Identification and determination of α-dicarbonyl compounds formed in the degradation of sugars

Article abstract of DOI:10.1271/bbb.70229

The α-dicarbonyl compounds formed in the degradation of glucose and fructose were analyzed by HPLC using 2,3-diaminonaphthalene as derivatizing reagent, and identified as glucosone (GLUCO), 3-deoxyglucosone (3DG), 3-deoxyxylosone (3DX), tetrosone (TSO), triosone (TRIO), 3-deoxytetrosone (3DT), glyoxal (GO), and methylglyoxal (MGO). The results suggest that α-dicarbonyl compounds were formed from glucose via non-oxidative 3-deoxyglucosone formation and oxidative glucosone formation in glucose degradation. In addition, TRIO, GO, and MGO were also formed from glyceraldehyde as intermediate. The α-dicarbonyl compounds might be formed from glucose via these pathways in diabetes.

Full text of DOI:10.1271/bbb.70229

Biosci. Biotechnol. Biochem., 71 (10), 2465–2472, 2007
Identification and Determination of ꢀ-Dicarbonyl Compounds
Formed in the Degradation of Sugars
Teruyuki USUI,1;2 Satoshi YANAGISAWA,1 Mio OHGUCHI,1 Miku YOSHINO,1 Risa KAWABATA,1
Junko KISHIMOTO,1 Yumi ARAI,1 Kaoru AIDA,1 Hirohito WATANABE,3 and Fumitaka HAYASE
y
1;
1Department of Agricultural Chemistry, Faculty of Agriculture, Meiji University,
1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan
2Department of Health Care Nutrition, Showagakuin Junior College,
2-17-1 Higashisugano, Ichikawa, Chiba 272-0823, Japan
3Department of Life Sciences, Faculty of Agriculture, Meiji University,
1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan
Received April 17, 2007; Accepted July 15, 2007; Online Publication, October 7, 2007
[doi:10.1271/bbb.70229]
The ꢀ-dicarbonyl compounds formed in the degrada-
tion of glucose and fructose were analyzed by HPLC
using 2,3-diaminonaphthalene as derivatizing reagent,
and identified as glucosone (GLUCO), 3-deoxyglucosone
(3DG), 3-deoxyxylosone (3DX), tetrosone (TSO), trio-
sone (TRIO), 3-deoxytetrosone (3DT), glyoxal (GO), and
methylglyoxal (MGO). The results suggest that ꢀ-
dicarbonyl compounds were formed from glucose via
non-oxidative 3-deoxyglucosone formation and oxida-
tive glucosone formation in glucose degradation. In
addition, TRIO, GO, and MGO were also formed from
glyceraldehyde as intermediate. The ꢀ-dicarbonyl com-
pounds might be formed from glucose via these path-
ways in diabetes.
identified as the 2,3-diaminonaphthalene (DAN) adduct
in diabetic and uremic plasma.5) 3-Deoxyglucosone
(3DG), glyoxal (GO), and methylglyoxal (MGO) have
been identified as benzo[g]quinoxaline derivatives using
DAN reagent, but other carbonyls have not been
identified. In this study, we identified eight ꢀ-dicarbonyl
compounds (including 3DG, MGO, and GO) as ben-
zo[g]quinoxaline derivatives, formed in the degradation
of glucose and fructose, and quantitatively determined
each carbonyl formed from glucose in the physiological
pH condition.
Materials and Methods
Chemicals. 2,3-Diaminonaphthalene (DAN) was ob-
tained from Tokyo Chemical Industry (Tokyo). Cyclo-
hexane-1,2-dione was from Nacalai Tesque (Kyoto,
Japan). D-Glucose, lysozyme, glyoxal, methylglyoxal,
ꢀ-cyano-4-hydroxy-cinnamic acid (CHCA), and angio-
tensin I were from Sigma-Aldrich (St. Louis, MO). D-
Xylose, benzaldehyde, acetic acid, CD3OD, and aceto-
nitrile (HPLC grade) were from Kanto Chemical
(Tokyo). p-Toluidine and 2,4-dinitrophenylhydrazine
(DNP) were from Wako Pure Chemical Industries
(Osaka, Japan). All other chemicals used in this study
were of analytical reagent grade.
Key words: ꢀ-dicarbonyl compounds; 2,3-diaminonaph-
thalene; Maillard reaction; glycation; glu-
cose degradation products
ꢀ-Dicarbonyl compounds such as 3-deoxyglucosone
(3DG) are reactive intermediates formed in the Maillard
reaction, degradation of sugars, metabolic diseases, and
so on.1–3) The carbonyls increase in diabetes,4,5) and they
are a potential precursor of advanced glycation products
(AGE) as a post-translational modification of protein.6)
The carbonyls and AGE are thought to be involved in
the progression of chronic diseases such as diabetic
microangiopathy.
Various techniques have been reported for the analy-
sis of ꢀ-dicarbonyl compounds, and the pre-labeled
HPLC method is the most common. Yamada et al.
reported that 3-deoxyglucosone (3DG) was detected and
Synthesis of glucosone and 3-deoxyglucosone. D-
Glucosone (GLUCO) and 3-deoxyglucosone (3DG)
were synthesized by the method previously describ-
ed.7–9)
y
To whom correspondence should be addressed. Fax: +81-44-934-7902; E-mail: fumi@isc.meiji.ac.jp
Abbreviations: AGE, advanced glycation end products; ARA, arabinose; CHCA, ꢀ-cyano-4-hydroxy-cinnamic acid; 1,2CHD, cyclohexane-1,2-
dione; DA, dihydroxyacetone; DAN, 2,3-diaminonaphthalene; DTPA, diethylenetriaminepentaacetic acid; ERY, erythrose; FFL, furfural; GDPs,
glucose degradation products; 3,4DGE, 3,4-dideoxyosone-3-ene; 3,4DXE, 3,4-dideoxyxylosone-3-ene; 3DG, 3-deoxyglucosone; 3DX, 3-
deoxyxylosone; GA, glycolaldehyde; GLA, glyceraldehyde; GLUCO, glucosone; HMF, 5-hydroxymethylfurfral; TR, triosereductone; TSO,
tetrosone; TRIO, triosone; 3DT, 3-deoxytetrosone; GO, glyoxal; MGO, methylglyoxal

2466
T. USUI et al.
Synthesis of 3-deoxyxylosone. 3-deoxyxylosone
A
(3DX) was synthesized a method similar to that for
3DG. D-xylose (7.6 g) and p-toluidine (4.4 g) were
refluxed in a mixture of 95% ethanol (180 ml) and acetic
acid (8.8 ml) at 110 ꢀC. After 10 min, benzoylhydrazine
(13.2 g) was added to the solution, which was refluxed
for 2 h. The mixture was cooled at room temperature and
then filtered. The residue was washed with methanol
(3 ꢁ 50 ml), diethylether (3 ꢁ 50 ml), and ethanol (3 ꢁ
50 ml). It was obtained as 3-deoxyxylosone-bis-benzoyl-
hydrazone (3-DX-bis).
N
N
R1
NH2
NH2
O
O
R1
R2
+
R2
B
P6
P11
P1
P2
3-DX-bis (6.5 g) and distilled benzaldehyde (10.2 ml)
were refluxed in a mixture of 95% ethanol (200 ml) and
acetic acid (8.8 ml) at 110 ꢀC for 2 h. The mixture was
cooled to room temperature, and water (50 ml) was
added to the solution. The solution was evaporated to
about 40 ml. The concentrate was cooled to 4 ꢀC over-
night. The solution was filtered, and the filtrate was
washed with diethylether (10 ꢁ 50 ml). The aqueous
layer was put on an Amberlite IR-120(Hþ) (Rohm and
Haas, Philadelphia, PA) column, and fractionated. The
3DX-containing fractions were detected with DNP
reagent (0.4% 2,4-dinitrophenylhydrazine in 2 N HCl).
Then they were lyophilized as crude-3DX.
P7
P5
P4
P8
P9
P3
10
P10
P12
0
20
30
40
50
60
70
Retention time (min)
P5
C
P1
P2
P4
P6
P7
P11
P8
P10
P9
Crude-3DX (0.4 g) was dissolved in a small amount
of the solvent as ethylacetate–methanol–water (6:1:1).
The solution was put on a silica gel 60 (Merck KGaA,
Darmstadt, Germany) column equilibrated with the
same solvent. The 3DX-containing fractions were con-
centrated under reduced pressure. The residue was
dissolved in water, and the solution was lyophilized as
3DX.
P12
0
10
20
30
40
50
60
70
Retention time (min)
Fig. 1. Detection of ꢀ-Dicarbonyl Compounds Formed in the
Degradation of Glucose and Fructose.
Glucose and fructose solutions were incubated at 50 ꢀC for 1
week. The ꢀ-dicarbonyl compounds were detected as benzo[g]qui-
noxaline derivatives using 2,3-diaminonaphthalene as derivatizing
reagent. The reaction scheme is shown in A. Typical HPLC profiles
of ꢀ-dicarbonyl compounds are shown in B (glucose) and C
(fructose). Major peaks are named P1–P12.
Sample preparation. Glucose (200 mM) or fructose
(200 mM) was dissolved in 200 mM sodium phosphate
buffered solution (pH 7.4), and then incubated with
or without diethylenetriaminepentaacetic acid (DTPA,
5.5 mM) at 37 ꢀC or 50 ꢀC for one week. Glucosone, 3-
deoxyglucosone, 3-deoxyxylosone and glyceraldehyde
were incubated in the same manner.
Identification of ꢀ-dicarbonyls. Benzo[g]quinoxaline
derivatives as the carbonyl-DAN adduct were isolated
by preparative reversed phase HPLC. The sample was
put on a mBondasphere C18 column (150 ꢁ 19 mm I.D.,
Waters), and eluted with an isocratic of 20% acetonitrile
at a flow rate of 7 ml/min. Absorbance was monitored at
268 nm, and eight benzo[g]quinoxaline derivatives were
collected. After lyophilization, they were identified by
MS and NMR.
HPLC analyses of ꢀ-dicarbonyls. ꢀ-Dicarbonyl com-
pounds were analyzed as 2,3-diaminonaphthalene
(DAN)-adduct on reversed phase HPLC. The method,
originally reported by Yamada et al.5) was modified. A
sample was added to an equal volume of DTPA (5.5
mM) containing 1,2-cyclohexanedione (1 mM) as the
internal standard. The mixture was combined with an
equal volume of 2 mM DAN (dissolved in hot water at
80 ꢀC, and then cooled to room temperature), and
incubated at 50 ꢀC for 1 h. The reaction mixture was
filtered with a hydrophilic LCR (PTFE) membrane filter
unit (Millex-LH, Millipore, Billerica, MA), and then put
on a Puresil C18 column (150 ꢁ 4:6 mm I.D., Waters,
Milford, MA). The column temperature was controlled
at 40 ꢀC. The sample was eluted with a linear gradient of
14.5–31.0% acetonitrile from 0 to 70 min at a flow rate
of 1 ml/min, and absorbance was monitored at 268 nm.
Apparatus. HPLC Systems (Shimadzu, Kyoto, Japan)
consisted of HPLC Pump LC-10AS, UV–VIS Spectro-
photomeric Detector SPD-10AV, Column Oven CTO-
10A, and a C-R6A Chromatopac. FAB-MS was record-
ed with tandem mass spectrometer SX102 (JEOL,
Tokyo) with glycerol as the matrix and polyethylene-
glycol 400 as the mass standard. MALDI-TOF-MS was
recorded with a Voyager DE Pro (Applied Biosystems,
Foster City, CA) with CHCA as the matrix and angio-
tensin I as the mass standard. NMR were recorded by the
ECP-500 system (500 MHz, JEOL) in CD3OD.

Determination of ꢀ-Dicarbonyl Compounds
2467
6
9
5
5
6
9
A
B
C
D
5a 4a
4a
5a
9a
N
N
N
3
3
7
8
7
8
11
HC O
C O
CH2
HC OH
H2C OH
HC O
C O
HC OH
H2C OH
11
HC
C O
HO CH
HC OH
HC OH
H2C OH
HC O
C O
CH2
HC OH
HC OH
H2C OH
O
N
CH2
CHOH
CH2OH
2
9a
2
10a
10a
10
P7
5
10
CHOH
CH2OH
12
12
13
P8
5
6
9
6
9
4a
5a
5a
4a
3
N
N
N
N
3
7
7
E
F
G
H
11
11
CH2OH
8
8
CH2
2
10a
10
9a
2
9a 10a
10
HC O
C O
CH2
HC O
HC O
HC O
C O
H2C OH
HC O
C O
CH3
CH2OH
12
P9
P10
H2C OH
Fig. 2. Chemical Structure of P7, P8, P9, and P10.
Fig. 3. Chemical Structures of ꢀ-Dicarbonyl Compounds Formed in
the Degradation of Glucose and Fructose.
P7, P8, P9, and P10 were identified as 2-(1,2-dihydroxyethyl)-
benzo[g]quinoxaline formed from tetrosone (TSO), 2-(2,3-dihydro-
propyl)-benzo[g]quinoxaline from 3-deoxyxylosone (3DX), 2-hy-
droxymethyl-benzo[g]quinoxaline from triosone (TRIO), and 2-(2-
hydroxyethyl)-benzo[g]quinoxaline from 3-deoxytetrosone (3DT)
respectively.
A, glucosone; B, 3-deoxyglucosone; C, tetrosone; D, 3-deoxy-
xylosone; E, triosone; F, 3-deoxytetrosone; G, glyoxal; H, methyl-
glyoxal.
Table 1. NMR Chemical Shift (ppm) of Benzo[g]-Quinoxaline Derivatives Formed from Carbonyls
P8 P9
P7
P10
No.a
13C-ꢁ
1H-ꢁ
No.a
13C-ꢁ
1H-ꢁ
No.a
13C-ꢁ
1H-ꢁ
No.a
13C-ꢁ
1H-ꢁ
12
11
—
5
65.55
74.27
—
3.97–4.02 (m)
5.02–5.04 (m)
—
11
13
12
5
40.15
65.82
71.52
126.4
126.7
126.9
127
3.13–3.18 (m)
3.66 (d)
4.21–4.23 (m)
8.60 (s)
11
—
—
5
64.62
—
—
4.89 (s)
11
12
—
5
39.92
61.19
—
3.18–3.20 (t)
4.01–4.03 (t)
—
—
—
126.88
127.01
126.88
127.01
128.10
128.17
133.87
134.20
137.79
138.04
145.37
157.53
8.68 (s)
7.62–7.64 (m)
8.68 (s)
7.62–7.64 (m)
8.17 (m)
8.17 (m)
—
127.5
127.7
127.8
127.9
128.9
129
8.53 (s)
7.52–7.54 (m)
8.57 (s)
7.52–7.54 (m)
8.06 (m)
8.06 (m)
—
127.2
127.5
127.70
127.8
128.9
129
8.53 (s)
7.52–7.52 (m)
7.52–7.53 (s)
7.52–7.54 (m)
8.06 (m)
8.06 (m)
—
7
7
7.60–7.62 (m)
8.64 (s)
7
7
10
8
10
8
10
8
10
8
7.60–7.62 (m)
8.14–8.16 (m)
8.14–8.16 (m)
—
6
6
128.1
128.1
133.59
134.12
137.46
138.24
147.82
156.34
6
6
9
9
9
9
5a
9a
4a
10a
3
5a
9a
4a
10a
3
5a
9a
4a
10a
3
134.6
135
5a
9a
4a
10a
3
134.4
135
—
—
—
—
—
—
—
—
138.5
138.7
146.2
157.6
138.3
139.1
148.3
157.3
—
9.11 (s)
—
—
8.88 (s)
—
—
8.94 (s)
—
—
8.79 (s)
—
2
2
2
2
aNumbers represent assigned positions as shown in Fig. 2.
Statistical analysis. The concentrations of glucosone
(GLUCO), 3-deoxyglucosone (3DG), 3-deoxyxylosone
(3DX), glyoxal (GO), and methylglyoxal (MGO) were
calculated by regression analysis using standards. The
tetrosone (TSO), triosone (TRIO), and 3-deoxytetrosone
(3DT) concentrations were calculated by regression
analysis using 3-deoxyglucosone as standard.
promotes oxidation, DTPA suppresses oxidation.
Figure 1B and C show the ꢀ-dicarbonyl formed by the
incubation of glucose and fructose. Twelve major
compounds were generated from glucose (Fig. 1B) and
fructose (Fig. 1C) in the physiological pH condition, and
named P1–P12. The UV spectrum of benzo[g]quinoxa-
line showed a maximum peak at 268 nm. The UV
spectra of P3, P5, P7, P8, P9, P10, P11, and P12 proved
to be benzo[g]-quinoxaline derivatives (the UV spectra
are not shown), and P6 was identified as DAN.
Results and Discussion
Detection of ꢀ-dicarbonyls
Benzo[g]quinoxaline derivatives of ꢀ-dicarbonyl
compounds were detected by HPLC analysis. As shown
in Fig. 1A, benzo[g]quinoxaline derivatives were form-
ed from ꢀ-dicarbonyl compounds and 2,3-diaminonaph-
thalene (DAN) derivatizing reagent. In this study, metal
chelator DTPA was used in the derivatization procedure.
Since the trace metal (included in phosphate salt)
Identification of P3, P5, P11, and P12
P3, P5, P11, and P12 were compared with synthesized
carbonyls (as standard) on HPLC and FAB-MS analysis.
The FAB-MS measured for P3, P5, P11, and P12
showed the ½M þ Hꢂþ ion at m=z 301, 285, 181, and
195 respectively. The carbonyls were identified as 2-
(1,2,3,4-tetrahydroxybutyl)-benzo[g]quinoxaline (P3)

2468
T. USUI et al.
200
150
100
50
30
A
GO
GLUCO
3DG
3DX
B
20
10
0
0
1
2
4
7
14
1
2
4
7
14
Incubation period (d)
Incubation period (d)
10
5
30
200
150
100
50
: with DTPA
D
C
TSO 3DT MGO TRIO
20
10
0
0
0
1
2
4
7
14
GO
3DT
3DX
3DG
TSO
Incubation period (d)
MGO TRIO
GLUCO
Fig. 4. Determination of ꢀ-Dicarbonyl Compounds Generated in the Degradation of Glucose.
A, GO concentration; B, GLUCO, 3DG, and 3DX concentrations; C, TSO, 3DT, MGO, and TRIO concentrations; D, concentrations of
carbonyls formed with DTPA (open circle) and without DTPA (closed circle).
1
formed from glucosone (GLUCO), 2-(2,3,4-trihydrox-
ybutyl)-benzo[g]quinoxaline (P5) from 3-deoxygluco-
sone (3DG), benzo[g]quinoxaline (P11) from glyoxal
(GO), and 2-methyl-benzo[g]quinoxaline (P12) from
methylglyoxal (MGO).
P9 showed an ½M þ Hꢂþ ion at m=z 211. The H-NMR
(500 MHz, CD3OD) ꢁ and 13C-NMR (CD3OD) ꢁ are
shown in Table 1. These assignments were also verified
by H-detected H–13C COSY, H–1H COSY, and 13C
1
1
1
DEPT spectra.
Identification of P7
Identification of P10
P7 was analyzed by FAB-MS and NMR (including
1H–1H COSY and 13C DEPT spectra). The carbonyl
compound was identified as 2-(1,2-dihydroxyethyl)-
benzo[g]quinoxaline, formed from tetrosone (TSO).
The chemical structure of P7 is shown in Fig. 2. The
spectral data were as follows: FAB-MS (m=z): 241
P10 was identified as 2-(2-hydroxyethyl)-benzo[g]-
quinoxaline, formed from 3-deoxytetrosone (3DT), by
NMR (including 1H–1H COSY, 1H–13C-COSY, and the
13C-DEPT method). The chemical structure of P10 was
shown in Fig. 2. MALDI-TOF-MS for P10 showed an
½M þ Hꢂþ ion at m=z 225. 1H-NMR (500 MHz, CD3OD)
ꢁ and 13C-NMR (CD3OD) ꢁ are shown in Table 1.
þ
1
(½M þ Hꢂ ). H-NMR (500 MHz, CD3OD) ꢁ and 13C-
NMR (CD3OD) ꢁ were shown in Table 1.
Determination of ꢀ-dicalbonyls
Identification of P8
In this study, eight carbonyls (glucosone, GLUCO;
3-deoxyglucosone, 3DG; 3-deoxyxylosone, 3DX; tetro-
sone, TSO; triosone, TRIO; 3-deoxytetrosone, 3DT;
glyoxal, GO; methylglyoxal, MGO) were identified as
glucose and fructose degradation products. The chemi-
cal structures of the carbonyls are shown in Fig. 3.
Subsequently, eight carbonyls formed with or without
DTPA in the glucose degradation in the physiological
pH condition were determined to be benzo[g]quinoxa-
line derivatives. Since the trace metal promotes oxida-
tion, the presence of the metal chelator DTPA indicates
a non-oxidative condition and absence of DTPA
indicates an oxidative condition.
P8 was identified as 2-(2,3-dihydropropyl)-benzo[g]-
quinoxaline, formed from 3-deoxyxylosone (3DX).
Figure 2 shows the chemical structure of P8. The
FAB-MS for P8 showed an ½M þ Hꢂþ ion at m=z 255.
The 1H-NMR (500 MHz, CD3OD) ꢁ and 13C-NMR
(CD3OD) ꢁ are shown in Table 1. These assignments
1
were verified by H–1H COSY and 13C DEPT spectra.
Furthermore, the carbonyl-DAN adduct from synthe-
sized 3DX showed the same data on HPLC analysis.
Identification of P9
P9 was identified as 2-hydroxymethyl-benzo[g]qui-
noxaline, formed from triosone (TRIO). The chemical
structure of P9 is shown in Fig. 2. MALDI-TOF-MS for
As shown in Fig. 4A and B, GLUCO, 3DG, 3DX, and
especially GO were generated at an early stage of

Determination of ꢀ-Dicarbonyl Compounds
2469
8
4
0
8
GO
3DT
MGO
A
GO
3DT
(with DTPA)
MGO
B
4
0
1
2
4
7
14
6
1
2
4
7
14
Incubation Period (d)
Incubation Period (d)
D
3DT
TSO
TRIO
MGO
GO
150
100
50
C
3
3DX
3DX (with DTPA)
0
6
0.5
E
1
2
4
7
14
Incubation period (d)
3DT
TSO
TRIO
MGO
GO
(with DTPA)
0
0.5
1
2
4
7
14
3
0
Incubation period(d)
0.5
1
2
4
7
14
Incubation period (d)
15
10
5
15
10
5
GO
GO
MGO
3DT
F
G
MGO
3DT
(with DTPA)
0
0
1
2
4
7
14
1
2
4
7
14
Incubation period (d)
Incubation period (d)
Fig. 5. Determination of ꢀ-Dicarbonyl Compounds Formed from 3-Deoxyglucosone, Glucosone, and 3-Deoxyxylosone.
Each carbonyl was formed from 3DG without DTPA (A) and with DTPA (B). 3DX was formed from GLUCO with or without DTPA, and
carbonyls were formed from GLUCO without DTPA (D) and with DTPA (E). Carbonyls were formed from 3DX without DTPA (F) and with
DTPA (G).
glucose degradation. Subsequently, MGO and TRIO
were formed in the reaction, and then TSO and 3DT
were generated in the reaction mixture (Fig. 4C). MGO,
TRIO, TSO, and 3DT might have been formed from the
intermediates. The metal chelator DTPA inhibited
GLUCO, 3DX, TSO, TRIO, 3DT, GO, and MGO
formation, but did not inhibit the 3DG formation
(Fig. 4D). 3DG might be formed under non-oxidative
conditions.
GLUCO, and 3DX degradation. 3DT, MGO, and
especially GO were formed from 3DG (Fig. 5A). The
formation of GO was suppressed by DTPA, but DTPA
hardly affected the generation of 3DT and MGO
(Fig. 5B).
3DX, MGO, GO, TRIO, and TSO were formed from
GLUCO (Fig. 5C and D). 3DX especially was generated
in the GLUCO degradation. Since 3DX formation
increased with addition of DTPA (Fig. 5C), 3DX might
have been formed in the non-oxidative condition. In
Moreover, ꢀ-dicarbonyls were analyzed in 3DG,

2470
T. USUI et al.
DAN
A
IS
TRIO
MGO
GO
0
10
20
30
40
50
60
70
Retention time (min)
GO
3
2
1
0
37°C
TRIO
MGO
B
(with DTPA)
3h 6h 12h 1d 2d 4d 7d
3h 6h 12h 1d 2d 4d 7d
Incubation period
3
2
1
0
50°C
C
TRIO
MG
GO
(with DTPA)
3h 6h 12h 1d 2d 4d 7d
3h 6h 12h 1d 2d 4d 7d
Incubation period
Fig. 6. Determination of ꢀ-Dicarbonyl Compounds Formed from Glyceraldehyde.
A, typical HPLC profile (IS, internal standard); B, carbonyls from GLA with and without DTPA at 37 ꢀC; C, carbonyls from GLA with and
without DTPA at 50 ꢀC.
addition, 3DT was newly formed with the addition of
DTPA.
level (with DTPA) at 37 ꢀC was higher than at 50 ꢀC
(Fig. 6C). TRIO might be converted to glycolaldehyde
(GA). GO and MGO were also formed from GLA. The
formation of GO was repressed by DTPA. Perhaps GO
is formed by oxidation. MGO was generated from GLA
and from TR by dehydration. MGO was increased by the
metal chlator, because MGO was transformed to the
oxidant of MGO by oxidation.
3DT, MGO, and especially GO were formed from
3DX (Fig. 5F), and the formation of GO was suppressed
by DTPA (Fig. 5G). In addition, the time-dependent loss
of MGO was repressed by DTPA. DTPA might suppress
the oxidation of MGO.
Figure 6 shows the concentration of ꢀ-dicarbonyls
generated from GLA. A typical chromatogram is shown
in Fig. 6A. TRIO, MGO, and GO were detected as
GLA-derived ꢀ-dicarbonyls. As shown in Fig. 6B, the
TRIO level increased rapidly in GLA degradation, and
then TRIO gradually decreased. The formation of TRIO
was affected by DTPA. TRIO might have been formed
from triose reductone (TR), which is formed by the
oxidation of glyceraldehyde. The TRIO level (without
DTPA) at 50 ꢀC was higher than at 37 ꢀC, but the TRIO
Formation of ꢀ-dicarbonyls
Recent study suggests that ꢀ-dicarbonyl compounds
induce a progression of diabetic complications. In this
study, we investigated ꢀ-dicarbonyl formation in glu-
cose degradation. The proposed pathway of ꢀ-dicarbon-
yl formation formed in glucose degradation is summa-
rized in Fig. 7.
3DG is generated from glucose by dehydration. It has

Determination of ꢀ-Dicarbonyl Compounds
2471
HC O
C O
CH2
HC OH
HC OH
H2C OH
HC O
C O
CH
CH
HC OH
H2C OH
OH
CHO
- H2O
- H2O
3DG
HOH2C
O
HC O
HC OH
HO CH
HC OH
HC OH
H2C OH
HOH2C
CHO
O
3,4DGE
HMF
GO
- H2O
GLC
H2C OH
C O
H2C OH
HC O
C O
H2C OH
HC O
C OH
HC OH
HC O
HC O
HC OH
C OH
HO CH
TR
- 2H
DA
TRIO
HC OH
HC OH
H2C OH
- 2H
HC O
HC OH
H2C OH
HC O
C O
CH3
HC OH
C OH
H2C OH
HC O
H2C OH
- H2O
1,2-endiol
GA
1,2-endiol
MGO
GLA
- 2H
HC O
C O
HO CH
HC OH
HC OH
H2C OH
CHO
FFL
HC O
C O
CH2
HC O
HC OH
HC OH
HC O
C O
HC OH
O
O
- H2O
- 2H
- H2O
H2C OH
H2C OH
H2C OH
OH
CHO
ERY
3DT
TSO
GLUCO
HC O
C OH
HO C
HC OH
HC OH
H2C OH
HC O
HO CH
HC OH
HC OH
H2C OH
HC OH
HO C
HC OH
HC OH
H2C OH
HC O
C O
CH2
HC OH
H2C OH
HC O
C O
CH
CH
H2C OH
- H2O
- H2O
1,2-endiol
3,4DXE
ARA
3DX
Fig. 7. Proposed Formation Pathway of ꢀ-Dicarbonyl Compounds Generated in the Degradation of Glucose.
been reported that 3DG is related to diabetic micro-
angiopathy,5) inhibits cell proliferation,10) and induces
apoptosis in cells.11,12) 3,4-Dideoxyosone-3-ene
(3,4DGE) and 5-hydroxymethylfurfral (HMF) as inter-
mediates were formed from 3DG by dehydration. The
intermediates are known as cytotoxic substances. The
3,4DGE intermediate has been reported to be a major
toxic product in peritoneal dialysis solution as a glucose-
containing medicines.13) HMF is also known as a
cytotoxic substance in glucose-containing medicines
such as parental solution.14) It is utilized as a glucose
degradation marker for the product quality of each
medicine. It is formed from 3,4DGE by dehydration.
The intermediates are formed under the non-oxidative
condition, and the formation shows temperature depend-
ence in sterilization and storage. Moreover, the carbon-
yls in glucose-containing medicines are often called
glucose degradation products (GDPs).15) GDPs are
quality markers in medicines and are thought to affect
cell function.
On the other hand, GLUCO was generated from
glucose by oxidation. Arabinose (ARA) was formed
from GLUCO by ꢀ-dicarbonyl cleavage, and then 3DX
was formed from ARA by dehydration. Then 3,4-
dideoxyxylosone-3-ene (3,4DXE) was formed from
3DX by dehydration, and furfural (FFL) was formed
from 3,4DXE by dehydration. In addition, 3DT and TSO
might have been formed from ERY in GLUCO
degradation. 3DT was also formed from 3DG and 3DX.
Furthermore, we have reported that GLA is generated
in glucose degradation.9) GLA might be formed from
glucose, 3DG, and GLUCO by the retro-aldol reaction.
In this case, TRIO, GO, and MGO were formed from
GLA intermediate. MGO was also formed from 3DG
and 3DX, and GO were formed from 3DG, 3DX, and
GA respectively. GA was generated from GLC and
TRIO. The GLA-related pathway is one of major
pathways in ꢀ-dicarbonyl formation.
In this study, we investigated whether ꢀ-dicarbonyl
compounds would be generated from glucose via non-

2472
T. USUI et al.
pp. 421–423 (1963).
oxidative 3DG formation, oxidative GLUCO formation,
GLA formation, and other pathways. It is suggested that
ꢀ-dicarbonyl compounds are formed in hyperglycemia
from diabetes, and by the infusion of glucose-containing
medicines such as peritoneal dialysis solution. Carbon-
yls might cause the progression of metabolic diseases.
8) Kato, H., Chuyen, N. V., Shinoda, T., Seiya, F., and
Hayase, F., Metabolism of 3-deoxyglucosone, an inter-
mediate compound in the Maillard reaction, adminis-
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Acta, 1035, 71–76 (1990).
9) Usui, T., Yoshino, M., Watanabe, H., and Hayase, F.,
Determination of glyceraldehyde formed in the glucose
degradation and glycation. Biosci. Biotechnol. Biochem.,
71, 2162–2168 (2007).
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