Structural Identification by Mass Spectrometry of Oxidized Phospholipids in Minimally Oxidized Low Density Lipoprotein That Induce Monocyte/Endothelial Interactions and Evidence for Their Presence in Vivo*

(Received for publication, November 22, 1996, and in revised form, March 21, 1997)

Andrew D. Watson Dagger §, Norbert Leitinger Dagger , Mohamad Navab Dagger , Kym F. Faull , Sohvi Hörkkö par , Joseph L. Witztum par , Wulf Palinski par , Dawn Schwenke **, Robert G. Salomon Dagger Dagger , Wei Sha Dagger Dagger , Ganesamoorthy Subbanagounder Dagger Dagger , Alan M. Fogelman Dagger and Judith A. Berliner Dagger §§

From the Dagger  Department of Medicine, Psychiatry and Biobehavioral Sciences, the  Neuropsychiatric Institute, and the §§ Department of Pathology, University of California, Los Angeles, California 90095-1679, the par  Department of Medicine, University of California, San Diego, California 92093-0682, the ** Department of Pathology, Bowman Gray University School of Medicine at Wake Forest, Winston-Salem, North Carolina 27157-1072, and the Dagger Dagger  Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106-7027

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Entry of monocytes into the vessel wall is an important event in atherogenesis. Previous studies from our laboratory suggest that oxidized arachidonic acid-containing phospholipids present in mildly oxidized low density lipoproteins (MM-LDL) can activate endothelial cells to bind monocytes. In this study, biologically active oxidized arachidonic acid-containing phospholipids were produced by autoxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) and analyzed by liquid chromatography and electrospray ionization mass spectrometry in conjuction with biochemical derivatization techniques. We have now determined the molecular structure of two of three molecules present in MM-LDL and Ox-PAPC that induce monocyte-endothelial interactions. These lipids were identified as 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine (m/z 594.3) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (m/z 610.2). These two molecules were produced by unambiguous total synthesis and found to be identical by analytical techniques and bioactivity assays to those present in MM-LDL and Ox-PAPC. Evidence for the importance of all three oxidized phospholipids in vivo was suggested by their presence in fatty streak lesions from cholesterol-fed rabbits and by their immunoreactivity with natural antibodies present in ApoE null mice. Overall, these studies suggest that specific oxidized derivatives of arachidonic acid-containing phospholipids may be important initiators of atherogenesis.


INTRODUCTION

Atherosclerosis is a chronic inflammatory disease responsible for profound human morbidity and mortality (1). The atherosclerotic lesion typically develops from a clinically silent fatty streak lesion, characterized by the presence of lipid-laden monocyte-macrophages in the subendothelial space, to an advanced fibro-fatty lesion that can rupture, resulting in acute thrombosis and vascular occlusion (2, 3). There is a growing body of evidence suggesting that oxidative modification of low density lipoprotein (LDL)1 is involved in the development and progression of atherosclerosis (4-9). The process by which LDL undergoes free radical-induced oxidation in vitro can be described as having three stages (10-13): the lag phase, during which lipophilic antioxidants (vitamin E, beta -carotene, ubiquinol) are depleted; the propagation phase, during which polyunsaturated fatty acids and cholesterol become oxidized to become lipid hydroperoxides and oxycholesterol, respectively; and the decomposition phase, during which oxidized lipids undergo fragmentation to form aldehydes capable of modifying amino groups on proteins and phospholipids. Minimally modified LDL (MM-LDL) is LDL that has undergone antioxidant depletion and oxidation of arachidonic acid-containing phospholipids, much less linoleic acid oxidation, with little or no protein modification (14, 15). MM-LDL has unique biological properties that are not associated with native LDL or highly oxidized LDL, which may be involved in the recruitment and entry of monocytes into the vessel wall and their subsequent maturation into macrophages (14, 16-18). We have previously shown that oxidized phospholipids isolated from MM-LDL, as well as a mixture of products formed from the autooxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC), mimic the biological activity of MM-LDL (19). There is also evidence that high density lipoproteins may inhibit monocyte-endothelial interactions induced by MM-LDL by altering the molecular structure of these biologically active oxidized phospholipids (15, 19). In this report we combine liquid chromatography with electrospray tandem mass spectrometry (MS/MS) to elucidate the structural identification of biologically active phospholipids in Ox-PAPC and confirm their presence in MM-LDL. In addition, we present evidence for the presence of these oxidized phospholipids in animal models susceptible to fatty streak formation and atherosclerosis.


EXPERIMENTAL PROCEDURES

Materials

Tissue culture media, serum, and supplements were obtained from sources previously reported (14, 17, 20). Acetonitrile, chloroform, hexane, methanol, 2-propanol, triethylamine, and water (all Optima or HPLC grade) were obtained from Fisher. Gelatin (endotoxin-free, tissue culture grade), methoxyamine hydrochloride, sodium borohydride, N,N-diisopropylethylamine, pentafluorobenzyl bromide, and butylated hydroxytoluene (BHT) were obtained from Sigma. Authentic L-alpha -1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL) or Sigma, and L-alpha -1-palmitoyl-2-[1-14C]-arachidonoyl-sn-glycero-3-phosphocholine was purchased from DuPont NEN. Aminopropyl solid phase extraction columns were obtained from J. T. Baker Inc.

Endothelial Cell Cultures

Human aortic endothelial cells at passages 4-7 were cultured as described (14, 17, 20) in Medium 199 supplemented with fetal bovine serum.

Rabbits and Diets

Rabbits were fed for 24 weeks with a semipurified atherogenic diet containing 20% protein (casein), 46% carbohydrate (sucrose), and 11% fat (butter). The diets also contained 0.09% cholesterol, 1% corn oil to provide essential fatty acids, 14% cellulose, and levels of vitamins and minerals similar to those used previously in similar diets (21). For some rabbits the diet was supplemented with the antioxidants vitamin E (147 IU alpha -tocopherol acetate/day/rabbit) or probucol (0.41 g/day/rabbit). After completion of the dietary regimen the rabbits were euthanized and the aortae were removed for the assessment of the extent of atherosclerosis. Immediately after sacrifice, the entire aorta from the aortic arch to just distal of the iliac bifurcation was removed and washed free of blood with 0.15 M phosphate buffer (pH 7.4). The aortae were freed from adventitial debris, opened longitudinally, pinned flat, and photographed. The aortic arch was separated from the descending thoracic aorta 1-2 mm distal to the ductus scar, and the descending thoracic aorta was separated from the abdominal aorta 1-2 mm proximal to the celiac artery. Aortic specimens were weighed, placed in a test tube, covered with argon or nitrogen, and frozen at -70 °C protected from light prior to analysis.

Lipoprotein and Lipid Modification

LDL (1.019-1.069 g/ml) was isolated from the sera of normal blood donors by density gradient ultracentrifugation as described (22) and stored at 4 °C until use within 1-2 weeks of isolation. Lipoprotein concentration is expressed in terms of protein content throughout this report. MM-LDL was produced by dialysis at 5 mg protein/ml in 5 µM FeSO4·7H2O in phosphate-buffered saline (diluted 1:9 with sterile water) + 0.15 M NaCl, pH 6.8, for ~48 h at room temperature. Oxidation was terminated by the addition of EDTA and BHT (100 and 0.3 µM, respectively). The concentration of bacterial endotoxin in medium containing each agonist was <20 pg/ml (determined by chromogenic assay), which is approximately 100-fold less than that required to induce monocyte binding to endothelial cells (data not shown). PAPC was oxidized by transferring 1 mg in 100 µl of chloroform to a clean 16 × 125 mm glass test tube and evaporating the solvent under a stream of nitrogen. The lipid residue was allowed to autoxidize while being exposed to air for 24-48 h. The extent of oxidation was monitored by positive ion flow injection electrospray mass spectrometry.

Lipid Extraction

Lipids were extracted from rabbit aortic tissue with chloroform/methanol (2:2, v/v) (23, 24). An aliquot of this extract was supplemented with 20 µmol BHT/ml, covered with argon, sealed, and stored protected from light. These extracts were supplemented with dimyristoyl phosphatidylcholine (50 ng/mg tissue) as an internal standard for the quantitative analysis of phospholipids (25). Phospholipids, free fatty acids, and neutral lipids were separated by the method of Kaluzny using aminopropyl solid phase extraction chromatography (26). Phospholipid recovery typically ranged between 95 and 98% as determined by including 1-palmitoyl-2-[1-14C]-arachidonoyl-sn-glycero-3-phosphocholine as an internal standard in the lipid-purification procedure. Lipid fractions not used immediately were dried under nitrogen, resuspended in chloroform containing 0.01% BHT, covered with argon, and stored at -44 °C.

Lipid Derivatization

Oxidized phospholipids of interest were semipurified by preparative normal phase HPLC and treated with either methoxyamine hydrochloride, pentafluorobenzyl bromide, or sodium borohydride, and then lipids were extracted with chloroform/methanol (27). Methoxyamine derivatives were produced by resuspending 250 µg of Ox-PAPC in a solution of 0.921 mM methoxyamine hydrochloride in phosphate-buffered saline (pH 4.0). The solution was incubated at 37 °C for 45 min, after which 3.5 ml of chloroform/methanol (2:1, v/v) and 0.5 ml of water was added. The solution was mixed thoroughly and centrifuged at 2,000 × g for 15 min. The chloroform phase was transferred to a clean glass test tube and 0.5 ml of methanol, 0.5 ml of water, and 40 µl of formic acid was added. The solution was again mixed thoroughly and centrifuged at 2,000 × g for 15 min. The chloroform phase was then collected for analysis. The second extraction step, which included formic acid, was necessary to displace salts (mostly sodium) that associated with phospholipids during incubation with phosphate-buffered saline. Without this step both the free phospholipids and phospholipid salts were detected by positive ion electrospray mass spectrometry. Pentafluorobenzyl ester derivatives of carboxylic acids were produced by resuspending 250 µg of Ox-PAPC in 50 µl of a 10% solution of pentafluorobenzyl bromide in acetonitrile and 50 µl of a 20% solution of N,N-diisopropylethylamine in acetonitrile. The solution was mixed thoroughly and incubated for 45 min at room temperature. The solvent was dried under argon, and the lipids were re-extracted by addition of 3.5 ml of chloroform/methanol (2:1) and 0.5 ml of water. The solution was mixed thoroughly and centrifuged at 2,000 × g for 15 min. The chloroform phase was transferred to a clean glass test tube and dried under nitrogen prior to lipid analysis. Ox-PAPC was reduced by resuspending 250 µg of lipid residue in 0.5 ml of a 0.1 M borate buffer (pH 8.0) and adding ~10 µg of sodium borohydride. The lipids were reduced instantaneously and then extracted by addition of 3.5 ml of chloroform/methanol (2:1, v/v). Salts were displaced by re-extraction in the presence of formic acid as described above.

High Performance Liquid Chromatography

Normal phase high performance liquid chromatography (NP-HPLC) was performed by injecting isolated phospholipids (resuspended in chloroform) onto a silica column and eluting isocratically with a mobile solvent of acetonitrile/methanol/water (77:8:15, v/v/v). NP-HPLC analysis was performed using an analytical column (Spherisorb, 150 mm × 4.6 mm, 5 µm; Alltech Associates, Inc.) at a flow rate of 1.0 ml/min or a preparative column (Adsorbosphere, 250 mm × 10 mm, 5 µm; Alltech Associates, Inc.) at a flow rate of 5.0 ml/min. UV absorbance was detected with a diode array detector (L-3000, Hitachi, Ltd., Tokyo, Japan) scanning from 200 to 350 nm. In some cases, fractions were collected from the column under sterile conditions, dried to a residue under nitrogen at 37 °C, and either resuspended in tissue culture medium for monocyte adhesion assays or used as a semipure preparation for further modification and/or analysis by mass spectrometry. Reversed phase HPLC (RP-HPLC) was performed using an ODS column (Adsorbosphere HS, C18, 250 mm × 4.6 mm, 5 µm, Alltech Associates, Inc.). Phospholipids were eluted with a mobile phase of 90% methanol, which was changed linearly over a period of 30 min to 100% methanol, at a flow rate of 1 ml/min. Fractions were collected at 1-min intervals. For a near homogeneous preparation of an oxidized phospholipid of interest, fractions obtained from preparative NP-HPLC were further purified by analytical RP-HPLC.

Electrospray Mass Spectrometry

Electrospray mass spectrometry was performed with a API III triple-quadrupole biomolecular mass analyzer (Perkin-Elmer) fitted with an articulated, pneumatically assisted nebulization probe and an atmospheric pressure ionization source. The tuning and calibration solution consisted of a mixture of polypropylene glycol 425, 1000, and 2000 (3.3 × 10-5, 1 × 10-4, and 2 × 10-4 M, respectively) in H2O/methanol (50:50, v/v) containing 2 mM ammonium formate and 0.1% acetonitrile. Calibration across the m/z range 10-2400 was effected by multiple ion monitoring of eight polypropylene glycol positive ion solution signals (typically the singly charged ions at m/z 58.99, 326.25, 334.30, 906.67, 1254.92, 1545.13, 1863.34, 2010.47, and the doubly charged ion at m/z 520.4). The ion spray voltage was typically operated at 4.5 kV in positive mode and 3.5 kV in negative mode with 40 p.s.i. "zero"-grade air produced by a Zero Air Generator (Peak Scientific, Chicago, IL) at 0.6 liter/min. A curtain gas of N2 produced from the vapors of liquid N2 was used at a flow rate of 0.8 liter/min. Normal and precursor ion mass spectra were acquired by scanning quadrupole Q1. Daughter ion spectra were obtained in the negative ion mode by colliding the quadrupole Q1-selected pseudo-parent ion of interest produced at an orifice voltage of -105 with a mixture of 5.2% nitrogen in argon in quadrupole Q2 and scanning quadrupole Q3 to analyze the fragment ion products. Normal scan spectra were recorded at instrument conditions sufficient to resolve the isotopes of the polypropylene glycol/NH4+ singly charged ion at m/z 906 (40% valley definition). For precursor ion tandem MS/MS the analyzing quadrupole (Q3) and the mass selecting quadrupole (Q1) were operated at a resolution of about unit m/z (using a 35% peak valley definition at low m/z and a 60% peak valley definition at high m/z). Reconstructed selected ion chromatograms were produced by software supplied by Perkin-Elmer. Phospholipids were introduced into the electrospray mass spectrometer by flow injection analysis or liquid chromatography (LC). For flow injection analysis (FIA), 20 µg of phospholipid was resuspended in 20 µl of FIA solvent consisting of acetonitrile/water/formic acid (50:50:0.1, v/v/v) and injected into FIA solvent flowing at a rate of 25 µl/min into the ion source of the mass spectrometer. The mass spectrometer was set to scan from m/z 450-950 with an orifice voltage of +65, a step size of 0.3, a dwell time of 3 ms, and a scan speed of ~4 s. For negative ion electrospray tandem mass spectrometry (step size = 1.0) a solvent of 100% methanol with 1 mM ammonium acetate was used. Quantitative analysis of phospholipids was performed essentially as described by Han et al. (25) using dimyristoylphosphatidylcholine as an internal standard. Phospholipids were quantitated based on their ion intensity relative to the internal standard using positive ion FIA-ESI-MS. The equimolar ion intensities of different species of phosphatidylcholines typically varied by less than 5% (25).

Phospholipid Synthesis

The procedure of Huckstep (28) was modified. Amberlyst-15 resin (4 g) was added to a magnetically stirred solution of 5-pentanolide (2.16 g, 21.6 mmol) in dry methanol (40 ml). The reaction mixture was boiled under reflux under dry nitrogen for 18 h, then filtered through florisil. The solvent was removed under reduced pressure to produce methyl 5-hydroxypentanoate (2.8 g, 99%), which, without further purification, was oxidized with pyridinium chlorochromate as described previously (28). Flash chromatography on silica gel with ethyl acetate/hexane (1:3, v/v) delivered 5-oxopentanoate (1.94 g, 69%). The 1H NMR spectra of methyl 5-hydroxypentanoate and methyl 5-oxopentanoate were identical with those reported previously (28). To a magnetically stirred solution containing ammonium nitrate (80 mg, 1 mmol) in dry methanol (11 ml) was added freshly distilled trimethyl orthoformate (2.96 g, 28 mmol, 4 eq) and methyl 5-oxopentanoate (907 mg, 7 mmol). The reaction mixture was stirred under nitrogen and monitored by TLC. The reaction was stopped at 24 h, and solvent was removed under reduced pressure. The residue was titrated with ether (30 ml), and the ether solution was filtered to remove ammonium nitrate. Solvent was removed under reduced pressure. Flash chromatography on a column (50 mm × 180 mm) of silica gel (40 µm, J. T. Baker Inc.) with ethyl acetate/hexane (1:3, v/v) yielded methyl 5,5-dimethoxypentanoate (883 mg, 78.6%). The 1H NMR spectrum of methyl 5,5-dimethoxypentanoate was identical with that reported previously (29). The methyl ester (142 mg, 0.81 mmol) was stirred with a solution of sodium hydroxide (161.4 mg, 4 mmol, 5 eq) in 4 ml of water/methanol/tetrahydrofuran (2:5:3, v/v/v) at room temperature. After 1.5 h, the reaction mixture was acidified to pH 3 with ice-cold 0.1 N HCl and then extracted with ethyl acetate (3 × 15 ml). The combined organic extracts were dried over MgSO4 and filtered, and the solvent was removed under reduced pressure to provide 5-dimethoxypentanoic acid (129 mg, 99%). The 1H NMR spectrum of 5-dimethoxypentanoic acid was identical with that reported previously (29). Esterification of lysophosphatidylcholine with 5-dimethoxypentanoic acid was accomplished by the general method of Hebertt et al. (30). Traces of moisture were removed from a mixture of acid (86.5 mg, 0.53 mmol, 3.1 eq) and 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (82.2 mg, 0.17 mmol) by azeotropic distillation with toluene (3 × 5 ml) under reduced pressure at 20 °C. Freshly distilled dry CHCl3 (10 ml), dicyclohexylcarbodiimide (105 mg, 0.51 mmol, 3 eq), and p-(N,N-dimethylamino)pyridine (62 mg, 0.51 mmol, 3 eq) were added, and then the flask was flushed with nitrogen and protected from light. The reaction was monitored by TLC and was stopped at 26 h, and the solvent was removed under reduced pressure. The resulting solid was flash chromatographed on a column (20 mm × 180 mm) of silica gel (40 µm, J. T. Baker Inc.) with CHCl3/MeOH/H2O (40:50:10, v/v/v) to produce 1-palmitoyl-2-(5-dimethoxypentanoyl)-sn-glycero-3-phosphocholine (199 mg, 93%). 1H NMR: d 5.21 (dd, 1H, J = 4 Hz, 4 Hz), 4.4-4.3 (4H), 4.1 (dd, 1H, J = 4 Hz, 7 Hz), 3.97 (t, 2H, J = 7 Hz), 3.79 (m, 2H), 3.35 (s, 9H), 3.28 (s, 6H), 2.32 (t, 2H, J = 8 Hz), 2.25 (t, 2H, J = 9 Hz), 1.62 (m, 2H), 1.24 (24H), 0.86 (t, 3H, J = 8 Hz). Acetal hydrolysis was catalyzed by an acidic resin (31). Thus, 1-palmitoyl-2-(5-dimethoxypentanoyl)-sn-glycero-3-phosphocholine (30 mg) was dissolved in 6 ml of acetone/water (6:1, v/v) and stirred magnetically with Amberlyst-15 resin (15 mg). The reaction was monitored by TLC and was stopped at 6.5 h. The solvent was removed under reduced pressure, and the crude product was flash chromatographed on a column (10 mm × 150 mm) of silica gel (40 µm, J. T. Baker Inc.) with CHCl3/MeOH/H2O (40:50:10, v/v/v) to yield 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine (POVPC, 21 mg, 76%). 1H NMR: d 9.78 (s, 1H), 5.23 (m, 1H), 3.85-4.45 (8H), 3.37 (s, 9H), 2.57 (t, 2H, J = 7 Hz), 2.39 (m, 2H), 2.28 (t, 2H, J = 7 Hz), 1.92 (t, 2H, J = 7 Hz), 1.58 (m, 2H), 1.26 (24H), 0.88(t, 3H, J = 7 Hz). This spectrum agrees closely with that reported previously (32).

For synthesis of 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (50 mg, 0.1 mmol) in a 15-ml flask was dried by azeotropic distillation with toluene (3 × 5 ml) under reduced pressure and then was connected to high vacuum (0.25 mm Hg) for 3 h. After adding glutaric anhydride (33.7 mg, 0.3 mmol), p-(N,N-dimethylamino)pyridine (18.5 mg, 0.15 mmol), and 3.5 ml of freshly distilled chloroform, the above flask was flushed with argon and the resulting solution was stirred at room temperature. The progress of the reaction was monitored by TLC (CHCl3/MeOH/H2O, 70:26:4, v/v/v). After 21 h the reaction mixture was acidified to pH 3.0 with cold 0.1 N HCl, extracted with CHCl3/MeOH (2:1, v/v, 3 × 30 ml), and washed with MeOH/H2O (pH 3.0, 1:1, v/v, 30 ml). The combined organic phase was evaporated under reduced pressure, and the resulting mixture was applied to a silica gel column (15 mm inner diameter × 200 mm), which was eluted with CHCl3/MeOH/H2O (40:50:10, v/v/v) to produce PGPC. 1-Palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine:1H NMR (in CHCl3): delta  5.25 (m, 1H), 3.75-4.35 (m, 8H), 3.32 (s, 9H), 2.38 (m, 2H), 2.27 (t, 4H, J = 8 Hz), 1.87 (m, 2H), 1.58 (m, 2H), 1.26 (m, 24H), 0.87 (t, 3H) J = 8 Hz).

Monocyte Adhesion Assays

These studies were performed essentially as described previously (14). Briefly, for monocyte adhesion assays human aortic endothelial cells were incubated with test medium for 4 h at 37 °C. A suspension of human monocytes was added for 12-15 min, and nonadherent monocytes were removed. Bound monocytes were counted and expressed as monocytes/microscopic field. Data were analyzed using model 1 analysis of variance.

Immunoreactivity with ApoEØ Monoclonal Autoantibodies

Monoclonal autoantibodies that recognized oxidation-specific epitopes of oxidized LDL were cloned from hybridomas generated from apolipoprotein E-deficient (ApoEØ) mice that had not been immunized exogenously as described (33). The supernatants from hybridomas produced by fusing B-lymphocytes obtained from the spleens of two ApoEØ mice with a myeloma cell line were screened for binding to either highly oxidized LDL, 4-hydroxynonenal-LDL, or malondialdehyde-modified LDL. Fractions obtained from LC-MS analysis of Ox-PAPC were dried under nitrogen at 37 °C and resuspended in 500 µl of absolute ethanol, and 25 µl of a 1:100 dilution in ethanol was added to each well of a 96-well white round bottomed MicroFluor (Dynatech) microtitration plate. Absolute ethanol was added to blank wells. The ethanol was evaporated to dryness over 10-15 min under a gentle stream of nitrogen. Purified murine monoclonal autoantibodies were diluted into Tris-buffered saline buffer with 3% bovine serum albumin, 0.27 mM EDTA, and 20 µM BHT (dilution buffer). After adding 50 µl of solutions containing the primary antibody per well (10 µg/ml), the plates were incubated at room temperature for 1 h. The wells were washed four times with washing buffer (Tris-buffered saline containing 0.27 mM EDTA, 0.02% NaN3, and 0.001% aprotinin), and the amount of antibody bound was measured with alkaline phosphatase-labeled goat anti-mouse IgM (Sigma) by incubating for 1 h at room temperature. After four more washes with washing buffer, plates were washed four times with distilled water. To each well was then added 25 µl of a 50% solution of LumiPhos 530, and the plates were incubated for 1 h at room temperature in the dark. Luminescence was determined using a Lucy 1 Luminometer and WINLCOM software (Anthos Labtec Instruments, Salzburg, Austria). Binding to native LDL and modified LDL were determined as described (34). Data are expressed as total flashes of light measured over the indicated times.

Other Procedures

Measurements of cell protein content and those of lipoproteins were performed by microtiter plate assay based on the method of Lowry et al. (35).


RESULTS

HPLC and Spectral Characteristics of Unoxidized and Oxidized PAPC

PAPC was analyzed by normal phase HPLC before and after autoxidation by exposure to air for 48 h. The eluant was passed through the flow cell of a diode array detector that was scanning between 200 and 360 nm. Unoxidized PAPC eluted at ~14 min and possessed maximal absorbance in the 200-205-nm range typical of polyunsaturated hydrocarbon chains (Fig. 1A). Autoxidation of PAPC resulted in the formation of a complex array of compounds with differing chromatographic and spectral characteristics (Fig. 1B). A peak of absorbance at 200 nm near 14 min (Fig. 1B, asterisk) suggested the presence of residual unoxidized PAPC. Several peaks were detected after autoxidation that had different HPLC retention times and absorbed at wavelengths greater than 215 nm (Fig. 1B).


Fig. 1. HPLC chromatogram of unoxidized PAPC (A) and Ox-PAPC (B). After autoxidation 3 mg of Ox-PAPC was injected onto a preparative normal phase HPLC column and eluted isocratically with acetonitrile/methanol/water (77:8:15, v/v/v) containing 1 mM ammonium acetate (pH 5.0) at 5 ml/min. UV absorbance was monitored using a diode array detector between 200 and 360 nm. Absorbance of Ox-PAPC is shown at 200 nm (---), 235 nm (--- - ---), 250 nm (·····), and 270 nm (--- - - ---). Full scale absorbance (100% relative absorbance) was 0.900 absorbance unit, 0.370 absorbance unit, 0.370 absorbance unit, and 0.370 absorbance unit, respectively.
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Electrospray Ionization Mass Spectrometry of Oxidized PAPC

Analysis of unoxidized PAPC by FIA-ESI-MS in the positive ion mode revealed a single predominant ion corresponding to the protonated molecule (M+H+) at m/z 782.7 with few impurities (Fig. 2A). Positive ion FIA-ESI-MS of Ox-PAPC confirmed the HPLC analysis showing that a variety of oxidation products derived from PAPC were present in Ox-PAPC (Fig. 2B). These derivatives included molecules that had a larger molecular mass than PAPC (m/z > 782.7), presumably due to the addition of oxygen atoms to the arachidonoyl moiety of PAPC, and molecules that had a smaller molecular mass than PAPC (m/z < 782.7), presumably due to oxidative fragmentation of PAPC. To verify that the observed ions were not fragmentation artifacts produced by the ionization process, Ox-PAPC was analyzed while the orifice voltage was increased from 60 to 120 at 20-unit increments. As the orifice voltage increased there was a uniform decrease in the abundance of all ions except for 1-palmitoyl-lysophosphatidylcholine (M+H+ = 496) and phosphocholine (M+H+ = 183), which increased proportionally to orifice voltage (data not shown). This was presumably due to nonspecific fragmentation at the sn-2 and sn-3 positions, respectively, by the ionization energy. In addition, fast atom bombardment mass spectrometry was performed and gave essentially identical ions in terms of m/z and relative abundance as those seen with positive ion FIA-ESI-MS (data not shown).


Fig. 2. Positive ion FIA-ESI-MS of unoxidized PAPC (A) and Ox-PAPC (B). Unoxidized PAPC (250 µg/ml) was introduced into the mass spectrometry ion source dissolved in flow injection solvent composed of acetonitrile/water/formic acid (50:50:0.1, v/v/v) at 20 µl/min. Ox-PAPC, produced by autoxidation in air for 24-72 h, was resuspended in flow injection solvent (1 mg/ml), and 20 µl (20 µg) was analyzed as described for unoxidized PAPC.
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Biological Activity of Lipids Isolated from Ox-PAPC

Our laboratory has previously shown that endothelial cells treated for 4 h with Ox-PAPC bound significantly more human monocytes than did endothelial cells treated with unoxidized PAPC (19). A LC-MS system was assembled that provided simultaneous diode array detection, mass spectrometry, and fraction collection of oxidized phospholipids isolated by HPLC. Ox-PAPC (3 mg) was injected onto this system using a preparative NP-HPLC column, and each 1-min fraction was collected and dried under a stream of nitrogen at 37 °C. For biological activity analysis, fractions were resuspended by vortexing for 1 min in tissue culture medium that contained 5% serum and then added to endothelial cells for 4 h at 37 °C. After incubation, the lipids were removed from the endothelial cells and human monocytes were added for 12-15 min at 37 °C. Nonadherent monocytes were removed, and adherent monocytes were quantitated by light microscopy. Endothelial cells treated with oxidized phospholipids collected in fractions 16, 18, 19, 20, and 22 bound significantly more monocytes than did sham-treated endothelial cells (Fig. 3A). Biological activity was closely associated with the elution of oxidized phospholipids with m/z 594-595, 610-611, and 828-829 as determined by reconstructed selected ion chromatograms (RSIC) for these ions (Fig. 3B).


Fig. 3. Normal phase liquid chromatography/mass spectrometry of biologically active oxidized phospholipids in Ox-PAPC and MM-LDL. Ox-PAPC (3 mg) was analyzed by preparative LC-MS, and the fractions eluting between 12 and 24 min were collected, dried under argon, resuspended in tissue culture medium, and tested for their ability to induce human aortic endothelial cells to bind monocytes (A). p values were obtained by comparison to control wells treated without exogenous lipids (n = 9). Biological activity was expressed as monocytes bound to endothelial cells above control as an average of three separate experiments, and p values are shown compared with the number of monocytes bound to untreated endothelial cells. A RSIC was prepared showing the ions that colocalized with biological activity (m/z 828.6, 610.2, and 594.3) (B). The 100% ion intensity for each RSIC is indicated in parentheses under each ion label. Phospholipids isolated from MM-LDL by solid phase extraction columns were analyzed by LC-MS in the same manner as above, and RSIC of m/z 828.6, 610.2, and 594.3 are shown (C).
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We then analyzed phospholipids extracted from MM-LDL using LC-MS to determine if the same biologically active phospholipids were present. Phospholipids with similar mass and retention time were found in phospholipids isolated from MM-LDL compared with those that possessed biological activity in Ox-PAPC (Fig. 3C). In addition, the molecule(s) with m/z 828-829 eluted as a bimodal peak at ~13.3 and 15 min from both Ox-PAPC and MM-LDL (Fig. 3, B and C). The molecules with m/z 594.3, 610.2, and 828.6 were purified to near homogeneity by preparative normal phase LC-MS followed by analytical reversed phase LC-MS (RP-LC-MS) for use in monocyte binding assays (Fig. 4A). Purification of each molecule was based on ions observed in each fraction using positive ion FIA-ESI-MS. Endothelial cells treated with purified m/z 594.3, 610.2, and 828.6 bound significantly more monocytes than did cells treated with medium alone (Fig. 4B). Dose-response curves for individual molecules were difficult to obtain because of the variability in the response of endothelial cells and monocytes from different donors. However, in most endothelial cell isolates all three molecules induced maximal response at concentrations of about 20-30 µg/ml (data not shown). Dilution experiments showed that m/z 594.3 and 610.3 were capable of inducing a significant increase in monocyte adhesion to endothelial cells at a concentration as low as 1-3 µg/ml. The molecule with m/z 828.6 was capable of inducing monocyte binding as low as 400 ng/ml, if it is assumed that this molecule was exclusively responsible for the biological activity of the fraction. However, contaminating ions present in this fraction (Fig. 4A) were more abundant in fractions with less biological activity (data not shown).


Fig. 4. Induction of monocyte binding by purified oxidized phospholipids. Fractions collected after sequential normal and reversed phase HPLC were dried under nitrogen, resuspended in tissue culture medium, and incubated with human aortic endothelial cells. After 4 h of incubation the medium was removed and human monocytes were added for 15 min. Unbound monocytes were removed after incubation, and adherent monocytes were quantitated by light microscopy. The mean number of monocytes bound in control wells (n = 9) was subtracted from all values, which are plotted as mean ± S.E.
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Electrospray Mass Spectrometry of Derivatized Ox-PAPC

Ox-PAPC (250 µg) was added to each of four test tubes and the solvent dried under nitrogen. The lipid residue in each tube was treated with either buffer alone, sodium borohydride, methoxyamine hydrochloride, or a mixture of pentafluorobenzyl bromide and diisopropylethylamine to derivatize particular functional groups such as hydroperoxides, aldehydes, ketones, and carboxylic acids that may have been present in our Ox-PAPC preparations. The lipids from each sample were re-extracted with chloroform/methanol (2:1, v/v) as described under "Experimental Procedures" and analyzed by positive ion FIA-ESI-MS. It has been well documented that sodium borohydride can reduce hydroperoxides, epoxides, and carbonyls to hydroxides. Reduction of hydroperoxides to hydroxides results in a net decrease of 16 Da due to the loss of a single oxygen atom. Reduction of aldehydes and ketones to hydroxides results in a net increase of 2 Da due to the addition of two hydrogen atoms to the carbonyl-containing carbon.

Sodium Borohydride Derivatization

The mass spectrum of Ox-PAPC treated with buffer alone (Fig. 5A) was similar to the mass spectrum of Ox-PAPC that had not been incubated with buffer (see Fig. 2B). The mass spectrum of Ox-PAPC treated with the reducing agent, sodium borohydride (Fig. 5B), was significantly different from that of Ox-PAPC treated with buffer alone (Fig. 5A). The ions observed at m/z 594.3, 620.4, 650.4, 828.6, 846.6, 862.5, and 878.7 were diminished, and the ions observed at m/z 596.4, 622.5, 652.5, 832.5, 848.7, 864.6, and 880.5 became predominant (Fig. 5B). This result indicated that many components of Ox-PAPC contain reducible functional groups. We suspect that the increase in 1-palmitoyl-lysophosphatidylcholine (m/z 496.2) was due to nonspecific hydrolysis of the fatty acid at the sn-2 position during the workup procedure. This may account for the decrease of some ions after derivatization, such as m/z 732.6 (Fig. 5). With regard to the molecules in Ox-PAPC that possessed biological activity (m/z 594.3, 610.2, and 828.6), the ions at m/z 594.3 and 828.6 were diminished and the ion at m/z 610.2 was not altered significantly (Fig. 5B). It was concluded that the former ions possessed sodium borohydride-reducible groups and the latter ion did not. These data suggested that the molecule with m/z 594.3 was reduced to m/z 596.4 (presumably due to the reduction of an aldehyde or ketone) and the molecule with m/z 828.6 was reduced to 832.5 (presumably due to the reduction of two carbonyl groups). Alternatively, the m/z 832.5 ion could have been produced by the reduction of a molecule with m/z 846.6 that possessed a carbonyl group and a hydroperoxide. Reduction of a hydroperoxyl group (-16 Da) and a carbonyl group (+2 Da) would also produce an ion near m/z 832.5 (846.6 - 16.0 + 2.0 = 832.6).


Fig. 5. Reduction of Ox-PAPC. Ox-PAPC was incubated with sodium borohydride (20 µg/ml) in a 0.1 M borate buffer (pH 8.0) or borate buffer alone for 15 min at room temperature. After treatment the lipids were extracted and sham-treated (A) and reduced (B). Ox-PAPC was analyzed by positive ion flow injection electrospray mass spectrometry.
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Pentafluorobenzyl Derivatization

Based on the molecular mass of the m/z 610.2 ion we hypothesized that it was an oxidatively fragmented phospholipid with a 5-carbon moiety at the sn-2 position containing a terminal carboxylic acid (glutaric acid) that would produce a M+H+ ion at m/z 610.2 (Fig. 6A, structure). To test this hypothesis, the molecule with m/z 610.2 was partially purified by NP-LC-MS (Fig. 6A) and then treated with pentafluorobenzyl bromide and N,N-diisopropylethylamine, which are commonly used to derivatize carboxylic acids to pentafluorobenzyl esters. After treatment, positive ion FIA-ESI-MS revealed that the ion at m/z 610.2 had diminished and a novel ion at m/z 790.2 had appeared (Fig. 6B). This observation was consistent with the addition of a pentafluorobenzyl group (180 Da) to the ion at m/z 610.2 (Fig. 6B, structure). The attachment of the pentafluorobenzyl group to the 5-carbon group at the sn-2 position was confirmed by FIA-ESI-MS/MS in the negative ion mode. The ion (M+H+) observed at m/z 790.2 by positive ion FIA-ESI-MS produced a pseudo-ion (M-15) at m/z 774.2 by negative ion FIA-ESI-MS due to loss of a methyl group from the choline moiety of the molecule (36). Prominate daughter fragments produced by collision-induced dissociation of this pseudo-parent ion (m/z 774.2) was the M-15 ion of 1-palmitoyl-lysophosphatidylcholine (m/z 480), the carboxylate anion of palmitic acid (m/z 255), and a third fragment with m/z 311 (Fig. 6C). The latter fragment (m/z 311) corresponded to the calculated molecular mass of the carboxylate anion of a 5-carbon pentafluorobenzyl ester that was predicted to be in the sn-2 position of this molecule. Therefore, the molecular structure of one biologically active oxidized phospholipid in Ox-PAPC and MM-LDL was confirmed to be 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) (Fig. 6A, structure). This assignment was confirmed by comparative analysis of PGPC prepared by unambiguous total synthesis, which had properties identical to those of the identified molecule when analyzed by positive ion FIA-ESI-MS, NP-LC-MS, and RP-LC-MS.


Fig. 6. Pentafluorobenzyl derivatization of phospholipid with m/z 610.2. Ox-PAPC (3 mg) was analyzed by preparative normal phase LC-MS as described under "Experimental Procedures." The fraction enriched in the phospholipids with m/z 610.2 was dried and treated with buffer alone (A) or pentafluorobenzyl bromide and diisopropylethylamine (B) and analyzed by positive ion electrospray MS. Negative ion electrospray collision-induced dissociation-MS/MS was also performed on the pseudo-ion at m/z 774 (M-15) produced from the derivatized molecule with M+H+ = 790.2 (C). Proposed molecular structures and fragmentation patterns are inset in each panel.
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Methoxyamine Derivatization

To determine the molecular structure of the molecule with m/z 594.3, the oxidized phospholipid was semipurified by NP-LC-MS (Fig. 7A) and treated with methoxyamine hydrochloride. After treatment, analysis by positive ion FIA-ESI-MS revealed that the ion at m/z 594.3 was diminished and a novel ion at m/z 623.4 was observed (Fig. 7B). This result was consistent with the addition of a methoxyamine group (29 Da) to the m/z 594.3 ion. We hypothesized, based on molecular mass and presence of a carbonyl group, that the molecule that produced the ion at m/z 594.3 was an oxidatively fragmented phospholipid containing palmitic acid in the sn-1 position and a 5-carbon chain with a terminal aldehyde (5-oxovaleric acid) in the sn-2 position (Fig. 7A, structure). To test this, negative ion FIA-ESI-MS/MS was performed to determine daughter fragments of the methoxyamine-derivatized molecule that produced the ion at m/z 623.1 (M+H+) by positive ion FIA-ESI-MS and the pseudo-ion at m/z 607 (M-15) by negative ion FIA-ESI-MS. Daughter fragments of the pseudo-parent ion at m/z 607 included the M-15 ion for 1-palmitoyl-lysophosphatidylcholine (m/z 480), the carboxylate anion from palmitic acid (m/z 255), and a fragment at m/z 144 that represented the carboxylate anion of the methoxyamine-derivatized 5-carbon moiety in the sn-2 position. The fragment at m/z 549 represented the pseudo-parent ion after the loss of the methoxime group with the terminal carbon (HC=N-O-CH3, 607-58 Da). The fragment at m/z 113 represented the derivatized carboxylate anion from the sn-2 position after the loss of the methoxy group (O-CH3) (144-31 Da). Thus, we conclude that the molecular structure of a second molecule present in Ox-PAPC and MM-LDL that induces endothelial cells to bind monocytes in vitro is POVPC. This assignment was confirmed by comparative analysis to 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) prepared by unambiguous total synthesis, which had properties identical to those of the identified molecule when analyzed by positive ion FIA-ESI-MS, NP-LC-MS, and RP-LC-MS.


Fig. 7. Methoxyamine derivatization of phospholipids with m/z 594.3. Ox-PAPC (3 mg) was analyzed by preparative normal phase LC-MS as described under "Experimental Procedures." The fraction enriched in the phospholipids with m/z 594.3 was dried and treated with buffer alone (A) or methoxyamine hydrochloride (B) and analyzed by positive ion electrospray MS. Negative ion electrospray collision-induced dissociation-MS/MS was also performed on the pseudo-parent ion at m/z 607 (M-15) produced from the derivatized molecule with M+H+ = 623.1 (C). Proposed molecular structures and fragmentation patterns are inset in each panel.
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Biological Activity of Synthesized Oxidized Phospholipids

Totally synthetic POVPC and PGPC were dried, resuspended in tissue culture medium, and analyzed for their ability to induce endothelial cells to bind monocytes. As did the same molecules produced by oxidation of PAPC, the synthesized phospholipids caused a significant increase in monocyte binding (Fig. 8). The dose-response curves for both synthetic and HPLC-isolated compounds were similar when identical endothelial cells and monocytes were used (data not shown).


Fig. 8. Biological activity of synthetically manufactured oxidized phospholipids. The phospholipids 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine (synthetic POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (synthetic PGPC) were synthesized and tested for the ability to induce endothelial cells to bind monocytes.
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Reactivity of Oxidized Phospholipids with Monoclonal Antibodies

LDL, MM-LDL, PAPC, Ox-PAPC, and HPLC-isolated fractions from Ox-PAPC were tested for reactivity to antibodies produced from hybridomas generated from apoE null mice. Of the nine autoantibodies tested, four (EØ-1, EØ-3, EØ-7, and EØ-14) displayed specific reactivity to Ox-PAPC compared with unoxidized PAPC and bovine serum albumin (Fig. 9). Antibody EØ-6, which has been shown to recognize an oxidized phospholipid epitope (34), also had increased binding to Ox-PAPC as well as to unoxidized PAPC and bovine serum albumin (data not shown), presumably due to epitopes already present. In general, the same antibodies that reacted specifically with Ox-PAPC also recognized epitopes on MM-LDL and not the native LDL from which it was derived (Fig. 9, inset). In control experiments, reactivity of these antibodies with bovine serum albumin was essentially the same as reactivity with unoxidized LDL (data not shown). Two of these antibodies (EØ-1 and EØ-3) were used in further studies to determine immunoreactivity of HPLC-isolated fractions collected from Ox-PAPC. Fractions were collected from Ox-PAPC (3 mg) during LC-MS analysis on a preparative normal phase HPLC column. After collection the fractions were dried under nitrogen at 37 °C and resuspended in chloroform prior to analysis. Immunoreactivity of each fraction that contained lipid (fractions 9-30) was determined and expressed in terms of flashes/100 ms (Fig. 10). After the level of reactivity of each fraction was determined the RSIC of the molecules with m/z 594-595, 610-611, and 828-829 were superimposed on the graph. In a separate experiment, EØ-6 and EØ-7 were shown to react with the fractions containing ions at m/z 828.6 and 610.2 (equivalent to fractions 13-19 in Fig. 3) but not fractions containing the ion at m/z 594.3. 


Fig. 9. Reactivity of LDL, MM-LDL, and Ox-PAPC with ApoE monoclonal autoantibodies. Nine monoclonal antibodies obtained from hybridoma lines obtained from ApoE null mice were tested for reactivity with oxidized PAPC, Ox-PAPC, or bovine serum albumin (control), which were adhered to the bottom of 96-well microtiter dishes. Inset, antibodies EØ-1, EØ-3, and EØ-7 were tested for reactivity with MM-LDL (black bars) and the LDL preparations from which the MM-LDL was derived by mild oxidation (white bars). Values are plotted as mean flashes of light/100 ms ± S.D.
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Fig. 10. Reactivity of isolated oxidized phospholipids from Ox-PAPC with ApoE autoantibodies. Ox-PAPC (3 mg) was analyzed by normal phase LC-MS as described under "Experimental Procedures." Fractions containing lipids (fractions 9-30) were dried under argon, resuspended in ethanol, and added to the bottom of a 96-well microtiter plate. The solvent was evaporated, and the lipids were treated with autoantibodies (EØ-1 and EØ-3) produced by immortalized lymphocyte obtained from ApoE null mice that reacted most specifically with Ox-PAPC in Fig. 11. A goat anti-mouse secondary antibody with a fluorescent tag was used to quantitate the amount of primary antibody attached to the lipids. Values are plotted as mean fluorescent flashes/ms ± S.D. RSIC were produced for m/z 828.6 (·····), m/z 610.2 (--- - ---), and m/z 594.3 (---) from the same LC-MS run.
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Presence of Biologically Active Oxidized Phospholipids in Atherosclerotic Lesions

Phospholipids extracted from the aortae of rabbits on atherogenic diets with or without supplementation with antioxidants were analyzed by positive ion FIA-ESI-MS. The aortae from rabbits that were not supplemented with antioxidants had a higher concentration of phospholipids with characteristics identical to those of the biologically active oxidized phospholipids found in Ox-PAPC and MM-LDL compared with aortae from rabbits that were on a chow diet (control) or rabbits that were fed an atherosclerotic diet that was supplemented with antioxidants (Fig. 11).


Fig. 11. Presence of biologically active oxidized phospholipids in atherosclerotic lesions. Aortae were obtained from rabbits fed a normal chow diet (Control) or an atherogenic diet with or without supplementation with the antioxidants vitamin E or probucol. Lipids were extracted with chloroform/methanol and phospholipids purified by solid phase extraction chromatography after addition of the internal standard, dimyristoylphosphatidylcholine. The phospholipid fraction was analyzed by positive ion electrospray mass spectrometry, and the abundance of the ions with m/z 594.3, 610.2, and 828.6 in aortae from rabbits fed the control diet (white bars, n = 10), rabbits feed an atherogenic diet (black bars, n = 10), and rabbits fed an atherogenic diet supplemented with antioxidants (hatched bars, n = 14) were plotted in terms of ng/mg tissue. * = p < 0.01 compared with white bars; dagger  = p < 0.05 compared with black bars.
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DISCUSSION

Our previous studies have suggested that the ability of MM-LDL to induced monocyte-endothelial interactions is due to the oxidation of arachidonic acid-containing phospholipids in LDL. The focus of these studies was to identify the molecular structure of these lipids using oxidized PAPC as a surrogate lipid. The products derived from the autoxidation of PAPC were remarkably reproducible (Fig. 2). Only slight variations were observed between preparations with regard to the time necessary for oxidation, the retention time by normal and reversed phase HPLC, and the relative abundance of each ion by positive ion FIA-ESI-MS.

Although numerous oxidation products of PAPC were present in preparations of Ox-PAPC (Fig. 2B) we concentrated on three ions, m/z 594.3, 610.2, and 828.6, based on the observation that NP-HPLC fractions that contained these species possessed biological activity in monocyte binding assays. All three of these species were present in MM-LDL. RSIC produced from normal phase LC-MS analysis of phospholipids extracted from MM-LDL showed a similar pattern and retention time to the oxidized phospholipids found in Ox-PAPC (Fig. 4, B and C). These lipids were also found in preparations of MM-LDL produced by incubation of LDL with cocultures of human endothelial cells and smooth muscle cells (data not shown).

We have observed that the presence of these particular species of oxidized phospholipids appear transiently during the oxidation of LDL. They appear to be most abundant during the late antioxidant-depletion phase and the early propagation phase of LDL oxidation (15, 19). This may help to explain the transient ability of oxidized LDL to induce monocyte-endothelial interactions, since the appearance of these oxidized phospholipids and the presence of biological activity occur in the same "window" of oxidation (9). We hypothesize that during the late propagation and decomposition phase of LDL oxidation the accumulation of end products of lipid oxidation such as malondialdehyde, 4-hydroxynonenal, cholesterol oxides, and lysophosphatidylcholine become the principle "biological activators." It is these latter products that appear to be responsible for protein modification (37-39), cell toxicity (40-42), and intimal proliferation (43, 44) associated with highly oxidized LDL.

The identification of the molecular structures of two of the oxidized phospholipids present in Ox-PAPC (m/z 594.3 and 610.2) was relatively straightforward. The molecule with m/z 594.3 was reduced by sodium borohydride (Fig. 6) and was derivatized by methoxyamine (Fig. 8). Based on the molecular mass of this ion and the fragmentation pattern by negative ion FIA-ESI-MS/MS the molecule was identified as POVPC. The molecule with m/z 610.2 was not reduced by sodium borohydride (Fig. 5) but was derivatized by pentafluorobenzyl bromide (Fig. 6). Based on the molecular mass of this ion and the fragmentation pattern by negative ion FIA-ESI-MS/MS the molecule was identified as PGPC. Both of these assignments were confirmed by total unambiguous synthesis of POVPC and PGPC. The synthetic molecules had identical analytical and biological properties compared with the molecules isolated from Ox-PAPC (Fig. 8).

Tokumura et al. have reported that PGPC, as part of a lipid extract from bovine brain (45) and as a pure compound (46), possessed hypotensive properties. Others have reported the presence of 5-oxovaleric acid esterified to phospholipids and cholesteryl esters in LDL (47, 48). In addition, POVPC has been shown to induce smooth muscle cell proliferation and neutrophil activation in vitro (49, 50). This molecule may contribute to the platelet aggregation effects associated with oxidized LDL, since it has structural similarity to platelet-activating factor (51). Interestingly, the biological activity of Ox-PAPC was decreased by 60-70% when treated with sodium borohydride (data not shown). This suggests that particular functional groups were necessary for the biological activity of Ox-PAPC. The structural identification of the molecule with m/z 828.6 was found to be more difficult. The molecular composition of this complex oxygenated phospholipid is currently under investigation.

There are several lines of evidence suggesting that the oxidized phospholipids with m/z 594.3, 610.2, and 828.6 play a role in atherosclerosis in vivo. First, there is an association between the extent of atherosclerotic lesions and the abundance of these lipids in an animal model of atherosclerosis (Fig. 11). Second, the same lipids that induced endothelial cells to bind monocytes are recognized by naturally occurring autoantibodies immunoreactive with oxidized LDL epitopes. In addition, the same murine autoantibodies that recognized several of the oxidation products of PAPC also immunostain atherosclerotic lesions of rabbits and humans (33). Oxidized LDL is immunogenic, and autoantibodies recognizing epitopes of highly oxidized LDL have been described in plasma and in atherosclerotic lesions of several species (52, 53). Moreover, higher titers of these autoantibodies are present in patients with carotid atherosclerosis, coronary artery disease, diabetes, peripheral vascular disease, hypertension, and pre-eclampsia (54-61). These observations suggest that under conditions of oxidative stress epitopes are produced that induce an immune response. It is not clear, however, whether the epitopes that are recognized by the autoantibodies are intrinsic to the oxidized phospholipids alone or are formed by oxidized lipid-protein adducts. The significance of these antibodies in limiting the development of atherosclerosis is shown by the observation that injection of malondialdehyde-modified LDL (62) or oxidized LDL (63) into rabbits before the initiation of cholesterol feeding caused a reduction in fatty streak lesion formation. Thus, the results of our studies together with previous observations cited above suggest that the active derivatives of PAPC that we have identified may play an important role in vivo in fatty streak formation.

The mechanism by which these oxidized phospholipids induced monocyte binding is not known. We have hypothesized that all three molecules may produce a common motif as part of a lipid monolayer or bilayer. Thermodynamic considerations suggest that polar functional groups such as hydroperoxides, hydroxides, and carbonyls associated with the fatty acid moieties of phospholipids may engage in hydrogen bonding with the aqueous medium in which the membrane is suspended. The oxygen-containing acyl group at the sn-2 position of these molecules has the potential to protrude into the aqueous medium. This has been suggested to be the mechanism by which phospholipase A2 preferentially hydrolyzes oxidized phospholipids (64, 65). We are investigating the possibility that this motif is also responsible for interaction with particular receptors on the endothelial cell surface.

The use of Ox-PAPC as a surrogate for MM-LDL greatly facilitates the study of monocyte-endothelial interactions induced by oxidized phospholipids. The active derivatives that we have identified probably do not represent all of the active phospholipid oxidation products present in MM-LDL or in the vessel wall. The synthesis of biologically active oxidized phospholipids in conjunction with the powerful analytical techniques of chromatography combined with mass spectrometry should provide methods by which the metabolism of oxidized phospholipids may be carefully dissected. Oxidized phospholipids have been implicated in the pathogenesis of several chronic inflammatory diseases such as rheumatoid arthritis (66, 67), inflammatory bowel disease (68), antiphospholipid antibody syndrome (34), and multiple sclerosis (69, 70). We are investigating the possibility that the biologically active phospholipids reported in this paper may play a fundamental role in the pathogenesis of these diseases.


FOOTNOTES

*   This work was supported by U.S. Public Health Services Grants HL 30568, HL 07386, and RR 865 and the Laubisch, Rachel Israel Berro, and M. K. Gray Funds (to A. D. W., N. L., M. N., A. M. F., and J. A. B.); a grant from the H. M. Keck Foundation (to K. F. F.); U.S. Public Health Services Grant HL 14197 (to J. L. W., S. H., W. P.); and the Academy of Finland (to S. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed: Dept. of Medicine/Cardiology, Center for the Health Sciences, Rm. 47-123, UCLA Medical Center, Los Angeles, CA 90095-1679. Tel: 310-825-2436; Fax: 310-206-9133; E-mail: adwatson{at}ucla.edu.
1   The abbreviations used are: LDL, low density lipoprotein; MM-LDL, mildly oxidized low density lipoprotein; Ox-PAPC, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; MS/MS, tandem mass spectrometry; NP, normal phase; RP, reversed phase; LC, liquid chromatography; FIA, flow injection analysis; ESI, electrospray ionization; MeOH, methanol; ApoEØ, apolipoprotein E-deficient; BHT, butylated hydroxytoluene; HPLC, high performance liquid chromatography; PAPC, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; PGPC, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine; POVPC, 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine; RSIC, reconstructed selected ion chromatogram.

ACKNOWLEDGEMENTS

The authors thank Drs. George Popják, Linda L. Demer, A. Jake Lusis, Farhad Parhami, Peter A. Edwards, Jason D. Morrow, Sampath Parthasarathy, Tom McIntyre, Christopher Fielding, Richard Havel, John Taylor, and Michael Reidy for valuable discussions and suggestions and Alan C. Wagner and Susan Hama for discussions and expert technical assistance.


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