Vasiliki Dritsa

*National Technical University of Athens, NTUA Greece* 

#### **1. Introduction**

270 Infrared Spectroscopy – Life and Biomedical Sciences

Moore, R.E., Smith, C.K., Bailey, C.S., Voelkel , E.F. & Tashijian, A.H.( 1993).

Mythili, J., Sastry, T.P. & Subramarian, M. (2000). Preparation and characterization of a new

Petra, M., Anastassopoulou, J., Theologis, T. & Theophanides T. (2005). Synchrotron micro-

Pissaridi, K., Dritsa, V., Mamarelis., I, Koutoulakis, E., Kotulas, Ch. & Anastassopoulou, J.

Takeuchi, T., Tsuboi, T., Arai, M. & Togani, A. (2000). Adrenergic stimulation of

NATO Advanced Study Institute, D Reidel Publishing Co, Dodrecht. Theophanides, T., Anastassopoulou J. & Fotopoulos N. (1993). Vibrational Circular

Shier, D., Butler, J. & Lewis, R. (1996). *Hole's Human Anatomy*. McGraw-Hill, pp. 184-197. Suzuki, A., Palmer, G., Bonjour, J.P. & Caverzasio, J. (1998). Catecholamines stimulate the

organ culture. *Bone Miner* , Vol.23, pp.301–315.

*Molecular Structure*, Vol. 78, pp. 101-116

*Biol.Med*, Vol.11 , pp. 219-224.

*Bone*, Vol. 23, pp.197–203.

32, pp. 155-159.

Characterization of beta-adrenergic receptors on rat and human osteoblastlike cells and demonstration that beta-receptor agonists can stimulate bone resorption in

bioinorganic composite: collagen and hydroxyapatite. *Biotechnol Appl Biochem,* Vol.

FT-IR spectroscopic evaluation of normal paediatric human bone. *Journal of* 

(2011). The role of Molybdenum on atheromatic plaque formation. *Metal Ions* 

proliferation and alkaline phosphatase activity of MC3T3–E1 osteoblast-like cells.

osteoclastogenesis mediated by expression of osteoclasts differentiation factor in MC3T3-E1 osteoblast-like cells, Biochem. Pharmacology, Vol. 61, pp. 579- 586Theophanides, T. (1978). *Infrared and Raman spectroscopy of biological molecules.*

Dichroism of Proteins in H2O Solution, *Fifth International Conference on the Spectroscopy of Biological Molecules,* Kluwer Academic Publishers, The Netherlands. Veis, A. & Schlueter, R.J. The macromolecular Organization of Dentine matrix Collagen I. Characterization of Dentine Collagen. Biochemistry, Vol. 3, pp:1650-1657.

Infrared spectroscopy has been widely applied for the characterisation of various substances. Due to its sensitivity to the chemical information and architecture of the molecule, infrared spectroscopy can play an important role in new applications such as in the life-science field and not only in the traditional fields of physics and chemistry. Spectroscopic techniques are simple, reproducible, non-destructive without particular sample preparation. As a result, they provide information for the functional groups, bonds and molecular structure.

Herschel discovered the existence of infrared radiation when he tried to measure the heat produced by separate colors of a rainbow spectrum in 1800. He noted that the highest temperature fell beyond the red end of the spectrum, implying the existence of invisible light beyond the red. Herschel termed this light *calorifi*c *rays*. Infrared spectra originate on the vibrational motions of atoms in chemical bonds within molecules. When a beam of light containing the IR radiation band is passed through a sample, light energy from the photons is absorbed by the chemical bonds and excites the vibrational motions. As a molecule absorbs radiation at a specific frequency, it produces a band in the infrared spectrum at the corresponding wavenumber. The approximate position of an infrared absorption band is determined by the vibrating masses and the chemical bonds (single, double, triple). Τhe exact position of the band depends also on electron withdrawing or donating effects of the intra- and intermolecular environment and coupling with other vibrations. The strength of absorption increases with increasing polarity of the vibrating atoms. The modes of vibration in a molecule that can absorb infrared radiation are many and increase with increasing complexity of the molecule. The vibrations that contribute to the spectrum are bending and stretching vibrations between atoms and rocking, twisting and wagging of a functional group (Theophanides, 1984; Goormaghtigh et al., 1999).

Fourier transform infrared spectroscopy is preferred over dispersive or filter methods of infrared spectroscopy due to the sensitivity and the rapid data collection. The FT-IR spectrometer uses an interferometer to modulate the wavelength from a broadband infrared source. Light emitted from the infrared source is split by a beam splitter. Half of the light is reflected towards a fixed mirror and from there reflected back towards the beamsplitter where about 50% passes to reach the detector. The other half of the initial light intensity passes the beam splitter on its first encounter, is reflected by the moving mirror back to the beamsplitter where 50% of it is reflected towards the detector (Figure 1).When the two

FT-IR Spectroscopy in Medicine 273

particularly in the biomedical sciences. High quality spectra can be obtained from cell suspensions containing l0-50.000 cells, depending upon the size of the cells. In the case of tissues, such measurements generally require a sample size of 1 mm3 (Legal, 1991). Additionally, ATR has been established as a method of choice of analyzing samples that are either too thick or too strongly absorbing to analyse by other transmitting techniques. The use of these techniques have become a great potential over other diagnostic methods for the determination of the chemical components of tissues at various diseases states, due to the rapid and reagent free procedure. An advantage of ATR-FTIR to study the structure of biomembranes is that the membrane can be deposited on the surface of the internal reflection element (IRE) as a thin film of highly oriented membranes by evaporation of the water. Variations in spectral signatures arising from nucleic acids, proteins and lipids can

In 1949 Blout, Mellors and Woernley in 1952 reported that infrared spectra of human and animal tissues could provide information on the molecular structure of tissues. These studies met with limited success due to non-developed instrumentation available and little knowledge of spectroscopic properties of biological molecules and the complexity of the samples. At the same time, Elliot and Ambrose (1950) proposed empirical correlations between peptide structure and the Amide I and Amide II bands. The development of sensitive and high throughput spectrometers led to a wide field of medical applications of FT-IR spectroscopy. The rapid experimental and theoretical development took place in

FT-IR has been extensively applied for the determination of a biochemical metabolite in biological fluids. The improved sensitivity and data processing capability of new instruments, the presence of water is no longer a serious obstacle in the analysis of fluids. Current enzymatic methods require frequent calibration controls and reagents, and this is very costly. FT –IR spectroscopy has been used for the determination of glucose, total protein, urea, triglyceride, cholesterol, chylomicron and very low density lipoproteins in plasma and serum, in order to replace commonly used ultracentrifugation techniques (Deleris and Petibois, 2003; D. Krilov et al., 2009).The differences in the size, lipid composition and apolipoprotein structure in particular classes of lipoproteins are reflected

FTIR has received much attention as a promising tool for non-destructive characterisation of the molecular features of atherosclerosis due to the fact that vibrational spectra are sensitive

1970s, where Fourier Transform interferometers interfaced to digital computers.

Fig. 2. Diagram of the ATR setup.

provide important information in a number of disease states.

in the characteristic spectral bands of lipid and protein moiety.

**1.1 Medical applications of FT-IR spectroscopy** 

beams recombine, they interfere and there will be constructive or destructive interference depending on the optical path difference. A detector measures the intensity of transmitted or reflected light as a function of its wavelength. The signal obtained from the detector is an interferogram, which is analyzed by a computer using Fourier transforms to obtain a singlebeam infrared spectrum (Barth, 2007).

Fig. 1. Schematic function of an FT-IR set-up.

Fourier transform infrared (FT-IR) spectroscopy has proven to be a fundamental and valuable technique in biology and medicine due to its high sensitivity to detecting changes in the functional groups belonging to tissue components such as lipids, proteins and nucleic acids. Each of these tissue components can be detected and characterized by their characteristic absorption bands at specific wavelengths within a single spectrum. For biological spectroscopy, the important vibrations occur in the mid-infrared region (4000 to 400 cm-1) where most organic molecules show characteristic spectral features (Theophanides, 1978). The domain of biological applications of infrared spectroscopy encompasses a wide range of very different molecular structures. Biological molecules are categorized into proteins, nucleic acids, lipids, membranes, blood tissues. Different biomolecules interact among themselves, comprising electrostatic interactions, hydrogen bondings and van der Waals interactions, which can be readily studied by infrared spectroscopy.

For complex samples that transmit infrared radiation poorly or no changes must take place in their specimen, attenuated total reflection (ATR) is applied. The technique was developed by Harrick (1960) and Fahrenfort (1961). ATR is a specialised sampling technique, where the sample is placed on ATR crystal. An infrared beam is passed through the ATR crystal, reflects of the interface of the crystal and the sample, and is passed through to the detector. During the reflection, an evanescent wave extends beyond the crystal into the sample, which enables the absorption of energies corresponding to infrared frequencies by the sample. The penetration depth of the evanescent wave is a function of wavelength with deeper penetration at longer wavelengths. This may lead to distortions in the relative intensities of infrared peaks if sample thickness is insufficient for complete coverage of the evanescent wave (Goormaghtigh et al., 1999). Figure 2 shows the diagram of a basic ATR set up.

Attenuated total reflection (ATR) coupled with FT-IR, can obtain the infrared spectrum of solid or liquid samples in their native state. The resultant FT-IR spectra provide molecular information of samples. Samples with minimal size can be non-destructively analyzed,

beams recombine, they interfere and there will be constructive or destructive interference depending on the optical path difference. A detector measures the intensity of transmitted or reflected light as a function of its wavelength. The signal obtained from the detector is an interferogram, which is analyzed by a computer using Fourier transforms to obtain a single-

Fourier transform infrared (FT-IR) spectroscopy has proven to be a fundamental and valuable technique in biology and medicine due to its high sensitivity to detecting changes in the functional groups belonging to tissue components such as lipids, proteins and nucleic acids. Each of these tissue components can be detected and characterized by their characteristic absorption bands at specific wavelengths within a single spectrum. For biological spectroscopy, the important vibrations occur in the mid-infrared region (4000 to 400 cm-1) where most organic molecules show characteristic spectral features (Theophanides, 1978). The domain of biological applications of infrared spectroscopy encompasses a wide range of very different molecular structures. Biological molecules are categorized into proteins, nucleic acids, lipids, membranes, blood tissues. Different biomolecules interact among themselves, comprising electrostatic interactions, hydrogen bondings and van der Waals interactions, which can be readily studied by infrared

For complex samples that transmit infrared radiation poorly or no changes must take place in their specimen, attenuated total reflection (ATR) is applied. The technique was developed by Harrick (1960) and Fahrenfort (1961). ATR is a specialised sampling technique, where the sample is placed on ATR crystal. An infrared beam is passed through the ATR crystal, reflects of the interface of the crystal and the sample, and is passed through to the detector. During the reflection, an evanescent wave extends beyond the crystal into the sample, which enables the absorption of energies corresponding to infrared frequencies by the sample. The penetration depth of the evanescent wave is a function of wavelength with deeper penetration at longer wavelengths. This may lead to distortions in the relative intensities of infrared peaks if sample thickness is insufficient for complete coverage of the evanescent

wave (Goormaghtigh et al., 1999). Figure 2 shows the diagram of a basic ATR set up.

Attenuated total reflection (ATR) coupled with FT-IR, can obtain the infrared spectrum of solid or liquid samples in their native state. The resultant FT-IR spectra provide molecular information of samples. Samples with minimal size can be non-destructively analyzed,

beam infrared spectrum (Barth, 2007).

Fig. 1. Schematic function of an FT-IR set-up.

spectroscopy.

Fig. 2. Diagram of the ATR setup.

particularly in the biomedical sciences. High quality spectra can be obtained from cell suspensions containing l0-50.000 cells, depending upon the size of the cells. In the case of tissues, such measurements generally require a sample size of 1 mm3 (Legal, 1991). Additionally, ATR has been established as a method of choice of analyzing samples that are either too thick or too strongly absorbing to analyse by other transmitting techniques. The use of these techniques have become a great potential over other diagnostic methods for the determination of the chemical components of tissues at various diseases states, due to the rapid and reagent free procedure. An advantage of ATR-FTIR to study the structure of biomembranes is that the membrane can be deposited on the surface of the internal reflection element (IRE) as a thin film of highly oriented membranes by evaporation of the water. Variations in spectral signatures arising from nucleic acids, proteins and lipids can provide important information in a number of disease states.

#### **1.1 Medical applications of FT-IR spectroscopy**

In 1949 Blout, Mellors and Woernley in 1952 reported that infrared spectra of human and animal tissues could provide information on the molecular structure of tissues. These studies met with limited success due to non-developed instrumentation available and little knowledge of spectroscopic properties of biological molecules and the complexity of the samples. At the same time, Elliot and Ambrose (1950) proposed empirical correlations between peptide structure and the Amide I and Amide II bands. The development of sensitive and high throughput spectrometers led to a wide field of medical applications of FT-IR spectroscopy. The rapid experimental and theoretical development took place in 1970s, where Fourier Transform interferometers interfaced to digital computers.

FT-IR has been extensively applied for the determination of a biochemical metabolite in biological fluids. The improved sensitivity and data processing capability of new instruments, the presence of water is no longer a serious obstacle in the analysis of fluids. Current enzymatic methods require frequent calibration controls and reagents, and this is very costly. FT –IR spectroscopy has been used for the determination of glucose, total protein, urea, triglyceride, cholesterol, chylomicron and very low density lipoproteins in plasma and serum, in order to replace commonly used ultracentrifugation techniques (Deleris and Petibois, 2003; D. Krilov et al., 2009).The differences in the size, lipid composition and apolipoprotein structure in particular classes of lipoproteins are reflected in the characteristic spectral bands of lipid and protein moiety.

FTIR has received much attention as a promising tool for non-destructive characterisation of the molecular features of atherosclerosis due to the fact that vibrational spectra are sensitive

FT-IR Spectroscopy in Medicine 275

peripheral vessels of young adults (Steinberg and Witztum, 1999). Fatty streaks are widely considered to be the initial lesion leading to the development of complex atherosclerotic lesions (Figure 3). The progression requires an additional stimulus, i.e. risk factor for the development of atherosclerosis. Smooth muscle cells secrete extracellular-matrix components (proteoglycans), increasing the retention and aggregation of lipids to

Atherosclerosis is a complex process and the behaviour of vulnerable atherosclerotic plaques is believed to be closely related to plaque composition. Knowledge of the composition and physical chemistry of atherosclerotic plaques is essential for understanding how these plaques originate and mature and how reversal of the pathological process may be achieved (Insull, 2009). It is therefore important to develop an effective technique for examining plaque constituent properties. FT-IR provides information on the molecular and structural composition directly in the untreated, unfixed, and unstained whole tissue, thus preserving the integrity of the original cells. In this work, Fourier transform infrared spectroscopy using attenuated total reflectance (FTIR-ATR) has been used to assess and analyze the biochemical properties of human atherosclerotic plaques. Additionally, Scanning electron microscopy (SEM) has been used to provide valuable information on the general characteristics of the morphology and structure of carotid and coronary arteries. SEM allows the scanning of large area in the atheromatous plaque and the use of large magnification provides a detailed view. Human tissues were viewed directly without any

monocytes (Stary, 1994).

Fig. 3. Formation of atherosclerotic plaque.

conductive coatings.

**1.3 Study of atheromatous plaques by FT-IR** 

to structures of biological molecules and their changes with the diseased state. Additionally, FTIR-spectroscopy on biological samples was pioneered in the medical sciences where it is used as a clinical tool to distinguish between malignant and healthy human cells. Acquired spectra of cells/ tissues give a detailed biochemical fingerprint that varies dependent on the clinical status. It has been successfully applied in the study of various human tissues such as mineralized tissue (Kolovou and Anastasopoulou, 2007), skin (McIntosh, 1999), colon (Conti et al., 2008), breast (Anastassopoulou et al., 2009), arteries (Mamarelis et al., 2010), cartilage(Petra et al., 2005), the urinary tract (prostate, bladder) (Gazi etal., 2003), lung(Yano et al., 2000), liver(Li et al., 2004), heart and spleen (Chua-anusorn and Webb, 2000; Gough et al., 2003).

According to a wide range of studies, it has been proved that FT-IR spectroscopy has been a significant clinical technique, which provides detailed information of the chemical components of the tissues (proteins, lipids, carbohydrates, DNA). By analysing chemical and biochemical changes, specific spectral features are to be considered for a diagnostic evaluation. In this chapter, it is discussed FT-IR spectroscopy in the field of atherosclerosis in carotid and coronary arteries. Experimental studies are summarized demonstrating the possibilities and prospects of these methods to detect and characterize the disease.

#### **1.2 Atherosclerosis**

Atherosclerosis, the most common form of cardiovascular diseases, is a leading cause of death affecting almost one third of humans in developed countries. Atherosclerosis is the usual cause of heart attacks, strokes, and peripheral vascular disease. Ross and Glomset (1973) were the first who introduced that atherosclerosis forms as a result of damage of endothelium. Multiple factors contribute to atherosclerosis, such as hypertension, smoking, diabetes mellitus, obesity, hypercholesterolemia and genetic predisposition. The major characteristics of human atherosclerosis are based on studies of coronary and carotid artery lesions. Atherosclerosis is a chronic inflammatory disease characterised by a stenotic lesion of arterial walls. Atherosclerotic lesions can cause stenosis with potentially lethal distal ischemia or can trigger thrombotic occlusion of major conduit arteries to the heart, brain, legs and other organs. The extracellular and intracellular accumulation of lipids from the circulating blood results in the thickening of the inner layer of the arterial wall. Analyses of human lesions by modern computer methods and biomechanical testing have established the probable link between this characteristic morphology and the actual rupture event (Lee and Libby, 1997).

During atherogenesis, the structure of the arterial wall changes in the intimal layer. The disease process consists of the intimal smooth muscle cell proliferation, the formation of large amounts of connective tissue matrix by the proliferated smooth muscle and the deposition of lipids within the cells and in the connective tissues surrounding them.

In the early stages of atherogenesis, fatty deposition occurs.

Atherogenic lipoproteins such as low-density lipoproteins (LDLs) enter the intima, where they are modified by oxidation or enzymatic activity and aggregate within the extracellular intima. Monocytes are transformed into macrophages, take up lipoproteins and become foam cells. The accumulation of foam cells leads to the formation of fatty streaks, which are often present in the aorta of children, the coronary arteries of adolescents, and other

to structures of biological molecules and their changes with the diseased state. Additionally, FTIR-spectroscopy on biological samples was pioneered in the medical sciences where it is used as a clinical tool to distinguish between malignant and healthy human cells. Acquired spectra of cells/ tissues give a detailed biochemical fingerprint that varies dependent on the clinical status. It has been successfully applied in the study of various human tissues such as mineralized tissue (Kolovou and Anastasopoulou, 2007), skin (McIntosh, 1999), colon (Conti et al., 2008), breast (Anastassopoulou et al., 2009), arteries (Mamarelis et al., 2010), cartilage(Petra et al., 2005), the urinary tract (prostate, bladder) (Gazi etal., 2003), lung(Yano et al., 2000), liver(Li et al., 2004), heart and spleen (Chua-anusorn and Webb, 2000; Gough et

According to a wide range of studies, it has been proved that FT-IR spectroscopy has been a significant clinical technique, which provides detailed information of the chemical components of the tissues (proteins, lipids, carbohydrates, DNA). By analysing chemical and biochemical changes, specific spectral features are to be considered for a diagnostic evaluation. In this chapter, it is discussed FT-IR spectroscopy in the field of atherosclerosis in carotid and coronary arteries. Experimental studies are summarized demonstrating the

Atherosclerosis, the most common form of cardiovascular diseases, is a leading cause of death affecting almost one third of humans in developed countries. Atherosclerosis is the usual cause of heart attacks, strokes, and peripheral vascular disease. Ross and Glomset (1973) were the first who introduced that atherosclerosis forms as a result of damage of endothelium. Multiple factors contribute to atherosclerosis, such as hypertension, smoking, diabetes mellitus, obesity, hypercholesterolemia and genetic predisposition. The major characteristics of human atherosclerosis are based on studies of coronary and carotid artery lesions. Atherosclerosis is a chronic inflammatory disease characterised by a stenotic lesion of arterial walls. Atherosclerotic lesions can cause stenosis with potentially lethal distal ischemia or can trigger thrombotic occlusion of major conduit arteries to the heart, brain, legs and other organs. The extracellular and intracellular accumulation of lipids from the circulating blood results in the thickening of the inner layer of the arterial wall. Analyses of human lesions by modern computer methods and biomechanical testing have established the probable link between this characteristic morphology and the actual rupture event (Lee

During atherogenesis, the structure of the arterial wall changes in the intimal layer. The disease process consists of the intimal smooth muscle cell proliferation, the formation of large amounts of connective tissue matrix by the proliferated smooth muscle and the

Atherogenic lipoproteins such as low-density lipoproteins (LDLs) enter the intima, where they are modified by oxidation or enzymatic activity and aggregate within the extracellular intima. Monocytes are transformed into macrophages, take up lipoproteins and become foam cells. The accumulation of foam cells leads to the formation of fatty streaks, which are often present in the aorta of children, the coronary arteries of adolescents, and other

deposition of lipids within the cells and in the connective tissues surrounding them.

In the early stages of atherogenesis, fatty deposition occurs.

possibilities and prospects of these methods to detect and characterize the disease.

al., 2003).

**1.2 Atherosclerosis** 

and Libby, 1997).

peripheral vessels of young adults (Steinberg and Witztum, 1999). Fatty streaks are widely considered to be the initial lesion leading to the development of complex atherosclerotic lesions (Figure 3). The progression requires an additional stimulus, i.e. risk factor for the development of atherosclerosis. Smooth muscle cells secrete extracellular-matrix components (proteoglycans), increasing the retention and aggregation of lipids to monocytes (Stary, 1994).

Fig. 3. Formation of atherosclerotic plaque.

### **1.3 Study of atheromatous plaques by FT-IR**

Atherosclerosis is a complex process and the behaviour of vulnerable atherosclerotic plaques is believed to be closely related to plaque composition. Knowledge of the composition and physical chemistry of atherosclerotic plaques is essential for understanding how these plaques originate and mature and how reversal of the pathological process may be achieved (Insull, 2009). It is therefore important to develop an effective technique for examining plaque constituent properties. FT-IR provides information on the molecular and structural composition directly in the untreated, unfixed, and unstained whole tissue, thus preserving the integrity of the original cells. In this work, Fourier transform infrared spectroscopy using attenuated total reflectance (FTIR-ATR) has been used to assess and analyze the biochemical properties of human atherosclerotic plaques. Additionally, Scanning electron microscopy (SEM) has been used to provide valuable information on the general characteristics of the morphology and structure of carotid and coronary arteries. SEM allows the scanning of large area in the atheromatous plaque and the use of large magnification provides a detailed view. Human tissues were viewed directly without any conductive coatings.

FT-IR Spectroscopy in Medicine 277

Fig. 5. FT-IR adsorption spectra obtained from human tissues (a) carotid artery, (b) coronary

chains (e.g. linolenic, arachidonic, etc.), and/or due to lipid peroxidation. For this reason,

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

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

the intensity of this band can be used as a diagnostic band of LDL.

artery in the region of 4000-400 cm-1.

Theophanides, 1990).
