© 1997 American Heart Association, Inc.
Articles |
Prostaglandin F2-Like Compounds, F2-Isoprostanes, Are Present in Increased Amounts in Human Atherosclerotic Lesions
From the Institute of Biochemistry, University Clinics Charité, Humboldt University, Hessische Str 3–4, D-10115 Berlin, F.R. Germany (C.G., J.D.M., H.K.), and the Division of Clinical Pharmacology, Departments of Medicine and Pharmacology, Vanderbilt University, Nashville, Tenn (L.J.R.).
Correspondence to Dr Hartmut Kühn, Institute for Biochemistry, University Clinics (Charité), Humboldt University, Hessische Str. 3–4, 10115 Berlin, F.R. Germany. E-mail hartmut.kuehn{at}rz.hu-berlin.de
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Abstract Oxidative modification of LDL is believed to play a major role in atherogenesis. As major lipid peroxidation products oxygenated linoleic acid derivatives and oxysterols have been described in human atherosclerotic lesions. Here we report that human lesions contain isoprostanes as peroxidation products of arachidonic acid at a level of 27.1±21.2 pg/mg wet weight (n=10), which corresponds to 75.9±59.3 pg/mg dry weight. n contrast, human umbilical veins (n=10), which were used as nonatherosclerotic control vessels, contain much smaller amounts of isoprostanes (1.4±0.7 pg/mg wet weight, which corresponds to 11.7±6.2 pg/mg dry weight), and there are significant differences between the two types of vessels. As major products of linoleic acid oxidation, racemic hydroxy linoleate isomers were detected in the lesional ester lipids. In human lesions, the hydroxy linoleic acid/linoleic acid ratio was about 0.5%, a result indicating that 5 out of 1000 linoleate residues are present as hydroxylated derivatives. In umbilical veins, no hydroxy linoleic acid could be detected.
These data show that human atherosclerotic lesions contain increased amounts of hydroxy linoleic acid isomers and isoprostanes when compared with nonatherosclerotic vessel wall and suggest a link between local lipid peroxidation and progression of atherosclerosis. For evaluation of the degree of lipid peroxidation, the determination of the hydroxy linoleic acid/linoleic acid ratio appears to be more suitable than the isoprostane content.
Key Words: atherogenesis • hydroxy fatty acids • lipid peroxidation • LDL modification • cholesterol esters
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Introduction |
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Oxidative modification of LDL is believed to play a major role in atherogenesis1–3 because LDL is converted to an atherogenic form that is rapidly taken up by macrophages via scavenger receptor mediated pathways.4 Since these pathways are not feedback controlled, lipids may be taken up excessively. These lipids are deposited inside macrophages, which thereby develop into lipid-laden foam cells.5 In vitro, enzymatic6,7 and nonenzymatic8,9 oxidation of LDL has been studied in detail, but the mechanisms of the in vivo oxidative processes remain unclear. Analysis of oxidized lipids found in atherosclerotic lesions of cholesterol-fed rabbits10 and humans11–13 indicated that esterified hydroxy derivatives and keto components of linoleic acid are the major lipid peroxidation products. Surprisingly, only small amounts of the corresponding oxidation products of arachidonic acid, such as 5-, 12-, and/or 15-HETE, were detected,12 despite the fact that arachidonic acid is one of the major fatty acids in human LDL14 and in the vessel wall. These data suggest that arachidonic acid may be either protected from oxidation or oxidized to other products that have not been looked for so far. Since arachidonic acid contains three doubly allylic methylene groups, double and/or triple oxygenation is possible, leading to a complex pattern of more polar oxidation products, including diH(P)ETE isomers15,16 and/or lipoxins derivatives.15,17 In addition, cyclization products such as isoprostanes may be formed when arachidonic acid–containing ester lipids undergo nonenzymatic lipid peroxidation. Isoprostanes are prostaglandin-like compounds derived from free radical mediated peroxidation of arachidonic acid. It is important to note that their biosynthesis is cyclooxygenase independent.18 These compounds possess potent biological activity and have been suggested as indicators of in vivo oxidative stress in both animal model systems and human disease.19 In addition, it has been shown that in humans who smoke cigarettes heavily and who are at increased risk for atherosclerosis, circulating levels of isoprostane are markedly increased.20
In this study, we examined whether lipid extracts of human atherosclerotic lesions contain isoprostanes and other stable end products of lipid peroxidation. Comparison of the isoprostane content of atherosclerotic lesions with control nonatherosclerotic vessels (umbilical veins) indicated significant differences, a result suggesting that lipid peroxidation products other than hydroxylated fatty acids may also play a role in atherogenesis.
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Methods |
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Chemicals
The chemicals used were from the following sources: 9S-HODE, 13S-HODE, 15S-HETE, linoleic acid, arachidonic acid, and docosahexaenoic acid from Cayman Chem; dimethylformamide and undecane from Aldrich; N, O-bis(trimethylsilyl)trifluoracetamide from Supelco. For isoprostane analysis, [2H4]-PGF2
(Cayman Chem) was used as internal standard. All solvents were of HPLC
grade and were purchased from Serva (FRG).
Human Vessels
Human atherosclerotic lesions were prepared from arteries (a.
femoralis, a. tibialis anterior, a. poplitea, aorta, and a.
vertebralis) that were removed during surgery. The patients (seven
males and three females) were 55 to 78 years of age and were selected
for surgery because of clinical symptoms. In all patients, a severe
stenosis of the artery was diagnozed by angiography.
Immediately after removal, the samples were frozen on dry ice and
stored at -30°C until workup. Macroscopic inspection of the lesions
and HPLC analysis of their lipid content indicated moderate
lipid deposition. In some lesions, signs of calcification were seen.
Before lipid extraction, the lesional area was freed from surrounding
tissue, keeping all layers of the vessel wall. Blood clots were
removed, and the tissue was washed in ice-cold PBS.
Human umbilical cords were removed from the placenta immediately after delivery. The blood was rinsed out of the umbilical vessels, and the umbilical veins were prepared and stored at -30°C.
Lipid Extraction
Extraction of the vessel lipids was carried out with a modified
Bligh/Dyer method.21 About 500 mg (wet weight) of
vessel wall were homogenized for 30 seconds with an
Ultraturax homogenizer in 5 mL of a
methanol/chloroform/water mixture (2:1:1 by volume). To minimize
artificial lipid peroxidation during the extraction procedure, ice-cold
solvents were used that were freed from oxygen by bubbling extensively
with argon gas. After addition of 1.25 mL of chloroform and 1.25 mL of
water, the extraction mixture was vortexed for 1 minute and then
centrifuged for phase separation. The lower chloroform phase
containing the total lipids was recovered, the solvents were
evaporated, and the residual lipids were reconstituted in 1 mL of
chloroform. This clear solution was split into two 0.5-mL portions, one
of which was used for HPLC analysis of the
cholesterol content and of the hydroxy linoleic acid
derivatives. The other half of the sample was used for GC/MS
quantification of the isoprostanes.
Analytical Protocols
RP-HPLC analysis of cholesterol
derivatives22 was performed on a Nucleosil C-18
column (Macherey/Nagel; KS-system, 250x4 mm, 5-µm particle
size). Aliquots of the crude nonhydrolyzed lipid extracts were
injected, and the compounds were eluted at 45°C with the solvent
system 2-propanol/acetonitrile (25/75 by volume) and a flow rate
of 1 mL/min. The absorbances at 210 nm (free cholesterol
and cholesterol esters) and 235 nm (oxidized
cholesterol esters) were recorded
simultaneously.
For HPLC analysis of the oxygenated linoleic acid derivatives, the extracted lipids were hydrolyzed under alkaline conditions. For this purpose, the chloroform solution of the lipids was concentrated to a final volume of about 0.04 mL. Then 0.4 mL of oxygen-free methanol and 0.06 mL of oxygen-free potassium hydroxide solution (40% in water) was added, and the lipids were hydrolyzed for 30 minutes at 60°C under argon atmosphere. After cooling down on ice, 0.06 mL of glacial acetic acid were added, precipitates were removed by centrifugation, and aliquots of the hydrolysis mixture were injected to HPLC analysis. Reverse-phase HPLC was carried out on Nucleosil C-18 column (Macherey/Nagel; KS-system, 250x4 mm, 5-µm particle size). The analytes were eluted isocratically with a solvent system consisting of methanol/water/acetic acid (85/15/0.1 by volume) at a flow rate of 1 mL/min. The absorbances at 235 nm (conjugated dienes of the hydroxy linoleic acid isomers) and 210 nm (nonoxygenated polyenoic fatty acids) were recorded simultaneously by using a Hewlett Packard diode array detector. The oxygenated and nonoxygenated polyenoic fatty acids were quantified by peak areas. Calibration curves (six-point calibration) for 13-HODE, linoleic acid, and arachidonic acid were established. For this purpose, known amounts of lipids were injected to HPLC, and the peak area was quantified for each amount. The fractions containing oxygenated fatty acids (retention volume, 4.5 to 6.5 mL) were collected, and the solvent was evaporated. The remaining lipids were reconstituted in 0.2 mL of hexane, and aliquots were injected to SP-HPLC for analysis of the hydroxy fatty acid positional isomers. SP-HPLC was carried out on a Nucleosil column (Macherey/Nagel; KS-system, 250x4 mm, 10-µm particle size) with a solvent system of n-hexane/2-propanol/acetic acid (100/2/0.1; by volume) and a flow rate of 1 mL/min. The absorbance at 235 nm (conjugated dienes) was recorded.
The isoprostane content of the lipid extracts were determined by negative ion chemical ionization GC/MS as described before.18 Briefly, lipid extracts from the vessel walls were subjected to alkaline hydrolysis, after which the F2-isoprostanes were purified, derivatized, and quantified as described elsewhere.18–20
Miscellaneous Methods
Since atherosclerotic plaques and human umbilical veins have a
different water content, the analytical data required correction.
Unfortunately, it was impossible to determine the dry weight of each
sample before lipid extraction, since we observed a massive lipid
peroxidation during the drying procedure. Therefore, we selected three
atherosclerotic lesions and four umbilical veins for the determination
of the wet weight/dry weight ratio and calculated the dry weights of
each tissue sample from its wet weight, using these ratios. The tissues
were dried in a stream of warm air until weight constancy. For
atherosclerotic lesions and for the umbilical veins, dry weight/wet
weight ratios of 0.36±0.10 (mean±SD) and 0.12±0.05, respectively,
were determined. Significance calculations (Student's t
test) were performed with the StatWorks TM1.2 program on a Power
Macintosh 7600/120.
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Results |
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The formation of atherosclerotic lesions is characterized, inter alia, by smooth muscle cell hyperproliferation and lipid deposition in the vessel wall.2 We analyzed the lipid extracts of human lesions and of human umbilical veins with respect to their content of cholesterol derivatives and found that the free cholesterol content in the lesions was one order of magnitude higher when normalized to the wet weight (Fig 1
, traces A and C). Normalization to the
dry weight indicated a free cholesterol content in the
lesions that was three times higher
(Table). Cholesterol esters
were found only in the human lesions but were virtually absent in
umbilical veins. By recording the chromatogram at 235 nm (Fig 1, trace B), cholesterol esters containing an oxidized
fatty acid residue were detected in human lesions. These compounds were
characterized by a conjugated diene chromophore
(R1
HOCHCHCHCHR2,
absorbance maximum at 235 nm) and by a conjugated ketodiene chromophore
((R1OCHCHCH
CHR2, absorbance maximum at 270 nm). It should
be stressed that peak (a), which is labeled oxidized
cholesterol esters, represents a mixture of several
molecular species of oxygenated cholesterol
esters. In these species, the cholesterol skeleton is
esterified with oxygenated polyenoic fatty acid
derivatives, predominantly with various hydroxy linoleic acid isomers
(13-hydroxy or 9-hydroxy linoleic acid) or with the corresponding
9-keto and 13-keto compounds.
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In umbilical veins, oxidized cholesterol esters have not
been detected (Fig 1
, trace D).
Lipid peroxidation has been implicated in the pathogenesis of
atherogenesis and previous analysis of the pattern of
oxygenated lipids suggested that enzymatic and nonenzymatic
processes may be involved.10–13 We
analyzed the hydrolyzed lipid extracts of human lesions by
RP-HPLC recording the absorbance at 235 nm (Fig 2A, traces I) and detected
oxygenated fatty acids comigrating with an authentic
standard of 13-hydroxy linoleic acid (13-HODE). In nonatherosclerotic
human vessels (umbilical veins), oxygenated polyenoic fatty
acids have not been found (Fig 2B, traces III). When the chromatograms
were recorded at 210 nm, the nonoxygenated polyenoic
fatty acids were analyzed (Fig 2A and B, traces II and IV).
Quantification of the chromatograms at 235 nm and 210 nm allowed the
calculation of the hydroxy linoleic acid/linoleic acid ratio, which
appears to be a suitable measure for the degree of oxidation of the
tissue lipids.
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Since the different positional isomers of hydroxy fatty acids such as
9-HODE and 13-HODE as well as their geometric double bond isomers are
poorly resolved in RP-HPLC the isomeric composition of the
oxygenated fatty acids were further analyzed by
SP-HPLC (Fig 3). By recording the
chromatogram at 235 nm, the positional and geometric isomers of hydroxy
linoleic acid were separated. In all samples, 13-HODE(Z,E) and
9-HODE(E,Z) were found to be the major oxygenation
products. Smaller amounts of the corresponding all-trans isomers
were also detected. Hydroxylated arachidonic acid
isomers were found only in trace amounts. Analysis of the
enantiomer composition of the major hydroxy linoleic acid isomers
(13-HODE and 9-HODE) revealed racemic mixtures (data not shown). These
data suggest that the majority of the oxygenation
products were formed via nonenzymatic lipid peroxidation
reactions.
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The lack of hydroxylated arachidonic acid isomers and
the fact that in human LDL, arachidonic acid is one of
the major polyenoic fatty acid prompted us to look for other
arachidonate oxidation products, particularly for
isoprostanes. A representative chromatogram of
isoprostane analysis is shown in Fig 4. By recording the chromatogram
at the m/z ratio of 569, the endogenous
F2-isoprostane isomers extracted from the
vascular tissue were analyzed (upper trace). The lower trace
recorded at a m/z ratio of 573, which shows a single peak
representing the deuterated standard used for quantification of
the endogenous isoprostane content. For this particular
sample of a human lesion, a F2-isoprostane level
of 51 pg/mg wet weight was determined.
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Quantification and statistical analysis of the analytical data
are summarized in the Table. The tissue content of free and esterified
cholesterol was significantly higher in the human lesions
than in the umbilical veins, a finding reflecting lipid deposition in
the vessel wall. As compared with the umbilical veins the free
cholesterol content of the lesions was about three times
higher and the cholesterol ester content was about 20-fold
higher, if the values were normalized to the tissue dry weight. These
data suggest that during lesion development, both free
cholesterol and esterified cholesterol are
deposited in the lesional area. This conclusion is in line with earlier
data obtained in cholesterol-fed
rabbits10 and in postmortem samples of human
aortic lesions.12 In the latter study, a strong
correlation between free and esterified cholesterol in
advanced human aortic lesions was observed.12 It
should be mentioned that the total cholesterol content
(free and esterified cholesterol) of the human lesions was
considerably lower than that reported in a previous study on the lipid
composition of human atheroma.23 This
difference may in part be due to the fact that in the present
study, only cholesteryl linoleate and cholesteryl
arachidonate were quantified. Other cholesterol
esters, such as cholesteryl oleate or cholesterol stearate,
that significantly contribute to the total cholesterol
content of human lesions have not been analyzed because they do
not contain polyenoic fatty acids and thus may be of less importance
for lipid peroxidation.
The isoprostane content in the atherosclerotic lesions (27.1±21.4
pg/mg wet weight) was also significantly higher
(P=.001) than that of the umbilical veins (1.37±0.7
pg/mg wet weight). When the samples were normalized to the dry
weight, the isoprostane content was about seven times higher in the
atherosclerotic lesions than in nonatherosclerotic vessel wall
(75.9±59.3 pg/mg dry weight versus 11.7±6.2 pg/mg dry
weight; P=.003). However, normalization to both wet and dry
weight may not be useful, since atherosclerotic lesions contain more
unsaturated lipids than normal vessel wall, and thus, the absolute
amount of isoprostanes is expected to be higher even if the degree of
oxygenation of the tissue lipids would be comparable.
Thus, we normalized the isoprostane content to the concentration of
arachidonic acid that constitutes the in vivo substrate
of isoprostane formation. As indicated in the Table, the
isoprostane/arachidonic acid ratio of atherosclerotic
lesions was about fourfold higher than the corresponding ratio for
umbilical veins and a significant difference (P=.009)
between both groups of vessel wall was observed. Taking into account
the small molecular weight difference between isoprostanes and
arachidonic acid, one may conclude that in
atherosclerotic lesions, approximately 15 isoprostane molecules are
present per 106 arachidonic
acid residues. For comparison, we determined the hydroxy linoleic
acid/linoleic acid ratio (HODE/LA-ratio) as second
parameter for the oxidation degree of the tissue lipids and
found that about five hydroxylated linoleate residues were present
per 1000 nonoxygenated linoleic acid molecules. In other
words, 0.5% of the linoleic acid residues were present as
hydroxylated derivatives in the lesion lipids. If this measure is used,
the difference between atherosclerotic lesions and umbilical veins was
more pronounced than that for the isoprostane/arachidonate
ratio.
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Discussion |
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The formation of atherosclerotic lesions is accompanied by lipid peroxidation at the site of lesion development. As major lipophilic oxidation products, ester lipids (phospholipids and cholesterolesters) containing oxygenated polyenoic fatty acids,10–13,23 in particular hydroxy and keto derivatives of linoleic acids, as well as oxysterols23–25 have been described. Here we report for the first time that isoprostanes are present in human atherosclerotic lesions in increased amounts. In contrast, in nonatherosclerotic vessel wall (umbilical veins), much smaller amounts of isoprostanes and virtually no hydroxy fatty acids were detected.
Because of the instability of the fatty acid hydroperoxides, lipid peroxidation in animal tissues is usually quantified by measuring secondary lipid peroxidation products, such as thiobarbituric acid reactive material,26 short-chain aldehydes,27,28 alkanes,29,30 hydroxylated polyenoic fatty acids,11–13,23 or isoprostanes.18–20 Each of these methods may be used as indicator that lipid peroxidation has occurred, but none of them is suitable to quantify the overall extent of lipid peroxidation in biological tissues. Calculation of the hydroxy linoleic acid/linoleic acid ratio (HODE/LA ratio) appears to be a suitable measure for quantification of the oxidation degree of the tissue lipids because it normalizes a rather stable oxidation product to its nonoxygenated parent fatty acid. Since both oxygenated and nonoxygenated fatty acids can be quantified in a single RP-HPLC run, this rather simple method may be used in each laboratory in which a HPLC system is available. The isoprostane/arachidonic acid ratio also normalizes a fairly stable oxidation product (isoprostane) to its nonoxygenated parent fatty acid. However, for isoprostane analysis, more specialized analytical tools (mass spectrometer with chemical ionization) are required that may not be available in many laboratories. Moreover, a second analytical method (GC or HPLC) for the quantification of the arachidonic acid content and several steps of derivatization are necessary. On the other hand, the sensitivity of the GC/MS method for isoprostane detection (lower detection limit of about 5 pg isoprostane/sample) is much higher than the HPLC method for the determination of hydroxy fatty acids (about 50 ng HODE/analytical run). (This detection limit was determined for the HP diode array detector 1040A. With modern fixed-wavelength detectors, which are more sensitive, the detection limit may be considerably lower.). High sensitivity of an assay system is certainly an advantage, but it also opens the question about the physiologic consequences of an oxidation process that oxidizes 15 out of 106 arachidonic acid molecules. In vitro studies on artificial model membranes suggested that structural and functional membrane alterations may only occur when the oxidation degree of the membrane phospholipids exceeds 3% to 5%. Such a degree of oxidation has been reported for mitochondrial membranes of rabbit reticulocytes31 that are degraded during the maturation of red blood cells.
Like any method, the determination of the hydroxy linoleic acid/linoleic acid ratio has its limits. For instance, when only small amounts of hydroxy fatty acids are present (hydroxy linoleic acid/linoleic acid ratio <0.05%), exact quantification of the hydroxy fatty acids becomes problematic. In such cases, impurities coeluting with hydroxy fatty acids in RP-HPLC become increasingly disturbing. However, in advance human lesions, the degree of oxidation varies between 0.1% and 1% (this study); therefore, impurities may be of only minor importance. However, to obtain reliable analytical data, several points must be considered: (1) The lesional area must be prepared carefully. Surrounding tissue should be dissected, and thrombi must be removed. We found that the hydroxy linoleic acid/linoleic acid ratio is impaired when nonatherosclerotic tissue (normal vessel wall) is included for sample workup. (2) Ultrapure solvents should be used for all steps of lipid extraction and HPLC. Since the lipid extracts are concentrated from larger volumes between the different HPLC steps, contamination in the solvents will be concentrated and then may disturb HPLC quantification. (3) We usually use new HPLC columns or clean up used columns before starting a new analytical series. This is of particular importance when crude lipid extracts from other sources were analyzed before on these columns. In such cases, we often observed ghost peaks, some of which interfered with quantification of the conjugated dienes. (4) Alkaline hydrolysis must be carried out under strictly anaerobic conditions to avoid artificial fatty acid oxidation.
For investigations of the dysregulation of lipid metabolism in human atherogenesis, control vessels are required to which the results of plaque analysis can be related. For this study, we used human umbilical veins as nonatherosclerotic control vessels. Umbilical veins are easily accessible and are likely to show minimal oxidative modification. It should be stressed, however, that they may not be considered artery equivalents because they do have a different histologic overall structure. However, since lipid deposition and lipid peroxidation in umbilical veins are very unlikely, these vessels may constitute suitable negative controls.
In earlier investigations, we analyzed composition of oxidized lipids of human lesions prepared from autopsy samples of human aorta12 and found a higher oxidation degree of the lesion lipids (a hydroxy linoleic acid/linoleic acid ratio of about 3%). In these studies, we observed a positive correlation between the oxidation degree of the lesion lipids (hydroxy linoleic acid/linoleic acid ratio) and the cholesterol content. A similar correlation was not observed in the present study, a result that may be partly due to the relatively low n-numbers and the large biological variability. However, since the cholesterol content of our lesions was considerably lower than that of the aortic atheromas, the relative low oxidation degree (0.5%) of the lesion lipids becomes plausible.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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Financial support for this study was provided by Deutsche Forschungsgemeinschaft (Ku 961/1 to 2), by the European Community (PL 93/1740) and by NHI (grants DK 48831 and GM 42096).
Received March 11, 1997; accepted August 31, 1997.
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J. D. Morrow Quantification of Isoprostanes as Indices of Oxidant Stress and the Risk of Atherosclerosis in Humans Arterioscler Thromb Vasc Biol, February 1, 2005; 25(2): 279 - 286. [Abstract] [Full Text] [PDF]
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J. V. Higdon and B. Frei Obesity and Oxidative Stress: A Direct Link to CVD? Arterioscler Thromb Vasc Biol, March 1, 2003; 23(3): 365 - 367. [Full Text] [PDF]
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H.-Y. Huang, L. J Appel, K. D Croft, E. R Miller III, T. A Mori, and I. B Puddey Effects of vitamin C and vitamin E on in vivo lipid peroxidation: results of a randomized controlled trial Am. J. Clinical Nutrition, September 1, 2002; 76(3): 549 - 555. [Abstract] [Full Text] [PDF]
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Y. Tintut, F. Parhami, A. Tsingotjidou, S. Tetradis, M. Territo, and L. L. Demer 8-Isoprostaglandin E2 Enhances Receptor-activated NFkappa B Ligand (RANKL)-dependent Osteoclastic Potential of Marrow Hematopoietic Precursors via the cAMP Pathway J. Biol. Chem., April 12, 2002; 277(16): 14221 - 14226. [Abstract] [Full Text] [PDF]
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J. Huber, H. Boechzelt, B. Karten, M. Surboeck, V. N. Bochkov, B. R. Binder, W. Sattler, and N. Leitinger Oxidized Cholesteryl Linoleates Stimulate Endothelial Cells to Bind Monocytes via the Extracellular Signal-Regulated Kinase 1/2 Pathway Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 581 - 586. [Abstract] [Full Text] [PDF]
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T. Cyrus, D. Pratico, L. Zhao, J. L. Witztum, D. J. Rader, J. Rokach, G. A. FitzGerald, and C. D. Funk Absence of 12/15-Lipoxygenase Expression Decreases Lipid Peroxidation and Atherogenesis in Apolipoprotein E-Deficient Mice Circulation, May 8, 2001; 103(18): 2277 - 2282. [Abstract] [Full Text] [PDF]
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S. R. Thomas, S. B. Leichtweis, K. Pettersson, K. D. Croft, T. A. Mori, A. J. Brown, and R. Stocker Dietary Cosupplementation With Vitamin E and Coenzyme Q10 Inhibits Atherosclerosis in Apolipoprotein E Gene Knockout Mice Arterioscler Thromb Vasc Biol, April 1, 2001; 21(4): 585 - 593. [Abstract] [Full Text] [PDF]
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A. C. Staff, T. Ranheim, T. Henriksen, and B. Halvorsen 8-Iso-Prostaglandin F2{alpha} Reduces Trophoblast Invasion and Matrix Metalloproteinase Activity Hypertension, June 1, 2000; 35(6): 1307 - 1313. [Abstract] [Full Text] [PDF]
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J. I. Heller, J. R. Crowley, S. L. Hazen, D. M. Salvay, P. Wagner, S. Pennathur, and J. W. Heinecke p-Hydroxyphenylacetaldehyde, an Aldehyde Generated by Myeloperoxidase, Modifies Phospholipid Amino Groups of Low Density Lipoprotein in Human Atherosclerotic Intima J. Biol. Chem., March 31, 2000; 275(14): 9957 - 9962. [Abstract] [Full Text] [PDF]
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J. A. Lawson, J. Rokach, and G. A. FitzGerald Isoprostanes: Formation, Analysis and Use As Indices of Lipid Peroxidation in Vivo J. Biol. Chem., August 27, 1999; 274(35): 24441 - 24444. [Full Text] [PDF]
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A. D. Watson, G. Subbanagounder, D. S. Welsbie, K. F. Faull, M. Navab, M. E. Jung, A. M. Fogelman, and J. A. Berliner Structural Identification of a Novel Pro-inflammatory Epoxyisoprostane Phospholipid in Mildly Oxidized Low Density Lipoprotein J. Biol. Chem., August 27, 1999; 274(35): 24787 - 24798. [Abstract] [Full Text] [PDF]
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M. Reza Mehrabi, C. Ekmekcioglu, F. Tatzber, A. Oguogho, R. Ullrich, A. Morgan, F. Tamaddon, M. Grimm, H. D Glogar, and H. Sinzinger The isoprostane, 8-epi-PGF2{alpha}, is accumulated in coronary arteries isolated from patients with coronary heart disease Cardiovasc Res, August 1, 1999; 43(2): 492 - 499. [Abstract] [Full Text] [PDF]
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A. C Carr and B. Frei Toward a new recommended dietary allowance for vitamin C based on antioxidant and health effects in humans Am. J. Clinical Nutrition, June 1, 1999; 69(6): 1086 - 1107. [Abstract] [Full Text] [PDF]
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S. Voutilainen, J. D. Morrow, L. J. Roberts II, G. Alfthan, H. Alho, K. Nyyssonen, and J. T. Salonen Enhanced In Vivo Lipid Peroxidation at Elevated Plasma Total Homocysteine Levels Arterioscler Thromb Vasc Biol, May 1, 1999; 19(5): 1263 - 1266. [Abstract] [Full Text] [PDF]
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J. Huber, H. Boechzelt, B. Karten, M. Surboeck, V. N. Bochkov, B. R. Binder, W. Sattler, and N. Leitinger Oxidized Cholesteryl Linoleates Stimulate Endothelial Cells to Bind Monocytes via the Extracellular Signal-Regulated Kinase 1/2 Pathway Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 581 - 586. [Abstract] [Full Text] [PDF]
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