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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:740-747

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:740-747.)
© 1995 American Heart Association, Inc.

Articles

Inhibition of the Oxidative Modification of LDL by Nitecapone

Markku O. Pentikäinen; Ken A. Lindstedt; Petri T. Kovanen

From the Wihuri Research Institute, Helsinki, Finland.

Correspondence to Petri T. Kovanen, Wihuri Research Institute, Kalliolinnantie 4, SF-00140 Helsinki, Finland.


*    Abstract
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 *Abstract
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 down arrowResults
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Abstract We studied in vitro the ability of nitecapone, 3-[(3,4-dihydroxy-5-nitrophenyl)methylene]-2,4-pentanedione, a novel water-soluble compound with antioxidative properties, to inhibit the LDL oxidation promoted by copper ions, the aqueous free radical generator 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH), and mouse peritoneal macrophages. In these three oxidation systems, the extent of LDL oxidation was determined by measuring the formation of conjugated dienes, the formation of thiobarbituric acid–reactive substances, the change in the electrophoretic mobility of LDL, and the uptake of LDL by macrophages. When LDL oxidation was promoted by copper ions, the reaction was found to be inhibited by nitecapone added in a three- to five-molar excess of the concentration of copper ions. The mechanism by which nitecapone exerted its antioxidative effect in copper-mediated LDL oxidation depended on binding and redox inactivation of the copper ions. Moreover, nitecapone released LDL-bound copper ions and so rendered the LDL particles more resistant to oxidation. In contrast to a water-soluble {alpha}-tocopherol analogue that was rapidly consumed during the oxidative process, nitecapone retained its inhibitory effect for at least 2 days. Using immobilized metal ion affinity chromatography, we showed that nitecapone binds both copper and iron ions, whereas its affinity for zinc ions is low. Nitecapone also inhibited LDL oxidation in the free radical–mediated oxidation system (AAPH). In this system, nitecapone showed synergistic antioxidative action with ascorbic acid. Finally, nitecapone inhibited macrophage-mediated LDL oxidation. Accordingly, nitecapone appears to have a unique antioxidative profile in that it both selectively chelates pro-oxidative transition metals and scavenges free radicals. Moreover, nitecapone has the potential of protecting LDL from oxidation in more complex biological in vitro systems in which multiple modes of oxidative stress act simultaneously, suggesting that this new compound, already tested in humans for its ability to inhibit catechol-O-methyltransferase activity, could potentially be used as an antioxidant drug.


Key Words: nitecapone • LDL • lipid peroxidation • atherosclerosis • macrophages


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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Accumulating evidence strongly suggests that the oxidation of LDL is an important causative factor in atherogenesis.1 The oxidative process is thought to occur in the arterial intima through the action of the local intimal cells and to lead to increased uptake of LDL by macrophages via scavenger receptors,2 3 with subsequent formation of foam cells.4

LDL oxidation was noticed when LDL was incubated with endothelial cells in the presence of small amounts of transition metals.2 Since then, a number of different cell types have been shown to be capable of oxidizing LDL in the presence of copper or iron ions.3 5 6 LDL is also oxidized when incubated with a high concentration of copper ions in the absence of cells.7 Similarly, ferric ions promote LDL oxidation in the absence of cells, although less effectively than copper ions.8 LDL can also be oxidized in the absence of transition metals by using the aqueous free radical generator 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH), which decomposes at 37°C and produces peroxyl radicals at a constant rate.9

LDL particles contain endogenous antioxidants, such as lipid-soluble vitamins (vitamin E), ß-carotene, lycopene,10 and ubiquinol,11 all of which may protect the LDL particles from oxidative stress. However, as oxidation of LDL occurs in vivo,12 13 14 15 16 this endogenous line of defense appears not to be sufficient under conditions of increased oxidative stress. Indeed, in vitro experiments have shown that during LDL oxidation the endogenous antioxidants of LDL are consumed rapidly, their protective antioxidative effect being lost.17 18

The capacity of LDL particles to resist oxidation can be increased or even regained by increasing the amount of endogenous antioxidants in the particles.19 20 21 22 Another possible way to protect LDL against oxidative stress would be to increase the concentration of antioxidants in the different tissue fluids. Thus, ascorbate (vitamin C), a water-soluble antioxidant, protects LDL from oxidation apparently by maintaining the lipophilic endogenous antioxidants in the reduced state.10 19 23 24 Furthermore, the oxidation products of ascorbate prevent the oxidation of LDL by binding covalently to the apoB-100 component of LDL particles, thus inhibiting interaction between this LDL component and copper ions.25 The ability of LDL particles to resist oxidation can also be increased pharmacologically. Thus, incubation of LDL with probucol, a potent synthetic lipophilic antioxidant that is transported in the lipid core of LDL, prevents LDL oxidation by both transition metals and macrophages in vitro.26 27 In addition, in animal models with high plasma LDL levels, such as the Watanabe heritable hyperlipidemic rabbit, probucol retards the rate of development of atherosclerosis in vivo.28 29 30

The present study was aimed at clarifying the antioxidative effect of nitecapone (Fig 1Down) on copper ion–, AAPH-, and macrophage-mediated oxidation of LDL. Nitecapone, 3-[(3,4-dihydroxy-5-nitrophenyl)methylene]-2,4-pentanedione, is a novel, water-soluble, synthetic drug originally designed to inhibit the activity of catechol-O-methyltransferase.31 This drug also has gastroprotective properties32 and has already been used in humans.33 In humans, fast and dose-dependent gastrointestinal absorption of nitecapone has been observed.34 35 Since reactive oxygen species have been implicated in gastrointestinal damage,36 37 the gastroprotective effect of nitecapone has been related to its newly discovered antioxidative effects.38 39 40 In this article we develop the idea of nitecapone as an antioxidant drug and demonstrate that it protects LDL from copper ion–, AAPH-, and macrophage-mediated oxidation in vitro.



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  		Figure 1. Diagram showing chemical structure of nitecapone 3-[(3,4-dihydroxy-5-nitrophenyl)methylene]-2,4-pentanedione.


*    Methods
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 *Methods
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Materials and Animals
Nitecapone (unlabeled and 14C-labeled) was provided byOrion-Farmos, Orion Research. [U-14C]Sucrose (>350 Ci/mmol) was from Amersham International; bovine serum albumin was from Sigma; Dulbecco's phosphate-buffered saline (PBS) and RPMI-1640 culture medium were from GIBCO; Na2EDTA, copper (II)–sulfate pentahydrate, and iron (III)–chloride pentahydrate were from Merck; zinc chloride was from Riedel-de Haën; chelating Sepharose was from Pharmacia LKB Biotechnology; Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was from Roche; and AAPH was from Polysciences. Female NMRI mice were purchased from Helmi Poikkijoki, a licensed animal breeder.

Preparation of LDL
Human LDL (d=1.019 to 1.050 g/mL) was isolated from plasma by sequential ultracentrifugation41 and labeled with [U-14C]sucrose.42 To obtain EDTA-free LDL, LDL was dialyzed against two changes (5 L each) of buffer A (150 mmol/L NaCl and 5 mmol/L Tris-chloride, pH 7.4). For comparing the susceptibility of different LDL preparations to oxidation, plasma from six healthy donors was collected in EDTA-containing tubes, and LDL was isolated by using single-step gradient ultracentrifugation according to Redgrave et al.43 The gradients were centrifuged at 272 000g for 18 hours at 4°C in an SW-40 rotor (Beckman), and the individual LDL preparations, which appeared as single bands, were collected from the top with a syringe. The LDL preparations were then desalted over a Sephadex G-25 minicolumn equilibrated in PBS, and their purity was tested by cellulose acetate electrophoresis. The amounts and concentrations of LDL are expressed in terms of protein.

Copper Ion–Mediated Oxidation of LDL
In a standard experiment, 50 µg EDTA-free LDL was incubated in 300 µL PBS containing 5 µmol/L CuSO4 and the indicated concentrations of nitecapone or Trolox. After incubation at 37°C for 3 hours, the extent of LDL oxidation was determined by measuring the amount of thiobarbituric acid–reactive substances (TBARS), electrophoretic mobility, and the macrophage uptake of LDL (see below).

Measurement of TBARS
The amount of TBARS was measured essentially as described by Hessler et al.44 Briefly, 1.0 mL of 20% trichloroacetic acid was added to 300 µL of a solution containing 50 µg LDL protein, and the mixture was vortexed. One percent thiobarbituric acid (1.0 mL) was then added, and the mixture was vortexed, incubated at 100°C for 30 minutes, cooled, and centrifuged at 1000g for 20 minutes. The absorbance of the supernatant at 532 nm was determined by using a Spectronic 601 spectrophotometer. The amount of TBARS is expressed as malondialdehyde (MDA) equivalents, using 1,1,3,3-tetramethoxypropane as the standard.

Measurement of Diene Formation
In a standard experiment, 200 µg EDTA-free LDL was incubated in 2 mL PBS containing 5 µmol/L CuSO4 and the indicated concentrations of nitecapone or Trolox. Diene formation was measured at A234 every fifth minute for 9 hours with a Pharmacia LKB Biochrom 4060 spectrophotometer equipped with an automatic seven-cell position changer. The lag phase was determined as described by Esterbauer et al.10

Electrophoretic Mobility of LDL
LDL was electrophoresed at 100 V for 30 minutes in 0.5% agarose gels (Beckman Lipo) with 0.05 mol/L barbital buffer, pH 8.6, in a Helena Laboratories electrophoresis system. The agarose gels were then fixed, stained, and destained according to the manufacturer's instructions, and the electrophoretic mobility of LDL was measured with a ruler from enlarged pictures of the gels. The electrophoretic mobility is expressed in relation to the mobility of native LDL.

Immobilized Metal Ion Affinity Chromatography
Copper (CuSO4), zinc (ZnCl2), and iron (FeCl3) ions were coupled to 400 µL chelating Sepharose Fast Flow (Pharmacia LKB Biotechnology) according to the manufacturer's instructions and packed into a 1-mL syringe.45 [14C]Nitecapone (56 000 dpm) was applied to the metal ion–Sepharose columns equilibrated in buffer B (0.02 mol/L Na2HPO4 and 0.5 mol/L NaCl, pH 7.2), eluted with the same buffer at room temperature, and collected in fractions of 1 mL. The material without affinity for the metal ion–Sepharose matrix was collected in fractions 1 through 10, and the material with affinity for the metal ion–Sepharose matrix (fractions 11 through 30) was then eluted with a pH gradient extending from 7.2 to 3.5. The radioactivity in the fractions was measured with a 1215 Rackbeta liquid scintillator (LKB Wallac). A column containing the same amount of chelating Sepharose but no bound metal ions was used as a control.

Isolation of Mouse Peritoneal Macrophages
Macrophages were harvested from unstimulated NMRI mice in PBS containing 1 mg/mL bovine serum albumin.46 The macrophages were plated in 24-well plastic culture plates (1.5 to 2x106/well) in RPMI-1640 containing 10% heat-inactivated fetal calf serum, 100 IU/mL penicillin, and 100 µg/mL streptomycin. The cells were incubated overnight in a humidified CO2 incubator (5% CO2 in air) at 37°C. After incubation, nonadherent cells were removed by rinsing with PBS.

Macrophage-Mediated Oxidation of LDL
In a standard experiment, 1x106 macrophages were incubated in 300 µL RPMI-1640 cell culture medium containing 100 µg EDTA-free LDL and 300 nmol/L copper sulfate. After incubation overnight in a humidified CO2 incubator (5% CO2 in air) at 37°C, the supernatants were removed, and the cells were washed once with 300 µL PBS. The macrophages were dissolved in 500 µL of 0.2 mol/L NaOH, and a sample was taken for analysis of the protein concentration. Other samples were taken for measuring the extent of lipid peroxidation in LDL,44 the electrophoretic mobility of LDL, and the uptake of LDL by fresh macrophage monolayers (see below).

Uptake of Oxidized LDL by Macrophages
In a standard assay, [14C]sucrose-LDL (1000 dpm/µg protein) was oxidized either with 5 µmol/L copper sulfate or with macrophages in the presence of 300 nmol/L copper sulfate as described above. The oxidation of LDL was terminated by the addition of 20 µmol/L butylated hydroxytoluene and 100 µmol/L EDTA. Aliquots of the incubation medium (150 µL) containing oxidized LDL were then combined with 150 µL RPMI-1640 containing 10% heat-inactivated fetal calf serum, 100 IU/mL penicillin, and 100 µg/mL streptomycin and added to fresh cultures of mouse peritoneal macrophages. The cells were incubated for 5 hours in a humidified CO2 incubator (5% CO2 in air) at 37°C, the supernatants were removed, and the cells were washed twice with 300 µL PBS. The macrophages were dissolved in 500 µL of 0.2 mol/L NaOH, and samples were taken for analysis of 14C radioactivity and protein concentration.

AAPH-Mediated Oxidation of LDL
In a standard experiment, 50 µg EDTA-free LDL was incubated in 300 µL PBS containing 25 mmol/L AAPH and the indicated concentrations of nitecapone or Trolox. After incubation at 37°C for 1 hour, the extent of lipid peroxidation was determined by measuring the amount of TBARS expressed as MDA equivalents.

Other Assays
The concentration of copper ions in LDL was determined by atomic absorption spectrophotometry (Perkin Elmer AAS-5000) by using the graphite furnace method according to the manufacturer's recommendations. Protein was determined by the procedure of Lowry et al47 with bovine serum albumin as standard.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Copper-Mediated Oxidation of LDL
The effect of nitecapone on copper ion–mediated oxidation of LDL was studied by incubating LDL with micromolar concentrations of copper ions. A water-soluble {alpha}-tocopherol analogue, Trolox, which has previously been introduced as a reference antioxidant compound,48 was included as a control. The early peroxidative changes in the lipids of LDL during copper ion–mediated oxidation were determined by measuring changes in absorbance at 234 nm, ie, the formation of conjugated dienes.10 Nitecapone retarded the onset of LDL oxidation (ie, increased the lag time), reflecting delayed initial steps of the oxidative process (Fig 2ADown). Furthermore, nitecapone strongly decreased the maximal rate of oxidation, which could also be demonstrated in the first derivative of diene formation ({Delta}A/{Delta}t), giving the rates of diene formation as a function of time (Fig 2BDown). This is in contrast to Trolox, which increased the lag time but did not affect the maximal rate of oxidation.



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  		Figure 2. Time course plot showing effects of nitecapone and Trolox on the copper-mediated oxidation of LDL as measured by the formation of conjugated dienes. LDL (200 µg) was incubated in phosphate-buffered saline (2 mL) containing 5 µmol/L CuSO4 and the indicated concentrations of nitecapone or Trolox. A, Diene formation measured at A234. B, First derivative of diene formation giving the rate of LDL oxidation as a function of time. C indicates control incubation, in which LDL was incubated with 5 µmol/L CuSO4 in the absence of antioxidants.

We also studied the formation of TBARS, ie, the appearance of aldehydes in the apoB-100 moiety of LDL, during the decomposition of lipid peroxides. The appearance of apoB-100–bound aldehydes is held to be one of the final steps in the oxidative modification of LDL leading to the recognition by scavenger receptors.49 When the concentration of copper ions was 5 µmol/L, nitecapone inhibited the formation of TBARS in a concentration-dependent manner, 90% inhibition being achieved at a nitecapone concentration of 25 µmol/L (Fig 3ADown). The inhibitory effect of nitecapone could be overcome by increasing the concentration of copper ions from 5 to 15 µmol/L (Fig 3BDown), indicating that the antioxidative effect of nitecapone was limited either because its copper ion–binding capacity was exceeded or because nitecapone itself was oxidized. To test this, we incubated LDL and copper ions with nitecapone for various lengths of time and measured the formation of TBARS. Nitecapone (25 µmol/L) strongly inhibited copper ion–mediated oxidation for at least 48 hours, whereas the ability of Trolox to inhibit the formation of TBARS was gradually lost during incubation, reaching control values (no antioxidant) within 12 hours (Fig 4Down). These results suggested that the inhibitory effect of nitecapone depended on its ability to chelate and redox inactivate copper ions without itself being consumed rather than on its radical scavenging properties.



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  		Figure 3. Plots showing the effects of nitecapone and Trolox on the copper-mediated oxidation of LDL as measured by the formation of thiobarbituric acid–reactive substances (TBARS). LDL (50 µg) was incubated at 37°C for 3 hours in phosphate-buffered saline (300 µL) containing (A) 5 µmol/L CuSO4 and the indicated concentrations of nitecapone or Trolox or (B) 25 µmol/L of either nitecapone or Trolox and the indicated concentrations of CuSO4. Formation of TBARS was measured as described in "Methods." Data points are means of triplicate incubations. The mean of the deviations of the individual data points was 1.95 nmol malondialdehyde (MDA)/mg LDL protein.



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  		Figure 4. Time course plot showing the effects of nitecapone and Trolox on the copper-mediated oxidation of LDL. LDL (50 µg) was incubated in phosphate-buffered saline (300 µL) containing 5 µmol/L CuSO4 in the presence or absence of either 25 µmol/L nitecapone or Trolox. Formation of thiobarbituric acid–reactive substances (TBARS) was measured as described in "Methods." Data points are means of duplicate incubations. The mean of the deviations of the individual data points was 0.73 nmol malondialdehyde (MDA)/mg LDL protein.

The ability of nitecapone to bind transition metal ions (copper, iron, and zinc) was tested by using immobilized metal ion affinity chromatography. Nitecapone was found to have an affinity for Sepharose-chelated copper ions, since it was retained in the column, and eluted when the pH of the elution buffer was lowered from 7.2 to 3.5 (Fig 5Down). Similarly, the compound was shown to have a strong affinity for chelated iron ions but little or no affinity for chelated zinc ions. Incubation of LDL-copper complexes with nitecapone also led to release of LDL-bound copper ions (TableDown).



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  		Figure 5. Plot showing the affinity of [14C]nitecapone for transition metal ions using immobilized metal ion affinity chromatography. [14C]Nitecapone was applied to a chelating Sepharose column complexed with ions of zinc ({circ}), copper ({bullet}), or iron ({triangleup}) and eluted as described in "Methods." Recovery of nitecapone from the columns containing Sepharose ({blacktriangleup}), zinc-Sepharose, and copper-Sepharose was 100%. No nitecapone could be eluted from the iron-Sepharose column.


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  		Table 1. Measurement of LDL-Bound Copper Ions

A commonly used biological test for the oxidative modification of LDL has been measurement of the uptake of modified LDL particles by macrophages. An increase in the uptake of the LDL particles reflects an increase in their net negative charge, which allows recognition of LDL by scavenger receptors. The increase in the net negative charge of LDL can also be measured from the increase in its electrophoretic mobility. Nitecapone inhibited the copper ion–mediated increase both in electrophoretic mobility (Fig 6ADown) and in the uptake of LDL particles by mouse peritoneal macrophages (Fig 6BDown).



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  		Figure 6. Plots showing the effects of nitecapone and Trolox on the copper-mediated oxidation of LDL as measured by (A) the relative electrophoretic mobility (REM) of LDL and (B) the uptake of radiolabeled LDL by macrophages. [14C]Sucrose LDL (50 µg) was incubated in phosphate-buffered saline (300 µL) containing 5 µmol/L CuSO4 and the indicated concentration of nitecapone or Trolox at 37°C for 3 hours. The degree of LDL oxidation was measured by agarose gel electrophoresis and uptake of LDL by isolated mouse peritoneal macrophages as described in "Methods." Data points in B are means of duplicate incubations. The mean of the deviations of the individual data points was 0.16 µg/mg cell protein.

Macrophage-Mediated Oxidation of LDL
Since intimal cells are known to cause LDL oxidation, we wanted to investigate the ability of nitecapone to affect cell-mediated oxidation of LDL. For this purpose, LDL was incubated with mouse peritoneal macrophages with increasing amounts of nitecapone in the presence of nanomolar concentrations (300 nmol/L) of copper ions, sufficient to permit oxidative modification of LDL in the presence of macrophages. In the absence of macrophages 300 nmol/L copper sulfate induced only slight oxidation of LDL. Incubations of LDL in the absence of macrophages yielded 4 nmol MDA/mg LDL protein (not shown), whereas in the presence of macrophages the above concentration of copper sulfate yielded 21 nmol MDA/mg LDL protein45 (Fig 7ADown). Nitecapone strongly inhibited the macrophage-mediated formation of TBARS (Fig 7ADown) and the increase in electrophoretic mobility of LDL (Fig 7BDown). We also added macrophage-treated LDL to fresh macrophage monolayers and found that the uptake of such LDL by macrophages was strongly inhibited if nitecapone had been present during the preincubation period (Fig 7CDown). Notably, with all three methods inhibition was almost complete at 5 µmol/L nitecapone.



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  		Figure 7. Plots showing the effects of nitecapone and Trolox on the modification of LDL by macrophages. [14C]Sucrose LDL (100 µg) was incubated overnight with isolated mouse peritoneal macrophages in RPMI-1640 culture medium (300 µL) containing 300 nmol/L CuSO4 and the indicated concentrations of nitecapone or Trolox. The degree of LDL oxidation was measured as (A) the formation of thiobarbituric acid–reactive substances (TBARS), (B) the change in relative electrophoretic mobility (REM), or (C) the uptake of LDL by fresh mouse peritoneal macrophages as described in "Methods." Data points (A and C) are means of duplicate incubations. The means of the deviations of the individual data points were 0.56 nmol malondialdehyde (MDA)/mg LDL protein and 0.16 µg/mg cell protein, respectively.

AAPH-Mediated Oxidation of LDL
Finally, we studied whether the antioxidant properties of nitecapone are limited to binding and redox inactivation of transition metal ions. This was done by using the free radical generator AAPH, which can oxidize LDL independently of transition metal ions. Indeed, nitecapone was found to be an effective antioxidant in this system also, where it protected LDL from oxidation by free radicals (Fig 8Down). Because nitecapone reacts with oxidation products of ascorbate,39 we studied whether nitecapone can act synergistically with ascorbate in the AAPH-mediated LDL oxidation system. Various concentrations of nitecapone and ascorbate were used alone and in combination to find the concentrations that inhibited AAPH-mediated LDL oxidation by 50%. The concentrations plotted in Fig 9Down show that the combined antioxidant effect of nitecapone and ascorbate is stronger than would be expected from a mere additive effect (dashed line).



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  		Figure 8. Plot showing the effects of nitecapone and Trolox on the 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH)–mediated oxidation of LDL. LDL (50 µg) was incubated in phosphate-buffered saline (300 µL) containing 25 mmol/L AAPH and the indicated concentrations of nitecapone or Trolox. Formation of thiobarbituric acid–reactive substances (TBARS) was measured as described in "Methods." Data points are means of duplicate incubations. The mean of the deviations of the individual data points was 1.0 nmol malondialdehyde (MDA)/mg LDL protein.



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  		Figure 9. Plot showing the synergistic antioxidative action of nitecapone and ascorbate on 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH)–mediated oxidation of LDL (—). LDL (50 µg) was incubated in phosphate-buffered saline (300 µL) containing 25 mmol/L AAPH and a series of concentrations of nitecapone and ascorbate at 37°C for 1 hour. Formation of thiobarbituric acid–reactive substances was measured as described in "Methods." Data points represent combinations of nitecapone and ascorbate concentrations that inhibited AAPH-mediated LDL oxidation by 50%. The original incubations were performed in triplicate. Dashed line indicates results expected from an additive effect only.

Effect of Nitecapone on LDL Obtained From Different Donors
Since different LDL preparations have different susceptibilities to oxidation,50 we wanted to examine whether different LDL preparations need different concentrations of nitecapone to be protected from oxidation. For this purpose, we isolated LDL from six healthy donors by using single-step gradient ultracentrifugation (see "Methods") and examined both the oxidation resistance of LDL and the effect of nitecapone on LDL oxidation. The oxidation resistance of LDL was determined by measuring the formation of conjugated dienes and determining the length of the lag phase. To test the effect of nitecapone on the copper-mediated oxidation of LDL, the drug was added in various concentrations to each individual LDL preparation, and the concentration of nitecapone that inhibited the formation of TBARS by 50% (IC50) was determined. The various LDL preparations differed in their susceptibility to oxidation, as reflected by the varying lengths of the lag time (range, 97 to 148 minutes) (Fig 10Down). Similarly, the IC50 values of nitecapone during a 3-hour incubation varied. Notably, there was a negative correlation between the lag-phase values of LDL and the IC50 value of nitecapone.



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  		Figure 10. Plot showing the correlation between the lag time of oxidation and the amount of nitecapone needed to inhibit the formation of thiobarbituric acid–reactive substances (TBARS) by 50% (IC50) during a 3-hour incubation. LDL from six healthy donors was isolated by single-step gradient ultracentrifugation. The resistance of LDL to oxidation was determined by measuring the formation of conjugated dienes and determining the length of the lag phase as described in "Methods." The IC50 was determined by incubating LDL (50 µg) at 37°C for 3 hours in phosphate-buffered saline (300 µL) containing 5 µmol/L CuSO4 in the presence of varying concentrations of nitecapone. Formation of TBARS was measured as described in "Methods."


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The present study showed that nitecapone is capable of inhibiting copper ion–mediated oxidation of LDL. In this in vitro system, in which only copper sulfate is added to LDL, the oxidative modification of LDL results from interaction between the copper ions and traces of lipid peroxides already present in the LDL particles.50 We showed here that the mechanism by which nitecapone prevents LDL oxidation in this simple system is due to binding and redox inactivation of copper ions. Moreover, we showed that nitecapone also deprives LDL of its copper ions. This latter ability of nitecapone is likely to be of importance, since apoB-100–bound copper ions appear to catalyze oxidation of LDL more readily than free copper ions.51 Since most of the copper ions in body fluids are protein bound, and some of them remain redox active, the strong affinity of nitecapone for copper, which enables nitecapone to release and redox inactivate copper from proteins, probably would be beneficial in attempts to prevent LDL oxidation in vivo. This is supported by the finding that one of the copper ions bound to ceruloplasmin, the physiological carrier of copper, is redox active and may induce oxidation of LDL.52

In addition to copper ions, nitecapone also binds and redox inactivates iron ions. Since both redox-active copper and redox-active iron have been found in "gruel" samples obtained from human aortic atheromas,53 the ability of nitecapone to inactivate the two metal ions is to be regarded as advantageous. Similarly, the observed lack of affinity of nitecapone for zinc ions is beneficial, since zinc is a transition metal with antioxidative rather than pro-oxidative properties.54

We showed that nitecapone protects LDL from the free radicals generated by AAPH, which accords with the demonstrated antioxidative properties of nitecapone.38 39 40 Nitecapone is a potent inhibitor of lipid peroxidation because of its ability to scavenge superoxide radicals (O2-), hydroxyl radicals (HO · ), and peroxyl radicals (ROO · ) both in solution and in membranes. In addition, nitecapone participates in the recycling of vitamin E by restoring the antioxidative capacity of ascorbate and inhibits xanthine oxidase, a potent producer of superoxide radicals. This broad antioxidative spectrum of nitecapone should aid in preventing LDL oxidation under more physiological conditions, eg, during cellular oxidation of LDL, in which secretion of oxygen radicals also plays a role. Indeed, in the present study we showed that nitecapone completely inhibited the macrophage-mediated oxidation of LDL. In addition, we have used the endothelial cell line EA.hy 926 to oxidize LDL, and nitecapone effectively inhibited LDL oxidation in this system also (data not shown). Although the mechanisms of copper ion– and cell-mediated oxidation of LDL in vitro are quite different in the two systems, oxidation requires the presence of copper ions in both, at micromolar concentrations in the former and at nanomolar concentrations in the latter. Thus, redox inactivation of copper ions by nitecapone is an antioxidative mechanism common to these two systems. However, cells produce several redox reagents that can either react with LDL directly or reduce any transition metal ions present, thus facilitating decomposition of lipid peroxides and chain peroxidation. The effect of nitecapone as an antioxidant in a more complex system such as cell-mediated oxidation of LDL is likely to depend on its ability to act both as a chelator of transition metal ions and as a scavenger of free radicals.

Nitecapone is a water-soluble compound, but it also possesses a hydrophobic domain (pentanedione; Fig 1Up) that could aid nitecapone in associating loosely with LDL. However, using 14C-labeled nitecapone, we were not able to detect any binding of nitecapone to LDL. Thus, with respect to the protection of LDL from the peroxidative attack of macrophages, the effect of nitecapone resembles that of the water-soluble antioxidant vitamin C. We also showed that 14C-labeled nitecapone was not significantly taken up by macrophages and that pretreatment of macrophages in culture for 18 hours with nitecapone had no significant effect on the rate of macrophage-mediated oxidation (data not shown). In this respect, nitecapone clearly differs from the lipid-soluble probucol, which is taken up by macrophages along with LDL and also has direct effects on macrophages.27

In humans, epidemiological and clinical studies have been performed and are in progress using either natural antioxidants (vitamin E, ß-carotene, and vitamin C) or drugs, notably probucol.55 The results of two large-scale prospective studies have shown that the use of large doses of vitamin E supplements is associated with a significantly decreased risk of coronary heart disease.56 57 In addition, the preliminary results of an ongoing clinical study showed that men with angina pectoris who were randomly assigned to receive ß-carotene had fewer subsequent cardiovascular episodes than those assigned to placebo.58 However, negative results with regard to protective effects of antioxidants have also been reported. Thus, neither vitamin E nor ß-carotene was shown to reduce mortality from ischemic heart disease in a recent large-scale prospective trial.59 Hence, it appears that experience with the various antioxidants in the prevention of atherogenesis is as yet insufficient to allow any final conclusions to be drawn about their value in this disease. The clinical field of antioxidative research is still open for new candidate molecules.49 60 61 62 Nitecapone, a drug with well-defined antioxidative properties in vitro and which has already been tested in humans, is clearly one such candidate.


*    Acknowledgments
 
The excellent technical assistance of Päivi Hiironen is gratefully acknowledged.

Received January 9, 1995; accepted March 14, 1995.


*    References
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 *References
 

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