Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : November 11, 2021
Accepted : December 1, 2021
Published online : December 31, 2021
doi:10.24659/gsr.8.4_171

Original article

Conventional evaluation of glycation with plate reader and its correlation
with CML formation
Sayuri Kato 1), Ryoji Nagai 1, 2)
1) Laboratory of Food and Regulation Biology, Graduate School of Agriculture, Tokai University
2) Laboratory of Food and Regulation Biology, Department of Bioscience, School of Agriculture, Tokai University, Kumamoto, Japan,

Abstract

The Maillard reaction is a reaction between the carbonyl group of reducing sugars and the amino group of amino acids,
which proceeds not only in foods but also in the body. In the early stage of this reaction, Amadori rearrangement products
are formed, followed by oxidation and condensation reactions, and finally forming advanced glycation end products (AGEs).
In this study, AGEs, which are still difficult to quantify, were simply measured using a plate reader and compared with an
ELISA using an antibody against N ε-(carboxymethyl) lysine (CML). It was evaluated how the formation of these AGEs
changed with different types (ribose, fructose, glucose) and concentrations (5 mM, 30 mM) of carbohydrates that arepredicted to alter AGE formation. In the results, changes were observed depending on the incubation period and the type ofcarbohydrate. Furthermore, the effect was more pronounced with higher concentrations of carbohydrates. The correlation
between the fluorescence measurement and the CML measurement using antibodies was confirmed, suggesting that the plate
reader method can be used to evaluate glycation conveniently.

KEY WORDS: advanced glycation end products (AGEs), glycation, fluorescence, N ε-(carboxymethyl) lysine (CML)

Introduction

A variety of sugars, amino acids, peptides, and proteinsare present in foods and the body. The Maillard reaction,a browning reaction, occurs when the carbonyl group of asugar reacts with the amino group of an amino acid. Thisreaction can be divided into early and late stage reactions.
In the early stage, the carbonyl group and the amino groupdehydrate and condense to form Amadori rearrangement
products (Amadori compounds) via Schiff bases, fromwhich various dicarbonyl compounds are also formed. These
Amadori compounds are relatively stable, but in the latestage, advanced glycation end-products (AGEs) are formedby oxidation and dehydration reactions. Factors that affectthe progress of this reaction include the type of sugar andamino acids, and temperature 1).
The Maillard reaction occurs not only in the heatingof food but also in vivo, and its progression is slower than in
the cooking of food, but in diabetic patients, the progression
accelerates and is faster than healthy subjects. This is because

the Maillard reaction progresses more rapidly in diabetics
due to higher blood glucose levels than in healthy subjects,
and also because carbonyl compounds formed by metabolic
pathways and inflammatory reactions rapidly generate AGEs
in vivo 2).
Some AGEs are fluorescent, and they are generally
detected at an excitation wavelength of 370 nm and afluorescence wavelength of 440 nm 3). Methods for assessing
AGE content include measuring fluorescence intensity,
enzyme-linked immune-sorbent assay (ELISA) using antiAGE antibodies, and instrumental analysis, however, AGE
measurement by instrumental analysis is still difficult inmany cases because of multiple pretreatment steps.
In this study, we evaluated AGEs by fluorescenceintensity using a microplate reader and compared it with
ELISA using antibodies against CML 4, 5), which is known tobe formed by oxidation reactions. In addition, we compared
the effects of the type and concentration of sugars known
to affect AGE formation by fluorescence and ELISA. Since

Address correspondence to: Ryoji Nagai, Ph.D.
Laboratory of Food and Regulation Biology, Department of Bioscience,
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: Kato S, 0cnfm005@mail.u-tokai.ac.jp

Glycative Stress Research 2021; 8 ( 4 ) : 171-174(c) Society for Glycative Stress Research_ 171 _Conventional Evaluation of Glycation with Plate Reader

fluorescent AGEs and non-fluorescent CML are measured
in different ways, different methods are currently used
when evaluating the formation or inhibition of these AGEs.
In this study, we examined whether there was a correlation
between fluorescent AGE and CML formation when theconditions for AGE formation were varied.

Methodsof horseradish peroxidase-conjugated secondary antibody,Goat antibody Mouse IgG (KPL, Milford, MA, USA),was added to the plate and stored at room temperaturefor one hour, followed by color development usingo-phenylenediamine dihydrochloride (OPD; Wako, Osaka,Japan). After 6 minutes of color development, the reaction
was stopped with 2 M sulfuric acid, followed by measuring
the absorbance with Tecan infinite M PLEX at 492 nm.

Sample preparation

Statistical analysis

Bovine serum albumin (BSA) (2 mg/mL) (Sigma,Darmstadt, Germany) was added to glucose, fructose (KantoChemical, Tokyo, Japan) or ribose (Wako, Osaka, Japan) inphosphate buffer saline (PBS) so that the concentration ofsugar was 5 mM or 30 mM. After incubation at 37 °C for 0,3, 7, and 14 days, the samples were stored at −20 °C. AGEs
were measured by the fluorescence method and ELISA at alater date. Three samples of each sugar, concentration, andincubation time were prepared.

Bonferroni’s multiple analysis was performed using thefree statistical software EZR (Easy R) 7) and a significancetest was performed.

Fluorescence measurement
Each incubated sample was diluted 10-fold with
distilled water, and 200 µL were added to a 96-well plate(Tecan Greiner 96 Flat Transparent, Greiner Bio-One,
Frickenhausen, Germany). The fluorescence was measured
with a fluorescence plate reader (Tecan infinite M PLEX,Tecan Japan, Kanagawa, Japan) at an excitation wavelengthof 370 nm and a fluorescence wavelength of 470 nm.

ELISA

ResultComparison of AGE formation by fluorescence
The results of fluorescence measurements showedthat the formation of fluorescent AGEs increased morein the pentose, ribose, than in the hexose, glucose andfructose, and that the increase was concentration- and timedependent (Fig. 1-a, b). Furthermore, fructose showed higher
fluorescence intensity than glucose in the same hexose. As aresult of multiple testing by Bonferroni for each sample on
day 14, there was a significant difference in the fluorescenceintensity between ribose and fructose, ribose and glucose,and fructose and glucose groups at 5 mM concentration; at30 mM concentration, the fluorescence intensity of ribose
was significantly higher than that of fructose and glucose.

Comparison of CML formation

The incubated samples were diluted to 1 µg/mL of BSA
using PBS and added to a 96 well plate (Clear Flat-Bottom
Immuno Nonsterile 96-Well Plates, Thermo, Massachusetts,USA), and CML was measured according to the standard
method 6). In practice, the antigen was kept refrigerated for
one day, then blocked with 0.5 % gelatin (Sigma, Darmstadt,Germany), and 100 µL of monoclonal anti-CML antibody(0. 5 µg/mL) was added to the plate as the primary antibody
and stored at room temperature for one hour. Then, 100 µL

At a sugar concentration of 5 mM, CML formation
increased more in the pentose ribose than in the hexose
glucose and fructose, and the same result was obtained at 30mM ( Fig. 2-a, b). The results of Bonferroni's multiple tests
in each sample on day 14 showed that there was a significant
difference in CML formation between ribose and fructose,ribose and glucose at 5 mM, and the same significant
difference was also observed at 30 mM.

a)b)

fluorescence
fluorescence(5 mM)(5 mM)

intensity / a.u.
intensity / a.u.

2500
2500

riboseribose3000
3000

glucoseglucose fructose
fructose

2500
2500

intensity / a.u.
intensity / a.u.

3000
3000
2000
2000

2000
2000

* * *

1500
1500

1000
1000
00 0

1500
1500

* *
5510 10dayday* * *riboseriboseglucoseglucosefructosefructose

1000
1000

5005000fluorescence
fluorescence(30(30mM)
mM)

500500015 15n =n3= 3* < *0.01< 0.0100 05510 10dayday

15 15

n =n3= 3* < *0.01< 0.01Fig. 1. Measurement of fluorescent AGEs in fluorescence assay.
a) Fluorescence AGE intensity of 5 mM ribose, 5 mM glucose, 5 mM fructose; b) Fluorescence AGE intesnsity of 30 mM ribose, 30 mMglucose, 30 mM fructose. Bar indicates standard deviation. * p < 0.01 by Bonferroni’s multiple test, n = 3, performed on each sample at day
14 using EZR software. AGEs, advanced glycation end products.

_ 172 _Comparison between the formation of fluorescence
AGEs and CML

The correlation between fluorescent AGE intensity
and CML formation was evaluated using samples incubatedfor 14 days. A strong correlation was observed for ribose
and fructose at both 5 mM and 30 mM (Fig. 3-a, b, e, f ). A
stronger correlation was noted for higher concentrations ofribose and fructose. In contrast, no correlation was observed
for glucose ( Fig. 3-c, d ).

a)b)

daydayELISAELISA(30 mM)(30 mM)
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

2.2 ribose ribose
2
1.8 glucoseglucose
1.6 fructosefructose
1.4
1.2
10.8
0.6
0.4
0.2
000
5510

absorbance at 492 nm/a.u.

02.2 ribose ribose
glucoseglucose
fructosefructose
21.8
1.6
1.4
1.2
* * * *
1
0.8
0.6
0.4
0.2
00
510
51015n 15= 3 n = 3

absorbance at 492 nm/a.u.

2.2
2
1.8
1.6
1.4
1.2
10.8
0.6
0.4
0.2
0ELISAELISA(5 mM)(5 mM)

absorbance at 492 nm/a.u.

absorbance at 492 nm/a.u.

Glycative Stress Researchday* < 0.01* < 0.01

day10* * * *15n15= 3 n = 3* < 0.01* < 0.01Fig. 2. CML measurement in ELISA.
a) CML formation of 5 mM ribose, 5 mM glucose and 5 mM fructose; b) CML formation of 30 mM ribose, 30 mM glucose and 30 mMfructose. Bar indicates standard deviation. * p < 0.01 by Bonferroni’s multiple test, n = 3, performed on each sample at day 14 using EZRsoftware. CML, N ε -(carboxymethyl) lysine; ELISA, enzyme-linked immuno sorbent assay.

5000150050002d)

30000.1

1500
1000
500
00.2 00.30.10.40.2

0.50.30.4

absorbance at 492
absorbance
nm / a.u. at 492 nm / a.u.

1500

1000
500

0.2

0.4 0.2

0.6 0.4

absorbance at 492
absorbance
nm/ a.u. at 492 nm/ a.u.

0.6

00.5

1500

1000
500

01 0

1.50.52 1

2.51.5

2

2.5

0.4

0.5

absorbance at 492
absorbance
nm / a.u. at 492 nm / a.u.

3000

2500

R² = 0.5591
2500

2000

2000

1500
1000
500
00.1

R² = 0.5591

1500
1000
500
00.20

0.30.1

0.40.20.50.3absorbance at 492
absorbance
nm / a.u. at 492 nm / a.u.

f)

30 mM fructose
30 mM fructose

3000
2500

3000

2000

2500
R² = 0.9684
2000

1000

1000

1500

500
00intensity / a.u.

500

2000

30 mM glucose
30 mM glucosef) f )

00

1000

0

R² = 0.862

2000

1500

3000

0.55 mM fructose5 mM fructose

2000

R² = 0.9931d)d)

R² = 0.1448

3000
R² = 0.862
2500

1000

1.55 mM glucose5 mM glucosee)e)2000

1 22500

0intensity / a.u.

1500

2500

0.51.5absorbance at 492
absorbance
nm / a.u. at 492 nm / a.u.

1000

3000

0 12500
R² = 0.1448
2000

2000

000.5c)c)

2500

500

intensity / a.u.

intensity / a.u.

3000

01000

30 mM ribose30 mM ribose
3000
R² = 0.9931
2500

intensity / a.u.

500

1500

3000

intensity / a.u.

1000

R ² = 0.9866b)
intensity / a.u.

2000

intensity / a.u.

2500
1500b) b)

5 mM ribose 5 mM ribose

2000c)

intensity / a.u.

a)

3000
R ² = 0.9866
2500

0e)

a)

intensity / a.u.

intensity / a.u.

3000

intensity / a.u.

a)0R² = 0.9684

1500

500
000.50.51

absorbance at 492absorbance
nm/ a.u. at 492 nm/ a.u.

Fig. 3. Correlation between results of fluorescent AGEs and CML.
a) 5 mM ribose; b) 30 mM ribose; c) 5 mM glucose; d) 30 mM glucose; e) 5 mM fructose; f) 30 mM fructose. Samples incubated for 14
days with each sugar and concentration shown in Fig. 1 and Fig. 2 were used for evaluation. AGEs, advanced glycation end products; CML,N ε -(carboxymethyl) lysine.

_ 173 _1

Conventional Evaluation of Glycation with Plate Reader

Discussion
When measuring AGEs, it is common to use HPLC
equipped with a fluorescent detector or mass spectrometry.
However, the maintenance cost of the equipment isnecessary and sample pretreatment is required in multiple
steps. In contrast, the use of a plate reader made it possible
to measure multiple samples of fluorescent AGEs at oncewith low cost and in a simple manner.
Since the fluorescence intensity increases with protein
glycation, it is common practice to evaluate AGE content
using a fluorescent detector. However, the conventional
fluorescent measurement requires transferring about 3 mLof sample to a cuvette and measuring it with a fluorescentdetector. In the present study, we measured the fluorescentintensity using 0.2 mL of the 0.2 mg/mL reaction solution,and confirmed that the fluorescence intensity increased
in a time-dependent manner, suggesting that AGEs could
be evaluated in the plate reader. The correlation between
results of the fluorescence intensity and the ELISA using
anti-CML antibody was also confirmed, indicating thatthe present method can evaluate AGE formation in a moreconvenient manner than the conventional method. Thismethod is expected to be useful in the search for inhibitors
of AGE formation.
Fluorescence measurements showed that AGE
formation was accelerated depending on sugar concentration
and reaction time, and that AGE formation was increasedin pentose rather than in hexose. Results by ELISA showedthat CML formation increased in a concentration- and timedependent manner for ribose, glucose, and fructose. That is
to say, as with fluorescent AGEs, CML formation increasedin pentose rather than in hexose.

Our data showed that the higher the sugar concentration,
the stronger the correlation. Therefore, it is highly likelythat fluorescent AGEs correlate with the amount of CML
formation in hyperglycemic conditions such as diabetes.
Furthermore N δ -(5-hydro - 5 - methyl- 4 -imidazolone - 2yl) - ornithine (MG-H1) was also tested by ELISA using
antibodies, but not much change was observed (data notshown).
In summary, it was confirmed that fluorescent AGEs
can be easily evaluated in a 96-well plate reader andcorrelated with CML formation. This method will be usefulin the search for inhibitors of AGEs formation, which
requires the measurement of a large number of samples.

Conflicts of Interest
All authors state that they have no conflicts of interest.

Acknowledgments
This work was supported by a JSPS KAKENHI grant(grant no. 20K05895) provided to Ryoji Nagai.

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