Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : December 11, 2021
Accepted : December 20, 2021
Published online : December 31, 2021
doi:10.24659/gsr.8.4_201

Original article

Identification of acetaldehyde-modified lysine
Ikuho Ban 1), Shiori Sakake 1), Naoto Kushida 2), Noriyoshi Manabe 2), Yoshiki Yamaguchi 2),
Ryoji Nagai 1)
1) Laboratory of Food and Regulation Biology, Graduate School of Agriculture, Tokai University, Kumamoto, Japan
2) Division of Structural Glycobiology, Institute of Molecular Biomembrane and Glycobiology,
Tohoku Medical and Pharmaceutical University, Sendai, Miyagi, Japan.

Abstract

Intermediate carbonyl compounds produced by abnormalities in glucose and lipid metabolism in vivo induce amino carbonyl
reactions that modify proteins in a non-enzymatic manner. This reaction is further accelerated with the progression of lifestylerelated diseases, eventually leading to the formation of advanced glycation end-products (AGEs). Also, alcoholic beverages
are consumed as a luxury item. Acetaldehyde (AA), which is produced as an intermediate metabolite of alcohol metabolism,
has a highly reactive carbonyl group and may modify proteins in the same manner as AGEs are produced. In this study, we
attempted to identify the structure of AA-modified amino acids (AA-A) and measure their amount by immunochemical
methods using monoclonal antibodies in order to detect the modification of proteins by AA.
The spleens of mice immunized with AA-keyhole limpet hemocyanin (AA-KLH) were fused with myeloma cells, andantibody-producing cells reactive with AA-bovine serum albumin (AA-BSA) were isolated. The amino acid analysis of AABSA showed that lysine (Lys) and histidine were highly modified. Focusing on Lys with amino groups on its side chain, AALys was isolated from the product generated from N α-benzyloxycarbonyl-L-lysine (N α-Cbz-Lys) and AA by HPLC and thestructure was analyzed by nuclear magnetic resonance (NMR) and mass spectrometry. As a result, m/z 385 and m/z 411 weredetected, and the structure was identified by NMR. The reactivity of these three fractions with antibodies against monoclonalanti-AA-modified proteins was evaluated and the structures were analyzed. In addition, the three fractions in AA-modified
BSA incubated in phosphate buffer (pH 7.4) at 37 °C for 7 days were evaluated by ELISA. As a result, the structures of thethree fractions were identified, and the highest reactivity with antibodies was observed in fraction 2. These increased in aconcentration-dependent manner with AA-modified BSA. These results indicate that three new AA-derived Lys structures
have been identified and are formed under physiological conditions, and that this structure will be one of the tools to evaluate
protein modification by AA.

KEY WORDS: acetaldehyde, alcohol, post-translational modification

Introduction

Ethyl-alcohol is contained in many foods to enhanceflavor and shelf life. Drinking alcohol is an essential part ofadult life as a communication tool and recreational activity,and moderate drinking is said to increase appetite and relieve
fatigue, making the population of drinkers extremely large 1).
On the other hand, an increase in blood alcohol concentration
causes not only acute alcohol intoxication, but also hangover,alcoholic liver disease, diabetes, and other conditions causedby alcohol metabolites. In recent years, it has also beenreported to be involved in the pathogenesis of Alzheimer's
disease, ischemic heart disease and so on 2).
The Ministry of Health, Labor and Welfare reports thatthe percentage of people who drink alcohol in amounts that
increase the risk of lifestyle-related diseases is 11.6 %, andthe percentage of people who drink five or more days a week
is 22.9 %. Alcohol from drinking is mainly metabolized anddetoxified in the liver. After ingested alcohol (ethanol) istaken up by liver cells, it is metabolized into acetaldehyde

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, Kumamoto 862-8652, Japan.
TEL & FAX: +81-96-386 -2692 e-mail: nagai-883@umin.ac.jp
Co-authors: Ban I, b.ikh.bad.0218@gmail.com; Sakake S, waterbear_42@yahoo.co.jp;
Kushida N, 21411221@is.tohoku-mpu.ac.jp; Manabe N, manabe@tohoku-mpu.ac.jp;
Yamaguchi Y, yyoshiki@tohoku-mpu.ac.jp  

_ 2 01 _Glycative Stress Research 2021; 8 (4 ) : 201-210(c) Society for Glycative Stress ResearchIdentification of Acetaldehyde-modified Lysine(AA) by alcohol dehydrogenase (ALD), into acetic acidby aldehyde dehydrogenase (ALDH), and after circulating
throughout the body by blood, it is broken down into waterand carbon dioxide. It is ultimately released from the bodythrough sweat, urine, and exhaled air.
The AA produced in this process is a reactive aldehyde
and is thought to proceed in a similar manner to the Maillard
reaction. In this reaction, glucose, a reducing sugar, reacts
non-enzymatically with the amino groups of proteins to
produce advanced glycation end-products (AGEs), which are
post-translational modifications 3, 4). AGEs increase withthe onset of diabetes mellitus 5), and their accumulation isenhanced with the complications of diabetes mellitus 6). In
fact, N ε-(carboxymethyl)lysine (CML) 7) and N δ-(5-hydro-5methyl-4-imidazolone-2 -yl) -ornithine (MG-H1) 8), a type ofAGEs, have been reported to present in the plasma of patientswith renal disease. It has been reported to accumulate in theplasma of patients with renal disease 9). Therefore, inhibition
of AGE formation is expected to prevent or delay the onset
and progression of diabetes and its complications.
It is also important to determine and evaluate thespecific AGE structure because of the different factors
involved, such as oxidative stress in CML 7) and abnormal
glucose metabolism in MG-H1 10). Hayashi N et al 11) reported
that AA modifies the amino group of bovine serum albumin(BSA) as well as glucose, AA-BSA decreases hepatocyte
viability, and AA-derived AGEs accumulate in the liver of
ethanol-treated rats. Currently, alanine transaminase (ALT)
is used as a marker of liver dysfunction. If the specific AGE
structure produced by AA is clarified, it will be possible
to evaluate the accumulation of AA-derived AGEs due
to alcohol consumption, and it is expected to become amarker for pathological conditions such as alcoholic liverdisease. Therefore, in this study, we evaluated the novel AGE
structures produced by AA.

Methods

Preparation of AA-BSA
AA-modified BSA (AA-BSA) was prepared byincubating 600 mM AA (Merck Millipore, Billerica, MA,
USA) and 2 mg/mL BSA in sodium phosphate buffer (NaPB;
pH 7.2) at 37 °C for 7 days. Subsequently, bicinchoninic acid
(BCA; Thermo Scientific, Waltham, MA, USA) was used forprotein quantification of AA-BSA.

Reactivity of antibodies to AA-A
Balb/c mice were immunized with AA-BSA (0.1 mg/
mouse) three times every 2 weeks and monoclonal antibodies
against AA-A were produced according to the conventional
method 12), BSA modified with methylglyoxal (MG), glyoxal(GO) were measured by enzyme-linked immunosorbent
assay (ELISA) as previously described 13). Briefly, in the noncompetitive ELISA, each well of a 96-well immunoplate(Thermo Fisher Scientific) was coated with 0.1 mL of 1 µg/mL
sample dissolved in phosphate-buffered saline (PBS) andblocked with 0.5 % gelatin hydrolysate dissolved in PBS. The
wells were blocked with 0.5% gelatin hydrolysate dissolved in
PBS. The wells were incubated for one hour with 0.1 mL of
AA-BSA antibody diluted 8 times in triplicate from 1 µg/mL.

Antibodies bound to the wells were detected using ahorseradish peroxidase-conjugated anti-mouse IgG antibody(Thermo Fisher Scientific). Then, 100 µL of 500 μg/mL of
O-phenylenediamine dihydrochloride (Fujifilm Wako Pure
Chemicals, Osaka, Japan) in citrate-phosphate buffer (pH 5.0)
containing 5.9 mM hydrogen peroxide was added, followed
by developing for 3 minutes. The reaction was terminatedwith 100 µL of 2.0 M sulfuric acid and the absorbance wasmeasured at 492 nm using a Sunrise Rainbow Thermo RC
system (Tecan, Männedorf, Switzerland).

Competitive ELISA of monoclonal anti-AA-A
antibody against AA-BSA and each fractional
component
Prepared AA-BSA samples and each fractional sample
prepared by high performance liquid chromatography(HPLC) were measured by competitive ELISA. In thecompetitive ELISA, each well of a 96-well immunoplate wascoated with 0.1 mL of 1 µg/mL sample dissolved in PBS andblocked with 0.5 % gelatin hydrolysate dissolved in PBS. To
these wells, 0.05 mL of AA-BSA diluted in triplicate from
1 µg/mL in seven successive step dilutions and BSA diluted
in the same manner as the control group and fractions 1, 2,
and 3 were added as competitors and well mixed for 10 min
with AA-BSA antibody, followed by one-hour incubation.
The antibodies bound to the wells were detected using
horseradish peroxidase-conjugated anti-mouse IgG antibody.
Subsequently, 100 µL of 500 µg/mL O-phenylenediamine
dihydrochloride in citrate -phosphate buffer ( pH 5.0)
containing 5.9 mM hydrogen peroxide was added for 3 minof developing. The reaction was terminated with 100 µL of
2.0 M sulfuric acid and the absorbance was measured at 492
nm using the Sunrise Rainbow Thermo RC system.

Amino acid analysis of AA-BSA
AA-BSA prepared with 100 mM AA was dissolvedin 1 mL of 6 M HC l in the presence of 14mM 2 - inercapioethdnol for hydrolysis; after 18 h, the sample wascollected and dried in a centrifugal concentrator, thenredissolved in 0.02 M HCl and injected into an amino acidanalyzer (L-8900, Hitachi, Tokyo, Japan) 14).

Measurement and preparative isolation of
AA-Cbz-Lys by HPLC

600 mM AA and 100 mM N α -benzyloxycarbonylL-lysine (Nα- Cbz-Lys; Sigma, St. Louis, MO, USA) wereincubated in 200 mM NaPB at 37 °C for 4 days (AA-CbzLys). The sample (50 µL) was dissolved in H 2 O (950 µL) andthe entire volume was run through the Sep-pack column.
The column was then washed with H 2 O (3 mL), followed
by 50 % MeOH (2 mL), and the fractions were collected for
purification. After complete drying of the generated sample, it
was dissolved in 100 µL of 20 % acetonitrile (ACN), filteredthrough a 0.45 µm polytetrafluoroethylene membrane filter(Merck Millipore) and injected into UV-HPLC. Thereafter,
AA-modified N α-Cbz-lysine will be referred to as AA- CbzLys. In preparative HPLC (Shimazdu, Kyoto, Japan: System
Controller CBM-20A, Pump LC-20AD, UV Detector SPD20A, Autosampler SIL-20A, Column Oven CTO-20AC),_ 202 _Glycative Stress Research

30 L of AA-Cbz-Lys was prepared as injected into a Cosmosil
Packed Column 5C18-AR-II (20 x 250 mm, Nacalai TesqueCo., Ltd., Kyoto, Japan). The column was maintained at
40 °C. The mobile phase was 0.1% TFA (Fujifilm Wako Pure
Chemicals), and ACN was gradient in two steps. Solution A
was 10 % ACN and solution B was 60 % ACN. The gradientwas (B: 0 - 40 min, 45-50 % ACN; B: 41- 46 min, 100 %ACN; B: 47-52 min, 45 % ACN ) at a flow rate of 5 mL/min.
The effluent was monitored by UV at 270 nm and separated
into three fractions (Fr. 1: 21-22 min, Fr. 4: 33-34 min, Fr. 5:35-36. 52 min). These fractions were repeated 30 times andeach fraction was lyophilized. The lyophilized samples wereredissolved in 50 % ACN. Based on the molecular weight
determined by mass spectrometry, the concentration of each
Fr fraction; concentrations were 17.4 mM, 9 mM, and 20.6
mM, respectively. These fractions were again measured by
HPLC and the fractions were collected. Each Fr fraction was
diluted to 100 µM with H2O and 10 µL was injected into the
UV-HPLC. The measurement conditions were carried out inthe same way.

Structural analysis by LC-ESI-QTOF
Each of the preparative Fr fractions was diluted to 10µM in 20 % ACN with 0.1% formic acid (FA) and measured
by liquid chromatography-quadrupole time-of-flight mass
spectrometry (LC-ESI-QTOF) 15). For simplicity, the LC
was maintained in an oven at 40 °C. The measurements werecarried out by flow injection mode. The mobile phase was
0.1 % (FA) with a two-step gradient of ACN (B: 0 -2 min,
90 % ACN; B: 2 -16 min, 90 -10 % ACN; B: 16 -19 min, 10 %ACN). The conditions were as follows: flow rate, 0.2 mL/min;injection volume, 5 μL; ionization source temperature, 200 °C;
capillary voltage, 4.5 kV. Collision-induced dissociation
was performed using nitrogen, with collision energy andpressure set to 20 eV and 1.6 bar, respectively. Data wereacquired in the conserved mass range of m/z 50-1000. The
compositional formulae of the detected ions were analyzed
using Smart Formula.

Nuclear magnetic resonance (NMR)

Compound 1, 2 and 3 was characterized by NMR using
a JNM-ECZ600R/S1 spectrometer equipped with a 5-mm
ROYAL probe (JEOL, Tokyo, Japan). The probe temperature
was set to 303 K. Each sample was dissolved in 750 μL ofacetonitrile-d 3 (99.96 atom % D), and 1 H and 13 C chemical
shifts were reported relative to the internal standard oftetramethylsilane. 15 N chemical shifts were indirectly
referenced using 15 N- 1 H chemical shift referencing ratio of
0.1013291444 16). NMR signals were assigned by 1D 1 H,
1D 13 C, 2 D 1 H - 1 H DQF- COSY, 1 H- 13 C HSQC, 1 H- 15 N
HSQC, 1 H- 13 C HMBC, and 1 H - 15 N HMBC. NMR dataprocessing, analysis and spectral simulation were performed
using Mnova software (version 14.1.2; Mestrelab Research,Santiago de Compostela, Spain).

Statistical analysis
All data were expressed as mean ± standard deviation(± SD). Fig. 2 was examined for statistical significance using
one-way analysis of variance (ANOVA) and Bonferroni's

post-test. Statistical analysis was performed using the EZR
software package (Oxfordshire, United Kingdom) 17).

Results

Reactivity of Monoclonal Anti-AA-A Antibodies

In the present study, monoclonal anti-AA-A antibodies(2F10, 3G4, 4B11), which were produced by immunizing
mice with AA-BSA in our laboratory, were used as antibodies
against AA modifications. First, a test was conductedto elucidate the reactivity of the antibodies with AABSA. The results of ELISA using the prepared AA-BSA
and monoclonal anti-AA-A antibodies showed that three
antibodies reacted with AA-BSA compared to unmodified
BSA in the control group. Among the three antibodies,
2F10 was found to be the most active (Fig. 1-a). Therefore,
we performed a competitive ELISA with AA-BSA using
2F10. The results showed that 2F10 reacted with AA-BSA
in a competitor concentration-dependent manner compared
to unmodified BSA (Fig. 1-b). Furthermore, the antibody
showed no reactivity with various aldehyde-modified BSA,
but reacted specifically with AA-BSA (Fig. 1-c).

Evaluation of the formation of
AA-modified amino acids
AA-BSA prepared at concentrations from 0 to 600
mM of AA was evaluated by ELISA using 2F10, and it was
confirmed that AA-modified amino acids were produced in aconcentration-dependent manner. In addition, the amount of
AA-modified product reached the upper limit when preparedwith 30 mM AA (Fig. 2-a). Next, AA-BSA prepared with 10
mM AA was evaluated at different temperature retention
times. The results showed a time-dependent formation of
AA-modified products (Fig. 2-b).

Evaluation of AA-modified amino acids
Since it was confirmed that AA reacts with proteinsto produce AA-modified products, AA-BSA was preparedto evaluate which amino acids in proteins react with thecarbonyl group of AA, and the variation of amino acidcontent was confirmed by amino acid analysis. As a result,
Lys, which is known to be involved in the formation ofAGEs, was found to be highly reactive with AA, whilearginine (Arg), which is also involved in the formation ofAGEs, was found to be less reactive with AA. ( Table 1)

Preparation of AA-Cbz-Lys generated by AA
From Table 1, it was confirmed that AA has highreactivity with Lys. Therefore, AA-A was generated bymixing AA and Lys, and was preparative. Since some of theAGEs structures are not fluorescent, we used the protecting
group Cbz-Lys to identify the structure by HPLC in thisstudy. The prepared AA-Cbz-Lys was detected at a UV
wavelength of 270 nm, and a chromatogram containing Fr
fractions 1-3 was obtained (Fig. 3-a). Fr fractions 1, 2, and3 were separated from each other and measured again by
HPLC under the same conditions, and single peaks weredetected ( Fig. 3-b, c, d ).

_ 203 _

Identification of Acetaldehyde-modified Lysine

a)

b)

3

0.5

Absorbance at 492 nm

Absorbance 492 nm

3.5

2.5
2
1.5
1
0.5
0
0.0001

0.001

0.01

0.1

1

0.4
0.3
0.2
0.1
0
0.0001

10

Antibody concentration (µg/mL)!"#$%"&'()*&+*,-.*'/
Absorbance at 492 nm

c)

0.001

0.01

0.1

1

10

Competitor conc. (µg/mL)

012
0.8
013
0.6
01,
0.4
01.
0.2
0
AA

-BS

A

GA

-BS

A

Gly

c-B

SA

MG

-BS

A

GO

-BS

A

BS

A

Antigen conc. (0.1 µg/mL)!'+45)'*($'(1*6017*859/:;

Fig. 1. Reactivity of monoclonal anti-AA-A antibody.

The reactivity of the prepared AA-BSA with monoclonal anti-AA-A antibodies (2F10, 3G4, 4B11) was evaluated by ELISA. a) Reactivityof AA-BSA with each antibody by non-competitive ELISA. b) Reactivity of AA-BSA with 2F10 by competitive ELISA. c) Reactivity of
BSA modified with AA, GA, Glyc, MG, GO and 2F10 by non-competitive ELISA. Data are presented as mean (± SD). Unmodified BSA
was used as control group. AA, acetaldehyde; AA-A, AA-modified amino acids; BSA, bovine serum albumin; ELISA, enzyme-linked
immuno sorbent assay; GA, glycolaldehyde; Glyc, glyceraldehyde; MG, methylglyoxal; GO, glyoxal; SD, standard deviation.

Fig 1

a)

b)

2.4

Absorbance at 492 nm

Absorbance at 492 nm

2.8

2
1.6
1.2
0.8
0.4
0

0

1

5

10

30

100

200

600

Acetaldehyde (mM)

2
1.6
1.2
0.8
0.4
0

0

11

2
2

44

77

7

Time (days)

Fig. 2. Evaluation of AA-A production.

The production of AA-A was evaluated by non-competitive ELISA at different concentrations of AA and different retention times.
a) Evaluation of the amount of AA-A produced by changing the concentration of AA. b) Evaluation of the amount of AA-A
produced by AA-BSA prepared with AA (10 mM) at different retention times. Data are shown as mean (± S.D.), a) * p < 0.01,
5 vs 0 mM, 10 vs 5 mM, 30 vs 10 mM, by Bonferroni test; b) # p < 0.05, 2 vs 0 days, 4 vs 2 days, 7 vs 4 days by Bonferroni test.
AA, acetaldehyde; AA-A, AA-modified amino acids; ELISA, enzyme-linked immuno sorbent assay; SD, standard deviation.

_ 204 _Glycative Stress Research

Table 1.
Asp
2.6 ± 0.7Thr
4.2 ± 0.7Ser
3.7 ± 0. 5

Glu
3.4 ± 0.6Gly
4.9 ± 0.6Ala
2.2 ± 0.8

Cys
0± 0

Val
5. 2 ± 0.6

Met10.3 ± 1.0

Ile6.4 ± 0.3

Leu
3. 5 ± 0.7

Tyr8.4 ± 0.6Phe
2.0 ± 0.8

Lys
81.3 ± 0.05

His
70.7 ± 0.4

Arg
10. 3 ± 0.6

a)Intensity

350000Fr 1250000Fr 2 Fr 3

15000050000-50000

10

15

20

25

30

35

40

Retention time (min)

c)
-5300

Fr. 1

-5800

Intensity

Intensityb)- 6300- 6800
10

15

20

25

30

35

40

Retention time (min)Intensity

d)

-5600

Fr. 2

-5800
- 6000- 6200

10

15

20

25

30

35

40

Retention time (min)

-5400
-5600

Fr. 3

-5800

- 6000- 6200

10

15

20

25

30

35

40

Retention time (min)Fig. 3. Preparation of AA-Cbz-Lys produced by AA.
a) Chromatogram of AA-Cbz-Lys. b) Re-measurement chromatogram of Fr fraction 1. c) Re-measurement chromatogram of Fr fraction 2.
d) Re-assay chromatogram of Fr fraction 3. AA, acetaldehyde; Cbz-Lys; benzyloxycarbonyl-L-lysine.

a)
b) c)
Fig 3
d)
Structural analysis by LC-ESI-QTOF
LC-ESI-QTOF analysis was performed for each Fr
fraction, which were separated as single peaks. The mass-tocharge ratio (m/z) of 385 was detected in Fr fraction 1 ( Fig.
4-a ), and the same m/z of 411 was detected in Fr fractions 2
and 3 (Fig. 4-b, c).

Structural analysis by NMR
NMR analyses were performed to determine the structureof 1, 2, and 3. The NMR signals were assigned by a series ofone- and two-dimensional experiments and the assignments
are shown in Table 2. 1D - 1 H NMR spectrum of 1 gave threesignals at 7.7, 8.2, and 8. 5 ppm, which were assigned topyridine protons ( Fig. 5-a, left). 2D-1 H-15 N HMBC spectrum
shows the correlation between pyridine nitrogen andneighboring protons (Fig. 5-a, right). Furthermore, thepyridine ring is modified with an ethyl group at C-5 and amethyl group at C-2. From the NMR analysis, compound 1
is identified as (S)-1- (5- (((benzyloxy) carbonyl) amino)-5carboxypentyl)-5-ethyl-2-methylpyridin -1- ium ( Table 2 ).
Compounds 2 and 3 also share a pyridine unit, however theside chains are more extended to have a C = C double bond.

_ 205 _

Identification of Acetaldehyde-modified Lysine

Compounds 2 and 3 are structural isomers, and the isomerism
originates from cis or trans configuration. From the 3 J
coupling data and spectral simulations, compound 2 is
identified as (S, Z)-1- (5- (((benzyloxy) carbonyl) amino) - 5carboxypentyl) -5 - (but-2 - en-1-yl) -2 -methylpyridin-1-iumwith cis configuration, and compound 3 is identified as (S, E)1-(5-(((benzyloxy) carbonyl) amino)-5-carboxypentyl)-5-(but-2-en-1-yl)-2-methylpyridin-1-ium with trans configuration(Table 2, Fig. 5-b, c).
The m/z of each fraction was calculated from the NMRestimated structure of each fraction using Smart Formulaa)Intens.
x10 6(Table 3 ), and the m/z was consistent with that of the peakdetected in Fig. 4.

Reactivity of monoclonal anti-AA-A antibody witheach fraction
We elucidated whether the structures detected in eachfraction reacted with monoclonal anti-AA-A antibody, which
specifically reacts with AA-BSA. As a result, competition
with 2F10 was observed in each fraction ( Fig. 6 ).

1_Fr1_1-77_01_23734.d: +MS, 0.2min #11

1+
385.2129

5
4
3
2
1
0

b)

100

200

300

Intens.
x10 6

400

500

600

700

800

900

m/z

2_Fr4_1-78_01_23736.d: +MS, 0.2min #12

1+
411.2272

1.5
1.0
0.5
0.0

c)

100

200

300

Intens.
x10 6

400

500

700

800

900

m/z

3_Fr5_1-79_01_23738.d: +MS, 0.2min #10

1+
411.2270

2.0

600

1.5
1.0
0.5
0.0

100

200

300

400

500

Fig. 4. Structural analysis by LC-ESI-QTOF.

600

700

800

900

m/z

Each Fr fraction was measured by LC-ESI-QTOF. a) Detection peak from Fr fraction 1. b) Detection peak from Fr fraction 2.
c) Detection peak from Fr fraction 3. LC-ESI-QTOF, liquid chromatography– quadrupole time-of-flight mass spectrometry.

_ 206 _Glycative Stress Researcha)50Compound 1

100

15 N

150

H-3
8.5

b)

7.0

4.0

2.5

1.0

ppm

10.0

7.0
7.0

5.5
5.5

H-6

8.0

6.0

4.0
4.0

2.5
2.5

8.5
8.5

1.0
1.0
ppm
ppm

Compound 2
Compound
2

7.0
7.0

5.5
5.5

H8
H8
Real
Real

Real
Real

5.7
5.7

5.5
5.5

H9
H9

ppm
ppm

H8
H8

5.9
5.9

5.7
5.7

7.3 Hz85.5
5.5 ppm
ppm

9

5.9

Simulated
Simulated

Simulated
Simulated

7

250

ppm

2.0

0.0
ppm

4.0
4.0

2.5
2.5

1.0
1.0
ppm
ppm

Compound 3
Compound
3

H9
H9

5.9

1H

4.0

200

Compound 3
Compound3

Compound 22
Compound

8.5
8.5

c)

5.5

Pyridine
nitrogen C-2 Me

10

5.9
5.9

H8
H8

5.7
5.7

5.5
5.5

6.6 Hz

8

5.5
5.5

9

15.2 Hz10.7 Hz

ppm
ppm

H8
H8

H9
H9

5.7
5.7

7
6.8 Hz

H9
H9

ppm
ppm

6.3 Hz10trans-type

cis-type

Fig. 5. NMR analysis of compounds 1, 2, and 3.
a) 1D 1 H and 2D 1 H-15 N HMBC spectra of compound 1. b) 1D 1 H-NMR spectra of compound 2 and 3. c) Expanded 1 H-NMR spectra (olefin
region) of compound(c)2 and 3 (upper) and simulated NMR spectra of the corresponding region (lower).

_ 207 _

Identification of Acetaldehyde-modified Lysine

Table 2.

10

7

4

3

5
+1

2

N

8

3

6

7

4
5
+1

2

N

ɛ

6

CH3

8

H

9

3

H

cis-type

5
+1

2

O

Structure

H
8

9

H

HO
O

1

N
H

O
HO

O

O

2

structure

1

H -3
H- 4
H-6
H -7, H -7’
H-8
H-9
H -10
Lys H

7.7
8.2
8. 5
2.8, 2. 8
1. 3
4. 4

C -2
C -3
C- 4
C -5
C-6
C -7
C-8
C-9
C -10

153.7
130.9
145. 9143. 814 4.9
26. 0
14.7
-

CH3
trans-type

O
O

10

ɛ

O
N
H

6

N

ɛ

HO

7

4

N
H

O

3
2

3

7.7
8.1
8.4
3.5, 3.6
5.5 (J = 10.7 Hz)
5.8
1.7
4.4
13 C chemical shift [ppm]153. 0131. 014 6.2
141.2
145.0
30.0
126.4
129.1
18.0

7.7
8.1
8.4
3.4, 3.4
5.6 (J = 15.2 Hz)
5.7
1.7
4.4

1 H chemical shift [ppm]Table 3.

154.0
130.9
146. 3
141.1
145.2
30.0
127.6
130.7
18.0

Fr No.

Compositional
formulae

Mass-to-charge ratio
(m/z)

1

C 22 H 29 N 2 O 4 +385. 2122

2

C 24 H 31 N 2 O 4 +411. 2278

3

C 24 H 31 N 2 O 4 +411. 2278

Absorbance at 492 nm

0.6
Cbz

0.5

Fr. 1

Fr. 2

Fr. 3

0.4
0.3
0.2
0.1
0
0.0001

0.001

0.01

0.1

1

10

Competitor conc. (µg/mL)

Fig. 6. Reactivity of each Fr fraction with monoclonal antiAA-A antibody.
The reactivity of each Fr fraction with monoclonal anti-AA-A
antibody was evaluated by competitive ELISA. AA, acetaldehyde;
AA-modified amino acids; ELISA, enzyme-linked
FigAA-A,6
immuno sorbent assay.

_ 208 _Glycative Stress Research

Discussion
The consumption of ethyl alcohol through alcoholic
beverages is widely incorporated into dietary habits for a richdiet. However, excessive intake of ethyl alcohol has negativeside effects, such as poisoning by its metabolic intermediate,AA, and increased risk of developing Alzheimer's disease
with prolonged and excessive intake 18). While AA- induced
tissue damage has been considered, its molecular mechanism
remains unclear. As with carbonyl compounds, modified
amino acids are thought to be generated from AA, but theirstructures are not well understood. In this study, in order toelucidate the biotoxicity of AA at the molecular level, we
identified the structure of modified amino acids derived
from AA and investigated a system that can easily measure
the structure using monoclonal antibodies.
The monoclonal anti-AA-A antibody, which was
prepared in this study, reacted with AA-BSA in aconcentration-dependent manner, indicating that thisantibody recognizes AA-modified proteins. In addition,
this antibody reacted only with AA-BSA among various
aldehyde-modified proteins, indicating that this antibody is
specific for the structure formed by the reaction with AA,
even though the aldehyde compound has a carbonyl moiety.
Human susceptibility to alcohol fluctuates on a daily
basis depending on physical condition, even in the same
individual. Currently, only the detection of alcohol in thebreath is used to evaluate the amount of alcohol intake, andthe concentration of AA, which causes sickness, cannot be
evaluated only by the concentration of alcohol in the breath.
If the AA-A identified in this study can be easily measured
using immunochromatography and other methods, it will
be possible to quickly evaluate the "level of intoxication"
when drinking. In today's society, traffic accidents caused by
drunk drivers are one of social issues. However, when testing
the alcohol concentration in the breath, there are cases where

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Declaration of Conflict of Interest
All authors declare that they have no conflicts of interest.

Acknowledgments
Authors would like to express our deepest gratitude to
the members of my laboratory for discussions during the
conduct of this research. This work was supported by a JSPS
KAKENHI grant (grant no. 20K05895) provided to Nagai R.

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