Probucol Protects Marek
Lipoprotein(a)
Naruszewicz,
Elizabeth
Selinger,
Against Oxidative Robert
Dufour,
Modification
and Jean Davignon
Probucol, which decreases cholesterol levels and has antioxidant properties, was administered orally to patients with familial combined hyperlipidemia and high plasma lipoprotein(a) [Lp(a)] levels. The drug had no effect on Lp(a) concentrations after 4 weeks, but was found to be distributed in both Lp(a) and low-density lipoprotein (LDL). Before treatment, in each case LDL and Lp(a) isolated from the same individual were readily oxidized by copper, resulting in increased electrophoretic mobility and enhanced uptake and degradation by macrophages of both lipoproteins. After probucol treatment, both lipoproteins acquired resistance to in vitro oxidation by copper. Furthermore, probucol prevented their enhanced uptake and degradation by the macrophages. It is surmised that oxidized Lp(a) may carry an atherogenic potential that could be opposed by probucol administration. Copyright D 1992 by W.E. Saunders Company
S
EVERAL CASE-CONTROL studies indicate that increased plasma concentrations of lipoprotein(a) [Lp(a)] are associated with an increased risk for coronary artery disease.’ However, it is as yet unknown whether this adverse cardiovascular association is related to an effect of Lp(a) on atherogenesis (ie, formation of foam cells). thrombogenesis (ie, inhibition of fibrinolysis), or both.? The recent observation that Lp(a) is present in human atheromatous plaques’ indicates that it is involved in atherogenesis. Since Lp(a) is detected in the arterial wall in concentrations that correlate with plasma Lp(a) levels,4 it is likely that it could be oxidatively modified locally by endothelial cells, smooth muscle cells, and macrophages, as is :he case for low-density lipoprotein (LDL) accessing the subendothelial space.5 In fact, we have shown that Lp(a) could be oxidatively modified when incubated in vitro with human mononuclear cells having CU’+.~ This modification caused marked changes in the structure and biological properties of Lp(a). Relative to native Lp(a), oxidized particles showed increased negative charge, protein fragmentation, and an increased ability to aggregate. They were taken up and degraded readily by macrophages via scavenger receptors and in part by phagocytosis, inducing cholesterol ester accumulation. Our findings regarding oxidation of Lp(a) are consistent with those of Jiirgens et al7 and Sattler et al,x and are in line with the observations of Haberland et al for Lp(a) modified by malondialdehyde.” It is well known that probucol, a widely used drug in the management of hypercholesterolemia, also has antioxidant properties. When administered orally, it has the ability to enter LDL and protect these particles against oxidative modification.‘” It is likely that by this mechanism probucol inhibits atheroma formation in the hypercholesterolemic, LDL receptor-deficient, Watanabe Heritable Hyperlipidemic rabbit, independent of its cholesterol-lowering effect.“.” The present study was undertaken to determine whether probucol administration can protect Lp(a) particles against oxidation. This report documents for the first time that administration of probucol to patients with very high levels of plasma Lp(a) results in the presence of this drug within Lp(a) particles. This protects Lp(a) particles against both oxidatibe modification in vitro and subsequent uptake by macrophages. Metabolism,
Vol 41, No 11 (November),
1992:
pp 1225.1228
SUBJECTS AND METHODS
Subjects Probucoi was administered (500 mg twice a day) to three outpatients (two women and one man) with familial combined hyperlipidemia, aged 52. 60, and 38 years, respectively. These patients were selected for their high levels of Lp(a) (0.7 to 1 g/L) and LDL cholesterol so that oxidative modifications of both lipoprotein fractions could be performed in the same individual. They were maintained on an American Heart Association phase-II diet before and during 1 month of probucol treatment. The patients received no supplemental vitamin E or p-carotene in tablet form. One patient (B.F.) had been treated 4.5 months with a bile acid sequestrant (cholestyramine, 4 g twice a day), which was maintained during the treatment with probucol. All patients were informed of the purpose of the study. which was approved by our institutional ethics review board. This study was designed without a wash-out period after probucol treatment, because of the long terminal-phase elimination half-life of this drug (47 days).
Methods After an overnight fast, blood samples were taken by venipuncture into tubes containing disodium EDTA (1.5 mg/mL), and the plasma was separated in a cold centrifuge. LDL (1.019 to 1.055 g/mL) and Lp(a) (1.055 to 1.090 g/mL) fractions were isolated by sequential ultracentrifugation from each individual plasma sample before and after probucol treatment. Lp(a) was further purified by column chromatography over Bio-Gel A-5m (Bio-Rad Laboratories, Richmond, CA) and heparin-sepharose13 to exclude contamination of the Lp(a) fraction with LDL. Lipoproteins were labeled with carrier-free Na12s1 and the water-insoluble iodination agent, Iodo-gen (Pierce. Rockford, IL). Specific activity ranged from 60 to 110 cpm/ng protein. Mouse peritoneal macrophages were harvested from unstimulated Swiss Webster mice. The degradation of labeled lipoproteins was measured by determining the trichloroacetic acid-soluble, iodide-free
From the Hyperlipidemia and Atherosclerosis Research Group, Clinical Research Insritute of Montreal, Afiliated with the University of Montreal and the Hotel-Dieu Hospital of Montreal, Canada. Supported in part by grants from the Medical Research Council of Canada ICIBA-Geigy Universityllndustyprogram (VI 0029), Succession J.A. De&e, and Marion Merrell Dow Research Institute. Address reprint requests IO Marek Naruszewicz, PhD. Visiting Professor, Hyperlipidemia and Atherosclerosis Research Group, Clinical Research Institute of Montreal. 110 Pine Ave. W, hfontreal. Quebec, Canada H2WIR7. Copyright Q 1992 by W B. Saunders Compare> 0026049519214111-0013$03.00/O 1225
1226
NARUSZEWICZ
Table 1. Effect of 4
Weeks’ Probucol
Treatment
ET AL
on Plasma Lipid, Lipoprotein, Vitamin E, and p-Carotene Levels Lipoprotein Fractions
TG (mmollL)
Patients
Cholesterol (mmol/L)
Vitamin E (pmol/L)
LPb)
(s/L)
p-carotene (pmol/L)
VLDL-C (lnnlol/L)
LDL-C (mmol/L)
HDL-C (mmol/L)
C.M. Before
2.84
6.15
1 .oo
25.6
0.57
1.25
4.18
0.72
After
2.91
4.87
0.96
23.4
0.54
1.33
2.97
0.57
Before
1.77
5.39
0.70
19.2
0.46
0.81
3.44
1.14
After
1.46
5.66
0.72
19.8
0.45
0.67
4.20
0.79
C.N.
B.F. Before
1.48
6.58
0.70
24.3
0.49
0.68
4.99
0.91
After
1.66
5.64
0.68
22.0
0.49
0.76
4.25
0.63
Abbreviation: *Values
TG, triglycerides.
obtained
after a 2.4-kg
weight
loss on diet. Basal levels on a mitigated
lz51appearing in the medium. Lipoproteins (250 ug protein/ml) were oxidized by exposure to 10 umol/L CU*+ at 37°C for 1 to 5 hours (using CuClr).’ The extent of lipid peroxidation was expressed in terms of thiobarbituric acid-reactive substances (TBARS) released in the medium, and was expressed in malondialdehyde (MDA) equivalents. The concentration of probucol in LDL and Lp(a) was determined by Wisconsin Analytical Research Services (Madison, WI) by high-performance liquid chromatography.r4 Plasma cholesterol, triglyceride, LDL, and high-density lipoprotein cholesterol levels were determined according to the Lipid Research Clinics Protocol. Very-low-density lipoprotein cholesterol was measured directly in the 1.006 g/mL fraction isolated by ultracentrifugation. Plasma Lp(a) concentration was measured using a selective bi-site enzyme-linked immunosorbent assay.15 cY-Tocopherol and S-carotene levels were determined in plasma and in lipoprotein fractions by high-performance liquid chromatography according to the method of Miller et a1.16
As shown in Table 1, probucol had no effect on plasma Lp(a) levels. In two patients, total plasma cholesterol
60
120
180
TIME (min)
240
300
0
cholesterol
and 4.92 mmol/L
for LDL-C.
concentrations decreased by 21% and 14% and LDL cholesterol levels decreased by 29% and lS%, respectively. One patient (C.N.) lost 2.4 kg on diet alone and showed an increase in plasma total cholesterol and LDL cholesterol levels after 4 weeks on probucol when his weight had stabilized; even so, his plasma LDL cholesterol level was 14.6% lower than at baseline. In all patients, HDL cholesterol concentrations decreased by 21% to 31%. The concentration of lipid-soluble antioxidants, vitamin E and p-carotene, did not change in plasma (Table 1) or in lipoproteins after probucol treatment (data not shown). Probucol was found to be distributed in both Lp(a) and LDL, but, relative to cholesterol (in molar ratio), the probucol content of Lp(a) (4.9 5 0.72 x 10-3) was 48% lower than that of LDL (9.4 * 1.3 x 10-3). The time-course of the formation of TBARS during oxidation of Lp(a) and LDL isolated from the plasma of each individual before and after probucol treatment is shown in Fig 1. Both lipoproteins in equal protein concen-
RESULTS
0
diet were 6.83 mmol/Lfor
60
120
180
240
TIME (min)
300
Fig 1. Time-dependentformetion of TBARS during oxidation of Lp(a) and LDL isolated from individual plasma of patients before [opened symbols) and after probucol treatment (closed symbols). (A) Lp(e); (B) LDL. Patients: CM. (a); C.N. (0); B.F. (0). Lipoproteins (250 pg protein) were oxidized by exposure to 10 pmol/L CuCI, at 37°C.
PROBUCOL PROTECTS Lp(a) FROM OXIDATIVE
oxidized ment.
after
4 weeks’
treat-
2
I
3
trations were subjected to oxidation in the presence of 10 kmol/L Cu*+, and TBARS levels were measured every 60 minutes, from 0 to 300 minutes. This study shows that after probucol treatment, the oxidation of Lp(a) and LDL was markedly inhibited. At all time points, a progressive decrease in TBARS generation was observed, and after 300 minutes of oxidation it was decreased 75% (range, 69% to 76%) for Lp(a) and 86% (range, 82% to 88%) for LDL after treatment. Before treatment, both oxidized lipoproteins showed an increased negative charge, causing a faster mobility on agarose gel electrophoresis; this effect disappeared when probucol was present in both fractions after probucol treatment (Fig 2). Since Lp(a) and LDL are markedly protected against oxidative modification by probucol, it was postulated that their uptake and degradation by macrophages in vitro could also be inhibited. This was confirmed by the experiment illustrated in Fig 3. After probucol administration, the degradation of oxidized Lp(a) by mouse peritoneal macrophages decreased by 63% and that of
I
II
m
E
1227
MODIFICATION
P
PT
Fig 3. Effect of 4 weeks’ probucol treatment on rates of degradation of 1*51-labeied native and oxidized lipoproteins by mouse peritoneal macrophages. (I) Native Lp(a); (II) oxidized Lp(aJ; (III) oxidized Lpla) after probucol; (IV) native LDL; (V) oxidized LDL; and (VI) oxidized LDL after pfobucol. Each bar represents the mean f SD calculated from dupliaates of three independent experiments (one for each patient). (a) Rates of degradation of lipoproteins filtrated by Millipore 0.22-Cm filter (without aggregates).
4
I
2
3
4
oxidized LDL decreased by 80%, as compared with degradation of these respective oxidized fractions before treatment. It must be noted that after oxidation Lp(a) contained a significant amount of aggregates (27% to 31% of radioactivity pelleted after centrifugation at 10,000 x g); when oxidized, LDL contained significantly fewer aggregates (7% to 11% pelleted). After probucol treatment, the ability of oxidized Lp(a) to aggregate was markedly lower (12% to 15% pelleted). Since we have shown previously that these aggregates are taken up by phagocytosis,h we also performed experiments using lipoproteins without aggregates (filtrated by 0.22~pm membrane filter [Millipore]). As shown in Fig 3, the differences between rates of degradation of oxidized Lp(a) with and without aggregates (white area inside bars) were decreased after probucol administration, which indicated reduced uptake of Lp( a) particles by phagocytosis. DISCUSSION
This study was made possible by the selection of hypercholesterolemic patients with very high levels of Lp(a) in whom administration of probucol was indicated: this happened to be a relatively rare occurrence. After 4 weeks of probucol treatment, its lack of effect on Lp(a) levels was observed. This confirms previously reported results.t7,r8 However, Lp(a) of patients treated with probucol resisted oxidation by copper in vitro to an extent similar to that observed for LDL isolated from the same plasma sample; this was related to the fact that the drug was present in both lipoproteins. Since Lp(a) consists of an LDL-like particle, disulphide bonded to ape(a), it might be expected that the concentrations of probucol in Lp(a) and LDL would be similar. However, Lp(a) contains less probucol relative to cholesterol than LDL. In fact, we found that this difference is in good agreement with our previous observation that lipid-soluble antioxidant levels are much lower in Lp(a) than in LDL.6 According to Greener and Kostner,‘” choles-
1228
NARUSZEWICZ
teryl ester transfer between HDL and Lp(a) occurs at approximately half the rate of exchange of cholesteryl ester between HDL and LDL. This slower lipid-exchange process might account for the limited transfer of lipophilic p-carotene, vitamin E, and probucol to Lp(a) as compared with LDL. It is of interest to determine why Lp(a) containing fewer antioxidants and probucol than LDL had a similar degree of resistance to oxidation. As shown recently by Sattler et a1,8 this could be due to a high concentration of N-acetyloneuraminic acid in ape(a), which in part protects Lp(a) particles against oxidation (perhaps by blocking free amino groups). Our findings and the presence of modified Lp(a) in human atheromatous 1esions20 lend support to the atherogenie potential of oxidized Lp(a), but further studies are needed to assess the relative importance of oxidized Lp(a) in atherogenesis. A recent study using selective coronary angiography indicates that Lp(a) may be predictive of the
ET AL
severity of coronary atherosclerosis.*l In familial hypercholesterolemia, in which the turnover of LDL is markedly reduced, Seed et a12*found that an elevated plasma level of Lp(a) is a strong independent risk factor for coronary artery disease. One may speculate that oxidized Lp(a) accounts for this effect, since native Lp(a) is not readily taken up and degraded by macrophage@ and is unlikely to contribute to foam-cell formation. However, it is possible that these stagnant lipoproteins with a slow turnover rate are more likely to be oxidized in the arterial wall. Although many drugs are effective at decreasing LDL concentrations, so far only nicotinic acid, 23stanozo101,Z4and apheresis25 appear to be of any value in reducing plasma levels of Lp(a). This report demonstrates that, although it has no effect on plasma Lp(a) levels, probucol protects both LDL and Lp(a) against both oxidative modification and further enhanced uptake by macrophages.
REFERENCES
1. Scanu AM, Fless GM: Lipoprotein(a)-Heterogeneity
and biological relevance. J Clin Invest 85:1709-1715,199O 2. Scanu AM: Lipoprotein (a), a potential bridge between the fields of atherosclerosis and thrombosis. Arch Patho! Lab Med 112:1045-1047,1988 3. Simon DJ, Schoen FJ, Fless GM, et al: Lipoprotein(a) is uniformly detectable in coronary atherectomy specimens obtained from primary and restenotic lesions after PTCA. Arteriosclerosis 10:812a, 1990 (abstr) 4. Rath M, Niendorf A, Reblin T, et al: Detection and quantitation of lipoprotein(a) in the arterial wall of 107 coronary bypass patients. Arteriosclerosis 9:579-692,1989 5. Steinberg D, Parthasarathy S, Carew TE, et al: Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med 320:915-924, 1989 6. Naruszewicz M, Selinger E, Davignon J: Oxidative modification of Lp(a) and the effect of B-carotene. Metabolism 41:1-9,1992 7. Jurgens G, Ashy A, Esterbauer H: Detection of new epitopes formed upon oxidation of low-density lipoprotein, lipoprotein (a) and very-low density lipoprotein. Use of an antiserum against 4-hydroxynonenal-modified low-density lipoproteins. Biochem J 265:605-608,199O 8. Sattler W, Kostner GM, Waeg G, et al: Oxidation of lipoprotein Lp(a). A comparison with low-density lipoproteins. Biochim Biophys Acta 1081:65-74, 1991 9. Haberland ME, Fless G, Scanu AM, et al: Modification of Lp(a) by malondialdehyde leads to avid uptake by human monocytemacrophages. Arteriosclerosis 9:700a, 1989 (abstr) 10. Parthasarathy S, Young SG, Witztum JL, et al: Probucol inhibits oxidative modification of low density lipoprotein. J Clin Invest 77:641-644,1986 11. Kita T, Nagano Y, Yokode M, et al: Probucol prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia. Proc Nat! Acad Sci USA 84:.5928-5931,1987 12. Carew TE, Schwenke DC, Steinberg DC: Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: Evidence that antioxidants in vivo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit. Proc Nat! Acad Sci USA 84:77257729,1987 13. Armstrong VW, Walli AK, Seidel D: Isolation, characterization, and uptake in human fibroblasts of an ape(a)-free lipoprotein
obtained on reduction of lipoprotein(a). J Lipid Res 26:1314-1323, 1985 14. Satonin DK, Coutant JE: Comparison of gas chromatography and high performance liquid chromatography for the analysis of probucol in plasma. J Chromatogr 380:401-406, 1986 15. Vu Dac N, Mezdour H, Parra HJ, et al: A selective bi-site immunoenzymatic procedure for human Lp(a) lipoprotein quantification using monoclonal antibodies against ape(a) and apoB. J Lipid Res 30:1437-1443,199O 16. Miller KW, Lorr NA, Yang CS: Simultaneous determination of plasma retinol, o-tocopherol, lycopene. o-carotene and B-carotene by high performance liquid chromatography. Anal Biochem 138:340-345,1984 17. Maeda S, Okuno M, Abe A, et al: Lack of effect of probucol on serum lipoprotein(a) levels. Atherosclerosis 79:267-269, 1989 18. Noma A, Maeda S, Okuno M, et al: Reduction of serum lipoprotein(a) levels in hyperlipidaemic patients with a-tocopheryl nicotinate. Atherosclerosis 84:213-217, 1990 19. Groener JEM. Kostner GM: Lipid transfer proteincatalyzed exchange of cholesteryl ester between high density lipoproteins and apoB-containing lipoproteins. J Lipid Res 28:10531056,1987 20. O’Neil JA. Pepin JM, Smejkal G, et al: Structural characteristics of Lp(a) extracted from human atherosclerotic plaques. Arteriosclerosis 10:812a, 1990 (abstr) 21. Yamazaki T, Katoh K, Nakanishi S, et al: Lp(a) predicts the severity of coronary artery sclerosis. Arteriosclerosis 10:820a, 1990 (abstr) 22. Seed M, Hoppichler F, Reavely D, et al: Relation of serum lipoprotein(a) concentration and apolipoprotein(a) phenotype to coronary heart disease in patients with familial hypercholesterolemia. N Engl J Med 322:1494-1499, 1990 23. Carlson LA, Hamsten A, Asplund A: Pronounced lowering of serum levels of lipoprotein Lp(a) in hyperlipidaemic subjects treated with nicotinic acid. J Intern Med 226:271-276, 1989 24. Albers JJ, Taggart HM, Applebaum-Bowden D, et al: Reduction of lecithin-cholesterol acyl transferase, apolipoprotein D and the Lp(a) lipoprotein with the anabolic steroid stanozolol. Biochim Biophys Acta 795:293-296, 1984 25. Armstrong VW, Schleef J, Thiery J, et al: Effect of HELPLDL-apheresis on serum concentrations of human lipoprotein(a): Kinetic analysis of the post-treatment return to baseline. Eur J Clin Invest 19:235-240,1989
Lipoprotein(a)
Naruszewicz,
Elizabeth
Selinger,
Against Oxidative Robert
Dufour,
Modification
and Jean Davignon
Probucol, which decreases cholesterol levels and has antioxidant properties, was administered orally to patients with familial combined hyperlipidemia and high plasma lipoprotein(a) [Lp(a)] levels. The drug had no effect on Lp(a) concentrations after 4 weeks, but was found to be distributed in both Lp(a) and low-density lipoprotein (LDL). Before treatment, in each case LDL and Lp(a) isolated from the same individual were readily oxidized by copper, resulting in increased electrophoretic mobility and enhanced uptake and degradation by macrophages of both lipoproteins. After probucol treatment, both lipoproteins acquired resistance to in vitro oxidation by copper. Furthermore, probucol prevented their enhanced uptake and degradation by the macrophages. It is surmised that oxidized Lp(a) may carry an atherogenic potential that could be opposed by probucol administration. Copyright D 1992 by W.E. Saunders Company
S
EVERAL CASE-CONTROL studies indicate that increased plasma concentrations of lipoprotein(a) [Lp(a)] are associated with an increased risk for coronary artery disease.’ However, it is as yet unknown whether this adverse cardiovascular association is related to an effect of Lp(a) on atherogenesis (ie, formation of foam cells). thrombogenesis (ie, inhibition of fibrinolysis), or both.? The recent observation that Lp(a) is present in human atheromatous plaques’ indicates that it is involved in atherogenesis. Since Lp(a) is detected in the arterial wall in concentrations that correlate with plasma Lp(a) levels,4 it is likely that it could be oxidatively modified locally by endothelial cells, smooth muscle cells, and macrophages, as is :he case for low-density lipoprotein (LDL) accessing the subendothelial space.5 In fact, we have shown that Lp(a) could be oxidatively modified when incubated in vitro with human mononuclear cells having CU’+.~ This modification caused marked changes in the structure and biological properties of Lp(a). Relative to native Lp(a), oxidized particles showed increased negative charge, protein fragmentation, and an increased ability to aggregate. They were taken up and degraded readily by macrophages via scavenger receptors and in part by phagocytosis, inducing cholesterol ester accumulation. Our findings regarding oxidation of Lp(a) are consistent with those of Jiirgens et al7 and Sattler et al,x and are in line with the observations of Haberland et al for Lp(a) modified by malondialdehyde.” It is well known that probucol, a widely used drug in the management of hypercholesterolemia, also has antioxidant properties. When administered orally, it has the ability to enter LDL and protect these particles against oxidative modification.‘” It is likely that by this mechanism probucol inhibits atheroma formation in the hypercholesterolemic, LDL receptor-deficient, Watanabe Heritable Hyperlipidemic rabbit, independent of its cholesterol-lowering effect.“.” The present study was undertaken to determine whether probucol administration can protect Lp(a) particles against oxidation. This report documents for the first time that administration of probucol to patients with very high levels of plasma Lp(a) results in the presence of this drug within Lp(a) particles. This protects Lp(a) particles against both oxidatibe modification in vitro and subsequent uptake by macrophages. Metabolism,
Vol 41, No 11 (November),
1992:
pp 1225.1228
SUBJECTS AND METHODS
Subjects Probucoi was administered (500 mg twice a day) to three outpatients (two women and one man) with familial combined hyperlipidemia, aged 52. 60, and 38 years, respectively. These patients were selected for their high levels of Lp(a) (0.7 to 1 g/L) and LDL cholesterol so that oxidative modifications of both lipoprotein fractions could be performed in the same individual. They were maintained on an American Heart Association phase-II diet before and during 1 month of probucol treatment. The patients received no supplemental vitamin E or p-carotene in tablet form. One patient (B.F.) had been treated 4.5 months with a bile acid sequestrant (cholestyramine, 4 g twice a day), which was maintained during the treatment with probucol. All patients were informed of the purpose of the study. which was approved by our institutional ethics review board. This study was designed without a wash-out period after probucol treatment, because of the long terminal-phase elimination half-life of this drug (47 days).
Methods After an overnight fast, blood samples were taken by venipuncture into tubes containing disodium EDTA (1.5 mg/mL), and the plasma was separated in a cold centrifuge. LDL (1.019 to 1.055 g/mL) and Lp(a) (1.055 to 1.090 g/mL) fractions were isolated by sequential ultracentrifugation from each individual plasma sample before and after probucol treatment. Lp(a) was further purified by column chromatography over Bio-Gel A-5m (Bio-Rad Laboratories, Richmond, CA) and heparin-sepharose13 to exclude contamination of the Lp(a) fraction with LDL. Lipoproteins were labeled with carrier-free Na12s1 and the water-insoluble iodination agent, Iodo-gen (Pierce. Rockford, IL). Specific activity ranged from 60 to 110 cpm/ng protein. Mouse peritoneal macrophages were harvested from unstimulated Swiss Webster mice. The degradation of labeled lipoproteins was measured by determining the trichloroacetic acid-soluble, iodide-free
From the Hyperlipidemia and Atherosclerosis Research Group, Clinical Research Insritute of Montreal, Afiliated with the University of Montreal and the Hotel-Dieu Hospital of Montreal, Canada. Supported in part by grants from the Medical Research Council of Canada ICIBA-Geigy Universityllndustyprogram (VI 0029), Succession J.A. De&e, and Marion Merrell Dow Research Institute. Address reprint requests IO Marek Naruszewicz, PhD. Visiting Professor, Hyperlipidemia and Atherosclerosis Research Group, Clinical Research Institute of Montreal. 110 Pine Ave. W, hfontreal. Quebec, Canada H2WIR7. Copyright Q 1992 by W B. Saunders Compare> 0026049519214111-0013$03.00/O 1225
1226
NARUSZEWICZ
Table 1. Effect of 4
Weeks’ Probucol
Treatment
ET AL
on Plasma Lipid, Lipoprotein, Vitamin E, and p-Carotene Levels Lipoprotein Fractions
TG (mmollL)
Patients
Cholesterol (mmol/L)
Vitamin E (pmol/L)
LPb)
(s/L)
p-carotene (pmol/L)
VLDL-C (lnnlol/L)
LDL-C (mmol/L)
HDL-C (mmol/L)
C.M. Before
2.84
6.15
1 .oo
25.6
0.57
1.25
4.18
0.72
After
2.91
4.87
0.96
23.4
0.54
1.33
2.97
0.57
Before
1.77
5.39
0.70
19.2
0.46
0.81
3.44
1.14
After
1.46
5.66
0.72
19.8
0.45
0.67
4.20
0.79
C.N.
B.F. Before
1.48
6.58
0.70
24.3
0.49
0.68
4.99
0.91
After
1.66
5.64
0.68
22.0
0.49
0.76
4.25
0.63
Abbreviation: *Values
TG, triglycerides.
obtained
after a 2.4-kg
weight
loss on diet. Basal levels on a mitigated
lz51appearing in the medium. Lipoproteins (250 ug protein/ml) were oxidized by exposure to 10 umol/L CU*+ at 37°C for 1 to 5 hours (using CuClr).’ The extent of lipid peroxidation was expressed in terms of thiobarbituric acid-reactive substances (TBARS) released in the medium, and was expressed in malondialdehyde (MDA) equivalents. The concentration of probucol in LDL and Lp(a) was determined by Wisconsin Analytical Research Services (Madison, WI) by high-performance liquid chromatography.r4 Plasma cholesterol, triglyceride, LDL, and high-density lipoprotein cholesterol levels were determined according to the Lipid Research Clinics Protocol. Very-low-density lipoprotein cholesterol was measured directly in the 1.006 g/mL fraction isolated by ultracentrifugation. Plasma Lp(a) concentration was measured using a selective bi-site enzyme-linked immunosorbent assay.15 cY-Tocopherol and S-carotene levels were determined in plasma and in lipoprotein fractions by high-performance liquid chromatography according to the method of Miller et a1.16
As shown in Table 1, probucol had no effect on plasma Lp(a) levels. In two patients, total plasma cholesterol
60
120
180
TIME (min)
240
300
0
cholesterol
and 4.92 mmol/L
for LDL-C.
concentrations decreased by 21% and 14% and LDL cholesterol levels decreased by 29% and lS%, respectively. One patient (C.N.) lost 2.4 kg on diet alone and showed an increase in plasma total cholesterol and LDL cholesterol levels after 4 weeks on probucol when his weight had stabilized; even so, his plasma LDL cholesterol level was 14.6% lower than at baseline. In all patients, HDL cholesterol concentrations decreased by 21% to 31%. The concentration of lipid-soluble antioxidants, vitamin E and p-carotene, did not change in plasma (Table 1) or in lipoproteins after probucol treatment (data not shown). Probucol was found to be distributed in both Lp(a) and LDL, but, relative to cholesterol (in molar ratio), the probucol content of Lp(a) (4.9 5 0.72 x 10-3) was 48% lower than that of LDL (9.4 * 1.3 x 10-3). The time-course of the formation of TBARS during oxidation of Lp(a) and LDL isolated from the plasma of each individual before and after probucol treatment is shown in Fig 1. Both lipoproteins in equal protein concen-
RESULTS
0
diet were 6.83 mmol/Lfor
60
120
180
240
TIME (min)
300
Fig 1. Time-dependentformetion of TBARS during oxidation of Lp(a) and LDL isolated from individual plasma of patients before [opened symbols) and after probucol treatment (closed symbols). (A) Lp(e); (B) LDL. Patients: CM. (a); C.N. (0); B.F. (0). Lipoproteins (250 pg protein) were oxidized by exposure to 10 pmol/L CuCI, at 37°C.
PROBUCOL PROTECTS Lp(a) FROM OXIDATIVE
oxidized ment.
after
4 weeks’
treat-
2
I
3
trations were subjected to oxidation in the presence of 10 kmol/L Cu*+, and TBARS levels were measured every 60 minutes, from 0 to 300 minutes. This study shows that after probucol treatment, the oxidation of Lp(a) and LDL was markedly inhibited. At all time points, a progressive decrease in TBARS generation was observed, and after 300 minutes of oxidation it was decreased 75% (range, 69% to 76%) for Lp(a) and 86% (range, 82% to 88%) for LDL after treatment. Before treatment, both oxidized lipoproteins showed an increased negative charge, causing a faster mobility on agarose gel electrophoresis; this effect disappeared when probucol was present in both fractions after probucol treatment (Fig 2). Since Lp(a) and LDL are markedly protected against oxidative modification by probucol, it was postulated that their uptake and degradation by macrophages in vitro could also be inhibited. This was confirmed by the experiment illustrated in Fig 3. After probucol administration, the degradation of oxidized Lp(a) by mouse peritoneal macrophages decreased by 63% and that of
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Fig 3. Effect of 4 weeks’ probucol treatment on rates of degradation of 1*51-labeied native and oxidized lipoproteins by mouse peritoneal macrophages. (I) Native Lp(a); (II) oxidized Lp(aJ; (III) oxidized Lpla) after probucol; (IV) native LDL; (V) oxidized LDL; and (VI) oxidized LDL after pfobucol. Each bar represents the mean f SD calculated from dupliaates of three independent experiments (one for each patient). (a) Rates of degradation of lipoproteins filtrated by Millipore 0.22-Cm filter (without aggregates).
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oxidized LDL decreased by 80%, as compared with degradation of these respective oxidized fractions before treatment. It must be noted that after oxidation Lp(a) contained a significant amount of aggregates (27% to 31% of radioactivity pelleted after centrifugation at 10,000 x g); when oxidized, LDL contained significantly fewer aggregates (7% to 11% pelleted). After probucol treatment, the ability of oxidized Lp(a) to aggregate was markedly lower (12% to 15% pelleted). Since we have shown previously that these aggregates are taken up by phagocytosis,h we also performed experiments using lipoproteins without aggregates (filtrated by 0.22~pm membrane filter [Millipore]). As shown in Fig 3, the differences between rates of degradation of oxidized Lp(a) with and without aggregates (white area inside bars) were decreased after probucol administration, which indicated reduced uptake of Lp( a) particles by phagocytosis. DISCUSSION
This study was made possible by the selection of hypercholesterolemic patients with very high levels of Lp(a) in whom administration of probucol was indicated: this happened to be a relatively rare occurrence. After 4 weeks of probucol treatment, its lack of effect on Lp(a) levels was observed. This confirms previously reported results.t7,r8 However, Lp(a) of patients treated with probucol resisted oxidation by copper in vitro to an extent similar to that observed for LDL isolated from the same plasma sample; this was related to the fact that the drug was present in both lipoproteins. Since Lp(a) consists of an LDL-like particle, disulphide bonded to ape(a), it might be expected that the concentrations of probucol in Lp(a) and LDL would be similar. However, Lp(a) contains less probucol relative to cholesterol than LDL. In fact, we found that this difference is in good agreement with our previous observation that lipid-soluble antioxidant levels are much lower in Lp(a) than in LDL.6 According to Greener and Kostner,‘” choles-
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teryl ester transfer between HDL and Lp(a) occurs at approximately half the rate of exchange of cholesteryl ester between HDL and LDL. This slower lipid-exchange process might account for the limited transfer of lipophilic p-carotene, vitamin E, and probucol to Lp(a) as compared with LDL. It is of interest to determine why Lp(a) containing fewer antioxidants and probucol than LDL had a similar degree of resistance to oxidation. As shown recently by Sattler et a1,8 this could be due to a high concentration of N-acetyloneuraminic acid in ape(a), which in part protects Lp(a) particles against oxidation (perhaps by blocking free amino groups). Our findings and the presence of modified Lp(a) in human atheromatous 1esions20 lend support to the atherogenie potential of oxidized Lp(a), but further studies are needed to assess the relative importance of oxidized Lp(a) in atherogenesis. A recent study using selective coronary angiography indicates that Lp(a) may be predictive of the
ET AL
severity of coronary atherosclerosis.*l In familial hypercholesterolemia, in which the turnover of LDL is markedly reduced, Seed et a12*found that an elevated plasma level of Lp(a) is a strong independent risk factor for coronary artery disease. One may speculate that oxidized Lp(a) accounts for this effect, since native Lp(a) is not readily taken up and degraded by macrophage@ and is unlikely to contribute to foam-cell formation. However, it is possible that these stagnant lipoproteins with a slow turnover rate are more likely to be oxidized in the arterial wall. Although many drugs are effective at decreasing LDL concentrations, so far only nicotinic acid, 23stanozo101,Z4and apheresis25 appear to be of any value in reducing plasma levels of Lp(a). This report demonstrates that, although it has no effect on plasma Lp(a) levels, probucol protects both LDL and Lp(a) against both oxidative modification and further enhanced uptake by macrophages.
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and biological relevance. J Clin Invest 85:1709-1715,199O 2. Scanu AM: Lipoprotein (a), a potential bridge between the fields of atherosclerosis and thrombosis. Arch Patho! Lab Med 112:1045-1047,1988 3. Simon DJ, Schoen FJ, Fless GM, et al: Lipoprotein(a) is uniformly detectable in coronary atherectomy specimens obtained from primary and restenotic lesions after PTCA. Arteriosclerosis 10:812a, 1990 (abstr) 4. Rath M, Niendorf A, Reblin T, et al: Detection and quantitation of lipoprotein(a) in the arterial wall of 107 coronary bypass patients. Arteriosclerosis 9:579-692,1989 5. Steinberg D, Parthasarathy S, Carew TE, et al: Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med 320:915-924, 1989 6. Naruszewicz M, Selinger E, Davignon J: Oxidative modification of Lp(a) and the effect of B-carotene. Metabolism 41:1-9,1992 7. Jurgens G, Ashy A, Esterbauer H: Detection of new epitopes formed upon oxidation of low-density lipoprotein, lipoprotein (a) and very-low density lipoprotein. Use of an antiserum against 4-hydroxynonenal-modified low-density lipoproteins. Biochem J 265:605-608,199O 8. Sattler W, Kostner GM, Waeg G, et al: Oxidation of lipoprotein Lp(a). A comparison with low-density lipoproteins. Biochim Biophys Acta 1081:65-74, 1991 9. Haberland ME, Fless G, Scanu AM, et al: Modification of Lp(a) by malondialdehyde leads to avid uptake by human monocytemacrophages. Arteriosclerosis 9:700a, 1989 (abstr) 10. Parthasarathy S, Young SG, Witztum JL, et al: Probucol inhibits oxidative modification of low density lipoprotein. J Clin Invest 77:641-644,1986 11. Kita T, Nagano Y, Yokode M, et al: Probucol prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia. Proc Nat! Acad Sci USA 84:.5928-5931,1987 12. Carew TE, Schwenke DC, Steinberg DC: Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: Evidence that antioxidants in vivo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit. Proc Nat! Acad Sci USA 84:77257729,1987 13. Armstrong VW, Walli AK, Seidel D: Isolation, characterization, and uptake in human fibroblasts of an ape(a)-free lipoprotein
obtained on reduction of lipoprotein(a). J Lipid Res 26:1314-1323, 1985 14. Satonin DK, Coutant JE: Comparison of gas chromatography and high performance liquid chromatography for the analysis of probucol in plasma. J Chromatogr 380:401-406, 1986 15. Vu Dac N, Mezdour H, Parra HJ, et al: A selective bi-site immunoenzymatic procedure for human Lp(a) lipoprotein quantification using monoclonal antibodies against ape(a) and apoB. J Lipid Res 30:1437-1443,199O 16. Miller KW, Lorr NA, Yang CS: Simultaneous determination of plasma retinol, o-tocopherol, lycopene. o-carotene and B-carotene by high performance liquid chromatography. Anal Biochem 138:340-345,1984 17. Maeda S, Okuno M, Abe A, et al: Lack of effect of probucol on serum lipoprotein(a) levels. Atherosclerosis 79:267-269, 1989 18. Noma A, Maeda S, Okuno M, et al: Reduction of serum lipoprotein(a) levels in hyperlipidaemic patients with a-tocopheryl nicotinate. Atherosclerosis 84:213-217, 1990 19. Groener JEM. Kostner GM: Lipid transfer proteincatalyzed exchange of cholesteryl ester between high density lipoproteins and apoB-containing lipoproteins. J Lipid Res 28:10531056,1987 20. O’Neil JA. Pepin JM, Smejkal G, et al: Structural characteristics of Lp(a) extracted from human atherosclerotic plaques. Arteriosclerosis 10:812a, 1990 (abstr) 21. Yamazaki T, Katoh K, Nakanishi S, et al: Lp(a) predicts the severity of coronary artery sclerosis. Arteriosclerosis 10:820a, 1990 (abstr) 22. Seed M, Hoppichler F, Reavely D, et al: Relation of serum lipoprotein(a) concentration and apolipoprotein(a) phenotype to coronary heart disease in patients with familial hypercholesterolemia. N Engl J Med 322:1494-1499, 1990 23. Carlson LA, Hamsten A, Asplund A: Pronounced lowering of serum levels of lipoprotein Lp(a) in hyperlipidaemic subjects treated with nicotinic acid. J Intern Med 226:271-276, 1989 24. Albers JJ, Taggart HM, Applebaum-Bowden D, et al: Reduction of lecithin-cholesterol acyl transferase, apolipoprotein D and the Lp(a) lipoprotein with the anabolic steroid stanozolol. Biochim Biophys Acta 795:293-296, 1984 25. Armstrong VW, Schleef J, Thiery J, et al: Effect of HELPLDL-apheresis on serum concentrations of human lipoprotein(a): Kinetic analysis of the post-treatment return to baseline. Eur J Clin Invest 19:235-240,1989
