role of lipid peroxidation and antioxidant in LDL

Free Radical Biology & Medicine, Vol. 13, pp. 341-390, 1992 0891-5849/92 $5.00 + .00 Printed in the USA. All rights reserved. Copyright © 1992 Pergamon Press Ltd.

Review Article

THE ROLE OF LIPID PEROXIDATION AND ANTIOXIDANTS IN OXIDATIVE MODIFICATION OF LDL

HERMANN ESTERBAUER,* JANUSZ GEBICKI, t HERBERT PUHL,* a n d GUNTHER J(IRGENS ¢

*Institute of Biochemistry, University of Graz, Schubertstrasse 1, A-8010 Graz, Austria; *School of Biological Sciences, Macquarie University, Sydney, Australia; and *Institute of Medical Biochemistry,

University of Graz, Harrachgasse 21, A-8010 Graz, Austria

(Received 3 December 1991; Revised and Accepted 30 March 1992)

Abstract - -The purpose of this study is to provide a comprehensive survey on the compositional properties of LDL (e.g., lipid classes, fatty acids, antioxidants) relevant for its susceptibility to oxidation, on the mechanism and kinetics of LDL oxidation, and on the chemical and physico-chemical properties of LDL oxidized by exposure to copper ions. Studies on the occurrence of oxidized LDL in plasma, arteries, and plaques of humans and experimental animals are discussed with particular focus on the use of poly- and monoclonal antibodies for immunochemical demonstration of apolipoprotein B modifications characteristic for lipid peroxidation. Apart from uptake of oxidized LDL by macrophages, studies describing biological effects of heavily or minimally oxidized LDL are only briefly addressed, since several reviews dealing with this subject were recently published. This article is concluded with a section on the role of natural and synthetic antioxidants in protecting LDL against oxidation, as well as some previously unpublished material from our laboratories.

Keywords--Low density lipoprotein, LDL, Lipid peroxidation, Free radicals, Antioxidants, Vitamin E, Atherosclerosis

INTRODUCTION

Atherosc le ros i s is no t a t r ivial or rare disease: A b o u t half of all people enjoying a Western lifestyle are

Address correspondence to: Prof. Dr. Hermann Esterbauer. Hermann Esterbauer, PhD, is a Professor of Biochemistry at the

University of Graz, Austria. He was trained in chemistry and biology at the Universities of Vienna and Graz, and graduated with a PhD in 1963. He did postdoctoral work (1973-1974) at the Univer- sity of Pittsburgh and at the Michigan State University and was visiting Professor at the Universities of Turin (1984-1988) and Siena (1989) and at the Brunel University (1987-1991).

Janusz Gebicki, PhD, is Associate Professor in Biology at the Macquarie University, Sydney. He studied chemistry at the Univer- sity of London, where he gained the BSc and PhD degrees. He subsequently worked at McMaster University, Hamilton, Canada, at the Washington University School of Medicine in St. Louis, Mis- souri, and at the Brookhaven National University, near New York. He was appointed to Macquarie University, Sydney, after a period as Research Fellow at the Australian National University.

Herbert Puhl, PhD, is a postdoctoral fellow at the Institute of Biochemistry, University of Graz. He studied biology and chemistry and gained his PhD in 1992 from the University of Graz.

Giinther Jiirgens, PhD, is Associate Professor of Biochemistry at the Institute of Medical Biochemistry, Medical School, University of Graz. He studied chemistry, performed his thesis in physical chemistry and biochemistry, and graduated with a PhD at the Uni- versity of Graz in 1974.

currently dying of myocardial infarcts or strokes caused by sudden damming of arteries narrowed by atherosclerotic plaques. Until recently, only the man- ifestations of the disease and its consequences have been studied extensively, with the underlying biochemical mechanism of atherogenesis largely unknown. However, a series of separate excellent studies carried out mainly in the last decade (Table 1) have provided the background allowing the formulation of a new reasonable theory of atherogenesis which has focused much of current research on tests of its validity.

The bare bones of this recent postulate is that atherosclerotic plaques form from cells engorged with lipids supplied by blood lipoproteins, modified by a free radical process. Pathological, microscopic, histochemical, and biochemical studies have shown that the oc- clusions and plaques which form in the intima regions of the major arteries are mainly made up of cells so altered in appearance by internalized lipids that they are known as foam cells. 1-~° Foam cells were identified as macrophages derived from monocytes circulating in the blood 7"s'13 and smooth muscle cells proliferating in the region of the plaque. 4'13 Their gross alter-

341

342 H. ESTERBAUER ~'t al.

Table 1. Observations which Provided the Basis of the Theory of Atherogenesis

Year Observation Reference

1910 1952 1954 1961

1971 1973

1973 1976

1976 1977 1979

1980

1981 1983 1983 1983 1984 1984 1984 1984 1985 1985 1986 1986 1986 1987 1987 1987 1988 1988 1988 1988 1989 1989

1989 1989 1989 1989 1989 1989 1989

1989 1989

1990

1990 1990 1990 1990 1990 1991 1992

Cholesterol found in atherosclerotic plaques Oxidized lipids in plaques High LDL levels in accelerated atherosclerosis Lipid laden plaque cells are mainly smooth muscle cells (SMC) and macrophages (MPH)

Plaques contain foam cells LDL is main supplier of cell cholesterol

Oxidized lipids in plaques not produced by enzymic oxidation Some foam cells are MPH derived from monocytes

LDL apo B protein found in plaques Normal LDL uptake by human fibroblasts is receptor regulated Acetylated and maleylated LDL uptake by MPH and other cells via scavenger pathway gives

massive cholesterol deposition Uptake of malonaldehyde-modified LDL by MPH leads to cholesterol deposition--lipid peroxides

may be the cause Endothelial cells (EC) modify LDL to a form (mod-LDL) recognised by MPH acetyl-LDL receptor Modification of apo B lysine allows binding to acetyl-LDL receptor MPH and neutrophils oxidize LDL by a free radical mechanism LDL oxidized by free radicals is toxic to human skin fibroblasts Effect of hydroperoxides on uptake of LDL by SMC SMC produce mod-LDL in presence of metals SMC and EC produce mod-LDL by free radical oxidation of lipid Modification of LDL by EC-involves lipid peroxidation Activated monocytes and neutrophils produce oxidized LDL (oLDL) Phospholipase A 2 and free radicals from EC produce mod-LDL Superoxide free radical from SMC gives mod-LDL LDL oxidized by MPH is recognised by their scavenger receptors EC modification of LDL requires viable cells, H202 or superoxide radicals not involved Inverse relation between plasma antioxidants and IHD Oxidaton of LDL alters particle composition and loss of antioxidants Free radicals from thiols produce oLDL Mod-LDL found in human plasma Lipoxygenase with phospholipase A 2 produce mod-LDL oLDL produced by superoxide from EC and SMC Lipid peroxidation and EC injury (review) Monoclonal antibodies reveal oLDL in rabbit aorta lesions Different uptake of acetylated LDL and oLDL by MPH

Involvement of EC lipoxygenase in formation of oLDL Lipid peroxidation products derivatize apo B Cigarette smoking renders LDL susceptible to oxidation Cytotoxic oLDL produced by superoxide from activated monocytes LDL modified by HNE phagocytosed by MPH LDL protected by lipophilic antioxidants Oxidized LDL produced by hydroxyl and perhydroxyl, but not superoxide free radicals, not

recognized by MPH scavenger receptor Monoclonal antibodies to oLDL or HNE-LDL recognize material in plaques Plasma lipid peroxide levels elevated in ischemic heart and peripheral arterial diseases

Staining of lesions with several antibodies against epitopes characteristic for oLDL

Antibody to HNE-LDL recognizes copper-oxidized LDL, VLDL, and Lp(a) Lipid free radicals found during copper oxidation of LDL 15-1ipoxygenase and oLDL co-localized in plaques Minimally oxidized LDL stimulates leukocyte endothelial interaction Characterization of MPH scavenger receptor Kupfer cells have an oLDL receptor Titers of autoantibodies against oLDL correlate with progression of carotid atherosclerosis

Windaus (12) Glavind (33) Gofman (1, 2) Geer (4), Ross (13) Cookson (9) Bailey (3), Goldstein (10) Harland (16) Adams (5), Schaffner (6), Gerrity (7, 8) Goldstein (10) Goldstein (10) Goldstein ( 11 )

Fogelman (14)

Henriksen (15) Brown (18) Morel (19) Morel (20) Nishigaki (31 ) Heinecke (21 ) Morel (22) Steinbrecher (23) Cathcart (24) Parthasarathy (25) Heinecke (26) Parthasarathy (27) van Hinsbergh (28) Gey (48) Esterbauer (29) Parthasarathy (30) Avogaro (32) Sparrow (34) Steinbrecher (35) Hennig (47) Boyd (37) Arai (36), Sparrow (45) Parthasarathy (44) Steinbrecher (46) Harats (42) Cathcart (38) Hoff(39) Esterbauer (40) Bedwell (41)

Palinski (43) Stringer (105), Domagala (109) Palinski (51 ) Rosenfeld (136) Jtirgens (49) Kalyanaraman (50) Yl~i-Herttuala (53) Berliner (271 ) Rohrer (52) van Berkel (55) Salonen (54)

Oxidation of LDL 343

ation is mainly caused by the entry of lipids (e.g., lipoprotein particles modified in or near the artery). These particles bypass the normal tight control exer- cised by the cells' surface receptors and enter the cells by a different, scavenger pathway, which has no such control, t o,1 l,~a There is much evidence (for review see Ref. 57) that the principal lipoproteins susceptible to the modification leading to foam cell formation are low density lipoproteins (LDL). Since LDL is the main carrier of free and esterified cholesterol in the body, these lipids are the predominant components of the foam cells. This brief summary covers the knowledge of likely atherogenic events derived from studies completed before about 1980.

The more recent research addressed the nature of modification of the LDL rendering it capable of producing foam cells and the events which could produce such modifications in vivo. It turned out ultimately that the most physiologically probable LDL modification is derivarization of its constituent apolipoprotein B (apo B) by breakdown products of lipid peroxides, while the lipid peroxidation is probably caused by an oxidizing agent in the vascular system. Thus, the currently favored chemical common denominator of the new hypothesis of an atherosclerotic plaque formation in vivo is formation of oxidized LDL (oLDL) by mechanisms involving free radicals and/or lipoxy- genases.

The observations and correlations germane to this summary are listed in Table 1. The list is not exhaus- tive and the chronology may not always be precise, because it is often not possible to establish the date of a significant statement or its priority. Rather, the purpose of the table is to provide references useful in doc- umenting findings which contributed in a major way to the development of the current theory of atherogenesis. Additional details can be found in several recent reviews. 56-62'261

METABOLISM, STRUCTURE, AND COMPOSITION

OF HUMAN LDL

The liver assembles triglyceride-rich, very low density lipoproteins (VLDL) and secretes them into the circulation. The main biological function of VLDL is to supply the peripheral tissue with fatty acids. Lipo- protein lipases on the surface of the vascular endothelial cells hydrolyze the VLDL triglycerides to free fatty acids, which are then taken up by adipose tissue and muscle cells. With increasing hydrolysis, the VLDL loses most of its triglycerides and progressively changes into lipoproteins with intermediate density (IDL) and finally to the cholesterol-rich low density

lipoprotein. VLDL and IDL have a short half life and are removed from the circulation within hours, whereas the LDL particles have a rather long life and circulate in the blood for about 2 d before they are cleared. In normolipidemic persons, the serum LDL concentration is about 3 mg/mL, and typically this LDL carries about 60% of the total serum cholesterol. 63 The uptake of LDL by cells occurs via a receptor-mediated pathway (B/E receptor) and by nonspe- cific endocytosis. The LDL first binds to the B/E receptor (LDL receptor) present on the surface of most cells and is then endocytosed. The highest concentration of LDL receptors is found in the liver, and about three quarters of the LDL is removed from the bloodstream by the liver, although the suprarenal gland and the ovary are comparably rich in LDL receptors. The liver converts most of the LDL cholesterol to bile acids, which are secreted into the duodenum. A reduction of the reabsorption of bile acids by bile-acid- binding drugs (e.g., ion exchange resins, cholesteryl- amine) is one possibility to reduce serum cholesterol levels. The cholesterol of LDL is of course also used by all cells as a building block for cell membranes and in specialized cells for biosynthesis of steroid hor- mones. The endogenous and exogenous (cholesterol from the diet) pathways of cholesterol are intimately fine tuned by several control mechanisms so that the serum cholesterol level in normolipidemic persons is maintained constant and in a narrow range of 160- 200 mg/100 mL (Ref. 64).

One of the most intensively studied (for review see Ref. 65) imbalances in cholesterol homeostasis is fa- miliar hypercholesterolemia (FHC). Patients with this heritable disease have a defect in the gene coding for the LDL receptor, and the deficiency of this receptor dramatically reduces the clearance rate of the LDL. The net result is a very high plasma LDL and cholesterol level. Very frequently used experimental animal models are Watanabe heritable hyperlipidemic rabbits (WHHL), which have a defect in the LDL receptor and develop severe atherosclerosis. 66

Human LDL is defined as the population of lipoproteins which can be isolated by ultracentrifugation within a density range of 1.019 to 1.063 g/mL. By equilibrium density-gradient ultracentrifugation and several other techniques, LDL can be further separated into two or more subfractions differing somewhat in density, size, and molecular weigh t . 63 LDL molecules are large spherical particles with a diameter of 19-25 nm and molecular weights between 1.8 and 2.8 million. The mean chemical composition calculated from values obtained from various sources is given in Table 2. Taking 2.5 million as mean molecu-

344 H. ESTERBAUER et al.

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lar weight of LDL, each LDL particle would contain about 1600 molecules ofcholesterylester and 170 molecules of triglycerides, which form a central lipophilic core. This core is surrounded by a monolayer of about 700 phospholipid molecules (mainly phosphatidylcholine with minor amounts of sphingomyelin and lysophophatidylcholine) and 600 molecules of free cholesterol. The polar heads of the phospholipids are located at the surface of the LDL particle and contribute to the solubility of LDL in an aqueous phase. Em- bedded in the outer layer is a large protein termed apolipoprotein B (apo B). This protein does not sit like a cap on the LDL (as schematically shown in some models) but should rather be seen like an "octo- pus" embracing the whole surface of the LDL. The apo B is an exceptionally large protein consisting of 4536 amino acids. The amino acid sequence has in part been determined directly on the protein and fully deduced from the cDNA (for review see Ref. 73). The molecular weight of apo B based on the amino acid composition is 512,937. The number of amino acid residues per apo B are: Ala 266, Asp + Asn 478, Arg 148, Cys 25, Glu + Gin 529, Gly 207, His 115, Ile 288, Leu 523, Lys 356, Met 78, Phe 223, Pro 169, Ser 393, Thr 298, Trl0 37, Tyr 152, Val 251. From the 25 cysteine residues, 4 have the SH group free; the re- mainder form SS bridges or thiol esters.

Apo B is glycosylated (the carbohydrate components are mannose, galactose, glucosamine, and sialic acid); the total carbohydrate content can amount to 8-10 weight % of the total apolipoprotein B. The molecular weight of the glycosylated apo B determined by gel electrophoresis is 550 kDa. With this value and the mean lipid composition given in Table 2, the average molecular weight of LDL would be 2.4 million, which is very close to the value determined by neutron small-angle scattering (2.32 + 0.2 million), v° For convenience and in accordance with our previous publications, all molar ratios are based in this review on a LDL molecular weight of 2.5 million.

It seems important to note that various options exist in determining the amount of LDL in a sample isolated from plasma by ultracentrifugation. Depend- ing on the method used, the results of an analysis of native or oLDL are expressed by different laboratories in different ways. Over many years, the group in Graz has determined the total dry mass of the samples (after removal of all salts by dialysis). Although this is rather tedious, it is in fact the only way to obtain the absolute amount of LDL present in a sample. It seems worthwhile to make a brief historical remark on this point. As early as 1957, Oncley et al. 3~° reported that the Sf 3-9 lipoprotein fraction (which corresponds to

Oxidation of LDL

Table 3. Lipid Composition of Native and Oxidized LDL

345

Native LDL

nmol/mg LDL Protein mol/mol LDL Mean _+ SD Mean

Oxidized LDL

Total phospholipids 1300 +_ 227 700 Phosphatidylcholine 818 450 Lysophosphatidylcholine 145 80 Sphingomyelin 336 185

Triglycerides 304 _+ 140 170 Free cholesterol 1130 +_ 82 600 Cholesteryl ester 2960 _+ 220 1600 Total cholesterol 4090 2200 Free fatty acids 48 26 Oxysterols 0 0 Oxodienes Not detectable Conjugated dienes Not detectable (72, 75) lodometric LOOH 18.6 _+ 9.4 (77,78) 10 Defined LOOH Not detectable (81) Hydroxy and hydroperoxy 18:2 Not detectable (82) Hydroxy and hydroperoxy 20:4 Not detectable (82) Prostanoid-like substances Not detectable (313)

No significant change of P content (71, 72) Decrease to 65-55% (28, 72) Increase to 250-300% (28, 71, 72) No significant change (28) Decrease to 76-52% (28, 71, 72) Decrease to 90% (71, 72) or increase to 150% (28) Decrease to 48% (28) Decrease to 78-60% (71, 72) Increase to 170% (28) Increase to 33 ug (28) or to 120-240/~g/mg protein (215) Strong increase (72) Strong increase to 190-350 mol/mol LDL (75-77) Strong increase to 190-550 mol/mol LDL (77-80, Figs. 5, 6) Increase (81 ) Strong increase to 30-200 mol/mol LDL (82) Increase to 20 mol/mol LDL (82) Material cross-reacting with antibodies to PGE2 (313)

If not otherwise indicated, values for native LDL are calculated from mean of Table 1. Values for oLDL are from the following sources: Steinbrecher e t al . , 71 LDL oxidized by endothelial cells, 20 h, 37°C or by 5 uM Cu *÷ in PBS, 20 h, 37°C; Barenghi e t a l . , 72 LDL oxidized by 5 ~M Cu ÷÷, 29 h, 37°C; van Hinsbergh et al., 28 LDL oxidized by 25 t~M Cu ÷÷ in PBS, 48 h, 4°C; our laboratory, 75-78 LDL oxidized by 1.6 #M Cu ÷÷ in PBS for 3-8 h at 25 °C; Jessup et al. ,79,80 LDL oxidized by macrophages or 100 ~M Cu ++ in F- I 0; and Jialal et al.,2~s LDL oxidized with 2.5 ~M Cu *÷ PBS for 24 h at 37°C.

LDL) contains 22.4% phospholipids and 21.9% protein based on experimentally determined dry weight. These values are very close to those found by us (Ta- ble 2). In most of our previous publications, data on composition of LDL (e.g., fatty acids, antioxidants, aldehydes) were given per milligram of total LDL. Other groups working on oxidative modification of LDL report its composition and changes during oxidation per milligram of LDL protein or per milligram of total cholesterol. The necessary conversion factors from one unit into others can be deduced from the data in Table 2. The good agreement on the chemical composition of LDL as determined by different laboratories (Table 2) indicates that conversion between the different reference systems used by different laboratories is justified. To facilitate comparison of data, we express in this review LDL analysis in nanomoles per milligram of LDL protein and, where appropriate, in mol/mol LDL.

The average distribution oflipids and of individual fatty acids is given in Tables 3 and 4. The total number of fatty acid molecules bound in the different lipid classes of an LDL molecule is 2700 on average. Of these, about half are polyunsaturated fatty acids (PUFAs), mainly linoleic acid with minor amounts of arachidonic acid and docosahexaenoic acid. The fatty acid content of LDL and their distribution pattern can vary considerably from donor to donor, probably due to different dietary habits. In the subjects which

we have investigated, the linoleic acid content varied (e.g., from about 1200 to 2400 nmol /mg LDL protein). Such a variation in the PUFA content is likely to have a significant effect on the oxidation behavior of different LDL samples. Parthasarathy et al. 89 showed recently that feeding rabbits an oleic-acid-rich diet results in an oleate-rich LDL, which is remarkably resistant toward oxidation.

The PUFAs in LDL are protected against free radical attack and oxidation by a number of lipophilic antioxidants listed in Table 4. Representative chro- matograms showing their separations are given in Fig- ure 1A, 1B, and 2. On a molar base, by far the major antioxidant is a-tocopherol; the amount of 11.58 nmol /mg LDL protein equals about 6 molecules a-tocopherol per LDL particle. All other antioxidants (i.e., gamma-tocopherol, carotenoids, oxycaroten- oids, and ubiquinol-10) are present in much smaller amounts. The antioxidant content of LDL varies, similarly to the PUFAs, significantly between individuals. For instance, the lowest and highest amount of a-tocopherol found by us among 87 (not vitamin E supplemented) donors were 3 and 15 mol/mol LDL. The frequency histogram of LDL a-tocopherol is shown in Figure 3. The vitamin E content of LDL increases with its PUFA content with a correlation of y = 0.0034x + 1.98, where y is mol a-tocopherol/mol LDL and x is mol PUFA/mol LDL. 163 The value of 0.29 mol/3-carotene/mol LDL (Table 4) means that

346 H. ESTERBAUER ~"/al.

Table 4. Fatty Acids and Antioxidants in Native and Oxidized LDL

Native LDL Oxidized LDL

nmol/mg LDL Protein tool/tool LDI Mean _+ SD (n) Mean

Palmitic aicd 1260 + 375 (19) 693 Weak decrease to 98-73% Palmitoleic acid 80 _+ 44 (19) 44 Weak decrease Stearic acid 260 _+ 118 (19) 143 Decrease to 96-79% Oleic acid 825 _+ 298 (19) 454 Decrease to 80-46% Linoleic acid 2000 _+ 541 (31 ) 1100 Strong decrease to 15-3% Arachidonic acid 278 _+ 100 (31 ) 153 Complete consumption Docosahexaenoic acid 53 _+ 31 (15) 29 Complete consumption a-tocopheroP 11.58 _+ 3.34 (87) 6.37 Complete consumption 3,-tocopherol 0.93 + 0.36 (88) 0.51 Complete consumption /3-carotene 0.53 _+ 0.47 (122) 0.29 Complete consumption a-carotene 0.22 _+ 0.25 (28) 0.12 Complete consumption Lycopene 0.29 _+ 0.20 (136) 0.16 Complete consumption Cryptoxanthin 0.25 _+ 0.23 (114) 0.14 Complete consumption Cantaxanthin 0.04 _+ 0.07 (53) 0.02 Complete consumption Lutein + zeaxanthin 0.07 _+ 0.05 (113) 0.04 Complete consumption Phytofluene 0.09 _+ 0.05 ( 1 O) 0.05 Complete consumption Ubiquinol-10 0.18 _+ 0.18 (7) 0.10 Complete consumption Total PUFAs (mean) 2332 1283 Total antioxidants (mean) 14.2 7.8

The values for native LDL are an updated version from previous reports 5s's3.s4 and a recent review. 85 Values for oxidized LDL (1.6 uM Cu ++, 3-8 h) are from Refs. 69,7L85-87; n gives the number of different LDL samples analyzed.

a nmol c~-tocopherol/mg protein reported by others are 12.8 _+ 4.3, n = 14, Babiy et al.SS; 7.4 _+ 4.3, n = 5, Jessup et al.79; 9.3 _+ 1.1, n = 5, Sattler et al. 69.

this antioxidant is present in only about one third of the LDL molecules. It has been argued by Halliwell et al. 9° that this suggests that o~-tocopherol is the only significant antioxidant in LDL and that the carotenoids play no or only a minor role in protecting LDL against oxidation. The same is likely to hold for ubiquinol-10. Recently it has been shown by Stocker et al. 8~ that ubiquinol-10 is contained in LDL and it was proposed that it acts as an even more powerful antioxidant than a-tocopherol. The concentration of ubiquinol-10 in LDL given by these authors is similar to that of/3-carotene and agrees with values determined independently by us (Table 4). Since plasma contains a great variety of water-soluble and lipid-soluble antioxidants (e.g., more than 20 different carotenoids have been r e p o r t e d , 93 it seems reasonable to us that LDL isolated from plasma may contain several other lipid- and/or water-soluble antioxidants in addition to those listed in Table 4. Since all of them are likely to affect the oxidation of LDL in vitro, it would be important to investigate this further. Ethanolamine- plasmalogens, for example, have antioxidant activity, and the presence of such plasmalogens in LDL may well contribute to its oxidation resistance. 226

The adsorption and transport of vitamin E in human subjects has been studied in detail with deuterium labelled c~-tocopheryl a c e t a t . 227'311 Consistent with earlier studies, 3~2 newly absorbed c~-tocopherol

increased most rapidly in chylomicrones, then in VLDL followed by LDL and HDL, and finally in red blood cells. This sequence of appearance and distribution strongly suggests that vitamin E is first incorporated into chylomicrones, transported in chylomicron remnants to the liver, and delivered into the circulation again in VLDL. The vitamin E molecules contained in LDL stem therefore primarily from VLDL. The majority of vitamin E appears to enter the cells with the uptake of the intact LDL by the LDL receptor. 92 Additional uptake may also occur from chylomicrones and VLDL by the action of lipoprotein li- pase (for review see Ref. 227).

In vitro, the a-tocopherol molecules of LDL un- dergo a rapid intermolecular exchange as well as exchange with other lipoproteins (VLDL, HDL) and blood cells; the estimated half times for this spontaneous a-tocopherol transfer are in the range of 20-70 min, which is about two to three times slower than cholesterol transfer. 9~ On the other hand, the spontaneous exchange (in vitro) of ~-carotene is very slow, and no equilibration occurs within 18 h. 91

DO PLASMA OR PLASMA LIPOPROTEINS FROM

HEALTHY OR ATHEROSCLEROTIC HUMAN

SUBJECTS CONTAIN LIPID PEROXIDATION PRODUCTS?

TO a n s w e r t h i s q u e s t i o n , i t is e s s e n t i a l t o e x c l u d e

a r t e f a c t u a l o x i d a t i o n o f L D L d u r i n g i ts i s o l a t i o n a n d

A

Oxidation of LDL

B

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Fig. 1. HPLC determination of carotenoids (A) and tocopherols (B) in LDL. A sample containing 0.5 mg total LDL was mixed with 0.2 mL ethanol and extracted with 1 mL hexane; the extract was dried with N2 and the residue was dissolved in the solvent (0.1 mL) used for HPLC separation. (A) acetonitrile/dichloromethane/methanol 67/19/14 as solvent, HPLC column ODS-2, flow 1.3 mL/min, UV detection at 436 nm, 20 t~L injected. 1 : lutein/zeaxanthin; 2: cantaxanthin; 3: cryptoxanthin; 4: lycopene; 5: cis-lyco~ne; 6: c~-carotene; 7: /3-carotene; 8: 15-cis-13-carotene. (B) Methanol as solvent, HPLC column ODS-2, flow 1 mL/min, fluorimetric detection at 335 nm with 292 nm excitation, 20 uL injected. 1 = gamma-tocopherol, 2 = a-tocopherol.

subsequent handling. It was recognized quite early by Ray et a l . 9 4 that oxidative degradation of isolated LDL may occur during prolonged dialysis if traces of copper ions are present. Interestingly, it was already noted in this early study that other transition metal ions, including Fe ÷+ and Fe +++, had no or only minimal degradative effects. A systematic investigation was made by Schuh et al., 17 who showed that a more or less complete oxidative degradation of L D L with concomitant formation of thiobarbi tur ic acid reactive substances (TBARS) occurs if it is dialyzed at 4°C for 24 h against phosphate-buffered saline without protection, which could be provided by ethylenediamine- tetraacetic acid (EDTA), butylated hydroxytoluene (BHT), or by nitrogen gassing.

Lee 95 pointed out that freshly prepared h u m a n

plasma contains only low levels of TBARS in the range of 0.22 to 0.4 nmo l /mL, but storage of plasma at 4°C in the absence of protecting agents (e.g., EDTA) led to a cont inuous increase of the TBARS at a rate of 0.15 _+ 0.14 nmo l /mL, week. The rate of TBARS format ion showed a strong individual variation, which could be indicative of different antioxidant contents of the p lasma samples. It was further shown by Lee that lipids of all l ipoproteins classes contributed to the format ion of TBARS during storage of plasma. F rom these and other investigations, it is clear that p roof of the existence of low levels of peroxidation products in native L D L requires a very careful preparat ion and handling. It is difficult to judge if that has been considered in all studies. Most researchers are now aware of the problems and spike

348 H. ESTERBAUER et al,

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Fig. 2. HPLC determination of tocopherols, ubiquinol-10, and carotenoids in LDL with electrochemical detection. A sample containing 0.5 mg total LDL was mixed with 0.2 mL ethanol and extracted with 1 mL hexane. The extract was dried with N2 and dissolved in 0.1 mL methanol. A volume of 20 uL was injected into the HPLC and separated on an ODS-2 column with methanol/ethanol 1/1 containing 12mM LiCIO4 and 1 g/L glacial acetic acid, flow 1 mL/min, with amperometric detection (HP-Electrochemical De- tector) at 0.55 volt. Detector response was 2 nano ampere for full scale, h zeaxanthin, 2: gamma-tocopherol, 3: a-tocopherol, 4: lycopene, 5: ubiquinol-10, 6: carotenes.

the freshly donated blood immediately with EDTA (1 mg/mL), and EDTA is then present at this concentration throughout all steps until the LDL sample is har- vested from the ultracentrifuge tube. Some researchers also include BHT in the isolation medium. According to our experience, EDTA alone is sufficient to completely block oxidation of LDL during its isolation. The various evidence supporting this has been discussed, 96 the most convincing being the observation that a freshly isolated EDTA containing LDL sample fully withstands oxidation at 35°C for at least 48 h, as evident by the unchanged content of vitamin E, carotenoids, TBARS, fatty acids, and 430 nm fluorescent chromophores. 96,97,3~3 Another important, yet often neglected, point is the methodology in reference to the question of whether the methods

applied are indeed specific and sensitive enough to detect low levels of lipid peroxidation products in lipoproteins.

The method most frequently used to assess the degree of lipid peroxidation in LDL is the thiobarbituric acid assay, which is known to have a low specificity. A number of studies were made to eludicate if patients with atherosclerosis have increased plasma TBARS 98-1°6'284 (Table 5). Although the absolute values for TBARS reported by various laboratories differ significantly, they all show the same trend of increased plasma TBARS in patients with atherosclerosis or myocardial infarctions. So far, only a few groups have made a systematic comparison of the TBARS in the different lipoproteins of human plasma (Table 6). The group led by Yagi presented three studies since 1981,1°2'~°7'j°8 none of which, however, addressed specifically the TBARS in lipoproteins of atherosclerotic subjects. In these studies, serum or isolated lipoproteins were precipitated with phosphotungstic acid, the sediment was reacted with TBA, and the formed chromogen was extracted into butanol and measured fluorimetrically at 535 nm with 515 nm excitation. The results are expressed as "lipid peroxides," although the term TBARS would be more appropriate for this assay. TBARS were found in VLDL, LDL, and HDL, but the LDL fraction always contained the highest proportion. This finding is the likely origin of the belief that LDL is the main carrier of lipid peroxides, because it was very frequently cited in later studies. The group of Szczeklik 99'~°9 used a modified TBA assay (trichloroacetic acid [TCA] precipitate heated 30 min at 100°C in 0.05 M sulfuric acid TBA reagent containing 2 M sodium sulfate, which is said to in-

O e -

"-I o"

i2

{

0 0 3

i!i:!ii

6 9 t2

alpha-tocopherol, mol/mol LDL

~5

Fig. 3. Frequency histogram ofLDL a-tocopherol. Frequency gives the number of subjects found in a given group of a-tocopherol, Sample size: 95.

Oxidation of LDL

Table 5. Serum TBARS in Normal and Atherosclerotic Human Subjects

349

Author(s) Study subject Mean _+ SD

Satoh, 1978 (98)

Szczeklik et al., 1980 ~ (99)

Goto, 1982 (100)

Aznar et al., 1983 (101)

Hagihara et al., 1984 a (102)

Ledwozyw et al., 1986 (103)

Schimke et al., 1986 (104)

Stringer et al., 1989 b (105)

Yalcin et al., 1989 (106)

35 normal subjects, 50-60 years 3.7 _+ 0.68 32 patients with infarction, 50-60 years 4.4 ___ 0.74 13 patients with hemorrhages, 50-60 years 4.4 ___ 1.04 17 normal subjects 2.9 +__ 0.1 6 hyperlipoproteinemia type V 3.7 _+ 0.3 4 hyperlipoproteinemia type Ila 3.3 -+ 0.1

- - normal subjects 5.0 _+ 0.5 - - patients with atheroselerosis 8.0 _+ 0.5 95 normal subjects 47.2 +_ 6.9 26 acute mycardial infarction < 61 50 normal subjects under 40 years 3.49 _+ 0.62 52 normal subjects over 40 years 3.96 +_+_ 0.79 15 normal subjects 0.94 _+ 0.09 15 patients with severe atherosclerosis 4.20 _+ 0.16 20 normal subjects 3.6 +_ 0.8 27 patients with atherosclerosis 4.1 _+ 1 . 2

57 patients, 12-24 h after myocardial infarction 8.1 _+ 4.2 75 normal subjects 3.65 (3.29-3.89) 50 patients with ischemic heart disease 4.37 (3.85-5.75) 50 patients with peripheral arterial occlusion 4.37 (3.88-5.21) 25 normal subjects 3.4 +_ 0.2 25 hyperlipidemic patients 4.6 _+ 0.5

The values are nmol TBARS/mL plasma or serum except Goto, who measured nmol TBARS/mL blood. Lipoprotein TBARS, see Table 6.

b The values are medians, interquartile range in bracket.

hibit the formation of TBARS ,from sialic acid) and found in serum similar levels of TBARS to those reported by Yagi's group; however, the TBARS in the LDL fraction were about 10-fold higher. According to these investigations, hyperlipoproteinemia is associated with a very strong increase of TBARS in VLDL, whereas TBARS in LDL are only slightly increased. The sum of TBARS determined in the separated lipoprotein fractions by far exceeded the TBARS of the parent plasma samples, and it was assumed 99 that in whole plasma certain components (e.g., HDL) inhibited the chemical reaction of TBA with the precursors of TBARS. This shows very clearly the problems and limitations of the TBA assay; two similar methods give values differing by one

order of magnitude. It should be considered that heat- ing in a hot acid, as applied in the TBA assay, is a very harsh condition for PUFAs, and it might well be that most if not all TBARS are formed during the assay itself by autooxidation of PUFAs. This could be avoided by inclusion of EDTA and BHT in the assay, but this seems to be done only rarely (e.g., Ref. 313). Also, in our investigations most of the freshly prepared LDL samples gave a weak absorption at 535 nm in the TBA assay, which corresponds to an apparent concentration of about 0.5 to 3 nmol TBARS/mg LDL protein, with a mean of 3.6 + 1.0 nmol/mg. Occasionally we have also recorded the full spectrum (400-600 nm) of the chromogen produced by the reaction of native LDL (TCA supernatant, TCA precipi-

Table 6. TBARS in Human Lipoproteins

Author(s) Study Subject Chylomicrons VLDL LDL HDL

Szczeklik et al., 1980 a (99)

Nishigaki et al., 1981 (107)

Maseki et al., 1981 (108)

Hagihara et al., 1984 a (102)

17 normal subjects 6 patients hyperlipoproteinemia V 4 patients hyperlipoproteinemia lla

32 normal subjects 31 diabetic patients 19 female, not pregnant 22 pregnant female 50 normal subjects, under 40 y 52 normal subjects, over 40 y

Not determined 0.9 _+ 0.6 9.0 + 0.6 < 0.5 8.4 _+ 2.4 7.3 +__ 1.0 10.1 ___ 1.2 < 0.5 0.6+__0.2 3 .5+ 1.2 12.8 + 0.1 <0 .5

Not determined 0.64 + 0.30 1.18 _+ 0.33 0.68 +_ 0.16 Not determined 0.68 +_ 0.34 1.26 + 0.35 1.07 + 0.40 Not determined 0.45 + 0.11 0.81 + 0.20 0.63 _+ 0.12 Not determined 1.49 _+ 0.45 1.86 + 0.52 0.95 _ 0.25 Not determined 0.43 +_ 0.30 0.84 _+ 0.25 0.66 _+ 0.13 Not determined 0.55 _+ 0.31 1.09 + 0.31 0.66 _+ 0.16

The values are in nmol TBARS/mL plasma, mean +_ SD. a Serum TBARS, see Table 5.

95'

0.20

0.16

~ 0.12

@ 0.08

0.04


, , , , , , , , , ,

400 500 600

wavelength, nm

Fig. 4. Spectra of TBARS in native LDL (0 min) and LDL oxidized with Cu ++ for 35, 70, and 95 min. 0.5 mL LDL solution (1.5 mg total LDL/mL in PBS + 10 uM CuC12) were mixed with 3 mL 1% H3PO4 and 1 mL TBA reagent and heated 45 min on a boiling water bath. The color was extracted into 4 mL butanol and the spectra were recorded.

tate) with TBA and found only a broad and uncharac- teristic absorption without a maximum at 535 nm, the peak absorption of the MDA-TBA complex (Fig. 4). Since in our assay 1 nmol TBARS/mg LDL protein would have given a detectable 535 nm maximum, we assume that the concentration of TBARS in native LDL from healthy subjects, if they exist at all, must be below that level which corresponds to 0.5 mol TBARS/mol LDL. The authors cited in Table 6 reported the LDL TBARS in terms of nmol /mL plasma. With the assumption that 3 mg L D L / m L are contained in plasma, the values of Nishigaki et al. 1°7 and Hagihara et al. ~°2 correspond to 0.7 to 1.0 mol TBARS/mol LDL, whereas the values of Szczeklik et al. 99 correspond to 7.5 to 11 tool TBARS/mol LDL. Heinecke et al. 2~ reported for native human LDL 0.8 + 2.3 nmol TBARS/mg protein; Harats et al. 42 found in LDL from smokers 0.6 _ 0.028 and in LDL from nonsmokers 0.55 8 ___ 0.020 nmol TBARS/mg protein. The potential diagnostic value of malonaldehyde determination by the TBA-test has recently be carefully reviewed by Janero. 258

The determination of lipid hydroperoxides by the iodometric methods also gives for most samples of native LDL a positive result corresponding about 10

to 30 nmol "lipid peroxides"/mg LDL protein 77"79"8°'88 (Table 3). But this is again at the borderline of the detection limit of the iodometric assays and does not substantiate the existence of lipid hydroperoxides in freshly prepared LDL. Stocker et al. 81 have recently reported that LDL isolated from healthy subjects was free from detectable amounts of cholesterol ester hydroperoxides, phospholipid hydroperoxides, and triglyceride hydroperoxides as measured by high-perfor- mance liquid chromatography (HPLC) postcolumn chemiluminescence detection. This method is incom- parably more specific than the TBA or iodometric assay, and from its sensitivity it can be concluded that the concentration of lipid hydroperoxides in LDL, if they are present at all, must be below 0.5 tool/tool LDL. In agreement with that is the report that none of the isomeric linoleic acid or arachidonic acid hydroperoxides found in oLDL were detectable by gas chromatography/mass spectroscopy (GC/MS) in native LDL. 82 A recently developed modified iodometric assay, ~59 which takes into account interfering phenom- ena leading to false results, gave for the total amount of lipid hydroperoxides esterified in the plasma lipoproteins of healthy humans a value of 4.0 _+ 1.7 #M; with the assumption that about half of that is contained in LDL, the lipid peroxide content in LDL would be 3.03 + 1.28 nmol/mg LDL protein (= 1.66 mol/mol LDL).

Miyzawa ~0 used a chemiluminescence HPLC assay to measure phosphatidylcholine hydroperoxides (PCOOH) in a large number of human plasma samples and found concentrations of 0.0 ! to 0.5 uM. Sam- ples from unhealthy subjects contained much higher concentrations in the range of 0.5 to 9 uM. In diabetic patients the PCOOH were mainly contained in VLDL and LDL.

Another possibility of searching for remnants of in vivo lipid peroxidation in LDL would be the measurement of defined aldehydic lipid peroxidation products. By HPLC we found traces of 4-hydroxynonenal (HNE) in some samples, but in others this aldehyde was undetectable. 96 The mean value, given in Table 9, would correspond to 0.5 mol HNE/mol LDL. Using the GC method developed by van Kuijk et al., 1~ HNE was undetectable in native LDL. 87 Hexanal, the major aldehyde in oLDL, was undetectable by HPLC or GC in native LDL. Thus, these analyses indicate again that native LDL of healthy individuals is devoid of free aldehydic lipid peroxidation products.

Several other attempts were made to demonstrate oxidation remnants in LDL of healthy subjects. The apolipoprotein B of in vitro oxidized LDL, for example, shows a very strong fluorescence at 430 nm with


excitation at 355 nm.l 7,29,56,112,113 This chromophore most likely results from reaction of aldehydic lipid peroxidation products with free amino groups of apo B. Apo B from native LDL analyzed by three-dimensional fluorescence spectroscopy showed always the presence of a small amount of a chromophore with exactly the same spectral characteristics, which could be indicative of subtle oxidative alterations of apo B which had occurred already in vivo)13,114 The relative fluorescence intensity at 430 nm showed strong individual variations. Dobretsov et al., 115 using conventional fluorescence spectroscopy, did not find the 430 nm fluorescent chromophore in plasma LDL, which is not surprising since it requires three-dimensional measurements to resolve it from the 13 different fluorophors present in LDL. 114

Avogaro's group 32'116'117'322 reported in 1988 (Ref. 32) that LDL collected from 18 different healthy humans contained a subtraction (about 5 to 20%) which could be separated from the LDL bulk by ion exchange chromatography. This subfraction was more electronegative, contained more conjugated dienes, had apo B aggregates, and led to a higher accumulation of cholesterol esters in cultured macrophages than normal LDL. It was concluded that this subfraction resulted from in vivo oxidation of LDL. The high proportion of the modified LDL together with the methodology first caused some doubts on the validity of this finding, but with an improved method (ion exchange HPLC) the more negatively charged LDL (now termed LDL-) was found again in LDL collected from normal subjects, 1 ~7 though the content of LDL- in total LDL given by the revised method was only 3.9% (range 0.5 to 9.8%, 32 normal male subjects aged 30 to 60). The LDL- content of LDL was negatively correlated with its vitamin E content and positively correlated with its TBARS. The TBARS content of LDL- was on average 7.3 mol/mol LDL, which is threefold higher than in normal LDL. In sodium dodecyl sulfate (SDS) polyacrylamide electrophoresis, the apo B from LDL- showed higher molecular weight peptides, just as they are occasionally also observed in LDL exposed to aldehydes) 18'1~9 Re- cently Shimano et al) 2° isolated a minor LDL fraction, only less than 1% of total LDL, by ion exchange chromatography on DEAE-Sepharose 6B. This minor fraction was more negative and had a higher density than normal LDL. In disagreement with Avogaro's findings, however, it did not exhibit properties indicative of mild oxidation and it was not recognized by macrophage scavenger receptors. However, the minor fraction was more labile to Cu ++ stimulated oxidation in vitro. In view of the possibility that such minor and

readily oxidizable LDL subfractions are associated with an increased risk of atherosclerosis, it would be important to improve the methods for their analysis and to ensure that the findings can be reproduced by others.

Monoclonal antibodies directed against oxidatively modified human LDL 37'43 or against malonaldehyde (MDA) or HNE conjugated LDL 43 did not show a noteworthy crossreaction with freshly isolated human LDL, and one must therefore assume that native LDL does not have epitopes typically found in oLDL.

The occurrence of heavily oxidized LDL as a subfraction in LDL of healthy human subjects is also not supported by studies with autoantibodies. Parums et al) 21 studied the incidence of serum autoantibodies against LDL, oLDL, and ceroid in 100 individuals. None of them had autoantibodies to native human LDL samples isolated by conventional ultracentrifugation. Since Avogaro's LDL- would have been present in such samples, it is clear that LDL- does not act as an immunogen and that LDL- is not a form of LDL which is recognized by antibodies directed against oLDL. Serum autoantibodies recognizing ar- tificially oxidized LDL were not present in young controls but were found in about 50% of elderly individuals and in most patients with chronic periaortitis. These autoantibodies probably developed against oxidatively modified LDL formed within arteriosclerotic plaques 121 and are not indicative of the presence of oLDL in serum. Recently autoantibodies against modified LDL were considered as a nonlipid factor of blood plasma that stimulates foam cell formation) 22 The literature on the relationship between atherosclerosis and circulating immune complexes containing LDL was recently reviewed by Orekhov. 325 For human placental blood the presence of an acetyl-like modified LDL has been shown by an ELISA assay. ~23

OCCURRENCE OF OXIDIZED LDL IN ARTERIES AND

ATHEROSCLEROTIC LESIONS OF HUMANS

AS early as 1952, Glavind et al. 33 reported that a chloroform extract of atherosclerotic lesions from human aortas (postmortem material) contains lipid peroxides and that the peroxide content is positively correlated with the extension of the atheromatas. The highest peroxide values found were in the range of about 10 to 20 milli-equivalent per kilogram of fat, which is equivalent to 10 to 20 nmol lipid peroxides/ mg lipid; for comparison LDL oxidized 24 h with Cu +÷ ions contains about 50 nmol peroxides/mg lipid

352 H. ESTERBAUER el aL

(Table 9). A later r e p o r t 16 showed that the peroxides probably formed in the period between death and tissue extraction, through cessation of enzymatic processes normally converting hydroperoxides to hydroxy-octadecadienoic cholesterol esters. It was also pointed out that the hydroxy-esters isolated from dis- eased arteries during surgery had structures inconsistent with lipoxygenase oxidation of the lipids forming the atherosclerotic deposits. Several other early re- p o r t s 314-316 also suggest the presence of oxidized cholesterol and oxidized cholesteryl esters in human aorta (for review see Ref. 319). Piotrowski et al. ~z4 found that the lipid extract (Folch) of aortic human tissue contains fluorochromes with emission maximum at 435 nm (Ex 355 nm) indicative of lipid peroxidation products. Copper or cell oLDL exhibits also a very strong 430 n m f l u o r e s c e n c e . 17,29,56,112A13 The amount of fluorescent lipid material was about 30% higher in atherosclerotic tissue than in normal aortic tissue (4.15 vs. 3.08 arbitrary units/g tissue). Kana- zawa et al. 125 separated the lipids of lesions by conventional thin-layer chromatography (TLC) and found an unknown spot (spot x) which is probably an oxidized form of cholesteryl esters, not present in native LDL, but formed if LDL was dialyzed over long periods (a condition known to induce lipid peroxidation) or if the LDL was oxygenated for 20 rain only. Ledwozyw et al. 1°3 determined for the first time TBARS in buffer extracts of the human arterial wall from patients suffering from atherosclerosis who had their limb amputated and found that it contained twice as much TBARS as extracts from normal subjects (7.38 vs. 3.42 nmol TBARS/g artery). A positive correlation (r = .790) also existed between TBARS in plasma and arterial wall TBARS.

In several studies the properties of LDL extracted from the arterial wall tissue were compared with plasma LDL (Table 7). Hoffand Gaubatz 68 compared the chemical composition of human LDL from plasma, normal intima, and fibrous plaques. No significant differences were seen in the percent distribution of protein, phospholipids, free cholesterol, cholesterol esters, and triglycerides. Some remarkable differences existed in the fatty acid distribution; thus the arachidonic acid and linoleic acid ofcholesteryl esters and triglycerides were strongly decreased in aorta-derived LDL, which would be consistent with the high susceptibility of these PUFAs toward oxidation. That aorta LDL has a lower content of linoleic acid than plasma LDL was also found by Camejo et al. ~26 and Yl~-Herttuala et al.; 67 moreover, the aorta LDL shows an increased electrophoretic mobility as compared to normal plasma LDL, a property shown also by oLDL.

An LDL with increased relative electrophoretic mobility (REM) was also found in human interstitial fluid. 129 It was also shown 68'126 that aorta LDL is associated with about 2 to 4 ug glycosaminoglycans (mainly chondroitin sulfate and dermatan sulfate). This is in accordance with the assumption that the LDL deposited in the intima-media is also retained extraceUularly by association with strongly negatively charged glycosamino-glycans. In vitro experiments 2zs'229 showed that subpopulations of LDL bind to human arterial chondroitin sulfate and proteoglycans. This binding reduces the thermal stability of the surface and core in LDL and leads to exposure oflysine- and arginine-rich segments of the apo B. LDL subclasses complexed with such proteoglycans exhibit in vitro an increased uptake by mouse peritoneal macrophages. Additional differences between aorta and plasma LDL include decrease in sphingomyelin, tendency to aggregate, increased particle diameter, and possible fragmentation of apo B in the former. Most importantly, the aorta LDL is more rapidly taken up by macrophages than plasma LDL. A complement activating lipid complex (vesicles with 100-500 nm in size) containing cholesterol and phospholipids can be extracted with saline from atherosclerotic lesions of human aorta. 23° The lesion lipid complement might be responsible for the inflammation in the atherosclerotic lesion. It would be worthwhile to investigate whether these vesicles also contain oxidized lipids. Belkner et al. 255 recently analyzed by HPLC hydroxy fatty acids in the lipids of pieces of thoratic aortas of five men who suffered from chronic ischemic heart disease and died from acute heart failure and found a peak indicative for oxygenated cholesteryl esters (probably cholesteryl linoleate). The amount varied between 17 and 55 mg/g wet weight for the five samples. About 12 to 21% of the cholesteryl linoleate was present as oxygenated metabolites. "Healthy"-look- ing parts of the same aortas also contained this material, but the amount was much smaller (i.e., 0.3-4.5 mg/g wet weight or 5.8-9.5% of total cholesteryl linoleate). The authors assumed that this oxygenated cholesteryl linoleate was formed by a 15-1ipoxygenase activity. In human plasma incubated with reticulocyte lipoxygenase, 13-HODE (main product), 9-HODE, and 15-HETE esterified with cholesterol were formed.

The physico-chemical (REM, fluorescence) compositional (less PUFAs, more peroxides and TBARS) and functional (increased uptake by macrophages) properties of aorta LDL and aorta lipids in atherosclerosis support the hypothesis that LDL deposited in the arteries is partly oxidized.


Table 7. Properties of LDL Isolated from the Aorta in Comparison to Plasma LDL

Difference Between Aorta LDL and Plasma LDL References

Humans Increased electrophoretic mobility 68, 127, Decreased content of linoleic and arachidonic acid 68, 126, Moderately changed (increase, decrease) of cholesteryl ester 68, 126, Increased content of stearic and oleic acid 68, 126 Decreased sphingomyelin content 67 Associated with glycosamino-glycans 68, 126 Increased tendency to aggregate 68, 126 Additionally to apo B band, several lower molecular weight bands 127, 67 LDL particle diameter increased from 22 to 25 nm 67 Increased uptake by macrophages scavenger receptor 127, 67

WHHL Rabbit Increased electrophoretic mobility 128 More cholesteryl ester, more sphingomyelin 128 Decreased diameter of LDL particle 128 Significantly increased TBARS and macrophage uptake 128 Contains apo B fragments with MDA and HNE modified Lysine residues (Western blot) 43

67 67 127,67

Table was compiled from reports by Hoff and Glaubatz, 1982, 68 Camejo et al., 1985,126 Shaikh et al,, 1988) 27 Daugherty et al., 1988, ~28 Yl~i-Herttuala et al., 1988, 67 Palinski et al., 198943 .

EXPERIMENTAL ANIMAL STUDIES

The highest difference in plasma TBARS likely to be ever seen comes from experimental animal studies with genetically defective nonlaying hens) 3° These animals have extremely high cholesterol (450 mg/dL) and triglyceride values (12.000 mg/dL) as compared to normal laying hens, which have 84 mg/dL cholesterol and 1.100 mg/dL triglycerides. The TBARS value in plasma of nonlaying hens is about 14 times higher than in laying hens (76 vs. 5.6 n m o l /m L plasma). The large increase also persisted in TBARS normalized to cholesterol. In a later study Smith et al) 3~ showed that the plasma TBARS value, but not the total plasma cholesterol or lipids, correlated with the development of atherosclerosis and intimal thickening in this animal model. In cholesterol-fed rabbits serum TBARS are 1.6-fold higher than in controls (i.e., 1.47 vs. 0.91 nmo l /mL serumS°°). The lipopro- rein fraction containing VLDL + LDL obtained from rats made diabetic by streptozotoxin injection is highly oxidized and contains about 25 nmol TBARS/ mg cholesterol compared to about 2.5 nmol in normal rats) 32 The H D L of the diabetic rats had TBARS values in the range of controls. In this study it was also reported that the VLDL + LDL of diabetic rats was highly toxic to proliferating fibroblasts and that vitamin E or probucol treatment of the rats inhibited oxidation of the lipoproteins in vivo and prevented formation of cytotoxicity in l ipoproteins) 32

Daugherty et al.t28 investigated LDL isolated from the vascular tissue of W H H L rabbits and found that it

was oxidized and contained about eight times more TBARS than plasma LDL ( 15.3 vs. 2.0 nmol /mg protein) and that it also showed an increased REM and was more readily taken up by macrophages than plasma LDL. Wang and Powell TM recently reported that the lipids derived from aorta or from plasma LDL of cholesterol-fed New Zealand White rabbits contain increased amounts of esterified and unesteri- fled hydroxy linoleic acid (9-HODE, 13-HODE) and hydroxy arachidonic acid (1 I-HETE, 12-HETE, 15- HETE). The amount ofhydroxy fatty acids compared to total polyunsaturated fatty acids (2332 nmol /mg LDL protein, Table 4) is very low. After 15 weeks cholesterol feeding, that total amount of esterified hydroxy fatty acids in rabbit LDL was only amount 0.35 nmol /mg LDL protein with 80% H O D E and 20% HETE. About 0.15 nmol hydroxy fatty acids (HODE + HETE) were also present in control rabbits LDL. The analysis of such trace amounts is only possible with GC/MS.

Segments from normal rabbit aortas incubated with arachidonic acid produce 12-HETE as the principal lipoxygenase product, 323 but aortic segments from cholesterol-fed rabbits or W H H L rabbits produce 15-HETE, suggesting an increased 15-1ipoxy- genase activity in atherosclerotic arteries. 323'324

IMMUNOLOGICAL STUDIES

A monoclonal antibody raised against MDA-modi- fled LDL (MDA-LDL) was used by Gonen et al) 33 to investigate human autopsy samples. No reaction was

354 H. ESTERBAUER el al.

Table 8. Summary of Studies with Antisera (as) or Monoclonal Antibodies (mAb) Recognizing LDL Modified by Cu ÷÷ Oxidation (oLDL) or by Treatment with Malonaldehyde (MDA-LDL), 4-Hydroxynonenal (HNE-LDL), or Other 2-Alkenals

Antibody Type of Type of Authors Code Antibody Immunogen Major Findings

Gonen et al. 1987 (133) EB 7-3 Mouse mAb Mouse MDA-LDL ELISA failed to show any MDA-LDL in extracts of 14 autopsy samples from aorta

Salmon et al. 1987 (134) - - Rabbit as Rabbit MDA-LDL MDA-lysine conjugates in Cu ++ oxidized LDL Haberland et al. 1988 (135) MDA-lys Mouse mAb Human MDA-LDL Stains proteins in atheroma of WHHL rabbits;

stain co-localizes with extracellular deposits of apo B

MAL-2 Guinea pig as All three antibodies stain the same area of HNE-6 Guinea pig as lesions in WHHL rabbits, immunostain OLF4-3CI0 Mouse mAb mostly intracellular, stained area rich in

macrophages Mouse mAb The two antibodies stain certain lesions in Mouse mAb WHHL aorta; no staining in aorta of normal

rabbits

Palinski et al. 1989 (43)

Boyd et al. 1989 (37) OXL-41.1 OXL-22.4

Guinea pig MDA-LDL Guinea pig HNE-LDL Mouse oLDL

Human Cu +÷ oLDL Human Cu +÷ oLDL

found with aortic intimal or medial extracts with an ELISA technique. However, a monoclonal antibody prepared against the float-up fraction of atherosclerotic arterial homogenate from WHHL rabbits was highly reactive with peroxidized LDL and MDA- LDL, but not with native or acetylated LDL.~37

Very impressive evidence for the oxidation theory comes from several recent immunohistochemical studies. A series of polyclonal and monoclonal antibodies to various forms of oLDL and aldehyde-modified LDL were raised (Table 8) and used for immuno- staining of lesions and lesion-free areas of arteries from WHHL rabbits. 37'43'135A36'231 Briefly stated, the findings were as follows: All antibodies stained the same areas of fatty streaks rich in macrophages, the stain was predominantly macrophage associated, and the staining of extracellular material occurred only in advanced lesions. 136 The staining was confined to atherosclerotic tissues; in some cases the adventitia of nonlesioned WHHL rabbits also showed staining, but no staining was observed in normal arteries of New Zealand White rabbits. ~36 Yl/i-Herttuala et al. 264 re-

c e n t l y reported that IgG isolated from rabbit (WHHL) and human atherosclerotic lesions recognizes MDA modified LDL and copper-oxidized LDL but not native LDL.

Atherosclerotic lesions from control and probucol- treated WHHL rabbits showed equivalent immuno- staining with a monoclonal antibody against oLDL (OXL 41.1), although the lesions were significantly smaller in the probucol treated animals. TM In the probucol-treated animals, immunoreactive oLDL was predominantly present in smooth muscle cells, whereas in control WHHL rabbits oLDL was found to be mainly associated with m a c r o p h a g e s . 136,231 Co- cultures ofmacrophages and smooth muscle cells pre-

pared from aortas of atherosclerotic cholesterol-fed rabbits were shown to avidly metabolize modified forms of LDL (tested was ac-LDL) and thereby accumulate cholesteryl esters. 235 In additional s tud i e s , 43'51

it was proven that the antisera or antibodies did not bind to the native LDL but only to the LDL which had been modified either by copper oxidation or by treatment with MDA or 4-hydroxynonenal (HNE). The a u t h o r s 43'51'j36 therefore assumed that the antibodies recognize epitopes in LDL which are specific for oxidative modification. In case of the antibodies against MDA-modified LDL (i.e., MDA-lys, 135 MAL- 2, 43 MDA-2 ,136 it was assumed that the recognized structure is a "MDA-lysine adduct," since the antibodies also bind to albumin, hemoglobin, polylysine, and e-amino caproic acid previously reacted with MDA. To prepare the immunogenic MDA-LDL (or other MDA conjugates), LDL is incubated at 37°C for 3 h, pH 7.4, with 0.5 M MDA prepared by acid hydrolysis from MDA-bisdimethyl-acetal. Excess MDA is then removed by dialysis. Under these conditions, about 77% of the ~-amino group oflysine residues were modified as assessed by the trinitrobenzene sulfonic acid (TNBS) assay. From many other studies it is now well established (for review see Ref. 138) that concentrated MDA solutions as used in these studies are heavily contaminated with dimeric, trimeric, and polymeric forms of MDA and that in many cases these forms are considerably more reactive with amino groups than monomeric MDA. Moreover, the reaction of MDA with amino groups does not only lead to amino- imino-propene structures but also to aminopropenals and dihydropyridine derivates and possibly to other not yet defined products. The authors working with antibodies assumed that the lysine adducts recognized possess the amino-imino-propene structure. From the

Oxidation of L D L

Table 8. Continued.

355

Antibody Type of Type of Authors Code Antibody Immunogen Major Findings

Steinbrecher et al. 1989 (46) Guinea pig as Guinea pig acrolein-LDL Only LDL modified with aldehydes in

J0rgens et al. 1990 (49)

Rosenfeld et al. 1990 (136) Palinski et al. 1990 (51)

Guinea pig as Guinea pig crotonal-LDL the presence ofcyanoboron hydride Guinea pig as Guinea pig 2-pentenal-LDL is immunogen. Antisera were used Guinea pig as Guinea pig 2-heptenal-LDL to characterize epitopes on Co ++ Guinea pig as Guinea pig 2-nonenal-LDL oxidized human LDL; only a slight

immuno reactivity with Cu ÷+ oxidized LDL was found with these antisera. MAL-2 and HNE-6 also showed only weak reactivity (solid phase antibody binding).

Rabbit as Antiserum reacts strongly with HNE- LDL and with Cu ÷* oxidized LDL, VLDL, and lipoprotein (a), but not with LDL treated with MDA, hexanal, heptadienal, hydroxyhexenal.

MAL-2 The epitopes recognized by the two HNE-6 antisera and three antibodies were MDA-2 characterized by Palinski et al., 5~ NA 59 1990. Used for immunosatining of OLF4-3C l0 atherosclerotic lesions of varying

Guinea pig as Guinea pig as Mouse mAb Mouse mAb Mouse mAb

severity from WHHL rabbits. Staining predominantly cell associated with macrophages, in advanced lesions increasing extracellular staining.

Atherosclerotic lesions of humans and rabbits contain IgG recognizing MDA-LDL and Cu ++ oxidized LDL.

Yla-Herttuala (264) Human lgG Rabbit IgG

Human HNE-LDL

Guinea pig MDA-LDL Guinea pig HNE-LDL Mouse MDA-LDL Mouse HNE-LDL Mouse Cu ÷+ oLDL

Likely in vivo oxidized LDL

complex chemistry of MDA, however, it seems clear that additional work will be necessary to verify the chemical structure of the epitopes. The situation is similar with antibodies against HNE-modified LDL. To prepare this immunogen, LDL (2 mg/mL) was reacted with 5 mM HNE at pH 9.0 in the presence of NaCNBH3 at 37°C for 24 h and then d i a l y z e d . 43'51'136 It was assumed that the HNE formed a SchitTs base with e-amino groups (R-CH--N-protein), which was reduced by the NaCNBH3 to the corresponding stable secondary amine R-CH2-NH-protein). This is supported by the fact that the HNE-conjugate formed under nonreducing conditions was a weak immunogen, probably because it dissociated again in the re- versible reaction (R-CH----N-protein ,--, R-CHO + NH:-protein). Another very likely reaction occurring with HNE is the nucleophilic addition of amino groups to the CC double bond (R-CHOH-CH(NH- protein)-CHE-CHO), which yields a saturated aldehyde with the amino group attached to the carbon atom 3. This saturated aldehyde could further react to a SchilTs base. 138 Which reactions in fact are favored and occur if LDL is treated with HNE was not yet been investigated. Preliminary work from our labora-

tory (unpublished) with N-acetyl lysine as model compound suggests that the reaction is much more complex than normally assumed. Jiirgens et al. 49 reacted human LDL with HNE under nonreducing (i.e., without NaCNBH3) conditions and found it to be a strong immunogen in rabbits. This is clearly different to Palinski's observation 43's~ that HNE-conjugated mouse or Guinea pig LDL is immunogenic only if conjugation is carried out under reducing conditions. The antiserum prepared by Jtirgens et al. 49 is apparently very specific for HNE epitopes, since no noteworthy cross-reaction occurred with LDL modified under identical conditions with MDA, 4-hydroxy- hexanal, hexanal, or 2.4-heptadienal. Cross-reactions, however, occurred with HNE-treated human VLDL and lipoprotein (a) and Cu ÷÷ oxidized human LDL. Rosenfeld et al. 136 showed by Western blot analysis and radioimmunoassay (RIA) that MDA-lysine and HNE-lysine residues derived from apo B are present in Cu ++ oxidized LDL as well as in the LDL extract- able from the arterial wall of WHHL rabbits, which strongly suggests that such aldehyde-modified epitopes of apo B are in fact formed in vitro and in vivo by oxidative modification of LDL. Solid-phase corn-


petition RIA revealed 5~ that the antisera and monoclonal antibodies against MDA-modified LDL strongly bind also to other MDA-treated proteins (human serum albumin, hemoglobin, transferrin) and polylysine, tBOC-lysine, and e-amino caproic acid treated with MDA. The antibodies against MDA-LDL did not react with HNE-modified LDL or native LDL but with copper-oxidized LDL. In similar assays, the specificity of the antisera and monoclonal antibodies against HNE-modified LDL was tested, 43,5~ when again it was found that the antibodies also bound HNE-treated hemoglobin, transferrin, polylysine, tBoc-lysine, and copper-oxidized LDL. LDL treated with MDA, hexanal, or butyraldehyde showed no binding with these antibodies against HNE-modified LDL. The monoclonal antibodies against oLDL (OLF4-3C10) reacted with apo B from delipidated copper-oxidized LDL, and a weak reaction occurred with MDA-modified LDL, but no reaction was obtained with HNE-modified LDL. The antibody appears to be not specific for oxidatively modified apo B, since strong cross-reactions also occurred with copper-oxidized HDL. Even if all the work with antibodies directed against oLDL or aldehyde-modified LDL is still at its early stage, the conclusions which can be made are, first, that the apo B is modified by MDA and HNE when LDL is oxidized by copper ions, and second, that MDA and HNE-modified proteins (most likely derived from apo B) are indeed present in atherosclerotic lesions of WHHL rabbits. It remains, however, to be established whether such modifications also occur in lesions of humans. Furthermore, it should be emphasized that the chemical structure of the conjugate formed by MDA or HNE is not yet clear. Macrophage-derived foam cells, which were isolated by Rosenfeld et al. 139 from the artery of New Zealand white rabbits, made atherosclerotic (balloon deendothelialization, cholesterol feeding) as well as macrophages within sections of the lesions show im- munostaining with the polyclonal and monoclonal antibodies against MDA-LDL and HNE-LDL. 139 Since in this animal model predominantly VLDL is elevated (in WHHL rabbits only LDL), these results do suggest that not only oLDL but also oxidized VLDL can play a role in lesion development. Zawadzki et al. 14° showed with three monoclonal antibodies against epitopes localized in different parts of apo B from native LDL that the immunoreactivity steadily decreased during oxidation, but another epitope located at the C-terminus ofapo B chain exhibited with one of the antibodies (Bsol 7) significantly enhanced immunoreactivity during the first 6 h of copper oxidation, which then gradually decreased again. Similar

changes were observed during LDL aging, a condition known to be associated with mild oxidation. The immunoreactivity of the Bsol 7 epitope was assumed to be one of the most sensitive parameter of LDL oxidation. 232 Salonen et al. 54 recently reported the occurrence of autoantibodies to MDA-LDL in sera of atherosclerotic Finnish men; the titer of these autoantibodies was an independent predicator of the progression of carotid atherosclerosis.

UPTAKE OF OXIDIZED LDL BY MACROPHAGES:

FORMATION OF FOAM CELLS

Early atherosclerosis lesions are characterized by the presence of fatty streaks, which are composed of so-called foam cells. These cells have accumulated large amounts of lipids, predominantly cholesteryl esters. Foam cells are derived from smooth muscle cells and monocyte-macrophages. In the last decade, it became evident from animal experiments (for review see Refs. 141,261) that in a first step monocytes in- vade from the bloodstream into the subendothelial space and become resident macrophages. They take up lipids and lipoproteins, infiltrated and deposited in those regions. However, cultured mouse peritoneal macrophages (MPM) neither took up native LDL to a significant degree nor did they accumulate cholesteryl esters when exposed to even high concentrations for a long period, as shown by Goldstein and BrownJ ° On the one hand this was probably due to the fact that macrophages only express a very small number of LDL receptors, and on the other hand the expression of these receptors is finely tuned to prevent an intracellular accumulation of cholesterol and its esters.

A chemical modification achieved by treatment of LDL with acetic anhydride led to a form of LDL, which entered the cells via a receptor-mediated endocytosis not under feedback control, leading to massive cholesterol accumulation within the macrophages. 18 One of the steps of this modification, probably of crucial importance for the recognition of this modified form of LDL (acetylated-LDL = ac-LDL), is the blockage of the E-amino group of the lysine residues of apolipoprotein B, resulting in an increase in the negative charge of ac-LDL. Once the lysine residues are blocked, ac-LDL does not bind to the LDL receptor but becomes recognized by another type of receptor, namely the ac-LDL or scavenger receptor(s) on mouse peritoneal macrophages. The ac-LDL receptor(s) is also expressed on monocytes freshly isolated from the blood but increases as much as 20-fold upon cultivation. Furthermore, this receptor was found and studied on peritoneal macrophages from rats and


dogs, Kupffer cells from rats and guinea pigs, tumour cell lines of the mouse (J774 and P388), as well as on endothelial cells. By treatment of smooth muscle cells and fibroblasts from the rabbit with phorbol esters, an upregulation of the ac-LDL receptor could also be achieved. Normally, these cells do not express this type of receptor. 142

The structure of the type I and type II macrophage scavenger receptors were just recently deduced by complementary DNA cloning by the group of M. Krieger. 52'~43 Both receptor types share five identical domains. However, the 1 10-amino-acid cyteine-rich domain VI of receptor type I is replaced by a six-residue C-terminus in receptor type II. 52't43

Other treatments of LDL, such as acetoacetyla- tion,145 malelylation,11 succinylation,~46 carbamylation, ~47 and incubation with malondialdehyde (MDA) or glutaraldehyde, 14 also transform LDL to a species which is readily recognized by the ac-LDL receptor(s). The ac-LDL receptor(s) is also able to recognize ligands, which are not necessarily lipoproteins but negatively charged polyanions or malelylated albumin. 18 However, the enhanced negative charge of modified LDL itself is probably not the only factor being responsible for recognition by the ac-LDL receptor(s); likewise the density of negative charges in certain regions of the macromolecule might be of importance.148 Another aspect is the recognition of car- bamylated LDL by the ac-LDL receptor(s); this was proportional to the degree of carbamylation, whereas in modification of LDL by MDA, recognition of MDA-LDL started only when 16.3% of the lysine residues on apo B had been modified by this aldehyde. ~49

Concomitantly with the exploration of the ac-LDL receptor, the question arose regarding the exact nature of the modification affecting LDL in vivo responsible for its recognition and unregulated uptake by macrophages. We want to point out that the group of Fogelman and Haberland 14'146'148349 studied extensively the modification of LDL by MDA. These authors assumed that during aggregation of thrombo- cytes a reasonable amount of MDA could be set free, which in turn would modify LDL particles nearby. 14,149 Comparing the capacity of modification by MDA with that of 4-hydroxynonenal (HNE), another aldehydic endproduct of lipid peroxidation of 18:2 or 20:4 PUFAs, it was shown that HNE is an even stronger modifier of LDL when compared on equal molar basis. ~18 Apart from lysine, HNE also modifies other amino acid residues such as tyrosine, serine, and histidine. ~8 Modification of LDL with low concentrations of HNE also reduced binding and uptake of the modified lipoprotein by the LDL-recep-

tor on fibroblasts. 15° This agreed with the observation that modification of LDL by MDA at lower concentrations hampered the recognition by the LDL-receptor. 149 A more extensive modification of LDL with HNE leads to the formation of aggregates of this lipoprotein. 39'~18 These aggregates are taken up by cultivated macrophages 0774), giving them a foamy appearance. This uptake was not mediated by the ac- LDL receptor(s), but facilitated via phagocytosis. 39 However, another study, modifying LDL concomitantly with MDA and HNE, demonstrated that the presence of MDA prevented formation of aggregates of LDL. At a constant level of HNE, an increasing amount of MDA led to an enhanced uptake of LDL by macrophages. Competition studies with labeled ac- LDL receptor(s). TM LDL modified by water-soluble products derived from autooxidation of unsaturated fatty acids, avoiding oxidation of LDL during the modification procedure, was rapidly degraded by cultured macrophages by means of the ac-LDL receptor(s). 46 To characterize the compounds eventually responsible for the recognition of LDL modified that way by the ac-LDL receptor(s), LDL was incubated with acrolein, crotonaldehyde, pentenal, heptenal, and nonenal. 46 Only incubation with nonenal in the presence of the reducing agent NaCNBH3 modified LDL to a form which stimulated its degradation in mouse peritoneal macrophages at rates comparable to oLDL. 46

The investigation on the modification of LDL by MDA or other aldehydes, 29'56 modifications which could be of physiological relevance in contrast to acet- ylation, were paralleled by studies of LDL modified in presence of cells. Henriksen et al. reported in 1981 ~ 5 that incubation of LDL with endothelial cells led to a modified form of LDL which was recognized by the ac-LDL receptor(s). Soon afterward it was shown that a free-radical-induced peroxidation of lipids made LDL cytotoxic 2° and that lipid peroxidation was a prerequisite for the uptake of LDL by macrophages. 23 Furthermore, it was demonstrated that solubilized fractions of apo B, after delipidation of oLDL, were responsible for the recognition of oLDL by the ac- LDL receptor(s). 152 Apart from an endothelial cell line, smooth muscle cells, 21'26'153 monocytes, 24'38'154 two myelomonocytic cell lines, 155 and macrophages ~ 56 were shown to be capable of oxidizing LDL. In all these studies the oxidized LDL was shown to be recognized and taken up by the ac-LDL receptor(s).

Studies on the intracellular processing of oLDL by macrophages showed that, differently to ac-LDL, only about 50% of the apo B is degraded by lysosomal proteases (cathepsins) to low-molecular-weight, tri-


chloroacetic-acid-soluble p r o d u c t s . 45,233 Thus significant amounts of nondegraded oLDL accumulate in macrophages. The resistance of oLDL to proteolytic cathepsin degradation is probably a consequence of the modification of the apo B by lipid peroxidation products. TM Another difference in the intracellular macrophagal processing between ac-LDL and oLDL is that the later yields significantly less cholesteryl esters, probably because oLDL has a reduced cholesterol content (Table 3) and some oxysterols formed by the oxidation process inhibit ACAT activity. 233

Recently receptors were detected on cultivated mouse peritoneal macrophages which did not recognize ac-LDL but recognized LDL incubated and oxidized by endothelial cells. 45 Another study revealed the existence of three classes of receptors on cultivated mouse peritoneal macrophages: a common one for ac-LDL and oLDL, one for ac-LDL solely, and one which specifically recognized and bound copper-oxidized L D L . 36

LDL was not the only class of lipoproteins shown to be modified by oxidation to a form which led to an enhanced uptake by macrophages./3-VLDL, a lipoprotein fraction occurring in cholesterol-fed animals and humans, was shown to be internalized by cultured rabbit aortic smooth muscle cells.~57 This inter- nalization was enhanced when /3-VLDL was incubated with bovine aortic endothelial cells. During this incubation, the /3-VLDL was oxidatively modified. The interaction of both /3-VLDL and oxidized /3- VLDL with cultured rabbit aortic smooth muscle cells was only in part mediated by the apo B/E receptor. ~57 Wiklund et al. 296 recently reported on the uptake of native and acetylated LDL in foam cells present in atherosclerotic rabbit aorta as measured by an in vitro perfusion system. They found that native LDL was taken up by the same mechanism as acetylated LDL. Addition of vitamin E (0.1 mg/mL) to the incubation medium prevented uptake of native LDL into the foam cells, suggesting that local oxidative modification of LDL plays a role in the uptake of LDL by foam cells.

Although there is an increasing evidence that oxidation of lipoproteins, especially LDL, will create a form of the lipoprotein which is taken up by certain cells in an unregulated fashion, it must be stated that other forms of modification of lipoproteins (e.g., in vitro by nonenzymic glycosylation or treatment of lipoproteins with proteases) can also lead to lipid loading of certain cells by the modified lipoproteins. An update of the latest results in this field was given in a recent review.~58

Another mechanism which may lead to foam cells

in vivo is the phagocytosis of LDL immune complexes by macrophages via the Fc r e c e p t o r . 236"237 Such immuncomplexes are likely to be a consequence of the autoimmune response to oLDL 54'~2''122 and possibly also to native L D L . 236-238

Several reports show that patients with severe atherosclerosis have LDL-immune complexes in the circulation, 239'24° which indicates that autoantibodies against oLDL and/or LDL were indeed produced) 4 LDL-immune complexes adsorbed to human red blood cells are rapidly phagocytosed by activated human monocyte-macrophages, and the uptake leads to intracellular cholesteryl ester accumulation. 23s Our group recently showed 24~ that severe atherosclerosis is associated with an up to 1 0-fold elevated serum neopterin level (neopterin is an index for activated macrophages).

MECHANISM AND KINETICS OF OXIDATION OF LDL

BY CELLS OR COPPER IONS

In the experiments 23 which led to the discovery that cells can oxidize LDL, the LDL was incubated with cultured rabbit aortic endothelial cells in Ham's F-10 medium (containing 3 #M iron, 0.01 #M copper ions) for 24 h. After incubation, the medium contained TBARS and the cell-conditioned LDL was more rapidly taken up and degraded by macrophages than native LDL. Since no modification of LDL took place in the presence of EDTA or in Dulbecco's modified Eagles (DME) medium, which is nominally free of iron or copper, it was concluded that transition metal ions are crucial for oxidative modification of LDL. It was also shown in this early investigation that LDL incubated 24 h in plain cell-free F-10 medium supplemented with 5 #M Cu ++ became oxidized and exhibited chemical (TBARS, REM, increased lysophosphatides) and biological properties (macrophage uptake) similar if not identical to cell-modified LDL.

Since then, many researchers working with oLDL use Cu ++ oxidation instead of modification by cells (for review see Ref. 62). The most frequently used procedure for preparing oLDL for biological experiments now is an incubation for 8 h or more in one of the cell culture media (F-10, DME) or plain phosphate-buffered saline (PBS) supplemented with Cu ++ ions in the range of 5 to 100 #M; in most studies the molar ratio of LDL to Cu ++ is about 1:10 to 1:20. With few exceptions, 21 ferrous or ferric ions were found to be weak prooxidants which do not lead to modifications recognized by macrophages. Recently Kuzuya et al.242 reported that concentrations of 10


~M FeSO4 or FeCI 3 induced oxidation of LDL comparable to 10 uM CuSO4, provided that the LDL was dissolved in 0.15 M NaC1 instead of 10 mM phosphate buffer. The authors assumed that phosphate buffer complexes iron (but not copper ions) and thus prevents formation of an iron-LDL complex necessary for initiation of oxidation. In the meantime we have repeated these experiments (unpublished) but could not reproduce them, under otherwise identical conditions (LDL: ion ratio 1:34, 37°C, 0.15 M NaCI, 0.15 mg LDL protein/mL). Oxidation of LDL by Fe ++ or Fe ÷++ after 3 to 5 h incubation was less than 20% of that produced by Cu ÷+. This agrees with our previous findings 58 and is supported by reports from several other groups.72'79' 161 Balla et al.327 recently suggested hemin as a possible physiological mediator for LDL oxidation and reported that in vitro hemin oxi- dizes LDL, and the heine-mediated oxidation is strongly accelerated by traces of H202 or lipid hydroperoxides. The somewhat inconsistent results regarding effÉciency of iron and copper ions as well as the degree of oxidative modification produced by a certain concentration of copper ions alone are likely to be due to differences in media composition. The F- 10 and DME medium contain amino acids capable of complexing Fe ÷+ or Cu ++ (e.g., histidine), and such metal ion complexes probably differ in their prooxidant capacity from free metal ions in plain PBS. To improve the comparability of results from different laboratories, it would be important in future work to standardize the conditions for oxidizing LDL in cell- free systems.

From experiments in which both TBARS and macrophage uptake were measured, one can conclude that TBARS must reach a threshold value of at least 25 mol/mol LDL (45 nmol/mg protein) in order to make the modified LDL cytotoxic, chemotactic, and degradable by macrophages. 4°'s8 Depending on the conditions (absence or presence of cells, cell type, medium, Cu ÷+ concentration), such a degree of oxidation is reached after about 12 to 24 h of incubation. Up to now, only two systematic investigations were made on the time course of cell-mediated oxidation of LDL.79' 156 In these experiments LDL was conditioned with mouse peritoneal macrophages in Ham's F-10 medium for periods up to 24 h. It was found that the LDL first lost its vitamin E within about 3 h; thereafter the lipid hydroperoxide content rapidly increased reaching a maximum of about 450 mol/mol LDL at 10-12 h incubation time. Afterward the peroxide content decreased again (Fig. 5, left panel). This time course fully agrees with that observed by Esterbauer et al. 4°'58'75 and EI-Saadani et al. 77 in Cu ++ stimulated

oxidation of LDL (Fig. 6). But more importantly, it was shown in these cell oxidation studies that the rate of formation of an LDL recognized and taken up by the macrophage scavenger receptor is maximal and temporarily linked with the decomposition of the lipid hydroperoxides. No measurable modification into high-uptake forms occurred during the period when the LDL became depleted of vitamin E, and modification was also minimal during the phase when the lipid hydroperoxides increased to the maximum value. Comparable results (Fig. 5, fight panel) were obtained with Cu ++ oxidation; here too, high- uptake forms of LDL were mainly generated at the late phase when the lipid hydroperoxides decomposed. 79 Ferrous sulfate in the absence of cells (Fig. 5, middle panel) led to some oxidation of LDL; the rate, however, was much lower than in the presence of cells. Moreover, the peroxides in the Fe ++ conditioned LDL were not decomposed and the LDL was also not degraded by macrophages. This clearly indicates that decomposition of lipid hydroperoxides is a necessary prerequisite to generation of epitopes on apo B recognized by the scavenger receptor. That the presence of lipid hydroperoxides in LDL is per se not sufficient for a rapid uptake by the macrophage scavenger receptor is also supported by oxidation of LDL with selected oxygen radicals. Hydroxyl and hydro- peroxyl radicals led to extensive oxidation of the PUFAs in LDL, yet the radical oxidized LDL was not taken up by macrophages. 4~''62 LDL oxidized by gamma-irradiation 88 was also not a good substrate for the scavenger receptor, but it could readily be converted into a high-uptake form when it was additionally treated with Cu ++, a condition known to decom- pose lipid hydroperoxides.

Details of the mechanism of metal-induced oxidation of LDL are not clear. Direct oxidation of pure lipids by free or complexed iron or copper under physiologically plausible conditions has not been demonstrated. Most authors faced with the need to explain metal-assisted peroxidation assume the occurrence of reactions analogous to processes leading to the reduction of H202. However, these proceed at reasonable rates only when the metal is in the reduced form: The rate constant of reaction Fe ++ + H202 is only about 70 M -~ s -l, but already this is almost 2 × 107 times greater than the corresponding rate constant for the Fe +++ form. 243 The reduction of H202 by Cu + is be- lieved to be quite fast, T M but again the reduction of H202 by Cu ++ is extremely slow and thermodynamically not favored.

It appears therefore that peroxidation of lipids in


macrophage uptake

lipid hydroperoxides ;/C~-Tocopherol

30. 6oo- 1 6 1 / FIO + 3/~M F,

+ m a e r o p h a ~ 25- 5OO

V " 2O- 4oo

t5" 3100 ~ - T / / /

10" 200 /

5" 100 14 I / -

J , .

1 . z-z II

i 0 12

F IO + ~ FeSO 4 F IO + IOOMh4 CuSO 4

o~-T 7 k / , /e,. .-----~---

LOCr~ LOOH / \

\ \

\

• I

12 2 4 0 1'2 2 4 0 2 4

incubation time, hours

Fig. 5. Temporal relationship between degree of LDL oxidation and its uptake by macrophages (79). LDL was incubated with macrophages in Ham's F-10 medium containing 3 #M FeSO4 (left panel) or in cell-free F-10 medium containing 3 #M FeSO4 (middle panel) or 100 uM CuSO4 (right panel). At the indicated time points the LDL was separated from the medium and its a-tocopherol (aT) and lipid hydroperoxide (LOOH) content and the uptake by macrophages (Mq) was determined, c~T and LOOH are in mol/mol LDL, Mq is in ug LDL protein degraded/mg cell protein in 20 h. Adapted with permission from Jessup, W.; Rankin, S. M.; De Whalley, C. V.; Hoult, J. R. S.; Scott, J.; Leake, D. S. c~-Tocopherol consumption during low-density lipoprotein oxidation. Biochem Z 265:399-405; 1990. Copyright 1990 The Biochemical Society and Portland Press.

isolated LDL by Cu ++ requires either the presence of preformed peroxides or agents or conditions capable of converting the ion to the active reduced form. The first requirement is likely to be met by the invariable presence of traces of peroxides in isolated lipid sam- pies. 245 Possibilities for the formation of the initial Cu + are more speculative. There is little doubt that the catalytically active ions are bound to the LDL particle. Both proteins 246 and phospholipids 247 readily bind many metals, including copper, and such catalytically active binding has been demonstrated in lipid micelles, 248 liposomes and microsomes, 249'25° LDL, 69 and phage particles. TM The ions need to be reduced before reaction with lipid peroxides, but once some Cu + form, more can be generated during subsequent peroxide decomposition. An interesting possible initial Cu + formation could be achieved in a process analogous to the reduction of Fe +÷+ by a-tocopherol incorporated in phospholipid liposomes. 25° In that process, the antioxidant was lost and the ion reduced. This resulted in rapid oxidation of further lipid molecules. Since part of the surface of the LDL particle is made up of exposed phospholipids which readily attract Cu++, 247 a similar reaction between the metal and a-tocopherol could occur. This possibility has not been tested experimentally. Taking into account the requirement of preformed lipid hydroperoxides

(LOOH) and a reducing agent, the principal step of Cu ÷÷ initiated LDL oxidation could be formulated as follows:

unknown reducing agent

I LOOH + Cu ÷ ~ LO. + OH- + Cu ++

initiation of lipid peroxidation

We have previously assumed 4° that the unknown reducing agent might be the preformed LOOH itself (LOOH + Cu ++ - LOO + Cu + + H+), and this mechanism was recently again proposed by Thomas and JacksonY 2 However, such a reaction is thermodynamically extremely unfavored (W. Koppenol, pri- vate communication) and therefore unlikely. The importance of traces of preformed lipid peroxides was recently clearly demonstrated 252 by experiments with ebselen, a synthetic Se containing compound with peroxidase-like activity. In the presence of glutathione, ebselen reduces LOOH to LOH. When LDL was first pretreated with ebselen plus glutathione and then reagents were removed (by dialysis) before oxidation, they totally prevented Cu ++ dependent oxidation as


._1 C3 _J

c~ E

E t -

160

140

120

100

80

60

40

20

° /j!

~ ~ ° ~ x / / /

- - x ~ \ Peroxides

/ o

!

, \ I / O ~ o

, \ o !

ncubation time (h) Fig. 6. Kinetics of formation of lipid hydroperoxides during Cu +÷ stimulated oxidation of LDL in PBS. O and x were LDL from two different donors. Reprinted with permission from Esterbauer, H.; Rotherneder, M.; Striegl, G.; et al. Vitamin E and other lipophilic antioxidants protect LDL against oxidation. Fat. Sci. Technol. 91: 316-324; 1989. Copyright 1989 FETT Wissenschaft E. V.

measured by TBARS and REM. This appears to indicate clearly that Cu +÷ oxidation has an absolute requirement for the presence of traces of LOOH in the LDL. Interestingly, oxidation of LDL by macrophages was also largely prevented if the culture medium was supplemented with ebselen + glutathione, which suggests that in this system lipid hydroperoxides are essential for LDL oxidation.

Speculation on the mechanism of LDL oxidation by cells in the presence of copper or iron ions presents few difficulties, because the system is complex enough to offer several alternatives. Tables summarizing cell oxidation experiments can be found in Refs. 62 and 40. The cells can produce a range of redox reagents which can react with the LDL directly or, more probably, reduce any transition metal ions present, facilitat- ing lipid peroxide decomposition and chain peroxidation. Recent studies suggest that a 15-1ipoxygenase activity forms the initiating peroxides when LDL is incubated with mouse peritoneal macrophages. 259 The involvement of a 15-1ipoxygenase in LDL oxidation is also suggested by the finding that aortic segments from cholesterol-fed rabbits and WHHL rabbits convert arachidonic acid to 15-HETE. 323'324 On

the other hand, 5-1ipoxygenase has been shown to be not essential for LDL oxidation by macrophages) 67 Several studies show that purified soybean lipoxygenase alone 26° or with phospholipase A 2 (Ref. 34) can oxidize isolated LDL and convert it into a form which is cytotoxic 26° and taken up by macrophages) 4 Inter- estingly, Cathcart et al.260 observed in their experiments that superoxide anion inhibited soybean lipoxygenase catalyzed LDL oxidation, whereas hydrogen peroxide appeared to be essential. A feature of the kinetics of oLDL formation during incubation with cell cultures is the long period required. Under such conditions, the chemical processes responsible for generation of oLDL are difficult to identify. It is clear, however, that oxidation of LDL is significantly accelerated by metal ions and that the process is inhibited by chelating agents, either in the absence or in presence of cells.

In agreement with this background, we have shown 58'69 that Cu ++ strongly binds to LDL, and we have proposed that LDL has at least two distinct copper-binding sites crucial for the initiation of lipid peroxidation. ESR measurements showed that Cu ÷+ binds to the LDL protein: ° Fluorescence spectroscopy investigations ~61 suggest that the copper ions bind in the vicinity of the lipid phase of LDL and lead to a preferential degradation oftryptophan residues in apo B; only 60 rain after addition of Cu ++ to LDL (0.2 mg protein/mL, 10 ~M Cu ++, 37°C) 28 of the 37 tryptophan residues contained in apo B were destroyed. The possibility should therefore be considered that the initiating radicals are formed site specifically at or near a tryptophan-Cu ÷+ complex. That binding of Cu ++ to the LDL is essential for the initiation of lipid peroxidation is clearly proven by the inhibitory effect of EDTA, which, if present in sufficient high concentration, prevents binding of Cu +÷ to LDL. Another free radical process which might have biological relevance in diabetes is the autooxidative glycosyla- t ion. 257'265 If LDL is incubated with high concentrations of glucose, trace amounts of transition metal ions generate free radicals, H202, and ketoaldehydes from glucose, and glycosylation and lipid peroxidation occurs concomitantly in LDL.

Whichever mechanism will ultimately prove to be involved in initiation of oxidative modification of LDL, it seems clear that the subsequent processes following initiation are always the same--that is, loss of antioxidants, lipid peroxidation, and decomposition of lipid hydroperoxides to aldehydes and other products (Fig. 7). An LDL at the late phase of decomposition will have more or less similar biological and chemical properties, regardless of how the oxidation


I INITIATION J I ipoxidase or

preexistin 9 LDOH

LDOH M n+ LOOH

LO" + OH" ~ Mtn+ m'~ H'+ LO0" ~ Oxidized / Antioxidants Antioxidsnts

X" LO" LDO" , 41k

LH L" LDD" LDDH

IC A,N-SamCH,NOI

LO" + LOO" e LDOH

/ ~ Lysophosphatides

Rearrangement and =L Aldehydes and other products consecutive products FragVmentstion JV (Hydrox~, ks=>, keto-hydroxy-, of apo B Covalent binding to apo B epoxJ-hydroxy productsl

Fig. 7. Scheme showing the major events occurring during LDL oxidation. LH is a lipid containing a PUFA; the main LH species in LDL is cholesteryl linoleate. X" is any reactive radical able to abstract a hydrogene atom from LH; L" is a carbon-cen- tered lipid radical (e.g.,-CH----CH-CH=CH-'CH-); LOO" and LO" are lipid peroxyl radicals and lipid alkoxyl radicals; LOOH are lipid hydroperoxides; in their decomposition to LO" and LOO" metal ions in both valency states (e.g., Cu++/Cu ÷ or Fe+÷+/Fe ÷+) can take part, but the reaction with Cu ÷ or Fe ÷÷ is thermodynamically favored.

was initiated. Vedie et al. 326 reported that LDL oxidized by Cu ++ for various times (up to 48 h) can be separated by anion-exchange chromatography on a Mono Q HR 5/5 column into five fractions, differing in degree of oxidation and macrophage uptake.

The sequence of the major events accompany- ing oxidation of LDL by Cu ÷+ in plain PBS or Ham's F-10 medium was studied in detail by us 40'58,83'85'86'96,163-166 and s o m e others, s°'79,80'167'168,256

The time course of oxidation can be followed by measuring the increase of TBARS, lipid hydroperoxides, conjugated dienes, 430 nm fluorescence 256 (Fig. 8), and aldehydes (Fig. 9). Other possibilities are the measurement of disappearance of antioxidants (Fig. 10) or PUFAs, the fragmentation of apo B (Fig. 1 1), and the increase of the relative electrophoretic mobility (Fig. 12). Each of these procedures alone gives only one aspect of the oxidation stage, and only a combina- tion of two or more time-related analyses allows us to predict the stage of oxidation and possible causal in-

terrelationships between the different events. It is clear from Figures 7 and 8 that the measurement of one single parameter at one time point is not sufficient to conclude whether LDL oxidation is in its early or late phase. This is particularly relevant to cell- induced LDL oxidation, where typically a single measurement is taken after a long incubation, making comparisons of results dependent on degree of LDL oxidation quite unreliable.

Based on many different time-dependent analyses, the chronology of LDL oxidation by Cu ÷+ ions can be divided into three consecutive time phases: lag phase, propagation phase, and decomposition phase. It is important to note that each subject's LDL exhibits its own characteristic kinetics and that sample-to-sample variation can therefore be a serious problem if kinetic data from different experiments with different LDL preparation need to be compared. A solution to this problem is to use in each experiment the diene versus time profile as time marker for the length of the lag


1 2 3

• - - - - - - - - - . ~ ' ~ . 1 / _ . / / / / . I T.A,s "ox'oEs , ,,- . . . . .-...~....

L.t__., I

10 15

incubat ion t ime (h)

Fig. 8. Kinetics of Cu +÷ stimulated oxidation of LDL measured by consumption of vitamin E, change of 430 nm fluorescence, lipid hydroperoxides, conjugated dienes, and TBARS. The numbers 1, 2, 3 on top give the length of the lag, propagation, and decomposition phases. Adapted from Esterbaur et al. 4°'85 with permission.

and propagation phase. Conjugated dienes develop in LDL through the oxidation of PUFAs with isolated double bonds to PUFA-hydroperoxides with conjugated double bonds (= dienes), with a UV-absorption maximum at 234 nm. Since LDL is fully soluble in aqueous phase and remains in solution during oxidation, the diene measurement does not require extraction of the lipids but can be performed directly with the oxidizing LDL sample, provided that the concentration is in a suitable range (0.1-0.5 mg total LDL/ mL) and that the solvent is sufficiently transparent at 234 nm. If LDL is dissolved in a cell culture medium (F-10, DME) instead of PBS, the content of aromatic

£3 ._1

0 E c

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Fig. 9. Kinetics of the formation of aldehydes during Cu ÷+ stimulated oxidation ofLDL. LDL (1 mg/mL) in PBS was incubated with 6.7 uM CuCI2. MDA = malonaldehyde, HNE = 4-hydroxynonenal. The arrows in the upper part represent the length of the lag, propagation, and the decomposition phases.

amino acids absorbing at 234 nm can impede the direct diene measurement. Our laboratory measures routinely the diene versus time profile by continuously recording the change of 234 nm absorption of the LDL solution (0.25 mg total LDL/mL = 0.1 uM, 1.66 uM Cu ++, PBS) in a 1-cm quartz cuvette. 75 Typi- cal examples are shown in Figure 13.

During the lag and propagation phase and the early part of the decomposition phase, the time course of the diene formation fully reflects the lipid hydroperoxides time profile (i.e., lag phases are identical and peroxide and diene maxima coincide temporally75). This has also been found for pig LDL. 76 The second increase of 234 nm absorption occurring shortly after the transit through the maximum is not due to newly formed peroxides but must be attributed to an increase of degradation products (e.g., u-¢t unsaturated carbonyls) absorbing in the 234 nm range. If this stage of oxidation is reached, peroxides of course no longer correlate with the dienes.

During the lag phase the LDL becomes progressively depleted of its antioxidants, with a-tocopherol as the first and/7-carotene as the last one (Fig. 10). During this period, only minimal lipid peroxidation occurs in LDL as evidenced by the measurement of fatty acids, TBARS, lipid hydroperoxides, or conjugated dienes. The approximate rate of diene formation during the lag phase is 0.3 nmol/mg protein/min, which means that one molecule of conjugated lipid hydroperoxide is formed on average in each LDL particle every 6 rain. A slow formation of lipid hydroperoxides during the lag phase is not unusual but indeed a consequence of the antioxidant effect of vitamin E


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Fig. 10. Temporal relationship between consumption of antioxidants and onset of lipid peroxidation in copper-stimulated oxidation of LDL. To an LDL solution (1 mg LDL/mL) in oxygen-saturated PBS (pH 7.4), CuC12 was added (6.7 #M final concentration). At the indicated time points, the antioxidants were determined by HPLC, and the degree of oxidation was determined by the conjugated diene absorption at 234 nm. The lower panel is an expansion showing the sequence during the first 60 min. Reprinted with permission from Esterbauer, H.; Puhl, H.; Waeg, G.; Krebs, A.; Dieber-Rotheneder, M. The role of vitamin E in lipoprotein oxidation. In: Packer, L.; Fuchs, J., eds. Vitamin E." Biochemistry and clinical application. By courtesy of Marcel Dekker, Inc., NY, 1992, pp. 649-671.

contained in LDL. Vitamin E scavenges lipid-peroxyl radicals (LOO") formed in LDL during the lag phase (LOO" + vitamin E --* L O O H + vitamin E radical). The tocopheroxyl radical was detected in LDL during Cu +÷ initiated oxidation. 5° Stoichiometric studies in other systems suggest that one vitamin E molecule scavenges two lipid-peroxyl radicals. 169 If this stoichi- ometry is applicable for LDL, the seven vitamin E molecules (a + gamma-tocopherol) contained on

average in LDL (Table 4) could scavenge 14 LOO" radicals yielding 14 lipid hydroperoxides and 7 oxidized vitamin E molecules-- that is, 14 LOO" + 7 vitE --* 14 L O O H + 7 vitE quinone. (We present this here in a somewhat simplified form; actually, one half of the LOO" gives LOOH, and the other half gives LOO-vitamin E adducts, but both would contribute to the diene or peroxide content of LDL. We also do not include the antioxidative effects of carotenoids,


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which act by largely unknown mechanisms. 321 There is also a suggestion 317,318 that vitamin E quinone can act as an antioxidant through a quinone/semiquinone redox cycle possibly driven by superoxide anion, which complicates predictions of the overall stoichi- ometry of the vitamin E reaction. At the end of the lag

100

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Fig. 12. Kinetics of the increase of the relative electrophoretic mobility (REM) of LDL during oxidation. LDL (1.5 mg/mL) in PBS was incubated with 10 uM CuCI2. The dienes (given as nmol/mg total LDL) were determined from the 234 nm absorbance. The electrophoretic mobility was determined by electrophoresis. Given is the change relative to native LDL (= 1.0). Reprinted with permission from Esterbauer, H.; Puhl, H.; Waeg, G.; Krebs, A.; Dieber-Roth- eneder, M. The role of vitamin E in lipoprotein oxidation. In: Packer, L.; Fuchs, J., eds. Vitamin E: Biochemistry and clinical application. By courtesy of Marcel Dekker, Inc., NY, 1992, pp. 649-671.

phase, the amount of lipid hydroperoxides measured experimentally from the diene absorption is about 20 mol /mol LDL, which is in rather good agreement with the value predicted by the scavenging effect of vitamin E. Analysis si of the lipid hydroperoxides contained in LDL oxidized by AAPH to about 4.6 mol LOOH/mol LDL by HPLC postcolumn chemiluminescence detection revealed the presence ofhydroper- oxides of cholesterylester (64%), phospholipids (12%), and triglycerides (24%).

When the LDL is depleted from its antioxidant it is, as expressed by Brown and Goldstein, i7° left to the mercy of oxygen. Or in other words, it is subjected to autocatalytic chain reactions of lipid peroxidation (= propagation phase). The rate of lipid peroxidation rapidly accelerates (see Fig. 13, lower part) in an exponential manner to a max imum equal to about three molecules lipid hydroperoxides formed in each LDL particle every minute.

The transit from the lag into the propagation phase and the exponential increase of the oxidation rate is mediated by the copper ions (which at this stage are probably released from the site where they were ini- tially bound) catalyzing the decomposit ion of the seed-lipid hydroperoxides formed during the lag phase to lipid radicals (LO' , LOO" ), which initiate by chain branching new series of free radical chain reactions. Using a-phenyl-t-butylnitrone (PBN) or 2- methyl-2-nitrosopropane (MNP) as spin traps, the formation of LDL-lipid radicals was observed by ESR

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rain. Fig. 13. Continuous measurement of LDL oxidation by the diene absorption. LDL samples (0.25 mg total LDL/mL in PBS) from two donors were supplemented with CuCI2 (1.67 uM) and the change of the 234 nm absorption was recorded in a spectrophotome- ter (LKB Ultrospec II) in l-cm cuvettes with automatic cuvette changing in 30-s intervals. The upper plot shows the diene vs. time profile; the lower figure is the first derivative (AA/At) giving the rate vs. time plot. The end of the lag phase is defined as the intercept at the abcissa in the diene vs. time plot (see arrows). After the end of the lag phase the rate increases exponentially (lower plot).

in Cu ++ or lipoxygenase oxidation, s°'253 At this time the structure of the radicals (L', LO', LOO') is not clear. EDTA added at any time point during the lag or propagation phase complexes Cu ++ and thereby prevents chain branching and further oxidation of LDL (unpublished from the authors' laboratory). The lipid hydroperoxides generated in the LDL particle during the propagation phase are labile intermediary products; their concentration rises within about 60-80 min to a maximum value. In case of Cu ÷+ stimulated oxidation, about 70-80% of the PUFAs are oxidized at the peroxide maximum (Table 9). Thereafter decomposition reactions become predominant, and consequently the lipid hydroperoxides or conjugated dienes start to decrease again. We define the time point of the diene maximum as end of the propagation and beginning of the decomposition phase. One should keep in mind, however, that both phases temporarily overlap and cannot be fully dissociated from each other. This is clearly evident from the fact that changes becoming prominent during the decomposi-

tion phase commence shortly after the onset of the propagation phase. Such changes with known kinetics include increase of aldehydic lipid peroxidation products (Fig. 9), generation of fluorescent chromophores (EX/EM 360/430 nm) in the apo B and the LDL lipids (Fig. 8), increase of the negative surface charge as measured by the REM (Fig. 12), fragmentation (Fig. 11), and modification ofapo B (Fig. 5). Other changes which probably have similar kinetics are formation of lysophosphatides and oxydienes 72 and loss of free amino groups in apo B. 71'112'168

The decomposition of LOOH to aldehydes is a gen- eral phenomenon during lipid peroxidation in biological systems (for review see Refs. 138, 171), and many findings suggest that these aldehydes (e.g., MDA, HNE) do act as "second toxic messengers." The amount of aldehydes which can be measured in oLDL strongly depends on the experimental conditions. LDL oxidized 24 h in the absence of Cu ++ in a dialysis bag contained 17.8 mol aldehyde/mol LDL; 29 LDL oxidized 3 h in the presence of 1.6 uM Cu +÷ contained 100 mol aldehyde/mol LDL, with 42% MDA, 12.5% HNE, 25% hexanal, and 20.5% others. 96 If LDL samples were oxidized in open vials, the concentration of aldehydes (except TBARS) decreased during the decomposition phase, most likely because of their volatility. However, if LDL was oxidized in closed vials with sufficient oxygen, the concentration of the aldehydes strongly increased during the decomposition phase (Fig. 9), by factors of 1.3 (MDA) to about 4 (hexanal, HNE), and the total amount of free aldehydes detectable at the end of the decomposition phase was about 300 mol/mol LDL (= 120 nmol/mg total LDL) (Table 9). Most of these aldehydes (except MDA) are lipophilic and remain associated with the LDL particlefl 9 Taking the lipid phase (= 80% of the LDL mass) as solvent, the aldehydes in the LDL particle reach a remarkably high concentration of about 120 raM. It has been shown 29 that at least 90% of the TBARS measured in oLDL by the conventional TBA assay is in fact free MDA. Because MDA is hydro- philic, it is, in contrast to most other aldehydes, released from the LDL into the aqueous phase. 29

Various lines of research suggest that a number of important changes occurring in oLDL during the decomposition phase result from aldehydes and their reactions with amino acid residues in apo B (for review see Refs. 56, 58, 138, 172). The strong increase of the 430 nm fluorescence ofapo B and the loss of free amino groups is, for example, likely to be caused by reactions of aldehydes with ~-amino groups of lysine residues. Similarly, the strong increase of the negative

Oxidat ion o f LDL 367

Table 9. Aldehydes in Native and Cu ÷+ Oxidized LDL

nmol per m g LDL Protein

Cu +÷ oxidized LDL

Native LDL 2 h 4 h 24 h

Total aldehydes 5.7 + 3.1 14 209 545 Iodometric determined peroxides 18.6 ___ 9.4 72 1000 227 Conjugated dienes - - 36 486 - - PUFAs consumed 0 91 1680 2310

Aldehydes, mol % of total aldehydes determined

Hexanal 0 51 25 42 Malonaldehyde (TBARS) 3.6 + 1.0 26 41 21 4-hydroxynoneal 1.1 + 1.3 10 12 21 4-hydroxyhexenal 0 0 4 9 Nonana l 0 13 5 5 Octanal 0 0 0.7 1 Pentanal 7 + 6 - - 2.2 - - Butanal 2.6 + 1.3 - - 1.6 w Propanal 1.9 _+ 1.4 - - 2.6 - - 2.4-hepdadienal 0 - - - - - - 4-hydroxyoctenal 0 - - 3.5 - -

Table compiled from data in Refs. 29, 77, 96, 166. The values for native LDL are m e a n _+ SD from at least three experiments. The values for Cu ++ oxidized LDL are f rom a single experiment, where L DL (0.055 m g pro te in /mL PBS) was oxidized with 1.66 z M CuCI2 in a tightly closed vial to avoid evaporation loss o f aldehydes. The linoleic and arachidonic acid content o f the L DL used for oxidation were 2168 and 168 n m o l / m g protein. Dash ( - - ) means not measured.

surface charge of LDL (Fig. 12) is most likely due to loss of positively charged amino groups through Schiff's base formation (R-CHO + protein-NH3 + --~ R - C H i N - p r o t e i n + H20 + H ÷) or formation of Mi- chael adducts with a,/3-aldehydes.138 However, it was shown recently by means of electron paramagnetic resonance (EPR) that additionally new negatively charged groups are formed on the surface of LDL oxidized by Cu++. ~73 The covalent binding of aldehydes to apo B is probably also, at least in part, involved in the formation of the characteristic epitopes which are recognized in oLDL by the macrophage scavenger receptor. In LDL containing phosphatidylcholine with 14-C arachidonic acid, a fraction of the radioactivity was associated with apo B after Cu ÷÷ oxidation. H2 The occurrence of aldehyde or carbonyl functions in apo B was demonstrated by reacting oLDL with 2,4- dinitrophenylhydrazine followed by separation of the apo B from the lipids. ~8 The isolated apo B was yellow colored and contained 68 nmol aldehyde functions/ mg protein (= 37 mol/mol LDL). Such aldehyde functions could result from 2-alkenals, which have reacted by Michael addition with nucleophiles (XH) according to R C H - - C H - C H O + XH -~ R C H X - CH2-CHO. Alternatively, some of the carbonyl functions could also be produced by free radical (LO',

LOO') mediated degradation of amino acids in a p o B. 174

Lipid-alkoxyl radicals are probably the cause of fragmentation of apo B. That apo B (MW 550 kD) is fragmented during LDL oxidation was first observed by Schuh et al.17 and later confirmed in several studies. 69'175'176 The time course of the loss of intact apo B is an exact mirror image of the increase of the dienes (Fig. 11). Apo B first breaks down into two distinct large peptides with molecular sizes of 260 and 232 kDa. With increasing lipid peroxidation, the remain- ing apo B and the 260 and 232 kDa fragments are further degraded to such an extent that SDS-PAGE shows only a smear of bands in a molecular range below 100 kDa (Ref. 69).

Many of the chemical (loss of NH2 groups), physico-chemical (increased fluorescence and REM), and biological (uptake by macrophages, cytotoxicity) properties exhibited by cell- or copper-oxidized LDL can be reproduced in full or in part by treatment ofunoxi- dized LDL with aldehydes (MDA, HNE, hexanals, alkadienals) or aldehyde m i x t u r e s . 39,56'1s0'172 This further supports the hypothesis that aldehydes act as ulti- mate damaging agents. As discussed previously, antibodies prepared against MDA- or HNE-treated native LDL react also with copper- or cell-oxidized LDL.


Table 10. Main Features, by which Oxidized LDL is Different from Native LDL

Chemical and Physicochemical Properties * Complete loss of antioxidants * More or less complete loss of PUFAs * Loss of phosphatidyl choline and cholesteryl ester * Increased content of lysophosphatidyl choline and oxysterols * Increased content of hydroxy- and hydroperoxy-PUFAs * Increased content of conjugated dienes * Increased content ofMDA, hexanal, HNE, and other aldehydes * Strong fluorescence at 430 nm with excitation at 360 nm * Partial loss of free amino groups in apo B * Fragmentation of apo B to smaller peptides * MDA and HNE epitopes on apo B recognized by specific antibodies * Increased electrophoretic mobility and increased density (1.06-1.08) * Increased tendency to aggregate, heterogeneity in size. * Conformational rearrangement of apo B structure and phospholipid monolayer (266) Biological Properties * Increased uptake and degradation by macrophages * Cytotoxic to most cells (19, 20, 34, 38, 132, 202, 278, 285) * Chemotactic for monocytes (267) and smooth muscle cells (268) * Inhibits NO activation ofguanylate cyclase (269) * Inhibits in isolated smooth muscle strips relaxation induced by acetyl choline, nitric oxide, and nitroglycerin (279-282) * Increases in cultured endothelial cells tissue factor activity (TF) and suppresses protein C (which increases thromboresistance) activity (275) * Suppresses the production of PDGF-mRNA and PDGF secretion by monocyte-macrophages (276) * Increases in macrophages glutathione content about twofold; HNE has a similar effect (283) * Systemic administration into hamsters causes immediate leukocyte adhesion to capillary endothelium (277) * Treatment of cultured endothelial cells with MM-LDL stimulates production of a number of biological active factors, such as monocyte

chemotactic factor, MCP- 1 (270); monocyte binding molecules (so-called endothelial-leukocyte-adhesion-molecules, ELAMs) (261, 271 ); growth factors for monocytes, M-CSF, and granulocytes, G-CSF (272), and tissue factors (TF) essential for coagulation (273).

* MM-LDL injected into mice increases in serum and tissue levels of MCP-1 and CSF (274). * MM-LDL inhibits mitogenesis in SMC and stimulates (low concentration) or inhibits PGI2 synthesis in SMC (307-309)

With the exception, of minimally modified LDL (MM-LDL), the altered properties are characteristic for LDL oxidized by Cu ++ for at least 4 h or by cells (endothelial cells, macrophages, smooth muscle cells) for about 24 h. References are only given for findings which are not explicitely discussed in the text.

This clearly indicates that these aldehydic lipid peroxidation products are indeed covalently bound to the apo B when LDL is oxidized by cells or Cu ÷÷ ions. MDA- and HNE-modified proteins (most probably apo B) have also been detected by immunohis tochem- ical methods in atherosclerotic lesions of Watanabe heritable hyperlipidemic rabbits (see Table 8). More- over, autoantibodies directed against MDA- or HNE- modified proteins are present in the serum of rabbits and humans, 43'54 All this underlines the theory that aldehydic lipid peroxidation products indeed play a significant role in oxidative modification of LDL, both in vitro as well as in vivo.

The aldehydes detected so far in LDL and listed in Table 9 are all (except MDA) derived from the methyl terminus of the PUFAs. The counterparts, where the aldehyde function is at the acyl chain, linked to the parental lipid molecule (i.e., phospholipids, cholesteryl esters) should also be present in comparable amounts in oLDL. Since these aldehydes also cer- tainly contribute to the altered properties of oLDL, it would be important to give more attention to their analysis. Steinbrecher et al., 46 for example, concluded from studies on modification of LDL by autooxidized

PUFAs that the recognition o f o L D L by the scavenger receptor is mediated by derivatisation of the apo B by lipid peroxidation products which are more complex than simple short- or medium-chain aldehydes (e.g., 2-alkenal). The complex chemistry of the numerous compounds formed by lipid peroxidation in biological systems was reviewed in Refs. 138, 171, and 177.

Recently it was shown by gas-chromatography 215'233'262 that LDL oxidized by Cu ÷+ or macrophages contains significant amounts of cholesterol oxidation products, with 7-ketocholesterol compris- ing about one half to two thirds of the total cholesterol. 7-Ketocholesterol was also identified in serum of cholesterol-fed rabbits. 3°4 According to these studies, 215 oxysterols are present in oLDL both in free and esterified form, indicating that both forms of cholesterol are susceptible to oxidation. From the graphs shown in this work, z15 it appears that after 24-h Cu ++ oxidation a high proportion of about 70% of cholesterol was converted to oxysterol. The increase ofoxy- sterols closely paralleled the increase in electrophoretic mobility. Other oxidized sterols likely present in Cu ÷+ oxidized LDL are 7-hydroxy cholesterol and 25- hydroxy cholesterol. 233 Thomas and Jackson 252 found


in Cu ++ oxidized LDL (5 #M Cu ++, 4 h) 13-HODE, 9-HODE, and 13-HPODE; macrophage-oxidized LDL contained per milligram LDL protein about 16 nmol 13-HODE and 10 nmol 9-HODE, or about 1% of the linoleic acid content of LDL (Table 4). Car- penter et al. 2~6 reported that cultures of human monocytes incubated with a complex of cholesteryl-linoleate and serum albumin (serves as artificial lipoprotein) produce cholest-5-en-3fl,7/3-diol.

Plasma treated with Cu ++ and H20 2 produces large amounts of oxysterols, the major product being cho- lesta-3.5-diene-7-one; other products identified by gas-chromatography are cholesterol a- and/3-epoxide, 7-ketocholesterol, and 25-hydroxy cholesterol. TM

Under otherwise identical conditions, plasma from diabetic patients produced about 30 times more oxysterols than control plasma. It is interesting to note that several sterol oxidation products, including cho- lesta-3.5-diene-7-one, were found in fatty materials isolated from the human aorta (reviewed in Ref. 319).

CHEMICAL, PHYSICO-CHEMICAL, AND BIOLOGICAL PROPERTIES OF OXIDIZED LDL

From the sequential changes occurring in LDL during oxidation by cells or copper ions, it is clear that the chemical, physico-chemical, functional, and biological properties of LDL change continuously during the lag, propagation, and decomposition phases. Due to this dynamic process, an oLDL with a defined constant composition does in fact not (at least in the classical chemical sense) exist. It is therefore very surprising that numerous laboratories appear to have samples of oLDL with reproducible biological properties. We conclude from that most of the biological studies were performed with a fully oxidized LDL which has reached the end of the decomposition phase, where most of the chemical reactions must come to a stop because no reactive structures are left in the LDL lipids or apo B. An analyst attempting to study the numerous oxidized lipids and fragments from apo B has an enormously difficult job. The classical methods developed for the analysis of lipids are not at all or only in part applicable to oxidized lipids. 2~5 For example, the enzymatic lipid assays were developed for determination of free and esterified cholesterol or triglycerides in serum or native lipoproteins, but not for oxidized lipoproteins. Completely wrong results for oLDL are obtained, for example, by conventional cholesterol assays based on cholesterol oxidase (makes H202) through interfering lipid hydroperoxides, so that some of the values for free or esterified cholesterol in oLDL reported in the literature (see Table 3)

should be regarded with reservations. Similarly, the unchanged phosphorous content (by which the amount of phospholipids is calculated) in oLDL should not be misinterpreted as unchanged phospholipids. All our attempts (unpublished) to determine the amount of unchanged residual cholesterol, cholesterylester, triglyceride, or phospholipids in oLDL with classical methods failed, through disturbances in- troduced by the oxidized products. An additional in- herent problem is that reference compounds necessary to establish structures of unknown oxidized lipids are rare. Taking all this together, it is understandable that the knowledge on the chemical composition of oLDL is rather limited; indeed, much more is known now about its biological properties. Much additional work will be required in the future to bridge this gap. In Tables 3, 4, 7, and 9 we have made an attempt to compare the presently available published data on the chemical composition of native and oLDL. Most analysis stem from copper-oxidized LDL, with comparable data on cell-oxidized LDL being rare and restricted to simple analysis such as TBARS, REM, amino groups, total peroxides, and lipid classes.

Table 10 summarizes the major chemical, physico- chemical, and biological properties, by which oLDL differs from native LDL. Recently it was discov- ered 27°-274 that a minimally modified LDL (MM- LDL) also exhibits a number of biological activities, which may play an important role for the pathogenesis of atherosclerosis. The biological effects of minimally oxidized LDL were recently reviewed by Leake. 328 Minimally modified LDL is prepared 273 by prolonged (3- to 6-month) storage of LDL (solutions in PBS containing 0.01% EDTA) at 4°C or by short incubation with 1 uM Fe ++. Such a mild oxidation leads to about 3 to 5 nmol TBARS/mg cholesterol. 272'273 This is equivalent to 4.7-7.8 nmol TBARS/mg protein or 2.6 to 4.3 mol TBARS/mol LDL. For comparison, fully oxidized LDL (oLDL) contains about l0 times more TBARS than MM- L D L Based on our kinetic experiments with Cu ++ oxidation, we would assume that MM-LDL has lost all antioxidants and represents a form which is in transit from the lag to the propagation phase. Such an LDL would be primed for rapid subsequent oxidation. The uptake of MM-LDL by cells occurs via the LDL receptor. At present it is, in our opinion, not evident whether the biologically active substances (oxidation products?) are already present in MM- LDL itself, or whether they are formed during the subsequent cell incubation, which usually lasts for at least 4 h.

370 H. ESTERBAUER ¢l a/.

The topic of the biological effects of the minimally modified LDL is largely outside the scope of this review but needs to be mentioned because of increasing attention it is receiving. CornweU and his collabora- tors 3°7'3°8 have shown that LDL with low levels of TBARS (9 to 11 nmol TBAR/mg cholesterol, prepared by incubation of LDL at 37°C in 96% air, 4% CO2) has profound effects on smooth muscle cells in culture: lowering of the mitotic index, changes in prostanoid synthesis, viability and thymidine incorporation. Some of these were prevented or reversed by antioxidants. It may well be that such subtle changes can precede gross cytotoxic effects of heavily oxidized LDL and contribute to the formation of foam cells. Another role for LDL with low TBARS in atherogenesis may lie in its ability to affect the synthesis ofpros- tacyclin PGI 2 and thus influence the progression of atherosclerosis. Recent work has resolved a contro- versy on the role of LDL in this process by showing that low-TBAR LDL stimulate and high-TBAR LDL suppress the synthesis of PGI 2 (Ref. 309).

EFFECTS OF ANTIOXIDANTS ON OXIDATIVE

MODIFICATION OF LDL

Since oxidative modification of LDL mediated by cells or occurring in cell-free medium results from lipid peroxidation, water- and lipid-soluble antioxidants should have a prominent effect in retarding or preventing the modification. This has in fact been confirmed in numerous studies with a variety of antioxidants. Steinbrecher et al. 23 showed in their classical study that inclusion of a high content of vitamin E ( 100 #M equal to 900 nmol/mg LDL protein) into the culture medium prevented oxidative modification of LDL by endothelial cells, as measured by TBARS and macrophage uptake. The dose of vitamin E used in this investigation was extremely high and about 80 times the amount of the endogenous vitamin E contained in native LDL, so that it is not surprising that LDL oxidation was retarded for up to 24 h. A lower dose of 20 gM (equal to 60 nmol vitamin E/mg LDL protein), as used by Morel et al., 22 reduced but did not completely prevent oxidation of LDL (increase of TBARS, REM, and cytotoxicity) in a 48-h smooth muscle cell or endothelial cell culture.

The protective effect of vitamin E against cell-mediated oxidation of LDL was repeatedly confirmed and can be considered as unequivocally proven. Pro- tection by vitamin E was found not only in cultures of e n d o t h e l i a l ce l ls 22'23'28 and smooth muscle cells 22 but also in monocyte-macrophage cultures. 24,ss,79 Other chain-breaking lipid-soluble antioxidants which were

shown to be potent inhibitors for cell-mediated LDL oxidation are butylated hydroxytoluene 21,22'24,27,2s,178 and probucol. 179,287 Water-soluble compounds, which prevented LDL oxidation by cultured cells, are glutathione and ascorbate. 22,~4'2~5 Breugnot et alfl s7 reported that inclusion of about 10 to 50 t~M phenothiazines (chlorpromazine, trifluoperazine) into the F-10 medium inhibits oxidation of LDL induced by endothelial cells or Cu ++ as assessed by TBARS, REM, and degradation by J744 macrophages. The protective effect of phenothiazines was similar to the effects obtained with comparable concentrations of probucol, BHT, or vitamin E. Later the same group 2s8 reported that inclusion of calcium antagonists (the most effective drug was flunarizine) into the medium also prevent oxidative modification of LDL by human monocytes or endothelial cells and by Cu ++.

The most potent inhibitors of LDL oxidation are agents complexing copper and/or iron ions, provided that they are present in sufficiently high concentrations. A complete blockage of cell-mediated LDL oxidation over 24 h and more can, for example, be achieved by inclusion of 50-100 #M EDTA or 10- 100 ~M desferrioxamine into the cell culture medium (for review see Ref. 62). The strong inhibitory effect EDTA is also used to protect LDL against oxidation during its isolation. For that, blood is drawn into EDTA-containing tubes, and the final EDTA concentration in plasma and in the concentrated LDL stock sample obtained by ultracentrifugation is 1 mg/mL (3 raM). Such high EDTA concentration would, of course, completely block LDL oxidation in subsequent experiments. To get rid of EDTA, most laboratories dialyze the LDL stock solution overnight at 4 °C against EDTA free buffer, made oxygen free by nitrogen gassing. We have substituted this lenghtly dialysis by separating LDL from EDTA on small Sephadex columns. 84 The group of D. Steinberf 3'25'27'57 pro- ceeds somewhat differently and dialyzes LDL against PBS containing 0.01% EDTA (to prevent any oxidation); in this case the dialyzed LDL stock solution still contains 300 uM EDTA. For oxidation experiments, this stock solution is then further diluted about 100- fold, which means that the final incubation mixture still contains about 3 uM EDTA that is equal to the iron ion concentration in F-10 medium (the exact EDTA concentration depends on the dilution factor). From that, it appears that low EDTA concentrations do not inhibit LDL oxidation by endothelial cells in F-10 medium. This is supported by a report from Heinecke et al. 26 that EDTA in equimolar concentration to Fe ++ (10 #M) stimulated oxidation of LDL in cultures of smooth muscle cells. Similar prooxidative


effects of EDTA-Fe ÷+ complexes have been reported for microsomal lipid peroxidation t8° and attributed to the capability of the iron complex to enter into the lipid phase. However, if oxidation is carried out in cell-free medium supplemented with Cu ÷+, residual EDTA severely interferes when its concentration is comparable to the Cu ÷+ concentration. This can be shown in kinetic experiments where LDL is incubated with a fixed amount of Cu +÷ (5 uM) and increasing concentrations of EDTA (0-1 0 uM). As the molar ratio of EDTA to copper approaches unity, the lag phase strongly increases; and if the EDTA is in excess, no oxidation occurs at all. 2~8 The reproducibil- ity of oxidation experiments with EDTA containing LDL samples might therefore be low unless the EDTA concentration is clearly defined and Cu ÷÷ is added in sufficient excess to overcome EDTA protection. In cases where the LDL stock solution contains 0.0 1% EDTA, a copper concentration of 5 #M, as used frequently for oxidation of the diluted LDL sample, is likely to be close to the borderline.

It has been pointed o u t 3°9 that BHT may be a more appropriate agent preventing LDL oxidation in tissue cultures than EDTA, which may have other actions, such as inhibition of PGi2 synthesis. BHT can block LDL oxidation at concentrations which are apparently harmless to smooth muscle cells.

Much work has been devoted to clarify the protective role of the endogenous vitamin E and other antioxidants contained in LDL itself. We s h o w e d 29 that autooxidation (in a dialysis bag, without addition of Cu +÷) of LDL as measured by the decrease of PUFAs or increase of aldehydes only occurs when LDL is largely depleted of its endogenous vitamin E and 13- carotene. When the loss of the endogenous antioxidants was followed during experiments with Cu ÷+ stimulated o x i d a t i o n , 4°'75J63 the same sequence was always found, as shown in Figure 10. The antioxidants disappearing first were ~- and gamma-tocopherol; thereafter the carotenoids decreased to zero, with cryptoxanthine first and H-carotene last. A fluorescent compound disappearing with the carotenoids was previously assumed to be retinyl stearate; ~64 later it was found that the compound is mainly phytofluene. ~63 Shortly after the time point when the LDL was depleted of antioxidants, a propagating lipid peroxidation chain reaction commenced, as indicated by rapid increase of the diene absorption. This finding was confirmed in several other studies. Steinbrecher et al . , 59 for example, showed that the increase of the REM of LDL is preceded by a lag phase during which the LDL-vitamin E and/3-carotene decreased to zero. J e s s u p et al. 79 showed that oxidative modification of

1 5 0

U)

C 1 0 0

~ 5 O Q

k = 1 . 5 7

o a = 5 8 . 9

o o ° o o

o~%o o ~ o o ~ o O

00°8 o o

r 2 = 0 , 0 4 3

( N.S. : n = 7 8 )

Q 4 8 1 2 1 6 2 0

tool dx - - tocophero l /mo l LDL

Fig. 14. Scatterplot showing the correlation between lag phase and c~-tocopherol content of LDL from not vitamin E-supplemented donors. The relationship is y = 1.57x + 58.9, r z = .043; n = 78. The statistical mean of x is 6.37 mol a-tocopherol/mol LDL (Table 4).

LDL (increase of lipid hydroperoxides, uptake by macrophages) by cultured macrophages or Cu ÷÷ ions does not occur unless it is depleted from its a-tocopherol.

This sequence of events suggests that the lag phase and hence the oxidation resistance of LDL is mainly determined by the antioxidant content; or, in other words, the lag phase should be predictable from the antioxidant status of the LDL sample. With our highly reproducible Cu ÷÷ oxidation assay (i.e., diene vs. time profile), the lag phase of a large number of LDL samples from different non-vitamin-E-supplemented donors was measured and correlated to the antioxidant content. 8sJ64'165 Much to our surprise, no clearly significant correlations were obtained, neither for the lag phase vs. a-tocopherol nor for lag phase vs. total antioxidants (i.e., vitamin E + all carotenoids) (Fig. 14). This indicates that at least for our study group, the antioxidant status is by itself not predictive for the oxidation resistance of any given individual LDL. This is in agreement with observations made by others, who found essentially no correlation between the vitamin E content of LDL and its oxidizability by gamma-irradiation 88 or by macrophages. 79'8°

This weak statistical correlation cannot, however, be interpreted as argument against the protective role of endogenous antioxidants, but rather as evidence that the oxidation resistance depends on more than one (i.e., antioxidant content) variable. Further investigations from our laboratory 85'~6s revealed that the oxidation resistance (y = lag phase in Cu ÷+ stimulated oxidation) of each subject's LDL depends on the vitamin E content (x), a subject-specific efficiency constant (k) of vitamin E, and a vitamin E independent variable (a) (Fig. 15). To assess the validity of this relationship and to estimate the respective subject- specific values for k and a, LDL samples from individuals differing in the vitamin E content must be avail-

372 H. ESTERBAUER el a[.

efficacy constant I ( alpha- tocopherol independent of alpha-tocopherol variable in minutes

y = k x + a

lag time alpha - tocopherol in minutes in mol/mol LDL

Fig. | 5. Equation describing the relationship between a-tocopherol and lag phase for the LDL ofa ~ven donor. Note that k and a vary from donor to donor. Reprinted with permission from Esterbauer, H.; Puhl, H.; Waeg, G.; Krebs, A.; Dieber-Rotheneder, M. The role of vitamin E in lipoprotein oxidation. In: Packer, L.; Fuchs, J., eds. Vitamin E: Biochemistry and clinical application. By courtesy of Marcel Dekker, Inc., NY, 1992, pp. 649-671.

able. A good method for loading in vitro one subject's LDL with vitamin E proved to be a 3-h preincubation of the plasma with increasing concentrations (125 to 1000 #M) of a-tocopherol prior to the conventional isolation of LDL by ultracentrifugation. '63 By that procedure, about 1 to maximally 10% of the added vitamin E became incorporated into the LDL, and consequently the isolated LDL samples had significantly increased contents of a-tocopherol. A dose of 1000 #M a-tocopherol added to plasma (this is about 40 times the normal plasma vitamin E level) increased the LDL a-tocopherol about two- to fourfold above the basal value, depending on the plasmaJ 63

When LDL samples from one subject loaded in such a way with different amounts of a-tocopherol were subjected to Cu ÷÷ oxidation, the lag phases always increased strictly linearly with the a-tocopherol content according to the equation in Figure 15, with correlation coefficients r 2 = 0.95 to 0.99 and significant levels o fp < 0.001 (for details see Refs. 85, 165).

Representative examples for two subjects are shown in Figure 16. The insert shows the linear de- pendency of the lag phase on the t~-tocopherol content. The different slopes and intercepts indicate that the efficiency of a-tocopherol (k) and the vitamin E independent variable (a) were different in the two subjects. In order to determine the interindividual variability of k and a, the investigations were ex- tended to a larger number of subjects. Both parameters varied unexpectedly over an extremely wide range, the mean + SD for k being 5.19 _+ 5.61 min (range 0.7 to 34.2, n = 40), and the mean ___ SD for a 40.9 _+ 29.9 min (range -49.3 to +97.3, n = 40). This explains why the vitamin E content is by itself not predictive of the oxidation resistance of a given LDL sample, since additionally the particular values for k and a must be known.

Still another possibility of changing the LDL vitamin E content of individuals is oral supplementa-

tion. 83,84 Although this takes a lot more trouble than the in vitro loading, we felt that such an ex vivo study is an essential supplement to the in vitro study. Clini- cally healthy volunteers took daily placebos over three weeks or 150, 225, 800, and 1200 iu RRR-a- tocopherol. The plasma and LDL antioxidant status (a- and gamma-tocopherol, all carotenoids) and the oxidation resistance of LDL (diene vs. time profile) were measured prior, during, and after supplementation as shown in a typical protocol in Table 11. The complete lipoprotein status (cholesterol, triglycerides, HDL cholesterol, LDL cholesterol) of all subjects par- ticipating in the study was determined prior to and after supplementation. The main findings of this study were briefly as follows: 83-85'165

1. All participants taking vitamin E capsules had higher plasma and LDL a-tocopherol levels compared to the initial levels measured prior supplementation (Table 12). Seven days after termination of vitamin E intake, the a-tocopherol was decreased again and very close to the initial value. The plasma and LDL carotenoids did not change significantly during the supplementation, but the gamma-tocopherol strongly decreased both in plasma and LDL. The increase of plasma a-tocopherol seen in our study group is in accordance with an early report by McCormick et al. 312 It also con- firms a study by Kitagawa and Mino, 'sl who found that oral intake of 900 IU RRR-a-tocopherol increases plasma a-tocopherol about 2.5- to 3-fold above the baseline value. Dimitrov et al.zs9 recently reported the results from a phase I study that used single and multiple doses of d, 1-a- tocopherol. Administration of a single of dose of 400, 800, or 1200 IU resulted in an elevation of plasma a-tocopherol with a peak between 12 and 24 h. The chronic administration of the same doses for 28 d led to a plateau by days 4 to 5, where


l donor

0.80

0.60 ' I

< °'°t J J/J/ Oo : r 1 = O . g 6

0.20

0,00 / ~ i tool oti-T/rno, LDL

0 60 120 180 240

time, minutes

E

CO Cq

<

1.00

0.80

0.60

0.40

0.20

0.00 0

, i ~ m ~ , donor E~

~¢°o , ~ , rZ --O'g~ o

tool ~--T/rt~l LI~_

60 120 180 240 300

time, minutes

Fig. 16. Determination of the efficacy of a-tocopherol to increase the resistance of LDL from two subjects against Cu ÷÷ oxidation. The LDL samples with different a-tocopherol contents were prepared by adding increasing concentrations of a-tocopherol to plasma samples of the donors prior to isolation of LDL. LDL (0.25 mg/mL) in PBS was oxidized with 1.67 ~M Cu ÷÷. For donor A, the relationship is y = 5.4 Ix + 6.48 (r z = 0.96); for donor B, y = 3.24x + 50.1 (r ~ = 0.98). Reprinted with permission from Esterbauer, H4 Puhl, H.; Dieber-Rotheneder, M.; Waeg, G.; Rabl, H. Effect ofantioxidants on oxidative modification of LDL. Annals Med. 23:573- 581; 1991. Copyright 1991 The Finnish Medical Society.

.

dation resistance as compared to the values determined prior to or 7 d after termination of the vitamin E intake. The average increases of the lag phase were 18, 56, 35, and 75% for doses of 150, 225,800, and 1200 IU (Table 12). For each person taking vitamin E, the temporal change of the oxidation resistance of LDL resem- bled very closely the temporal change of the a-tocopherol content in LDL (i.e., lag phase increased when a-tocopherol increased and vice versa; Fig. 17). The plots of measured lag phases versus the associated LDL a-tocopherol according to equation in Figure 15 gave for all subjects straight lines with correlation coefficients of r 2 = 0.5 tO 0.9 and significant levels o f p < 0.001. This proves that the protective effect of vitamin E in LDL from single donors can be described by the same linear relationship (y = k x + a), regardless of whether loading of the LDL with vitamin E is performed by adding it to plasma or by oral intake. The individual values for k (range 1.4 to 10.0 rain, mean ___ SD 4.66 +_ 2.5) and a (range 31.8 to 64.4 min, mean _+ SD 35.9 _+ 26.1) determined for the subjects participat- ing in the vitamin E study were in the same range as found by supplementing the plasma. This suggests that both types of supplementation lead essentially to the same results regarding the protective effect of vitamin E. The linear relationship between lag phase and a-tocopherol seen during the three weeks of supplementation further shows that k and a did not change during this period. This suggests that k and a are indeed characteristic subject-specific constants by which the oxidation resistance of the respective LDL is determined. At present, however, we do not know whether a person's constants change with age or living and dietary habits.

the average increase was about 80% and similar for all groups. A high-fat intake significantly increased the resorption of vitamin E and vitamin E levels in plasma.

2. The total plasma cholesterol levels showed no significant change, neither in the placebo nor in the experimental group. The plasma triglycerides in the vitamin E group were, except in one case, slightly (_<20%) elevated after termination of the supplementation. FarreU and Bieri t82 also found a slight increase in serum lipids (triglycerides and cholesterol) in healthy adults who received daily 800 IU of vitamin E.

3. The LDL isolated from plasma during the vitamin E supplementation period exhibited a higher oxi-

If the many data (n --- 206) for lag phases and LDL a-tocopherol levels determined by us so far (i.e., basal values, LDL loaded in vitro with vitamin E, oral intake of vitamin E) are treated statistically, a highly significant positive correlation is obtained with y = 2.94x + 52.4, r 2 = 0.46; p < 0.001; Fig. 18). Statisti- cally only about one third of the lag phase can be attributed to vitamin E, whereas two thirds are due to the vitamin E independent variable a. Our first suspi- cion that this variable mainly reflects the variable ca- rotenoid content of LDL could not be verified with certainty. Linear regression analysis for the correlation of the lag phase and the total LD L antioxidants (vitamin E + carotenoids) gave more or less the same result as shown in Figure 18 for a-tocopherol alone.


Table 11. Protocol for Oral Supplementation with Vitamin E

Day of study - 3 3 5 10 12 18 26

c~-tocopherol, mol/mol 8.40 12.03 16.93 15.93 20.6 26.08 8.83 Gamma-tocopherol, mol/mol 0.75 0.13 0.18 0.18 0.18 0.15 0.45 r-carotene, mol/mol 0.45 0.30 0.25 0.20 0.25 0.25 0.30 Cryptoxanthine, mol/mol 0.48 0.33 0.33 0.28 0.25 0.30 0.38 Lycopene, mol/mol 0.15 0.10 0.10 0.08 0.13 0.10 0.33 Zeaxanthin + lutein, mol/mol 0.08 0.05 0.05 0.05 0.05 0.05 0.10 Lag phase, min 75 118 132 120 141 170 103

The subject received 1200 IU RRR-a-tocopherol/day for 21 d; the indicated analyses of LDL were performed 3 d prior to the start of vitamin E intake (day 3), 5 times during vitamin E intake, and 5 d after termination (day 26).

To dissociate the potentially protective effects ofcarot- enoids and possibly ubiquinol-10 from vitamin E, it would be necessary to load LDL with these antioxidants.

Our statistical results (Table 13; Figs. 18, 19, 20) are still based on a rather small sample size and are all obtained from subjects living in the same area and having more or less the same dietary and living habits. It would be worthwhile to investigate if other study groups in other countries exhibit comparable rela- tionships. In our study group, the bulk of vitamin E values (Fig. 3) and k and a values (Figs. 19, 20) were within a Gaussian frequency distribution, but there were also persons whose LDL gave k and a values clearly outside this distribution. It is intriguing to spec- ulate that a high vitamin E intake and high LDL vitamin E levels are crucial for those persons having a low or even negative value for the vitamin E independent variable a. For such persons vitamin E is, at least in in vitro oxidation, the only protective component in LDL. On the other hand, for persons where the efficiency (k) of vitamin E is low, even megadoses of vitamin E may bring only a minimal additional protective effect. The factors determining the respective efficiency of vitamin E and the vitamin E independent protection are not known. Important contributions

Table 12. Effect of Dietary Vitamin E on the a-Tocopherol Content of Plasma and LDL and the Oxidation Resistance (lag

phase) of LDL

Dose a-tocopherol a-tocopherol Oxidation IU n in Plasma in LDL Resistance

0 20 95+ 11 98+ 17 97_+30 150 10 146+18 138_+12 118_+17 225 10 165 _+ 15 158 _+ 32 156 _+ 22 800 10 183 _+ 23 144 _+ 12 135 _+ 23 1200 10 248 _+ 53 215 _+ 47 175 _+ 21

Volunteers took daily for three weeks the indicated doses of RRR-a-tocopherol. The values are mean percentage _+ SD compared to the initial value at day - 3 (= 100%). n is the number of analyses.

might come from the amount of PUFAs, the ratio of PUFAs to saturated fatty acids, the cholesterol content, preformed peroxides, mobility of vitamin E, structure of apo B, or amounts of other antioxidants, such as plasmalogens. 226 Although these factors are not yet known, we feel that the measurement ofk and a for individual persons provides valuable additional information regarding their antioxidant status and might be of considerable medical interest in treatment of patients with vitamin E. De Graaf 286 separated human LDL (1 1 healthy volunteers) into three subfractions differing in density--LDL~ (very light), LDL 2 (light), and LDL 3 (dense)--and determined their oxidation resistance by measuring the diene versus time profile in Cu ++ stimulated oxidation. The dense LDL fractions (LDL2, LDL3) were clearly more susceptible to oxidation (as evidenced by the reduced lag phase) than the light LDL~; also, LDL2 and LDL3 contained more conjugated dienes after 4 h oxidation, probably because of their higher content in PUFAs. Chait et al. 263 recently studied the oxidation susceptibility in six LDL subfractions prepared by equilibrium density ultracentrifugation. The fractions were oxidized by 1.67 gM Cu ++ and the diene versus time profile was recorded. The denser LDL subfractions (fractions 3, 4, 5) had the lowest lag time and the highest rate of oxidation (= increase ofdienes during propagation). This indicates that the denser LDL particles (which are predominant in atherogenic lipoprotein phenotype subjects with subclass pattern B) are significantly more susceptible to oxidation.

The protective effect of several other lipophilic antioxidants was determined by us similarly to vitamin E by preincubation of plasma from a single donor with different concentrations of the agent to be tested. In the isolated LDL samples, the amount of the respective substance as well as the resistance toward Cu ++ oxidation (diene vs. time profile) was then determined. Figures 21 and 22 show the effects of a-tocotrienol and probucol. Both compounds gave a concentration-dependent linear increase of the lag phase.


180 ~ ' ~ p h a 30 db -J

se _ o

.c_ E E 120 20 ~_

0

/ / ~ / _ \ G - T 0 _C

cz 60 \ o • 10 o

o ~ o T 2o tool - / tool L[~_ ~ 0

0 . . . . . . . . . . . . . . , . . . . , . . . . . . . . 0 E - 5 0 5 10 15 20 25 30

c ~ - t o c o p h e r o l

gig. 17. Plot showing the effect of oral intake of vitamin E on o~-tocopherol content and oxidation resistance (lag phase) of LDL. The subject received daily 1200 IU RRR-a-tocopherol for 3 weeks; baseline values were determined 3 d prior (day -3 ) and 5 d after termination of supplementation (day 26). • a4ocopherol; B lag phase. The correlation in the insert is y = 4.44x + 52.4, r 2 = .868.

Probucol was more efficient than a-tocopherol or tocotrienol. Barnhart et al. 2~7 reported that in the presence of 0.6 mol % probucol relative to phospholipids (corresponds to 4.2 mol/mol LDL), the time required for half-maximal LDL oxidation (3 #M Cu ÷÷) increased from 130 to 170 min. Our attempts to load LDL in vitro in a similar way to that described for vitamin E with r-carotene failed, since the solubility of r-carotene in the polar organic solvents used to supplement plasma (ethanol, DMSO) was too low. Jialal et al. 29° reported that a specially prepared solution of/3-carotene (i.e., r-carotene first dissolved in hexane at 80°C followed by stepwise dilution with

3 0 0

(/3 ¢

4-J

c-

E

d) ~3

_C {3_

Q~

2 0 0

1 0 0

0

0

k = 2 . 9 4 o

a = 5 2 . 4 o

0%0 ° o o ~ go #o

o2OO,Ooo o o . . . .

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°~ - ~ (p < 0 . 0 0 1 ; n = 2 0 6 )

i i i i

1 0 2 0 3 0 4 0 5 0

moL c~-tocopherol/mol LDL

Fig. 18. Statistical correlation between lag phase and a-tocopherol content ofLDL: y = 2.94x + 52.4, fl = 0.46, p < .001, n = 206. This plot contains all baseline values (Fig. 14) and all values from a-tocopherol in vitro or in vivo supplementation. This is an updated version from Refs. 83 and 85 with a larger number of subjects.

ethanol) added to the LDL (final concentration of ~3- carotene 0.5 to 2 ~M; the amount of r-carotene incorporated into LDL was not determined) significantly inhibited the oxidative modification of LDL by cultured human monocyte macrophages in Ham's F-10 as well as by Cu ++ in PBS. In this study, oxidative modification was assessed by TBARS, REM, conjugated dienes, and degradation by macrophages.

The effect of water-soluble antioxidants was tested by adding different amounts of aqueous solution to the assay immediately before initiating the oxidation w i t h C H + + . 4 0 ' 1 6 4 Ascorbate at micromolar concentrations prolonged the lag phase in a concentration-dependent fashion and exhibited a sparing effect on vitamin E and carotenoids. At 10 uM externally added ascorbate, for example, the endogenous antioxidants of LDL remained virtually unchanged for 90 min. 163J64 During this time, the ascorbate decreased to zero; thereafter the endogenous antioxidants decreased, with a-tocopherol first and r-carotene last. Thereafter the lipid peroxidation entered into the propagation phase with the same rate and kinetics as observed in the absence of ascorbate. Jialal et a1.144'291 also compared the effect of ascorbate (40-60 uM) and probucol (10 ~M) on oxidative modification of LDL by Cu ++ in PBS and in cultured human monocyte macrophages in Ham's F-10 medium. Both substances inhibited oxidation of LDL to a similar degree as assessed by TBARS, REM, and degradation by macrophages. In agreement with US, 163'164 it was also found that ascorbate had a sparing effect on the endogenous antioxidants (a- and gamma-tocopherol, r-car-


Table 13. The Oxidation Resistance of LDL (y = Lag Phase in Minutes in Cu ÷÷ Stimulated Oxidation) is Correlated with the LDL c~-Tocopherol (x = mol a-tocopherol/mol LDL) According to the Equation y = k~c + a

n Mean _+ SD Median Minimum Maximum

Oxidation resistance of LDL, y 76 67.5 + 15.1 66.0 34 114 a-tocopherol content ofLDL, x 95 6.49 _+ 1.90 6.22 2.9 14.9 Efficiency constant of a-tocopherol, k 55 4.39 _+ 3.05 3.79 0.7 17.14 Vitamin E independent variable, a 55 35.99 _+ 35.86 41.3 -68.6 108.6

The table gives means, medians, and ranges; n is the number of subjects studied.

otene) contained in the LDL; in contrast, probucol even in high concentrations (10-80 uM) did not protect the endogenous antioxidants. Frei et al. 297 recently reported that exposure of human plasma to gas phase oxidants of cigarette smoke induces lipid peroxidation and a slight increase of the REM of LDL. These effects were temporarily correlated with the complete consumption of endogenous ascorbate. In a similar study, Yokode et al. 298 previously found that a cigarette smoke extract (cigarette smoke passed through PBS at 4°C) modifies LDL into a form which has an increased REM and is about 12-fold more rapidly taken up by macrophages than normal LDL. This modification was, however, not associated with lipid peroxidation.

By analogy to other lipid peroxidation systems, it is reasonable to assume that the protective effect of ascorbate in Cu ÷÷ oxidation of LDL relies on its capability of reactivating vitamin E radicals in LDL according to vitamin E" + ascorbate --~ vitamin E + ascorbyl radical. This reaction most likely occurs at the surface of the LDL particle, where the chromanol- ring of vitamin E faces the aqueous, ascorbate-containing medium.

5 . . . . . . . . . . . . . . . . . . . . . . .

i2

o "" 9

- 6

,')i

, , . ,

::':::: !i::ii::i

-5 0 iO 15

k - v a l u e s

Fig. 19. Frequency histogram of the a-tocopherol efficacy constant k. Frequency gives the number of subjects found in a given group of k values.

A very powerful antioxidant for Cu ÷+ stimulated LDL oxidation is urate, which also led to a linear and concentration-dependent prolongation of the lag phase.40,163,164 A protective effect of urate was also observed by Sato et al.) 83 but the mechanism is not clear at all. Since urate produces a prolongation of the lag phase but has no effect on the rate of the subsequent propagation phase, it can be assumed that it does not act as a preventative (e.g., complexing of Cu ÷÷) but as chain-breaking antioxidant. Other water-soluble antioxidants which were tested by us are glutathione and trolox. Glutathione exhibited a sigmoidal dose effect curve with a plateau of maximum inhibition at about 7.5 #M. The dose effect curve of trolox, on the other hand, showed a hyperbolic shape with a decrease of inhibition above l0 uM.

The capacity of various agents to delay the propagation phase in a dose-dependent manner in Cu ÷÷ stimulated LDL oxidation was also studied by Jessup et al., 167 Huber et al.) 84 and Barnhart et a i r 17 These authors added the agents directly to the assay. Of the more lipophilic compounds, probably only a fraction distributed into the LDL, which may explain the somewhat lower activity of probucol compared to our

o 6 e-

o- 4

iOl .................. i

-tO0 -~) -20 20 60 i~ 140

a - v a l u e s

Fig. 20. Frequency histogram of the a-tocopherol independent variable a. Frequency gives the number of subjects found in a given group of a values.


E c-

Ch od

<

0.70

0.60

0.50

0.40

0.30

0.20

0.10

0.00

- 0 . 1 0 i I i I i I i I i I ,

0 60 120 180 2 4 0 3 0 0 3 6 0

t i m e , m i n u t e s

Fig. 21. Effect of a-tocotrienol on the oxidation of resistance of LDL. The LDL samples with different a-tocotrienol contents were prepared by adding increasing concentrations of a-tocotrienol to plasma samples of one donor prior isolation of LDL. The tocotrienol content of LDL was determined by HPLC. LDL (0,25 mg/mL) in PBS was oxidized with 1.67 #M Cu ++. Adapted with permission from Esterbauer, H.; Puhl, H.; Waeg, G.; Krebs, A.; Dieber-Rotheneder, M. The role of vitamin E in lipoprotein oxidation. In: Packer, L.; Fuchs, J., eds. Vitamin E: Biochemistry and clinical application. By courtesy of Marcel Dekker, Inc., NY, 1992, pp. 649-671.

results based on probucol actually present in LDL. Lean and H a g e m a n 213'214 loaded LDL in vitro with probucol by incubation of 0.2 mg protein/mL with 10 #M probucol for l h at 37°C and reported that all the probucol was incorporated into the LDL, so the LDL must therefore have contained about 25 mol probucol/tool LDL. This dose of probucol inhibited copper

oxidation (5 #M Cu ++, 37°C) for 24 h, as judged by circular dichroism spectroscopy, increase of 430 nm fluorescence, and decrease of reactive NH2 groups. LDL has a phase transition temperature around 35 to 40°C, and in vitro incorporation of lipophilic substances into the LDL is probably markedly facilitated if performed above the phase transition temperature.

E (-

Oh Od

<::

0.80

0.60

0.40

0.20

0.00 0 200 4 0 0 6 0 0 8 0 0

t i m e , m i n u t e s Fig. 22. Effect of probucol on the oxidation resistance of LDL. 2ts Loading with probucol and oxidation as in Figure 21, the probucol content of LDL was determined by HPLC.


Table 14. Effects of Antioxidants on Lag Phase in Cu ++ Stimulated LDL Oxidation

Prolongation of lag phase (minutes)

Agent added to the assay Ascorbate (40, 164) 1.2 _+ 0.1 Urate (40, 164) 6.9 _+ 0.5 Glutathione (218) 2.3 +_ 0.5 Trolox (40, 164) 4.2 _+ 0.6 c~-tocopherol (40) 0.5 _+ 0.2 Butylated hydroxytoluene, BHT (167) 13.0 _+ 3.6 Probucol (167) 3.3 _+ 0.3 Probucol (184) 8.0 lonox 220 (167) 11.7 _+ 1.5 Nordihydroguaiaretic acid, NDEA (167) 13.7 _+ 4.6 Eicosatetraynoic acid, ETYA (167) 0 Testosterone (184) 0 17-/3-estradiol (184) 12.0 _+ 4.0 Retinylstearate (40) 3.9 _+ 1.1

Agent incorporated into LDL a-tocopherol (164, 165) 4.6 _+ 3.3 a-tocotrienol (165) 5.4 _+ 1.0 Probucol (218) 11.7 _+ 1.5

Given is the prolongation of the lag phase in minutes produced by 1 mol agent/mol LDL. Table compiled from data reported by Esterbauer e t al. , 4°'164'165 Jessup et al., 167 Huber et al., ~s4 and unpublished results from Puhl et al. 2~s

An objective comparison of the effectiveness of lipophilic antioxidants requires exact knowledge of the conditions and the amount of the respective agents actually present in LDL. Table 14 lists those compounds for which dose effect curves of the antioxidant effect were measured under largely comparable conditions. Of course, a number of additional studies ex- ists, in which it was reported that a single, large dose of an antioxidant prevents cell or copper oxidation of LDL for a certain time.

Stocker et al. 81 reported recently that ubiquinol-10 protects LDL more efficiently against lipid peroxidation than a-tocopherol. This conclusion was based on the kinetics of the disappearance of antioxidants and the appearance of the first detectable traces of defined classes of lipid hydroperoxides (measured by HPLC postcolumn chemiluminescence detection) in LDL exposed to the radical generators AAPH, AMVN, or human PMN. They found that ascorbate disappeared first, followed by ubiquinol- 10, after which a-tocopherol, carotenoids, and urate started to decrease. The onset of detectable lipid peroxidation corresponded closely with the consumption of ubiquinol-10. The experimental conditions and approach used in this study differed in many respects from those used by us and many others (e.g., LDL concentration was 10- fold higher; the isolated LDL was contaminated by ascorbate, urate, and albumin; oxidation was initiated

by AAPH or AMVN). The most significant difference was that the LDL was oxidized only to such a low extent that it contained not more than about 0.7 to 3 mol LOOH/mol LDL. In addition, the rate of lipid peroxidation was extremely low with about 0.05 mol LOOH formed per LDL particle every minute. This has to be compared with the amount of 200 to 400 mol LOOH contained in fully oxidized LDL (end of propagation phase) and with the maximum rate of 3 LOOH formed every minute in an LDL molecule during the propagation phase (Figs. 5, 6; Table 9). We think that what Stocker et al. s~ measured was indeed the early part of the lag phase, yet not the onset of the propagating chain reaction occurring in LDL when it is depleted from all endogenous antioxidants. That LOOH are formed even though a-tocopherol is present fully agrees with the theory that vitamin E scavenges LOO" radicals. As outlined previously, the 7 mol vitamin E/mol LDL give by that scavenging at least 7 mol LOOH/mol LDL. Still another reason why ubiquinol-10 is not likely to be an important antioxidant is its low concentration, with only a small fraction of the LDL molecules containing ubiquinol- 10, while the others are free of this compound (Table 4).

AAPH-induced oxidation of LDL was also studied by Sato et al. 183 In contrast to the work of Stocker et al., sl it was found that the onset of rapid lipid peroxidation coincides with the depletion of vitamin E and that ascorbate and urate inhibit vitamin E consumption in LDL oxidation. These researchers also reported for the first time that the kinetic chain length during the propagation phase is about 10. Mino et al. 219 oxidized LDL and H D L with AAPH and found a strict linear correlation (r = .977) between lag phase (expressed as T inhibition) and the a-tocopherol content. The use of AAPH instead of Cu ÷+ to initiate oxidation in LDL could have several advantages if mechanistic aspects (e.g., kinetic chain length) are investigated. Figure 23 shows the diene versus time pro- files recorded by us for LDL samples with different o~-tocopherol contents exposed to AAPH (the LDL samples were those from donor A for which Cu +÷ oxidation is shown in Fig. 16). The initial more or less linear increase of the 234 nm absorption in the AAPH samples occurred with the same rate also in the absence of the LDL and is therefore not due to conjugated LOOH. Taking this into account, the kinetics of diene formation in the AAPH samples exhibit a lag phase and propagation phase similar to Cu ÷+ induced oxidation. As expected, the two prooxidants give, however, different lengths of the lag phases. The important point is that vitamin E delayed, as found by Mino


E ( - ,¢ o~ o4

<::

1.50

1.20

0.90

0.60

0.30

0 .00

0

l a g . rain

~ , h , i , i

60 120 180 2 4 0

t i m e , m i n u t e s

Fig. 23. Determination of the efficacy of a-tocopherol to increase the resistance of LDL against AAPH-induced oxidation. 2~g The LDL samples were those from donor A used in the Cu ++ oxidation shown in Fig. 16. LDL (0.25 mg/mL) in PBS was oxidized with 100 uM AAPH. The relationship is y = 6.23x + 25.2 (r z = .96).

e t al. , z~9 the onset of the propagation phase in the AAPH system in a concentration-dependent fashion, with an efficiency (k) very close to the value observed in the Cu ++ oxidation system.

The capacity of ascorbate and urate to delay in vitro a propagating lipid peroxidation in LDL is likely to have significant biological implications. Human plasma contains about 10-50 uM ascorbate and 200- 400 #M urate, and these agents should prevent depletion of the LDL vitamin E and oxidation of LDL in blood to forms causing foam cell formation or possess- ing chemotactic and cytotoxic properties. Where, then, can oLDL be formed? It is reasonable to assume that in the arterial wall local destruction of water-soluble antioxidants can occur (e.g., by oxygen radicals released from inflammatory cells). LDL infiltrated and present in such an oxidizing environment would solely depend on protection by its endogenous antioxidants, and under such circumstances sufficient levels of vitamin E as well as other antioxidants are crucial.

Does an elevated plasma vitamin E content reduce the risk of atherosclerosis, and can antioxidant treatment reduce oxidation of LDL in vivo and inhibit progression of atherosclerosis? A remarkable contri- bution relevant to this question comes from epide- miological studies (WHO/Monica project) in the European population. 48'185'1s6 Essential plasma antioxidants (a-tocopherol, ascorbate, vitamin A, carotenoids, selenium) were determined in 16 European study populations which differed sixfold in mortality from ischemic heart disease. The incidence of ischemic heart disease mortality showed a strong inverse correlation (r 2 = .63, p = .002) with the level of plasma t~-tocopherol. It was suggested by Gey 1s6 "that

the relative risk due to a low vitamin E status may have a greater quantitative importance than that of the classical risk factors such as total cholesterol and blood pressure." A low plasma level of a-tocopherol and ascorbate has also been identified as a risk factor in early angina pectoris.~ s7 Kok et al.292 compared selenium, PUFAs, and a-tocopherol in subjects with varying degrees of coronary atherosclerosis. Levels ofa-tocopherol and PUFAs were similar in the cases and in controls. However, the ratio Se/PUFAs was significantly lower in the cases, which suggests that a higher PUFA level with insufficient antioxidant protection might indicate a higher risk. Short-term supplementation of humans with vitamin C (l g/d, 2 weeks) or vitamin E (600 mg/d, 3 weeks) has a protective effect on in vivo oxidation of LDL (as measured by the content of TBARS in isolated LDL) induced by acute cigarette smoking. 42 Supplementation of elderly people with vitamin E led to a significant lowering of the plasma lipid peroxides ( T B A R S ) ) 9° Salonen et al. tgl

reported recently that the common carotid intima thickening is greater in men with high serum copper (this suggests that Cu ++ could act as a prooxidant in vivo) and low serum selenium. Supplementing men (39 subjects) with a low antioxidant status with 600 mg ascorbate, 300 mg a-tocopherol, 27 mg 3-carotene, and 75 #g selenium daily over 5 months resulted in a reduction of serum lipid peroxides, platelet aggregation, and platelet-produced thromboxane B2 (Ref. 300). Fruchart et al. 3°1 reported on in vitro oxidizability of LDL isolated from hypercholesterolemic men treated with vitamin E (1 g/d) or fenofribrate + vitamin E or placebo for two months. Copper-treated LDL samples from the patients receiving vitamin E showed a significant reduced uptake by mouse peritoneal macrophages as compared to oxidized LDL of the placebo group. Gaziano et a1.192 presented preliminary findings from the USA Physician Health Study, according to which the group receiving 50 mg 3-carotene every second day had a significant (44%) reduction of all major coronary events (such as cardiac death) and a 49% reduction of all major vascular events (such as stroke or cardiovascular death). All these findings suggest, but of course do not prove, that antioxidant therapy could lower the risk of atherosclerosis and ischemic heart disease. A controlled atherosclerosis intervention study with the classical antioxidants (vitamin E, vitamin C) or antioxidant drugs (e.g., probucol) has so far not been conducted or at least has not been published as a full paper. Short- term treatment with vitamin E appears to have no effect on the progression of the disease. In 75 heart patients, treatment with 200 mg all-rac a-tocopheryl-

380 H. ESTERBAUER ~ al.

nicotinate for 4-6 weeks gave no significant difference between the treated (38 patients) and the placebo group (37 patients).194 In angina pectoris patients supplemented with 1600 IU tocopheryl-succinate for 6 months (52 patients) or 3200 IU tocopheryl-succinate for 9 weeks (18 treated, 18 placebo), no difference in clinical parameters was seen in comparison with the placebo group. 195'196 Hata reported at a recent confer- ence 3°5 a secondary prevention trial on cerebrovascular disease with d, 1-a-tocopherol nicotinate. In total, 2358 patients with cerebral infarction (CI) were ran- domly allocated either tocopherol nicotinate (600 mg daily) or conventional treatment. The incidence of recurrent cerebral vascular infarction was significantly (p < .05) lower (125 cases) in the tocopherol nicotinate group than in the group on conventional therapy (172 cases).

A number of animal experiments related to antioxidant therapy of atherosclerosis were published. Ver- langieri et al . 193'197 presented data showing that daily doses of 108 IU of vitamin E/day significantly reduced symptoms of atherosclerosis in primates fed an atherogenic diet. This is in agreement with an earlier report 19s that atherosclerosis of triglyceride-fed rabbits can be prevented by vitamin E, and that vitamin E and vitamin A supplements reduce spontaneous atherosclerosis in hens during the egg-laying per iod . 199

Smith et al. z2° also reported on the protective effect of dietary vitamin E on atherogenesis in nonlaying hens. It has been reported that chronic deficiency of vitamin C or vitamin E is associated with atherosclerosis- like lesions in rodents, pigs, and primates. 2°°'2°~ Morel et al. 132 and Chisholm et al. 2°2 showed that treatment of rats suffering experimental streptozotocin diabetes with vitamin E (450 mg/d, 4 weeks) or probucol inhibits oxidation of LDL in vivo (as measured by TBARS of isolated LDL); moreover, the LDL from the treated rats was not cytotoxic in contrast to the LDL from the diabetic untreated group. Very high doses of vitamin E (10 g/kg diet, 14 weeks) were reported to potentiate in rabbits formation of lesions in aortas of cholesterol-fed rabbits that were mechani- cally deendothelialized. 2°3 Bj6rkhem et al. 3°4 reported about the effect of BHT on plasma lipids and development ofatherosclerotic lesions in rabbits fed a 1% cholesterol diet for 12 weeks. The addition of 1% BHT to the diet gave higher levels of cholesterol, triglycerides, and LDL, but despite that the degree of atherosclerosis of the aortic surface was considerably reduced as compared to the control group fed a cholesterol diet only. These authors also found that BHT led to a significant reduction of serum cholesterol oxidation products (7-ketocholesterol, cholesterol 5a, 6a- epoxide).

A number of studies have addressed the question of whether probucol (4.4'(isopropylidenedithio)-bis- (2.6-di-t-butylphenol) prevents progression of atherosclerosis, at least in part, by its antioxidant effects. Probucol was originally developed as antioxidant for manufacturing plastic and rubber. In 1969 and 1970 the first reports a p p e a r e d 2°4'2°5 that probucol lowers serum cholesterol in human subjects 2°4 and in mice, rats, dogs, and monkeys. 2°5 Probucol is now a widely used drug for treatment of hypercholesterolemia in people (for review see Ref. 206). In 1984 Naruszewicz suggested that it might limit oxidation o f L D L , 2°7 and in 1986 Patharasarathy et al. 2°8 presented evidence that probucol indeed inhibits in vitro oxidative modification of LDL (TBARS) by endothelial cells and copper ions and proposed that probucol, in addition to the lipid-lowering effect, may reduce progression of atherosclerosis by its antioxidant properties.

Kita et al. 2°9 showed that probucol treatment (1% in diet, 6 month) of WHHL rabbits significantly reduced the formation of atheromatas in the thoratic aorta. Carew et al. TM found that the formation ofmac- rophage-rich fatty streak lesions in WHHL rabbits was inhibited by probucol to a much higher extent than expected from its cholesterol-lowering effect. Probucol does not affect lipoprotein metabolism in macrophages of WHHL rabbits. 212 The plasma probucol concentration of the treated animals was 83 uM, and roughly two thirds was associated with LDL. This means that probucol must have reached a concentration of about 40-50 mol/mol LDL. 2t° Serum of treated patients contains about 25-100 #M probu- CO1. 210'213 As expected, LDL isolated from probucol- treated patients or rabbits was highly resistant to cell or Cu ++ oxidation in vitro. 2°9 Since the first reports, 209'211 several other studies were published which confirmed that probucol in the food (about 0.5 to 1%) reduces atherosclerotic lesions in rabbits fed a stan- dard c h o w 209"211'221-223 o r a c h o l e s t e r o l chow. 224 In

contrast to these studies, Stein et al. 225 found no protective effect of probucol (1% in chow) in New Zea- land albino rabbits fed a 1% cholesterol chow.

This article has focussed mainly on the role of antioxidants in conferring oxidation resistance to LDL. In the last 2 or 3 years, a number of experimental results have accumulated which strongly suggest that vitamin E and possibly also probucol exhibit a much broader spectrum of biological activities than expected just from their ability to act as chain-breaking antioxidants in lipid peroxidation. Azzi's group, ~ 88,189 for example, reported that 50-100 #M of a-tocopherol (added as an ethanol solution) inhibits proliferation of cultured smooth muscle cells, an effect not


associated with the antioxidant activity but mediated by a modulation of the protein kinase-C activity. As proliferation of smooth muscle cells is an important event during plaque formation, the beneficial effects of vitamin E in vivo may therefore be dual and not solely relying on its antioxidant properties. However, some aspects of the experimental protocol adapted in this study may require critical testing. The inhibition of cell proliferation may have been caused by the detergent effect of the added a-tocopherol, which would be absent with the acetate. Such an unphysiological detergent effect would correspond to the observations reported for free fatty acids. In a series of studies, Cornwell's group (for review see Ref. 320) investigated the inhibitory effects of polyunsaturated fatty acids on cell proliferation and developed the concept that PUFA-derived lipid peroxidation products inhibit smooth muscle cell proliferation, whereas antioxidants, such as a-tocopherol (optimal at 1 #M) or dipyridamole, promote proliferation by preventing lipid peroxidation. It was also proposed by Cornwell's g r o u p 97 that, in vivo, there might be an optimal level of minimally oxidized LDL, where proliferation of smooth muscle cells is inhibited whereas PGi2 synthesis is enhanced, a-Tocopherol likely also has a dual effect in the synthesis and secretion of collagen. 293,294 In cultured human fibroblasts, lipid peroxidation (induced, for example, by ascorbate and traces of iron) markedly enhances the collagen gene expression, by preventing lipid peroxidation, a-tocopherol indirectly prevents enhanced collagen synthesis. 293 Addition- ally, a-tocopherol decreases in fibroblasts the consti- tutive basal transcription of the procollagen al (I) gene. 294 Enhanced formation of collagen fibres is involved in intima thickening. That vitamin E can mod- ulate gene expression is also evident from the studies of Akeson et al. 29s who showed that a-tocopherol and probucol inhibit production of the cytokine interleu- kin- 1 (IL- 1 a) by preventing the activation of expression of the IL-I gene. IL-1 promotes a number of effects involved in the early phase of plaque formation (e.g., stimulation of smooth muscle cell proliferation and adherence of leukocytes to the endothelial lin- ing). Pretreatment of cells with vitamin E has a cyto- protective effect and protects cells against cytotoxicity of UV-oxidized L D L . 299 In an in vitro perfusion system, it was s h o w n 296 that uptake of native and acetylated LDL in foam cells in atherosclerotic tissue can be inhibited by adding vitamin E (0.1 mg/mL) to the perfusion medium. Finally, Steiner recently reported 3°6 that a-tocopherol is a potent inhibitor of platelet adhesion. Platelet adhesion was studied in 12 normal individuals supplemented with vitamin E up to 1200 IU/day. The a-tocopherol content in platelets

was correlated with the reduction in platelet adhesive- ness, with a maximal effect at 400 IU/day.

From all these studies, we conclude that vitamin E has, in addition to its antioxidant function in LDL, a great potential in preventing other deleterious events involved in the pathogenesis of atherosclerosis.

The therapeutic potential of vitamin E in various diseases, including those associated with oxidative stress, was recently reviewed by Janero 3°2 and C h o w . 303

Acknowledgement-- The authors' work has been supported by the Association for International Cancer Research (AICR), U.K., and by the Austrian Science Fondation, Project No. 8271-Med. We gratefully acknowledge the critical evaluation of this manuscript by Dr. David G. Cornwell and thank him for many valuable sugges- tions.

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ABBREVIATIONS

A A P H - - 2 , 2 ' - a z o b i s ( 2 - a m i d i n o p r o p a n e ) d i h y d r o - ch lor ide

a c - L D L - - a c e t y l a t e d L D L A M V N - - 2 , 2 ' - a z o b i s (2 ,4 -d imethy lva le ron i t f i l e ) apo B - - a p o l i p o p r o t e i n B E L I S A - - e n z y m e - l i n k e d i m m u n o s o r b a n t assay G C / M S - - g a s c h r o m a t o g r a p h y c oup l e d with mass

spec t roscopy H D L - - h i g h dens i ty l i popro te in H E T E - - h y d r o x y e icosa te t r aeno ic ac id H N E - - 4 - h y d r o x y n o n e n a l H N E - L D L - - 4 - h y d r o x y n o n e n a l mod i f i ed L D L H O D E ~ h y d r o x y oc t a de c a d i e no i c ac id H P L C - - h i g h - p e r f o r m a n c e l iqu id c h r o m a t o g r a p h y H P O D E - - h y d r o p e r o x y oc t a de c a d i e no i c ac id L D L - - l o w dens i ty l i pop ro t e in L O O H - - a l ip id h y d r o p e r o x i d e M D A - - m a l o n a l d e h y d e M D A - L D L - - m a l o n a l d e h y d e mod i f i ed L D L M M - L D L p m i n i m a l l y mod i f i ed L D L o L D L - - o x i d i z e d low dens i ty l i pop ro t e in P B S ~ p h o s p h a t e - b u f f e r e d sal ine P U F A - - p o l y u n s a t u r a t e d fat ty ac id R E M - - r e l a t i v e e l ec t rophore t i c m o b i l i t y T B A - - t h i o b a r b i t u f i c ac id T B A R S - - t h i o b a r b i t u f i c ac id react ive subs tances V L D L - - v e r y low dens i ty l i pop ro t e in W H H L - - W a t a n a b e her i tab le h y p e r l i p i d e m i c rabbi t s

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