[6,30], these analyses were only carried out over the time frame prior to the formation of significant levels of lipid and protein oxidation products i.e.. Minor increases in electrophore
Trang 1Glycation and glycoxidation of low-density lipoproteins by glucose and low-molecular mass aldehydes
Formation of modified and oxidized particles
Heather M Knott*, Bronwyn E Brown, Michael J Davies and Roger T Dean†
The Heart Research Institute, Camperdown, Australia
Patients with diabetes mellitus suffer from an increased
incidence of complications including cardiovascular disease
and cataracts; the mechanisms responsible for this are not
fully understood One characteristic of such complications
is an accumulation of advanced glycation end-products
formed by the adduction of glucose or species derived from
glucose, such as low-molecular mass aldehydes, to proteins
These reactions can be nonoxidative (glycation)or oxidative
(glycoxidation)and result in the conversion of low-density
lipoproteins (LDL)to a form that is recognized by the
scavenger receptors of macrophages This results in the
accumulation of cholesterol and cholesteryl esters within
macrophages and the formation of foam cells, a hallmark of
atherosclerosis The nature of the LDL modifications
required for cellular recognition and unregulated uptake are
poorly understood We have therefore examined the nature,
time course, and extent of LDL modifications induced by
glucose and two aldehydes, methylglyoxal and
glycolalde-hyde It has been shown that these agents modify Arg, Lys
and Trp residues of the apoB protein of LDL, with the extent
of modification induced by the two aldehydes being more rapid than with glucose These processes are rapid and unaffected by low concentrations of copper ions In contrast, lipid and protein oxidation are slow processes and occur to a limited extent in the absence of added copper ions No evi-dence was obtained for the stimulation of lipid or protein oxidation by glucose or methylglyoxal in the presence of copper ions, whereas glycolaldehyde stimulated such reac-tions to a modest extent These results suggest that the earliest significant events in this system are metal ion-independent glycation (modification)of the protein component of LDL, whilst oxidative events (glycoxidation or direct oxidation of lipid or proteins)only occur to any significant extent at later time points This carbonyl-stress may facilitate the forma-tion of foam cells and the vascular complicaforma-tions of diabetes Keywords: AGE; atherosclerosis; glycolaldehyde; methyl-glyoxal; protein modification
The correlation between diabetes and cardiovascular disease
(CVD)has been well established [1], although the precise
mechanisms that facilitate the many complications
associ-ated with diabetes, including CVD and cataracts, are poorly
understood Uncontrolled plasma glucose concentrations and the ability of glucose to either oxidatively or nonoxid-atively (covalently)modify proteins have been proposed to
be instrumental in the development of CVD in both the insulin-deficient and insulin-resistant forms of diabetes [2–4] Both free and protein-bound glucose are known to undergo nonenzymatic and enzymatic modifications which can result in the formation of low molecular mass aldehydes such as methylglyoxal (MG), glyoxal, and glycolaldehyde (GA) These aldehydes form adducts with Lys and Arg residues resulting in Schiff base formation, Amadori rearrangements, and the formation of advanced glycation end products (AGEs)[5–9] Thus, reaction of GA with Lys results in the formation of the well characterized AGE carboxymethyllysine whilst reaction of MG with Lys gives carboxyethyllysine [10,11] The levels of these small reactive a-dicarbonyls are known to be elevated in diabetics [12–14] and the accumulation of AGEs has been implicated in the pathogenesis of diabetes and ageing [4,7]
Modification of low-density lipoprotein (LDL)can lead
to alteration of the apoB protein to the extent that it is no longer recognized by the regulated cholesterol-feedback receptors [15] Instead, this modified LDL is taken up via scavenger receptors leading to cholesterol and cholesteryl ester loading of macrophages [16]; this is believed to be the primary step in foam cell formation and the development of
Correspondence to Heather M Knott, Centenary Institute of Cancer
Medicine and Cell Biology, Locked Bag Number 6, Newtown,
Sydney, NSW 2042, Australia.
Fax: +61 2 9565 6101, Tel.: +61 2 9565 6156,
E-mail: h.knott@centenary.usyd.edu.au
Abbreviations: CVD, cardiovascular disease; AGE, advanced
gly-cation end-products; GA, glycolaldehyde; MG, methylglyoxal; LDL,
low-density lipoprotein; DOPA, 3,4-dihydroxyphenylalanine;
m-Tyr, m-tyrosine (3-hydroxyphenylalanine); o-Tyr, o-tyrosine
(2-hydroxyphenylalanine); PTQ, phenanthrenequinone; TOH,
a-tocopherol; FC, free cholesterol; CA, cholesteryl arachidonate;
CL, cholesteryl linoleate; 7K, 7-ketocholesterol; apoB, apolipoprotein
B100; CEO(O)H, cholesteryl ester hydro(pero)xides; REM,
relative electrophoretic mobility.
*Present address: Centenary Institute of Cancer Medicine and Cell
Biology, Newtown, Sydney, Australia.
Present address: University of Canberra ACT 2601 Australia.
(Received 17 February 2003, revised 19 June 2003,
accepted 8 July 2003)
Trang 2atherosclerosis [17] Furthermore, the two- to threefold
increase in nonenzymatic glycosylation of serum albumin in
hyperglycaemia has been suggested to alter the antioxidant
(radical scavenging)role of this protein which may, in
concert with increased levels of redox-active copper and iron
levels [18], contribute to the complications of diabetes [19]
In this study the nature, time course, and extent of the
covalent and oxidative changes that occur on LDL particles
exposed to glucose, GA, and MG have been quantified in
order to determine the significance of glycation and
glycoxidation to the pathogenesis of atherogenic
complica-tions related to diabetes
Materials and methods
Materials
All solutions were prepared with nanopure water (Milli Q
system, Millipore-Waters)and treated with washed
Chelex-100 (Bio-Rad)to remove transition metals prior to use [20]
PD-10 columns (Sephadex G-25M)were from Amersham
Biosciences Pre-cast 1% agarose gels were from Helena
Laboratories (Mt Waverly, VIC, Australia) Fatty
acid-free BSA, fluorescamine, MG, GA, glucose, arginine, and
lysine were all from Sigma-Aldrich All other chemicals
were of analytical grade and all solvents were of HPLC
grade
Isolation of LDL
LDL (density 1.019–1.063 gÆmL)1)was prepared from
plasma of fasted, healthy volunteers by density gradient
ultracentrifugation as described previously [21] After
iso-lation, LDL was dialysed against four to five 1 L changes of
degassed NaCl/Pi containing 0.1 mgÆmL)1
chlorampheni-col, filter sterilized, and used immediately in most cases
When necessary, dialysis buffers were supplemented with
1 mgÆmL)1EDTA and the LDL stored until required In
the latter case, EDTA was removed immediately prior to
use by either dialysis (as above)or by passage of LDL
(<2 mL)through two PD-10 columns as per the
manu-facturer’s instructions; NaCl/Pi was used as both the
equilibration and elution buffer Removal of the EDTA
was confirmed by carrying out a small-scale oxidation of
0.4 mgÆmL)1LDL with 10 lMCuSO4for 2–3 h at 37C
After this incubation, a wavelength difference spectrum was
acquired using the control (no copper ion)sample as a
blank Gross changes in the spectrum, particularly the loss
of the carotenoid peak at 450 nm and changes in the
absorbance at 234 nm, were accepted as indicative of
susceptibility to oxidation [22] If such changes were not
observed, the LDL was subject to further dialysis or
rechromatographed Oxidation assays were only performed
using LDL tested in this manner
Data analysis
For the lipid and protein oxidation data, the variation in
absolute data obtained from LDL extracted from different
donors, containing different levels of antioxidants and
pre-existing lipid peroxides and hence different lag phases [22],
made collation of all data impractical This variation results
in differing absolute kinetics, though the trends with each donor were the same Therefore, although all data were analysed, we present here that from a single experiment representative of all with each data point representing the average of two samples and the error bars representing standard deviation (half-range) The protein modification data are presented as mean ± SEM of data from four samples and normalized to the control (no added modifier)
at each time point after initial assessment indicated minimal variation in the zero time raw data Statistics were performed by one-way analysis of variance (ANOVA)with Tukey’s multiple comparison post-test analysis using
GRAPHPAD PRISM (version 3.0a for MacIntosh, GraphPad Software, San Diego, California, USA) For each compar-ison, statistical significance was set at P < 0.05 unless stated otherwise For each of the protein modification assays, control experiments were performed to exclude the possibility that direct interference between the fluorescamine
or phenanthrequinone and either aldehyde contributed to the changes in fluorescence observed
Protein assays LDL protein concentration was measured by the bicin-choninic acid (BCA; Pierce)method using 0.4 mgÆmL)1 BSA as a standard, with incubations performed at 60C for 45–60 min
Glycoxidation of LDL Glucose, MG, and GA stock solutions were prepared immediately prior to use and filter-sterilized EDTA-free LDL was diluted to 350–450 lgÆmL)1 in filter-sterilized NaCl/Picontaining the required concentration of aldehyde and/or copper ions and incubated at 37C under 5% (v/v)
CO2 The screw caps on the incubation vials were loosely fastened to enable oxygen ingress without compromising the sterility of the sample
Lipid extraction and analysis
At the required times, 0.1 mL samples were collected aseptically into tubes containing 10 lL 20 mM EDTA,
10 lL 0.2 mM butylated hydroxytoluene (BHT), and
400 lL nanopure water (or alternatively 0.2 mL sample/
20 lL EDTA/20 lL BHT/300 lL water)and the samples mixed immediately One mL methanol and 5 mL hexane were added and the samples were mixed thoroughly Extracts were stored at )20 C until use Samples were centrifuged at 2060 g for 5–10 min, 4 mL of the hexane layer was removed and evaporated to dryness and the residue was resuspended in 200 lL of isopropanol Lipid content was analysed by reversed-phase HPLC on a Shimadzu system with a Supelco ODS LC 18 column (25· 0.46 cm, 5 lm particle size)and a 2 cm Pelliguard guard column The mobile phase, ethanol/methanol/iso-propanol at 19.7 : 6 : 1 (v/v/v), was degassed immediately prior to use Lipophilic components were separated iso-cratically at 1 mLÆmin)1 over 30 min with the eluent monitored using a diode array UV detector (PDA-M10AVP)set at 205 and 234 nm and a RF-10AXL fluorescence detector (for TOH; k 290 nm and k
Trang 3330 nm) Metabolites were quantified by comparison with
standards and expressed as nmolÆmg)1apoB protein
Protein isolation and analysis
Samples (0.5 mL)of the LDL incubations were collected
aseptically into 50 lL 20 mM EDTA and 50 lL 0.2 mM
BHT and mixed thoroughly Samples were subsequently
stored at)20 C before further processing Protein samples
were reduced by addition of 10 lL 10 mgÆmL)1NaBH4and
incubation at room temperature for >30 min The samples
were delipidated by the addition of 100 lL 0.3% (w/v)
sodium deoxycholate, precipitated using 50 lL 50% (w/v)
trichloroacetic acid, and centrifuged at 4000 g for 2 min
After removal of the supernatant, the protein pellet was
washed twice with ice-cold acetone and once with diethyl
ether, with the samples centrifuged (6610 g, 1 min)and the
supernatant discarded after each addition The samples
were then evaporated to dryness and subjected to gas-phase
hydrolysis in Pico-Tag vessels (Waters)containing 1 mL
6MHCl and 50 lL 2-mercaptoacetic acid The vessels were
sealed under vacuum and incubated at 110C overnight
After hydrolysis, the samples were dried, resuspended in
200 lL nanopure water, filtered through 0.22 or 0.45 lm
membranes, and analysed by HPLC
Analysis of oxidized amino acids was performed by
reversed-phase HPLC using UV and fluorescence
detec-tion as described previously [23] Quantificadetec-tion was
performed by integration of peak areas and comparison
with standards Oxidized amino acid levels are expressed
as lmol per mol parent amino acid to account for any
losses during sample preparation o-Tyr data was
con-verted from lmolÆmol)1Tyr to lmolÆmol)1Phe using the
abundance of these amino acids in the apoB100 molecule
(152 Tyr/223 Phe) Preliminary experiments that
quanti-fied the consumption of Tyr under the incubation
conditions used indicated that the (low)loss of Tyr had
negligible impact on this calculation (data not shown)
Previous studies using this methodology have demonstrated
minimal artefactual oxidation during such sample
hand-ling and analysis [23,24]
Relative electrophoretic mobility gels
Samples (10–15 lL)were removed from the LDL
incuba-tions and loaded onto precast 1% (w/v)agarose gels Native
and acetylated LDL (Ac-LDL, prepared as described
previously [25])were loaded as negative and positive
controls, respectively, and the samples were run at 90 V
for 45 min Gels were fixed in 100% methanol (1 min),
stained for 5–10 min with Fat Red 7B (Sigma-Aldrich),
destained for 5–10 min with 70% methanol, and dried at
60C The relative electrophoretic mobility (REM)is
defined as the ratio of the distances travelled by modified
LDL and native LDL
Tryptophan consumption
Samples (0.2 ml)were removed from the LDL incubations,
0.1 vol each of 20 mM EDTA and 0.2 mM BHT were
added, and the volume adjusted to 1 mL with NaCl/Pi Trp
fluorescence was measured on a Perkin Elmer
Lumines-cence LS50B Spectrometer with kex 280 nm and kem
335 nm
Lysine consumption Samples (80 lL)were removed from the LDL incubations and 0.1 vol each of 20 mMEDTA and 0.2 mM BHT and
670 lL borate buffer (pH > 8)were added During vortex mixing, 250 lL 0.15 mgÆmL)1fluorescamine dissolved in acetone was added The fluorescence of these samples, representative of free amine groups, was measured using kex
390 nm and kem475 nm and expressed as a percentage of the zero time value for the control (no added aldehyde/ glucose)samples [26]
Arginine consumption One-tenth vol each of 20 mM EDTA and 0.2 mM BHT were added to 0.25–0.50 mL of the LDL incubation and the volume adjusted to 1 mL with NaCl/Pi Three millilitres
120 lM 9,10-phenanthrenequinone (PTQ; in absolute eth-anol)was added and the reaction initiated by addition of 0.5 mL 2M NaOH with the samples then incubated at
60C for 3 h At both t ¼ 0 and t ¼ 3 h, 0.5 mL samples were removed and the reaction stopped by addition of an equal volume of 1.2MHCl The fluorescence was recorded using kex312 nm and kem392 nm with the Arg-dependent fluorescence calculated as the difference between the t¼ 0 and t¼ 3 h samples [27]
Results
Glucose-mediated oxidation of LDL Lipid oxidation Six different parameters of lipid peroxi-dation were measured (Fig 1A–F) Three concentrations
of glucose representing normal (5 mM), pathological (25 mM), and suprapathological (100 mM)levels with
1 lMCu2+were examined, as well as samples containing
1 lMCu2+alone, 100 mMglucose only, and no additions The zero time levels of all lipids and products correlate well with published plasma data (e.g [28])and are not statistically different between experiments The oxidation
of the lipid moieties of LDL in the presence of glucose and 1 lM Cu2+proceeded slowly with complete destruc-tion of a-tocopherol (TOH; Fig 1A)occurring between
1 and 2 w These results correlate with loss of free cholesterol (Fig 1B, FC), cholesteryl arachidonate (Fig 1C, CA), and cholesteryl linoleate (Fig 1D, CL)
by 2 weeks as well as the accumulation of the cholesterol oxidation product, 7-ketocholesterol (Fig 1E, 7K), to a maximum of 200 nmolÆmg)1 apoB at 4 w Cholesteryl ester hydroxide and hydroperoxide accumulation (Fig 1F, CEO(O)H) was not detected for the Cu2+-containing samples; this presumably reflects the production and subsequent rapid degradation of these unstable products With 100 mM glucose alone and with the incubated control (no addition)samples, oxidation proceeded more slowly than in any of the samples containing 1 lMCu2+
A small acceleration of the rate of oxidation was detected
in the 100 mM glucose samples compared with the controls at 2 and 4 weeks in all the parameters measured,
Trang 4with the exception of FC and 7K In these samples, no
conversion of FC to 7K was detected, whilst TOH was
not fully depleted until 7 weeks in the presence of 100 mM
glucose, with a corresponding 84% loss in the control
Due to the lower rate of oxidation of these samples,
accumulation of CEO(O)H was detected and reached a
maximum (170 nmolÆmg)1 apoB)by 4 weeks The small
increases in the rate of oxidation observed in the presence
of 100 mM glucose compared with the controls is
consistent with a very low rate of radical formation in
the absence of added metal ions such as Cu2+ No additive effect of the various glucose concentrations over the rate of oxidation induced by Cu2+ alone was discernible
Protein oxidation and modification The formation of the Tyr and Phe oxidation products, DOPA and o-Tyr, were examined in identical incubations to those described above The levels of these products are shown in Fig 1G and H Zero time levels of DOPA (900 lmolÆmol)1Tyr)and o-Tyr (550 lmolÆmol)1Phe)are in accord with literature data for plasma [23] and not statistically different between prepara-tions By week 2, accumulation of both DOPA (Fig 1G, four- to sixfold increase)and o-Tyr (Fig 1H, seven- to eightfold increase)was evident in all samples containing added Cu2+, independent of the glucose concentration with
no statistically significant change in the two controls The levels of DOPA detected tended towards a decrease at longer incubation times, consistent with the further oxida-tion of this material to undetected indolic materials [29]
To complement the above markers of oxidative damage
to apoB, analysis of the potential loss of other amino acid side chains (Trp, Lys and Arg), that would be expected to be targets of glycation reactions (i.e covalent modification), was quantified The loss of each of these side chains was examined using fluorescence spectroscopy and in each case changes were measured relative to control (no addition) samples Care was taken to eliminate any possible inter-ference from the added reagents As previous data has suggested that glycation reactions are rapid (e.g [6,30]), these analyses were only carried out over the time frame prior to the formation of significant levels of lipid and protein oxidation products (i.e up to 2 weeks, see above)
No evidence was obtained for significant consumption of Trp or Lys residues under any of the incubation conditions studied (data not shown)indicating that neither glucose nor low levels of Cu2+induce significant Trp or Lys modifica-tion Furthermore the absence of any peak shifts in the Trp fluorescence spectra imply only minor, if any, structural changes in the vicinity of these residues, as the fluorescence
of this residue is environment dependent Changes in the level of Arg were detected with high, but not low, concentrations of glucose with this being independent of the presence of added Cu2+ Thus a loss of 67% of Arg residues was detected after 14 days’ incubation with
100 mMglucose when compared with incubated controls;
no significant loss of Trp or Lys residues was detected under these conditions (data not shown)
The relative electrophoretic mobility of the modified LDL particles was also examined using agarose gels, as this technique yields information on the overall net positive charge on the particle (i.e the total contribution of Arg, Lys, and protonated His residues together with the N terminus, relative to Glu, Asp, and the C terminus) Minor increases
in electrophoretic mobility were evident for the samples incubated with low concentrations of glucose (data not shown), though these were only 1.2–1.3-fold greater than native (nonincubated)LDL, and similar changes were detected with the incubated controls These minor changes were Cu2+-independent and consistent with minor extents
of modification of Lys and Arg residues With samples incubated with 100 m glucose for 14 days a small increase
Fig 1 Glucose-mediated oxidation of the lipid and protein moieties of
LDL LDL ( 0.4 mg proteinÆmL)1)was incubated with varying
concentrations of glucose and/or copper ions At the indicated times,
0.2 mL samples were removed and extracted with 1 mL methanol and
5 mL hexane Four millilitres of the hexane fraction was then dried to
completion, resuspended in 200 lL isopropanol, and the levels
(expressed as nmolÆmg LDL protein)1)of TOH (A) , FC (B) , CA (C) ,
CL (D), 7-KC (E), and CEO(O)H (F) were determined by HPLC (see
Materials and methods) Concurrently, 0.5 mL samples were removed,
delipidated, hydrolysed, and the resulting oxidized and parent amino
acids quantified by reversed-phase HPLC The levels of the oxidized
amino acids DOPA (G)and o-Tyr (H)are expressed relative to their
parent amino acids, Tyr and Phe, respectively Each data point
rep-resents the mean (± SD)of two replicates from a single experiment
representative of several Black bars, 100 m M glucose + 1 l M Cu 2+ ;
horizontal striped bars, 25 m M glucose + 1 l M Cu2+; dark stippled
bars, 5 m M glucose + 1 l M Cu 2+ ; light stippled bars, 100 m M glucose
only; white bars, 1 l M Cu 2+ only; diagonal striped bars, control (no
addition)incubations Asterisks indicate the first time point at which
the data becomes statistically different when compared with the t ¼ 0
value.
Trang 5in REM was detected but this was not significantly different
from the incubated controls (data not shown) Acetylated
LDL samples run under identical conditions as positive
controls gave REM values of 3–4
MG-mediated oxidation of LDL
Lipid oxidation LDL was incubated with a wide range of
MG concentrations (10 lM)100 mM)in both the absence
and presence of low concentrations of added Cu2+(1 lM)
No evidence for lipid oxidation was obtained up to the
longest time period studied (17 days, data not shown) That
is, there was no change in the level of TOH, no loss of
cholesterol or the cholesteryl esters examined (CA, CL), and
no accumulation of CEO(O)H or 7K (data not shown)
Protein oxidation and modification As the above lipid
data was negative, the generation of DOPA and o-Tyr,
which are radical-mediated products [31,32], was not
examined Protein modification by MG was examined by
quantifying the loss of Trp, Lys, and Arg residues and the
changes in REM using the same incubation conditions as
described above Fig 2 shows the data obtained, expressed
as a percentage of control incubations (no added MG) At
the initial time point there was a significant decrease in the
measurable level of Trp in the presence of 100 mM MG
(P < 0.001)while there was no statistical difference
between any of the other conditions No statistically
significant changes in Trp levels were detected with low
concentrations of MG (10 and 100 lM) With higher
concentrations (10 and 100 mMMG)a significant decrease
in the level of this residue was observed by day 5 (Fig 2A);
this decrease occurred in the absence of added Cu2+but
was dependent on the MG concentration (by 1 day the
difference between 100 mM and 10 mM is significant to
P< 0.01) The concentration of Lys residues was not
significantly different at zero time Significant Lys loss
(Fig 2B)was detected by 1 day in the samples containing
either 100 or 10 mMMG (P < 0.001 relative to all other
samples)and at this time has occurred to a greater extent in
the 100 mMthan in the 10 mMMG samples (P < 0.001),
indicating that that the loss of this residue is dependent on
the concentration of MG In the experiments using 100 lM
MG, loss of lysine was only seen in the samples containing
1 lMCu2+(P < 0.05 relative to other samples)and not
until day 14 (P < 0.01 relative to previous time points)
Whilst this is suggestive of a copper-ion dependence it is not
supported by any of the other data
Modification of Arg residues by MG has also been
quantified and the data obtained is presented in Fig 2C
(expressed relative to control samples at each time point,
with the latter set to 100%) With low concentrations of
MG (10 lM or 100 lM)no significant changes were
observed in the presence or absence of added copper ions,
except with 100 lMMG in the absence of Cu2+from day 5
onwards With higher (millimolar)concentrations, rapid
concentration-dependent loss of this side-chain was detected
even in the absence of Cu2+(P < 0.05 by 1 day for both 10
and 100 mMMG); this is in accord with a previous report
[33] Loss of Arg was particularly rapid, with significant
losses observed immediately after mixing (i.e in the t¼ 0
samples)with the 100 m MG samples (P < 0.01 relative
to other samples at time zero) No additional loss of Arg was observed at later time points For the samples containing 10 mM MG, a significant (P < 0.01)loss of arginine was seen at 1 day with, again, no further losses during the remainder of the time course
As expected on the basis of the above data, changes in the REM of LDL incubated with MG (conditions as above) were detected (Fig 2D) For the sake of clarity, data for native LDL (REM set to 1)and AcLDL (REM > 3)are not shown With 10 lMMG, minor changes occurred after
5 days of incubation and increased at subsequent time points (approximately twofold relative to native LDL by
14 days) More pronounced and more rapid changes were detected with higher concentrations of MG in the absence of added Cu2+
GA-mediated oxidation of LDL Lipid oxidation LDL was incubated with 0, 0.1, or 1 mM
GA with and without added 1 lMcopper ions Fig 3 shows typical data; similar trends were observed with other
Fig 2 MG-mediated modification of apoB LDL ( 0.4 mg proteinÆmL)1)was incubated with 10 or 100 l M MG with and without added 1 l M Cu2+or LDL ( 1 mg proteinÆmL)1)was incubated with
10 or 100 m M MG in the absence of Cu 2+ Trp residues (A)were quantified by fluorescence (k ex 280 nm, k em 335 nm)after dilution in NaCl/P i Lys residues (B)were quantified by fluorescence (k ex 390 nm,
k em 475 nm)after dilution in borate buffer (pH > 8)and derivatiza-tion with fluorescamine Arg residues (C)were quantified by fluores-cence (k ex 312 nm, k em 392 nm)after derivitization with 9,10-phenanethrequinone For further details see Materials and methods Data are means (n ¼ 4)± SEM For REM analysis (D), 10–15 lL samples were loaded onto precast 1% (w/v)agarose gels, subjected to electrophoresis, and the distance migrated relative to native LDL (REM set to 1)calculated Data are expressed as a per-centage of control incubations with no added MG Black bars,
100 m M MG; horizontal striped bars, 10 m M MG; dark stippled bars,
100 l M MG + 1 l M Cu 2+ ; light stippled bars, 100 l M MG; white bars, 10 l M MG + 1 l M Cu2+; diagonal striped bars, 10 l M MG Asterisks indicate the first time point at which the data becomes statistically different when compared with the t ¼ 0 value.
Trang 6concentrations of GA (data not shown) No significant
differences were seen for any of the lipid peroxidation
parameters between treated and nontreated samples at the
zero time point By 15 days, the most rapid lipid
peroxi-dation occurred in those samples containing 1 mM GA
supplemented with 1 lM copper ions In this case, TOH
(Fig 1A)and CA (Fig 1C)were completely consumed and
CL (Fig 1D)partly consumed ( 70% and 20%,
respectively)with a concomitant accumulation of
CEO(O)H (Fig 1E) (loss of TOH significant at 7 days,
loss of CA and accumulation of CEO(O)H significant at
12 days, and loss of CL significant at 15 days The two
other Cu2+-containing conditions showed significant
changes in these parameters at day 15, except in the case
of consumption of CL which did not decline at any time
point In no case was there any statistically significant
change in the level of FC (Fig 1B)nor any accumulation of
7K (data not shown) No statistically significant changes
were observed with the Cu2+-free conditions, suggesting
that Cu2+ acts as a catalyst for GA-mediated LDL
oxidation Experiments were performed to exclude the
possibility of GA and Cu2+competing for the same sites on
LDL and therefore interfering with the ability of each agent
to initiate oxidation In these experiments, incubations were
carried out as normal but either the GA or Cu2+were left
out initially and then added after 1 h (an arbitrary time
frame anticipated to enable binding of either oxidant to the
surface of the LDL) In these experiments, the order of preincubation had no impact on the rate or extent of oxidation (data not shown)suggesting that competition for particular sites on the LDL particle was not affecting the extent of lipid oxidation
Protein oxidation and modification As the above experi-ments demonstrated little lipid oxidation at short incuba-tion times, it was expected that protein oxidaincuba-tion might also
be modest This was confirmed by the examination of DOPA and o-Tyr formation as a result of the incubation of LDL with either 0.1 or 1 mMGA with and without added
Cu2+ over 4 days; no significant generation of either material was detected under these conditions (data not shown)
Protein modification was examined in LDL samples incubated with 1, 10, and 100 mM GA alone (with
1 mgÆmL)1 LDL)and 1 mM GA with added Cu2+ (1 lM) The data obtained are presented in Fig 4 and expressed as percentage of control samples (no added GA)
A rapid and extensive loss of Trp fluorescence was detected with high concentrations of GA that was time- and concentration-dependent (Fig 4A)and these changes occurred in the absence of Cu2+; for 10 and 100 mMGA loss of Trp was significant at 1 day, with higher loss of Trp
at this time point occurring in the presence of 100 mMGA For the lower concentrations of GA, losses did not become significant until 7 days when compared with time zero
Fig 3 GA-mediated oxidation of LDL lipids and alpha-tocopherol.
LDL ( 0.4 mgÆmL protein)1)was incubated with 0, 0.1 or 1 m M GA
with and without 1 l M Cu 2+ ; control samples were incubated with
1 l M Cu2+alone At the indicated time points, 0.2 mL samples were
removed and the levels of TOH (A) , FC (B) , CA (C) , CL (D) , and
CEO(O)H (E) quantified as indicated in the legend to Fig 1 Each data
point represents the mean (± SD)of two replicates from a single
experiment representative of several Black bars, 1 m M GA + 1 l M
Cu 2+ ; horizontal striped bars, 1 m M GA; dark stippled bars, 0.1 m M
GA + 1 l M Cu2+; light stippled bars, 0.1 m M GA; white bars,
1 l M Cu2+ Asterisks indicate the first time point at which the data
becomes statistically different when compared with the t ¼ 0 value.
0 1 7 14
Time (days)
0 20 40 60 80 100 120
0 1 7 14
Time (days)
0 20 40 60 80 100 120
0 1 7 14
Time (days)
0 20 40 60 80 100 120
140
0 1 7 14
Time (days)
0 1 2 3 4
5 6 140
A
*
*
*
C
*
B
D
*
*
*
*
*
Fig 4 GA-mediated modification of apoB LDL ( 0.4 mg proteinÆ
mL)1)was incubated with 1 m M GA ± 1 l M copper ions or LDL ( 1 mg proteinÆmL)1)was incubated with 1, 10 and 100 m M GA Quantification of Trp (A), Lys (B)and Arg (C)residues was carried out as described in the legend to Fig 2 The data are mean (n ¼ 4)± SEM REM analysis (D)was carried out as described in the legend to Fig 2 Black bars, 1 mg LDL proteinÆmL)1with 100 m M
GA; horizontal striped bars, 1 mg LDL proteinÆmL)1with 10 m M
GA; dark stippled bars, 1 mg LDL proteinÆmL)1with 1 m M GA; light stippled bars, 0.4 mg LDL proteinÆmL)1 with 1 m M GA + 1 l M
Cu 2+ ; white bars, 0.4 mg LDL proteinÆmL)1with 1 m M GA only Asterisks indicate the first time point at which the data becomes sta-tistically different when compared with the t ¼ 0 value.
Trang 7Similar behaviour was observed for Lys (Fig 4B) In this
case, an effect of Cu2+could be seen) the high
concentra-tion GA samples, and those containing Cu2+, each showed
statistically significant (relative to zero time)loss of Lys
residues by day 1, while those samples containing 1 mMGA
with no added Cu2+did not show significant loss of Lys
until day 7 The effect of GA on the Arg residues of apoB
is shown in Fig 4C; these data are expressed relative to the
controls at each time point and zero time values of all
samples (data not shown)were compared to exclude the
possibility of interference by GA with the assay In the
presence of 100 mMGA, 50% of the Arg residues were
observed to be lost in the samples examined immediately
after mixing (t¼ 0 samples)and no further loss was
observed at longer time points In none of the other samples
was any statistically significant loss of Arg residues detected
Fig 4D shows the changes in REM for analogous LDL
incubations Native LDL and AcLDL were also examined
but these data are not shown for reasons of clarity; the
values obtained for these materials were within the expected
range (see above) Incubations with 10 or 100 mM GA
showed maximal changes in REM as early as 24 h (up to
4.8-fold increase), with little increase after this time, while
the lower concentrations of GA facilitated small increases
by 24 h ( 1.8-fold)increasing to 2.7-fold by 14 days
These changes in LDL mobility also occurred in the absence
of added copper ions
Discussion
Although there is a direct parallel between increased
blood sugar and the clinical state of diabetes, and
extensive support for the theory that increased oxidative
stress is involved in this pathology [4,7], detailed studies
on the processes of glycation and glycoxidation have not
provided a conclusive causative mechanism (or
mech-anisms)for the damage induced by high glucose
concen-trations It has been clearly established that products of
metal ion catalysed oxidation of glucose and
protein-bound glucose (i.e glycoxidation)can accumulate at
elevated levels on proteins from diabetic patients, as do
products of direct covalent modification (glycation)
[3,4,7] Whether one or both of these processes is
causative in the development of the complications of
diabetes, such as atherosclerosis, is less well established
[4] It is therefore pertinent to establish the relative roles
of both oxidative processes and direct glycation (covalent,
nonoxidative)reactions in the development of
atheroscle-rosis, in particular the modification of LDL which might
promote the formation of lipid-laden (foam)cells in the
artery wall; a hallmark of early atherogenesis Several
studies have reported elevated levels of oxidized- and/or
AGE-modified LDL in diabetic subjects [34–36] and in
human atherosclerotic lesions [37] It has also been
reported that glycoxidized and peroxidized LDL
colocal-ize with the macrophage scavenger receptor [38]
indicat-ing a plausible involvement of the accumulation of AGEs
and increased oxidative stress in this pathology
Further-more, protein-bound sugars have been demonstrated to
generate free radicals (particularly in the presence of
metal ions [39]), which could potentiate further damage,
including lipid peroxidation [40–42], while the
suscepti-bility of LDL to Cu2+-induced oxidation has been shown
to increase in the presence of glucose [43,44]
In addition to the hyperglycaemia seen in poorly controlled diabetic patients, and its potential involvement
in the accumulation of AGEs, a number of studies have examined the role of low molecular mass aldehydes such as glyoxal, MG, and GA These materials are formed both as a consequence of oxidative processes and AGE modifications
of proteins as well as by a variety of nonrelated metabolic processes [45,46] Evidence has been presented for an elevated level of these materials (or products arising from them)in diabetics [47,48] and the presence of antibodies to MG-derivatized proteins in corneal collagen and plasma proteins [49] We have therefore carried out a comprehen-sive analysis of the relative efficacies of glucose and two aldehydes (MG and GA)in inducing lipid and protein oxidation and antioxidant depletion of LDL particles as well as glycation of the apoB protein by measuring specific parameters of these processes As previous workers [44,50,51] have presented data demonstrating that lipid peroxidation and protein modification of LDL by glucose can be dependent on the presence of transition metal ions, studies were also carried out in the presence of low levels of
Cu2+ This transition metal ion dependence may indicate a cooperative effect of glucose and Cu2+as glucose has been reported to increase Cu2+-induced LDL oxidation without affecting oxidation by aqueous peroxyl radicals [52] Glu-cose- and transition metal ion-dependent protein oxidation has also been detected in studies on rat tail collagen as measured by accumulation of the specific protein side chain oxidation products DOPA, m-Tyr, di-Tyr, and Leu and Val alcohols [53]
In the current study it has been shown that the time course of oxidation of antioxidants, lipids, and protein side chains in LDL is slow in the presence of glucose alone, though marginally faster than in control samples with no additions The time course of oxidation was much more rapid in the presence of low concentrations of Cu2+, compared with its absence, but there were no significant differences between the samples treated with Cu2+alone compared with those containing Cu2+ plus any of the glucose concentrations This suggests that the observed reactions are primarily due to oxidation catalysed by Cu2+ alone and that glucose does not play a major role in these reactions over the concentration range studied, the highest
of which is well in excess of that observed even in very poorly controlled diabetics It has been shown that the oxidation of LDL by Cu2+is saturable, due to the limited number of high-affinity Cu2+-binding sites on the LDL particle [54], but the experiments performed here were carried out with Cu2+: LDL ratios ( 1.2 : 1)that are well below the lower threshold of 5–6 postulated by the these workers, and hence the absence of any stimulatory effect of glucose cannot be ascribed to a saturation effect This marginal effect of glucose is in accord with some previous reports which have shown that elevated levels of glucose failed to potentiate the accumulation of the protein oxidation product o-Tyr in skin collagen [55] and urine [56] from diabetics compared with nondiabetics, suggesting that the accumulation of this oxidation product is
unaffect-ed by the level of hyperglycaemia The absence of any effect
of glucose on Cu2+-stimulated oxidation is, however, in
Trang 8contrast to another recent study which has reported a
stimulatory effect of glucose on Cu2+-mediated LDL
oxidation [44] The latter study used Cu2+: LDL ratios
which were higher that those used in the current study (3 : 1
and 31 : 1)which may account for the observed differences
in behaviour; the lower ratios used in the current study are
likely to be the more physiologically relevant
A similar absence of any stimulatory effect on the
oxidation of the lipid, protein, and antioxidants of LDL,
above that seen with Cu2+alone, was observed with MG
Within this system, even the presence of Cu2+ alone
induced only very limited oxidation In contrast GA, which
is more chemically reactive than either glucose or MG, did
induce the oxidation of lipids and consumption of
a-tocopherol in LDL with this process being GA
concen-tration-dependent although GA in the absence of Cu2+had
little effect No protein oxidation was detected in the Cu2+
plus GA system, suggesting that such oxidation occurs after
the induction of lipid oxidation and potentially as a result of
damage transfer from the oxidized lipids to the protein
component; this possibility was not investigated further
Overall, the oxidation of lipids and protein side chains in
LDL and the depletion of a-tocopherol by glucose and the
two aldehydes examined appears to proceed slowly, even in
the presence of low concentrations of Cu2+
In contrast with the above processes, covalent
modifica-tion (glycamodifica-tion)of LDL has been shown to occur rapidly
with the aldehydes, occur in the absence of added metal
ions, and depend on both the structure of the compound
and its concentration Of the two aldehydes examined,
modification of Trp and Lys was more rapid with GA than
with MG and the loss of Lys appears to occur earlier than
that of Trp in the MG system With GA the loss of these
two amino acids occured too rapidly to determine an order
of reaction Loss of Arg occurred prior to either of these
other amino acids as evidenced by the significant loss of this
residue in the time zero samples that were analysed
immediately after mixing A more extensive loss of Arg
was detected with GA compared with MG at this time point
but again the reactions were too rapid to analyse
statisti-cally Thus with MG the order of depletion of these residues
is Arg > Lys > Trp which is in accord with previous
studies on the reaction of MG with LDL [33], and GA and
MG with Lys and Arg residues on other proteins [6,9,57–
59] Adducts formed with both residues have been identified
on plasma proteins [10,58]
The mechanisms of modification of the Lys and Arg
residues, and the products formed, have not been examined;
it is likely that these reactions proceed by the pathways
outlined previously (e.g [6,9]) The loss of Trp fluorescence
was observed to occur in the absence of added Cu2+
Whether this reflects conversion of Trp to products (e.g via
the kynurenine pathway)or alteration in the local
environ-ment of these (hydrophobic)residues cannot be clearly
differentiated from the current data [60] The observed
alteration of Lys and Arg residues, which are major
contributors (with protonated His side-chains)to the overall
charge of the LDL particle, are mirrored in the observed
changes in the relative electrophoretic mobility of the
modified particles
In contrast with the behaviour of GA and MG, no
modification of either Trp or Lys residues was detected even
with the highest levels of glucose used (100 mM)in either the absence or presence of Cu2+ Limited modification of Arg residues was however detected with high concentrations of glucose with and without added Cu2+ These observations are in accord with a more rapid rate of modification of Arg residues over Lys residues, particularly given the higher concentration of Lys residues over Arg in apoB (356 Lys vs
148 Arg; Swiss-Prot file P04114, residues 28–4563) Fur-thermore, the absence of significant levels of Lys and Trp modification in systems where Cu2+was added, and where significant levels of lipid oxidation were detected (cf the data obtained after 2 weeks of incubation in Fig 1), suggests that modification of these residues by lipid oxidation products does not occur to any significant extent under the conditions used This is in contrast to previous reports which have suggested that lipid oxidation is a major route to the modification of protein side chains such as Lys residues on LDL [50] In accordance with these measure-ments, only small changes were detected in the REM of the glucose-treated LDL particles except with both supra-pathological levels of glucose and long incubation times
It has been established that neither glucose nor MG are effective catalysts of oxidation of the major components of LDL particles in either the presence or absence of added
Cu2+; this inability of MG to initiate LDL oxidation agrees with a previous report [33] In contrast, GA can facilitate lipid peroxidation induced by Cu2+ but is relatively ineffective in the absence of such metal ions These results indicate that the glycoxidation of LDL, as has been suggested previously [52], is transition metal ion-dependent Interestingly, GA, but not MG, can promote oxidation and this is likely to be due to the oxidizable b-hydroxyaldehyde function [– CH(OH)-C(O)-] on this molecule which is not present in MG which contains the corresponding oxidized a,b-dicarbonyl function [i.e (– C(O)-C(O)-)] Reaction of the b-hydroxyaldehyde function with Cu2+is believed to result in the formation of the reduced metal ion Cu+and a radical anion species from the GA (e.g [61]) Further reactions of one or both of these species may give rise to the observed induction of lipid peroxidation Similar reactions cannot occur with MG but are likely to occur, albeit at a much lower rate, with the open-chain form of glucose; the low concentration of this latter species in equilibrium mixtures of glucose anomers is likely to be at least partially responsible for the slow reaction kinetics detected when compared with equimolar amounts of GA [61] In contrast with such oxidative reactions, covalent modification (gly-cation)appears to be a much more rapid and potentially more significant process, particularly as there is controversy regarding the presence of significant concentrations of reactive transition metal ions in the artery wall and in developing atherosclerotic lesions [62–64]
The loss (derivatization, covalent modification)of Lys and Arg residues may be highly pertinent to the biological effects
of modified LDL particles as it has been shown that modification of Lys residues promotes recognition of LDL
by the macrophage scavenger receptor [65,66] The effect of specific modification of Arg residues on LDL particle recog-nition is less well established Preliminary data (B E Brown,
H M Knott, R T Dean and M J Davies, unpublished results)on the uptake of LDL particles modified by GA,
MG, and glucose prepared under similar conditions to
Trang 9those used in the studies reported here are consistent with the
recognition of GA- (and possibly MG-)modified LDL
particles, but not glucose-modified species, by receptors
present on mouse macrophage-like cells (cf data with other
modified proteins [59,67])and the subsequent accumulation
of lipids (cholesterol and cholesteryl esters, as measured by
HPLC)within these cells These data support a previous
suggestion [68] that covalent modification (glycation), in the
absence of lipid or protein oxidation or antioxidant
consumption, is sufficient for the formation of foam
cells Such carbonyl stress may play a significant role in
the modification of LDL particles both in plasma and in the
intima of the artery wall and may therefore contribute to the
elevated (two- to threefold)levels of atherogenesis observed
in diabetic patients compared with nondiabetic patients
Acknowledgements
This work was supported by the National Health and Medical
Research Council, the Australian Research Council, the Juvenile
Diabetes Foundation International, Diabetes Australia Research Trust,
and the Wellcome Trust B.E Brown gratefully acknowledges receipt
of an Australian Postgraduate Award, administered through The
University of Sydney.
References
1 Kannel, W.B & McGee, D.L (1979)Diabetes and cardiovascular
disease: the Framingham Study Diabetes 241, 2035–2038.
2 Thorpe, S & Baynes, J.W (1996)Role of the Maillard
Reaction in Diabetes Mellitus and disease of aging Drugs Aging 9,
69–77.
3 Baynes, J.W & Thorpe, S.R (1999)Role of oxidative stress in
diabetic complications: a new perspective on an old paradigm.
Diabetes 48, 1–9.
4 Baynes, J.W & Thorpe, S.R (2000)Glycoxidation and
lipoxi-dation in atherogenesis Free Radic Biol Med 28, 1708–1716.
5 Wolff, S.P., Jiang, A.Y & Hunt, J.V (1991)Protein glycation and
oxidative stress in diabetes mellitus and ageing Free Radic Biol.
Med 10, 339–352.
6 Lo, T.W., Westwood, M.E., McLellan, A.C., Selwood, T &
Thornalley, P.J (1994)Binding and modification of proteins by
methylglyoxal under physiological conditions A kinetic and
mechanistic study with N-alpha-acetylarginine,
N-alpha-acetyl-cysteine, and N-alpha-acetyllysine, and bovine serum albumin.
J Biol Chem 269, 32299–32305.
7 Brownlee, M (1995)Advanced protein glycosylation in diabetes
and aging Annu Rev Med 46, 223–234.
8 Fu, M.-X., Requena, J.R., Jenkins, A.J., Lyons, T.J., Baynes, J.W.
& Thorpe, S.R (1996)The advanced glycation end product
N e -(carboxymethyl)lysine is a product of both lipid peroxidation
and glycoxidation reactions J Biol Chem 271, 9982–9986.
9 Degenhardt, T.P., Thorpe, S.R & Baynes, J.W (1998)Chemical
modification of proteins by methylglyoxal Cell Mol Biol 44,
1139–1145.
10 Nagaraj, R.H., Shipanova, I.N & Faust, F.M (1996)Protein
cross-linking by the Maillard reaction Isolation, characterization,
and in vivo detection of a lysine-lysine cross-link derived from
methylglyoxal J Biol Chem 271, 19338–19345.
11 Glomb, M.A & Monnier, V.M (1995)Mechanism of protein
modification by glyoxal and glycolaldehyde, reactive intermediates
of the Maillard reaction J Biol Chem 270, 10017–10026.
12 Atkins, T.W & Thornally, P.J (1989)Erythrocyte glyoxalase
activity in genetically obese (ob/ob)and streptozotocin diabetic
mice Diabetes Res 11, 125–129.
13 Odani, H., Shinzato, T., Matsumoto, Y., Usami, J & Maeda, K (1999)Increase in three a,b-dicarbonyl compound levels in human uremic plasma: specific in vivo determination of intermediates in advanced Maillard reaction Biochem Biophys Res Commun.
256, 89–93.
14 Reichard, G.A., Jr, Skutches, C.L., Hoeldtke, R.D & Owen, O.E (1986)Acetone metabolism in humans during diabetic keto-acidosis Diabetes 35, 668–674.
15 Brown, M.S & Goldstein, J.L (1986)A receptor-mediated pathway for cholesterol homeostasis Science 232, 34–47.
16 Araki, N., Higashi, T., Mori, T., Shibayama, R., Kawabe, Y., Kodama, T., Takahashi, K., Shichiri, M & Horiuchi, S (1995) Macrophage scavenger receptor mediates the endocytic uptake and degradation of advanced glycation end products of the Maillard reaction Eur J Biochem 230, 408–415.
17 Suzuki, H., Kurihara, Y., Takeya, M., Kamada, N., Kataoka, M., Jishage, K., Ueda, O., Sakaguchi, H., Higashi, T., Suzuki, T., Takashima, Y., Kawabe, Y., Cynshi, O., Wada, Y., Honda, M., Kurihara, H., Aburatani, H., Doi, T., Matsumoto, A., Azuma, S., Noda, T., Toyoda, Y., Itakura, H., Yazaki, Y & Kodama, T (1997)A role for macrophage scavenger receptors in athero-sclerosis and susceptibility to infection Nature 386, 292–296.
18 Dean, R.T (1991)Protein damage and repair In Oxidative Damage and Repair (Davies, K.J.A., ed), pp 341–347 Pergamon Press, Oxford.
19 Bourdon, E., Loreau, N & Blacher, D (1999)Glucose and free radicals impair the antioxidant properties of serum albumin FASEBJ 13, 233–244.
20 van Reyk, D.M., Brown, A.J., Jessup, W & Dean, R.T (1995) Batch-to-batch variation of Chelex-100 confounds metal-cata-lysed oxidation Leaching of inhibitory compounds from a batch
of Chelex-100 and their removal by a pre-washing procedure Free Radic Res 23, 533–535.
21 Jessup, W., Simpson, J.A & Dean, R.T (1993)Does superoxide radical have a role in macrophage-mediated oxidative modifica-tion of LDL? Atherosclerosis 99, 107–120.
22 Esterbauer, H., Striegl, G., Puhl, H & Rotheneder, M (1989) Continuous monitoring of in vitro oxidation of human low density lipoprotein Free Radic Res Commun 6, 67–75.
23 Fu, S., Davies, M.J., Stocker, R & Dean, R.T (1998)Evidence for roles of radicals in protein oxidation in advanced human athero-sclerotic plaque Biochem J 333, 519–525.
24 Bruce, D., Fu, S., Armstrong, S & Dean, R.T (1999)Human apo-lipoprotein B from normal plasma contains oxidised peptides Int J Biochem Cell Biol 31, 1409–1420.
25 Kritharides, L., Upston, J., Jessup, W & Dean, R.T (1998) Accumulation and metabolism of low density lipoprotein-derived cholesteryl linoleate hydroperoxide and hydroxide by macro-phages J Lipid Res 39, 2394–2405.
26 Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber,
W & Weigele, M (1972)Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the pico-mole range Science 178, 871–872.
27 Smith, R.E & MacQuarrie, R (1978)A sensitive fluorometric method for the determination of arginine using 9,10-phenan-threnequinone Anal Biochem 90, 246–255.
28 Knott, H.M., Baoutina, A., Davies, M.J & Dean, R.T (2002) Comparative time-courses of copper-ion-mediated protein and lipid oxidation in low-density lipoprotein Arch Biochem Biophys.
400, 223–232.
29 Armstrong, S.G & Dean, R.T (1995)A sensitive fluorometric assay for protein-bound DOPA and related products of radical-mediated protein oxidation Redox Report 1, 291–298.
30 Fu, M.X., Wells-Knecht, K.J., Blackledge, J.A., Lyons, T.J., Thorpe, S.R & Baynes, J.W (1994)Glycation, glycoxidation, and cross-linking of collagen by glucose Kinetics, mechanisms, and
Trang 10inhibition of late stages of the Maillard reaction Diabetes 43,
676–683.
31 Davies, M.J., Fu, S., Wang, H & Dean, R.T (1999)Stable
markers of oxidant damage to proteins and their application in the
study of human disease Free Radic Biol Med 27, 1151–1163.
32 Davies, M.J & Dean, R.T (1997) Radical-Mediated Protein
Oxidation: from Chemistry to Medicine Oxford University Press,
Oxford.
33 Schalkwijk, C.G., Vermeer, M.A., Stehouwer, C.D., te Koppele,
J., Princen, H.M & van Hinsbergh, V.W (1998) Effect of
methylglyoxal on the physico-chemical and biological properties
of low-density lipoprotein Biochim Biophys Acta 1394, 187–198.
34 Bucala, R., Makita, Z., Koschinsky, T., Cerami, A & Vlassara, H.
(1993)Lipid advanced glycosylation: Pathway for lipid oxidation
in vivo Proc Natl Acad Sci USA 90, 6434–6438.
35 Bucala, R., Makita, Z., Vega, G., Grundy, S., Koschinsky, T.,
Cerami, A & Vlassara, H (1994)Modification of low density
lipoprotein by advanced glycation end products contributes to the
dyslipidemia of diabetes and renal insufficiency Proc Natl Acad.
Sci USA 91, 9441–9445.
36 Frye, E.B., Degenhardt, T.P., Thorpe, S.R & Baynes, J.W (1998)
Role of the Maillard reaction in aging of tissue proteins Advanced
glycosylation end product-dependent increase in imidazolium
cross-links in human lens proteins J Biol Chem 273, 18714–
18719.
37 Sakata, N., Imanaga, Y., Meng, J., Tachikawa, Y., Takebayashi,
S., Nagai, R., Horiuchi, S., Itabe, H & Takano, T (1998)
Immunohistochemical localization of different epitopes of
advanced glycation end products in human atherosclerotic lesions.
Atherosclerosis 141, 61–75.
38 Sakata, N., Uesugi, N., Takebayashi, S., Nagai, R., Jono, T.,
Horiuchi, S., Takeya, M., Itabe, H., Takano, T., Myint, T &
Taniguchi, N (2001)Glycoxidation and lipid peroxidation of
low-density lipoprotein can synergistically enhance atherogenesis.
Cardiovasc Res 49, 466–475.
39 Sajithlal, G.B., Chithra, P & Chandrakasan, G (1998)The role of
metal-catalyzed oxidation in the formation of advanced glycation
end products: an in vitro study on collagen Free Radic Biol Med.
25, 265–269.
40 Hunt, J.V., Smith, C.C.T & Wolff, S.P (1990)Autoxidative
glycosylation and possible involvement of peroxides and free
radicals in LDL modification by glucose Diabetes 39, 1420–1424.
41 Sakurai, T., Kimura, S., Nakano, M & Kimura, H
(1991)Oxi-dative modification of glycated low density lipoprotein in the
presence of iron Biochem Biophys Res Commun 177, 433–439.
42 Mullarkey, C.J., Edelstein, D & Brownlee, M (1990)Free radical
generation by early glycation products: a mechanism for
accelerated atherogenesis in diabetes Biochem Biophys Res.
Commun 173, 932–939.
43 Kobayashi, K., Watanabe, J., Umeda, F & Nawata, H (1995)
Glycation accelerates the oxidation of low density lipoprotein by
copper ions Endocrinol J 42, 461–465.
44 Leoni, V., Albertini, A., Passi, A., Abuja, P.M., Borroni, P.,
D’Eril, G.M & de Luca, G (2002)Glucose accelerates
copper-and ceruloplasmin-induced oxidation of low-density lipoprotein
and whole serum Free Radic Res 36, 521–529.
45 Thornalley, P.J (1996)Pharmacology of methylglyoxal:
forma-tion, modification of proteins and nucleic acids, and enzymatic
detoxification – a role in pathogenesis and antiproliferative
chemotherapy Gen Pharmacol 27, 565–573.
46 Thornalley, P.J., Langborg, A & Minhas, H.S (1999)Formation
of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation
of proteins by glucose Biochem J 344, 109–116.
47 McLellan, A.C., Phillips, S.A & Thornalley, P.J (1992)The assay
of methylglyoxal in biological systems by derivatization with
1,2-diamino-4,5-dimethoxybenzene Anal Biochem 206, 17–23.
48 McLellan, A.C., Thornalley, P.J., Benn, J & Sonksen, P.H (1994) Glyoxalase system in clinical diabetes mellitus and correlation with diabetic complications Clin Sci 87, 21–29.
49 Shamsi, F.A., Partal, A., Sady, C., Glomb, M.A & Nagaraj, R.H (1998)Immunological evidence for methylglyoxal-derived modi-fications in vivo Determination of antigenic epitopes J Biol Chem 273, 6928–6936.
50 Kawamura, M., Heinecke, J.W & Chait, A (1994)Pathophy-siological concentrations of glucose promote oxidative modifica-tion of low density lipoprotein by a superoxide-dependent pathway J Clin Invest 94, 771–778.
51 Millican, S.A., Schultz, D., Bagga, M., Coussons, P.J., Mu¨ller, K.
& Hunt, J.V (1998)Glucose-modified low density lipoprotein enhances human monocyte chemotaxis Free Radic Res 28, 533–542.
52 Mowri, H., Frei, B & Keaney, J.F (2000)Glucose enhancement
of LDL oxidation is strictly metal ion dependent Free Radic Biol Med 29, 814–824.
53 Fu, S.-L., Fu, M.-X., Baynes, J.W., Thorpe, S.R & Dean, R.T (1998)Presence of dopa and amino acid hydroperoxides in pro-teins modified with advanced glycation end products (AGEs): amino acid oxidation products as a possible source of oxidative stress induced by AGE proteins Biochem J 330, 233–239.
54 Gieseg, S.P & Esterbauer, H (1994)Low density lipoprotein is saturable by pro-oxidant copper FEBS Lett 343, 188–194.
55 Wells-Knecht, M.C., Lyons, T.J., McCance, D.R., Thorpe, S.R & Baynes, J.W (1997)Age-dependent increase in ortho-tyrosine and methionine sulfoxide in human skin collagen is not accelerated in diabetes Evidence against a generalized increase in oxidative stress
in diabetes J Clin Invest 100, 839–846.
56 Krapfenbauer, K., Birnbacher, R., Vierhapper, H., Herkner, K., Kampel, D & Lubec, G (1998)Glycoxidation, and protein and DNA oxidation in patients with diabetes mellitus Clin Sci 95, 331–337.
57 Oya, T., Hattori, N., Mizumo, Y., Miyata, S., Maeda, S., Osawa,
T & Uchida, K (1999)Methylglyoxal modification of proteins Chemical and immunohistochemical characterization of methyl-glyoxal-arginine adducts J Biol Chem 274, 18495–18502.
58 Westwood, M.E & Thornalley, P.J (1995)Molecular character-istics of methylglyoxal-modified bovine and human serum albu-mins Comparison with glucose-derived advanced glycation endproduct-modified serum albumins J Prot Chem 14, 359–372.
59 Nagai, R., Matsumoto, K., Ling, X., Suzuki, H., Araki, T & Horiuchi, S (2000)Glycolaldehyde, a reactive intermediate for advanced glycation end products, plays an important role in the generation of an active ligand for the macrophage scavenger receptor Diabetes 49, 1714–1723.
60 Yang, C.-Y., Gu, Z.-W., Yang, M., Lin, S.-N., Siuzdak, G & Smith, C.V (1999)Identification of modified tryptophan residues
in apolipoprotein B-100 derived from copper ion-oxidized low-density lipoprotein Biochemistry 38, 15903–15908.
61 Thornalley, P.J (1985)Monosaccharide autoxidation in health and disease Environ Health Perspect 64, 297–307.
62 Evans, P.J., Smith, C., Mitchinson, M.J & Halliwell, B (1995) Metal ion release from mechanically-disrupted human arterial wall Implications for the development of atherosclerosis Free Radic Res 23, 465–469.
63 Smith, C., Mitchinson, M.J., Aruoma, O.I & Halliwell, B (1992) Stimulation of lipid peroxidation and hydroxyl-radical generation
by the contents of human atherosclerotic lesions Biochem J 286, 901–905.
64 Swain, J & Gutteridge, J.M.C (1995)Prooxidant iron and cop-per, with ferroxidase and xanthine oxidase activities in human atherosclerotic material FEBS Lett 368, 513–515.
65 Haberland, M.E., Olch, C.L & Fogelman, A.M (1984)Role
of lysines in mediating interaction of modified low density