Oxidative deamination of lysine residue in plasma protein
of diabetic rats
Novel mechanism via the Maillard reaction
Mitsugu Akagawa, Takeshi Sasaki and Kyozo Suyama
Department of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural Science,
Tohoku University, Japan
The levels of a-aminoadipic-d-semialdehyde residue, the
oxidative deamination product of lysine residue, in plasma
protein from streptozotocin-induced diabetic rats were
evaluated. a-Aminoadipic-d-semialdehyde was converted to
a bisphenol derivative by acid hydrolysis in the presence of
phenol, and determined by high performance liquid chro-
matography. Analysis of plasma proteins revealed three
timeshigherlevelsofa-aminoadipic-d-semialdehyde in dia-
betic subjects compared with normal controls. Furthermore,
we explored the oxidative deamination via the Maillard
reaction and demonstrated that the lysine residue of bovine
serum albumin is oxidatively deaminated during the incu-
bation with various carbohydrates in the presence of Cu
2+
at a physiological pH and temperature. This experiment
showed that 3-deoxyglucosone and methylglyoxal are the
most efficient oxidants of the lysine residue. When the
reaction was initiated from glucose, a significant amount of
a-aminoadipic-d-semialdehyde was also formed in the
presence of Cu
2+
. The reaction was significantly inhibited by
deoxygenation, catalase, and a hydroxyl radical scavenger.
The mechanism we propose for the oxidative deamination is
the Strecker-type reaction and the reactive oxygen species-

of glucose, the degradation of glycated proteins, and lipid
peroxidation [12]. In addition, increased levels of 3-DG,
MG, and GO are found in blood from diabetic patients and
streptozotocin (STZ)-induced diabetic rats [11–14]. In the
advanced stages of the Maillard reaction, these a-dicarbonyl
compounds irreversibly modify lysine and arginine residues
in proteins at physiological conditions, leading to the
formation of various AGEs in vitro, which are also identified
in vivo [1,5,7–10,15,16]. Therefore, a-dicarbonyl compounds
have been recognized as the major intermediates and
precursors in AGEs formation in vivo. Recently it has been
proposed that a-dicarbonyl stress is among the major
factors in the pathogenesis of diabetic complications
[1,7,11,16].
The oxidative degradation of a-amino acid by a-dicar-
bonyls is known as the so-called Strecker degradation in
food science. In the Strecker degradation [17,18], a number
of carbohydrate-derived a-dicarbonyls as well as glucose are
able to degrade a-amino acids at high temperatures, thus
generating an aldehyde with one carbon atom less than
a-amino acids. On the other hand, o-quinone compounds,
which have an a-dicarbonyl group, are known to catalyze
the oxidative deamination of primary amines to form the
corresponding aldehydes under physiological conditions
Correspondence to K. Suyama, Department of Applied Bioorganic
Chemistry, Division of Life Science, Graduate School of Agricultural
Science, Tohoku University, Tsutsumidori-Amamiyamachi,
Aobaku, Sendai 981–8555, Japan.
Fax: +81 22 717 8820, Tel.: +81 22 717 8818,
E-mail:

30] and the formation of some AGEs has been shown to
require oxygen free radicals [30–32]. Therefore, the oxygen
free radicals derived from the Maillard reaction in vivo may
also serve as adventitious oxidants of lysine residues.
a-Aminoadipic-d-semialdehyde residue is known as a pre-
cursor of cross-links in elastin and collagen [21–23], thus
implying the formation of cross-links, i.e. AGEs.
In the present study, we measured the a-aminoadipic-d-
semialdehyde in plasma protein. Analysis of rat plasma
proteins by RP-HPLC revealed significantly higher levels of
a-aminoadipic-d-semialdehyde residues in STZ-induced
diabetic rats compared with normal controls. Furthermore,
we explored the oxidative-deamination reaction via the
Maillard reaction and demonstrated the occurrence of
the oxidative deamination of the lysine residues in BSA via
the Maillard reaction at a physiological pH and tempera-
ture. Based on these findings, we propose a novel mechan-
ism for the oxidative modification of proteins in diabetes,
namely the oxidative deamination of the lysine residue via
Maillard reaction.
MATERIALS AND METHODS
Materials
Methanol was of HPLC grade from Nacalai Tesque Co.,
Kyoto, Japan.
D
-Ribose and catalase from bovine liver were
from Tokyo Kasei Co, Tokyo, Japan. STZ was from Sigma
Chemical Co, St. Louis, MO. Biuret reagent was from
Wako Pure Chemical Industries Co, Osaka, Japan. 3-DG
was from Dojindo Laboratories Co, Kumamoto, Japan. All

After 14 days of administration, blood was drawn from the
abdominal aorta of rats under light anesthesia with diethyl
ether and placed into heparinized tubes. Plasma was
immediately prepared by centrifugation at 1500 g for
20 min. The concentration of protein in the plasma samples
was measured by the Biuret reaction using BSA as reference
protein. Each plasma sample (500 lL) in a Pyrex test tube
with a Teflon-lined screw cap was treated with 2 mL of cold
10% (w/v) trichloroacetic acid. All subsequent steps were
performed in these tubes, and all samples were kept on ice
during processing. After 5 min, the mixture was centrifuged
at 2000 g for 30 min, and the resulting pellet of precipitated
protein was separated. The pellet was washed with 2 mL of
cold 5% (w/v) trichloroacetic acid. Then the resulting
protein was hydrolyzed for RP-HPLC analysis as described
below.
Detection of a-aminoadipic-d-semialdehyde by RP-HPLC
a-Aminoadipic-d-semialdehyde was derivatized to a
bisphenol derivative, 1-amino-1-carboxy-5,5-bis-p-hydroxy-
phenylpentane (ACPP), and determined by a modification
of the previous method [33,34] as follows. The protein in a
Pyrex test tube with a Teflon-lined screw cap was
hydrolyzed in a conventional manner for 48 h at 110 °C
with 4 mL of 6
M
HCl containing 3% (v/v) phenol. The
hydrolysate was extracted twice with 2.0 mL of diethyl
ether, and the water layer was dried by rotary evaporation
in vacuo followed by reconstitution in 500 lL of distilled
water. A Sep–Pak plus C

maintained at 40 °C. ACPP was eluted at 15.4 min using a
flow rate of 1.0 mLÆmin
)1
. Quantification of ACPP was
performed by calculating the peak area of the HPLC
absorbance profile (at 278 nm) of purified ACPP and
comparing it with those of samples.
Selective reduction of a-aminoadipic-d-semialdehyde
in plasma protein
Human plasma from a nondiabetic patient was dialyzed for
24 h at 4 °C against phosphate buffered saline. For
reduction with sodium borohydride (NaBH
4
), plasma
sample (500 lL) in a Pyrex test tube with a Teflon-lined
screw cap was diluted with 3.0 mL of 0.1
M
sodium borate
buffer (pH 9.0) followed by the addition of NaBH
4
(25 mg,
0.66 mmol). For reduction with sodium cyanoborohydride
(NaBH
3
CN), plasma sample (500 lL) in a Pyrex test tube
with a Teflon-lined screw cap was diluted with 3.0 mL of
0.1
M
sodium phosphate buffer (pH 6.0) followed by the
addition of NaBH

reaction mixtures containing 10 lL of toluene were incu-
bated at 37 °C with shaking in the dark. After incubation,
the mixture was treated with 2 mL of cold 10% (w/v)
trichloroacetic acid in ice bath. After 5 min, the mixture was
centrifuged at 2000 g for 30 min, and the resulting pellet of
precipitated protein was separated. The pellet was washed
with 2 mL of cold 5% (w/v) trichloroacetic acid. Then the
resulting protein was hydrolyzed for RP-HPLC analysis as
described above.
Incubation under nitrogen. The test tube was tightly fitted
with a silicone rubber cap. The tube was evacuated and then
filled with N
2
gas through a hypodermic needle. After
another hypodermic needle was inserted in the tube to serve
as an outlet port, gas was passed through the incubation
mixture for 10 min and charged until the pressure of
0.05 MPa inside the tube was reached. Then the reaction
mixture was incubated at 37 °C for 3 weeks with shaking in
the dark.
Statistical analysis
The significance of changes in the experimental variables
measured was assessed by Student’s t-test. We considered a
change with a P-value < 0.05 statistically significant. The
STATVIEW
program (StatView J-4.5, Abacus Concepts,
Berkeley, CA) was used for the analysis.
RESULTS
Detection of a-aminoadipic-d-semialdehyde
in rat plasma protein

HPLC with detection at 278 nm. (A) Chromatogram of ACPP
standard. (B) Chromatogram of hydrolyzate of plasma protein from
diabetic rat. Details are shown in the Experimental procedures section.
Ó FEBS 2002 Oxidative deamination via the Maillard reaction (Eur. J. Biochem. 269) 5453
procedure (Fig. 2). The mean ± SD of a-aminoadipic-
d-semialdehyde concentration was 3.21 ± 0.88 nmolÆmg
)1
proteinindiabetic(n ¼ 7) and 0.99 ± 0.29 nmolÆmg
)1
protein in control subjects (n ¼ 10). The 3.2-fold increase in
a-aminoadipic-d-semialdehyde was statistically significant
by Student’s t-test (P <0.001).
Selective reduction of plasma protein
We examined whether the a-aminoadipic-d-semialdehyde
residue exists as aldehyde, or Schiff base, or a mixture of the
two structures in vivo by selective reduction. Human plasma
was reduced, and then a-aminoadipic-d-semialdehyde was
analyzed by RP-HPLC. Figure 3 shows HPLC chromato-
grams of the plasma protein (A) and the NaBH
4
-reduced
plasma protein (B). The ACPP peak is abolished by the
reduction with NaBH
4
. On the other hand, reduction of
plasma protein with NaBH
3
CN, which is a selective
reductant toward Schiff base at pH 6–7 [35], only resulted
in a 5% decrease in the a-aminoadipic-d-semialdehyde peak

2+
did not increase a-aminoadipic-d-semialdehyde content
(Fig. 4A). As shown in Fig. 4A, in the presence of 5 l
M
Cu
2+
, a significant amount of a-aminoadipic-d-semialde-
hyde was produced by the reaction with glucose. There was
a time-dependent increase in the concentration of
a-aminoadipic-d-semialdehyde throughout the incubation
period (3 weeks).
We also evaluated various carbohydrates as possible
oxidants of the lysine residue. The formation of a-amino-
adipic-d-semialdehyde in BSA after incubation with various
sugars for 3 weeks in the presence of Cu
2+
is summarized in
Table 1. In the case of aldose, pentoses were more effective
oxidants than hexoses. A marked increase was observed
with an ascorbic acid/Cu
2+
system that generates reactive
oxygen species. This result is consistent with a recent report
by Stadtman et al. [26]. Furthermore, a significant increase
was found with a low concentration (1.0 m
M
)ofMGand
Fig. 3. Selective reduction of a-aminoadipic-d-semialdehyde residue in
plasma protein. Plasma protein was reduced with NaBH
4

)1
)
was incubated in 50 m
M
phosphate buffer with 1.0 m
M
of
each a-dicarbonyl under a physiological pH and tempera-
ture (pH 7.4, 37 °C). As shown in Fig. 4B, in the presence
of Cu
2+
, a significant amount of aldehyde was produced by
the reaction with 3-DG but not in the absence of Cu
2+
.MG
oxidatively deaminated the lysine residue in the presence
and absence of Cu
2+
(Fig. 4C). The oxidation was appar-
ently stimulated by the addition of Cu
2+
.
Effect of scavengers on the oxidative deamination
of BSA
The presence of oxygen plays an important role in the
Maillard reaction [36], and, actually, oxygen is required for
the formation of some AGEs [30,37]. To assess for the
participation of oxygen in the reaction, BSA was incubated
with glucose, 3-DG, and MG in the presence of Cu
2+

strated that the a-aminoadipic-d-semialdehyde level in
STZ-induced diabetic rat plasma is significantly higher than
Fig. 4. Time course of oxidative deamination of BSA by glucose, 3-DG,
and MG. BSA (10 mgÆmL
)1
) was incubated with 100 m
M
glucose (A),
1.0 m
M
3-DG (B), or 1.0 m
M
MG(C)in50m
M
phosphate buffer
(pH 7.4) in the presence or absence of 5 l
M
Cu
2+
at 37 °C. After the
reaction was terminated, a-aminoadipic-d-semialdehyde was measured
by RP-HPLC.
Table 1. Formation of a-aminoadipic-d-semialdehyde by incubation of
BSA with various carbohydrates. BSA (10 mgÆmL
)1
)wasincubated
with 5 l
M
Cu
2+

Glyoxal 1 0.07
Ó FEBS 2002 Oxidative deamination via the Maillard reaction (Eur. J. Biochem. 269) 5455
that in normal rat plasma. Analysis of selectively reduced
plasma protein suggested that the a-aminoadipic-d-semial-
dehyde residue exists primarily as the free aldehyde form
in vivo. Furthermore, we explored the oxidative-deamination
reaction via the Maillard reaction, and demonstrated the
occurrence of the oxidation of the lysine residue of BSA in
the incubation with various carbohydrates in the presence of
Cu
2+
at a physiological pH and temperature. This experi-
ment showed that 3-DG and MG are the most efficient
oxidant of the lysine residue. When the reaction was
initiated from glucose, a significant amount of a-aminoad-
ipic-d-semialdehyde was also formed in the presence of
Cu
2+
. We have also determined the effects of oxygen and
scavenger on the oxidative deamination. The formation of
a-aminoadipic-d-semialdehyde by glucose, 3-DG, and MG
was inhibited by deoxygenation, catalase, and dimethylsulf-
oxide. From these results we propose the Strecker-type
reaction by a-dicarbonyls and the reactive oxygen species-
mediated oxidation for the oxidative deamination mechan-
ism via the Maillard reaction. The proposed mechanism of
the formation of a-aminoadipic-d-semialdehyde from the
lysine residue by the Strecker-type reaction is summarized in
Fig. 5. The formation of a-dicarbonyls is induced through
the autoxidation of glucose and the degradation of Amadori

reaction may subsequently degrade, in a transition metal-
catalyzed process, to yield H
2
O
2
, reactive oxidants and
further protein-reactive aldehydes [28]. The production of
Table 2. Effect of O
2
and scavengers on the formation of a-aminoadipic-d-semialdehyde in BSA by glucose, 3-DG, and MG. BSA (10 mgÆmL
)1
)was
incubatedwith5l
M
Cu
2+
and each of indicated carbohydrate in 50 m
M
sodium phosphate buffer (pH 7.4) at 37 °Cfor3 weeks.a-Aminoadipic-d-
semialdehyde was quantitated by RP-HPLC as described in Experimental procedures. The values are shown as mean ± SEM (n ¼ 3). Native BSA
contained 0.06 ± 0.01 nmolÆmg
)1
protein of a-aminoadipic-d-semialdehyde.
a-Aminoadipic-d-semialdehyde (nmolÆmg protein
)1
)
Condition Glucose (50 m
M
) 3-DG (1 m
M

is also
consistent with a metal ion-catalyzed mechanism for the
production of hydroxyl radicals, probably through the
intermediary of superoxide and H
2
O
2
. Recently it has been
demonstrated that lysine residue is oxidatively deaminated
to a-aminoadipic-d-semialdehyde residue by reactive oxy-
gen species [24–26]. In addition, we have found that various
primary amines are converted to the corresponding alde-
hydes in the presence of H
2
O
2
and transition metal ions, and
the oxidation is effectively prevented by catalase and
dimethylsulfoxide [25]. Therefore, the hydroxyl radical
generated by the Fenton-type reaction is also likely to
contribute to the oxidative deamination via the Maillard
reaction. Based on these findings, we propose a novel
mechanism for the oxidative modification of proteins in
diabetes, namely the oxidative deamination of the lysine
residues via the Maillard reaction. Our proposed mechan-
ism of oxidation may also be the case in vivo.Infact,
increased levels of 3-DG, MG, and GO are found in blood
from diabetic patients and STZ-induced diabetic rats [11–
14]. These a-dicarbonyls are likely to react with the lysine
residue to form Schiff base adducts in the first step of protein

in plasma. The potential pathobiological role for a-amino-
adipic-d-semialdehyde residue in diabetes is still speculative.
The conversion of lysine residues to aldehydes might reflect
changes in protein conformation as a result of the continu-
ously decreasing loss of positive charge, and then the protein
will be inactivated. Lysyl oxidase, a copper-containing amine
oxidase, catalyzes the oxidative deamination of certain lysine
residues in elastin and collagen to form a-aminoadipic-
d-semialdehyde, which participates in cross-linking reactions
in these connective tissue proteins [20]. Once generated,
a-aminoadipic-d-semialdehydes condense with each other
via aldol condensation or with lysine residue via Schiff base
formation to form various inter- and intramolecular cross-
links spontaneously [41–46]. Therefore, a-aminoadipic-
d-semialdehyde residues may be candidates of a precursor
for the formation of protein cross-links, i.e. AGEs, in
diabetes although a-aminoadipic-d-semialdehyde derived
cross-links are not found in plasma protein. The protein
cross-linking leads to increasing resistance to removal by
proteolytic means as well as impeding function. Further-
more, increases in the level of the aldehyde residue may play
an important role in the cumulative modification of proteins
in tissues through cross-links. Thus, the oxidative deamina-
tion of the lysine residue may be implicated in the develop-
ment of diabetic complications at the molecular level, as
speculated for AGEs.
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