Dicarbonyl compounds such as glyoxal and
methylglyoxal are reactive dicarbonyl intermediates in the
nonenzymatic browning and cross-linking of proteins during the Maillard
reaction. We describe here the quantification of glyoxal and
methylglyoxal-derived imidazolium cross-links in tissue proteins. The
imidazolium salt cross-links, glyoxal-lysine dimer (GOLD) and
methylglyoxal-lysine dimer (MOLD), were measured by liquid
chromatography/mass spectrometry and were present in lens protein at
concentrations of 0.02-0.2 and 0.1-0.8 mmol/mol of lysine,
respectively. The lens concentrations of GOLD and MOLD correlated
significantly with one another and also increased with lens age. GOLD
and MOLD were present at significantly higher concentrations than the
fluorescent cross-links pentosidine and dityrosine, identifying them as
major Maillard reaction cross-links in lens proteins. Like the
N-carboxy-alkyllysines
N
-(carboxymethyl)lysine and
N
-(carboxyethyl)lysine, these
cross-links were also detected at lower concentrations in human skin
collagen and increased with age in collagen. The presence of GOLD and
MOLD in tissue proteins implicates methylglyoxal and glyoxal, either
free or protein-bound, as important precursors of protein cross-links
formed during Maillard reactions in vivo during aging and
in disease.
 |
INTRODUCTION |
The Maillard reaction is a complex series of reactions between
reducing sugars and amino groups on proteins, which lead to browning,
fluorescence, and cross-linking of protein (1, 2). Advanced glycation
end products, formed during later stages of the Maillard reaction,
accumulate in long lived tissue proteins, such as tissue collagens and
lens crystallins, and may contribute to the development of
complications in aging, diabetes, and atherosclerosis (3, 4). Glyoxal
(GO),1 methylglyoxal (MGO),
and deoxyglucosones belong to a series of dicarbonyl compounds,
identified as intermediates in the Maillard reaction. GO is formed on
autoxidation of glucose under physiological conditions (5) and also as
a product of lipid peroxidation (6). MGO is formed nonenzymatically by
spontaneous decomposition of triose phosphate intermediates in
glycolysis (7) and by amine-catalyzed sugar fragmentation reactions (8,
9). It is also a product of metabolism of acetone (10) and threonine (11). Both GO and MGO are detoxified by the
glutathione-dependent glyoxalase pathway, yielding
hydroxyacetic acid and D-lactate, respectively (12-14).
MGO can also be detoxified by the NADPH-dependent enzyme
aldose reductase, yielding 1,2-propanediol (15). The concentration of
MGO is elevated in the blood of diabetic patients in vivo
(16, 17), and the metabolites of MGO detoxification, acetol and
1,2-propanediol, are also increased in blood during diabetic
ketoacidosis (10).
GO and MGO are reactive toward amino, guanidino, and sulfhydryl
functional groups in protein (18, 19), leading to browning, denaturation, and cross-linking of proteins. Besides unidentified brown
and fluorescent products, the reaction of GO and MGO with lysine and
arginine residues in protein yields well characterized compounds, such
as the N-(carboxyalkyl)lysines,
N
-(carboxymethyl)lysine (CML) (20) and
N
-[1-(1-carboxy)ethyl]lysine (CEL)
(21), and imidazolones and dehydroimidazolones (19, 22, 23).
Imidazolones have been detected in tissue proteins only by
immunological methods, whereas CML and CEL have been measured by gas
chromatography/mass spectometry (GC/MS) and increase in skin collagen
and lens protein with age (21, 24-26). Two other products of the
reaction of GO or MGO with lysine, glyoxal-lysine dimer (GOLD) and
methylglyoxal-lysine dimer (MOLD) (Fig.
1), were originally characterized in
reactions of GO or MGO, respectively, with hippuryllysine (27, 28). Nagaraj and colleagues (29) recently detected and measured MOLD in
human serum proteins by reverse phase high performance liquid chromatography assay (RP-HPLC) and showed that cross-linking of serum
proteins by MOLD was increased in diabetes. In the present study, we
describe the reaction of MGO with the model protein bovine pancreatic
RNase and quantify the role of MGO in cross-linking of the protein.
Using a liquid chromatography/mass spectrometry (LC/MS) assay with
15N-labeled GOLD and MOLD internal standards, we also
measure the levels of GOLD and MOLD in lens protein and show that the
concentrations of both cross-links increase in concert with
chronological age in lens protein and skin collagen. Quantitative
analysis indicates that MOLD is the major chemically characterized
cross-link formed in lens protein during the Maillard reaction.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Unless otherwise indicated, all chemical reagents
were of the highest quality obtainable from Sigma, including MGO (40%
aqueous solution) and RNase A (type XII-A).
(Carboxymethyl)trimethylammonium chloride hydrazide (Girard's Reagent
T) was obtained from Aldrich. SupelClean LC-18 cartridges were obtained
from Supelco (Bellefonte, PA), and C-18 preparative material was
obtained from the Waters Corporation (Milford, MA).
15N2-L-Lysine·HCl was
obtained from Cambridge Isotope Laboratories Inc. (Andover, MA).
Reaction of N
-Hippuryllysine with
MGO--
For identification of products formed in reactions of MGO
with the
-amino groups of lysine, mixtures of 100 mM
hippuryllysine and 100 or 200 mM MGO were incubated in 0.2 M phosphate buffer at pH 7.4 and 37 °C. Aliquots were
removed at various time points and analyzed by RP-HPLC.
Preparative Procedures--
MOLD and GOLD were prepared by
incubating 100 mM hippuryllysine with 200 mM
MGO or GO in aqueous 200 mM formaldehyde. The reaction of
MGO and hippuryllysine was conducted at 37 °C, and the reaction of
GO and hippuryllysine was conducted at 65 °C. Additional MGO or GO
(100 mM) and formaldehyde (100 mM) were added 8 times at hourly intervals and the reaction continued overnight. The pH
dropped from 6 initially to 2.5 at the end of the reaction. The
solutions were applied to a 0.75 × 10-cm column of C-18
preparative resin and eluted with a gradient of increasing
concentrations of acetonitrile in water. Fractions were analyzed for
absorbance at 228 nm, and those containing hippuryl-MOLD or -GOLD,
detected by RP-HPLC, were pooled, dried, and finally cleaned by
semi-preparative HPLC, using the analytical RP-HPLC system described
below. To obtain free MOLD or GOLD, the hippuryl group was removed by
hydrolysis in 6 N HCl for 4 h at 110 °C.
For preparation of the heavy labeled cross-links
15N4-MOLD and
15N4-GOLD,
15N2-L-N
-formyllysine
(100 mM), prepared from
15N2-L-lysine as described by
Hofmann et al. (30), was incubated with MGO or GO (200 mM) and formaldehyde (200 mM) in deionized water. The reactions were conducted for 30 h as described above. Deformylated MOLD and GOLD were obtained by hydrolysis in 6 N HCl for 2 h at 110 °C. The hydrolysate was dried
by centrifugal evaporation (Speed-Vac, Savant Instruments, Holbrook,
NY) and reconstituted in water, and brown products were removed by
applying the product to a Supelclean-LC18 column (3 ml) in water. The
yield determined by cation exchange chromatography was approximately 60% for 15N4-MOLD and 50% for
15N4-GOLD.
Reaction of RNase with MGO--
RNase (20 mg/ml, 1.5 mM) was incubated with MGO (15 or 30 mM MGO at
molar ratios to lysine of 1:1 or 2:1, respectively) in 0.2 M phosphate buffer, pH 7.4, at 37 °C. Aliquots were
removed at various times and reduced with NaBH4 as
described above. After quenching the reaction with acetic acid, the
reduced protein was dialyzed against deionized water, concentrated by
centrifugal evaporation, and hydrolyzed in 6 N HCl at
110 °C for 22 h. The hydrolyzed protein was dried and applied
to a Supelclean LC-18 column (1 ml) to remove brown products. The dried
protein, dissolved in 5 ml of deionized water, was applied to
sulfopropyl-cation exchange gel (SP-Sephadex C-25; 1.2 ml). Neutral and
acidic amino acids were eluted with 25 ml of 0.05 N HCl and
basic amino acids, including MOLD, with 7 ml of 1 N HCl.
For phenylisothiocyanate (PITC) derivatization, the sample was dried,
dissolved in 30 µl of coupling buffer (water:ethanol:triethylamine,
2:2:1), and dried. PITC derivatization was conducted with 50 µl of a
mixture of ethanol, water, triethylamine, and PITC (7:1:1:1) as
described by Bidlingmeyer et al. (31). The derivatization
mixture was evaporated by centrifugal evaporation, and the residue was
dissolved in deionized water. Excess derivatization reagent was
extracted with n-heptane, discarding the organic layer. The
phenylthiocarbamoyl (PTC) amino acids were dissolved in solvent A for
RP-HPLC. Histidine, which was unaltered during reaction of the protein
with MGO and was recovered quantitatively during this procedure, was
used as the internal standard for quantitation of Lys, Arg, and
MOLD.
HPLC Procedures--
Amino acid analysis was performed using a
Pickering sodium cation exchange column (25 cm × 4.6 mm) and
sodium buffers (Pickering Laboratories Inc., Mountain View, CA), as
described by the manufacturers. Amino acids were quantified by
post-column derivatization with o-phthalaldehyde and
fluorescence detection (20, 32). Hippuryl-amino acids were analyzed by
RP-HPLC either on a 25 cm × 4.6 mm Zorbax SB C-18 HPLC column
(MAC-MOD Analytical Inc., Chadds Ford, PA) or on a 15 cm × 3 mm-Waters C-18 Symmetry column (Waters Corporation) using a detection
wavelength of 228 nm. The gradient consisted of solvent A (0.05%
acetic acid, 0.05% formic acid, and 0.1% triethylamine in water) and
solvent B (75% solvent A in acetonitrile). The gradient program for
the Zorbax column was: 0-50 min, 0-5% solvent B; 50-160 min,
5-100% solvent B; 160-190 min, wash with 100% acetonitrile, at a
flow rate of 1 ml/min; and for the Symmetry column: 0-30 min, 0-10%
solvent B; 30-100 min, 10-100% solvent B; 100-105 min, wash with
100% acetonitrile, at a flow rate of 0.5 ml/min. PTC amino acids were
analyzed on a 15 cm × 4.6 mm-inner diameter 218TP54 protein and
peptide C-18 column (VYDAC/The Separations Group, Hesperia, CA) using a
detection wavelength of 246 nm. The mobile phase consisted of solvent A
(12.5 mM sodium phosphate, pH 6.2) and solvent B (30%
solvent A and 70% acetonitrile). The gradient was programmed as
follows: 0-2 min, 0% solvent B; 2-40 min, 0-50% solvent B; 40-50
min, 50-100% solvent B; this was followed by a 10-min washing step
with 100% acetonitrile, flow rate 1.2 ml/min.
Measurement of MGO Concentrations--
MGO was measured using
Girard's Reagent T in 0.5 M formic acid, pH 2.9, as
described by Mitchel and Birnboim (33), using absorbance at 292 nm for
quantitation.
Electrophoresis--
SDS-PAGE was conducted under reducing
conditions using a 4% stacking gel and a 15% separating gel as
described by Laemmli (34).
Preparation of Human Lens Protein and Skin Collagen for Analysis
of MOLD and GOLD--
Human lenses were obtained from the South
Carolina Lions Eye Bank (Columbia, SC) and stored at
70 °C until
used. Lenses were decapsulated, homogenized and dialyzed against
deionized water, as described previously (26, 35). Total lens protein
was hydrolyzed in 6 N HCl at 110 °C for 24 h. The
hydrolysate was dried and then applied to an SP-Sephadex cation
exchange resin to recover basic amino acids, washed, and eluted, as
described above. The eluent was dried by centrifugal evaporation and
reconstituted in buffer A (sodium eluent, pH 3.15) for amino acid
analysis. Fractions in the time interval including lysine, MOLD, and
GOLD (15-25 min, 6 ml total volume) were collected. These pools were
diluted with deionized water to 50 ml, acidified with 4 N
HCl to pH 1-2, applied to a 2 ml-DOWEX-50W cation (sodium form), and
desalted by washing with 70 ml of 0.5 N HCl; then the basic
amino acids were eluted with 4 N HCl. The eluent was dried
and derivatized for PTC amino acid analysis as described above.
Human skin collagen was isolated by full thickness biopsy from the
upper buttock, as described previously in detail (24). Briefly, the
skin was scraped to remove adventitious tissue, extracted sequentially
for 24 h with 1 M NaCl in 10 mM phosphate
buffer, pH 7.4, and with 0.5 M acetic acid to remove
soluble proteins, and then extracted with chloroform:methanol (2:1) to
remove any residual lipid. The collagen was hydrolyzed and analyzed as
described above for lens proteins.
LC/MS Analysis of GOLD and MOLD in Human Lens Proteins and Skin
Collagen--
For LC/MS analysis about 2 mg of lens protein was mixed
with 3 nmol of 15N4-MOLD and 2.5 nmol of
15N4-GOLD or 4 mg of young (18 years) and old
(85 years) pools of human skin collagen with 1.5 nmol of
15N4-MOLD and 1.25 nmol of
15N4-GOLD. The samples were hydrolyzed,
processed, and derivatized with PITC, as described above. Gradient
HPLC/MS analysis was performed using a 100 × 1-mm Hypersil ODS
column (Keystone Scientific Inc., Bellefonte, PA) with a 2 × 1-mm
guard column. The gradient consisted of solvent A (90% water, 10%
methanol, and 0.3% glacial acetic acid) and solvent B (20% water,
80% methanol, and 0.3% glacial acetic acid). The gradient was: 100%
solvent A for 5 min, then to 100% solvent B at 15 min, and hold at
100% solvent B for 20 min; the flow rate was 50 µl/min. The ion
source was an Analytica of Branford Inc. electrospray model 103443 (Branford, CT), operating with the following settings: cylinder
voltage,
2600 V; end cap voltage,
3700 V; capillary voltage,
4800
V; current, 4 × 10
8 A; source temperature,
275 °C; needlegas pressure, 38 p.s.i.; lens 1-6 voltages,
147.8, 25.6, 25.4, 2.7, 0, and
49.1 V. The mass spectrometer was a VG
TRIO triple quadrupole mass analyzer (Beverly, MA) operating at: dwell
time, 150 ms; delay time, 20 ms; and photomultiplier voltage, 600 V. The masses monitored were 417 amu for PTC-Lys+H+, 597 amu
for (PTC)2-GOLD, 601 amu for
(PTC)2-15N4-GOLD, 611 amu for
(PTC)2-MOLD, and 615 amu for
(PTC)2-15N4-MOLD. All assays were
analyzed in a single batch to exclude interassay variation. Results of
single analyses of each sample are shown. The intra-assay coefficients
of variation for assay of GOLD and MOLD, measured at the mid-range of
the samples, were 14 and 12%, respectively (n = 5).
 |
RESULTS |
Characterization of Imidazolium Cross-links--
As reported
previously, GOLD and MOLD were originally isolated from reactions of GO
or MGO, respectively, with the model peptide N
-hippuryllysine (27, 28). RP-HPLC
analysis of reactions of MGO with hippuryllysine (MGO:Lys, 2:1) yielded
two major products (Fig. 2A)
subsequently identified as
N
-hippuryl-CEL and
(N
-hippuryl)2-MOLD. Acid
hydrolysis and amino acid analysis also yielded two major products, CEL
and MOLD (Fig. 2B). The identity of CEL was confirmed by its
elution time on HPLC analysis and by GC/MS analysis of its
trifluoroacetyl methyl ester derivative (M+ = 438 amu)
(21). ESI mass spectrometry was used for identification of
(N
-hippuryl)2-MOLD and
MOLD (M+ = 663 and 341 amu, respectively, before and after
acid hydrolysis) (28). Yields were ~12% CEL and ~32% MOLD, based
on original N
-hippuryllysine. Similar
results were obtained from reactions of GO with
N
-hippuryllysine, yielding ~33% CML
and ~32% GOLD (27) (data not shown). In numerous reactions of GO or
MGO with N
-hippuryllysine or proteins
(RNase or albumin) at a variety of concentration ratios, both
carboxyalkyllysines and imidazolium salt compounds were always formed
together. Yields and relative yields varied with absolute
concentrations and concentration ratios, as described for RNase in Fig.
3. Browning was more rapid and intense in
reactions containing MGO, indicating a higher yield of melanoidins from
MGO, compared with GO.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2.
Chromatographic identification of MOLD in the
reactions of MGO with N -hippuryllysine.
A, RP-HPLC chromatogram of a reaction mixture of 200 mM MGO and 100 mM hippuryllysine in 200 mM phosphate buffer for 24 h at 37 °C. Products
were identified as N -hippuryl-CEL and
(N -hippuryl)2-MOLD (73 and
120 min, respectively). B, cation exchange amino acid
analysis of the same reaction mixture after acid hydrolysis (6 M HCl, 24 h at 110 °C). Products were detected by
fluorescence following reaction with o-phthalaldehyde (20,
32). CEL (24 min) eluted in the region of isoleucine-methionine and
MOLD (58 min) as a basic amino acid.
|
|

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 3.
Detection of MOLD in MGO-modified RNase by
RP-HPLC. RNase was incubated with MGO (MGO:Lys = 2:1) in 0.2 M phosphate buffer, pH 7.4, for 24 h at 37 °C.
Basic amino acids were isolated from the protein hydrolysate by
chromatography on SP-Sephadex as described under "Experimental
Procedures" and then derivatized with PITC for HPLC analysis. The
inset shows the enlarged view of the elution time frame of
MOLD.
|
|
Formation of Imidazolium Cross-links during the Reaction of MGO
with RNase--
Because MGO is present in higher concentrations in
biological systems than GO (14), we concentrated on the reactions of MGO with protein. Reactions of MGO with the model protein RNase were
studied under physiological conditions (pH 7.4, 37 °C). MGO was
reacted with RNase, which has 10 lysine residues, at molar ratios of
MGO:Lys, 1:1 and 2:1. As shown in Fig. 3, MOLD was readily detectable
in MGO- modified RNase by RP-HPLC of the PTC derivative. The identity
of the MOLD was confirmed by co-elution with an authentic standard and
by its molecular mass of (PTC)2-MOLD (611 amu) measured by
ESI-MS. The kinetics of the formation of MOLD described in Fig.
4A were consistent with the
kinetics of the disappearance of MGO from the reaction mixture (Fig.
4B). The half-life of MGO in the presence of the protein was
~4 h at both concentrations, whereas in phosphate buffer alone it
disappeared with a half-life of ~30 h (Fig. 4B). The yield
of MOLD increased 4-5-fold on doubling of the MGO concentration, an
observation confirmed in two independent experiments, consistent with
either a rate-limiting second order process or the requirement for 2 mol of MGO/mol of MOLD formed. In contrast, lysine and arginine
decreased to similar extents at both MGO concentrations, yielding
maximal modification of 5-6 of 10 lysine and 3 of 4 arginine residues
in RNase (Fig. 4, C and D). The possibility that
some free lysine and arginine were generated during acid hydrolysis
cannot be excluded; however, the extent of the lack of reactivity of
3-4 lysine residues was confirmed by reaction with
trinitrobenzenesulfonic acid (data not shown). Although
50% of
lysine residues were modified in these reactions with formation of 20 or 95 mmol MOLD, only 1 or 3.6%, respectively, of the lysine loss
could be accounted for as MOLD. CEL, measured by GC/MS (data not
shown), accounted for an additional 1.3 or 1.6% of the total lysine
modification (21). Thus, MOLD and CEL accounted for
5% of the
modification of lysine residues in the protein. Browning,
cross-linking, and formation of fluorescent products also occurred,
indicating that other unidentified products were formed. As shown in
Fig. 5, the time course of cross-linking of RNase, estimated by SDS-PAGE, was consistent with the rate of
formation of MOLD (Fig. 4A).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 4.
Kinetics of the reaction of MGO with
RNase. MGO and RNase were incubated in 0.2 M phosphate
buffer, pH 7.4, at molar ratios of MGO:Lys, 1:1 ( ) or 2:1 ( ),
respectively. MOLD, lysine, and arginine were measured as their PTC
derivatives. MGO was measured using Girard's Reagent T. A,
kinetics of the formation of MOLD; B, kinetics of the
consumption of MGO at MGO:Lys, 2:1 ( ) and at the same concentration
in phosphate buffer in absence of RNase ( ). C and
D, kinetics of the modification of lysine and arginine
residues in RNase.
|
|

View larger version (96K):
[in this window]
[in a new window]
|
Fig. 5.
SDS-PAGE analysis of cross-linking of RNase
during reaction with MGO. Lanes 1 and 9, molecular mass markers, cytochrome c (12,400 Da), carbonic anhydrase
(29,000 Da), bovine serum albumin (66,000 Da), and alcohol
dehydrogenase (150,000 Da); lane 2, native RNase;
lanes 3-8, RNase, reacted with MGO (MGO:Lys, 1:1) at 0, 1, 2, 8, 48, and 96 h.
|
|
MGO-modified RNase was fractionated into monomer, dimer, and polymer by
chromatography on Sephadex G-75 (Fig. 6).
Fractions were pooled, as indicated in the figure, and then analyzed
for their MOLD content. As shown in the inset to Fig. 6, the
MOLD content of RNase increased with the extent of polymerization of the protein. These data indicate that MOLD contributes to both inter-
and intramolecular cross-linking of lysine residues in RNase; however,
the yield of MOLD in the RNase dimer fraction (Fig. 6,
inset) accounted for only about 5% of intermolecular cross-links, indicating that other cross-links were also formed.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 6.
Gel permeation chromatography of RNase
following reaction with MGO. RNase was pooled from reactions with
MGO at 18 h, as described in Fig. 4, and then applied to a
Sephadex G-75 column (75 × 1.5 cm; 18 mg of protein). The column
was eluted with phosphate-buffered saline, and fractions of 1.7 ml were
collected. The protein was pooled into monomer, dimer, and trimer
fractions, as shown. Aliquots of these pools were hydrolyzed and
analyzed by RP-HPLC as described above. Results of analyses are shown
in the table (inset).
|
|
Detection of MOLD and GOLD in Vivo--
Human lens proteins were
analyzed for their MOLD and GOLD content using the RP-HPLC analytical
procedure described in Fig. 3. The lens proteins were analyzed as a
function of age, because both CEL and CML accumulate in these proteins
with age (21, 26). As shown in Fig. 7,
both MOLD and GOLD were detectable by RP-HPLC in the hydrolysate of
lens protein. The peak identities were confirmed by co-elution with an
authentic standard, but the reliability of the assay was questionable
because of the low levels of MOLD and GOLD in lens proteins and their
possible co-elution with other trace compounds in protein. To obtain
more reliable results, we applied an LC/MS procedure to analyze lens
protein hydrolysates that were derivatized with PITC. Heavy labeled
MOLD and GOLD were prepared from 15N2-lysine
for use as internal standards. Nonlabeled PTC2-MOLD and
PTC2-GOLD were detected at the masses 611 and 597 amu,
respectively, and their heavy labeled counterparts at 615 and 601 amu,
respectively. Fig. 8 shows an
RP-HPLC/SIM-ESI chromatogram of PITC-derivatized lens protein,
confirming detection of both GOLD and MOLD in lens protein. The
standard curves (inset) were prepared by adding increasing amounts of natural GOLD and MOLD to a fixed amount of internal standard.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 7.
Detection of imidazolium cross-links in human
lens proteins by RP-HPLC. Basic amino acids were isolated by
cation exchange chromatography then derivatized with PITC and analyzed
by RP-HPLC. MOLD was identified by co-elution of an authentic standard.
Trace levels of GOLD were also detectable in lens proteins.
|
|

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 8.
Determination of MOLD and GOLD in human lens
proteins by LC/MS. Heavy labeled standards were added to lens
protein prior to acid hydrolysis. The protein was hydrolyzed, basic
amino acids isolated, and amino acid derivatized with PITC, as
described under "Experimental Procedures." Using HPLC-ESI-MS, the
masses recorded were 597 amu for GOLD, 601 amu for
15N4-GOLD, 611 amu for MOLD, and 615 for
15N4-MOLD. Standard curves were prepared by
adding known amounts of GOLD and MOLD (597 and 611 amu) to constant
amounts of internal standards (601 and 615 amu)
(insets).
|
|
Using internal standardization with heavy labeled MOLD and GOLD, these
compounds were measured in a group of lens proteins by HPLC-ESI-SIM-MS.
As shown in Fig. 9, both MOLD and GOLD
correlated significantly with age in human lens protein
(r2 = 0.69 and 0.75, respectively;
p < 0.001). The MOLD and GOLD content of lens protein
also correlated with each other (r2 = 0.69, p < 0.001). The concentration of MOLD in lens protein was approximately 5 times higher on average than that of GOLD. Measurements of MOLD in lens protein, determined by the LC/MS assay,
were consistent with the results of RP-HPLC analysis, as described in
Fig. 7, but concentrations measured by LC/MS were 25-80% of those
estimated by RP-HPLC, consistent with errors in quantification or
interference by co-eluting compounds in the HPLC assay. GOLD could not
be measured in lens proteins by HPLC alone.
In preliminary experiments, MOLD and GOLD were also detected in human
skin collagen. Traces of both cross-links (<0.02 mmol/mol of Lys) were
detectable in a pool of collagen from young donors (18 years old), but
MOLD and GOLD were present at concentrations of 0.38 and 0.04 mmol/mol
of Lys, respectively, in a pool from older donors (85 years old). The
lower level of cross-links in collagen is consistent with the
correspondingly lower levels of CML and CEL in skin collagen, compared
with lens proteins (21, 24).
 |
DISCUSSION |
In these and other studies, we have identified CML and GOLD (27)
and CEL and MOLD (28) as products of the reaction of GO and MGO with
hippuryllysine and with RNase, respectively. Our studies on reactions
with MGO confirm the observations of Nagaraj et al. (29) who
described the formation of an MGO-derived imidazolium cross-link (MOLD,
imidazolysine) in model systems and detected MOLD in human serum
proteins by an HPLC method. Skovsted et al. (36) have also
described another imidazolium cross-link, termed DOLD, which is formed
by reaction of 3-deoxyglucosone with protein, although this compound
has not yet been detected in tissue proteins. Dicarbonyl compounds,
such as GO, MGO, and 3-deoxyglucosone, are also known to form
imidazolones and dehydroimidazolones on reaction with arginine residues
in proteins (19, 22, 23); however, the relative reactivity of Lys and
Arg residues with dicarbonyl compounds has not been rigorously
evaluated, in part because of the lack of appropriately sensitive and
specific analytical methods for (dehydro)imidazolones.
In the present study, we describe an LC/MS assay for specific,
accurate, and simultaneous measurement of both MOLD and GOLD in tissue
proteins and show that both compounds increase in concert with one
another in human lens protein during normal aging. Levels of MOLD
measured by this technique were significantly lower than levels
measured by HPLC alone, undoubtedly because of the greater specificity
of the HPLC-ESI-SIM-MS technique, and in our hands, the HPLC technique
was not reliable for estimation of the lower concentrations of GOLD in
tissue proteins. MOLD and GOLD together account for only 0.2% of
chemical modification of lysine residues in a senescent lens (~0.2
mmol of GOLD + 0.8 mmol of MOLD/mol of Lys at age 80 × 2 mol of
Lys/cross-link). In contrast CML and CEL are present at nearly 10-fold
higher concentrations (~4 mmol of CML + 4 mmol of CEL/mol of lysine),
representing 0.8% of lysine modification (21). Regardless of their low
concentrations, MOLD and GOLD are present at significantly higher
concentrations in lens proteins than the fluorescent cross-links
pentosidine and dityrosine. At age 80, MOLD, GOLD, pentosidine, and
dityrosine represent approximately 800, 200, 4, and 3 µmol/mol of
lysine in lens protein (37, 38). In our 85-year-old skin collagen pool,
MOLD and GOLD are present at 400 and 40 µmol/mol lysine, respectively, compared with 40 µmol pentosidine/mol lysine (24). At
100 mol of Lys/mol of triple stranded collagen, MOLD is present at 0.04 mol/mol of collagen (0.08% of lysine residues). In contrast, enzymatically formed cross-links are much more abundant (1-5 mol/mol of collagen) (39). Thus, it is unlikely that GOLD or MOLD would have a
significant impact on collagen cross-linking in aging, unless they are
present at critical sites in the collagen molecule.
Our proposed mechanism for imidazolium cross-link formation includes
the initial reaction of the dicarbonyl with two lysine molecules
forming a labile Schiff base, diimine cross-link, and then recruitment
of a second molecule of MGO for the cyclization reaction (27, 28).
Glomb and Monnier (40) have described the formation of a diimine
structure in reactions of protein with GO in vitro. Yim
et al. (41) also propose that the reaction of dicarbonyls
with amino groups yields radical intermediates, including a dicarbonyl
dialkylamine radical cation and a dicarbonyl radical anion, which then
react to form the stable advanced glycation end products. Although it
is possible that GOLD and MOLD were among the major advanced glycation
end products formed in these and our in vitro studies in the
presence of an excess of dicarbonyl compounds, it is likely that in
biological systems at low GO and MGO concentrations, other aldehydes,
including formaldehyde, acetaldehyde, glucose, or glycolytic
intermediates would serve as carbon donors for the cyclization
reaction. Thus, the pathway to formation of GOLD and MOLD in
vivo may be unusually complex and difficult to unravel. As we and
others have shown, there are many possible routes and precursors of CML
and CEL in vitro (5, 21, 37, 40, 42), and it is possible
that other precursors, e.g. ascorbate or polyunsaturated
fatty acids in lipids, are more significant sources of GOLD and MOLD
in vivo.
In our studies on the cross-linking of RNase by MGO, we show that MOLD
is involved in both intermolecular and intramolecular cross-linking
reactions. MOLD accounts for only a small percentage (~5%) of the
cross-links in the dimer fraction of RNase (Fig. 6), indicating that
there are other unidentified cross-links, possibly involving amino
acids other than lysine. The ratio of carboxyalkyllysines:imidazolium
salts and of intramolecular:intermolecular cross-links by imidazolium
salts undoubtedly varies with protein structure and the packing density
of protein monomers. RNase has three lysine residues (1, 7, 41)
clustered in or near the active site of the protein, which are also
major sites for modification of the protein by glucose (43). The
adjacency of these lysine amino groups may favor intramolecular
cross-linking of the protein. In other proteins, glycation occurs
preferably at lysine-lysine sequences (44), which should favor
intramolecular cross-linking, or at lysine residues in other basic
amino acid sequences (45, 46) and at lysine residues apposed to acidic amino acids (46, 47), which may favor intermolecular cross-linking. Proteins on membrane surfaces, which are packed more densely than soluble proteins, may also be more susceptible to intermolecular cross-linking reactions. The close packing may also accelerate the
age-dependent, intermolecular cross-linking, aggregation, and insolubilization of lens proteins during the Maillard reaction. Studies on the distribution of GOLD and MOLD in various lens protein fractions and on the specificity of carboxyalkylation and cross-linking of proteins by GO and MGO should provide insight into the role of these
reactions in alteration of tissue proteins in aging and disease.
We thank Dr. William E. Cotham, University of
South Carolina Mass Spectrometry Laboratory for conducting the mass
spectrometry analyses.