atherosclerosis zinc

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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 inhibitslipid 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

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.
http://dx.doi.org/10.1016/j.freeradbiomed.2006.11.024http://dx.doi.org/10.1016/j.freeradbiomed.2006.11.024http://dx.doi.org/10.1016/j.freeradbiomed.2006.11.024http://dx.doi.org/10.1016/j.freeradbiomed.2006.11.024


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ACCEPTED MANUSCRIPTZinc Supplementation Inhibits Atherosclerosis

1

Zinc supplementation inhibits lipid peroxidation and the development of atherosclerosis in rabbits

fed a high cholesterol diet

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

and Barry Halliwell1

1Department of Biochemistry, 3Department of Pharmacology, Yong Loo Lin School of Medicine and

2Department of Physics, Centre for Ion Beam Applications, National University of Singapore,

Singapore 117597, [email protected]

Acknowledgements - We are grateful to the BMRC Singapore for support through the grant

04/1/21/19/328

Corresponding author: Dr Andrew Jenner

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: [email protected].

Running Title : Zinc Supplementation Inhibits Atherosclerosis


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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

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

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

same sample.

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

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

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- and cholesterol epoxides, 7-hydroxycholesterol, 7-ketocholesterol) in

aorta and total F2-isoprostanes in plasma and aorta of rabbits fed cholesterol diet were significantly

decreased by zinc supplementation.

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

free radical reactions.

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

Abbreviations

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

product,


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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

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

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

sample and time, we have adapted our F2-isoprostane extraction procedure [49] to include the extraction

of cholesterol and COPs prior to analysis.

Materials and methods

Materials

Pentafluorobenzylbromide (PFBBr), N,N-diisopropylethylamine (DIPEA), cholesterol, 7-

ketocholesterol, 7-OH cholesterol, cholesterol 5,6-epoxide, cholesterol 5,6-epoxide and 5-

cholestane were purchased from Sigma-Aldrich-Fluka (St. Louis, MO, USA) and of at least 98% purity.

7-Keto cholesterol-d7, and 7-OH cholesterol-d7 were purchased from C/D/N isotopes (Quebec,

Canada). Arachidonic acid, arachidonic acid-d8, 8-Iso-PGF2 (iPF2-III or 15-F2t-IsoP), 8-iso-PGF2-d4

(iPF2-III-d4) and iPF2-VI-d4 (an 8-F2-isoprostane ) were purchased from Cayman Chemical (Ann

Arbor, MI USA). Formic acid (Lancaster, England), ammonium hydroxide, potassium hydroxide,

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

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

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

Lipid extraction and hydrolysis

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

(CHCl3/methanol 2:1 v/v + 0.005% BHT) at 4C. 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.

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

(0.5ug), COPs (20 ng of 7-keto cholesterol-d7 and 7-OH cholesterol-d7) and 20 ug 5-cholestane and

then sealed under N2.

After hydrolysis at 23C for 2 h in the dark, the sample was cooled and 0.5 ml methanol added, then

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.

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 l 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 40C 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

thickness 0.33 m) (Ultra2, Agilent, J and W). The quadrupole and ion source of the MS weremaintained at 150C and 230C respectively. For cholesterol quantitation, derivatized samples (1 l)

were injected with a 25:1 split into the GC injection port (280C). Column temperature was increased

from 240C to 300C at 25C/min after 1 min at 240C, and then held at 300C for 4 min. Cholesterol

(retention time 4.25 min) was monitored using m/z 329 as target ion and m/z 458, 453 as qualifier ions

and 5-cholestane was monitored as internal standard (retention time = 3.45 min, target ion =m/z 357,

qualifier ions = m/z 372, 232). For COP quantitation, derivatized samples (1 l) were injected splitless

into the GC injection port (280C). Column temperature was increased from 160C to 300C at

40C/min after 1 min at 160C, then held at 300C 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

compounds mass spectrum to optimize sensitivity and specificity. Quantification of oxysterols was

calculated by comparison with their heavy isotopes except for the 5,6-cholesterol epoxides which used

7-OH cholesterol d7 . Cholesterol was calculated using 5-cholestane as internal standard. Relative

molar response factors for each analyte were calculated from calibration curves constructed from 5

different concentrations in triplicate of cholesterol (10 to 500 ug) as well as oxysterols (2-500 ng), and

showed good linearity (r2

> 0.98).

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 23C 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 23C, 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 280C and 300C, 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 m), (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 l) were injected splitless

into the GC injection port. Column temperature was increased from 170C to 275C at 30C/min after 1

min at 170C, then held at 275C for 14 min Finally temperature was raised to 300C at 30C/min and

held at 300C for 2 min. Selected-ion monitoring was performed using the negative chemical ionization

(NCI) mode at 70eV using methane as the reagent gas (2 ml/min) with the ion source maintained at150C and the quadrupole at 106C. Selected ion monitoring was performed to monitor the carboxylate

ion (M-181: loss of CH2C6F5) at ions m/z 569 for iPF2-III and iPF2-VI and at m/z 573 for their

corresponding isotopic d4 labelled internal standards. Selected ion monitoring at a much reduced

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

achieved by relating the total peak area of the F2-isoprostanes with its corresponding internal standard

peak.Statistical analysis

One-way ANOVA and unpaired Student's t-test were performed and associations were studied using

Pearson correlation coefficients. The coefficient of variation (CV) for each analyte was calculated by

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

with heavy standards of identical concentration not subjected to hydrolysis or solid phase extraction

(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

cholesterol epoxides was estimated using 7-OH cholesterol- d7as 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

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

Alkaline hydrolysis for 2 h under argon at 23C has been reported to eliminate artifactual oxidation of

cholesterol and decomposition of COPs that were observed in other lipid hydrolysis protocols [50].

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-

isoprostane levels in identical plasma and tissue samples hydrolysed at 23C for 2 h and 37C for 30

min (conditions used previously by us [49]) illustrated no significant effect of hydrolysis conditions on

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

spiking arterial sample homogenates with analytical standards and calculation of CV (Table 1). Levels

of total F2-isoprostanes in plasma and atherosclerosed arterial wall were significantly elevated in rabbits

fed high cholesterol diet compared to non treated animals (Fig 2A and B), but this was significantly

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

plasma F2-isoprostanes expressed as umol/mol arachidonic acid were also significantly elevated

(p


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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.

Discussion

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

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

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.

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

of aorta rather than isolated atherosclerotic lesions and consequently levels might be an underestimate,

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

F2-isoprostane levels. Expression of biomarkers relative to their target lipid molecule indicates that the

increase of arterial wall COPs 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

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

cholesterol levels also did not correlate with higher levels of plasma F2-isoprostanes measured in human

hypercholesterolemics [14,51].

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,

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

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

reduces the plasma level of circulating F2-isoprostanes is unclear at present and also whether this may be

a factor involved in zincs 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

circulating F2-isoprostanes may come out of the atherosclerotic lesion into plasma. If this is the case,

then the decrease in lesion formation by zinc may explain the decrease in plasma F 2-isoprostane levels

as well. Since zinc is not redox active it may not itself act directly as a scavenging antioxidant [38]. For

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 zincs

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.

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

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

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

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

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

lipid peroxidation, supporting the hypothesis that zinc acts as an indirect antioxidant, possibly by

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

supplementation of zinc in the diet of humans at risk of atherosclerosis should be considered as a strong

candidate for further clinical investigation.

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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

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 7-OH cholesterol d7as a calibration standard)

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

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

zinc carbonate] (cholesterol + Zn, n = 5). Statistical significance was calculated using one-wayANOVA

and Student's t-test. * p


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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


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