Oxidation of High Density Lipoproteins
I. FORMATION OF METHIONINE SULFOXIDE IN APOLIPOPROTEINS AI AND AII IS AN EARLY EVENT THAT ACCOMPANIES LIPID PEROXIDATION AND CAN BE ENHANCED BY alpha -TOCOPHEROL*

Brett Garner, Paul K. Witting, A. Reginald Waldeck, Julie K. Christison, Mark RafteryDagger , and Roland Stocker§

From the Biochemistry and Dagger  Immunology Groups, The Heart Research Institute, Sydney New South Wales 2050, Australia

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The lipids of high density lipoproteins (HDL) are initially oxidized in preference to those in low density lipoprotein when human plasma is exposed to aqueous peroxyl radicals. In this work we report on the relative susceptibility of HDL protein and lipid to oxidation and on the role HDL's alpha -tocopherol (alpha -TOH) plays in modulating protein oxidation. Exposure of isolated HDL to either low fluxes of aqueous peroxyl radicals, Cu2+ ions, or soybean lipoxygenase resulted in the oxidation of apoAI and apoAII during the earliest stages of the reaction, i.e. after consumption of ubiquinol-10 and in the presence of alpha -TOH. Hydro(pero)xides of cholesteryl esters and phospholipids initially accumulated together with specific oxidized forms of apoAI and apoAII, separated by high pressure liquid chromatography. The specific oxidized forms of apoAI were 16 and 32 mass units heavier than those of the native apolipoproteins and contained 1 and 2 methionine sulfoxide residues per protein, respectively. The third methionine residue in apoAI, as well as Trp residues, remained unoxidized during the earliest stages of HDL oxidation examined. Exposure of isolated apoAI to peroxyl radicals, Cu2+, or soybean lipoxygenase resulted in nonspecific (for peroxyl radicals) or no discernible protein oxidation (Cu2+ and soybean lipoxygenase). This indicated that the formation of the specific oxidized forms of apoAI observed with native HDL was not the result of direct reaction of these oxidants with the apolipoprotein. In vitro and in vivo enrichment of HDL with alpha -TOH resulted in a dose-dependent increase in the extent of peroxyl radical-induced formation of HDL cholesteryl ester hydroperoxides (r = 0.96) and cholesteryl ester hydroxides (r = 0.92), as well as the loss of apoAI (r = 0.96) and apoAII (r = 0.94). alpha -TOH enrichment also enhanced HDL lipid and protein oxidation induced by Cu2+ or soybean lipoxygenase. These results indicate that the earliest stages of HDL oxidation are accompanied by the oxidation of specific methionine residues in apoAI and apoAII and that in the absence of co-antioxidants, alpha -TOH can promote this process.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Plasma levels of high density lipoprotein (HDL)1 cholesterol and apolipoprotein AI (apoAI) inversely correlate with the risk of developing coronary artery disease (1). An important anti-atherogenic activity postulated to underlie the beneficial property of high HDL levels is the removal of cholesterol from peripheral tissues and its transport to the liver for excretion, a process known as reverse cholesterol transport (2). Other potentially anti-atherogenic properties of HDL also exist. For example, HDL preferentially transports oxidized cholesteryl esters to the liver for excretion into bile (3, 4). HDL also inhibits Cu2+- or endothelial cell-induced oxidation of low density lipoprotein (LDL) (5); there is strong evidence that LDL oxidation contributes to atherogenesis in humans (6).

HDL is the major carrier of extremely low concentrations of lipid hydroperoxides in human plasma, and initially, HDL lipids are oxidized in preference to those in LDL when human plasma is exposed to aqueous peroxyl radicals (ROO·) (7). The oxidation of specific Met residues on apoAI has also been reported in isolated human HDL (8). Furthermore, HDL can accept oxidized cholesteryl esters from LDL, a process mediated by cholesteryl ester transfer protein (9). These observations are of potential physiological significance as lipid oxidation products derived from LDL can lead to cross-linkage of apoAI (10), which can impair the interaction of HDL with lecithin:cholesterol acyltransferase (11), and increase the clearance of HDL from plasma (12). Oxidation of HDL also reduces its ability to accept cholesterol from cell membranes, a crucial step in reverse cholesterol transport (13). Therefore, it is important to understand the biochemical mechanisms that lead to the oxidation of apoAI and apoAII and the possible role of HDL's antioxidants on this.

Reports on the relative susceptibility to oxidation of HDL's protein versus lipid and antioxidants during the earliest stages of oxidation are lacking. In addition, it is not known whether or not alpha -tocopherol (alpha -TOH) can protect HDL apolipoproteins from oxidation. Previous studies have shown that alpha -TOH acts as a pro-oxidant for lipid peroxidation in LDL at low radical fluxes but as an antioxidant at high fluxes (14). Described initially for LDL and ROO·, tocopherol-mediated peroxidation (TMP) has subsequently been confirmed as a general model for lipid peroxidation in lipid emulsions and lipoproteins and extended to other 1-electron oxidants as well as conditions that give rise to radical reactions (14-17). This pro-oxidant activity of alpha -TOH is prevented by co-antioxidants that eliminate alpha -tocopheroxyl radical (18), which otherwise propagates lipoprotein lipid peroxidation (14). It follows that alpha -TOH makes lipoproteins more reactive toward radical oxidants, and this can, depending on the conditions, lead to increased oxidation of lipoprotein lipids and, in principle, apolipoproteins.

The aim of the present study was to compare the susceptibility of HDL's antioxidants, polar and neutral lipids, and apoAI and apoAII to oxidation, using controlled and low fluxes of radical oxidants favoring TMP. The results obtained show that following consumption of ubiquinol-10 (CoQ10H2), oxidation of HDL induced by ROO·, Cu2+, or soybean lipoxygenase (SLO) resulted in the oxidation of apoAI and apoAII which occurred concomitantly with alpha -TOH consumption and lipid peroxidation. The oxidation of apoAI and apoAII was initially targeted toward specific Met residues in the early stages of the reaction. Supplementation of HDL with alpha -TOH resulted in a greater degree of both lipid and specific apolipoprotein oxidation, independent of the oxidant used. In the accompanying article (19) we investigate the mechanism of apoAI- and apoAII-Met oxidation and provide evidence for Met oxidation by HDL-associated lipid hydroperoxides.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- 2,2'-Azo-bis(2-amidinopropane) dihydrochloride (AAPH) was purchased from Wako (Osaka, Japan); EDTA and SDS were from Sigma; potassium bromide and dimethyl sulfoxide (Me2SO) were from BDH (Poole, UK) and Merck (Kilsyth, Victoria, Australia), respectively. PD-10 Sephadex G-25 M columns were from Pharmacia Biotech Inc. (Uppsala, Sweden); guanidine hydrochloride (ultra pure) was from Life Technologies, Inc. (Paisley, UK), and trifluoroacetic acid (HPLC grade) was from Pierce. Cholesteryl (9-hydroxy)linoleate was from Cayman Chemicals (Ann Arbor, MI), and hydroperoxides of cholesteryl linoleate and soybean phosphatidylcholine (both Sigma) were prepared as described (21 and references therein). Ubiquinone-10 (CoQ10) and alpha -TOH were from Mitsubishi Gas Chemicals (Tokyo, Japan) and Henkel Corp. (Sydney, Australia), respectively. Ethanol and t-butyl alcohol were from BDH and Rhône-Poulenc (Paris, France), respectively. All other organic solvents were from Mallinckrodt (Clayton, Australia). Buffers were prepared from the highest quality materials available and using nanopure water (MODULAB).

Isolation of HDL and ApoAI-- Human HDL was isolated rapidly from freshly obtained EDTA plasma using a two-step density gradient and ultracentrifugation in a TL 100.4 rotor (Beckman Instruments, Palo Alto, CA) (20). HDL was isolated directly by needle aspiration after 4 h centrifugation at 100,000 rpm. Immediately prior to use in experiments, low molecular weight compounds were removed by size exclusion chromatography (PD-10 column), and the HDL solution was supplemented with 1 mM EDTA. HDL protein concentrations were estimated using the bicinchoninic acid method (Sigma) with bovine serum albumin (Sigma) as a standard; the HDL particle concentration was calculated by cholesterol determination, assuming an average of 35 molecules of free cholesterol per HDL particle. ApoAI, isolated by a standard procedure (21) with minor modifications (22), typically contained <5% of the apoprotein as apoAI+32 (see below).

Enrichment of HDL with alpha -Tocopherol-- Human HDL was enriched in vitro and in vivo with alpha -TOH. For in vitro enrichment, EDTA plasma was incubated for 5 h at 37 °C in the presence of 528 µM alpha -TOH dissolved in Me2SO (final Me2SO concentration <= 3 volume %). Control plasma, treated with Me2SO only, was incubated in parallel for 5 h at 37 °C. Following incubation, HDL was isolated as described above. For in vivo enrichment, alpha -TOH capsules (335 mg, Blackmores, Balgowlah, Australia) were taken 3 times daily with meals for 4 days, before HDL isolation. A plasma sample was also collected immediately prior to the 1st day of supplementation and stored under argon in the dark at 4 °C in a sterile environment to serve as source for non-enriched, control HDL (18).

Oxidation of HDL and Isolated ApoAI-- Isolated HDL (1.5-2.0 mg of protein/ml) was oxidized in phosphate-buffered saline (PBS, pH 7.4) containing 1 mM EDTA by aerobic incubation at 37 °C in the presence of either AAPH (1.75-4 mM) which produces ROO· in a controlled and quantitative manner, CuSO4 (molar ratio of 1.5:1 with respect to HDL particle concentration), or SLO (EC 1.13.11.12, Sigma) at a final concentration of 4 × 103 units/ml. Where indicated, isolated lipid-free apoAI (3.9 mg/ml) was incubated under air and at 37 °C in the presence of either AAPH (5 mM final concentration), Cu2+ (208 µM), or soybean lipoxygenase (103 units/ml). At the times indicated, aliquots of the reaction mixtures were removed and analyzed for antioxidants, lipids, lipid oxidation products, and apolipoprotein oxidation by HPLC as described below.

Analysis of Antioxidants, Lipids, and Lipid Oxidation Products-- Aliquots (100 µl) of the HDL samples were extracted into 1 ml of methanol containing 0.02% (v/v) acetic acid and 5 ml of hexane; 4 ml of the hexane phase was evaporated and resuspended in 200 µl of isopropyl alcohol. An aliquot of this was then analyzed by HPLC for neutral unoxidized lipids, antioxidants, and cholesteryl ester hydroperoxides (CE-OOH, principally those of cholesteryl linoleate and cholesteryl arachidonate) using UV210 nm, electrochemical, and post-column chemiluminescence detection, respectively (20). The organic extract was also analyzed for cholesteryl linoleate hydroperoxides and cholesteryl linoleate hydroxides (referred to as CE-OH) (23). The aqueous methanol phase (0.5 ml) was filtered (0.2 µm) and analyzed for phosphatidylcholine hydroperoxides by HPLC with chemiluminescence detection (20).

HPLC Analysis of HDL Apolipoproteins-- Aliquots (50 µl) of the reaction mixture were removed, added to 150 µl of 8 M guanidine hydrochloride on ice, and analyzed by HPLC using a 5-µm, 25 × 0.46 cm C18 protein and peptide column (Vydac, Hesperia, CA) with a 300-Å pore size, eluted with an acetonitrile/H2O gradient containing 0.1% trifluoroacetic acid at 1 ml/min at 22 °C, and detected at 214 nm as described (24, 25), with the following modifications. The gradient was formed starting with 40% acetonitrile and 60% H2O. The content of acetonitrile was first increased linearly to 65% over 25 min, then to 90% over 5 min, and finally decreased to 40% over 10 min.

For isolation and subsequent mass spectrometry (MS) and amino acid analyses of the different forms of apoAI, a more shallow gradient and a decreased flow rate were used. Thus, the gradient was started at 1.0 ml/min and 40% acetonitrile. After 5 min the flow rate was reduced to 0.5 ml/min and the content of acetonitrile increased linearly to 53% over 6 min and then to 58% over 24 min. Following this, the flow was increased to 1.0 ml/min, and the content of acetonitrile increased to 90% over 5 min, and finally decreased to 40% over 10 min.

Characterization of HDL by Polyacrylamide Gel Electrophoresis-- Aliquots (50 µl) of the reaction mixtures were removed, diluted 1/5 in sample buffer (0.06 M Tris-HCl, pH 6.8, 10% glycerol (v/v), 2% SDS (w/v), 0.025% bromphenol blue), and analyzed by SDS-PAGE using 10% gels and a Mini Protean II (Bio-Rad) electrophoresis cell. Dithiothreitol was added to samples run under reducing conditions. Protein bands were visualized by Coomassie staining.

Characterization of Oxidized ApoAI by Mass Spectrometry-- AAPH-oxidized HDL was subjected to HPLC, and the fractions of oxidized and unoxidized apoAI were collected, pooled, and analyzed by electrospray ionization MS using a single quadrupole mass spectrometer equipped with an electrospray ionization source (Platform, VG-Fisons Instruments, Manchester, UK). Samples (10 µl) were injected into a moving solvent (10 µl/min; H2O:acetonitrile 1:1 v/v, 0.05% trifluoroacetic acid) coupled directly to the ionization source via a fused silica capillary interface (50 µm × 40 cm). The source temperature was 50 °C, and N2 was used as the nebulizer gas. Sample droplets were ionized at a positive potential of approx 3 kV, transferred to the mass spectrometer with a cone voltage of 60 V, and the peak width at half-height of 1 Da. Spectra were scanned over the mass range of 700 to 1800 Da in 5 s and calibrated with horse heart myoglobin (Sigma).

Amino Acid Analysis of ApoAI-- Fractions of unoxidized and oxidized apoAI, collected as described above for the MS analysis, were dried under reduced pressure before 100 µl of a 50 mM CNBr solution in 100% acetonitrile, and 400 µl of formic acid were added. The mixture was top gassed with N2, sealed, and incubated in the dark for 18 h at 22 °C. H2O (5 volumes) was then added, the sample dried under reduced pressure, hydrolyzed in gaseous 6 M HCl containing 1.0% phenol (v/v), 0.01% mercaptoacetic acid (v/v), and analyzed for amino acids after derivatization with o-phthalaldehyde (26). Trp loss was estimated by serial UV210 nm and fluorescence (Ex280 nm/Em350 nm) monitoring of apoAI following HPLC separation and calculated from the UV/fluorescence ratio. Loss of endogenous fluorescence was also used as an index of Trp oxidation in intact human HDL (27). For this, aliquots (400 µl) of oxidizing HDL were added to 500 µl of PBS containing 1% (v/v) SDS, and the fluorescence was measured (Hitachi F-4010 fluorescence spectrophotometer) with Ex280 nm/Em350 nm.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Oxidation of HDL Lipids and Antioxidants-- To define the temporal relationship between the consumption of HDL's CoQ10H2 and alpha -TOH and the accumulation of oxidized lipids, isolated HDL was subjected to a constant low flux of ROO· at 37 °C. The HDL particle concentration in these experiments was 14 ± 4 µM (mean ± SD, n = 4). Prior to oxidation, HDL contained alpha -TOH and total coenzyme Q10 at 0.56 ± 0.10 and 0.012 ± 0.005 molecules/particle, consistent with previous observations (7). Approximately 50% of HDL's coenzyme Q10 was present as CoQ10H2 (Fig. 1), indicating that relatively little adventitious oxidative damage to HDL had occurred during its isolation. Upon initiation of oxidation, HDL's CoQ10H2 was oxidized to CoQ10 within 30 min (Fig. 1), and this was followed by a gradual, linear loss of CoQ10. alpha -TOH was consumed in a time-dependent manner from the onset of oxidation and was below the limit of detection after 3-4 h incubation (Fig. 1). Fig. 1 also shows the kinetics of formation of phosphatidylcholine hydroperoxides, CE-OOH and CE-OH in AAPH-oxidizing HDL. The concomitant formation of CE-OOH and CE-OH confirms previous observations (28) of HDL's CE-OOH-reducing activity. The formation of CE-OOH and CE-OH was accompanied by an approximately stoichiometric loss of cholesteryl linoleate (51 µM) and cholesteryl arachidonate (16 µM) over the 7-h time course in the four oxidation experiments performed. Qualitatively similar results were obtained when AAPH was replaced with Cu2+ or SLO as the oxidant (see below).


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Fig. 1.   Consumption of antioxidants and formation of lipid hydroperoxides in isolated HDL oxidized by AAPH. HDL was oxidized by AAPH (4 mM) at 37 °C in PBS supplemented with 1 mM EDTA. At the time points indicated, lipids and lipophilic antioxidants were analyzed as described under "Experimental Procedures." A shows depletion of CoQ10H2 (bullet ) and alpha -TOH (black-square) and accumulation of CoQ10 (open circle ). B shows formation of CE-OH (triangle ), CE-OOH (black-triangle), and phosphatidylcholine hydroperoxides (square ). Data shown are means ± S.E. of four separate experiments.

Oxidation and Characterization of HDL Apolipoproteins-- To compare HDL lipid versus protein oxidation, we adapted an HPLC method that separates native apoAI and apoAII from oxidized forms containing specifically oxidized Met residues (24). Fig. 2A shows a representative chromatogram of apolipoproteins in native HDL; apoAI and apoAII were the major apolipoproteins identified, in agreement with previous observations (8). Oxidized forms of apoAI and apoAII were not detected in freshly isolated HDL. ApoCs eluted before apoAs (Fig. 2; see also Refs. 8 and 29). The mass of apoAI in native HDL was 28,079.5 ± 1.1 Da (mean ± SD, n = 3), in agreement with that predicted from its amino acid sequence (i.e. 28,078.7).


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Fig. 2.   HPLC chromatograms of HDL apolipoproteins during oxidation initiated by AAPH. HDL was oxidized by AAPH (4 mM) at 37 °C. At the time points indicated below, samples were taken and apolipoproteins analyzed as described under "Experimental Procedures." The major forms of apoAI and apoAII are labeled. The molecular masses of the various forms of apoAI, determined by MS, are given in parentheses; values are means from three experiments. The arrows indicate the appearance of oxidized forms of apoAI, designated apoAI+16 (see arrow a) and apoAI+32 (see arrow b). A through F represent samples taken at 0 (i.e. directly after AAPH addition) and 2-6 h of incubation, respectively. Data are from one experiment representative of four. Note the difference in scale between the y ordinates of A and B and C-F.

As oxidation progressed the content of HDL's apoAI and apoAII decreased time-dependently. Representative chromatograms are shown for AAPH-induced oxidation (Figs. 2, B-F, and 3A), with qualitatively similar results being obtained with Cu2+ or SLO as alternative oxidants (data not shown). Concomitant with the loss of unoxidized apoAI and apoAII, new peaks were detected (Fig. 2, B-F). The peak eluting between apoAI and apoAII has been designated as apoAIIa and is known to contain one of the two Met26 residues in apoAII dimer as Met sulfoxide (Met(O)) (8). In addition, a peak eluting with a retention time of 0.85 relative to apoAI increased in a time-dependent fashion (Fig. 2, B and F). The fraction corresponding to this peak was collected, and the mass of the compound was determined to be 28,111.9 ± 0.6 (mean ± S.D., n = 3), i.e. 32 mass units greater than that of unoxidized apoAI. This oxidized form of apoAI will be referred to as apoAI+32. Formation of apoAI+32 is consistent with a previous study on proteolytic peptides derived from oxidized apoAI which suggested that the compound contained two (Met112 and Met148) of the three Met residues as Met(O) (24).

In addition to apoAIIa and apoAI+32, oxidation of HDL with AAPH consistently resulted in the formation of a further product eluting close to apoAI (relative retention time of 0.97) (Fig. 2, B and F). During the early stages of oxidation this compound appeared as a leading shoulder on the apoAI peak (see arrow in Fig. 2B). As oxidation progressed, the compound became partially resolved from apoAI. By using a more shallow acetonitrile gradient (see "Experimental Procedures") better separation was obtained, and a relatively pure preparation of this form of apoAI was collected. Upon re-chromatography of the collected fraction, a single peak was observed (not shown), the molecular mass of which was 28,095.9 ± 1.8 Da (mean ± S.D., n = 3), i.e. 16 Da greater than that of native apoAI; the compound was assigned apoAI+16. The increased mass of 16 Da suggested introduction of one additional atom of oxygen, and the slight decrease in hydrophobicity was consistent with one of the three Met residues of apoAI being converted to Met(O). Amino acid analysis confirmed that approximately approx 33% of the Met residues in apoAI+16 were depleted, whereas Met(O) levels were approx 50% of those found in apoAI+32, which contained 2 Met(O) (Table I). Amino acids other than Met were not oxidized in apoAI+16 (Table I), consistent with the molecular mass obtained. From this we conclude that apoAI+16 is a previously unrecognized oxidized product of apoAI, formed during the earliest stages of HDL oxidation.

                              
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Table I
Amino acid content of apoAI in native and AAPH-oxidized HDL
Fractions of native and oxidized apoAI were collected and analyzed for amino acid content as described under "Experimental Procedures." The data shown are means ± S.D. of triplicate determinations, with values expressed as percentages of that in native apoAI, defined as 100% in all cases except for Met(O) and Trp where the Met(O) content of apoAI+32 and the ratio of (Ex280 nm/Em350 nm) fluorescence to UV absorbance of apoAI, respectively, are defined as 100%. Human apoAI does not contain Cys or Ilc. Pro is not detected by the method used.

Fig. 3 shows the time-dependent changes in the levels of apoAI, apoAII, and their oxidized forms apoAI+16, apoAI+32, and apoAIIa during AAPH-induced oxidation of HDL. Protein oxidation was clearly detected after 1 h of incubation, i.e. after complete consumption of CoQ10H2, yet in the presence of alpha -TOH (cf. Figs. 1 and 3). By 2 h, approx 35 and 40% of apoAI and apoAII, respectively, were oxidized (Fig. 3), although alpha -TOH was still detectable (Fig. 1). At advanced stages of oxidation (>4 h), apoAI+16 and apoAIIa also decreased, suggesting that these oxidized apolipoproteins are temporary products and that oxidation in addition to Met(O) formation occurred. Also, as oxidation progressed, a decreasing proportion of the apoAI and apoAII was detected as oxidized forms, suggesting that oxidation products were formed that were no longer resolved by the HPLC column. Consistent with the latter, SDS-PAGE analysis of AAPH-oxidized HDL showed that at later stages of oxidation high molecular weight complexes were formed (Fig. 4).


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Fig. 3.   Changes in HDL's apoAI, apoAII, and their oxidized forms during the course of oxidation initiated by AAPH. HDL was oxidized by AAPH (4 mM) at 37 °C. At the time points indicated, apolipoproteins were analyzed as described in the legend to Fig. 2. A shows the loss of apoAI (bullet ) and apoAII (open circle ). B shows formation of oxidized forms of apoAI (apoAI+32, black-square; apoAI+16, black-triangle;) and apoAII (apoAIIa, square ). Data, represented as percent of maximum peak area detected, show means ± S.E. of four separate experiments.


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Fig. 4.   Characterization of oxidized HDL by SDS-PAGE. HDL was oxidized by AAPH (7 mM) at 37 °C. Samples were taken at 0, 2, 5, and 22 h (indicated at the top) and subjected to PAGE (6 µg of protein per well); + denotes the presence of dithiothreitol. Molecular weight standards are shown in the margins. The 28-kDa band originates from apoAI, and the 16-kDa (- dithiothreitol) and 8-kDa (+ dithiothreitol) bands from apoAII dimer and monomer, respectively. As oxidation progressed, apoAI and apoAII decreased while several faint bands appeared in the approx 35- to 45-kDa molecular mass region. At 22 h, a series of faint bands were detected at >67 kDa. The 67-kDa band is due to albumin contamination of the isolated HDL (20).

Loss of endogenous Trp fluorescence, previously used as a marker of oxidative damage to HDL apolipoproteins (27), largely reflects damage to apoAI as human apoAII does not contain Trp (30). Only 25-30% of the initial Trp fluorescence was lost over 7 h during AAPH-induced oxidation of HDL (Fig. 5). This together with the data presented in Table I confirm that in the initial stages of AAPH-induced oxidation of HDL, apoAI+16 and apoAI+32 are formed selectively.


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Fig. 5.   Loss of HDL endogenous Trp fluorescence during the course of oxidation initiated by AAPH. HDL was oxidized by AAPH (4 mM) at 37 °C. At the time points indicated, samples were taken and mixed with 0.8 volume of PBS (containing SDS (1% (v/v)), and fluorescence was measured as described under "Experimental Procedures." Data shown are means ± S.E. of four separate experiments. Extensively oxidized HDL (4 mM AAPH for 60 h) possessed remaining endogenous fluorescence of approx 250 fluorescence units, suggesting that a proportion of the grossly oxidized Trp products were also fluorescent or that some Trp remained unoxidized.

Lack of Selective Oxidation of ApoAI Met by Direct Oxidation of Isolated ApoAI-- To assess whether the above described changes to apoAI (and apoAII) were due to direct interaction of the apolipoproteins with the oxidation-initiating species, we first exposed isolated, lipid-free apoAI to AAPH. This resulted in a general broadening of the apoAI peak on HPLC chromatography without selective formation of apoAI+16 and apoAI+32 (Fig. 6), consistent with AAPH oxidizing several different amino acids in proteins (31), in addition to giving rise to Met(O) (32). Exposure of isolated apoAI to Cu2+ or SLO at 37 °C for up to 48 h also failed to result in specific formation of apoAI+16 and apoAI+32, as indicated by the unaltered ratio of apoAI+32 to total apoAI (Fig. 7). Under these conditions oxidation of apoAI did not occur, the small amounts of apoAI+32 detected (Fig. 7) being present in the isolated apoAI, i.e. before addition of the oxidants (see "Experimental Procedures"). Together, these data rule out that the observed specific formation of Met(O) in apoAI and apoAII of HDL is not due to direct oxidation of the apolipoproteins by ROO·, Cu2+, or SLO.


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Fig. 6.   Oxidation of isolated human apoAI by AAPH. Purified (22) lipid-free human apoAI (3.9 mg/ml) was oxidized by AAPH (5 mM) under air and at 37 °C. At various time points, aliquots of the reaction mixture were removed and analyzed for apolipoprotein oxidation as described under "Experimental Procedures." The results shown are representative of three separate experiments with two different preparations of apoAI and show the apoAI peak eluting at 16.6 min before (A) and after 24 (B) and 48 h of incubation (C). Note that small amounts (typically < 5%) of apoAI+32 (eluting at 14.1 min in A) are present in the starting material, indicating that some apolipoprotein oxidation occurs during the isolation procedure.


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Fig. 7.   Oxidation of isolated human apoAI by Cu2+ and SLO. Purified (22) lipid-free human apoAI (3.9 mg/ml, corresponding to 139 µM in HDL particle concentration) was incubated under air and at 37 °C in the absence (bullet ) or presence of either Cu2+ (208 µM, square ) or SLO (103 units/ml, open circle ). At various time points, aliquots of the reaction mixture were analyzed for apolipoprotein oxidation as described under "Experimental Procedures." The results shown are representative for two separate experiments obtained with two different preparations of apoAI. ApoAI oxidation is expressed as the area ratio of apoAI+32 to total apoAI (oxidized plus unoxidized apoAI), the latter of which did not change throughout the incubation. Discernible formation of apoAI+16 was not observed.

Role of alpha -TOH in HDL Apolipoprotein Oxidation-- As oxidation of apoAI and apoAII was observed even when nearly normal levels of alpha -TOH were present (Figs. 1 and 3), the vitamin appeared not to protect HDL apolipoproteins from oxidative damage. Alternatively, the observed oxidation of apoAI and apoAII could have reflected events occurring in a subpopulation of HDL devoid of alpha -TOH since, on average, only one in two HDL particles contained alpha -TOH. To distinguish between these two possibilities, HDL was enriched with alpha -TOH prior to oxidation. Such enrichment resulted in HDL which, on average, contained >1 molecule of alpha -TOH per particle (Table II). In alpha -TOH-enriched HDL exposed to AAPH, there was a striking increase in the extent of both loss of apoAI and apoAII and formation of oxidized apolipoproteins (Table II). This pro-oxidant effect of alpha -TOH was observed with in vivo and in vitro enriched HDL (Table II) and correlated directly with the amount of HDL's alpha -TOH (r = 0.96 and 0.94 for alpha -TOH content versus loss of apoAI and apoAII, respectively). The extent of formation of CE-OOH and CE-OH also increased with increasing alpha -TOH enrichment (r = 0.96 and 0.92 for CE-OOH and CE-OH, respectively), consistent with HDL lipid peroxidation proceeding via TMP. The loss of Trp fluorescence also increased in alpha -TOH-enriched HDL although this effect was less pronounced than that observed for the formation of apoAI+16 and apoAI+32 (Table II).

                              
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Table II
HDL enriched with alpha -tocopherol exhibits increased sensitivity of cholesteryl esters and Met residues of apoAI and apoAII to AAPH-induced oxidation
HDL enriched with alpha -TOH both in vivo (Experiment 1) and in vitro (Experiments 2-4) was incubated at 37 °C in PBS containing 1 mM EDTA in the presence of 2 mM AAPH. After 3 h the samples were analyzed for CE-OOH, CE-OH, and oxidized apoAI and AII as described under "Experimental Procedures." The values shown are the ratio of the amount of the analyte in the alpha -TOH-enriched HDL/non-enriched HDL. Initial alpha -TOH levels in native HDL were as follows: 0.36, 0.45, 0.32, and 0.45 molecules of alpha -TOH per HDL for experiments 1-4, respectively.

To rule out the possibility that the pro-oxidant effect of alpha -TOH was a peculiarity associated with AAPH-induced oxidation, we also oxidized HDL with Cu2+ and SLO. Similar to the situation with ROO·, supplementation of HDL with alpha -TOH increased the extent of CE-O(O)H formation regardless of the oxidant employed (Fig. 8). It has been shown previously that under the conditions employed, Cu2+ and SLO oxidize lipoprotein lipids via TMP (17, 33). In all cases, increased lipid peroxidation was paralleled closely by increased levels of the Met(O)-containing forms of apoAI and AII in the alpha -TOHsupplemented HDL (Fig. 8).


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Fig. 8.   Oxidation of HDL by Cu2+ or SLO. HDL was oxidized at 37 °C by SLO (A and C, 4 × 103 units/ml) or Cu2+ (B and D, at a 1.5:1 molar ratio with HDL) and lipids and apolipoproteins analyzed as described in the legends to Figs. 1 and 2. Analyses were performed on in vitro alpha -TOH-enriched (open circle ) and non-enriched HDL (bullet ), which contained 32 ± 5 and 5 ± 1 µM alpha -TOH (mean ± S.E.), respectively, before oxidation. CE-O(O)H (A and B) refers to CE-OOH plus CE-OH. Apolipoprotein oxidation (C and D) is expressed as the ratio of the peak area of the oxidized apolipoproteins (i.e. apoAI+32, apoAI+16, and apoAIIa) divided by the total peak area of oxidized and native apolipoproteins present in each sample. Data shown are means ± S.E. of three separate experiments.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Previous studies have shown that lipids in HDL can become oxidized before those in LDL (7) and that oxidation of HDL by Cu2+ or lipid oxidation products derived from LDL can affect HDL functions related to reverse cholesterol transport (13). Mechanistic studies on the oxidation of HDL lipids and its relationship to apolipoprotein oxidation are therefore of potential physiological significance. The present study demonstrates that specific oxidation of HDL's apoAI and apoAII accompanies lipid peroxidation, occurs during the early alpha -TOH-containing stages of oxidation, is independent of direct reaction with the oxidants added, and can be promoted by alpha -TOH. The results demonstrate, for the first time, that in the absence of co-antioxidants, alpha -TOH can exert a pro-oxidant effect on proteins and that apolipoprotein oxidation represents an early event, even when mild oxidizing conditions are employed.

The HPLC method used for the measurements of apoAI and apoAII oxidation is based on a report by von Eckardstein et al. (24). These authors suggested that two (Met112 and Met148) of the three Met residues in isolated apoAI are susceptible to oxidation and that neither or both of these two Met residues are oxidized (24). However, in the present study, using intact HDL rather than isolated apoAI, we detected a distinct modified form of apoAI with a molecular mass consistent with the addition of one oxygen atom to the native protein, i.e. one Met(O) as evidenced by amino acid analysis. Therefore, the previous result (24) that neither or both of the oxidation-susceptible Met residues in isolated apoAI become oxidized does not appear to hold when apoAI is oxidized in intact HDL. Future studies may reveal which of the Met residues is initially oxidized in apoAI or may confirm that both residues are equally susceptible to oxidation. It may be that oxidation of one of the Met residues in apoAI renders the second residue more susceptible to oxidation. Of possible significance, amino acid substitutions in apoAI peptides are known to affect Met oxidizability (24).

A previous study suggested that oxidized forms of apoAI are present in isolated human HDL (8); however, we have found no evidence for this in HDL isolated rapidly (20) from non-fasted normolipidemic volunteers (n = 9) (see e.g. Fig. 2A). The differences between these studies could be due to the isolation procedures employed; in vitro storage of HDL is known to produce modified apolipoproteins (34). The data in Table I suggest that approx 10% of the Met in native apoAI may already be present as Met(O). However, the method used for Met determination relies on the conversion of Met to homoserine and its lactone by CNBr, so that upon hydrolysis of the sample under reducing conditions, Met(O) is detected as Met. The reaction of Met with CNBr is known to be less efficient where Met residues are located adjacent to Ser, due to an N- to O-acyl shift (35). Since Met86 is adjacent to Ser87 in apoAI, the conversion of Met86 to homoserine may not be complete and the remaining Met erroneously assigned as Met(O), thereby overestimating the content of the latter in native apoAI. In support of this, it is difficult to create CNBr peptides of canine apoAI, where Met86 is the sole Met residue of the protein flanked by Ser87 (36). Thus the amount of Met(O)-containing apoAI present in circulating HDL has yet to be defined unequivocally.

An important finding of the present work is that apoAI and apoAII oxidation proceeds while HDL's content of alpha -TOH remains largely intact. It was not possible to assess whether protein oxidation occurred in the presence of CoQ10H2, as the detection of protein oxidation by UV absorbance is much less sensitive than the electrochemical detection of CoQ10H2. However, since <1% of circulating HDL contain CoQ10H2, this antioxidant does not likely constitute a major defense against HDL apolipoprotein oxidation. It remains to be shown whether apolipoproteins in in vivo CoQ10H2-supplemented HDL are more resistant to oxidation. In any case, alpha -TOH did not protect lipids or apolipoproteins in isolated HDL from the oxidative damage initiated by either ROO·, Cu2+, or SLO under the mild oxidizing conditions used here. In fact, alpha -TOH enrichment increased the extent of oxidation of HDL's lipids and apolipoproteins. The observed parallel increase in alpha -TOH content and lipoprotein lipid oxidizability is consistent with previous reports of lipid peroxidation proceeding via TMP (14, 17, 33). Increasing the alpha -TOH content increases the reactivity of HDL particles toward 1-electron oxidants and, hence, the likelihood of formation of alpha -tocopheroxyl radical. Once present in CoQ10H2-free HDL, alpha -tocopheroxyl radical promotes lipid peroxidation under conditions of low radical fluxes (14).

Several observations argue against a direct oxidation of HDL's apolipoproteins by the oxidants employed. Foremost, ROO·-induced oxidation of isolated apoAI did not result in specific oxidation (Fig. 6), and Cu2+ and SLO failed to oxidize isolated apoAI (Fig. 7). These findings are in sharp contrast to the situation with intact HDL where specific formation of apoAI+16 and apoAI+32 is observed with the same oxidants at comparable oxidant to protein ratios (Figs. 2, 3, and 8, Table I). Although not investigated here, it is likely that the same is true for the selective oxidation of the single Met residue in HDL's apoAII. The implied lipid peroxidation-dependent oxidation of Met residues in HDL's apolipoproteins is supported further by the increased formation of oxidized apoAI and apoAII in alpha -TOH-enriched HDL observed under conditions where the vitamin promotes lipid peroxidation (Table II, Fig. 8).

Met(O) is the primary oxidation product of Met formed by 2-electron oxidants (such as lipid hydroperoxides), whereas 1-electron oxidants (such as ROO· and Cu2+) would be expected to yield ethylene rather than Met(O) (37). Also, Met112 and Met148 of apoAI and Met26 of apoAII (i.e. the Met residues susceptible to oxidation) lie within the hydrophobic regions of class A amphipathic helices and hence are not expected to be exposed to HDL's surface (38) for direct reaction with aqueous oxidants. For these reasons, apoAI+16, apoAI+32, and apoAIIa are likely the result of reaction of the apolipoproteins with product(s) formed during radical-induced HDL oxidation. We propose that CE-OOH and other lipid hydroperoxides, formed during the oxidation of HDL, react with oxidation-susceptible Met residues of apoAI and apoAII to form Met(O) (Reaction 1). The accompanying paper (19) provides further evidence for this proposal.
<UP>     CE-OOH</UP>+<UP>Met</UP><SUB><UP>apoA</UP></SUB> →<UP> CE-OH</UP>+<UP>Met</UP>(<UP>O</UP>)<SUB><UP>apoA</UP></SUB> (Reaction 1)
As the observed pro-oxidant activity of alpha -TOH for Met residues in HDL's apoAI and apoAII is most likely indirect (19), future studies are required to examine if the present results can be extrapolated to Met and/or other amino acid residues in other proteins.

    ACKNOWLEDGEMENTS

We thank Ingrid Gelissen for the purified apoAI and Dr. Mike Davies for helpful comments on the manuscript.

    FOOTNOTES

* This work was supported by Grant G95S4337 from the National Heart Foundation of Australia (to R. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Biochemistry Group, Heart Research Institute, 145 Missenden Rd., Camperdown, Sydney, New South Wales 2050, Australia. Fax: 61(2) 9550-3302; E-mail: r.stocker{at}hri.edu.au.

1 The abbreviations used are: HDL, high density lipoproteins; AAPH, 2,2'-azo-bis(2-amidinopropane) dihydrochloride; apoAI, apolipoprotein AI; apoAI+16, oxidized apoAI 16 mass units greater than apoAI; apoAI+32, oxidized apoAI 32 mass units greater than apoAI; apoAII, apolipoprotein AII; CE-OOH, cholesteryl ester hydroperoxides; CE-OH, cholesteryl ester hydroxides; CoQ10, ubiquinone-10; CoQ10H2, ubiquinol-10; LDL, low density lipoprotein; Met(O), methionine sulfoxide; PBS, phosphate-buffered saline; ROO·, peroxyl radical; SLO, soybean lipoxygenase; alpha -TOH, alpha -tocopherol; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; MS, mass spectrometry; TMP, tocopherol-mediated peroxidation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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