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Free Radical Biology & Medicine, Vol. 13, pp. 341-390, 1992 0891-5849/92 $5.00 + .

Printed in the USA. All rights reserved. Copyright © 1992 Pergamon Press Ltd.

Review Article


*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as some previously unpublished material from our laboratories.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

caused by sudden damming of arteries narrowed by
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 atherosclerotic plaques. Until recently, only the man-
half of all people enjoying a Western lifestyle are ifestations of the disease and its consequences have
been studied extensively, with the underlying bio-
chemical mechanism of atherogenesis largely un-
Address correspondence to: Prof. Dr. Hermann Esterbauer. known. However, a series of separate excellent studies
Hermann Esterbauer, PhD, is a Professor of Biochemistry at the carried out mainly in the last decade (Table 1) have
University of Graz, Austria. He was trained in chemistry and biol-
ogy at the Universities of Vienna and Graz, and graduated with a provided the background allowing the formulation of
PhD in 1963. He did postdoctoral work (1973-1974) at the Univer- a new reasonable theory of atherogenesis which has
sity of Pittsburgh and at the Michigan State University and was focused much of current research on tests of its va-
visiting Professor at the Universities of Turin (1984-1988) and
Siena (1989) and at the Brunel University (1987-1991). lidity.
Janusz Gebicki, PhD, is Associate Professor in Biology at the The bare bones of this recent postulate is that ath-
Macquarie University, Sydney. He studied chemistry at the Univer-
sity of London, where he gained the BSc and PhD degrees. He erosclerotic plaques form from cells engorged with lip-
subsequently worked at McMaster University, Hamilton, Canada, ids supplied by blood lipoproteins, modified by a free
at the Washington University School of Medicine in St. Louis, Mis- radical process. Pathological, microscopic, histochem-
souri, and at the Brookhaven National University, near New York.
He was appointed to Macquarie University, Sydney, after a period ical, and biochemical studies have shown that the oc-
as Research Fellow at the Australian National University. clusions and plaques which form in the intima regions
Herbert Puhl, PhD, is a postdoctoral fellow at the Institute of of the major arteries are mainly made up of cells so
Biochemistry, University of Graz. He studied biology and chemis-
try and gained his PhD in 1992 from the University of Graz. altered in appearance by internalized lipids that they
Giinther Jiirgens, PhD, is Associate Professor of Biochemistry at are known as foam cells. 1-~° Foam cells were identi-
the Institute of Medical Biochemistry, Medical School, University fied as macrophages derived from monocytes circu-
of Graz. He studied chemistry, performed his thesis in physical
chemistry and biochemistry, and graduated with a PhD at the Uni- lating in the blood 7"s'13and smooth muscle cells prolif-
versity of Graz in 1974. erating in the region of the plaque. 4'13Their gross alter-

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 343

ation is mainly caused by the entry of lipids (e.g., lipo- lipoprotein. VLDL and IDL have a short half life and
protein particles modified in or near the artery). are removed from the circulation within hours,
These particles bypass the normal tight control exer- whereas the LDL particles have a rather long life and
cised by the cells' surface receptors and enter the cells circulate in the blood for about 2 d before they are
by a different, scavenger pathway, which has no such cleared. In normolipidemic persons, the serum LDL
control, to,1l,~a There is much evidence (for review see concentration is about 3 mg/mL, and typically this
Ref. 57) that the principal lipoproteins susceptible to LDL carries about 60% of the total serum choles-
the modification leading to foam cell formation are terol. 63 The uptake of LDL by cells occurs via a recep-
low density lipoproteins (LDL). Since LDL is the tor-mediated pathway (B/E receptor) and by nonspe-
main carrier of free and esterified cholesterol in the cific endocytosis. The LDL first binds to the B/E re-
body, these lipids are the predominant components of ceptor (LDL receptor) present on the surface of most
the foam cells. This brief summary covers the knowl- cells and is then endocytosed. The highest concentra-
edge of likely atherogenic events derived from studies tion of LDL receptors is found in the liver, and about
completed before about 1980. three quarters of the LDL is removed from the blood-
The more recent research addressed the nature of stream by the liver, although the suprarenal gland and
modification of the LDL rendering it capable of pro- the ovary are comparably rich in LDL receptors. The
ducing foam cells and the events which could produce liver converts most of the LDL cholesterol to bile
such modifications in vivo. It turned out ultimately acids, which are secreted into the duodenum. A re-
that the most physiologically probable LDL modifica- duction of the reabsorption of bile acids by bile-acid-
tion is derivarization of its constituent apolipoprotein binding drugs (e.g., ion exchange resins, cholesteryl-
B (apo B) by breakdown products of lipid peroxides, amine) is one possibility to reduce serum cholesterol
while the lipid peroxidation is probably caused by an levels. The cholesterol of LDL is of course also used
oxidizing agent in the vascular system. Thus, the by all cells as a building block for cell membranes and
currently favored chemical common denominator of in specialized cells for biosynthesis of steroid hor-
the new hypothesis of an atherosclerotic plaque for- mones. The endogenous and exogenous (cholesterol
mation in vivo is formation of oxidized LDL (oLDL) from the diet) pathways of cholesterol are intimately
by mechanisms involving free radicals and/or lipoxy- fine tuned by several control mechanisms so that the
genases. serum cholesterol level in normolipidemic persons is
The observations and correlations germane to this maintained constant and in a narrow range of 160-
summary are listed in Table 1. The list is not exhaus- 200 mg/100 mL (Ref. 64).
tive and the chronology may not always be precise, One of the most intensively studied (for review see
because it is often not possible to establish the date of Ref. 65) imbalances in cholesterol homeostasis is fa-
a significant statement or its priority. Rather, the pur- miliar hypercholesterolemia (FHC). Patients with this
pose of the table is to provide references useful in doc- heritable disease have a defect in the gene coding for
umenting findings which contributed in a major way the LDL receptor, and the deficiency of this receptor
to the development of the current theory of athero- dramatically reduces the clearance rate of the LDL.
genesis. Additional details can be found in several re- The net result is a very high plasma LDL and choles-
cent reviews. 56-62'261 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-
The liver assembles triglyceride-rich, very low den- proteins which can be isolated by ultracentrifugation
sity lipoproteins (VLDL) and secretes them into the within a density range of 1.019 to 1.063 g/mL. By
circulation. The main biological function of VLDL is equilibrium density-gradient ultracentrifugation and
to supply the peripheral tissue with fatty acids. Lipo- several other techniques, LDL can be further sepa-
protein lipases on the surface of the vascular endothe- rated into two or more subfractions differing some-
lial cells hydrolyze the VLDL triglycerides to free fatty what in density, size, and molecular w e i g h t . 63 LDL
acids, which are then taken up by adipose tissue and molecules are large spherical particles with a diameter
muscle cells. With increasing hydrolysis, the VLDL of 19-25 nm and molecular weights between 1.8 and
loses most of its triglycerides and progressively 2.8 million. The mean chemical composition calcu-
changes into lipoproteins with intermediate density lated from values obtained from various sources is
(IDL) and finally to the cholesterol-rich low density 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
some models) but should rather be seen like an "octo-
pus" embracing the whole surface of the LDL. The
apo B is an exceptionally large protein consisting of
4536 amino acids. The amino acid sequence has in
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.
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-

~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
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- we have investigated, the linoleic acid content varied
tein based on experimentally determined dry weight. (e.g., from about 1200 to 2400 n m o l / m g L D L pro-
These values are very close to those found by us (Ta- tein). Such a variation in the P U F A content is likely
ble 2). In most of our previous publications, data on to have a significant effect on the oxidation behavior
composition of L D L (e.g., fatty acids, antioxidants, of different L D L samples. Parthasarathy et al. 89
aldehydes) were given per milligram of total LDL. showed recently that feeding rabbits an oleic-acid-rich
Other groups working on oxidative modification of diet results in an oleate-rich LDL, which is remark-
L D L report its composition and changes during oxi- ably resistant toward oxidation.
dation per milligram of L D L protein or per milligram The PUFAs in L D L are protected against free radi-
of total cholesterol. The necessary conversion factors cal attack and oxidation by a n u m b e r of lipophilic
from one unit into others can be deduced from the antioxidants listed in Table 4. Representative chro-
data in Table 2. The good agreement on the chemical matograms showing their separations are given in Fig-
composition of L D L as determined by different labo- ure 1A, 1B, and 2. On a molar base, by far the major
ratories (Table 2) indicates that conversion between antioxidant is a-tocopherol; the a m o u n t of 11.58
the different reference systems used by different labo- n m o l / m g L D L protein equals about 6 molecules a-to-
ratories is justified. To facilitate comparison of data, copherol per L D L particle. All other antioxidants
we express in this review L D L analysis in nanomoles (i.e., gamma-tocopherol, carotenoids, oxycaroten-
per milligram of L D L protein and, where appropriate, oids, and ubiquinol-10) are present in m u c h smaller
in mol/mol LDL. amounts. The antioxidant content of L D L varies, sim-
The average distribution oflipids and o f individual ilarly to the PUFAs, significantly between individ-
fatty acids is given in Tables 3 and 4. The total num- uals. For instance, the lowest and highest a m o u n t of
ber of fatty acid molecules b o u n d in the different lipid a-tocopherol found by us a m o n g 87 (not vitamin E
classes of an L D L molecule is 2700 on average. O f supplemented) donors were 3 and 15 m o l / m o l LDL.
these, about half are polyunsaturated fatty acids The frequency histogram of L D L a-tocopherol is
(PUFAs), mainly linoleic acid with m i n o r a m o u n t s of shown in Figure 3. The vitamin E content o f L D L
arachidonic acid and docosahexaenoic acid. The fatty increases with its P U F A content with a correlation of
acid content of L D L and their distribution pattern y = 0.0034x + 1.98, where y is mol a-tocopherol/mol
can vary considerably from d o n o r to donor, probably L D L and x is mol P U F A / m o l LDL. 163 The value of
due to different dietary habits. In the subjects which 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 increased most rapidly in chylomicrones, then in
the LDL molecules. It has been argued by Halliwell et VLDL followed by LDL and HDL, and finally in red
al. 9° that this suggests that o~-tocopherol is the only blood cells. This sequence of appearance and distribu-
significant antioxidant in LDL and that the carot- tion strongly suggests that vitamin E is first incorpo-
enoids play no or only a minor role in protecting LDL rated into chylomicrones, transported in chylomicron
against oxidation. The same is likely to hold for ubi- remnants to the liver, and delivered into the circula-
quinol-10. Recently it has been shown by Stocker et tion again in VLDL. The vitamin E molecules con-
al. 8~that ubiquinol-10 is contained in LDL and it was tained in LDL stem therefore primarily from VLDL.
proposed that it acts as an even more powerful antiox- The majority of vitamin E appears to enter the cells
idant than a-tocopherol. The concentration of ubi- with the uptake of the intact LDL by the LDL recep-
quinol-10 in LDL given by these authors is similar to tor. 92 Additional uptake may also occur from chylo-
that of/3-carotene and agrees with values determined micrones and VLDL by the action of lipoprotein li-
independently by us (Table 4). Since plasma contains pase (for review see Ref. 227).
a great variety of water-soluble and lipid-soluble an- In vitro, the a-tocopherol molecules of LDL un-
tioxidants (e.g., more than 20 different carotenoids dergo a rapid intermolecular exchange as well as ex-
have been r e p o r t e d , 93 it seems reasonable to us that change with other lipoproteins (VLDL, HDL) and
LDL isolated from plasma may contain several other blood cells; the estimated half times for this spontane-
lipid- and/or water-soluble antioxidants in addition ous a-tocopherol transfer are in the range of 20-70
to those listed in Table 4. Since all of them are likely min, which is about two to three times slower than
to affect the oxidation of LDL in vitro, it would be cholesterol transfer. 9~ On the other hand, the sponta-
important to investigate this further. Ethanolamine- neous exchange (in vitro) of ~-carotene is very slow,
plasmalogens, for example, have antioxidant activity, and no equilibration occurs within 18 h. 91
and the presence of such plasmalogens in LDL may
well contribute to its oxidation resistance. 226 DO PLASMA OR PLASMA LIPOPROTEINS FROM
The adsorption and transport of vitamin E in hu- HEALTHY OR ATHEROSCLEROTIC H U M A N
man subjects has been studied in detail with deute- SUBJECTS CONTAIN LIPID PEROXIDATION PRODUCTS?
rium labelled c~-tocopheryl a c e t a t . 227'311 Consistent 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
with earlier studies, 3~2 newly absorbed c~-tocopherol 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






(9 ffl

/ "6

8 "~

G) 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 plasma contains only low levels of T B A R S in the
Ray et a l . 9 4 that oxidative degradation of isolated range of 0.22 to 0.4 n m o l / m L , but storage of plasma
L D L m a y occur during prolonged dialysis if traces of at 4°C in the absence of protecting agents (e.g.,
copper ions are present. Interestingly, it was already E D T A ) led to a continuous increase of the T B A R S at
noted in this early study that other transition metal a rate of 0.15 _+ 0.14 n m o l / m L , week. The rate of
ions, including Fe ÷+ and Fe +++, had no or only mini- T B A R S formation showed a strong individual varia-
mal degradative effects. A systematic investigation tion, which could be indicative of different antioxi-
was m a d e by Schuh et al., 17 who showed that a m o r e dant contents of the plasma samples. It was further
or less complete oxidative degradation of L D L with shown by Lee that lipids o f all lipoproteins classes
concomitant formation ofthiobarbituric acid reactive contributed to the formation of T B A R S during stor-
substances (TBARS) occurs if it is dialyzed at 4°C for age of plasma. F r o m these and other investigations, it
24 h against phosphate-buffered saline without pro- is clear that p r o o f of the existence of low levels of
tection, which could be provided by ethylenediamine- peroxidation products in native L D L requires a very
tetraacetic acid (EDTA), butylated hydroxytoluene careful preparation and handling. It is difficult to
(BHT), or by nitrogen gassing. judge if that has been considered in all studies. Most
Lee 95 pointed out that freshly prepared h u m a n 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-
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
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
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
searchers also include BHT in the isolation medium. "-I
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
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 order of magnitude. It should be considered that heat-
found in serum similar levels of TBARS to those re- ing in a hot acid, as applied in the TBA assay, is a very
ported by Yagi's group; however, the TBARS in the harsh condition for PUFAs, and it might well be that
LDL fraction were about 10-fold higher. According to most if not all TBARS are formed during the assay
these investigations, hyperlipoproteinemia is asso- itself by autooxidation of PUFAs. This could be
ciated with a very strong increase of TBARS in avoided by inclusion of EDTA and BHT in the assay,
VLDL, whereas TBARS in LDL are only slightly in- but this seems to be done only rarely (e.g., Ref. 313).
creased. The sum of TBARS determined in the sepa- Also, in our investigations most of the freshly pre-
rated lipoprotein fractions by far exceeded the pared LDL samples gave a weak absorption at 535 nm
TBARS of the parent plasma samples, and it was as- in the TBA assay, which corresponds to an apparent
sumed 99 that in whole plasma certain components concentration of about 0.5 to 3 nmol TBARS/mg
(e.g., HDL) inhibited the chemical reaction of TBA LDL protein, with a mean of 3.6 + 1.0 nmol/mg.
with the precursors of TBARS. This shows very Occasionally we have also recorded the full spectrum
clearly the problems and limitations of the TBA as- (400-600 nm) of the chromogen produced by the reac-
say; two similar methods give values differing by one 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 H. ESTERBAUERet al.

95' 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
0.16 reported that L D L isolated from healthy subjects was
free from detectable amounts of cholesterol ester hy-
droperoxides, phospholipid hydroperoxides, and tri-
~ 0.12 glyceride hydroperoxides as measured by high-perfor-
mance liquid chromatography (HPLC) postcolumn
chemiluminescence detection. This method is incom-
@ parably more specific than the TBA or iodometric
0.08 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
0.04 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
400 500 600 LDL. 82 A recently developed modified iodometric as-
say, ~59 which takes into account interfering phenom-
wavelength, nm ena leading to false results, gave for the total a m o u n t
Fig. 4. Spectraof TBARS in native LDL (0 min) and LDL oxidized o f lipid hydroperoxides esterified in the plasma lipo-
with Cu++ for 35, 70, and 95 min. 0.5 mL LDL solution (1.5 mg proteins o f healthy humans a value of 4.0 _+ 1.7 #M;
total LDL/mL in PBS + 10 uM CuC12)were mixed with 3 mL 1% with the assumption that about half of that is con-
H3PO4and 1mL TBA reagentand heated 45 min on a boilingwater
bath. The color was extracted into 4 mL butanol and the spectra tained in LDL, the lipid peroxide content in LDL
were recorded. would be 3.03 + 1.28 nmol/mg LDL protein (= 1.66
mol/mol LDL).
tate) with TBA and found only a broad and uncharac- Miyzawa ~0 used a chemiluminescence HPLC as-
teristic absorption without a m a x i m u m at 535 nm, say to measure phosphatidylcholine hydroperoxides
the peak absorption of the MDA-TBA complex (Fig. (PCOOH) in a large number of human plasma sam-
4). Since in our assay 1 nmol T B A R S / m g L D L pro- ples and found concentrations of 0.0 ! to 0.5 uM. Sam-
tein would have given a detectable 535 n m maxi- ples from unhealthy subjects contained much higher
mum, we assume that the concentration of TBARS in concentrations in the range of 0.5 to 9 uM. In diabetic
native L D L from healthy subjects, if they exist at all, patients the P C O O H were mainly contained in
must be below that level which corresponds to 0.5 V L D L and LDL.
mol TBARS/mol LDL. The authors cited in Table 6 Another possibility of searching for remnants o f in
reported the L D L TBARS in terms o f n m o l / m L vivo lipid peroxidation in L D L would be the measure-
plasma. With the assumption that 3 mg L D L / m L are ment of defined aldehydic lipid peroxidation prod-
contained in plasma, the values o f Nishigaki et al. 1°7 ucts. By H P L C we found traces of 4-hydroxynonenal
and Hagihara et al. ~°2 correspond to 0.7 to 1.0 mol (HNE) in some samples, but in others this aldehyde
TBARS/mol LDL, whereas the values of Szczeklik et was undetectable. 96 The mean value, given in Table 9,
al. 99 correspond to 7.5 to 11 tool TBARS/mol LDL. would correspond to 0.5 mol H N E / m o l LDL. Using
Heinecke et al. 2~ reported for native h u m a n L D L 0.8 the GC method developed by van Kuijk et al., 1~
+ 2.3 nmol TBARS/mg protein; Harats et al. 42 found H N E was undetectable in native LDL. 87 Hexanal, the
in L D L from smokers 0.6 _ 0.028 and in L D L from major aldehyde in oLDL, was undetectable by HPLC
nonsmokers 0.55 8 ___0.020 nmol TBARS/mg protein. or GC in native LDL. Thus, these analyses indicate
The potential diagnostic value o f malonaldehyde de- again that native LDL of healthy individuals is devoid
termination by the TBA-test has recently be carefully o f free aldehydic lipid peroxidation products.
reviewed by Janero. 258 Several other attempts were made to demonstrate
The determination o f lipid hydroperoxides by the oxidation remnants in L D L of healthy subjects. The
iodometric methods also gives for most samples o f apolipoprotein B of in vitro oxidized LDL, for exam-
native L D L a positive result corresponding about 10 ple, shows a very strong fluorescence at 430 nm with
Oxidation of LDL 351

excitation at 355 nm.l 7,29,56,112,113 This chromophore readily oxidizable LDL subfractions are associated
most likely results from reaction of aldehydic lipid with an increased risk of atherosclerosis, it would be
peroxidation products with free amino groups of apo important to improve the methods for their analysis
B. Apo B from native LDL analyzed by three-dimen- and to ensure that the findings can be reproduced by
sional fluorescence spectroscopy showed always the others.
presence of a small amount of a chromophore with Monoclonal antibodies directed against oxida-
exactly the same spectral characteristics, which could tively modified human LDL 37'43 or against malonal-
be indicative of subtle oxidative alterations of apo B dehyde (MDA) or HNE conjugated LDL 43 did not
which had occurred already in vivo)13,114 The relative show a noteworthy crossreaction with freshly isolated
fluorescence intensity at 430 nm showed strong indi- human LDL, and one must therefore assume that na-
vidual variations. Dobretsov et al., 115 using conven- tive LDL does not have epitopes typically found in
tional fluorescence spectroscopy, did not find the 430 oLDL.
nm fluorescent chromophore in plasma LDL, which The occurrence of heavily oxidized LDL as a sub-
is not surprising since it requires three-dimensional fraction in LDL of healthy human subjects is also not
measurements to resolve it from the 13 different supported by studies with autoantibodies. Parums et
fluorophors present in LDL. 114 al) 21 studied the incidence of serum autoantibodies
Avogaro's group 32'116'117'322 reported in 1988 (Ref. against LDL, oLDL, and ceroid in 100 individuals.
32) that LDL collected from 18 different healthy hu- None of them had autoantibodies to native human
mans contained a subtraction (about 5 to 20%) which LDL samples isolated by conventional ultracentrifu-
could be separated from the LDL bulk by ion ex- gation. Since Avogaro's LDL- would have been pres-
change chromatography. This subfraction was more ent in such samples, it is clear that LDL- does not act
electronegative, contained more conjugated dienes, as an immunogen and that LDL- is not a form of
had apo B aggregates, and led to a higher accumula- LDL which is recognized by antibodies directed
tion of cholesterol esters in cultured macrophages against oLDL. Serum autoantibodies recognizing ar-
than normal LDL. It was concluded that this subfrac- tificially oxidized LDL were not present in young
tion resulted from in vivo oxidation of LDL. The high controls but were found in about 50% of elderly indi-
proportion of the modified LDL together with the viduals and in most patients with chronic periaortitis.
methodology first caused some doubts on the validity These autoantibodies probably developed against oxi-
of this finding, but with an improved method (ion datively modified LDL formed within arteriosclerotic
exchange HPLC) the more negatively charged LDL plaques 121 and are not indicative of the presence of
(now termed LDL-) was found again in LDL col- oLDL in serum. Recently autoantibodies against
lected from normal subjects, 1~7though the content of modified LDL were considered as a nonlipid factor of
LDL- in total LDL given by the revised method was blood plasma that stimulates foam cell formation) 22
only 3.9% (range 0.5 to 9.8%, 32 normal male subjects The literature on the relationship between atheroscle-
aged 30 to 60). The LDL- content of LDL was nega- rosis and circulating immune complexes containing
tively correlated with its vitamin E content and posi- LDL was recently reviewed by Orekhov. 325 For hu-
tively correlated with its TBARS. The TBARS con- man placental blood the presence of an acetyl-like
tent of LDL- was on average 7.3 mol/mol LDL, modified LDL has been shown by an ELISA assay. ~23
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- AS early as 1952, Glavind et al. 33 reported that a
tion, only less than 1% of total LDL, by ion exchange chloroform extract of atherosclerotic lesions from hu-
chromatography on DEAE-Sepharose 6B. This minor man aortas (postmortem material) contains lipid per-
fraction was more negative and had a higher density oxides and that the peroxide content is positively
than normal LDL. In disagreement with Avogaro's correlated with the extension of the atheromatas. The
findings, however, it did not exhibit properties indica- highest peroxide values found were in the range of
tive of mild oxidation and it was not recognized by about 10 to 20 milli-equivalent per kilogram of fat,
macrophage scavenger receptors. However, the minor which is equivalent to 10 to 20 nmol lipid peroxides/
fraction was more labile to Cu ++ stimulated oxidation mg lipid; for comparison LDL oxidized 24 h with
in vitro. In view of the possibility that such minor and Cu +÷ ions contains about 50 nmol peroxides/mg lipid

(Table 9). A later r e p o r t 16 showed that the peroxides An LDL with increased relative electrophoretic mobil-
probably formed in the period between death and tis- ity (REM) was also found in human interstitial
sue extraction, through cessation of enzymatic pro- fluid. 129It was also shown 68'126that aorta LDL is asso-
cesses normally converting hydroperoxides to hy- ciated with about 2 to 4 ug glycosaminoglycans
droxy-octadecadienoic cholesterol esters. It was also (mainly chondroitin sulfate and dermatan sulfate).
pointed out that the hydroxy-esters isolated from dis- This is in accordance with the assumption that the
eased arteries during surgery had structures inconsis- LDL deposited in the intima-media is also retained
tent with lipoxygenase oxidation of the lipids forming extraceUularly by association with strongly nega-
the atherosclerotic deposits. Several other early re- tively charged glycosamino-glycans. In vitro experi-
p o r t s 314-316 also suggest the presence of oxidized cho- ments 2zs'229 showed that subpopulations of LDL bind
lesterol and oxidized cholesteryl esters in human to human arterial chondroitin sulfate and proteogly-
aorta (for review see Ref. 319). Piotrowski et al. ~z4 cans. This binding reduces the thermal stability of the
found that the lipid extract (Folch) of aortic human surface and core in LDL and leads to exposure ofly-
tissue contains fluorochromes with emission maxi- sine- and arginine-rich segments of the apo B. LDL
mum at 435 nm (Ex 355 nm) indicative of lipid per- subclasses complexed with such proteoglycans exhibit
oxidation products. Copper or cell oLDL exhibits also in vitro an increased uptake by mouse peritoneal mac-
a very strong 430 n m f l u o r e s c e n c e . 17,29,56,112A13 The rophages. Additional differences between aorta and
amount of fluorescent lipid material was about 30% plasma LDL include decrease in sphingomyelin, ten-
higher in atherosclerotic tissue than in normal aortic dency to aggregate, increased particle diameter, and
tissue (4.15 vs. 3.08 arbitrary units/g tissue). Kana- possible fragmentation of apo B in the former. Most
zawa et al. 125separated the lipids of lesions by conven- importantly, the aorta LDL is more rapidly taken up
tional thin-layer chromatography (TLC) and found by macrophages than plasma LDL. A complement
an unknown spot (spot x) which is probably an oxi- activating lipid complex (vesicles with 100-500 nm in
dized form of cholesteryl esters, not present in native size) containing cholesterol and phospholipids can be
LDL, but formed if LDL was dialyzed over long pe- extracted with saline from atherosclerotic lesions of
riods (a condition known to induce lipid peroxida- human aorta. 23° The lesion lipid complement might
tion) or if the LDL was oxygenated for 20 rain only. be responsible for the inflammation in the atheroscle-
Ledwozyw et al. 1°3 determined for the first time rotic lesion. It would be worthwhile to investigate
TBARS in buffer extracts of the human arterial wall whether these vesicles also contain oxidized lipids.
from patients suffering from atherosclerosis who had Belkner et al. 255 recently analyzed by HPLC hydroxy
their limb amputated and found that it contained fatty acids in the lipids of pieces of thoratic aortas of
twice as much TBARS as extracts from normal sub- five men who suffered from chronic ischemic heart
jects (7.38 vs. 3.42 nmol TBARS/g artery). A positive disease and died from acute heart failure and found a
correlation (r = .790) also existed between TBARS in peak indicative for oxygenated cholesteryl esters
plasma and arterial wall TBARS. (probably cholesteryl linoleate). The amount varied
In several studies the properties of LDL extracted between 17 and 55 mg/g wet weight for the five sam-
from the arterial wall tissue were compared with ples. About 12 to 21% of the cholesteryl linoleate was
plasma LDL (Table 7). Hoffand Gaubatz 68compared present as oxygenated metabolites. "Healthy"-look-
the chemical composition of human LDL from ing parts of the same aortas also contained this mate-
plasma, normal intima, and fibrous plaques. No sig- rial, but the amount was much smaller (i.e., 0.3-4.5
nificant differences were seen in the percent distribu- mg/g wet weight or 5.8-9.5% of total cholesteryl lino-
tion of protein, phospholipids, free cholesterol, choles- leate). The authors assumed that this oxygenated cho-
terol esters, and triglycerides. Some remarkable differ- lesteryl linoleate was formed by a 15-1ipoxygenase ac-
ences existed in the fatty acid distribution; thus the tivity. In human plasma incubated with reticulocyte
arachidonic acid and linoleic acid ofcholesteryl esters lipoxygenase, 13-HODE (main product), 9-HODE,
and triglycerides were strongly decreased in aorta-de- and 15-HETE esterified with cholesterol were
rived LDL, which would be consistent with the high formed.
susceptibility of these PUFAs toward oxidation. That The physico-chemical (REM, fluorescence) com-
aorta LDL has a lower content of linoleic acid than positional (less PUFAs, more peroxides and TBARS)
plasma LDL was also found by Camejo et al. ~26 and and functional (increased uptake by macrophages)
Yl~-Herttuala et al.; 67 moreover, the aorta LDL shows properties of aorta LDL and aorta lipids in atheroscle-
an increased electrophoretic mobility as compared to rosis support the hypothesis that LDL deposited in
normal plasma LDL, a property shown also by oLDL. 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
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.,

EXPERIMENTAL ANIMAL STUDIES 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-
The highest difference in plasma TBARS likely to tein) and that it also showed an increased REM and
be ever seen comes from experimental animal studies was more readily taken up by macrophages than
with genetically defective nonlaying hens) 3° These plasma LDL. Wang and Powell TM recently reported
animals have extremely high cholesterol (450 mg/dL) that the lipids derived from aorta or from plasma
and triglyceride values (12.000 mg/dL) as compared L D L of cholesterol-fed New Zealand White rabbits
to normal laying hens, which have 84 mg/dL choles- contain increased amounts of esterified and unesteri-
terol and 1.100 mg/dL triglycerides. The TBARS fled hydroxy linoleic acid (9-HODE, 13-HODE) and
value in plasma of nonlaying hens is about 14 times hydroxy arachidonic acid (1 I-HETE, 12-HETE, 15-
higher than in laying hens (76 vs. 5.6 n m o l / m L HETE). The a m o u n t o f h y d r o x y fatty acids compared
plasma). The large increase also persisted in TBARS to total polyunsaturated fatty acids (2332 n m o l / m g
normalized to cholesterol. In a later study Smith et L D L protein, Table 4) is very low. After 15 weeks
al) 3~ showed that the plasma TBARS value, but not cholesterol feeding, that total a m o u n t of esterified hy-
the total plasma cholesterol or lipids, correlated with droxy fatty acids in rabbit L D L was only a m o u n t 0.35
the development of atherosclerosis and intimal thick- n m o l / m g LDL protein with 80% H O D E and 20%
ening in this animal model. In cholesterol-fed rabbits HETE. About 0.15 nmol hydroxy fatty acids ( H O D E
serum TBARS are 1.6-fold higher than in controls + HETE) were also present in control rabbits LDL.
(i.e., 1.47 vs. 0.91 n m o l / m L serumS°°). The lipopro- The analysis of such trace amounts is only possible
rein fraction containing V L D L + L D L obtained from with GC/MS.
rats made diabetic by streptozotoxin injection is Segments from normal rabbit aortas incubated
highly oxidized and contains about 25 nmol TBARS/ with arachidonic acid produce 12-HETE as the prin-
mg cholesterol compared to about 2.5 nmol in nor- cipal lipoxygenase product, 323 but aortic segments
mal rats) 32 The H D L of the diabetic rats had TBARS from cholesterol-fed rabbits or W H H L rabbits pro-
values in the range of controls. In this study it was also duce 15-HETE, suggesting an increased 15-1ipoxy-
reported that the V L D L + L D L of diabetic rats was genase activity in atherosclerotic arteries. 323'324
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 A monoclonal antibody raised against MDA-modi-
Daugherty et al.t28 investigated L D L isolated from fled L D L (MDA-LDL) was used by G o n e n et al) 33 to
the vascular tissue of W H H L rabbits and found that it 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 Type of

Authors Code Antibody Immunogen Major Findings

Gonen et al. 1987 (133) EB 7-3 Mouse mAb Mouse MDA-LDL ELISA failed to show any MDA-LDL in
extracts of 14 autopsy samples from aorta
Salmon et al. 1987 (134) -- Rabbit as Rabbit MDA-LDL MDA-lysine conjugates in Cu ++ oxidized LDL
Haberland et al. 1988 (135) MDA-lys Mouse mAb Human MDA-LDL Stains proteins in atheroma of WHHL rabbits;
stain co-localizes with extracellular deposits
of apo B
Palinski et al. 1989 (43) MAL-2 Guinea pig as Guinea pig MDA-LDL All three antibodies stain the same area of
HNE-6 Guinea pig as Guinea pig HNE-LDL lesions in WHHL rabbits, immunostain
OLF4-3CI0 Mouse mAb Mouse oLDL mostly intracellular, stained area rich in
Boyd et al. 1989 (37) OXL-41.1 Mouse mAb Human Cu +÷ oLDL The two antibodies stain certain lesions in
OXL-22.4 Mouse mAb Human Cu +÷ oLDL WHHL aorta; no staining in aorta of normal

found with aortic intimal or medial extracts with an pared from aortas of atherosclerotic cholesterol-fed
ELISA technique. However, a monoclonal antibody rabbits were shown to avidly metabolize modified
prepared against the float-up fraction of atheroscle- forms of LDL (tested was ac-LDL) and thereby accu-
rotic arterial homogenate from WHHL rabbits was mulate cholesteryl esters. 235 In additional s t u d i e s , 43'51
highly reactive with peroxidized LDL and MDA- it was proven that the antisera or antibodies did not
LDL, but not with native or acetylated LDL.~37 bind to the native LDL but only to the LDL which
Very impressive evidence for the oxidation theory had been modified either by copper oxidation or by
comes from several recent immunohistochemical treatment with MDA or 4-hydroxynonenal (HNE).
studies. A series of polyclonal and monoclonal anti- The a u t h o r s 43'51'j36 therefore assumed that the anti-
bodies to various forms of oLDL and aldehyde-modi- bodies recognize epitopes in LDL which are specific
fied LDL were raised (Table 8) and used for immuno- for oxidative modification. In case of the antibodies
staining of lesions and lesion-free areas of arteries against MDA-modified LDL (i.e., MDA-lys, 135MAL-
from WHHL rabbits. 37'43'135A36'231 Briefly stated, the 2, 43 M D A - 2 , 1 3 6 it was assumed that the recognized
findings were as follows: All antibodies stained the structure is a "MDA-lysine adduct," since the antibod-
same areas of fatty streaks rich in macrophages, the ies also bind to albumin, hemoglobin, polylysine, and
stain was predominantly macrophage associated, and e-amino caproic acid previously reacted with MDA.
the staining of extracellular material occurred only in To prepare the immunogenic MDA-LDL (or other
advanced lesions. 136The staining was confined to ath- MDA conjugates), LDL is incubated at 37°C for 3 h,
erosclerotic tissues; in some cases the adventitia of pH 7.4, with 0.5 M MDA prepared by acid hydrolysis
nonlesioned WHHL rabbits also showed staining, but from MDA-bisdimethyl-acetal. Excess MDA is then
no staining was observed in normal arteries of New removed by dialysis. Under these conditions, about
Zealand White rabbits. ~36 Yl/i-Herttuala et al. 264 re- 77% of the ~-amino group oflysine residues were mod-
c e n t l y reported that IgG isolated from rabbit ified as assessed by the trinitrobenzene sulfonic acid
(WHHL) and human atherosclerotic lesions recog- (TNBS) assay. From many other studies it is now well
nizes MDA modified LDL and copper-oxidized LDL established (for review see Ref. 138) that concentrated
but not native LDL. MDA solutions as used in these studies are heavily
Atherosclerotic lesions from control and probucol- contaminated with dimeric, trimeric, and polymeric
treated WHHL rabbits showed equivalent immuno- forms of MDA and that in many cases these forms are
staining with a monoclonal antibody against oLDL considerably more reactive with amino groups than
(OXL 41.1), although the lesions were significantly monomeric MDA. Moreover, the reaction of MDA
smaller in the probucol treated animals. TM In the pro- with amino groups does not only lead to amino-
bucol-treated animals, immunoreactive oLDL was imino-propene structures but also to aminopropenals
predominantly present in smooth muscle cells, and dihydropyridine derivates and possibly to other
whereas in control WHHL rabbits oLDL was found not yet defined products. The authors working with
to be mainly associated with m a c r o p h a g e s . 136,231 Co- antibodies assumed that the lysine adducts recognized
cultures ofmacrophages and smooth muscle cells pre- 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) 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).
J0rgens et al. 1990 (49) 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,
Rosenfeld et al. 1990 (136) MAL-2 Guinea pig as Guinea pig MDA-LDL The epitopes recognized by the two
Palinski et al. 1990 (51) 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.
Yla-Herttuala (264) Human lgG Likely in vivo oxidized LDL Atherosclerotic lesions of humans and
Rabbit IgG rabbits contain IgG recognizing
MDA-LDL and Cu++ oxidized

complex chemistry of MDA, however, it seems clear tory (unpublished) with N-acetyl lysine as model
that additional work will be necessary to verify the compound suggests that the reaction is much more
chemical structure of the epitopes. The situation is complex than normally assumed. Jiirgens et al. 49
similar with antibodies against HNE-modified LDL. reacted human LDL with HNE under nonreducing
To prepare this immunogen, LDL (2 mg/mL) was (i.e., without NaCNBH3) conditions and found it to
reacted with 5 mM HNE at pH 9.0 in the presence of be a strong immunogen in rabbits. This is clearly dif-
NaCNBH3 at 37°C for 24 h and then d i a l y z e d . 43'51'136 ferent to Palinski's observation 43's~ that HNE-conju-
It was assumed that the HNE formed a SchitTs base gated mouse or Guinea pig LDL is immunogenic only
with e-amino groups (R-CH--N-protein), which was if conjugation is carried out under reducing condi-
reduced by the NaCNBH3 to the corresponding stable tions. The antiserum prepared by Jtirgens et al. 49 is
secondary amine R-CH2-NH-protein). This is sup- apparently very specific for HNE epitopes, since no
ported by the fact that the HNE-conjugate formed noteworthy cross-reaction occurred with LDL modi-
under nonreducing conditions was a weak immuno- fied under identical conditions with MDA, 4-hydroxy-
gen, probably because it dissociated again in the re- hexanal, hexanal, or 2.4-heptadienal. Cross-reactions,
versible reaction (R-CH----N-protein ,--, R-CHO + however, occurred with HNE-treated human VLDL
NH:-protein). Another very likely reaction occurring and lipoprotein (a) and Cu ÷÷ oxidized human LDL.
with HNE is the nucleophilic addition of amino Rosenfeld et al. 136 showed by Western blot analysis
groups to the CC double bond (R-CHOH-CH(NH- and radioimmunoassay (RIA) that MDA-lysine and
protein)-CHE-CHO), which yields a saturated alde- HNE-lysine residues derived from apo B are present
hyde with the amino group attached to the carbon in Cu ++ oxidized LDL as well as in the LDL extract-
atom 3. This saturated aldehyde could further react to able from the arterial wall of WHHL rabbits, which
a SchilTs base. 138 Which reactions in fact are favored strongly suggests that such aldehyde-modified epi-
and occur if LDL is treated with HNE was not yet topes of apo B are in fact formed in vitro and in vivo
been investigated. Preliminary work from our labora- by oxidative modification of LDL. Solid-phase corn-
356 H. ESTERBAUERel al.

petition RIA revealed5~that the antisera and monoclo- changes were observed during LDL aging, a condition
nal antibodies against MDA-modified LDL strongly known to be associated with mild oxidation. The im-
bind also to other MDA-treated proteins (human munoreactivity of the Bsol 7 epitope was assumed to
serum albumin, hemoglobin, transferrin) and polyly- be one of the most sensitive parameter of LDL oxida-
sine, tBOC-lysine, and e-amino caproic acid treated tion. 232 Salonen et al. 54 recently reported the occur-
with MDA. The antibodies against MDA-LDL did rence of autoantibodies to MDA-LDL in sera of ath-
not react with HNE-modified LDL or native LDL but erosclerotic Finnish men; the titer of these autoanti-
with copper-oxidized LDL. In similar assays, the spec- bodies was an independent predicator of the
ificity of the antisera and monoclonal antibodies progression of carotid atherosclerosis.
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 Early atherosclerosis lesions are characterized by
binding with these antibodies against HNE-modified the presence of fatty streaks, which are composed of
LDL. The monoclonal antibodies against oLDL so-called foam cells. These cells have accumulated
(OLF4-3C10) reacted with apo B from delipidated large amounts of lipids, predominantly cholesteryl es-
copper-oxidized LDL, and a weak reaction occurred ters. Foam cells are derived from smooth muscle cells
with MDA-modified LDL, but no reaction was ob- and monocyte-macrophages. In the last decade, it be-
tained with HNE-modified LDL. The antibody ap- came evident from animal experiments (for review
pears to be not specific for oxidatively modified apo see Refs. 141,261) that in a first step monocytes in-
B, since strong cross-reactions also occurred with cop- vade from the bloodstream into the subendothelial
per-oxidized HDL. Even if all the work with antibod- space and become resident macrophages. They take
ies directed against oLDL or aldehyde-modified LDL up lipids and lipoproteins, infiltrated and deposited in
is still at its early stage, the conclusions which can be those regions. However, cultured mouse peritoneal
made are, first, that the apo B is modified by MDA macrophages (MPM) neither took up native LDL to a
and HNE when LDL is oxidized by copper ions, and significant degree nor did they accumulate cholesteryl
second, that MDA and HNE-modified proteins (most esters when exposed to even high concentrations for a
likely derived from apo B) are indeed present in ath- long period, as shown by Goldstein and BrownJ ° On
erosclerotic lesions of W H H L rabbits. It remains, how- the one hand this was probably due to the fact that
ever, to be established whether such modifications macrophages only express a very small number of
also occur in lesions of humans. Furthermore, it LDL receptors, and on the other hand the expression
should be emphasized that the chemical structure of of these receptors is finely tuned to prevent an intra-
the conjugate formed by MDA or HNE is not yet cellular accumulation of cholesterol and its esters.
clear. Macrophage-derived foam cells, which were A chemical modification achieved by treatment of
isolated by Rosenfeld et al. 139 from the artery of New LDL with acetic anhydride led to a form of LDL,
Zealand white rabbits, made atherosclerotic (balloon which entered the cells via a receptor-mediated endo-
deendothelialization, cholesterol feeding) as well as cytosis not under feedback control, leading to massive
macrophages within sections of the lesions show im- cholesterol accumulation within the macrophages. 18
munostaining with the polyclonal and monoclonal an- One of the steps of this modification, probably of cru-
tibodies against MDA-LDL and HNE-LDL. 139 Since cial importance for the recognition of this modified
in this animal model predominantly VLDL is ele- form of LDL (acetylated-LDL = ac-LDL), is the
vated (in W H H L rabbits only LDL), these results do blockage of the E-amino group of the lysine residues of
suggest that not only oLDL but also oxidized VLDL apolipoprotein B, resulting in an increase in the nega-
can play a role in lesion development. Zawadzki et tive charge of ac-LDL. Once the lysine residues are
al. 14° showed with three monoclonal antibodies blocked, ac-LDL does not bind to the LDL receptor
against epitopes localized i n different parts of apo B but becomes recognized by another type of receptor,
from native LDL that the immunoreactivity steadily namely the ac-LDL or scavenger receptor(s) on
decreased during oxidation, but another epitope lo- mouse peritoneal macrophages. The ac-LDL recep-
cated at the C-terminus ofapo B chain exhibited with tor(s) is also expressed on monocytes freshly isolated
one of the antibodies (Bsol 7) significantly enhanced from the blood but increases as much as 20-fold upon
immunoreactivity during the first 6 h of copper oxida- cultivation. Furthermore, this receptor was found and
tion, which then gradually decreased again. Similar studied on peritoneal macrophages from rats and
Oxidation of LDL 357

dogs, Kupffer cells from rats and guinea pigs, tumour tor on fibroblasts. 15° This agreed with the observation
cell lines of the mouse (J774 and P388), as well as on that modification of LDL by MDA at lower concen-
endothelial cells. By treatment of smooth muscle cells trations hampered the recognition by the LDL-recep-
and fibroblasts from the rabbit with phorbol esters, an tor. 149 A more extensive modification of LDL with
upregulation of the ac-LDL receptor could also be HNE leads to the formation of aggregates of this lipo-
achieved. Normally, these cells do not express this protein. 39'~18 These aggregates are taken up by culti-
type of receptor. 142 vated macrophages 0774), giving them a foamy ap-
The structure of the type I and type II macrophage pearance. This uptake was not mediated by the ac-
scavenger receptors were just recently deduced by LDL receptor(s), but facilitated via phagocytosis.39
complementary DNA cloning by the group of M. However, another study, modifying LDL concomi-
Krieger. 52'~43 Both receptor types share five identical tantly with MDA and HNE, demonstrated that the
domains. However, the 1 10-amino-acid cyteine-rich presence of MDA prevented formation of aggregates
domain VI of receptor type I is replaced by a six-resi- of LDL. At a constant level of HNE, an increasing
due C-terminus in receptor type II. 52't43 amount of MDA led to an enhanced uptake of LDL
Other treatments of LDL, such as acetoacetyla- by macrophages. Competition studies with labeled ac-
tion,145 malelylation,11 succinylation,~46 carbamyla- LDL receptor(s). TM LDL modified by water-soluble
tion, ~47 and incubation with malondialdehyde products derived from autooxidation of unsaturated
(MDA) or glutaraldehyde,14 also transform LDL to a fatty acids, avoiding oxidation of LDL during the
species which is readily recognized by the ac-LDL re- modification procedure, was rapidly degraded by cul-
ceptor(s). The ac-LDL receptor(s) is also able to recog- tured macrophages by means of the ac-LDL recep-
nize ligands, which are not necessarily lipoproteins tor(s). 46To characterize the compounds eventually re-
but negatively charged polyanions or malelylated al- sponsible for the recognition of LDL modified that
bumin. 18 However, the enhanced negative charge of way by the ac-LDL receptor(s), LDL was incubated
modified LDL itself is probably not the only factor with acrolein, crotonaldehyde, pentenal, heptenal,
being responsible for recognition by the ac-LDL re- and nonenal. 46 Only incubation with nonenal in the
ceptor(s); likewise the density of negative charges in presence of the reducing agent NaCNBH3 modified
certain regions of the macromolecule might be of im- LDL to a form which stimulated its degradation in
portance.148 Another aspect is the recognition of car- mouse peritoneal macrophages at rates comparable to
bamylated LDL by the ac-LDL receptor(s); this was oLDL. 46
proportional to the degree of carbamylation, whereas The investigation on the modification of LDL by
in modification of LDL by MDA, recognition of MDA or other aldehydes,29'56 modifications which
MDA-LDL started only when 16.3% of the lysine resi- could be of physiological relevance in contrast to acet-
dues on apo B had been modified by this aldehyde. ~49 ylation, were paralleled by studies of LDL modified in
Concomitantly with the exploration of the ac-LDL presence of cells. Henriksen et al. reported in 1981 ~5
receptor, the question arose regarding the exact na- that incubation of LDL with endothelial cells led to a
ture of the modification affecting LDL in vivo respon- modified form of LDL which was recognized by the
sible for its recognition and unregulated uptake by ac-LDL receptor(s). Soon afterward it was shown that
macrophages. We want to point out that the group of a free-radical-induced peroxidation of lipids made
Fogelman and Haberland 14'146'148349 studied exten- LDL cytotoxic2° and that lipid peroxidation was a pre-
sively the modification of LDL by MDA. These au- requisite for the uptake of LDL by macrophages. 23
thors assumed that during aggregation of thrombo- Furthermore, it was demonstrated that solubilized
cytes a reasonable amount of MDA could be set free, fractions of apo B, after delipidation of oLDL, were
which in turn would modify LDL particles responsible for the recognition of oLDL by the ac-
nearby. 14,149 Comparing the capacity of modification LDL receptor(s). 152 Apart from an endothelial cell
by MDA with that of 4-hydroxynonenal (HNE), an- line, smooth muscle cells, 21'26'153 monocytes,24'38'154
other aldehydic endproduct of lipid peroxidation of two myelomonocytic cell lines, 155 and macro-
18:2 or 20:4 PUFAs, it was shown that HNE is an phages ~56were shown to be capable of oxidizing LDL.
even stronger modifier of LDL when compared on In all these studies the oxidized LDL was shown to be
equal molar basis. ~18 Apart from lysine, HNE also recognized and taken up by the ac-LDL receptor(s).
modifies other amino acid residues such as tyrosine, Studies on the intracellular processing of oLDL by
serine, and histidine. ~8 Modification of LDL with macrophages showed that, differently to ac-LDL,
low concentrations of HNE also reduced binding and only about 50% of the apo B is degraded by lysosomal
uptake of the modified lipoprotein by the LDL-recep- proteases (cathepsins) to low-molecular-weight, tri-

chloroacetic-acid-soluble p r o d u c t s . 45,233 Thus signifi- in vivo is the phagocytosis of LDL immune com-
cant amounts of nondegraded oLDL accumulate in plexes by macrophages via the Fc r e c e p t o r . 236"237 Such
macrophages. The resistance of oLDL to proteolytic immuncomplexes are likely to be a consequence of
cathepsin degradation is probably a consequence of the autoimmune response to oLDL 54'~2''122 and possi-
the modification of the apo B by lipid peroxidation bly also to native L D L . 236-238
products. TM Another difference in the intracellular Several reports show that patients with severe ath-
macrophagal processing between ac-LDL and oLDL erosclerosis have LDL-immune complexes in the cir-
is that the later yields significantly less cholesteryl es- culation, 239'24° which indicates that autoantibodies
ters, probably because oLDL has a reduced choles- against oLDL and/or LDL were indeed produced) 4
terol content (Table 3) and some oxysterols formed by LDL-immune complexes adsorbed to human red
the oxidation process inhibit ACAT activity.233 blood cells are rapidly phagocytosed by activated hu-
Recently receptors were detected on cultivated man monocyte-macrophages, and the uptake leads to
mouse peritoneal macrophages which did not recog- intracellular cholesteryl ester accumulation. 23s Our
nize ac-LDL but recognized LDL incubated and oxi- group recently showed24~that severe atherosclerosis is
dized by endothelial cells. 45 Another study revealed associated with an up to 10-fold elevated serum neop-
the existence of three classes of receptors on cultivated terin level (neopterin is an index for activated macro-
mouse peritoneal macrophages: a common one for phages).
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- In the experiments23 which led to the discovery
protein fraction occurring in cholesterol-fed animals that cells can oxidize LDL, the LDL was incubated
and humans, was shown to be internalized by cul- with cultured rabbit aortic endothelial cells in Ham's
tured rabbit aortic smooth muscle cells.~57 This inter- F-10 medium (containing 3 #M iron, 0.01 #M copper
nalization was enhanced when /3-VLDL was incu- ions) for 24 h. After incubation, the medium con-
bated with bovine aortic endothelial cells. During this tained TBARS and the cell-conditioned LDL was
incubation, the /3-VLDL was oxidatively modified. more rapidly taken up and degraded by macrophages
The interaction of both /3-VLDL and oxidized /3- than native LDL. Since no modification of LDL took
VLDL with cultured rabbit aortic smooth muscle place in the presence of EDTA or in Dulbecco's modi-
cells was only in part mediated by the apo B/E recep- fied Eagles (DME) medium, which is nominally free
tor. ~57 Wiklund et al. 296 recently reported on the up- of iron or copper, it was concluded that transition
take of native and acetylated LDL in foam cells pres- metal ions are crucial for oxidative modification of
ent in atherosclerotic rabbit aorta as measured by an LDL. It was also shown in this early investigation that
in vitro perfusion system. They found that native LDL incubated 24 h in plain cell-free F-10 medium
LDL was taken up by the same mechanism as acety- supplemented with 5 #M Cu ++ became oxidized and
lated LDL. Addition of vitamin E (0.1 mg/mL) to the exhibited chemical (TBARS, REM, increased lyso-
incubation medium prevented uptake of native LDL phosphatides) and biological properties (macrophage
into the foam cells, suggesting that local oxidative uptake) similar if not identical to cell-modified LDL.
modification of LDL plays a role in the uptake of Since then, many researchers working with oLDL
LDL by foam cells. use Cu ++ oxidation instead of modification by cells
Although there is an increasing evidence that oxi- (for review see Ref. 62). The most frequently used
dation of lipoproteins, especially LDL, will create a procedure for preparing oLDL for biological experi-
form of the lipoprotein which is taken up by certain ments now is an incubation for 8 h or more in one of
cells in an unregulated fashion, it must be stated that the cell culture media (F-10, DME) or plain phos-
other forms of modification of lipoproteins (e.g., in phate-buffered saline (PBS) supplemented with Cu ++
vitro by nonenzymic glycosylation or treatment of li- ions in the range of 5 to 100 #M; in most studies the
poproteins with proteases) can also lead to lipid load- molar ratio of LDL to Cu ++ is about 1:10 to 1:20.
ing of certain cells by the modified lipoproteins. An With few exceptions,21 ferrous or ferric ions were
update of the latest results in this field was given in a found to be weak prooxidants which do not lead to
recent review.~58 modifications recognized by macrophages. Recently
Another mechanism which may lead to foam cells Kuzuya et al.242 reported that concentrations of 10
Oxidation of LDL 359

~M FeSO4 or FeCI3induced oxidation of LDL compa- oxidation of LDL (Fig. 6). But more importantly, it
rable to 10 uM CuSO4, provided that the LDL was was shown in these cell oxidation studies that the rate
dissolved in 0.15 M NaC1 instead of 10 mM phos- of formation of an LDL recognized and taken up by
phate buffer. The authors assumed that phosphate the macrophage scavenger receptor is maximal and
buffer complexes iron (but not copper ions) and thus temporarily linked with the decomposition of the
prevents formation of an iron-LDL complex neces- lipid hydroperoxides. No measurable modification
sary for initiation of oxidation. In the meantime we into high-uptake forms occurred during the period
have repeated these experiments (unpublished) but when the LDL became depleted of vitamin E, and
could not reproduce them, under otherwise identical modification was also minimal during the phase
conditions (LDL: ion ratio 1:34, 37°C, 0.15 M NaCI, when the lipid hydroperoxides increased to the maxi-
0.15 mg LDL protein/mL). Oxidation of LDL by mum value. Comparable results (Fig. 5, fight panel)
Fe ++ or Fe ÷++ after 3 to 5 h incubation was less than were obtained with Cu ++ oxidation; here too, high-
20% of that produced by Cu ÷+. This agrees with our uptake forms of LDL were mainly generated at the
previous findings58 and is supported by reports from late phase when the lipid hydroperoxides decom-
several other groups.72'79'161Balla et al.327 recently sug- posed. 79 Ferrous sulfate in the absence of cells (Fig. 5,
gested hemin as a possible physiological mediator for middle panel) led to some oxidation of LDL; the rate,
LDL oxidation and reported that in vitro hemin oxi- however, was much lower than in the presence of
dizes LDL, and the heine-mediated oxidation is cells. Moreover, the peroxides in the Fe ++ condi-
strongly accelerated by traces of H202 or lipid hydro- tioned LDL were not decomposed and the LDL was
peroxides. The somewhat inconsistent results regard- also not degraded by macrophages. This clearly indi-
ing effÉciency of iron and copper ions as well as the cates that decomposition of lipid hydroperoxides is a
degree of oxidative modification produced by a cer- necessary prerequisite to generation of epitopes on
tain concentration of copper ions alone are likely to apo B recognized by the scavenger receptor. That the
be due to differences in media composition. The F- 10 presence of lipid hydroperoxides in LDL is per se not
and DME medium contain amino acids capable of
sufficient for a rapid uptake by the macrophage scav-
complexing Fe ÷+ or Cu ++ (e.g., histidine), and such
enger receptor is also supported by oxidation of LDL
metal ion complexes probably differ in their prooxi-
with selected oxygen radicals. Hydroxyl and hydro-
dant capacity from free metal ions in plain PBS. To
peroxyl radicals led to extensive oxidation of the
improve the comparability of results from different
PUFAs in LDL, yet the radical oxidized LDL was not
laboratories, it would be important in future work to
taken up by macrophages. 4~''62 LDL oxidized by
standardize the conditions for oxidizing LDL in cell-
gamma-irradiation88was also not a good substrate for
free systems.
the scavenger receptor, but it could readily be con-
From experiments in which both TBARS and mac-
verted into a high-uptake form when it was addition-
rophage uptake were measured, one can conclude
ally treated with Cu ++, a condition known to decom-
that TBARS must reach a threshold value of at least
pose lipid hydroperoxides.
25 mol/mol LDL (45 nmol/mg protein) in order to
make the modified LDL cytotoxic, chemotactic, and Details of the mechanism of metal-induced oxida-
degradable by macrophages. 4°'s8 Depending on the tion of LDL are not clear. Direct oxidation of pure
conditions (absence or presence of cells, cell type, me- lipids by free or complexed iron or copper under phys-
dium, Cu ÷+ concentration), such a degree of oxida- iologically plausible conditions has not been demon-
tion is reached after about 12 to 24 h of incubation. strated. Most authors faced with the need to explain
Up to now, only two systematic investigations were metal-assisted peroxidation assume the occurrence of
made on the time course of cell-mediated oxidation of reactions analogous to processes leading to the reduc-
LDL.79'156In these experiments LDL was conditioned tion of H202. However, these proceed at reasonable
with mouse peritoneal macrophages in Ham's F-10 rates only when the metal is in the reduced form: The
medium for periods up to 24 h. It was found that the rate constant of reaction Fe ++ + H202 is only about 70
LDL first lost its vitamin E within about 3 h; thereaf- M -~ s-l, but already this is almost 2 × 107 times
ter the lipid hydroperoxide content rapidly increased greater than the corresponding rate constant for the
reaching a maximum of about 450 mol/mol LDL at Fe +++ form. 243 The reduction of H202 by Cu + is be-
10-12 h incubation time. Afterward the peroxide lieved to be quite fast,T M but again the reduction of
content decreased again (Fig. 5, left panel). This time H202 by Cu ++ is extremely slow and thermodynami-
course fully agrees with that observed by Esterbauer et cally not favored.
al. 4°'58'75 and EI-Saadani et al. 77 in Cu ++ stimulated It appears therefore that peroxidation of lipids in
360 H. ESTERBAUERel al.

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


t5" 3100 ~-T ///
10" 200 /

z-z II \
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 (LOOH) and a reducing agent, the principal step of
preformed peroxides or agents or conditions capable Cu ÷÷ initiated LDL oxidation could be formulated as
of converting the ion to the active reduced form. The follows:
first requirement is likely to be met by the invariable
presence of traces of peroxides in isolated lipid sam- unknown reducing agent
pies. 245 Possibilities for the formation of the initial
Cu + are more speculative. There is little doubt that
LOOH + Cu ÷ ~ LO. + OH- + Cu ++
the catalytically active ions are bound to the LDL par-
ticle. Both proteins 246 and phospholipids247 readily
initiation of lipid
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 We have previously assumed 4° that the unknown
before reaction with lipid peroxides, but once some reducing agent might be the preformed LOOH itself
Cu + form, more can be generated during subsequent (LOOH + Cu ++ - L O O + Cu + + H+), and this mech-
peroxide decomposition. An interesting possible ini- anism was recently again proposed by Thomas and
tial Cu + formation could be achieved in a process anal- JacksonY 2 However, such a reaction is thermody-
ogous to the reduction of Fe +÷+ by a-tocopherol incor- namically extremely unfavored (W. Koppenol, pri-
porated in phospholipid liposomes. 25° In that process, vate communication) and therefore unlikely. The im-
the antioxidant was lost and the ion reduced. This portance of traces of preformed lipid peroxides was
resulted in rapid oxidation of further lipid molecules. recently clearly demonstrated252 by experiments with
Since part of the surface of the LDL particle is made ebselen, a synthetic Se containing compound with
up of exposed phospholipids which readily attract peroxidase-like activity. In the presence of glutathi-
C u + + , 247 a similar reaction between the metal and a-to- one, ebselen reduces LOOH to LOH. When LDL was
copherol could occur. This possibility has not been first pretreated with ebselen plus glutathione and then
tested experimentally. Taking into account the re- reagents were removed (by dialysis) before oxidation,
quirement of preformed lipid hydroperoxides they totally prevented Cu ++ dependent oxidation as
Oxidation of LDL 361

160 the other hand, 5-1ipoxygenase has been shown to be

140 °
/j! 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
120 is cytotoxic26° and taken up by macrophages) 4 Inter-
~~°~ x /// estingly, Cathcart et al.260 observed in their experi-
100 ments that superoxide anion inhibited soybean lipox-
C3 - - x~ \ Peroxides
_J ygenase catalyzed LDL oxidation, whereas hydrogen
E peroxide appeared to be essential. A feature of the
/ o
kinetics of oLDL formation during incubation with
E cell cultures is the long period required. Under such
60 ! conditions, the chemical processes responsible for gen-
, \ eration of oLDL are difficult to identify. It is clear,
40 however, that oxidation of LDL is significantly accel-
I/ O~o
erated by metal ions and that the process is inhibited
, \ by chelating agents, either in the absence or in pres-
! o
20 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-
ncubation time (h)
oxidation. ESR measurements showed that Cu ÷+
Fig. 6. Kinetics of formation of lipid hydroperoxides during Cu +÷ binds to the LDL protein: ° Fluorescence spectros-
stimulated oxidation of LDL in PBS. O and x were LDL from two
different donors. Reprinted with permission from Esterbauer, H.; copy investigations ~61 suggest that the copper ions
Rotherneder, M.; Striegl, G.; et al. Vitamin E and other lipophilic bind in the vicinity of the lipid phase of LDL and lead
antioxidants protect LDL against oxidation. Fat. Sci. Technol. 91: to a preferential degradation oftryptophan residues in
316-324; 1989. Copyright 1989 FETT Wissenschaft E. V.
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-
measured by TBARS and REM. This appears to indi- tophan residues contained in apo B were destroyed.
cate clearly that Cu +÷ oxidation has an absolute re- The possibility should therefore be considered that
quirement for the presence of traces of LOOH in the the initiating radicals are formed site specifically at or
LDL. Interestingly, oxidation of LDL by macro- near a tryptophan-Cu ÷+ complex. That binding of
phages was also largely prevented if the culture me- Cu ++ to the LDL is essential for the initiation of lipid
dium was supplemented with ebselen + glutathione, peroxidation is clearly proven by the inhibitory effect
which suggests that in this system lipid hydroperox- of EDTA, which, if present in sufficient high concen-
ides are essential for LDL oxidation. tration, prevents binding of Cu +÷ to LDL. Another
Speculation on the mechanism of LDL oxidation free radical process which might have biological rele-
by cells in the presence of copper or iron ions presents vance in diabetes is the autooxidative glycosyla-
few difficulties, because the system is complex enough tion. 257'265 If LDL is incubated with high concentra-
to offer several alternatives. Tables summarizing cell tions of glucose, trace amounts of transition metal
oxidation experiments can be found in Refs. 62 and ions generate free radicals, H202, and ketoaldehydes
40. The cells can produce a range of redox reagents from glucose, and glycosylation and lipid peroxida-
which can react with the LDL directly or, more proba- tion occurs concomitantly in LDL.
bly, reduce any transition metal ions present, facilitat- Whichever mechanism will ultimately prove to be
ing lipid peroxide decomposition and chain peroxida- involved in initiation of oxidative modification of
tion. Recent studies suggest that a 15-1ipoxygenase ac- LDL, it seems clear that the subsequent processes fol-
tivity forms the initiating peroxides when LDL is lowing initiation are always the same--that is, loss of
incubated with mouse peritoneal macrophages. 259 antioxidants, lipid peroxidation, and decomposition
The involvement of a 15-1ipoxygenase in LDL oxida- of lipid hydroperoxides to aldehydes and other prod-
tion is also suggested by the finding that aortic seg- ucts (Fig. 7). An LDL at the late phase of decomposi-
ments from cholesterol-fed rabbits and W H H L rab- tion will have more or less similar biological and
bits convert arachidonic acid to 15-HETE. 323'324 On chemical properties, regardless of how the oxidation
362 H. ESTERBAUERel al.

I ipoxidase or
preexistin9 L D O H

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




/ ~ Lysophosphadites

Rearrangementand =L Aldehydesand otherproducts

consecutiveproducts FragVmentstion JV
(Hydrox~,ks=>,keto-hydroxy-, ofapoB Covalentbindingto apoB
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- terrelationships between the different events. It is
dized by Cu ++ for various times (up to 48 h) can be clear from Figures 7 and 8 that the measurement of
separated by anion-exchange chromatography on a one single parameter at one time point is not suffi-
Mono Q H R 5/5 column into five fractions, differing cient to conclude whether LDL oxidation is in its
in degree of oxidation and macrophage uptake. early or late phase. This is particularly relevant to cell-
The sequence of the major events accompany- induced LDL oxidation, where typically a single mea-
ing oxidation of LDL by Cu ÷+ in plain PBS or surement is taken after a long incubation, making
Ham's F-10 medium was studied in detail by comparisons of results dependent on degree of LDL
us 40'58,83'85'86'96,163-166 and s o m e others, s°'79,80'167'168,256 oxidation quite unreliable.
The time course of oxidation can be followed by mea- Based on many different time-dependent analyses,
suring the increase of TBARS, lipid hydroperoxides, the chronology of LDL oxidation by Cu ÷+ ions can be
conjugated dienes, 430 nm fluorescence256 (Fig. 8), divided into three consecutive time phases: lag phase,
and aldehydes (Fig. 9). Other possibilities are the mea- propagation phase, and decomposition phase. It is
surement of disappearance of antioxidants (Fig. 10) important to note that each subject's LDL exhibits its
or PUFAs, the fragmentation of apo B (Fig. 1 1), and own characteristic kinetics and that sample-to-sample
the increase of the relative electrophoretic mobility variation can therefore be a serious problem if kinetic
(Fig. 12). Each of these procedures alone gives only data from different experiments with different LDL
one aspect of the oxidation stage, and only a combina- preparation need to be compared. A solution to this
tion of two or more time-related analyses allows us to problem is to use in each experiment the diene versus
predict the stage of oxidation and possible causal in- 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
, ,,- . . . . .-...~....

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 amino acids absorbing at 234 nm can impede the di-
LDL through the oxidation of PUFAs with isolated rect diene measurement. Our laboratory measures
double bonds to PUFA-hydroperoxides with conju- routinely the diene versus time profile by continu-
gated double bonds (= dienes), with a UV-absorption ously recording the change of 234 nm absorption of
maximum at 234 nm. Since LDL is fully soluble in the LDL solution (0.25 mg total LDL/mL = 0.1 uM,
aqueous phase and remains in solution during oxida- 1.66 uM Cu ++, PBS) in a 1-cm quartz cuvette. 75 Typi-
tion, the diene measurement does not require extrac- cal examples are shown in Figure 13.
tion of the lipids but can be performed directly with During the lag and propagation phase and the early
the oxidizing LDL sample, provided that the concen- part of the decomposition phase, the time course of
tration is in a suitable range (0.1-0.5 mg total LDL/ the diene formation fully reflects the lipid hydroper-
mL) and that the solvent is sufficiently transparent at oxides time profile (i.e., lag phases are identical and
234 nm. If LDL is dissolved in a cell culture medium peroxide and diene maxima coincide temporally75).
(F-10, DME) instead of PBS, the content of aromatic 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
60 formed peroxides but must be attributed to an in-
1 2 3 . _ . ~
crease of degradation products (e.g., u-¢t unsaturated
carbonyls) absorbing in the 234 nm range. If this stage
£3 40 of oxidation is reached, peroxides of course no longer
._1 correlate with the dienes.
During the lag phase the LDL becomes progres-
0 sively depleted of its antioxidants, with a-tocopherol
c 20 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
O: fatty acids, TBARS, lipid hydroperoxides, or conju-
0 8 16 24 gated dienes. The approximate rate of diene forma-
tion during the lag phase is 0.3 nmol/mg protein/min,
time (hours) which means that one molecule of conjugated lipid
Fig. 9. Kinetics of the formation of aldehydes during Cu ÷+ stimu- hydroperoxide is formed on average in each LDL par-
lated oxidation ofLDL. LDL (1 mg/mL) in PBS was incubated with ticle every 6 rain. A slow formation of lipid hydroper-
6.7 uM CuCI2. MDA = malonaldehyde, HNE = 4-hydroxynon-
enal. The arrows in the upper part represent the length of the lag, oxides during the lag phase is not unusual but indeed
propagation, and the decomposition phases. a consequence of the antioxidant effect of vitamin E
364 H. ESTERBAUERet al.

100: 0.50

80 0.40 E
_J c
d increase in~nes
60 0.30 C~
4-J ¢
c c
40 0.20
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
> 60 0.30 04

40' 0.20
. ~ / ~ Cryptoxanthin~ m In _(3
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 average in L D L (Table 4) could scavenge 14 LOO"
radicals (LOO") formed in L D L during the lag phase radicals yielding 14 lipid hydroperoxides and 7 oxi-
(LOO" + vitamin E --* L O O H + vitamin E radical). dized vitamin E molecules--that is, 14 LOO" + 7
The tocopheroxyl radical was detected in L D L during vitE --* 14 L O O H + 7 vitE quinone. (We present this
Cu +÷ initiated oxidation. 5° Stoichiometric studies in here in a somewhat simplified form; actually, one half
other systems suggest that one vitamin E molecule o f the LOO" gives L O O H , and the other half gives
scavenges two lipid-peroxyl radicals. 169 If this stoichi- LOO-vitamin E adducts, but both would contribute
ometry is applicable for LDL, the seven vitamin E to the diene or peroxide content of LDL. We also do
molecules (a + gamma-tocopherol) contained on not include the antioxidative effects of carotenoids,
Oxidation of LDL 365



\/\o,...s -0.6

50- .t3

m" i

8. -0.2 D

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 phase, the a m o u n t o f lipid hydroperoxides measured
is also a suggestion 317,318that vitamin E quinone can experimentally from the diene absorption is about 20
act as an antioxidant through a quinone/semiquinone mol/mol LDL, which is in rather good agreement
redox cycle possibly driven by superoxide anion, with the value predicted by the scavenging effect of
which complicates predictions of the overall stoichi- vitamin E. Analysis si of the lipid hydroperoxides con-
ometry of the vitamin E reaction. At the end of the lag 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-
100 nescence detection revealed the presence ofhydroper-
oxides of cholesterylester (64%), phospholipids (12%),
and triglycerides (24%).
n 75 When the L D L is depleted from its antioxidant it

is, as expressed by Brown and Goldstein, i7° left to the
33 mercy o f oxygen. Or in other words, it is subjected to
~ 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

1.2 tion phase commence shortly after the onset of the

propagation phase. Such changes with known kinetics
E include increase of aldehydic lipid peroxidation prod-
E 0.8 ucts (Fig. 9), generation of fluorescent chromophores
93 (EX/EM 360/430 nm) in the apo B and the LDL lip-
ids (Fig. 8), increase of the negative surface charge as
< measured by the REM (Fig. 12), fragmentation (Fig.
11), and modification ofapo B (Fig. 5). Other changes
which probably have similar kinetics are formation of
0 60 120 180 240 300 lysophosphatides and oxydienes 72 and loss of free
>[ min. 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
-0 findings suggest that these aldehydes (e.g., MDA,
< 0.01 HNE) do act as "second toxic messengers." The
-0 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
0 60 120 180 240 300 LDL oxidized 3 h in the presence of 1.6 uM Cu +÷
rain. contained 100 mol aldehyde/mol LDL, with 42%
MDA, 12.5% HNE, 25% hexanal, and 20.5% others. 96
Fig. 13. Continuous measurement of LDL oxidation by the diene
absorption. LDL samples (0.25 mg total LDL/mL in PBS) from If LDL samples were oxidized in open vials, the con-
two donors were supplemented with CuCI2 (1.67 uM) and the centration of aldehydes (except TBARS) decreased
change of the 234 nm absorption was recorded in a spectrophotome- during the decomposition phase, most likely because
ter (LKB Ultrospec II) in l-cm cuvettes with automatic cuvette
changing in 30-s intervals. The upper plot shows the diene vs. time of their volatility. However, if LDL was oxidized in
profile; the lower figure is the first derivative (AA/At) giving the rate closed vials with sufficient oxygen, the concentration
vs. time plot. The end of the lag phase is defined as the intercept at of the aldehydes strongly increased during the decom-
the abcissa in the diene vs. time plot (see arrows). After the end of
the lag phase the rate increases exponentially (lower plot). 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
in Cu ++ or lipoxygenase oxidation, s°'253 At this time
phase was about 300 mol/mol LDL (= 120 nmol/mg
the structure of the radicals (L', LO', LOO') is not
total LDL) (Table 9). Most of these aldehydes (except
clear. EDTA added at any time point during the lag or
propagation phase complexes Cu ++ and thereby pre- MDA) are lipophilic and remain associated with the
vents chain branching and further oxidation of LDL LDL particlefl 9 Taking the lipid phase (= 80% of the
(unpublished from the authors' laboratory). The lipid LDL mass) as solvent, the aldehydes in the LDL parti-
hydroperoxides generated in the LDL particle during cle reach a remarkably high concentration of about
the propagation phase are labile intermediary prod- 120 raM. It has been shown 29 that at least 90% of the
ucts; their concentration rises within about 60-80 TBARS measured in oLDL by the conventional TBA
min to a maximum value. In case of Cu ÷+ stimulated assay is in fact free MDA. Because MDA is hydro-
oxidation, about 70-80% of the PUFAs are oxidized philic, it is, in contrast to most other aldehydes, re-
at the peroxide maximum (Table 9). Thereafter de- leased from the LDL into the aqueous phase. 29
composition reactions become predominant, and Various lines of research suggest that a number of
consequently the lipid hydroperoxides or conjugated important changes occurring in oLDL during the de-
dienes start to decrease again. We define the time composition phase result from aldehydes and their
point of the diene maximum as end of the propaga- reactions with amino acid residues in apo B (for re-
tion and beginning of the decomposition phase. One view see Refs. 56, 58, 138, 172). The strong increase of
should keep in mind, however, that both phases tem- the 430 nm fluorescence ofapo B and the loss of free
porarily overlap and cannot be fully dissociated from amino groups is, for example, likely to be caused by
each other. This is clearly evident from the fact that reactions of aldehydes with ~-amino groups of lysine
changes becoming prominent during the decomposi- 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
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 LOO') mediated degradation of amino acids in
loss of positively charged amino groups through a p o B . 174
Schiff's base formation ( R - C H O + protein-NH3 + --~ Lipid-alkoxyl radicals are probably the cause of
R - C H i N - p r o t e i n + H20 + H ÷) or formation of Mi- fragmentation of apo B. That apo B (MW 550 kD) is
chael adducts with a,/3-aldehydes.138 However, it was fragmented during LDL oxidation was first observed
shown recently by means of electron paramagnetic by Schuh et al.17 and later confirmed in several stud-
resonance (EPR) that additionally new negatively ies. 69'175'176 The time course of the loss of intact apo B
charged groups are formed on the surface of LDL oxi- is an exact mirror image of the increase of the dienes
dized by Cu++. ~73 The covalent binding of aldehydes (Fig. 11). Apo B first breaks down into two distinct
to apo B is probably also, at least in part, involved in large peptides with molecular sizes of 260 and 232
the formation of the characteristic epitopes which are kDa. With increasing lipid peroxidation, the remain-
recognized in oLDL by the macrophage scavenger re- ing apo B and the 260 and 232 kDa fragments are
ceptor. In LDL containing phosphatidylcholine with further degraded to such an extent that SDS-PAGE
14-C arachidonic acid, a fraction of the radioactivity shows only a smear of bands in a molecular range
was associated with apo B after Cu ÷÷ oxidation. H2 below 100 kDa (Ref. 69).
The occurrence of aldehyde or carbonyl functions in Many of the chemical (loss of NH2 groups), phy-
apo B was demonstrated by reacting oLDL with 2,4- sico-chemical (increased fluorescence and REM), and
dinitrophenylhydrazine followed by separation of the biological (uptake by macrophages, cytotoxicity) prop-
apo B from the lipids. ~8The isolated apo B was yellow erties exhibited by cell- or copper-oxidized LDL can
colored and contained 68 nmol aldehyde functions/ be reproduced in full or in part by treatment ofunoxi-
mg protein (= 37 mol/mol LDL). Such aldehyde dized LDL with aldehydes (MDA, HNE, hexanals,
functions could result from 2-alkenals, which have alkadienals) or aldehyde m i x t u r e s . 39,56'1s0'172 This fur-
reacted by Michael addition with nucleophiles (XH) ther supports the hypothesis that aldehydes act as ulti-
according to R C H - - C H - C H O + X H -~ R C H X - mate damaging agents. As discussed previously, anti-
CH2-CHO. Alternatively, some of the carbonyl func- bodies prepared against MDA- or HNE-treated native
tions could also be produced by free radical (LO', LDL react also with copper- or cell-oxidized LDL.

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

Chemical and Physicochemical Properties

* Complete loss of antioxidants
* More or less complete loss of PUFAs
* Loss of phosphatidyl choline and cholesteryl ester
* Increased content of lysophosphatidyl choline and oxysterols
* Increased content of hydroxy- and hydroperoxy-PUFAs
* Increased content of conjugated dienes
* Increased content ofMDA, hexanal, HNE, and other aldehydes
* Strong fluorescence at 430 nm with excitation at 360 nm
* Partial loss of free amino groups in apo B
* Fragmentation of apo B to smaller peptides
* MDA and HNE epitopes on apo B recognized by specific antibodies
* Increased electrophoretic mobility and increased density (1.06-1.08)
* Increased tendency to aggregate, heterogeneity in size.
* Conformational rearrangement of apo B structure and phospholipid monolayer (266)
Biological Properties
* Increased uptake and degradation by macrophages
* Cytotoxic to most cells (19, 20, 34, 38, 132, 202, 278, 285)
* Chemotactic for monocytes (267) and smooth muscle cells (268)
* Inhibits NO activation ofguanylate cyclase (269)
* Inhibits in isolated smooth muscle strips relaxation induced by acetyl choline, nitric oxide, and nitroglycerin (279-282)
* Increases in cultured endothelial cells tissue factor activity (TF) and suppresses protein C (which increases thromboresistance) activity (275)
* Suppresses the production of PDGF-mRNA and PDGF secretion by monocyte-macrophages (276)
* Increases in macrophages glutathione content about twofold; HNE has a similar effect (283)
* Systemic administration into hamsters causes immediate leukocyte adhesion to capillary endothelium (277)
* Treatment of cultured endothelial cells with MM-LDL stimulates production of a number of biological active factors, such as monocyte
chemotactic factor, MCP- 1 (270); monocyte binding molecules (so-called endothelial-leukocyte-adhesion-molecules, ELAMs) (261, 271 );
growth factors for monocytes, M-CSF, and granulocytes, G-CSF (272), and tissue factors (TF) essential for coagulation (273).
* MM-LDL injected into mice increases in serum and tissue levels of MCP-1 and CSF (274).
* MM-LDL inhibits mitogenesis in SMC and stimulates (low concentration) or inhibits PGI2 synthesis in SMC (307-309)

With the exception,of minimally 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- PUFAs that the recognition o f o L D L by the scavenger
idation products are indeed covalently bound to the receptor is mediated by derivatisation of the apo B by
apo B when L D L is oxidized by cells or Cu ÷÷ ions. lipid peroxidation products which are more complex
MDA- and HNE-modified proteins (most probably than simple short- or medium-chain aldehydes (e.g.,
apo B) have also been detected by immunohistochem- 2-alkenal). The complex chemistry o f the numerous
ical methods in atherosclerotic lesions of Watanabe compounds formed by lipid peroxidation in biologi-
heritable hyperlipidemic rabbits (see Table 8). More- cal systems was reviewed in Refs. 138, 171, and 177.
over, autoantibodies directed against MDA- or HNE- Recently it was shown by gas-chromatogra-
modified proteins are present in the serum o f rabbits phy 215'233'262 that L D L oxidized by Cu ÷+ or macro-
and humans, 43'54 All this underlines the theory that phages contains significant amounts o f cholesterol
aldehydic lipid peroxidation products indeed play a oxidation products, with 7-ketocholesterol compris-
significant role in oxidative modification o f LDL, ing about one half to two thirds of the total choles-
both in vitro as well as in vivo. terol. 7-Ketocholesterol was also identified in serum
The aldehydes detected so far in L D L and listed in of cholesterol-fed rabbits. 3°4 According to these stud-
Table 9 are all (except MDA) derived from the methyl ies, 215 oxysterols are present in o L D L both in free and
terminus o f the PUFAs. The counterparts, where the esterified form, indicating that both forms of choles-
aldehyde function is at the acyl chain, linked to the terol are susceptible to oxidation. From the graphs
parental lipid molecule (i.e., phospholipids, choles- shown in this work, z15 it appears that after 24-h Cu ++
teryl esters) should also be present in comparable oxidation a high proportion of about 70% of choles-
amounts in oLDL. Since these aldehydes also cer- terol was converted to oxysterol. The increase o f o x y -
tainly contribute to the altered properties o f oLDL, it sterols closely paralleled the increase in electropho-
would be important to give more attention to their retic mobility. Other oxidized sterols likely present in
analysis. Steinbrecher et al., 46 for example, concluded Cu ÷+ oxidized L D L are 7-hydroxy cholesterol and 25-
from studies on modification o f L D L by autooxidized hydroxy cholesterol. 233 T h o m a s and Jackson 252 found
Oxidation of LDL 369

in Cu ++ oxidized LDL (5 #M Cu ++, 4 h) 13-HODE, should be regarded with reservations. Similarly, the
9-HODE, and 13-HPODE; macrophage-oxidized unchanged phosphorous content (by which the
LDL contained per milligram LDL protein about 16 amount of phospholipids is calculated) in oLDL
nmol 13-HODE and 10 nmol 9-HODE, or about 1% should not be misinterpreted as unchanged phospho-
of the linoleic acid content of LDL (Table 4). Car- lipids. All our attempts (unpublished) to determine
penter et al. 2~6reported that cultures of human mono- the amount of unchanged residual cholesterol, choles-
cytes incubated with a complex of cholesteryl-lino- terylester, triglyceride, or phospholipids in oLDL
leate and serum albumin (serves as artificial lipopro- with classical methods failed, through disturbances in-
tein) produce cholest-5-en-3fl,7/3-diol. troduced by the oxidized products. An additional in-
Plasma treated with Cu ++ and H20 2 produces large herent problem is that reference compounds neces-
amounts of oxysterols, the major product being cho- sary to establish structures of unknown oxidized lip-
lesta-3.5-diene-7-one; other products identified by ids are rare. Taking all this together, it is
gas-chromatography are cholesterol a- and/3-epoxide, understandable that the knowledge on the chemical
7-ketocholesterol, and 25-hydroxy cholesterol. TM composition of oLDL is rather limited; indeed, much
Under otherwise identical conditions, plasma from more is known now about its biological properties.
diabetic patients produced about 30 times more oxy- Much additional work will be required in the future to
sterols than control plasma. It is interesting to note bridge this gap. In Tables 3, 4, 7, and 9 we have made
that several sterol oxidation products, including cho- an attempt to compare the presently available pub-
lesta-3.5-diene-7-one, were found in fatty materials lished data on the chemical composition of native and
isolated from the human aorta (reviewed in Ref. 319). 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.
From the sequential changes occurring in LDL Table 10 summarizes the major chemical, physico-
during oxidation by cells or copper ions, it is clear that chemical, and biological properties, by which oLDL
the chemical, physico-chemical, functional, and bio- differs from native LDL. Recently it was discov-
logical properties of LDL change continuously during ered 27°-274 that a minimally modified LDL (MM-
the lag, propagation, and decomposition phases. Due LDL) also exhibits a number of biological activities,
to this dynamic process, an oLDL with a defined con- which may play an important role for the pathogene-
stant composition does in fact not (at least in the clas- sis of atherosclerosis. The biological effects of mini-
sical chemical sense) exist. It is therefore very surpris- mally oxidized LDL were recently reviewed by
ing that numerous laboratories appear to have sam- Leake. 328 Minimally modified LDL is prepared 273 by
ples of oLDL with reproducible biological properties. prolonged (3- to 6-month) storage of LDL (solutions
We conclude from that most of the biological studies in PBS containing 0.01% EDTA) at 4°C or by short
were performed with a fully oxidized LDL which has incubation with 1 uM Fe ++. Such a mild oxidation
reached the end of the decomposition phase, where leads to about 3 to 5 nmol TBARS/mg choles-
most of the chemical reactions must come to a stop terol. 272'273 This is equivalent to 4.7-7.8 nmol
because no reactive structures are left in the LDL lip- TBARS/mg protein or 2.6 to 4.3 mol TBARS/mol
ids or apo B. An analyst attempting to study the nu- LDL. For comparison, fully oxidized LDL (oLDL)
merous oxidized lipids and fragments from apo B has contains about l0 times more TBARS than MM-
an enormously difficult job. The classical methods de- L D L Based on our kinetic experiments with Cu ++
veloped for the analysis of lipids are not at all or only oxidation, we would assume that MM-LDL has lost
in part applicable to oxidized lipids. 2~5 For example, all antioxidants and represents a form which is in
the enzymatic lipid assays were developed for determi- transit from the lag to the propagation phase. Such an
nation of free and esterified cholesterol or triglycer- LDL would be primed for rapid subsequent oxida-
ides in serum or native lipoproteins, but not for oxi- tion. The uptake of MM-LDL by cells occurs via the
dized lipoproteins. Completely wrong results for LDL receptor. At present it is, in our opinion, not
oLDL are obtained, for example, by conventional cho- evident whether the biologically active substances
lesterol assays based on cholesterol oxidase (makes (oxidation products?) are already present in MM-
H202) through interfering lipid hydroperoxides, so LDL itself, or whether they are formed during the
that some of the values for free or esterified choles- subsequent cell incubation, which usually lasts for at
terol in oLDL reported in the literature (see Table 3) least 4 h.
370 H. ESTERBAUER¢l a/.

The topic of the biological effects of the minimally shown to be potent inhibitors for cell-mediated LDL
modified LDL is largely outside the scope of this re- oxidation are butylated hydroxytoluene21,22'24,27,2s,178
view but needs to be mentioned because of increasing and probucol. 179,287Water-soluble compounds, which
attention it is receiving. CornweU and his collabora- prevented LDL oxidation by cultured cells, are gluta-
tors 3°7'3°8 have shown that LDL with low levels of thione and ascorbate. 22,~4'2~5 Breugnot et alfls7 re-
TBARS (9 to 11 nmol TBAR/mg cholesterol, pre- ported that inclusion of about 10 to 50 t~M phenothi-
pared by incubation of LDL at 37°C in 96% air, 4% azines (chlorpromazine, trifluoperazine) into the
CO2) has profound effects on smooth muscle cells in F-10 medium inhibits oxidation of LDL induced by
culture: lowering of the mitotic index, changes in endothelial cells or Cu ++ as assessed by TBARS,
prostanoid synthesis, viability and thymidine incorpo- REM, and degradation by J744 macrophages. The
ration. Some of these were prevented or reversed by protective effect of phenothiazines was similar to the
antioxidants. It may well be that such subtle changes effects obtained with comparable concentrations of
can precede gross cytotoxic effects of heavily oxidized probucol, BHT, or vitamin E. Later the same group 2s8
LDL and contribute to the formation of foam cells. reported that inclusion of calcium antagonists (the
Another role for LDL with low TBARS in atherogen- most effective drug was flunarizine) into the medium
esis may lie in its ability to affect the synthesis ofpros- also prevent oxidative modification of LDL by hu-
tacyclin PGI 2 and thus influence the progression of man monocytes or endothelial cells and by Cu ++.
atherosclerosis. Recent work has resolved a contro- The most potent inhibitors of LDL oxidation are
versy on the role of LDL in this process by showing agents complexing copper and/or iron ions, provided
that low-TBAR LDL stimulate and high-TBAR LDL that they are present in sufficiently high concentra-
suppress the synthesis of PGI 2 (Ref. 309). 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
Since oxidative modification of LDL mediated by EDTA is also used to protect LDL against oxidation
cells or occurring in cell-free medium results from during its isolation. For that, blood is drawn into
lipid peroxidation, water- and lipid-soluble antioxi- EDTA-containing tubes, and the final EDTA concen-
dants should have a prominent effect in retarding or tration in plasma and in the concentrated LDL stock
preventing the modification. This has in fact been sample obtained by ultracentrifugation is 1 mg/mL (3
confirmed in numerous studies with a variety of an- raM). Such high EDTA concentration would, of
tioxidants. Steinbrecher et al. 23 showed in their classi- course, completely block LDL oxidation in subse-
cal study that inclusion of a high content of vitamin E quent experiments. To get rid of EDTA, most labora-
( 100 #M equal to 900 nmol/mg LDL protein) into the tories dialyze the LDL stock solution overnight at 4 °C
culture medium prevented oxidative modification of against EDTA free buffer, made oxygen free by nitro-
LDL by endothelial cells, as measured by TBARS and gen gassing. We have substituted this lenghtly dialysis
macrophage uptake. The dose of vitamin E used in by separating LDL from EDTA on small Sephadex
this investigation was extremely high and about 80 columns. 84 The group of D. Steinberf 3'25'27'57 pro-
times the amount of the endogenous vitamin E con- ceeds somewhat differently and dialyzes LDL against
tained in native LDL, so that it is not surprising that PBS containing 0.01% EDTA (to prevent any oxida-
LDL oxidation was retarded for up to 24 h. A lower tion); in this case the dialyzed LDL stock solution still
dose of 20 gM (equal to 60 nmol vitamin E/mg LDL contains 300 uM EDTA. For oxidation experiments,
protein), as used by Morel et al., 22 reduced but did not this stock solution is then further diluted about 100-
completely prevent oxidation of LDL (increase of fold, which means that the final incubation mixture
TBARS, REM, and cytotoxicity) in a 48-h smooth still contains about 3 uM EDTA that is equal to the
muscle cell or endothelial cell culture. iron ion concentration in F-10 medium (the exact
The protective effect of vitamin E against cell-me- EDTA concentration depends on the dilution factor).
diated oxidation of LDL was repeatedly confirmed From that, it appears that low EDTA concentrations
and can be considered as unequivocally proven. Pro- do not inhibit LDL oxidation by endothelial cells in
tection by vitamin E was found not only in cultures of F-10 medium. This is supported by a report from
e n d o t h e l i a l cells 22'23'28 and smooth muscle cells 22 but Heinecke et al. 26 that EDTA in equimolar concentra-
also in monocyte-macrophage cultures. 24,ss,79 Other tion to Fe ++ (10 #M) stimulated oxidation of LDL in
chain-breaking lipid-soluble antioxidants which were cultures of smooth muscle cells. Similar prooxidative
Oxidation of LDL 371

effects of EDTA-Fe ÷+ complexes have been reported U)
for microsomal lipid peroxidation t8° and attributed to o
the capability of the iron complex to enter into the C
100 o o° o o
lipid phase. However, if oxidation is carried out in o~%o o~
cell-free medium supplemented with Cu ÷+, residual
EDTA severely interferes when its concentration is ~
~ o O 00°8


comparable to the Cu ÷+ concentration. This can be r 2 = 0,043
( N.S. : n = 78)
shown in kinetic experiments where LDL is incu-
Q 4 8 12 16 20
bated with a fixed amount of Cu +÷ (5 uM) and in-
creasing concentrations of EDTA (0-1 0 uM). As the tool d x - - t o c o p h e r o l / m o l LDL
molar ratio of EDTA to copper approaches unity, the Fig. 14. Scatterplot showing the correlation between lag phase and
lag phase strongly increases; and if the EDTA is in 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
excess, no oxidation occurs at all. 2~8The reproducibil- statistical mean of x is 6.37 mol a-tocopherol/mol LDL (Table 4).
ity of oxidation experiments with EDTA containing
LDL samples might therefore be low unless the
EDTA concentration is clearly defined and Cu ÷÷ is LDL (increase of lipid hydroperoxides, uptake by
added in sufficient excess to overcome EDTA protec- macrophages) by cultured macrophages or Cu ÷÷ ions
tion. In cases where the LDL stock solution contains does not occur unless it is depleted from its a-tocoph-
0.0 1% EDTA, a copper concentration of 5 #M, as erol.
used frequently for oxidation of the diluted LDL sam- This sequence of events suggests that the lag phase
ple, is likely to be close to the borderline. and hence the oxidation resistance of LDL is mainly
It has been pointed o u t 3°9 that BHT may be a more determined by the antioxidant content; or, in other
appropriate agent preventing LDL oxidation in tissue words, the lag phase should be predictable from the
cultures than EDTA, which may have other actions, antioxidant status of the LDL sample. With our
such as inhibition of PGi2 synthesis. BHT can block highly reproducible Cu ÷÷ oxidation assay (i.e., diene
LDL oxidation at concentrations which are appar- vs. time profile), the lag phase of a large number of
ently harmless to smooth muscle cells. LDL samples from different non-vitamin-E-supple-
Much work has been devoted to clarify the protec- mented donors was measured and correlated to the
tive role of the endogenous vitamin E and other an- antioxidant content. 8sJ64'165 Much to our surprise, no
tioxidants contained in LDL itself. We s h o w e d 29 that clearly significant correlations were obtained, neither
autooxidation (in a dialysis bag, without addition of for the lag phase vs. a-tocopherol nor for lag phase vs.
Cu +÷) of LDL as measured by the decrease of PUFAs total antioxidants (i.e., vitamin E + all carotenoids)
or increase of aldehydes only occurs when LDL is (Fig. 14). This indicates that at least for our study
largely depleted of its endogenous vitamin E and 13- group, the antioxidant status is by itself not predictive
carotene. When the loss of the endogenous antioxi- for the oxidation resistance of any given individual
dants was followed during experiments with Cu ÷+ LDL. This is in agreement with observations made by
stimulated o x i d a t i o n , 4°'75J63 the same sequence was others, who found essentially no correlation between
always found, as shown in Figure 10. The antioxi- the vitamin E content of LDL and its oxidizability by
dants disappearing first were ~- and gamma-tocoph- gamma-irradiation88 or by macrophages. 79'8°
erol; thereafter the carotenoids decreased to zero, with This weak statistical correlation cannot, however,
cryptoxanthine first and H-carotene last. A fluorescent be interpreted as argument against the protective role
compound disappearing with the carotenoids was pre- of endogenous antioxidants, but rather as evidence
viously assumed to be retinyl stearate; ~64 later it was that the oxidation resistance depends on more than
found that the compound is mainly phytofluene. ~63 one (i.e., antioxidant content) variable. Further inves-
Shortly after the time point when the LDL was de- tigations from our laboratory85'~6s revealed that the
pleted of antioxidants, a propagating lipid peroxida- oxidation resistance (y = lag phase in Cu ÷+ stimulated
tion chain reaction commenced, as indicated by rapid oxidation) of each subject's LDL depends on the vita-
increase of the diene absorption. This finding was min E content (x), a subject-specific efficiency con-
confirmed in several other studies. Steinbrecher et stant (k) of vitamin E, and a vitamin E independent
al., 59 for example, showed that the increase of the variable (a) (Fig. 15). To assess the validity of this
REM of LDL is preceded by a lag phase during which relationship and to estimate the respective subject-
the LDL-vitamin E and/3-carotene decreased to zero. specific values for k and a, LDL samples from individ-
J e s s u p et al. 79 showed that oxidative modification of 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

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 tion. 83,84Although this takes a lot more trouble than
LDL with vitamin E proved to be a 3-h preincubation the in vitro loading, we felt that such an ex vivo study
of the plasma with increasing concentrations (125 to is an essential supplement to the in vitro study. Clini-
1000 #M) of a-tocopherol prior to the conventional cally healthy volunteers took daily placebos over
isolation of LDL by ultracentrifugation. '63 By that three weeks or 150, 225, 800, and 1200 iu RRR-a-
procedure, about 1 to maximally 10% of the added tocopherol. The plasma and LDL antioxidant status
vitamin E became incorporated into the LDL, and (a- and gamma-tocopherol, all carotenoids) and the
consequently the isolated LDL samples had signifi- oxidation resistance of LDL (diene vs. time profile)
cantly increased contents of a-tocopherol. A dose of were measured prior, during, and after supplementa-
1000 #M a-tocopherol added to plasma (this is about tion as shown in a typical protocol in Table 11. The
40 times the normal plasma vitamin E level) in- complete lipoprotein status (cholesterol, triglycerides,
creased the LDL a-tocopherol about two- to fourfold H D L cholesterol, LDL cholesterol) of all subjects par-
above the basal value, depending on the plasmaJ 63 ticipating in the study was determined prior to and
When LDL samples from one subject loaded in after supplementation. The main findings of this
such a way with different amounts of a-tocopherol study were briefly as follows:83-85'165
were subjected to Cu ÷÷ oxidation, the lag phases al-
ways increased strictly linearly with the a-tocopherol 1. All participants taking vitamin E capsules had
content according to the equation in Figure 15, with higher plasma and LDL a-tocopherol levels com-
correlation coefficients r 2 = 0.95 to 0.99 and signifi- pared to the initial levels measured prior supple-
cant levels o f p < 0.001 (for details see Refs. 85, 165). mentation (Table 12). Seven days after termina-
Representative examples for two subjects are tion of vitamin E intake, the a-tocopherol was de-
shown in Figure 16. The insert shows the linear de- creased again and very close to the initial value.
pendency of the lag phase on the t~-tocopherol con- The plasma and LDL carotenoids did not change
tent. The different slopes and intercepts indicate that significantly during the supplementation, but the
the efficiency of a-tocopherol (k) and the vitamin E gamma-tocopherol strongly decreased both in
independent variable (a) were different in the two plasma and LDL. The increase of plasma a-tocoph-
subjects. In order to determine the interindividual erol seen in our study group is in accordance with
variability of k and a, the investigations were ex- an early report by McCormick et al. 312 It also con-
tended to a larger number of subjects. Both parame- firms a study by Kitagawa and Mino, 'sl who found
ters varied unexpectedly over an extremely wide that oral intake of 900 IU RRR-a-tocopherol in-
range, the mean + SD for k being 5.19 _+ 5.61 min creases plasma a-tocopherol about 2.5- to 3-fold
(range 0.7 to 34.2, n = 40), and the mean ___SD for a above the baseline value. Dimitrov et al.zs9 re-
40.9 _+ 29.9 min (range -49.3 to +97.3, n = 40). This cently reported the results from a phase I study
explains why the vitamin E content is by itself not that used single and multiple doses of d, 1-a-
predictive of the oxidation resistance of a given LDL tocopherol. Administration of a single of dose of
sample, since additionally the particular values for k 400, 800, or 1200 IU resulted in an elevation of
and a must be known. plasma a-tocopherol with a peak between 12 and
Still another possibility of changing the LDL vita- 24 h. The chronic administration of the same
min E content of individuals is oral supplementa- 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-

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,
' I 225,800, and 1200 IU (Table 12).
. For each person taking vitamin E, the temporal

< °'°t J J/J/

0,00 /
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
tool ~--T/rt~lLI~_ of the L D L with vitamin E is performed by adding
Cq it to plasma or by oral intake. The individual val-
< 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
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

the average increase was about 80% and similar for If the many data (n --- 206) for lag phases and L D L
all groups. A high-fat intake significantly increased a-tocopherol levels determined by us so far (i.e., basal
the resorption of vitamin E and vitamin E levels in values, L D L loaded in vitro with vitamin E, oral in-
plasma. take of vitamin E) are treated statistically, a highly
2. The total plasma cholesterol levels showed no sig- significant positive correlation is obtained with y =
nificant change, neither in the placebo nor in the 2.94x + 52.4, r 2 = 0.46; p < 0.001; Fig. 18). Statisti-
experimental group. The plasma triglycerides in cally only about one third of the lag phase can be
the vitamin E group were, except in one case, attributed to vitamin E, whereas two thirds are due to
slightly (_<20%) elevated after termination of the the vitamin E independent variable a. Our first suspi-
supplementation. FarreU and Bieri t82 also found a cion that this variable mainly reflects the variable ca-
slight increase in serum lipids (triglycerides and rotenoid content of L D L could not be verified with
cholesterol) in healthy adults who received daily certainty. Linear regression analysis for the correla-
800 IU of vitamin E. tion o f the lag phase and the total L D L antioxidants
3. The L D L isolated from plasma during the vitamin (vitamin E + carotenoids) gave more or less the same
E supplementation period exhibited a higher oxi- 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
se _
.c_ E
E 120 20 ~_

_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

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 =

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-
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
E to zero; thereafter the endogenous antioxidants de-
0%0 ° o o ~ creased, with a-tocopherol first and r-carotene last.
go #o Thereafter the lipid peroxidation entered into the
_C 100
o2OO,Ooo o o . . . .
propagation phase with the same rate and kinetics as
o ~5 ~ "~ - r2 = 0.46
observed in the absence of ascorbate. Jialal et a1.144'291
°~- ~ (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 A very powerful antioxidant for Cu ÷+ stimulated
even in high concentrations (10-80 uM) did not pro- LDL oxidation is urate, which also led to a linear and
tect the endogenous antioxidants. Frei et al. 297 re- concentration-dependent prolongation of the lag
cently reported that exposure of human plasma to gas phase.40,163,164 A protective effect of urate was also ob-
phase oxidants of cigarette smoke induces lipid perox- served by Sato et al.) 83but the mechanism is not clear
idation and a slight increase of the REM of LDL. at all. Since urate produces a prolongation of the lag
These effects were temporarily correlated with the phase but has no effect on the rate of the subsequent
complete consumption of endogenous ascorbate. In a propagation phase, it can be assumed that it does not
similar study, Yokode et al. 298previously found that a act as a preventative (e.g., complexing of Cu ÷÷) but as
cigarette smoke extract (cigarette smoke passed chain-breaking antioxidant. Other water-soluble an-
through PBS at 4°C) modifies LDL into a form which tioxidants which were tested by us are glutathione and
has an increased REM and is about 12-fold more rap- trolox. Glutathione exhibited a sigmoidal dose effect
idly taken up by macrophages than normal LDL. This curve with a plateau of maximum inhibition at about
modification was, however, not associated with lipid 7.5 #M. The dose effect curve of trolox, on the other
peroxidation. hand, showed a hyperbolic shape with a decrease of
By analogy to other lipid peroxidation systems, it is inhibition above l0 uM.
reasonable to assume that the protective effect of The capacity of various agents to delay the propaga-
ascorbate in Cu ÷÷ oxidation of LDL relies on its capa- tion phase in a dose-dependent manner in Cu ÷÷ stimu-
bility of reactivating vitamin E radicals in LDL ac- lated LDL oxidation was also studied by Jessup et
cording to vitamin E" + ascorbate --~ vitamin E + al., 167 Huber et al.) 84 and Barnhart et a i r 17 These au-
ascorbyl radical. This reaction most likely occurs at thors added the agents directly to the assay. Of the
the surface of the LDL particle, where the chromanol- more lipophilic compounds, probably only a fraction
ring of vitamin E faces the aqueous, ascorbate-con- distributed into the LDL, which may explain the
taining medium. somewhat lower activity of probucol compared to our

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


"" 9 ,')i oe- 6

- 6 4

-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



c- 0.40
Ch 0.30
< 0.20


-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. oxidation (5 #M Cu ++, 37°C) for 24 h, as judged by
Lean and H a g e m a n 213'214 loaded LDL in vitro with circular dichroism spectroscopy, increase of 430 nm
probucol by incubation of 0.2 mg protein/mL with 10 fluorescence, and decrease of reactive NH2 groups.
#M probucol for l h at 37°C and reported that all the LDL has a phase transition temperature around 35 to
probucol was incorporated into the LDL, so the LDL 40°C, and in vitro incorporation of lipophilic sub-
must therefore have contained about 25 mol probu- stances into the LDL is probably markedly facilitated
col/tool LDL. This dose of probucol inhibited copper if performed above the phase transition temperature.



Oh 0.40

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
Oxidation of LDL 379

1.50 the relative risk due to a low vitamin E status may

lag. rain
have a greater quantitative importance than that of
1.20 the classical risk factors such as total cholesterol and
blood pressure." A low plasma level of a-tocopherol
,¢ 0.90 and ascorbate has also been identified as a risk factor
o~ in early angina pectoris.~ s7 Kok et al.292compared sele-
0.60 nium, PUFAs, and a-tocopherol in subjects with vary-
ing degrees of coronary atherosclerosis. Levels ofa-to-
0.30 copherol and PUFAs were similar in the cases and in
controls. However, the ratio Se/PUFAs was signifi-
0.00 ~ , h , i , i
cantly lower in the cases, which suggests that a higher
0 60 120 180 240
PUFA level with insufficient antioxidant protection
time, minutes might indicate a higher risk. Short-term supplementa-
tion of humans with vitamin C (l g/d, 2 weeks) or
Fig. 23. Determination of the efficacy of a-tocopherol to increase
the resistance of LDL against AAPH-induced oxidation. 2~g The vitamin E (600 mg/d, 3 weeks) has a protective effect
LDL samples were those from donor A used in the Cu ++ oxidation on in vivo oxidation of LDL (as measured by the con-
shown in Fig. 16. LDL (0.25 mg/mL) in PBS was oxidized with 100 tent of TBARS in isolated LDL) induced by acute
uM AAPH. The relationship is y = 6.23x + 25.2 (r z = .96).
cigarette smoking. 42 Supplementation of elderly peo-
ple with vitamin E led to a significant lowering of the
the onset of the propagation phase in the
e t al., z~9 plasma lipid peroxides ( T B A R S ) ) 9° Salonen et al. tgl
AAPH system in a concentration-dependent fashion, reported recently that the common carotid intima
with an efficiency (k) very close to the value observed thickening is greater in men with high serum copper
in the Cu ++ oxidation system. (this suggests that Cu ++ could act as a prooxidant in
The capacity of ascorbate and urate to delay in vi- vivo) and low serum selenium. Supplementing men
tro a propagating lipid peroxidation in LDL is likely (39 subjects) with a low antioxidant status with 600
to have significant biological implications. Human mg ascorbate, 300 mg a-tocopherol, 27 mg 3-caro-
plasma contains about 10-50 uM ascorbate and 200- tene, and 75 #g selenium daily over 5 months resulted
400 #M urate, and these agents should prevent deple- in a reduction of serum lipid peroxides, platelet aggre-
tion of the LDL vitamin E and oxidation of LDL in gation, and platelet-produced thromboxane B2 (Ref.
blood to forms causing foam cell formation or possess- 300). Fruchart et al. 3°1 reported on in vitro oxidizabil-
ing chemotactic and cytotoxic properties. Where, ity of LDL isolated from hypercholesterolemic men
then, can oLDL be formed? It is reasonable to assume treated with vitamin E (1 g/d) or fenofribrate + vita-
that in the arterial wall local destruction of water-solu- min E or placebo for two months. Copper-treated
ble antioxidants can occur (e.g., by oxygen radicals LDL samples from the patients receiving vitamin E
released from inflammatory cells). LDL infiltrated showed a significant reduced uptake by mouse perito-
and present in such an oxidizing environment would neal macrophages as compared to oxidized LDL of
solely depend on protection by its endogenous antiox- the placebo group. Gaziano et a1.192presented prelimi-
idants, and under such circumstances sufficient levels nary findings from the USA Physician Health Study,
of vitamin E as well as other antioxidants are crucial. according to which the group receiving 50 mg 3-caro-
Does an elevated plasma vitamin E content reduce tene every second day had a significant (44%) reduc-
the risk of atherosclerosis, and can antioxidant treat- tion of all major coronary events (such as cardiac
ment reduce oxidation of LDL in vivo and inhibit death) and a 49% reduction of all major vascular
progression of atherosclerosis? A remarkable contri- events (such as stroke or cardiovascular death). All
bution relevant to this question comes from epide- these findings suggest, but of course do not prove, that
miological studies (WHO/Monica project) in the antioxidant therapy could lower the risk of atheroscle-
European population. 48'185'1s6 Essential plasma an- rosis and ischemic heart disease. A controlled athero-
tioxidants (a-tocopherol, ascorbate, vitamin A, carot- sclerosis intervention study with the classical antioxi-
enoids, selenium) were determined in 16 European dants (vitamin E, vitamin C) or antioxidant drugs
study populations which differed sixfold in mortality (e.g., probucol) has so far not been conducted or at
from ischemic heart disease. The incidence of isch- least has not been published as a full paper. Short-
emic heart disease mortality showed a strong inverse term treatment with vitamin E appears to have no
correlation (r 2 = .63, p = .002) with the level of effect on the progression of the disease. In 75 heart
plasma t~-tocopherol. It was suggested by Gey 1s6 "that patients, treatment with 200 mg all-rac a-tocopheryl-
380 H. ESTERBAUER~ al.

nicotinate for 4-6 weeks gave no significant differ- A number of studies have addressed the question
ence between the treated (38 patients) and the placebo of whether probucol (4.4'(isopropylidenedithio)-bis-
group (37 patients).194 In angina pectoris patients sup- (2.6-di-t-butylphenol) prevents progression of athero-
plemented with 1600 IU tocopheryl-succinate for 6 sclerosis, at least in part, by its antioxidant effects.
months (52 patients) or 3200 IU tocopheryl-succinate Probucol was originally developed as antioxidant for
for 9 weeks (18 treated, 18 placebo), no difference in manufacturing plastic and rubber. In 1969 and 1970
clinical parameters was seen in comparison with the the first reports a p p e a r e d 2°4'2°5 that probucol lowers
placebo group. 195'196 Hata reported at a recent confer- serum cholesterol in human subjects2°4 and in mice,
ence 3°5 a secondary prevention trial on cerebrovascu- rats, dogs, and monkeys.2°5 Probucol is now a widely
lar disease with d, 1-a-tocopherol nicotinate. In total, used drug for treatment of hypercholesterolemia in
2358 patients with cerebral infarction (CI) were ran- people (for review see Ref. 206). In 1984 Naruszewicz
domly allocated either tocopherol nicotinate (600 mg suggested that it might limit oxidation o f L D L , 2°7 and
daily) or conventional treatment. The incidence of in 1986 Patharasarathy et al. 2°8 presented evidence
recurrent cerebral vascular infarction was signifi- that probucol indeed inhibits in vitro oxidative modi-
cantly (p < .05) lower (125 cases) in the tocopherol fication of LDL (TBARS) by endothelial cells and
nicotinate group than in the group on conventional copper ions and proposed that probucol, in addition
therapy (172 cases). to the lipid-lowering effect, may reduce progression of
A number of animal experiments related to antiox- atherosclerosis by its antioxidant properties.
idant therapy of atherosclerosis were published. Ver- Kita et al. 2°9 showed that probucol treatment (1%
langieri et al. 193'197 presented data showing that daily
in diet, 6 month) of WHHL rabbits significantly re-
doses of 108 IU of vitamin E/day significantly re- duced the formation of atheromatas in the thoratic
duced symptoms of atherosclerosis in primates fed an aorta. Carew et al. TM found that the formation ofmac-
atherogenic diet. This is in agreement with an earlier
rophage-rich fatty streak lesions in WHHL rabbits
report 19s that atherosclerosis of triglyceride-fed rab-
was inhibited by probucol to a much higher extent
bits can be prevented by vitamin E, and that vitamin
than expected from its cholesterol-lowering effect.
E and vitamin A supplements reduce spontaneous ath-
Probucol does not affect lipoprotein metabolism in
erosclerosis in hens during the egg-laying p e r i o d . 199
macrophages of WHHL rabbits. 212 The plasma pro-
Smith et al. z2° also reported on the protective effect of
bucol concentration of the treated animals was 83
dietary vitamin E on atherogenesis in nonlaying hens.
uM, and roughly two thirds was associated with LDL.
It has been reported that chronic deficiency of vita-
This means that probucol must have reached a con-
min C or vitamin E is associated with atherosclerosis-
centration of about 40-50 mol/mol LDL. 2t° Serum of
like lesions in rodents, pigs, and primates. 2°°'2°~Morel
et al. 132 and Chisholm et al. 2°2 showed that treatment treated patients contains about 25-100 #M probu-
CO1.210'213 As expected, LDL isolated from probucol-
of rats suffering experimental streptozotocin diabetes
with vitamin E (450 mg/d, 4 weeks) or probucol in- treated patients or rabbits was highly resistant to cell
hibits oxidation of LDL in vivo (as measured by or Cu ++ oxidation in vitro. 2°9 Since the first re-
ports, 209'211 several other studies were published which
TBARS of isolated LDL); moreover, the LDL from
the treated rats was not cytotoxic in contrast to the confirmed that probucol in the food (about 0.5 to 1%)
LDL from the diabetic untreated group. Very high reduces atherosclerotic lesions in rabbits fed a stan-
doses of vitamin E (10 g/kg diet, 14 weeks) were re- 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
ported to potentiate in rabbits formation of lesions in contrast to these studies, Stein et al. 225 found no pro-
aortas of cholesterol-fed rabbits that were mechani- tective effect of probucol (1% in chow) in New Zea-
cally deendothelialized.2°3 Bj6rkhem et al. 3°4 reported land albino rabbits fed a 1% cholesterol chow.
about the effect of BHT on plasma lipids and develop- This article has focussed mainly on the role of an-
ment ofatherosclerotic lesions in rabbits fed a 1% cho- tioxidants in conferring oxidation resistance to LDL.
lesterol diet for 12 weeks. The addition of 1% BHT to In the last 2 or 3 years, a number of experimental
the diet gave higher levels of cholesterol, triglycerides, results have accumulated which strongly suggest that
and LDL, but despite that the degree of atherosclero- vitamin E and possibly also probucol exhibit a much
sis of the aortic surface was considerably reduced as broader spectrum of biological activities than ex-
compared to the control group fed a cholesterol diet pected just from their ability to act as chain-breaking
only. These authors also found that BHT led to a sig- antioxidants in lipid peroxidation. Azzi's group, ~88,189
nificant reduction of serum cholesterol oxidation for example, reported that 50-100 #M of a-tocoph-
products (7-ketocholesterol, cholesterol 5a, 6a- erol (added as an ethanol solution) inhibits prolifera-
epoxide). tion of cultured smooth muscle cells, an effect not
Oxidation of LDL 381

associated with the antioxidant activity but mediated was correlated with the reduction in platelet adhesive-
by a modulation of the protein kinase-C activity. As ness, with a maximal effect at 400 IU/day.
proliferation of smooth muscle cells is an important From all these studies, we conclude that vitamin E
event during plaque formation, the beneficial effects has, in addition to its antioxidant function in LDL, a
of vitamin E in vivo may therefore be dual and not great potential in preventing other deleterious events
solely relying on its antioxidant properties. However, involved in the pathogenesis of atherosclerosis.
some aspects of the experimental protocol adapted in The therapeutic potential of vitamin E in various
this study may require critical testing. The inhibition diseases, including those associated with oxidative
of cell proliferation may have been caused by the de- stress, was recently reviewed by Janero 3°2 and
tergent effect of the added a-tocopherol, which would C h o w . 303
be absent with the acetate. Such an unphysiological
detergent effect would correspond to the observations Acknowledgement-- The authors' work has been supported by the
reported for free fatty acids. In a series of studies, Association for International Cancer Research (AICR), U.K., and
by the Austrian Science Fondation, Project No. 8271-Med. We
Cornwell's group (for review see Ref. 320) investi- gratefully acknowledge the critical evaluation of this manuscript by
gated the inhibitory effects of polyunsaturated fatty Dr. David G. Cornwell and thank him for many valuable sugges-
acids on cell proliferation and developed the concept tions.
that PUFA-derived lipid peroxidation products in-
hibit smooth muscle cell proliferation, whereas an-
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