© 1996 American Heart Association, Inc.
Articles |
Selective Resistance of LDL Core Lipids to Iron-Mediated Oxidation
Implications for the Biological Properties of Iron-Oxidized LDL
Department of Molecular and Nuclear Medicine, Life Science Division, Lawrence Berkeley National Laboratory, University of California, Berkeley.
Correspondence to Diane L. Tribble, Lawrence Berkeley National Laboratory, Donner Laboratory, Room 465, University of California, Berkeley, CA 94720.
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Although the nature and consequences of oxidative changes in the chemical constituents of low density lipoproteins (LDLs) have been extensively examined, the physical dynamics of LDL oxidation and the influence of physical organization on the biological effects of oxidized LDLs have remained relatively unexplored. To address these issues, in the present studies we monitored surface- and core-specific peroxidative stress relative to temporal changes in conjugated dienes (CDs), particle charge (an index of oxidative protein modification), and LDL-macrophage interactions. Peroxidative stress in LDL surface and core compartments was evaluated with the site-specific, oxidation-labile fluorescent probes parinaric acid (PnA) and PnA cholesteryl ester (PnCE), respectively. When oxidation was initiated by Cu2+, oxidative loss of the core probe (PnCE) closely followed that of the surface probe (PnA), as indicated by the time to 50% probe depletion (t1/2; 15.5±7.8 and 30.4±12 minutes for PnA and PnCE, respectively). Both probes were more resistant in LDL exposed to Fe3+ (t1/2, 53.2±8.1 and 346.7±155.4 minutes), although core probe resistance was much greater with this oxidant (PnCE t1/2/PnA t1/2, 5.8 vs 2.0 for Cu2+). Despite differences in the rate and extent of oxidative changes in Cu2+- versus Fe3+-exposed LDLs, PnCE loss occurred in close correspondence with CD formation and appeared to precede changes in particle charge under both conditions. Exposure of LDLs to hemin, a lipophilic Fe3+-containing porphyrin that becomes incorporated into the LDL particle, resulted in rapid loss of PnCE and simultaneous changes in particle charge, even at concentrations that yielded increases in CDs and thiobarbituric acid–reactive substances similar to those obtained with free Fe3+. These results suggest that oxidation of the LDL hydrophobic core occurs in conjunction with accelerated formation of CDs and may be essential for LDL protein modification. In accordance with the known effects of oxidative protein modifications on LDL receptor recognition, exposure of LDLs to Cu2+ and hemin but not Fe3+ produced particles that were readily processed by macrophages. Thus, the physical site of oxidative injury appears to be a critical determinant of the chemical and biological properties of LDLs, particularly when oxidized by Fe3+.
Key Words: lipid peroxidation • parinaric acid • copper • iron • hemin
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Introduction |
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Free radical–mediated oxidation alters the chemical and biological properties of LDLs, and these alterations are postulated to play a role in the pathogenesis of atherosclerosis.1 Among the properties of oxidized LDLs that may be related to atherogenesis are their recognition and uptake by macrophages via scavenger receptors,2 3 their ability to alter the chemotactic and adhesion properties of monocytes,4 5 and their effects on the function and viability of endothelial and smooth muscle cells.6 7 8 9 10 11 12 13
The biological properties of oxidized LDLs differ depending on the nature and degree of oxidative modification. Under a variety of oxidizing conditions that have been used in vitro, modification of lipid components, including peroxidation of polyunsaturated fatty acids, occurs early in the oxidation process. mm-LDL particles, which are characterized by low levels of lipid peroxides, are capable of evoking alterations in the biological behavior of endothelial cells.10 11 12 However, because apo B is unaffected, mm-LDL particles are still recognized by the native LDL receptor. Propagation of injury among lipids leads to loss of mm-LDL biological activity, possibly owing to decomposition of critical phospholipid oxidation products.14 As oxidized lipids decompose, injury is "transferred" to the apo B component, which is believed to occur primarily through derivatization of lysyl residues by lipid peroxide fragmentation products3 as well as by direct reaction with lipid-derived radicals. The resultant changes in apo B charge and/or conformation lead to loss of recognition by the native LDL receptor and increased recognition by macrophage scavenger receptors, a property crucial to the ability of oxidized LDLs to promote macrophage cholesterol engorgement.
Although attention has focused primarily on the chemical nature of LDL oxidation and the association between chemical and biological changes (as partially described above), recent studies have suggested that LDL physical organization influences the transmission of oxidation events within the LDL particle and through these effects may determine the biological properties of oxidized LDLs. Using site-specific, oxidation-labile fluorescent probes to monitor the physical dynamics of LDL oxidation, we recently reported that the surface monolayer is the initial site of peroxidative stress and that the properties of this compartment influence overall particle oxidative susceptibility in the presence of some external oxidants (eg, Cu2+).15 Notably, peroxidative changes in surface constituents appeared well before marked changes in CDs, suggesting that measures of bulk lipid peroxidation are relatively insensitive to LDL surface oxidation events.15 16 LDL susceptibility to oxidation by Cu2+ is also influenced by properties of the hydrophobic core, as indicated in the recent study of Schuster et al,17 which showed that the lag time before accelerated lipid peroxidation is related to the thermal transition temperature of LDL core lipids. Collectively, these results suggest that oxidative injury is initiated within the LDL surface monolayer and is subsequently transferred to the LDL core, possibly associated with the accelerated phase of lipid peroxidation.
The purpose of the present studies was to better understand the influence of LDL physical organization on the physical and chemical dynamics and biological effects of LDL oxidation. We hypothesized that at any stage in the oxidation process, differences in the degree of involvement of surface and core lipids are important in determining LDL chemical and biological properties. Surface- and core-specific peroxidation events were evaluated with the oxidation-labile fluorescent probes PnA and PnCE, which preferentially localize within these respective domains.15 18 Parinaroyl probe oxidative loss was analyzed relative to temporal changes in CDs, particle charge, and LDL-macrophage interactions. Oxidation was initiated with Cu2+, Fe3+, or hemin-Fe3+, which have been shown to exert different effects on the kinetics and consequences of LDL oxidation.
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Materials
cis-PnA and cis-PnCE were obtained from Molecular Probes; stock solutions of PnA in ethanol and PnCE in chloroform were stored at -80°C. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was obtained from Aldrich Chemical Co. EDTA; BHT; cumOOH; CuCl2; FeCl3; hemin; 1,1,3,3-tetraethoxypropane; DMEM; and gentamicin sulfate were from Sigma Chemical Co. CETP was a gift from Genentech Inc. Fetal bovine serum and PenStrep were from the University of California at San Francisco Cell Culture Facility. [125I]NaI (15 mCi/µg) was from Amersham Corp.
J774A.1 mouse monocytes/macrophages, from the American Type Culture Collection, were used after the ninth or 10th passage. Cells were grown in DMEM with 10% fetal bovine serum and PenStrep. For experiments, cells were seeded at a density of 106 cells per well in six-well plates and used after 7 days.
Isolation and Oxidation of LDL
LDLs (d=1.019 to 1.063 g/mL) were isolated by sequential ultracentrifugation by standard methods.19 Protein concentrations were determined by the method of Lowry et al that was modified to include SDS.20 Preparations were dialyzed for 48 hours to remove EDTA and Trolox before oxidation. Dialysis and oxidation studies were performed in normal (0.9%) saline (pH 7.4) rather than PBS because the latter has been suggested to induce formation of an iron complex that is incapable of initiating oxidation.21 LDLs were incubated at 0.1 mg protein per milliliter at 37°C, and oxidation was initiated by adding CuCl2 (5 µmol/L), FeCl3 (5 µmol/L), or hemin (0.25 or 1.0 µmol/L, as indicated). cumOOH (10 µmol/L) was added to facilitate heme degradation, a prerequisite for hemin-induced LDL oxidation.22 We previously reported that cumOOH has no independent effects on LDL oxidation kinetics, as indicated by CD formation and parinaroyl probe oxidation.15 Incubations were carried out with 1 to 2 mL LDLs in covered cuvettes or foil-wrapped, capped, glass tubes.
Measurement of CDs
CD formation was determined as a measure of endogenous LDL lipid peroxidation by monitoring the increase in absorbance at 234 nm in a Shimadzu UV2101-PC scanning spectrophotometer equipped with a thermostatted six-position automatic sample changer. Initial absorbance was set at zero and recorded every 2 minutes for up to 12 hours. Lag times (minutes) were derived from slope intercepts of the slow and rapid phases of CD formation.
LDL Incorporation and Measurement of Parinaroyl Probes
Surface- and core-specific peroxidative changes were evaluated by monitoring the loss of fluorescence of LDL-associated PnA and PnCE, which were incorporated separately into LDLs. As reported previously,18 these incorporation procedures do not have major effects on the chemical composition and oxidative behavior of LDLs.
PnA was added directly to LDLs as an ethanolic solution to a final concentration of 0.85 µmol/L (
4 to 5 molecules per LDL particle). This concentration had been found to be optimal for quantum yield and probe partitioning into LDLs.16 Because PnA does not fluoresce in aqueous solutions due to self-quenching in micelles, its incorporation into LDLs was ascertained by the appearance of a fluorescence signal. The mixture was allowed to equilibrate (as indicated by signal stability) before addition of the oxidant.
PnCE was incorporated into LDLs by transfer from PnCE-containing microemulsions (phosphatidylcholine/triolein/cholesteryl oleate) prepared according to a modification18 of the method of Bisgaier et al.23 In brief, stock solutions of 1-palmitoyl-2-oleoyl-phosphatidylcholine and triolein in ethanol and of cholesteryl oleate and PnCE (15 mol %) in chloroform were combined in the appropriate proportions, dried under N2, and dissolved in DMSO (40 nmol lipid per microliter). This solution was heated to 37°C and slowly injected (4 to 20 µL/mL) from a Hamilton syringe (26-gauge needle) below the liquid surface of 50 mmol/L Tris, 150 mmol/L NaCl, and 2 mmol/L EDTA, pH 7.4, in a glass scintillation vial. PnCE was transferred from microemulsions to LDLs (3.5 to 4.0 mg/mL protein) in the presence of recombinant CETP (25 µg/mg protein) for 17 hours at 37°C in 50 mmol/L Tris buffer containing 2 mmol/L EDTA. PnCE-containing LDLs were separated from microemulsions and CETP by fast protein liquid chromatography on two Superose 6 columns connected in series.
Parinaroyl probe oxidation was monitored by following fluorescence loss on a Shimadzu RF-5000 spectrofluorophotometer equipped with a thermostatically controlled cuvette and magnetic stirrer. Excitation wavelength was set at 324 nm (slit width, 1.5 nm) and emission wavelength at 413 nm (slit width, 20 nm), and fluorescence was monitored for up to 17 hours at 37°C. Non-probe–containing LDLs were run under similar conditions in order to assess changes in background fluorescence, which was subtracted from total fluorescence values.
Measurement of Cu+ and Fe2+
The ability of LDLs to reduce Cu2+ and Fe3+ to pro-oxidant forms Cu+ and Fe2+, respectively, was evaluated by monitoring the formation of Cu+ and Fe2+ after addition of CuCl2 and FeCl3. Concentrations of Cu+ and Fe2+ were assessed by the indicator molecules bathocuproine disulfonate and bathophenanthroline disulfonate, respectively, according to procedures described by Lynch and Frei.24 These agents bind the reduced but not the oxidized forms of these metals, and the extent of binding is indicated by a change in absorbance characteristics.
Measurement of TBARS
TBARS, used as a measure of the lipid peroxide fragmentation product malondialdehyde, were determined according to published procedures25 using freshly diluted malondialdehyde as the standard.
Agarose Gel Electrophoresis
Anionic electrophoretic mobility was determined after nondenaturing electrophoresis on agarose gels and staining with Sudan Black, as previously described.26
Macrophage Uptake and Processing of Oxidized LDLs
Sterile-filtered LDLs were iodinated according to published procedures27 and reisolated on a desalting column. After dialysis to remove all noncovalently bound 125I, virtually all counts (>97%) were found within the trichloroacetic acid–precipitable (protein) compartment. Oxidation of 125I-LDLs was performed as described above and arrested after 4 or 12 hours by addition of EDTA (100 µmol/L) and BHT (5 µmol/L). Oxidized 125I-LDLs were diluted in DMEM with 0.2% BSA, and 20 µg LDL protein in a total volume of 0.9 mL was added to confluent J774A.1 macrophage cultures in six-well plates (0.9 mL per well). Lipoprotein-cell mixtures were incubated for 5 hours at 37°C. After incubation, the medium was removed and combined with a 1-mL PBS-BSA wash. After six more washes with PBS, cells were solubilized with 2x1 mL of 0.1N NaOH for 30 minutes. The radioactivity in the incubation medium and that associated with the washed cells was counted in a Packard 800 auto 
-counter. Lipoprotein degradation products were measured as trichloroacetic acid–soluble counts in the incubation medium. Cell-free degradation was found to be minimal (<5% of total counts) and was subtracted from total degradation.
Statistical Analyses
Differences in PnA and PnCE fluorescence loss were analyzed by comparing time to 50% probe oxidation by paired t tests (PnA versus PnCE) and ANOVA (PnA or PnCE loss in Cu2+- versus Fe3+- versus hemin-Fe3+–induced LDL oxidation). Differences in CD formation, TBARS content, and LDL macrophage degradation were evaluated by ANOVA (Cu2+ versus Fe3+ versus hemin). All significance levels were based on two-tailed tests.
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CD Formation and Parinaroyl Probe Oxidation in Cu2+- and Fe3+-Exposed LDLs
We used Cu2+ (CuCl2) and Fe3+ (FeCl3) for these studies because both agents are believed to initiate oxidation by hydroperoxide scission at or near the particle surface but differ in their ability to promote alterations in the chemical composition and biological activity of LDLs. Cu2+ promotes rapid and extensive chemical modifications and produces LDLs that are highly active in promoting macrophage cholesterol engorgement. Fe3+ elicits milder modifications that involve oxidation of phospholipids but not apo B and under appropriate conditions produces particles that exhibit the biological activity of mm-LDLs.9 10 11 12
Fig 1
shows differences in the kinetics of CD formation (234-nm–absorbing species) in LDLs oxidized by Cu2+ and Fe3+. Consistent with numerous previous observations, Cu2+ induced a biphasic response characterized by an initial lag phase followed by a rapid phase that led to marked accumulation of CDs. Fe3+-exposed LDLs exhibited an initial minor increase in absorbance but subsequently maintained a slow rate of change for hours. This was followed much later by a rapid phase, leading to CD accumulations similar to those observed for Cu2+. Differences in lag times for Cu2+- versus Fe3+-exposed LDLs were significant at P<.001. Notably, an initial increase and leveling off were also observed when oxidation was initiated by very low concentrations of Cu2+ (eg, <1 µmol/L; see Fig 2), indicating that this response is not unique to Fe3+-induced LDL oxidation. Rather, we propose that this behavior occurs under conditions of very mild oxidation and may reflect greater stability of CDs that are formed during the initial stages of oxidation.
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Parinaroyl probe oxidative behavior also differed in LDLs exposed to Cu2+ versus Fe3+. The nature and magnitude of these differences depended on probe identity (Fig 3). Oxidative loss of PnA occurred almost immediately after Cu2+ was added and was quickly followed by oxidative loss of PnCE. PnA loss occurred over a longer period in Fe3+-exposed LDLs (240 minutes in the present example). However, there was a marked delay in PnCE response, which was still present 600 minutes after Fe3+ was added. As shown in Fig 4, when the results from four LDL preparations were compared, PnA loss was found to be threefold to fourfold slower and PnCE loss 10-fold slower in Fe3+- than Cu2+-exposed LDLs (P<.001). Given that PnA is present almost exclusively within the LDL surface monolayer15 and PnCE within the hydrophobic core, these results suggest that the extent of involvement of core lipids is markedly reduced in Fe3+ compared with Cu2+-exposed LDLs.
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The increase in CDs exhibited some temporal correspondence with PnCE loss but showed no such relationship with PnA loss (see Fig 3
). Specifically, in Cu2+-exposed LDL, PnCE underwent rapid loss, and this event immediately preceded (and slightly overlapped) the increase in CDs, whereas in Fe3+-exposed LDLs, PnCE underwent a slow steady loss that essentially mirrored the slow, steady increase in CDs (ie, during the prolonged "lag phase"). In contrast, in both Cu2+- and Fe3+-exposed LDLs, CDs had increased to only 25% (or less) of their maximum values by the time PnA loss was complete. Thus, the change in absorbance at 234 nm appears to reflect peroxidative alterations that involve LDL core lipids but not peroxidative alterations that involve LDL surface lipids. This may be partially explained by the fact that core lipids constitute a much greater portion of the total LDL lipids. The more rapid response of PnCE in Cu2+-exposed LDLs may reflect the fact that PnCE is the most susceptible of the core fatty acids, whereas CD formation reflects the collective behavior of all oxidizable fatty acids.
Reductive Activation of Cu2+ and Fe3+ by LDLs
Differences in the responsiveness of LDL to Cu2+ and Fe3+ have been suggested to arise in large part from differences in the extent of their binding and reductive activation (to Cu+ and Fe2+, respectively).24 28 29 In the present studies, we examined differences in the extent of LDL reduction of Cu2+ and Fe3+ by comparing concentrations of Cu+ and Fe2+ for up to 60 minutes after their respective metal salts were added to LDL (final concentrations, 2.5 to 30 µmol/L). Cu+ and Fe2+ were detected almost immediately and reached maximal levels within 30 minutes. As shown in Fig 5 (upper panel), LDLs were fairly effective in reducing Cu2+, with a maximum of 6 nmol Cu+ formed per 0.1 mg LDL protein. LDLs were much less effective in reducing Fe3+, with a maximum of 2.5 nmol Fe2+ formed per 0.1 mg LDL protein (values were significantly lower than for Cu2+ at P<.05). Thus, in accordance with the findings of Lynch and Frei,24 our results suggest that differences in the ability of LDLs to reduce Cu2+ and Fe3+ are likely to account for differences in the effectiveness of these agents in oxidizing LDLs. This does not appear to be the entire explanation, however, since differences in CD formation were still apparent when similar concentrations of the pro-oxidant forms were compared (Fig 5, lower panel). Given the relationship between CD formation and PnCE loss, we suggest that differences in the physical dynamics of Cu2+- and Fe3+-mediated oxidation may be involved. Of particular importance may be the resistance of core lipids to Fe3+-mediated oxidation.
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CD Formation and Surface and Core Probe Oxidation in Hemin-Exposed LDLs
In a previous study involving the use of PnA and its methyl ester as probes of peroxidative stress in the surface and outer core of LDL, we showed that the lipophilic Fe3+-containing porphyrin hemin is taken up by and initiates oxidation from within the LDL particle. In the current study, we evaluated the effects of hemin to assess whether the site of injury initiation and the accessibility of the core lipid pool influence the ability of Fe3+ to oxidize LDLs. In accordance with our previous report,15 hemin was found to produce rapid changes in CDs, even at concentrations as low as 0.25 µmol/L (data not shown). This is particularly noteworthy, given that this concentration is lower than that of the reduced form of Fe3+ in LDL exposed to 5 µmol/L FeCl3 (Fig 5, upper panel). The magnitude of the CD response increased with increasing hemin concentrations and at 1 µmol/L hemin approached (but did not attain) values observed with Cu2+. In contrast to results for Fe3+, hemin produced marked changes in the fluorescence of LDL-associated PnCE (Fig 6). Thus, despite the fact that Fe3+ is the active component, hemin is effective in promoting rapid LDL lipid peroxidation and in oxidizing core-localized LDL constituents.
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Changes in TBARS and Anionic Electrophoretic Mobility in Cu2+-, Fe3+-, and Hemin-Exposed LDLs
Alterations in apo B charge and/or conformation, an important effect of LDL oxidation, are suggested to occur secondary to lipid peroxidation and to involve lysyl derivatization by aldehydic fragmentation products of lipid peroxidation (eg, malondialdehyde).3 The relative inability of Fe3+ compared with Cu2+ to promote apo B modification has been suggested to reflect differences in the extent of lipid peroxidation. To test whether differences in the site of injury initiation and the physical dynamics of LDL oxidation might also be involved, we examined changes in particle charge (anionic electrophoretic mobility, used as an index of apo B modification) in LDLs exposed to Cu2+, Fe3+, or hemin. Two concentrations of hemin were used: 0.25 µmol/L, which promotes elevations in TBARS that are similar to those obtained with Fe3+, and 1 µmol/L, which promotes elevations in TBARS that are closer to those obtained with Cu2+ (see Fig 7).
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As shown in Fig 8, Cu2+ produced a stepwise increase in LDL particle charge from 0 to 6 hours, whereas Fe3+ had no discernible effect over the same period. Hemin, in contrast to both, produced an increase within 2 hours but had little additional effect thereafter. We previously reported that the oxidative response of LDLs to hemin leveled off after an initial rapid change similar to that observed here but that subsequent addition of Cu2+ or hemin led to further rapid oxidation.15 We suggested that hemin-derived iron may be converted to iron oxides or other compounds incapable of initiating further oxidation. Notably, an effect on LDL particle charge was observed with both 0.25 and 1.0 µmol/L hemin, indicating that differences relative to Fe3+ were not solely due to differences in the extent of generation of aldehydes.
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Macrophage-Mediated LDL Degradation in Cu2+-, Fe3+-, and Hemin-Exposed LDL
Oxidative alterations in apo B charge and/or conformation are responsible for alterations in LDL receptor interactions, namely, reduced recognition by the native LDL receptor and increased recognition by macrophage scavenger receptor(s).1 To assess whether the observed differences in LDL particle charge (protein modification) were associated with differences in LDL-macrophage interactions, we examined the extent of LDL-macrophage association and macrophage-mediated LDL degradation in relation to oxidant identity (Cu2+, Fe3+, or hemin) and the time of exposure to oxidants (4 or 12 hours). 125I-labeled LDLs were oxidized for either 4 or 12 hours and then incubated with J774A.1 macrophages for 5 hours. Fig 9 shows results for each oxidation condition. All three oxidants produced time-dependent increases in LDL-macrophage interactions. Fe3+ had a more modest effect, which was particularly apparent at 4 hours. At this time, macrophage-associated counts were significantly lower for Fe3+- exposed than for Cu2+- or hemin-exposed LDLs (P<.05), and TCA-soluble counts were significantly lower for Fe3+-exposed than for hemin-exposed LDLs. By 12 hours, Fe3+- and hemin-exposed LDLs looked almost identical, but macrophage-associated counts were almost twofold lower than for Cu2+-exposed LDL (P<.05). These results suggest that the ability of Fe3+ to produce LDL particles that interact with macrophages is markedly affected by the form in which it is introduced, specifically as this relates to its ability to gain access to LDL core components. This is particularly important during the early stages of oxidation but becomes insignificant at later stages (ie, 12 hours), probably because Fe3+-induced oxidation has enveloped the core and produced changes in particle charge by this time.
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Discussion |
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The present studies show that Cu2+, Fe3+, and hemin differ in their effects on oxidative behavior and biological activity of LDLs and that these differences are related to their relative ability to deliver oxidative injury to the LDL core. This is best exemplified in comparisons of Fe3+ and hemin, which possess the same intrinsic reactivity but differ in their sites of interaction with the LDL particle. Fe3+-induced injury is initiated and spreads within the surface monolayer of LDLs but remains largely confined to this compartment. This behavior is associated with modest changes in CDs, particle charge, and LDL-macrophage interaction. Hemin, in contrast to free Fe3+, is taken up into the LDL lipid phase. Degradation of the heme ring, either directly by LDLs or after addition of H2O222 or cumOOH (Reference 15 and the present studies), causes the release of free Fe3+ into the LDL particle. As shown herein, when introduced in this manner (ie, in the form of hemin), Fe3+ is highly effective in oxidizing LDL core components and promoting oxidative changes in LDL constituents. Even at concentrations
1 µmol/L, hemin produces rapid CD formation and changes in particle charge (protein modification) and increases LDL-macrophage interactions. On the basis of these results, we suggest that the ability of oxidants to promote modifications in LDL core constituents is a major determinant of their effects on the chemical and biological properties of LDLs. Among the primary factors suggested to contribute to differences in the responsiveness of LDL to Cu2+ and Fe3+ are differences in the relative extent of their binding and reductive activation.24 28 29 Lynch and Frei showed that LDLs are capable of reducing Cu2+ in impressive quantities but have virtually no effects on Fe3+.24 Using similar methodology, we confirmed their findings for Cu2+ but noted some activation of Fe3+. Nonetheless, steady-state concentrations of Fe2+ were threefold lower than those of Cu+. Differences in the extent of reductive activation did not appear to completely explain the reduced effectiveness of Fe3+, however, since differences in LDL oxidative behavior were still noted when identical concentrations of Fe2+ and Cu+ (the pro-oxidant forms) were compared. On the basis of the reduced effect of Fe3+(Fe2+) on core lipids, we suggest that one contributing factor may be reduced accessibility of Fe3+(Fe2+) to reactive regions of the LDL particle, possibly due to reduced penetration of binding sites for Fe3+(Fe2+) versus Cu2+(Cu+).
The reduced responsiveness of core lipids to free Fe3+ compared with Cu2+, due to differences either in direct interactions of these metals with core lipids (as suggested above) or in the transfer rate of injury from surface lipids, was further supported by experiments with surface- and core-localized fluorescent probes PnA and PnCE, respectively. Parinaroyl probes undergo a stoichiometric loss in fluoresence after oxidation and are useful for directly monitoring peroxidative stress after their introduction into lipid environments. This approach provides information somewhat analogous to that obtained by following the peroxidative loss of other fatty acids within LDL but has the advantage of technical simplicity and potential for describing the site specificity of peroxidative changes. With regard to the latter, we observed that with Cu2+ and Fe3+, marked peroxidative injury first occurs within the surface compartment (PnA). With Cu2+, however, injury quickly envelopes the core (PnCE), whereas with Fe3+ the core appears to be relatively resistant to oxidative modification. Hemin, in contrast to both Cu2+ and Fe3+, promotes rapid and overlapping changes in surface and core compartments (PnA and PnCE, respectively; data not shown for PnA).
To evaluate the association between site-specific peroxidative stress and other chemical changes, we examined the temporal relationship between PnA and PnCE oxidation and changes in CDs and particle charge. Oxidative loss of PnA preceded changes in all other measures, whereas the course of PnCE loss was more like that of CD formation under all three conditions (CD data not shown for hemin; see Reference 15). On the basis of these results, we suggest that measures of bulk lipid peroxidation (eg, CDs) are relatively unresponsive to surface oxidation events and that movement of injury from the surface to the core is associated with the transition from the lag to the rapid phase of LDL lipid peroxidation. The LDL oxidation lag phase has generally been regarded as an antioxidant-protected period, after which injury can propagate relatively unabated.30 Results of the present studies suggest that in addition to antioxidants, particle organization may play a key role in determining the biphasic character of LDL lipid peroxidation through effects on the relative susceptibility of surface and core lipid pools.
The apparent insensitivity of LDL core lipids to Fe3+-induced oxidation produces discrete stages in the physical dynamics of LDL oxidation. We hypothesize that LDL particles in which oxidative injury is confined almost exclusively to the particle surface may possess the biological properties of mm-LDLs (see Fig 10). This idea is supported by observations that mm-LDLs are characterized by low levels of oxidation products,9 10 11 12 that phospholipid oxidation products are implicated in mm-LDL stimulatory activity,14 and that Fe3+ generates both active mm-LDLs9 10 11 12 and surface-oxidized LDLs (present studies). Moreover, we suggest that movement of injury into the LDL core is associated with a loss in mm-LDL activity and a corresponding increase in the biological properties of fully oxidized LDLs, including changes in the apo B constituent that lead to increased uptake by macrophage scavenger receptor(s). Indeed, we show herein that the latter events are enhanced in Cu2+- and hemin-exposed but not Fe3+-exposed LDLs and that particle charge (apo B modifications) and LDL-macrophage interactions increase in conjunction with or shortly after the oxidation of core lipids.
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The association of LDL core lipid oxidation with particle charge changes and particularly with increased LDL interactions with macrophages may indicate that modifications of core-localized apo B residues are principally involved in mediating these events. Alternatively, these events may occur in parallel but independently as a result of some common third process; eg, radical propagation may be required for the movement of injury into the LDL core and for generation of fragments capable of derivatizing apo B. In support of the former notion, Zawadzki et al31 and Gandjini et al32 showed that oxidant-mediated immunological changes in apo B are greater for epitopes associated with the hydrophobic core regions of LDLs. Likewise, using spin-labeled LDLs, Singh et al33 showed that nitroxide labels located within the hydrophobic environment are predominantly degraded after Cu2+-mediated oxidation. Nonetheless, changes in particle charge reflect alterations in surface-exposed charges. Moreover, oxidative alterations in particle charge, protein conformation, and receptor recognition3 34 35 are attributed primarily to derivatization of apo B–associated lysyl groups, and most (up to 60% to 70%) of these residues are estimated to be surface exposed.33 34 35 As our results suggest, however, the site of oxidative injury is an important determinant of the extent of changes in particle charge and LDL receptor recognition, with reduced effects for modifications that are primarily confined to the particle surface. In this regard, we observed that concentrations of hemin (ie, 0.25 µmol/L) that promoted changes in CDs and TBARS similar to those obtained with Fe3+ led to more extensive loss of PnCE and increases in particle charge. Furthermore, concentrations of hemin (ie,1.0 µmol/L) that produced changes in CDs, TBARS, and particle charge that were much lower than those obtained with Cu2+ were as effective in promoting LDL-macrophage interactions.
In conclusion, we report that the ability of iron (Fe3+) to oxidize LDLs appears to depend on its ability to promote the oxidation of core lipids, which is determined in part by its site of interaction with the LDL particle. Free Fe3+ elicits modifications in LDL surface components but has only modest effects on LDL core components. As a result, exposure of LDLs to Fe3+ leads to production of a particle that exhibits predominantly surface modifications, modest changes in CDs, and virtually no changes in particle charge. We hypothesize that the lesser interaction of Fe3+ with LDL core lipids explains the unique biological properties of LDLs that are exposed to Fe3+, including mm-LDL stimulatory activity and reduced recognition by macrophage scavenger receptors.
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Acknowledgments |
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This research was supported by National Institutes of Health (Bethesda, Md) grants HL27059 and DK32094 and a grant from the National Dairy Promotion and Research Board administered in cooperation with the National Dairy Council (to R.M.K.) and was conducted at Lawrence Berkeley National Laboratory through the US Department of Energy under contract No. DE-AC03-76SF00098. The authors wish to thank Patrick M. Thiel for technical assistance and Drs Alex V. Nichols and Sean M. Lynch for helpful discussions.
Received February 16, 1996;
revision received May 29, 1996;
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References |
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