Influence of Antioxidant Vitamins on LDL Oxidation" I. JIALAL AND S . M . GRUNDY Center f o r Human Nutrition and Departments of Internal Medicine, Pathology, and Clinical Nutrition University of Texas Southvtlestern Medical Center 5323 Harry Hines Boirlevard DUIln s , TPXU s 75235-9052 Evidence continues to accumulate supporting the hypothesis that the oxidative modification of LDL is a key step in the genesis of the atherosclerotic lesion. Dietary micronutrients with antioxidant properties, such as ascorbate, alphatocopherol, and beta-carotene, levels of which can be favorably manipulated by dietary measures without generally resulting in side effects, could prove a safe approach in inhibiting LDL oxidation and consequently preventing atherosclerosis progression. Hence this review will focus on the role of antioxidant vitamins on LDL oxidation and atherosclerosis. First the role of oxidized LDL (Ox-LDL) in atherosclerosis will be discussed. Then the relationship between antioxidant vitamins and atherosclerosis will be reviewed. Finally the studies focusing on the effect of antioxidant vitamins on LDL oxidation will be presented.
OXIDIZED LDL AND ATHEROSCLEROSIS An increased level of plasma LDL cholesterol is a major risk factor for premature atherosclerosis. The mechanism(s), however, by which LDL promotes the development of the early fatty streak lesion with its characteristic lipid-laden macrophages remains to be elucidated. Uptake of cholesterol by way of the classical LDL receptor cannot result in appreciable cholesterol accumulation because the LDL receptor is subject to feedback inhibition by the cellular cholesterol content.' Certain modified forms of LDL, for example, acetyl-LDL, are taken up by way of the scavenger receptor mechanism, resulting in substantial cholesterol accumulation and foam cell formation in macrophages because the scavenger receptor is not regulated by the cellular cholesterol content.' The most plausible and biologically relevant modification of LDL that appears to occur is oxidation.2 The free radical peroxidation of LDL lipids results in numerous structural changes, all depending on a common initiating event: the peroxidation of the polyunsaturated fatty acids on LDL.3-SLDL can be oxidatively modified in the presence of transi~ - ~ all the major cells tion metals (iron and copper) in a cell-free e n ~ i r o n m e n t . Also of the arterial wall (endothelial cells, macrophages, and smooth muscle cells) can oxidatively modify LDL. Both forms of oxidized LDL are taken up more avidly a Work cited in this review was supported by the American Heart Association (Texas Affiliate), Hoffmann-La Roche, and the Bristol-Meyers Squibb/Mead Johnson Nutrition Research Grant. 231
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238 TABLE 1.
Properties of Oxidatively Modified LDL Decreased content of polyunsaturated fatty acids Enrichment in lipid peroxides Increased negative charge Increased lysolecithin content Increased content of oxysterols Fragmentation of apoprotein B-100 Decreased uptake by LDL receptor Increased uptake by scavenger receptor
by macrophages than LDL and appear to be processed by the scavenger receptor mechanism.'-'Some of the more important properties of oxidized LDL are shown in TABL E I . The biological effects of oxidized LDL reported to date could contribute to initiation and progression of the atherosclerotic process (FIG.1). The cytotoxicity of Ox-LDL may be important in inducing endothelial cell dysfunction and/ or promoting the evolution of the fatty streak to a more complex and advanced lesion.?-' Ox-LDL is a potent chemoattractant for the circulating monocyte but not neutrophils.2-5In fact, it has been postulated that in the early phase of oxidation there are formed in the subendothelial space minimally modified LDL (MM-LDL) that are mildly oxidized.',' Once formed MM-LDL could induce the endothelium to express adhesion molecules for monocytes, and to secrete monocyte chemotactic protein (MCP-I ) and macrophage colony-stimulating factors.',' These molecular events result in monocyte binding to the endotheliun and its subsequent migration
A
R T E R > Y W A L L
Foam Cell
FIGURE 1. Schema depicting the relationship between oxidized LDL and the genesis of the fatty streak lesion. (Based on the studies of Fogelman et and Steinberg et a / . ? ) ~
1
.
~
3
~
JIALAL & GRUNDY: LDL OXIDATION
239
into the subendothelial space, where MM-LDL promotes differentiation into tissue macrophages. The macrophages then further modify MM-LDL to a more oxidized form.8 This Ox-LDL can then be processed by way of the scavenger receptor mechanism leading to cholesterol ester accumulation. Because Ox-LDL is a potent
(A)
OR% 1
2
3
5
4
6
L
ox-LC + Vit
_ -
T
-
I
ox-LC L OX-LDL OX-LDL + Vit 1 + Vit C + Vit C
FIGURE 2. Effect of ascorbate and alpha-tocopherol on the 24th oxidative modification of LDL. LDL (200 pg/mL) was subjected to a 24-h oxidation with 2.5 pM copper in PBS in the absence and presence of alpha-tocopherol and ascorbate at the concentrations shown. Thereafter the reaction was stopped, and the samples were subjected to agarose gel electro~ permission from phoresis (A) and assayed for TBARS activity (B). (Jialal el ~ 1 . ’With Atherosclerosis .)
inhibitor of macrophage motility it could also promote retention of macrophages in the arterial Furthermore, several lines of evidence support the in vivo existence of oxidized LDL.l0-l2Data have been presented for the occurrence of a modified form of LDL with many physical, chemical, and biological properties of Ox-LDL in arterial lesions. Also, antibodies against epitopes on Ox-LDL recognize material in athero-
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sclerotic lesions, but not normal arteries, and circulating antibodies against epitopes of Ox-LDL have been demonstrated in the plasma of Wantanabe heritable hyperlipidemic (WHHL) rabbits and humans. Additional support for the role of Ox-LDL in atherogenesis is the observation that antioxidants such as probucol and biitylated hydroxytoluene can inhibit the development of atherosclerotic lesions in WHHL-rabbits and cholesterol-fed rabbits.’3-” These agents, however, are not free from side effects, and their utility for prevention of atherosclerosis in human populations may be limited. ’‘.I7 Furthermore, because probucol has other effects. such as an inhibition of interleukin-I release and increasing the activity of cholesterol ester transfer protein, one cannot ascribe its antiatherogenic effect . ’ ~ the role of the dietary micronutrients solely io its antioxidant p r ~ p e r t y . ’ ~Hence with antioxidant properties such as ascorbate, alpha-tocopherol, and beta-carotene assumes great significance inasmuch as levels can be favorably manipulated with dietar), measures without resulting in any systemic side effects.
ANTIOXIDANT VITAMINS AND ATHEROSCLEROSIS There is limited data supporting a relationship between the antioxidant vitamins (ascorbate, alpha-tocopherol, and beta-carotene) and the development of atherosclerosis. Ascorbate is a water soluble chain-breaking antioxidant. It reacts directly with superoxide, hydroxyl radicals and singlet oxygen.’” Furthermore, it interacts with the tocopheroxy radical, resulting in the generation of tocopherol.*’ Also ic has been shown that ascorbate is the most effective antioxidant in plasma incubared with a water soluble radical initiator.:? In an epidemiological study, Gey ot ( / I . have shown a significant inverse correlation between plasma ascorbate and coronary artery disease mortality.?3Also, ascorbate levels are significantly lower in the aortas of patients with atherosclerotic vascular disease compared to control^.?^ Diabetics, smokers, and patients with coronary artery disease have lower levels of ;iscorbate.”-” Thus, all these findings raise the possibility that lower levels of riscorbate, especially in the arterial wall, play a role in the development of atherosclerosis. Alpha-tocopherol is the most active and abundant isomer of the vitamin E family. It is the principal lipid-soluble chain-breaking antioxidant in tissues and plasma and is the predominant antioxidant in the L D L parti~le.’~.’~ It functions by trapping peroxyl free radicals. In a cross-sectional study in 16 European populations, the investigators documented a significant inverse correlation between the lipid-standardized plasma alpha-tocopherol levels and coronary artery disease mortality.” Furthermore. a recent case control study showed that plasma vitamin E levels were independently and inversely related to the risk of angina p e c t ~ r i s . ~ ~ ) In addition, some studies in animal models suggest that dietary vitamin E can retard the progression of atherosclerosis.31Thus, all of the above findings would appeal to support a role for lower alpha-tocopherol levels in the genesis of the atherosclerotic lesion. Beta-carotene, a plant-derived hydrocarbon carotenoid is an efficient quencher of singlet oxygen; it can also function as a radical-trapping antioxidant at low oxygen pressures.’? The relationship between beta-carotene and atherosclerosis development has not been addressed in any detail. Smoking, a major risk factor for coronary artery disease, results in decreased levels of beta-carotene.26 Recently, it was reported in preliminary form that beta-carotene supplementation significantly reduced all major vascular events in a group of male subjects.33It is tempting to
JIALAL & GRUNDY: LDL OXIDATION
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speculate that this benefical effect of beta-carotene on atherosclerosis progression was being mediated in part by its inhibitory effect on LDL oxidation.
EFFECT OF ANTIOXIDANT VITAMINS ON LDL OXIDATION In a series of experiments we have documented that ascorbate in concentrations ranging from 40 to 80 pM can have a significant and substantial inhibitory effect
Origin
+ 1 2 3 4 5 6
30 -
40 c
c ‘5
ig $2 sz
8E
20-
10-
I-0
on LDL oxidation.34 As shown in FIGURE 2 , ascorbate inhibited the oxidative modification of LDL as evidenced by the decreased electrophoretic mobility and thiobarbituric acid reacting substances (TBARS) content of LDL.34By inhibiting LDL oxidation, ascorbate decreased subsequent uptake and degradation of
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242
Effect of Ascorbate and Probucol on the Antioxidant Content of LDL during OxidationJs
TABLE 2.
LDL
OX-LDL
OX-LDL plus ascorbate
plus probucol
OX-LDL
(50 uM)
(10 uM)
TBARS
(nmolimg protein) 1.6 37.0 3.3 Electrophoretic mobility" I 5 .o I .3 Alpha-iocopherol 3.9 N D ~ 2.1 (nmolimg protein) Gamma-tocopherol (nmolimg protein) 2.4 0.3 2. I Beta-carotene (pmolimg protein) 28.2 ND 26.9 " Electrophoretic mobility is expressed as migration relative to native LDL. ' Not detectable.
7.5 I .6
ND ND ND
[12'I]Ox-LDLby monocyte macrophages by 93 percent.34In further experiments ascorbate was found to be as potent as probucol in inhibiting copper-catalyzed LDL oxidation and its subsequent processing by macrophages.3' In a biological system of oxidation (coincubation of LDL with human macrophages). probucol and ascorbate were equipotent in inhibiting LDL oxidation (FIG.3) and its subsequent uptake by a second set of macrophages.3' Previously, it has been shown in other systems that ascorbate can reduce the tocopheroxyl radical and hence regenerate tocopherol.?' To investigate if such a situation, obtained with respect to the antioxidant effect of ascorbate on LDL, the effect of both ascorbate and probucol on the endogenous antioxidants present in the LDL particle was determined during copper-catalyzed oxidation of LDL. Oxidative modification of LDL in this system, as evidenced by the increased lipid-peroxide content, resulted in an inability to detect alpha-tocopherol and beta-carotene, and an 87.5% reduction in gamma-tocopherol content of LDL, compared to the control LDL (TABLE2 ) . Coincubation of LDL with ascorbate during similar oxidative conditions inhibited LDL oxidalion, as evidenced by the decreased TBARS activity and electrophoretic mobility, and resulted i n a preservation of the endogenous antioxidants in the LDL particle (beta-carotene 9595, gamma-tocopherol 8856, and alpha-tocopherol 69% of control). Probucol(l0 pM),however, also inhibited the oxidation of LDL to a similar degree as ascorbate but failed to preserve the endogenous antioxidants in LDL.3' This action cannot be attributed only to the ability of ascorbate to reduce the tocopheroxy radical to tocopherol, because both the tocopherols and betacarotene were preserved. Therefore, ascorbate seemingly acts at a proximal step in the oxidation process, scavenging free radical(s) and protecting the LDL particle from significant oxidative attack. In other words, ascorbate would appear to behave as a sacrificial antioxidant. In the only other study that examined the effect of ascorbate on LDL oxidation, Esterbauer et showed that ascorbate in concentrations between 2.5 and 10 pM prolonged the lag phase of LDL oxidation. Inasmuch as the concentrations of ascorbate used by these workers was at the level ai which vitamin C deficiency is diagnosed, one can speculate that if their studies had been performed with higher concentrations of ascorbate, their findings would have been more dramatic. Recently, in an in uiuo study, it was shown that vitamin C feeding decreases the susceptibility of LDL from smokers to oxidation.J7
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243
Previous studies have failed to show that beta-carotene inhibits LDL oxidaAn important point that must be considered with in uitro testing with betacarotene is its varying solubility in different solvents. Although it is readily soluble in hexane and chloroform, it is sparingly soluble in ethanol. In one of the previous studies, ethanol was used to dissolve the beta-carotene. Because these workers did not quantify the concentration or purity of their beta-carotene solution, it is possible that the beta-carotene was not adequately prepared. In our recently reported study, the beta-carotene was dissolved in both hexane and ethanol and
(A) TBARS ACTIVITY
I
35 .F 30 Ia c 25
5
0 LDL
Ox-LDL
-
0.5
1.0
2.0
Vehicle
f&Carotene (IJW
(B) ELECTROPHORESIS
0
FIGURE 4. Effect of beta-carotene on copper-catalyzed oxidation of LDL. ['251]LDL(200 pg/mL) was oxidized with 2.5 pM copper in PBS at 37" C for 24 h in the presence of beta-carotene at the concentrations shown. The reaction was stopped, and an aliquot was subjected to agarose gel electrophoresis and TBARS activity.
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244
E
0
3
W
0.40
LDL LDL + Beta-Carotene
0.35 0.30 0.25
f/J 0.10 P
LDL
a 0.05 2
4
8
8
10
12
14
18
18
20
Fraction No. FIGURE 5. Absorbance (4.50 nm) of column chromatography fractions of LDL incubated in the ahsence and presence of beta carotene. LDL ( 1 rngimL) was incubated in PBS at 37°C for 24 h in the absence and presence of beta-carotene (5.0 pM).Thereafter the samples were subjectcd 10 Sephadex G-2.5 column chromatography and the fractions monitored at 450 nM.
3.O 2.5
2.0 t
U 4
1.5 1.o
0.5
0.0
OX-LDL
OX-LDL
+
OX-LDL
+
AA
t?T ( 5 0 ~ M ) (40pM) FIGURE 6. Effect of ascorbate and alpha-tocopherol on LDL oxidation. LDL (200 pg/mL) was suh,jected to oxidation with 2.5 pM copper in PBS in the absence and presence of ascorbate and alpha-tocopherol for 24 h. Thereafter oxidation was stopped and the absorbance oft he samples read at 234 nM. A A?34denotes increase in absorbance from native LDL.
JIALAL & GRUNDY: LDL OXIDATION
245
its purity checked by HPLC.40As shown in FIGURE 4, beta-carotene in the concentrations shown clearly inhibits LDL oxidation. By inhibiting LDL oxidation, betacarotene prevented its subsequent uptake by macro phage^.^^ To determine if the beta-carotene was associated with the LDL particle, LDL was incubated with and without beta-carotene for 24 h, and the absorbance of the fractions was eluted from a Sephadex G-25 column monitored at 280 and 450 nM. As is evident from FIGURE 5, the beta-carotene appeared to be associated with the LDL particle (protein peak). Also in this study, beta-carotene inhibited macrophage modification of LDL s ~ b s t a n t i a l l yIn . ~a~ recent report, Navab er a/.'' have shown that preincubation of cocultures of aortic endothelial cells and aortic smooth muscle cells with beta-carotene and alpha-tocopherol prevented LDL modification and its induction of monocyte transmigration. In the in vitro studies examining the effect of alpha-tocopherol on LDL oxidation (FIG.2), we have shown that alpha-tocopherol (40pM)inhibits LDL oxidation only partially and is not as potent as a ~ c 0 r b a t e .This j ~ was also evident when the formation of conjugated dienes were used as an index of LDL oxidation (FIG. 6). In these studies alpha-tocopherol decreased the uptake of oxidized LDL by macrophages by 45 percent.j4 In previous studies, workers have found a more substantial inhibitory e f f e ~ t . j * .A~ 'reasonable explanation for this discrepancy is that because the previous studies used either dialysis at 4" C or human endothelial cells to modify LDL, these systems produce a milder degree ofoxidation and hence are more susceptible to the antioxidant effect of a l p h a - t o ~ o p h e r o l .Supporting ~~~~' evidence for this has recently been obtained in our laboratory in that alphatocopherol exerts a substantial inhibitory effect on macrophage modification of LDL . In a series of experiments using the prolongation of the lag phase as an index of LDL oxidation, Esterbauer r t 01.~' have shown that supplementation of plasma with alpha-tocopherol prior to isolation of LDL resulted in a fourfold enrichment of LDL with alpha-tocopherol. This enrichment of LDL resulted in a proportional increase in the lag phase of oxidation and hence increased the oxidation resistance of the LDL.42 Also in two studies in which subjects were fed alpha-tocopherol, the LDLs from these subjects were more resistant to oxidative m ~ d i f i c a t i o n . ~ ' . ~ ~ In conclusion, all three dietary micronutrients with antioxidant properties exert an inhibitory effect on LDL oxidation. Thus, ascorbate, alpha-tocopherol, and beta-carotene could have a major role in future strategies for atherosclerosis prevention.
ACKNOWLEDGMENTS The authors wish to express their gratitude to Drs. H. Bhagavan and E. L. Norkus for their discussions and scientific assistance and to R. Kent and A . Cossi for technical assistance and manuscript preparation.
REFERENCES
1983. Annu. Rev. Biochem. 52: 223-261. 1. BROWN,M. S . & J. L. GOLDSTEIN. D., S. PARTHASARATHY, T. CAREW,J . KHOO& .I.WITZTUM.1989. N . 2, STEINBERG, Engl. J . Med. 320: 915-924.
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& H. ESTERBAUER. 1987. Chem. Phys. Lipids45 3 . JIIRGENS, G., H. HOFF,G . CHISOLM 315-336. Free Radical Biol. Med. 9: 4. STEINBRECHER, U . P., H. XHANG& M. LOUGHEED. 155- 168. 1991. J. Clin. Invest. 88: 1785-1792. J. L. & D. STEINBERG. 5 . WITZTUM, A., VALENTE,M. TERRITO. M. NAVAB,F. PARHAMI, R. , J. BERLINER 6. C U S H I N GS., G F K K i i Y . C . SCHWARTZ & A. L. FOGELMAN. 1990. Proc. Natl. Acad. Sci. USA 87: 5134-5138. A. SEVANIAN, S. R A M I NJ., KIM, B. BAMSHAD, M. 7. B K R L I N E RJ ,. A , , M. TERRITO, 1990. J. Clin. Invest. 85: 1260-1266. ESTERSON & A. L. FOGELMAN. , S. IMES, S. HAMA,G. HOUGH,L. Ross, R. BORK, A . VALENTE, J. 8. N ~ V A BM., & A. L. FOGELMAN. 1991. J. Clin. Invest. 88: Bmi.iNER, D. DRINKWATER, H. LAKS 7039-2046. & D. STEINBERG. 1985. Proc. Natl. Acad. Sci. 9. Q U I N N ,M. T., S. PARTHASARATHY USA 82: 5949-5953. M.. D , D. FONG& L. CHENG.1988. Science 241: 215-218. 10. H ~ B E R L A N 1 1 . P\LlNSKI. w . , M. ROSENFELD, s. YLA-HARTTUALAL, G. CURTHER, s. SOCHER, s. BUTLER, s. PARTHASARATHY, T. C A R E W . D. STEINBERG & J. WITZTUM. 1989. ProC. Narl. Acad. Sci. USA 86: 1372-1376. 12. Y1.A-HERTTUALA. s.,w.PALINSKI, M. ROSENFELD, s. PARTHASARATHY, T. CAREW, S. BUTLER,J. WITZTUM & D. STEINBERG. 1989. J. C h . Invest. 2 8 4 1806-1095. H. YOSHIDA& 13. K I T A . ' r . , Y . NAGANO.M. YOKODE,K. ISHII, K . KUME.A . OOSHIMA, C. KAWAI.1987. Proc. Natl. Acad. Sci. USA 84: 5928-5931. T., E.. D. SCHWENKE & D. STEINBERG. 1987. Proc. Natl. Acad. Sci. USA84: 14. C ~ R E W 7725-7729. 15. B I O R L H A M , I . . A. HENRICKSSON-FREYSCHUSS, 0 . BREUER, v. DICZFALUSY, L. & P. HENRICKSON. 1991. Arteriosclerosis Thrombosis. 11: 15-22. BERGL.UND 1989. Drugs 37: 761-800. 16. f 3 I I C K L E Y . M., K. GOA,A. PRICE& E. BROGDEN. 17. HIROSE, M., M. SHIBATA. A. H A G I W A RK. A , IMAIDA& N. ITO. 1981. Food Cosrnet. Toxicol. 19: 147-151. 18. F K A N C E S C H I N I . G.. M . SIRTORI, v. VACCARINO. G . GIANFRANCESCHI, L. REZZONICO & G. C. SIRTOKI. 1989. Arteriosclerosis 9: 462-469. L. SCHMIDT & R. JACKSON.1990. FASEB J. 4: 1645-1653. 19. K C J ,G . . N . S . DOHERTY. , BURTON& D. WAYNER. 1986. Adv. 20. H I . N D I C H . A , . L. MACHLIN.0 . ~ C A N D U R R AG. Free-Radical Biol. Med. 2: 419-444. 21. NiKI. E. 1987. Chem. Phys. Lipids 44: 227-253. 22. FKEI.H., L. E N G L A N D B &. N. AMES. 1989. Proc. Natl. Acad. Sci. USA86: 6377-6381. 23. Gcu, K. F., P. PUSKA,P. JORDON& U. MOSER. Am. J. Clin. Nutr. 53: 3263334s. 24. DLJBICK. M., G. H U N T E RS., CASEY & C . K E E N .1987. Proc. Soc. Exp. Biol. Med. 184: 138-141. 25. S I A N K O V AL., , M. RIDDLE,J. L A R N E DK. , B U R R YD. , MENASHE, J. HART& R. BIGLEY. 1984. Metabolism 33: 347-353. 26. Ctiow, C. K . , C. CHANGCHIT, R . BRIDGES, S. R E H N J, . H U M B L& E J. T U R B E K1986. . J. Am. Coll. Nutr. 5: 305-312. 27. RkMIREZ, J. & N . FLOWERS.1990. Am. J. Chn. Nutr. 33: 2079-2087. 28. M A C H L I N . L. 1984. In HandbookofVitamins. L. Machlin. Ed.: 99-145. Marcel Dekker Inc. New York. 29. ECTERBAUER, H., M . ROTHENEDER, G . STIEtiL, G. WAEG,A. ASHY, SATTLER & G . JURGENS. 1989. Fat Sci Technol. 91: 316-324. R. ELTON,K. F. GEY& M. F. OLIVER. 30. R I E M E R S M A , R., D. WOOD, C. MACINTYRE, 1991. Lancet 337: 1-5. 31. JANERO, D. R. 1991. Free Rad. Biol. Med. 11: 129-144. 3 2 . KHINSKY. N . 1. 1989. Free Radical Biol. Med. 7: 617-635. J., J . MANSON, J. RIDKER, J. BURLING & C . H. HENNENKENS. 1990. Circula33. G.\ZIANO. tion 82: suppl 11. 201. 34. JI41.AI.. I . G . VEGA& S. M. GRUNDY.1990. Atherosclerosis 82: 185-191. 35. J 1 4 L A L , I. & S. M. GRUNDY.1991. J. Clin. Invest. 87: 597-601.
w.
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36. ESTERBAUER, H., G. STRIEGL, H. PUHL& M. ROTHENEDER. 1989. Free. Radical Res. Commun. 6 67-75. Y. DABACH.G . HOLLANDER, E. HAVIVA, 0. STEIN& Y. 37. HARATS,D., M. BEN-NAIM, STEIN.1990. Atherosclerosis 85: 47-54. 38. MOREL,D., J. HESSLER& G. M. CHISOLM.1983. J. Lipid Res. 24: 1070-1076. 39. VAN HINSBERG, V. W., M. SCHEFFER,L. HAVEKES & H. KEMPEN.1986. Biochim. Biophys. Acta 878: 49-64. 40. JIALAL,I., E. NORKUS,L. CRISTOL& S. M. GRUNDY.1991. Biochim. Biophys. Acta 1086 134-138. 41. STEINBRECHER. U. P.. S. PARTHASARATHY, D. LEAKE,J . WITZTUM& D. STEINBERC. 1984. Proc. Natl. Acad. Sci. USA 81: 3883-3887. 42. ESTERBAUER. H., M. ROTHENEDER, G . STRIEGL& G. WAEG.1991. Am. J. Clin. Nutr. 53: 314s-321s. 43. DIEBER-ROTHENEDER, M.. H. PUHL,G. WAEG,G. STRIEGL & H. ESTERBAUER. 1991. J. Lipid Res. 32: 1325-1332. 4.JIALALI. & S. M. GRUNDY.1992. J. Lipid Res. 33: 899-906.
DISCUSSION C. H. HENNEKENS (Harvard Medical School, Bosron, MA): Before opening the paper up for questions, Dr. Matthias Rath of the Linus Pauling Institute wants to say a few words about his recent work on vitamin C, LP(a), and cardiovascular disease. M. RATH (Linvs Pnuling Institirtr, Palo Alto, C A ) : There are a number of quesitons that are not answered by the hypothesis that oxidized LDL may be the initial event in human atherosclerosis. Above all is the question, Why does, in a vascular surface of about 1000 square meters, the system fail in very defined sites of less than a square foot-actually the coronary arteries, and cervical and cerebral arteries? If oxidized LDL is the culprit because of cytotoxicity to the endothelium, then one would postulate that the noxious effect of this particle would be particularly detrimental at the sites where the contact time between this particle and the endothelium is long and intense. This would be in the periphery of the arterial system. If LDL oxidation were the main mechanism for the initiation of human atherosclerosis, we would find peripheral vascular disease as the primary manifestation of human cardiovascular disease. We think that this mechanism may be very valid in certain cases, for example, in type-three hyperlipidemia where triglyceride-rich lipoproteins are particularly susceptible to oxidation, and also in smokers. These conditions are characterized by peripheral disease. But the general form of human cardiovascular disease, heart disease and stroke, has a different underlying pathomechanism. LP(a), is very similar to LDL but has an additional glycoprotein; we found that LP(a), is the predominant form of lipoprotein deposition in the human vascular wall. In 400 slices of coronary arteries and aortas of human beings, wherever we found the structural protein of LDL, which is Apo B, we also found the colonization in the exact same area for the Apo A, suggesting that the extracellular deposition of lipoprotein A is the predominant mechanism for human cardiovascular disease. We recently published a paper with the rather unassuming title, “Solution to the Puzzle of Human Cardiovascular Disease,” where we say that the primary cause is a vitamin C deficiency leading to the deposition of LP(a), and fibrinogen
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and fibrin in the vascular wall. What is the evolutionary advantage of LP(a), in human beings'! Why is this thing kept in human beings, especially if it is so injurious? The answer is pretty surprising. If the collagen in the blood vessel wall is weakened by vitamin C deficiency, LP(a), infiltrates the vascular wall at predisposed sites and binds to fibrinogen and fibrin there, thus strengthening the wall and preventing the increasing permeability, which is a necessary sequence of vitamin C deficiency. So, we suddenly understand why during evolution having LP(a), was an important particle. Now what's the role of vitamin C? Vitamin C lowers LP(a),, and vitamin C, above all, prevents the deposition of LP(a), in the vascular wall by stabilizing collagen in the vascular wall. In summary, we think that the stabilization of the vascular wall is much more important than most of the plasma risk factors. The fact that human beings suffer from heart attack and stroke underlines this idea. These are the places where the instability of the vascular wall is primarily unmasked: the signs of increased systolic pressure, turbulence, pulsatile blood flow in the coronary arteries and subsequently the development of cardiovascular disease occurs in very defined organs at very defined locations. HENNEKENS:Your proposed mechanism is certainly attractive and plausible, and if proven, in my view, would be complementary and not at all competing with those proposed by Dr. Jialal. I. JIALAL (.Soirth,cvstern Medical Center, D d m , T X ) :Exactly. Nobody doubts today that increased LDL cholesterol is the major mechanism for premature atherosclerosis. And, yet, we don't understand the biological mechanism. I stated that oxidation is one plausible and biologically relevant modification of LDL. In fact, there might he other modifications; for example, in the diabetic you get glycosylation of the LDL. LP(a), is also an atherogen. What we're trying to understand today is the biology by which LDL gets onto the arterial wall and causes foam cell formation. What the evidence is showing is that antioxidants might he able to interrupt this crucial step in atherogenesis. LP(a), is a new molecule, and we've got to learn a lot more about it. To the best of my knowledge, we have no measures to normalize (LP(a), in humans at this point. J. ( ' L A U S E N (Uiiiuersity ofRoskildc, D e n m n r k ) : In your supplementation study your correlation coefficient was 0.64. I think we should be very careful to conclude statistical significance with this level of correlation. Do you think we can make conclu\ions with the degree of correlation? JIALAL: The correlation between the LDL and plasma levels alpha-tocopherol was 0.79. Hermann Esterbauer got the same kind of correlation, suggesting that the lipid-standardized plasma alpha-tocopherol is a practical surrogate for LDL alpha-tocopherol. Our intervention focused on alpha-tocopherol, but numerous factors could dictate the propensity of an LDL to oxidation.
OXIDIZED LDL AND ATHEROSCLEROSIS An increased level of plasma LDL cholesterol is a major risk factor for premature atherosclerosis. The mechanism(s), however, by which LDL promotes the development of the early fatty streak lesion with its characteristic lipid-laden macrophages remains to be elucidated. Uptake of cholesterol by way of the classical LDL receptor cannot result in appreciable cholesterol accumulation because the LDL receptor is subject to feedback inhibition by the cellular cholesterol content.' Certain modified forms of LDL, for example, acetyl-LDL, are taken up by way of the scavenger receptor mechanism, resulting in substantial cholesterol accumulation and foam cell formation in macrophages because the scavenger receptor is not regulated by the cellular cholesterol content.' The most plausible and biologically relevant modification of LDL that appears to occur is oxidation.2 The free radical peroxidation of LDL lipids results in numerous structural changes, all depending on a common initiating event: the peroxidation of the polyunsaturated fatty acids on LDL.3-SLDL can be oxidatively modified in the presence of transi~ - ~ all the major cells tion metals (iron and copper) in a cell-free e n ~ i r o n m e n t . Also of the arterial wall (endothelial cells, macrophages, and smooth muscle cells) can oxidatively modify LDL. Both forms of oxidized LDL are taken up more avidly a Work cited in this review was supported by the American Heart Association (Texas Affiliate), Hoffmann-La Roche, and the Bristol-Meyers Squibb/Mead Johnson Nutrition Research Grant. 231
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238 TABLE 1.
Properties of Oxidatively Modified LDL Decreased content of polyunsaturated fatty acids Enrichment in lipid peroxides Increased negative charge Increased lysolecithin content Increased content of oxysterols Fragmentation of apoprotein B-100 Decreased uptake by LDL receptor Increased uptake by scavenger receptor
by macrophages than LDL and appear to be processed by the scavenger receptor mechanism.'-'Some of the more important properties of oxidized LDL are shown in TABL E I . The biological effects of oxidized LDL reported to date could contribute to initiation and progression of the atherosclerotic process (FIG.1). The cytotoxicity of Ox-LDL may be important in inducing endothelial cell dysfunction and/ or promoting the evolution of the fatty streak to a more complex and advanced lesion.?-' Ox-LDL is a potent chemoattractant for the circulating monocyte but not neutrophils.2-5In fact, it has been postulated that in the early phase of oxidation there are formed in the subendothelial space minimally modified LDL (MM-LDL) that are mildly oxidized.',' Once formed MM-LDL could induce the endothelium to express adhesion molecules for monocytes, and to secrete monocyte chemotactic protein (MCP-I ) and macrophage colony-stimulating factors.',' These molecular events result in monocyte binding to the endotheliun and its subsequent migration
A
R T E R > Y W A L L
Foam Cell
FIGURE 1. Schema depicting the relationship between oxidized LDL and the genesis of the fatty streak lesion. (Based on the studies of Fogelman et and Steinberg et a / . ? ) ~
1
.
~
3
~
JIALAL & GRUNDY: LDL OXIDATION
239
into the subendothelial space, where MM-LDL promotes differentiation into tissue macrophages. The macrophages then further modify MM-LDL to a more oxidized form.8 This Ox-LDL can then be processed by way of the scavenger receptor mechanism leading to cholesterol ester accumulation. Because Ox-LDL is a potent
(A)
OR% 1
2
3
5
4
6
L
ox-LC + Vit
_ -
T
-
I
ox-LC L OX-LDL OX-LDL + Vit 1 + Vit C + Vit C
FIGURE 2. Effect of ascorbate and alpha-tocopherol on the 24th oxidative modification of LDL. LDL (200 pg/mL) was subjected to a 24-h oxidation with 2.5 pM copper in PBS in the absence and presence of alpha-tocopherol and ascorbate at the concentrations shown. Thereafter the reaction was stopped, and the samples were subjected to agarose gel electro~ permission from phoresis (A) and assayed for TBARS activity (B). (Jialal el ~ 1 . ’With Atherosclerosis .)
inhibitor of macrophage motility it could also promote retention of macrophages in the arterial Furthermore, several lines of evidence support the in vivo existence of oxidized LDL.l0-l2Data have been presented for the occurrence of a modified form of LDL with many physical, chemical, and biological properties of Ox-LDL in arterial lesions. Also, antibodies against epitopes on Ox-LDL recognize material in athero-
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sclerotic lesions, but not normal arteries, and circulating antibodies against epitopes of Ox-LDL have been demonstrated in the plasma of Wantanabe heritable hyperlipidemic (WHHL) rabbits and humans. Additional support for the role of Ox-LDL in atherogenesis is the observation that antioxidants such as probucol and biitylated hydroxytoluene can inhibit the development of atherosclerotic lesions in WHHL-rabbits and cholesterol-fed rabbits.’3-” These agents, however, are not free from side effects, and their utility for prevention of atherosclerosis in human populations may be limited. ’‘.I7 Furthermore, because probucol has other effects. such as an inhibition of interleukin-I release and increasing the activity of cholesterol ester transfer protein, one cannot ascribe its antiatherogenic effect . ’ ~ the role of the dietary micronutrients solely io its antioxidant p r ~ p e r t y . ’ ~Hence with antioxidant properties such as ascorbate, alpha-tocopherol, and beta-carotene assumes great significance inasmuch as levels can be favorably manipulated with dietar), measures without resulting in any systemic side effects.
ANTIOXIDANT VITAMINS AND ATHEROSCLEROSIS There is limited data supporting a relationship between the antioxidant vitamins (ascorbate, alpha-tocopherol, and beta-carotene) and the development of atherosclerosis. Ascorbate is a water soluble chain-breaking antioxidant. It reacts directly with superoxide, hydroxyl radicals and singlet oxygen.’” Furthermore, it interacts with the tocopheroxy radical, resulting in the generation of tocopherol.*’ Also ic has been shown that ascorbate is the most effective antioxidant in plasma incubared with a water soluble radical initiator.:? In an epidemiological study, Gey ot ( / I . have shown a significant inverse correlation between plasma ascorbate and coronary artery disease mortality.?3Also, ascorbate levels are significantly lower in the aortas of patients with atherosclerotic vascular disease compared to control^.?^ Diabetics, smokers, and patients with coronary artery disease have lower levels of ;iscorbate.”-” Thus, all these findings raise the possibility that lower levels of riscorbate, especially in the arterial wall, play a role in the development of atherosclerosis. Alpha-tocopherol is the most active and abundant isomer of the vitamin E family. It is the principal lipid-soluble chain-breaking antioxidant in tissues and plasma and is the predominant antioxidant in the L D L parti~le.’~.’~ It functions by trapping peroxyl free radicals. In a cross-sectional study in 16 European populations, the investigators documented a significant inverse correlation between the lipid-standardized plasma alpha-tocopherol levels and coronary artery disease mortality.” Furthermore. a recent case control study showed that plasma vitamin E levels were independently and inversely related to the risk of angina p e c t ~ r i s . ~ ~ ) In addition, some studies in animal models suggest that dietary vitamin E can retard the progression of atherosclerosis.31Thus, all of the above findings would appeal to support a role for lower alpha-tocopherol levels in the genesis of the atherosclerotic lesion. Beta-carotene, a plant-derived hydrocarbon carotenoid is an efficient quencher of singlet oxygen; it can also function as a radical-trapping antioxidant at low oxygen pressures.’? The relationship between beta-carotene and atherosclerosis development has not been addressed in any detail. Smoking, a major risk factor for coronary artery disease, results in decreased levels of beta-carotene.26 Recently, it was reported in preliminary form that beta-carotene supplementation significantly reduced all major vascular events in a group of male subjects.33It is tempting to
JIALAL & GRUNDY: LDL OXIDATION
241
speculate that this benefical effect of beta-carotene on atherosclerosis progression was being mediated in part by its inhibitory effect on LDL oxidation.
EFFECT OF ANTIOXIDANT VITAMINS ON LDL OXIDATION In a series of experiments we have documented that ascorbate in concentrations ranging from 40 to 80 pM can have a significant and substantial inhibitory effect
Origin
+ 1 2 3 4 5 6
30 -
40 c
c ‘5
ig $2 sz
8E
20-
10-
I-0
on LDL oxidation.34 As shown in FIGURE 2 , ascorbate inhibited the oxidative modification of LDL as evidenced by the decreased electrophoretic mobility and thiobarbituric acid reacting substances (TBARS) content of LDL.34By inhibiting LDL oxidation, ascorbate decreased subsequent uptake and degradation of
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242
Effect of Ascorbate and Probucol on the Antioxidant Content of LDL during OxidationJs
TABLE 2.
LDL
OX-LDL
OX-LDL plus ascorbate
plus probucol
OX-LDL
(50 uM)
(10 uM)
TBARS
(nmolimg protein) 1.6 37.0 3.3 Electrophoretic mobility" I 5 .o I .3 Alpha-iocopherol 3.9 N D ~ 2.1 (nmolimg protein) Gamma-tocopherol (nmolimg protein) 2.4 0.3 2. I Beta-carotene (pmolimg protein) 28.2 ND 26.9 " Electrophoretic mobility is expressed as migration relative to native LDL. ' Not detectable.
7.5 I .6
ND ND ND
[12'I]Ox-LDLby monocyte macrophages by 93 percent.34In further experiments ascorbate was found to be as potent as probucol in inhibiting copper-catalyzed LDL oxidation and its subsequent processing by macrophages.3' In a biological system of oxidation (coincubation of LDL with human macrophages). probucol and ascorbate were equipotent in inhibiting LDL oxidation (FIG.3) and its subsequent uptake by a second set of macrophages.3' Previously, it has been shown in other systems that ascorbate can reduce the tocopheroxyl radical and hence regenerate tocopherol.?' To investigate if such a situation, obtained with respect to the antioxidant effect of ascorbate on LDL, the effect of both ascorbate and probucol on the endogenous antioxidants present in the LDL particle was determined during copper-catalyzed oxidation of LDL. Oxidative modification of LDL in this system, as evidenced by the increased lipid-peroxide content, resulted in an inability to detect alpha-tocopherol and beta-carotene, and an 87.5% reduction in gamma-tocopherol content of LDL, compared to the control LDL (TABLE2 ) . Coincubation of LDL with ascorbate during similar oxidative conditions inhibited LDL oxidalion, as evidenced by the decreased TBARS activity and electrophoretic mobility, and resulted i n a preservation of the endogenous antioxidants in the LDL particle (beta-carotene 9595, gamma-tocopherol 8856, and alpha-tocopherol 69% of control). Probucol(l0 pM),however, also inhibited the oxidation of LDL to a similar degree as ascorbate but failed to preserve the endogenous antioxidants in LDL.3' This action cannot be attributed only to the ability of ascorbate to reduce the tocopheroxy radical to tocopherol, because both the tocopherols and betacarotene were preserved. Therefore, ascorbate seemingly acts at a proximal step in the oxidation process, scavenging free radical(s) and protecting the LDL particle from significant oxidative attack. In other words, ascorbate would appear to behave as a sacrificial antioxidant. In the only other study that examined the effect of ascorbate on LDL oxidation, Esterbauer et showed that ascorbate in concentrations between 2.5 and 10 pM prolonged the lag phase of LDL oxidation. Inasmuch as the concentrations of ascorbate used by these workers was at the level ai which vitamin C deficiency is diagnosed, one can speculate that if their studies had been performed with higher concentrations of ascorbate, their findings would have been more dramatic. Recently, in an in uiuo study, it was shown that vitamin C feeding decreases the susceptibility of LDL from smokers to oxidation.J7
JIALAL & GRUNDY: LDL OXIDATION
243
Previous studies have failed to show that beta-carotene inhibits LDL oxidaAn important point that must be considered with in uitro testing with betacarotene is its varying solubility in different solvents. Although it is readily soluble in hexane and chloroform, it is sparingly soluble in ethanol. In one of the previous studies, ethanol was used to dissolve the beta-carotene. Because these workers did not quantify the concentration or purity of their beta-carotene solution, it is possible that the beta-carotene was not adequately prepared. In our recently reported study, the beta-carotene was dissolved in both hexane and ethanol and
(A) TBARS ACTIVITY
I
35 .F 30 Ia c 25
5
0 LDL
Ox-LDL
-
0.5
1.0
2.0
Vehicle
f&Carotene (IJW
(B) ELECTROPHORESIS
0
FIGURE 4. Effect of beta-carotene on copper-catalyzed oxidation of LDL. ['251]LDL(200 pg/mL) was oxidized with 2.5 pM copper in PBS at 37" C for 24 h in the presence of beta-carotene at the concentrations shown. The reaction was stopped, and an aliquot was subjected to agarose gel electrophoresis and TBARS activity.
ANNALS NEW YORK ACADEMY OF SCIENCES
244
E
0
3
W
0.40
LDL LDL + Beta-Carotene
0.35 0.30 0.25
f/J 0.10 P
LDL
a 0.05 2
4
8
8
10
12
14
18
18
20
Fraction No. FIGURE 5. Absorbance (4.50 nm) of column chromatography fractions of LDL incubated in the ahsence and presence of beta carotene. LDL ( 1 rngimL) was incubated in PBS at 37°C for 24 h in the absence and presence of beta-carotene (5.0 pM).Thereafter the samples were subjectcd 10 Sephadex G-2.5 column chromatography and the fractions monitored at 450 nM.
3.O 2.5
2.0 t
U 4
1.5 1.o
0.5
0.0
OX-LDL
OX-LDL
+
OX-LDL
+
AA
t?T ( 5 0 ~ M ) (40pM) FIGURE 6. Effect of ascorbate and alpha-tocopherol on LDL oxidation. LDL (200 pg/mL) was suh,jected to oxidation with 2.5 pM copper in PBS in the absence and presence of ascorbate and alpha-tocopherol for 24 h. Thereafter oxidation was stopped and the absorbance oft he samples read at 234 nM. A A?34denotes increase in absorbance from native LDL.
JIALAL & GRUNDY: LDL OXIDATION
245
its purity checked by HPLC.40As shown in FIGURE 4, beta-carotene in the concentrations shown clearly inhibits LDL oxidation. By inhibiting LDL oxidation, betacarotene prevented its subsequent uptake by macro phage^.^^ To determine if the beta-carotene was associated with the LDL particle, LDL was incubated with and without beta-carotene for 24 h, and the absorbance of the fractions was eluted from a Sephadex G-25 column monitored at 280 and 450 nM. As is evident from FIGURE 5, the beta-carotene appeared to be associated with the LDL particle (protein peak). Also in this study, beta-carotene inhibited macrophage modification of LDL s ~ b s t a n t i a l l yIn . ~a~ recent report, Navab er a/.'' have shown that preincubation of cocultures of aortic endothelial cells and aortic smooth muscle cells with beta-carotene and alpha-tocopherol prevented LDL modification and its induction of monocyte transmigration. In the in vitro studies examining the effect of alpha-tocopherol on LDL oxidation (FIG.2), we have shown that alpha-tocopherol (40pM)inhibits LDL oxidation only partially and is not as potent as a ~ c 0 r b a t e .This j ~ was also evident when the formation of conjugated dienes were used as an index of LDL oxidation (FIG. 6). In these studies alpha-tocopherol decreased the uptake of oxidized LDL by macrophages by 45 percent.j4 In previous studies, workers have found a more substantial inhibitory e f f e ~ t . j * .A~ 'reasonable explanation for this discrepancy is that because the previous studies used either dialysis at 4" C or human endothelial cells to modify LDL, these systems produce a milder degree ofoxidation and hence are more susceptible to the antioxidant effect of a l p h a - t o ~ o p h e r o l .Supporting ~~~~' evidence for this has recently been obtained in our laboratory in that alphatocopherol exerts a substantial inhibitory effect on macrophage modification of LDL . In a series of experiments using the prolongation of the lag phase as an index of LDL oxidation, Esterbauer r t 01.~' have shown that supplementation of plasma with alpha-tocopherol prior to isolation of LDL resulted in a fourfold enrichment of LDL with alpha-tocopherol. This enrichment of LDL resulted in a proportional increase in the lag phase of oxidation and hence increased the oxidation resistance of the LDL.42 Also in two studies in which subjects were fed alpha-tocopherol, the LDLs from these subjects were more resistant to oxidative m ~ d i f i c a t i o n . ~ ' . ~ ~ In conclusion, all three dietary micronutrients with antioxidant properties exert an inhibitory effect on LDL oxidation. Thus, ascorbate, alpha-tocopherol, and beta-carotene could have a major role in future strategies for atherosclerosis prevention.
ACKNOWLEDGMENTS The authors wish to express their gratitude to Drs. H. Bhagavan and E. L. Norkus for their discussions and scientific assistance and to R. Kent and A . Cossi for technical assistance and manuscript preparation.
REFERENCES
1983. Annu. Rev. Biochem. 52: 223-261. 1. BROWN,M. S . & J. L. GOLDSTEIN. D., S. PARTHASARATHY, T. CAREW,J . KHOO& .I.WITZTUM.1989. N . 2, STEINBERG, Engl. J . Med. 320: 915-924.
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& H. ESTERBAUER. 1987. Chem. Phys. Lipids45 3 . JIIRGENS, G., H. HOFF,G . CHISOLM 315-336. Free Radical Biol. Med. 9: 4. STEINBRECHER, U . P., H. XHANG& M. LOUGHEED. 155- 168. 1991. J. Clin. Invest. 88: 1785-1792. J. L. & D. STEINBERG. 5 . WITZTUM, A., VALENTE,M. TERRITO. M. NAVAB,F. PARHAMI, R. , J. BERLINER 6. C U S H I N GS., G F K K i i Y . C . SCHWARTZ & A. L. FOGELMAN. 1990. Proc. Natl. Acad. Sci. USA 87: 5134-5138. A. SEVANIAN, S. R A M I NJ., KIM, B. BAMSHAD, M. 7. B K R L I N E RJ ,. A , , M. TERRITO, 1990. J. Clin. Invest. 85: 1260-1266. ESTERSON & A. L. FOGELMAN. , S. IMES, S. HAMA,G. HOUGH,L. Ross, R. BORK, A . VALENTE, J. 8. N ~ V A BM., & A. L. FOGELMAN. 1991. J. Clin. Invest. 88: Bmi.iNER, D. DRINKWATER, H. LAKS 7039-2046. & D. STEINBERG. 1985. Proc. Natl. Acad. Sci. 9. Q U I N N ,M. T., S. PARTHASARATHY USA 82: 5949-5953. M.. D , D. FONG& L. CHENG.1988. Science 241: 215-218. 10. H ~ B E R L A N 1 1 . P\LlNSKI. w . , M. ROSENFELD, s. YLA-HARTTUALAL, G. CURTHER, s. SOCHER, s. BUTLER, s. PARTHASARATHY, T. C A R E W . D. STEINBERG & J. WITZTUM. 1989. ProC. Narl. Acad. Sci. USA 86: 1372-1376. 12. Y1.A-HERTTUALA. s.,w.PALINSKI, M. ROSENFELD, s. PARTHASARATHY, T. CAREW, S. BUTLER,J. WITZTUM & D. STEINBERG. 1989. J. C h . Invest. 2 8 4 1806-1095. H. YOSHIDA& 13. K I T A . ' r . , Y . NAGANO.M. YOKODE,K. ISHII, K . KUME.A . OOSHIMA, C. KAWAI.1987. Proc. Natl. Acad. Sci. USA 84: 5928-5931. T., E.. D. SCHWENKE & D. STEINBERG. 1987. Proc. Natl. Acad. Sci. USA84: 14. C ~ R E W 7725-7729. 15. B I O R L H A M , I . . A. HENRICKSSON-FREYSCHUSS, 0 . BREUER, v. DICZFALUSY, L. & P. HENRICKSON. 1991. Arteriosclerosis Thrombosis. 11: 15-22. BERGL.UND 1989. Drugs 37: 761-800. 16. f 3 I I C K L E Y . M., K. GOA,A. PRICE& E. BROGDEN. 17. HIROSE, M., M. SHIBATA. A. H A G I W A RK. A , IMAIDA& N. ITO. 1981. Food Cosrnet. Toxicol. 19: 147-151. 18. F K A N C E S C H I N I . G.. M . SIRTORI, v. VACCARINO. G . GIANFRANCESCHI, L. REZZONICO & G. C. SIRTOKI. 1989. Arteriosclerosis 9: 462-469. L. SCHMIDT & R. JACKSON.1990. FASEB J. 4: 1645-1653. 19. K C J ,G . . N . S . DOHERTY. , BURTON& D. WAYNER. 1986. Adv. 20. H I . N D I C H . A , . L. MACHLIN.0 . ~ C A N D U R R AG. Free-Radical Biol. Med. 2: 419-444. 21. NiKI. E. 1987. Chem. Phys. Lipids 44: 227-253. 22. FKEI.H., L. E N G L A N D B &. N. AMES. 1989. Proc. Natl. Acad. Sci. USA86: 6377-6381. 23. Gcu, K. F., P. PUSKA,P. JORDON& U. MOSER. Am. J. Clin. Nutr. 53: 3263334s. 24. DLJBICK. M., G. H U N T E RS., CASEY & C . K E E N .1987. Proc. Soc. Exp. Biol. Med. 184: 138-141. 25. S I A N K O V AL., , M. RIDDLE,J. L A R N E DK. , B U R R YD. , MENASHE, J. HART& R. BIGLEY. 1984. Metabolism 33: 347-353. 26. Ctiow, C. K . , C. CHANGCHIT, R . BRIDGES, S. R E H N J, . H U M B L& E J. T U R B E K1986. . J. Am. Coll. Nutr. 5: 305-312. 27. RkMIREZ, J. & N . FLOWERS.1990. Am. J. Chn. Nutr. 33: 2079-2087. 28. M A C H L I N . L. 1984. In HandbookofVitamins. L. Machlin. Ed.: 99-145. Marcel Dekker Inc. New York. 29. ECTERBAUER, H., M . ROTHENEDER, G . STIEtiL, G. WAEG,A. ASHY, SATTLER & G . JURGENS. 1989. Fat Sci Technol. 91: 316-324. R. ELTON,K. F. GEY& M. F. OLIVER. 30. R I E M E R S M A , R., D. WOOD, C. MACINTYRE, 1991. Lancet 337: 1-5. 31. JANERO, D. R. 1991. Free Rad. Biol. Med. 11: 129-144. 3 2 . KHINSKY. N . 1. 1989. Free Radical Biol. Med. 7: 617-635. J., J . MANSON, J. RIDKER, J. BURLING & C . H. HENNENKENS. 1990. Circula33. G.\ZIANO. tion 82: suppl 11. 201. 34. JI41.AI.. I . G . VEGA& S. M. GRUNDY.1990. Atherosclerosis 82: 185-191. 35. J 1 4 L A L , I. & S. M. GRUNDY.1991. J. Clin. Invest. 87: 597-601.
w.
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36. ESTERBAUER, H., G. STRIEGL, H. PUHL& M. ROTHENEDER. 1989. Free. Radical Res. Commun. 6 67-75. Y. DABACH.G . HOLLANDER, E. HAVIVA, 0. STEIN& Y. 37. HARATS,D., M. BEN-NAIM, STEIN.1990. Atherosclerosis 85: 47-54. 38. MOREL,D., J. HESSLER& G. M. CHISOLM.1983. J. Lipid Res. 24: 1070-1076. 39. VAN HINSBERG, V. W., M. SCHEFFER,L. HAVEKES & H. KEMPEN.1986. Biochim. Biophys. Acta 878: 49-64. 40. JIALAL,I., E. NORKUS,L. CRISTOL& S. M. GRUNDY.1991. Biochim. Biophys. Acta 1086 134-138. 41. STEINBRECHER. U. P.. S. PARTHASARATHY, D. LEAKE,J . WITZTUM& D. STEINBERC. 1984. Proc. Natl. Acad. Sci. USA 81: 3883-3887. 42. ESTERBAUER. H., M. ROTHENEDER, G . STRIEGL& G. WAEG.1991. Am. J. Clin. Nutr. 53: 314s-321s. 43. DIEBER-ROTHENEDER, M.. H. PUHL,G. WAEG,G. STRIEGL & H. ESTERBAUER. 1991. J. Lipid Res. 32: 1325-1332. 4.JIALALI. & S. M. GRUNDY.1992. J. Lipid Res. 33: 899-906.
DISCUSSION C. H. HENNEKENS (Harvard Medical School, Bosron, MA): Before opening the paper up for questions, Dr. Matthias Rath of the Linus Pauling Institute wants to say a few words about his recent work on vitamin C, LP(a), and cardiovascular disease. M. RATH (Linvs Pnuling Institirtr, Palo Alto, C A ) : There are a number of quesitons that are not answered by the hypothesis that oxidized LDL may be the initial event in human atherosclerosis. Above all is the question, Why does, in a vascular surface of about 1000 square meters, the system fail in very defined sites of less than a square foot-actually the coronary arteries, and cervical and cerebral arteries? If oxidized LDL is the culprit because of cytotoxicity to the endothelium, then one would postulate that the noxious effect of this particle would be particularly detrimental at the sites where the contact time between this particle and the endothelium is long and intense. This would be in the periphery of the arterial system. If LDL oxidation were the main mechanism for the initiation of human atherosclerosis, we would find peripheral vascular disease as the primary manifestation of human cardiovascular disease. We think that this mechanism may be very valid in certain cases, for example, in type-three hyperlipidemia where triglyceride-rich lipoproteins are particularly susceptible to oxidation, and also in smokers. These conditions are characterized by peripheral disease. But the general form of human cardiovascular disease, heart disease and stroke, has a different underlying pathomechanism. LP(a), is very similar to LDL but has an additional glycoprotein; we found that LP(a), is the predominant form of lipoprotein deposition in the human vascular wall. In 400 slices of coronary arteries and aortas of human beings, wherever we found the structural protein of LDL, which is Apo B, we also found the colonization in the exact same area for the Apo A, suggesting that the extracellular deposition of lipoprotein A is the predominant mechanism for human cardiovascular disease. We recently published a paper with the rather unassuming title, “Solution to the Puzzle of Human Cardiovascular Disease,” where we say that the primary cause is a vitamin C deficiency leading to the deposition of LP(a), and fibrinogen
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and fibrin in the vascular wall. What is the evolutionary advantage of LP(a), in human beings'! Why is this thing kept in human beings, especially if it is so injurious? The answer is pretty surprising. If the collagen in the blood vessel wall is weakened by vitamin C deficiency, LP(a), infiltrates the vascular wall at predisposed sites and binds to fibrinogen and fibrin there, thus strengthening the wall and preventing the increasing permeability, which is a necessary sequence of vitamin C deficiency. So, we suddenly understand why during evolution having LP(a), was an important particle. Now what's the role of vitamin C? Vitamin C lowers LP(a),, and vitamin C, above all, prevents the deposition of LP(a), in the vascular wall by stabilizing collagen in the vascular wall. In summary, we think that the stabilization of the vascular wall is much more important than most of the plasma risk factors. The fact that human beings suffer from heart attack and stroke underlines this idea. These are the places where the instability of the vascular wall is primarily unmasked: the signs of increased systolic pressure, turbulence, pulsatile blood flow in the coronary arteries and subsequently the development of cardiovascular disease occurs in very defined organs at very defined locations. HENNEKENS:Your proposed mechanism is certainly attractive and plausible, and if proven, in my view, would be complementary and not at all competing with those proposed by Dr. Jialal. I. JIALAL (.Soirth,cvstern Medical Center, D d m , T X ) :Exactly. Nobody doubts today that increased LDL cholesterol is the major mechanism for premature atherosclerosis. And, yet, we don't understand the biological mechanism. I stated that oxidation is one plausible and biologically relevant modification of LDL. In fact, there might he other modifications; for example, in the diabetic you get glycosylation of the LDL. LP(a), is also an atherogen. What we're trying to understand today is the biology by which LDL gets onto the arterial wall and causes foam cell formation. What the evidence is showing is that antioxidants might he able to interrupt this crucial step in atherogenesis. LP(a), is a new molecule, and we've got to learn a lot more about it. To the best of my knowledge, we have no measures to normalize (LP(a), in humans at this point. J. ( ' L A U S E N (Uiiiuersity ofRoskildc, D e n m n r k ) : In your supplementation study your correlation coefficient was 0.64. I think we should be very careful to conclude statistical significance with this level of correlation. Do you think we can make conclu\ions with the degree of correlation? JIALAL: The correlation between the LDL and plasma levels alpha-tocopherol was 0.79. Hermann Esterbauer got the same kind of correlation, suggesting that the lipid-standardized plasma alpha-tocopherol is a practical surrogate for LDL alpha-tocopherol. Our intervention focused on alpha-tocopherol, but numerous factors could dictate the propensity of an LDL to oxidation.
