Comparison of effects of probucol versus vitamin E on ex vivo oxidation susceptibility of lipoproteins in hyperlipoproteinemia

PREVENTIVE CARDIOIBGY

Comparison of Effects of Proha/ Versus Vitamin Eon Ex Vivo Oxidation Susceptibility of Lipoproteins in Hyperlipoproteinemia Carlos A. Dujovne, MD, William S. Harris, PhD, Linda L. Colle Gerrond, MD, Junyuan Fan, BS, and Fulvio Muzio, MD Oxidative modiRcaUon of lowdensity Ii-in (UK)cMesterolappearstoconthbuteto~ gelw!si% Probucol reduces LDL cholesterol oxida tion susceptibilRy, but the consistency, dose, and time course BCBnot well described. Twelve bypep lipidemic pathts were given probucol for 4 cllohterol reduction weeksattbeusualdosefor (l,ooo mivdw, or one Mf (mww or one quarter (250 mg/day) the usual dose. Lipoprotein oxidath susceptibility of apolipoprotein 54~~ usingarapid tainhg Ilpopmteins was asses~I test in which LDL cholesterol and vefy-lowdensity lipoprotein precipitated with dextran sulfate and magnesium ions, redissolved, incubated with copperionsfor3hcurs,andtestedforulii turic acSm&ive substance% ResuRs are expressed as nmoles of malonyldialdehyde (MDA) m per mb @ml MDA/mg) n-MNsity lipopmtein cboksterol. Lipoproteins from ~~beeo~resistatitO0~

dationwithapredrugvalueof85~19,andd~ creasingto3+lnmolMDA/mgaf&erdmga& ministmtion (p eO.001). Both ahaP and 'full" doses were effective in lowering lipoprotein oxk dation susceptibilii by 95%. The ‘quarter” dose was less effective. Oxidation inMition was maximixed within 2 weeks, returning to baseline 4 to 6 weeks after discontinuing probucol. Four pa tieots were subsequently aossed over to vitamin E (1,200 W/day). Vtimin E had a milder, less pre+ dictaMe mtiixidant effa& lowering lipoprotein oxidation susceptibilii by a mean of 24%. In COI, elusion, probud treatment Mectively and pre dictabiy reduces plasma lipoprotein susceptibilii to 8x vivo, coppdnduced oxidath. This clinC tally ~licable test may mvide quantitation of antioxidanteff6CtSOfprobuCOlWotherantiOxi-

dants and thus faciliie dose adjustments and conwlath with anti-ic effect% (AmJCardid 1994;7498-42) From the Lipid and Arteriosclerosis Prevention Clinic, Division of Clinical Pharmacology,Departmentof Medicine, University of Kansas Medical Center,KansasCity, Kansas.This study was supportedin part by a grant from Marion Merrell Dow, Kansas City, Missouri. Manuscript received August 27,1993; revised manuscriptreceived and acceptedDecember 17, 1993. Address for reprints: Linda L. Colle Gerrond, MD, 1348 Kansas University Hospital, 3901 Rainbow Boulevard, Kansas City, Kansas 66160-7374. Dr. Muzio was a ResearchFellow in Clinical Pharmacology from Santa Corona Hospital, GarnagnateMilanese, Milan, Italy. 38 THE AMERICANJOURNALOF CARDIOLOGY VOLUME74

ipoprotein oxidation in atherogenesishas come under intense scrutiny following reports that oxidation may play a role in the development and progression of atherosclerosis.’ Oxidative modification of low-density lipoprotein (LDL) cholesterol leadsto enhanced and unregulated LDL cholesterol uptake by arterial macrophages, and thus enhanced production of subendothelial foam cells2 Patients with clinically significant atherosclerosishave increasedlevels of lipid peroxides,3and those with hypercholesterolemiahave LDL cholesterol particles that are more readily oxidized than those from normal subjects4 Antioxidants such as probucol, MDL 29,311 (a probucol analog with no cholesterol-lowering properties), and butylated hydroxytoluene protect LDL cholesterol from oxidation and prevent atherosclerosis in hyperlipidemic animal models.5-8Although naturally occurring plasma antioxidants such asascorbic acid, a-tocopherol, and p-carotenehave somecapacity to reduce LDL cholesterol oxidization,%” as do diets rich in oleic acid,12the most potent currently marketedantioxidant drug appearsto be probucol.” Our objectives in this study were (1) to determine if lower dosesof probucol than those recommendedto achieve a cholesterol-lowering effect could protect serum lipoproteins againstex vivo oxidation, and (2) to develop a rapid assayfor routine clinical testing of such effect.

L

METHODS Patlents: Twelve patients participated in this trial. All had LDL cholesterol levels above the 95th percentile for their age and sex before treatment, but most had reduced their levels to less than the 75th percentile by lifestyle intervention as well as single or combined treatment with blood lipid-regulating drugs (i.e., statins, gemftbrozil, niacin, or bile acid-binding resins, or any combination of these). None of these drugs are known to have any pro- or antioxidant properties. They were administered without a changein dosagethroughout the trial. No patient was taking probucol or pharmacologic doses of nutritional antioxidants such as Vitamin C, E, or p-carotene.Informed consentwas obtained from each patient before entry into the study, which had been approved by the Human Subjects Committee of the University of Kansas Medical Center. Protocol: Two baseline samples were obtained before starting the 4-week probucol intervention period. Sevenof the patients were given the “full” doseof probuco1 (500 mg twice daily), and 5 others were given the half dose (250 mg twice daily). After completing this protocol, 7 subjects were given the “quarter” dose of probucol (250 mg/day). After a washout period without

JULY 1, 1994

probucol of 1 to 3 months, during which time LDL cholesterol oxidation susceptibility had returned to baseline, 4 patients were given 1,200 IU/day of d-o-tocopherol (vitamin E). Blood was drawn weekly for 4 weeks and again 4 weeks after discontinuation of probucol therapy. In the 4 patients whose therapy was switched to vitamin E, blood measurementswere obtained before and after 1 week of vitamin therapy. bIboratory TEINS: Blood

methods:

PLASMA LIPIDS AND LIPOPRO-

was obtained with minimal stasis into ethylenediaminetetraaceticacid (1 mg/ml) with the patient in the sitting position after a 1Zhour overnight fast. The profile included a determination of cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol as previously described14;LDL cholesterol was estimated by the Friedewald equation.15These analyses were performed using a Cobas Mira with reagentsfrom Boehringer Mannheim (cholesterol) and Sigma (triglycerides). Quality control was maintained through participation in the CDC Lipid Standardization Program.16 Lipoprotein oxidation susceptibilii: SoLuTIoNsDEXTRANSLJLFATE/MAG~SIUM~I~~RIDE(PRBCIPITATI~N REAGENT): Equal volumes of a 20 g/liter solution of dex-

tran sulfate (molecular weight = 50,000; Genzyme Inc., Cambridge, Massachusetts) and a 1 M MgCl,-6H,O were combined. The pH of the linal mixture was adjusted to 7. PRECIPITATE WASHING SOLUTION: Bovine serum albumin, 60 g/liter (Fraction V, Sigma no. A7906), was stored at 4’C in the dark and prepared fresh weekly. SALINE (4%): 0.68 M sodium chloride. COPPER SOLUTION: 0.5 I&! CuC12-2H,O. THIOBARBITURICACID-REAcTIVESUBSTAhQ.SREiAGENT:

200 ml of a solution containing 26 mM thiobarbituric acid and 0.92 M trichloroacetic acid in 0.25 M hydrochloride was prepared. The thiobarbituric acid was added ftrst, heated, and stirred. After it was dissolved, the trichloroacetic acid was added, and the solution brought to volume with hydrochloride. The reagentwas storedin a dark bottle at room temperature. In the routine assay for HDL cholesterol, the verylow-density lipoprotein (VLDL) and LDL cholesterol

FIGURE 1. The effecte of 1 month of probucol treatment and 1 month of washout on lipoprotein oxidation euua coptibilRy in hypercholeeterolemic pb tients full dose q 1,000 mg/day; half dose = SO0 mg/day; quarter doee = 250 mg/day. No waehout valuer were obtained after quarter does therapy. *p
are precipitated from 500 pL of plasma by adding 50 FL of the precipitation reagent, incubating and centrifuging as described.l7 The supematant HDL cholesterol is then measured.For the lipoprotein oxidation susceptibility assay,after the supematantwas removed, the precipitate (which is normally discarded) was washedby resuspensionin 1 ml of the precipitate washing solution and 100 ~.LLof the precipitation reagent. The suspension was vortexed briefly and the precipitate reisolated by centrifugation as previously described. After discarding the supematant,the pellet was dissolved in 2.5 ml of 4% saline solution, giving a final fivefold dilution of the lipoproteins. A volume of redissolved precipitate containing 100 pg of non-HDL cholesterol (calculated from the total minus HDL cholesterol level) was mixed with sufficient 4% sodium chloride to give 500 p,L of total solution in a screw-top glass test tube (16 X 100 mm). Copper solution, 50 pL, was added to this volume. The final concentration of copper was 10 p,M. The mixture was incubated at 37°C for 3 hours in a shaking water bath. After incubation, the solution was assayedfor thiobarbituric acid-reactive substancesas an index of oxidation. The thiobarbituric acid-reactive substanceassaywas conducted by adding 2 ml of the thiobarbituric acid substance reagent to the 550 pL incubation mixture and heating it in a boiling water bath for 15 minutes. After removing the tubes from the water bath and cooling, 2.5 ml of n-butanol was added. After shaking briefly, the phases were separatedby centrifugation at 1,500 g for 15 minutes. The pink organic layer was read in a spectrophotometer at 532 nmoles against a blank containing 4% saline solution instead of the lipoprotein suspension that was carried through the entire oxidation and thiobarbituric acid substances procedure. The nmoles of malonyldialdehyde (MDA) present in the sample were estimatedby comparison with a standardcurve prepared from 1,1,3,3-tetraethoxypropane (0.5 to 16 nmol/ml) which was treated like the blank above. Based on the amount of non-HDL cholesterol in 0.5 ml of plasma and the amount of MDA generatedfrom the same volume, the linal results were expressedas nmoles MDA equiv-

0 Before probucol n After 4 weeks of probucol 4 weeks after discontinuing probucol

I

Full Dose n-7

Half Dose n-5

Quarter Dose n-7

OXIDATIONSUSCEPTIBILITY WITHPROBUCOLAND VITAMINE 39

dividual variability resulted in no statistically significant change. Results from 2 representativepatients, 1 receiving the full dose and the other the half dose, are shown in Figure 2. The rapid institution of the antioxidant effect and washout rate are clearly seen.The patient receiving the full doserapidly achieved a nearly complete antioxidant effect, but washout also occurred more rapidly than in the patient receiving the half dose. This tendency for washout to occur more rapidly with the full than with the half dose in patients was confirmed in the group data (Figure 1). RESULTS Probucol had a modest effect on plasma lipid and Probucol dosagesof 1,000 and 500 mg/day were remarkably effective in almost completely preventing cop- lipoprotein levels (Figure 3). The half dose(500 mg/day) per-induced oxidation of VLDL + LDL cholesterol (Fig- had virtually no effect on LDL cholesterol levels and deure 1). The average percent inhibition was 95% (p creasedthe total cholesterol by only 8% (comparedwith ~0.01). Lipoprotein oxidation susceptibility values re- an 18% decreasewith full dose). HDL cholesterol deturned to or approached baseline during the 4-week creasedby similar amountswith both full and half doses. washout period. Although not statistically different from The quarter dose (250 mg/day) did not significantly rebaseline values, a complete washout (i.e., return to base- duce total, LDL, or HDL cholesterol levels. Therapy in 4 patients was subsequentlycrossedover line values) did not appearto be achieved in 1 month in most instances.Although 250 mg/day lowered the mean to vitamin E, and the lipoprotein oxidation susceptibillipoprotein oxidation susceptibility by 50%, marked in- ity value decreasedby an average of 24% (Figure 4). alents generated/mgnon-HDL cholesterol. The coefficient of variation of the assayis 4%. St~tistlcal evaluation: Becauseeach subject served as his/her own control for 3 observations (baseline, during drug treatment period, and during the postdrug or washout periods), the data were analyzed by analysis of variance with repeatedmeasuresfollowed by the Newman-Keull multiple comparisonstest. Data are presented as mean f SD, and a 2-tailed p value ~0.05 was required for statistical significance.

150Patient 2 - Half Dose

0 Patient l- Full Dose

E

l

FIGURE 2. lhe effects of 1 month of probucol treatment and 1 month of washout on Ilpoprotein oxidation sus ceptibillty values in 2 hypercboieo terdemk patie* 1 given the full dose of probucol (1,000 mg/day) and the other half that dose. Abbrevib tions as in FIgwe 1. weeks

-1

0

Baseline

1

2

3

4

5

Probucol

6

7

8 s

Washout

300

17 Baseline n Probucol RGURES.l'heeffectsoflmonthof pfobucol treatment with 1,000 mcvday (full dose), SO0 m&/day (haH dose), and 2SO mg/day (quarter dose) on serum llpld Ievek In hypwcholeeterolemk patiewts (n = 7, S, and 7, respectively). *p *O.OB; LGL q low density Ilpoprotein; other abhrevie tkns as in Flgure 1.

180

60

Full

Half Quarter

Total Chol

40

Full

Half Quarter

LDL Chol

THE AMERICANJOURNALOF CARDIOLOGY VOLUME74

Full

Half Quarter

HDL Chol

JULY 1,1994

Two patients had good responses (decreasesof about probucol at dosesmuch lower than those recommended 45%), but the other 2 had little response (decreasesof for lowering serum cholesterol levels. ~5%). Plasma lipid and lipoprotein levels were not alThe antioxidant effect of vitamin E has been known tered by vitamin E. for years, but has only recently been investigated relative to preventing LDL cholesterol oxidation. Vitamin E DISCUSSION (800 to 1,600 ID/day) can reduce the susceptibility of In this study, in vivo administration of probucol pro- LDL cholesterol to ex vivo oxidation by about 30% aftected serum lipoproteins from ex vivo oxidation by cop- ter at least 1 month of treatment.22-24In our study, 1 per ions. Lipoproteins from patients taking both full and week after giving vitamin E 1,200 IU/day, the oxidation half doses were almost completely protected from ex susceptibility decreasedby a mean of 24%, suggesting vivo copper-induced oxidation 1 or 2 weeks after start- that the effects of vitamin E may appear rather quickly. ing probucol. These effects were consistent through 4 Whether the antioxidant properties of vitamin E relate weeks of therapy. A quarter dose was not as consistently to the reduced risk of congestive heart failure observed effective. Thesedata agreewith both in vitro and ex vivo in subjects taking vitamin E supplements25*26 is not yet studies in which probucol reduced thiobarbituric acid clear. substancesformation, slowed LDL cholesterol apolipoThe safety of full dose probucol has been demonprotein B degradation, and attenuated accumulation of strated over many years of clinical use. The half dose cholesteryl esters in macrophages.i3 therapy effectively inhibits LDL cholesterol oxidation. Reaven et al’* reported that both full and quarter Consideration should be given to recommending that padosesof probucol achieved significant protection against tients with known atherosclerotic vascular disease be oxidation in hypercholesterolemic patients. However, in treated with probucol at 500 mg/day in addition to whattheir study, it took about 2 and 6 months for the full and ever other lipid-regulating drugs they may need for conquarter doses,respectively, to achieve a 40% reduction trol of dyslipidemia. The potential benefitsof such a regin oxidation susceptibility. In our study,a 95% reduction imen generally outweigh the minimal risks involved with in oxidation susceptibility was achieved within 2 weeks. using probucol (especially at half its usual dose), alHDL cholesterol levels were reduced even by the 250 though, of course, individual assessmentof each patient is needed. mg/day dose in their study. A preliminary presentation at the European AtheroCristol et all9 reported that after 4 months of probuco1 250 mg/day, LDL cholesterol oxidation susceptibil- sclerosis Society meeting (Jerusalem,September 1993) ity significantly decreased,but HDL cholesterol levels of the recently completed Probucol Quantitative Redid not. Probucol 250 mg/day decreasedserum HDL gression Swedish Trial reported no effects of probucol cholesterol levels in Reaven’s study but not in Cristol’s; on progression or regression of peripheral artery atherhowever,baseline HDL cholesterol levels were 57 mg/dl osclerosis as measuredby serial ultrasound evaluations in Reaven’s and 38 mg/dl in Cristol’s. No data suggest of the femoral artery. Because these results are not yet that the probucol-induced reduction in HDL cholesterol published, full analysis and interpretation are not possiis proatherogenic. Yamamoto et a120showed a strong ble. correlation between the HDL cholesterol-lowering effect and regression of tendon xanthomasin patients with hoAcknowledgment: We thank Sheryl Windsor, MT, mozygous familial hypercholesterolemia taking probu- ASCP, for her assistancein the lipoprotein/nutrition labcol. oratory, and Patricia McFarland for her help in typing Cristol and Reaven and their co-workers isolated and editing the manuscript. LDL cholesterol by ultracentrifugation, oxidized it either with copper or by exposure to cultured endothelial cells, and then assessedthe degree of oxidation by thio- I barbituric acid substancesassay and macrophage LDL cholesterol degradation rates. We precipitated LDL and ( 2507 VLDL cholesterol together,exposedthem to copper, and measured thiobarbituric acid substancesgenerated.Recent work has shown that when LDL cholesterol is complexed (in a disaggregated form) with arterial chondroitin sulfate proteoglycans, glycosaminoglycans, or heparin, it becomesmore susceptible to copper-induced oxidation.21 In the subendothelial space, LDL cholesterol is very likely to be bound to such “ground substance” speciesand may thus be more susceptibleto oxidation. Dextran sulfate was used in our assay to 0’ 1 I precipitate the apolipoprotein B-containing lipoproteins, Pre Vitamin E Post Vitamin E which may make our assaymore sensitive and more representative of the in vivo situation. This difference in FIGURE 4. Lipoprotein oxidation susceptibility in 4 pb technique may also explain why probucol seemedto be tients given 1,200 W/day of vitamin E for 1 week. Mean much more potent and fast-acting in our assay.All 3 f SD values are men but the differences are not SW studies support the lipoprotein antioxidant capacity of tistically si@dfkant. Abbreviations as in Rgure 1. OXIDATIONSUSCEPTIBILITY WITHPROBUCOLAND VITAMINE 41

l. Steinberg D, Pathasaratby S, Carew TE, Khoo JC, Witzum JL. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med 1989;320:915-924. 2. Quinn MT, Parthasamthy S, Fong LG, Steinberg D. Oxidatively modified low density lipoproteins: a potential role in recruitment and retention of monocytef macrophages during atherogenesis. Proc Narl Acad Sci USA 1987;84:2995-2998. %. Stringer MD, Gorog PG, Freeman A, Kakkar VV. Lipid peroxides and atherosclerosis. Br Med J 1989;298:281-284. 4. Lavy A, Brook GJ, Dankner G, Amotz AB, Avimm M. Enhanced in vitro oxidation of plasma lipoproteins derived from hypercholesterolemic patients. Metabolism 1991;40:794-799. b. Bjorkhem I, Hemiksson-Freyschuss A, Breuer 0, Diczfalusy U, Berglund L, Hem&son P. The antioxidant butylated hydroxytoluene protects against atherosclerosis. Arterioscler Thromb 1991; 1l:lS-22. S. Chew TE, Schwenke D, Steinberg D. Antiathcrogenic effect of probucol “ruelated to its hypocholesterolemic effects. ProcNatl AcadSci USA 1987;84:7725-7729. 7. Mao SJT, Yates MT, Parker RA, Chi EM, Jackson RL. Attenuation of atherosclerosis in a modified strain of hypercholesterolemic Watanabe rabbits with use of a probucol analogue (MDL 29,311) that does not lower serum cholesterol. Arterioscler Thromb 1991;11:12661275. 8. Kita T, Nagano Y, Yokode M, Ishii K, Kume N, Ooshima A, Yoshida H, Kawai C. Probucol prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia. Proc h’arl Acad Sci USA 1987;84:5928-5931. 9. Babiy AV, Gebicki JM, Sullivan DR. Vitamin E content and low density lipoprotein oxidizability induced by free radicals. Atherosclerosis 1990;81: 175-181. 10. Esterbauer H, Dieber-Rotbeneder M, Striegl G, Waeg G. Role of vitamin E in preventing the oxidation of low density lipoprotein. Am J Chin Nufr 1991;53:314S -321s. fl. JialaJ I, Vega GL, Gmndy SM. Physiologic levels of ascorbate inhibit the oxidative modification of low density lipoprotein. Atherosclerosis 1990;82: 185-191. l2. Parthasamthy S, Khoo JC, Miller E, Bamett J, Witzum JL, Steinberg D. Low density lipoprotein rich in oleic acid is protected against oxidative modification: implications for dietary prevention of atherosclerosis. Proc Natl Acad Sci USA 1990;87:3894-3898. la. Regnstrom 3, Walldius G, Carlson LA, Nilsson J. Effect of probucol treatment on the susceptibility of low density lipoprotein isolated from hypercholesterolemic

42 THE AMERICANJOURNALOF CARDIOLOGY VOLUME74

patients to become oxidatively modified in vitro. Atherosclerosis 1990;82:43-51. l4. Harris WS, Dujovne CA, Zucker M, Johnson B. Effects of a low satorated fat, low cholesterol fish oil supplement in hypemiglyceridemic patients: a placeb-o-controlled trial. Ann Intern Med 1988; 109:465-470. 1s. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low density lipoprotein cholesterol in plasma without use of the preparative ultracentrifuge. Clin Chem 1972;19:499-502. 19. Allain CC, Peon LS, Ghan CSG, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. C/in Chem 1974;20:470-475. 17. Bachorik PS, Albers JJ. Precipitation methods for quantification of lipoproteins. Methods Enzymol 1986;129:78-100. 58. Reaven PD, Panhasarathy S, Beltz WF, Witzum JL. Effect of probucol dosage and plasma lipid and lipoprotein levels and on protection of low density lipoprotein against in vitro oxidation in humans. Arrerioscler Thromb 1992;12:318-324. 19. Cristol LS, Jialal I, Gmndy SM. Effect of low-dose probucol therapy on LDL oxidation and the plasma lipoprotein profile in male volunteers. Atherosclerosis 1992;97:1 l-20. 20. Yamamoto A, Matsuzaqwa Y, Yokoyama S, Funahasbi T, Yamamura T, Kishino B. Effects of probucol on xantbomata regression in familial hypercholesterolemia. Am J Cardiol 1986;57:29H-35H. 21. Comejo G, Hurt-Comejo E, Rosengren B, Wiklund 0, Lopez F, Bondjers G. Modification of copper-catalyzed oxidation of low density lipoprotein by proteoglycans and glycosaminoglycans. J Lipid Res 1991;32:1983-1991. 22. Raven PD, Khouw A, Beltz WA, Pathasarathy S, Witzum JL. Effect of dietary antioxidant combinations in humans: protection of LDL by vitamin E but not by @-carotene. Arterioscler Thromb 1993; 13:59@600. 23. Dieber-Rotbeneder M, Pohl H, Wang G, Sbiegl G, Esterbauer H. Effect of oral supplementation with d-a-tocopherol on the vitamin E content of human low density lipoproteins and resistance to oxidation. J Lipid Res 1991;32:1325-1332. 24. Jialal I, Gnmdy SM. Effect of dietary supplementation with a-tocopherol on the oxidative modification of low density lipoprotein. JLipidRes 1992;33:899-906. 21. Stampfer MJ, Hennekens CH, Manson JE, Colditz GA, Rosner B, WiIlett WC. Vitamin E consumption and the risk of coronary disease in women. N En@ J Med 1993;328:1444-1449. 29. Rimm EB, Stampfer MJ, Ascherio A, Giovannucci E, Cold& GA, Willett WC. Vitamin E consumption and the risk of coronary disease in men. N EngI J Med 1993;328:1450-1456.

JULY 1,1994