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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1616-1624.)
© 1995 American Heart Association, Inc.

Articles

Human Atherosclerotic Plaque Contains Both Oxidized Lipids and Relatively Large Amounts of -Tocopherol and Ascorbate

Cacang Suarna; Roger T. Dean; James May; Roland Stocker

From the Biochemistry (C.S., R.S.) and Cell Biology (R.T.D.) Groups, The Heart Research Institute, and the Department of Vascular Surgery (J.M.), Royal Prince Alfred Hospital, Camperdown, Australia.

Correspondence to Dr Roland Stocker, The Heart Research Institute, 145 Missenden Rd, Camperdown, NSW 2050, Australia.

	Abstract

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Abstract We assessed the antioxidant status and contents of unoxidized and oxidized lipids in freshly obtained, homogenized samples of both normal human iliac arteries and carotid and femoral atherosclerotic plaque. Optimal sample preparation involved homogenization of human atherosclerotic plaque for 5 minutes, which resulted in recovery of most of the unoxidized and oxidized lipids without substantial destruction of endogenous vitamins C and E and 87% and 43% recoveries of added standards of -tocotrienol and isoascorbate, respectively. The total protein, lipid, and antioxidant levels obtained from human plaque varied among donors, although the reproducibility of replicates from a single sample was within 3%, except for ubiquinone-10 and ascorbate, which varied by 20% and 25%, respectively. Plaque samples contained significantly more ascorbate and urate than control arteries, with no discernible difference in the vitamin C redox status between plaque and control materials. The concentrations of -tocopherol and ubiquinone-10 were comparable in plaque samples and control arteries. However, approximately 9 mol percent of plaque -tocopherol was present as -tocopherylquinone, whereas this oxidation product of vitamin E was not detectable in control arteries. Coenzyme Q₁₀ in plaque and control arteries was only detected in the oxidized form ubiquinone-10, although coenzyme Q₁₀ oxidation may have occurred during processing. The most abundant of all studied lipids in plaque samples was free cholesterol, followed by cholesteryl oleate and cholesteryl linoleate (Ch18:2). Approximately 30% of plaque Ch18:2 was oxidized, with 17%, 12%, and 1% present as fatty acyl hydroxides, ketones, and hydroperoxides, respectively. In comparison, 7-ketocholesterol was detected at an 75-fold lower concentration. Normal arteries contained similar levels of protein as atherosclerotic arteries, much less free cholesterol, and no detectable amounts of unoxidized or oxidized cholesteryl esters. Together, these results demonstrate the coexistence in human plaque of large amounts of oxidized cholesteryl esters with significant concentrations of ascorbate and vitamin E in their reduced, antioxidant-active form. We conclude that compared with healthy human arteries, advanced atherosclerotic plaques are not deficient in the antioxidant vitamins C and E, despite the occurrence of massive lipid oxidation.

Key Words: antioxidants • atherosclerosis • oxidative stress • 7-ketocholesterol • vitamin E

	Introduction

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In recent years the oxidation theory of atherogenesis has been influential. This theory envisages oxidation of LDL as a key and early event responsible for the loading of lipid into atherosclerotic foam cells and possibly for many other features of atherogenesis (for review, see References 1 through 3^{1 2 3} ). Because plasma contains a multitude of antioxidant defenses,^{4 5} and there is little direct evidence of oxidized LDL in the circulation,⁶ it is generally considered that LDL oxidation occurs in the intimal space. In the light of this hypothesis, it is critical to obtain a clear knowledge of the state of oxidation of proteins, lipids, and antioxidants in plaque.

Because modification of the protein moiety of LDL, apoB-100, is essential for the generation of at least some high-uptake forms of LDL, several studies have considered the status of apoB in lesion-free aortic intima and atherosclerotic plaque. Ylä-Herttuala and coworkers^{7 8} have shown that mildly extracted LDL from lesion-free aortas has an increased electrophoretic mobility in agarose gels, with no fragmentation of apoB. In contrast, whereas LDL isolated from minced aortas containing advanced atherosclerotic plaques also demonstrates increased electrophoretic mobility, this LDL showed degradation of apoB.^{8 9 10 11 12} Lesion LDL also cross-reacted with antibodies raised against malondialdehyde- and 4-hydroxynonenal–modified apoB, was chemotactic for monocytes, and showed increased degradation by macrophages,^{8 10 11} thus sharing properties with in vitro copper-oxidized LDL. There is some controversy regarding the extent of oxidative modification of apoB of lesion LDL. While earlier studies provide evidence supporting uptake of at least some lesion LDL by the scavenger receptor,^{8 9 10} Steinbrecher and Lougheed¹¹ found that the extent of oxidative modification of aortic-lesion LDL apoB as assessed by electrophoretic mobility and protein fragments was less than that required for scavenger receptor–mediated uptake. However, neither an increased electrophoretic mobility of LDL nor increased apoB fragmentation are direct proof of oxidative modification of the (lipo)protein.

It is commonly assumed that LDL lipid oxidation precedes¹ and, in fact, can contribute to oxidative modification of apoB¹³ (see, however, Reference 14¹⁴ ). Many early studies have demonstrated oxidized lipids in human plaque samples. Probably the first was that of Glavind et al,¹⁵ who identified (unspecified) lipid hydroperoxides in human atherosclerotic aorta. Subsequently, isomers of hydroxy and hydroperoxy cholesterol derived from cholesterol oxidation were detected in esterified and free forms.^{16 17 18 19 20 21 22} Similarly, free and esterified oxidized fatty acids have been detected in human atherosclerotic plaque,^{18 21 22 23 24 25 26} and there are some indications that (lipid) hydroperoxides may increase in parallel with the extent of disease progression¹⁵ (see, however, Reference 27²⁷ ). Studies of oxidized lipid composition of plaque from atherosclerotic rabbits concur with these observations.²⁸

It is well recognized that LDL contains a number of nonproteinaceous antioxidants that attenuate the oxidative modification of its lipids and protein.^{3 29} In addition, LDL is surrounded by extracellular fluid that likely contains many different antioxidants in the form of metal-chelating proteins, enzymes, and water-soluble nonproteinaceous antioxidants.^{4 5} Although one early study reported AA levels in human aortic diseased tissue to be similar to those in plasma,³⁰ data on the status of lipid-soluble antioxidants in human plaque are, surprisingly, totally lacking. From in vitro studies on radical-mediated oxidation of isolated LDL or human plasma one would expect that significant lipid oxidation does not proceed as long as ascorbate and ubiquinol-10 (the reduced form of coenzyme Q₁₀) are present.^{4 5 6 29 31 32 33 34 35 36} After these antioxidants are depleted (LDL) lipid oxidation can occur even in the presence of -tocopherol, the most active form of vitamin E.^{29 31 33 34 35 36}

We therefore investigated the status of the major lipid- and water-soluble antioxidants in freshly obtained normal human arteries and advanced atherosclerotic lesions, first optimizing the sample workup procedure to avoid artifactual oxidation of antioxidants. As this potential problem has not been adequately addressed in studies on the characterization of plaque lipids yet could conceivably result in artifactual lipid oxidation, particularly if endogenous antioxidants were depleted, we also examined the extent of oxidation of Ch18:2, the major single substrate for oxidation in isolated LDL. Our results show that human plaque contains large amounts of oxidized Ch18:2 that are not detectable in normal arteries even though human plaque samples are not deficient in the antioxidant vitamins C and E when compared with healthy arteries.

	Methods

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Materials
CoQ₁₀, (all-E)-lycopene and -carotene, and D--tocopherol and -tocopherol were generously given to us by Mitsubishi Gas Chemicals, Hoffmann-La Roche, and Henkel, respectively. -TQ was purchased from Kodak, and ß-carotene (type IV) was from Aldrich. FC, Ch18:2, cholesteryl arachidonate, cholesteryl oleate, cholesteryl linolenate, D-isoascorbate, Dulbecco's PBS, butylated hydroxytoluene, and EDTA were from Sigma. Dodecyltriethylammonium phosphate (Q₁₂ ion-pairing cocktail) was obtained from Regis and acetic acid for HPLC from Rhône-Poulenc. Ch18:2-OH was obtained from Cayman Chemicals, and Ch18:2-OOH was prepared.³⁷ -T-3 was isolated from Palmvitee supplied by the Palm Oil Research Institute of Malaysia.³⁸ All organic solvents were of HPLC quality and obtained from Mallinckrodt or were of the highest grade available. Hexane was washed with nanopure water prior to its use. Isotonic PBS, pH 7.4, was prepared in nanopure water and stored overnight over Chelex-100 (Bio-Rad) to remove contaminating transition metals. The efficiency of this treatment was routinely verified by the ascorbate oxidation method.³⁹ Glass homogenizers (30 and 55 mL) lined with polytetrafluoroethylene were purchased from Wheaton.

Preparation of Intima and Intimal Homogenate
Normal iliac arteries were obtained from liver transplant donors and human plaques from patients undergoing carotid endarterectomy or surgery of the superficial or common femoral artery. The normal subjects were 17 to 29 years of age (mean, 23 years); all were accident victims, and their arteries showed no macroscopic evidence of atherosclerosis. The patients undergoing endarterectomy were 57 to 80 years of age; their sites of clinical atherosclerosis (as assessed from medical records) are summarized in Table 1. As expected for endarterectomy specimens, all 11 samples represented advanced fibrofatty lesions; 4 were calcified, and 5 contained attached thrombus. Aortas with aneurysms were excluded. All samples were obtained by qualified hospital staff, and all procedures were approved by the local human ethics committee.

View this table: [in this window] [in a new window]   Table 1. Description of Subjects and Plaque Samples

Immediately after the vessels were removed, samples were placed in Chelex 100–treated and argon-flushed PBS and processed within 0.5 to 1 hour of surgical removal. Intimas were dissected from adventitia and media under a dissecting microscope, rinsed from the loosely adherent blood components, blotted with Whatman filter paper, and weighed. PBS was then added, and the intima was chopped into small pieces by using a pair of stainless steel scissors before isoascorbate (final concentration, 5 µmol/L) and -T-3 (1.0 µmol/L) as water- and lipid-soluble external standards, respectively, and butylated hydroxytoluene (100 µmol/L) and EDTA (1 mmol/L) as antioxidant and metal-complexing reagent, respectively, were added. The mixtures containing 15 mg wet tissue/mL PBS were then homogenized at 4°C by using a newly purchased polytetrafluoroethylene-lined glass homogenizer with the piston rotating at 200 to 500 rpm. On the basis of careful investigation (see "Results") a 5-minute homogenization period under these conditions was found to be optimal for the efficient extraction of lipids without substantial destruction of antioxidants. The intimal homogenates obtained were then used within 3 to 5 minutes for analysis of total protein, apoB (for plaque samples only), lipids, and antioxidants.

Total protein in the homogenates obtained was determined.⁴⁰ For apoB determination, the plaque homogenates were centrifuged (4000g for 3 minutes), and the resulting supernatant was diluted with PBS (1:10, vol/vol) and subjected to slot-blot analysis. For the analysis of lipids and lipid-soluble antioxidants, the intimal homogenate (200 µL) was extracted by using a mixture of cold methanol (2 mL) and hexane (10 mL), vortexed, and centrifuged at 1000g for 3 minutes. Of the hexane layer, 9 mL was evaporated to dryness, and the solutes were resuspended in 180 µL ethanol and subjected to HPLC analyses for lipids and lipid-soluble antioxidants. For analysis of water-soluble AA and urate, plaque homogenate was added to 5% metaphosphoric acid (1:1, vol/vol) and mixed vigorously; the mixture was centrifuged (10 000g for 1 minute), and the resulting supernatant (150 µL) was diluted with 0.25 mol/L potassium phosphate buffer, pH 5.8 (43 µL), and mobile phase (7 µL) (see below) before analysis.

HPLC Analyses of Lipids and Antioxidants
Unoxidized lipids (FC and cholesteryl esters) were analyzed by reversed-phase HPLC by using a C₁₈ column (25x0.46 cm, 5-µm particle size with 5-cm guard column; Supelco) with methanol/tert-butyl alcohol (1:1, vol/vol) at 1 mL/min as the mobile phase monitored at 210 nm.³⁷ Unesterified 7-KC was determined by reversed-phase HPLC.⁴¹ This method was also used to measure esterified forms of 7-KC, which eluted with the following retention times (in minutes): 7-KC linoleate, 11.3; 7-KC oleate, 14.4; 7-KC palmitate, 15.1; and 7-KC stearate, 18.7 (A.J. Brown, R.T. Dean, W. Jessup, unpublished data, 1995).

The reversed-phase HPLC method of Kritharides et al⁴¹ was also used to determine Ch18:2[O(H)], defined here as a group of compounds coeluting with an authentic standard of Ch18:2-OH under these chromatographic conditions. Separate rechromatography of plaque Ch18:2[O(H)] on normal-phase HPLC (silica column, 25x0.46 cm, 5-µm particle size with 2-cm guard column; Supelco) using n-heptane/diethyl ether/isopropanol (100:0.5:0.175, vol/vol/vol) as the eluant at 2 mL/min and monitoring at 234 and 270 nm showed that it contained four isomers each of Ch18:2-OH and Ch18:2=O. Under these conditions the four isomers of Ch18:2=O (monitored at 270 nm) eluted at 4.3, 4.8, 4.9, and 5.7 minutes, whereas those of Ch18:2-OH (monitored at 234 nm) eluted at 12.0, 13.9, 19.0, and 21.1 minutes. The details of the separation and characterization of these oxidized forms of Ch18:2 and esterified 7-KC will be reported separately.

-Tocopherol, -T-3, -tocopherol, -TQ,^{38 42} CoQ₁₀, ubiquinol-10, lycopene, and ß-carotene⁴³ concentrations were determined by reversed-phase HPLC with reductive and oxidative electrochemical detection. AA and urate levels were determined by an ion-paired, reversed-phase HPLC with oxidative electrochemical detection by using a C₁₈ column (25x0.46 cm, 5-µm particle size with 5-cm guard column; Supelco) and 40 mmol/L sodium acetate, 0.54 mmol/L EDTA, and 1.5 mmol/L Q₁₂ containing 7.5% (vol/vol) methanol and adjusted to pH 4.75 with acetic acid as the eluant at a flow rate of 0.9 mL/min.⁴⁴ Electrochemical detection was performed by using a BAS LC-4C system equipped with a glassy carbon electrode (Bioanalytical Systems) with applied potentials of -300 and 500 mV versus the silver/silver chloride reference electrode for reductive and oxidative detection, respectively. For integration, the LC-4C system was connected to a Shimadzu C-R4A computer.

The recovery of AA was determined by adding a known amount of isoascorbate to the chopped tissue prior to homogenization of plaque, assuming that its loss is equivalent to that of AA.⁴⁵ The ratio of AA to isoascorbate does not change during storage at 4°C for 16 hours, even though AA is oxidized under these conditions.⁴⁵ The overall recovery for isoascorbate from plaque samples was 43.4±15.4% (mean±SD, n=6), indicating some donor-to-donor variation. In experiments designed to optimize the homogenization procedure itself, isoascorbate correction was not applied. Recoveries of urate and lipid-soluble antioxidants from plaque samples were not corrected, as urate is significantly more stable than AA, and added -T-3 was recovered almost completely (ie, 86.5±8.5%, n=6). Recoveries of AA and -T-3 from normal arteries did not differ from those of plaque samples.

	Results

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To study the levels of nonproteinaceous antioxidants such as AA and ubiquinol-10 in biological samples, it is imperative that fresh material is used, as these compounds are extremely labile and lost rapidly upon tissue storage. As we were interested in examining human intima, the only fresh material we could obtain readily and reliably were control, healthy vessels from transplantation donors and advanced lesions derived from patients undergoing endarterectomy, ie, samples representing the two ends of the spectrum of the disease. To obtain information about the overall content of lipid- and water-soluble antioxidants in control and lesion intima, we deemed it essential to use intimal homogenates and develop a homogenization protocol so that our extracts would represent the tissue as a whole.

Optimization of Plaque Homogenization Procedure
Most reports on the composition of human plaque have not included information on the efficacy and reproducibility of the extraction procedure. We therefore first examined the effect of homogenization time on extraction efficacy by using plaque samples (5 and 6 in Table 1). Maximal recovery for protein, FC, Ch18:2 (Fig 1A), and Ch18:2-OOH (Fig 1B) were obtained within 10 minutes of homogenization. By using this procedure, we detected 34 to 100 µg apoB protein/g wet tissue in the homogenates (data not shown), in agreement with the results of Cushing et al⁴⁶ in tissue segments from coronary bypass vein grafts and Rath et al⁴⁷ in a supernatant of coronary bypass homogenate, although somewhat lower than those of Pepin et al.⁴⁸

View larger version (19K): [in this window] [in a new window]   Figure 1. Line graphs showing time-dependent recoveries of protein and unoxidized lipids (A) and oxidized lipids (B) during homogenization. Intimas of plaque were homogenized for the times indicated and analyzed as described in "Methods" for protein (), FC (), Ch18:2 (), Ch18:2-OOH (), and Ch18:2[O(H)] (). All recoveries shown here were expressed as percentage of the maximum value of each individual sample and are not corrected for the recoveries of the internal standards. Results represent mean±SD of six independent experiments performed with human atherosclerotic plaque from six different donors. Since the time of maximal value for a given parameter varied slightly between experiments, none of the average values shown are necessarily 100%. The absolute values from 5-minute homogenizations, chosen as optimal, are shown in Tables 3 and 4.

Time-dependent recoveries of reduced (Fig 2A) and two-electron oxidized forms of small-molecular-weight (Fig 2B) antioxidants revealed increasing amounts of the former with longer homogenization times (15 to 20 minutes). An important exception to this was AA and DHA, the recoveries of which decreased substantially with homogenization times beyond 5 and 10 minutes, respectively (Fig 2A and 2B). In agreement with this, when we added isoascorbate prior to homogenization we found that it was lost to an extent similar to AA, but we recovered no dehydroisoascorbic acid (data not shown). To prevent substantial loss of AA while obtaining acceptable recoveries (80%) for all other compounds analyzed, we chose a homogenization period of 5 minutes as the optimal and standard condition. During the homogenization procedure itself the external standards -T-3 and isoascorbate were recovered with 95.6±17.7% and 66.1±24.4% efficiency (mean±SD, n=6), respectively. As noted already, the overall recovery of -T-3 was almost complete, whereas in the case of AA, losses besides those due to homogenization occurred, as the overall recovery of added isoascorbate was only 43% (see "Methods"). This is not unexpected considering the very labile nature of AA. Recoveries of all compounds, including AA, were not significantly different for plaque and control artery samples (data not shown).

View larger version (21K): [in this window] [in a new window]   Figure 2. Line graphs showing time-dependent recoveries of reduced (A) and oxidized (B) antioxidants during homogenization. Intimas or plaque were homogenized for the times indicated and analyzed as described in "Methods" for AA (), urate (\), -tocopherol (-T) (), DHA (), -TQ (), and CoQ10 (). The results are expressed as for Fig 1 and represent mean±SD of six independent experiments performed with human atherosclerotic plaque from different donors. The absolute values from 5-minute homogenizations, chosen as optimal, are shown in Table 2.

Reproducibility of Plaque Homogenization
To determine the reproducibility of the plaque homogenization procedure, two arteries from different donors were cut longitudinally into three and two pieces, respectively. The resulting portions were homogenized independently in singular, duplicate, or triplicate before analysis. While we observed the expected donor-to-donor variation, the reproducibility of a single sample worked up in triplicate was good (generally within 3%), except for CoQ₁₀ and AA (after isoascorbate correction) for which the amounts recovered varied by 20% and 25%, respectively (data not shown). In addition, there was a distinct difference (up to 50%) in some of the parameters between different longitudinal sections of the same plaque sample. These results highlight the heterogeneity within and between plaque samples.

Contents of Antioxidants
Table 2 summarizes the contents of lipid- and water-soluble antioxidants in the homogenates prepared from normal and diseased arteries. All lipid-soluble antioxidants were standardized to the content of FC. As expected, -tocopherol was quantitatively the single most important lipid-soluble antioxidant, with plaque homogenates on average containing 50% more lipid-standardized -tocopherol than homogenates prepared from normal arteries. Because the FC/protein ratio is 15 times higher in diseased than healthy arteries (Table 3), this means that when expressed per unit of protein, plaque homogenate contains some 20 times more -tocopherol than homogenate from normal arteries. In plaque homogenates 9.1±6.6% (mean±SD, n=11) of the -tocopherol was present as -TQ, the two-electron oxidized form of vitamin E, which could not be detected in normal arteries (Table 2). In contrast to -tocopherol, coenzyme Q₁₀ was detected only in its oxidized form, CoQ₁₀, in both plaque and normal arteries (Table 2). Whether this truly reflects the in vivo situation is not clear, as ubiquinol-10 autoxidizes readily to CoQ₁₀; this may have occurred during the sample workup procedure, and we did not try to avoid this. Homogenized human plaque but not normal arteries also contained detectable amounts of -tocopherol, lycopene, and ß-carotene (Table 2).

View this table: [in this window] [in a new window]   Table 2. Lipid- and Water-Soluble Antioxidants in Normal Human Arteries and Atherosclerotic Plaque Samples

View this table: [in this window] [in a new window]   Table 3. Contents of Protein and Free and Esterified Cholesterol in Homogenates Prepared From Human Normal and Atherosclerotic Plaque Intimas

Homogenates derived from human plaque contained some 10 and 30 times more AA and urate, respectively, than those from normal arteries (Table 2). In fact, the concentrations of AA and urate in plaque homogenates (1.3 and 3.3 nmol/mg protein, respectively) were comparable to those in normal human plasma.³¹ In contrast, the levels of both AA and urate in normal vessel samples were significantly lower than in plasma, suggesting that the intima may be relatively poorly protected by these aqueous-phase antioxidants. In a few cases, the urate concentrations in plaque samples were so high that some could have originally been present as crystals. The mean ratios of urate to (corrected) AA were 2.6 and 1 for plaque and normal homogenate, respectively (Table 2), whereas in human plasma the ratio is 6. Importantly, the redox statuses of vitamin C, ie, the relative contribution of DHA to total vitamin C, in the plaque (n=8) and normal arteries (n=4) were 11±7.3% and 22±19.4%, respectively. This indicates that the redox state of this important aqueous antioxidant was not different in the two samples and that the vitamin was present primarily in its reduced, antioxidant-active form, as it is in the blood plasma of healthy humans.⁴⁹

Contents and Redox Status of Lipids
The total protein concentration in the whole intimal homogenates varied between 41 to 67 and 47 to 79 µg/mg wet weight for plaque and normal arteries, respectively (Table 3). The quantitatively most abundant of all lipids in plaque and normal arteries was FC, although the former contained, on average, some 14 times more FC than control arteries. Plaque homogenate but not normal arteries also contained significant amounts of cholesteryl oleate, Ch18:2, and cholesteryl arachidonate (Table 3). Cholesteryl oleate refers to a mixture of cholesteryl oleate plus cholesteryl palmitate, consisting of 67% and 33% peak composition, respectively, as judged by rechromatographing samples from eight donors by using a less polar reversed-phase HPLC method.⁴¹ An additional unsaturated cholesteryl ester in homogenized human plaques was cholesteryl linolenate, detected at 0.04±0.03 mol/mol FC (not shown). Although cholesteryl linolenate coeluted with an authentic standard, its detailed characterization has not been undertaken, and the possibility that other components coelute cannot be excluded. For reason(s) not understood, the relative concentrations of the various cholesteryl esters in the different plaque samples varied greatly (data not shown).

Despite extensive literature on the presence of oxidized lipids in human plaques, we deemed it important to analyze and directly compare the contents of oxidized lipids and antioxidants within the same tissue samples, particularly as we processed the samples with as little artifactual oxidation of readily oxidizable materials as possible. Among the lipid oxidation products, we analyzed primarily for those of Ch18:2, quantitatively the most important single readily oxidizable lipid in human LDL. We initially analyzed for Ch18:2-OOH and hydroxides of Ch18:2 (Ch18:2-OH) by using a recently developed reversed-phase HPLC method with UV detection.⁴¹ As judged by coelution with authentic standards, some 19% and 1% of the Ch18:2 detected in plaque samples were present as Ch18:2-OH and Ch18:2-OOH, respectively (Table 4), indicating that at least 20% of Ch18:2 present in plaque was oxidized. Neither of these two oxidation products of Ch18:2 was detected in homogenates prepared from normal arteries.

View this table: [in this window] [in a new window]   Table 4. Contents of Oxidized Ch18:2 and Cholesterol in Human Atherosclerotic Plaques

Using normal-phase HPLC for a more detailed characterization of the Ch18:2-OH peak of plaque (defined here as Ch18:2[O(H)]), which coeluted with authentic Ch18:2-OH on reversed-phase HPLC, revealed that it contained four isomers of Ch18:2-OH and Ch18:2=O as well as small amounts (as judged by peak area) of four presently unidentified compounds (data not shown). The concentration of the Ch18:2-OH and Ch18:2=O isomers as determined by the normal-phase HPLC assay were 171±140 and 121±69 mmol/mol Ch18:2, respectively (Table 4). Thus, in total some 30% of plaque Ch18:2 was present as either Ch18:2-OH, Ch18:2=O, or Ch18:2-OOH.

In contrast to the oxidation products of Ch18:2, plaque homogenates contained 7-KC at a comparatively low concentration of 3 to 100 nmol/g wet tissue or 0.6±0.4 mmol/mol FC or 4.3±5.0 mmol/mol Ch18:2 (Table 4). 7-KC could not be detected in homogenates from normal arteries (data not shown).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We report the first detailed study of antioxidants and oxidized lipids in human atherosclerotic plaque and normal arteries, analyzing homogenates prepared from whole intimas of the two samples under conditions optimized for maximal recoveries of lipids and minimal artifactual loss of antioxidants due to inadvertent oxidation. Since we included isoascorbate and -T-3 as external standards and followed their recoveries during sample processing, our results may be the first reliable assessment of AA levels as well as the first observations on lipophilic antioxidants, particularly -tocopherol. Our results show that the levels of -tocopherol and AA in homogenates of advanced human atherosclerotic plaques are respectively comparable to and higher than those in normal arteries. Despite this apparent lack of deficiency in the antioxidant vitamins E and C, lipids in plaques but not normal arteries were substantially oxidized.

AA is a most important antioxidant that effectively protects extracellular lipids, including those of LDL, from most radical oxidants.^{31 32} AA acts without becoming continuously associated with lipoproteins, and hence there is no alternative to the use of whole homogenates to assess its concentration in normal and diseased intimas. Calculations based on the reported recoveries of cells per gram of human plaque⁵⁰ and the assumption that average intracellular AA in the cells of plaque is no more than 10 mmol/L show that >90% of recovered AA in plaque homogenates must be extracellular in origin. This is consistent with the relatively high plaque concentrations of urate, which is located almost exclusively in the extracellular space. The observations that AA is rapidly converted into its oxidation product DHA during sample workup (Fig 2), that only small and comparable amounts of the vitamin C in plaque and normal arteries were present as DHA, and that plaque contained about seven times more AA than normal arteries all argue against the redox status of vitamin C in plaque being deficient in any way. Our findings, based on selective HPLC-electrochemical detection, agree with an early study by Willis and Fishman,³⁰ who reported AA levels as measured by a spectrophotometric assay in aortic plaque samples obtained from sudden death victims, including those who suffered myocardial infarction.

-Tocopherol, quantitatively the major lipid-soluble antioxidant in human blood plasma,⁵¹ was also the major lipid-soluble antioxidant in homogenates of human plaque and normal arteries. Even when standardized to FC, plaque contained at least as much -tocopherol as control arteries (Table 2). Expressing -tocopherol per unit of polyunsaturated fatty acid is perhaps the most pertinent criterion of its antioxidant sufficiency. In human plasma and LDL, the single largest polyunsaturated fatty acid component is Ch18:2. Thus it is appropriate to consider the [-tocopherol]/[Ch18:2] ratio: in plaque the ratio was 34 (in millimoles per mole), two to three times higher than that in plasma and LDL^{3 29} of healthy subjects. Unfortunately, we could not compare plaque and autologous plasma samples, which will be desirable in the future. However, there is clearly no evidence from our data of -tocopherol deficiency in these advanced plaque samples.

Should it hold true for plaque LDL that [-tocopherol]/[Ch18:2] is comparable to what we have measured in plaque homogenates, then there are some fascinating possible implications. During in vitro radical-mediated oxidation of LDL isolated from peripheral blood, there are stages at which substantial formation of oxidized Ch18:2 can occur as a result of tocopherol-mediated peroxidation,^{29 36} ie, when -tocopherol is consumed at relatively slow rates. It is not known whether this mechanism also operates during a putative in vivo radical-mediated oxidation of LDL. If so, we would have to assume that for whatever reason(s), this process is not inhibited efficiently by the AA detected in the advanced lesions studied here, perhaps because of its transient consumption. What also remains unclear is whether ubiquinol-10 is present in plaque (see "Results"). Ubiquinol-10 is important, as it makes -tocopherol a more efficient antioxidant, at least in LDL isolated from blood.^{35 52} Alternatively, intimal LDL lipid oxidation could occur via enzymic processes⁵³ not affected by antioxidants.

The observation that almost 10% of the -tocopherol in plaque but not normal arteries is present as -TQ is compatible with the idea that oxidation is ongoing in plaque. Furthermore, an organ subjected to oxidative attack may respond by elevating both proteinaceous (enzymatic) and sacrificial (aqueous or lipophilic) antioxidants. This could explain the coexistence in plaque but not normal artery of a vast enhancement of the aqueous-phase antioxidant shield, in many respects the first-line antioxidant defense, and yet large quantities of lipid oxidation products.

Because of the limitations in most studies of postmortem tissue and of properly restricting and controlling for artifactual oxidation during processing, we also examined the contents of oxidized lipids in plaque and normal artery. We observed a remarkably large proportion (30%) of the total fatty acyl moiety of Ch18:2 in plaque in oxidized forms (these were undetectable in normal arteries), with Ch18:2-OH and Ch18:2=O being the predominant forms (Table 4). The contribution of esterification subsequent to formation of hydroxy and keto fatty acids to this accumulation is not known. Our results are in remarkable agreement with the findings of Kühn et al²⁶ and Belkner et al,⁵⁴ who have demonstrated hydroxy and keto fatty acids in human plaque and suggest that this indicates an involvement of 15-lipoxygenase in the in vivo oxidation of intimal lipid. However, these authors did not determine precisely to what extent the oxidized fatty acyl chains were esterified. Although several studies have demonstrated oxidized lipids in plaque samples, few have determined lipid hydroperoxides, in contrast to the more stable lipid hydroxides. We found that 1% of the total Ch18:2 was present as Ch18:2-OOH. By using a spectrophotometric method, Harland et al²⁵ have shown a substantially lower Ch18:2-OOH value, ie, 0.01 to 0.03 mol percent of Ch18:2 in postmortem samples. In addition to possible artifacts due to extraction, enzymic and/or nonenzymic reduction of hydroperoxides to the corresponding hydroxides could have occurred after death. Besides hydroxy, keto, and hydroperoxy derivatives of Ch18:2, other oxidation products, such as octadecadienediols, have been identified as oxidized free fatty acids in hydrolyzed samples of aortic plaque obtained from humans postmortem.²⁵

Oxysterols are well known to occur in plaque, and in this study we determined levels of 7-KC, often a major oxysterol formed during in vitro oxidation of LDL with transition metals³⁵ (A.J. Brown, unpublished data, 1995). 7-KC was present at 75-fold lower concentrations than the fatty acyl oxidation products of Ch18:2 (Table 4). Preliminary analyses (data not shown) indicated that esterified forms of 7-KC predominate over the free oxysterol. However, even when added together, the various forms of 7-KC seem to constitute only a small fraction of the oxidized lipids present in plaque. The implied preferential oxidation of the fatty acyl chain over the cholesterol moiety is consistent with the comparatively 100-fold higher oxidizability of the former^{55 56} ; acyl chains of cholesteryl esters are also oxidized well before the cholesterol moiety during metal-catalyzed oxidation of LDL in vitro.⁴¹ This may be the major factor determining the product distribution we observed in plaque, although other factors such as differences in product transport and mode of origin may contribute.

The presence of oxidized lipids in lesions could conceivably lead to a number of biological effects that may affect atherogenesis. For example, fatty acid hydroperoxides resulting from hydrolysis of Ch18:2-OOH are potentially cytotoxic and/or may cause vasoconstriction^{57 58} or inactivation of endothelium-derived relaxing factor, as has been shown for oxidized LDL and the lipids extracted from it.⁵⁹ In contrast to the known biological activities of lipid hydroperoxides and oxysterols,²² relatively little is known about potential activities of keto and hydroxy derivatives of fatty acids. Considering their presence in large quantities, studies of the biological activities of keto and hydroxy fatty acids in both free and esterified forms seem warranted.

In studying isolated human plaque, one has access only to a single time point in what is a complicated disease process with ongoing, stage-dependent events. Thus, it is very difficult to discern the possible sequence of factors contributing to oxidative stress and damage. Despite this restriction, our results are striking. On the one hand, the nonproteinaceous antioxidant shield seems to be largely intact in plaque, a fact that has not been appreciated previously. Our observations of a greater proportion of oxidized -tocopherol in plaque than normal artery may nevertheless indicate that modest oxidative events routinely occur. On the other hand, large quantities of oxidatively damaged lipids are present. This may indicate that such ongoing oxidation results in a gross accumulation of oxidized lipids in the intima, presumably indicating also that the removal of the relatively stable hydroxy and keto fatty acids is not capable of keeping pace with their generation. What is clear from our results, however, is the heterogeneity within and between plaque samples and the significance of in vitro artifactual oxidation.

It is interesting to consider at what location the oxidation occurs. It is easiest to assume, in line with current conventional thought, that this oxidation most probably occurs in the intima, though we have no direct evidence for this. But we find it noteworthy that the argument used to suggest that oxidative damage to LDL must occur in the intima rather than plasma (ie, that plasma has an overwhelming antioxidative capacity) is undermined by our observations, which suggest that commonly, even in advanced plaque, the intima has a comparably overwhelming antioxidant shield as well. Further studies suggested by our data will be required to resolve these issues.

	Selected Abbreviations and Acronyms

 

AA = ascorbic acid Ch18:2 = cholesteryl linoleate Ch18:2-OH = cholesteryl linoleate hydroxides Ch18:2-OOH = cholesteryl linoleate hydroperoxides Ch18:2=O = cholesteryl linoleate ketones Ch18:2[O(H)] = compounds coeluting with authentic standard of Ch18:2-OH in reversed-phase HPLC CoQ10 = ubiquinone-10 DHA = dehydroascorbic acid FC = free cholesterol HPLC = high-performance liquid chromatography 7-KC = 7-ketocholesterol PBS = phosphate-buffered saline -T-3 = -tocotrienol -TQ = -tocopherylquinone

	Acknowledgments

 
This work was supported by the National Heart Foundation of Australia (grants G92S3527 and G94S4061 to Drs Dean and Stocker). We are indebted to Drs T. McGahan, K. Shannon, G. White, and J. Harris and nurses from the Department of Vascular Surgery, Royal Prince Alfred Hospital, who helped us in providing the patient consents and plaque material and Drs G. Kyd and A.G.R. Sheil for normal arteries. We thank Drs L. Kritharides, P. Bannon, W. Lau, and D. Brieger for discussions of the specimens, Drs A. Zammit and A. Brown for help in performing the apoB and esterified 7-KC analyses, and Drs A. Brown, L. Kritharides, W. Jessup, and U. Steinbrecher for critically reading the manuscript and interesting discussions.

Received January 25, 1995; accepted June 29, 1995.

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				A. C. Carr, M. R. McCall, and B. Frei Oxidation of LDL by Myeloperoxidase and Reactive Nitrogen Species : Reaction Pathways and Antioxidant Protection Arterioscler Thromb Vasc Biol, July 1, 2000; 20(7): 1716 - 1723. [Abstract] [Full Text] [PDF]

				R. A. Memon, I. Staprans, M. Noor, W. M. Holleran, Y. Uchida, A. H. Moser, K. R. Feingold, and C. Grunfeld Infection and Inflammation Induce LDL Oxidation In Vivo Arterioscler Thromb Vasc Biol, June 1, 2000; 20(6): 1536 - 1542. [Abstract] [Full Text] [PDF]

				S. Tsimikas, B. P. Shortal, J. L. Witztum, and W. Palinski In Vivo Uptake of Radiolabeled MDA2, an Oxidation-Specific Monoclonal Antibody, Provides an Accurate Measure of Atherosclerotic Lesions Rich in Oxidized LDL and Is Highly Sensitive to Their Regression Arterioscler Thromb Vasc Biol, March 1, 2000; 20(3): 689 - 697. [Abstract] [Full Text] [PDF]

				A. J. Brown, E. L. Mander, I. C. Gelissen, L. Kritharides, R. T. Dean, and W. Jessup Cholesterol and oxysterol metabolism and subcellular distribution in macrophage foam cells: accumulation of oxidized esters in lysosomes J. Lipid Res., February 1, 2000; 41(2): 226 - 237. [Abstract] [Full Text]

				A. Baoutina, R. T. Dean, and W. Jessup Macrophages Can Decrease the Level of Cholesteryl Ester Hydroperoxides in Low Density Lipoprotein J. Biol. Chem., January 21, 2000; 275(3): 1635 - 1644. [Abstract] [Full Text] [PDF]

				G. J. Schroepfer Jr. Oxysterols: Modulators of Cholesterol Metabolism and Other Processes Physiol Rev, January 1, 2000; 80(1): 361 - 554. [Abstract] [Full Text] [PDF]

				H. Massaeli, J. A. Austria, and G. N. Pierce Chronic Exposure of Smooth Muscle Cells to Minimally Oxidized LDL Results in Depressed Inositol 1,4,5-Trisphosphate Receptor Density and Ca2+ Transients Circ. Res., September 17, 1999; 85(6): 515 - 523. [Abstract] [Full Text] [PDF]

				I. C. Gelissen, K.-A. Rye, A. J. Brown, R. T. Dean, and W. Jessup Oxysterol efflux from macrophage foam cells: the essential role of acceptor phospholipid J. Lipid Res., September 1, 1999; 40(9): 1636 - 1646. [Abstract] [Full Text]

				X. Niu, V. Zammit, J. M. Upston, R. T. Dean, and R. Stocker Coexistence of Oxidized Lipids and {alpha}-Tocopherol in All Lipoprotein Density Fractions Isolated From Advanced Human Atherosclerotic Plaques Arterioscler Thromb Vasc Biol, July 1, 1999; 19(7): 1708 - 1718. [Abstract] [Full Text] [PDF]

				M. H. Alderman, H. Cohen, S. Madhavan, and S. Kivlighn Serum Uric Acid and Cardiovascular Events in Successfully Treated Hypertensive Patients Hypertension, July 1, 1999; 34(1): 144 - 150. [Abstract] [Full Text] [PDF]

				J. M. Letters, P. K. Witting, J. K. Christison, A. W. Eriksson, K. Pettersson, and R. Stocker Time-dependent changes to lipids and antioxidants in plasma and aortas of apolipoprotein E knockout mice J. Lipid Res., June 1, 1999; 40(6): 1104 - 1112. [Abstract] [Full Text]

				J. F. KEANEY JR., D. I. SIMON, and J. E. FREEDMAN Vitamin E and vascular homeostasis: implications for atherosclerosis FASEB J, June 1, 1999; 13(9): 965 - 975. [Abstract] [Full Text]

				J. M. UPSTON, A. C. TERENTIS, and R. STOCKER Tocopherol-mediated peroxidation of lipoproteins: implications for vitamin E as a potential antiatherogenic supplement FASEB J, June 1, 1999; 13(9): 977 - 994. [Abstract] [Full Text]

				P. K. WITTING, K. PETTERSSON, A.-M. ÖSTLUND-LINDQVIST, C. WESTERLUND, A. W. ERIKSSON, and R. STOCKER Inhibition by a coantioxidant of aortic lipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E and low density lipoprotein receptor gene double knockout mice FASEB J, April 1, 1999; 13(6): 667 - 675. [Abstract] [Full Text]

				L. Chancharme, P. Therond, F. Nigon, S. Lepage, M. Couturier, and M. J. Chapman Cholesteryl Ester Hydroperoxide Lability Is a Key Feature of the Oxidative Susceptibility of Small, Dense LDL Arterioscler Thromb Vasc Biol, March 1, 1999; 19(3): 810 - 820. [Abstract] [Full Text] [PDF]

				L. Kritharides, J. Upston, W. Jessup, and R. T. Dean Accumulation and metabolism of low density lipoprotein-derived cholesteryl linoleate hydroperoxide and hydroxide by macrophages J. Lipid Res., December 1, 1998; 39(12): 2394 - 2405. [Abstract] [Full Text]

				B. Karten, H. Boechzelt, P. M. Abuja, M. Mittelbach, K. Oettl, and W. Sattler Femtomole analysis of 9-oxononanoyl cholesterol by high performance liquid chromatography J. Lipid Res., July 1, 1998; 39(7): 1508 - 1519. [Abstract] [Full Text]

				D. Lapenna, S. de Gioia, G. Ciofani, A. Mezzetti, S. Ucchino, A. M. Calafiore, A. M. Napolitano, C. Di Ilio, and F. Cuccurullo Glutathione-Related Antioxidant Defenses in Human Atherosclerotic Plaques Circulation, May 19, 1998; 97(19): 1930 - 1934. [Abstract] [Full Text] [PDF]

				B. Garner, A. R. Waldeck, P. K. Witting, K.-A. Rye, and R. Stocker Oxidation of High Density Lipoproteins. II. EVIDENCE FOR DIRECT REDUCTION OF LIPID HYDROPEROXIDES BY METHIONINE RESIDUES OF APOLIPOPROTEINS AI AND AII J. Biol. Chem., March 13, 1998; 273(11): 6088 - 6095. [Abstract] [Full Text] [PDF]

				S. Lehto, L. Niskanen, T. Ronnemaa, and M. Laakso Serum Uric Acid Is a Strong Predictor of Stroke in Patients With Non–Insulin-Dependent Diabetes Mellitus Stroke, March 1, 1998; 29(3): 635 - 639. [Abstract] [Full Text] [PDF]

				J. Neuzil, J. K. Christison, E. Iheanacho, J.-C. Fragonas, V. Zammit, N. H. Hunt, and R. Stocker Radical-induced lipoprotein and plasma lipid oxidation in normal and apolipoprotein E gene knockout (apoE-/-) mice: apoE-/- mouse as a model for testing the role of tocopherol-mediated peroxidation in atherogenesis J. Lipid Res., February 1, 1998; 39(2): 354 - 368. [Abstract] [Full Text]

				A. Martin and B. Frei Both Intracellular and Extracellular Vitamin C Inhibit Atherogenic Modification of LDL by Human Vascular Endothelial Cells Arterioscler Thromb Vasc Biol, August 1, 1997; 17(8): 1583 - 1590. [Abstract] [Full Text]

				J. Neuzil, P. K. Witting, and R. Stocker alpha -Tocopheryl hydroquinone is an efficient multifunctional inhibitor of radical-initiated oxidation of low density lipoprotein lipids PNAS, July 22, 1997; 94(15): 7885 - 7890. [Abstract] [Full Text] [PDF]

				C. J. Fielding, A. Bist, and P. E. Fielding Caveolin mRNA levels are up-regulated by free cholesterol and down-regulated by oxysterols in fibroblast monolayers PNAS, April 15, 1997; 94(8): 3753 - 3758. [Abstract] [Full Text] [PDF]

				S. Goulinet and M. J. Chapman Plasma LDL and HDL Subspecies Are Heterogenous in Particle Content of Tocopherols and Oxygenated and Hydrocarbon Carotenoids: Relevance to Oxidative Resistance and Atherogenesis Arterioscler Thromb Vasc Biol, April 1, 1997; 17(4): 786 - 796. [Abstract] [Full Text]

				B. Garner, D. van Reyk, R. T. Dean, and W. Jessup Direct Copper Reduction by Macrophages. ITS ROLE IN LOW DENSITY LIPOPROTEIN OXIDATION J. Biol. Chem., March 14, 1997; 272(11): 6927 - 6935. [Abstract] [Full Text] [PDF]

				S. R. Thomas, J. Neuzil, and R. Stocker Cosupplementation With Coenzyme Q Prevents the Prooxidant Effect of {alpha}-Tocopherol and Increases the Resistance of LDL to Transition Metal–Dependent Oxidation Initiation Arterioscler Thromb Vasc Biol, May 1, 1996; 16(5): 687 - 696. [Abstract] [Full Text]

				H. M. Abu-Soud and S. L. Hazen Nitric Oxide Is a Physiological Substrate for Mammalian Peroxidases J. Biol. Chem., November 22, 2000; 275(48): 37524 - 37532. [Abstract] [Full Text] [PDF]

				A. C. Terentis, S. R. Thomas, J. A. Burr, D. C. Liebler, and R. Stocker Vitamin E Oxidation in Human Atherosclerotic Lesions Circ. Res., February 22, 2002; 90(3): 333 - 339. [Abstract] [Full Text] [PDF]

				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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