(Received for publication, July 13, 1995; and in revised form, September 27, 1995)
From the
The formation of -dicarbonyl compounds seems to be an
important step for cross-linking proteins in the glycation or Maillard
reaction. To elucidate the mechanism for the cross-linking reaction, we
studied the reaction between a three-carbon
-dicarbonyl compound,
methylglyoxal, and amino acids. Our results showed that this reaction
generated yellow fluorescent products as formed in some glycated
proteins. In addition, three types of free radical species were also
produced, and their structures were determined by EPR spectroscopy.
These free radicals are 1) the cross-linked radical cation, 2) the
methylglyoxal radical anion as the counterion, and 3) the superoxide
radical anion produced only in the presence of oxygen. The generation
of the cross-linked radical cations and the methylglyoxal radical
anions does not require metal ions or oxygens. These results indicate
that dicarbonyl compounds cross-link free amino groups of protein by
forming Schiff bases, which donate electrons directly to dicarbonyl
compounds to form the cross-linked radical cations and the
methylglyoxal radical anions. Oxygen can accept an electron from the
radical anion to generate a superoxide radical anion, which can
initiate damaging chain reactions. Time course studies suggest that the
cross-linked radical cation is a precursor of yellow fluorescent
glycation end products.
Free amino groups of proteins react slowly with reducing sugars
such as glucose by the glycation or Maillard reaction to form poorly
characterized brown fluorescent compounds(1) . This process is
initiated by the condensation reaction of reducing sugars with free
amino groups to form Schiff bases (Fig. 1A), which
undergo rearrangement to form the relatively stable Amadori
products(2, 3) . The Amadori products subsequently
degrade into -dicarbonyl compounds,
deoxyglucosones(4, 5) . These compounds are more
reactive than the parent sugars with respect to their ability to react
with amino groups of proteins to form cross-links, stable end products
called advanced Maillard products or advanced glycation end products
(AGEs)(
)(6) . AGEs are irreversibly formed and found
to accumulate with aging, atherosclerosis, and diabetes mellitus,
especially associated with long-lived proteins such as
collagens(7, 8) , lens
crystallines(9, 10) , and nerve
proteins(11, 12) . It was suggested that the formation
of AGEs not only modifies protein properties, but also induces
biological damage in
vivo(13, 14, 15, 16, 17, 18) .
For example, AGEs deposited in the arterial wall could themselves
generate free radicals capable of oxidizing vascular wall lipids and
accelerate atherogenesis in hyperglycemic diabetic
patients(17, 18) .
Figure 1:
A, general scheme of the glycation or
Maillard reaction; B, several advanced glycation end products
or Maillard products: pentosidine (structure a), pyrrole
derivatives (structure b), and pyrazine derivatives (structure c). CML, N-carboxymethyllysine.
The molecular structures of some
AGEs have been identified as pentosidines (Fig. 1B, structure
a)(19, 20, 21, 22) , pyrrole
derivatives (structure b)(23) , pyrazine derivatives (structure
c)(24, 25, 26, 27) , and N-carboxymethyllysine (Fig. 1A, CML)(28, 29, 30, 31, 32) .
In the presence of molecular oxygen, the formation of these products
from sugars is catalyzed by transition metal ions via glycoxidation,
which oxidizes Amadori products to N
-carboxymethyllysine (28, 29) , and the autoxidation of glucose, which
produces superoxide radical anions (O
),
H
O
, and
-ketoaldehydes (33, 34, 35, 36, 37) . The
major pathways of glycation reaction-mediated damage to macromolecules
therefore involve both nonoxidative and oxidative processes. Their
individual contributions to biological damage, however, are not well
understood.
The formation of -dicarbonyl compounds seems to be
an essential step for the cross-linking reaction, which leads to the
formation of AGEs. To elucidate the mechanism for the cross-linking
reaction (Fig. 1A, reaction 5), we studied the
reaction between three-carbon
-dicarbonyl methylglyoxals and L-alanines. Our results showed that the yellow fluorescent
products, formed in some glycated proteins, were generated by this
reaction. In addition, we found that three types of free radical
species were generated, and their structures were identified by EPR
spectroscopy and other methods. These radicals are a cross-linked
radical cation (the methylglyoxal dialkylimine radical cation or its
protonated cation), the methylglyoxal radical anion, and the superoxide
radical anion (which formed in the presence of oxygen molecules). The
generation of the cross-linked radical cation and the methylglyoxal
radical anion does not require metal ions or oxygens, suggesting that
they are formed by a direct 1-electron transfer process.
The generation of
O in the aerobic reaction mixture was
determined by NBT reduction and inhibition of its reduction by
superoxide dismutase(18, 38, 39) . A 1-ml
reaction mixture, which initially contained equal concentrations of
methylglyoxal and alanine, was added at 30 s after initiation of the
reaction to a 2-ml aliquot of 0.25 mM NBT in 100 mM
carbonate buffer (pH 9.5). The reduction rate was determined as the
increase in absorbance at 540 nm for 10 min at 30 °C. Electronic
absorption and fluorescence spectra were obtained with a diode array
spectrometer (Hewlett-Packard 8452A) and an LS-100 fluorescence
spectrometer (Photon Technology International), respectively.
Figure 2:
EPR spectra obtained from the reaction
mixture containing methylglyoxal (0.2 M) and various
isotope-enriched L-alanines (0.2 M) in carbonate
buffer (0.5 M) at pH 9.5 (upper spectra) and
simulated spectra (lower spectra). A, methylglyoxal (MG) and natural-abundance L-alanine; B,
methylglyoxal and L-[N]alanine; C, methylglyoxal and L-[2-
C]alanine. The hyperfine coupling
constants used for the simulation are listed under
``Results.'' The spectrometer settings for the spectral
acquisition are described under ``Experimental
Procedures.''
The
spectrum in Fig. 2C and the spectra in Fig. 3(B and C) were obtained with the
reaction mixture containing L-[2-C]alanine, L-[1-
C]alanine, and L-[3-
C]alanine in place of
natural-abundance alanine, respectively. They exhibited extra hyperfine
interactions due to
C (I = 1/2) nuclei: two
2-
C (
-carbons) with A
Figure 3:
EPR spectra obtained from the reaction
mixture containing methylglyoxal (0.2 M) and various
isotope-enriched L-alanines (0.2 M) in carbonate
buffer (0.5 M) at pH 9.5 (upper spectra) and
simulated spectra (lower spectra). A, methylglyoxal (MG) and natural-abundance L-alanine; B,
methylglyoxal and L-[1-C]alanine; C, methylglyoxal and L-[3-
C]alanine. The hyperfine coupling
constants used for the simulation are listed under
``Results.'' The spectrometer settings for the spectral
acquisition are described under ``Experimental
Procedures.''
Although we have obtained a large number of hfc
constants from this cross-linked radical, exact structural assignment
for this radical is difficult because of asymmetry in hfc constants.
Two equivocal structures are shown in Fig. 4(structures a-1 and a-2). In the case of structure a-1, the asymmetric
nature of the spin distribution may be caused by the effect of the
methyl group of methylglyoxal on the singly occupied molecular orbital,
most likely the -orbital, which includes two N=C bonds. On
the basis of experimental hfc constants, structure a-1` is assigned to
the conformations of two alkyl groups with respect to the p-orbitals of the two nitrogen atoms. In this conformation,
carboxyl,
-, and methyl carbons will have one small and one large
C hfc constant in each carbon group because of the
different dihedral angles to the p-orbitals of the nitrogen
atoms (one carbon is close to
= 90°, and the other is
closer to 0°) and the cos
dependence of these hfc
constants. The assignment of
C hfc constants to individual
carbons will be as follows: for the N-1 side, 8.52 G for
1-
C, 4.10 G for 2-
C, and 0.3 G for
3-
C; and for the N-2 side, 0.3 G for 1-
C, 0.2
G for 2-
C, and 3.0 G for 3-
C. This assignment
gives a ratio of 3.7 for the total carbon spin densities between the
N-1 and N-2 sides. This value is closest, among several possible
assignments, to the value of 3.2, the ratio of spin densities on N-1
and N-2 atoms (A
/A
). The other possible
structure of this radical is shown in Fig. 4(structure
a-2). A protonation of a nitrogen in the cross-linked Schiff base
will produce a triene-type compound, which may lose an electron to form
the cross-linked radical cation. In this structure, the observed large
C hfc constants will originate entirely from one alanine
molecule in the cross-linked radical. In addition, we expect to detect
two sets of methyl hydrogen hfc constants if the radical has this
structure, in contrast to the experimental observation of only one set
of methyl hydrogen hfc constants. It may be possible, however, that
A
(3) of one set is smaller than the line width, which may
arise from the canceling effects of spin delocalization
(hyperconjugation) and spin polarization in the spin transfer to the s-orbitals of the methyl hydrogens from the delocalized
-center. Although we prefer structure a-1 as the structure of the
cross-linked radical, structure a-2 cannot be ruled out at this time.
It is certain, however, that the radical formed due to the
cross-linking reaction contains two amino acids and one methylglyoxal.
Figure 4: Chemical structures of the cross-linked radical cation and the radical anion observed in this reaction and identified by EPR.
To find whether the Schiff base is the precursor of this radical,
the base was reduced with NaCNBH, which is known to reduce
Schiff bases selectively and to inhibit the subsequent reactions. When
NaCNBH
(1.0 M) was added to the reaction mixture,
the EPR signal of the cross-linked radical and the yellow color were
not detected. The effect of NaCNBH
may indicate that
methylglyoxal dialkylimine,
O
C(CH
)-
HCN=C(CH
)HC=NCH(CH
)CO
,
is the intermediate for the formation of this cross-linked radical.
Figure 5:
EPR spectra obtained from the reaction
mixture containing methylglyoxal (0.2 M) and various
isotope-enriched L-alanines (0.2 M) in carbonate
buffer (0.5 M) at pH 9.5. Spectrum A, spectrum of
methylglyoxal and natural-abundance L-alanine recorded at 5
min after starting the reaction. Spectrum B, spectrum of
methylglyoxal and 2,3,3,3-D-alanine in carbonate
buffer solution prepared in D
O. The spectrum was recorded
at 5 min after initiating the reaction. The resonance lines marked by asterisks belong to another species as explained under
``Results.'' Spectrum C, spectrum recorded at 15 min
with the same sample used for spectrum B. Spectrum D,
simulated spectrum obtained by using the hyperfine coupling constants
listed under ``Results.''
Figure 6:
Effects of oxygen (A) and
transition metal ions (B) on the amplitude of the EPR signal
of the cross-linked radical cation. All samples contain 0.2 M methylglyoxal and 0.2 ML-alanine in 0.5 M carbonate buffer at pH 9.5. Additional treatments were as
follows: A, aerobic conditions () and anaerobic
conditions (
); B, no addition (
), 0.1 mM
FeCl
(
), and 1 mM DTPA (
). Reactions
were started by injecting methylglyoxal. arb. unit, arbitrary
unit.
These results together indicate that the cross-linked radical cation and the methylglyoxal radical anion are generated from the direct electron transfer between methylglyoxal (MG) and a Schiff base, probably methylglyoxal dialkylimine (MGDI) or its protonated species, as shown in .
On-line formulae not verified for accuracy
Previous investigations of the reactions of methylglyoxal with protein or methylamine also suggested that a condensation product, which was not well characterized, served as the electron donor and that methylglyoxal acted as an electron acceptor (41, 42, 43) .
Figure 7: Generation of the superoxide radical anion in the methylglyoxal and alanine reaction. A, reduction rates of NBT were measured by increasing concentrations of reaction products. The reaction products (1 ml) were added to 2 ml of 0.25 mM NBT in 100 mM carbonate buffer at pH 9.5. The absorbance changes were monitored at 540 nm for 10 min at 30 °C. B, the effect of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) on the reduction rate of NBT is shown. The concentrations of methylglyoxal and alanine were both 40 mM.
These results demonstrate that
although the initiation of the cross-linking reaction does not require
molecular oxygen, the reaction products generate superoxide radical
anions in the presence of oxygen. The methylglyoxal radical anion is
most likely responsible for O generation by its electron transfer reaction to oxygen via .
On-line formulae not verified for accuracy
This result is consistent with the observation that the EPR signal amplitude of the methylglyoxal anion was higher under anaerobic conditions than under aerobic conditions.
Figure 8: Optical absorbance and cross-linked radical cation formation. A: the reaction mixtures contained 40 mM alanine and 40 mM methylglyoxal in 0.5 M carbonate buffer at pH 9.5. The spectra were recorded at 3-min intervals. The arrows indicate the direction of absorbance change with time. B: curve a, the absorbances at 334 nm were obtained with diluents of 0.2 M methylglyoxal and 0.2 M alanine in 0.5 M carbonate buffer at pH 9.5. A 10-µl portion of reaction mixture was diluted with 1 ml of 3 M HCl. Curve b, the signal amplitude of EPR spectra is plotted. The EPR sample contained 0.2 M methylglyoxal and 0.2 M alanine in 0.5 M carbonate buffer at pH 9.5. arb. unit, arbitrary unit.
The late stage of the glycation reaction between
deoxyglucosones and free amino groups of proteins shown in Fig. 1A (step 5) was studied using a model
system of methylglyoxal and L-alanine. The results are
consistent with the reaction scheme shown in Fig. 9. We detected
three free radical species: the cross-linked methylglyoxal dialkylimine
radical cation ( Fig. 9(structure a) and 4 (structure a-1)) or its protonated species (Fig. 4, structure a-2), the enediol radical anion of methylglyoxal (Fig. 9, structure b), and the superoxide radical anion (Fig. 9, structure c). The addition of NaCNBH inhibited the formation of the cross-linked radical and the
yellow color. This result indicates that the formation of the Schiff
bases is an essential step (Fig. 9, steps i and ii) for cross-linking. Furthermore, transition metal ions and
oxygen were not required for the generation of the cross-linked radical
cations or the methylglyoxal radical anions (Fig. 6, A and B). These results together suggest that a direct
1-electron transfer between a Schiff base methylglyoxal dialkylimine
(or its protonated form) and methylglyoxal is responsible for the
generation of the cross-linked radical cation and the radical
counteranion of methylglyoxal (Fig. 9, step iii). Under
aerobic conditions, molecular oxygen can then accept an electron from
the methylglyoxal anion to generate the superoxide radical anion (Fig. 9, step iv). The time course studies (Fig. 8) indicate that the cross-linked radical cations are
precursors of the yellow end products.
Figure 9: Partial reaction scheme between methylglyoxal (MG) and amino acids (AA).
Previous investigations have shown that N,N`-dialkylpyrazine radical cations are formed by the addition of ascorbate in the reaction mixture containing glucose and amine or amino acid in water at 80 °C(24, 25, 26, 27) . The intermediate for the free radical was glyoxal dialkylimine, which was formed immediately after glycosylamine formation, prior to Amadori rearrangement. It was proposed that the formation of the dialkylpyrazine radical cation from the dialkylimine was via the reverse aldol condensation reaction, which requires acid hydrolysis followed by reduction(26, 27) . In the absence of reducing agents, two-carbon and three-carbon fragments in the form of glyoxal and methylglyoxal were formed from glucose, depending on where the fragmentation occurred(26) . It appears, on the basis of our results, that methylglyoxal, once formed, is able to act as a reducing agent as well as a cross-linker under mild conditions. In addition, the structure of the cross-linked radical cation is different from that obtained with glyoxal.
Glycation is believed to be
modulated by oxidative stress. Wolff and co-workers (34, 35, 36, 37) demonstrated that
reducing sugars can undergo oxidation in the presence of oxygen and
transition metal ions, which generates HO
,
oxygen radicals, and
-ketoaldehydes. This reaction leads to
protein browning, conformational changes, and fragmentation. In
addition, the formation of N
-carboxymethyllysine (Fig. 1, step
4) and pentosidine also requires transition metal ions and
oxygen(22, 28, 29) . Therefore, AGEs in
vivo are products of the combined processes of glycation and
oxidative modification. Our results also suggest that the formation of
-ketoaldehydes or deoxyglucosones is a critical step that leads to
protein cross-linking, formation of radical cation sites on the
cross-linked proteins, and generation of radical counteranions.
O
and H
O
generated from radical counteranions can initiate free radical chain
reactions including lipid peroxidation. The cross-linked radical
cations, which have an extensively delocalized unpaired electron, are
quite stable. These radical sites in cross-linked proteins may be more
persistent and could be a reactive site for putative reducing (A) and
oxidizing (B) molecules, which produce free radicals for a long
duration. One of many possible reaction schemes could be as shown in .
On-line formulae not verified for accuracy
This kind of reaction by long-lived glycated protein may contribute to the increased peroxidation of lipids when glycated protein was added in vitro and may also contribute to accelerating oxidative modification of vascular wall lipid in diabetes and atherosclerosis.