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
-tocopherol (
-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
-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
-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).
-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,
-TOH can promote this
process.
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INTRODUCTION |
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
-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·, 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 (CoQ10H2), oxidation of HDL
induced by ROO·, Cu2+, 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.
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EXPERIMENTAL PROCEDURES |
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
-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
-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 µM
-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,
-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
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.
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RESULTS |
Oxidation of HDL Lipids and Antioxidants--
To define the
temporal relationship between the consumption of HDL's
CoQ10H2 and
-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
-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.
-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 ( ) and -TOH ( ) and
accumulation of CoQ10 ( ). B shows formation
of CE-OH ( ), CE-OOH ( ), and phosphatidylcholine hydroperoxides
( ). Data shown are means ± S.E. of four separate
experiments.
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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.
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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
33% of the Met residues in
apoAI+16 were depleted, whereas Met(O) levels were
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.
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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
-TOH (cf. Figs. 1 and 3). By 2 h,
35
and 40% of apoAI and apoAII, respectively, were oxidized (Fig. 3),
although
-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 ( ) and apoAII
( ). B shows formation of oxidized forms of apoAI
(apoAI+32, ; apoAI+16, ;) and apoAII
(apoAIIa, ). 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
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).
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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
250 fluorescence units, suggesting that a proportion of the grossly
oxidized Trp products were also fluorescent or that some Trp remained
unoxidized.
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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
( ) or presence of either Cu2+ (208 µM,
) or SLO (103 units/ml, ). 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.
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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 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 >1 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 = 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 = 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+16 and apoAI+32 (Table II).
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Table II
HDL enriched with -tocopherol exhibits increased sensitivity of
cholesteryl esters and Met residues of apoAI and apoAII to AAPH-induced oxidation
HDL enriched with -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 -TOH-enriched HDL/non-enriched HDL. Initial -TOH
levels in native HDL were as follows: 0.36, 0.45, 0.32, and 0.45 molecules of -TOH per HDL for experiments 1-4, respectively.
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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 Cu2+ and SLO. Similar to the situation with
ROO·, 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, 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
-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
-TOH-enriched ( ) and non-enriched HDL ( ), which contained
32 ± 5 and 5 ± 1 µM -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.
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DISCUSSION |
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
-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
co-antioxidants,
-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
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
-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,
-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,
-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 CoQ10H2-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·-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
-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.
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(Reaction 1)
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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.
We thank Ingrid Gelissen for the purified
apoAI and Dr. Mike Davies for helpful comments on the manuscript.