From the
The role of glyoxal and glycolaldehyde in protein cross-linking
and N
The reaction between reducing sugars and amino structures in
amino acids or proteins (also called Maillard reaction) has been shown
to proceed in living systems and to correlate with age and severity of
diabetes
(1) . Unlike organic syntheses, it does not result in
one or few well defined products but proceeds through complex reaction
pathways resulting in a large number of structures
(2) . After
the initial formation of a Schiff base adduct between the carbonyl and
the amine moeity, the aldimine rearranges to a more stable ketoamine or
Amadori product. Enolization, dehydration, cyclization, fragmentation,
and oxidation reactions form reactive intermediates that ultimately
lead to stable end products.
Although enormous effort was devoted to
elucidate the nature of these protein modifications, so far only two
structures besides the Amadori product have been successfully
established in vivo, pentosidine and
N
The slow progress in elucidating
the structure of the major Maillard cross-link in proteins together
with the finding of a C-2-glucose fragmentation product in form of CML,
suggested to us that the pathway proposed by Namiki
(9) may
play an important role in CML formation and cross-linking. In this
pathway, glyoxal and glycolaldehyde were detected as Maillard
fragmentation products in heated model systems of sugars and
alkylamines
(10, 11) . According to Namiki, the Schiff
base adduct 1 undergoes retro aldol condensation at
C-2-C-3 to yield fragment 2 (Fig. 1). Compound
2 is the Schiff base adduct of the amine with glycolaldehyde,
which on one hand could hydrolyze to release glycolaldehyde or
rearrange to form aldoamine 3. Condensation with a second
molecule 3 was proposed to result in pyrazine 4,
which has been shown to be easily oxidized and fragmented to give, for
example, di-Schiff base adduct 5 or to release glyoxal
(12) . Condensation with a second amine compound would yield
imine 7. Thus, the formation of glyoxal and glycolaldehyde in
this pathway are linked together, and both, upon reaction with amines,
would result in common structures. Both molecules are highly reactive
intermediates and have been reported to modify and cross-link proteins,
although structures were only proposed
(13, 14, 15) .
In the first part of this study,
we investigated the relationship between glyoxal/glycolaldehyde and
presumed C-2-imine cross-links 5/ 7 in proteins
incubated with these carbonyl compounds and various sugars. In the
second part, we studied the mechanism of CML formation from these
reagents. In addition, we evaluated the contribution of glyoxal
versus oxidative cleavage of the Amadori product on CML
formation and the relative importance of the ``Namiki
pathway'' during incubations with reducing sugars.
Reagents Reagents of highest quality available were obtained from Sigma, Fisher,
and Aldrich, unless otherwise indicated. Spectra
N
N
For GC/MS analysis of amino acids, proper
fractions were collected from HPLC without postcolumn derivatization
(HPLC system 5 for 6, HPLC system 6 for CML
and 9, 9`, 9"), solvents were removed to
absolute dryness, and material obtained was derivatized as
trifluoroacetylmethylester derivatives. First, up to 10 mg of
material was dissolved in 1 ml of 0.1 M thionylchloride
(Fluka) solution in anhydrous methanol and heated for 1 h at 110
°C. Solvents were evaporated to absolute dryness. The residue was
taken up in 300 µl of pyridine and 100 µl of trifluoroacetic
anhydride (Aldrich), and the reaction mixture was heated for 10 min at
65 °C. After complete removal of solvents, the residue was
dissolved in ethyl acetate and passed over a short silica gel column
(eluent ethyl acetate). Proper fractions were combined and subjected to
GC/MS.
3-Amino-1,2,4-triazine was extracted from incubations
containing aminoguanidine with ethyl acetate. Solvents were evaporated
after drying the combined organic layers over anhydrous sodium sulfate.
The residue was derivatized using a 1:1 (v/v) ratio of pyridine and
N, O-bistrimethyl silylacetamide (Fluka), and the
reaction mixture was analyzed with GC/MS.
Amadori product
concentration in incubations with
N
Because of the instability of the C-2-imines 5 and
7 (Fig. 1) during acid hydrolysis, their common reduced
form 6, 2,15-diamino-7,10-diaza-hexadecane-1,16-dioic acid,
was synthesized from N
Structure 6 was found to be stable under the
conditions used for acid hydrolysis of proteins. To confirm the nature
of 6 in hydrolysates, two different HPLC systems with
o-phthaldialdehyde-postcolumn derivatization and fluorescence
detection were developed, and the absence of the corresponding peak in
nonreduced samples was confirmed throughout all investigations.
Additionally, in selected cases, the material was collected by HPLC,
derivatized, and applied to GC/MS.
In BSA incubations, the formation
of diimine 5 monitored after reduction to 6 was
dependent on the glyoxal concentration and began to level off above
10-20 mM (Fig. 2 A). We chose 20
mM to investigate cross-link structures 5 and 7 in BSA incubated with glyoxal or glycolaldehyde, respectively.
Time course experiments ranging from 5 min to 170 h showed that the
formation quickly reached a maximum at about 0.5 h followed by
degradation (Fig. 2 B). Remarkably, in the case of
glycolaldehyde, the initial peak was much smaller, but the steady level
reached later was higher than with glyoxal. These findings were
correlated with the rate of cross-linking using SDS-polyacrylamide gel
electrophoresis after dialysis of the reduced and unreduced reaction
mixtures (Fig. 2 B, inset). Reduced and
unreduced samples showed no visible difference (data not shown).
Cross-linking increased with time. With glyoxal, the trimer became
faintly visible at 20 mM, and more intramolecular
modifications could be observed in contrast to glycolaldehyde that had
significant intermolecular reactions.
Additional
experiments with 5 mM aminoguanidine as a trapping reagent
specific for
The
data so far implicate unequivocally glyoxal and glycolaldehyde in CML
formation and raise the question of the mechanisms by which CML forms
from higher sugars, as demonstrated in and by others
(19, 27) . In our own experiments (), CML
formation rates in BSA incubations generally followed the anomerization
rates of the sugar except for D-ribose, which was a potent
generator of CML.
The key question concerning CML synthesis from
higher sugars is whether formation rates are primarily dependent on
sugar autoxidation or Schiff base fragmentation as proposed by Wolff
(28) and Namiki
(10) , respectively, or whether the major
source stems from oxidative breakdown of the Amadori product of the
sugar
(19) . Clarification of this question is problematic since
all molecules are simultaneously present in sugar-amine incubation
systems.
As previously reported
(18) , CML formation from
glucose and N
Investigations into
the origin of carbon atoms involved in CML formation utilizing C-1 and
C-6 radiolabeled sugars revealed surprising findings. By comparison of
CML measured by HPLC using radioactivity with total CML using
postcolumn derivatization 74% (5 days)/68% (10 days) of CML was
attributed to the C-1 and 29%/31% to the C-6 portion of
N-(1-deoxyfructos-1-yl)propylamine. Starting the incubation
with glucose only 38% (5 days)/32% (10 days) for the C-1 and 22%/36%
for the C-6 region were recovered, suggesting thereby that about 30% of
CML is stemming from the C-2-C-5 region of the original carbon
backbone.
The purpose of this study was to clarify the mechanism of
formation of structures incorporating a C-2 fragment of the original
sugar during the Maillard reaction of reducing sugars and proteins
in vivo. Clarification of this question is important because
the major advanced Maillard product found in vivo so far is
CML. Its levels increase with age in human skin
(29) and in the
presence of diabetes, and the levels can be correlated with severity of
diabetic complications
(30) . In addition, CML was detected in
human lens, where it is thought to originate from ascorbate
(4, 27) . Previous mechanistic studies by Dunn et
al. (19) suggested that CML forms from the Amadori product
of glucose or threose through metal-catalyzed oxidative fragmentation.
The data in this study show unequivocally that, under physiological
conditions at 37 °C and pH 7.4, glyoxal and glycolaldehyde are
immediate precursors in the formation of CML and that they are involved
in formation of C-2-imine cross-link 5, 7 (Fig. 1) and other more important, but yet unknown,
structures. Formation of CML and 5/ 7 during Maillard
reaction from higher sugars like glucose is much more complex because
all three potential sources, i.e. the free sugar, the Schiff
base adduct, and the Amadori product, are present together as soon as
the reaction proceeds. Additionally, our findings raise questions of
the importance of glyoxal and glycolaldehyde in the reaction pathways
leading to CML and cross-linking.
When the reaction was initiated
from the Amadori product of glucose, very large quantities of CML were
formed that were almost totally stalled by deaerated conditions
(Fig. 7 B). The fact, that aminoguanidine had no
suppressive effect until 100 h strongly favors a mechanism involving
direct oxidative cleavage of the Amadori product. In contrast, when
starting the reaction from glucose in the presence of
N
The key question therefore is what
is the major source of glyoxal/glycoladehyde production leading to CML.
Fig. 7B shows at 170 h that only 15% of total CML
formation from the Amadori product can be attributed to C-2
intermediates. Indeed, the amount of glyoxal-triazine generated in the
presence of 42 mM Amadori product was only 2.8 mmol/mol of
initial sugar concentration. Thus, less than 0.3% of the original
Amadori product was transformed into glyoxal (). As the
Amadori product concentration in a
glucose/ N
Thus, in an
incubation system starting from glucose in the presence of
amines, the major pathway leading to glyoxal is not autoxidation of
glucose or degradation of the Amadori product but degradation of an
amine-assisted oxidative step prior to formation of the Amadori
product, presumably the Schiff base adduct (Fig. 8).
Incubations conducted in the presence of boric acid support the
findings with aminoguanidine. Boric acid forms a stable chelating
complex with the coplanar cis diol groups of the furanoic structure of
1-deoxyfructos-1-yl-amines
(31) and thus totally blocks CML
formation from Amadori products via oxidative cleavage and
glyoxal/glycolaldehyde (Fig. 7 B). Based on the
experiments with aminoguanidine, this should result in a decrease of
57% CML synthesis in glucoseamine systems. The actual decrease of 37%
was found to be smaller and is probably due to accelerated CML
synthesis linked to increased saltconcentrations
(18) .
Since
aminoguanidine itself is an amine compound, it could compete with the
target amine of the system used and contribute to the generation of
reactive intermediates. The possibility of generating
glyoxal/glycolaldehyde solely by this reaction pathway was discounted
based on the detection of CML in incubations of the Amadori product of
propylamine and glucose with
N
Other independent evidence for glyoxal and
glycolaldehyde generation from higher sugars was gained from the
abundance of imine-intermediates 2 and 3 as
glycolaldehyde and 8 as glyoxal derivatives (Figs. 1 and 6) in
incubation mixtures with proteins. After reduction, 9 as their
common product was detected in incubations with glucose and in much
higher levels also with ribose. Use of a deuterated reduction reagent
made it possible to distinguish between the
N
Based on these considerations, and the finding that Amadori products
are present in significant concentrations in tissues, the source of CML
formation in vivo is expected to follow the sequence of
precursor reactivity: Amadori product (via oxidative cleavage) >
Schiff base adduct (via glyoxal/glycolaldehyde) >>> glucose
(via autoxidation). Indeed, when using the data of CML and
glyoxal-triazine formation to calculate the contribution for CML
formation under severe diabetic conditions, i.e. 30
mM glucose and 2 mM Amadori product
(32) ,
most would originate from the Amadori product via oxidative cleavage,
but glyoxal from Amadori product and glucose-amine interaction would
also account for 17% (I).
The fact that addition of a
heterologous Amadori product to
N
Hayashi et al. (10) reported the formation of glyoxalalkyldiimines in heated
reaction systems. The data shown in this paper establish the formation
of analogue lysine derivative diimine 5 and also imine 7 (Fig. 1) under physiological conditions. Both structures
could be detected in protein incubations with their immediate
precursors glyoxal and glycolaldehyde, as well as from various sugars.
Although the C-2-imine cross-links are formed in vitro, they
are not stable and, as such, of minor importance as permanent
cross-links. For this reason, no attempt was made to distinguish
between 5 and 7. The concentrations formed were very
small compared with other modifications like CML and were strictly
dependent on the amount of glyoxal and glycolaldehyde present in the
system. Since the expected steady-state levels of both carbonyl
compounds formed in living systems are extremely low, and neither 5 nor 7 accumulate, this cross-link may therefore be
present only in trace amounts in vivo and not contribute to
protein cross-linking during aging or diabetes. Thus, it is not
surprising that preliminary analysis of human skin and aorta tissue and
serum samples showed no evidence for the formation of 5 and
7.
In summary, the generation of glyoxal and glycolaldehyde
from higher sugars under physiological conditions and the tight
relationship between these carbonyl compounds and CML formation became
apparent in this study. In addition the in vitro data of
protein cross-linking by us and others
(33) provide new
insights in the possible nature and mechanisms of sugar-mediated
protein modification in vivo. Mechanistic studies in vivo are now necessary to probe the significance of the proposed
pathway for CML formation and cross-linking. It is conceivable that
some answers may come from in vivo determination of the type
of triazines formed during the aminoguanidine therapy
(34) .
We thank Dr. Douglas Gage at the Michigan State
University Mass Spectrometry Facility for performing high resolution
fast atom bombardment spectral analysis. This facility is supported, in
part, by a grant (DRR-00480) from the Biotechnology Resources Branch,
Division of Research Resources, National Institutes of Health.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-(carboxymethyl)lysine (CML) formation
during Maillard reaction under physiological conditions was
investigated. Incubation of bovine serum albumin with these reagents
lead to rapid formation of C-2-imine cross-links and CML. Initial CML
formation rate from glyoxal was not dependent on oxidation, suggesting
an intramolecular Cannizzaro reaction. CML formation from
glucose/lysine or Amadori product of both was strongly dependent on
oxidation. Blocking of Amadori product by boric acid totally suppressed
CML formation from Amadori product, but only by 37% in the
glucose/lysine system. Trapping of glyoxal with aminoguanidine hardly
suppressed CML formation from Amadori product, whereas it blocked 50%
of CML production in the glucose/lysine system. While these results
would support a significant role for glucose autoxidation in CML
formation, the addition of lysine to a glucose/aminoguanidine
incubation system catalyzed glyoxal-triazine formation 7-fold, thereby
strongly suggesting that glucose autoxidation is not a factor for
glyoxal-mediated CML formation. Based on these results, it can be
estimated that approximately 50% of the CML forming in a glucose/lysine
system originates from oxidation of Amadori product, and 40-50%
originates from a pre-Amadori stage largely independent from glucose
autoxidation. This step may be related to the so-called Namiki pathway
of the Maillard reaction.
-carboxymethyllysine (CML)
(
)(3, 4) . A third structure, pyrraline, was
found in tissue matrix by immunochemical and chromatographic methods
(5, 6, 7) , but its existence in vivo is controversial
(8) .
Figure 1:
Reaction
scheme for the formation of CML and imine cross-links during the
Maillard reaction.
Based on these preliminary
observations and the finding that carboxymethylation of amino acids can
occur with glyoxal in heated reaction mixtures
(16, 17) , we hypothesize that a mechanism involving
glyoxal/glycolaldehyde could contribute to CML formation and protein
cross-linking under physiological conditions. This hypothesis departs
from the previous notion that CML originates only from the Amadori
product
(18, 19) .
H and
C nuclear magnetic resonance (NMR)
spectra (Me
Si (CDCl
),
3-(trimethylsilyl)propionic-2,2,3,3- d
-acid
(D
O) as internal standards) were recorded with a Varian 300
MHz spectrometer Gemini-300 (Varian Associates, Inc. Palo Alto, CA).
High resolution fast atom bombardment mass spectral data were obtained
at Michigan State University Mass Spectrometry Facility, and high
resolution mass spectra were obtained with a Kratos MS 25 RFA dual
beam, double focussing, magnetic sector mass spectometer (direct probe
insertion, EI 20 eV). High Performance Liquid Chromatography HPLC was performed on a Waters gradient system (Waters Chromatography
Division, Milford, MA) equipped with two model 510 pumps and a model
470 fluorescence detector. Amino acids were derivatized postcolumn with
o-phthaldialdehyde (Aldrich)
(20) . System 1
for detection of CML consisted of water (eluent A) and 70% methanol in
water (eluent B), both with 0.01 M heptafluorobutyric acid
(Aldrich), C18 column (0.4
25 cm, 5 µm, VYDAC 218TP54,
Hesperia, CA), flow rate 1 ml/min, gradient 2% B for 20 min and then in
5 min to 100% B. System 2 for detection of CML and
glucitolyl-lysine was as follows: column as system 1, with 5%
propanol (eluent A) and 60% propanol in water (eluent B), both with 3 g
of SDS (Fluka)/liter, 1 g of monobasic sodium phosphate
monohydrate/liter, and adjusted to pH 2.8 with phosphoric acid,
gradient 15% B
22% B in 30 min
40% B in 20 min
100% B in 5 min and flow rate 1 ml/min. System 3 for detection
of 6 was as follows: column, flow and eluents like system
1, gradient 15% B
40% B in 25 min
50% B in 22
min
100% B in 3 min. System 4 for detection of 6 was as
follows: column and flow like system 2, eluent A 5% propanol
and eluent B 60% propanol in water, both with 3 g of SDS/liter, 1.5 g
of monobasic sodium phosphate monohydrate/liter and adjusted to pH 7
with potassium hydroxide, gradient 15% B
25% B in 30 min
35% B in 5 min
100% B in 5 min. System 5 for
preparative isolation of 6 was as follows: eluents like system
1, C18 column (2.2
25 cm, 10 µm, VYDAC 218TP1022),
gradient 15% B
50% B in 50 min
100% B in 25 min and flow
8 ml/min. System 6 for preparative isolation of CML and 9 was as follows: eluents, column and flow like system 5,
gradient 2% B for 40 min then to 100% B in 5 min. System 7 for
detection of 9 was as follows: column, flow, and eluents like
system 2, gradient 20% B
28% B in 40 min
100% B
in 5 min. Coupled Gas Chromatography-Mass Spectrometry GC/MS was performed on a Hewlett Packard 5890 series II
chromatograph (Wilmington, DE), quartz capillary column (25 m, inner
diameter 0.2 mm, Ultra 5 (Hewlett Packard), 0.33 µm, He, 12 psi,
26.26 cm/s, constant flow program on), injection port 270 °C,
interface 280 °C, temperature program 100 °C
200
°C/5 °C
min
to 200 °C
270
°C/10 °C
min
to 10 min isothermal 270
°C; connected to Hewlett Packard 5971 series mass selective
detector, EI and positive CH
-CI mode. Chromatography Silica gel 60 F
Merck 5554 (EM Separations, Gibbstown, NJ)
was used for thin-layer chromatography (TLC) and silica gel
(63-200 µm) (Alltech, Deerfield, IL) was used for column
chromatography. Chromatography solvents were all ACS grade. From the
individual chromatographic fractions, solvents were evaporated under
reduced pressure. Synthetic Procedures
Ethylenedihexylamine
A solution of 0.5 g (4.2 mmol) of
2,3-dihydroxy-1,4-dioxane (Fluka) and 1.25 g (12.5 mmol) of hexylamine
(Fisher) in 5 ml of anhydrous methanol was stirred at 25 °C for 30
min, 380 mg of sodium borohydride (Fluka) was added, and after 15 min
of stirring, the solvents were evaporated to dryness. The residue was
taken up in 1 N NaOH and extracted with ethyl acetate. The
combined organic layers were dried over anhydrous sodium sulfate and
the solvents were evaporated. The residue was dissolved in 10 ml of
anhydrous pyridine, and 5 ml of trifluoroacetic anhydride (Aldrich)
were slowly added at 0 °C. The solution was stirred for 5 min at 0
°C and then for 55 min at ambient temperature. The solvents were
evaporated to dryness, and the resulting brown oil was purified by
column chromatography (eluent CHCl
). Fractions
containing
N, N`-di-(trifluoroacetyl)ethylenedihexylamine
( R
0.56, TLC (same solvent)) were combined and
evaporated to dryness (300 mg (17%), colorless oil, GC
t
23.21 min). CI-GC/MS, m/ z 421 (M + 1;100) 307
(10) 224
(13) 210
(3) 198
(6) 154
(5) . To remove the trifluoroacetyl
groups, 300 mg of
N, N`-di(trifluoroacetyl)ethylenedihexylamine was
dissolved in 0.5% potassium hydroxide solution in methanol and stirred
for 45 min at 80 °C. TLC (solvent as above) showed complete absence
of trifluoroacetyl derivative. Solvents were evaporated and the residue
was taken up in 1 N NaOH and extracted with diethylether. The
combined organic layers were dried over anhydrous sodium sulfate, and
solvents were evaporated to yield ethylenedihexylamine (150 mg (overall
16%) of colorless oil, TLC R
0.49 (solvent
butanol/water/acetic acid, 25:5:3), GC t
19.02 min). CI-GC/MS, m/ z 229 (M + 1;100)
227
(91) 157
(40) 128
(51) 114
(53) .
H NMR(CDCl
),
(ppm) 0.88 (t, 6H, J = 6.9 Hz) 1.28 (m, 12H) 1.48 (m, 4H) 2.59 (t, 4H, J = 7.5 Hz) 2.71 (s, 4H).
2,15-Diamino-7,10-diaza-hexadecane-1,16-dioic acid
6
A suspension of 0.25 g (2.1 mmol) of
2,3-dihydroxy-1,4-dioxane, 1.28 g (5.2 mmol) of
N- t-Boc-lysine (Bachem, Torrance, CA)
and three potassium hydroxide pellets in 5 ml of anhydrous methanol was
stirred for 1 h at room temperature. Following addition of 190 mg (5
mmol) of sodium borohydride, the solution was stirred for another 15
min, and the pH was then adjusted to 7 with 1 N HCl. TLC
(solvent butanol/water/acetic acid, 25:5:3) revealed a new spot at
R
0.10. Eluents were evaporated to dryness,
protecting groups were removed by treatment with 3 N HCl for
30 min, and the solution was then freeze dried. The lyophilisate was
taken up in water and subjected to preparative HPLC system 5.
Fractions containing 6 were collected and freeze dried (65 mg
(10%), colorless amorphous hygroscopic powder, HPLC system 5,
t
63 min; system 3,
t
47.5 min; system 4,
t
30.3 min; GC ( 6 as its
trifluoroacetylmethylester derivative) t
32.81 min). EI(70 eV)-GC/MS, m/ z 378
(M
-352;24) 356
(9) 319
(13) 309
(35) 305
(54) 194
(29) 180
(100) 178
(48) 152
(15) 140
(98) 126
(19) 114
(9) 96
(10) 84
(10) 67
(40) .
H
NMR(D
O),
(ppm) 1.38 (m, 4H), 1.62 (p, 4H, J = 7.7 Hz) 1.80 (m, 4H), 2.99 (t, 4H, J =
7.7 Hz), 3.28 (s, 4H), 3.81 (t, 2H, J = 6.3 Hz).
C NMR(D2O),
(ppm) 23.85, 27.56, 31.85, 45.39, 50.17,
55.68, 175.26. High resolution fast atom bombardment mass spectrometry,
319.4217 calculated 319.4233 for C14 H31 N4 O4. Elementary analysis
showed that material obtained was the 6
4
heptafluorobutyrate salt.
N
246
mg (1 mmol) of N-(Carboxymethyl)lysine
- t-Boc-lysine and 186
mg (1 mmol) of iodoacetic acid (Aldrich) were dissolved in 10 ml of
phosphate buffer, pH 10, and stirred overnight at ambient temperature.
The solution was freeze dried and purified by column chromatography
(solvent methanol/ethyl acetate, 2:1). Fractions containing material
with R
0.69 (TLC/solvent methanol) were
evaporated, taken up in 3 N HCl, and stirred for 60 min at
room temperature. Solvents were removed by freeze drying, and the
lyophilisate was subjected to preparative HPLC system 6.
Fractions containing CML were combined and freeze dried (45 mg (22%),
colorless amorphous powder, HPLC system 6 t
22.1 min, system 1
t
9.4 min, system 2
t
26.3 min, GC (CML as its
trifluoroacetylmethylester derivative) t
22.03 min, spectroscopic data compliant with the literature
(4) , elementary analysis showed that the material obtained was
the CML
2 heptafluorobutyrate salt).
N
A solution of 0.6 g (3.3
mmol) of glucose (Sigma), 0.82 g (3.3 mmol) of
N-(Glucitolyl)lysine (reduced Amadori
product of glucose and lysine)
- t-Boc-lysine, and 0.21 g (3.3 mmol)
of sodium cyanoborohydride (Aldrich) in 8.5 ml of anhydrous methanol
was stirred overnight, 2 g of silica gel was added, solvents were
evaporated, and the resulting powder was fractionated by column
chromatography (eluent methanol/ethyl acetate, 1:2). Fractions
containing
N
- t-Boc- N
-(glucitolyl)lysine
( R
0.12, TLC, eluent butanol/water/acetic acid,
25:5:3) were evaporated. The residue was taken up in 3 N HCl,
stirred for 60 min at room temperature, and freeze dried.
N
-(glucitolyl)lysine was obtained as colorless
crystals (125 mg (12%), HPLC system 2
t
39.1 min, spectroscopic data compliant
with the literature
(21) ).
N
273 mg (1.1 mmol) of
N-(2-Hydroxyethyl)lysine
9
- t-Boc-lysine, 66 mg (1.1 mmol) of
glycolaldehyde, and 69 mg (1.1 mmol) of sodium cyanoborohydride were
dissolved in 7 ml of anhydrous methanol and stirred overnight. Solvents
were evaporated, and the resulting oil was applied to column
chromatography (eluent methanol/ethyl acetate, 1.75:1) to remove
inorganic salts. Fractions containing material with R
0.37 (TLC solvent n-butyl alcohol/water/acetic acid,
25:5:3) were combined, eluents were removed, and the residue was taken
up in 3 N HCl and stirred for 60 min at room temperature.
Solvents were removed by freeze drying, and the lyophilisate was
subjected to preparative HPLC system 6. Fractions with 9 were combined and freeze dried. (75 mg (35%), colorless amorphous
powder, HPLC system 6 t
37 min,
system 1 t
15.5 min, system
7 t
37.1 min, GC ( 9 as
its trifluoroacetylmethylester derivative) t
20.31 min, elementary analysis showed that the material obtained
was the HEL
2 heptafluorobutyrate salt). EI(70 eV)-GC/MS,
m/ z 460 (M
-32;14) 433
(6) 395
(6) 379
(9) 346
(12) 319
(50) 266
(35) 180
(59) 141
(100) 69
(50) . High
resolution mass spectrometry, m/ z 492.0948
(M
°), calculated 492.0943 for C
H
F
N
O
,
m/ z 460.0636, calculated 460.0591 for C
H
F
N
O
,
m/ z 266.0254, calculated 266.0256 for C
H
F
N
O
.
-(2-Hydroxy-1-D-ethyl)
( 9`) and
N
-(2-hydroxy-1,2- d
-ethyl)lysine
( 9") were synthesized like nonlabeled 9 but with the
use of sodium cyanoborodeuteride ( 9`) or sodium
cyanoborodeuteride (Aldrich) and glyoxal ( 9"). EI(70
eV)-GC/MS, 9` (trifluoroacetylmethylester derivative),
m/ z 461 (M
°-32;14) 434
(5) 396
(5) 380
(8) 347
(9) 320
(59) 267
(45) 180
(63) 142
(100) 69
(71) . EI(70 eV)-GC/MS 9" (trifluoroacetylmethylester derivative), m/ z 462
(M
°-32;11) 435
(4) 397
(5) 381
(6) 348
(10) 321
(48) 268
(31) 180
(47) 143
(100) 69
(51) .
Radioactive Labeled
N-(1-Deoxyfructos-1-yl)propylamine
Unlabeled glucose was added
to 250 µCi of 1-C- and 6-C
-labeled
glucose (Amersham Corp.) to adjust the specific activity to 10.8
mCi/mmol. After complete removal of water, the residue was taken up in
50 µl of anhydrous methanol and 150 µl of a solution of 4.2
µl of propylamine/100 µl of methanol, and the solution was
heated for 45 min at 65 °C in a closed system. Solvents were
evaporated, 100 µl of a solution of 2.94 mg of oxalic
acid
2H
O/100 µl of methanol were added, and the
mixture was heated for 15 min at 65 °C in a closed system. The
methanol was removed, 0.5 ml of 1 N NaOH were added, and the
solution was extracted 5 times with methylenechloride. After
neutralization with 1 N HCl, the aqueous layer was freeze
dried, and the lyophilisate was taken up in 400 µl of water and
applied to column chromatography (Dowex 50W-X4-400, Aldrich, 3 cm
height in Pasteur pipette). To remove unreacted glucose, the column was
first washed with 6 ml of water; the Amadori product was then eluted
with 0.2 N pyridine formate, pH 5.5, and proper fractions were
combined (control by TLC (eluent n-butyl alcohol/water/acetic
acid, 25:5:3, R
0.16) and radioactivity TLC
scanner (Berthold, Germany)) and freeze dried.
- t-Boc-N
-(1-deoxyfructos-1-yl)lysine
and
N
- t-Boc- N
-(1-deoxyribulos-1-yl)lysine
were synthesized mainly according to Ref. 22. N-(1-Deoxyfructos-1-yl)propylamine and 3-amino-1,2,4-triazine (GC t
8.40
min (triazine as its trimethylsilyl derivative)) according to Refs. 23
and 24, respectively. Incubations All incubations were conducted at 37 °C in a shaker incubator after
sterile filtration (0.2-µm filter, Gelman Science, Ann Arbor, MI),
followed by reduction prior to hydrolysis. Deaerated conditions were
achieved by the presence of 1 mM phytic acid (Sigma), 1
mM diethylenetriaminepentaacetic acid (Sigma), and gassing for
3 min with argon. Phosphate-buffered saline (pH 7.4, unless otherwise
indicated) was used in all glyoxal/glycolaldehyde experiments; in all
other cases, 0.2 M phosphate buffer, pH 7.4, was used. Samples
were reduced for 1 h at room temperature in the presence of a 1.25-fold
(glyoxal/glycolaldehyde) and a 5.3-fold (all other) molar excess of
sodium borohydride to the carbonyl compounds used. In protein
incubations, BSA (Sigma) concentration was 0.1 mM. After
reduction, the protein was precipitated and washed twice with
trichloroacetic acid (Sigma) (10, 5, and 5%). The resulting precipitate
was dried and subjected to acid hydrolysis (6 N HCl) for 20 h
at 110 °C under argon. For incubations with radioactive labeled
sugars (specific activity, 10.8 mCi/mmol) concentrations of 77.3
µCi/500 µl were used. Analytical Procedures Imines 5 and 7 were monitored as their common
reduction product 6. For HPLC analysis, hydrolysates were
evaporated in a Savant Speed-Vac concentrator (Hicksville, NY) and
taken up in water. 9 was determined with HPLC system 7 after material had been collected from HPLC system 1 ( t
13.5-18.0 min) without
postcolumn derivatization. CML from incubations with radioactive
labeled sugars was first collected from HPLC system 1 without
postcolumn detection ( t
6-11.5 min)
and then quantified on HPLC system 2 by counting the
radioactivity of proper fractions (LS6000, Beckman, Fullerton, CA) or
postcolumn derivatization.
- t-Boc-lysine was determined prior
to acid hydrolysis after reduction and removal of the protecting groups
by 3 N HCl as N
-(glucitolyl)lysine.
Solvents were removed in a Savant Speed-Vac concentrator, and the
residue was taken up in water and subjected to HPLC system 2. SDS-Polyacrylamide Gel Electrophoresis For estimation of the extent of cross-linking mediated by
glyoxal/glycolaldehyde at various time points, RNase (0.5 mM,
Sigma) incubations were reduced, dialyzed overnight at 4 °C against
phosphate-buffered saline, pH 7.4, and analyzed by SDS-gel
electrophoresis on a 12% acrylamide gel. The amounts of RNase loaded
onto the gel was 20 µg in each lane. The gel was stained with
Coomassie Blue for 1 h and destained in a mixture of 40% methanol and
10% acetic acid in water.
- t-Boc-lysine
and 2,3-dihydroxy-1,4-dioxane and purified by preparative HPLC. The
structure was unequivocally established from its spectroscopical data
as described under ``Experimental Procedures.'' To optimize
conditions, the synthesis of the hexylamine derivative of 6 was studied first. Best conditions were obtained using
2,3-dihydroxy-1,4-dioxane as a water-free glyoxal substitute
(25) , strong alkaline reaction conditions, and subsequent
reduction with sodium borohydride rather than the use of commercially
available glyoxal/water solutions under neutral conditions in the
presence of sodium cyanoborohydride. Product formation was assessed by
coupled GC/MS.
Figure 2:
A, effect of glyoxal concentration on the
formation of C-2-imine 5 in BSA incubations. B,
time-dependent formation of C-2-imines 5 and 7 in BSA
incubations with 20 mM glyoxal () and 20 mM
glycolaldehyde (▾). B, inset, formation of
polymers during incubation of RNase with 20 mM glyoxal
( 8/5 h, 7/170 h), 200 mM glyoxal
( 6/5 h, 5/170 h), and 20 mM glycolaldehyde
( 4/5 h, 3/170 h), monitored by SDS-polyacrylamide gel
electrophoresis. Lane 1 shows low molecular mass
standard (14.4-97.4 kDa), and lane 2 shows
control incubation (170 h) without carbonyl compounds
added.
C-2-imines 5/ 7 were also detected in other sugar-BSA incubations ().
The rate of formation was time-dependent and roughly followed the
reactivity of the sugar. Glucose protein mixtures showed no cross-link
formation under the same conditions (detection limit of this
experiment, 0.1 mmol/mol of lysine). In a detailed time course
experiment, the cross-links formed progressively during incubation with
ribose until a peak was reached at about 120 h with 0.28 mmol/mol of
lysine followed by degradation. Retention times and mass spectra of
derivatized collected HPLC material from glyoxal/glycolaldehyde/ribose
incubation mixtures were identical to the data of the synthesized
authentic product 6 (Fig. 3).
Figure 3:
Identification of C-2-imines 5 and 7 as their reduction product 6 in BSA
incubations with 20 mM glyoxal (B), 20
mM glycolaldehyde ( C) and 200 mM ribose
( D). Mass spectra obtained after derivatization are identical
to the one of synthesized authentic amino acid 6 (A).
The data presented so
far suggested that a significant amount of 5 and 7 was further degraded into one or several more stable products. One
known product, which contains a C-2 fragment of the original sugar, is
CML. Incubations of BSA with both glyoxal and glycolaldehyde revealed
indeed large quantities of CML formed (Fig. 4). The identity of
CML detected by HPLC was confirmed after collection and derivatization
by GC/MS (Fig. 5). For comparison, authentic CML was synthesized.
Deaerated conditions and presence of transition metal ion chelator
altered the rate of formation from both carbonyl compounds.
Figure 4:
Time-dependent formation of CML in BSA
incubations with () 20 mM glyoxal and (▾) 20
mM glycolaldehyde. Full symbols indicate
aerated conditions; open symbols indicate deaerated
conditions. Inset shows same experiment up to 5
h.
Figure 5:
Identification of CML formed in BSA
incubations with 20 mM glyoxal ( B) and 20 mM
glycolaldehyde ( C) by GC/MS. Mass spectra obtained after
derivatization are identical to the one of authentic CML
( A).
However,
the role of oxidation in CML formation from glyoxal and glycolaldehyde
is complex since the initial formation rate of CML from glyoxal is not
dependent on oxygen, whereas it is totally suppressed under anaerobic
conditions for glycolaldehyde (Fig. 4, inset). This
suggests that CML formation from glyoxal can occur through an
intramolecular Cannizzaro reaction without oxidation (Fig. 6,
intermediate 8). In support of this possibility, pH studies
showed that CML synthesis from glyoxal was favored by alkaline
conditions, pH 9, but totally suppressed at pH 5 (data not shown).
Figure 6:
Proposed mechanism for formation of CML
from glyoxal and glycolaldehyde.
Whereas oxidation of Amadori-rearranged glycolaldehyde adduct 3 (Fig. 6) is a necessary step for initial CML formation, the
finding that CML formation during long term incubation of BSA with
glycolaldehyde progresses in spite of deaerated conditions suggests a
possible intermolecular Cannizzaro reaction of aldoamine 3 into acid CML and alcohol
N-(2-hydroxyethyl)lysine 9. In order
to clarify the significance of this pathway, authentic 9 was
synthesized and detected in high levels upon trapping the Schiff base
2 with sodium cyanoborohydride. On the other hand, only small
levels were recovered without reducing reagent ( i.e. 4.5
compared with 29.2 mmol/mol of lysine of CML at 170 h). Since even
lower levels were detected under deaerated conditions (2.0 mmol/mol of
lysine), intermolecular Cannizzaro reaction is excluded as a
significant source of CML from glycolaldehyde.
-dicarbonyl compounds
(24) showed that
whereas only 1.16% (aeration) and 0.95% (deaeration) of glycolaldehyde
was transformed into glyoxal, as measured as by the corresponding
triazine, as much as 37% CML formation was suppressed. The formation of
an aromatic ring structure represents such gain of stability that not
only free glyoxal will react, but hidden glyoxal in Schiff bases like
8 or aminals
(26) will also react. Thus, detection of
the glyoxal-triazine and significant inhibition of CML formation
suggests glyoxal or glyoxal derivatives 8 as major
intermediates for CML synthesis in glycolaldehyde incubations.
- t-Boc-lysine was
strongly dependent on presence of oxygen and free metals
(Fig. 7). Aminoguanidine and boric acid were used as tools to
dissect the relative importance of Amadori product versus glucose as a precursor of CML in incubations with
N
- t-Boc-lysine. Only when the
reaction was started from glucose itself did aminoguanidine have an
important impact, as evidenced by almost 50% reduction in CML
formation. In presence of Amadori product, incubations with
aminoguanidine showed almost no effect up to 100 h, although the
absolute amount of inhibition after 170 h was larger compared with
glucose experiments. Formation of the triazine derived from glyoxal and
aminoguanidine measured by GC/MS was strictly dependent on oxidation
(). After 170 h, the Amadori product produced about 4
times more glyoxal than the sugar. In addition, 7-fold catalytic effect
of N
- t-Boc-lysine on glyoxal
formation as the amine component in glucose systems is evident. In
contrast to aminoguanidine, 10 times excess boric acid completely
inhibit CML formation from the Amadori compound, but only by about 37%
when the reaction was initiated by glucose. This suggests an additional
mechanism, which is not dependent on the Amadori product. Indeed,
whereas in presence of Amadori product, only as much as 40% degradation
within 170 h yielded 2.8 mmol of glyoxal triazine/mol of lysine
(), only 0.7% Amadori product were actually detected in
the glucose/ N
- t-Boc-lysine system
after the same time (100% refers to the initial Amadori concentration
in Amadori product only incubations).
Figure 7:
Time dependent formation of CML in
incubations of ( A) glucose (42.2 mM) and
N- t-Boc-lysine (42.2 mM)
( B) Amadori product of glucose and
N
- t-Boc-lysine. Incubations were
conducted under (▾) aerated, aerated in presence of
aminoguanidine (5 mM,
), and in the presence of boric
acid (420 mM,
), and (
) deaerated
conditions.
Two different approaches were
utilized to establish the formation of glyoxal and glycolaldehyde in
incubations of higher sugars without the use of trapping reagent.
First, the formation of
N-(2-hydroxyethyl)lysine 9 in reduced
incubations of glucose and ribose was confirmed reaching concentrations
of 0.1 and 7.5 mmol/mol of lysine, respectively, after 170 h. In the
case of ribose, the nature of the original C-2 carbonyl intermediates
could be elucidated by reducing with sodium borodeuterate. Isotopic
distribution of characteristic fragments in the mass spectrum, as 461
for N
-(2-hydroxy-1- d-ethyl)lysine
9` and 462 for N
-(2-hydroxy-1,
2- d
-ethyl)lysine 9`', revealed a
ratio of 4:5 of 9` to 9" through comparison with the
spectra of authentic synthesized compounds 9, 9`, and
9". Signals at 461 and 462 represent loss of methanol, which
is a common fragmentation pattern for methylesters. Second, in
incubations of the Amadori product (0.5 mM) of glucose and
propylamine with N
- t-Boc-lysine (42
mM), the formation of CML was confirmed and found to increase
with time (0.47 mmol/mol of lysine at 170 h).
- t-Boc-lysine, 50% inhibition of CML
formation by aminoguanidine indicates approximately a 50% participation
of the C-2 intermediates glyoxal/glycolaldehyde and 50% of CML
originating from the Amadori product via oxidative cleavage, which is
not influenced by aminoguanidine.
- t-Boc-lysine system reaches
only 0.7% of 42 mM, this source contributes only minor
quantities CML by C-2 intermediates. Based on Fig. 7 B,
it can be estimated at approximately 7%. This leaves 43% of total CML
originating from other sources via glyoxal/glycolaldehyde
(Fig. 8).
Figure 8:
Formation of CML from various sugars
during the Maillard reaction. Contribution of oxidative cleavage
versus glyoxal is calculated on data in Fig.
7.
Utilizing the data on 3-amino-1,2,4-triazine
formation, glucose autoxidation, as proposed by Wolff et al. (28) , can be excluded as a significant source of glyoxal
generation (). We recovered small quantities of the
triazine from glucose incubated in absence of amine. However, about 7
times higher levels were formed when the identical experiment was
carried out in the presence of
N- t-Boc-lysine.
- t-Boc-lysine. The Amadori product
will primarily form carboxymethylated propylamine via oxidative
cleavage and glyoxal generation, but part of the glyoxal will also
react with lysine to give carboxymethylated lysine or CML
(Fig. 8).
-(2-hydroxy-1- d-ethyl)lysine 9` for reduced 2 and 3 and
N
-(2-hydroxy-1,
2- d
-ethyl)lysine 9" for reduced 8 in ribose incubations. This experiment unequivocally proves the
existence of glycolaldehyde and glyoxal as their imine derivatives in
incubations under physiological conditions and therefore emphasizes
their role as potent contributors of CML formation from higher sugars.
- t-Boc-lysine induced CML formation
led to investigations about the origin of the C-2 fragments with
radiolabeled sugars. Incubating glucose with protein, about 30% of
total CML incorporate the C-1 and about 30% incorporate the C-6 region,
representing a C-2-C-3 and C-4-C-5 split of the sugar
backbone. This indicates that significant amounts of C-2 fragments
originate in between C-1 and C-6 and are therefore not accounted for by
radioactivity. About 70% originate from the C-1 and about 30% from the
C-6 part, when CML was induced by labeled Amadori product of glucose
and propylamine. In this case, only glyoxal/glycolaldehyde transfer to
the
-amino lysine residues can generate CML. Thus, other reaction
pathways in addition to the one proposed by Namiki contribute to CML
formation. These may not be in contradiction with the importance of the
Schiff base as a major contributor to C-2 fragmentation as evidenced
above, because activation of the sugar by the imine bond may result in
enolization through the whole molecule and facilitated fragmentation in
multiple sites
(2) .
Table:
C-2-imine/CML formed in various sugar-BSA
incubations
Table:
3-Amino-1,2,4-triazine formed after 170
h in various sugar/amine-incubations (all 42.2 mM) in the
presence of 5 mM aminoguanidine
Table:
Calculated CML and glyoxal formation after 170
h of incubation mimicking severe diabetic conditions, based on results
presented in Table II and Fig. 7
-(carboxymethyl)lysine; GC/MS, coupled gas
chromatography-mass spectrometry; HPLC, high performance liquid
chromatography; BSA, bovine serum albumin;
N
-(glucitolyl)lysine, reduced Amadori product
of glucose and lysine; TLC, thin-layer chromatography; t-Boc,
t-butoxycarbonyl.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.