Published online before print March 4, 2004, doi: 10.1161/01.ATV.0000124892.90999.cb
© 2004 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Direct Detection and Quantification of Transition Metal Ions in Human Atherosclerotic Plaques: Evidence for the Presence of Elevated Levels of Iron and Copper
From The Heart Research Institute, Sydney, Australia.
Correspondence to Michael J. Davies, The Heart Research Institute, 145 Missenden Road, Camperdown, Sydney, NSW 2050, Australia. E-mail m.davies{at}hri.org.au
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Abstract |
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Objective— The involvement of transition metals in atherosclerosis is controversial. Some epidemiological studies have reported a relationship between iron (Fe) and cardiovascular disease, whereas others have not. Experimental studies have reported elevated levels of iron and copper (Cu) in diseased human arteries but have often used methods that release metal ions from proteins.
Methods and Results— In this study, we have used the minimally invasive technique of electron paramagnetic resonance (EPR) spectroscopy and inductively coupled plasma mass spectroscopy (ICPMS) to quantify iron and copper in ex vivo healthy human arteries and carotid lesions. The EPR spectra detected are characteristic of nonheme Fe(III) complexes. Statistically elevated levels of iron were detected in the intima of lesions compared with healthy controls (0.370 versus 0.022 nmol/mg tissue for EPR, 0.525 versus 0.168 nmol/mg tissue by ICPMS, P<0.05 in each cases). Elevated levels of copper were also detected (7.51 versus 2.01 pmol/mg tissue, lesion versus healthy control, respectively, P<0.05). Iron levels did not correlate with the gender or age of the donor, or tissue protein or calcium levels, but cholesterol levels correlated positively with iron accumulation, as measured by EPR.
Conclusions— These data support the hypothesis that iron accumulates in human lesions and may contribute to disease progression.
Key Words: iron • copper • atherosclerosis • oxidation • free radicals • EPR
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Introduction |
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Atherosclerosis is a multifactorial disease, and defining the role of individual risk factors has proven problematic. Oxidation, particularly oxidative modification of low-density lipoproteins (LDL) within the artery wall and its subsequent unregulated uptake by macrophages, has been postulated to be an important in disease development.1 A range of species can oxidize LDL in vitro, but the nature of the oxidants in vivo is controversial.1
Evidence has been presented for a role for lipoxygenase,2 peroxynitrite,3 hypochlorous acid,4,5 and metal ion-mediated oxygen–radical formation.6 Products arising from the reactions of HOCl (3-chlorotyrosine), peroxynitrite (3-nitrotyrosine), lipoxygenase (oxidized lipids), and metal ions (hydroxylated amino acids) have been detected in human plaques.1,7 Plaques have been reported to contain hypochlorite-oxidized proteins, active myeloperoxidase (the enzymatic source of HOCl), lipoxygenase protein and mRNA, and redox-active metal ions.8–14 However, 3-nitrotyrosine can be generated by species other than peroxynitrite; the participation of (cytosolic) lipoxygenase in extracellular processes has been questioned, as has the presence of metal ions on the basis of the invasive methods used to detect these species.1 The present study focuses on the quantification of transition metal ions in lesions, because recent studies have demonstrated the presence of elevated levels of specific protein oxidation products in advanced human lesions and identified metal ions as possible catalysts for these species.6,7
Epidemiological studies have sought to link various measures of iron (Fe) and copper (Cu) with the incidence of cardiovascular disease, after the suggestion that the development of disease is linked to iron stores, with iron-deficiency offering protection.15 These data are equivocal, with positive associations detected in some studies but not others.16–18 Decreasing body iron levels have been reported to be protective, and iron administration can be detrimental.16–18 Thus, some data support an association between high levels of iron and copper and disease incidence.
The presence of metal ions in human plaques has been examined using a number of approaches, but these techniques are often invasive and destructive (eg, homogenisation and digestion), preventing information on the nature and reactivity of the metal ion from being obtained.8–14 The capacity of lesion materials to catalyze damage has been used to implicate metal ions,9,11,19 but mechanical disruption (homogenisation) of normal artery samples can release equal amounts of metal ions.20 Recent studies have used silver staining to examine the presence of redox-active iron in postmortem human arteries, and elevated levels were detected in macroscopically evident lesions;14,21 however, this method is not specific for iron.
Overall, these data suggest that elevated levels of metal ions may be present in advanced atherosclerotic lesions, but there is a paucity of quantitative information on metal ion concentrations, and the nature of these species is uncertain. We have used a minimally invasive technique, electron paramagnetic resonance (EPR) spectroscopy, together with inductively coupled plasma mass spectroscopy (ICPMS), to identity and quantify iron and copper in washed (but otherwise intact) normal and diseased human carotid artery samples ex vivo. The nondestructive nature of EPR has also allowed, for the first time to our knowledge, the quantification of cholesterol, calcium, and protein in the same samples. A direct correlation between iron levels and cholesterol accumulation has been detected, suggesting that these 2 processes are temporally, although not necessarily causatively, linked.
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Methods |
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Reagents
High-grade Suprasil EPR tubes (4-mm outside diameter, 706-PQ-9.50) were obtained from Wilmad (Buena, NJ). These tubes showed minimal metal ion contamination. The 2,6-di-tert-butyl-4-methylphenol (BHT), FeCl3, and desferrioxamine were from Sigma-Aldrich Chemical Company (Milwaukee, Wis). High-purity nitric acid (69%; low in iron and copper) was from BDH (Poole, England). All solutions were treated with washed Chelex-100 resin (Bio-Rad, Calif) to remove transition metals, with the exception of the Fe(III)-desferrioxamine complex.
Human Artery Samples
Diseased human artery samples where obtained, after informed consent, from patients (age 50 to 84 years, average 67.9±8.2 years) undergoing carotid endarterectomy at the Royal Prince Alfred Hospital (Sydney, Australia), with the approval of the hospital ethics committee. Healthy artery specimens (aortae, mammary, and radial) were obtained from patients undergoing heart bypass and transplantation operations. Immediately after surgery, tissue samples were rinsed, then placed in ice-cold PBS containing antioxidants (1 mmol/L EDTA, 0.1 mmol/L BHT). The intimal layer was detached and dissected into regions of similar morphology; these samples were either examined immediately or stored in the aforementioned buffer at –80°C until analyzed. In some cases, healthy artery samples from which the intima had been removed (designated as "smooth muscle" from here onward) were also examined. Lesions were graded according to AHA guidelines.22
Electron Paramagnetic Resonance Spectroscopy
Tissue samples (150 to 300 mg) were blotted dry, inserted in open-ended EPR tubes, frozen in liquid N2, and inserted into a liquid N2 Dewar within the EPR spectrometer cavity (EMX X-band spectrometer; Bruker Biospin GmbH, Rheinstetten, Germany) equipped with 100-KHz modulation and a cylindrical ER 4103TM cavity. Typical acquisition parameters were: gain 1x104, modulation amplitude 2 Gauss, time constant 164 ms, scan time 844 seconds, conversion time 82 ms, resolution 1024 points, power 2.5 mW, and frequency 9.43 GHz, with 20 scans averaged. Multiple spectra were acquired for each sample and for empty tubes. The spectra were baseline- and background-corrected using the Bruker WINEPR program. The resulting data were imported in Origin 7.0 (OriginLab Corporation, Mass), double-integrated, and quantified, as previously reported,23,24 by comparison with standard curves generated using known concentrations of Fe(III)-desferrioxamine (1:1 complex, generated from the addition of known concentrations of FeCl3 to desferrioxamine) under identical conditions.
Inductively Coupled Plasma Mass Spectroscopy
Tissue sections were dissected, blotted, weighed, and digested (24 hours, room temperature) in 500 µL HNO3 (69%) in washed Eppendorf tubes. Samples were then diluted to 10 mL with Milli Q water; control samples were prepared in an identical manner. All glassware used was acid-washed in HNO3 before use, and all plastic-ware was washed with Milli Q water. Analysis of Fe (54Fe and/or 56Fe; both isotopes gave values that were not statistically different), Cu (63Cu), and Ca (44Ca) was performed by Dr Jim Keegan (University of Technology, Sydney) using a Perkin Elmer SCIEX ELAN 5000 apparatus (Thornhill, Ontario, Canada) equipped with a concentric nebulizer and a conical spray chamber (Glass Expansion Ltd., Australia). Instrumental operation conditions were: plasma flow 15 L/min, nebulizer flow 0.95 L/min, auxiliary flow 0.8 L/min, RF power 1200 W, and sample uptake 0.8 mL/min.
Cholesterol Quantification
Cholesterol analysis was performed as described previously.25 Tissues were thawed, blotted dry, weighed, minced, and homogenized in sodium carbonate buffer at 4°C in an Ultra-Turrax T8 homogenizer (3000 to 5000 rpm, 2 to 3 minutes; IKA Labortechnik, Janke and Kunkel GmbH, Staufen, Germany). Aliquots were extracted with 1 mL of methanol and 5 mL of hexane, vortexed, then centrifuged (1000g, 4°C, 5 minutes). Then 4 mL of the hexane layer was removed, dried, and resuspended in isopropanol for high-performance liquid chromatography analysis.25
Protein Quantification
Protein concentrations were determined on homogenates (as mentioned) using the bicinchoninic acid assay with bovine serum albumin as standard, according to the manufacturers instructions, except with 30-minute incubation at 60°C.
Statistics
Student t test, or one-way ANOVA with Tukey multiple comparison tests (for analysis of data from >2 groups) were used (Prism Version 4 for Macintosh; GraphPad Software Inc., San Diego, Calif). Correlations were calculated using the linear least-squares function in Microsoft Excel. P<0.05 was considered significant.
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Results |
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Iron and copper concentrations were measured in intimal samples from healthy arteries and advanced human carotid atherosclerotic lesions by EPR spectroscopy and ICPMS. The former technique has been performed on intact intimal sections at liquid nitrogen temperatures, whereas ICPMS was performed on acid-digested tissue.
EPR Detection and Quantification of Metal Ions in Artery Samples
Typical EPR spectra obtained from intimal sections of a carotid plaque and a healthy artery are shown in Figure 1A. The intense absorption peak detected in the plaque at g
4 is characteristic of the presence of high-spin, rhombic, mononuclear Fe(III) complexes.26 This signal was not present in the empty EPR tubes and was only detected at low levels in healthy human intima samples (Figure 1A). Similar spectra were detected with healthy intima and smooth muscle samples from pig arteries (data not shown). These absorption peaks are distinct from those observed from Fe(III) in heme proteins, which typically give absorption peaks at g6.26 No significant absorptions from Cu(II) (g2.126) were detected. Similar behavior was observed with pig artery samples (data not shown). Additional EPR absorption lines were detected at g2, which are characteristic of organic radicals or iron–sulfur clusters.26 These absorptions were only detected in the tissue samples, but the nature and concentration of these species have not been investigated further. Similar species have been detected in other tissues.27,28 The g4 Fe(III) species was quantified, and although the levels varied (Figure 1B), the mean value for all the plaques examined is significantly elevated over that detected in healthy intima samples when expressed per milligram of tissue (Mann-Whitney test, P=0.0001). Similar signals were detected with intact artery tissue, although at lower intensities (data not shown), and no significant differences were observed in signal intensity between plaque samples that were examined immediately after removal from the donor or after storage for extended periods at –80°C (data not shown).
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The concentration of this EPR-detectable iron species at g4 was subsequently plotted against lesion type (Figure 1C), with the lesions crudely categorized into "clean," "calcified," (on the basis of ICPMS calcium measurements of 
300 nmol Ca/mg tissue), and "complex" (presence of macroscopically evident thrombus, with or without calcification) lesions. Significantly lower levels of iron were detected in the "clean" compared with "calcified" and "complex" lesions. Statistically elevated levels were also detected for the calcified and complex lesions when compared with healthy intima and for calcified versus clean lesions.
ICPMS Quantification of Metal Ions in Artery Samples
Because EPR spectroscopy does not detect all iron ions [eg, signals are not detected from some Fe(II) complexes, or multinuclear Fe(III) complexes, such as ferritin26], ICPMS was used to quantify total iron levels; total copper and calcium were also assessed in some cases. Statistically elevated levels of total iron were detected in diseased intima when compared with healthy intima and healthy smooth muscle samples (Figure 2A). Statistically elevated levels of total copper were detected, although at lower levels than for total iron, in diseased intima samples when compared with healthy intima (7.51 versus 2.01 pmol/mg tissue, respectively), but not healthy smooth muscle (5.17 pmol/mg tissue).
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Comparison of Iron Levels Detected by EPR Spectroscopy and ICMPS
Because EPR spectroscopy is nondestructive, some samples were analyzed by both techniques. The levels of Fe(III) detected by EPR spectroscopy were 70% of the total iron value detected by ICPMS, and a good correlation was observed between the 2 techniques (r2=0.26; Figure 3).
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Correlation of Metal Ion Levels With Lesion Parameters
No significant differences were detected in the levels of EPR-detectable Fe(III) or total (ICPMS) iron with sex or age of the donors (Figure 4). Insufficient data were obtained to allow a similar analysis for copper.
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The iron and copper levels in the lesions did not correlate with total protein concentration (which might include iron- and copper-binding species) in the intimal samples (Figure 5A). The level of EPR-detectable Fe(III) correlated positively with cholesterol levels measured in the same samples (Figure 5B; r2=0.2233). When a single outlying point (indicated in Figure 5B) is removed from the analysis, a much stronger correlation is observed (r2=0.5444). The total iron levels detected by ICPMS did not correlate with cholesterol levels (Figure 5C).
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As some ligands (eg, carboxylic acids) bind both calcium and iron, both metal ions were quantified in some samples, but no correlation was detected between these parameters (Figure 5D).
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Discussion |
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Metal ions have been proposed as causative agents in a number of diseases, including atherosclerosis.18 Although some epidemiological studies have reported positive correlations between iron levels and cardiovascular disease, others have been negative.16–18 Previous experimental data on metal ion levels in human lesions have been criticized as a result of the methods used to quantify these materials.20,29 In the current study, we used a novel minimally invasive method, EPR spectroscopy, to quantify metal ion levels in washed, but otherwise intact, intimal samples from healthy and diseased arteries ex vivo. Because this methodology is nondestructive, and because the samples are kept at –196°C under an atmosphere of nitrogen during the experimental measurements (thereby preventing sample deterioration), both metal ion and cholesterol measurements could be made on the same samples, thereby eliminating tissue inhomogeneity as a confounding factor.
Statistically elevated levels of iron were detected in advanced human carotid lesions when compared with normal healthy intima samples by both techniques. These 2 measurements correlate well. The values obtained by EPR, which measures only Fe3+ complexes and not multinuclear complexes such as ferritin and hemosiderin, accounted for 70% of the total iron present. The elevation in iron levels is localized to the intima, with only low levels of iron detected in smooth muscle samples from the medial layer. The levels of iron measured by ICPMS are similar in magnitude to those reported for other human artery samples.30 The increase in total iron levels detected in the current human study (3.3-fold) is lower than that detected in rabbits fed a high-fat diet to promote atherosclerosis (7- to 8-fold increase), with this iron deposition reported to occur at the onset of lesion formation.12,31,32 Elevated levels of total copper were also detected by ICPMS, although at much lower concentrations than iron. The absolute concentrations of copper are lower than, but of similar magnitude to, those detected in rabbits exposed to high copper levels.33
The difference in iron levels detected by EPR and ICPMS is ascribed to contributions from EPR-silent Fe(II) complexes (particularly heme proteins) and multinuclear iron complexes such as ferritin. The Fe(III) signals detected by EPR have been assigned to high-spin rhombic species and are not caused by typical heme proteins.26 These signals are similar to those reported for poorly defined low-molecular-weight iron complexes detected in yeast, bacteria,23,24 and mammalian tissues.27,28 This iron pool may arise from the presence of elevated heme protein levels in the lesions (although no evidence was obtained for this), with subsequent degradation or damage to these species and iron release, either as a result of oxidative events or through the action of heme oxygenase on released heme. Previous studies have reported iron release from oxidized heme proteins34 and for elevated levels of heme oxygenase mRNA and protein in lesions.35,36
The greater increase in iron levels detected by EPR (16.7-fold) when compared with ICMPS (3.3-fold) may be of particular significance, because this reflects species that are not present within ferritin or hemosiderin (which do not give rise to EPR signals because of exchange effects24) or heme. The elevated levels of ferritin gene expression detected in lesions may be a response to these elevated iron levels.37 This nonferritin, nonheme iron pool may be redox-active and catalyze oxidative events within the artery wall, although this has not been investigated here. A recent report has suggested that iron may accumulate in lesions from intraplaque hemorrhage,38 and this would be consistent with the presence of these iron complexes in macrophage-derived cells, as proposed previously.14
The correlation between the EPR-detectable iron and cholesterol levels in advanced lesions is consistent with the accumulation of these species being inter-related. Whether this elevated nonferritin, nonheme iron pool contributes to low-density lipoprotein oxidation and the formation of macrophage-derived foam (lipid-laden) cells requires further study; both iron and copper can promote such processes in vitro.39–41
Iron and copper can promote oxidative damage to extracellular matrix components.42,43 These elevated metal ions levels may therefore affect plaque stability and propensity to rupture. Such damage may occur independently of, or synergistically with, that induced by matrix-degrading enzymes (eg, matrix metalloproteinases). Metalloproteinases are released as inactive pro forms, and oxidation can activate these species44 and inactivate inhibitors.45 Previous oxidative damage can also enhance matrix degradation by proteolytic enzymes.46,47 Studies with rupture-prone lesion types (ie, those with thin fibrous caps, low levels of smooth muscle cells, large numbers of macrophages, and lipid-rich48,49) would therefore seem warranted. The elevated metal ion levels are also consistent with the detection of elevated levels of protein oxidation products ascribed to metal ion catalyzed reactions detected in lesion proteins.6 Recent studies have shown that the majority of these species are present on matrix-associated materials, consistent with the aforementioned hypothesis.50
Overall, the data obtained in the current study are consistent with the hypothesis that high iron and copper levels may contribute to atherosclerosis and its sequelae as one factor in a multifactorial disease.
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Acknowledgments |
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Acknowledgments
The authors thank the National Health and Medical Research Council and the Australian Research Council for financial support. We thank the surgeons of Royal Prince Alfred Hospital for the provision of the artery samples.
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Footnotes |
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N.S. and R.A.L. contributed equally to this work.
Current affiliation for R.A.L. is Proteome Systems Ltd, North Ryde, Sydney, Australia.
Received November 11, 2003; accepted January 29, 2004.
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