Oxidative deamination of lysine residue in plasma proteinof diabetic rats Novel mechanism via the Maillard reaction Mitsugu Akagawa, Takeshi Sasaki and Kyozo Suyama Department of Applied
Trang 1Oxidative 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
times higher levels of a-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 Cu2+
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 Cu2+ 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-mediated oxidation Based on these findings, we propose a novel mechanism for the oxidative modification of proteins
in diabetes, namely the oxidative deamination of the lysine residue via the Maillard reaction
Keywords: Maillard reaction; glycation; reactive oxygen species; a-aminoadipic-d-semialdehyde; oxidative deamina-tion
The Maillard reaction (nonenzymatic glycation) is thought
to contribute to the pathogenesis of diabetic complications
and the ageing process The first step in this reaction is the
formation of a Schiff base between a reducing sugar and an
amino group in proteins, followed by an Amadori
rear-rangement to yield a relatively stable ketoamine adduct
Subsequently, the adducts (Amadori products) are further
degraded to form a variety of structurally diverse
com-pounds known as advanced glycation end products (AGEs)
which frequently have chromophores, fluorophores, and
protein cross-links [1] Recent researches have demonstrated
that the formation and accumulation of AGEs in plasma
and tissue are associated with aging [1–5] and the long-term
complications of diabetes [1,6–10]
In the process of the Maillard reaction, several reactive
a-dicarbonyl compounds are found in vitro and in vivo
3-Deoxyglucosone (3-DG) is produced from the multiple dehydration in the early stage Maillard reaction and by the fragmentation of fructose 3 phosphate [11,12] Methylgly-oxal (MG) is mainly formed by amine-catalyzed sugar fragmentation reactions and by spontaneous decomposition
of triose phosphate intermediates in glycolysis [12] Glyoxal (GO) is formed by the spontaneous oxidative degradation
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: suyama@bios.tohoku.ac.jp
Abbreviations: ACPP,
1-amino-1-carboxy-5,5-bis-p-hydroxyphenyl-pentane; AGE, advanced glycation end-product; 3-DG,
3-deoxy-glucosone; LTQ, lysine tyrosylquinone; MG, methylglyoxal;
GO, glyoxal; STZ, streptozotocin.
Enzyme: lysyl oxidase (EC 1.4.3.13).
(Received 14 May 2002, revised 4 September 2002,
accepted 9 September 2002)
Trang 2[19] Lysyl oxidase (EC 1.4.3.13), a copper-containing
amine oxidase, catalyzes the oxidative deamination of the
e-amino group of lysine residue of elastin and collagen,
which are connective tissue proteins, to form
a-aminoadi-pic-d-semialdehyde [20] Lysyl oxidase has a lysine
tyrosyl-quinone (LTQ) cofactor consisting of an amino-o-tyrosyl-quinone
skeleton at the active site [21,22], and the LTQ cofactor itself
catalyzes the amine oxidation [23] The a-dicarbonyls
derived from the Maillard reaction exist as free compounds
in vivo and thus potentially may serve as adventitious
oxidants of amines possibly including protein lysine residues
and, in the course of the oxidative deamination, generate
a-aminoadipic-d-semialdehyde
On the other hand, it was recently found that
a-aminoadipic-d-semialdehyde is produced from protein
lysine residue by the attack of oxygen-derived free radicals
[24–26] In addition, the aldehyde residue has been identified
in serum albumin collected from some mammalian species
[24] The Maillard reaction can give rise to oxygen free
radicals in the presence of O2and transition metal ions [27–
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
M A T E R I A L S A N D M E T H O D S
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
other chemicals were from Nacalai Tesque Co
Diabetic model rats
Animal experiments were carried out according to a
protocol approved by the Animal Care Committee of
Tohoku University Male Wistar rats (8 weeks old, Japan
SLC Co., Shizuoka, Japan), weighing 180–200 g, were used
for the disease model study Experimental diabetes was
induced by a single i.p dose of STZ (55 mgÆkg)1) STZ was
dissolved in 0.1Mcitrate buffer (pH 4.5) The animals were
fasted overnight prior to STZ administration Two weeks
after STZ administration, all animals with plasma glucose level > 400 mgÆdL)1 were considered diabetic and were included in the study Plasma glucose levels were measured using the commercial kit, Glucose CII Test Wako (Wako Pure Chemical Industries Co) Control animals received 0.1Mcitrate buffer (pH 4.5) During the experiment the rats were housed in groups of two or three per cage Tap water and pelleted standard diet Laboratory MR (Nihon Nosan Kogyo Co, Yokohama, Japan) were available ad libitum The rats were housed in temperature- (23 ± 1C), humi-dity- (55 ± 5%), and light- (8.00–20.00 h) controlled room Plasma protein
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 110C with 4 mL of 6M 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 vacuofollowed by reconstitution in 500 lL of distilled water A Sep–Pak plus C18 environmental cartridge (Waters Co, Milford, MA, USA) was used for prepurifi-cation as follows The Sep–Pak cartridge was flushed with methanol (10 mL) and then distilled water (10 mL), and the sample (500 lL) was put onto the cartridge After the cartridge was washed with 20.0 mL of distilled water, ACPP was eluted with 4.0 mL of distilled water/methanol (1 : 1, v/v) The eluate was evaporated to dryness in vacuo, and then reconstituted in 500 lL of distilled water After filtration with a poly(vinylidene difluoride) (PVDF) syringe filter (0.45 lm pore size, Whatman Co, Clifton, NJ), 40-lL portion of it was injected into an HPLC apparatus with a C-18 reversed phase column (COSMOSIL 5C18-AR-II,
250· 4.6 mm, Nacalai Tesque Co) The RP-HPLC ana-lysis was performed on a Perkin Elmer Liquid Chroma-tograph Integral 4000 system (Norwalk, CT, USA) Solvent A was 10% methanol, and solvent B was methanol The solvents were degassed by sonication and then continuously bubbled with a slow stream of helium during chromatography The column was equilibrated with
Trang 3solvent A The elution started with a linear gradient from 0
to 15% B in 20 min Then solvent B was increased to 95%
over the following 15 min Finally, the eluting solvent was
changed linearly to 100% A over a period of 20 min The
column was reequilibrated for 15 min with solvent A
before the next run was started The column oven was
maintained at 40C 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 4C against phosphate buffered saline For
reduction with sodium borohydride (NaBH4), plasma
sample (500 lL) in a Pyrex test tube with a Teflon-lined
screw cap was diluted with 3.0 mL of 0.1Msodium borate
buffer (pH 9.0) followed by the addition of NaBH4(25 mg,
0.66 mmol) For reduction with sodium cyanoborohydride
(NaBH3CN), plasma sample (500 lL) in a Pyrex test tube
with a Teflon-lined screw cap was diluted with 3.0 mL of
0.1M sodium phosphate buffer (pH 6.0) followed by the
addition of NaBH3CN (50 mg, 0.80 mmol) The mixture
was incubated for 24 h at 37C with shaking in the dark
After incubation, each plasma sample was treated with
5 mL of acetonitrile The mixture was centrifuged at 2000 g
for 30 min, and the resulting pellet of precipitated protein
was separated The pellet was washed with 5 mL of
acetonitrile and 2 mL of cold 5% (w/v) trichloroacetic acid
Then the resulting protein was hydrolyzed for RP-HPLC
analysis as described above
Incubation of BSA with carbohydrates
General procedure.Reaction mixtures (2.0 mL) in a Pyrex
test tube with a Teflon-lined screw cap contained
10.0 mgÆmL)1BSA, 5 lMCuSO4, each carbohydrate, and
50 mMsodium phosphate buffer (pH 7.4) In some
experi-ments, we added dimethylsulfoxide (50 mM) or catalase
(200 UÆmL)1) to the mixture Carbohydrate concentrations
are given in the respective legends to figures and tables The
reaction mixtures containing 10 lL of toluene were
incu-bated at 37C 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 N2 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 37C 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
R E S U L T S
Detection of a-aminoadipic-d-semialdehyde
in rat plasma protein Rat plasma protein from STZ-induced diabetic and control subjects was investigated for the presence of a-aminoadipic-d-semialdehyde Protein hydrolysis with 6MHCl contain-ing 3% phenol converts a-aminoadipic-d-semialdehyde to a bisphenol derivative (ACPP) which is a condensation product of one a-aminoadipic-d-semialdehyde residue and two phenol molecules by Baeyer’s reaction [33,34] After hydrolysis, ACPP was measured by RP-HPLC using a linear gradient solvent system Figure 1 shows the HPLC chromatograms of the ACPP standard and the hydrolysate
of diabetic rat plasma protein followed by detection at
278 nm with a diode array detector ACPP was eluted in a
tRof 15.4 min (Fig 1A) and observed in the hydrolyzate of diabetic rat plasma protein (Fig 1B) We identified the peak
as ACPP either by cochromatography with an authentic standard or by comparing UV spectra with the standard using a diode array detector (data not shown) The concentration of a-aminoadipic-d-semialdehyde in rat plasma protein from STZ-induced diabetic and control subjects was determined using the RP-HPLC analytical
Fig 1 Determination of a-aminoadipic-d-semialdehyde in diabetic rat plasma protein by RP-HPLC After the protein was hydrolyzed with
6 M HCl containing 3% phenol at 110 C for 48 h, ACPP, a bisphenol derivative of a-aminoadipic-d-semialdehyde, was measured by RP-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.
Trang 4procedure (Fig 2) The mean ± SD of
a-aminoadipic-d-semialdehyde concentration was 3.21 ± 0.88 nmolÆmg)1
protein in diabetic (n¼ 7) and 0.99 ± 0.29 nmolÆmg)1
protein in control subjects (n¼ 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 NaBH4-reduced
plasma protein (B) The ACPP peak is abolished by the
reduction with NaBH4 On the other hand, reduction of
plasma protein with NaBH3CN, 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
(Fig 3C) This result indicates that the
a-aminoadipic-d-semialdehyde residue exists primarily as the free aldehyde
in vivo
Oxidative deamination of lysine residue
in BSA by carbohydrates
The oxidative deamination of the e-amino groups of lysine
residue via the Maillard reaction was assessed by the
reaction of BSA with glucose BSA (10.0 mgÆmL)1) was
incubated with 100 mM glucose in 50 mM sodium
phos-phate buffer (pH 7.4) in the presence and absence of 5 lM
Cu2+at 37C After glucose-incubated BSA was
hydro-lyzed with 6M HCl containing 3% phenol, ACPP was measured by RP-HPLC Figure 4A shows the formation of a-aminoadipic-d-semialdehyde with the incubation of BSA with glucose Native BSA also contained 0.06 nmolÆmg)1 protein of a-aminoadipic-d-semialdehyde but the content remained constant during the incubation without carbohy-drates The incubation with glucose in the absence of Cu2+ did not increase a-aminoadipic-d-semialdehyde content (Fig 4A) As shown in Fig 4A, in the presence of 5 lM
Cu2+, 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 Cu2+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/Cu2+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 ) of M G and
Fig 3 Selective reduction of a-aminoadipic-d-semialdehyde residue in plasma protein Plasma protein was reduced with NaBH 4 or NaBH 3 CN as described in the Experimental procedures section After the protein was hydrolyzed with 6 M HCl containing 3% phenol at
110 C for 48 h, ACPP was measured by RP-HPLC with detection at
278 nm (A) Intact plasma protein (B) NaBH 4 -reduced plasma pro-tein (C) NaBH 3 CN-reduced plasma protein.
Fig 2 a-Aminoadipic-d-semialdehyde levels in rat plasma protein
de-rived from STZ-induced diabetic and control subjects After plasma
protein was hydrolyzed with 6 M HCl containing 3% phenol at 110 C
for 48 h, ACPP, a bisphenol derivative of
a-aminoadipic-d-semialde-hyde, was measured by RP-HPLC as described in the Experimental
Procedures section Mean ± SD was 3.21 ± 0.88 and 0.99 ±
0.29 nmolÆmg)1 protein in STZ-induced diabetic and controls,
respectively; P < 0.0001 by Student’s t-test.
Trang 53-DG but not GO Figure 4B,C shows the formation of
a-aminoadipic-d-semialdehyde in BSA by MG and 3-DG,
respectively, with the advance of time BSA (10 mgÆmL)1)
was incubated in 50 mMphosphate buffer with 1.0 mMof
each a-dicarbonyl under a physiological pH and
tempera-ture (pH 7.4, 37C) As shown in Fig 4B, in the presence
of Cu2+, a significant amount of aldehyde was produced by the reaction with 3-DG but not in the absence of Cu2+ M G oxidatively deaminated the lysine residue in the presence and absence of Cu2+(Fig 4C) The oxidation was appar-ently stimulated by the addition of Cu2+
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 Cu2+under nitrogen atmosphere Indeed, the reaction under a nitrogen atmosphere almost completely inhibited the oxidative deamination by glucose, clearly illustrating the involvement
of oxygen in the oxidative deamination (Table 2) In addition, the deoxygenation caused a significant decrease
in the production of a-aminoadipic-d-semialdehyde by 3-DG and MG
Further, we investigated the participation of reactive oxygen species in the reaction BSA and Cu2+ were incubated with glucose, 3-DG, and M G in the presence of catalase and dimethylsulfoxide, which is a hydroxyl radical scavenger As shown in Table 2, addition of catalase (100 UÆmL)1) markedly inhibited the formation of a-aminoadipic-d-semialdehyde by glucose and significantly inhibited it by 3-DG and MG The oxidation of BSA by glucose, 3-DG, and MG was also significantly inhibited in the presence of 50 mM dimethylsulfoxide These results suggest that the hydroxyl radical is produced by the Maillard reaction and is responsible for the a-aminoadi-pic-d-semialdehyde formation
D I S C U S S I O N
In the present study, we identified the a-aminoadipic-d-semialdehyde residue in rat plasma protein and demon-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 M G (C) in 50 m M phosphate buffer
(pH 7.4) in the presence or absence of 5 l M Cu2+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) was incubated with 5 l M Cu 2+ and each indicated carbohydrate in 50 m M sodium phosphate buffer (pH 7.4) at 37 C for 3 weeks a-Aminoadipic-d-semialdehyde was quantitated by RP-HPLC as described in Experi-mental procedures.
Carbohydrate
Concentration (m M )
a-Aminoadipic-d-semialdehyde (nmolÆmg protein)1)
L -Ascorbic acid 50 2.04 3-Deoxyglucosone 1 0.26
Trang 6that in normal rat plasma Analysis of selectively reduced
plasma protein suggested that the
a-aminoadipic-d-semial-dehyde residue exists primarily as the free ala-aminoadipic-d-semial-dehyde 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
Cu2+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
Cu2+ 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 mechanmechan-ism 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
products or Schiff base adduct by metal ion-catalysis [12]
Subsequently, the resulting MG and 3-DG could condense
with protein lysine residues to form the Schiff base adduct,
an iminoketone (I) Then the e-proton of the lysine moiety
would be abstracted by basic media, and the enolization
might give an iminoenaminol (II) In the electron transfer
process, it is assumed that Cu2+serves as the electron-pair
acceptor and stabilized II through the formation of a
coordination complex because the requirement of Cu2+was
observed in model studies Finally, spontaneous hydrolysis
of II can lead to the release of an enaminol (III) and
the formation of a-aminoadipic-d-semialdehyde (IV) The
mechanism of the Strecker-type reaction is not accompanied
by decarboxylation and is consistent with the
o-quinone-mediated mechanism previously proposed [23] Although
the incubation of BSA with GO produced a very small
amount of a-aminoadipic-d-semialdehyde, GO may
prefer-entially form stable inter- and intramolecular cross-links or
carboxymethyllysine during the incubation with BSA
because of its high reactivity [38,39] Another possible
pathway of a-aminoadipic-d-semialdehyde formation via
the Maillard reaction is reactive oxygen species-mediated
oxidative deamination The fact that the aerobic glycation
of proteins in the presence of transition metals is
accom-panied by radical-generating reactions supports this possi-bility [27] Firstly, protein-bound products of the Amadori reaction may subsequently degrade, in a transition metal-catalyzed process, to yield H2O2, 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 incubated with 5 l M Cu2+and each of indicated carbohydrate in 50 m M sodium phosphate buffer (pH 7.4) at 37 C for 3 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)1protein of a-aminoadipic-d-semialdehyde.
a-Aminoadipic-d-semialdehyde (nmolÆmg protein)1)
a Incubation under N 2 atmosphere.
Fig 5 Proposed mechanism of oxidative deamination of lysine residue
by the Strecker-type reaction.
Trang 7oxidants by the oxidation of glucose-protein adducts has
been termed glycoxidation Furthermore, 3-DG, MG, and
GO also produce superoxides during the reaction with
lysine and arginine [29] In this study, catalase and
dimethylsulfoxide significantly inhibited the oxidative
deamination by glucose, 3-DG, and MG, indicating the
participation of hydroxyl radicals Dependency of the
oxidative deamination on both oxygen and Cu2+ is also
consistent with a metal ion-catalyzed mechanism for the
production of hydroxyl radicals, probably through the
intermediary of superoxide and H2O2 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 H2O2and 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 In fact,
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
modification in vivo Therefore, the Strecker-type oxidative
deamination may contribute to the formation of
a-amino-adipic-d-semialdehyde in diabetes Oxidative stress has the
potential for causing gross oxidative damage to biological
molecules, which is mainly induced by reactive oxygen
species, and is apparently responsible for diabetic
complica-tions [40] Diabetes is associated with increased free radical
formation and malondialdehyde, which is the most
exten-sively studied marker in lipid peroxidation Recently,
a-aminoadipic-d-semialdehyde is thought to be a biomarker
of oxidative damage to proteins in vivo because
a-aminoad-ipic-d-semialdehyde originates from the lysine residue by the
attack of oxygen-derived free radicals [24,26] When
oxida-tive stress is induced in rats by treatment with tert-butyl
hydroperoxide, which induces oxidative stress as a
conse-quence of the production of reactive oxygen species, this
aldehyde is found to be significantly higher compared with
control rats [24] Furthermore, the content of
a-aminoadipic-d-semialdehyde in plasma proteins shows a positive
corre-lation with the rat’s age Therefore, the formation of oxygen
free radicals during the Maillard reaction is also a possible
source of the formation of a-aminoadipic-d-semialdehyde in
diabetes It has been observed that blood Cu2+levels are
higher than normal in diabetic individuals, although it is not
clear whether this is caused by an increase in ceruloplasmin
or an increase in the pool of copper associated with albumin
or low molecular weight chelates [27] In diabetes, copper
may play a significant role in the oxidation of lysine residues
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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