**2. Lipoproteins**

### **2.1. Classes of lipoproteins in plasma and CNS**

Lipoproteins are classified based on their density, ranging from high density lipoproteins (HDL), low density lipoproteins (LDL), intermediate density lipoproteins (IDL), very low density lipoproteins (VLDL) and, chylomicrons (CM), Figure 1. They can also be classified based on their mobility during electrophoresis on an agarose gel as α-, pre-β- and βlipoproteins, which correspond to HDL, VLDL and LDL, respectively. Lipoproteins vary significantly in particle diameter and density, protein and lipid components and composition. In general, the particle diameter is inversely related to the density.

© 2012 Narayanaswami et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Figure 1.** Lipoprotein classes. The classification of the major types of lipoproteins is based on their densities obtained by flotation ultracentrifugation analysis. The density range for each class is shown, in addition to the lipid (red) and protein (blue) content. The diagram is not to scale.

One of the main functions of lipoproteins is to transport hydrophobic factors in the highly aqueous vascular system. The CM is composed of lipids of dietary origin and is synthesized by the intestines. The VLDL is synthesized and secreted by the liver, and plays a role in distribution of lipids synthesized by the liver to the peripheral tissues. In the blood stream, the VLDL undergoes particle re-modulation to IDL and LDL, during which process the LDL become enriched in cholesterylesters. LDL plays a predominant role in delivery of cholesterol to the peripheral tissues and to the liver, with the cell surface-localized LDL receptor (LDLr) family of proteins playing a role in the cellular uptake and internalization of lipoproteins in target cells. The HDL may be synthesized by the liver and intestines or derived from other lipoproteins. In addition, peripheral tissues such as the macrophages are an important source of HDL, which are formed from cellular cholesterol efflux, and eventually transported to the liver. This process is called reverse cholesterol transport (RCT), which is an important mechanism for removal of peripheral cholesterol to the liver for eventual disposal.

Much less is known about lipoprotein metabolism in the CNS. Studies on lipoproteins secreted by astrocytes and those isolated from the cerebrospinal fluid (CSF) indicate the presence of only HDL-sized particles; large triglyceride-containing lipoprotein particles have not been detected in the CNS. The CNS maintains autonomy in terms of cholesterol synthesis and metabolism, right from the time when the blood brain barrier is established during development. One of the main functions of HDL secreted by astrocytes appears to be cholesterol delivery to the neurons via the LDLr family of proteins, for eventual use in the process of synaptogenesis.

#### **2.2. Lipid and protein components of lipoproteins**

384 Lipoproteins – Role in Health and Diseases

for eventual disposal.

process of synaptogenesis.

**Figure 1.** Lipoprotein classes. The classification of the major types of lipoproteins is based on their densities obtained by flotation ultracentrifugation analysis. The density range for each class is shown, in

One of the main functions of lipoproteins is to transport hydrophobic factors in the highly aqueous vascular system. The CM is composed of lipids of dietary origin and is synthesized by the intestines. The VLDL is synthesized and secreted by the liver, and plays a role in distribution of lipids synthesized by the liver to the peripheral tissues. In the blood stream, the VLDL undergoes particle re-modulation to IDL and LDL, during which process the LDL become enriched in cholesterylesters. LDL plays a predominant role in delivery of cholesterol to the peripheral tissues and to the liver, with the cell surface-localized LDL receptor (LDLr) family of proteins playing a role in the cellular uptake and internalization of lipoproteins in target cells. The HDL may be synthesized by the liver and intestines or derived from other lipoproteins. In addition, peripheral tissues such as the macrophages are an important source of HDL, which are formed from cellular cholesterol efflux, and eventually transported to the liver. This process is called reverse cholesterol transport (RCT), which is an important mechanism for removal of peripheral cholesterol to the liver

Much less is known about lipoprotein metabolism in the CNS. Studies on lipoproteins secreted by astrocytes and those isolated from the cerebrospinal fluid (CSF) indicate the presence of only HDL-sized particles; large triglyceride-containing lipoprotein particles have not been detected in the CNS. The CNS maintains autonomy in terms of cholesterol synthesis and metabolism, right from the time when the blood brain barrier is established during development. One of the main functions of HDL secreted by astrocytes appears to be cholesterol delivery to the neurons via the LDLr family of proteins, for eventual use in the

addition to the lipid (red) and protein (blue) content. The diagram is not to scale.

In general lipoproteins are spherical in shape with a monolayer of amphipathic lipids (for example phospholipids, cholesterol and sphingolipids) and proteins encircling a core of neutral lipids (such as triglycerides and cholesterylesters), **Figure 2**. The lipid composition and content vary significantly in the different lipoproteins: in general, the larger lipoproteins (CM, VLDL and IDL) are enriched in triglycerides, while the smaller lipoproteins (LDL and HDL) are enriched in cholesterylesters and cholesterol. Unesterified or free cholesterol is esterified to cholesterylester by the action of lecithin-cholesterol acyltransferase (LCAT) on HDL. The phospholipids serve as the donor of the fatty acyl chains (especially 18:1 or 18:2 fatty acids) utilized in the esterification process. The fatty acid composition of triglycerides in the fasted state is dominated by 16:0 and 18:1 fatty acids.

**Figure 2.** Schematic representation of a generic lipoprotein particle. Lipoproteins have a spherical geometry with a monolayer of amphipathic lipids and proteins encircling a core of neutral lipids. ApoB-100 (pale blue) is a single large polypeptide and is the non-exchangeable component of lipoproteins such as VLDL and LDL. The molecular mass of exchangeable lipoproteins (yellow cylinders) varies from 8-50 kDa. The exchangeable apolipoproteins have the ability to exist in lipid-free and lipid-bound states.

The protein composition and content of the lipoproteins also vary significantly from one particle to another. There are two types of apolipoproteins: non-exchangeable and exchangeable. The non-exchangeable apolipoproteins, apolipoprotein B-100 (apoB-100) and apoB-48, are present as a single copy per lipoprotein particle: apoB-100 on VLDL, IDL or LDL and apoB-48 on CM and CM remnants. ApoB-100 is 4536 residues long that is synthesized in the liver, while apoB-48, represents the N-terminal 48% of apoB-100, and is synthesized in the intestines. Both apoB-100 and apoB-48 are integral to the structure and stability of the lipoprotein particle. There are different types of exchangeable apolipoproteins, Table 1, which undergo a reversible association with lipoproteins depending on the metabolic state.


1 Adapted with permission from the AOCS Lipid Library, "Plasma Lipoproteins: Composition, Structure and Biochemistry," Table 3, 'The main properties of apoproteins, in http://lipidlibrary.aocs.org/lipids/lipoprot/index.htm, accessed June 12, 2012

**Table 1.** Major apolipoprotein components of lipoproteins and associated functions1

The exchangeable apolipoproteins have the ability to exist in lipid-free and lipid-bound states, and undergo a large conformational change upon transitioning from one state to the other. Exchangeable apolipoproteins are characterized by an abundance of amphipathic αhelices that are folded into a helix bundle. For example, in the case of apoE, the protein is composed of a series of α-helices that are folded into an N-terminal (NT) and a C-terminal (CT) domain. The NT domain is comprised of a 4-helix bundle bearing the LDLr binding sites on helix 4, which has an abundance of basic residues, while the CT domain harbors high-affinity lipid binding sites and apoE self-association sites. A similar arrangement was noted for apoAI; however, apoAI does not have the capability to interact with LDLr.

In summary, lipoprotein particles offer several targets that are vulnerable to attack by oxidative species. Oxidative modification is expected to compromise the structure and function of the protein and lipid components.

#### **3. Oxidative stress**

386 Lipoproteins – Role in Health and Diseases

**Apolipoprotein Lipoprotein Function**

ApoB-48 Chylomicron and chylomicron remnants

ApoE2 VLDL, IDL, chylomicrons,

ApoE3 HDL, VLDL, IDL, chylomicron

ApoE4 VLDL, HDL, IDL, chylomicron

accessed June 12, 2012

chylomicron remnants

remnants (higher binding preference for HDL over VLDL)

remnants (higher binding preference for VLDL over HDL)

ApoAII HDL Inhibits LCAT

ApoB-100 VLDL, IDL and LDL Binds to LDLr ApoCI VLDL, HDL Activates LCAT

ApoCII VLDL, IDL, chylomicrons Activates lipoprotein lipase ApoCIII VLDL, IDL, chylomicrons Inhibits lipoprotein lipase

ApoD HDL Carrier proteins family (lipocalins)

ApoM HDL Transports sphingosine-1-phosphate Apo(a) Lipoprotein(a) (Lp(a)) Linked to apoB-100 via disulfide

The exchangeable apolipoproteins have the ability to exist in lipid-free and lipid-bound states, and undergo a large conformational change upon transitioning from one state to the other. Exchangeable apolipoproteins are characterized by an abundance of amphipathic αhelices that are folded into a helix bundle. For example, in the case of apoE, the protein is composed of a series of α-helices that are folded into an N-terminal (NT) and a C-terminal (CT) domain. The NT domain is comprised of a 4-helix bundle bearing the LDLr binding sites on helix 4, which has an abundance of basic residues, while the CT domain harbors high-affinity lipid binding sites and apoE self-association sites. A similar arrangement was

1 Adapted with permission from the AOCS Lipid Library, "Plasma Lipoproteins: Composition, Structure and Biochemistry," Table 3, 'The main properties of apoproteins, in http://lipidlibrary.aocs.org/lipids/lipoprot/index.htm,

noted for apoAI; however, apoAI does not have the capability to interact with LDLr.

**Table 1.** Major apolipoprotein components of lipoproteins and associated functions1

ApoAI HDL activates LCAT; promotes ABCA1-

ApoAIV Chylomicrons, HDL Activates LCAT, cholesterol clearance

mediated cholesterol efflux in RCT

Cholesterol clearance and transport;

lacks LDLr binding sites

Poor LDLr binding activity; associated with Type III

hyperlipoproteinemia and CVD

Binds to LDLr family of proteins with high affinity; significant role in cholesterol efflux and RCT in

Binds to LDLr family of proteins with high affinity; associated with CVD

and transport

atherosclerosis

and Alzheimer's disease

bond; similar to plasminogen

Reactive oxygen species (ROS) generally refer to oxygen free radicals with one or more unpaired electrons, and other highly reactive oxygen-containing molecules. They may be generated by the cells as products of normal cellular metabolism, or derived from exogenous sources including environmental pollutants, which could in turn, trigger further release of ROS. Whereas the ROS may be beneficial at low concentrations and play key physiological roles, their deleterious effect at high concentrations contributes to the etiology of several disease states. Organisms have developed an exquisite arsenal of defense mechanisms to combat the harmful effects of ROS, thereby maintaining a redox homeostasis. However, when the ROS overcome the cellular defense mechanism, there is a dysregulation of the redox balance, which leads to the state of oxidative stress (Esterbauer et al., 1991).

#### **3.1. ROS**

The high reactivity of free radicals is attributed to the unpaired electron(s) on the oxygen molecule. The primary ROS is the superoxide anion, which is generated by the addition of one electron to molecular oxygen during mitochondrial electron transport (Equation 1). This in turn gives rise to hydrogen peroxide following the action of antioxidant enzymes, superoxide dismutases (SOD) (Equation 2). The superoxide anions also give rise to the hydroxyl radicals in the presence of trace amounts of Fe2+ (Fenton reaction) (Equation 3); the hydroxyl radical is an extremely deleterious species that is capable of causing indiscriminate damage in the immediate vicinity of its formation.

$$\text{O}\_2 + \text{e}^- \longrightarrow \text{\textbullet O}\_2^- \tag{1}$$

$$\bullet \mathrm{O}\_{2}^{-} + 2\mathrm{H}^{+} \xrightarrow{\mathrm{SO}} \mathrm{H}\_{2}\mathrm{O}\_{2} + \mathrm{O}\_{2} \qquad\qquad\text{Hydrogen peroxide}\tag{2}$$

$$\bullet \bullet \mathrm{O}\_{2}^{-} + \mathrm{H}\_{2}\mathrm{O}\_{2} \xrightarrow{\mathrm{Fe^{2+}}} \bullet \mathrm{OH} \\ \bullet \mathrm{OH}^{\cdot} \bullet \mathrm{O}\_{2} \quad \text{Hydroxyl radical} \tag{3}$$

SOD catalyzes the dismutation of superoxide anion into oxygen and hydrogen peroxide, thereby affording protection to the cell. Three forms of SOD exist in humans and other mammals: SOD1 is located in the cytoplasm, SOD2 in the mitochondria, while SOD3 is extracellular. Mutations in SOD lead to diseases commonly associated with high oxidative stress such as familial amyotrophic lateral sclerosis, Parkinson's Disease, and cardiovascular disease (Fukai et al., 2002; Noor et al., 2002; Tórsdóttir et al., 2006). Overexpression of SOD inhibits LDL oxidation by endothelial cells (Fang et al., 1998). Higher SOD levels were demonstrated to be protective against LDL oxidation *in vitro* (Laukkanen et al., 2000).

Red blood cells (RBC) are particularly susceptible to ROS and free radical stress due to its constant interactions with oxygen. Despite their lack of mitochondria, RBC are under tremendous oxidative stress, due to the abundance of hemoglobin. Hemoglobin is prone to oxidation through either exogenous or endogenous sources, which results in superoxide production. Therefore, the RBC contains many SOD enzymes to convert the superoxides to hydrogen peroxide. Furthermore, hydrogen peroxide can combine with hemoglobin to form ferrylhemoglobin, a strong oxidizing agent (Rifkind et al, 2002). The RBCs are also equipped with other antioxidant enzymes such as catalase and glutathione peroxidase to overcome the harmful effects of ROS.

#### **3.2. Reactive aldehydes**

It is well established that polyunsaturated fatty acids (PUFA) undergo lipid peroxidation that is initiated by ROS, which generate a 'spectrum' of reactive aldehyde species. The reactive aldehydes formed in biological systems are more complex than those formed in simple systems like purified lipid preparations and their physiological concentrations are believed to be lower. They play a crucial role in amplifying the free radical-initiated reaction by generating a complex array of toxic end products. Although considered as end products of oxidative damage of lipids, the aldehydic products display reactivity with a wide variety of biological molecules under cellular conditions, thereby enhancing the pathogenesis of the diseases. They are therefore considered as toxic second messengers of oxidative stress and lipid peroxidation. Protein-bound aldehydes have been proposed as potential markers of oxidative stress as evidenced by immunohistochemical analysis of atherosclerotic lesions (Uchida et al., 1998a). Numerous α,β-unsaturated aldehydes have been reported in literature: we will focus on acrolein, 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), **Figure 3.**

**Figure 3.** Major aldehydic products of lipid peroxidation

#### *3.2.1. Acrolein*

Acrolein is an acrid smelling environmental pollutant that is formed during combustion of organic and plastic substances (Beauchamp et al., 1985). It is present as one of the major components in the gaseous phase of tobacco smoke (up to 140 μg/cigarette) (Witz, 1989). It is also generated as a natural metabolite during oxidative stress mediated lipid peroxidation (Uchida et al., 1998b). In addition, oxidation of threonines by myeloperoxidase (MPO) gives rise to acrolein (Anderson, et al., 1997; Savenkova et al., 1994). Acrolein is the most reactive of all α,β-unsaturated aldehydes; it causes oxidative modification of proteins by reacting with the sulfhydryls of cysteines, ε-amino groups of lysines and imidazole group of histidines (Witz, 1989; Esterbauer et al., 1991). Some possible products formed upon reaction of acrolein with lysine side chains include aldimine adducts (Schiff base formation), propanal adducts (Michel addition) and Nε-(3-formyl-3,4-dehydropiperidino)lysine (FDPlysine), **Figure 4***.*

#### *3.2.2. 4-HNE*

388 Lipoproteins – Role in Health and Diseases

the harmful effects of ROS.

**3.2. Reactive aldehydes** 

(MDA), **Figure 3.**

*3.2.1. Acrolein* 

**Figure 3.** Major aldehydic products of lipid peroxidation

Red blood cells (RBC) are particularly susceptible to ROS and free radical stress due to its constant interactions with oxygen. Despite their lack of mitochondria, RBC are under tremendous oxidative stress, due to the abundance of hemoglobin. Hemoglobin is prone to oxidation through either exogenous or endogenous sources, which results in superoxide production. Therefore, the RBC contains many SOD enzymes to convert the superoxides to hydrogen peroxide. Furthermore, hydrogen peroxide can combine with hemoglobin to form ferrylhemoglobin, a strong oxidizing agent (Rifkind et al, 2002). The RBCs are also equipped with other antioxidant enzymes such as catalase and glutathione peroxidase to overcome

It is well established that polyunsaturated fatty acids (PUFA) undergo lipid peroxidation that is initiated by ROS, which generate a 'spectrum' of reactive aldehyde species. The reactive aldehydes formed in biological systems are more complex than those formed in simple systems like purified lipid preparations and their physiological concentrations are believed to be lower. They play a crucial role in amplifying the free radical-initiated reaction by generating a complex array of toxic end products. Although considered as end products of oxidative damage of lipids, the aldehydic products display reactivity with a wide variety of biological molecules under cellular conditions, thereby enhancing the pathogenesis of the diseases. They are therefore considered as toxic second messengers of oxidative stress and lipid peroxidation. Protein-bound aldehydes have been proposed as potential markers of oxidative stress as evidenced by immunohistochemical analysis of atherosclerotic lesions (Uchida et al., 1998a). Numerous α,β-unsaturated aldehydes have been reported in literature: we will focus on acrolein, 4-hydroxynonenal (4-HNE) and malondialdehyde

Acrolein is an acrid smelling environmental pollutant that is formed during combustion of organic and plastic substances (Beauchamp et al., 1985). It is present as one of the major components in the gaseous phase of tobacco smoke (up to 140 μg/cigarette) (Witz, 1989). It is also generated as a natural metabolite during oxidative stress mediated lipid peroxidation Discovered more than 50 years ago, alkenals have been under intense scrutiny in terms of their chemistry, biochemistry, toxicology and as oxidative stress agents. Amongst these alkenals, 4-HNE was determined to be the most toxic. It was discovered to be a product of lipid peroxidation, particularly of n-6 PUFA such as linoleic acid and arachidonic acid. Since then, 4-HNE has been implicated in a number of diseases, including atherosclerosis, Alzheimer's Disease, Parkinson's Disease, liver cirrhosis, and cancer (Zarkovic, 2003).

**Figure 4.** Modifications of lysine side chains by acrolein. The major lysine modification products of acrolein are shown, along with the expected increments in molecular weight during mass spectral analysis. Adapted from Furuhata et al, 2003.

From a chemical perspective, the reactivity of 4-HNE is conferred by the carbonyl group at C1 position, and a C=C bond between the second and third carbons which provide a partial positive charge to C3 (Schaur, 2003). This results in 4-HNE being highly reactive towards thiol and amino groups of proteins. 4-HNE reacts with thiols via a Michael addition, in which a nucleophile such as the sulfhydryl of cysteine or glutathione reacts with the C3 of HNE to form a covalent adduct. In addition, amino compounds such as lysine, ethanolamine, guanine, and the imidazole group of histidine are capable of forming Michael addition adducts with 4-HNE. 4-HNE can also undergo reactions involving a reduction or an epoxidation of the double bond. 4-HNE has been shown to react *in vivo* with common biomolecules, and can often have an inhibitory role with enzymes (Schaur, 2003; Korotchkina et al, 2001). Cross-reactivity of anti-4-HNE antibody with oxidized LDL (Ox-LDL) suggests that 4-HNE may contribute to the overall process of atherosclerotic plaque formation (Uchida et al., 1993).

## *3.2.3. MDA*

In fresh samples, MDA is formed mainly from PUFA such as arachidonic acid (Esterbauer et al., 1991). Initially determined by the thiobarbituric acid (TBA) assay as TBA reactive substances (TBARS), the presence of pre-existing MDA or protein-bound MDA needs to be confirmed by other sensitive assays as well. Under physiological conditions, MDA readily modifies protein side chains forming stable cross-linked adducts with the ε-amino groups of lysines. Other side chains that may be modified include those of histidine, tyrosine, arginine and methionine.

While the levels of acrolein, 4-HNE, MDA and other aldehydic compounds *per se* are thought to be too low for detection, their role in mediating oxidative damage has been confirmed by the presence of autoantibodies against aldehyde-modified proteins. It provides evidence for the *in vivo* occurrence of lipid peroxidation products and their role in disease progression (Steinberg et al, 1989). We have a better understanding of the association between lipid peroxidative products, oxidative stress and disease progression, in part due to the development of antibodies directed against oxidized lipids and protein-bound aldehydes in the past two decades, Table 2. Currently, we have a repertoire of antibodies directed against different types of oxidatively modified lipids and proteins, **Table 2**, that serve as powerful tools for ELISA, immunohistochemical and immunoblotting analyses.

### **3.3. Sources of ROS**

In addition to the mitochondrial electron transport, there are enzymatic sources of ROS that contribute to the redox status in biological systems. Located in the blood stream, they are of relevance to lipoprotein oxidation.

#### *3.3.1. MPO*

MPO is an enzyme found abundantly in neutrophil granulocytes and to some extent in macrophages. It is a lysosomal protein stored in the azurophilic granules in neutrophils and can be identified by its characteristic green heme pigment. During the respiratory burst of the neutrophil, MPO catalyses the production of hypochlorous acid (HOCl) from hydrogen peroxide and chloride anion (Equation 4). MPO is also responsible for generation of 3 chlorotyrosine and 3-nitrotyrosine; individuals with coronary artery disease show elevated levels of these two products in their blood and HDL. MPO has been shown to play a role in promoting atherosclerotic lesions through oxidative modification of apoAI (Shao et al., 2012) (described under apoAI).

$$\text{H}\_2\text{O}\_2 + \text{Cl}^- + \text{H}^+ \rightarrow \text{HOCl} + \text{H}\_2\text{O} \tag{4}$$


aUnpublished

390 Lipoproteins – Role in Health and Diseases

formation (Uchida et al., 1993).

*3.2.3. MDA* 

and methionine.

**3.3. Sources of ROS** 

(described under apoAI).

*3.3.1. MPO*

relevance to lipoprotein oxidation.

biomolecules, and can often have an inhibitory role with enzymes (Schaur, 2003; Korotchkina et al, 2001). Cross-reactivity of anti-4-HNE antibody with oxidized LDL (Ox-LDL) suggests that 4-HNE may contribute to the overall process of atherosclerotic plaque

In fresh samples, MDA is formed mainly from PUFA such as arachidonic acid (Esterbauer et al., 1991). Initially determined by the thiobarbituric acid (TBA) assay as TBA reactive substances (TBARS), the presence of pre-existing MDA or protein-bound MDA needs to be confirmed by other sensitive assays as well. Under physiological conditions, MDA readily modifies protein side chains forming stable cross-linked adducts with the ε-amino groups of lysines. Other side chains that may be modified include those of histidine, tyrosine, arginine

While the levels of acrolein, 4-HNE, MDA and other aldehydic compounds *per se* are thought to be too low for detection, their role in mediating oxidative damage has been confirmed by the presence of autoantibodies against aldehyde-modified proteins. It provides evidence for the *in vivo* occurrence of lipid peroxidation products and their role in disease progression (Steinberg et al, 1989). We have a better understanding of the association between lipid peroxidative products, oxidative stress and disease progression, in part due to the development of antibodies directed against oxidized lipids and protein-bound aldehydes in the past two decades, Table 2. Currently, we have a repertoire of antibodies directed against different types of oxidatively modified lipids and proteins, **Table 2**, that serve as powerful

In addition to the mitochondrial electron transport, there are enzymatic sources of ROS that contribute to the redox status in biological systems. Located in the blood stream, they are of

MPO is an enzyme found abundantly in neutrophil granulocytes and to some extent in macrophages. It is a lysosomal protein stored in the azurophilic granules in neutrophils and can be identified by its characteristic green heme pigment. During the respiratory burst of the neutrophil, MPO catalyses the production of hypochlorous acid (HOCl) from hydrogen peroxide and chloride anion (Equation 4). MPO is also responsible for generation of 3 chlorotyrosine and 3-nitrotyrosine; individuals with coronary artery disease show elevated levels of these two products in their blood and HDL. MPO has been shown to play a role in promoting atherosclerotic lesions through oxidative modification of apoAI (Shao et al., 2012)


tools for ELISA, immunohistochemical and immunoblotting analyses.

bShibata et al. (2009) Acta Histochem. Cytochem. 42, 197; c Shibata et al. (2011) JBC 277, 10459; dUchida et al. (1998) PNAS 95, 4882; e Ichihashi et al. (2002) JBC 276, 23903; f Ishino et al. (2010) JBC 285, 15302; gYamada et al. (2001) JLR 42, 1187; hYamada et al. (2004) JLR 45, 626; i Itakura et al. (2003) BBRC 308, 452; j Toyokuni et al. (1995) FEBS Lett. 359, 189; kItakura et al. (2000) FEBS Lett. 473, 249; l Hashimoto et al. (2003) JBC 278, 5044; mHashimoto et al. (1995) JBC 278, 5044; nShimozu et al. (2011) JBC 286, 29313; oShibata et al. (2011) JBC 286, 19943; pKawai et al. (2003) JBC 278, 21040; qKawai et al. (2003) JBC 278, 50346

**Table 2.** Lipid peroxidation-specific monoclonal antibodies

#### *3.3.2. NADPH oxidase*

NADPH oxidase is an enzyme complex that is normally found in the plasma membranes of neutrophils and monocytes. Like MPO, it is activated during the respiratory burst of the neutrophil. Its main role is to generate superoxide by transferring electrons from NADPH to molecular oxygen (Equation 5). The superoxide is then used to destroy phagocytosed bacteria or pathogens. NADPH oxidase and its product, superoxide, are major contributory factors for foam cell formation in atherosclerosis (Meyer & Schmitt, 2000).

$$\text{2O}\_2 + \text{NADPH} \rightarrow \text{2} \bullet \text{O}\_2^\cdot + \text{NADP}^\cdot + \text{H}^+ \tag{5}$$

#### *3.3.3. Lipoxygenase*

Lipoxygenases are iron-containing enzymes that catalyze the dioxygenation of PUFA in lipids. Specifically, they form hydroperoxides from fatty acids and molecular oxygen. 15 lipoxygenase is the main protein in this family; it is involved in the metabolism of eicosanoids (prostaglandins, leukotrienes), which function as secondary messengers. 15 lipoxygenase has been shown to be involved in LDL oxidation (Bailey et al., 1995), with considerably greater 15-lipoxygenase activities in atherosclerotic compared to normal aortas (Hiltunen et al., 1995).
