©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Reduction of Copper, but Not Iron, by Human Low Density Lipoprotein (LDL)
IMPLICATIONS FOR METAL ION-DEPENDENT OXIDATIVE MODIFICATION OF LDL (*)

(Received for publication, December 5, 1994)

Sean M. Lynch Balz Frei (§)

From the Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 02118

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Cell-mediated oxidative modification of human low density lipoprotein (LDL), most likely an important early step in atherosclerosis, requires redox active metal ions such as copper or iron. We have previously shown that iron-dependent, in contrast to copperdependent, oxidative modification of LDL requires superoxide, a physiological reductant. In the present study, we sought to explain these discrepant results. LDL was incubated at 37 °C with Cu (10 µM) and bathocuproine (BC, 360 µM), an indicator molecule which specifically complexes Cu, but not Cu. In a time- and concentration-dependent manner, LDL reduced Cu to Cu. An LDL concentration as low as 10 µg of protein/ml (about 20 nM) reduced about 7 µM Cu within 1 h of incubation. Complexation of the Cu formed under these conditions with BC significantly inhibited oxidative modification of LDL, as assessed by agarose gel electrophoresis. Preincubation of LDL with N-ethylmaleimide had no effect on the rate and extent of Cu reduction nor LDL oxidation, indicating that free sulfhydryl groups associated with apolipoprotein B are not involved. Addition of either superoxide dismutase or catalase or increasing the alpha-tocopherol content of LDL from 11.8 ± 3.0 to 24.4 ± 2.8 nmol/mg of protein also had no significant effect on the kinetics of Cu reduction by LDL. In contrast, incubation of LDL with Fe-citrate (10 µM) and the indicator bathophenanthroline (BP, 360 µM) resulted in no significant Fe formation, even at LDL concentrations as high as 200 µg of protein/ml. However, incubation of LDL with Fe-citrate and an enzymatic source of superoxide led to rapid formation of Fe and consequent oxidative modification of LDL. Addition of BP inhibited iron-mediated LDL oxidation under these conditions. Our results indicate that reduced metal ions are important mediators of LDL oxidation, and that LDL specifically reduces Cu, but not Fe. These data, therefore, help explain why copper, in addition to being chemically more reactive, is more potent than iron at mediating LDL oxidation.


INTRODUCTION

A primary event in early atherosclerosis is the unregulated uptake of low density lipoprotein (LDL) (^1)by monocyte-macrophages within the arterial wall to generate lipid-laden foam cells characteristic of the fatty streak lesion(1) . However, in vitro experiments have demonstrated that incubation of monocyte-macrophages with native LDL does not result in foam cell genesis(2, 3) , and that chemical modification such as acetylation or conjugation with certain aldehydic species is necessary to render the LDL particle atherogenic(3, 4, 5) . Although the in vivo nature of this chemical modification awaits unequivocal identification, there is considerable evidence that oxidative modification of LDL involving lipid peroxidation with subsequent lysine residue modification in apolipoprotein B is important(6, 7, 8) . Regardless of the precise mechanisms, numerous in vitro studies have demonstrated that copper and iron are essential for cell-mediated atherogenic modification of LDL(9, 10, 11, 12, 13, 14, 15, 16) . Cell-free incubation of LDL with cupric ions (Cu) also results in generation of a lipoprotein particle with chemical and physiological characteristics identical with those of cell-modified LDL(17) .

In a recent study(18) , we reported that copper-dependent oxidative modification of human LDL was independent of a source of superoxide, hydrogen peroxide, or aqueous hydroxyl radicals and occurred despite the absence of detectable amounts of pre-existing lipid hydroperoxides in the lipoprotein particle. Oxidative modification of LDL induced by ferric (Fe)-citrate also was independent of hydrogen peroxide, aqueous hydroxyl radicals, and pre-existing lipid hydroperoxides, but required a source of superoxide(18) . Although it had previously been suggested that either pre-existing lipid hydroperoxides in the LDL particle (16, 19, 20) or hydroxyl radicals generated via a superoxide-driven Fenton reaction mechanism (21) serve as initiators of metal ion-dependent lipid peroxidation, our in vitro studies showed that neither is required (18) for LDL oxidation. However, the observation that superoxide, a known Fe reductant(22) , was needed for hydroxyl radical-independent iron-mediated LDL oxidation suggested that reduction of Fe to ferrous ion (Fe) was essential for this process. Interestingly, Aust and co-workers(22, 23, 24, 25, 26) have demonstrated that both Fe and Fe must be present to initiate lipid peroxidation in liposomes. We therefore investigated the hypothesis that copper- and iron-mediated oxidative modification of human LDL depend upon generation of the reduced metal ions. Furthermore, since copper-dependent oxidative modification of LDL occurs in the absence of an exogenous reductant, we also tested whether some redox activity intrinsic to the lipoprotein particle itself could mediate cuprous ion (Cu) formation.


EXPERIMENTAL PROCEDURES

Materials

Sodium heparin Vacutainers were purchased from Becton-Dickinson, Acrodisc LC13 syringe filters from Gelman Sciences, Sephadex G-25M PD-10 columns from Pharmacia (Uppsala, Sweden), Lipo gels for agarose gel electrophoresis of LDL from Beckman, and Chelex-100 resin from Bio-Rad. All chemicals were purchased from Sigma and were of the highest purity available.

LDL Isolation

Blood from a healthy, normolipidemic male volunteer was collected after an overnight fast in a Vacutainer tube (286 USP units of sodium heparin/15 ml of blood) and centrifuged (500 times g for 20 min at 7 °C) to obtain plasma. LDL was isolated from fresh plasma by single vertical spin discontinuous density ultracentrifugation(18) . Contaminating metal ions were removed from the isolated LDL by addition of a small quantity (about 0.1 g/ml) of Chelex-100 resin with gentle mixing. The resin was removed by centrifugation (500 times g for 1 min at 7 °C) and the supernatant, containing LDL, was filtered (Gelman 0.2 µm Acrodisc filter). KBr and potential low molecular weight contaminants (e.g. ascorbate) were removed from LDL preparations by passage through a Sephadex G-25M PD-10 gel filtration column according to the manufacturer's instructions. In contrast to our previous experiments (18) in which isolated LDL was treated with the synthetic peroxidase Ebselen and reduced glutathione to remove possible pre-existing lipid hydroperoxides, this procedure was omitted in the present study in order to eliminate possible trace contamination of the LDL preparations with reduced glutathione, which could have interfered with our measurements of metal ion reduction. However, the absence of pre-existing lipid hydroperoxides (<10 pmol/mg of protein, corresponding to <0.005 molecule per LDL particle(18) ) in LDL preparations was confirmed by a sensitive and specific HPLC procedure with postcolumn chemiluminescence detection(27, 28) . LDL protein was determined by a modification (29) of the Lowry method(30) . Incubations were performed in 10 mM phosphate-buffered saline (154 mM NaCl), pH 7.4 (PBS).

Measurement of Cu and Fe

Cu and Fe were quantitated using bathocuproine disulfonate (BC) and bathophenanthroline disulfonate (BP), respectively. These indicator molecules specifically bind the reduced (Cu, Fe), but not the oxidized (Cu, Fe), forms of copper and iron(31, 32) . The CubulletBC and FebulletBP complexes thus formed exhibit absorbance maxima at 480 nm ( = 9,058 M cm) and 535 nm ( = 22,522 M cm), respectively (see Fig. 1and Fig. 2), permitting specific quantitation of Cu and Fe.


Figure 1: Reduction of Cu by LDL. Absorbance spectra of samples containing various combinations of BC (360 µM), LDL (20 µg of protein/ml), and either Cu or Cu (10 µM each) in PBS are shown following incubation at 37 °C for 60 min. Incubations contained BC (bullet), BC with Cu (circle), BC with Cu (X), BC with LDL (), or BC with LDL and Cu (). Cu was prepared by direct reduction of a Cu solution (10 µM) with ascorbic acid (1 mM). For each incubation, a single spectrum representative of four independent experiments is shown.




Figure 2: Lack of Fe reduction by LDL. Absorbance spectra of samples containing various combinations of BP (360 µM), LDL (20 µg of protein/ml), and either Fe-citrate or Fe-citrate (10 µM each) in PBS are shown following incubation at 37 °C for 60 min. Incubations contained BP (bullet), BP with Fe-citrate (circle), BP with Fe-citrate (X), BP with LDL (), or BP with LDL and Fe-citrate (). For each incubation, a single spectrum representative of three independent experiments is shown.



Oxidative Modification of LDL

Oxidative modification of LDL was assessed as an increase in the electronegativity of the lipoprotein particle (see (8) ). Aliquots of LDL (approximately 2 µg of protein) were loaded on 0.5% agarose gels (Beckman Lipo gels) and electrophoresed at 100 V for 30 min with 0.05 M barbital buffer, pH 8.6, in a Beckman Paragon electrophoresis system. Gels were fixed, stained with Sudan Black B, and destained according to the manufacturer's instructions.

Vitamin E Concentration in LDL

Vitamin E was extracted into hexane and stored at -20 °C before quantitation by HPLC with electrochemical detection(33) .


RESULTS

Fig. 1and Fig. 2show the results of experiments in which Cu or Fe (10 µM) were incubated with LDL (20 µg of protein/ml) at 37 °C for 60 min in the presence of indicator molecules (BC and BP, respectively; 360 µM each) specific for the reduced forms of these metal ions. Incubation of Cu with LDL for 60 min resulted in reduction to Cu as indicated by formation of a peak with maximal absorbance at 480 nm characteristic of the BCbulletCu complex (Fig. 1). In contrast, following incubation of Fe with LDL for 60 min, no increase in the absorbance at 535 nm was observed, i.e. no BPbulletFe complex was formed (Fig. 2). Increasing the LDL concentration 10-fold to 200 µg of protein/ml also did not result in significant Fe reduction in this experimental system (Fig. 3). Thus, LDL specifically reduced Cu to Cu, but was unable to reduce Fe to Fe. Furthermore, complexation of the newly formed Cu with BC in a redox inactive form (31, 34, 35) during incubation of LDL (200 µg of protein/ml) with Cu (10 µM) significantly protected LDL from oxidative modification, as assessed by electrophoretic mobility of LDL on an agarose gel (Fig. 4).


Figure 3: Reduction of Fe by the MFO system. Fe-citrate (10 µM) and LDL (200 µg of protein/ml) were incubated in PBS at 37 °C with BP (360 µM) in the absence (bullet) or presence (circle) of the hypoxanthine (1.5 mM)-xanthine oxidase (0.02 unit/ml) MFO system. Superoxide dismutase (1000 units/ml, X) or catalase (1,000 units/ml, ) was added to incubations containing the MFO system. Fe formation was monitored as the increase in absorbance at 535 nm. Each point represents the mean of at least two independent experiments.




Figure 4: Effect of BC on copper-dependent oxidative modification of LDL. LDL (200 µg of protein/ml) was incubated in PBS at 37 °C for 6 h without(-) or with (+) Cu (10 µM) in the absence(-) or presence (+) of BC (360 µM). Electrophoretic mobility of LDL was measured on an agarose gel.



In contrast to the inability of LDL to reduce Fe, when a standard mixed function oxidase system (MFO, 1.5 mM hypoxanthine with 0.02 unit of xanthine oxidase/ml) was added as a source of superoxide(18) , rapid reduction of Fe to Fe was observed (Fig. 3). As shown in Fig. 5, LDL became oxidatively modified under these conditions, in contrast to LDL incubated with Fe in the absence of MFO. Addition of BP to the LDL incubation containing MFO and Fe completely inhibited LDL oxidation (Fig. 5). Reduction of Fe by MFO was dependent on superoxide, but not hydrogen peroxide, as addition of superoxide dismutase, but not catalase, significantly inhibited Fe reduction (Fig. 3). In contrast, reduction of Cu by LDL was not inhibited by superoxide dismutase, and addition of catalase also was without effect (Fig. 6).


Figure 5: Effect of BP on iron-dependent oxidative modification of LDL. LDL (200 µg of protein/ml) was incubated in PBS at 37 °C for 6 h with Fe-citrate (10 µM) in the absence(-) or presence (+) of the hypoxanthine (1.5 mM)-xanthine oxidase (0.02 unit/ml) MFO system, and BP (360 µM). Electrophoretic mobility of LDL was measured on an agarose gel.




Figure 6: Effects of superoxide dismutase and catalase on Cu reduction by LDL. Cu (10 µM) and LDL (20 µg of protein/ml) were incubated in PBS at 37 °C with BC (360 µM) in the absence (bullet) or presence of either superoxide dismutase (1000 units/ml, circle) or catalase (1000 units/ml, X). Cu formation was monitored as the increase in absorbance at 480 nm. Each point represents the mean of two independent experiments.



To investigate the dependence of Cu reduction on LDL concentration, 10 µM Cu was incubated for 60 min at 37 °C with BC (360 µM) and varying concentrations of LDL (0-30 µg of protein/ml). Maximal levels of Cu (approximately 7.3 µM) were observed at LDL concentrations as low as 10 µg of protein/ml (Fig. 7). Below this concentration, LDL reduced Cu in a dose-dependent manner (Fig. 7). To determine if the Cu reducing ability of LDL was saturable, LDL (10 µg of protein/ml) was incubated for 60 min at 37 °C with BC (360 µM) and varying concentrations of Cu (0-80 µM). Independent of the initial Cu concentration, incubation with LDL resulted in reduction of about 65% of the added Cu (Fig. 8). Thus, a linear relationship between initial Cu concentration and Cu formed was observed, indicating that under the experimental conditions used LDL's ability to reduce Cu was not saturated.


Figure 7: Dependence of Cu reduction on LDL concentration. LDL (0-30 µg of protein/ml) was incubated in PBS at 37 °C for 60 min with Cu (10 µM) and BC (360 µM). Cu formation was monitored as the increase in absorbance at 480 nm. Each point represents the mean of at least two independent experiments.




Figure 8: Effect of initial Cu concentration on Cu formation by LDL. LDL (10 µg of protein/ml) was incubated in PBS at 37 °C for 60 min with BC (360 µM) and Cu (0-80 µM). Cu formation was monitored as the increase in absorbance at 480 nm.



The significance of free sulfhydryl groups on apolipoprotein B as possible Cu reductants was investigated by preincubating LDL (470 µg of protein/ml) with N-ethylmaleimide (6.3 mM) for 30 min at 37 °C before exposure to Cu. This treatment had no effect on either the ability of LDL (20 µg of protein/ml) to reduce Cu (10 µM) (Table 1) or Cu-induced LDL oxidation (Fig. 9). Similarly, increasing the alpha-tocopherol content of LDL from 11.8 ± 3.0 to 24.4 ± 2.8 nmol/mg of protein by preincubating plasma with alpha-tocopherol prior to LDL isolation (36) also had no significant effect on the lipoprotein's ability to reduce Cu (Table 1).




Figure 9: Effect of N-ethylmaleimide on copper-dependent oxidative modification of LDL. LDL (470 µg of protein/ml) was preincubated in PBS at 37 °C without(-) or with (+) N-ethylmaleimide (6.3 mM) for 30 min. These LDL preparations were diluted with PBS (final LDL concentration = 200 µg of protein/ml), incubated at 37 °C with Cu (10 µM), and aliquots were removed at intervals (0-4 h). The reaction was terminated by addition of the metal ion chelator diethylenetriaminepentaacetic acid (1 mM), and the electrophoretic mobility of LDL was measured on an agarose gel. Numbers indicate incubation time (h).




DISCUSSION

The results of this investigation demonstrate that Cu, but not Fe, is specifically reduced by LDL in vitro ( Fig. 1and Fig. 2). These findings have significance for our previous observations (18) that Cu alone can oxidatively modify LDL, while iron-dependent LDL oxidation requires a source of superoxide, a known Fe reductant (22) . Our data suggest that reduced metal ions are an important factor mediating oxidative modification of LDL. Consistent with this hypothesis, chelation of nascent Cu or Fe by BC or BP, respectively, significantly inhibited metal ion-dependent LDL oxidation ( Fig. 4and Fig. 5). Although it has been argued that pre-existing lipid hydroperoxides in isolated LDL are required for initiation of lipid peroxidation by reduced metal ions(16, 19, 20) , our previous (18) and present data do not support this hypothesis. The LDL preparations used in the experiments reported here were routinely analyzed for pre-existing lipid hydroperoxides by a sensitive and specific HPLC procedure(27, 28) , and none were detected. The low detection limit of this assay (1 lipid hydroperoxide molecule in 200 LDL particles) makes it highly unlikely that pre-existing lipid hydroperoxides were an important factor in the initiation of lipid peroxidation in LDL or the observed reduction of Cu.

Heinecke et al.(37) and, more recently, Sparrow and Olszewski (38) have shown that extracellular cysteine or other sulfhydryl compounds are essential for cell-mediated oxidation of LDL in media containing redox active metal ions. Since cysteine is a physiological reductant capable of reducing metal ions, we investigated the possibility that free sulfhydryl groups in apolipoprotein B could mediate the observed reduction of Cu by LDL. Preincubation of LDL with an excess of the sulfhydryl group alkylating agent N-ethylmaleimide had no effect upon subsequent reduction of Cu (Table 1) nor copper-mediated oxidative modification of LDL (Fig. 9). Moreover, addition of superoxide dismutase did not affect Cu reduction by LDL (Fig. 6), whereas cysteine-dependent oxidation of LDL by smooth muscle cells was inhibited by superoxide dismutase(37) . These data indicate that free sulfhydryl groups associated with apolipoprotein B are not an important factor mediating copper-dependent LDL oxidation.

Recently, some investigators have presented evidence that under certain conditions alpha-tocopherol may function as a pro-oxidant, rather than an antioxidant(39, 40, 41, 42, 43) . Bowry et al.(39) have observed that the initial rate of lipid peroxidation in LDL is directly related to its alpha-tocopherol content. Maiorino et al.(42) have shown that alpha-tocopherol in a detergent dispersion is rapidly oxidized by Cu, and that phospholipids added to these dispersions undergo lipid peroxidation at a rate determined by the initial alpha-tocopherol concentration. It has been suggested that lipid peroxidation observed in these systems results from a direct interaction between Cu and alpha-tocopherol with generation of Cu(42, 43) :

where alpha-TocH denotes alpha-tocopherol, and alpha-Tocbullet the alpha-tocopheroxyl radical. In the presence of pre-existing lipid hydroperoxides (LOOH), Cu then can initiate lipid peroxidation via formation of lipid alkoxyl radicals (LObullet):

where LH denotes a lipid molecule with a polyunsaturated fatty acid side chain containing a doubly allylic hydrogen, LOH a lipid hydroxide, and Lbullet a carbon-centered polyunsaturated fatty acid radical. The latter then can reinitiate the radical chain reaction of lipid peroxidation(19, 43) . However, as discussed above, we could not detect pre-existing lipid hydroperoxides in our LDL preparations. It is, therefore, tempting to speculate that it is not Cu plus lipid hydroperoxides, but the alpha-tocopheroxyl radical formed in that initiates lipid peroxidation in LDL, as has been suggested by Stocker and colleagues(39, 40, 41) :

This is an attractive hypothesis, as it would not only explain how Cu initiates lipid peroxidation in LDL devoid of pre-existing lipid hydroperoxides, but also why LDL can reduce Cu in excess over its alpha-tocopherol content. For example, we observed that LDL (20 µg of protein/ml) containing 11.8 nmol of alpha-tocopherol/mg of protein (i.e. 0.24 µM alpha-tocopherol) can reduce approximately 7.6 µM Cu (Table 1), i.e. a 30-fold molar excess of Cu over the LDL-associated alpha-tocopherol (see also below). This phenomenon could be explained by the regeneration of alpha-tocopherol in , so that the reducing equivalents for Cu reduction () would ultimately be provided by the polyunsaturated fatty acids in LDL, the concentration of which is much greater than the concentration of alpha-tocopherol(19) .

However, there are at least two lines of evidence that seem inconsistent with the above hypothesis. First, measuring alphatocopherol consumption, Yoshida et al.(43) estimated that the apparent rate constant for the interaction of endogenous alphatocopherol in LDL with Cu () is 2.9 M s. Based on this rate constant, the predicted rate of Cu reduction by alpha-tocopherol in LDL under the conditions described in Table 1would be 0.42 nM/min; the observed rate, however, was almost 3 orders of magnitude higher (0.27µM/min). Second, doubling of LDL's alpha-tocopherol content had no significant effect on either the rate or extent of Cu reduction by LDL (Table 1). However, it is conceivable that under the conditions employed the alpha-tocopherol content of LDL was not rate-limiting. Thus, the potential significance of LDL-associated alpha-tocopherol as the specific Cu reductant is presently unresolved.

Regardless of the mechanism of Cu reduction by LDL, it is obvious from Fig. 7that this process is highly effective, as maximal reduction of 10 µM Cu was observed during 60 min of incubation with LDL at a concentration as low as 10 µg of protein/ml (approx20 nM LDL). The maximum Cu concentration observed under these conditions was approximately 7.3 µM, corresponding to reduction of about 360 Cu ions by each LDL particle. At LDL concentrations greater than 10 µg of protein/ml, no further increase in Cu reduction was observed, indicating maximal activity of the Cu reducing system under these conditions (Fig. 7). Results from other experiments in which the LDL concentration was held constant (10 µg of protein/ml) and the initial Cu concentration was varied (Fig. 8) indicate that under the conditions employed the Cu reducing activity of LDL is nonsaturable with respect to copper: a linear relationship between Cu formation and initial Cu concentration was observed up to the maximum Cu concentration tested (80 µM, corresponding to approximately a 4,000-fold molar excess of Cu over LDL).

The observation of a Cu specific reducing activity of LDL may be related to the ability of LDL to bind Cu(44, 45, 46, 47, 48, 49) . Thus, Cu reduction may occur at specific binding sites proximal to the reducing moieties within the lipoprotein particle. Interestingly, Kuzuya et al.(44) have reported that while both Cu and Fe may bind to LDL in 0.15 M NaCl, Fe binding is significantly decreased in phosphate-containing buffer. This may explain the Cu specific reducing activity of LDL observed in the experiments reported here, which were performed in PBS. Although LDL did not reduce Fe, incubation with an enzymatic source of superoxide resulted in rapid Fe reduction (Fig. 3). This process was not affected by catalase, indicating that hydrogen peroxide also generated by the enzymatic MFO system used was without effect on Fe formation. Addition of superoxide dismutase, however, significantly inhibited Fe reduction, demonstrating the importance of superoxide in this reaction. Consistent with these data, we reported previously that iron-dependent LDL oxidation requires the presence of MFO, and that addition of superoxide dismutase, but not catalase, inhibits LDL oxidation(18) . In the studies reported here we have also shown that addition of BP to LDL incubations containing MFO and Fe completely inhibits LDL oxidation (Fig. 5) indicating that iron-mediated LDL oxidation requires nonchelated Fe. Using an enzymatic system similar to our MFO system, Miller et al.(22) reported that lipid peroxidation in liposomes is initiated directly by iron, probably via an FebulletFe complex. It is conceivable that a similar complex is responsible for iron-dependent initiation of lipid peroxidation in LDL, and, by analogy, that copper-dependent oxidative modification of LDL requires formation of a CubulletCu complex.

In summary, our results indicate that reduced metal ions (Cu and Fe) are important factors mediating oxidative modification of human LDL. Under the experimental conditions used, LDL specifically reduced Cu to Cu, but could not reduce Fe to Fe. This metal ion specificity may explain the ability of Cu to oxidize LDL and may be related to binding of Cu, but not Fe, to LDL under these conditions. Reduction of Cu by LDL is unlikely to be mediated by pre-existing lipid hydroperoxides in the lipoprotein particle and is also independent of superoxide and free sulfhydryl groups associated with apolipoprotein B. Although reduction of Cu by LDL occurred much faster than expected based on the known reaction rate between Cu and alpha-tocopherol, and alpha-tocopherol supplementation of LDL had no effect on lipoprotein-mediated Cu reduction, the potential significance of alpha-tocopherol as the LDL-associated Cu reductant is presently unclear. In contrast to copper, iron-dependent oxidative modification of LDL requires an exogenous reductant such as superoxide to facilitate reduction of Fe to Fe. Other physiological reductants may also be important for iron-mediated oxidative modification of LDL.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL-49954 and ES-06593 and Grant 3412 from the Council for Tobacco Research-USA, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Whitaker Cardiovascular Institute, Boston University School of Medicine, 80 East Concord St., W 601, Boston, MA 02118. Tel.: 617-638-4896; Fax: 617-638-4066.

(^1)
The abbreviations used are: LDL, low density lipoprotein; BC, bathocuproine disulfonate; BP, bathophenanthroline disulfonate; MFO, mixed function oxidase (1.5 mM hypoxanthine with 0.02 unit of xanthine oxidase/ml); PBS, 10 mM phosphate-buffered saline (154 mM NaCl), pH 7.4; HPLC, high performance liquid chromatography.


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