Identification and quantification of the regioisomeric cholesteryl linoleate hydroperoxides in oxidized human low density lipoprotein and high density lipoprotein

Article abstract of DOI:10.1021/tx9600098

Oxidation of human LDL is implicated as an initiatior of atherosclerosis. Isolated low density lipoprotein (LDL) and high density lipoprotein (HDL₂) were exposed to aqueous radicals generated from the thermolabile azo compound 2,2'-azobis(2-amidinopropane) dihydrochloride. The primary nonpolar lipid products formed from the autoxidation of LDL and HDL were the regioisomeric cholesteryl linoleate hydroperoxides. In LDL oxidations, 9- and 13- hydroperoxides with trans,cis conjugated diene were formed as the major oxidation products if endogenous α-tocopherol was present in the LDL. After extended oxidation of LDL, at the time when endogenous α-tocopherol was consumed, the two trans,cis conjugated diene hydroperoxides began to disappear and the 9- and 13-hydroperoxides with trans,trans conjugated diene appeared. At very long oxidation times, none of the primary products, the conjugated diene hydroperoxides, were present. In HDL₂, which has only very low levels of antioxidants, both the 9- and 13-hydroperoxides with trans,cis conjugated diene and the 9- and 13-hydroperoxides with trans,trans conjugated diene were formed at early stages of oxidation. The corresponding alcohols were also formed in the HDL₂ oxidations. A mechanistic hypothesis consistent with these observations is presented.

Full text of DOI:10.1021/tx9600098

Chem. Res. Toxicol. 1996, 9, 737-744
737
Id en tifica tion a n d Qu a n tifica tion of th e Regioisom er ic
Ch olester yl Lin olea te Hyd r op er oxid es in Oxid ized
Hu m a n Low Den sity Lip op r otein a n d High Den sity
Lip op r otein
J ames A. Kenar,† Christine M. Havrilla,† Ned A. Porter,*,† J ohn R. Guyton,‡
Spencer A. Brown,‡ Keith F. Klemp,‡ and Elizabeth Selinger‡
Department of Chemistry, Paul M. Gross Laboratories, Duke University, and Department of
Medicine, Duke University Medical Center, Durham, North Carolina 27708
Received J anuary 19, 1996X
Oxidation of human LDL is implicated as an initiatior of atherosclerosis. Isolated low density
lipoprotein (LDL) and high density lipoprotein (HDL2) were exposed to aqueous radicals
generated from the thermolabile azo compound 2,2′-azobis(2-amidinopropane) dihydrochloride.
The primary nonpolar lipid products formed from the autoxidation of LDL and HDL were the
regioisomeric cholesteryl linoleate hydroperoxides. In LDL oxidations, 9- and 13-hydroperoxides
with trans,cis conjugated diene were formed as the major oxidation products if endogenous
R-tocopherol was present in the LDL. After extended oxidation of LDL, at the time when
endogenous R-tocopherol was consumed, the two trans,cis conjugated diene hydroperoxides
began to disappear and the 9- and 13-hydroperoxides with trans,trans conjugated diene
appeared. At very long oxidation times, none of the primary products, the conjugated diene
hydroperoxides, were present. In HDL2, which has only very low levels of antioxidants, both
the 9- and 13-hydroperoxides with trans,cis conjugated diene and the 9- and 13-hydroperoxides
with trans,trans conjugated diene were formed at early stages of oxidation. The corresponding
alcohols were also formed in the HDL2 oxidations. A mechanistic hypothesis consistent with
these observations is presented.
Understanding the earliest stages of LDL modification,
in particular, lipid peroxidation and the protective role
of antioxidants, is of fundamental importance since the
clues found here may provide means to increase the
resistance of LDL to oxidation and ultimately prevent
atherosclerosis. The lipid-soluble antioxidant, R-toco-
pherol, is the major antioxidant associated with plasma
and LDL, although minor amounts of γ-tocopherol,
carotenoids, retinol, and ubiquinol-10 (COQ10H2) are also
present (4). The protective role that these antioxidants
play in lipoprotein oxidation has been extensively stud-
ied, although not fully understood (15-17). Lipoprotein
oxidation is initiated by transition metals such as copper
and iron (18), γ-radiation (19), and cultured cells that
produce active oxygen (6, 20). Unfortunately, initiation
of free radical chains by these methods is not reproduc-
ible, and this leads to ambiguities in the oxidation studies
of the lipoproteins. The recent use of thermolabile water-
soluble azo initiators as a radical source, although not
biologically relevant, generates a constant flux of free
radicals with known rate constants, and provides a
valuable tool in which to study LDL oxidation (21, 22).
In tr od u ction
Low density lipoprotein (LDL)1 is the major carrier of
cholesteryl esters in human blood plasma (1). Free
radical-mediated modification of LDL may play a crucial
role in the development of atherosclerosis, and much
effort has been devoted to the study of lipoprotein
oxidation and its prevention over the last decade (2, 3).
Peroxidation of the LDL’s lipids affords hydroperoxides
mainly from cholesteryl linoleate (Ch18:2), the predomi-
nant lipid component in the lipoprotein core (4). Hydro-
peroxide formation is generally thought to precede and
result in the modification of apoprotein B-100, the single
protein associated with LDL (5-7). These hydroperox-
ides can undergo decomposition and give a variety of
secondary breakdown products, including reactive alde-
hydes that are known to react with the lysine residues
of the apoprotein (8). Once modified, the apoprotein is
taken up by scavenger receptors of monocyte-derived
macrophages (9-12). Internalization of lipids in this
manner is uncontrolled and leads to intracellular lipid
accumulation and the appearance of lipid-laden foam
cells similar to those found in early atherosclerotic lesions
(13, 14).
In contrast to LDL, there are relatively few studies on
the oxidation of high density lipoprotein (HDL). Recent
reports suggest that HDL is more prone to radical-
mediated oxidation and carries most of the cholesteryl
ester hydroperoxides detectable in freshly obtained hu-
man plasma (23). Although oxidation studies of LDL and
HDL have revealed hydroperoxide formation among the
various lipid classes, i.e., cholesteryl esters, phospholip-
ids, and free cholesterol, no studies have identified in
detail the molecular species Ch18:2-OOH regioisomers
and the role antioxidants may have on their distribution
(24, 25). There is one literature report concerning the
* Please address correspondence to this author. Phone: (919) 660-
1550; email: porter@chem.duke.edu.
† Department of Chemistry, Duke University.
‡ Department of Medicine, Duke University Medical Center.
X Abstract published in Advance ACS Abstracts, May 1, 1996.
1
Abbreviations: LDL, low density lipoproteins; HDL, high density
lipoproteins; CoQ10H2, ubiquinol-10; PBS, phosphate buffered saline;
AAPH, 2,2′-azobis(2-amidinopropane) dihydrochloride; DTBN, di-tert-
butyl hyponitrite; 18:2, linoleic acid; 20:4, arachidonic acid; Ch18:2,
cholesteryl linoleate; Ch18:2-OH, cholesteryl linoleate alcohol; Ch18:
2-OOH, cholesteryl linoleate hydroperoxide; Ch20:4, cholesteryl arachi-
donate.
S0893-228x(96)00009-4 CCC: $12.00 © 1996 American Chemical Society
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738 Chem. Res. Toxicol., Vol. 9, No. 4, 1996
Kenar et al.
I and II. Peaks III and IV could not be separated and were
isolated as a mixture.
chemo-enzymatic synthesis and identification of choles-
teryl 13-hydroperoxyoctadeca-cis-9,trans-11-dienoate, one
of the Ch18:2-OOH isomers (24), and analyses of choles-
terol ester hydroperoxides by the use of a chiral HPLC
column have also been reported (25). In an effort to gain
further insight into the formation and breakdown of lipid
hydroperoxides in the early stages of LDL and HDL
oxidation, we have isolated and completely characterized
the major regioisomeric hydroperoxides and alcohols
formed in the peroxidation of Ch18:2. In addition, we
have established methodology based on high performance
liquid chromatography (HPLC) to directly assay for the
regioisomeric cholesteryl linoleate hydroperoxides and
alcohols in oxidized LDL and HDL, and we have moni-
tored oxidations of LDL and HDL initiated by a water-
soluble azo initiator. Herein, we report the details and
results of our investigations.
Ch olester yl 13-Hyd r op er oxyocta d eca -cis-9,tr a n s-11-d i-
en oa te (1). Analytical HPLC fraction I, tR 15.0 min; TLC Rf
0.48; 1H NMR δ 0.67 (s, 3H), 0.8-2.60 (66H), 4.38 (m, 1H), 4.50-
4.70 (m, 1H), 5.36 (m, 1H), 5.40-5.65 (m, 2H), 6.00 (dd, J )
10.6, 6.6 Hz, 1H), 6.57 (dd, J ) 15.3, 11.2 Hz, 1H), 7.83 (s, 1H).
Ch olester yl 13-Hyd r op er oxyocta d eca -tr a n s-9,tr a n s-11-
d ien oa te (2). Analytical HPLC fraction II, tR 17.5 min; TLC
1
Rf 0.48; H NMR δ 0.67 (s, 3H), 0.8-2.60 (66H), 4.33 (m, 1H),
4.50-4.70 (m, 1H), 5.36 (m, 1H), 5.47 (dd, J ) 15.1, 8.4 Hz, 1H),
5.75 (dt, J ) 15.0, 7.0, 7.5 Hz, 1H), 6.05 (dd, J ) 14.9, 10.5 Hz,
1H), 6.27 (dd, J ) 15.2, 10.2 Hz, 1H), 7.71 (s, 1H).
Mixtu r e of Ch olester yl 9-Hyd r op er oxyocta d eca -tr a n s-
10,cis-12-d ien oa te (3) a n d Ch olester yl 9-Hyd r op er oxyoc-
ta d eca -tr a n s-10,tr a n s-12-d ien oa te (4). Analytical HPLC
fraction III, tR 20.1 min: IV, tR 20.5 min; TLC Rf 0.48; 1H NMR
δ 0.67 (s, 3H), 0.80-2.50 (66H), 4.28-4.41 (m, 1H), 4.50-4.70
(m, 1H), 5.30-5.60 (m, 2H, vinyl H’s 3; 1H, vinyl H 4; 1H, vinyl
ring H), 6.58 (dd, J ) 15.3, 11.7 Hz, 1H, vinyl H 3), 6.27 (dd, J
) 15.2, 10.3 Hz, 1H, vinyl H 4), 6.04 (m, 1H, vinyl H 3; 1H,
vinyl H 4), 5.75 (dt, J ) 15.3, 7.7, 6.7 Hz, 1H, vinyl H 4), 7.71
(s, 1H, OOH 4), 7.73 (s, 1H, OOH 3).
Red u ction of Ch olester yl Lin olea te Hyd r op er oxid es. A
sample of the purified hydroperoxides (ca. 10-20 mg) was
dissolved in 500 µL of diethyl ether along with triphenylphos-
phine (1.2 equiv) and allowed to stir (28). Thin-layer chroma-
tography showed the disappearance of peroxidic products, and
the reaction was continued until no peroxides were detected,
approximately 30 min. The crude product was concentrated in
vacuo, and preparative HPLC afforded pure samples of the four
alcohols.
Ma ter ia ls a n d Meth od s
Gen er a l Meth od s. Thin layer chromatography was per-
formed with the use of Whatman 250 µm silica plates (5 × 20
cm). The solvent system used was 15% ethyl acetate in hexane.
1H NMR spectra were recorded on a Varian XL 300 MHz
spectrometer in CDCl3. Chemical shifts are reported in ppm
(δ) with respect to the residual H signal in CDCl3 (δ ) 7.26 ppm),
and coupling constants (J values) are given in hertz. Multiplici-
ties are indicated by s (singlet), d (doublet), t (triplet), and m
(multiplet).
Analytical HPLC was conducted on a Waters Model 590
HPLC instrument with a Hewlett Packard Multiwavelength
1050 detector and a Hewlett Packard 3396 Series III integrator.
The HPLC was equipped with two tandem Beckman 5 µm
Ultrasphere columns (4.6 mm × 25 cm), 0.5% 2-propanol in
hexane, and a delivery rate of 1 mL/min. Preparative HPLC
was conducted on a Waters Model 600E HPLC instrument, with
a Waters Model 481 variable wavelength detector operating at
234 nm and connected to a chart recorder. A Dynamax-60A Si
83-141-C column purchased from Rainin Instrument Co.
(Woburn, MA), 0.66% 2-propanol in hexane, and a solvent
delivery rate of 25 mL/min were used for the separations.
Ch em ica ls. Phosphate buffered saline (PBS, pH 7.4, 50 mM)
was stored over Chelex-100 at least 24 h to remove transition
metal contaminants. All chemicals were purchased from Ald-
rich Chemical Co. (Milwaukee, WI) and used without further
purification, unless otherwise noted. Methyl linoleate was
purchased from Nu-Chek Prep (Elysian, MN). The free radical
initiator, 2,2′-azobis(2-amidinopropane) dihydrochloride, (AAPH)
was obtained from Polyscience, Inc. (Warrington, MA). The free
radical initiator di-tert-butyl hyponitrite (DTBN) was synthe-
sized prior to use (26, 27). Organic solvents such as 2-propanol,
hexane, benzene, and ethyl acetate were HPLC quality and were
purchased from Mallinckrodt, Inc. (St. Louis, MO). The stan-
dard, methyl 13-hydroxyoctadeca-trans-9,trans-11-dienoate, was
prepared by autoxidation of methyl linoleate followed by reduc-
tion with triphenylphosphine, and purification by preparative
HPLC as described previously (28). The antioxidants, R-toco-
pherol and δ-tocopherol, were purchased from Sigma Chemical
Co. (St. Louis, MO).
Oxid a tion of Ch olester yl Lin olea te. A round bottom flask
was charged with 300 mg of cholesteryl linoleate and diluted to
0.20 M with freshly distilled benzene. The sealed flask was
heated to 37 °C under an oxygen atmosphere, and after a brief
period of equilibration, one crystal of DTBN was added and the
solution stirred magnetically. Thin-layer chromatography showed
the formation of peroxidic products, and the oxidation was
continued until enough peroxide products were generated for
convenient analysis. Analytical HPLC indicated that four major
peaks were present in the product mixture with the following
relative percentages: I, tR 15.0 min, 17.2%; II, tR 17.5 min,
32.0%; III, tR 20.1 min, 11.1%; and IV, tR 20.5 min, 39.7%.
Preparative HPLC was used to isolate pure fractions of peaks
Ch olester yl 13-Hydr oxyoctadeca-cis-9,tr a n s-11-dien oate
(5). TLC Rf 0.39; Analytical HPLC tR 18.0 min; 1H NMR δ 0.67
(s, 3H), 0.80-2.50 (66H), 4.16 (m, 1H), 4.55-4.65 (m, 1H), 5.30-
5.50 (m, 2H), 5.66 (dd, J ) 15.2, 6.8 Hz, 1H), 5.97 (dd, J ) 11.1,
6.0 Hz, 1H), 6.48 (dd, J ) 15.2, 11.1 Hz, 1H).
Ch olest er yl 13-H yd r oxyoct a d eca -tr a n s-9,tr a n s-11-d i-
en oa te (6). TLC Rf 0.35; Analytical HPLC tR 21.1 min; 1H NMR
δ 0.67 (s, 3H), 0.80-2.5 (66H), 4.10 (m, 1H), 4.55-4.65 (m, 1H),
5.37 (m, 1H), 5.56 (dd, J ) 15.0, 7.1 Hz, 1H), 5.70 (dt, J ) 15.0,
7.5, 6.8 Hz, 1H), 6.02 (dd, J ) 14.8, 10.4 Hz, 1H), 6.17 (dd, J )
15.0, 10.3 Hz, 1H).
Ch ole st e r yl 9-H yd r oxyoct a d e ca -t r a n s-10,cis-12-d i-
en oa te (7). TLC Rf 0.32; Analytical HPLC tR 27.0 min; 1H NMR
δ 0.67 (s, 3H), 0.80-2.50 (66H), 4.15 (m, 1H), 4.50-4.70 (m, 1H),
5.30-5.50 (m, 2H), 5.66 (dd, J ) 15.2, 6.8 Hz, 1H), 5.97 (dd, J
) 11.1, 6.0 Hz, 1H), 6.48 (dd, J ) 15.3, 11.1 Hz, 1H).
Ch olest er yl 9-H yd r oxyoct a d eca -tr a n s-10,tr a n s-12-d i-
en oa te (8). TLC Rf 0.32; Analytical HPLC tR 29.5 min; 1H NMR
δ 0.67 (s, 3H), 0.80-2.50 (66H), 4.10 (m, 1H), 4.50-4.70 (m, 1H),
5.37 (m, 1H), 5.56 (dd, J ) 15.0, 7.0 Hz, 1H), 5.70 (dt, J ) 15.0,
7.4, 6.0 Hz, 1H), 6.02 (dd, J ) 14.8, 6.0 Hz, 1H), 6.17 (dd, J )
15.1, 10.3 Hz, 1H).
Lip op r otein Isola tion . Whole blood from fasting, normo-
lipidemic healthy subjects was collected in a 440 mL ACD blood
collection bag (Baxter) containing the following: 2 g of dextrose
monhydrate; 1.66 g of sodium citrate dihydrate; 188 mg of
anhydrous citric acid; 140 mg of monobasic sodium phosphate
monohydrate; and 17.3 mg of adenine. Tubes containing blood
were centrifuged at 2000g for 30 min at 10 °C. The lipoproteins,
LDL (1.019 < d < 1.063 g/mL) and HDL2 (1.063 < d < 1.125
g/mL), were isolated from plasma over 3 days by density
gradient sequential ultracentrifugation at 14 °C using a Beck-
man L 70 (Optima) centrifuge and a 50.2 Ti rotor. Each spin
was performed at 100000g for 18 h. Lipoproteins were dialyzed
extensively against 50 mM PBS, sterilized by passage through
a Millex-HA 0.45 µM filter, and stored at 4 °C under argon. All
protein concentrations of the lipoprotein preparations were
determined by the method of Lowry (29).
Oxid a tion a n d Extr a ction of Lip op r otein s. The lipopro-
tein concentrations were adjusted to 1.5 mg of protein/mL with
PBS, allowed to equilibrate to 37 °C for 5 min in a round bottom
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Lipoprotein Autoxidation
Chem. Res. Toxicol., Vol. 9, No. 4, 1996 739
F igu r e 1. Products formed in the autoxidation of cholesteryl linoleate.
flask. To the magnetically stirred solution was added AAPH
(100 mM stock solution) to give a final AAPH concentration of
1 mM. Immediately after the addition of AAPH (time zero), and
at various time intervals, two 200 µL and a 400 µL (LDL only)
aliquots were removed for analysis. To both 200 µL aliquots
were added 20 µL of BHT (25.5 mM in ethanol) and a known
amount of the internal standard, methyl 13-hydroxyoctadeca-
trans-9,trans-11-dienoate (10). One of the 200 µL aliquots was
reduced with triphenylphosphine and analyzed for C18:2-OH
after extraction. The 400 µL aliquots taken for the R-tocopherol
analyses had 50 µL of BHT (25.5 mM in ethanol) and a known
amount of the internal standard δ-tocopherol added. Extraction
of all the aliquots was performed immediately with (5 volumes
of) ice-cold methanol and (25 volumes of) hexane in sequence,
vortexed vigorously after the addition of each solvent (ca. 15 s)
and then centrifuged at 1700 rpm for approximately 1 min with
the use of an Adams analytical centrifuge. The hexane phase
was removed by pipet and concentrated under argon, and the
resulting organic residue was resuspended in 50 µL of HPLC
solvent and immediately analyzed by analytical HPLC for Ch18:
2-OOH and Ch18:2-OH. The 400 µL aliquots were stored in
dilute hexane solutions at -120 °C until analysis could be
performed (usually within 4 days of sample preparation). The
hexane was removed under a stream of argon, and the organic
residue was resuspended in 50 µL of hexane and immediately
assayed for R-tocopherol by analytical HPLC (30).
Resu lts
F igu r e 2. HPLC chromatograms (0.5% 2-propanol in hexane,
λ ) 234 nm) from the autoxidation of cholesteryl linoleate with
DTBN at 37 °C. (A) Isomeric Ch18:2-OOH’s: I ) 1; II ) 2; III
) 3; IV ) 4. (B) Isomeric Ch18:2-OHs obtained from Ch18:2-
OOH’s reduction with the use of PPh3: V ) 5; VI ) 6; VII ) 7;
VIII ) 8.
Oxid a tion of Ch olester yl Lin olea te. The bond
dissociation energy of a bis allylic carbon-hydrogen bond
(CdCCH2CdC) is substanitially lower than that of an
allylic carbon-hydrogen bond (CdCCH2) (31). For this
reason, autoxidation of cholesteryl linoleate should give
peroxidic products predominantly from linoleate side
chain oxidation. Indeed, the azo-initiated bulk phase
oxidation of 0.2 M cholesteryl linoleate, in benzene at 37
°C, generated four major peroxidic products, 1-4, in the
ratio 1.5:2.9:1.0:3.6 (Figure 1).
The four alcohols gave two sets of essentially identical
1H NMR spectra; presumably, the cis,trans isomers 1 and
3 formed one set, while the trans,trans isomers 2 and 4
formed the other set. The geometry of the conjugated
double bonds in the linoleate side chain was determined
from the vinyl H’s coupling constants (Figure 3). Thus,
the vinyl region of spectrum A is consistent with the
cis,trans double bond geometry found in 1 and 3 and
consists of signals at δ 5.54 [d,t, HD, (buried in the vinyl
H of the cholesterol ring)], 5.66 [d,d, HA, J A,B ) 15.2 Hz,
J A,CH ) 6.8 Hz)], 5.97 [d,d, HC, J C,D ) 11.1 Hz (cis, C,
D)], and 6.48 [d,d, HB, J B,A ) 14.8 Hz (trans B, A)].
Likewise, the vinyl region of spectrum B is consistent
with the trans,trans double bond geometry in 2 and 4
and consists of signals at δ 5.56 [d,d, HA, J A,B ) 15.0 Hz,
A typical HPLC analysis (λ ) 234 nm detection) of the
Ch18:2-OOH isomeric product mixture is shown in Figure
2 (panel A). As can be seen, the two later eluting peaks
III and IV coelute. Preparative HPLC cleanly isolated
peaks I and II, while peaks III and IV were isolated as a
mixture. Because the hydroperoxides readily decompose,
the isolated peroxide fractions were immediately reduced
with the use of triphenylphosphine to give their corre-
sponding alcohols, 5-8 (Figure 1). By HPLC, the alco-
hols obtained from the isolated hydroperoxides are
resolved from one another, Figure 2 (panel B). Prepara-
tive HPLC provided purified alcohol samples from peaks
V-VIII, which were subsequently identified by 1H NMR
and chemical transformations.
J A,CH ) 7.0 Hz], 5.70 [d,t, HD, J D,C ) 15.0 Hz, J D,CH
)
2
7.4 and 6.0 Hz], 6.02 [d,d, HC, J C,D ) 14.8 Hz (trans C,
D)], and 6.17 [d,d, HB, J B,A ) 15.1 Hz (trans B, A)].
Unfortunately, it was not possible to distinguish between
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740 Chem. Res. Toxicol., Vol. 9, No. 4, 1996
Kenar et al.
F igu r e 3. Vinyl region of 1H NMR spectra of (A) cholesteryl 13-hydroxyoctadeca-cis-9,trans-11-dienoate (5) and cholesteryl
9-hydroxyoctadeca-trans-10,cis-12-dienoate (7); and (B) cholesteryl 13-hydroxyoctadeca-trans-9,trans-11-dienoate (6) and cholesteryl
9-hydroxyoctadeca-trans-10,trans-12-dienoate (8). R ) cholesterol.
the 9 and 13 position of the hydroxyl functionality by
NMR; therefore, the position of the hydroxyl group was
determined through chemical transformations.
Since the 9-trans,cis and 9-trans,trans Ch18:2-OOH
isomers (3 and 4) coelute, analysis of their corresponding
alcohols derived from Ch18:2-OOH reduction gives a
more accurate picture of the hydroperoxide product
distributions. Accordingly, aliquots from various times
of the LDL oxidation were immediately reduced with the
use of triphenylphosphine to give their corresponding
isomeric Ch18:2-OH’s, which were then analyzed by
HPLC. A typical HPLC analysis presented in Figure 4
(panel C) shows that, in the initial stages of oxidation,
the cis,trans alcohols 5 and 7 predominate. For example,
after 120 min of oxidation with 1 mM AAPH, the
cis,trans/trans,trans [(5 + 7)/(6 + 8)] ratio is 14.7:1.0.
Formation of 5 and 7 maximizes after approximately 8
h of oxidation under these conditions of intiation and then
begins to decline as the oxidation continues. Meanwhile,
production of the trans,trans Ch18:2-OHs, 6 and 8,
increases and eventually exceeds 5 and 7 after 16 h of
oxidation. It is of interest to note that by 16 h nearly all
of the R-tocopherol has been consumed.
The isolated Ch18:2-OHs, 5-8, were transesterified
with the use of NaOMe in MeOH to afford the hydroxy
linoleate fatty acid Me esters 9-12. These Me esters
have been previously characterized (28), and their HPLC
elution order is known. Comparison of the Me esters
obtained from the transesterification reaction to an
authentic mixture allowed assignment of the -OH posi-
tion in each Ch18:2-OH isomer. The elution order of
alcohols 5-8 is shown in Figure 2 (panel B): peak V, 5;
peak VI, 6; peak VII, 7; peak VIII, 8. The identities of
the hydroperoxides 1-4, Figure 2 (panel A), were as-
signed by reduction to their alcohol derivatives: peak I,
1; peak II, 2; peak III, 3; peak IV, 4.
Oxid a tion of LDL. With the ability to identify the
isomeric Ch18:2-OOH and Ch18:2-OHs by HPLC, analy-
sis of freshly extracted LDL samples by HPLC indicated
no detectable traces of Ch18:2-OOH or Ch18:2-OH (UV
detection at 234 nm). Exposure of isolated LDL to
aqueous radicals, generated from the water-soluble azo
initiator AAPH, results in the time-dependent formation
of Ch18:2-OOH’s 1-4, and Ch18:2-OH’s 5-8, as shown
from a representative oxidation experiment (Figure 4).
Panel A in Figure 4 shows the concomitant formation
of Ch18:2-OOH and Ch18:2-OH’s during the early stages
of LDL oxidation. As can be seen, there is an initial 1 h
lag time in which hydroperoxide formation is suppressed,
after which time substantial amounts of Ch18:2-OOH
begin to form, although approximately 90% of the R-to-
copherol remains. Small amounts of isomeric Ch18:2-
OH’s are also produced during the initial stages of LDL
oxidation, which are represented by the 9-trans,cis and
trans,trans Ch18:2-OH’s in Figure 4. Unfortunately, the
chromatographic region where the 13-Ch18:2-OH isomers
elute has adjacent peaks that make accurate analysis
difficult; therefore, we do not report the 13-Ch18:2-OHs
(5 and 6) in Figure 4, although we assume that they
are formed with equal proclivity as the 9-Ch18:2-OH’s
(7 and 8).
At extended LDL oxidation times, there is a substantial
time-dependent decrease in the amount of all Ch18:2-
OOH formed, presumably from the various hydroperoxide
decomposition pathways. Interestingly, Ch18:2-OH was
not produced as a result of these processes.
The cholesteryl linoleate hydroperoxides obtained from
oxidized LDL were also analyzed by reverse phase HPLC
with 50% acetonitrile in 2-propanol and UV detection at
210 nm (data not shown) (32). The general lipid classes,
cholesterol, Ch20:4, Ch18:2, and Ch18:1, were observed
under these conditions in freshly extracted samples of
LDL. No detectable traces of Ch18:2-OOH and Ch18:2-
OH were observed. As the LDL was oxidized, peaks
corresponding to the formation of Ch18:2-OOH, Ch18:2-
OH, and Ch20:4-OOH were observed along with the
simultaneous depletion of the Ch20:4 and Ch18:2 peaks.
Oxid a tion of HDL
2. An HPLC analysis for Ch18:2-
OOH and Ch18:2-OH similar to that used to monitor the
LDL oxidation reported above was performed with the
use of HDL2 as the lipid substrate. Freshly extracted
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Lipoprotein Autoxidation
Chem. Res. Toxicol., Vol. 9, No. 4, 1996 741
F igu r e 5. Free radical oxidation of HDL2 (1.5 mg of protein/
mL) initiated by 1 mM AAPH at 37 °C. At the time points
indicated, samples were withdrawn, extracted, and analyzed by
HPLC with UV detection (λ ) 234 nm) as described in the
Materials and Methods. trans,cis-13-OOH Ch18:2 (1, 9); tran-
s,trans-13-OOH Ch18:2 (2, 2); cis,trans-9-OOH Ch18:2 and
trans,trans-9-OOH Ch18:2 (3 and 4, O); trans,cis-13-OH Ch18:2
(5, *); trans,trans-13-OH Ch18:2 (6, b); cis,trans-9-OH Ch18:2
(7, )); trans,trans-9-OH Ch18:2 (8, 3). A and B, analysis before
PPh3 reduction; C, analysis after PPh3 reduction.
F igu r e 4. Free radical oxidation of LDL (1.5 mg of protein/
mL) initiated by 1 mM AAPH at 37 °C. At the time points
indicated, samples were withdrawn, extracted, and analyzed by
HPLC with UV detection (λ ) 234 nm) as described in the
Materials and Methods. trans,cis-13-OOH Ch18:2 (1, 9); tran-
s,trans-13-OOH Ch18:2 (2, 2); cis,trans-9-OOH Ch18:2 and
trans,trans-9-OOH Ch18:2 (3 and 4, O); trans,cis-13-OH Ch18:2
(5, *); trans,trans-13-OH Ch18:2 (6, b); cis,trans-9-OH Ch18:2
(7, )); trans,trans-9-OH Ch18:2 (8, 3); R-tocopherol (+). A and
B, analysis before PPh3 reduction; C, analysis after PPh3
reduction.
Figure 5 (panel C). In HDL2, the cis,trans/trans,trans
[(5 + 7)/(6 + 8)] ratio is only 2.6:1.0 after 120 min of
oxidation with 1 mM AAPH (in LDL oxidations, this ratio
was 14.7:1.0). As the oxidation proceeds, 6 and 8 formed
concomitantly with alcohols 5 and 7, although at slightly
lower levels. After 6 h, 6 and 8 production surpassed 5
and 7 formation (6 and 8 surpassed 5 and 7 after 16 h of
LDL oxidation). Since fewer antioxidants are associated
with HDL2 relative to LDL, any antioxidants present are
probably consumed sooner in HDL2, which allows 6 and
8 to form earlier. HDL2 also shows a time-dependent
samples of HDL2 showed no detectable amounts of Ch18:
2-OOH or Ch18:2-OH isomers by HPLC (UV detection
at 234 nm). Oxidation of HDL2 with aqueous radicals
generated from AAPH results in the time-dependent
formation of Ch18:2-OOH’s 1-4 and Ch18:2-OH’s 5-8
as shown from a typical experiment (Figure 5).
As in the case of LDL, triphenylphosphine reduction
of the isomeric Ch18:2-OOH’s gave their corresponding
alcohols, which were then analyzed by HPLC to ac-
curately determine hydroperoxide product distributions,
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742 Chem. Res. Toxicol., Vol. 9, No. 4, 1996
Kenar et al.
Sch em e 1
decrease in the amount of all hydroperoxides without the
formation of Ch18:2-OH, presumably from various hy-
droperoxide decomposition pathways.
The results from this study demonstrate that peroxi-
dation of isolated LDL and HDL2 induced by chemically
generated aqueous radicals results in the formation of
significant amounts of Ch18:2-OOH and lower levels of
Ch18:2-OH after an initial 1 h lag time. After the lag
time, substantial amounts of hydroperoxides begin to
form, although approximately 90% of the R-tocopherol is
still present in LDL. Recent results by Stocker et al. have
shown ubiquinol-10, a minor endogenous antioxidant
present in LDL and HDL, is a more effective antioxidant
than R-tocopherol, and no hydroperoxides form until the
ubiquinol-10 is consumed (4).
Discu ssion
Mechanistically, the four hydroperoxide products, 1-4,
from cholesteryl linoleate autoxidation are formed as
shown in Scheme 1 (33-35). First, hydrogen atom
abstraction from C-11 of the linoleate side chain leads
to the pentadienyl radical 13. Subsequent oxygen addi-
tion to 13 generates one of two trans,cis peroxyl radicals,
14 or 15, either of which has three possible options.
Considering just the 9-substituted radical 14, hydrogen
atom abstraction from a donor such as an antioxidant or
another linoleate side chain generates 9-cis,trans hydro-
peroxide 3, a kinetic product.
The other two mechanistic options involve â-fragmen-
tation of the intermediate peroxyl radical. Nonproductive
â-fragmentation gives the original pentadienyl radical 13.
Alternatively, if bond rotation about C9-C10 precedes
â-fragmentation, then a new pentadienyl radical 16 is
formed. This radical has trans rather than cis geometry
about the partial double bond ∆-9,10. Oxygen addition
at C-13 then leads to a peroxyl radical that gives the 13-
trans,trans hydroperoxide 2, the thermodynamic product.
The same processes can occur with the initially formed
13-substituted peroxyl radical 15. Lipid peroxidation
product ratios are, therefore, determined by the competi-
tion between hydrogen atom abstraction from hydrogen
donor molecules, like antioxidants, R-tocopherol, and
ubiquinol-10, to give the cis,trans kinetic products and
â-fragmentation to give the trans,trans thermodynamic
products. In addition to the role antioxidants may play
in preventing lipid peroxidation products, their presence
should also efficiently trap any peroxyl radicals formed
during the initial stages of oxidation as the kinetic 13-
and 9-cis,trans Ch18:2-OOH products, 1 and 3.
Notably, in the early phases of LDL oxidation, we
observe the exclusive formation of 13- and 9-cis,trans
Ch18:2-OOH, 1 and 3. Apparently, the antioxidants
present in LDL, and to a lesser extent in HDL2, ef-
fectively trap the initially formed cis,trans peroxyl radi-
cals 14 and 15 to give the kinetic Ch18:2-OOH’s 1 and
3. Only after depletion of R-tocopherol, the main anti-
oxidant present in LDL, do â-fragmentation and read-
dition reactions occur to give 2 and 4, the thermodynamic
13- and 9-trans,trans Ch18:2-OOH’s.
The kinetic/thermodynamic (cis,trans/trans,trans) Ch18:
2-OOH product ratio in oxidized LDL and HDL2 was
determined from the Ch18:2-OH’s obtained from Ch18:
2-OOH reduction. After 120 min of oxidation with 1 mM
AAPH, there was approximately 14.7 times as much
cis,trans Ch18:2-OH as trans,trans Ch18:2-OH in LDL,
while in HDL2, only 2.6 times as much of the cis,trans
Ch18:2-OH formed relative to the 13-trans,trans Ch18:
2-OH.
In the absence of antioxidants, as in the LDL case
when R-tocopherol is depleted after 16 h, â-fragmentation
of the initially formed radicals (14 and 15) that lead to
13- and 9-cis,trans Ch18:2-OOH 1 and 3 can occur, and
readdition of molecular oxygen leads to the 13- and
9-trans,trans Ch18:2-OOH, 2 and 4. It is also possible
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Lipoprotein Autoxidation
Chem. Res. Toxicol., Vol. 9, No. 4, 1996 743
that the trans,trans Ch18:2-OOH isomers observed in the
early oxidation time points are formed by rearrangement
of the cis,trans isomers even in the presence of antioxi-
dants (35).
effects, acting over decades in concert with other cellular
and molecular processes, may eventually lead to overt
atherosclerotic disease. In addition, the oxidation prod-
ucts derived from arachidonic acid are known to generate
prostaglandins, leukotrienes, and hydroxyeicosatetraeno-
ic acids, which themselves initiate various cellular sig-
naling cascades (43). Therefore, since cholesteryl arachi-
donate (Ch20:4) is also present in LDL and HDL, we have
undertaken the identification and quantification of Ch20:4
oxidation products found in oxidized LDL and HDL. The
results of this study will be reported in due course.
In HDL2, 2 and 4 formed concomitantly, although at
slightly lower levels, with 1 and 3. Since fewer antioxi-
dants are associated with HDL2 relative to LDL, the
antioxidants present in HDL2 are probably consumed
sooner in the oxidation, allowing â-fragmentation to
occur, and generating trans,trans Ch18:2-OOHs. In any
case, one expects the product distibution of isomeric
hydroperoxides to depend on the H atom donating
propensity of the medium of oxidation, and the antioxi-
dant-rich LDL should give more kinetic products (the 13-
and 9-cis,trans Ch18:2-OOH 1 and 3) than the antioxi-
dant-poor HDL.
Ack n ow led gm en t. Support for this research by NIH
HL17921 is gratefully acknowledged. C.M.H. acknowl-
edges support from an NIEHS Toxicology Training Grant.
Extended oxidation times showed a time-dependent
decrease in the amount of Ch18:2-OOH in LDL and
HDL2, presumably from various hydroperoxide decom-
position pathways. Large amounts of the cholesteryl
linoleate alcohols were not produced as a result of these
processes.
Refer en ces
(1) Horton, A. A., and Fairhurst, S. (1987) Lipid peroxidation and
mechanisms of toxicity. CRC Crit. Rev. Toxicol. 18, 27-79.
(2) Esterbauer, H., and Cheeseman, K. H., Eds. (1987) Lipid
Peroxidation: Part I. Biochemical and Biophysical Aspects. Chem.
Phys. Lipids 44, 2-4.
(3) Steinberg, D., Parhasarathy, S., Carew, T. E., Khoo, J . C., and
Witztum, J . L. (1989) Beyond cholesterol. Modifications of low-
density lipoprotein that increase its atherogenicity. N. Engl. J .
Med. 320, 915-924 and references therein.
(4) Stocker, R., Bowry, V. W., and Frei, B. (1991) Ubiquinol-10
protects human low density lipoprotein more efficiently against
lipid peroxidation than does R-tocopherol. Proc. Natl. Acad. Sci.
U.S.A. 88, 1646-1650.
(5) J essup, W., Rankin, S. M., De Whalley, C. V., Hoult, J . R. S., Scott,
J ., and Leake, D. S. (1990) Alpha-tocopherol consumption during
low-density-lipoprotein oxidation. Biochem. J . 265, 399-405.
(6) Esterbauer, H., Dieber-Rotheneder, M., Striegl, G., and Waeg, G.
(1991) Role of vitamin E in preventing the oxidation of low-density
lipoprotein. Am. J . Clin. Nutr. 53, 314s-321s.
(7) Suarna, C., Hood, R. L., Dean, R. T., and Stocker, R. (1993)
Comparative antioxidant activity of tocotrienols and other natural
lipid-soluble antioxidants in a homogeneous system, and in rat
and human lipoproteins. Biochim. Biophys. Acta 1166, 163-170.
(8) Steinbrecher, H. P., Lougheed, M., Kwan, W. C., and Dirks, M.
(1989) Recognition of oxidized low density lipoprotein by the
scavenger receptor of macrophages results from derivatization of
apolipoprotein B by products of fatty acid peroxidation. J . Biol.
Chem. 264, 15216-15223.
(9) Henriksen, T., Mahoney, M. E., and Steinberg, D. (1981) En-
hanced macrophage degradation of low density lipoprotein previ-
ously incubated with cultured endothelial cells: Recognition by
receptors for acetylated low density lipoproteins. Proc. Natl. Acad.
Sci. U.S.A. 78, 6499-6503.
(10) Morel, D. W., DiCorleto, P. E., and Chilsom, G. M. (1984)
Endothelial and smooth muscle cells alter low density lipoprotein
in vitro by free radical oxidation. Arteriosclerosis 4, 357-364.
(11) Steinbrecher, U. P., Parthasarathy, S., Leake, D. S., Witztum, J .
L., and Steinberg, D. (1984) Modification of low density lipoprotein
by endothelial cells involves lipid peroxidation and degradation
of low density lipoprotein phospholipids. Proc. Natl. Acad. Sci.
U.S.A. 81, 3883-3887.
Recently, Stocker et al. reported Ch18:2-OH were
formed simultaneously with Ch18:2-OOH in the early
stages of LDL and HDL oxidation (36). After 180 min of
oxidation with 2 mM AAPH, these workers found that
the Ch18:2-OOH/Ch18:2-OH ratio ranged from 2.7 to 9
for LDL and from 1.0 to 3.5 for HDL. In our experiments,
low levels of alcohols were also produced. For example,
after 120 min of oxidation with 1 mM AAPH, the overall
hydroperoxide/alcohol ratio was approximately 3.8:1.0 for
LDL, while we determine a ratio of 2.6:1.0 for HDL2, in
agreement with Stocker’s results. In addition, we ob-
served only the formation of the 13- and 9-cis,trans Ch18:
2-OH, 5 and 7, in the early stages of the oxidation. As
the oxidation continued, products 6 and 8 were formed
in greater amounts than 5 and 7 (approximately 22 h
for LDL, and 11 h for HDL2).
The thermolabile AAPH used in this study as a radical
generator initiates lipid peroxidation along the conven-
tional chain pathway, which does not include significant
formation of lipid hydroxides. In addition, we note that
Ch18:2-OOH decomposition during the course of oxida-
tion does not form significant amounts of Ch18:2-OH.
Therefore, Ch18:2-OH formation was probably not pro-
duced via these pathways, which suggests an alternative
pathway for their production. Stocker et al. have implied
the involvement of phospholipid hydroperoxide glu-
tathione peroxidase (PxGSH) which can use Ch18:2-OOH
as a substrate (37).
The role, if any, the cis,trans Ch18:2-OOH and Ch18:
2-OH play in the development of atherosclerotic plaques
relative to their trans,trans isomers is presently unclear
and warrants further investigation (38). Recently, 9-hy-
droxy Ch18:2 and free 9-hydroxy 18:2 were found to
stimulate the release of interleukin 1, an inflammatory
cytokine, from cultured human macrophages (39). Cho-
lesteryl esters in atherosclerotic lesions are subject to
enzymatic cleavage of the ester bond, producing free fatty
acids that diffuse readily into cells. Enhancement of cell
proliferation induced by epidermal growth factor was
shown after administration of linoleic acid derivatives,
13-OOH and 13-OH, to cultured cells (40). The free 18:
2-OOH’s were also found to injure endothelial cells,
stimulate collagen production, modulate production of
matrix metalloproteinases by vascular cells, and activate
endothelial phospholipase D (41, 42). These and other
(12) Steinbrecher, U. P., Witztum, J . L., Parthasarathy, S., and
Steinberg, D. (1987) Decrease in reactive amino groups during
oxidation or endothelial cell modification of LDL. Correlation with
changes in receptor-mediated catabolism. Arteriosclerosis 7, 135-
143.
(13) Gerrity, R. G. (1981) The role of the monocyte in atherogenesis:
I. Transition of blood-borne monocytes into foam cells in fatty
lesions. Am. J . Pathol. 103, 181-190.
(14) Faggiotto, A., Ross, R., and Harker, L. (1984) Studies of hyper-
cholesterolemia in the nonhuman primate. I. Changes that lead
to fatty streak formation. Arteriosclerosis 4, 323-340.
(15) For a discussion of the antioxidant effects of R-tocopherol, see:
Burton, G. W., and Ingold, K. U. (1986) Vitamin E: Application
of the principles of physical organic chemistry to the exploration
of its structure and function. Acc. Chem. Res. 19, 194-201. For a
discussion of the pro-oxidant effects of R-tocopherol, see refs 16
and 17.
(16) Bowry, V. W., Ingold, K. U., and Stocker, R. (1992) Vitamin E in
human low-density lipoprotein: When and how this antioxidant
becomes a pro-oxidant. Biochem. J . 288, 341-344.
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+

744 Chem. Res. Toxicol., Vol. 9, No. 4, 1996
Kenar et al.
(17) Ingold, K. U., Bowry, V. W., Stocker, R., and Walling, C. (1993)
Autoxidation of lipids and antioxidation by R-tocopherol and
ubiquinol in homogeneous solution and in aqueous dispersions
of lipids: Unrecognized consequences of lipid particle size as
exemplified by the oxidation of human low density lipoprotein.
Proc. Natl. Acad. Sci. U.S.A. 90, 45-49.
(18) Esterbauer, H., Striegl, G., Puhl, H., and Rotheneder, M. (1989)
Continuous monitoring of in vitro oxidation of human low density
lipoprotein. Free Radical Res. Commun. 6, 67-75.
(19) Bedwell, S., Dean, R. T., and J essup, W. (1989) The action of
defined oxygen-centered free radicals on human low-density
lipoprotein. Biochem. J . 262, 707-712.
(20) Esterbauer, H., Gebicki, J ., Puhl, H., and J u¨rgens, G. (1992) The
role of lipid peroxidation and antioxidants in oxidative modifica-
tion of LDL. Free Radical Biol. Med. 13, 341-390.
(21) Sato, K., Niki, E., and Shimasaki, H. (1990) Free radical-mediated
chain oxidation of low density lipoprotein and its synergistic
inhibition by vitamin E and vitamin C. Arch. Biochem. Biophys.
279, 402-405.
(22) Bowry, V. W., and Stocker, R. (1993) Tocopheryl-mediated per-
oxidation. The prooxidant effect of vitamin E on radical-initiated
oxidation of human low-density lipoprotein. J . Am. Chem. Soc.
115, 6029-6044.
(23) Bowry, V. W., Stanley, K. K., and Stocker, R. (1992) High density
lipoprotein is the major carrier of lipid hydroperoxides in human
blood plasma from fasting donors. Proc. Natl. Acad. Sci. U.S.A.
89, 10316-10320.
(24) Baba, N., Tahara, S., Nakajima, S., Iwasa, J ., Kaneko, T., and
Matsuo, M. (1992) Syntheses of cholesteryl 13-hydroperoxyocta-
decadienoate and its derivative with lipoxygenase. Biosci. Biotech.
Biochem. 56, 540-541.
(31) Fort, R., Hrovat, D., and Borden, W. (1993) Ab Initio Calculations
of the Stabilization Energy of Pentadienyl Fadical from Rotational
Barriers and from Lowering of Bond Dissocation Energies. J . Org.
Chem. 58, 211-216.
(32) Sattler, W., Mohr, D., and Stocker, R. (1994) Rapid isolation of
lipoproteins and assessment of their peroxidation by high per-
formance liquid chromatography postcolumn chemiluminescence.
Methods Enzymol. 233, 469-489.
(33) Porter, N. A., Lehman, L. S., Weber, B. A., and Smith, K. J . (1981)
A Unified Mechanism for Polyunsaturated Fatty Acid Autoxida-
tion: Competition of Peroxy Radical Hydrogen Atom Abstraction,
â-Scission, and Cyclization. J . Am. Chem. Soc. 103, 6447-6455.
(34) Porter, N. A. (1986) Mechanisms for the Autoxidation of Polyun-
saturated Lipids. Acc. Chem. Res. 19, 262-268.
(35) Porter, N. A., Caldwell, S. E., and Mills, K. A. (1995) Mechanisms
of Free Radical Oxidation of Unsaturated Lipids. Lipids 30, 277-
290.
(36) Sattler, W., Christison, J ., and Stocker, R. (1995) Cholesterylester
hydroperoxide reducing activity associated with isolated high- and
low-density lipoproteins. Free Radical Biol. Med. 18, 421-429.
(37) Sattler, W., Maiorino, M., and Stocker, R. (1994) Reduction of
HDL- and LDL-associated cholesteryl-ester and phospholipid
hydroperoxides by phospholipid hydroperoxide glutathione per-
oxidase and ebselen (PZ 51). Arch. Biochem. Biophys. 309, 214-
221.
(38) Hajjar, D., and Nicholson, A. C. (1995) Atherosclerosis. Am. Sci.
83, 460-467 and references therein.
(39) Ku, G., Thomas, C. E., Akeson, A. L., and J ackson, R. L. (1992)
Induction of interleukin 1-â expression from human peripheral
blood monocyte-derived macrophages by 9-hydroxyoctadecadienoic
acid. J . Biol. Chem. 267, 14183-14188.
(25) Folcik, V. A., Nivar-Aristy, R. A., Krajewski, J . P., and Cathcart,
M. K. (1995) Lipoxygenase Contributes to the Oxidation of Lipids
in Human Atherosclerotic Plaques. J . Clin. Invest. 96, 504-510.
(26) Traylor, T. G., and Kiefer, H. (1966) Di-t-Butyl hyponitrite. A
convenient source of t-butoxy radicals. Tetrahedron Lett. 49,
6163-6168.
(27) Mendenhall, G. D. (1983) The Lewis acid catalyzed reaction of
trans-hyponitrite ion with alkyl halides. Tetrahedron Lett. 24,
451-452.
(40) Eling, T. E., and Glasgow, W. C. (1994) Cellular proliferation and
lipid metabolism: Importance of lipoxygenases in modulating
epidermal growth factor-dependent mitogenesis. Cancer Metasta-
sis Rev. 13, 397-410.
(41) Sasaguri, Y., Kakita, N., Murahashi, N., Kato, S., Hiraoka, K.,
and Morimatsu, M. (1993) Effect of linoleic acid hydroperoxide
on production of matrix metalloproteinases by human aortic
endothelial and smooth muscle cells. Atherosclerosis 100, 189-
196.
(28) Porter, N. A., and Wujek, D. G. (1984) Autoxidation of polyun-
saturated fatty acids, an expanded mechanistic study. J . Am.
Chem. Soc. 106, 2626-2629.
(29) Lowry, O. H., Rosebrough, N. J ., Farr, A. L., and Randall, R. J .
(1951) Protein measurement with folin phenol reagent. J . Biol.
Chem. 193, 265-275.
(30) Burton, G. W., Webb, A., and Ingold, K. U. (1985) A mild rapid,
and efficient method of lipid extraction for use in determining
vitamin E/lipid ratios. Lipids 20, 29-39.
(42) Natarajan, V., Taher, M. M., Roehm, B., Parinandi, N. L., Schmid,
H. H. O., Kiss, Z., and Garcia, J . G. N. (1993) Activation of
endothelial cell phospholipase D by hydrogen peroxide and fatty
acid hydroperoxide. J . Biol. Chem. 268, 930-937.
(43) Honn, K. V., Tang, D. G., Gao, X., Butovich, I. A., Liu, B., Timar,
J ., and Hagmann, W. (1994) 12-Lipoxygenases and 12(S)-HETE:
Role in cancer metastasis. Cancer Metastasis Rev. 13, 365-396.
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