©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
-Tocopherol as a Reductant for Cu(II) in Human Lipoproteins
TRIGGERING ROLE IN THE INITIATION OF LIPOPROTEIN OXIDATION (*)

(Received for publication, December 1, 1995; and in revised form, February 14, 1996)

Anatol Kontush (1)(§) Stefanie Meyer (1) Barbara Finckh (2) Alfried Kohlschütter (2) Ulrike Beisiegel (1)

From the  (1)From Medizinische Klinik and (2)Kinderklinik, Universitätskrankenhaus Eppendorf, 20246 Hamburg, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Initiation of lipid peroxidation by Cu(II) requires reduction of Cu(II) to Cu(I) as a first step. It is unclear, however, whether this reaction occurs in the course of lipoprotein oxidation. It is also unknown which reductant, if any, can drive the reduction of Cu(II) in this case. We found that Cu(II) was rapidly reduced to Cu(I) by all major human lipoproteins (high, low, and very low density lipoproteins (HDL, LDL, and VLDL), and chylomicrons). Cu(II)-reducing activity was associated with a lipid moiety of the lipoproteins. The rates of Cu(II) reduction by different lipoproteins were similar when the lipoproteins were adjusted to similar alpha-tocopherol concentrations. Enriching lipoproteins with alpha-tocopherol considerably increased the rate of Cu(II) reduction. Cu(II) reduction by alpha-tocopherol-deficient LDL isolated from a patient with familial inherited vitamin E deficiency was found to occur much slower in comparison with LDL isolated from a donor with a normal plasma level of alpha-tocopherol. Initial rate of Cu(II) reduction by alpha-tocopherol-deficient LDL was found to be zero. Enriching LDL with ubiquinol-10 to concentrations close to those of alpha-tocopherol did not influence the reaction rate. When LDL was treated with ebselen to eliminate preformed lipid hydroperoxides, the reaction rate was also not changed significantly. Cu(II) reduction was accompanied by a consumption of lipoprotein alpha-tocopherol and accumulation of conjugated dienes in the samples. Increasing alpha-tocopherol content in lipoproteins slightly decreased the rate of conjugated diene accumulation in LDL and HDL and considerably increased it in VLDL. The results suggest that alpha-tocopherol plays a triggering role in the lipoprotein oxidation by Cu(II), providing its initial step as follows: alphaTocH + Cu(II) alphaToc + Cu(I) + H. This reaction appears to diminish or totally eliminate the antioxidative activity of alpha-tocopherol in the course of lipoprotein oxidation.


INTRODUCTION

There is strong evidence that the oxidation of low density lipoprotein (LDL) (^1)plays an important role in atherogenesis(1, 2) . LDL oxidation appears to occur in the arterial wall, giving rise to the formation of early atherosclerotic lesions. The mechanisms by which LDL oxidation is initiated in vivo, however, remain unclear.

In vitro, LDL can be oxidized by all major groups of arterial wall cells, namely macrophages, smooth muscle cells, endothelial cells, and lymphocytes(3, 4) . A number of agents, such as transition metal ions, lipoxygenases, peroxidases, and heme proteins, are capable in the absence of cells of oxidizing LDL(5, 6) . Of these oxidants, transition metal ions seem to be most likely involved in LDL oxidation in vivo because they are essential for oxidizing LDL in vitro in the presence of cells(3, 5, 6, 7) . In order to be oxidized by transition metal ions, LDL presumably must initially contain traces of lipid hydroperoxides(8, 9) . The decomposition of these hydroperoxides by metal ions provides free radicals that can directly oxidize LDL components. Current knowledge suggests that, in order to decompose lipid hydroperoxides to free radicals, transition metal ions must be reduced to the highly active low valency state(10, 11) . In the case of LDL oxidation, it means that the metal ions, which are tightly bound to the LDL surface(12, 13) , must be reduced by a reductant present in the LDL particle or in the aqueous environment. Such a reaction is expected to represent the initial step of LDL oxidation by transition metals. It has recently been shown that Cu(II) is reduced to Cu(I) by incubation with human LDL(14) . However, it is currently unknown which reductant drives this reaction in the course of LDL oxidation. The occurrence of this reaction in the course of the oxidation of other plasma lipoproteins has also not been demonstrated.

The LDL particle contains several reducing agents which may potentially be involved in the reduction of transition metal ions. They include antioxidants alpha-tocopherol and ubiquinol-10(10, 15) , surface SH groups of apolipoprotein B(15, 16) , and preformed lipid hydroperoxides (17) . It has been suggested by Esterbauer et al.(10) that alpha-tocopherol, the major, on a molar base, antioxidant in LDL, can provide initial reduction of Cu(II) in the course of LDL oxidation by this ion. Recently, it has been shown that alpha-tocopherol can be oxidized by Cu(II) in homogeneous solution (11) as well as in phospholipid (18) and detergent (11) micelles. A similar reaction between alpha-tocopherol and Fe(III) has been described earlier(19, 20) . These data correspond well with the absence of any significant correlation found between oxidizability by Cu(II) and alpha-tocopherol content of native LDL unsupplemented with this antioxidant(21, 22, 23, 24, 25) . Prooxidative activity of alpha-tocopherol as a reductant for Cu(II) might explain the weakening of its activity toward oxidation found in these experiments. In agreement with these findings, we have recently shown that alpha-tocopherol does not significantly contribute to LDL oxidizability by Cu(II)(26, 27) . We have also demonstrated that the antioxidant efficiency of LDL supplementation with alpha-tocopherol is much weaker than, for example, with ubiquinol-10(28) . In the present study we report on a prominent Cu(II)-reducing activity of all major human lipoproteins, specifically very low, low, and high density lipoproteins (VLDL, LDL, and HDL, respectively) and chylomicrons, and show that alpha-tocopherol plays a central role in the Cu(II) reduction.


MATERIALS AND METHODS

Chemicals

Bovine serum albumin was obtained from Pierce (Pierce, Oud-Beijerland, Netherlands). Ebselen was a generous gift from Dr. Roland Stocker (Sydney, Australia). All other chemicals and solvents were from Sigma (Deisenhofen, Germany) or Merck (Darmstadt, Germany).

Isolation and alpha-Tocopherol Enrichment of the Lipoproteins

VLDL, LDL, and HDL were isolated from plasma of a fasted healthy normolipidemic male donor aged 33 years who was not on a special antioxidant or fatty acid diet. The blood was taken on 5 different days within a period of 3 months into EDTA-containing tubes (Sarstedt, Nümbrecht, Germany). Plasma was obtained by centrifugation of the blood at 4 °C for 10 min. To enrich the lipoproteins with alpha-tocopherol, the plasma samples were incubated for 3 h at 4 °C with alpha-tocopherol, added as a 100 mM solution in Me(2)SO to the final concentrations of 0.5 and 1.0 mM(29) . Control incubation contained a corresponding amount of Me(2)SO (1% v/v) which was the same in all plasma samples used to isolate lipoproteins. To enrich LDL with ubiquinol-10, the plasma samples were incubated under identical conditions with ubiquinol-10, added as 2.9 mM solution in Me(2)SO to the final concentrations of 39, 77, and 193 µM. All incubations contained the same amount of Me(2)SO (6.7% v/v). Following the incubation, VLDL, LDL, and HDL were isolated by density gradient ultracentrifugation of the plasma for 20 h at 4 °C in the presence of 1.0 mM EDTA(30) . Chylomicrons were obtained from EDTA plasma of a 25-year-old male patient with a familial hypertriglyceridemia by a single ultracentrifugation step for 45 min at 4 °C as described elsewhere(31) . The patient was homozygote for a defect in the apoC-II gene. The resulting EDTA-containing lipoprotein suspensions were stored at 4 °C under nitrogen in the dark and used for experiments within 7 days.

In separate experiments, LDL was isolated from a 27-year-old male patient with familial isolated vitamin E deficiency (FIVE)(32, 33) . The patient was homozygotic for a defect in the alpha-tocopherol transfer protein gene (33) and required regular vitamin E supplements (1800 mg daily). In experiment A, he was completely withdrawn from his vitamin E supplementation 5 days before taking his first blood sample (day 0). Then, he was resupplemented with increasing amounts of vitamin E (400, 1200, and 1800 mg on the 1st, 2nd, and 3rd days of the resupplementation). Blood was taken from the patient on the first 3 days after resuming the supplementation (days 1, 2, and 3). In experiment B, the patient was withdrawn from his regular vitamin E supplementation for 5 days (days 1-5) and simultaneously supplemented with ubiquinone-10. Amounts of antioxidants taken by the patient were 1800 mg of vitamin E and 90 mg of ubiquinone-10 on day 0 and 180 mg of ubiquinone-10 on days 1-5. All patient's blood samples were taken after an overnight fast into the EDTA-containing tubes and centrifuged. The plasma obtained from the patient was frozen at -80 °C under nitrogen immediately after isolation and stored under these conditions until use. LDL was isolated from the plasma as described above and used for experiments within 2 h.

Characterization of the Lipoproteins

alpha-Tocopherol in the healthy donor's lipoproteins and in chylomicrons was quantified by reversed-phase high performance liquid chromatography (HPLC) with fluorescent detection(28) . Ubiquinol-10 and alpha-tocopherol in the FIVE patient's LDL were quantified immediately after LDL isolation by reversed-phase HPLC with electrochemical detection as described elsewhere(34) . Ubiquinol-9, ubiquinone-7, and -tocotrienol were used as internal standards. Fatty acid composition of LDL was characterized by capillary gas chromatography(35) . Tricosanoic acid and t-butylhydroxytoluene solutions in ethanol were used as an internal standard and antioxidant, respectively. Fatty acid methyl esters were chromatographed on a Hewlett Packard 5890 Series II gas chromatograph (Hewlett Packard, Palo Alto, CA) equipped with a flame ionization detector and a 30 m HP-5MS column (Hewlett Packard) with an internal diameter of 0.25 mm. The column temperature was held at 170 °C for 2 min, increased to 220 °C at a rate of 4 °C/min, held at 220 °C for 4 min, and increased to 310 °C at a rate of 15 °C/min. Plasma and LDL content of polyunsaturated fatty acids was calculated as a sum of their content of linoleic, linolenic, -linolenic, eicosadienoic, eicosatrienoic, arachidonic, eicosapentaenoic, docosadienoic, docosatetraenoic, docosapentaenoic, and docosahexaenoic acids.

Total cholesterol, free cholesterol, triglyceride and phospholipid content of lipoproteins were determined by commercially available enzymatic tests (Boehringer Mannheim, Mannheim, Germany). Lipoprotein cholesterol esters were calculated from total and free cholesterol values using data on average lipoprotein content of these lipids(10, 36) , on the assumption that fatty acid moiety of cholesterol esters contributes to 6.0%, 17.1%, and 6.3% of the total lipoprotein weight in VLDL, HDL, and LDL, respectively. Total protein was determined as described by Lowry et al.(37) using bovine serum albumin as a standard. To recalculate the values obtained into mol/mol lipoproteins, approximate HDL, LDL, and VLDL molecular masses of 0.25, 2.5, and 25 MDa, respectively, were used(10, 36, 38) .

Reduction of Cu(II) by the Lipoproteins

Immediately before experiments, EDTA and KBr were removed from the lipoprotein suspensions by gel filtration on Sephadex PD-10 columns (Sephadex G-25M, Pharmacia Fine Chemicals, Uppsala, Sweden). To obtain stock lipoprotein suspensions, the EDTA-free lipoproteins were diluted with phosphate-buffered saline (PBS), pH 7.4, to the final total cholesterol concentration of 0.87 mg/ml for LDL and 0.60 mg/ml for HDL or to the final triglyceride concentration of 1.20 mg/ml for VLDL. These lipoprotein concentrations were chosen to obtain similar final alpha-tocopherol concentrations, when alpha-tocopherol-unsupplemented lipoproteins were used (on the average 6.35, 8.30, and 11.1 µM (23.9, 23.1, and 28.3 nmol/mg total lipids) for VLDL, LDL, and HDL, respectively). Chylomicrons were diluted to 1.70 mg of triglycerides/ml (3.00 µM alpha-tocopherol). All experiments on Cu(II) reduction and lipoprotein oxidation were performed at room temperature (21 ± 2 °C) and completed within 12 h from the time of removal of EDTA.

The amount of Cu(II) reduced by lipoproteins was measured as the amount of generated Cu(I) using bathocuproinedisulfonic acid (BCSA) which is known to form a colored complex with Cu(I)(39) . To measure Cu(I) accumulation, tandem cuvettes (Hellma, Müllheim, Germany) were used(40) . This enabled us to mix reagents in one compartment of the reaction cuvette and separate them in two compartments of the reference cuvette. 200-µl EDTA-free stock lipoprotein suspensions were mixed with 50 µl of 5.0 mM BCSA solution and necessary volume of PBS. The reaction was started by adding freshly prepared aqueous solution of CuSO(4) to a final concentration of 100 µM and final volume of 1.0 ml, in order to achieve the same final dilution of 1:5 for all stock lipoprotein suspensions. Formation of the BCSA-Cu(I) complex was followed at 480 nm(39) , starting 30 s after addition of Cu(II). Presence of BCSA did not influence the course of the reaction between Cu(II) and alpha-tocopherol, since similar time courses were recorded in the presence and in the absence of the chelator in experiments on Cu(II) reduction by alpha-tocopherol incorporated into sodium dodecyl sulfate micelles. (^2)

To assess the contribution of lipoprotein SH-groups to Cu(II) reduction, the SH-groups were blocked by addition of p-chloromercuriphenylsulfonic acid (PCMPS) to a final concentration of 50 µM(16) . To characterize the contribution of lipoprotein lipids and proteins to Cu(II) reduction, they were separated using chloroform/methanol mixture. 400-µl lipoprotein suspensions were mixed with 3 ml of chloroform/methanol mixture (2:1 v/v). Chloroform and methanol phases were separated, collected, and evaporated under nitrogen. The residues obtained were resuspended in 0.9% NaCl (lipids) or 50 µM SDS (proteins) and assayed for Cu(II) reduction at a Cu concentration of 50 µM. Sodium borohydride was added to the lipoprotein/chloroform/methanol mixture before separation of the fraction containing lipoprotein lipids to remove lipid hydroperoxides. Such a treatment was found to efficiently remove high amounts of hydroperoxides from linoleic acid dissolved in the chloroform/methanol mixture.^2

In some experiments, ebselen was used to remove lipid hydroperoxides from isolated LDL(41) . 2 ml of LDL (0.79 mg of total cholesterol/ml) was mixed with 12.5 µl of 10 mM ebselen solution in ethanol. After adding 50 µl of 30 mM freshly prepared aqueous glutathione solution, the LDL was incubated at 37 °C for 30 min. Ebselen was removed from the LDL using gel filtration on Sephadex PD-10 columns (repeated twice). This procedure has been shown to efficiently reduce all lipid hydroperoxides present in the sample into corresponding hydroxides(41) .

Consumption of the Lipoprotein alpha-Tocopherol

To measure the alpha-tocopherol consumption, the EDTA-free stock lipoprotein suspensions were oxidized without further dilution at a Cu(II) concentration of 500 µM, in order to keep the same Cu(II)/lipoprotein ratio as in the experiments on Cu(II) reduction and lipoprotein oxidation. The reaction was stopped after 1 min by addition of EDTA and butylhydroxytoluene to a final concentration of 270 µM and 220 µM, respectively. alpha-Tocopherol was measured in the oxidized samples by HPLC with fluorescent detection as described above.

Accumulation of Conjugated Dienes in the Lipoproteins

Immediately after removing EDTA, the EDTA-free lipoprotein suspensions (final total cholesterol concentration of 0.17 mg/ml for LDL and 0.12 mg/ml for HDL or final triglyceride concentration of 0.24 mg/ml for VLDL) were oxidized by adding freshly prepared solution of CuSO(4) to the final concentration of 100 µM. The level of lipoprotein oxidation was evaluated as an accumulation of conjugated dienes in the samples. Conjugated dienes were measured according to Esterbauer et al.(42) as an absorption increase at 234 nm.

Cu(II) Binding to the Lipoproteins

Cu(II) binding was measured in PBS, pH 7.4, at a Cu(II) concentration of 10, 20, 50, and 100 µM and lipoprotein concentration of 160 mg of total lipids/dl (corresponding to triglyceride concentrations of 158 mg/dl for chylomicrons and 114 mg/dl for VLDL or total cholesterol concentrations of 63 mg/dl for LDL and 44 mg/dl for HDL). The lipoproteins (final volume 500 µl) were incubated with Cu(II) for 5 min at 37 °C, precipitated with 100 µl of methanol and 350 µl of HDL-cholesterol precipitant (Boehringer Mannheim, Mannheim, Germany), and centrifuged for 10 min at 13,000 rpm. 450 µl of supernatant was mixed with 510 µl of water, 50 µl of 2.5 mM BCSA, and 10 µl of 0.1 M NaBH(4). The absorbance of the solution at 480 nm was measured. The concentration of unbound Cu(II) was calculated from the values obtained using a calibration performed without adding lipoproteins. The amount of Cu(II) bound was calculated as a difference between added and unbound Cu(II).


RESULTS

It was found that all major human lipoproteins rapidly reduced Cu(II) to Cu(I) (Fig. 1). The reaction occurred rapidly and usually reached saturation after about 5 min of incubation. The maximal amount of Cu(II) reduced was similar in different lipoproteins, if they were preliminarily diluted to a similar final alpha-tocopherol concentration (Fig. 1).


Figure 1: Cu(II) reduction to Cu(I) by human VLDL (bullet), LDL (), and HDL () from a healthy donor and by chylomicrons (+) from an apoC-II-deficient patient. Cu(I) accumulation was monitored as the increase in absorbance at 480 nm. Initial concentrations of the reagents: Cu(II), 100 µM; BCSA, 250 µM; VLDL, 0.24 mg of triglyceride/ml (1.20 µM alpha-tocopherol); LDL, 0.17 mg of total cholesterol/ml (1.80 µM alpha-tocopherol); HDL, 0.12 mg of total cholesterol/ml (1.54 µM alpha-tocopherol); chylomicrons, 0.34 mg of triglyceride/dl (0.60 µM alpha-tocopherol). The results shown are representative of three separate experiments performed with three different plasma samples from the healthy donor and of three separate experiments performed with one plasma sample from the patient.



When fractions containing lipoprotein lipids and proteins were separated by extraction with a chloroform/methanol mixture, only the fraction containing lipids retained the ability to reduce Cu(II) (Fig. 2). The results shown here for VLDL are representative of similar experiments performed with LDL and HDL (data not shown). Micelles formed of the fraction containing lipoprotein lipids reduced Cu(II) even more rapidly than corresponding native lipoproteins, despite the fact that the lipoproteins were treated with sodium borohydride to remove preformed lipid hydroperoxides. Denaturated apoproteins showed no significant Cu(II)-reducing activity (1.16 ± 2.16 µM Cu(II) reduced after 5 min of incubation, averaged for 3 experiments with each VLDL, LDL, and HDL apoprotein). In accordance with this finding, blocking protein SH-groups with PCMPS did not suppress Cu(II) reduction by native lipoproteins (Fig. 2). The slight acceleration of the reaction actually observed could be due to the weak reductive activity of PCMPS itself. Such an availability of Cu(II) to exogeneous reductants was confirmed by a considerable acceleration of Cu(II) reduction by native LDL after addition of cysteine (4.85, 12.4, and 70.9 µM Cu(II) reduced in the presence of 0, 10, and 100 µM cysteine, respectively, after a 5-min incubation, means of 2 independent experiments).


Figure 2: Cu(II) reduction to Cu(I) by different fractions of human VLDL from a healthy donor. Fractions containing lipids () and proteins () from VLDL (0.12 mg of triglyceride/ml) were assayed for Cu(II) reduction after their separation as described under ``Materials and Methods.'' Cu(II) reduction by native VLDL (0.12 mg of triglyceride/ml) in the absence (circle) and in the presence (bullet) of PCMPS (50 µM) is also shown. Cu(I) accumulation was monitored as the increase in absorbance at 480 nm. Initial concentrations of the reagents: Cu(II), 50 µM; BCSA, 125 µM.



When the plasma from a healthy donor was incubated with increasing amounts of alpha-tocopherol, the lipoproteins were enriched with the antioxidant. Such an enrichment considerably increased both the initial rate of Cu(II) reduction and the maximal amount of Cu(II) reduced by the lipoproteins (Fig. 3). The initial rate of Cu(II) reduction by the lipoproteins was related to their alpha-tocopherol content, increasing with the latter (Fig. 3, insets). The maximal amount of produced Cu(I) was similarly related to the amount of alpha-tocopherol present, when Cu(II) was in excess to alpha-tocopherol. In this case, the amount of Cu(I) produced was always slightly higher than the amount of alpha-tocopherol present. When the initial concentration of alpha-tocopherol in the reaction mixture exceeded that of Cu(II) (in the alpha-tocopherol-enriched VLDL), nearly all Cu(II) present was reduced. When LDL from a healthy donor was treated with ebselen to eliminate preformed lipid hydroperoxides, the reaction rate was not changed significantly (23.1 and 24.2 mol of Cu(II) reduced/mol of LDL for native and ebselen-treated LDL, respectively, after a 5-min incubation, means of 2 independent experiments).


Figure 3: Cu(II) reduction to Cu(I) by human VLDL, LDL, and HDL enriched with alpha-tocopherol. To perform the enrichment, the plasma from a healthy donor was incubated for 3 h at 4 °C with alpha-tocopherol, added as a solution in Me(2)SO to the final concentration of 0.5 () and 1.0 () mM. Control incubation was performed with 1% Me(2)SO (circle). Following the incubation, HDL, LDL, and VLDL were isolated by density gradient ultracentrifugation. Cu(I) accumulation was monitored as the increase in absorbance at 480 nm. Initial concentrations of the reagents were as described in the legend to Fig. 1. Numbers at the curves denote lipoprotein content of alpha-tocopherol (mol/mol lipoprotein). The results shown are representative of three separate experiments performed with three different plasma samples. Insets show dependences of the initial rate of Cu(II) reduction by lipoproteins measured after a 30-s incubation on their initial alpha-tocopherol content for all samples studied.



When the FIVE patient was given vitamin E after his withdrawal from his regular vitamin E supplementation (experiment A), the LDL isolated from the patient's plasma was enriched with alpha-tocopherol. Enriching LDL with alpha-tocopherol considerably increased the rate of Cu(II) reduction (Fig. 4A). Noteworthy was that the LDL isolated on day 0 of the experiment, which contained a negligibly low level of alpha-tocopherol (0.37 mol/mol of LDL), reduced Cu(II) at a very low rate. No Cu(I) production was found in this LDL incubated with Cu(II) for 2 min (Fig. 4A), implying that the initial rate of Cu(II) reduction by alpha-tocopherol-deficient LDL equaled 0. When the FIVE patient was supplemented with ubiquinone-10 instead of vitamin E (experiment B), the LDL isolated from the patient's plasma was enriched with ubiquinol-10 and depleted in alpha-tocopherol. Such a modification considerably decreased the rate of Cu(II) reduction by LDL (Fig. 4B), demonstrating that the enrichment with ubiquinol-10 (from 0.05 to 0.83 mol/mol LDL) could not overcome the effect of much more pronounced depletion in alpha-tocopherol (from 7.04 to 0.49 mol/mol) on Cu(II) reduction. Enriching LDL from a healthy donor with higher amounts of ubiquinol-10 (up to 3.2 mol/mol LDL) by incubating the plasma with this antioxidant also did not significantly influence the reaction rate (16.9, 16.9, and 16.1 mol of Cu(II) reduced/mol of LDL for 0.16, 2.6, and 3.2 mol of ubiquinol-10/mol of LDL, respectively, after a 5-min incubation, means of 2 independent experiments).


Figure 4: Cu(II) reduction to Cu(I) by human LDL from the FIVE patient supplemented with vitamin E (A) and ubiquinone-10 (B). A and B correspond to experiments A and B described under ``Materials and Methods.'' Numbers at the curves denote LDL content of alpha-tocopherol and ubiquinol-10 (in parentheses) expressed as mol/mol LDL. Cu(I) accumulation was monitored as the increase in absorbance at 480 nm. Initial concentrations of the reagents: Cu(II), 100 µM; BCSA, 250 µM; LDL, 0.17 mg of total cholesterol/ml. Each point represents the mean of two independent experiments.



Cu(II) reduction was accompanied by a consumption of lipoprotein alpha-tocopherol in most samples obtained from a healthy donor (Fig. 5). No systematic decrease in alpha-tocopherol content was found in the experiments with alpha-tocopherol-enriched VLDL (probably due to its highly increased alpha-tocopherol level, complicating accurate determinations). The rate of alpha-tocopherol consumption was generally lower than the rate of Cu(I) accumulation in the same samples. This was in accordance with data previously reported for human LDL(14) .


Figure 5: alpha-Tocopherol consumption in human VLDL, LDL, and HDL enriched with alpha-tocopherol and incubated with Cu(II). The enrichment was performed as described in the legend to Fig. 3. Lipoprotein content of alpha-tocopherol before and after a 1-min incubation is shown with open and closed bars, respectively. alpha-Tocopherol consumption in the control lipoproteins and lipoproteins isolated from the plasma incubated with 0.5 and 1.0 mM alpha-tocopherol are shown from left to right. Initial concentrations of the reagents were as described in the legend to Fig. 1. The results shown are representative of three separate experiments performed with three different plasma samples.



Cu(II) reduction was also accompanied by an accumulation of conjugated dienes in the lipoproteins (Fig. 6). Increasing lipoprotein content of alpha-tocopherol slightly decreased the rate of conjugated diene accumulation in LDL and HDL. Much more pronounced increases in the alpha-tocopherol content found in VLDL resulted in considerable increases in the conjugated diene accumulation in this lipoprotein.


Figure 6: Conjugated diene accumulation in human VLDL, LDL, and HDL enriched with alpha-tocopherol and incubated with Cu(II). The enrichment was performed as described in the legend to Fig. 3. Conjugated diene accumulation in the control lipoproteins (circle) and lipoproteins isolated from the plasma incubated with 0.5 () and 1.0 () mM alpha-tocopherol was monitored as the increase in absorbance at 234 nm. Initial concentrations of the reagents were as described in the legend to Fig. 1. Numbers at the curves denote lipoprotein content of alpha-tocopherol (mol/mol lipoprotein). The results shown are representative of three separate experiments performed with three different plasma samples.



All plasma lipoproteins studied were found to bind Cu(II). The amount of bound Cu(II) increased with increasing amounts of added Cu(II) and was found to be saturable (data not shown). The relationship between bound and added Cu(II) was linear, when expressed in reciprocal coordinates, permitting calculation of binding parameters (Table 1). Both parameters calculated (binding constant and maximal binding capacity) were lowest for the lipoprotein with the lowest protein content studied (chylomicrons) and highest for that with the highest protein (HDL), when measured at the same concentration of total lipids in the reaction mixture and expressed as µM. On the contrary, maximal binding capacity decreased from chylomicrons to HDL, when expressed as mol/mol of lipoprotein. The maximal amount of Cu(II) reduced by native lipoproteins was much lower than their maximal Cu(II) binding capacity (cf. Fig. 3and Table 1). In contrast, the maximal amount of Cu(II) reduced by alpha-tocopherol-enriched lipoproteins was lower than their maximal Cu(II) binding capacity only in LDL and HDL. alpha-Tocopherol-enriched VLDL reduced more Cu(II) than its native counterpart could bind, suggesting that such an enrichment considerably changed the lipoprotein structure.




DISCUSSION

All major human lipoproteins (HDL, LDL, VLDL, and chylomicrons) were found to possess Cu(II)-reducing activity. Lipoprotein particles contain several reducing agents which may potentially be involved in Cu(II) reduction. Our results indicate that lipophilic antioxidant alpha-tocopherol plays a central role in Cu(II) reduction by lipoproteins. This was concluded from the following findings: (i) Cu(II)-reducing activity was associated with the lipid moiety of the lipoproteins; (ii) the rate of Cu(II) reduction by different lipoproteins was similar when they were diluted to a similar alpha-tocopherol concentration; (iii) enriching lipoproteins with alpha-tocopherol considerably increased the rate of Cu(II) reduction; (iv) Cu(II) reduction by alpha-tocopherol-deficient LDL isolated from the FIVE patient was found to occur much more slowly in comparison with LDL isolated from a donor with a normal plasma level of alpha-tocopherol, and (v) initial rate of Cu(II) reduction by alpha-tocopherol-deficient LDL was found to be 0. All other potential candidates (ubiquinol-10, SH groups of apoproteins, and preformed lipid hydroperoxides) did not contribute substantially to Cu(II) reduction. Enriching LDL with ubiquinol-10 to the concentrations exceeding its physiological range, blockage of SH groups of apoproteins by PCMPS, or eliminating lipid hydroperoxides from LDL with ebselen had no influence on the rate of Cu(II) reduction. Denaturated apoproteins also did not reveal any significant Cu(II)-reducing activity. However, alpha-tocopherol may not represent the only factor determining the rate of Cu(II) reduction by lipoproteins, as judged by quite different rates of Cu(I) production found in different LDL with a similar alpha-tocopherol content (cf. Fig. 3and Fig. 4, A and B). This suggests that some other unknown factors, such as lipoprotein Cu(II) binding capacity, might be important for this reaction.

Cu(II) reduction was accompanied by oxidation of alpha-tocopherol, suggesting that a redox reaction between these reagents occurred. The reaction between alpha-tocopherol and Cu(II) in micellar solution presumably has a stoichiometry of 1:1 (11) and proceeds as follows: Cu(II) + alpha-TocH Cu(I) + alpha-TocH + H where alpha-TocH and alpha-Toc denote alpha-tocopherol and alpha-tocopheroxyl radical, respectively. Our results suggest that alpha-tocopherol reduces Cu(II) by the same reaction in human lipoproteins. This scheme corresponds with the hypothetical mechanism of interaction between Cu(II) and alpha-tocopherol in human LDL proposed in earlier studies(10, 11, 14, 18) .

Cu(II) reduction by alpha-tocopherol is expected to occur on the lipoprotein surface, where bound Cu(II) is located(12, 13) . Increases in both the binding constant and maximal amount of bound Cu(II) with increasing protein content in the lipoproteins suggest that Cu(II) is bound predominantly to the protein moiety of the lipoproteins. This protein-bound and surface-localized Cu(II) must be available to alpha-tocopherol to provide the reaction between them. This model is in good agreement with the location of alpha-tocopherol in the surface lipoprotein monolayer(43) . The surface location of alpha-tocopherol can also explain the slightly higher rate of Cu(II) reduction by micelles formed of the fraction containing lipoprotein lipids than by native lipoproteins, simply by a lower availability of Cu(II) to lipids and, hence, to alpha-tocopherol in the lipoproteins than in the micelles. The lack of Cu(II) reduction by ubiquinol-10 can accordingly be explained by a deeper location of this antioxidant in the lipoprotein particle (44) , hindering its direct interaction with Cu(II).

Reduction of Cu(II) by alpha-tocopherol found in our study suggests that this classical antioxidant plays a dual role in the lipoprotein oxidation by Cu(II). Our results indicate that alpha-tocopherol triggers the oxidation, reducing Cu(II) to Cu(I). Formation of Cu(I), a highly active prooxidant, by alpha-tocopherol implies that this antioxidant can develop a prooxidant activity in the course of lipoprotein oxidation. On the other hand, a part of alpha-tocopherol pool which is not directly oxidized by Cu(II) can function antioxidatively, scavenging free radicals according to the classical view(45) . We found that Cu(I) production was accompanied by accumulation of lipid hydroperoxides in the lipoproteins. Increasing lipoprotein content of alpha-tocopherol decreased the rate of conjugated diene accumulation in LDL and HDL and increased it in VLDL. This suggests that the antioxidant activity of alpha-tocopherol prevailed in LDL and HDL, while the prooxidant activity prevailed in VLDL. There are two major mechanisms by which alpha-tocopherol can behave as a prooxidant in the course of lipoprotein oxidation: reduction of transition metal ions to a highly active low valency state (11, 14, 18) and propagation of the oxidation via the alpha-tocopheroxyl radical(15, 46) . The alpha-tocopheroxyl radical can efficiently propagate lipid peroxidation exclusively under mild oxidative conditions (in comparison with alpha-tocopherol concentration) (15, 46) . The concentration of Cu(II) used to initiate oxidation in our study was much higher than that of alpha-tocopherol in almost all samples. The alpha-tocopherol-enriched VLDL represented the only exception due to its highly elevated alpha-tocopherol content. Decreased Cu(II)/alpha-tocopherol ratio (implying milder oxidative conditions relative to alpha-tocopherol) might result in increased propagation via alpha-tocopheroxyl radical and a net prooxidative action of alpha-tocopherol. Classical antioxidant activity known to prevail under strong oxidative conditions (15, 46) could be responsible for the inhibitory action of alpha-tocopherol on HDL and LDL oxidation.

These results indicate that a net effect of alpha-tocopherol on lipid peroxidation is determined by a subtle balance of its pro- and antioxidant activities. The relative importance of different components of this balance seems to depend on the kind of oxidative stress applied to lipids(15, 47, 48) . There is evidence that transition metal ions may be involved in the LDL oxidation in vivo(6) . Nevertheless, assays using solely Cu(II) to induce oxidation can overestimate the prooxidant activity of alpha-tocopherol, in comparison with its physiological role. This assumption can explain the lack of any significant correlation found between the oxidizability by Cu(II) and alpha-tocopherol content of native LDL under relatively strong oxidative conditions(21, 22, 23, 24, 25, 26, 27) . Exaggerated production of Cu(I) and alpha-tocopheroxyl radical may underlie the prooxidant activity of alpha-tocopherol in this case. Cu(I) might be expected to accelerate lipid peroxidation, when oxidizing lipids contain sufficient amounts of preformed lipid hydroperoxides (such as in LDL isolated by time-consuming procedures). alpha-Tocopheroxyl radical can initiate peroxidation in virtually ``hydroperoxide-free'' LDL(9, 14) , interacting directly with lipoprotein polyunsaturated fatty acids(15, 46) . Higher rates of Cu(I) production in comparison to the rates of alpha-tocopherol consumption found in our study imply that alpha-tocopherol is regenerated by unknown mechanisms during the oxidation. Interaction of alpha-tocopheroxyl radical with lipoprotein polyunsaturated fatty acids might represent such a mechanism of regeneration.

It is unclear at present whether the reaction between Cu(II) and alpha-tocopherol is important under physiological conditions. Cell-mediated oxidation of LDL in media containing transition metal ions has been shown to require extracellular cysteine or other sulfhydryl compounds(49, 50) . In accordance with this finding, considerable acceleration of Cu(II) reduction by LDL in the presence of cysteine was found in our study. These data suggest that the initial reduction of transition metal ions may represent an important step in the lipoprotein oxidation in vivo.


FOOTNOTES

*
This study was supported by Klinische Forschergruppe Grant Gr 258/10-1 from Deutsche Forschungsgemeinschaft. 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: Biochemisches Labor, Medizinische Kern- und Poliklinik, Universitätskrankenhaus Eppendorf, Martinistraße 52, 20246 Hamburg, Germany. Tel.: 49-40-4717-4449; Fax: 49-40-4717-4592.

(^1)
The abbreviations used are: LDL, low density lipoprotein; BCSA, bathocuproinedisulfonic acid; FIVE, familial isolated vitamin E deficiency; HDL, high density lipoprotein; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; PCMPS, p-chloromercuriphenylsulfonic acid; VLDL, very low density lipoprotein.

(^2)
S. Meyer and A. Kontush, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. David Evans for careful reading of the manuscript.


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