Oxidation of High Density Lipoproteins

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 α-tocopherol (α-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 α-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 α-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). α-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, α-TOH can promote this process.

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 antiatherogenic 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 Cu 2ϩ -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 ⅐ ) (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 ␣-tocopherol (␣-TOH) can protect HDL apolipoproteins from oxidation. Previous studies have shown that ␣-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 ⅐ , 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 ␣-TOH is prevented by co-antioxidants that eliminate ␣-tocopheroxyl radical (18), which otherwise propagates lipoprotein lipid peroxidation (14). It follows that ␣-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 (CoQ 10 H 2 ), oxidation of HDL induced by ROO ⅐ , Cu 2ϩ , or soybean lipoxygenase (SLO) resulted in the oxidation of apoAI and apoAII which occurred concomitantly with ␣-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 ␣-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.
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 Ͻ5% of the apoprotein as apoAI ϩ32 (see below).
Enrichment of HDL with ␣-Tocopherol-Human HDL was enriched in vitro and in vivo with ␣-TOH. For in vitro enrichment, EDTA plasma was incubated for 5 h at 37°C in the presence of 528 M ␣-TOH dissolved in Me 2 SO (final Me 2 SO concentration Յ3 volume %). Control plasma, treated with Me 2 SO only, was incubated in parallel for 5 h at 37°C. Following incubation, HDL was isolated as described above. For in vivo enrichment, ␣-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 ⅐ in a controlled and quantitative manner, CuSO 4 (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 ϫ 10 3 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), Cu 2ϩ (208 M), or soybean lipoxygenase (10 3 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 l) 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 l 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 UV 210 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 m) and analyzed for phosphatidylcholine hydroperoxides by HPLC with chemiluminescence detection (20).
HPLC Analysis of HDL Apolipoproteins-Aliquots (50 l) of the reaction mixture were removed, added to 150 l of 8 M guanidine hydrochloride on ice, and analyzed by HPLC using a 5-m, 25 ϫ 0.46 cm C18 protein and peptide column (Vydac, Hesperia, CA) with a 300-Å pore size, eluted with an acetonitrile/H 2 O 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% H 2 O. 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 Oxidized ApoAI by Mass Spectrometry-AAPHoxidized 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 l) were injected into a moving solvent (10 l/min; H 2 O:acetonitrile 1:1 v/v, 0.05% trifluoroacetic acid) coupled directly to the ionization source via a fused silica capillary interface (50 m ϫ 40 cm). The source temperature was 50°C, and N 2 was used as the nebulizer gas. Sample droplets were ionized at a positive potential of Ϸ3 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 l of a 50 mM CNBr solution in 100% acetonitrile, and 400 l of formic acid were added. The mixture was top gassed with N 2 , sealed, and incubated in the dark for 18 h at 22°C. H 2 O (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 UV 210 nm and fluorescence (Ex 280 nm /Em 350 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 l) of oxidizing HDL were added to 500 l of PBS containing 1% (v/v) SDS, and the fluorescence was measured (Hitachi F-4010 fluorescence spectrophotometer) with Ex 280 nm /Em 350 nm .

RESULTS
Oxidation of HDL Lipids and Antioxidants-To define the temporal relationship between the consumption of HDL's CoQ 10 H 2 and ␣-TOH and the accumulation of oxidized lipids, isolated HDL was subjected to a constant low flux of ROO ⅐ at 37°C. The HDL particle concentration in these experiments was 14 Ϯ 4 M (mean Ϯ SD, n ϭ 4). Prior to oxidation, HDL contained ␣-TOH and total coenzyme Q 10 at 0.56 Ϯ 0.10 and 0.012 Ϯ 0.005 molecules/particle, consistent with previous observations (7). Approximately 50% of HDL's coenzyme Q 10 was present as CoQ 10 H 2 (Fig. 1), indicating that relatively little adventitious oxidative damage to HDL had occurred during its isolation. Upon initiation of oxidation, HDL's CoQ 10 H 2 was oxidized to CoQ 10 within 30 min (Fig. 1), and this was followed by a gradual, linear loss of CoQ 10 . ␣-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).  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 Ϯ 1.1 Da (mean Ϯ SD, n ϭ 3), in agreement with that predicted from its amino acid sequence (i.e. 28,078.7).
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 Cu 2ϩ 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 Met 26 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 timedependent 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 Ϯ 0.6 (mean Ϯ S.D., n ϭ 3), i.e. 32 mass units greater than that of unoxidized apoAI. This oxidized form of apoAI will be referred to as apoAI ϩ32 . Formation of apoAI ϩ32 is consistent with a previous study on proteolytic peptides derived from oxidized apoAI which suggested that the compound contained two (Met 112 and Met 148 ) of the three Met residues as Met(O) (24).
In addition to apoAIIa and apoAI ϩ32 , 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 Ϯ 1.8 Da (mean Ϯ S.D., n ϭ 3), i.e. 16 Da greater than that of native apoAI; the compound was assigned apoAI ϩ16 . 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 Ϸ33% of the Met residues in apoAI ϩ16 were depleted, whereas Met(O) levels were Ϸ50% of those found in apoAI ϩ32 , which contained 2 Met(O) ( Table I). Amino acids other than Met were not oxidized in apoAI ϩ16 (Table I), consistent with the molecular mass obtained. From this we conclude that apoAI ϩ16 is a previously unrecognized oxidized product of apoAI, formed during the earliest stages of HDL oxidation. Fig. 3 shows the time-dependent changes in the levels of apoAI, apoAII, and their oxidized forms apoAI ϩ16 , apoAI ϩ32 , and apoAIIa during AAPH-induced oxidation of HDL. Protein oxidation was clearly detected after 1 h of incubation, i.e. after complete consumption of CoQ 10 H 2 , yet in the presence of ␣-TOH (cf. Figs. 1 and 3). By 2 h, Ϸ35 and 40% of apoAI and apoAII, respectively, were oxidized (Fig. 3), although ␣-TOH

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 (q) and apoAII (E). B shows formation of oxidized forms of apoAI (apoAI ϩ32 , f; apoAI ϩ16 , OE;) and apoAII (apoAIIa, Ⅺ). Data, represented as percent of maximum peak area detected, show means Ϯ S.E. of four separate experiments. was still detectable (Fig. 1). At advanced stages of oxidation (Ͼ4 h), apoAI ϩ16 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).
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 ϩ16 and apoAI ϩ32 are formed selectively.
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 ϩ16 and apoAI ϩ32 (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 Cu 2ϩ or SLO at 37°C for up to 48 h also failed to result in specific formation of apoAI ϩ16 and apoAI ϩ32 , as indicated by the unaltered ratio of apoAI ϩ32 to total apoAI (Fig. 7). Under these conditions oxidation of apoAI did not occur, the small amounts of apoAI ϩ32 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 ⅐ , Cu 2ϩ , or SLO.
Role of ␣-TOH in HDL Apolipoprotein Oxidation-As oxidation of apoAI and apoAII was observed even when nearly normal levels of ␣-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 ␣-TOH since, on average, only one in two 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 g of protein per well); ϩ denotes the presence of dithiothreitol. Molecular weight standards are shown in the margins. The 28-kDa band originates from apoAI, and the 16-kDa (Ϫ dithiothreitol) and 8-kDa (ϩ dithiothreitol) bands from apoAII dimer and monomer, respectively. As oxidation progressed, apoAI and apoAII decreased while several faint bands appeared in the Ϸ35to 45-kDa molecular mass region. At 22 h, a series of faint bands were detected at Ͼ67 kDa. The 67-kDa band is due to albumin contamination of the isolated HDL (20). HDL particles contained ␣-TOH. To distinguish between these two possibilities, HDL was enriched with ␣-TOH prior to oxidation. Such enrichment resulted in HDL which, on average, contained Ͼ1 molecule of ␣-TOH per particle (Table II). In ␣-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 ␣-TOH was observed with in vivo and in vitro enriched HDL (Table II) and correlated directly with the amount of HDL's ␣-TOH (r ϭ 0.96 and 0.94 for ␣-TOH content versus loss of apoAI and apoAII, respectively). The extent of formation of CE-OOH and CE-OH also increased with increasing ␣-TOH enrichment (r ϭ 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 ␣-TOH-enriched HDL although this effect was less pronounced than that observed for the formation of apoAI ϩ16 and apoAI ϩ32 (Table II).
To rule out the possibility that the pro-oxidant effect of ␣-TOH was a peculiarity associated with AAPH-induced oxidation, we also oxidized HDL with Cu 2ϩ and SLO. Similar to the situation with ROO ⅐ , supplementation of HDL with ␣-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, Cu 2ϩ 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 ␣-TOHsupplemented HDL (Fig. 8). DISCUSSION Previous studies have shown that lipids in HDL can become oxidized before those in LDL (7) and that oxidation of HDL by Cu 2ϩ 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 ␣-TOH-containing stages of oxidation, is independent of direct reaction with the oxidants added, and can be promoted by ␣-TOH. The results demonstrate, for the first time, that in the absence of coantioxidants, ␣-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 (Met 112 and Met 148 ) 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 ϭ 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 Ϸ10% 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 Met 86 is adjacent to Ser 87 in apoAI, the conversion of Met 86 to homoserine may not be complete and the remaining FIG. 7. Oxidation of isolated human apoAI by Cu 2؉ and SLO. Purified (22) lipid-free human apoAI (3.9 mg/ml, corresponding to 139 M in HDL particle concentration) was incubated under air and at 37°C in the absence (q) or presence of either Cu 2ϩ (208 M, Ⅺ) or SLO (10 3 units/ml, E). 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 ϩ32 to total apoAI (oxidized plus unoxidized apoAI), the latter of which did not change throughout the incubation. Discernible formation of apoAI ϩ16 was not observed.  (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 ␣-TOH remains largely intact. It was not possible to assess whether protein oxidation occurred in the presence of CoQ 10 H 2 , as the detection of protein oxidation by UV absorbance is much less sensitive than the electrochemical detection of CoQ 10 H 2 . However, since Ͻ1% of circulating HDL contain CoQ 10 H 2 , this antioxidant does not likely constitute a major defense against HDL apolipoprotein oxidation. It remains to be shown whether apolipoproteins in in vivo CoQ 10 H 2 -supplemented HDL are more resistant to oxidation. In any case, ␣-TOH did not protect lipids or apolipoproteins in isolated HDL from the oxidative damage initiated by either ROO ⅐ , Cu 2ϩ , or SLO under the mild oxidizing conditions used here. In fact, ␣-TOH enrichment increased the extent of oxidation of HDL's lipids and apolipoproteins. The observed parallel increase in ␣-TOH content and lipoprotein lipid oxidizability is consistent with previous reports of lipid peroxidation proceeding via TMP (14,17,33). Increasing the ␣-TOH content increases the reactivity of HDL particles toward 1-electron oxidants and, hence, the likelihood of formation of ␣-tocopheroxyl radical. Once present in CoQ 10 H 2 -free HDL, ␣-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 ⅐ -induced oxidation of isolated apoAI did not result in specific oxidation (Fig. 6), and Cu 2ϩ 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 ϩ16 and apoAI ϩ32 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 ␣-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 ⅐ and Cu 2ϩ ) would be expected to yield ethylene rather than Met(O) (37). Also, Met 112 and Met 148 of apoAI and Met 26 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 ϩ16 , apoAI ϩ32 , 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.
CE-OOH ϩ Met apoA 3 CE-OH ϩ Met(O) apoA (Reaction 1) As the observed pro-oxidant activity of ␣-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.