**3. Analysis and discussion of FT-IR spectra**

Two representative FT-IR absorption spectra of a carotid and coronary artery in the region 4000 -400 cm-1 are shown in Figure 5. The spectra provide distinct features for the determination of the chemical composition and the diagnostic classification of arterial wall.

The spectra in high frequency region, 4000–2500 cm−1, mainly consist of νΟΗ, νΝΗ, asymmetric and symmetric methyl (CH3) and methylene (CH2) stretching vibrations. Significant differences are observed among carotid and coronary artery. In the case of coronary artery, the shoulder observed at 3524 cm–1 is assigned to vOH vibration of hydroxyl groups produced by the hyperoxidation of lipids and proteins and by addition of hydroxyl (HO•) free radicals to the double bonds of the fatty acids. The band at 3282 cm–1 is assigned to vNH stretching of the peptide bond (–NHCO-) of proteins (Theophanides et al., 1988). According to the intensity of vNH band, it is estimated that the coronary artery has a higher damage in proteins compared to the carotid artery.

The olefinic band v=C-H at 3077 cm−1 arises from the unsaturated lipids. In coronary artery, the high intensity of the band indicates that the foam cells are rich in low density lipoproteins (LDL). It is known that unsaturated lipids are more prone to lipid peroxidation (Mamarelis et al., 2010). The integrated area of the olefinic CH band can be used as an index of relative concentration of double bonds in the lipid structure from unsaturated fatty acyl

20 samples from carotid and coronary arteries from patients (60-85 years old) who underwent endarterectomy were used for the study (Figure 4). Representative sections of the Carotid atheromatic plaques and coronary arteries were restored in formalin. The FT-IR spectra were obtained with a Nicolet 6700 thermoscientific spectrometer, connected to an attenuated total reflection, 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 the OMNIC 7.1 software was used for data analysis. All the spectra for each patient and region were obtained in the same way. The analysis of bacterial morphology was performed by Scanning Electron Microcopy –SEM using a Fei Co at an accelerating potential 25 kV. Uncoated freeze dried cells were examined with LFD and BSED detectors. Qualitive elemental data analysis of the samples was determined by EDX (Energy-dispersive X-ray

Fig. 4. Sample from carotid arteryA: atheromatous plaque, B: adventitia , C: intima

Two representative FT-IR absorption spectra of a carotid and coronary artery in the region 4000 -400 cm-1 are shown in Figure 5. The spectra provide distinct features for the determination of the chemical composition and the diagnostic classification of arterial wall. The spectra in high frequency region, 4000–2500 cm−1, mainly consist of νΟΗ, νΝΗ, asymmetric and symmetric methyl (CH3) and methylene (CH2) stretching vibrations. Significant differences are observed among carotid and coronary artery. In the case of coronary artery, the shoulder observed at 3524 cm–1 is assigned to vOH vibration of hydroxyl groups produced by the hyperoxidation of lipids and proteins and by addition of hydroxyl (HO•) free radicals to the double bonds of the fatty acids. The band at 3282 cm–1 is assigned to vNH stretching of the peptide bond (–NHCO-) of proteins (Theophanides et al., 1988). According to the intensity of vNH band, it is estimated that the coronary artery has a

The olefinic band v=C-H at 3077 cm−1 arises from the unsaturated lipids. In coronary artery, the high intensity of the band indicates that the foam cells are rich in low density lipoproteins (LDL). It is known that unsaturated lipids are more prone to lipid peroxidation (Mamarelis et al., 2010). The integrated area of the olefinic CH band can be used as an index of relative concentration of double bonds in the lipid structure from unsaturated fatty acyl

**3. Analysis and discussion of FT-IR spectra** 

higher damage in proteins compared to the carotid artery.

**2. Materials and methods** 

spectroscopy).

Fig. 5. FT-IR adsorption spectra obtained from human tissues (a) carotid artery, (b) coronary artery in the region of 4000-400 cm-1.

chains (e.g. linolenic, arachidonic, etc.), and/or due to lipid peroxidation. For this reason, the intensity of this band can be used as a diagnostic band of LDL.

The CH2 asymmetric (2929 cm−1) and symmetric (2851 cm−1) stretching vibrations give intense bands, while asymmetric CH3 stretching at 2955 cm−1 and symmetric stretching at 2865 cm−1 bands are seen as shoulders. The bands arise from lipids, phospholipids and membranes. The intensity of symmetric and asymmetric stretching vibrations of CH2 and CH3 reflect lipid hyperoxidation (Liu et al., 2002). The increase in the intensity of the bands in coronary artery shows that the environment is less lipophilic due to fragmentation of the lipoproteins and accumulation of free cholesterol and cholesterol esters in the atheromatous core, as a result membrane fluidity changes significantly (Anastassopoulou and Theophanides, 1990).

Significant changes are also observed in the infrared absorption bands in the region 1800- 1500cm–1, as it is shown in the spectra. The presence of cholesterol esters and other estercontaining compounds is also identified from the carboxyl ion (-O-C=O) stretching absorption at 1735 cm−1 apart from the C=C-H stretching band (3077 cm−1). This band confirms lipid hyperoxidation and the increased intensity of the band indicates increased LDL concentrations according to the blood analyses of the patient. All the patients who underwent coronary endarterectomy showed higher intensity in the specific band. The bands at 1735 and 3077 cm-1 can be used as indicators for LDL cholesterol of patients.

The Amide I absorption band, arises mainly from the C=O stretching vibration with minor contributions from the out-of-phase CN stretching vibration, the CCN deformation and the NH in-plane bend. The Amide I band is down-shifted near 1635 cm-1, approximately 20 cm-1 difference compared to the absorption of a normal tissue (1656 cm-1), suggesting a

FT-IR Spectroscopy in Medicine 279

Fig. 7. A: FT-IR spectra of carotid in the region 1800-1500 cm-1, B: Deconvolution of the

atheromatous plaque, which are probably connected to metal with high molecular weight. The broad band in the region 1750—1720 cm-1 appears to be the summation of two underlying components by deconvolution, giving one band at 1743cm-1 and a second one at 1721 cm-1. These bands are assigned to LDL cholesterol and are due to stretching vibrations of the carbonyl group involved in ester bonds. These bands arise from the interfacial region of the glycerolipid moiety and are responsive to changes in their environment, such as

The broad band at 1700–1600 cm−1 is Amide I band, constituted from the bands at 1691, 1674, 1654, 1629, and 1610 cm-1. The main amide I band at 1654 cm-1 is indicative of a high content of a-helical structure, although part of the absorption at this frequency also corresponds to random coil structure at 1630cm-1 and antiparallel β-sheet at 1691 cm-1 (Barth, 2007). Particularly, the band at 1691 cm-1 arises from the peptide bond of proteins and mainly from the vibration of C=O compared to NH group. The band at 1674 cm-1 is attributed to apolipoprotein ApoC-III, which resides in HDL and inhibits the lipolysis of triglyceride-rich lipoproteins. The decrease of a-helix absorbance band compared to random

The bands at the wavelengths 1592, 1570, 1547 and 1512, which are assigned to the groups of arginine, aspartic acid, glutaminic acid and tyrosine of apolipoproteins Apo A-I. The specific bands were revealed after the deconvolution treatment of spectra. The wide band at 1537 cm-1 is split into two bands, one in 1550cm-1, which is characteristic of Amide II absorption due to the stretching vibrations C-N and bending vibration N-H because of the influence of lipids. The apolipoproteins of Apo A-I and A-II, are components of HDL (High density Lipoproteins) and control HDL metabolism (Nara et al., 2002). The distinctive structures and properties of apoA-I and apoA-II, the two major HDL proteins, determine in different ways the thermodynamic stability of HDL - the former through its greater plasticity and the latter by its higher lipophilicity. Apo A-I protects phospholipids from oxidation due to a

spectra in the same region.

hydrogen bonding or polarity (Arrondo and Goni, 1998).

coil confirms the fragmentation due to free radical interactions.

conformational change in a –helixes (Anastassopoulou et al., 2009 ). The shifting of the Amide I band suggests that proteins lose their structure from a-helix to random coil due to fragmentation induced from free radical reactions. The exposure of proteins to free radicals induces secondary structural changes, since secondary structure is stabilized by hydrogen bonding of peptide backbone. Proteins are organized into a-helixes, but the hydrogen bond is damaged, so the chains opens and are more prone to free radicals, leading to the change of a-helix to random coil.

The change of dipole moment of peptide bond, as it is shown in equation [1] at resonance structures, leads to a change in the orientation of amino groups (NH) to the carbonyl group C=O, resulting in the destruction of alpha –helix and the secondary structure of proteins.

The band at 1537 cm-1 is attributed to the vibrations of Amide II. The amide II mode is the out-of-phase combination of the NH in plane bend and the CN stretching vibration with smaller contributions from the CO in plane bend and the NC stretching vibrations. The bands of Amide I and Amide II are representative of –NH-CO- vibrations of proteins (Theophanides et al., 1988). The analysis of the spectra by Fourier self-deconvolution was used to enhance resolution in the region 1800-1500 cm-1 (figure 6 and 7).

Fig. 6. A: FT-IR spectra of coronary artery in the region 1800-1500 cm-1, B: Deconvolution of the spectra in the same region.

As it is determined from the deconvolution in the spectra of the coronary artery of a patient, the band at 1781 and 1768 cm-1 is attributed to the carboxyl anions –COO- of the

conformational change in a –helixes (Anastassopoulou et al., 2009 ). The shifting of the Amide I band suggests that proteins lose their structure from a-helix to random coil due to fragmentation induced from free radical reactions. The exposure of proteins to free radicals induces secondary structural changes, since secondary structure is stabilized by hydrogen bonding of peptide backbone. Proteins are organized into a-helixes, but the hydrogen bond is damaged, so the chains opens and are more prone to free radicals, leading to the change

The change of dipole moment of peptide bond, as it is shown in equation [1] at resonance structures, leads to a change in the orientation of amino groups (NH) to the carbonyl group C=O, resulting in the destruction of alpha –helix and the secondary structure of proteins.

The band at 1537 cm-1 is attributed to the vibrations of Amide II. The amide II mode is the out-of-phase combination of the NH in plane bend and the CN stretching vibration with smaller contributions from the CO in plane bend and the NC stretching vibrations. The bands of Amide I and Amide II are representative of –NH-CO- vibrations of proteins (Theophanides et al., 1988). The analysis of the spectra by Fourier self-deconvolution was

Fig. 6. A: FT-IR spectra of coronary artery in the region 1800-1500 cm-1, B: Deconvolution of

As it is determined from the deconvolution in the spectra of the coronary artery of a patient, the band at 1781 and 1768 cm-1 is attributed to the carboxyl anions –COO- of the

used to enhance resolution in the region 1800-1500 cm-1 (figure 6 and 7).

(1)

of a-helix to random coil.

the spectra in the same region.

Fig. 7. A: FT-IR spectra of carotid in the region 1800-1500 cm-1, B: Deconvolution of the spectra in the same region.

atheromatous plaque, which are probably connected to metal with high molecular weight. The broad band in the region 1750—1720 cm-1 appears to be the summation of two underlying components by deconvolution, giving one band at 1743cm-1 and a second one at 1721 cm-1. These bands are assigned to LDL cholesterol and are due to stretching vibrations of the carbonyl group involved in ester bonds. These bands arise from the interfacial region of the glycerolipid moiety and are responsive to changes in their environment, such as hydrogen bonding or polarity (Arrondo and Goni, 1998).

The broad band at 1700–1600 cm−1 is Amide I band, constituted from the bands at 1691, 1674, 1654, 1629, and 1610 cm-1. The main amide I band at 1654 cm-1 is indicative of a high content of a-helical structure, although part of the absorption at this frequency also corresponds to random coil structure at 1630cm-1 and antiparallel β-sheet at 1691 cm-1 (Barth, 2007). Particularly, the band at 1691 cm-1 arises from the peptide bond of proteins and mainly from the vibration of C=O compared to NH group. The band at 1674 cm-1 is attributed to apolipoprotein ApoC-III, which resides in HDL and inhibits the lipolysis of triglyceride-rich lipoproteins. The decrease of a-helix absorbance band compared to random coil confirms the fragmentation due to free radical interactions.

The bands at the wavelengths 1592, 1570, 1547 and 1512, which are assigned to the groups of arginine, aspartic acid, glutaminic acid and tyrosine of apolipoproteins Apo A-I. The specific bands were revealed after the deconvolution treatment of spectra. The wide band at 1537 cm-1 is split into two bands, one in 1550cm-1, which is characteristic of Amide II absorption due to the stretching vibrations C-N and bending vibration N-H because of the influence of lipids. The apolipoproteins of Apo A-I and A-II, are components of HDL (High density Lipoproteins) and control HDL metabolism (Nara et al., 2002). The distinctive structures and properties of apoA-I and apoA-II, the two major HDL proteins, determine in different ways the thermodynamic stability of HDL - the former through its greater plasticity and the latter by its higher lipophilicity. Apo A-I protects phospholipids from oxidation due to a

FT-IR Spectroscopy in Medicine 281

Fig. 8. FT-IR spectra of carotid and coronary artery in the region 1500-1300 cm-1 and the

al., 2005). The calcified atherosclerotic plaque spectra are dominated by bands from calcified

Scanning electron microscopy (SEM) was used for imaging biological specimens, thus enabling rapid and high-resolution imaging of atherosclerotic lesions (Kamar et al., 2008). High resolution images can be obtained without gold coating, thereby enabling imaging of atherosclerotic lesions close to its original state. Significant structural alterations and mineral salts were observed in carotid and coronary arteries. The membrane morphology of carotid

The architecture of foam cells is heterogenous as well as the size of white stones. It was found that this region is rich in phosphorous. It has been found that initiation of atheromats 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 the oxidized phospholipids to lysophosphatydil choline and causes to atherogenesis (Gorelick, 2008; Parthasarathy et al., 2008). The ratio of [Ca]/[P] according to elemental

On the contrary, the coronary artery is rich in calcium but no phosphorus was detected (Figure 11). As a result, carbonated apatite is mainly formed in coronary arteries. This observation is in agreement with the results from FT-IR spectra. Many of the mineral salts

deconvolution of the band at 1454 cm-1 of coronary (a) and carotid (b) artery.

analysis (EDAX)demonstrates that the stones of carotid are hydroxyapatite.

minerals such as hydroxyapatite and carbonated apatite.

**4. Scanning electron microscopy** 

artery is shown in figure 10.

conformational constraint governed by adjacent amphiphatic a-helices located in C-terminal lipid-binding domain. Apo A-I is a potent inhibitor of lipid peroxidation, protecting the phospholipids from water-soluble and lipophilic free radical initiators (Bolanos-Garcia and Miguel, 2003). The reduction of Apo A-I and A-II and the increase of the characteristic band of LDL at 1735 cm-1 are connected to the blood analyses of the patients.

Relatively, the deconvolution in the carotid artery spectra of a patient as it is shown in figure 7, revealed the bands at 1776 and 1757 cm-1, which are attributed to the carboxyl anions – COO- of the atheromatous plaque. The lipid content is indicated by the lipid ester band at 1734 cm-1. The wide band of amide I is split to the bands at 1691, 1653, 1630 cm-1, which reveals the destruction of a-helical structure of proteins due to the free radicals reactions. The peptide bond appears another characteristic band at the region of amide II absorption, which is constituted from the bands at 1592, 1552, 1534 and 1513, which are attributed to arginine, a-helix of collagen, random coil and tyrosine.

In carotid and coronary artery, the deconvolution confirmed the peroxidation of lipids and lipoproteins. The intensity of the aldehydes due to peroxidation of LDL (1735 and 1742 cm-1) is higher in the case of coronary artery. Various small molecular weight aldehydes such as acrolein, malondialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE) are formed during lipid peroxidation as secondary or decomposition products. The main product of aldehydes in this region is malonaldehyde (MDA), which is an end product of lipid peroxidation that starts with abstracting a hydrogen atom from an unsaturated fatty acid chain, and this peroxidation spreads to the adjacent fatty acids continually. It has been proved that MDA inhibits the metabolism of high density lipoproteins (HDL), which are protecting factors of human organism. Thus, the increase of LDL, which is relative to the clinical condition of the patients, is associated with a higher risk of cardiovascular disease.

In figure 8, the wide band at 1454 cm-1 is constituted of two bands at at 1461 cm-1and 1443 cm-1 in the coronary artery, which arise from the carbon chain of lipids, combination of bending vibrations of δCH2 of carbon chains of lipids and δCOOH of non-ionic groups, respectively. The deconvolution of the band in the carotid artery revealed three bands, at 1467, 1454, 1443 cm-1, which are assigned to the bending vibrations of δasCH3 of lipids, stretching vibration of νCO3 2- and δCOOH of non-ionic groups, respectively. In the coronary artery, the intense increase of the band results in the decrease of lipophilic environment of membrane. This observation is in agreement with the increase of the stretching vibration bands in the region 3000-2870 cm-1.

The absorptions at 1238, 1173 and 1024 cm–1 matched the spectral patterns that arise from amide III (in plane N –H bending and C-N stretching vibrations) and the asymmetric and symmetric stretching modes of PO2 – in DNA or the phosphodiester groups of the phospholipids, cholesterol ester and –C-O-C- vibrations of fatty acids and ketals (Mamarelis et al., 2010), respectively, which are product of atheromatous plaque of coronary and carotid artery due to hyperoxidation of membranes (Figure 9).. The comparison of coronary and a carotid artery reveals that intense hyperoxidation has taken place in the coronary artery, as it is determined from the C-O-C vibrations of aldehyde groups. To the contrary, the vibrations of phosphate groups PO2 of phospholipids and DNA band together with the bending vibration *v*4CO3 2– at 874 cm-1 suggests that the atheromatic plaque is consisted from calcium carbonate (CaCO3) and that the foam cells are rich in calcium minerals(Petra et

conformational constraint governed by adjacent amphiphatic a-helices located in C-terminal lipid-binding domain. Apo A-I is a potent inhibitor of lipid peroxidation, protecting the phospholipids from water-soluble and lipophilic free radical initiators (Bolanos-Garcia and Miguel, 2003). The reduction of Apo A-I and A-II and the increase of the characteristic band

Relatively, the deconvolution in the carotid artery spectra of a patient as it is shown in figure 7, revealed the bands at 1776 and 1757 cm-1, which are attributed to the carboxyl anions – COO- of the atheromatous plaque. The lipid content is indicated by the lipid ester band at 1734 cm-1. The wide band of amide I is split to the bands at 1691, 1653, 1630 cm-1, which reveals the destruction of a-helical structure of proteins due to the free radicals reactions. The peptide bond appears another characteristic band at the region of amide II absorption, which is constituted from the bands at 1592, 1552, 1534 and 1513, which are attributed to

In carotid and coronary artery, the deconvolution confirmed the peroxidation of lipids and lipoproteins. The intensity of the aldehydes due to peroxidation of LDL (1735 and 1742 cm-1) is higher in the case of coronary artery. Various small molecular weight aldehydes such as acrolein, malondialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE) are formed during lipid peroxidation as secondary or decomposition products. The main product of aldehydes in this region is malonaldehyde (MDA), which is an end product of lipid peroxidation that starts with abstracting a hydrogen atom from an unsaturated fatty acid chain, and this peroxidation spreads to the adjacent fatty acids continually. It has been proved that MDA inhibits the metabolism of high density lipoproteins (HDL), which are protecting factors of human organism. Thus, the increase of LDL, which is relative to the clinical condition of the

In figure 8, the wide band at 1454 cm-1 is constituted of two bands at at 1461 cm-1and 1443 cm-1 in the coronary artery, which arise from the carbon chain of lipids, combination of bending vibrations of δCH2 of carbon chains of lipids and δCOOH of non-ionic groups, respectively. The deconvolution of the band in the carotid artery revealed three bands, at 1467, 1454, 1443 cm-1, which are assigned to the bending vibrations of δasCH3 of lipids, stretching vibration of νCO3 2- and δCOOH of non-ionic groups, respectively. In the coronary artery, the intense increase of the band results in the decrease of lipophilic environment of membrane. This observation is in agreement with the increase of the

The absorptions at 1238, 1173 and 1024 cm–1 matched the spectral patterns that arise from amide III (in plane N –H bending and C-N stretching vibrations) and the asymmetric and symmetric stretching modes of PO2 – in DNA or the phosphodiester groups of the phospholipids, cholesterol ester and –C-O-C- vibrations of fatty acids and ketals (Mamarelis et al., 2010), respectively, which are product of atheromatous plaque of coronary and carotid artery due to hyperoxidation of membranes (Figure 9).. The comparison of coronary and a carotid artery reveals that intense hyperoxidation has taken place in the coronary artery, as it is determined from the C-O-C vibrations of aldehyde groups. To the contrary, the vibrations of phosphate groups PO2 of phospholipids and DNA band together with the bending vibration *v*4CO3 2– at 874 cm-1 suggests that the atheromatic plaque is consisted from calcium carbonate (CaCO3) and that the foam cells are rich in calcium minerals(Petra et

of LDL at 1735 cm-1 are connected to the blood analyses of the patients.

arginine, a-helix of collagen, random coil and tyrosine.

patients, is associated with a higher risk of cardiovascular disease.

stretching vibration bands in the region 3000-2870 cm-1.

Fig. 8. FT-IR spectra of carotid and coronary artery in the region 1500-1300 cm-1 and the deconvolution of the band at 1454 cm-1 of coronary (a) and carotid (b) artery.

al., 2005). The calcified atherosclerotic plaque spectra are dominated by bands from calcified minerals such as hydroxyapatite and carbonated apatite.

#### **4. Scanning electron microscopy**

Scanning electron microscopy (SEM) was used for imaging biological specimens, thus enabling rapid and high-resolution imaging of atherosclerotic lesions (Kamar et al., 2008). High resolution images can be obtained without gold coating, thereby enabling imaging of atherosclerotic lesions close to its original state. Significant structural alterations and mineral salts were observed in carotid and coronary arteries. The membrane morphology of carotid artery is shown in figure 10.

The architecture of foam cells is heterogenous as well as the size of white stones. It was found that this region is rich in phosphorous. It has been found that initiation of atheromats 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 the oxidized phospholipids to lysophosphatydil choline and causes to atherogenesis (Gorelick, 2008; Parthasarathy et al., 2008). The ratio of [Ca]/[P] according to elemental analysis (EDAX)demonstrates that the stones of carotid are hydroxyapatite.

On the contrary, the coronary artery is rich in calcium but no phosphorus was detected (Figure 11). As a result, carbonated apatite is mainly formed in coronary arteries. This observation is in agreement with the results from FT-IR spectra. Many of the mineral salts

FT-IR Spectroscopy in Medicine 283

Fig. 11. A: SEM imaging of coronary membrane, region rich in mineral deposits

e O2 O2

The ubiquitous presence of oxygen in higher species and diatomic oxygen's ability to readily accept electrons has made oxygen-centered free radicals the most frequently encountered radical species, which are involved in the pathogenesis of atherosclerosis. The hydrogen peroxide molecules are intermediate products in the catalytic cycle of oxidation of P450

.

The main factors for the production of free radicals are the iron cations (Fe2+) of the hemoproteins and bivalent copper cations (Cu2+) from copper proteins(Halliwell & Gutteridge, 2000; Anastassopoulou & Dovas, 2007 ). The irons cations react with hydrogen

Lipids, usually polyunsaturated fatty acids react with the produced hydroxyl radicals by hydrogen abstraction leading to the formation of lipid free radicals, according to the

2H+

H2O2

+ OH-


ΟΗ), according to Fenton or Haber-Weiss like

(3)

(scale 50 μm), B: EDAX analysis of a white spot.

**5. Oxygen-centered free radicals** 

cytochrome according to the reaction [2]:

peroxide and produce hydroxyl free radicals (.

Fe2+ + H2O2 Fe3+ + ΗΟ.

reactions:

reaction [4]:

Fig. 9. FT-IR spectra of carotid and coronary artery in the region 1300-800 cm-1.

Fig. 10. A:SEM imaging of carotid membrane, region rich in mineral deposits (scale 500 μm), B: EDAX analysis of a white spot.

are not connected to the tissues while other are bound by chemical bonds. Additionally, an increased number of fibrils are presented that confirm the fact that free radicals play an important role in the development of atheromas. The architecture of surface is rich in spheres of LDL, as it was demonstrated from FT-IR spectra. The molecules of LDL obtain the shape of sphere that corresponds to the minimum energy.

Fig. 9. FT-IR spectra of carotid and coronary artery in the region 1300-800 cm-1.

Fig. 10. A:SEM imaging of carotid membrane, region rich in mineral deposits (scale 500 μm),

are not connected to the tissues while other are bound by chemical bonds. Additionally, an increased number of fibrils are presented that confirm the fact that free radicals play an important role in the development of atheromas. The architecture of surface is rich in spheres of LDL, as it was demonstrated from FT-IR spectra. The molecules of LDL obtain

B: EDAX analysis of a white spot.

the shape of sphere that corresponds to the minimum energy.

Fig. 11. A: SEM imaging of coronary membrane, region rich in mineral deposits (scale 50 μm), B: EDAX analysis of a white spot.

#### **5. Oxygen-centered free radicals**

The ubiquitous presence of oxygen in higher species and diatomic oxygen's ability to readily accept electrons has made oxygen-centered free radicals the most frequently encountered radical species, which are involved in the pathogenesis of atherosclerosis. The hydrogen peroxide molecules are intermediate products in the catalytic cycle of oxidation of P450 cytochrome according to the reaction [2]:

$$\bullet\_{\mathcal{O}\_2} \xrightarrow{\text{e}} \bullet\_{\mathcal{O}\_2} \text{:} \xrightarrow{\text{2H}^+} \bullet\_{\text{H}\_2\mathcal{O}\_2} \tag{2}$$

The main factors for the production of free radicals are the iron cations (Fe2+) of the hemoproteins and bivalent copper cations (Cu2+) from copper proteins(Halliwell & Gutteridge, 2000; Anastassopoulou & Dovas, 2007 ). The irons cations react with hydrogen peroxide and produce hydroxyl free radicals (. ΟΗ), according to Fenton or Haber-Weiss like reactions:

$$\mathrm{Fe^{2+} + H\_2O\_2 \longrightarrow Fe^{3+} + HO^{\cdot} + OH^{\cdot}} \tag{3}$$

Lipids, usually polyunsaturated fatty acids react with the produced hydroxyl radicals by hydrogen abstraction leading to the formation of lipid free radicals, according to the reaction [4]:

FT-IR Spectroscopy in Medicine 285

Peroxidized lipids decompose easily, generating both free and core aldehydes and ketones

These reactions explain the FT-IR spectra of coronary and carotid artery. In the region of 1800- 1600 cm-1, the band at 1735 cm-1 which was assigned to the carbonyl group of lipid esters and the presence of malonaldehyde is confirmed by the former reactions. Malondialdehyde (MDA) is frequently measured as indicator of lipid peroxidation and oxidative stress (Dotan et al.,

The non-destructive nature of FT-IR spectroscopy and the ability to directly probe biochemical changes lead to an understanding of the biochemical and structural changes associated with arteriosclerosis. The decrease in the intensity of the band at 1651 cm-1 and

2004). It is produced from lipid hydroperoxyl radical due to the following reactions:

according to the reaction [9] (Mamarelis et.al. 2010).

(8)

(9)

The produced lipid free radical is reproduced rapidly, leading to stable dimer products as it is shown in equation 5:

Lipid radicals react with each other, leading to the generation of one terminal double bond [6].

The former reaction explains the increasing of the intensity of the olefinic band at 3077 cm-1.

Taking into account that human lives under aerobic conditions, the oxygen in the form of double free radical (•O=O•) reacts rapidly with the lipid free radicals or other biomolecules such as collagen, elastin, resulting in the formation of lipid hydroperoxyl radical [7]. This step is the initiation of peroxidation.

The abstraction of hydrogen (adjacent lipids, thiols) from lipid hydroperoxyl radical leads to the generation of hydroperoxyl groups (–C-O-OH), which are non-ionic and lead to fixation of lipid damage.

(4)

(5)

(6)

(7)

284 Infrared Spectroscopy – Life and Biomedical Sciences

The produced lipid free radical is reproduced rapidly, leading to stable dimer products as it

Lipid radicals react with each other, leading to the generation of one terminal double bond

The former reaction explains the increasing of the intensity of the olefinic band at 3077 cm-1. Taking into account that human lives under aerobic conditions, the oxygen in the form of double free radical (•O=O•) reacts rapidly with the lipid free radicals or other biomolecules such as collagen, elastin, resulting in the formation of lipid hydroperoxyl radical [7]. This

The abstraction of hydrogen (adjacent lipids, thiols) from lipid hydroperoxyl radical leads to the generation of hydroperoxyl groups (–C-O-OH), which are non-ionic and lead to fixation

is shown in equation 5:

step is the initiation of peroxidation.

of lipid damage.

[6].

Peroxidized lipids decompose easily, generating both free and core aldehydes and ketones according to the reaction [9] (Mamarelis et.al. 2010).

$$\begin{array}{llll} \stackrel{\text{R}}{\text{R}}^{\text{R}}\stackrel{\text{R}'\text{O}}{\text{R}'\text{O}} & + \stackrel{\text{H}}{\text{H}\_2\text{O}} & \xrightarrow{\text{H}^+} & \stackrel{\text{H}^+}{\text{several steps}} & \text{R}\stackrel{\text{ii}}{\text{-C}}\text{-H} & + 2\text{R}'\text{OH} \\ \text{Acetal} & & & & \text{Aldebye} & \text{Alcohol} \end{array}$$

These reactions explain the FT-IR spectra of coronary and carotid artery. In the region of 1800- 1600 cm-1, the band at 1735 cm-1 which was assigned to the carbonyl group of lipid esters and the presence of malonaldehyde is confirmed by the former reactions. Malondialdehyde (MDA) is frequently measured as indicator of lipid peroxidation and oxidative stress (Dotan et al., 2004). It is produced from lipid hydroperoxyl radical due to the following reactions:

The non-destructive nature of FT-IR spectroscopy and the ability to directly probe biochemical changes lead to an understanding of the biochemical and structural changes associated with arteriosclerosis. The decrease in the intensity of the band at 1651 cm-1 and

FT-IR Spectroscopy in Medicine 287

Dotan, Y., Lichtenberg , D.& Pinchuk, I. (2004). Lipid peroxidation cannot be used as a

Elliot, A. & Ambrose, E. (1950). Structure of Synthetic Polypeptides. *Nature*, Vol.165, pp.921 Fahrenfort, J. (1961). Attenuated total reflection: A new principle for the production of

Gazi, E., Dwyer, J., Gardner, P., Ghanbari-Siakhani, A., Wde, A.P., Lockyer, N.P.,Vickerman,

Gorelick, P.B.(2008). Lipoprotein-Associated Phospholipase A2 and Risk of Stroke. *American* 

Goormaghtigh, E., Raussens, V. & Ruysschaert, J.-M. (1999). Attenuated total re£lection

Gough, K. M., Zelinski, D., Wiens, R., Rakand, M. & Dixon, M.C. (2003). Fourier transform

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the shifting to lower wavenumber (approximately 1630cm-1) justify the way of disappearance of the Amide I band and the change in the structure of proteins from alpha – helix to random coil due to free radicals reactions.

The bands at the region 1280 - 1170 cm-1 are attributed to the presence of O-C-C, O-C(O)-C groups due to the peroxidation of membranes. Thus, the presence of characteristic bands in the region 4000 -400 cm-1 confirms the peroxidation of membranes.
