INTRODUCTION

In contrast to low density lipoproteins (LDL),¹ high plasma concentrations of high density lipoproteins (HDL) are associated with a decreased risk for the development of coronary artery disease, an effect commonly attributed to their central role in reverse cholesterol transport (1). During this process, HDL is able to promote efflux of cholesterol from peripheral tissues, and the accepted cholesterol is (at least in part) esterified by the action of lecithin-cholesterol acyltransferase (2). Cholesteryl esters (CEs) formed by the lecithin-cholesterol acyltransferase reaction are then transferred from HDL to other lipoproteins mediated by the cholesterol ester transfer protein or are delivered to the liver for bilary secretion or reutilization during lipoprotein assembling (3).

Within the oxidative theory, HDL appears to be Janus-faced (4-6). The majority of lipid hydroperoxides (the first detectable products of lipoprotein oxidation) in plasma are transported in the HDL fraction (7), and HDL was suggested to act as a sink for preformed lipid hydro(pero)xides. HDL-associated cholesteryl ester hydroperoxides, which are probably transferred to HDL by the action of the cholesterol ester transfer protein (8), are preferentially catabolized over nonoxidized CEs; this was shown in HepG2 cells (9), in situ perfused rat liver (10), and intact rats (11). HDL was also shown to protect LDL from lipid peroxidation, inhibiting the formation of lipid hydroperoxides but not the formation of conjugated dienes (12). On the other hand, HDL is more easily oxidized than LDL (7), HDL-associated lecithin-cholesterol acyltransferase is modified by reactive short and long chain aldehydes (13), and the ability of oxidatively modified HDL to promote cellular cholesterol efflux is diminished (14-16), most probably due to alterations in its apolipoprotein moiety. During the acute phase response, up to 80% of apolipoprotein A-I (apoA-I), the major apolipoprotein of HDL, is displaced by serum amyloid A (17), and due to this exchange in the HDL apolipoprotein domain, native, anti-inflammatory HDL became proinflammatory during the acute phase response (18). The above mentioned results imply that HDL can protect LDL against oxidative modifications and could serve as a vehicle for detoxification of potentially (cyto)toxic lipid hydroperoxides. However, under certain circumstances many of the important physiological properties of HDL can be lost during oxidation/modification, transforming HDL into a proatherogenic lipoprotein particle.

Within the wide spectrum of oxidants available for the organism, hypochlorous acid (HOCl) is thought to play an important role during microbial killing and inflammatory tissue injury by neutrophils and monocytes (19). Hypochlorite (OCl), formed in vivo via the myeloperoxidase (MPO)/H₂O₂/halide system from activated neutrophils and/or monocytes, may react with a wide range of biological target molecules, including lipids, antioxidants, and proteins (20). Modification of LDL with HOCl in vitro has generated a modified particle, which was avidly taken up by macrophages (21). The importance of MPO as a potential in vivo oxidant is further underlined by the presence of enzymatically active MPO in human atherosclerotic lesions (22). Accordingly, the presence of HOCl-modified (lipo)proteins was recently demonstrated in advanced human atherosclerotic lesions (23) and inflammatory kidney tissues rich in MPO (24).

In the current study, we examined the consequences of NaOCl modification on the apolipoprotein and lipid domain of HDL₃, the concomitant changes in HDL₃-associated CE and holoparticle metabolism in macrophages, and the cholesterol efflux properties. We found that HDL₃-associated amino acids, unsaturated fatty acids, and cholesterol were modified by HOCl, either added as reagent or generated by the MPO/H₂O₂/Cl system. Presumably as a result of apolipoprotein modification, binding, internalization, and degradation of NaOCl and MPO modified HDL₃ by mouse peritoneal macrophages were greatly enhanced. We demonstrate further that NaOCl treatment of HDL₃ resulted in impaired ability to promote cholesterol efflux from the cellular plasma membrane of macrophages.

EXPERIMENTAL PROCEDURES

Materials

NaOCl, BF₃/MeOH, egg yolk lecithin, hexamethyldisilazane, trimethylchlorosilane, organic solvents, and potassium bromide were obtained from Sigma. MPO (isolated from human leukocytes) was from Alexis Biochemicals. Radiochemicals were purchased from NEN Life Science Products. Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum (FCS) were obtained from Boehringer Ingelheim Bioproducts. Plasticware used for tissue culture was obtained from Costar. All other chemicals were obtained from Merck, except where indicated.

Methods

Human apoE-free HDL₃ was prepared by discontinuous density ultracentrifugation of plasma obtained from normolipemic donors in a TL120 Beckman tabletop ultracentrifuge using a TLA100.4 rotor (Beckman) as described (25).

Modification of HDL₃ with OCl was performed as described previously (21, 26). One mg of HDL₃ protein/ml of phosphate-buffered saline (PBS) was incubated with NaOCl solution (added as single addition and gentle vortexing) at molar ratios of NaOCl to HDL₃ ranging from 7.5 (90 µM NaOCl) to 400 (5.3 mM NaOCl; 60 min, 37 °C, under argon) with the final pH adjusted to 7.4. The modified HDL₃ preparations were passed over a PD10 column to remove unreacted NaOCl. The concentration of the reagent NaOCl was determined spectrophotometrically using a molar absorption coefficient for OCl of 350 cm¹ at 292 nm (27). Depending on the type of experiment performed after NaOCl treatment, the modified HDL₃ preparations were used between 2 and 24 h after exposure to the oxidant.

HDL₃ modification in the presence of the MPO/H₂O₂/Cl system was performed at pH 7.4 or 4.5 to generate HOCl or Cl₂ as the oxidant (28, 29). Briefly, to the substrates (1 mg of HDL₃ protein/ml in PBS (50 mM, pH 7.4) or in sodium phosphate (50 mM, 100 µM diethylenetriamine pentaacetic acid, pH 4.5)) additions of 20 µM H₂O₂ were made at 10-min intervals at 37 °C until reaching a total of 15 additions (final concentration 300 µM; assuming quantitative conversion, a molar oxidant:lipoprotein ratio of ~20:1 could be expected). MPO (13 nM) was added at the start and subsequently at every second addition of H₂O₂. At alternate additions of H₂O₂, 2 µM ascorbate was added. To blank incubations, only H₂O₂ and ascorbate were added in the same concentrations and sequence as described above. The reaction mixture was incubated for 1 h (37 °C) and subsequently dialyzed against PBS (10 mM, 4 °C).

Iodination of HDL₃ was performed as described by Sinn et al. (30) using N-Br-succinimide as the coupling agent. Routinely, 1 mCi of ¹²⁵INa (Amersham Corp.) was used to label 5 mg of HDL₃ protein. This procedure resulted in specific activities between 300 and 450 dpm/ng of protein with less than 3% lipid-associated activity. No cross-linking or fragmentation of apoA-I due to the iodination procedure could be detected by SDS-polyacrylamide gel electrophoresis and subsequent autoradiography.

HDL₃ was labeled with [cholesteryl-1,2,6,7-³H]linoleate (NEN Life Science Products) by cholesterol ester transfer protein-catalyzed transfer from donor liposomes essentially as described (9). [³H]Ch18:2-labeled HDL₃ was reisolated in a TLX120 benchtop ultracentrifuge using a TLA100.4 rotor as described (25). This labeling procedure resulted in specific activities of 5-7 cpm/ng HDL₃ protein.

SDS-polyacrylamide gel electrophoresis of HDL₃ apolipoproteins was performed using 5-15% polyacrylamide gradient gels with electrophoresis at 150 V for 90 min in a Bio-Rad miniprotean chamber (Bio-Rad, Austria) (26). For Western blotting experiments, proteins were electrophoretically transferred to nitrocellulose membranes (150 mA, 4 °C, 90 min).

Immunochemical detection of NaOCl-modified apolipoproteins was performed with a monoclonal antibody clone 2D10G9 (Ref. 26; dilution 1:50) followed by peroxidase-conjugated goat anti-mouse IgG (Bio-Rad, 1:5000). Immunochemical detection of apoA-I in the same lipoprotein samples was performed with rabbit polyclonal anti-human apoA-I (Behring, Germany) as a primary antibody, followed by peroxidase-conjugated goat anti-rabbit IgGs as secondary antibodies. Detection was performed by the ECL method.

Dynamic light-scattering experiments were performed with native and NaOCl-modified HDL₃. The laboratory-built goniometer was equipped with an Argon⁺ laser (Spectra Physics, model 2060-55, P_max = 5 watts,  = 514.5 nm), single mode fiber detection and an ALV-5000 correlator (ALV, Germany). Measurements were carried out at a scattering angle of 90° at 25 °C and a laser power of 200 milliwatts. The time dependence of the scattering intensity, represented by its correlation function, provides information on diffusional motion of the scatterer. The diffusion coefficient is related to the apparent hydrodynamic radius, R_H, by the Stokes-Einstein equation, i.e. an equivalent sphere with radius R_H shows the same diffusion behavior as the particle under investigation and serves as a size parameter. Polydisperse systems give rise to a correlation function with a spectrum of different decay constants. A series expansion called cumulant fit (31) results in a mean value for the diffusion coefficient (first cumulant, c₁) and its variance (second cumulant, c₂). The ratio c₂/c₁² is called the polydispersity index. Any aggregation of HDL₃ particles will lead to an increase of the measured R_H values and the polydispersity index.

The integrated intensity (averaged over long times compared with the time scale of fluctuations in the dynamic experiment) is another, completely independent, parameter for aggregation phenomena. For particles that are small compared with the wavelength of the scattered radiation, this intensity is directly related to the mean mass or aggregation number of the particles and independent of its diffusion dynamics. Any increase in this intensity relative to the native control is equivalent to the degree of aggregation, i.e. dimerization would lead to a 2-fold increase of the integrated intensity.

Aliquots of native and NaOCl-modified HDL₃ (450 µg of protein) were lyophylized in 5-ml ampules and purged with nitrogen before hydrolysis in constant boiling 6 N HCl (24 h, 120 °C). Amino acid analysis was performed as described by Parks and Rudel (32) on a Biotronics analyzer.

Fatty acid analysis of HDL₃ lipids was performed as described (33). Separation of fatty acid methylesters (2-µl samples) was performed on a fused silica 25-m FFAP-CB column (0.32-mm inner diameter; Chrompack) using an HP 5890 gas chromatograph equipped with a flame ionization detector and a split/splitless injector (Hewlett-Packard Co.). Cholesterol analysis of HDL₃ lipids was performed as described by Sattler et al. (25).

Gas chromatography-mass spectrometry (GC-MS) analysis of NaOCl-modified HDL₃ lipids was performed essentially as described by van den Berg et al. (28, 34). The extracted HDL₃ lipids were transmethylated with BF₃/MeOH as described above. The fatty acid and chlorohydrin fatty acid methylesters were converted to the corresponding trimethylsilyl (TMS)-ether derivatives with pyridine/hexamethyldisilazane/trimethylchlorosilane (9:3:1; v/v/v) at 65 °C for 1 h. One-µl samples of the O-methylester-O-TMS-ether derivatives were analyzed by GC-MS without further extraction. Separation was performed on a DB-5 capillary column (15 m, 0.25-mm inner diameter, 0.25-µm film thickness from J & W Scientific, Folsom, CA) using helium as carrier gas. The column was kept at 120 °C (3 min) and programmed to 180 °C at a rate of 8 °C/min. A second ramp was programmed from 180 °C at 5 °C/min to 260 °C with an isothermal hold at 260 °C for 5 min. The quadrupole was operated in the electron impact mode at 70 eV and a source temperature of 200 °C.

Thioglycollate-elicited mouse peritoneal macrophages were plated on 6- or 12-well trays in DMEM containing 10% (v/v) FCS as described recently by Panzenboeck et al. (35). After 4 h, cells were washed three times with PBS and kept in DMEM (containing FCS) for 2 or 3 days. One day prior to the experiments, cells were preincubated in DMEM containing lipoprotein-depleted serum (LPDS, 10%, v/v). Lipid loading experiments of macrophages were performed with radioactively labeled (¹²⁵INa or [³H]Ch18:2) native HDL₃ and NaOCl-HDL₃ in the presence or absence of a 20-fold excess of unlabeled HDL₃. Radioactively labeled native or NaOCl-modified HDL₃ was added to a final concentration of 50 µg of protein/well, and cells were incubated in DMEM (10% FCS) at 37 °C for 6 h. Following this incubation, the cells were washed twice in Tris-buffered saline (TBS) containing bovine serum albumin (5%, w/v), followed by three washes in TBS. To release cell membrane-bound HDL₃, cells were incubated at 37 °C for 10 min in the presence of trypsin (0.05%, Cytosystems). The trypsin-releasable fraction is referred to as "bound" fraction. Cells were then lysed in NaOH (1 ml, 0.3 N, 1 h at 4 °C) to determine both the non-trypsin-releasable fraction ("internalized" fraction) and the cell protein in the lysate. Specific binding/internalization was calculated as the difference between total and nonspecific binding/internalization. Protein measurement was performed according to Lowry (36). On average, the protein content per well was 300-350 µg. Degradation of ¹²⁵I-HDL₃ or ¹²⁵I-NaOCl-HDL₃ by mouse peritoneal macrophages was estimated by measuring the non-trichloroacetic acid-precipitable radioactivity in the medium after precipitation of free iodine with AgNO₃ (37). To facilitate the comparison of results obtained with ¹²⁵I-HDL₃/¹²⁵I-NaOCl-HDL₃ and [³H]Ch18:2-HDL₃/[³H]Ch18:2-NaOCl-HDL₃, selective uptake of HDL₃-CE is expressed as apparent HDL₃ particle uptake (i.e. as the amount of apoA-I that would deliver the observed amount of tracer if uptake were solely mediated by holoparticle uptake) as suggested by Pittman et al. (38).

Thioglycollate-stimulated mouse peritoneal macrophages were harvested and plated as described above. The cells were incubated in the presence of DMEM containing LPDS (10%, v/v) and [³H]cholesterol (0.05 µCi/ml) for 2 days (39). Prior to the cholesterol efflux experiments the [³H]cholesterol-containing medium was aspirated, and the cells were washed twice in TBS (containing 5% (w/v) bovine serum albumin) and twice in TBS. Efflux experiments were then initiated by the addition of DMEM containing LPDS (10%, v/v) and HDL₃ or NaOCl-HDL₃ (0.5 mg of protein/ml). At the indicated time points, the medium was collected, and the cells were lysed in 0.3 N NaOH to estimate both the remaining radioactivity and the cellular protein content. The efflux of radioactive label to the medium was calculated as the percentage of radioactivity present in the cells prior to the addition of NaOCl-HDL₃- or native HDL₃-containing medium.

RESULTS

To investigate the effect of NaOCl on the amino acid composition of HDL₃-associated apoA-I, HDL₃ aliquots (containing a total HDL₃ mass of 900 µg) were treated with increasing concentrations of NaOCl (pH 7.4). The molar oxidant:lipoprotein ratio ranged between 7.5 and 300. The results of the amino acid analyses are shown in Table I. An increasing degree of NaOCl-modification of HDL₃ was paralleled by an increasing loss of total amino acids from 3 to 109 mol/mol HDL₃. Following the loss of individual amino acids as a function of increasing NaOCl concentration, three groups of amino acids with respect to their sensitivity toward NaOCl reactivity could be discriminated. The first and most sensitive group was composed of Cys, Met, and Tyr. Cys and Met were completely consumed at a molar NaOCl:HDL₃ ratio of 30 and 60, respectively. Tyr was consumed in a dose-dependent fashion by 5-95% at NaOCl:HDL₃ ratios of 7.5:1 and 300:1. The second group (less reactive toward NaOCl modification) is constituted by Phe, Lys, His, and Arg, which were consumed by 67, 55, 67, and 63%, respectively, at the highest NaOCl concentration used. Finally, the third group, relatively insensitive to NaOCl treatment, is composed of Asx, Thr, Ser, Glx, Pro, Gly, Ala, Val, Ile, and Leu. As can be seen from data shown in Table I, between 36 and 78% of NaOCl was consumed by reactions with HDL₃ apolipoprotein amino acids. In line with these findings, the relative electrophoretic mobility of NaOCl-modified HDL₃ as assessed by agarose gel electrophoresis increased to 1.02 (15:1), 1.1 (30:1), 1.2 (60:1), 1.6 (150:1), and 1.9 (300:1).

Table I. Amino acid analysis of native and NaOCl-modified HDL3 apolipoproteins ApoE-free HDL3 was prepared by discontinuous density ultracentrifugation and modified with increasing concentrations of NaOCl (7.5-300 molecules/HDL3 particle). Amino acid analysis was performed on hydrolysates (900 µg of total lipoprotein mass of native or NaOCl-modified HDL3) prepared in sealed tubes under vacuum in 6 N HCl at 105 °C for 24 h. Data shown represent mean values from one experiment performed in triplicate. Results are expressed in mol of amino acids/mol of HDL3. Amino acid Native NaOCl:HDL3 (ratio) 7.5 15 30 60 150 300 mol/mol Asxa 45.7 46.0 44.9 45.1 44.6 42.5 44.4 Thr 31.1 30.3 29.7 28.7 28.1 27.6 27.4 Ser 39.0 39.1 39.0 38.2 34.5 35.1 34.5 Glxb 110.1 110.4 109.9 110.7 107.9 106.3 109.0 Pro 24.2 24.4 24.5 24.5 24.2 23.5 23.6 Gly 22.3 22.5 21.4 21.8 21.2 20.3 20.5 Ala 40.5 40.4 40.9 41.8 41.4 39.3 39.5 Val 32.5 33.3 32.7 32.8 31.8 32.4 31.8 Cys 1.3 0.6 1.1 NTd NT NT NT Met 1.9 1.7 1.4 1.2 NT NT NT Ile 4.7 4.6 4.3 4.0 4.0 4.0 3.9 Leu 78.2 78.9 78.2 79.3 77.5 76.4 77.8 Tyr 22.7 21.6 20.0 16.9 13.4 4.9 0.3 Phe 20.3 20.0 19.4 19.4 19.4 19.1 6.7 Lys 55.3 53.7 52.5 48.9 38.1 30.6 25.2 His 8.7 8.5 8.4 7.8 7.6 4.8 2.9 Trp NDc ND ND ND ND ND ND Arg 29.0 28.7 28.5 27.9 27.4 23.2 10.8 Total 567.6 564.7 556.7 549.1 521.2 490.0 458.3 Lost 3 11 19 46 78 109 a Asp + Asn. b Glu + Gln. c ND, not determined. d NT, not detectable.

The effects of the MPO/H₂O₂/Cl system on the amino acid composition of HDL₃-associated apolipoproteins are shown in Table II. Incubations were performed at pH 7.4 and 4.5 to generate either HOCl or Cl₂ as the oxidant (28, 29). Assuming quantitative conversion of H₂O₂ by MPO, a molar oxidant:HDL₃ ratio of ~20-23 would be expected. Under these experimental conditions, we have observed pronounced Cys and Met consumption (60 and 40%), in line with data presented in Table I (NaOCl:HDL₃  15:1). Tyr, Phe, and Lys were also modified by the MPO/H₂O₂/Cl system, although to a lesser extent (17, 7, and 5%, respectively). Comparable modification rates were observed for Thr, Ala, and Val (Table II). With the exception of Thr, the modification rates for individual amino acids at pH 7.4 and 4.5 were quite similar. In summary, both HOCl and Cl₂ generated by the MPO/H₂O₂/Cl system resulted in modification of HDL₃-associated apolipoproteins.

Table II. Amino acid analysis of native and MPO-modified HDL3 apolipoproteins HDL3 was modified in the presence of the MPO/H2O2/Cl system at pH 7.4 and 4.5 to generate OCl and Cl2, respectively. MPO modification at pH 7.4 and 4.5 was performed as described under "Experimental Procedures." Amino acid analysis was performed on hydrolysates (1000 µg of total lipoprotein mass of native or MPO-modified HDL3) prepared in sealed tubes under vacuum in 6 N HCl at 105 °C for 24 h. Data shown represent mean values from one experiment performed in duplicate. Results are expressed in mol of amino acids/mol of HDL3. Amino acid Native MPO, pH 7.4 MPO, pH 4.5  Asxa 45.9 45.4 45.8 Thr 31.3 27.4 29.3 Ser 40.9 41.4 40.1 Glxb 112.7 109.9 112.0 Pro 25.9 25.1 24.4 Gly 25.1 26.6 26.3 Ala 44.8 42.4 42.2 Val 35.3 33.0 33.8 Cys 1.2 0.6 0.5 Met 4.7 2.8 2.9 Ile 6.0 5.3 6.1 Leu 82.7 82.3 82.5 Tyr 21.8 18.9 18.2 Phe 19.9 18.4 18.9 Lys 55.9 53.8 53.4 His 8.8 8.4 8.6 Trp NDc ND ND Arg 29.1 29.3 28.5 Total 591.8 571.0 573.5 Lost 21 18 a Asp + Asn. b Glu + Gln. c ND, not determined.

Next the modification of native apoA-I during treatment of HDL₃ with increasing NaOCl concentrations was followed by Western blotting experiments. Detection of apoA-I and NaOCl-modified apolipoproteins was performed with polyclonal rabbit anti-human apoA-I antiserum and a monoclonal antibody, specifically recognizing HOCl-modified proteins (26). An increasing molar oxidant:lipoprotein ratio resulted in a gradual loss of monomeric, 28-kDa apoA-I, with the concomitant formation of high molecular mass aggregates of apoA-I (Fig. 1A). At a molar oxidant:lipoprotein ratio of 50:1, immunoreactive bands with apparent molecular masses of ~45, 60, 90, and 140 kDa were formed. The appearance of high molecular mass apoA-I products (Fig. 1A, lanes 2-5) increased as a function of increasing NaOCl concentrations. The observed molecular masses suggest the formation of dimeric up to pentameric apoA-I products. While no NaOCl-modified epitopes were present in native apoA-I (Fig. 1B, lane 1), modification of HDL₃ with increasing NaOCl concentrations resulted in the generation of NaOCl-modified epitopes (Fig. 1B, lanes 2-5). Detection of immunoreactive bands by monoclonal antibody 2D10G9 revealed the formation of NaOCl-modified apoA-I cross-linked products with apparent molecular masses of approximately 40, 80, and 120-140 kDa, data similar to those shown in Fig. 1A.

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To investigate whether HDL₃ particles aggregated during NaOCl modification, native and NaOCl-modified lipoproteins were analyzed by dynamic and static light scattering. As can be seen from Fig. 2, NaOCl modification led to a dose-dependent increase in R_H values from 6.7 ± 0.3 nm (native) to 9.0 ± 0.8 nm (NaOCl:HDL₃ = 300:1). The polydispersity index was independent of the molar NaOCl:HDL₃ ratio, with a value of 0.1 ± 0.02. The mean scattering intensity increased in a dose-dependent fashion from 53.3 ± 1.2 (native HDL₃) to 75.7 kcps (NaOCl:HDL₃ = 300:1). This moderate increase indicates that only a few HDL₃ particles aggregate even at the highest NaOCl concentrations used. The mean aggregation number at a molar ratio of 30:1 was 1.1 (calculated from the integrated intensities shown in Fig. 2), and even at a 300-fold molar excess of NaOCl we have calculated aggregation numbers of only 1.4. Summarizing the light-scattering results shown in Fig. 2, we were able to demonstrate that NaOCl modification of HDL₃ does not lead to essential interparticle aggregation.

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The fatty acid composition of HDL₃ lipids before and after NaOCl treatment is shown in Table III. The fatty acid composition of the native sample is in good agreement with previous results (40). Treatment of HDL₃ by increasing concentrations of NaOCl resulted in gradual loss of unsaturated fatty acids, i.e. C16:1, C18:1, C18:2, C18:3, C20:4, and C22:6. At a 300-fold molar excess of NaOCl, unsaturated fatty acids were almost completely consumed. Under the experimental conditions described in Tables III and IV, no formation of lipid peroxidation products (measured as thiobarbituric acid-reactive substances) could be detected (data not shown).

Table III. Fatty acid composition of native and NaOCl-modified HDL3 lipids Apo E-free HDL3 was modified with NaOCl at a molar ratio of 7.5-300 molecules/HDL3 particle. Fatty acid analysis was performed by gas chromatography of fatty acid methylesters as described under "Experimental Procedures." Cholesterol was analyzed by high pressure liquid chromatography. Results represent the mean ± S.D. from two experiments performed in triplicate and are expressed in mol/mol of HDL3. Fatty acid Native NaOCl:HDL3 (ratio) 7.5 15 30 60 150 300 mol/mol 14:0 1.0  ± 0.08 1.0  ± 0.16 1.0  ± 0.05 0.9  ± 0.02 0.9  ± 0.01 0.9  ± 0.01 0.9  ± 0.10 16:0 36.3  ± 1.54 35.9  ± 2.06 35.1  ± 0.23 34.5  ± 1.18 34.3  ± 0.35 36.7  ± 0.36 36.5  ± 0.33 16:1 2.7  ± 0.11 2.6  ± 0.16 2.6  ± 0.02 2.6  ± 0.11 2.5  ± 0.12 2.1  ± 0.09 NTa 18:0 14.1  ± 0.58 13.6  ± 0.77 13.6  ± 0.07 13.3  ± 0.37 13.2  ± 0.10 13.0  ± 0.13 12.8  ± 0.01 18:1 (n-7) 22.0  ± 0.97 21.5  ± 1.08 21.4  ± 0.12 21.0  ± 0.64 20.9  ± 0.26 14.6  ± 0.09 2.0  ± 0.09 18:1 (n-9) 1.9  ± 0.10 1.9  ± 0.13 1.9  ± 0.07 1.8  ± 0.05 1.8  ± 0.07 2.6  ± 0.01 NT 18:2 39.9  ± 1.56 39.5  ± 2.29 38.7  ± 0.03 37.9  ± 1.10 36.4  ± 0.58 19.6  ± 0.14 NT 18:3 1.1  ± 0.04 1.1  ± 0.05 1.3  ± 0.07 1.2  ± 0.04 1.1  ± 0.06 0.6  ± 0.06 NT 20:4 18.9  ± 0.70 18.0  ± 1.03 17.6  ± 0.10 17.0  ± 0.50 15.1  ± 0.06 3.8  ± 0.05 NT 22:6 5.0  ± 0.11 1.5  ± 0.10 1.5  ± 0.02 1.4  ± 0.07 1.3  ± 0.02 NT NT Total 139.2  ± 5.8 136.6  ± 7.8 134.4  ± 0.8 131.6  ± 4.1 127.5  ± 1.6 93.9  ± 0.9 52.1  ± 0.5 UFAsb 88.0  ± 3.62 86.1  ± 4.84 84.9  ± 0.43 82.9  ± 2.51 79.1  ± 1.17 43.4  ± 0.44 2.0  ± 0.09 UFAs lost 0 2 3   5 9 45 86 Cholc 6 6 6 5.5 5  4  3 a NT, not detectable. b UFAs, unsaturated fatty acids. c Unesterified cholesterol.

Table IV. Fatty acid composition of native and MPO-modified HDL3 lipids HDL3 was modified in the presence of the MPO/H2O2/Cl system at pH 7.4 and 4.5 to generate OCl and Cl2, respectively. Incubations at pH 7.4 and 4.5 were performed as described under "Experimental Procedures." HDL3 lipids were extracted and transmethylated, and fatty acid methyl esters were analyzed by gas chromatography as described under "Experimental Procedures." Results represent the mean ± S.D. from one experiment performed in triplicate and are expressed in mol/mol of HDL3. Fatty acid Native H2O2 blank MPO (pH 7.4) MPO (pH 4.5) mol/mol 14:0 0.6  ± 0.12 0.6  ± 0.08 0.8  ± 0.09 0.6  ± 0.14 16:0 31.5  ± 2.31 29.5  ± 2.17 29.3  ± 2.75 30.9  ± 1.91 16:1 2.6  ± 0.17 2.5  ± 0.09 2.5  ± 0.22 2.0  ± 0.12 18:0 13.4  ± 0.79 13.0  ± 0.51 12.8  ± 2.31 13.0  ± 1.15 18:1 (n-7) 18.8  ± 1.85 16.1  ± 1.12 17.0  ± 0.21 15.3  ± 0.93 18:1 (n-9) 2.0  ± 0.19 2.1  ± 0.07 1.7  ± 0.12 1.6  ± 0.08 18:2 39.9  ± 2.2 37.3  ± 1.3 32.7  ± 0.29 32.6  ± 2.91 18:3 0.7  ± 0.13 NT NT NT 20:4 13.6  ± 1.94 NT NT NT 22:6 2.5  ± 0.16 NT NT NT Total 125.6  ± 9.86 101.1  ± 5.34 96.8  ± 5.99 96.0  ± 7.24 UFAsa 80.1  ± 6.64 58.0  ± 2.58 53.9  ± 5.99 51.5  ± 4.99 UFAs lost 0 22 26 28 Cholb 6  6  6  4 a UFAs, unsaturated fatty acids. b Unesterified cholesterol. c NT, not detectable.

MPO/H₂O₂/Cl-mediated Loss of Fatty Acids in HDL₃

These experiments were performed to clarify whether oxidants generated by the MPO/H₂O₂/Cl system could inflict fatty acid modification in the lipid moiety of HDL₃. Incubations of HDL₃ in the presence of H₂O₂ and the complete MPO/H₂O₂/Cl system were performed at pH 7.4 and 4.5, and the resulting fatty acid composition is presented in Table IV. It is evident that the addition of H₂O₂ in the absence of MPO led to complete oxidation of 18:3, 20:4, and 22:6. When HDL₃ was modified in the presence of the complete MPO/H₂O₂/Cl system at either pH 7.4 or 4.5, we observed a small increase in fatty acid consumption as compared with the H₂O₂ blank. The resulting fatty acid modification is very similar to data presented in Table III at NaOCl:HDL₃ ratios of 15:1. In line with data reported for LDL (29), the modification of cholesterol in HDL₃ exposed to the MPO/H₂O₂/Cl system occurred only at pH 4.5 (Table IV).

In the next series of experiments, we investigated whether consumption of HDL₃ fatty acids by NaOCl is accompanied by the formation of fatty acid chlorohydrins. Chlorohydrin formation was followed by GC-MS, essentially as described by van den Berg et al. (28, 34). The total ion current (TIC) trace of NaOCl-modified HDL₃ lipid extracts revealed the occurrence of two peak clusters with retention times of 28.02 and 28.51 min not present in native HDL₃ lipids (Fig. 3A, trace II). The mass spectrum of the peak eluting at 28.51 min (Fig. 3B) showed characteristic fragment ions at m/z values of 259 and 263 (relative abundance of 25 and 1.3%, respectively; base peak at m/z 73), corresponding to fragmentation next to the -O-TMS group of the [9-O-TMS,10-Cl] derivative of the chlorohydrin stearic acid methylester. As a comparison, the mass spectrum of a chlorohydrin stearic acid methylester obtained by NaOCl modification of oleic acid is shown in Fig. 3C. The fragment ions at m/z 215 and 307 (relative abundance of 17.9 and 0.96%, respectively; base peak at m/z 73) correspond to fragmentation next to the -O-TMS group of the 9-Cl,10-O-TMS stearic acid methylester. The peak cluster eluting at 28.02 min (Fig. 3A) contained several diagnostic mass fragments (m/z 173, 221, 259, 268, 307, and 403) indicative of the presence of 18:1 monochlorohydrins, which are formed by modification of linoleic acid with NaOCl (42). Although we could detect the M^·+-CH₃ fragment (m/z 403, Fig. 3A, trace VI), we consistently failed to detect the molecular ion at m/z 418 of 18:1 monochlorohydrins.

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The next sets of experiments were designed to study the turnover of native and NaOCl-modified HDL₃ by mouse peritoneal macrophages with emphasis on holoparticle and HDL₃-CE turnover. Holoparticle, total HDL₃-CE, and selective HDL₃-CE uptake was measured as described under "Experimental Procedures." In Fig. 4, a comparison of holoparticle (A), total HDL₃-CE (B), and selective HDL₃-CE uptake (C) of native and NaOCl-modified HDL₃ preparations is presented. In these experiments, we have not discriminated between bound and internalized fraction, i.e. uptake refers to the sum of bound and internalized fraction. The values for maximal uptake of native HDL₃ were 364 ± 54, 3554 ± 207, and 3191 ± 217 ng of HDL₃ protein/mg of cell protein for holoparticle, total [³H]Ch18:2, and selective [³H]Ch18:2 uptake, respectively. When HDL₃ was modified with increasing concentrations of NaOCl, we observed increased holoparticle, total [³H]Ch18:2, and selective [³H]Ch18:2 uptake. Holoparticle uptake was increased 1.2- and 2.4-fold (150 and 300 mol of NaOCl/mol of HDL₃; Fig. 4A). Total and selective [³H]Ch18:2 uptake was increased 2-fold (Fig. 4, B and C). Selective uptake exceeded particle uptake by a factor of 8.7 and 9.3 in native and NaOCl-HDL₃ (molar oxidant:lipoprotein ratio of 150), while the capacity for selective uptake decreased to 7.3 in HDL₃ treated with a molar NaOCl:lipoprotein ratio of 300 (Fig. 4C). Taken together, the results shown in Fig. 4 indicate that NaOCl modification of HDL₃ converts this lipoprotein particle into a "high uptake" form for mouse peritoneal macrophages, leading to intracellular cholesterol(ester) accumulation in these cells in further consequence.

HDL₃ holoparticle (A), total HDL₃-CE (B), and selective HDL₃-CE (C) uptake of native HDL₃ and two different NaOCl-modified HDL₃ preparations (molar NaOCl:HDL₃ ratio was 150 and 300) by mouse peritoneal macrophages. Macrophages were obtained from thioglycollate-elicited mice as described under "Experimental Procedures." Cells were seeded in DMEM containing 10% FCS and cultivated for 4 h. 12 h prior to the uptake experiments, the medium was switched to DMEM containing 10% LPDS. Cells were then incubated in the presence of the indicated concentrations of native and NaOCl-modified ¹²⁵I-labeled and [³H]Ch18:2-labeled HDL₃ preparations for 6 h at 37 °C. Subsequently, the cells were washed, and the total (i.e. bound and internalized) radioactivity was measured. ¹²⁵I uptake represents HDL₃ holoparticle cell association, while uptake of [³H]Ch18:2 represents cell association of HDL₃-associated CEs. Selective uptake was calculated as the difference between [³H]Ch18:2 and ¹²⁵I-labeled HDL₃ cell association. To allow the comparison of cellular uptake of HDL₃ tracers, uptake is shown in terms of apparent HDL₃ particle uptake (expressed as HDL₃ protein that would be necessary to account for the observed tracer uptake; see "Experimental Procedures"). Values shown (mean ± S.D. from triplicate dishes from one representative experiment) represent specific cell association calculated as the difference of activities measured in the absence or presence of a 20-fold excess of unlabeled HDL₃.

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The next series of experiments was designed to identify mechanisms responsible for increased uptake of NaOCl-modified HDL₃ over native HDL₃. During binding experiments at 4 °C, we have observed a pronounced increase in HDL₃ binding with increasing modification rates. In parallel, the ability of native HDL₃ to compete for binding of NaOCl-modified HDL₃ was gradually lost with increasing modification rates (data not shown), indicating that native and NaOCl-modified HDL₃ are bound by different receptors and/or binding proteins on mouse peritoneal macrophages.

To test whether NaOCl treatment of lipoprotein particles resulted in altered binding, internalization, and degradation rates by mouse peritoneal macrophages, the cells were incubated at 37 °C in the presence of 20 µg of protein/ml of iodinated (native and NaOCl-modified) HDL₃ for 6 h. Results of these experiments are shown in Fig. 5. NaOCl treatment of HDL₃ (NaOCl:HDL₃ = 300) increased the amount of trypsin-releasable material (i.e. bound HDL₃ particles) 1.6-fold. Lower NaOCl:HDL₃ molar ratios were without effect on steady state binding of HDL₃ holoparticles at 37 °C. As can be seen in Fig. 5, the effect of NaOCl modification on internalization and degradation was even more pronounced; internalization was increased in a dose-dependent manner 1.1-, 1.2-, and 1.7-fold (75, 150, and 300 mol of NaOCl/mol of HDL₃, respectively). Degradation rates for NaOCl-modified HDL₃ were also significantly enhanced over base-line values; while mouse peritoneal macrophages degraded 180 ± 9.0 ng of native HDL₃/mg of cell protein, these values increased to 287 ± 14.3, 765 ± 38, and 699 ± 35 ng/mg of cell protein (75-, 150-, and 300-fold molar excess of NaOCl, respectively). The data presented above demonstrated that modification of HDL₃ by NaOCl enhanced binding, internalization, and degradation of modified HDL₃ by mouse peritoneal macrophages up to 1.6-, 1.7-, and 4.3-fold in comparison with native HDL₃. These findings were also confirmed by fluorescence microscopy of macrophages incubated in the presence of fluorescently labeled native and NaOCl-modified HDL₃.²

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In this set of experiments, we have modified HDL₃ in the presence of MPO/H₂O₂/Cl (see "Experimental Procedures") at pH 7.4 and pH 4.5 to generate OCl and Cl₂ as the oxidant. Results of these experiments are shown in Fig. 6. Binding of MPO-modified HDL₃ at 4 °C was enhanced 2.5- and 1.5-fold (pH 7.4 and 4.5). At 37 °C, the bound and internalized fraction of MPO/H₂O₂/Cl-modified HDL₃ was 1.9-2.4-fold higher as compared with controls. MPO modification led to strongly increased degradation, exceeding control values by 14.7-fold (302 ± 7.7 versus 4429 ± 380 ng/mg of cell protein, modified at pH 7.4) and 17.7-fold (302 ± 7.7 versus 5352 ± 520.7 ng/mg cell protein, modified at pH 4.5). Incubation of HDL₃ in the presence of H₂O₂ alone did not cause increased degradation (253 ± 10.4 ng of HDL₃/mg of cell protein). Taken together, these results demonstrate that enzymatically active MPO can convert high density lipoproteins into a "high uptake form" for macrophages, ultimately resulting in lipid decomposition in these cells. It is important to note that despite relatively small modifications in the lipid and protein domain of HDL₃ particles, degradation of MPO-modified HDL₃ was approximately 4-fold higher as observed for HDL₃ modified in the presence of the highest molar NaOCl:HDL₃ ratios.

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We have been interested in whether modification of apoA-I by NaOCl may alter the cholesterol efflux efficiency of HDL₃. Results of a representative experiment are shown in Fig. 7. During the incubation with the radioactive tracer, cells acquired 66.000 ± 2.784 dpm/mg of cell protein (n = 3, 60% of the initial radioactivity added). Following a 24-h incubation period of macrophages in the presence of native HDL₃, the cellular cholesterol content decreased to 30 ± 2.7%. However, 39 ± 0.5% cholesterol remained in the cells when the efflux experiment was performed in the presence of NaOCl-modified HDL₃ (NaOCl:HDL₃ = 300). Accordingly, the radioactivity present in the medium (after a 24-h incubation) was 74 ± 0.8 and 61 ± 2.1% of the initial radioactivity when cells were cultivated in the presence of native and NaOCl-modified HDL₃, respectively. This became even more pronounced when the time necessary to remove 50% of the cell-associated [³H]cholesterol from the plasma membrane (/2) was calculated by nonlinear regression analysis; the calculated /2 values were 11.4 ± 0.5 and 19 ± 1.2 h for native and NaOCl-modified HDL₃, approaching /2 values observed with J774 macrophages (41), cells with particularly long efflux times. Taken together, the above mentioned results demonstrate that the efficacy of NaOCl-modified HDL₃ to promote cholesterol efflux was significantly diminished in comparison with native HDL₃.

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DISCUSSION

One important question arising from our study is whether HOCl concentrations that favor HDL₃ modification exist in vivo. Based on in vitro experiments (42, 43), the HOCl concentrations at sites of acute inflammation were calculated to be about 340 µM or greater (44). HDL₃ plasma concentrations of ~6-12 µM (and it is conceivable that subendothelial concentrations would be lower) would yield a minimum estimate of HOCl:HDL₃ ratios of approximately 30:1 to 50:1. Even at these low oxidant:lipoprotein ratios, we have observed apolipoprotein and fatty acid modification in the HDL₃ particle. In addition to these theoretical considerations, our results obtained with the MPO/H₂O₂/Cl system support the assumption that enzymatically active MPO, as expressed in atherosclerotic lesions (22), could transform HDL into a proatherogenic form that is avidly taken up by macrophages.

During the present study, we have observed modification of apoA-I amino acids and the formation of high molecular mass aggregates of intact apoA-I. ApoA-I cross-links were apparently not mediated by disulfide bridging, and in light of the observed tyrosine modification and other investigations it is conceivable that the formation of immunoreactive high molecular mass apoA-I could proceed via dityrosine formation (45, 46). In principle, apoA-I cross-linking can occur by intra- or interparticle cross-linking. Results obtained from our static and dynamic light-scattering experiments suggest that high molecular mass apoA-I products are formed predominantly by intraparticle reactions. This is supported by a rather moderate increase in R_H and aggregation number (from 1 to 1.4), even at the highest NaOCl concentrations used (Fig. 2). In line with results published for LDL modification (21), we have observed pronounced modification of apoprotein amino acids, independent of whether reagent NaOCl or HOCl formed via the MPO/H₂O₂/Cl system was used in these experiments. The varying sensitivities of individual amino acids toward NaOCl modification could be a reflection of their different location within amphipathic apoA-I helices (47).

One remarkable feature of HDL₃ modification was the fact that also fatty acids were modified by the MPO/H₂O₂/Cl system and by NaOCl. Fatty acid consumption occurred even at low and probably physiologically occurring NaOCl concentrations. These results are different from observations made during NaOCl-mediated LDL modification, where the majority of the oxidant was consumed by the apolipoprotein domain (21). These findings suggest that apoA-I protects the HDL₃ lipid domain against excessive modifications by NaOCl. However, in contrast to LDL, HDL₃ apolipoproteins could not provide absolute protection toward modification of cholesterol and fatty acids by NaOCl. GC-MS analyses performed during the present study (Fig. 3) have demonstrated the formation of new compounds with diagnostic ions compatible with the formation of 18:0 chlorohydrins and 18:1 monochlorohydrins as described for the reaction of fatty acid micelles with NaOCl (48). The chlorinating intermediate in the MPO/H₂O₂/Cl system is generally believed to be HOCl, although chlorine (Cl₂) production was demonstrated at acidic pH (29), and the formation of a dichlorinated cholesterol derivative was reported upon incubation of LDL with the MPO/H₂O₂/Cl system at pH 4.5 (49). In line with results reported in Ref. 29, cholesterol consumption was only observed when HDL₃ was modified in the presence of the MPO/H₂O₂/Cl system at pH 4.5, where Cl₂ is generated as the oxidant.