The effects of probucol on the levels, structure, composition and metabolism of plasma lipoprotein in rats

79

Biochimica et Biophysicu Actu, 1167 (1993) 79-84 0 1993

Elsevier Science Publishers B.V. All rights reserved 0005-2760/93/$06.00

BBALIP 54121

The effects of probucol on the levels, structure, composition and metabolism of plasma lipoprotein in rats Shlomo Eisenberg Lipid Research Laboratory, Department of Medicine, Hadassah University Hospital, Jerusalem (Israel) (Received

Key words:

Atherosclerosis;

Cholesteryl

ester; Probucol;

14 August

1992)

Apolipoprotein

A-I; Cholesterol-LDL;

Cholesterol-HDL-2

The effects of probucol administration on the levels, structure, composition and metabolism of plasma lipoproteins were determined in male rats. Probucol caused a 25% decrease of plasma cholesterol and a 20% decrease of plasma triacylglycerols. The effect was more pronounced on lipoproteins of density less than 1.063 g/ml (-35%) than on density greater than 1.063 g/ml ( - 21%). The density of LDL, HDL-1 and HDL-2 separated by ultracentrifugation on a zonal rotor was shifted towards a higher density. The content of cholesteryl esters increased in VLDL and LDL and decreased in HDL-1 and HDL-2. LDL, HDL-1 and HDL-2 were relatively enriched with proteins. The intravascular metabolism of ‘251-labeled apo A-I was identical in control and probucol-treated rats with a circulating half-lifetime of 9.5 h. The circulating half-lifetime of LDL labeled biosynthetically with [3H]cholesteryl esters was also identical, 6.5 h. The circulating half-lifetime of [3H]cholesteryl esters in HDL-2, in contrast, was 9.5 h in control rats and 7.5 h in probucol-treated animals. Plasma cholesterol ester transfer activity was almost undectectable in either control or probucol treated animals. The investigation demonstrates a pronounced effect of probucol on plasma lipid and lipoprotein levels in rats, on cholesterol ester distribution between lipoproteins and on lipoprotein transport rates in the plasma. These effects may contribute to the anti-atherogenetic action of probucol.

Introduction Probucol, a potent antioxident, has been used for treatment of hypercholesterolemia syndromes in humans for over a decade [l]. The drug has only minimal effect on LDL but effectively reduces HDL cholesterol

levels [2-41. In spite of this theoretical disadvantage, administration of probucol causes a remarkable decrease of xanthoma size in human subjects with familial hypercholesterolemia, especially homozygotes [5], and was shown to reduce and prevent atherosclerosis in the Watanabe heritable hyperlipidemic (WHHL) rabbit and cholesterol fed rabbits [6,71. These anti-atherosclerosis effects were ascribed to the antioxidant action of probucol [S-lo]. Alternatively, it has been suggested that probucol increases the transport of cholesterol from peripheral tissues, including the arterial wall to the liver, a process designated ‘reverse cholesterol transport’. Indeed, administration of probucol to human subjects is associated with an increase of plasma cholesterol ester transfer activity [4,111, a reaction me-

Correspondence to: S. Eisenberg, partment of Medicine, Hadassah Israel.

Lipid Research Laboratory, DeUniversity Hospital, Jerusalem,

diated by a specific plasma cholesterol ester transfer protein (CETP) [ 121. The metabolic mechanisms responsible for the effects of probucol on either the plasma lipoprotein system or the arterial wall are poorly understood. In mice, probucol administration is associated with a dramatic reduction of the HDL levels that is due, predominantly, to a decreased synthesis of apo A-I [13]. In rats, in contrast, it has been reported that probucol does not affect plasma cholesterol levels but decreases plasma triacylglycerol levels and hepatic low density lipoprotein (LDLl-receptor mRNA concentration [14]. A detailed investigtion on the effects of probucol on plasma lipoproteins in rats, however, has not been reported. To this end, we studied the effects of probuco1 on the structure, composition and metabolism of all major plasma lipoproteins in rats. Materials

and Methods

Animals

Male rats (100-140 g, 4-6 weeks old) of the Hebrew University strain on ad libitum rat chow diet [15] were used. After 1 week on pelleted chow, the diet was changed to ground powder chow with or without (control) probucol, 1% and 2% (w/w). The rats were used

for metabolic experiments 10-14 days after initiation of the control or probucol-containing diet. Rates of growth and consumption of food were identical in the different groups of animals. Preparation of lipoproteins, apoproteins, and [‘Hlcholesteryl ester labeled lipoproteins Blood plasma was obtained from the abdominal aorta under light ether anesthesia. Very low density lipoprotein WLDL) (d < 1.006 g/ml> and lipoproteins of density 1.006-1.085 g/ml and of density greater than 1.085 g/ml were separated by ultracentrifugation in appropriate salt densities using a Beckman 50-Ti rotor (Spinco Div., Palo Alto, CA) at 45000 rpm in an L5-50 ultracentrifuge. LDL and high density lipoprotein (HDL)-1 were prepared by zonal ultracentrifugation from the plasma lipoprotein fraction of density 1.006-1.085 g/ml on a linear NaBr (1.0-l .3 g/ml) gradient after 170 min centrifugation at 42000 rpm in a Ti-14 zonal rotor as previously described [16]. HDL-2 was prepared from the plasma fraction of density greater than 1.085 g/ml after a 22 h centrifugation at 41000 rpm in a Ti-14 zonal rotor and a 1.0-1.4 g/ml non-linear gradient [ 161. Lipoprotein fractions were identified, pooled, dialyzed against a 0.9% NaCI, 20 mM Tris (pH 7.4), 0.001% EDTA buffer, and concentrated to a volume of 2-4 ml by reverse dialysis. Rat plasma apolipoprotein A-I (apo A-I) was prepared by gel-filtration on Sephadex G-150 from total delipidated HDL apoproteins as previously described [17]. For the apo A-I, the HDL was preparation of ‘*“I-labeled radioiodinated prior to the gel filtration by the ICI technique [18] as modified for the labeling of lipoproteins [19]. The apo A-I and 12’I-labeled apo A-I were more than 98% pure. Rat plasma lipoproteins labeled biosynthetically with [ “Hlcholesteryl esters were prepared by zonal ultracentrifugation as above, from the plasma of control or probucol treated (2%, w/w) rats injected intravenously with [“HIcholesterol 6 h prior to exsanguination [20]. The [ “HIcholesterol labeled lipoproteins (more than 70% of the radioactivity in cholesteryl esters) were used within 3 days of preparation. In rlico tumoL$er of ‘251-labeled apo A-I and [“Hlcholesteryl ester labeled LDL and HDL-2 The plasma turnover of [‘251]apo A-I was determined in groups of control or probucol (2%, w/w) treated rats (approx. 200 g body weight) at the end of 14 days diet. Rats were injected during morning hours with 0.2 ml of [‘*‘I]apo A-I in 0.9% NaCl, 1.5% (w/v> bovine serum albumin solution through an exposed saphenous vein. Each rat received 0.1 mg of [1251]apo A-I (spec. act. 104. lo6 dpm/mg) while under light ether anesthesia. Two blood samples were obtained from the rats: the first of 0.3-0.4 ml through puncture

of the heart and the second during exsanguination through the abdominal aorta. Plasma was separated promptly at 4°C. The first blood sample was used to determine plasma radioactivity only. The second sample (usually 4-6 ml blood) for determination of plasma radioactivity, plasma lipid levels and separation of lipoproteins of density less and more than 1.063 g/ml. Blood samples were obtained 2 min after the injection (designated ‘zero time’) and 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after the injection. The plasma turnover of [‘HIcholesterol-labeled LDL and HDL, was determined by a procedure similar to that described for [‘*“I]apo A-I with the following modifications. Control rats (approx. 240 g body weight) were injected with 0.25-0.35 ml of 0.9% NaCI, 1.5% albumin solution containing either about 110 000 dpm [ ‘H]cholesterollabeled control LDL (0.1-0.2 mg protein) or 500000 dpm [3H]cholestero1-labeled HDL-2 (0.3-0.4 mg protein) prepared in control rats. Probucol-treated rats were injected in parallel with [‘HIcholesterol-labeled LDL or HDL-2 prepared in probucol-treated animals, 140 000 dpm (0.1-0.2 mg protein) or 430 000 dpm (0.30.4 mg protein) respectively. Plasma and lipoprotein radioactivity in unesterified and esterified cholesterol was determined 2 min, 1 h, 4 h and 8 h after the injection of labeled LDL, and 2 min, 1 h, 6 h and 24 h after the injection of labeled HDL-2. Cholesterol ester transfer a&city Cholesterol ester transfer activity was determined by transfer of [“Hlcholesteryl esters from human plasma HDL-3 to human VLDL. Labeled human plasma HDL-3 was prepared by the LCAT reaction as previously described [21]. The [“Hlcholesteryl ester labeled HDL, (0.3 mg cholesteryl esters, 100000 dpm) in 20 mM Tris (pH 7.4) and 0.9% NaCl buffer was incubated at 37°C in the absence or presence of 2.0 ml control or probucol-treated rat plasma fraction of density greater than 1.21 g/ml. Human plasma fraction of density greater than 1.21 g/ml (1 ml) served as a positive (active) control. Incubations were carried out for 4 h and the VLDL was isolated by ultracentrifugation at density of 1.019 g/ml. The amount of [‘Hlcholesteryl esters separated with the VLDL was determined by thin-layer chromatography of the VLDL lipids [20]. A mixture incubated without rat or human plasma and mixtures containing rat plasma fractions incubated on ice served to determine non-specific association of [“Hlcholesteryl esters with the VLDL. The amount of [“Hlcholesteryl esters in VLDL at the end of these incubations was less than 1% of the amount of [“Hlcholesteryl esters introduced with the HDL,. Analytical procedures Plasma cholesterol and triacylglycerol determined on a batch analyzer (Vitalab,

levels were The Nether-

81 TABLE I Effect

of probucol on plasma lipids and lipoprotein cholesterol levels

Data for plasma lipids are mean f S.D. of the number of experiments shown in parentheses. 4-6 experiments using pooled plasma samples.

Lipoprotein-cholesterol

Plasma lipid (mg/dl)

Control (n = 46) Probucol, 1% (n = 17) Probucol, 2% (n = 48)

Data for lipoprotein-cholesterol

are mean rt SD. of

(%)

cholesterol

triacylglycerol

d < 1.063 g/ml

d > 1.063 g/ml

57.5 + 7.8 42.925.6 42.3 + 6.6

147539 1261t35 114*35

42.5 + 6.5 _ 37.4 I 6.0

57.5 f 6.5

lands) as previously described [22]. Lipoprotein protein, triglyceride, free and esterified cholesterol and phospholipids were determined by standard procedures. Polyacrylamide gel electrophoresis in SDS was performed following the method of Weber and Osborne [23]. Labeled 3H-free and esterified cholesterol were separated by thin layer chromatography [20] and the amount of radioactivity in each lipid was determined in a liquid scintillation spectrometer (Packard, USA). Results

The effects of probucol on plasma lipid and lipoprotein cholesterol levels are shown in Table I. Probucol administration was associated with a 26% reduction of total plasma cholesterol and E-23% reduction of total plasma triacylglycerol levels. The effects appeared to be more pronounced on lipoproteins of density less than 1.063 g/ml (VLDL, LDL and HDL-1) than on higher density lipoproteins (HDL-1 and HDL-2). Similar effects were observed when 1% or 2% (w/w)

62.6 + 6.0

probucol were added to the diet. With lower concentrations of probucol (< OS%), no or only minimal effects were found. No effects were found on the body weight or the rates of growth of the animals (data not shown). The data shown in Table I were obtained after 10 days of probucol administration. Similar results were found after 7 days or 14 days of probucol. Lipoproteins of density of 1.006-1.085 g/ml and density greater than 1.085 g/ml were separated on a rate zonal centrifugation system. The zonal elution profile of lipoproteins from rats fed control or probucol-containing diet and the chemical composition of the lipoproteins are shown in Fig. 1 and Table II. LDL, HDL-1 and HDL-2 from probucol treated rats eluted at slightly higher volume in the zonal rotor (Fig. 11, implying a higher density of the lipoproteins. The chemical composition of VLDL remained unchanged after probucol treatment, except for a tendency to a shghtly higher cholesteryl ester content with 2% probucol. In LDL, the contribution of protein and cholesteryl esters to the total lipoprotein mass was increased, and that of tria~lglycerols, decreased. A similar effect was

TABLE If Effects of probucol treatment on the chemical composition of rat plasma lipoproteins

Data are mean + SD. of three different plasma pools, each containing samples from 3-6 rats. VLDL was separated at density less than 1.006 g/ml and LDL, HDL-1 and HDL-2 by centrifugation on a zonal rotor, as described in Materials and Methods. TG, triacylglycerol; FC, free cholesterol; CE, cholesteryl esters; PL, phospholipids. Lipoprotein

Diet

Composition (my/100 mg lipoprotein) TG

FC

VLDL

Control Probucoi, 1% Probucol, 2%

9.4kO.l 9.6*0.1 9.OkO.6

64.5 f 0.9 63.4 + 0.3 62.6k2.9

2.5rtO.l 2.1 f 0.2 1.9kO.3

1.6+0.1 1.6k0.2 3.0+1.1

22.1il.2 23.3 i 0.7 23.5 f 2.6

LDL

Control Probucol, 1% Probucol, 2%

28.4k4.1 30.8 f 1.7 33.0+2.1

21.3k3.3 16.3 f 1.9 14.5 * 1.3

5.8,0.8 5.OkO.7 4.3 + 0.6

23.2k5.0 26.1 k4.1 27.25 1.1

21.3 i 1.3 21.s+2.0 21.0 + 2.3

HDL-I

Control Probucol, 1% Probucol, 2%

34.5 + 4.1 40.0 + 2.2 43.1 f0.5

2.0 + 0.7 2.8k0.5 3.1 kO.6

6.3rl: 1.7 4.0 + 0.8 3.7* 1.2

24.3 k 5.0 22.0& 1.8 18.2 + 0.5

32.9 + 1.0 31.2*2.4 31.9 k 0.6

HDL-2

Control Probucol, 1% Probucol, 2%

42.7* 2.2 44.5 + 3.5 44.9+ 1.6

0.9 + 0.3 0.6kO.l OX+_0.4

1.75 1.1 1.5 f 1.1 1.4io.9

23.8 f 3.4 23.5 + 3.3 20.8+1.5

30.9 * 4.3 29.9 + 5.9 32.1+_3.1

Protein

CE

PL

82 0.03 t A Ll

0.02 ’ E 5;

0.01 ’

?a z;..oo, ::

_ 0

. 100

_

.

-

0.004 0

, 200

-

.

-

200

100

Zonai eMuent

. 300

-

.

-

300

. 400

. ’

400

volume (ml 1

Fig. 1. Effect of probucol administration (2%, w/w) on the zonal elution profile of rat plasma lipoproteins of density less than (A) and greater than (B) 1.085 g/ml. Thick lines represent lipoproteins from control rats and thin lines those from probucoi-treated rats.

found in HDL-1 and HDL-2 except for triacylglycerols that were very low at the end of either the control or probucol diet (Table 10. Probucol had no effect on the apo-protein profile of all rat plasma lipoproteins (data not shown). The decay from plasma of [1251]apo A-I in control and probucol-treated rats is shown in Fig. 2. The decay curves were virtually identical. In the different animals 89-97% of the radioactivity separated with lipoproteins of density greater than 1.063 g/ml, predominantly HDL-2. The circulating half-lifetime of [ 1251]apo A-I in either control or probucol treated animals, as calculated from the linear part of the decay curve, was

IO/. 0

8

6

4

2

10

TIME(h)

Fig. 3. The effects of probucol (2%, w/w) on. the decay of [“Hlcholesteryl ester labeled LDL from the plasma compartment. Data are mean i: S.D. of 3 or 4 determinations.

The plasma decay of [“Hlcholesteryl esters in biosynthetically labeled LDL and HDL-2 is shown in Figs. 3 and 4 for control and probucol-treated rats. In this study, animals were injected with labeled lipoproteins prepared from rats consuming the respective diets, i.e., control animals were injected with labeled lipoproteins prepared in control animals, and probucoi-treated animals, with lipoproteins prepared in rats fed the probucol-containing diet. During the first hour after the injection, [ 3H]cholesteryl esters in LDL disappeared slightly faster from the plasma of probucoltreated animals whereas HDL-2 disappeared slightly slower. Thereafter, however, the decay curves were 100

IO

1

4

8

12

16

20

24

Time

Fig. 2. ~*2SI]apolipoprotein A-I decay from the plasma of control and probucol-treated (2%, w/w) rats. Data are meanfS.D. of 4-6 determinations.

0

4

8

12

16

20

24

TIME(h)

Fig. 4. The effects of probucol (2%, w/w) on the decay of [ ‘H]cholesteryl ester labeled HDL-2 from the plasma compartment. Data are mean rt SD. of 3 or 4 determinations.

83 TAf3LE III Cholestetyl ester transfer activity in the plasma of control and probucol-treated rats

Data are mean 1 SD. of three determinations. The incubations were carried out at 37°C for 4 h as described in Methods. Transfer activity is expressed as% [3H]cholestetyl esters isolated with labeled VLDL (d < 1.019 g/ml) after incubation with [3H]cholesterol-iabeled HDL3. Plasma

Diet

[ 3H]CE transfer (%)

none control diet probucol, 1% probucol, 2% none

0.6kO.l 2.1ao.2 2.OztO.3 2.2rf:O.l 38.0f0.7

(d > 1.21 g/ml)

None Rat Rat Rat Human

parallel in the two groups of animals for LDL and slightly faster for HDL-2 in probucol treated animals. The circulating half-lifetime of ~3H]cboleste~l esters in LDL was 6.5 h in both groups and in HDL-2, 9.5 h in control rats and 7.5 h in probucol-treated animals. Cholesteryl ester transfer activity in the plasma of control and probucol-treated rats is shown in Table III. Transfer of [3H]cholesteryl esters in the presence of control rat plasma fraction of density greater than 1-21 g/ml was minimal, 2.1% as compared to 0.6% in the absence of plasma. Plasma from probucol-treated rats yielded the same low activity. A plasma fraction of density greater than 1.21 g/ml from a normal human subject, in contrast, induced transfer of 38.0% of the [3H]choleste~l esters from HDL to VLDL. Discussion Prevention of arteriosclerosis, the most common disease in humans, is a major challenge of medicine and biology for the 1990s. Therapy of common risk factors, including dyslipidemia syndromes, remains a cornerstone in the attempt to prevent atheroma formation [24]. Other approaches are, however, being sought. An exciting new approach is prevention of lipid oxidation in lipoproteins, a process that is assumed to play a critical role in atherogenesis j2.51. Probucol, a potent antioxidant [S-lo] is one of the few drugs that is used in humans for lowering high cholesterol levels. The drug, however, appears to lower the levels of both the atherogenic lipoprotein LDL and the anti-atherogenic lipoprotein, HDL [2-41. However, probucol administration is associated with regression of xanthomas and prevention of the formation of atherosclerotic plaques [5--31. Neither the mechanisms by which probucol affects plasma lipoprotein levels nor those that prevent atheroma formation are well understood. In the present investigation an attempt was made to clarify the

former in an experimental animal - the rat - that is devoid of cholesterol ester transfer activity. Probucol treatment caused a 26% reduction of plasma cholesterol and a 15-23% reduction of plasma triacylglycerol levels. Similar effects were found with 1% and 2% probucol in the diet and after 7, 10 or 14 days feeding. These findings differ from those reported by Staels et al. 1141,who did not observe any effect of probucol on plasma cholesterol in Wistar rats and from the dramatic 63% decrease of plasma cholesterol observed in mice [13]. This last observation is especially remarkable as the mice were treated for 14 days with only 0.2% (w/w) probucol. Possible explanations for these diverse observations are amount of probucol actually consumed in the different studies, e.g., mice vs. rats, and/or the fraction of probucol absorbed from the gut. It appears that these and other possible mechanisms should be taken into consideration in studies on probucol. A unique feature of the present investigation was the elucidation of the effects of probucol on the structure and composition of all major rat plasma lipoproteins. We showed previously that centrifugation in a zonal rotor is especially adequate to study the structure, composition and metabolism of the lipoprotein system in rats [161. With this technique, we demonstrate that probucol causes a slight shift of LDL, HDL-1 and HDL-2 towards a higher density. The chemical composition of these three lipoproteins, in particular the higher protein content implying a smaller and denser particle, support the obse~ations from the zonal runs. A mechanism that may be responsible for such effects is an increased neutral lipid (cholesteryl esters and triacylglycerols) heteroexchange between Iipoproteins followed by hydrolysis of the transferred triacylglycerols [12]. Probucol indeed increases the activity of the cholesterol ester exchange reaction in humans [ll]. We, however, failed to demonstrate any effect of probucol on the reaction in rats. Yet, in spite of a lack of effect of probucol on plasma cholesterol ester exchange we observed a small but definite effect of probucol on cholesteryl ester distribution among lipoproteins: paucity of these molecules in the cholesterolrich lipoproteins HDL-1 and HDL-2, and excess in the triacylglycerol-rich lipoproteins, VLDL and LDL. These considerations suggest the interesting possibility that probucol affects plasma cholesterol ester distribution among lipoproteins by a mechanisms(s) that differs from those discussed above. One potential mechanism is an effect of probucol on the other lipid transfer protein that is active in rat plasma and is responsible for phospholip’id exchange [26]. Other possibilities are an effect on cholesterol ester content of nascent lipoproteins secreted from the liver and the intestine or effects on lipoproteins - cell interaction, e.g., selective cholesterol ester tissue uptake [27,28].

84 The results of the metabolic experiments described here are simiiar to those reported in control and human apo A-I transgenic mice by Hayek et al. [13]. In both studies, probucol had no or only a minimal effect on apo A-I plasma catabolism and in both, an effect on HDL cholesteryl ester fractional catabolic rate was observed. This last effect, however, was considerably more pronounced in the mouse, where a dramatic decrease of the HDL cholesterol level was found. Hayek et al. concluded that the main effect of probucol on plasma apo A-I levels is due to a decreased transport rate, while the effect on HDL cholesterol is due to an increased cholesteryi ester catabolic rate, predominantly by the selective uptake pathway. Our results are compatible with these conclusions. Yet, it is important to note that neither Hayek et al. [13] nor Staels et al. [ 141have found any effects of probucol on tissue mRNA for apo A-I. Whether probucol may affect apo A-I mRNA translation rate or the transport of apo A-I from nascent lipoprotein particles to the plasma kinetic pool is unknown and remains to be investigated. Possibie effects of probucol on LDL metabolism in experimental animals have, to our knowledge, not been previously reported. In the present study we found that probucol administration caused a moderate reduction of LDL plasma levels without any significant effect on LDL cholesteryl ester turnover rate. These observations imply that probucol decreases the rate of production of LDL in the rat. Noteworthy is that as probucol was reported to decrease hepatic LDL receptor mRNA concentrations [14], a phenomenon that should increase LDL levels, the effects of probucol on LDL production may be considerably more pronounced than those inferred from the metabolic study reported here. The mechanisms responsible for a decreased production of LDL in probucol-treated rats were not clarified. However, since probucol does not affect the liver and intestinal apo B and apo E mRNA levels [14], it might be postulated that the drug increases VLDL remnant catabolism and thereby causes a decrease of the VLDL to LDL conversion process, the main pathway responsible for LDL formation [151. This same mechanism could also lead to the reduction of plasma triacylglycerol levels. The results of the present and previous studies 113,141indicate that in addition to its antio~dant properties, probucol also has profound effects on the structure, composition and metabolism of all major plasma lipoproteins. The drug appears to affect apoprotein and lipoprotein transport rates, plasma lipoprotein cholesteryl ester distribution and perhaps lipoproteincell interactions. Some or all of these metabolic effects of probucol may contribute to the anti-atherogenic action of the drug. In particular, the mechanism(s) by which probucol affect plasma apolipoprotein and cholesterol distribution and transport may be relevant

to processes that induce or prevent atherogenesis. Such mechanisms should be elucidated in future research.

Acknowledgements The excellent assistance of Ms. H. Lefkovitz is greatly acknowledged. This study was supported in part by a research grant from Otsuka Pharmaceutical Co. Ltd., Japan. References 1 Buckley, M.M., Goa, K.L., Price, A.H. and Brogden, R.N. (1989) Drugs 37, 761-800. 2 Mellies, M.J., Gartside, P.S. and GlatFelter, L. (1980) Metabolism 29, 956-964. 3 Matsuzawa, Y., Yamashita, S., Funahashi, T., Yamamoto, A. and Tarui, S. (19881 Am. J. Cardiol. 62, 668-728. 4 Franceschini, G.. Sirtori, M., Vaccarino, V., GianFranceshi, G., Rezzonico, L., Chiesa, G. and Sirtori, CR. (1989) Arteriosclerosis 9, 462-469. 5 Yamamoto, A., Matsuzawa. Y., Yokoyama, S., Funahashi, T., Yamamura, T. and Kichino, B. (1986) Am. J. Cardiol. 57, 29H35H. 6 Carew, T.E., Schwenke, D.C. and Steinberg, D. (1987) Proc. Natl. Acad. Sci. USA 84, 7125-7729. 7 Kita, T., Nagano, Y. and Yokode, M. (1987) Proc. Natl. Acad. Sci. USA 84, 5928-5931. 8 Parthasarathy, S., Young, S.G., Witztum, J.L., Pittman, R.C. and Steinberg, D. (1986) J. Clin. Invest. 77, 641-644. 9 McClean, L.R. and Hagaman, K.A. (1989) Biochemistry 28, 321327. 10 Barnhart, R.L., Bush, S.J. and Jackson, R.L. (1989) J. Lipid Res. 30, 1703-1710. R., Hogue, M., Mime, R.W., Tall. A.R. and Marcel, 11 McPherson, Y.L. (1991) Arterioscl. Thromb. 11, 476-481. 12 Tall, A.R. (1986) J. Lipid Res. 27, 359-365. T., Walsh, A., Azrolan, N. and Bresiow, 13 Hayek, T., Chajek-Shaul, J.L. (1991) Arterioscl. Thromb. 11, 1295-1302. 14 Staels, B., Van Tol, A., Jansen, H. and Auwerx, J. (1991) Biochim. Biophys. Acta 1085, 131-135. S. and Rachmilewitz, D. (1973) Biochim. Biophys. 15 Eisenberg, Acta 326, 378-390. 16 Oschry, Y. and Eisenberg, S. (1982) J. Lipid Res. 23, 1099-1106. 17 Brewer, H.B., Ronan, R., Meng, M. and Bishop C. (1986) Methods Enzymol. 128, 223-246. A.S. (1958) Nature 182, 53. 18 McFarlane, D.W., Eisenberg, S. and Levy R.L. (1972) Biochim. 19 Bilheimer, Biophys. Acta 260, 212-221. J. (1984) J. Lipid Res. 20 Eisenberg, S., Oschry, Y. and ~immer~n, 25. 121-128. 21 Gavish, D., Oschry, Y. and Eisenberg, S. (1987) J. Lipid Res. 28, 257-267. R. and Eisenberg, S. (19901 22 Raveh, D.. Israeli, A., Arnon, Atherosclerosis 82, 19-26. 23 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244,4406-4412. 24 The Expert Panel (1988) Arch. Intern. Med. 148, 36-69. D., Parthasarathy S., Carew, T.E., Khoo, J.C. and 25 Steinberg, Witztum, J.L. (1989) N. Engl. J. Med. 320, 911-924. 26 Eisenberg, S. (1978) J. Lipid Res. 19, 229-236. R.C., Weinstein, D.B. and Steinberg, D. 27 Glass, C.S., Pittman, (1983) Proc. Natl. Acad. Sci. USA 80.5435-5439. 28 Glass, C.K., Pittman, R.C., Civen. M. and Steinberg, D. (19851 J. Biol. Chem. 260, 744-750.