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INTRODUCTION |
There is significant evidence that the role of lipoproteins in
cardiovascular disease involves oxidation of the lipid-protein complex
(1). The oxidative susceptibility and products of the oxidation of low
density lipoprotein (LDL)1
have been the most intensively studied area to date (2, 3). However, it
is known that the other major lipoprotein complexes, the very low
density lipoproteins (4, 5) and the high density lipoproteins (HDL)
(6-8), can also undergo oxidation. Within nature, protection of
polyunsaturated fatty acids from oxidation has come in the form of
water-soluble antioxidant molecules, such as vitamin C, and
lipid-soluble anti-oxidant molecules, such as vitamin E. Goulinet and
Chapman (9) have shown that there is a marked gradient of the
tocopherol and carotenoid classes of lipid-soluble antioxidants among
the major lipoproteins. Very low density lipoproteins, LDL, and HDL
were shown to have an average of 43, 10, and 0.7 antioxidant molecules
per particle, respectively. On this basis alone, one would predict that
HDL would be the lipoprotein complex most susceptible to oxidation.
Indeed, HDL is as susceptible or more susceptible to
oxidation in vitro than is LDL (7). However, HDL has been
shown to protect LDL from oxidation in vitro (10). The
factors protecting against formation of lipid hydroperoxides in plasma
appear to be apoA-I (11-13) and the enzyme paraoxonase (PON-1) (10,
14).
Peroxynitrite, the product of nitric oxide and superoxide, is thought
to be an important biologically produced oxidant (15, 16). It is
relevant to cardiovascular disease, as its formation is enhanced by
inflammatory responses of macrophages and neutrophils, and in
conditions such as ischemia reperfusion (16, 17). During an
inflammatory response, acute phase HDL is formed, which itself becomes
proinflammatory, in contrast to the anti-inflammatory properties of
native HDL (18, 19). This acute phase HDL may be oxidatively modified
by peroxynitrite. Peroxynitrite can directly oxidize polyunsaturated
fatty acids (16), tocopherols (20-22), carotenoids (20), proteins,
carbohydrates, and DNA (16). As reviewed by Francis (7), a number of
oxidants have been used to modify HDL. These either primarily modify
HDL lipid or HDL proteins and most often impair known functions of HDL.
Oxidation of HDL by tyrosyl radical affects primarily HDL apoproteins
and enhances its activity in reverse-cholesterol transport (7). However, the oxidation of HDL by peroxynitrite has not been well characterized.
3-Morpholinosydnonimine (SIN-1) generates both nitric oxide and
superoxide simultaneously to form peroxynitrite (23). Therefore, it
mimics the environment that lipoproteins may be exposed to in the
vasculature (15).
We have compared the oxidation products of native HDL, trypsinized HDL,
and HDL lipid suspensions and phosphatidylcholine apoA-I
proteoliposomes. Our results show that apoA-I increases the formation
of phosphatidylcholine core aldehydes. In addition, we observed that
lysophosphatidylcholine was formed in significant amounts only during
oxidation of intact HDL, consistent with activation of a phospholipase
A2-like activity. We conclude that PON-1 has a
phospholipase A2-like activity toward phosphatidylcholine
core aldehydes.
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EXPERIMENTAL PROCEDURES |
Materials--
SIN-1,
N-bis(carboxymethylamino)-ethylglycinepentaacetic acid)
(DTPA), dipentadecanoylglycerophosphocholine,
1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC),
1-palmitoyl-2-arachidonoylglycero-3-phosphocholine (PAPC), and
dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were from
Avanti Inc. (Alabaster, AL); t-butyl-hydroperoxide and
trypsin (bovine pancreas) were purchased from Sigma. Polyclonal rabbit
anti-human apoA-I and anti-human apoA-II antibodies were prepared in
the laboratory (24), and anti-rabbit IgG alkaline phosphatase conjugate was purchased from Bio-Rad.
1-Palmitoyl-2-(5-oxo)valeroyl-sn-glycero-3-phosphocholine and
1-palmitoyl-2-(9-oxo)nonanoyl-sn-glycero-3-phosphocholine and the corresponding core acids were prepared in the laboratory by
reductive ozonization of PLPC and PAPC, as described previously (25).
Choline phospholipids were measured using the choline phospholipid
enzymatic assay kit from Roche Molecular Biochemicals. All solvents
used in liquid chromatography-mass spectrometry were HPLC grade. Other
solvents and chemicals were of reagent grade and were provided by local suppliers.
Isolation and Oxidation of HDL--
HDL was isolated from serum
of subjects fasted for 12-14 h, by ultracentrifugation between
densities 1.063 and 1.21 g/ml (26), and exhaustively dialyzed in 10 mM phosphate-buffered saline (PBS), pH 7.4. HDL (1 mg/ml
protein) (27) was oxidized by the peroxynitrite donor, SIN-1 (1 mM), for up to 20 h at 37 °C, in the presence of
DTPA (100 µM), a metal ion chelator (28). At each time
point, an aliquot was withdrawn; the oxidation was stopped by the
addition of 100 µM butylated hydroxytoluene (BHT), and
HDL lipid-soluble oxidation products were extracted with
chloroform/methanol 2:1 (v/v) (29). The yields of the oxidation
products were expressed as µg/mg of phosphatidylcholine at time 0.
SDS-PAGE--
SDS-PAGE was performed, in the presence of
dithiothreitol, using an 11% polyacrylamide gel (30). After
electrophoresis, proteins were either stained, using silver stain (Plus
One, Amersham Pharmacia Biotech), or transferred to polyvinylidine
fluoride membranes and immunoblotted using polyclonal rabbit anti-human apoA-I or anti-human apoA-II antibodies.
Liquid Chromatography/Electrospray Ionization/Mass
Spectrometry--
Lipid-soluble HDL oxidation products were extracted,
as described previously, after addition of an internal standard
(dipentadecanoyl glycerophosphocholine). The organic phase was
evaporated under nitrogen, and lipids were redissolved in 500 µl of
chloroform/methanol 2:1 (v/v). LC/ESI/MS analysis was performed using a
normal phase silica column (4.6 × 250 mm, Alltech Associates,
Deerfield, IL), in a Hewlett-Packard model 1050 liquid chromatograph,
connected to a Hewlett-Packard model 5989A Quadrupole Mass Spectrometer (MS), equipped with a nebulizer-assisted electrospray ionization (ESI)
interface (HP 59987A). The column was eluted with a linear gradient of
100% A (chloroform/methanol/30% ammonium hydroxide, 80:19.5:0.5 by
volume) to 100% B (chloroform/methanol/water/30% ammonium hydroxide,
60:34:5.5:0.5 by volume) for 14 min, followed by 100% B for 10 min, at
a flow rate of 1 ml/min (31). The effluent was split 1:50, resulting in
20 µl/min being admitted into the mass spectrometer. The retention
time for phosphatidylcholine was determined using standard 16:0/20:4
glycerophosphocholine. The capillary exit voltage was set at 150 V,
with electron multiplier at 1795 V. Positive ESI spectra were examined
in the mass range 450-1100 atomic mass units. The masses given in the
figures are nominal masses. The actual masses of the [M + 1]+ (positive ion mode) ions are 1 mass unit higher. The
molecular species of the oxidation products were identified based on
the molecular mass provided by ESI/MS, the knowledge of the fatty acid
composition of the phospholipid classes, and the relative chromatographic retention time of the phosphatidylcholine standard.
Tryptic Cleavage of HDL-bound Proteins--
Trypsin was
dissolved in PBS, pH 7.4, and added to HDL at an enzyme to HDL protein
ratio of 1:10. The mixture was incubated overnight at 37 °C. HDL was
diluted to 1 mg/ml using PBS and subjected to oxidation with SIN-1. At
specific times, 1 ml of the oxidation mixture was withdrawn, and the
reaction was stopped by the addition of 100 µM BHT,
followed by extraction of the HDL lipids.
Preparation of HDL Lipid Suspensions--
HDL was extracted with
chloroform/methanol (2:1, v/v) and the solvent removed under
N2. Buffer was added; the sample was vortexed, and the
lipid suspension was sonicated (model 1200 bath sonicator, Branson
Instruments) for 2 min. This preparation, referred to as HDL lipid
suspension, was oxidized with SIN-1 at 37 °C for up to 6 h,
under conditions similar to those used for HDL oxidation. Lipid-soluble
oxidation products were extracted with chloroform/methanol 2:1 (v/v),
and after addition of the internal standard, dipentadecanoyl glycerophosphocholine, specimens were prepared for LC/ESI/MS analysis.
Preparation of ApoA-I Proteoliposomes--
HDL was isolated from
serum taken from subjects fasted for 12-14 h, by ultracentrifugation
between densities 1.063 and 1.21 g/ml, and exhaustively dialyzed in
PBS, pH 7.4. ApoA-I was isolated by ion-exchange HPLC using an Aquapore
AX-300 column (3 cm × 4.6 mm, Pierce) at a flow rate of 1 ml/min,
using a gradient of 0.03 M Tris, pH 7.45, 40%
isopropyl alcohol to 0.3 M Tris, pH 7.45, 50%
acetonitrile, run over 15 min. 1-Palmitoyl-2-linoleoyl
glycerophosphocholine hydroperoxides were prepared by incubation of
PLPC with 10 mM t-butyl-hydroperoxide at
37 °C for 1 h, at which time the reaction was terminated by
addition of BHT to a final concentration of 100 µM (32).
Proteoliposomes enriched in PLPC hydroperoxides were prepared with PLPC
hydroperoxides/lysophosphatidylcholine/apoA-I in the molar ratio of
57:170:1 (33), and were incubated for up to 6 h at 37 °C in the
presence of 100 µM DTPA. Proteoliposomes to be used for
oxidation experiments, including controls, were prepared by cholate
dialysis of emulsions of apoA-I/PLPC/DMPC/cholesterol in a molar ratio
1:18:82:5 (34). ApoAI was also exposed to SIN-1 for 2 h and used
at the same molar ratio as described above. Proteoliposomes were
incubated with 1 mM SIN-1 for up to 6 h at 37 °C in
the presence of 100 µM DTPA, at a final amount of 150 µg of phosphatidylcholine. Liposomes (without protein) were also
prepared and oxidized by SIN-1. At each time point, for all
experiments, an aliquot was withdrawn; the oxidation was stopped by the
addition of BHT (final concentration, 100 µM), and
lipid-soluble oxidation products were extracted with
chloroform/methanol 2:1 (v/v). The lipid extract was dried under
nitrogen and redissolved in 500 µl of chloroform/methanol for
LC/ESI/MS analysis.
Preparation of Core Aldehyde
Proteoliposomes--
Proteoliposomes were prepared by cholate dialysis
(35). Briefly, apoA-I in 10 mM Tris-HCl buffer, pH 7.4, was
added to a dried lipid film containing equimolar quantities of
1-palmitoyl-2-(5-oxo)valeroyl-sn-glycero-3-phosphocholine and the corresponding carboxylic acid,
1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (C5 Ald
PC and C5 Acid PC) or
1-palmitoyl-2-(9-oxo)nonanoyl-sn-glycero-3-phosphocholine and the corresponding carboxylic acid 1-palmitoyl-2-azelaoyl
sn-glycero-3-phosphocholine (C9 Ald PC and C9 Acid PC), with
dimyristoylphosphatidylcholine and free cholesterol in molar ratio
0.8:30:30:190:12.5. The dispersed liposomes were dialyzed overnight
against 10 mM Tris-HCl, 0.15 M NaCl buffer, pH
7.4.
Serum PON-1 Purification--
PON-1 Q192 type was purified from
outdated human plasma through (pseudo) affinity chromatography using
Cibacron Blue 3GA (Sigma) and anion-exchange chromatography consisting
of two consecutive DEAE-Bio-Gel A (Bio-Rad) columns as described (36,
37). SDS-PAGE analysis of purified PON-1 showed one broad band at 45 kDa corresponding to PON-1. In order to check for other residual
protein contaminants such as PAF-AH or lecithin:cholesterol
acyltransferase, we analyzed the PON-1 preparation by reverse
phase-high pressure liquid chromatography using Supelcosil LC-8
(250 × 4.6 mm inner diameter, Supelco, Bellefonte, PA), in
Hewlett-Packard model 1090 liquid chromatograph equipped with
photodiode array detector (Waters 990). The UV absorbance detector was
set to 280 nm. The column was eluted with a linear gradient of 10% A
(0.1% trifluoroacetic acid/water) to 80% B (0.1% trifluoroacetic
acid/acetonitrile) in 20 min, followed by 80% B for 5 min, at a flow
rate 1 ml/min. Further confirmation of the purity of our PON-1
preparation was obtained by microcapillary electrospray LC/MS/MS.
Briefly, the equivalent of 1 µg of PON-1 in 100 mM
NH4HCO3, 1 mM CaCl2
buffer, pH 8.5, was digested by trypsin overnight at 37 °C with 2 µl of immobilized trypsin Poros beads (Perspective Biosystems). The
digested peptides were fractionated on a 7.5-cm (100 µm inner
diameter) reverse phase C18 capillary column, attached in-line to a
Finnigan LCQ-Deca ion trap mass spectrometer. The entire digested
sample was loaded as described by Gatlin et al. (38), and
the peptides were eluted by ramping a linear gradient from 2 to 60%
solvent B in 90 min. Solvent A consisted of 5% acetonitrile, 0.5%
acetic acid, and 0.02% HFBA and solvent B consisted of 80:20 (v/v)
acetonitrile/water, containing 0.5% acetic acid and 0.02% HFBA. The
flow rate at the tip of the needle was set to 300 nl/min by programming
the HPLC pump and use of a split line. The mass spectrometer cycled
through four scans as the gradient progressed. The first was a full
mass scan, followed by three tandem mass scans of the successive three
most intense ions. A dynamic exclusion list was used to limit
collection of tandem mass spectra for peptides that eluted over a long
period. All tandem mass spectra were searched using the SEQUEST
computer algorithm against the complete non-redundant protein data base (6/2000). Each high scoring peptide sequence was manually compared with
the corresponding tandem mass spectrum to ensure the match was correct.
Neither lecithin:cholesterol acyltransferase nor PAF-AH was detected.
PON-1 Arylesterase Activity--
PON-1 arylesterase was
determined using 1 mM phenyl acetate as the substrate, and
the product was monitored at 270 nm at 25 °C in 20 mM
Tris-Cl, pH 8.0, 1 mM CaCl2. Blanks were used
to correct for the spontaneous hydrolysis of phenyl acetate. PON-1
arylesterase activity was calculated as described (39).
Statistics--
All results are expressed as mean ± S.D.
Comparisons among treatments were tested for statistical significance
using repeated measures analysis of variance in Prism 3.0 (GraphPad
Software, Inc., San Diego, CA). Bonferroni post-t tests were
used for statistical comparison of individual time points.
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RESULTS |
Identification of Phosphatidylcholine Oxidation
Products--
Representitive total positive ion current profiles for
HDL, oxidized HDL, and oxidized HDL lipids are shown in Fig.
1. LC/ESI/MS analysis of HDL (Fig.
1A) showed phosphatidylcholine as the major component with
smaller amounts of sphingomyelin and lysophosphatidylcholine, which are
eluted in order of increasing polarity. An examination of the total ion
current averaged over the phosphatidylcholine peak from HDL showed that
the major components were 16:0/18:2 (m/z 758), 16:0/20:4
(m/z 782), 18:1/18:2 (m/z 784), 18:0/18:2 (m/z 786), and 18:0/20:4 (m/z 810)
glycerophosphocholines. A minor peak was seen for the ethanolamine
phospholipids, which are best determined in the negative ion mode.
Following a 6-h incubation with SIN-1, the phosphatidylcholine peak was
greatly reduced (Fig. 1B), whereas the peak for
lysophosphatidylcholine was greatly increased. Peaks were seen with
retention times corresponding to phosphatidylcholine hydroperoxides,
isoprostanes, and core aldehydes. The mass spectrum, averaged over the
lysophosphatidylcholine peak, indicated the presence of the 16:0
(m/z 494) and 18:0 (m/z 522) species as major
components in both native and oxidized HDL. In contrast to HDL,
phosphatidylcholine in the HDL lipid suspensions was much less affected
by SIN-1 (Fig. 1C), as seen from the limited accumulation of
phosphatidylcholine hydroperoxides, isoprostanes, core aldehydes, and
lysophosphatidylcholine.

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Fig. 1.
LC/ESI/MS analysis of HDL phospholipids
following oxidation with SIN-1. A, control HDL.
B, HDL oxidized for 6 h. C, HDL lipid
suspension oxidized for 6 h. Eluted peaks were identified by
relative retention times of standards and the molecular mass of the
components as follows: PE, phosphatidylethanolamine;
PC, phosphatidylcholine; SM, sphingomyelin;
LysoPC, lysophosphatidylcholine; PC OOH,
phosphatidylcholine hydroperoxides; isoP PC, isoprostane
glycerophosphocholines; Ald PC, phosphatidylcholine core
aldehydes. LC/ESI/MS conditions were as described under "Experimental
Procedures". The profile is representative of three separate
experiments.
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The mass spectra of the major components eluting from 16.5 to 18.0 min
of a typical profile for HDL after 6 h of oxidation with SIN-1 is
shown in Fig. 2. The major ions included
m/z 790 (34:2 monohydroperoxy glycerophosphocholine);
m/z 846 (36:4 dihydroperoxy glycerophosphocholine);
m/z 830 (36:4 E2/D2
isoprostanes), m/z 858 (38:4 E2/D2
isoprostanes), m/z 832, (36:4 F2 isoprostanes), and m/z 860 (38:4 F2 isoprostanes)
glycerophosphocholine (Fig. 2A). The major ions of a
representative profile of HDL oxidized for 6 h and averaged over
19.0-21.5 min are shown in Fig. 2B. The ions m/z
594 (16:0/5:0), m/z 650 (16:0/9:0), and m/z 678 (18:0/9:0) represent the molecular ions of core aldehydes of
glycerophosphocholines. There was also a small ion, m/z 622, representing the 18:0/5:0 core aldehyde glycerophosphocholine.

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Fig. 2.
Mass spectra of mono- and dihydroperoxides,
isoprostanes, and core aldehydes accumulated during 6 h of
oxidation of HDL with SIN-1. A, full mass spectrum
averaged over the elution times of the hydroperoxide and isoprostane
glycerophosphocholine peaks (16.5-18 min) from oxidation of HDL.
B, selected positive ion mass spectrum averaged over the
elution time of the phosphatidylcholine core aldehyde peaks (19-21.5
min) from oxidation of HDL. m/z 790, phosphatidylcholine monohydroperoxide; m/z 846, phosphatidylcholine dihydroperoxide; m/z 830, 16:0-E2/D2 isoprostanes glycerophosphocholine;
m/z 858, 18:0- E2/D2 isoprostanes
glycerophosphocholine; m/z 832, 16:0-F2
isoprostanes glycerophosphocholine; m/z 860, 18:0-F2 isoprostanes glycerophosphocholine; m/z
650, 16:0-9:0 aldehyde glycerophosphocholine; m/z 678, 18:0-9:0 aldehyde glycerophosphocholine. LC/ESI/MS conditions were as
described under "Experimental Procedures."
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The major mono- and dihydroperoxy phosphatidylcholines present
following a 2-h incubation of HDL with SIN-1 are shown in Fig. 3. The single positive ion mass
chromatograms showed the m/z 814 (36:4) and m/z
816 (36:2) monohydroperoxides and the m/z 846 (36:4), m/z 848 (36:3), and m/z 874 (38:4)
dihydroperoxide glycerophosphocholines. The monohydroperoxides eluted
from the normal phase column before the dihydroperoxides, regardless of
the length of the fatty acyl chains.

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Fig. 3.
Single positive ion mass chromatograms of the
mono- and dihydroperoxides of phosphatidylcholine in HDL lipid
suspension after a 2-h oxidation with SIN-1. Most of these ions
could be seen in the total mass spectrum of the 6-h oxidation of HDL
(Fig. 2A). Peaks are identified in the figure. 34:2,
36:2, 36:3, 36:4 and 38:4 represent the total number of
fatty acyl carbons and the number of double bonds. PC OOH
(1xOOH), phosphatidylcholine monohydroperoxide; PC
OOH (2xOOH), phosphatidylcholine dihydroperoxide; other
abbreviations are as given under "Results." LC/ESI/MS conditions
were as described under "Experimental Procedures."
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The major isoprostane derivatives of phosphatidylcholine present
following a 2-h incubation of HDL with SIN-1 are shown in Fig.
4. Single positive ion mass chromatograms
for the ions m/z 830 and 858 correspond to the 36:4 and 38:4
E2/D2 isoprostanes, whereas the ions
m/z 832 and m/z 860 correspond to the 36:4 and 38:4 F2 isoprostanes. The ion m/z 828 corresponds to an isoprostane-like product,
epoxy-isoprostanoylglycerophosphocholine. The isoprostanes eluted as
broad peaks, consistent with these compounds being a mixture of
regioisomers. The 18:0/F2 isoprostane
glycerophosphocholines were identified as homologues possessing 28 additional mass units.

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Fig. 4.
Single positive ion mass chromatograms of the
phosphatidylcholine isoprostanes accumulated in HDL after 2 h of
oxidation with SIN-1. Peaks are identified and structural formulae
given in the figure. Epoxy IsoP PC, epoxy-isoprostane
glycerophosphocholines; IsoP PC, isoprostane
glycerophosphocholines. 36:4 and 38:4 represent
total number of fatty acyl carbons and number of double bonds.
LC/ESI/MS conditions were as described under "Experimental
Procedures."
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The single positive ion mass chromatograms for the major core aldehydes
of phosphatidylcholine that were present following a 2-h incubation of
HDL with SIN-1 are shown in Fig. 5. The
ions at m/z 594 and m/z 622 correspond to the
16:0/5:0 aldehyde and 18:0/5:0 aldehyde glycerophosphocholines, and
ions at m/z 650 and m/z 678 correspond to the
16:0/9:0 aldehyde and 18:0/9:0 aldehyde glycerophosphocholines. The
ions at m/z 636 and m/z 676 were attributed to
the monohydroxy 16:0/9:1 aldehyde and the 18:1/9:0 aldehyde glycerophosphocholines, respectively. The aldehydes are eluted as sharp
peaks in order of increasing polarity (decreasing chain length).

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Fig. 5.
Single positive ion mass chromatograms of the
elution time of the phosphatidylcholine core aldehyde peaks (19-21.5
min) from oxidation of HDL after a 6-h incubation with SIN-1.
Peaks are identified in the figure. Carbon:double bond numbers are
indicated in figure. Ald PC, aldehyde glycerophosphocholine;
OH, Ald PC, hydroxy aldehyde glycerophosphocholine.
LC/ESI/MS conditions were as described under "Experimental
Procedures."
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The accumulation of the hydroperoxides of phosphatidylcholine (Fig.
6A) was most rapid and reached
the highest level during oxidation of native HDL, compared with HDL
lipid suspensions or trypsinized HDL (p < 0.0013). In
contrast, the accumulation of the isoprostane derivatives (Fig.
6B) was much more similar between native and trypsinized HDL
and HDL lipid suspensions, although the initial rate of accumulation
was highest for the native HDL (p < 0.035). The
accumulation of core aldehydes (Fig. 6C) paralleled that of
hydroperoxides and was higher for native HDL than trypsinized HDL or
HDL lipid suspensions. The accumulation of the lysophosphatidylcholine (Fig. 6D) was highest for native HDL (p < 0.0001) compared with HDL lipid suspensions or trypsinized HDL.

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Fig. 6.
Time course of accumulation of major
phosphatidylcholine oxidation products in HDL (open
squares), in HDL lipid suspensions (closed
squares), and in trypsinized HDL (open
circles) during oxidation with SIN-1.
Phosphatidylcholine hydroperoxides (A), phosphatidylcholine
isoprostanes (B), phosphatidylcholine core aldehydes
(C), and lysophosphatidylcholine (D). LC/ESI/MS
conditions were as described under "Experimental Procedures."
Values represent the mean ± S.D. of three separate
experiments.
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Apolipoprotein A-I Promotes Formation of Phosphatidylcholine Core
Aldehydes--
ApoA-I proteoliposomes were incubated with SIN-1 for up
to 6 h, and the formation of core aldehydes was measured. There
was a significantly greater increase in PLPC hydroperoxides in the presence of apoA-I (Fig. 7, lower
panel) parallel to the accumulation of core aldehydes compared
with control (p < 0.0001) (Fig. 7, upper
panel). The above experiments provided evidence that apoA-I promoted the formation of core aldehydes. However, to exclude the
possibility that reaction between phosphatidylcholine oxidation products and SIN-1 may have confounded the observations, we studied the
formation of core aldehydes in the absence of SIN-1 by using phosphatidylcholine hydroperoxides.

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Fig. 7.
Concentration of phosphatidylcholine core
aldehyde (upper panel), phosphatidylcholine
monohydroperoxide (middle panel), and
phosphatidylcholine hydroxide (lower panel) following
oxidation with SIN-1 in the absence (open symbols) or
presence (closed symbols) of apoA-I. PLPC
proteoliposomes were prepared by cholate dialysis. Conditions of
proteoliposome peroxidation and of LC/ESI/MS analysis are as described
under "Experimental Procedures". Values represent mean ± S.D.
of three separate experiments.
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The effect of apoA-I on the conversion of phosphatidylcholine
hydroperoxides into core aldehydes is shown in Fig.
8. Phosphatidylcholine hydroperoxides
showed a modest decrease in the presence of apoA-I, compared with
control incubations in the absence of apoA-I (Fig. 8, upper
panel). There was a 2-fold higher concentration of core aldehydes
in the presence of apoA-I, compared with the absence of apoA-I (Fig. 8,
lower panel, p < 0.001).

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Fig. 8.
Concentration of phosphatidylcholine
monohydroperoxides (upper panel) and
phosphatidylcholine core aldehydes (lower panel) in
the absence (open symbols) and the presence of
(closed symbols) apoA-I. PLPC hydroperoxide
proteoliposomes were prepared using lysophosphatidylcholine. Conditions
of proteoliposome incubation and of LC/ESI/MS analysis as described
under "Experimental Procedures." Values represent mean ± S.D.
of three separate experiments.
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PON-1 Has a Phospholipase A2-like Activity toward PC
Core Aldehydes and Acids--
ApoA-I proteoliposomes were prepared
with DMPC and either a mixture of C5 Ald PC and C5 Acid PC or a mixture
of C9 Ald PC and C9 Acid PC. PON-1 readily converted C5 Ald PC and C5
Acid PC proteoliposomes to lysophosphocholine (p < 0.0055 and p < 0.0001, respectively) (Fig.
9A) and also C9 Ald PC and C9
Acid PC proteoliposomes to lysophosphocholine (p < 0.001 and p < 0.0001, respectively) (Fig.
9B). The hydrolysis of C5 Ald PC was significantly greater than the hydrolysis of C9 Ald PC. C5 Acid PC and C9 Acid PC were hydrolyzed to a similar extent. Myristoyl lysoglycerophosphocholine was
not detected, consistent with PON-1 being specific for short or
intermediate length fatty acyl chains, but not the intact long chain
fatty acids in the sn-2 position of PC. This is also
consistent with the absence of the release of mono- or
dihydroperoxylinoleate in the previous experiments (data not shown).
The hydrolysis of the core aldehydes and acids by PON-1 in this
experiment suggests that the relatively high concentrations of
lysophosphatidylcholine during HDL oxidation by SIN-1 could be
accounted for by PON-1.

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Fig. 9.
The effect of PON-1 on the core aldehydes and
carboxylate proteoliposomes. A, hydrolysis of C5 Ald PC
(striped bar) and C5 Acid PC (open bar) by PON-1
to produce lysophosphatidylcholine (solid bar).
B, hydrolysis of C9 Ald PC (striped bar) and C9
Acid PC (open bar) by PON-1 to produce lyso PC (solid
bar). Preparation of proteoliposomes and LC/ESI/MS conditions were
as described under "Experimental Procedures." Values represent the
mean ± S.D. of three separate experiments.
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Effect of Oxidation on HDL Proteins--
The apoproteins of native
and oxidized HDL were analyzed by SDS-PAGE and showed that monomeric
apoA-I was the major component at all times. Bands with the molecular
weight of the apoA-I dimer, apoA-I trimer, and apoA-I:apoA-II dimer
were detectable by 1 h. The identity of these bands was confirmed
by immunoblot with polyclonal anti-apoA-I antibodies (Fig.
10) and anti-apoA-II antibodies (data not shown).

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Fig. 10.
SDS-electrophoresis of HDL apolipoproteins
showing the time course from 0 to 20 h during oxidation with
SIN-1. Left panel, silver stain of proteins;
Right panel, anti-apoA-I immunoblot.
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PON-1 Arylesterase Activity Is Reduced by Peroxidation--
HDL
PON-1 arylesterase activity was relatively stable under control
conditions (Fig. 11A,
open circles). PON-1 arylesterase activity of HDL declined
to about 60% of the original value within a few minutes of the
addition of SIN-1 and decreased to 20% of the original value after
4 h, being maintained at this level for up to 20 h (Fig.
11A, open squares). To address whether the loss of activity was due to SIN-1-derived peroxynitrite or lipid oxidation products, the arylesterase activity of native HDL was followed during
incubation with HDL lipid suspensions that had been oxidized by
incubation with SIN-1 for 4 h (Fig. 11A, closed
squares). This resulted in a significant decrease in arylesterase
activity, although it was not as large as that observed by oxidation of
HDL. This suggests that both peroxynitrite and lipid oxidation products are responsible for the large decrease in arylesterase activity.

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Fig. 11.
A, time course of changes in
HDL-associated PON-1 arylesterase activity. Native HDL was incubated
with SIN-1 (open squares), oxidized HDL lipids (closed
squares), or without additions (open circles). Values
represent the mean ± S.D. of three separate experiments.
B, PON-1 arylesterase activity during oxidation of PLPC
proteoliposomes by SIN-1, or C5 Ald PC, or C9 Ald PC proteoliposomes.
PON-1 was incubated with PLPC proteoliposomes alone (closed
circles) or with PLPC in the presence of SIN-1 (closed
squares). C5 Ald PC, open circles; and C9 Ald PC,
open squares. PON-1 arylesterase was reduced by 30%
compared with control (p < 0.03) during PLPC oxidation
by SIN-1. Values represent the mean ± S.D. of three separate
experiments.
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Loss of PON-1 Arylesterase Activity during PLPC Oxidation--
To
characterize the loss of PON-1 arylesterase activity during oxidation
of HDL with SIN-1, this activity was assayed over a period of 6 h
during the oxidation of apoA-I proteoliposomes with SIN-1 and compared
with its activity after incubation with SIN-1 or the PC oxidation
products, C5 Ald PC or C9 Ald PC (Fig. 11B). PON-1
arylesterase activity was reduced by 30%, compared with control
(p < 0.03) after 6 h when incubated with PLPC
exposed to SIN-1. In contrast, when PON-1 was incubated for 6 h
with C9 aldehyde- and C9 carboxylate PC or C5 aldehyde- and C5
carboxylate PC, there was minimal loss of PON-1 arylesterase activity
(15 and 5%, respectively) compared with control. This demonstrates that PON-1 arylesterase activity is not inhibited by PC aldehydes. Incubation of PON-1 with 1 mM SIN-1 for 6 h reduced
PON-1 arylesterase activity by 10%. Thus the marked loss of activity
of PON-1 that we observed during oxidation of HDL was due to the
additive effects of lipid oxidation products and SIN-1.
 |
DISCUSSION |
This study demonstrates that peroxynitrite is capable of oxidizing
HDL lipids to a variety of products, the content and composition of
which is modified by the presence of apoA-I and PON-1. The major
phosphatidylcholine oxidation products were the mono- and dihydroperoxides, core aldehydes, and isoprostanes. The high yield of
the phosphatidylcholine core aldehydes during peroxynitrite oxidation
of native HDL, in the presence of metal chelators is of special
interest, since this eliminates the potential confounding reactions of
metal ions, such as Cu2+ (40-42). Mashima et
al. (13) showed that apoA-I was capable of stoichiometric
conversion of phosphatidylcholine hydroperoxides to phosphatidylcholine
hydroxides. The efficiency of this conversion was found to be low,
compared with incubation of phosphatidylcholine hydroperoxides with
whole plasma, but the conversion to core aldehydes was not studied. Our
data suggest that the major product formed by the interaction of
phosphatidylcholine hydroperoxides with apoA-I is the
phosphatidylcholine core aldehydes. There may be differences in
experimental conditions that explain the different conclusions of
previous studies and the results presented here. However, none of the
previous studies used LC/ESI/MS to study the intact phosphatidylcholine
oxidation products, and thus they were not able to assess the formation
of phosphatidylcholine core aldehydes.
Studies by Gardner et al. (43) have indicated that chemical
decomposition of hydroperoxides to aldehydes requires alkaline conditions. However, Kanazawa and Ashida (44, 45) have recently reported that lipid hydroperoxides, liberated by intestinal lipases from triacylglycerols, decompose to aldehydes via epoxyketones and
hydroxides. The exact mechanism of this reaction is unknown, and it is
not known whether or not similar reactions take place with
hydroperoxides and hydroxides attached to the fatty acids of
glycerophospholipids. The observation that apoA-I can catalyze this
decomposition suggests that specific amino acid(s) or a cluster of
amino acids act as an active center in apoA-I that in the presence of
lipid hydroperoxides enhances the formation of aldehydes.
The oxidation of methionine to methionine sulfoxide by lipid
hydroperoxide is a two-electron oxidation (11, 12). The observation that both apoA-I and apoA-II can participate in this transformation suggests that the major requirement is for a methionine residue to be
in the hydrophobic environment of the hydroperoxy fatty acyl chains.
This contrasts with the one-electron oxidation that produces core
aldehydes via
-scission of the fatty acyl alkoxyl radical (46).
Oxidation-sensitive amino acids that may be involved would include
tyrosine, which is well known to form tyrosyl free radical (7, 47),
tryptophan, and histidine (48). Tyrosyl free radical has been shown to
modify apoA-I and apoA-II and promote apoA-I:apoA-II dimer formation in
HDL (7) and dityrosine formation in LDL (47). ApoA-I has seven tyrosine
residues, and a number of these are predicted to reside within the
phospholipid acyl chains in apoA-I proteoliposomes (49). Future studies
will address whether specific apoA-I tyrosine residues are involved in
this reaction.
The high proportion of lysophosphatidylcholine, formed during the
course of the oxidation of native HDL, suggests the presence of one or
more phospholipase A2-like enzymes. Since control
incubations of HDL, in the absence of free radical-generating systems,
yielded only minimal increases in lysophosphatidylcholine, it must be concluded that the(se) enzyme(s) became activated by the presence of
oxidized phospholipids or are specific for oxidized phospholipids. The
increased lysophosphatidylcholine content of oxidized LDL can be
accounted for by PAF-AH (50, 51). HDL, however, contains much lower
levels of PAF-AH than LDL, and MacPhee et al. (52) have
shown that inhibition of PAF-AH by SB-222657, an azetidinone derivative, has little effect on lysophosphatidylcholine generation during HDL oxidation by Cu2+. Our direct investigation of
PON-1 demonstrates that it fulfills the criteria to be the
phospholipase A2 in HDL that is active during lipid
oxidation. Our studies do not exclude the presence of additional
enzymes. A second candidate is paraoxonase-3, which has been shown to
be present in rabbit HDL (53). Paraoxonase-3 shares many of the same
substrates as PON-1, and to date no reagent or experimental condition
has been described to inhibit selectively PON-1 versus
paraoxonase-3. Further studies are needed to determine whether
additional enzymes are present in HDL that could also hydrolyze
phospholipid oxidation products.
The present study showed that incubation of HDL with SIN-1 led to an
80% reduction in the arylesterase activity of PON-1. This report is
consistent with Aviram et al. (54) in which they showed
PON-1 activity was inhibited by 31-65% (assayed as arylesterase) by
PAPC or oxidized cholesteryl linoleate (55). PON-1 can hydrolyze multiple substrates with different catalytic activities (56, 57),
including PAF-AH-like activity (58). Thus, although PON-1 arylesterase
activity is inhibited during HDL oxidation, it appears that its
phospholipase A2-like activity toward core aldehydes is maintained.
We measured the phosphatidylcholine isoprostanes of the
E2/D2 and F2 series over a wide
range of experimental conditions. The relatively similar yields of
these products suggest that their formation is determined by the
chemical reaction of peroxynitrite and arachidonic acid with little or
no influence due to the proteins of HDL.
Remarkably, the present study has shown that apoA-I increases the yield
of a class of oxidized phospholipids, the core aldehydes, that have
been identified as a major bioactive component of minimally modified
LDL (59). We propose that, in vivo, this function of apoA-I
is coupled with the activity of PAF-AH and PON-1 to effectively divert
phosphatidylcholine hydroperoxides to biologically inactive products.
However, in the context of a pro-inflammatory response, apoA-I would
maintain its function while the activity of both PAF-AH and PON-1
would decrease. This duality of function for HDL would be consistent
with the observations and hypothesis of van Lenten et al.
(18) that HDL can be converted from an anti-inflammatory lipoprotein to
a pro-inflammatory lipoprotein (19).
In summary, the present results show that peroxynitrite oxidation of
HDL phosphatidylcholine results in a broad spectrum of oxidation
products. However, the proportions of the types of oxidized phosphatidylcholines and lysophosphatidycholine are significantly affected by apoA-I and PON-1. The yields of the core aldehydes, which
are generated by cleavage of the peroxidized fatty acyl chains, are
probably underestimated as they are continuously degraded by the
phospholipase A2-like activity of the oxidized HDL.