glycation model with glyoxal.
Glycative Stress Res. 2017; 4: 104-108.

17) Kinoshita S, Sugawa H, Nanri T, et al. Trapa bispinosaRoxb. and lutein ameliorate cataract in type 1 diabetic rats.
J Clin Biochem Nutr. 2020; 66: 8-14.
18) Ma L, Lin XM. Effects of lutein and zeaxanthin on
aspects of eye health. J Sci Food Agric. 2010; 90: 2-12.
19) Gao S, Qin T, Liu Z, et al. Lutein and zeaxanthin
supplementation reduces H 2 O 2 -induced oxidative damage
in human lens epithelial cells. Mol Vis. 2011; 17: 31803190.
20) Di Carlo E, Augustin AJ. Prevention of the Onset of
Age-Related Macular Degeneration. J Clin Med. 2021; 10:3297.
21) Takeshita S, Yagi M, Uemura T, et al. Peel extract ofwater chestnut (Trapa bispinosa Roxb.) inhibits glycation,
degrades α-dicarbonyl compound, and breaks advanced
glycation end product crosslinks. Glycative Stress Res.
2015; 2: 72-79.
22) Ban I, Sugawa H, Nagai R. Protein Modification with
Ribose Generates N δ-(5-hydro-5-methyl-4-imidazolone2-yl)-ornithine. Int J Mol Sci. 2022; 23: 1224.
23) Nagai R, Unno Y, Hayashi MC, et al. Peroxynitrite induces
formation of N(epsilon)-(carboxymethyl) lysine by thecleavage of Amadori product and generation of glucosone
and glyoxal from glucose: novel pathways for protein
modification by peroxynitrite. Diabetes. 2002; 51: 28332839.
24) Koito W, Araki T, Horiuchi S, et al. Conventional antibody
against Nepsilon-(carboxymethyl)lysine (CML) shows
cross-reaction to Nepsilon-(carboxyethyl)lysine (CEL):
Immunochemical quantification of CML with a specificantibody. J Biochem. 2004; 136: 831-837.
25) Yamaguchi H, Nagai M, Sugawa H, et al. Developmentof a conventional immunochemical detection system fordetermination of N δ-(5-hydro-5-methyl-4-imidazolone2-yl)-ornithine in methylglyoxal-modified proteins.
Glycoconj J. 2021; 38: 293-301.
26) Katsuta N, Takahashi H, Nagai M, et al. Changes in S-(2succinyl)cysteine and advanced glycation end-products
levels in mouse tissues associated with aging. Amino
Acids. 2022; 54: 653-661.
27) Katsuta N, Nagai M, Saruwatari K, et al. Mitochondrial
stress and glycoxidation increase with decreased kidney
function. J Clin Biochem Nutr. 2023; 72: 147-156.
28) Postma M, Goedhart J. PlotsOfData-A web app forvisualizing data together with their summaries. PLoS Biol.
2019; 17: e3000202.
29) Kanda Y. Investigation of the freely available easy-touse software ‘EZR’ for medical statistics. Bone Marrow
Transplant. 2013; 48: 452-458.
30) Schleicher ED, Wagner E, Nerlich AG. Increased
accumulation of the glycoxidation product N(epsilon)(carboxymethyl)lysine in human tissues in diabetes andaging. J Clin Invest. 1997; 99: 457-468.
31) Ahmed N, Thornalley PJ, Dawczynski J, e t al.
Methylglyoxal-derived hydroimidazolone advanced
glycation end-products of human lens proteins. Invest
Ophthalmol Vis Sci. 2003; 44: 5287-5292.
32) Monnier VM, Kohn RR, Cerami A, et al. Accelerated agerelated browning of human collagen in diabetes mellitus.
Proc Natl Acad Sci U S A. 1984; 81: 583-587.

_ 25 _

Mass spectrometric and immunological evaluation of AGEs during incubation of ribose with gelatin

33) Tominaga Y, Sugawa H, Hirabayashi K, et al. Drosera

tokaiensis extract containing multiple phenolic compounds
inhibits the formation of advanced glycation end-products.
Arch Biochem Biophys. 2020; 693: 108586.
34) Nagai R, Nagai M, Shimasaki S, et al. Citric acid inhibits
development of cataracts, proteinuria and ketosis in
streptozotocin (type 1) diabetic rats. Biochem Biophys Res
Commun. 2010; 393: 118-122.
35) Sell DR, Monnier VM. Structure elucidation of asenescence cross-link from human extracellular matrix.
Implication of pentoses in the aging process. J Biol Chem.
1989; 264: 21597-21602.
36) Nakamura K, Hasegawa T, Fukunaga Y, et al. Crosslines
A and B as candidates for the fluorophores in age-anddiabetes-related cross-linked proteins, and their diacetates
produced by Maillard reaction of α-N-acetyl-L-lysinewith D-glucose. J Chem Soc Chem Commun. 1992; 14:992-994.
37) Lehman TD, Ortwerth BJ. Inhibitors of advanced
glycation end product-associated protein cross-linking.
Biochim Biophys Acta. 2001; 1535: 110-119.
38) Shimasaki S, Kubota M, Yoshitomi M, et al. N ε(carboxymethyl)arginine Accumulates in GlycatedCollagen and Klotho-deficient Mouse Skin. Anti-Aging
Medicine. 2011; 8: 82-87.

_ 26 _