Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : March 15, 2020
Accepted : April 28, 2020
Published online : June 30, 2020
doi:10.24659/gsr.7. 2_152

Original article

Comparison of α-dicarbonyl compound contents in alcoholic beverages.
Tatsushi Kimoto, Wakako Takabe, Masayuki Yagi, Mari Ogura, Yoshikazu Yonei
Anti-Aging Medical Research Center and Glycative Stress Research Center, Faculty of Life and Medical Sciences,
Doshisha University, Kyoto, Japan

Abstract

Purpose: In the process of producing advanced glycation end products (AGEs), α-dicarbonyl compounds (α-DCs) act asintermediates for AGEs. In this study, we measured the amount of 3-deoxyglucosone (3-DG), glyoxal (GO), and methylglyoxal(MGO) as α-DCs contained in alcoholic beverages.
Methods: As samples, 30 commercially available alcoholic beverages (18 brewages: Japanese sake, beer, red wine, white
wine, and sherry, 15 distilled spirits: whiskey, shochu, gin, vodka, and rum) were used. α-DCs in each sample were labeledwith 2, 3-diaminonaphthalene (DAN) and measured by reversed phase high performance liquid chromatography (HPLC).
Results: Concentrations of 3-DG and GO in alcoholic beverages were higher in brewages than in distilled spirits. The
concentration of 3-DG was the highest in brewages in the order of beer (1,048.7 ± 120.6 μg/mL), white wine (216.7 ± 98. 5
μg/mL), red wine (177.0 ± 59.8 μg/mL), sherry (107.3 ± 7.6 μg/mL), and sake (51.0 ± 4 4.8 μg/mL). The 3-DG concentrationof distilled spirits was 4.8 ± 13.8 μg/mL. The GO concentration of brewages was in the order of sherry (6.5 ± 3.2 μg/mL),beer (4.5 ± 0.8 μg/mL), red wine (3.3 ± 0.7 μg/mL), white wine (2.7 ± 1.9 μg/mL), and sake (0.9 ± 0. 3 μg/mL). The GOconcentration of the distilled spirits was 1.2 ± 1.8 μg/mL. Among brewages, α-DC concentration of sake was low.
Conclusions: Alcoholic beverages contained, 3-DG, GO, and MGO as α-DCs, the contents of which was low in distilled
spirits. In brewages, the content of 3-DG and GO in sake was low. Alcoholic beverages with less glycative stress could besake in distilled spirits or brewages, however, proper intake should be strictly observed.

KEY WORDS: advanced glycation endproducts (AGEs), 3-deoxyglucosone, glyoxal, methylglyoxal, Japanese sake
Introductiona) 3-DGb) GOc) MGOThe glycative reaction in the living body is caused by anirreversible reaction between reducing sugar and protein.
α-Dicarbonyl compounds (α-DCs) are generated by dehydration/hydrolysis of Amadori compounds and cleavage between
carbons, act as intermediates in the glycative stress-induced
reactions. α-DCs in vivo include glyoxal (GO), methylglyoxal(MGO), and 3- deoxyglucosone (3-DG), which have two
carbonyl bonds (C = O) as shown in Fig. 1. α-DCs react withamino acids when heated or exposed to ultraviolet light,
causing Strecker’s decomposition and producing carbon
dioxide, aldehydes, pyrazines, and carbonylated proteins 1, 2).
These compounds change to intermediates, structurally
have an aldehyde base (-CHO), and thus recognized as

CHO

CHO

CHO

C= O

C= O

C= O

CH 2

H

CH 3

(CHOH) 2
CH 2OH

Fig. 1. Structures of α-DCs; 3-DG, GO and MGO.
a) 3-deoxyglucosone (3-DG); MW, 162.14 g/mol; density, 1.406 g/mL.
b) Glyoxal (GO); MW, 58.04 g/mol; density, 1.270 g/mL. c) Methylglyoxal(MGO); MW. 72.06 g/mol; density, 1.046 g/mL. α-DCs, α-dicarbonylcompounds; MW, molecular weight.

Contact Address: Professor Masayuki Yagi, PhD
Anti-Aging Medical Research Center and Glycative Stress Research Center,
Faculty of Life and Medical Sciences, Doshisha University​
1- 3 Tatara Miyakodani, Kyotanabe, Kyoto, 610 - 0394 Japan
TEL / FAX: +81-774 - 65- 6394 e-mail: myagi@mail.doshisha.ac.jp

Co-authors: Kimoto T, ta.chan027@gmail.com;
Takabe W, wtakabe@mail.doshisha.ac.jp; Ogura M, m-ogura@po.kbu.ac.jp;
Yonei Y, yyonei@mail.doshisha.ac.jp_ 152 _

Glycative Stress Research 2020; 7 ( 2) : 152-161(c) Society for Glycative Stress ResearchGlycative Stress Researchaldehyde in vivo, resulting in being involved in the productionof advanced glycation end products (AGEs) 3). Accumulationof AGEs in living tissues is a factor that causes functional
deterioration of proteins and, therefore, has been reported to
be involved in the onset of diabetes, osteoporosis, or otherdegenerative disease 4).
Food has three functions related to energy source,
palatability, and biological regulation. Foods with strong
functions related to biological regulation are called functional
foods. In recent years, various functional foods have beendeveloped with various materials that act to suppress theglycative reaction 5, 6). Among these are fermented foods,
which are processed by fermenting food materials by theaction of microorganisms or chemical reactions. Japanesesake, one of the fermented foods, is a food product obtainedby milling raw rice and then fermenting it by adding kojimold and yeast. Sake has already been reported to havefunctionalities such as prevention of hypertension, preventionof osteoporosis, suppression of cholesterol absorption, andantioxidant effect 7). There is a saying in Japan that "liquor
is a hundred medicine", conveying a message that there is anadvantage if you drink a proper amount of alcohol. There
have been many reports on the identification of functional
components of sake and its mechanism of action 8, 9), whilealcoholic beverages cause various organ disorders when
their intake increases 10). As affairs, there are few reports on

α-DCs contained in alcoholic beverages 11, 12). In this study,
the amount of α-DCs contained in alcoholic beverages wasmeasured and the content was compared according to thetype of beverage.

Materials and methods
Materials
Alcoholic drinks used as samples, 33 items, werepurchased at liquor stores in Kyoto, Japan. The purchased
alcoholic beverages were classified into 18 brewages (sixsake, three beer, three red wine, three white wine, and threesherry) and 15 distilled spirits (three whiskey, three shochu,
three gin, three vodka, and three rum) as shown in Table 1.

Measurement of solid content
The solid content concentration (mg/mL) of the sample
was calculated by adding 5 mL of the sample to an aluminum
tray and measuring the weight before and after drying at
120 °C for 2 hours followed by calculating from the change
value.

Table 1. The sample list of alcoholic beverages
ID

Liquor
type

Classification

Samplename

Product name

Manufacturer

Alcohol
percentage

Solid
Sugar
content content（mg/mL） （%）

S5

Seishu A

Konteki(Jozo type)

Higashiyama Shuzo(Kyoto, Japan)

16%

46. 2

10.6

S7

Seishu B

Hana no Izanai(Ginjo type)

Shoutoku Shuzo(Kyoto, Japan)

12%

28.0

7. 5

Seishu C

Hana no Izanai(Summer limited edition)(Ginjo type)

Shoutoku Shuzo(Kyoto, Japan)

12%

33.0

8.3

Seishu D

Densho Yamahai Shikomi
Tamanohikari (Ginjo type)

Tamanohikari Shuzo(Kyoto, Japan)

16%

45. 5

10.9

Seishu E

Tomiou Tanshu Yamadanishiki (Ginjo type)

Kitagawa Honke(Kyoto, Japan)

15%

47.6

10.9

Seishu F

Eikun Koto Sen-nen(Ginjo type)

Saito Shuzo(Kyoto, Japan)

15%

52.3

11.0

Beer A

Ichiban Shibori

Kirin Brewery(Tokyo, Japan)

5%

167.1

6.4

Beer B

Super Dry

Asahi Breweries(Tokyo, Japan)

5%161. 5

6. 2

B3

Beer CBlack Label

Sapporo Breweries(Tokyo, Japan)

5%

168.9

6. 2

WR1

Red
wine A

World Selection

Mercian(Tokyo, Japan)

12%121. 5

7.0

WR2 wineRedwine B

Vina Maipo Cabernet
Sauvignon

Vina Maipo(Santiago, Chile)

12%

136.1

7.6

WR3

Red
wine C

Aires Andinos

Bodegas La Rosa
(Mendoza, Argentina)

12%

128.0

7. 5

WW1

White
wine A

Tree Bear

Lion(Sydney, Australia)

13%

143.9

7.7

White
wine B

Miramonte

Miramonte Sauvignon
Blanc-Semillon(Valle Central, Chile)

12%

92.0

6.9

White

Grande Polaire Okayama

S7s
S8Japanesesake(Seishu)

S11
S15

Brewages

B1
B2

Beer

Red

White

WW2 wine_ 153 _

Sapporo Breweries

B3

Beer CBlack Label(Tokyo, Japan)

5%

168.9

6. 2

WR1

Red
wine A

World Selection

Mercian(Tokyo, Japan)

12%121. 5

7.0

12%

136.1

7.6

WR2 wine

Compounds
Beverages
Vina Maipo
Vina Maipo
Cabernetin Alcoholic
Red α-Dicarbonyl
wine B(Santiago, Chile)
Sauvignon

WR3

Redwine C

Aires Andinos

Bodegas La Rosa
(Mendoza, Argentina)

12%

128.0

7. 5

WW1

White
wine A

Tree Bear

Lion(Sydney, Australia)

13%

143.9

7.7

WW2 wineWhitewine B

Miramonte

Miramonte Sauvignon
Blanc-Semillon(Valle Central, Chile)

12%

92.0

6.9

WW3

White
wine C

Grande Polaire Okayama
Muscatbailey

Sapporo Breweries(Tokyo, Japan)

11%241. 89.1

WS1

Sherry A

González Tio Pepe

González Byass(Jerez de la Frontera,Spain)

15%

54.6

7. 2

WS2 Sherry

Sherry B

Manzanilla La Sanluquena

Bodegas Teresa
Rivero (Sanlúcar deBarrameda, Spain)

15%

68.3

7.6

WS3

Sherry C

Sandeman Fino

Sandeman (Jerez dela Frontera, Spain)

15%

66.7

7.4

W1

Whisky A

Suntory Yamazaki

Suntory(Osaka, Japan)

4 0%

3. 5

15.1

Whisky B

Chita

Suntory(Osaka, Japan)

43%

2.9

15.8

W3

Whisky C

Jack Daniel's

Jack Daniel's(Lynchburg, USA)

4 0%

5. 2

15.1

J1

PotatoshochuKuro Kirishima

Kirishima Shuzo(Miyazaki, Japan)

25%

0. 2

10.0

BarleyshochuIichikoSanwa Shurui(Oita, Japan)

20%

0. 2

7.8

J3

Rice
shochuChonmaHakutaka(Nada, Hyogo, Japan)

25%

0.1

9.9

G1

Gin A

Beefeater

Beefeater(London, UK)

47%

0. 0

16.4

Gin B

Dry GinSuntory(Osaka, Japan)

4 0%

0. 0

14.8

G3

Gin C

Wilkinson

Nikka Whisky (Yoichi,
Hokkaido, Japan)

37%

0.1

14.0

V1

Vodka A

Skyy

Skyy Spirits(San Francisco, USA)

4 0%

0.0

14.7

Vodka B

Suntory Vodka

Suntory(Osaka, Japan)

4 0%

0.0

14.6

V3

Vodka C

Wilkinson

Nikka Whisky(Yoichi, Hokkaido)

4 0%

0.4

14.7

R1

Rum A

Myers’s Rum

Sazerac(Metairie, USA)

4 0%

13.9

14.9

Rum B

Dover White

Dover(Tokyo, Japan)

45%

0. 5

15.7

Rum C

Dover 45

Dover
(Tokyo, Japan)

45%

7. 4

15.8

Red

White

W2

J2

G2

V2

R2
R3

Brewages

WhiskyShochu

Gin

Vodka

Rum

Distilled
spirits

Measurement of sugar content
The sugar content of the sample was measured using a
pocket sugar content meter (PL-1: Atago, Tokyo, Japan).

α-DC measurement in alcoholic beverages
For α-DCs in alcoholic beverages, 3-DG, GO, andMGO concentrations were measured according to previous
reports 13). For sample pretreatment, add 100 µL of eachsample, 155 µL of purified water, and 75 µL of 200 mmol/L

phosphate buffer, then add 170 µL of 6 % perchloric acidfollowed by centrifuge at 15,000 rpm for 10 minutes. For
labeling α-DCs by 2, 3- diaminonaphthalene (DAN), 4 00 µLof the supernatant after centrifugation was mixed with 350 µL
of saturated sodium hydrogen carbonate solution, and then
50 µL of 50 % methanol containing 1 mg/mL DAN was
added and reacted at 4 °C for 22 hours. The labeled solution
was centrifuged at 4 °C, 15,000 rpm for 10 minutes, andthe supernatant was used as a sample solution for HPLC
analysis. Table 2 shows the analytical conditions for 3-DG,
GO, and MGO by HPLC.

_ 15 4 _Glycative Stress Research

Table 2. HPLC condition.
Column:Eluent:Gradient:
Detector:

Unison UK- Phenyl, 75ｘ3 mm I.D. (Imtakt, Kyoto, Japan)

A: 2.5 mmol/L phosphate, B: acetonitrile

0 - 30 min (0 -10 % B), 30 - 42 min (10% B), 42- 47 min (10 -70 % B)
Excitation: 271 nm, Enission: 503 nm

Flow rate: 1.0 mL/min

Column temperature: 40 ℃

Injection volume:

20 μ L

Statistical analysis
The measurement was performed 3 times for each sample(n = 3), and the results are expressed as the average value ±standard deviation. Statistical analysis was performed byt-test for comparison between two groups and Tukey-Kramer
multiple comparison test for comparison between three ormore groups. The significance level was determined by a
two-sided test to be significant with a level of less than 5 %.

Results
1. Solid content and sugar content
Table 1 shows the solid content and sugar content of
the alcoholic beverages used as samples. The solid content
of brewages (100.2 ± 60.6 mg/mL, n = 18) was 43.7 times
higher than that of distilled spirits (2.3 ± 3.9 mg/mL, n = 15,
p < 0.001). The solid content of brewages in the order fromthe highest is beer (165.8 ± 3.9 mg/mL, n = 3), white wine
(159.2 ± 76.1 mg/mL, n = 3), red wine (128.5 ± 7.3 mg/mL,
n = 3), sherry (63.2 ± 7. 5 mg/mL, n = 3), and sake (42.9 ±9.4 mg/mL, n = 6 ). The solid content of distilled spirits inthe order is rum ( 7. 3 ± 6.7 mg/mL, n = 3), whiskey (3.9 ± 1. 2
mg/mL, n = 3), shochu (0.2 ± 0.1 mg/mL, n = 3), gin (0.1 ±0.2 mg/mL, n = 3), and vodka (0.03 ± 0.1 mg/mL).
The sugar content was 1. 7 times higher in distilled
spirits (14.0 ± 2.6 %, n = 15) than in brewages (8.1 ± 1.7 %, n
= 18, p < 0.001). The sugar content of brewages in the orderis sake ( 9.9 ± 1.6%, n = 6), white wine ( 7.9 ± 1.1%, n = 3),
red wine ( 7.4 ± 0.3%, n = 3), sherry (7.4 ± 0.2%, n = 3),
and beer (6.3 ± 0.1%, n = 3). The sugar content of distilled
spirits in the order is whiskey (15.3 ± 0.4 %, n = 3), rum(15. 5 ± 0.5%, n = 3), gin (15.1 ± 1.2 %, n = 3), vodka (14.7± 0.1%, n = 3), and shochu (9.2 ± 1.2 %, n = 3).

2. 3-DG concentration in alcoholic beverages
3-D G concentrations differed among the samples,nevertheless the average value of brewages (275.3 ± 366.7µg/mL, n = 18) was 57.8 % higher than that of distilled
spirits (4.8 ± 13.8 µg/mL, n = 15, p < 0. 001, Fig. 2 ). The
concentration of 3-DG in brewages in the order was beer
(1,048.7 ± 120.6 µg/mL, n = 3), white wine (216.7 ± 98. 5µg/mL, n = 3), red wine (177.0 ± 59.8 µg/mL, n = 3), sherry(107.3 ± 7.6 µg/mL, n = 3), and sake (51.0 ± 44. 8 µg/mL,
n = 6). The 3-DG concentration of distilled spirits washigh in rum (19.4 ± 30.1 µg/mL, n = 3), whiskey (4.2 ± 2.1µg/mL, n = 3), and shochu (0.2 ± 0.3 µg/mL, n = 3), while
it was not noted in gin or vodka.

3. GO concentration in alcoholic beverages
The GO concentration was lower than that of 3-DG,
and the average value of brewages (3.1 ± 2.4 µg/mL, n = 18)
was higher than that of distilled liquor (1.2 ± 1.8 µg/mL,
n = 15), being 2.7 times higher (p < 0.05, Fig. 3 ). The GOconcentration of brewages in the order was sherry (6.5 ± 3. 2
µg/mL, n = 3), beer (4.5 ± 0.8 µg/mL, n = 3), red wine (3.3
± 0.7 µg/mL, n = 3), white wine (2.7 ± 1.9 µg/mL, n = 3) andsake (0.9 ± 0.3 µg/mL, n = 6). 3-DG of distilled spirits wererecognized in whiskey (3.4 ± 1.3 µg/mL, n = 3) and rum (2.4
± 2.5 µg/mL, n = 3), slight in shochu, but not noted in gin or
vodka.

4. MGO concentration in alcoholic beverages
The MGO concentration was lower than that of 3-DG,
similar to the concentration of GO, and there was no significant
difference in the mean values between brewages (3.7 ± 2.9µg/mL, n = 18) and distilled spirits (2.0 ± 3.5 µg/mL, n = 15,
Fig. 4 ). MGO concentration of brewages in the order was whitewine (6.7 ± 3.8 µg/mL, n = 3), red wine (5.8 ± 0.3 µg/mL,
n = 3), sherry (4.9 ± 2.6 µg/mL, n = 3), followed by beer
(2.6 ± 0. 5 µg/mL, n = 3), and sake (1.0 ± 0.4 µg/mL, n = 6).
MGO concentrations in distilled spirits were recognized inwhiskey (8. 2 ± 2.9 µg/mL, n = 3) and rum (1.3 ± 1.5 µg/mL,
n = 3), however, MGO was found only slightly in shochu (0.2
± 0.0 µg/mL, n = 3), gin (0.1 ± 0.1 µg/mL, n = 3), and vodka(0.1 ± 0.1 µg/mL, n = 3).

5. α-DC content by type of alcoholic beverage
The alcoholic beverages used as samples were classified
into 10 categories, and the concentrations of 3-DG, GO,
and MGO were compared for each category. The 3-DG
concentration of beer was higher than that of the other 9categories (p < 0.001, Fig. 5 ). White wine contained higher
3-DG than sake, whiskey, shochu, gin, vodka, and rum (p <0.001), while red wine has higher 3-DG than whiskey, shochu,
gin, vodka, and rum (p < 0.05). The 3-DG value of sake waslower than that of white wine (p < 0.001).
The GO concentration of sherry was higher than thatof sake, shochu, gin, vodka (p < 0.001), and rum (p < 0.05,
Fig. 6 ). Beer contained higher GO than sake (p < 0.001),
shochu, gin, and vodka (p < 0.05). The GO value of sake was_ 155 _

α-Dicarbonyl Compounds in Alcoholic Beverages

1,400

1,200

3-DG (μg/mL)

1,000

800

600

400

200

R1

R2

R3

R1

R2

R3

V2

V3

V1

G2

G3

J3

Brewages

G1

J1

J2

W3

W1

W2

WS 3

WS1

WS2

WW2

WW3

WR 3

WW1

WR 2

B3

WR1

B1

B2

S15

S8

S 11

S7

S7s

S5

0

Distilled spirits

Fig. 2. 3-DG content in alcoholic beverage.

Results are expressed as mean ± standard deviation in the triplet measurement. 3-DG, 3-deoxyglucosone.

12

10

6

4

2

Brewages

V2

V3

V1

G2

G3

J3

G1

J2

J1

W3

W1

W2

WS 3

WS1

WS2

WW2

WW3

WW1

WR 3

WR 2

B3

WR1

B1

B2

S15

S8

S11

S7

S7s

0
S5

GO (μg/mL)

8

Distilled spirits

Fig. 3. GO content in alcoholic beverage.

Results are expressed as mean ± standard deviation in the triplet measurement. GO, glyoxal.

_ 15 6 _Glycative Stress Research

16
14

10
8
6
4
2

Brewages

Distilled spirits

Fig. 4. MGO content in alcoholic beverage.

Results are expressed as mean ± standard deviation in the triplet measurement. MGO, methylglyoxal.

Fig. 5. Comparison of 3-DG content by a type of alcoholic beverage.

Results are expressed as mean ± standard deviation, ** p < 0.01 by Tukey-Kramer test, n = 3 eachexcept sake (n = 6). 3-DG, 3-deoxyglucosone.

_ 157 _

R3

R2

R1

V3

V1

V2

G2

G3

J3

G1

J1

J2

W2

W3

W1

WS 3

WS1

WS2

WW3

WW1

WW2

WR 3

WR 2

B3

WR1

B1

B2

S 11

S15

S8

S7s

S7

0
S5

MGO (μg/mL)

12

α-Dicarbonyl Compounds in Alcoholic Beverages

Fig. 6. Comparison of GO content by a type of alcoholic beverage.

Results are expressed as mean ± standard deviation, ** p < 0.01 by Tukey-Kramer test, n = 3 each except
sake (n = 6). GO, glyoxal.

Fig. 7. Comparison of MGP content by a type of alcoholic beverage.

Results are expressed as mean ± standard deviation, , ** p < 0.01 by Tukey-Kramer test, n = 3 each except
sake (n = 6). MGO, methylglyoxal.

_ 158 _Glycative Stress Researchlower than that of beer or sherry (p < 0.001).
MGO concentrations were higher in whiskey, white
wine, and red wine than in sake, shochu, gin, vodka, and rum(p < 0.05, Fig. 7 ). Sherry contained higher MGO than vodka(p < 0.05). The MGO value of sake was lower than red wine,
white wine, and whiskey (p < 0.001).

Discussion
1. Manufacturing method of alcoholic beverage,solid content, and sugar content
Generally, brewages are liquor obtained by alcoholic
fermentation of raw materials such as yeast. Distilled spirits
are liquor obtained by distilling brewed liquor (brewages). In
this study, the solid content was 43.7 times higher in brewages
than in distilled spirits (p < 0.001), while the sugar content of
brewages was 1.7 times lower than that of distilled spirits ( p <0.001). The solid content of brewages is considered to be
derived from sugars produced during the brewing process inwhich the material is alcohol-fermented with yeast, and thecomponents that are produced from the grain of the material
during the fermentation process. Typically, in distilled spirits,
as the distillation of the original raw liquor is repeated, thealcohol content increases and the solid content is reduced
due to removal of the sugars from the raw materials. In thismeasurement, the sugar content of distilled spirits was higher
than that of brewages. This may be due to the influence of
the alcohol concentration on the sugar content measurementwith a sugar content meter. As a principle, the sugar content
meter calculates the sugar concentration from the refractive
index of the sample solution. The alcohol content of distilled
spirits was 37.8 ± 0.08% (n = 15), which was 3.1 times
higher than that of brewages at 12.1 ± 0.04% (n = 18). As amatter of course, a positive correlation was noted between
the alcohol content and the sugar content of all samples ( y =24.245x + 4.999, r = 0.958, n = 33).

2. Aldehyde content of sake
There are some reports on the acetaldehyde content ofsake 11, 12). The acetaldehyde contained in sake is producedby the oxidation of ethanol produced during the brewing
process. That is, alcohol is sequentially changed from ethanol
to acetaldehyde, and finally to acetic acid by the oxidation
reaction. The content of aldehyde varies from 30 to 110 μL/mL,
which is more than three times different depending on thetype of sake 11). The amount of acetaldehyde contained insake was the highest for the second-class liquor, according tothe old type classification of sake, followed by the first-classliquor and special-grade liquor. The relationship between the
production method of sake and the amount of acetaldehyde
is as follows: Daiginjo sake (0.26 μL/mL), Old sake (0.475
μL/mL), Ginjo sake (0.96 μL/mL), Junmai sake (1.0 μL/mL),and Hon-jozo sake (1.2 μL/mL) 12). Acetic acid producedby the oxidation of alcohol reacts with ethanol to produce
ethyl acetate. Therefore, Daiginjo sake and Old sake have aunique taste and aroma because they have a small amount ofacetaldehyde and are rich in ethyl acetate. Since the brewing
method of Daiginjo sake keeps a high alcohol content, theproduced ethanol is easily oxidized and becomes rich in
ethyl acetate. As for Old sake, the amount of ethyl acetate
increases as the alcohol oxidizes due to aging after brewing.
3-DG concentration in sake
It has been reported that the 3-DG content of sake varies
from 0.137 mmol/L to 0.407 mmol/L (22.2 to 66.0 μg/mL),a difference of about 3.0 times, and Junmai sake tends tocontain a larger amount when compared between the types 11).
In this study, the 3-DG amount of sake samples (n = 6) was
the minimum: 16.9 to the maximum: 138.5 μg/mL (0.104 to
0.854 mmol/L), where an 8.2 times difference was observed.
In the brewing process of sake, sugar derived from rice starchused as a raw material reacts with 3-DG, thus producing alarge amount of furfural. The amount of furfural produced
depends on the sugar produced during brewing 13). The furfuralcontent was higher in the order of Daiginjo sake: 1. 5 μL/mL,
Ginjo sake: 1.0 μL/mL, Junmai sake 0.6 μL/mL, and Honjozo sake 0.4 μL/mL, indicating that the higher the ricepolishing rate, the higher the amount of furfural. Furfural isrich also in Old sake (1.0 μL/mL) and is known to give aunique taste and flavor 11, 13). 3-DG was contained in brewages
about 57.8 times as much as distilled spirits. Furfural and
3-DG may be the factors that bring out the unique flavor ofsake.
GO concentration in sakeGO was detected in all alcoholic beverages except gin
and vodka. The difference in GO concentration may berelated to the autoxidation of glucose and the aging period
in the barrel. Mainly oak (Quercus crispula) wood is used
for whiskey aging barrels, where the material contains
polyphenols such as ellagic acid. Polyphenols have beenreported to suppress the formation of α-DCs, i.e. 3-DG,
G O, and MGO 14), and also to have an antioxidant effect 15),thus probably being involved in the suppression of GO
production. One of the characteristics of sake is that theamount of GO is small.
MGO concentration in sake
The MGO content in alcoholic beverages was lower
than that of 3-DG, and was recognized in whiskey (8.2 ±2.9 μg/mL, n = 3), white wine (6.7 ± 3.8 μg/mL, n = 3), andred wine (5.8 ± 0.3 μg/mL, n = 3). In the whisky distillery,
the manufacturers use copper stills (distillation apparatus),
called pot stills. It is known that glycative reaction occurs
by heating in the distillation process, and β-damascenone
and fural are produced 16, 17). MGO in whiskey may be an
autoxidation product derived from glycated materials during
the distillation process. One of the characteristics of sake isthat the amount of MGO is small.

3. Regarding the safety of α-DCs contained infood and beverage
In this study, we found that alcoholic beverages
contained 3-DG, GO, and MGO as α-DCs.
Manuka honey and coffee are known to contain MGO.
Manuka honey is a honey collected by bees from the nectar of
Manuka (Leptospermum scoparium), native to New Zealand,
and has a unique flavor like a crude drug. Manuka means"resurrection tree" or "healing tree" in the words of native_ 159 _

α-Dicarbonyl Compounds in Alcoholic Beverages

Maori people. Manuka honey has already been reported tohave antibacterial action 18), antiviral action 19), wound healing
promotion 20), and plaque prevention action 21). Manuka honeycontains 80 mg/kg (80 μg/g) to 1,200 mg/kg (1,200 μg/g)
of MGO 22, 23). It is said that the higher the MGO content,
the better the quality of Manuka honey. On the other hand,coffee contains 50 to 70 μmol/L of MGO. MGO of coffee
differs depending on the type of beans and the extraction
method. Espresso contains 230.9 μmol/L (16.6 μg/mL) of
MGO 24). The concentration of MGO in alcoholic beverages
is 15 μg/mL or less, which is lower than that of manuka
honey or espresso.
Aldehydes and α-DCs such as GO 25), which are associatedwith glycative stress, may cause DNA damage and induce
genetic mutations and teratogenicity if the intake amount ishigh 26, 27). The generation of 3-DG in vivo increases the riskof teratogenicity 28). Excessive intake of alcoholic beverages
causes DNA damage in squamous cells and increases theonset risk of esophageal cancer 29), which becomes higher
particularly by the intake of the liquor with high alcohol
content.
There are various indicators for food safety evaluation.
The no observed adverse effect level (NOAEL) is the doselevel at which no adverse effects were observed in animal
toxicity studies. Generally, a 1/100 concentration (dose) of
NOAEL is approved as an acceptable daily intake (ADI) in
humans. ADI is the amount by which a person is considered
to have no adverse health effects if they continue to eat every
day for a lifetime. It is customary to take one-third of ADI
for safe daily intake.
GO is contained, as well as in alcoholic beverages, infermented foods such as miso and yogurt (0.63 to 4. 2 mg/kg),bakery foods such as bread (0.07 to 1.6 mg/kg), vegetable
food materials (3 to 14 mg/kg), and cooking oil (up to 6.5
mg/kg).
Estimating the medium-term toxicity of GO, NOAEL
is 100 ~ 130 mg/kg bw/day in Europe 30), ADI 1.0 ~ 1.3 mg/kg
bw/day, one-third of that amount is 0.3 ~ 0.4 mg/kg bw/day,

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Conclusion
Alcoholic beverages contained α-DCs, 3-DG, GO, and
MGO. Among them, distilled spirits were shown to have a
low α-DCs content, while, in brewages, the content of 3-DG
and GO in sake was low. The alcoholic beverage with a low
glycative stress could be distilled spirits or sake in brewages.
However, proper intake should be strictly observed.

Acknowledgment
The part of this study was presented at the 18th Meetingof Glycative Stress Research on August 31, 2019, Kyoto,
Japan.

Conflict of Interest Statement
The authors claim no conflict of interest in this study.

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