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 G U N T H E R 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as some previously unpublished material from our laboratories.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 currently dying of myocardial infarcts or strokes

A t h e r o s c l e r o s i s is n o t a trivial o r 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 biol-
ogy 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 chemis-
try 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.
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 bio-
chemical mechanism of atherogenesis largely un-
known. 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 va-
lidity.
The bare bones of this recent postulate is that ath-
erosclerotic plaques form from cells engorged with lip-
ids supplied by blood lipoproteins, modified by a free
radical process. Pathological, microscopic, histochem-
ical, 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 identi-
fied as macrophages derived from monocytes circu-
lating in the blood 7"s'13and smooth muscle cells prolif-
erating in the region of the plaque. 4'13Their gross alter-

341
342 H. ESTERBAUER~'t al.

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

Year Observation Reference

1910 Cholesterol found in atherosclerotic plaques Windaus (12)

1952 Oxidized lipids in plaques Glavind (33)
1954 High LDL levels in accelerated atherosclerosis Gofman (1, 2)
1961 Lipid laden plaque cells are mainly smooth muscle cells (SMC) and macrophages (MPH) Geer (4),
Ross (13)
1971 Plaques contain foam cells Cookson (9)
1973 LDL is main supplier of cell cholesterol Bailey (3),
Goldstein (10)
1973 Oxidized lipids in plaques not produced by enzymic oxidation Harland (16)
1976 Some foam cells are MPH derived from monocytes Adams (5),
Schaffner (6),
Gerrity (7, 8)
1976 LDL apo B protein found in plaques Goldstein (10)
1977 Normal LDL uptake by human fibroblasts is receptor regulated Goldstein (10)
1979 Acetylated and maleylated LDL uptake by MPH and other cells via scavenger pathway gives Goldstein ( 11)
massive cholesterol deposition
1980 Uptake of malonaldehyde-modified LDL by MPH leads to cholesterol deposition--lipid peroxides Fogelman (14)
may be the cause
1981 Endothelial cells (EC) modify LDL to a form (mod-LDL) recognised by MPH acetyl-LDL receptor Henriksen (15)
1983 Modification of apo B lysine allows binding to acetyl-LDL receptor Brown (18)
1983 MPH and neutrophils oxidize LDL by a free radical mechanism Morel (19)
1983 LDL oxidized by free radicals is toxic to human skin fibroblasts Morel (20)
1984 Effect of hydroperoxides on uptake of LDL by SMC Nishigaki (31 )
1984 SMC produce mod-LDL in presence of metals Heinecke (21 )
1984 SMC and EC produce mod-LDL by free radical oxidation of lipid Morel (22)
1984 Modification of LDL by EC-involves lipid peroxidation Steinbrecher (23)
1985 Activated monocytes and neutrophils produce oxidized LDL (oLDL) Cathcart (24)
1985 Phospholipase A2 and free radicals from EC produce mod-LDL Parthasarathy (25)
1986 Superoxide free radical from SMC gives mod-LDL Heinecke (26)
1986 LDL oxidized by MPH is recognised by their scavenger receptors Parthasarathy (27)
1986 EC modification of LDL requires viable cells, H202 or superoxide radicals not involved van Hinsbergh (28)
1987 Inverse relation between plasma antioxidants and IHD Gey (48)
1987 Oxidaton of LDL alters particle composition and loss of antioxidants Esterbauer (29)
1987 Free radicals from thiols produce oLDL Parthasarathy (30)
1988 Mod-LDL found in human plasma Avogaro (32)
1988 Lipoxygenase with phospholipase A2 produce mod-LDL Sparrow (34)
1988 oLDL produced by superoxide from EC and SMC Steinbrecher (35)
1988 Lipid peroxidation and EC injury (review) Hennig (47)
1989 Monoclonal antibodies reveal oLDL in rabbit aorta lesions Boyd (37)
1989 Different uptake of acetylated LDL and oLDL by MPH Arai (36),
Sparrow (45)
1989 Involvement of EC lipoxygenase in formation of oLDL Parthasarathy (44)
1989 Lipid peroxidation products derivatize apo B Steinbrecher (46)
1989 Cigarette smoking renders LDL susceptible to oxidation Harats (42)
1989 Cytotoxic oLDL produced by superoxide from activated monocytes Cathcart (38)
1989 LDL modified by HNE phagocytosed by MPH Hoff(39)
1989 LDL protected by lipophilic antioxidants Esterbauer (40)
1989 Oxidized LDL produced by hydroxyl and perhydroxyl, but not superoxide free radicals, not Bedwell (41)
recognized by MPH scavenger receptor
1989 Monoclonal antibodies to oLDL or HNE-LDL recognize material in plaques Palinski (43)
1989 Plasma lipid peroxide levels elevated in ischemic heart and peripheral arterial diseases Stringer (105),
Domagala (109)
1990 Staining of lesions with several antibodies against epitopes characteristic for oLDL Palinski (51 )
Rosenfeld (136)
1990 Antibody to HNE-LDL recognizes copper-oxidized LDL, VLDL, and Lp(a) Jtirgens (49)
1990 Lipid free radicals found during copper oxidation of LDL Kalyanaraman (50)
1990 15-1ipoxygenase and oLDL co-localized in plaques Yl~i-Herttuala (53)
1990 Minimally oxidized LDL stimulates leukocyte endothelial interaction Berliner (271 )
1990 Characterization of MPH scavenger receptor Rohrer (52)
1991 Kupfer cells have an oLDL receptor van Berkel (55)
1992 Titers of autoantibodies against oLDL correlate with progression of carotid atherosclerosis Salonen (54)
 Oxidation of LDL
ation is mainly caused by the entry of lipids (e.g., lipo-
protein 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, to,1l,~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 knowl-
edge 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 pro-
ducing foam cells and the events which could produce
such modifications in vivo. It turned out ultimately
that the most physiologically probable LDL modifica-
tion 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 for-
mation 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 pur-
pose 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 athero-
genesis. Additional details can be found in several re-
cent reviews. 56-62'261
METABOLISM, STRUCTURE, AND COMPOSITION
OF HUMAN LDL
The liver assembles triglyceride-rich, very low den-
sity 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 endothe-
lial 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
343
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 choles-
terol. 63 The uptake of LDL by cells occurs via a recep-
tor-mediated pathway (B/E receptor) and by nonspe-
cific endocytosis. The LDL first binds to the B/E re-
ceptor (LDL receptor) present on the surface of most
cells and is then endocytosed. The highest concentra-
tion of LDL receptors is found in the liver, and about
three quarters of the LDL is removed from the blood-
stream 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 re-
duction 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 choles-
terol level. Very frequently used experimental animal
models are Watanabe heritable hyperlipidemic rab-
bits (WHHL), which have a defect in the LDL recep-
tor and develop severe atherosclerosis. 66
Human LDL is defined as the population of lipo-
proteins 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 sepa-
rated into two or more subfractions differing some-
what in density, size, and molecular w e i g h 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 calcu-
lated from values obtained from various sources is
given in Table 2. Taking 2.5 million as mean molecu-
344 H . ESTERBAUER et al.

lar weight of LDL, each LDL particle would contain

+l +1 +1 +1 +1 +1 about 1600 molecules ofcholesterylester and 170 mol-
ecules of triglycerides, which form a central lipophilic
core. This core is surrounded by a monolayer of about
700 phospholipid molecules (mainly phosphatidyl-
choline with minor amounts of sphingomyelin and
+1+1+1+1 lysophophatidylcholine) and 600 molecules of free
cholesterol. The polar heads of the phospholipids are
located at the surface of the LDL particle and contrib-
8 ute to the solubility of LDL in an aqueous phase. Em-
7- 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
+l+l+l+l
some models) but should rather be seen like an "octo-
e~
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
o part been determined directly on the protein and fully
deduced from the cDNA (for review see Ref. 73). The
+1 +1 +1 +1 +1 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
0 +1 +1 +1 +l +l
393, Thr 298, Trl0 37, Tyr 152, Val 251. From the 25
cysteine residues, 4 have the SH group free; the re-
~ ~ 0 mainder form SS bridges or thiol esters.
0
Apo B is glycosylated (the carbohydrate compo-
nents are mannose, galactose, glucosamine, and sialic
eq on -- ~" 0 ~oOr.,.) e~ 0 acid); the total carbohydrate content can amount to
0 8-10 weight % of the total apolipoprotein B. The mo-
0

~a lecular 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 aver-
• -,=~ II o "~:1~ ¢',1
age molecular weight of LDL would be 2.4 million,
which is very close to the value determined by neu-
tron 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 ex-
ist 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
O O
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 345

Table 3. Lipid Composition of Native and Oxidized LDL

Native LDL Oxidized LDL

nmol/mg LDL Protein mol/molLDL

Mean _+SD Mean
Total phospholipids 1300 +_227 700 No significant change of P content (71, 72)
Phosphatidylcholine 818 450 Decrease to 65-55% (28, 72)
Lysophosphatidylcholine 145 80 Increase to 250-300% (28, 71, 72)
Sphingomyelin 336 185 No significant change (28)
Triglycerides 304 _+ 140 170 Decrease to 76-52% (28, 71, 72)
Free cholesterol 1130 +_ 82 600 Decrease to 90% (71, 72) or increase to 150%(28)
Cholesteryl ester 2960 _+220 1600 Decrease to 48% (28)
Total cholesterol 4090 2200 Decrease to 78-60% (71, 72)
Free fatty acids 48 26 Increase to 170%(28)
Oxysterols 0 0 Increase to 33 ug (28) or to 120-240/~g/mgprotein (215)
Oxodienes Not detectable Strong increase (72)
Conjugated dienes Not detectable (72, 75) Strong increase to 190-350 mol/mol LDL (75-77)
lodometric LOOH 18.6 _+9.4 (77,78) 10 Strong increase to 190-550 mol/mol LDL (77-80, Figs. 5, 6)
Defined LOOH Not detectable (81) Increase (81)
Hydroxy and hydroperoxy 18:2 Not detectable (82) Strong increase to 30-200 mol/mol LDL (82)
Hydroxy and hydroperoxy20:4 Not detectable (82) Increase to 20 mol/mol LDL (82)
Prostanoid-like substances Not detectable (313) Material cross-reactingwith 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 followingsources:
Steinbrecher et al., 71 LDL oxidized by endothelial cells, 20 h, 37°C or by 5 uM Cu*÷ in PBS, 20 h, 37°C; Barenghi et al., 72 LDL oxidized by 5
~M Cu÷÷, 29 h, 37°C; van Hinsbergh et al.,28LDL oxidized by 25 t~M Cu÷÷in PBS, 48 h, 4°C; our laboratory,75-78LDL oxidized by 1.6 #M
Cu÷÷in PBS for 3-8 h at 25°C; Jessup et al.,79,80 LDL oxidizedby macrophagesor 100 ~M Cu++in F- I0; and Jialal et al.,2~sLDL oxidizedwith
2.5 ~M Cu*÷ PBS for 24 h at 37°C.

LDL) contains 22.4% phospholipids and 21.9% pro-
tein 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 L D L (e.g., fatty acids, antioxidants,
aldehydes) were given per milligram of total LDL.
Other groups working on oxidative modification of
L D L report its composition and changes during oxi-
dation per milligram of L D L 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 L D L as determined by different labo-
ratories (Table 2) indicates that conversion between
the different reference systems used by different labo-
ratories is justified. To facilitate comparison of data,
we express in this review L D L analysis in nanomoles
per milligram of L D L protein and, where appropriate,
in mol/mol LDL.
The average distribution oflipids and o f individual
fatty acids is given in Tables 3 and 4. The total num-
ber of fatty acid molecules b o u n d in the different lipid
classes of an L D L molecule is 2700 on average. O f
these, about half are polyunsaturated fatty acids
(PUFAs), mainly linoleic acid with m i n o r a m o u n t s of
arachidonic acid and docosahexaenoic acid. The fatty
acid content of L D L and their distribution pattern
can vary considerably from d o n o r 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 n m o l / m g L D L pro-
tein). Such a variation in the P U F A content is likely
to have a significant effect on the oxidation behavior
of different L D L samples. Parthasarathy et al. 89
showed recently that feeding rabbits an oleic-acid-rich
diet results in an oleate-rich LDL, which is remark-
ably resistant toward oxidation.
The PUFAs in L D L are protected against free radi-
cal attack and oxidation by a n u m b e r 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 a m o u n t of 11.58
n m o l / m g L D L protein equals about 6 molecules a-to-
copherol per L D L particle. All other antioxidants
(i.e., gamma-tocopherol, carotenoids, oxycaroten-
oids, and ubiquinol-10) are present in m u c h smaller
amounts. The antioxidant content of L D L varies, sim-
ilarly to the PUFAs, significantly between individ-
uals. For instance, the lowest and highest a m o u n t of
a-tocopherol found by us a m o n g 87 (not vitamin E
supplemented) donors were 3 and 15 m o l / m o l LDL.
The frequency histogram of L D L a-tocopherol is
shown in Figure 3. The vitamin E content o f L D L
increases with its P U F A content with a correlation of
y = 0.0034x + 1.98, where y is mol a-tocopherol/mol
L D L and x is mol P U F A / m o l LDL. 163 The value of
0.29 mol/3-carotene/mol L D L (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 L D I

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 ( 1O) 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 carot-
enoids play no or only a minor role in protecting LDL
against oxidation. The same is likely to hold for ubi-
quinol-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 antiox-
idant than a-tocopherol. The concentration of ubi-
quinol-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 an-
tioxidants (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 hu-
man subjects has been studied in detail with deute-
rium 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 distribu-
tion strongly suggests that vitamin E is first incorpo-
rated into chylomicrones, transported in chylomicron
remnants to the liver, and delivered into the circula-
tion again in VLDL. The vitamin E molecules con-
tained 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 recep-
tor. 92 Additional uptake may also occur from chylo-
micrones 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 ex-
change with other lipoproteins (VLDL, HDL) and
blood cells; the estimated half times for this spontane-
ous 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 sponta-
neous 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 H U M A N
SUBJECTS CONTAIN LIPID PEROXIDATION PRODUCTS?
TO a n s w e r t h i s q u e s t i o n , it 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 its i s o l a t i o n a n d
 Oxidation of LDL 347

A B

e"

CO

U.I
E
t-"

(0
O3

(9 ffl
t-
O
0
Q.

5
/ "6
(
"0
"10

1
8 "~

"6
G) I

I
J t l l l l l l l l l l l

8 ' 1'2 5 10

retention time (minutes) retention time (minutes)

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~Linjected. 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
L D L m a y 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 mini-
mal degradative effects. A systematic investigation
was m a d e by Schuh et al., 17 who showed that a m o r e
or less complete oxidative degradation of L D L with
concomitant formation ofthiobarbituric acid reactive
substances (TBARS) occurs if it is dialyzed at 4°C for
24 h against phosphate-buffered saline without pro-
tection, 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 T B A R S in the
range of 0.22 to 0.4 n m o l / m L , but storage of plasma
at 4°C in the absence of protecting agents (e.g.,
E D T A ) led to a continuous increase of the T B A R S at
a rate of 0.15 _+ 0.14 n m o l / m L , week. The rate of
T B A R S formation showed a strong individual varia-
tion, which could be indicative of different antioxi-
dant contents of the plasma samples. It was further
shown by Lee that lipids o f all lipoproteins classes
contributed to the formation of T B A R S during stor-
age of plasma. F r o m these and other investigations, it
is clear that p r o o f of the existence of low levels of
peroxidation products in native L D L requires a very
careful preparation 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. ESTERBAUERet al,

3 4 6 applied are indeed specific and sensitive enough to

detect low levels of lipid peroxidation products in li-
poproteins.
I
The method most frequently used to assess the de-
gree 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
e-
o TBARS 98-1°6'284(Table 5). Although the absolute val-
ues for TBARS reported by various laboratories differ
significantly, they all show the same trend of in-
creased plasma TBARS in patients with atherosclero-
sis or myocardial infarctions. So far, only a few groups
"0
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 spe-
5 cifically the TBARS in lipoproteins of atherosclerotic
subjects. In these studies, serum or isolated lipopro-
I teins were precipitated with phosphotungstic acid, the
sediment was reacted with TBA, and the formed chro-
\ mogen was extracted into butanol and measured
fluorimetrically at 535 nm with 515 nm excitation.
r P F i I I l l : t l l i r p I
The results are expressed as "lipid peroxides," al-
5 10 15 2O though the term TBARS would be more appropriate
for this assay. TBARS were found in VLDL, LDL,
retention time (minutes) and HDL, but the LDL fraction always contained the
Fig. 2. HPLC determination of tocopherols, ubiquinol-10, and ca- highest proportion. This finding is the likely origin of
rotenoids in LDL with electrochemical detection. A sample con- the belief that LDL is the main carrier of lipid perox-
taining 0.5 mg total LDL was mixed with 0.2 mL ethanol and ex- ides, because it was very frequently cited in later stud-
tracted with 1 mL hexane. The extract was dried with N2 and dis-
solved in 0.1 mL methanol. A volume of 20 uL was injected into the ies. The group of Szczeklik 99'~°9 used a modified TBA
HPLC and separated on an ODS-2 column with methanol/ethanol assay (trichloroacetic acid [TCA] precipitate heated
1/1 containing 12mM LiCIO4 and 1 g/L glacial acetic acid, flow 1 30 min at 100°C in 0.05 M sulfuric acid TBA reagent
mL/min, with amperometric detection (HP-Electrochemical De-
tector) at 0.55 volt. Detector response was 2 nano ampere for full containing 2 M sodium sulfate, which is said to in-
scale, h zeaxanthin, 2: gamma-tocopherol, 3: a-tocopherol, 4: lyco-
pene, 5: ubiquinol-10, 6: carotenes.

the freshly donated blood immediately with EDTA (1

mg/mL), and EDTA is then present at this concentra-
tion throughout all steps until the LDL sample is har-
vested from the ultracentrifuge tube. Some re- O
e-
i2
searchers also include BHT in the isolation medium. "-I
o"
According to our experience, EDTA alone is suffi-
cient to completely block oxidation of LDL during its i!i:!ii
isolation. The various evidence supporting this has {
been discussed, 96 the most convincing being the ob-
servation that a freshly isolated EDTA containing
0
LDL sample fully withstands oxidation at 35°C for at
least 48 h, as evident by the unchanged content of 0 3 6 9 t2 ~5
vitamin E, carotenoids, TBARS, fatty acids, and 430 alpha-tocopherol, mol/mol LDL
nm fluorescent chromophores. 96,97,3~3 Another im-
Fig. 3. Frequency histogram ofLDL a-tocopherol. Frequency gives
portant, yet often neglected, point is the methodology the number of subjects found in a given group of a-tocopherol,
in reference to the question of whether the methods Sample size: 95.
 Oxidation of LDL 349

Table 5. Serum TBARS in Normal and Atherosclerotic H u m a n Subjects

Author(s) Study subject Mean _+ SD

Satoh, 1978 (98) 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
Szczeklik et al., 1980 ~ (99) 17 normal subjects 2.9 +__0.1
6 hyperlipoproteinemia type V 3.7 _+ 0.3
4 hyperlipoproteinemia type Ila 3.3 -+ 0.1
Goto, 1982 (100) -- normal subjects 5.0 _+ 0.5
-- patients with atheroselerosis 8.0 _+ 0.5
Aznar et al., 1983 (101) 95 normal subjects 47.2 +_ 6.9
26 acute mycardial infarction < 61
Hagihara et al., 1984 a (102) 50 normal subjects under 40 years 3.49 _+ 0.62
52 normal subjects over 40 years 3.96 +_+_0.79
Ledwozyw et al., 1986 (103) 15 normal subjects 0.94 _+ 0.09
15 patients with severe atherosclerosis 4.20 _+ 0.16
Schimke et al., 1986 (104) 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
Stringer et al., 1989 b (105) 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)
Yalcin et al., 1989 (106) 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 re-
ported by Yagi's group; however, the TBARS in the
LDL fraction were about 10-fold higher. According to
these investigations, hyperlipoproteinemia is asso-
ciated with a very strong increase of TBARS in
VLDL, whereas TBARS in LDL are only slightly in-
creased. The sum of TBARS determined in the sepa-
rated lipoprotein fractions by far exceeded the
TBARS of the parent plasma samples, and it was as-
sumed 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 as-
say; 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 pre-
pared 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 reac-
tion of native LDL (TCA supernatant, TCA precipi-

Table 6. TBARS in H u m a n Lipoproteins

Author(s) Study Subject Chylomicrons VLDL LDL HDL

Szczeklik et al., 1980 a (99) 17 normal subjects Not determined 0.9 _+ 0.6 9.0 + 0.6 < 0.5
6 patients hyperlipoproteinemia V 8.4 _+ 2.4 7.3 +__1.0 10.1 ___ 1.2 < 0.5
4 patients hyperlipoproteinemia lla 0.6+__0.2 3 . 5 + 1.2 12.8 + 0.1 <0.5
Nishigaki et al., 1981 (107) 32 normal subjects Not determined 0.64 + 0.30 1.18 _+ 0.33 0.68 +_ 0.16
31 diabetic patients Not determined 0.68 +_ 0.34 1.26 + 0.35 1.07 + 0.40
Maseki et al., 1981 (108) 19 female, not pregnant Not determined 0.45 + 0.11 0.81 + 0.20 0.63 _+ 0.12
22 pregnant female Not determined 1.49 _+ 0.45 1.86 + 0.52 0.95 _ 0.25
Hagihara et al., 1984 a (102) 50 normal subjects, under 40 y Not determined 0.43 +_ 0.30 0.84 _+ 0.25 0.66 _+ 0.13
52 normal subjects, over 40 y 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.
350
95'
0.20
0.16
~ 0.12
@ parably more specific than the TBA or iodometric
0.08
0.04
, , , , , , , , , ,
400 500
wavelength, nm
Fig. 4. Spectraof 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%
H3PO4and 1mL TBA reagentand heated 45 min on a boilingwater
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 m a x i m u m at 535 nm,
the peak absorption of the MDA-TBA complex (Fig.
4). Since in our assay 1 nmol T B A R S / m g L D L pro-
tein would have given a detectable 535 n m maxi-
mum, we assume that the concentration of TBARS in
native L D L 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 L D L TBARS in terms o f n m o l / m L
plasma. With the assumption that 3 mg L D L / m L are
contained in plasma, the values o f 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 h u m a n L D L 0.8
+ 2.3 nmol TBARS/mg protein; Harats et al. 42 found
in L D L from smokers 0.6 _ 0.028 and in L D L from
nonsmokers 0.55 8 ___0.020 nmol TBARS/mg protein.
The potential diagnostic value o f malonaldehyde de-
termination by the TBA-test has recently be carefully
reviewed by Janero. 258
The determination o f lipid hydroperoxides by the
iodometric methods also gives for most samples o f
native L D L a positive result corresponding about 10
H. ESTERBAUERet al.
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 L D L isolated from healthy subjects was
free from detectable amounts of cholesterol ester hy-
droperoxides, phospholipid hydroperoxides, and tri-
glyceride hydroperoxides as measured by high-perfor-
mance liquid chromatography (HPLC) postcolumn
chemiluminescence detection. This method is incom-
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 hydro-
peroxides found in o L D L were detectable by gas chro-
matography/mass spectroscopy (GC/MS) in native
600 LDL. 82 A recently developed modified iodometric as-
say, ~59 which takes into account interfering phenom-
ena leading to false results, gave for the total a m o u n t
o f lipid hydroperoxides esterified in the plasma lipo-
proteins o f healthy humans a value of 4.0 _+ 1.7 #M;
with the assumption that about half of that is con-
tained 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 as-
say to measure phosphatidylcholine hydroperoxides
(PCOOH) in a large number of human plasma sam-
ples 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 P C O O H were mainly contained in
V L D L and LDL.
Another possibility of searching for remnants o f in
vivo lipid peroxidation in L D L would be the measure-
ment of defined aldehydic lipid peroxidation prod-
ucts. By H P L C 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 H N E / m o l LDL. Using
the GC method developed by van Kuijk et al., 1~
H N E 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
o f free aldehydic lipid peroxidation products.
Several other attempts were made to demonstrate
oxidation remnants in L D L of healthy subjects. The
apolipoprotein B of in vitro oxidized LDL, for exam-
ple, shows a very strong fluorescence at 430 nm with
 Oxidation of LDL
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-dimen-
sional 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 indi-
vidual variations. Dobretsov et al., 115 using conven-
tional 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 hu-
mans contained a subtraction (about 5 to 20%) which
could be separated from the LDL bulk by ion ex-
change chromatography. This subfraction was more
electronegative, contained more conjugated dienes,
had apo B aggregates, and led to a higher accumula-
tion of cholesterol esters in cultured macrophages
than normal LDL. It was concluded that this subfrac-
tion 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 col-
lected from normal subjects, 1~7though 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 nega-
tively correlated with its vitamin E content and posi-
tively correlated with its TBARS. The TBARS con-
tent of LDL- was on average 7.3 mol/mol LDL,
which is threefold higher than in normal LDL. In so-
dium dodecyl sulfate (SDS) polyacrylamide electro-
phoresis, the apo B from LDL- showed higher molecu-
lar 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 frac-
tion, 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 indica-
tive 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
351
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 oxida-
tively modified human LDL 37'43 or against malonal-
dehyde (MDA) or HNE conjugated LDL 43 did not
show a noteworthy crossreaction with freshly isolated
human LDL, and one must therefore assume that na-
tive LDL does not have epitopes typically found in
oLDL.
The occurrence of heavily oxidized LDL as a sub-
fraction 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 ultracentrifu-
gation. Since Avogaro's LDL- would have been pres-
ent 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 indi-
viduals and in most patients with chronic periaortitis.
These autoantibodies probably developed against oxi-
datively 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 atheroscle-
rosis and circulating immune complexes containing
LDL was recently reviewed by Orekhov. 325 For hu-
man 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 hu-
man aortas (postmortem material) contains lipid per-
oxides 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. ESTERBAUERel aL
(Table 9). A later r e p o r t 16 showed that the peroxides
probably formed in the period between death and tis-
sue extraction, through cessation of enzymatic pro-
cesses normally converting hydroperoxides to hy-
droxy-octadecadienoic cholesterol esters. It was also
pointed out that the hydroxy-esters isolated from dis-
eased arteries during surgery had structures inconsis-
tent 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 cho-
lesterol 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 maxi-
mum at 435 nm (Ex 355 nm) indicative of lipid per-
oxidation 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. 125separated the lipids of lesions by conven-
tional thin-layer chromatography (TLC) and found
an unknown spot (spot x) which is probably an oxi-
dized form of cholesteryl esters, not present in native
LDL, but formed if LDL was dialyzed over long pe-
riods (a condition known to induce lipid peroxida-
tion) 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 sub-
jects (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 68compared
the chemical composition of human LDL from
plasma, normal intima, and fibrous plaques. No sig-
nificant differences were seen in the percent distribu-
tion of protein, phospholipids, free cholesterol, choles-
terol esters, and triglycerides. Some remarkable differ-
ences existed in the fatty acid distribution; thus the
arachidonic acid and linoleic acid ofcholesteryl esters
and triglycerides were strongly decreased in aorta-de-
rived 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 mobil-
ity (REM) was also found in human interstitial
fluid. 129It was also shown 68'126that aorta LDL is asso-
ciated 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 nega-
tively charged glycosamino-glycans. In vitro experi-
ments 2zs'229 showed that subpopulations of LDL bind
to human arterial chondroitin sulfate and proteogly-
cans. This binding reduces the thermal stability of the
surface and core in LDL and leads to exposure ofly-
sine- and arginine-rich segments of the apo B. LDL
subclasses complexed with such proteoglycans exhibit
in vitro an increased uptake by mouse peritoneal mac-
rophages. Additional differences between aorta and
plasma LDL include decrease in sphingomyelin, ten-
dency 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 atheroscle-
rotic 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 sam-
ples. About 12 to 21% of the cholesteryl linoleate was
present as oxygenated metabolites. "Healthy"-look-
ing parts of the same aortas also contained this mate-
rial, but the amount was much smaller (i.e., 0.3-4.5
mg/g wet weight or 5.8-9.5% of total cholesteryl lino-
leate). The authors assumed that this oxygenated cho-
lesteryl linoleate was formed by a 15-1ipoxygenase ac-
tivity. 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) com-
positional (less PUFAs, more peroxides and TBARS)
and functional (increased uptake by macrophages)
properties of aorta LDL and aorta lipids in atheroscle-
rosis support the hypothesis that LDL deposited in
the arteries is partly oxidized.
 Oxidation of LDL 353

Table 7. Properties of LDL Isolated from the Aorta in Comparison to Plasma LDL
DifferenceBetweenAorta LDL and Plasma LDL References
Humans
Increased electrophoreticmobility 68, 127, 67
Decreased content of linoleic and arachidonic acid 68, 126, 67
Moderately changed (increase,decrease)of cholesterylester 68, 126, 127,67
Increased content of stearic and oleic acid 68, 126
Decreased sphingomyelincontent 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 macrophagesscavengerreceptor 127, 67
WHHL Rabbit
Increased electrophoreticmobility 128
More cholesterylester, more sphingomyelin 128
Decreased diameter of LDL particle 128
Significantlyincreased TBARS and macrophage uptake 128
Contains apo B fragmentswith MDA and HNE modified
Lysine residues (Western blot) 43
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,~28Yl~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 choles-
terol 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 thick-
ening 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 n m o l / m L serumS°°). The lipopro-
rein fraction containing V L D L + L D L 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 nor-
mal 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 V L D L + L D L of diabetic rats was
highly toxic to proliferating fibroblasts and that vita-
min E or probucol treatment of the rats inhibited oxi-
dation o f the lipoproteins in vivo and prevented for-
mation of cytotoxicity in lipoproteins) 32
Daugherty et al.t28 investigated L D L 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 L D L ( 15.3 vs. 2.0 n m o l / m g pro-
tein) 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
L D L 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 a m o u n t o f h y d r o x y fatty acids compared
to total polyunsaturated fatty acids (2332 n m o l / m g
L D L protein, Table 4) is very low. After 15 weeks
cholesterol feeding, that total a m o u n t of esterified hy-
droxy fatty acids in rabbit L D L was only a m o u n t 0.35
n m o l / m g LDL protein with 80% H O D E and 20%
HETE. About 0.15 nmol hydroxy fatty acids ( H O D E
+ 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 prin-
cipal lipoxygenase product, 323 but aortic segments
from cholesterol-fed rabbits or W H H L rabbits pro-
duce 15-HETE, suggesting an increased 15-1ipoxy-
genase activity in atherosclerotic arteries. 323'324
IMMUNOLOGICAL STUDIES
A monoclonal antibody raised against MDA-modi-
fled L D L (MDA-LDL) was used by G o n e n et al) 33 to
investigate h u m a n autopsy samples. No reaction was
354 H. ESTERBAUERel 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
Authors Code Antibody
Gonen et al. 1987 (133) EB 7-3 Mouse mAb
Salmon et al. 1987 (134) -- Rabbit as
Haberland et al. 1988 (135) MDA-lys Mouse mAb
Palinski et al. 1989 (43) MAL-2 Guinea pig as
HNE-6 Guinea pig as
OLF4-3CI0 Mouse mAb
Boyd et al. 1989 (37) OXL-41.1 Mouse mAb
OXL-22.4 Mouse mAb
found with aortic intimal or medial extracts with an
ELISA technique. However, a monoclonal antibody
prepared against the float-up fraction of atheroscle-
rotic 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 anti-
bodies to various forms of oLDL and aldehyde-modi-
fied 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. 136The staining was confined to ath-
erosclerotic 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 recog-
nizes 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 pro-
bucol-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-
Type of
Immunogen Major Findings
Mouse MDA-LDL ELISA failed to show any MDA-LDL in
extracts of 14 autopsy samples from aorta
Rabbit MDA-LDL MDA-lysine conjugates in Cu ++ oxidized LDL
Human MDA-LDL Stains proteins in atheroma of WHHL rabbits;
stain co-localizes with extracellular deposits
of apo B
Guinea pig MDA-LDL All three antibodies stain the same area of
Guinea pig HNE-LDL lesions in WHHL rabbits, immunostain
Mouse oLDL mostly intracellular, stained area rich in
macrophages
Human Cu +÷ oLDL The two antibodies stain certain lesions in
Human Cu +÷ oLDL WHHL aorta; no staining in aorta of normal
rabbits
pared from aortas of atherosclerotic cholesterol-fed
rabbits were shown to avidly metabolize modified
forms of LDL (tested was ac-LDL) and thereby accu-
mulate cholesteryl esters. 235 In additional s t u d 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 anti-
bodies recognize epitopes in LDL which are specific
for oxidative modification. In case of the antibodies
against MDA-modified LDL (i.e., MDA-lys, 135MAL-
2, 43 M D A - 2 , 1 3 6 it was assumed that the recognized
structure is a "MDA-lysine adduct," since the antibod-
ies 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 mod-
ified 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 355

Table 8. Continued.

Antibody Type of Type of

Authors Code Antibody Immunogen Major Findings

Steinbrecher et al. 1989 (46)
J0rgens et al. 1990 (49)
Rosenfeld et al. 1990 (136)
Palinski et al. 1990 (51)
Yla-Herttuala (264)
Guinea pig as Guinea pig acrolein-LDL Only LDL modified with aldehydes in
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 Human HNE-LDL 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 Guinea pig as Guinea pig MDA-LDL The epitopes recognized by the two
HNE-6 Guinea pig as Guinea pig HNE-LDL antisera and three antibodies were
MDA-2 Mouse mAb Mouse MDA-LDL characterized by Palinski et al., 5~
NA 59 Mouse mAb Mouse HNE-LDL 1990. Used for immunosatining of
OLF4-3C l0 Mouse mAb Mouse Cu ÷+ o L D L atherosclerotic lesions of varying
severity from WHHL rabbits.
Staining predominantly cell
associated with macrophages, in
advanced lesions increasing
extracellular staining.
Human lgG Likely in vivo oxidized LDL Atherosclerotic lesions of humans and
Rabbit IgG rabbits contain IgG recognizing
MDA-LDL and Cu++ 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 sup-
ported by the fact that the HNE-conjugate formed
under nonreducing conditions was a weak immuno-
gen, 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 alde-
hyde 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 dif-
ferent to Palinski's observation 43's~ that HNE-conju-
gated mouse or Guinea pig LDL is immunogenic only
if conjugation is carried out under reducing condi-
tions. The antiserum prepared by Jtirgens et al. 49 is
apparently very specific for HNE epitopes, since no
noteworthy cross-reaction occurred with LDL modi-
fied 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 epi-
topes of apo B are in fact formed in vitro and in vivo
by oxidative modification of LDL. Solid-phase corn-
356 H. ESTERBAUERel al.
petition RIA revealed5~that the antisera and monoclo-
nal antibodies against MDA-modified LDL strongly
bind also to other MDA-treated proteins (human
serum albumin, hemoglobin, transferrin) and polyly-
sine, 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 spec-
ificity 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 ob-
tained with HNE-modified LDL. The antibody ap-
pears to be not specific for oxidatively modified apo
B, since strong cross-reactions also occurred with cop-
per-oxidized HDL. Even if all the work with antibod-
ies 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 ath-
erosclerotic lesions of W H H L rabbits. It remains, how-
ever, 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 an-
tibodies against MDA-LDL and HNE-LDL. 139 Since
in this animal model predominantly VLDL is ele-
vated (in W H H L 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 i n different parts of apo B
from native LDL that the immunoreactivity steadily
decreased during oxidation, but another epitope lo-
cated 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 oxida-
tion, which then gradually decreased again. Similar
changes were observed during LDL aging, a condition
known to be associated with mild oxidation. The im-
munoreactivity of the Bsol 7 epitope was assumed to
be one of the most sensitive parameter of LDL oxida-
tion. 232 Salonen et al. 54 recently reported the occur-
rence of autoantibodies to MDA-LDL in sera of ath-
erosclerotic Finnish men; the titer of these autoanti-
bodies 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 es-
ters. Foam cells are derived from smooth muscle cells
and monocyte-macrophages. In the last decade, it be-
came 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 intra-
cellular 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 endo-
cytosis not under feedback control, leading to massive
cholesterol accumulation within the macrophages. 18
One of the steps of this modification, probably of cru-
cial 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 nega-
tive 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 recep-
tor(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
 Oxidation of LDL
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-resi-
due C-terminus in receptor type II. 52't43
Other treatments of LDL, such as acetoacetyla-
tion,145 malelylation,11 succinylation,~46 carbamyla-
tion, ~47 and incubation with malondialdehyde
(MDA) or glutaraldehyde,14 also transform LDL to a
species which is readily recognized by the ac-LDL re-
ceptor(s). The ac-LDL receptor(s) is also able to recog-
nize ligands, which are not necessarily lipoproteins
but negatively charged polyanions or malelylated al-
bumin. 18 However, the enhanced negative charge of
modified LDL itself is probably not the only factor
being responsible for recognition by the ac-LDL re-
ceptor(s); likewise the density of negative charges in
certain regions of the macromolecule might be of im-
portance.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 resi-
dues 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 na-
ture of the modification affecting LDL in vivo respon-
sible for its recognition and unregulated uptake by
macrophages. We want to point out that the group of
Fogelman and Haberland 14'146'148349 studied exten-
sively the modification of LDL by MDA. These au-
thors 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), an-
other 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-
357
tor on fibroblasts. 15° This agreed with the observation
that modification of LDL by MDA at lower concen-
trations hampered the recognition by the LDL-recep-
tor. 149 A more extensive modification of LDL with
HNE leads to the formation of aggregates of this lipo-
protein. 39'~18 These aggregates are taken up by culti-
vated macrophages 0774), giving them a foamy ap-
pearance. This uptake was not mediated by the ac-
LDL receptor(s), but facilitated via phagocytosis.39
However, another study, modifying LDL concomi-
tantly 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 cul-
tured macrophages by means of the ac-LDL recep-
tor(s). 46To characterize the compounds eventually re-
sponsible 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 cytotoxic2° and that lipid peroxidation was a pre-
requisite 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 macro-
phages ~56were 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-
358 H. ESTERBAUERel aL
chloroacetic-acid-soluble p r o d u c t s . 45,233 Thus signifi-
cant 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 es-
ters, probably because oLDL has a reduced choles-
terol 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 recog-
nize ac-LDL but recognized LDL incubated and oxi-
dized 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-oxi-
dized 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 lipo-
protein fraction occurring in cholesterol-fed animals
and humans, was shown to be internalized by cul-
tured rabbit aortic smooth muscle cells.~57 This inter-
nalization was enhanced when /3-VLDL was incu-
bated 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 recep-
tor. ~57 Wiklund et al. 296 recently reported on the up-
take of native and acetylated LDL in foam cells pres-
ent 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 acety-
lated 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 oxi-
dation 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 li-
poproteins with proteases) can also lead to lipid load-
ing 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 com-
plexes 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 possi-
bly also to native L D L . 236-238
Several reports show that patients with severe ath-
erosclerosis have LDL-immune complexes in the cir-
culation, 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 hu-
man monocyte-macrophages, and the uptake leads to
intracellular cholesteryl ester accumulation. 23s Our
group recently showed24~that severe atherosclerosis is
associated with an up to 10-fold elevated serum neop-
terin level (neopterin is an index for activated macro-
phages).
M E C H A N I S M AND KINETICS OF OXIDATION OF LDL
BY CELLS OR COPPER IONS
In the experiments23 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 con-
tained 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 modi-
fied 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 lyso-
phosphatides) 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 experi-
ments now is an incubation for 8 h or more in one of
the cell culture media (F-10, DME) or plain phos-
phate-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
 Oxidation of LDL
~M FeSO4 or FeCI3induced oxidation of LDL compa-
rable to 10 uM CuSO4, provided that the LDL was
dissolved in 0.15 M NaC1 instead of 10 mM phos-
phate buffer. The authors assumed that phosphate
buffer complexes iron (but not copper ions) and thus
prevents formation of an iron-LDL complex neces-
sary 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 findings58 and is supported by reports from
several other groups.72'79'161Balla et al.327 recently sug-
gested 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 hydro-
peroxides. The somewhat inconsistent results regard-
ing effÉciency of iron and copper ions as well as the
degree of oxidative modification produced by a cer-
tain 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 prooxi-
dant 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 mac-
rophage 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, me-
dium, Cu ÷+ concentration), such a degree of oxida-
tion 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.79'156In 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; thereaf-
ter 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
359
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 maxi-
mum 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 decom-
posed. 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 ++ condi-
tioned LDL were not decomposed and the LDL was
also not degraded by macrophages. This clearly indi-
cates 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 scav-
enger 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-irradiation88was also not a good substrate for
the scavenger receptor, but it could readily be con-
verted into a high-uptake form when it was addition-
ally treated with Cu ++, a condition known to decom-
pose lipid hydroperoxides.
Details of the mechanism of metal-induced oxida-
tion of LDL are not clear. Direct oxidation of pure
lipids by free or complexed iron or copper under phys-
iologically plausible conditions has not been demon-
strated. Most authors faced with the need to explain
metal-assisted peroxidation assume the occurrence of
reactions analogous to processes leading to the reduc-
tion 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 thermodynami-
cally not favored.
It appears therefore that peroxidation of lipids in
360 H. ESTERBAUERel al.

macrophage uptake
lipid hydroperoxides
;/C~-Tocopherol
30. 6oo-1 61/
F I O + 3/~M F, FIO + ~ FeSO4 F I O + IOOMh4 CuSO4
+ maeropha~
25- 5OO
V"
2O- 4oo o~-T 7k /,/e,..-----~---

LOCr~

1.
t5" 3100 ~-T ///
LOOH/ \
10" 200 /

z-z II \
\
i
5" 100 14
I /- \
J ,. • I
0 12 240 12 24 0 1'2 24

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~-Tocopherolconsumption during low-density
lipoprotein oxidation. BiochemZ 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 par-
ticle. Both proteins 246 and phospholipids247 readily
bind many metals, including copper, and such catalyt-
ically 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 ini-
tial Cu + formation could be achieved in a process anal-
ogous to the reduction of Fe +÷+ by a-tocopherol incor-
porated 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
C u + + , 247 a similar reaction between the metal and a-to-
copherol could occur. This possibility has not been
tested experimentally. Taking into account the re-
quirement 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 ++ - L O O + Cu + + H+), and this mech-
anism was recently again proposed by Thomas and
JacksonY 2 However, such a reaction is thermody-
namically extremely unfavored (W. Koppenol, pri-
vate communication) and therefore unlikely. The im-
portance of traces of preformed lipid peroxides was
recently clearly demonstrated252 by experiments with
ebselen, a synthetic Se containing compound with
peroxidase-like activity. In the presence of glutathi-
one, 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

160
140 °
/j!
120
~~°~ x ///
._1
100
C3 - - x~ \ Peroxides
_J
c~
E peroxide appeared to be essential. A feature of the
80
/ o
E cell cultures is the long period required. Under such
t-
60 ! conditions, the chemical processes responsible for gen-
, \
40
I/
, \
! o
20
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 indi-
cate clearly that Cu +÷ oxidation has an absolute re-
quirement for the presence of traces of LOOH in the
LDL. Interestingly, oxidation of LDL by macro-
phages was also largely prevented if the culture me-
dium was supplemented with ebselen + glutathione,
which suggests that in this system lipid hydroperox-
ides 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 proba-
bly, reduce any transition metal ions present, facilitat-
ing lipid peroxide decomposition and chain peroxida-
tion. Recent studies suggest that a 15-1ipoxygenase ac-
tivity forms the initiating peroxides when LDL is
incubated with mouse peritoneal macrophages. 259
The involvement of a 15-1ipoxygenase in LDL oxida-
tion is also suggested by the finding that aortic seg-
ments from cholesterol-fed rabbits and W H H L rab-
bits convert arachidonic acid to 15-HETE. 323'324 On
Oxidation of LDL 361
the other hand, 5-1ipoxygenase has been shown to be
not essential for LDL oxidation by macrophages) 67
Several studies show that purified soybean lipoxygen-
ase alone 26° or with phospholipase A 2 (Ref. 34) can
oxidize isolated LDL and convert it into a form which
is cytotoxic26° and taken up by macrophages) 4 Inter-
estingly, Cathcart et al.260 observed in their experi-
ments that superoxide anion inhibited soybean lipox-
ygenase catalyzed LDL oxidation, whereas hydrogen
kinetics of oLDL formation during incubation with
eration of oLDL are difficult to identify. It is clear,
however, that oxidation of LDL is significantly accel-
O~o
erated by metal ions and that the process is inhibited
by chelating agents, either in the absence or in pres-
ence of cells.
In agreement with this background, we have
s h o w n 58'69 that Cu ++ strongly binds to LDL, and we
have proposed that LDL has at least two distinct cop-
per-binding sites crucial for the initiation of lipid per-
oxidation. ESR measurements showed that Cu ÷+
binds to the LDL protein: ° Fluorescence spectros-
copy 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 tryp-
tophan 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 concen-
tration, prevents binding of Cu +÷ to LDL. Another
free radical process which might have biological rele-
vance in diabetes is the autooxidative glycosyla-
tion. 257'265 If LDL is incubated with high concentra-
tions of glucose, trace amounts of transition metal
ions generate free radicals, H202, and ketoaldehydes
from glucose, and glycosylation and lipid peroxida-
tion 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 fol-
lowing initiation are always the same--that is, loss of
antioxidants, lipid peroxidation, and decomposition
of lipid hydroperoxides to aldehydes and other prod-
ucts (Fig. 7). An LDL at the late phase of decomposi-
tion will have more or less similar biological and
chemical properties, regardless of how the oxidation
362 H. ESTERBAUERel al.

IINITIATION J
I ipoxidase or
preexistin9 L D O H

LDOH M n+ LOOH
LO"+OH"~ Mtn+m'~ H'+LO0"~ Oxidized
/ Antioxidants Antioxidsnts
X" LO" LDO" , 41k

LH L" LDD" LDDH

A,N-SamCH,NOI
IC

LO" + LOO" e LDOH

/ ~ Lysophosphadites

Rearrangementand =L Aldehydesand otherproducts

consecutiveproducts FragVmentstion JV
(Hydrox~,ks=>,keto-hydroxy-, ofapoB Covalentbindingto apoB
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 oxi-
dized by Cu ++ for various times (up to 48 h) can be
separated by anion-exchange chromatography on a
Mono Q H R 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 mea-
suring the increase of TBARS, lipid hydroperoxides,
conjugated dienes, 430 nm fluorescence256 (Fig. 8),
and aldehydes (Fig. 9). Other possibilities are the mea-
surement 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 suffi-
cient 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 mea-
surement 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
 Oxidation of LDL 363

1 2 3

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

10 15

incubation t i m e (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 conju-
gated double bonds (= dienes), with a UV-absorption
maximum at 234 nm. Since LDL is fully soluble in
aqueous phase and remains in solution during oxida-
tion, the diene measurement does not require extrac-
tion of the lipids but can be performed directly with
the oxidizing LDL sample, provided that the concen-
tration 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
60
1 2 3 . _ . ~
Hexanel
£3 40
._1
0 sively depleted of its antioxidants, with a-tocopherol
E
c 20
O:
0 8 16
time (hours)
Fig. 9. Kinetics of the formation of aldehydes during Cu ÷+ stimu-
lated oxidation ofLDL. LDL (1 mg/mL) in PBS was incubated with
6.7 uM CuCI2. MDA = malonaldehyde, HNE = 4-hydroxynon-
enal. 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 di-
rect diene measurement. Our laboratory measures
routinely the diene versus time profile by continu-
ously 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 hydroper-
oxides 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 in-
crease 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 progres-
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 conju-
24 gated dienes. The approximate rate of diene forma-
tion 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 par-
ticle every 6 rain. A slow formation of lipid hydroper-
oxides during the lag phase is not unusual but indeed
a consequence of the antioxidant effect of vitamin E
364 H. ESTERBAUERet al.

100: 0.50

80 0.40 E
_J c
£3
d increase in~nes
0
>
60 0.30 C~
4-J ¢
0
c c
40 0.20
0
0
m
cl
20 0.10

0 0.00
0 60 120 180 240 300

time, minutes

100i ~ ~ a 1 0.50
rotene [

.J 80 Lutein/Zeaxanthin 0.40
63
/
CO
> 60 0.30 04

c
40' 0.20
o;
. ~ / ~ Cryptoxanthin~ m In _(3
20
T
0 ~ o . o o
0 10 20 30 40 50 60

time, minutes
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), CuC12was 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 showingthe 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 L D L during the lag phase
(LOO" + vitamin E --* L O O H + vitamin E radical).
The tocopheroxyl radical was detected in L D L 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 L D L (Table 4) could scavenge 14 LOO"
radicals yielding 14 lipid hydroperoxides and 7 oxi-
dized 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
o f the LOO" gives L O O H , 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,
 Oxidation of LDL 365

-0.8

100"

o"
75.
\/\o,...s -0.6
E
¢-

O4
>
G)
.0.4
t-
r~
50- .t3

o~
m" i

8. -0.2 D
25"

60 120 180 240

time (min)

Fig. 11. Temporal relationship between lipid peroxidation measured by dienes and fragmentation of apo B during Cu++
oxidation of LDL.

which act by largely unknown mechanisms. 321 There
is also a suggestion 317,318that 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
n 75
phase, the a m o u n t o f 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 con-
tained in L D L oxidized by AAPH to about 4.6 mol
L O O H / m o l L D L by H P L C postcolumn chemilumi-
nescence detection revealed the presence ofhydroper-
oxides of cholesterylester (64%), phospholipids (12%),
and triglycerides (24%).
When the L D L is depleted from its antioxidant it

5/'
..J
is, as expressed by Brown and Goldstein, i7° left to the
33 mercy o f oxygen. Or in other words, it is subjected to
Rq
~ 5o
E E autocatalytic chain reactions o f lipid peroxidation (=
propagation phase). The rate o f lipid peroxidation rap-
~t - 25 idly accelerates (see Fig. 13, lower part) in an exponen-
O tial m a n n e r to a m a x i m u m equal to about three mole-
cules lipid hydroperoxides formed in each L D L parti-
0 1
cle every minute.
0 8 16 24
The transit from the lag into the propagation phase
time (hours) and the exponential increase of the oxidation rate is
mediated by the copper ions (which at this stage are
Fig. 12. Kineticsofthe increaseofthe relativeelectrophoreticmobil-
ity (REM) of LDL during oxidation. LDL (1.5 mg/mL) in PBS was probably released from the site where they were ini-
incubated with 10 uM CuCI2. The dienes (given as nmol/mg total tially bound) catalyzing the decomposition of the
LDL) were determined from the 234 nm absorbance. The electro- seed-lipid hydroperoxides formed during the lag
phoretic mobility was determined by electrophoresis.Given is the
change relative to native LDL (= 1.0). Reprinted with permission phase to lipid radicals (LO', LOO" ), which initiate by
from Esterbauer, H.; Puhl, H.; Waeg, G.; Krebs, A.; Dieber-Roth- chain branching new series o f free radical chain reac-
eneder, M. The role of vitamin E in lipoprotein oxidation. In: tions. Using a-phenyl-t-butylnitrone (PBN) or 2-
Packer, L.; Fuchs, J., eds. Vitamin E: Biochemistry and clinical
application. By courtesy of Marcel Dekker, Inc., NY, 1992, pp. methyl-2-nitrosopropane (MNP) as spin traps, the
649-671. formation of LDL-lipid radicals was observed by ESR
366 H. ESTERBAUERet aL
1.2
E include increase of aldehydic lipid peroxidation prod-
E 0.8
93
L'q
0.4
< measured by the REM (Fig. 12), fragmentation (Fig.
0.0
0 60 120 180 240 300
>[ min.
0.03
0.02
-0
< 0.01
-0
0.00
I I I I
0 60 120 180 240 300
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 pre-
vents 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 prod-
ucts; 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 de-
composition 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 propaga-
tion and beginning of the decomposition phase. One
should keep in mind, however, that both phases tem-
porarily 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
ucts (Fig. 9), generation of fluorescent chromophores
(EX/EM 360/430 nm) in the apo B and the LDL lip-
ids (Fig. 8), increase of the negative surface charge as
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 biologi-
cal 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 condi-
tions. 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 con-
centration 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 decom-
position 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 parti-
cle 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, re-
leased from the LDL into the aqueous phase. 29
Various lines of research suggest that a number of
important changes occurring in oLDL during the de-
composition phase result from aldehydes and their
reactions with amino acid residues in apo B (for re-
view 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
 Oxidation o f L D L 367

Table 9. Aldehydes in Native a n d Cu ÷+ Oxidized L D L

n m o l per m g L D L Protein

Cu +÷ oxidized
LDL
Native L D L 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
Nonanal 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. T h e values for native L D L are m e a n _+
SD from at least three experiments. T h e values for C u ++ oxidized L D L are from a single
experiment, where L D L (0.055 m g p r o t e i n / m L PBS) was oxidized with 1.66 z M CuCI2 in a
tightly closed vial to avoid evaporation loss o f aldehydes. T h e linoleic a n d arachidonic acid
content o f the L D L used for oxidation were 2168 a n d 168 n m o l / m g protein. Dash ( - - )
m e a n s n o t measured.

surface charge of LDL (Fig. 12) is most likely due to
loss of positively charged amino groups through
Schiff's base formation ( R - C H O + 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 oxi-
dized 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 re-
ceptor. 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. ~8The 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 + X H -~ R C H X -
CH2-CHO. Alternatively, some of the carbonyl func-
tions 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 stud-
ies. 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), phy-
sico-chemical (increased fluorescence and REM), and
biological (uptake by macrophages, cytotoxicity) prop-
erties 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 fur-
ther supports the hypothesis that aldehydes act as ulti-
mate damaging agents. As discussed previously, anti-
bodies prepared against MDA- or HNE-treated native
LDL react also with copper- or cell-oxidized LDL.
368 H. ESTERBAUERel aL

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 modifiedLDL (MM-LDL),the altered propertiesare characteristicfor LDL oxidizedby Cu++for at least
4 h or by cells (endothelial cells, macrophages, smooth muscle cells) for about 24 h. Referencesare only given for findings which are not
explicitelydiscussed in the text.

This clearly indicates that these aldehydic lipid perox-
idation products are indeed covalently bound to the
apo B when L D L is oxidized by cells or Cu ÷÷ ions.
MDA- and HNE-modified proteins (most probably
apo B) have also been detected by immunohistochem-
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 o f rabbits
and humans, 43'54 All this underlines the theory that
aldehydic lipid peroxidation products indeed play a
significant role in oxidative modification o f LDL,
both in vitro as well as in vivo.
The aldehydes detected so far in L D L and listed in
Table 9 are all (except MDA) derived from the methyl
terminus o f the PUFAs. The counterparts, where the
aldehyde function is at the acyl chain, linked to the
parental lipid molecule (i.e., phospholipids, choles-
teryl esters) should also be present in comparable
amounts in oLDL. Since these aldehydes also cer-
tainly contribute to the altered properties o f oLDL, it
would be important to give more attention to their
analysis. Steinbrecher et al., 46 for example, concluded
from studies on modification o f L D L 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 o f the numerous
compounds formed by lipid peroxidation in biologi-
cal systems was reviewed in Refs. 138, 171, and 177.
Recently it was shown by gas-chromatogra-
phy 215'233'262 that L D L oxidized by Cu ÷+ or macro-
phages contains significant amounts o f cholesterol
oxidation products, with 7-ketocholesterol compris-
ing about one half to two thirds of the total choles-
terol. 7-Ketocholesterol was also identified in serum
of cholesterol-fed rabbits. 3°4 According to these stud-
ies, 215 oxysterols are present in o L D L both in free and
esterified form, indicating that both forms of choles-
terol 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 choles-
terol was converted to oxysterol. The increase o f o x y -
sterols closely paralleled the increase in electropho-
retic mobility. Other oxidized sterols likely present in
Cu ÷+ oxidized L D L are 7-hydroxy cholesterol and 25-
hydroxy cholesterol. 233 T h o m a s and Jackson 252 found
 Oxidation of LDL
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. 2~6reported that cultures of human mono-
cytes incubated with a complex of cholesteryl-lino-
leate and serum albumin (serves as artificial lipopro-
tein) 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 oxy-
sterols 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 bio-
logical properties of LDL change continuously during
the lag, propagation, and decomposition phases. Due
to this dynamic process, an oLDL with a defined con-
stant composition does in fact not (at least in the clas-
sical chemical sense) exist. It is therefore very surpris-
ing that numerous laboratories appear to have sam-
ples 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 lip-
ids or apo B. An analyst attempting to study the nu-
merous oxidized lipids and fragments from apo B has
an enormously difficult job. The classical methods de-
veloped 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 determi-
nation of free and esterified cholesterol or triglycer-
ides in serum or native lipoproteins, but not for oxi-
dized lipoproteins. Completely wrong results for
oLDL are obtained, for example, by conventional cho-
lesterol assays based on cholesterol oxidase (makes
H202) through interfering lipid hydroperoxides, so
that some of the values for free or esterified choles-
terol in oLDL reported in the literature (see Table 3)
369
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 phospho-
lipids. All our attempts (unpublished) to determine
the amount of unchanged residual cholesterol, choles-
terylester, 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 neces-
sary to establish structures of unknown oxidized lip-
ids 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 pub-
lished data on the chemical composition of native and
oLDL. Most analysis stem from copper-oxidized
LDL, with comparable data on cell-oxidized LDL be-
ing 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 pathogene-
sis of atherosclerosis. The biological effects of mini-
mally 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 choles-
terol. 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 oxida-
tion. 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
The topic of the biological effects of the minimally
modified LDL is largely outside the scope of this re-
view 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, pre-
pared 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 incorpo-
ration. 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 atherogen-
esis 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 antioxi-
dants should have a prominent effect in retarding or
preventing the modification. This has in fact been
confirmed in numerous studies with a variety of an-
tioxidants. Steinbrecher et al. 23 showed in their classi-
cal 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 con-
tained 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-me-
diated 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 cells 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
H. ESTERBAUER¢l a/.
shown to be potent inhibitors for cell-mediated LDL
oxidation are butylated hydroxytoluene21,22'24,27,2s,178
and probucol. 179,287Water-soluble compounds, which
prevented LDL oxidation by cultured cells, are gluta-
thione and ascorbate. 22,~4'2~5 Breugnot et alfls7 re-
ported that inclusion of about 10 to 50 t~M phenothi-
azines (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 hu-
man 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 concentra-
tions. A complete blockage of cell-mediated LDL oxi-
dation 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 concen-
tration 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 subse-
quent experiments. To get rid of EDTA, most labora-
tories dialyze the LDL stock solution overnight at 4 °C
against EDTA free buffer, made oxygen free by nitro-
gen 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 oxida-
tion); 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 concentra-
tion to Fe ++ (10 #M) stimulated oxidation of LDL in
cultures of smooth muscle cells. Similar prooxidative
 Oxidation of LDL
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 incu-
bated with a fixed amount of Cu +÷ (5 uM) and in-
creasing 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. 2~8The 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 protec-
tion. 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 sam-
ple, 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 appar-
ently harmless to smooth muscle cells.
Much work has been devoted to clarify the protec-
tive role of the endogenous vitamin E and other an-
tioxidants 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 antioxi-
dants 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 antioxi-
dants disappearing first were ~- and gamma-tocoph-
erol; thereafter the carotenoids decreased to zero, with
cryptoxanthine first and H-carotene last. A fluorescent
compound disappearing with the carotenoids was pre-
viously 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 de-
pleted of antioxidants, a propagating lipid peroxida-
tion 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
371
150
U)
o
k
a
=
=
1.57
58.9
C
100 o o° o o
o~%o o~
~
Q
5O
~ o O 00°8
o
o
o
o
r 2 = 0,043
( N.S. : n = 78)
Q 4 8 12 16 20
tool d x - - t o c o p h e r o l / m o 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-tocoph-
erol.
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-supple-
mented 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-irradiation88 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 inves-
tigations from our laboratory85'~6s revealed that the
oxidation resistance (y = lag phase in Cu ÷+ stimulated
oxidation) of each subject's LDL depends on the vita-
min E content (x), a subject-specific efficiency con-
stant (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 individ-
uals 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=kx+a
lag time alpha - tocopherol
in minutes in mol/mol LDL

Fig. |5.Equation describingthe relationshipbetween a-tocopherol and lagphase forthe L D L ofa ~ven donor. Note thatk 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 applica-
tion. 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 signifi-
cantly 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) in-
creased 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 al-
ways 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 signifi-
cant levels o f p < 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 con-
tent. 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 parame-
ters 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 vita-
min E content of individuals is oral supplementa-
tion. 83,84Although 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 supplementa-
tion as shown in a typical protocol in Table 11. The
complete lipoprotein status (cholesterol, triglycerides,
H D L 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 com-
pared to the initial levels measured prior supple-
mentation (Table 12). Seven days after termina-
tion of vitamin E intake, the a-tocopherol was de-
creased 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-tocoph-
erol 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 in-
creases plasma a-tocopherol about 2.5- to 3-fold
above the baseline value. Dimitrov et al.zs9 re-
cently 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
 Oxidation of LDL 373

l dation resistance as compared to the values deter-

donor
mined prior to or 7 d after termination of the vita-
0.80 min E intake. The average increases of the lag
phase were 18, 56, 35, and 75% for doses o f 150,
0.60
' I 225,800, and 1200 IU (Table 12).
. For each person taking vitamin E, the temporal

< °'°t J J/J/

0.20
0,00 /
0
~
60
i
120
Oo : r1 = O.g6

tool oti-T/rno, LDL

180 240
change of the oxidation resistance of L D L resem-
bled very closely the temporal change of the a-to-
copherol content in L D L (i.e., lag phase increased
when a-tocopherol increased and vice versa; Fig.
17). The plots o f measured lag phases versus the
associated L D L a-tocopherol according to equa-
time, minutes tion in Figure 15 gave for all subjects straight lines
with correlation coefficients of r 2 = 0.5 tO 0.9 and
1.00 significant levels o f p < 0.001. This proves that the
,i~ m ~ , donor E~ protective effect of vitamin E in L D L from single
0.80 donors can be described by the same linear rela-
E ~¢°o
,~, rZ--O'g~ tionship (y = k x + a), regardless of whether loading
o
0.60
tool ~--T/rt~lLI~_ of the L D L with vitamin E is performed by adding
CO
Cq it to plasma or by oral intake. The individual val-
0.40
< ues 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
0.20
35.9 _+26.1) determined for the subjects participat-
0.00 ing in the vitamin E study were in the same range
0 60 120 180 240 300 as found by supplementing the plasma. This sug-
gests that both types of supplementation lead es-
time, minutes sentially to the same results regarding the protec-
Fig. 16. Determination of the efficacyof a-tocopherol to increase tive effect of vitamin E. The linear relationship be-
the resistance of LDL from two subjects against Cu÷÷ oxidation. tween lag phase and a-tocopherol seen during the
The LDL samples with different a-tocopherol contents were pre- three weeks of supplementation further shows that
pared by adding increasing concentrations of a-tocopherol to
plasma samples of the donors prior to isolation of LDL. LDL (0.25 k and a did not change during this period. This
mg/mL) in PBS was oxidizedwith 1.67 ~M Cu÷÷.For donor A, the suggests that k and a are indeed characteristic sub-
relationshipis y = 5.4 Ix + 6.48 (rz = 0.96);for donor B, y = 3.24x + ject-specific constants by which the oxidation resis-
50.1 (r~ = 0.98). Reprinted with permission from Esterbauer, H4
Puhl, H.; Dieber-Rotheneder,M.; Waeg, G.; Rabl, H. Effectofan- tance of the respective L D L is determined. At pres-
tioxidants on oxidativemodificationof LDL. Annals Med. 23:573- ent, however, we do not know whether a person's
581; 1991. Copyright 1991 The Finnish Medical Society. 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 sig-
nificant 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 L D L isolated from plasma during the vitamin
E supplementation period exhibited a higher oxi-
If the many data (n --- 206) for lag phases and L D L
a-tocopherol levels determined by us so far (i.e., basal
values, L D L loaded in vitro with vitamin E, oral in-
take 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 L D L could not be verified with
certainty. Linear regression analysis for the correla-
tion o f the lag phase and the total L D L antioxidants
(vitamin E + carotenoids) gave more or less the same
result as shown in Figure 18 for a-tocopherol alone.
374 H. ESTERBAUERel al.

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- might come from the amount of PUFAs, the ratio of
enoids and possibly ubiquinol-10 from vitamin E, it PUFAs to saturated fatty acids, the cholesterol con-
would be necessary to load LDL with these antioxi- tent, preformed peroxides, mobility of vitamin E,
dants. structure of apo B, or amounts of other antioxidants,
Our statistical results (Table 13; Figs. 18, 19, 20) such as plasmalogens. 226 Although these factors are
are still based on a rather small sample size and are all not yet known, we feel that the measurement o f k and
obtained from subjects living in the same area and a for individual persons provides valuable additional
having more or less the same dietary and living habits. information regarding their antioxidant status and
It would be worthwhile to investigate if other study might be of considerable medical interest in treat-
groups in other countries exhibit comparable rela- ment of patients with vitamin E. De Graaf286 sepa-
tionships. In our study group, the bulk of vitamin E rated human LDL (1 1 healthy volunteers) into three
values (Fig. 3) and k and a values (Figs. 19, 20) were subfractions differing in density--LDL~ (very light),
within a Gaussian frequency distribution, but there LDL2 (light), and LDL 3 (dense)--and determined
were also persons whose LDL gave k and a values their oxidation resistance by measuring the diene ver-
clearly outside this distribution. It is intriguing to spec- sus time profile in Cu ++ stimulated oxidation. The
ulate that a high vitamin E intake and high LDL vita- dense LDL fractions (LDL2, LDL3) were clearly more
min E levels are crucial for those persons having a low susceptible to oxidation (as evidenced by the reduced
or even negative value for the vitamin E independent lag phase) than the light LDL~; also, LDL2 and LDL3
variable a. For such persons vitamin E is, at least in in contained more conjugated dienes after 4 h oxidation,
vitro oxidation, the only protective component in probably because of their higher content in PUFAs.
LDL. On the other hand, for persons where the effi- Chait et al. 263 recently studied the oxidation suscepti-
ciency (k) of vitamin E is low, even megadoses of vita- bility in six LDL subfractions prepared by equilib-
min E may bring only a minimal additional protec- rium density ultracentrifugation. The fractions were
tive effect. The factors determining the respective effi- oxidized by 1.67 gM Cu ++ and the diene versus time
ciency of vitamin E and the vitamin E independent profile was recorded. The denser LDL subfractions
protection are not known. Important contributions (fractions 3, 4, 5) had the lowest lag time and the high-
est rate of oxidation (= increase ofdienes during prop-
agation). This indicates that the denser LDL particles
Table 12. Effect of Dietary Vitamin E on the a-Tocopherol (which are predominant in atherogenic lipoprotein
Content of Plasma and LDL and the Oxidation Resistance (lag phenotype subjects with subclass pattern B) are signifi-
phase) of LDL
cantly more susceptible to oxidation.
Dose a-tocopherol a-tocopherol Oxidation The protective effect of several other lipophilic an-
IU n in Plasma in LDL Resistance tioxidants was determined by us similarly to vitamin
0 20 9 5 + 11 9 8 + 17 97_+30 E by preincubation of plasma from a single donor
150 10 146+18 138_+12 118_+17 with different concentrations of the agent to be tested.
225 10 165 _+ 15 158 _+ 32 156 _+ 22 In the isolated LDL samples, the amount of the re-
800 10 183 _+ 23 144 _+ 12 135 _+ 23
1200 10 248 _+ 53 215 _+ 47 175 _+ 21 spective substance as well as the resistance toward
Cu ++ oxidation (diene vs. time profile) was then de-
Volunteers took daily for three weeks the indicated doses of termined. Figures 21 and 22 show the effects of a-to-
RRR-a-tocopherol. The values are mean percentage _+ SD com-
pared to the initial value at day - 3 (= 100%). n is the number of cotrienol and probucol. Both compounds gave a con-
analyses. centration-dependent linear increase of the lag phase.
 Oxidation of LDL 375

180 ~ ' ~ p h a 30 db
-J
se _
o
.c_ E
E 120 20 ~_
0

/
_C /~/_ \G-T 0
cz 60 \• 10 oo

o ~o T 2o
tool - / tool L[~_~ 0
0 .............. , .... , ........ 0 E
-5 0 5 10 15 20 25 30

c~-tocopherol
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, r2 =
.868.

Probucol was more efficient than a-tocopherol or to- ethanol) added to the LDL (final concentration of ~3-
cotrienol. Barnhart et al. 2~7 reported that in the pres- carotene 0.5 to 2 ~M; the amount of r-carotene incor-
ence of 0.6 mol % probucol relative to phospholipids porated into LDL was not determined) significantly
(corresponds to 4.2 mol/mol LDL), the time required inhibited the oxidative modification of LDL by cul-
for half-maximal LDL oxidation (3 #M Cu ÷÷) in- tured human monocyte macrophages in Ham's F-10
creased from 130 to 170 min. Our attempts to load as well as by Cu ++ in PBS. In this study, oxidative
LDL in vitro in a similar way to that described for modification was assessed by TBARS, REM, conju-
vitamin E with r-carotene failed, since the solubility gated dienes, and degradation by macrophages.
of r-carotene in the polar organic solvents used to The effect of water-soluble antioxidants was tested
supplement plasma (ethanol, DMSO) was too low. by adding different amounts of aqueous solution to
Jialal et al. 29° reported that a specially prepared solu- the assay immediately before initiating the oxidation
tion of/3-carotene (i.e., r-carotene first dissolved in with C H + + . 4 0 ' 1 6 4 Ascorbate at micromolar concentra-

hexane at 80°C followed by stepwise dilution with tions prolonged the lag phase in a concentration-de-
pendent fashion and exhibited a sparing effect on vi-
300
tamin E and carotenoids. At 10 uM externally added
(/3 ascorbate, for example, the endogenous antioxidants
¢ k = 2.94
4-J o of LDL remained virtually unchanged for 90
a = 52.4 o
c- m i n . 163J64 During this time, the ascorbate decreased
200
E to zero; thereafter the endogenous antioxidants de-
0%0 ° o o ~ creased, with a-tocopherol first and r-carotene last.
d)
go #o Thereafter the lipid peroxidation entered into the
~3
_C 100
o2OO,Ooo o o . . . .
propagation phase with the same rate and kinetics as
{3_
o ~5 ~ "~ - r2 = 0.46
observed in the absence of ascorbate. Jialal et a1.144'291
Q~
°~- ~ (p < 0.001; n = 206) also compared the effect of ascorbate (40-60 uM) and
0 i i i i probucol (10 ~M) on oxidative modification of LDL
0 10 20 30 40 50 by Cu ++ in PBS and in cultured human monocyte
macrophages in Ham's F-10 medium. Both sub-
moL c~-tocopherol/mol LDL stances inhibited oxidation of LDL to a similar degree
Fig. 18. Statistical correlation between lag phase and a-tocopherol as assessed by TBARS, REM, and degradation by
content ofLDL: y = 2.94x + 52.4, fl = 0.46, p < .001, n = 206. This macrophages. In agreement with US, 163'164 it was also
plot contains all baseline values (Fig. 14) and all values from a-to-
copherol in vitro or in vivo supplementation. This is an updated found that ascorbate had a sparing effect on the endog-
version from Refs. 83 and 85 with a larger number of subjects. enous antioxidants (a- and gamma-tocopherol, r-car-
376 H. ESTERBAUERel al.

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 pro-
tect the endogenous antioxidants. Frei et al. 297 re-
cently reported that exposure of human plasma to gas
phase oxidants of cigarette smoke induces lipid perox-
idation 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. 298previously 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 rap-
idly 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 capa-
bility of reactivating vitamin E radicals in LDL ac-
cording 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-con-
taining medium.
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 ob-
served by Sato et al.) 83but 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 an-
tioxidants 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 propaga-
tion phase in a dose-dependent manner in Cu ÷÷ stimu-
lated LDL oxidation was also studied by Jessup et
al., 167 Huber et al.) 84 and Barnhart et a i r 17 These au-
thors 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

5 .......................
iOl .................. i

i2

o
"" 9 ,')i oe- 6
,,.,

o-
- 6 4
::'::::!i::ii::i

-5 0 iO 15 -tO0 -~) -20 20 60 i~ 140

k - values a - values
Fig. 19. Frequency histogram of the a-tocopherol efficacy constant Fig. 20. Frequency histogram of the a-tocopherol independent vari-
k. Frequency gives the number of subjects found in a given group of able a. Frequency gives the number of subjects found in a given
k values. group of a values.
 Oxidation of LDL 377

0.70

0.60

0.50
E
c- 0.40
Ch 0.30
od
< 0.20

0.10

0.00
-0.10 i I i I i I i I i I ,

0 60 120 180 240 300 360

time, minutes
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 probu-
col/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 sub-
stances into the LDL is probably markedly facilitated
if performed above the phase transition temperature.

0.80

0.60
E
(-

Oh 0.40
Od
<::
0.20

0.00
0 200 400 600 800

time, minutes
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.
378 H. ESTERBAUERet al.

Table 14. Effectsof Antioxidants on Lag Phase in Cu++ by AAPH or AMVN). The most significant difference
Stimulated LDL Oxidation
was that the L D L was oxidized only to such a low
Prolongation of extent that it contained not more than about 0.7 to 3
lag phase (minutes) mol L O O H / m o l LDL. In addition, the rate of lipid
Agent added to the assay peroxidation was extremely low with about 0.05 mol
Ascorbate (40, 164) 1.2 _+0.1 L O O H formed per LDL particle every minute. This
Urate (40, 164) 6.9 _+0.5 has to be compared with the a m o u n t of 200 to 400
Glutathione (218) 2.3 +_0.5
Trolox (40, 164) 4.2 _+0.6 mol L O O H contained in fully oxidized LDL (end of
c~-tocopherol(40) 0.5 _+0.2 propagation phase) and with the m a x i m u m rate of 3
Butylated hydroxytoluene,BHT (167) 13.0 _+3.6 L O O H formed every minute in an L D L molecule
Probucol (167) 3.3 _+0.3
Probucol (184) 8.0 during the propagation phase (Figs. 5, 6; Table 9). We
lonox 220 (167) 11.7 _+ 1.5 think that what Stocker et al. s~ measured was indeed
Nordihydroguaiareticacid, NDEA (167) 13.7 _+4.6 the early part of the lag phase, yet not the onset of the
Eicosatetraynoicacid, ETYA (167) 0
Testosterone (184) 0 propagating chain reaction occurring in LDL when it
17-/3-estradiol (184) 12.0 _+4.0 is depleted from all endogenous antioxidants. That
Retinylstearate (40) 3.9 _+ 1.1 L O O H are formed even though a-tocopherol is pres-
Agent incorporated into LDL
a-tocopherol (164, 165) 4.6 _+3.3 ent fully agrees with the theory that vitamin E scav-
a-tocotrienol (165) 5.4 _+ 1.0 enges LOO" radicals. As outlined previously, the 7
Probucol (218) 11.7 _+ 1.5 mol vitamin E/mol LDL give by that scavenging at
Given is the prolongation of the lag phase in minutes produced least 7 mol L O O H / m o l LDL. Still another reason
by 1 mol agent/mol LDL. Table compiled from data reported by why ubiquinol-10 is not likely to be an important an-
Esterbaueret al., 4°'164'165 Jessup et al.,167Huber et al.,~s4and unpub- tioxidant is its low concentration, with only a small
lished results from Puhl et al.2~s
fraction of the LDL molecules containing ubiquinol-
10, while the others are free of this compound
(Table 4).
An objective comparison of the effectiveness of lipo- AAPH-induced oxidation of LDL was also studied
philic antioxidants requires exact knowledge of the by Sato et al. 183 In contrast to the work of Stocker et
conditions and the a m o u n t of the respective agents al., sl it was found that the onset of rapid lipid peroxi-
actually present in LDL. Table 14 lists those com- dation coincides with the depletion of vitamin E and
pounds for which dose effect curves o f the antioxidant that ascorbate and urate inhibit vitamin E consump-
effect were measured under largely comparable con- tion in L D L oxidation. These researchers also re-
ditions. O f course, a n u m b e r of additional studies ex- ported for the first time that the kinetic chain length
ists, in which it was reported that a single, large dose o f during the propagation phase is about 10. Mino et
an antioxidant prevents cell or copper oxidation of al. 219 oxidized LDL and H D L with AAPH and found
L D L for a certain time. a strict linear correlation (r = .977) between lag phase
Stocker et al. 81 reported recently that ubiquinol-10 (expressed as T inhibition) and the a-tocopherol con-
protects L D L more efficiently against lipid peroxida- tent. The use of AAPH instead of Cu ÷+ to initiate
tion than a-tocopherol. This conclusion was based on oxidation in LDL could have several advantages if
the kinetics o f the disappearance of antioxidants and mechanistic aspects (e.g., kinetic chain length) are in-
the appearance o f the first detectable traces of defined vestigated. Figure 23 shows the diene versus time pro-
classes o f lipid hydroperoxides (measured by H P L C files recorded by us for L D L samples with different
postcolumn chemiluminescence detection) in L D L o~-tocopherol contents exposed to AAPH (the LDL
exposed to the radical generators AAPH, AMVN, or samples were those from donor A for which Cu +÷ oxi-
h u m a n PMN. They found that ascorbate disappeared dation is shown in Fig. 16). The initial more or less
first, followed by ubiquinol- 10, after which a-tocoph- linear increase of the 234 nm absorption in the AAPH
erol, carotenoids, and urate started to decrease. The samples occurred with the same rate also in the ab-
onset o f detectable lipid peroxidation corresponded sence o f the L D L and is therefore not due to conju-
closely with the consumption of ubiquinol-10. The gated LOOH. Taking this into account, the kinetics of
experimental conditions and approach used in this diene formation in the AAPH samples exhibit a lag
study differed in many respects from those used by us phase and propagation phase similar to Cu ÷+ induced
and many others (e.g., L D L concentration was 10- oxidation. As expected, the two prooxidants give, how-
fold higher; the isolated L D L was contaminated by ever, different lengths of the lag phases. The impor-
ascorbate, urate, and albumin; oxidation was initiated tant point is that vitamin E delayed, as found by Mino

1.50
lag. rain
1.20
E
(-
,¢ 0.90
o~
o4
0.60
<::
0.30
0.00 ~ , h , i ,
0 60 120
time, minutes
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).
the onset of the propagation phase in the
e t al., z~9
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 vi-
tro 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 deple-
tion 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-solu-
ble 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 antiox-
idants, 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 treat-
ment 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 an-
tioxidants (a-tocopherol, ascorbate, vitamin A, carot-
enoids, selenium) were determined in 16 European
study populations which differed sixfold in mortality
from ischemic heart disease. The incidence of isch-
emic 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
Oxidation of LDL 379
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.292compared sele-
nium, PUFAs, and a-tocopherol in subjects with vary-
ing degrees of coronary atherosclerosis. Levels ofa-to-
copherol and PUFAs were similar in the cases and in
controls. However, the ratio Se/PUFAs was signifi-
i
cantly lower in the cases, which suggests that a higher
180 240
PUFA level with insufficient antioxidant protection
might indicate a higher risk. Short-term supplementa-
tion 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 con-
tent of TBARS in isolated LDL) induced by acute
cigarette smoking. 42 Supplementation of elderly peo-
ple 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-caro-
tene, and 75 #g selenium daily over 5 months resulted
in a reduction of serum lipid peroxides, platelet aggre-
gation, and platelet-produced thromboxane B2 (Ref.
300). Fruchart et al. 3°1 reported on in vitro oxidizabil-
ity of LDL isolated from hypercholesterolemic men
treated with vitamin E (1 g/d) or fenofribrate + vita-
min E or placebo for two months. Copper-treated
LDL samples from the patients receiving vitamin E
showed a significant reduced uptake by mouse perito-
neal macrophages as compared to oxidized LDL of
the placebo group. Gaziano et a1.192presented prelimi-
nary findings from the USA Physician Health Study,
according to which the group receiving 50 mg 3-caro-
tene every second day had a significant (44%) reduc-
tion 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 atheroscle-
rosis and ischemic heart disease. A controlled athero-
sclerosis intervention study with the classical antioxi-
dants (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 differ-
ence between the treated (38 patients) and the placebo
group (37 patients).194 In angina pectoris patients sup-
plemented 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 cerebrovascu-
lar 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 signifi-
cantly (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 antiox-
idant 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 re-
duced symptoms of atherosclerosis in primates fed an
atherogenic diet. This is in agreement with an earlier
report 19s that atherosclerosis of triglyceride-fed rab-
bits can be prevented by vitamin E, and that vitamin
E and vitamin A supplements reduce spontaneous ath-
erosclerosis in hens during the egg-laying p e r i o d . 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 vita-
min 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 in-
hibits 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 re-
ported 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 develop-
ment ofatherosclerotic lesions in rabbits fed a 1% cho-
lesterol 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 atherosclero-
sis 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 sig-
nificant 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 athero-
sclerosis, 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 subjects2°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 modi-
fication 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 re-
duced 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 pro-
bucol concentration of the treated animals was 83
uM, and roughly two thirds was associated with LDL.
This means that probucol must have reached a con-
centration 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 re-
ports, 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 c h o w . 224 I n
contrast to these studies, Stein et al. 225 found no pro-
tective 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 an-
tioxidants 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 ex-
pected 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-tocoph-
erol (added as an ethanol solution) inhibits prolifera-
tion of cultured smooth muscle cells, an effect not
 Oxidation of LDL
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 de-
tergent 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) investi-
gated the inhibitory effects of polyunsaturated fatty
acids on cell proliferation and developed the concept
that PUFA-derived lipid peroxidation products in-
hibit smooth muscle cell proliferation, whereas an-
tioxidants, 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 synthe-
sis 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 (in-
duced, 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 in-
volved 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- 1a) by preventing the activation of expres-
sion of the IL-I gene. IL-1 promotes a number of ef-
fects 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 sys-
tem, it w a s s h o w n 296 that uptake of native and acety-
lated 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 re-
ported 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
381
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
AAPH--2,2'-azobis (2-amidinopropane) dihydro-
chloride
ac-LDL--acetylated LDL
AMVN--2,2'-azobis (2,4-dimethylvaleronitfile)
apo B--apolipoprotein 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 o u p l e d with m a s s
spectroscopy
HDL--high density lipoprotein
HETE--hydroxy eicosatetraenoic acid
HNE--4-hydroxynonenal
HNE-LDL--4-hydroxynonenal modified LDL
HODE~hydroxy octadecadienoic acid
HPLC--high-performance liquid chromatography
HPODE--hydroperoxy octadecadienoic acid
LDL--low density lipoprotein
L O O H - - a lipid h y d r o p e r o x i d e
MDA--malonaldehyde
MDA-LDL--malonaldehyde modified LDL
MM-LDLpminimally modified LDL
o L D L - - o x i d i z e d low d e n s i t y l i p o p r o t e i n
P B S ~ p h o s p h a t e - b u f f e r e d saline
P U F A - - p o l y u n s a t u r a t e d fatty a c i d
REM--relative electrophoretic mobility
TBA--thiobarbitufic acid
T B A R S - - t h i o b a r b i t u f i c a c i d reactive s u b s t a n c e s
V L D L - - v e r y low d e n s i t y l i p o p r o t e i n
WHHL--Watanabe heritable hyperlipidemic rabbits