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Zinc supplementation inhibits lipid peroxidation and the development of
atherosclerosis in rabbits fed a high cholesterol diet

Andrew Jenner, Minqin Ren, Reshmi Rajendran, Pan Ning, Benny Tan
Kwong Huat, Frank Watt, Barry Halliwell

PII: S0891-5849(06)00762-3
DOI: doi: 10.1016/j.freeradbiomed.2006.11.024
Reference: FRB 8822

To appear in: Free Radical Biology and Medicine

Received date: 5 July 2006


Revised date: 3 October 2006
Accepted date: 24 November 2006

Please cite this article as: Andrew Jenner, Minqin Ren, Reshmi Rajendran, Pan Ning,
Benny Tan Kwong Huat, Frank Watt, Barry Halliwell, Zinc supplementation inhibits
lipid peroxidation and the development of atherosclerosis in rabbits fed a high cholesterol
diet, Free Radical Biology and Medicine (2006), doi: 10.1016/j.freeradbiomed.2006.11.024

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Zinc Supplementation Inhibits Atherosclerosis

Zinc supplementation inhibits lipid peroxidation and the development of atherosclerosis in rabbits
fed a high cholesterol diet

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Andrew Jenner1, Minqin Ren,2 Reshmi Rajendran,2 Pan Ning,1 Benny Tan Kwong Huat3, Frank Watt2

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and Barry Halliwell1

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1Department of Biochemistry, 3Department of Pharmacology, Yong Loo Lin School of Medicine and
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2Department of Physics, Centre for Ion Beam Applications, National University of Singapore,
Singapore 117597, bchjam@nus.edu.sg
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Acknowledgements - We are grateful to the BMRC Singapore for support through the grant
04/1/21/19/328
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Corresponding author: Dr Andrew Jenner


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Yong Loo Linn School of Medicine Department of Biochemistry,


National University of Singapore, MD7, 8 Medical Drive, Singapore 117597;
Tel: +65-68743240;
Fax: +65-67791453;
Email: bchjam@nus.edu.sg.

Running Title : Zinc Supplementation Inhibits Atherosclerosis

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Zinc Supplementation Inhibits Atherosclerosis

Abstract
Developing atherosclerotic lesions in hypercholesterolemic rabbits are depleted in zinc, whilst iron
accumulates. This study examined the influence of zinc supplementation on the development of

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atherosclerosis and used isotope dilution gas chromatography-mass spectrometry techniques to measure
biomarkers of oxidative lipid damage in atherosclerotic rabbit aorta. Our previous method for F2-

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isoprostane measurement was adapted to include the quantitation of cholesterol oxidation products in the
same sample.

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Two groups of New Zealand White Rabbits were fed a high cholesterol (1% w/w) diet and one group
was also supplemented with zinc (1 g/kg) for 8 weeks. Controls were fed normal diet. Zinc

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supplementation did not significantly alter the increase in total plasma cholesterol levels observed in
animals fed high cholesterol. However, in cholesterol fed animals zinc supplementation significantly
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reduced the accumulation of total cholesterol levels in aorta which was accompanied by a significant
reduction in average aortic lesion cross sectional areas of the animals. Elevated levels of cholesterol
oxidation products (5,6- α and β cholesterol epoxides, 7β-hydroxycholesterol, 7-ketocholesterol) in
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aorta and total F2-isoprostanes in plasma and aorta of rabbits fed cholesterol diet were significantly
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decreased by zinc supplementation.


Our data indicate that zinc has an anti-atherogenic effect, possibly due to a reduction in iron-catalysed
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free radical reactions.


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Key words: Zinc, F2-isoprostanes, lipid peroxidation, atherosclerosis, cholesterol, oxysterol.


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Abbreviations

OH - hydroxy, GC-MS - gas chromatography-mass spectrometry, COP – cholesterol oxidation


product,

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Introduction
Hypercholesterolemia is a major risk factor for atherosclerosis and coronary heart disease [1]. The
etiology of atherosclerosis appears to be a multifactorial series of events, but it is widely believed that

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lipoprotein oxidation is an important early event in the pathogenesis of atherosclerosis [2]. It is well
established that lipid components and their oxidative damage products accumulate during atherosclerotic

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lesion formation, including cholesterol [1-3], cholesterol oxidation products (oxysterols or COPs) [4],
hydroxy- and hydroperoxy- fatty acids [5-9], aldehydes [10] and F2-isoprostanes [11-14]. Although

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several oxidation products of cholesterol and fatty acids are biologically active eg, oxysterols [15],
hydroxy fatty acids, [16,17] and 8-isoprostaglandin-F2α [18,19], it is unknown which (if any) of these

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biomarkers of lipid peroxidation may also act as mediators of atherosclerosis [20].
Human atherosclerotic plaques contain iron and copper that may catalyse free radical formation [21,22]
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and in vitro studies have demonstrated that transition metal ions are required for LDL oxidation by
cultured endothelial cells [23], macrophages [24,25] and smooth muscle cells [26]. Since some
techniques used to measure metal ions can be confounded by possible artefacts generated during tissue
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processing, more advanced and minimally invasive techniques have been used in recent studies to
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quantitate the accumulation of iron and other metals in atherosclerotic lesions, such as electron
paramagnetic resonance (EPR) [27] and also nuclear microscopy [28-31], a technique for mapping and
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measuring trace elements in tissue sections.


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The authors have previously demonstrated using nuclear microscopy that during early development of
atherosclerosis in rabbits fed a high cholesterol diet there is accumulation of iron in the aortic lesion,
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compared to lesion-free artery wall, that is also accompanied by a depletion of zinc [30]. Indeed, for
each individual animal, the lesion iron concentrations were positively correlated with the lesion depth,
whereas the zinc concentrations were inversely correlated with lesion depth [30]. Most recently we have
demonstrated that dietary zinc supplementation decreases the accumulation of lesion iron and the
development of atherosclerosis in such rabbits [31]
Zinc is an essential component of a large number (>300) of enzymes and molecular structures and is
present in all body fluids and tissues. Total human body zinc content has been estimated to be 30 mmol
(2 g) and its turnover appears to be under very close homeostatic control [32]. Deficiency of this
micronutrient in the diet has been widely studied and can lead to growth failure, neuropathy, diarrhoea,
dermatitis, hypotension and hyperthermia [32,33]. Several studies have demonstrated that zinc
supplementation can reduce oxidative damage [24,34-37], although since zinc is not redox active it

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probably does not act directly as an antioxidant [38]. Zinc has also been observed in several studies to
have an anti-atherosclerotic effect [31,37,39-45] and has been inversely associated in epidemiological
studies with cardiovascular disease [46-48], but its mechanism of action is unclear.
The aim of the present study was to examine the role of zinc on lipid peroxidation in relation to

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atherosclerosis. The influence of dietary zinc supplementation on lesion development was examined in
rabbits fed a high cholesterol diet, and levels of several different biomarkers of lipid peroxidation were

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measured in aorta using isotope dilution gas chromatography-mass spectrometry techniques. Since
analysis of different biomarkers often requires several different protocols and consequently more tissue

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sample and time, we have adapted our F2-isoprostane extraction procedure [49] to include the extraction
of cholesterol and COPs prior to analysis.

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Materials and methods MA
Materials
Pentafluorobenzylbromide (PFBBr), N,N-diisopropylethylamine (DIPEA), cholesterol, 7-
ketocholesterol, 7β-OH cholesterol, cholesterol 5α,6α-epoxide, cholesterol 5β,6β-epoxide and 5α-
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cholestane were purchased from Sigma-Aldrich-Fluka (St. Louis, MO, USA) and of at least 98% purity.
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7-Keto cholesterol-d7, and 7β-OH cholesterol-d7 were purchased from C/D/N isotopes (Quebec,
Canada). Arachidonic acid, arachidonic acid-d8, 8-Iso-PGF2α (iPF2α-III or 15-F2t-IsoP), 8-iso-PGF2α-d4
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(iPF2α-III-d4) and iPF2α-VI-d4 (an 8-F2-isoprostane ) were purchased from Cayman Chemical (Ann
Arbor, MI USA). Formic acid (Lancaster, England), ammonium hydroxide, potassium hydroxide,
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butylated hydroxytoluene (BHT), hydrochloric acid (Merck, Darmstadt, Germany), and hexane (Tedia,
OH, USA) were of analytical grade. Methanol (EM Science, Darmstadt, Germany) and ethyl acetate
(Fisher Scientific UK) were of HPLC grade. N,O-bis(trimethylsilyl)trifluoroacetamide +1%
trimethylchlorosilane (BSTFA+1% TMCS) was obtained from Pierce Chemicals (Rockford, IL USA).
Oasis Mixed Anion Exchange (MAX) cartridges were from Waters Corp. Distilled water passed through
a purification system (Elga, High Wycombe, Bucks.) was used to make up all solutions.
Animals
Animal treatment has been described previously [31]. Briefly New Zealand white rabbits (mean 2.5 kg)
were fed for 8 wk with either (1) normal diet [GPR] n=6, (2) high cholesterol diet [GPR + 1%
cholesterol] n=6, or (3) zinc-supplemented high cholesterol diet [GPR + 1% Cholesterol + 1000 ppm (1
g/kg of diet) zinc as zinc carbonate] n=5. The level of zinc in normal and high cholesterol diet are 60
and 50mg/kg diet respectively. After sacrifice the aortic arch was removed, cut into three segments,

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flushed with deionized water, and flash frozen in liquid nitrogen [31]. Blood samples were taken for
clinical chemical analysis. Plasma was prepared for F2-isoprostane assay as previously described [49]
and cholesterol measured by routine clinical enzymatic assay. This study was approved by the NUS
local Animal Care and Use committee. Analysis of artery and blood was carried out as previously

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described [31]. Aortic arch segments were sectioned, stained with hematoxylin and eosin and the lesion
areas analysed by light microscopy. Blood parameters included: hemoglobin, total red and white blood

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cells, platelet count, total cholesterol, total triglycerides, LDL, blood and plasma zinc.
Lipid extraction and hydrolysis

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Lipids were extracted from whole segments of aorta (approximately 4-6 mm long and 30-50 mg). Entire
artery was homogenised in 1 ml phosphate buffered saline (pH 7.4) and 4 ml organic solvent mixture

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(CHCl3/methanol 2:1 v/v + 0.005% BHT) at 4°C. After centrifugation at 1,000 g for 10 min the lower
organic layer was carefully transferred to a glass vial and dried under a stream of N2.
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1ml of 1M KOH (in pure methanol) was added to a glass vial containing either 1 ml plasma or the dried
lipid extract with 1ml PBS. Heavy isotopic F2-isoprostane internal standards (1 ng type VI-d4 and 0.5 ng
type III-d4 in 20 ul ethanol) were also added, together with heavy isotopic standards of arachidonic acid
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(0.5ug), COPs (20 ng of 7-keto cholesterol-d7 and 7β-OH cholesterol-d7) and 20 ug 5α-cholestane and
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then sealed under N2.


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After hydrolysis at 23°C for 2 h in the dark, the sample was cooled and 0.5 ml methanol added, then
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neutralised with 5 M HCl. Finally 2.7 ml of 40 mM (pH 4.0) formic acid was added and the mixture
centrifuged at 12,000 g for 10 min to pellet and remove any protein/precipitate before column loading.
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Solid phase extraction was carried out as described previously for F2-isoprostanes in urine/plasma [49]
with certain additional fractions being retained for cholesterol and COP analysis. Briefly a 60mg MAX
(mixed ion exchange, Waters) column was preconditioned with 2 ml methanol and then 2 ml 20 mM
formic acid (pH 4.0). The extract was then loaded and the column was washed with 2 ml 2% ammonium
hydroxide, followed by 2 ml methanol/20 mM formic acid pH 4.0 (40/60) and subsequently dried for 2
min. Cholesterol and oxidized sterols were eluted with 2 ml hexane followed by 2 ml ethyl
acetate/hexane (30/70) and 40 µl of the extract was aliquoted into a separate glass vial for cholesterol
analysis. Finally, arachidonic acid and F2-isoprostanes were eluted from the SPE column with 1.8 ml
ethyl acetate and then all extracts were dried under N2 gas.
GC-MS quantification of cholesterol and cholesterol oxidation products (COPs)
Cholesterol and COPs were derivatized at 40°C for 1 h with 40 ul pyridine plus 40 ul BSTFA+1%
TMCS to their trimethylsilyl ethers. Derivatives for COP analysis were dried under N2 gas and

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reconstituted in 40 ul undecane. Cholesterol and COPs were both analyzed by a Hewlett-Packard 5973
mass selective detector interfaced with a Hewlett-Packard 5890II gas chromatograph and equipped with
an automatic sampler and a computer workstation. Separations were carried out on a fused silica
capillary column (12 m x 0.2 mm i.d.) coated with cross-linked 5% phenylmethylsiloxane (film

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thickness 0.33 µm) (Ultra2, Agilent, J and W). The quadrupole and ion source of the MS were
maintained at 150°C and 230°C respectively. For cholesterol quantitation, derivatized samples (1 µl)

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were injected with a 25:1 split into the GC injection port (280°C). Column temperature was increased
from 240°C to 300°C at 25°C/min after 1 min at 240°C, and then held at 300°C for 4 min. Cholesterol

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(retention time 4.25 min) was monitored using m/z 329 as target ion and m/z 458, 453 as qualifier ions
and 5α-cholestane was monitored as internal standard (retention time = 3.45 min, target ion =m/z 357,

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qualifier ions = m/z 372, 232). For COP quantitation, derivatized samples (1 µl) were injected splitless
into the GC injection port (280°C). Column temperature was increased from 160°C to 300°C at
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40°C/min after 1 min at 160°C, then held at 300°C for 6 min. The carrier gas was helium with a flow
rate of 0.8 ml/min (average velocity = 55 cm/sec). Selected-ion monitoring was performed using the
electron ionization (EI) mode at 70 eV to monitor one target ion and 2 qualifier ions selected from each
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compound’s mass spectrum to optimize sensitivity and specificity. Quantification of oxysterols was
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calculated by comparison with their heavy isotopes except for the 5,6-cholesterol epoxides which used
7β-OH cholesterol d7 . Cholesterol was calculated using 5α-cholestane as internal standard. Relative
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molar response factors for each analyte were calculated from calibration curves constructed from 5
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different concentrations in triplicate of cholesterol (10 to 500 ug) as well as oxysterols (2-500 ng), and
showed good linearity (r2 > 0.98).
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GC-MS quantification of total F2-isoprostanes and arachidonic acid


For F2-isoprostane and arachidonic acid analysis the acid moiety was derivatized with 30 ul of
pentafluorobenzylbromide (PFBBr) [10% in acetonitrile] and 15 ul of N,N-diisopropylethylamine
(DIPEA) [10% in acetonitrile] at 23°C for 30 min. Excess reagents were evaporated under N2. The F2-
isoprostane PFBenzyl ester was derivatized with 15 ul acetonitrile plus 30 ul BSTFA+1% TMCS for 1 h
at 23°C, dried under N2 gas and reconstituted in 40 ul isooctane. The arachidonic acid PFBenzyl ester is
stable and does not undergo further derivatisation. Derivatized samples were analysed by a Hewlett-
Packard 5973/ 6890 GC-MS equipped with an automatic sampler and a computer workstation. The
injection port and GC-MS interface were kept at 280°C and 300°C, respectively. Separations were
carried out on a fused silica capillary column (30 m x 0.2 mm i.d.) coated with cross-linked 5%
phenylmethylsiloxane (film thickness 0.33 µm), (Agilent, J and W). Helium was the carrier gas with a

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flow rate of 1 ml/min (average velocity = 59cm/sec). Derivatized samples (2 µl) were injected splitless
into the GC injection port. Column temperature was increased from 170°C to 275°C at 30°C/min after 1
min at 170°C, then held at 275°C for 14 min Finally temperature was raised to 300°C at 30°C/min and
held at 300°C for 2 min. Selected-ion monitoring was performed using the negative chemical ionization

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(NCI) mode at 70eV using methane as the reagent gas (2 ml/min) with the ion source maintained at
150°C and the quadrupole at 106°C. Selected ion monitoring was performed to monitor the carboxylate

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ion (M-181: loss of CH2C6F5) at ions m/z 569 for iPF2α-III and iPF2α-VI and at m/z 573 for their
corresponding isotopic d4 labelled internal standards. Selected ion monitoring at a much reduced

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electron multiplier voltage was also performed to monitor the carboxylate ion at m/z 303 for arachidonic
acid and at m/z 311 for the corresponding isotopic d8 labelled internal standards. Quantitation was

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achieved by relating the total peak area of the F2-isoprostanes with its corresponding internal standard
peak.
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Statistical analysis
One-way ANOVA and unpaired Student's t-test were performed and associations were studied using
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Pearson correlation coefficients. The coefficient of variation (CV) for each analyte was calculated by
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analysing 8 aliquots of an identical tissue (50 mg) lipid extract. Recovery of each analyte after
hydrolysis and solid phase extraction was calculated by comparing heavy standard analytes in samples
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with heavy standards of identical concentration not subjected to hydrolysis or solid phase extraction
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(n=8). Limit of detection (LOD) was defined as the lowest concentration of heavy isotope standard that
could be detected in a tissue lipid extract by GC-MS with a peak > 5 signal:noise ratio. LOD for
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cholesterol epoxides was estimated using 7β-OH cholesterol- d7 as a calibration standard.

Results
Data regarding animal weight, blood levels of hemoglobin, triglycerides, lipoproteins, platelets, red and
white blood cells as well as levels of zinc and iron have been reported elsewhere [31]. In summary all
rabbits gained weight with no significant difference in food intake due to zinc supplementation. Blood
zinc levels were significantly raised in cholesterol fed animals when supplemented with zinc, but plasma
levels were not. Zinc supplementation had no significant effect on levels of LDL or plasma triglyceride,
which were significantly raised by high cholesterol diet. After 8 wk of a high cholesterol diet, levels of
total plasma cholesterol were greatly increased in the animals fed high cholesterol diet (Fig 1A). High
cholesterol rabbits supplemented with zinc had a trend to lower plasma cholesterol levels than the non-
supplemented hypercholesterolemic group, but this was not statistically significant. Similar to plasma,

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total arterial wall cholesterol levels were also significantly elevated in high cholesterol fed rabbits (Fig
1B), but in contrast to plasma levels supplementation with zinc significantly reduced this elevation.
Atherosclerotic lesions were only detected in rabbits fed the high cholesterol diet as expected from
previous studies [29]. The average lesion cross sectional areas of the zinc supplemented animals were

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significantly decreased compared with lesion areas of the animals fed only on the high cholesterol diet
(Fig 2A, data reproduced from [31] with permission).

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Alkaline hydrolysis for 2 h under argon at 23°C has been reported to eliminate artifactual oxidation of
cholesterol and decomposition of COPs that were observed in other lipid hydrolysis protocols [50].

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Analysis of cholesterol and COP standards in our study confirmed that our alkaline hydrolysis
conditions with nitrogen flushing, did not suffer any artifacts. Furthermore, comparison of F2-

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isoprostane levels in identical plasma and tissue samples hydrolysed at 23°C for 2 h and 37°C for 30
min (conditions used previously by us [49]) illustrated no significant effect of hydrolysis conditions on
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F2-isoprostane analysis and no formation of F2-isoprostane from arachidonic acid-d8 was detected.
Recovery of total tissue F2-isoprostane and cholesterol oxidation products was calculated to be 49.8%
and 91.4-97.1% respectively (Table 1). Sensitive and accurate analytical quantitation was verified by
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spiking arterial sample homogenates with analytical standards and calculation of CV (Table 1). Levels
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of total F2-isoprostanes in plasma and atherosclerosed arterial wall were significantly elevated in rabbits
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fed high cholesterol diet compared to non treated animals (Fig 2A and B), but this was significantly
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decreased by zinc supplementation. Arachidonic acid levels in artery wall were not significantly
changed in hypercholesterolemic rabbits (data not shown), but plasma levels were significantly elevated
in rabbits fed cholesterol both with or without Zn supplementation by 46 and 45% respectively. Total
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plasma F2-isoprostanes expressed as umol/mol arachidonic acid were also significantly elevated
(p<0.05) in rabbits fed high cholesterol diet (218 ± 97) compared to non treated animals (103 ± 59) and
this was significantly reduced (p<0.05) by zinc supplementation (70 ± 52)
Control levels of cholesterol oxidation products in complete arterial wall were also significantly elevated
in rabbits fed high cholesterol (Fig 3) and these increases were again prevented by zinc supplementation.
When expressed as umol COP/mol cholesterol only cholesterol 5ß,6ß-epoxide was significantly elevated
in hypercholesterolemic animals compared to controls, but zinc supplementation significantly reduced
all cholesterol oxidation products (Table2). Table 3 illustrates the strength and significance of
correlation calculated between different parameters of all rabbits from the 3 diet groups. Lesion area
analysis in hypercholesterolemic rabbits excluded a single extreme, outlying data point, where lesion
size was outside the range of ± 3.5 standard deviations. Lesion size was strongly correlated to

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cholesterol levels in plasma and to a lesser extent in artery wall, but was not significantly correlated to
COPs and F2-isoprostanes in either plasma or vessel wall. COPs and F2-isoprostanes were correlated to
each other and artery cholesterol oxidation was strongly correlated to cholesterol levels in plasma and
artery. Artery cholesterol was significantly correlated to F2-Isoprostanes in plasma, but not artery.

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Discussion

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F2-Isoprostanes, derived from oxidation of arachidonic acid are considered to be reliable markers of
oxidative stress in vivo and can be detected in biological fluids and tissues as biomarkers of lipid

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peroxidation [11-14]. By adapting our solid phase extraction procedure that was originally developed to
purify the F2-isoprostane fraction in human biological fluids [49], we were also able to extract

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arachidonic acid, cholesterol and COPs. Hence these different lipid oxidation biomarkers can be
measured sensitively and accurately by GC-MS conveniently from the same tissue lipid extract.
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In agreement with previous studies, our data demonstrate that cholesterol [1,2,15], cholesterol oxidation
products (COPs) [4,15], and F2-isoprostanes [11-14] accumulate in arterial walls during atherosclerotic
lesion formation. In this study, biomarkers of lipid peroxidation were measured in a complete segment
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of aorta rather than isolated atherosclerotic lesions and consequently levels might be an underestimate,
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since atherosclerotic lesions have been reported to contain much higher levels of extensively oxidized
lipid compared to healthy artery wall [4-15]. Levels of arterial wall COPs were elevated much more than
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F2-isoprostane levels. Expression of biomarkers relative to their target lipid molecule indicates that the
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increase of arterial wall COP’s in this study is largely influenced by the increase in cholesterol rather
than a significant increase in the proportion of cholesterol oxidized (Table 2), whereas F2-isoprostanes
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formation was due to significant increases in the proportion of arachidonic acid that was oxidized.
A previous study of oxidized arachidonic acid in human carotid plaques found that F2-isoprostanes
levels were much lower than hydoxyeicosatetraenoic acids (HETEs) [11]. The most abundant oxidation
products reported in human plaque and LDL are hydroxy- and oxo-octadecadienoic acids (HODEs and
oxoODEs) that are oxidation products of linoleic acid which makes up to 45% of LDL polyunsaturated
fatty acids [5-7]. Atherosclerotic plaques are dynamic and the amount and nature of lipid damage
changes during plaque development [2,3,8,11] so a true comparison of atherosclerotic lipid oxidation
biomarkers needs to be carried out in the same tissue samples as done here. It is well established that
elevated plasma cholesterol plays a dominant role in atherosclerosis and in the present study lesion
growth was strongly correlated to cholesterol levels in plasma and to a lesser extent in artery wall.
However, lesion size was not significantly correlated to any biomarker of lipid peroxidation, suggesting

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that in our animal model the development of early stage lesions may be more dependent on the
accumulation of cholesterol compared to lipid oxidation. However, our analysis involves only a small
dataset and other products of fatty oxidation products were not examined.
Few studies have compared different biomarkers of lipid damage in the same tissue sample and it is still
unknown whether lipid oxidation products are involved in the pathogenesis of atherosclerosis, or exist

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only as products of secondary oxidative events. Interestingly, in common with our study, serum

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cholesterol levels also did not correlate with higher levels of plasma F2-isoprostanes measured in human
hypercholesterolemics [14,51].

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Our data show that zinc supplementation significantly reduces both atheroma formation [31] and lipid
peroxidation in plasma and arterial wall during atherosclerosis in hypercholesterolemic rabbits,

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suggesting that the anti-atherogenic action of zinc may involve antioxidant actions. This is consistent
with previous studies of zinc supplementation in animal models of atherosclerosis that have observed a
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decrease in atherosclerotic lesion formation [37] or atherosclerotic markers [43] accompanied by a
decrease in plasma levels of lipid oxidation biomarkers. Although a decrease in plasma F2-isoprostane
levels was reported in the latter study no data were provided [43]. The mechanism by which zinc
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reduces the plasma level of circulating F2-isoprostanes is unclear at present and also whether this may be
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a factor involved in zinc’s anti-atherogenic activity. The fact that plasma F2-isoprostane levels correlate
strongly with levels of F2-isoprostanes in arterial wall suggests that a significant proportion of
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circulating F2-isoprostanes may come out of the atherosclerotic lesion into plasma. If this is the case,
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then the decrease in lesion formation by zinc may explain the decrease in plasma F2-isoprostane levels
as well. Since zinc is not redox active it may not itself act directly as a scavenging antioxidant [38]. For
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example zinc was unable to prevent liposome oxidation by a variety of oxidant species [36]. However,
iron induced lipid oxidation [33-35] and modification of LDL by macrophages in the presence of iron
ions [41] are inhibited by zinc, possibly due to the displacement of iron from negatively charged binding
sites in lipid or protein. These data indicate that zinc may act as an indirect antioxidant by competing
with pro-oxidant metals such as iron and copper for strategic binding sites and reducing their formation
of reactive oxygen species, which in turn decreases oxidative damage and delays atherosclerosis.
Expression of biomarkers relative to their target lipid molecule indicates that zinc significantly decreases
the proportion of cholesterol and arachidonic acid that is oxidized, accentuating its antioxidant activity.
Data from our study also demonstrate that during atherosclerosis zinc significantly reduces the
accumulation of cholesterol into the artery, but does not significantly alter elevated plasma levels of
cholesterol, LDL or triglycerides [31]. Reduced aortic cholesterol accumulation in the zinc
supplemented rabbits would also likely play a role in reducing lesion formation and inhibiting the rate of
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lipid damage. However, the relative decrease of cholesterol oxidation was far greater than that of arterial
cholesterol, suggesting that the inhibition of lipid oxidation processes may be more important for zinc’s
anti-atherogenic effect. It is not known whether zinc acts directly to reduce lesion formation or indirectly
by inhibiting accumulation of cholesterol and or oxidized lipid.

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Nuclear microscope analysis, illustrates that decreased atherosclerotic lesion formation in zinc
supplemented animals from this study was accompanied by a significant depletion in iron levels in both

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lesion and the adjacent smooth muscle wall [31], but with no obvious changes in zinc levels [31]. The
important role of iron in atherosclerosis and its balance against zinc is also highlighted by our previous

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studies demonstrating depletion of zinc in newly formed lesions compared with adjacent healthy artery
wall tissue, and that lesion formation is reduced by depletion of iron in the lesion, induced by either

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controlled anemia [29] or chelation treatment [52]. Currently not much is known about zinc homeostasis
except that it is closely regulated and there are no recognized tissue storage sites [33]. In this study zinc
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supplements 20 fold higher than normal were used, but we have not established the threshold
concentration of zinc in diet that is necessary to significantly reduce atherosclerosis.
Our research, indicates that zinc supplementation has an anti-atherosclerotic role and prevents arterial
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lipid peroxidation, supporting the hypothesis that zinc acts as an indirect antioxidant, possibly by
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displacing redox active metal ions and reducing iron-dependent lipid peroxidation. Since some groups of
the population have deficient dietary intake or lower tissue levels of zinc [32,33,45-48,53] reasonable
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supplementation of zinc in the diet of humans at risk of atherosclerosis should be considered as a strong
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candidate for further clinical investigation.


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[4] Iuliano, L.; Micheletta, F.; Natoli, S.; Ginanni Corradini, S.; Iappelli, M.; Elisei, W.; Giovannelli,
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Table and figure legends

Table 1. Sensitivity, variability and recovery of lipid peroxidation biomarkers in tissue (50 mg) lipid
extracts after hydrolysis and solid phase extraction using GC-MS. Coefficient of variation (CV) and

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recovery of each analyte were calculated from 8 samples. Limit of detection (LOD) was defined as GC-
MS peak > 5 signal:noise ratio. (* estimated using 7β-OH cholesterol d7 as a calibration standard)

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Figure 1 A and B. Total cholesterol levels were measured in plasma (A) and atherosclerosed arterial

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segments (B) of rabbits after 8 weeks diet manipulation. Rabbits were fed either control diet (non-
treated, n = 5), + 1% cholesterol (cholesterol, n = 6) or + 1% cholesterol + zinc [1000 ppm or 1g/kg

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zinc carbonate] (cholesterol + Zn, n = 5). Statistical significance was calculated using one-way ANOVA
and Student's t-test. * p<0.05 or p<0.01 compared to cholesterol fed animals.
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Figure 2A. Atherosclerotic lesion areas measured in aorta of rabbits after 8 weeks diet manipulation.
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Rabbits were fed either control diet (non-treated, n = 5), + 1% cholesterol (cholesterol, n = 6) or + 1%
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cholesterol + zinc [1000 ppm or 1g/kg zinc carbonate] (cholesterol + Zn, n = 5). Statistical significance
was calculated using one-way ANOVA and Student's t-test. * p<0.05 or **p<0.01 compared to
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cholesterol fed animals.


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Figure 2 B and C. Total F2-isoprostane levels were measured in plasma (B) and atherosclerosed arterial
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segments (C) of rabbits after 8 weeks diet manipulation. Rabbits were fed either control diet (non-
treated, n = 5), + 1% cholesterol (cholesterol, n = 6) or + 1% cholesterol + zinc [1000 ppm or 1g/kg
zinc carbonate] (cholesterol + Zn, n = 5). Statistical significance was calculated using one-way ANOVA
and Student's t-test. * p<0.05 or **p<0.01 compared to cholesterol fed animals.

Figure 3. Total levels of cholesterol oxidation products (COPs) were measured in atherosclerosed
arterial segments of rabbits after 8 weeks diet manipulation. Rabbits were fed either control diet (non-
treated, n = 5), + 1% cholesterol (cholesterol, n = 6) or + 1% cholesterol + zinc [1000 ppm or 1g/kg
zinc carbonate] (cholesterol + Zn, n = 5). Statistical significance was calculated using one-way ANOVA
and Student's t-test. * p<0.05 or **p<0.01 compared to cholesterol fed animals.

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Table 2. Total levels of cholesterol oxidation products (COPs) expressed as umol/mol cholesterol.
Rabbits were fed either control diet (non-treated, n = 5), + 1% cholesterol (cholesterol, n = 6) or + 1%
cholesterol + zinc [1000 ppm or 1g/kg zinc carbonate] (cholesterol + Zn, n = 5). Statistical significance
was calculated using one-way ANOVA and Student's t-test. * p<0.05 or **p<0.01 compared to

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cholesterol fed animals.

Table 3. Correlation between atherosclerotic lesion size, cholesterol and lipid oxidation biomarkers in

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plasma and atherosclerosed arterial segments of rabbits after 8 weeks diet manipulation. Correlation

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coefficient (r) and statistical significance (* p<0.05, **p<0.01 or ***p<0.001) was calculated using all
rabbits from the 3 diet groups. Atherosclerotic lesion area analysis in hypercholesterolemic rabbits

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excluded a single data point, where lesion size was outside the range of ± 2 standard deviations. Rabbits
were fed either control diet (non-treated, n = 5), + 1% cholesterol (cholesterol, n = 6) or + 1%
cholesterol + zinc [1000 ppm or 1g/kg zinc carbonate] (cholesterol + Zn, n = 5).
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