Báo cáo khoa học: Glycation and glycoxidation of low-density lipoproteins by glucose and low-molecular mass aldehydes Formation of modified and oxidized particles pot

Eur J Biochem 270, 3572–3582 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03742.x Glycation 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 glycolaldehyde 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 evidence was obtained for the stimulation of lipid or protein oxidation by glucose or methylglyoxal in the presence of copper ions, whereas glycolaldehyde stimulated such reactions 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 Ôcarbonyl-stressÕ may facilitate the formation of foam cells and the vascular complications of diabetes The correlation between diabetes and cardiovascular disease (CVD) has been well established [1], although the precise mechanisms that facilitate the many complications associated with diabetes, including CVD and cataracts, are poorly understood Uncontrolled plasma glucose concentrations and the ability of glucose to either oxidatively or nonoxidatively (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 9565 6101, Tel.: +61 9565 6156, E-mail: h.knott@centenary.usyd.edu.au Abbreviations: CVD, cardiovascular disease; AGE, advanced glycation 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 July 2003) Keywords: AGE; atherosclerosis; glycolaldehyde; methylglyoxal; protein modification Ó FEBS 2003 Glycation and glycoxidation of LDL (Eur J Biochem 270) 3573 atherosclerosis [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 complications related to diabetes Materials and methods Materials All solutions were prepared with nanopure water (Milli Q system, Millipore-Waters) and treated with washed Chelex100 (Bio-Rad) to remove transition metals prior to use [20] PD-10 columns (Sephadex G-25 M) were from Amersham Biosciences Pre-cast 1% agarose gels were from Helena Laboratories (Mt Waverly, VIC, Australia) Fatty acidfree 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 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 comparison, 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 bicinchoninic acid (BCA; Pierce) method using 0.4 mgỈmL)1 BSA as a standard, with incubations performed at 60 °C for 45–60 Glycoxidation of LDL )1 LDL (density 1.019–1.063 gỈmL ) was prepared from plasma of fasted, healthy volunteers by density gradient ultracentrifugation as described previously [21] After isolation, LDL was dialysed against four to five L changes of degassed NaCl/Pi containing 0.1 mgỈmL)1 chloramphenicol, filter sterilized, and used immediately in most cases When necessary, dialysis buffers were supplemented with mgỈmL)1 EDTA 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 (30 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 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, 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 mL M HCl and 50 lL 2-mercaptoacetic acid The vessels were sealed under vacuum and incubated at 110 °C 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 detection as described previously [23] Quantification 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 converted from lmolỈmol)1 Tyr to lmolỈmol)1 Phe using the abundance of these amino acids in the apoB100 molecule (152 Tyr/223 Phe) Preliminary experiments that quantified 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 handling and analysis [23,24] Samples (80 lL) were removed from the LDL incubations and 0.1 vol each of 20 mM EDTA and 0.2 mM BHT and 670 lL borate buffer (pH > 8) were added During vortex mixing, 250 lL 0.15 mgỈmL)1 fluorescamine dissolved in acetone was added The fluorescence of these samples, representative of free amine groups, was measured using kex 390 nm and kem 475 nm and expressed as a percentage of the zero time value for the control (no added aldehyde/ glucose) samples [26] Relative electrophoretic mobility gels Samples (10–15 lL) were removed from the LDL incubations 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 Gels were fixed in 100% methanol (1 min), stained for 5–10 with Fat Red 7B (Sigma-Aldrich), destained for 5–10 with 70% methanol, and dried at 60 °C 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 mL with NaCl/Pi Trp fluorescence was measured on a Perkin Elmer Lumines- 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 mL with NaCl/Pi Three millilitres 120 lM 9,10-phenanthrenequinone (PTQ; in absolute ethanol) was added and the reaction initiated by addition of 0.5 mL M NaOH with the samples then incubated at 60 °C for h At both t ¼ and t ¼ h, 0.5 mL samples were removed and the reaction stopped by addition of an equal volume of 1.2 M HCl The fluorescence was recorded using kex 312 nm and kem 392 nm with the Arg-dependent fluorescence calculated as the difference between the t ¼ and t ¼ h samples [27] Results Glucose-mediated oxidation of LDL Lipid oxidation Six different parameters of lipid peroxidation were measured (Fig 1A–F) Three concentrations of glucose representing normal (5 mM), pathological (25 mM), and suprapathological (100 mM) levels with lM Cu2+ were examined, as well as samples containing lM Cu2+ alone, 100 mM glucose 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 lM Cu2+ proceeded slowly with complete destruction of a-tocopherol (TOH; Fig 1A) occurring between and w These results correlate with loss of free cholesterol (Fig 1B, FC), cholesteryl arachidonate (Fig 1C, CA), and cholesteryl linoleate (Fig 1D, CL) by weeks as well as the accumulation of the cholesterol oxidation product, 7-ketocholesterol (Fig 1E, 7K), to a maximum of 200 nmolỈmg)1 apoB at 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 lM Cu2+ A small acceleration of the rate of oxidation was detected in the 100 mM glucose samples compared with the controls at and weeks in all the parameters measured, Ó FEBS 2003 Glycation and glycoxidation of LDL (Eur J Biochem 270) 3575 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 Fig Glucose-mediated oxidation of the lipid and protein moieties of LDL LDL ( 0.4 mg proteinỈmL)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 mL methanol and mL hexane Four millilitres of the hexane fraction was then dried to completion, resuspended in 200 lL isopropanol, and the levels (expressed as nmolỈmg 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 represents the mean (± SD) of two replicates from a single experiment representative of several Black bars, 100 mM glucose + lM Cu2+; horizontal striped bars, 25 mM glucose + lM Cu2+; dark stippled bars, mM glucose + lM Cu2+; light stippled bars, 100 mM glucose only; white bars, lM Cu2+ 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 value with the exception of FC and 7K In these samples, no conversion of FC to 7K was detected, whilst TOH was not fully depleted until 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 nmolỈmg)1 apoB) by 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 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 lmolỈmol)1 Tyr) and o-Tyr (550 lmolỈmol)1 Phe) are in accord with literature data for plasma [23] and not statistically different between preparations 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 oxidation 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 interference 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 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 modification 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 mM glucose 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 mM glucose for 14 days a small increase Ó FEBS 2003 3576 H M Knott et al (Eur J Biochem 270) in 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 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 mM MG) a significant decrease in the level of this residue was observed by day (Fig 2A); this decrease occurred in the absence of added Cu2+ but was dependent on the MG concentration (by 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 day in the samples containing either 100 or 10 mM MG (P < 0.001 relative to all other samples) and at this time has occurred to a greater extent in the 100 mM than in the 10 mM MG 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 lM Cu2+ (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 lM MG in the absence of Cu2+ from day 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 day for both 10 and 100 mM MG); 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 t ¼ samples) with the 100 mM MG samples (P < 0.01 relative Fig MG-mediated modification of apoB LDL ( 0.4 mg proteinỈmL)1) was incubated with 10 or 100 lM MG with and without added lM Cu2+ or LDL ( mg proteinỈmL)1) was incubated with 10 or 100 mM MG in the absence of Cu2+ Trp residues (A) were quantified by fluorescence (kex 280 nm, kem 335 nm) after dilution in NaCl/Pi Lys residues (B) were quantified by fluorescence (kex 390 nm, kem 475 nm) after dilution in borate buffer (pH > 8) and derivatization with fluorescamine Arg residues (C) were quantified by fluorescence (kex 312 nm, kem 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 percentage of control incubations with no added MG Black bars, 100 mM MG; horizontal striped bars, 10 mM MG; dark stippled bars, 100 lM MG + lM Cu2+; light stippled bars, 100 lM MG; white bars, 10 lM MG + lM Cu2+; diagonal striped bars, 10 lM MG Asterisks indicate the first time point at which the data becomes statistically different when compared with the t ¼ value 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 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 lM MG, minor changes occurred after 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 mM GA with and without added lM copper ions Fig shows typical data; similar trends were observed with other Ó FEBS 2003 Glycation and glycoxidation of LDL (Eur J Biochem 270) 3577 140 120 Lys (% of control) Trp (% of control) B A 120 100 80 * ** 60 * 40 * * 80 * 60 * 40 20 * 20 100 * 0 14 140 100 D 80 REM Arg (% of control) 14 60 40 * 20 0 Time (days) concentrations 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 peroxidation occurred in those samples containing mM GA supplemented with 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 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 h (an arbitrary time frame anticipated to enable binding of either oxidant to the C 120 Fig GA-mediated oxidation of LDL lipids and alpha-tocopherol LDL ( 0.4 mgỈmL protein)1) was incubated with 0, 0.1 or mM GA with and without lM Cu2+; control samples were incubated with lM 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 Each data point represents the mean (± SD) of two replicates from a single experiment representative of several Black bars, mM GA + lM Cu2+; horizontal striped bars, mM GA; dark stippled bars, 0.1 mM GA + lM Cu2+; light stippled bars, 0.1 mM GA; white bars, lM Cu2+ Asterisks indicate the first time point at which the data becomes statistically different when compared with the t ¼ value Time (days) Time (days) 14 14 Time (days) Fig GA-mediated modification of apoB LDL ( 0.4 mg proteinỈ mL)1) was incubated with mM GA ± lM copper ions or LDL ( mg proteinỈmL)1) was incubated with 1, 10 and 100 mM GA Quantification of Trp (A), Lys (B) and Arg (C) residues was carried out as described in the legend to Fig The data are mean (n ¼ 4) ± SEM REM analysis (D) was carried out as described in the legend to Fig Black bars, mg LDL proteinỈmL)1 with 100 mM GA; horizontal striped bars, mg LDL proteinỈmL)1 with 10 mM GA; dark stippled bars, mg LDL proteinỈmL)1 with mM GA; light stippled bars, 0.4 mg LDL proteinỈmL)1 with mM GA + lM Cu2+; white bars, 0.4 mg LDL proteinỈmL)1 with mM GA only Asterisks indicate the first time point at which the data becomes statistically different when compared with the t ¼ value 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 experiments demonstrated little lipid oxidation at short incubation times, it was expected that protein oxidation 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 mM GA with and without added Cu2+ over 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 mgỈmL)1 LDL) and mM GA with added Cu2+ (1 lM) The data obtained are presented in Fig 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 mM GA loss of Trp was significant at day, with higher loss of Trp at this time point occurring in the presence of 100 mM GA For the lower concentrations of GA, losses did not become significant until days when compared with time zero 3578 H M Knott et al (Eur J Biochem 270) Similar behaviour was observed for Lys (Fig 4B) In this case, an effect of Cu2+could be seen ) the high concentration 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 mM GA with no added Cu2+ did not show significant loss of Lys until day 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 mM GA,  50% of the Arg residues were observed to be lost in the samples examined immediately after mixing (t ¼ 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 mechanisms) for the damage induced by high glucose concentrations It has been clearly established that products of metal ion catalysed oxidation of glucose and proteinbound glucose (i.e glycoxidation) can accumulate at elevated levels on proteins from diabetic patients, as 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 atherosclerosis, 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 colocalize with the macrophage scavenger receptor [38] indicating a plausible involvement of the accumulation of AGEs and increased oxidative stress in this pathology Furthermore, 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- Ó FEBS 2003 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 comprehensive 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] Glucose- 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 unaffected by the level of hyperglycaemia The absence of any effect of glucose on Cu2+-stimulated oxidation is, however, in Ó FEBS 2003 Glycation and glycoxidation of LDL (Eur J Biochem 270) 3579 contrast 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 : 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 concentration-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 modification (glycation) 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 statistically 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 environment 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) Furthermore, 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 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 measurements, only small changes were detected in the REM of the glucose-treated LDL particles except with both suprapathological 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 (glycation) 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 recognition 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 3580 H M Knott et al (Eur J Biochem 270) those 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 Ôcarbonyl stressÕ 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 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A.M (1984) Role of lysines in mediating interaction of modified low density 3582 H M Knott et al (Eur J Biochem 270) lipoproteins with the scavenger receptor of human monocyte macrophages J Biol Chem 259, 11305–11311 66 Jinnouchi, Y., Sano, H., Nagai, R., Hakamata, H., Kodama, T., Suzuki, H., Yoshida, M., Ueda, S & Horiuchi, S (1998) Glycolaldehyde-modified low density lipoprotein leads macrophages to foam cells via the macrophage scavenger receptor J Biochem 123, 1208–1217 Ó FEBS 2003 67 Westwood, M.E., Argirov, O.K., Abordo, E.A & Thornalley, P.J (1997) Methylglyoxal-modified arginine residues – a signal for receptor-mediated endocytosis and degradation of proteins by monocytic THP-1 cells Biochim Biophys Acta 1356, 84–94 68 Colaco, C.A & Roser, B.J (1994) Atherosclerosis and glycation Bioessays 16, 145–147  ... 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. .. 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. .. accumulation of this oxidation product is unaffected by the level of hyperglycaemia The absence of any effect of glucose on Cu2+-stimulated oxidation is, however, in Ó FEBS 2003 Glycation and glycoxidation