Abstract
Atherosclerosis is a complex phenomenon which leads to sudden death. Fourier transform infrared (FT-IR) spectroscopy was used to study the pathogenic components of carotids that produce the atheromatic plaque at the molecular level, as well as the role of free radicals, which are developed during oxidative stress and their effect on plaque generation. The absorption infrared spectra reflected significant changes which were analogous to clinical data of each patient. The spectra contained signature bands of the biological molecules which were characteristic for the plaque components. The bands found at about 3080 cm−1 and 1736 cm−1 were proportional to low-density lipoprotein concentration for each patient, suggesting the hydroperoxidation of lipids due to free radicals, generated during oxidative stress. From scanning electron microscopy analysis, it was found that the carotid plaques contained calcium minerals, silicon and heavy metals, such as copper, silver, lead and titanium, which were related to the working environment of the patients.
Atherosclerosis is a complex phenomenon of plaque formation in blood vessels. The biochemical changes that take place metabolically in lipids generate an atheromatous plaque that thickens the lumen and decreases the blood flow, while the chemical components of the plaque can induce atheroembolic events (1). The risk factors that influence atheromatic plaque generation include hypertension, hypercholesterolemia, diabetes and smoking. Fourier transform infrared (FT-IR) spectroscopy is a non-destructive technique and is sensitive for evaluating the complicated systems of human tissues and cells (2-6). Using FT-IR spectroscopy, we have shown that it is possible to differentiate normal and premalignant cells (3-6). The infrared analysis of carotid plaques provides characteristic fingerprint bands of the tissues of each patient. In the present work, attenuated total reflection (ATR)-FT-IR spectroscopy was used to study the development of atheromatic plaque of carotid artery in patients who underwent carotid endarterectomy. In addition, scanning electron microscopy (SEM) was used to analyze the composition and architecture of the membrane surface and foam cells and the metals present in the atheroma of the patients.
Patients and Methods
Fifty samples (from 8 females and 42 males) from carotid atheromatic plaques from patients (60-85 years old) who underwent carotid endarterectomy were used for the study. All of the patients were smokers, 92% hypertensive, 95% hyperlipidemic, 33%, diabetic, 59% hyperuricaemic and 59% with coronary artery disease and/or peripheral vascular disease. Representative sections of the biopsies were restored in formalin and histological evaluation showed normal atheroma, with no evidence of metabolic or inflammatory disease. Figure 1 shows a carotid artery atheroma of a patient. One can see the yellow (foam cells), white (fibrotic tissues) and brown (with haemorrhage) regions.
The FT-IR spectra were obtained with a Nicolet 6700 thermoscientific spectrometer, connected to an ATR accessory. For each region a series of spectra were recorded and every spectrum consisted of 120 co-added spectra at a resolution of 4 cm−1 and OMNIC 7.1 software was used for data analysis. All the spectra for each patient and region were obtained in the same way. The SEM and the microanalyzer probe were from Fei Co, Eindhoven, the Netherlands.
Carotid artery with atheromatic plaque. The yellow (foam), white (fibrils) and brown (hemorrhagic) regions shown are the locations from where the spectra were obtained.
Results
The FT-IR spectra of three representative regions from a carotid artery atheroma with hemorrhage (a), rich in calcium carbonate minerals (b) and foam cells (c) in the mid infrared region 4000-400 cm−1 are shown in Figure 2. The shoulder observed at 3527 cm−1 is assigned to vOH vibration of hydroxyl groups produced most of lipids by addition of hydroxyl (HO•) free radicals to the double bonds of the fatty acids. The intensity of this band increases in the region of foam cells and also it is influenced by clinical characteristics of each patient.
The band at 3280 cm−1, which is assigned to vNH stretching of the peptide bond (−NHCO-) of proteins (7) decreases in the area of foam cells, leading to the conclusion that in foam cells there is a higher damage of the membranes. The band at 3082 cm−1 arises from the carbon hydrogen (C-H) stretching vibration of the fatty acids, and is assigned to the olefinic v=C-H carbon-hydrogen bond. From the high intensity of the band at 3082 cm−1, it is suggested that the foam cells are rich in low density lipoproteins (LDL), since only LDL contains the olefinic=C-H group. This was also observed in a separate environment as a concentration effect serving as a calibration experiment. The intensity of this band increases with increasing plasma level in patients and could be used as diagnostic band.
Significant variations were also observed in the region of 2970 to 2850 cm−1 vibrations that arise from the stretching vibrations of vCH3 and vCH2 groups of lipids, phospholipids and membranes (2, 8). The significant higher intensity of these bands in foam cells indicates that the environment became less lipophilic due to fragmentation of the lipoproteins (9-12), induced by hydroxyl free radical (HO•). Comparison between our different subgroups showed that diabetic and hyperuricemic patients revealed significantly different FT-IR bands in the region 2970-2850 cm−1.
The absorption at 1735 cm−1 is assigned to vC=O carbonyl stretching vibration of ester carbonyl groups (ROC=O) or to the carboxyl COOH of the atherogenic plaque (5-8, 11-13). This particular band is associated with LDL cholesterol concentration and is a sign that hyperoxidation of lipids through free radicals was taking place. Significant changes are also observed in the spectra of samples of patients in the region 1700-700 cm−1. The infrared absorption bands in the region 1700-1500 cm−1 are generated by the −vC=O stretching and the δNH bending of the amide I (-NHCO-), amide II and amide III modes of vibration in proteins (3-7). Upon shifting of these bands near 1630 cm−1, it was suggested that the proteins change their tertiary structure from α-helix to random coil due to fragmentation induced by free radical reactions (3-7). This particular band is in accordance with that at 3280 cm−1. The infrared spectra showed two minor bands at 1690 cm−1 and at 1620 cm−1 which are attributed to apo-B100 of LDL. It is known that LDL contains only apo-B100 apolipoprotein (12) and is characterized from their β-strand conformation. Indeed, for some patients there were greater changes in carotid FT-IR spectra due to several eliminations of proteins (apoprotein). From the intensity and shape of band near 1454 cm−1 (Figure 2, spectrum b) it is suggested that this band is not simple but it is a combination of bending vibrations of CH2 and stretching vibrations of the carbonate v3CO 2−3 anion near 1423 cm−1. This band together with the bending vibration v4CO 2−3 at 874 cm−1 suggests that the atheromatic plaque is composed of calcium carbonate (CaCO3) (5, 7) and that the foam cells are rich in calcium.
The absorptions at 1234, 1169 and 1029 cm−1 matched the spectral patterns that arise from amide III and the asymmetric and symmetric stretching modes of PO −2 in DNA or the phosphodiester groups of the phospholipids, cholesterol ester and −C-O-C-vibrations of fatty acids and ketals (13), respectively, which all fall in this region.
Discussion
It has been established that free radicals are produced in humans during metabolism or oxidative stress, which destroy many important biological molecules. The hydrogen peroxide molecules are intermediate products in the catalytic cycle of oxidation of P450 cytochrome according to the reaction (16):
[Eqtn 1]
The hydrogen peroxide molecules which are formed could react with bivalent iron cations (Fe2+) of the hemoproteins or with bivalent copper cations (Cu2+) from copper proteins, well as with toxic bivalent metal ions of transition metals, e.g. Co2+, Ni2+, Cr2+, producing hydroxyl free radicals (HO•) according to the following Fenton or Haber-Weiss [Eqtn 2] -like reactions (15, 16):
[Eqtn 2]
The hydroxyl free radicals that are produced then can react with lipids by hydrogen abstraction leading to lipid free radicals formation as follows:
[Eqtn 3]
Once lipid radicals are formed, they react with each other following the well known dismutation reaction (17, 18):
[Eqtn 4]
Through this reaction, the initial molecule (lipid) is reproduced together with one more molecule with one less hydrogen atom, leading to the generation of one terminal double bond. This reaction [Eqtn 4] explains the increasing of the intensity of the band at 3086 cm−1, which is assigned to the v=C-H group, very well. Since aerobic conditions predominate in humans, oxygen (O2), which is a double free radical (•O=O•), reacts rapidly with the above formed radical to generate a lipid hydroperoxyl radical:
[Eqtn 5]
The formed peroxyl radicals (C-O-O•) take up very fast mobile hydrogen atoms from compounds (donors) in the
environment, such as adjacent lipids, thiols, etc. and can finally produce hydroperoxyl groups (−C-O-OH), which are non-ionic. This reaction also leads to the fixation of lipid damage, induced by hydrogen abstraction from hydroxyl free radicals (18). Hydroperoxyl groups can also be produced by reaction of lipid radicals with hydroperoxyl radicals (HO •2) as follows (19):
[Eqtn 6]
Reactions shown in equations 5 and 6 explain the presence of the band near 1000 cm−1, which is assigned to −v-O-O-peroxyl stretching vibration. This finding allows us to suggest that the hydroxyl free radicals are required for the formation of lipid hydroperoxides and aldehydes according to the following general reaction (16):
[Eqtn 7]
It is accepted that peroxidized lipids decompose easily, generating both free and core aldehydes and ketones that covalently modify ε-amino groups of lysine residues of the protein moiety (20-23).
ATR-FT-IR spectra of carotid tissues from a patient in the range 4000-400 cm−1. a: Hemorrhage region, b: region rich in calcium carbonate minerals, and c: foam cells as detected by SEM.
SEM imaging of carotid membrane. A: Foam cells (scale 200 μm), B: region rich in mineral deposits (scale 1.0 mm), C: copper-rich region (scale 100 μm), and D: calcium carbonate-rich region (scale 200 μm).
Disulfide (S-S) bonds are found in the region 540-520 cm−1 resulting most likely from glutathiols (GSH) the natural protection factors of humans, which are oxidized in the first steps of oxidative stress. It is known that thiols act as protectors either as scavengers, reacting with hydroxyl radicals, or as H donors to restore the damage induced in important biological molecules. Finally, the carbon-sulphur (C-S) bond of thiols is found at about 640 cm−1.
Membrane morphology of the carotid is shown in Figure 3, taken with SEM. The imaging shows the heterogeneous architecture of foam cells (Figure 3A). Foam cells are rich in minerals with different size (shown Figure 3B).
This region of carotid atheroma was shown to be rich in phospholipids and phosphodiesters, as it was also shown by the FT-IR spectra in the region of 1200 to 900 cm−1, depending significantly on the clinical characteristics of the patients, such as hyperlipidemia, diabetes. It has been found that initiation of atheroma takes place in this region and thus, it is expected to be a region, which corresponds to atheromatic plaque rich in phospholipipases (Lp-PLA2). The enzyme Lp-PLA2 hydrolyses oxidized phospholipids to lysophosphatidy choline, which most likely causes the atherogenesis (19-21).
A region rich in copper (Cu) is shown in Figure 3C, while in Figure 3D the minerals are rich in calcium (Ca), as is shown in SEM analysis. Comparing these two images (Figure 3C and D) one can see that copper forms covalent bonds with the membrane, while calcium is different, forming ionic bonds, which do not absorb in the mid infrared. Regions rich in fibrils are shown in Figure 3D.
The presence of copper explains the initiation of free radical formation via Haber-Weiss reactions (15, 16) which leads to peroxidation of LDL. Silicon was present in almost all patients according to SEM analyses, but in different amounts. SEM analysis demonstrated the presence of heavy metals such as silver (Ag), lead (Pb) and titanium (Ti). The patient whose sample contained Ag was a photographer and the patient with Pb was a solderer using lead, while the patient with Ti had undergone coronary artery bypass graft surgery, using titanium sternal wires. This is an example of internal occupational pollution of the bodies of these patients.
The presence of such heavy metals and smoke of cigarettes has been related to the initiation of oxidative stress and cardiovascular diseases. However, to our knowledge, no epidemiological studies exist so far on this subject. This process results in inhibition of the normal functioning of the NO cycle regulation, which finally leads to cardiovascular disease (24).
Conclusion
The spectral analysis and the changes of infrared spectra at different sites of carotid atheromatic plaque showed the effect of hydroxyl free radicals on the infrared spectra of the carotid at a molecular level. It was suggested that peroxidation of lipids, phospholipids and membranes may take place during atherogenesis, which changes the structure of proteins from α-helix to random coil. Cigarette smoking and the heavy metals present in the carotids of the patients are high risk factors for production of reactive oxygen species, and most likely lead to development of atheromatic plaque. Furthermore, in the bypass patient who had sternal wires, titanium was detected by SEM analysis in his carotid. It is important to notice that the heavy metals silver and lead, which were found in the photographer and solderer patients, respectively, were related with their working environment and are known to induce oxidative stress (25, 26). The methodology of FT-IR and SEM used here could help find the mechanism of formation and generation of an atheromatic plaque. This result could lead to a new intravascular pre-diagnostic method, i.e., to detect and characterize chemically sites of atherosclerosis.
Footnotes
-
This work is part of the Ph.D. thesis of I. Mamarelis MD, at the National Technical University of Athens, 2010.
- Received June 30, 2010.
- Revision received August 13, 2010.
- Accepted August 23, 2010.
- Copyright © 2010 The Author(s). Published by the International Institute of Anticancer Research.
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