CHAPTER 1

INTRODUCTION

CHAPTER 1:

INTRODUCTION

1.1 Free radicals and disease

A free radical can be defined as any molecular species capable of independent existence which contains an unpaired electron in an orbital, such as CH 3 or Cl, the dot representing a single (unpaired) electron (Halliwell and Gutteridge, 1989; Kaul et al., 1993). This electronic configuration makes free radicals highly unstable and chemically very reactive. Gomberg (1900) was the first scientist to demonstrate the existence of organic free radicals, in the form of the triphenylmethyl radical. In 1954, Gerschman and co-workers (Gerschman et al., 1954) demonstrated the presence of oxygen-containing free radicals in a biological system by showing that compounds that protected animals against radiolysis also protected them against oxygen challenge. This result contributed to the free radical theory of ageing which was proposed by Harman in 1956 (Harman, 1956).

Free radicals are weakly attracted to a magnetic field and are described as paramagnetic. Electrons are more stable when paired together in an orbital: the two electrons in a pair have different directions of spin. Hence, radicals are generally less stable than non-radicals, although their activity can vary considerably. Once radicals are formed they can either react with another radical to form a non-radical species or with another molecule by various interactions (Cadogan, 1973). As noted before, most radicals are highly reactive and thus are short lived. They are therefore difficult to measure directly, and their activity is often determined by indirect measurements of tissue damage products or footprints (Halliwell and

Chirico, 1993). One direct technique for the detection and study of free radicals themselves is electron spin resonance (ESR) or electron paramagnetic resonance (EPR) spectroscopy. This technique exploits the absorption of microwave radiation of particular frequencies when the radical is located in a strong magnetic field (Kaur and Perkins, 1991).

The rate and selectivity of reactions undergone by radical species depends on the radical concentration, the extent to which delocalisation of the single electron of the radical occurs and on the absence of weak bonds in any other molecules present with which the radical could interact (Bensasson et al., 1993).

The most important free radicals in many disease states are oxygen derivatives due to the ubiquitous presence of oxygen in higher species and diatomic oxygen's ability to readily accept electrons. Oxygen plays an essential role in aerobic life, including the production of energy and the synthesis of a variety of important compounds. In many of these reactions, iron, copper and other transition metals play an essential role, usually bound in specific complexes within proteins. Any major disruption of these complex oxidative reactions rapidly leads to death (Olson, 1995). Molecular oxygen normally contains three unpaired electrons, and in this state is known as triplet oxygen. When triplet oxygen reacts with transition metals and other

compounds, partly reduced and highly active forms of oxygen may be produced, of which the hydroxyl radical (OH) is one of the most reactive (Cheeseman and Slater, 1993; Halliwell and Gutteridge, 1995). When the intermediates of oxygen reduction, i.e. reactive oxygen species (ROS), are not strictly regulated they are potentially problematic as they are more reactive than ground state molecular oxygen. A variety

of adverse consequences may occur including lipid, carbohydrate and nucleic acid oxidation, protein inactivation, disruption of membrane function and activation of pro-carcinogens and other xenobiotics (Thomas, 1994).

1.1.1

Sources of free radicals

There are several endogenous sources of free radicals. These are produced during normal metabolism in mitochondria during the process of oxidative phosphorylation as electrons are passed along the electron transport chain at the mitrochondrial inner membrane (Kehrer and Smith, 1994). During normal aerobic metabolism,

mitochondria consume molecular oxygen and reduce it sequentially to produce water. The inevitable by-products of this reaction are superoxide, hydrogen peroxide and the hydroxyl radical. It has been calculated that over 2 kg of superoxide are produced in the human body every year (Halliwell, 1996).

Peroxisomes, which contain fatty acyl CoA oxidase, dopamine -hydroxylase, urate oxidase and other oxidative enzymes, produce hydrogen peroxide during metabolism, which is then degraded by catalase. Some hydrogen peroxide escapes degradation and leaks into other cellular compartments and increases oxidative damage.

Other enzyme reactions are also important endogenous sources of radical production. The cytochrome P-450 mixed function oxidase system constitutes a primary defence against various xenobiotics and endogenous substances and enhances production of free radicals. Some superoxide is produced deliberately, for example by NADPH oxidase in phagocytic cells to aid the destruction of bacteria or virus-infected cells

with an oxidative burst of superoxide, hydrogen peroxide, hypochlorite and nitric oxide. There are also important exogenous drivers of free radical production such as a high intake of iron and copper, cigarette smoke, inhaled atmospheric pollutants, radiation (Kubow, 1993) and lipid peroxidation products in foods.

1.1.2

Reactive oxygen species (ROS)

ROS include all oxygen containing free radicals and related non-radical oxygen containing species, and are constantly formed in human body by various physiological processes and insults as noted previously (Figure 1.1).

Sources of ROS in vivo

Physiological (normal) production of ROS

Pathological (abnormal) production of ROS

Serving no purpose; always potentially damaging Purposeful, directed, useful, but potentially damaging if excessive, prolonged or uncontrolled e.g. superoxide and hypocholorous acid production during phaocytic respiratory burst; endothelial cell release of nitric oxide for maintenance of normal vascular tone. Accidental, potentially harmful e.g. leakage of electrons from cytochrome in the mitochondrial electron transport chain; exerciseinduced local ischaemia; autoxidation of catecholamines and other endogenous compounds. e.g. in food, cigarette smoke, pollutants, ozone; radiation-induced water splitting; radical forms of toxins and drugs from peroxisomal and cytochrome P-450 metabolism; as a consequence of reactions between peroxides and free iron or copper; during ischaemia and postischaemia reperfusion .

Figure 1.1

Sources of reactive oxygen species (adapted from Strain and Benzie, 1998)

Many of them serve useful physiological functions, but they can be toxic when generated in excess or in inappropriate environments, and this toxicity is usually aggravated by the presence of ions of such transition metals as iron or copper.

Table 1.1

Reactive oxygen species (ROS) of physiological interest Name Superoxide Hydrogen peroxide Hydroxyl R-yl

Radical O2 H2O2 OH R

Typical biological target Enzymes Unsaturated fatty acids All biomolecules Oxygen Unsaturated fatty acids Unsaturated fatty acids Unsaturated fatty acids H2O Several

RO

R-oxyl R-dioxyl (R-peroxyl) Hydroperoxide Singlet molecular oxygen Nitroxyl

ROO
1

ROOH O2 NO

(Muggli and Hoffmann, 1993) ROS can be divided into two main groups these being oxygen containing free radicals such as the superoxide anion, hydroxyl radical, peroxyl and alkoxyl radicals, and non-radical ROS such as hydrogen peroxide, singlet oxygen, hydroperoxides and hypochlorite.

The most important free radicals in many disease conditions are oxygen derivatives, specifically the superoxide anion (O2 ) and the hydroxyl radical (OH). Superoxide (O2 ) is produced in vivo by adding a single electron to the diatomic oxygen molecule.

O2 + e O2

In biological systems, the relatively short half-life of O2 limits its diffusion away from its site of production. Part of the O2 formed in vivo is a chemical accident (Halliwell, 1991). As an example, when mitochondria are functioning, some of the electrons passing through the respiratory chain 'leak' from the electron carriers and pass directly onto oxygen forming O2 (Fridovich, 1974). Other molecules such as adrenaline, several sugars, including glucose, can also oxidize in vivo to produce oxygen radicals. Superoxide is both a reducing and an oxidizing agent. In aqueous solutions, it can oxidise ascorbic acid. It can also reduce certain iron complexes such as cytochrome-C and ferric-ethylenediamine-tetraacetic acid (Fe-EDTA).

Superoxide dimutase (SOD) removes O2

by catalysing a dismutation reaction,

converting it to hydrogen peroxide (H2O2) and oxygen (O2) (Halliwell and Gutteridge, 1989).

2O2 + 2H+ H2O2 + O2

Although some of the O2 production in vivo may be accidental, much is functional as O2 production is important in allowing phagocytes to kill some of the bacterial strains which they can engulf. Also, in the presence of the enzyme myeloperoxidase the H2O2 produced by dismutation of O2 oxidizes chloride ions to hypochlorous acid (HOCl) which is powerful antibacterial agent usually found in household bleaches.

H2O2 + Cl HOCl + OH

A further example of the usefulness of O2 may be provided by the endothelial cells that line blood vessels. It is thought that endothelial cells produce nitric oxide (NO), a free radical that is identical to endothelium-derived relaxing factor (EDRF). Vascular endothelium also appears to produce small amounts of O 2

which can

inactivate NO, combining to give a non-radical product, the nitrate ion (NO3):

NO + O2 NO3

Thus, variations in the production of NO and O2 by endothelium may provide one mechanism for the regulation of vascular tone (Halliwell, 1989).

Hydrogen peroxide (H2O2) has a relatively long half-life, is membrane permeable, and may traverse considerable distances, causing damage at sites distant from its origin (Kaul et al., 1993). Although H2O2 is poorly reactive at low levels, at higher levels it can attack several cellular energy producing systems. For instance, it can inactivate the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. Hydrogen peroxide can sometimes be toxic to cells because it can give rise to OH which is highly reactive. Thus H2O2 acts as a conduit to transmit ROS induced damage across cell compartments and between cells (Young and Woodside, 2001).

Stohs and Bagchi (1995) suggested that there are several mechanisms by which OH may be produced, but the most important mechanism is likely to be the transition metal ion catalysed decomposition of O2 and H2O2. The most important transition metals in terms of OH generation are iron and copper. Fenton (1894) was the first

scientist to describe the involvement of transition metals in the formation of hydroxyl radicals:

Fe (II) + H2O2 Fe (III) + OH + OH

Biochemical studies of free radicals indicate that O2

is the main source of

hydrogen peroxide in vivo. In the presence of transition metal ions such as iron or copper, the reaction of O2 and H2O2 together can directly form a hydroxyl radical at rapid speed:

Fe (III) + O2 Fe (II) + O2 Fe (II) + H2O2 Fe (III) + OH + OH

This can be simplified as the Haber-Weiss reaction (Haber and Weiss 1932):

O2 + H2O2 OH + OH + O2

The hydroxyl radical (OH) is a highly reactive oxygen-centred radical that attacks all molecules in the human body, and is likely to be the final mediator of most free radical induced tissue damage (Loyd et al., 1997). It is the most reactive radical known, with an extremely short half-life and a very limited diffusion capacity. It can attack almost every molecule found in living cells and because it is a radical it can leave behind a legacy in the cell in the form of free-radical chain reactions. Thus, if OH attacks DNA, free-radical chain reactions occur within the DNA and lead to chemical alteration of the deoxyribose, purines and pyrimidines which in turn can

lead to mutations and DNA strand breakage. Imperfect repair of DNA damage can result in oncogene and carcinogenesis. Probably the most common biological

damage caused by OH is its ability to promote lipid peroxidation which occurs when OH is generated adjacent to the fatty acid side-chains of membrane lipids. Lipid hydroperoxides and their aldehyde decomposition products are toxic, and are responsible for the rancidity of peroxidized food material (Halliwell, 1991).

The actual reaction in the body may be more complicated than the described reactions, in that they may involve the formation of the ferryl or perferryl radical. The propensity of transition metal ions to drive OH formation means that it is important for these metals to be sequestered in forms that are not available to catalyse the final reaction (Lloyd et al., 1997).

The peroxyl (RO2) and alkoxyl radicals (RO) are organic radicals, typically encountered as intermediates during lipid peroxidation which will be discussed in more detail later.

Singlet oxygen (1O2) is an electronically excited, non-radical form of dioxygen and a highly reactive oxidizing agent. The major source of 1O2 is photosensitizing

reactions although small amounts are produced by self-reaction of peroxyl radicals during lipid peroxidation (Muggli and Hoffmann, 1993).

1.1.3

Consequences of excessive production of free radicals

Although free radicals are critical for the maintenance of normal physiological function, overabundance or uncontrolled chain reactions initiated as well as

10

propagated by free radicals are potentially lethal for the cell (Kaul et al., 1993). Excess generation of ROS within tissues can damage DNA, lipids, proteins, and carbohydrate. Which is the most important target of damage depends on the cell type subjected to the oxidative stress and how the stress is imposed. Biological systems respond to oxidative stress in a poorly understood fashion with a cascade of interacting reactions at the molecular, cellular and organ level (Figure 1.2; Muggli and Hoffmann, 1993). Free radicals have been implicated in more than one hundred disease conditions in humans, including atherosclerosis, arthritis, ischemia and reperfusion injury of many tissues, central nervous system injury, gastritis, tumour promotion and carcinogenesis, and AIDS (Pitot and Dragan, 1991; Kehrer, 1993).
OXIDATIVE STRESS HYPOTHESIS Oxidative stress

1O2

O2

H2O2

OH

Reactive oxygen species (ROS)

Lipids Proteins Receptors Chromosomes

Molecular reaction with biological substances

Enzymes

Membranes Nucleic acids Carbohydrates

Cellular disturbances

Tissue injuries

DISEASE

Figure 1.2.

Cascade of multiple derangements of cell metabolism by oxidative stress (from Muggli and Hoffmann, 1993)

11

Peroxidation of lipids is most likely the most extensively investigated of all the radical-mediated mechanisms.

1.2

Lipid oxidation and disease

Lipid oxidation may be defined as the process of oxidative deterioration of polyunsaturated fatty acids and is initiated and propagated by free radicals (Henning and Chow, 1988). In cellular systems, lipid oxidation is of absolute importance, particularly in membranes where most of the oxygenactivating enzymes are found and there is typically a relatively high concentration of polyunsaturated fatty acids, which are more susceptible to oxidation. Lipid oxidation in cell membranes can disintegrate the membrane structure and cause loss in the function of the cell organelles (Kappus, 1985).

Neurological tissue is particularly susceptible to oxidation because it consumes a high rate of oxygen, and has relatively low levels of antioxidant defences. In

addition, there is a high content of polyunsaturated fatty acids and transition metals ions. There are some examples of neurodegenerative disorders in which lipid

peroxidation is likely to be important including Parkinsons and Alzheimers diseases.

Myoglobin, the major heme protein in muscle tissues involved in transport and storage of oxygen, has been identified as a powerful catalyst able to initiate lipid oxidation. In its higher oxidation states, it has been shown to oxidise a wide range of biological substrates, including proteins, nicotinamide adenine dinucleotide (reduced

12

form) (NADPH), ascorbate, carotenoids, and plant polyphenols (Kanner and Harel, 1985). Under physiological conditions deoxymyoglobin has been found not to be pro-oxidative (Kanner and Harel, 1985). On the other hand, pro-oxidative activity has been identified under acidic conditions such as at inflammation and ischemic sites (Fantone et al., 1989). The catalytic activity of myoglobin in biological systems is expected to originate from its interaction with lipid peroxides and particularly with hydrogen peroxide (Kanner and Harel, 1985). Table 1.2 (from Jadhav et al., 1995) outlines range of diseases which can be caused by lipid oxidation in vivo.

Table 1.2. Disease

Lipid peroxidation induced diseases and effects Remarks Organ damage due to Fe overload leading to increased lipid oxidation. Selenium deficiency causes a decrease in glutathione peroxidase activity leading to increased lipid peroxidation. Due to Fe-induced lipid oxidation. Lipid peroxides and reaction products of lipid oxidation such as hydroxyalkenals alter low density lipoproteins, which is important in atherosclerotic lesions. Occurs during reperfusion injury of heart and brain. Also results in lipid peroxidation probably by transformation of xanthine dehydrogenase to xanthine oxidase and by the production of reactive oxygen species. May be due to lipid peroxidation, but has been confirmed in erthyrocytes. Wide speculation about the involvement of lipid peroxidation in carcionogensis. This is due to genotoxic effects of lipid peroxides

1. Haemochromatosis 2. Keshan disease

3. Rheumatoid arthritis 4. Atherosclerosis

5. Ischaemia

6. Ageing 7. Carcinogensis

(Jadhav et al., 1995) 13

1.2.1

Mechanism of lipid peroxidation

Lipid peroxidation has been extensively reviewed by a number of authors, including Labuza (1971), Burton and Ingold (1984), Halliwell and Chirico (1993) and Morrissey et al. (1994). The process of lipid peroxidation includes three main steps initiation, propagation and termination.

Initiation:

X + RH R + XH

(a)

R + O2 ROO

(b)

Propagation:

ROO + RH R ROOH +

(c)

2ROOH RO + ROO + H2O

(d)

Termination:

R , RO , ROO stable, non-propagating species

(e)

Figure 1.3.

Outline of the reactions involved in the autoxidation of unsaturated fatty acids (Coultate, 2002)

The initiation reactions result in the production of a small number of highly reactive fatty acids molecules with unpaired electrons, the free radicals denoted by R (Figure 1.3a). During the propagation reactions, atmospheric oxygen reacts with these radicals to form peroxy radicals, ROO (Figure 1.3b), which are also highly reactive and further react with other unsaturated fatty acids to generate hydroperoxides, ROOH, and another free radical (Figure 1.3c). The free radical can go round and repeat this process thereby forming a chain reaction (Figure 1.3b) but the hydroperoxide can break down to give other free radicals (Figure 1.3d) which can

14

also behave much as ROO did. Consequently, the ever increasing number of free radicals accumulate in the lipid which absorbs considerable quantities of oxygen from the air. Eventually, the free radical concentration reaches a point when they start to react with each other to produce stable end-products and these are known as the termination reactions (Figure 1.3e).

1.2.2

Lipoprotein oxidation

Within the human body the oxidation of lipoporotein is a key early stage in the development of atherosclerosis (Young and McEneney, 2001).

1.2.2.1

Lipoproteins

Lipids such as triglycerides and cholesterol are essential to the body and serve a number of causes. The triglycerides are required within the body as a source of energy, being composed of fatty acids and glycerol. Cholesterol is a structural component of cell membranes and nerve sheaths being required for the synthesis of steroid and adrenocortical hormones and bile acids. As these lipids are insoluble in water they are transported in the blood bound to proteins (apoproteins and apolipoproteins). lipoproteins. Thus they become water-soluble complexes and are termed

The lipoprotein complexes undergo metabolism by the body during which the initially large, lipid-filled, low-density complexes, which transport either diet derived or endogenously synthesised lipids, undergo gradual conversion to smaller, denser particles that are rich in protein and phospholipid prior to their utilisation or

15

excretion (Thomas, 2001). The lipoproteins are therefore classified by density, due to their change in lipid content during metabolism (Thomas, 2001):

1.2.2.1.1

Chylomicrons

These are the form in which lipids that are consumed in the diet are absorbed from the gastrointestinal tract. During circulation round the body, their triglyceride is gradually removed by skeletal muscle cells and adipose tissue under the influence of lipoprotein lipase. The remnants of the chylomicron are taken up by the liver and reassembled into new lipoproteins.

1.2.2.1.2

Very low-density lipoproteins (VLDL)

VLDL particles are constructed by the liver from chylomicron remnants and are comprised mainly of triglyceride. The VLDL maintain a supply of triglyceride for energy production to body tissues in the fasting state.

1.2.2.1.3

Intermediate-density lipoproteins (IDL)

IDL are derived from partially degraded VLDL and are short-lived intermediates.

1.2.2.1.4

Low-density lipoproteins (LDL)

LDL particles are derived from VLDL. As the triglyceride is removed by the body cells from VLDL, the remaining cholesterol is concentrated within LDL for transfer to the peripheral tissues. Approximately 60% of total circulating cholesterol is contained within LDL.

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1.2.2.1.5

High-density lipoproteins (HDL)

HDL are small, dense particles derived from the breakdown of the chylomicrons, and are composed of protein, cholesterol and phospholipid. HDL have an important role transporting cholesterol from cells back to the liver.

1.2.2.2

Oxidation of VLDL

VLDL is susceptible to oxidation because it contains high levels of triglycerides, cholesteryl esters, and phospholipids that harbour unsaturated fatty acids such as linoleic acid and arachidonic acid (Arai et al., 1999). Apolipoprotein (apoE), a component of VLDL, acts as a ligand for the LDL receptor and heparan sulfate proteoglycan on the cell surface and plays an important role in regulating lipoprotein metabolism (Weisgraber, 1994). In addition, apoE contributes to restoration and regeneration of nerve tissue (Ignatius et al., 1986). McEneny et al. (1996) found that oxidation of VLDL in vitro increases macrophage uptake and promotes foam cell formation. Recent studies suggest that both oxidised VLDL and HDL may play a role with LDL in the pathogenesis of atherosclerosis. Much work has been done on the oxidative modification of LDL, as will be discussed. However, a much smaller number of studies have been performed investigating the properties of oxidised VLDL and HDL. Mohr and Stocker, 1994) reported that radical-mediated lipid peroxidation proceeded via a similar mechanism in isolated human LDL and VLDL. A difference in the compositional structure of VLDL compared to other lipoproteins was reported by Bailey and Southon (1998), in that C18:1 (oleic acid) is more prominent in VLDL. In all sub-fractions of LDL and HDL, C18:2 (linoleic acid) is the most abundant long chain fatty acid, with C16:0 (palmitic acid) being the next most abundant. This may be important in relation to lipoprotein oxidation, as

17

monounsaturated fatty acids resist oxidation due to an absence of diene sites at which free radical initiation can take place, and hence conjugation (Yamamoto, 1990).

1.2.2.3

Oxidation of LDL

Increasing evidence indicates that oxidative modification of LDL is a crucial factor in the process of atherogenesis (Witzum and Steinberg, 1991; Esterbauer et al., 1992; Parthasarathy and Rankin, 1992). The initial step in the development of atherosclerosis is believed to be injury to the endothelium or lining of the artery. Where damage has occurred, macrophages ingest LDL and other atherogenic particles to form foam cells. Upon death of the macrophages, the lipid remains in the arterial wall, accumulating over time to form a lipid core or pool. The formation of this fatty streak is the first step in plague development which eventually leads to narrowing of the artery and inadequate supply of oxygen to the heart muscle and consequently coronary heart disease (Thomas, 2001). It is believed that modification of LDL within the arterial wall, particularly by oxidation as noted previously, is crucial to the cellular uptake of LDL in the first stages of plaque development (Young and McEneny, 2001).

Oxidation of LDL is initiated by free radicals or by one of several enzymes, such as lipoxygenase and myeloperoxidase, within the walls of arteries (Heinecke, 1997). It appears that following the initial attack by a free radical on the polyunsaturated fatty acids in the LDL particles, lipid peroxidation (as described previously) occurs. The lipid hydroperoxides fragment in the presence of catalytically active iron or copper to form a wide variety of aldehydes, which then combine covalently with the lysl and other amino acid residues of the protein moiety of LDL, apolipoprotin B-100 (Leake,

18

1995). The modified protein then becomes recognised by the scavenger receptors of macrophages. The bound LDL may then be internalised rapidly by the cells and deposit its cholesterol within them thereby giving rise to the foam cells that are characteristic of atherosclerotic lesions (Figure 1.4). Once initiated, oxidation of LDL is a free-radical-driven lipid peroxidation chain reaction.

Figure 1.4
(a)

Pathogenesis of atherosclerosis

(b) (c)

The initiating stages of lesion development are characterised by endothelial dysfunction when adhesion molecules are upregulated resulting in the attachment of leukocytes. The endothelium becomes more permeable facilitating the transmigration of cells and intimal accumulation of plasma lipoproteins. A fatty streak develops which consists of lipid loaded monocytes and macrophages (foam cells). This occurs in conjunction with smooth muscle cell migration and proliferation. Fatty streak progresses to an advanced lesion following the development of a fibrous cap. A necrotic core is formed as a result of cell apoptosis and necrosis, the proteolytic degradation of the extracelluar matrix, plaque calcification and the accumulation of extracellular lipid.

(Adapted from Ross, 1999)

Lipoprotein-like particles with oxidative damage have been isolated from atherosclerotic lesions (Witzum and Steinberg, 1991) and lipid oxidation products such as malondialdehyde have been immunohistochemically detected in human and animal atherosclerotic lesions (Esterbauer et al., 1992). The main reason it is

unlikely that oxidation of LDL occurs in the plasma is due to the presence of high antioxidant concentrations and proteins that chelate metal ions. Thus, oxidation of 19

LDL is likely to occur in the intima of large arteries as noted previously. Oxidation may be mediated by lipoxygenases and myeloperoxidase.

Lipoxygenases are cytosolic enzymes present in macrophages, endothelial cells, and smooth muscle cells, and they directly oxidise polyunsaturated fatty acids (Yamamoto, 1992), particularly those bound to phospholipids within LDL. Amounts of 12-lipoxygenase increase in cholesterol-loaded macrophages (Mather et al., 1985). Heinecke (1997) reported that 15-lipoxygenase may oxidise LDL in early stages of atherosclerotic lesions.

Phagocytes secrete hydrogen peroxide and the heme protein myeloperoxidase, which interact to generate antimicrobial toxins (Klebanoff, 1980). This enzyme uses

hydrogen peroxidase to convert chloride to hypochlorous acid (Harrison and Schultz, 1976) and to convert L-tyrosine to the tyrosyl radical. Myeloperoxidase, with its ability to generate a range of reactive species, may play a role in lipoprotein oxidation throughout atherosclerotic lesion development, and may therefore represent an important link between chronic inflammation and development of atherosclerotic plaques in the human artery wall (Daugherty et al., 1994).

1.2.2.4

Oxidation of HDL

Plasma HDL is a powerful negative risk factor for atherosclerotic cardiovascular disease (Rifkind, 1990) as it has been found to promotes reverse transport of cholesterol from peripheral cells to the liver. In addition, HDL protects LDL against oxidation, an effect mediated mainly by HDL associated enzymes such as paraoxonase, platelet activating factor acetylhydrolase (PAF-AH) and lecithin-

20

cholesterol acyltranferase (L-CAT) (Schnell et al., 2001). These enzymes have been shown to inhibit LDL oxidation in vitro and to convert bioactive lipid peroxidation products into inactive compounds. Oxidative modification of HDL results in deterioration in several biological functions critical to its role in reverse cholesterol transport (Schnell et al., 2001). Sakai et al. (1992) found that oxidative modification of HDL impairs its binding to cells thereby reducing its effectiveness in promoting cellular lipid efflux. Recent studies (Chait et al., 2004) have indicated that HDL is much more susceptible to oxidation than LDL.

1.2.2.5

Factors influencing lipoprotein oxidation

The oxidation of lipoprotein in vivo is influenced by the composition of the lipoprotein (intrinsic factors) and the microenvironment in which it is found (extrinsic factors). Among the intrinsic factors, the fatty acid composition of LDL is of primary importance as a high concentration of polyunsaturated fatty acids (PUFAs) will make the LDL significantly more susceptible to oxidation. Conversely, a high concentration of monounsaturated fatty acids such as oleic acid (C18:1) can protect against oxidation. Also of importance is the concentration of endogenous antioxidants in lipoproteins, such as -tocopherol, ubiquinol-10 and carotenoids, since the propagation phase of LDL oxidation begins after these have been consumed (Young and Woodside, 2001).

In terms of extrinsic factors susceptibility of LDL to oxidation in vivo is likely to be influenced by its microenvironment, including transition metal availability, pH, the presence of specific enzyme systems and local antioxidant concentration, the latter of which is now receiving significant attention.

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Antioxidant defence against disease

Nature has developed a variety of sophisticated defences against undesirable side effects of oxygen. To offset the undesirable effects a complex interactive network of antioxidants has evolved (Krinsky, 1992; Olson, 1995). The term antioxidant has two parts anti which means against and oxidant or an oxidising agent, which means any substance which accepts electrons and causes another reactant to be oxidised. A reductant or a reducing agent is a substance that donates electrons and, thereby, causes another reactant to be reduced. Reductant and oxidant are chemical terms, whereas antioxidant and pro-oxidant have meanings in the context of biological systems (Prior and Cao, 1999). Antioxidants may be defined as any substance which when found at low concentrations compared with those of an oxidizable substrate significantly prevents or delays a pro-oxidant initiated oxidation of the substrate. An antioxidant is a reductant, but a reductant is not necessarily an antioxidant. A prooxidant is a toxic substance that can cause oxidative damage to lipids, proteins, and nucleic acids, resulting in various pathological events or diseases.

Intracellular, extracellular, lipophilic and aqueous antioxidant mechanisms are found throughout the body and work together to: (i) (ii) (iii) prevent generation of ROS (preventative antioxidants); destroy or inactivate ROS which are formed (scavenging antioxidants); terminate chains of ROS-initiated peroxidation (chain-breaking antioxidants).

(Strain and Benzie, 1998).

22

Many endogenous and exogenous constituents of biological fluids have been shown to exhibit antioxidant activity in vitro. However, for an antioxidant to have a

physiological role, there are certain criteria that must be met: (i) the proposed antioxidant must be able to react with ROS found at sites in the body where the proposed antioxidant if found; (ii) upon reacting with the ROS, the proposed antioxidant must not be changed into a more reactive species than the original ROS. (iii) the proposed antioxidant must be found in sufficient quantity at the site of its presumed action in vivo for it to make an appreciable contribution to antioxidant defence; if its concentration is very low, there must be some recycling or replenishing mechanism in vivo. (Strain and Benzie, 1998).

Some of these antioxidant mechanisms are highly integrated enzymatic systems, including endogenous antioxidants such as catalase, superoxide dimutase, glutathione peroxidase and reductase. Exogenous antioxidants encompass a

comprehensive group of free radical scavengers including lipophilic antioxidants that protect membranes and lipoproteins. This type of antioxidant includes vitamin E, vitamin A and the carotenoids. The hydrophilic antioxidants that protect against free radicals in the aqueous phase include ascorbate and uric acid. The antioxidants can be conveniently divided into three groups these being

(i) antioxidant enzymes; (ii) transition metal binding proteins; and (iii) chain breaking antioxidants.

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1.3.1 1.3.1.1

Antioxidant enzymes Catalase

Catalases help prevent the accumulation of H2O2 within cells, and catalyse the following reaction:

2H2 O2 2H2O + O2

They are located in animal and plant tissues in sub-cellular organelles bound by a single membrane and known as peroxisomes (Halliwell and Gutteridge, 1989; Robinson, 1991). These organelles also contain some of the cellular H2O2 generating enzymes such as glycollate oxidase, urate oxidase, and flavoprotein dehydrogenases involved in the -oxidation of fatty acids.

Catalases were initially found in aerobic cells, whereas most anaerobic organisms do not contain the enzyme activity. In animals, catalase is present in all major body organs, being specifically accumulated in the liver and erythrocytes (Halliwell and Gutteridge, 1989). The brain, heart and skeletal muscle contain only low amounts, although the activity does vary between muscles and even among different regions of the same muscle.

Halliwell and Gutteridge (1989) reviewed the mechanism of dismutation of H2O2 into oxygen and water by catalase. The reaction proceeds in two steps:

Catalase-Fe (III) + H2O2 compound Compound I + H2O2 catalase-Fe (III) + H2O +O2

(k1) (k2)

24

The second order rate constants k1 and k2 for rate liver catalase have values of 1.7x107M-1 and 2.6x107M-1, respectively.

1.3.1.2

Glutathione peroxidases (GSH-Px) and glutathione reductase

Mills (1957) was the first scientist who discovered glutathione peroxidases (GSHPx) in animal tissues as an enzyme which protects red blood cells against haemoglobin oxidation and haemolysis. GSH-Px uses a simple tripeptide (Glu-CysGly; glutathione), as a co-factor, abbreviated to GSH in its reduced form. The oxidised form, GSSG, consists of two GSH molecules joined by a disulphide bridge. GSH is the predominant form of free glutathione in vivo. The enzyme GSH-Px catalyses the oxidation of GSH to GSSH at the expense of H 2O2 as in the following reaction:

H2O2 + 2GSH GSSG + 2H2O

The enzyme mechanism of GSH-Px is concluded in 3 main steps. The first step includes the oxidation of the co-factor by a peroxidase substrate and produces the corresponding alcohol. This is followed by two successive additions of GSH and release of GSSG (Flohe and Gunzler, 1974).

The ratios of GSH to GSSG in normal cells generally are kept high. In order to maintain this, GSSG must be reduced back to GSH, a reaction catalysed by glutathione reductase:

GSSG + NADPH + H+ 2GSH + NADP+

25

Glutathione reductase can also catalyse the reduction of certain mixed disulfides such as that between GSH and coenzyme-A (Halliwell and Gutteridge, 1989). NADPH is required for these reactions and is available in animal tissues via the oxidative pentose phosphate pathway. Glutathione reductase contains two protein sub-units, each of them having flavin FAD as its active site. During the catalytic cycle the NADPH reduces the FAD, which then passes its electrons on to a disulfide (-S-S-) between two cysteine residues in the protein. The twoSH groups so formed then interact with GSSG and reduce it to 2 GSH, therefore re-forming the protein disulfide.

1.3.1.3

Superoxide dimutase (SOD)

Mann and Keilin (1938) discovered superoxide dimutase (SOD), initially thinking that it played an important role in copper storage. In 1960 McCord and Fridovich (1960) demonstrated that the protein previously discovered catalyses the dismutation of superoxide radicals (O2) to hydrogen peroxide (H2O2) as described previously.

SOD is found widely in all oxygen-consuming organisms (McCord et al., 1971). All SODs are metalloproteins containing copper, iron or manganese at their active sites. There are three forms of SOD in mammalian tissues, each of which has a specific sub-cellular location and different tissue distribution. Copper zinc superoxide

dimutase (CuZn-SOD) is found in animals, plants and yeasts. Mammalian CuZnSODs are highly thermostable, resistant to protease attack, and tolerant to organic solvents.

26

Manganese superoxide dimutase (Mn-SOD) is found in the mitochondria of all cells (Weisiger and Fridovich, 1973). These are more susceptible to denaturation by heat or chemicals such as detergents (Halliwell and Gutteridge, 1989) although they are not affected by H2O2.

Iron superoxide dimutases are generally dimeric proteins, with each sub-unit having a molecular mass of about 22,000 kDa and containing a functional iron ion. Fe-SOD exhibits a very high degree of sequence homology with Mn-SODs. They have helical proteins, each monomer consisting of 6 basic helical segments and three strands of anti-parallel -sheet. Halliwell and Gutteridge (1989) claimed that the iron in the resting state is Fe(III) and that it probably oscillates between Fe(III) and Fe(II) states during the catalytic cycle.

1.3.2

Transition metal binding antioxidants

Transition metals bind proteins, that is, ferritin, transferrin, lactoferrin and caeruloplasmin, act as a crucial component of the antioxidant defence system by sequestering iron and copper so that they are not available to drive the formation of the hydroxyl radical.

The principle copper-binding protein, caeruloplasmin, may also function as an antioxidant enzyme that can catalyse the oxidation of divalent iron as follows:

4Fe(II) + O2 + 4H+ 4Fe(III) + 2H2O

27

Fe(II) is the form of iron that drive the Fenton reaction and the rapid oxidation of Fe(II) to the less reactive Fe(III) for is therefore an antioxidant effect (Young and Woodside, 2001).

1.3.3

Chain-breaking antioxidants

Chain-breaking antioxidants may be defined as small molecules that can receive an electron from a radical or donate an electron to a radical with the formation of stable by-products (Halliwell, 1995). Generally, the charge associated with the presence of an unpaired electron becomes dissociated over the scavenger and the resulting product will not readily accept an electron from or donate an electron to another molecule, thereby preventing further propagation of the chain reaction (Young and Woodside, 2001). Such chain breaking antioxidants can be divided into aqueous phase and lipid phase antioxidants.

1.3.3.1 1.3.3.1.1

Aqueous phase chain-breaking antioxidants Vitamin C

Vitamin C or ascorbic acid (ascorbate) is a water-soluble vitamin, known as antihaemorrhagic agent when its discovery was associated with curing scurvy. It is an essential nutrient with a number of specific functions as a nutrient such as collagen formation and it acts as an essential co-factor for several enzymes catalysing hydroxylation reactions. Fresh fruits and vegetables are the main sources of vitamin C, particularly citrus fruit, parsley, peppers, broccoli, spinach and tomatoes. It can be synthesised by most mammals, though not by humans, primates, guinea pigs and fruit bats. It is a non-thermostable vitamin and is affected by cooking.

28

Ascorbate is a chain-breaking antioxidant and is considered the most important antioxidant in extracellular fluids. It can efficiently scavenge superoxide, hydrogen peroxide, the hydroxyl radical, hypochlorous acid, aqueous peroxyl radicals, and singlet oxygen (Sies et al., 1992). Frei (1991) reported that ascorbic acid is

sufficiently reactive to intercept oxidants in the aqueous phase before they can attack and cause detectable oxidative damage to lipids.

The principal pathway of oxidation and turnover of ascorbic acid is thought to involve the successive removal of two electrons yielding firstly the ascorbate free radical (AFR) and then dehydroascorbate (Thurnham et al., 2000). Two molecules of AFR may react together to form one molecule of ascorbate or one of dehydroascorbate. Alternatively, AFR may be reduced by a microsomal NADHdependent enzyme, mono-dehydro-L-ascorbate reductase. It has been shown that AFR is less reactive than many other free radicals (Bielski et al., 1975) thus the reason for its protective role as a free-radical chain terminator. In addition, as ascorbate is readily soluble in aqueous fluids, it is easily dispersed through the tissues and in contact with probably all biological membranes where vitamin E is the principal antioxidant (Thurnham et al., 2000).

Vitamin C is capable of restoring oxidised vitamin E back to its original form. In other words, the unique ability of low concentrations of vitamin E to act as an efficient antioxidant in biological systems is due to its ability to be reduced from its chromanoxyl radical form to its native state by intracellular reductants such as ascorbate (Packer and Kagan, 1993).

29

Although ascorbate can play a key role in protecting cells against oxidative damage, in the presence of the transition metals Fe(III) or Cu(II), it can promote generation of the same ROS it is known to destroy (Stadtman, 1991). This ability to act as a prooxidant is due to the ability of ascorbate to reduce Fe(III) and Cu(II) to Fe(II) and Cu(I), respectively, and to reduce oxygen to the superoxide ion and hydrogen peroxide. Thus, ascorbate can play a dual role as a pro-oxidant and an antioxidant.

1.3.3.1.2

Uric acid

Uric acid is formed in the body by the breakdown of purines and by direct synthesis from 5-phosphoribosyl pyrophosphate (5-PRPP) and glutamine. In humans, uric acid is normally excreted in the urine, while in other mammals, it is further oxidized to allantoin prior to excretion. Uric acid can scavenge free radicals, being converted in the process to allantoin. It is particularly important in protecting against certain oxidising agents such as ozone (Cross et al., 1992). Ames et al. (1981) suggested that the increase in the life-span that has existed during human evolution could be partly explained by the protective effect of uric acid in human plasma. Urate can directly quench free radicals and contribute to the formation of stable non-reactive complexes with iron (Frei et al., 1988).

1.3.3.1.3

Albumin

Albumin is also an efficient radical scavenger when bound to bilirubin (Frei et al., 1988). Copinathan et al. (1994) suggested that albumin may play an important role in protecting the neonate from oxidative damage, because of lack of other chainbreaking antioxidants in the neonates. Albumin is the predominant protein in plasma and makes the major contribution to plasma sulphydryl groups although it also has

30

several other antioxidant properties. It contains 17 disulphide bridges and has a single remaining cysteine residue and this residue is responsible for antioxidant properties (Stocker and Frei, 1991). Albumin also has the capacity to bind copper ions and will inhibit copper-dependent lipid peroxidation and hydroxyl radical formation (Young and Woodside, 2001). It has also been shown to act as a powerful scavenger of hypocholorous acid, and provides the main plasma defence against this oxidant (Hu et al., 1993).

1.3.3.1.4

Reduced glutathione (GSH)

Glutathione (Glu-Cys-Gly) or GSH is the only significant electron donor substrate for the metabolite reactions involving glutathione peroxidase (GSH-Px), although the latter can use a variety of hydroperoxide acceptor substrates. scavenger of hydroxyl radicals and singlet oxygen. GSH is also a

Since it is present at high

concentrations in many cells, it may help protect against these radical species (Deshpande et al., 1995).

1.3.3.2 1.3.3.2.1

Lipid phase chain-breaking antioxidants Vitamin E

Vitamin E is the principal component of the secondary defence mechanism against free radical-mediated cellular injuries. It is the only natural physiological lipidsoluble antioxidant that can inhibit lipid peroxidation in cell membranes.

In 1922 Evans and Bishop discovered vitamin E as a substance in lettuce which prevented sterility in animals (Evans and Bishop, 1922). It was later isolated as a closely related family of compounds, designated as tocopherols. -Tocopherol was

31

thus known as an anti-sterility factor, the name being derived from the Greek words tokos and pherein, which mean to bring forth children. In 1936 the vitamin was isolated from wheatgerm oil (Evans et al., 1936). Vitamin E is a fat-soluble vitamin, a deficiency of which can lead to a group of symptoms in animals and poultry such as embryonic degeneration, liver necrosis, encephalomalacia and testicular degeneration, although deficiency in humans is rare. Vegetable oils, nuts and plant sources are good sources of the vitamin while animal sources such as lard and butter are less important. Vitamin E is absorbed from the gut with the aid of bile salts and the vitamin is not esterfied as is the case with retinol. It is transported to the blood stream via chylomicrons and distributed to the various tissues via lipoproteins.

Vitamin E refers to two groups of compounds, the tocopherols and the tocotrienols, and includes eight different forms, which differ greatly in their degree of biological activity. The tocopherols include , , , and -tocopherol and have a

chromanol ring and a phytyl tail, and differ in the number and position of the methyl groups on the ring. The tocotrienols, , , , and , are structurally similar but have unsaturated tails. (Horwitt, 1991). Both groups of compounds have antioxidant properties

Burton et al. (1983) reported that probably vitamin E is the most important lipid antioxidant. -Tocopherol (TOH) is quantitatively the most important antioxidant in plasma and LDL because it is present in concentrations at least 10-15 times higher than any of other lipid soluble antioxidants (Burton et al., 1983). It is an essential component of biological membranes as it has membrane-stabilising properties, the hydrophobic tail being the means by which TOH inserts into lipoproteins or anchors

32

into membranes next to unsaturated fatty acids (Diplock, 1985). The chromanol nucleus lies at the surface of a lipoprotein or at the surface of membranes, and it is the phenolic hydroxyl group that quenches free radical (Packer and Kagan, 1993). TOH is only active during the propagation phase of lipid peroxidation, that is, the consumption of this lipid soluble antioxidant is associated with the formation of lipid hydroperoxides. The hydrogen atom of the phenolic hydroxyl group on the

chromanol ring is very easy to remove. It therefore reacts readily with lipid peroxy and alkoxy radicals by donating the labile hydrogen to them, thereby terminating the chain reaction of peroxidation by scavenging chain-propagating radicals (Deshpande et al., 1995).

When the chromanol phenolic group of TOH encounters a peroxy radical (ROO ), it forms a hydroperoxide (ROOH) and in the process a tocopheroxyl radical (TO ) is formed:

TOH + ROO ROOH + TO

The tocopheroxyl radical (TO) generated is poorly reactive and is therefore unable to attack adjacent fatty acid side chains. TOH might be regenerated by reaction at the aqueous interface with ascorbate as noted previously, or another aqueous phase chain breaking antioxidants, such as glutathione or urate. On the other hand, two

tocopheroxyl radicals might combine to form a stable dimer, or the radical may be completely oxidised to form tocopherol quinone (Young and Woodside, 2001).

33

1.3.3.2.2

Ubiquinol-10

Ubiquinol-10, the reduced form of coenzyme-Q, has long been known to be a component of the mitochondrial respiratory chain (Crane et al., 1993). During the last three decades it has also been shown that in its reduced form it can serve as a potent antioxidant (Ernster and Forsmark, 1993) and protect membrane phospholipids (Takayanagi et al., 1980) and LDL cholesterol (Stocker and Frei, 1991). It has been pointed out (Ernster and Forsmark, 1993) that ubiquinol is the only known lipid soluble antioxidant that is synthesised de novo in animal cells and for which there exists enzymic mechanisms by which it can be maintained in the reduced form. The reduced form of coenzyme QH2 is also effective as a lipid soluble chain breaking antioxidant (Shi et al., 1999).

Ubiquinol-10 is found in lower concentrations than vitamin E but it can trap lipid peroxyl radicals more efficiently than vitamin E or -carotene and can also restore membrane bound vitamin E from the tocopheroxyl radical (Lass and Sohal, 1998). Thomas et al. (1996) reported that ubiquinol-10 is the first antioxidant consumed during oxidation of LDL cholesterol and this may suggest that it is of particular importance in preventing the propagation of lipid oxidation. Scientific work in this area has been hampered by the instability of ubinquinol-10 during sample handling or analysis.

1.3.3.2.3

Carotenoids and vitamin A

The carotenoids are a group of lipid soluble antioxidants based around an isoprenoid carbon skeleton, the most important being -carotene. Recently, there has been considerable interest in the antioxidant activity of carotenoids and their potential role

34

in the pathogenesis of several diseases. There are particularly efficient scavengers of singlet oxygen (Fukuzawa et al., 1998), although they can also trap peroxy radicals at low oxygen pressure with an efficiency at least as great as that of TOH. Thus, the carotenoids may play a role in preventing in vivo lipid peroxidation. The other important role of certain carotenoids is as precursors of vitamin A (retinol), the latter also having antioxidant properties (Keys and Zimmerman, 1999). As the carotenoids are part of the 'phytochemical' group of compounds, their protective effect in vivo will be discussed in more detail later in the chapter.

1.4 Phytochemicals

Fruits and vegetables contain a wide variety of low-molecular weight secondary metabolites which can (i) protect the plant against attack by predators and pathogens, (ii) function with physical barriers and some defensive enzymes to provide a system of defence against physiological stress and attack by other organisms, (iii) provide organoleptic or characteristics such as flavour compounds and colour pigments, and (iv) the reduction of characteristics with adverse acceptability properties such as tannins (Rhodes, 1996). Such potentially protective plant compounds are termed 'phytochemicals'. Many of these phyotnutrients are ubiquitous throughout the plant kingdom and are therefore present in the human diet. To date, approximately 30,000 phytochemicals have been identified, of which 5,000 to 10,000 are present in the food consumed in the human diet (Cassidy and Dalais, 2003).

Although they might not be considered as essential nutrients, interest in these 'phytonutrients', as they are otherwise known, as bioactive components is currently of

35

major interest due to increasing epidemiological evidence that they are involved in the maintenance of healthy tissues and organs in the body. For example, there is evidence that diets rich in fruit and vegetables could have a protective effect against a number of cancers (Block et al., 1992) and other chronic health conditions such as cardiovascular disease (John et al., 2002). According to Rice-Evans et al. (1997) many dietary polyphenolic compounds derived from plants are more effective antioxidants in vitro than vitamins E or C and therefore might contribute to the protective effects in vivo. It is though that these dietary phenolic compounds might make a contribution to total antioxidant capacity.

Phytochemicals may be classified as carotenoids, phytosterols, and sulphur containing compounds (sulfides and glucosinolates) and phenolic phytochemicals (tannins, stilbenes and lignans and the flavonoids).

1.4.1

Carotenoids

Carotenoids (Figure 1.5) are natural pigments present in plants and responsible for colours such as red, orange and yellow in various fruits and vegetables such as carrots, tomatoes, spinach, oranges and peaches. More than 600 different

compounds have been identified in various organisms. Carotenoids containing only carbon and hydrogen atoms are collectively referred to as carotenes ( -carotene, -carotene, and lycopene). However, most natural carotenoids contain at least one oxygen function such as keto (violerythrin), hydroxy (lutein), or epoxy groups (violaxanthin) and these are collectively referred to as xanthophylls or oxocarotenoids (Stahl and Sies, 1999).

36

Beta-carotene, -carotene and -cryptoxanthin have important nutritional roles in the body as they act as pro-vitamins for vitamin A. The non pro-vitamin A

compounds with implications for human health are lutein, zeaxanthin and lycopene. These together make up major dietary carotenoids, the major dietary sources being vegetables such as broccoli, carrots, greens, parsley, green and orange peppers, spinach, tomatoes, and various fruits including apricots, grapefruit and oranges (Mangels et al., 1993). They can also be found in various seafoods such as lobster and salmon (Liaaen-Jensen, 1990). On a commercial basis, the carotenoid

astaxanthin is used as a feed additive to obtain the typical colour of salmon flesh (Bjerkeng, 1992). Carotenoids such as crocetin and bixin are becoming increasingly popular as food colourants with many now officially sanctioned for use.

Although many hundreds of carotenoids have been identified, only a small number have been investigated for their effects on human health (Ottaway, 2001). Increasingly, there is evidence that a diet rich in carotenoids is associated with a reduction in risk for certain cancers and cardiovascular disease. Although early epidemiological studies suggested a protective effect of -carotene, in the 1990s it was reported that large doses of supplemental -carotene could increase the risk of lung cancer in heavy smokers and asbestos workers (ATBC Study Group, 1994; Albanes et al., 1996). There is evidence to suggest that lutein and zeaxanthin, the predominant carotenoids in human macula lutea, may protect against age-related macular degeneration (AMD), a leading cause of irreversible blindness among the 65+ age group (Seddon et al., 1994).

37

 -carotene

Lycopene

Lutein

Figure 1.5

Chemical structure of carotenoids

Lycopene, the major carotenoid in tomatoes, is one of the most effective scavengers of 'singlet oxygen' free radicals. Research has suggested that eating tomatoes every day will reduce the risk of developing a cancer in the upper aero-digestive tract, stomach, lungs or respiratory tract. However, observations suggest that it is not lycopene alone that is responsible for the protective effect of tomatoes but possibly other compounds present in the tomatoes such as -carotene, vitamins C and E, folates, phenolics and lignans. A number of studies have suggested a relationship

38

between lycopene and a risk reduction for prostate cancer, particularly in aggressive cases (Giovannucci et al., 1995). A protective effect of lycopene against pancreatic cancer has also been suggested (Burney et al., 1989).

1.4.2

Phytosterols

Phytosterols (Figure 1.6) have been reported in measurable amounts in over 250 plants (Cassidy and Dalais, 2003). These compounds have a similar structure to cholesterol but with some modifications to the side chain, including the addition of a double bond and/or methyl or ethyl group.

The most common dietary phytosterols are sitosterol, campesterol and stigmasterol representing <50% of the total intake of sterols in the Western diet, the remainder being cholesterol (Subbiah, 1971). The major dietary sources include legumes, nuts, seeds, soyabeans, and unrefined plant oils (Pennington, 2002). The daily diet in the UK contains an average of 200-400 mg plant sterols, although vegetarians may eat around 800 mg daily (Flora, 2003).

Interest in phytosterols has centred on their cholesterol lowering properties thereby offering protection from cardiovascular disease (Law, 2000). Numerous studies have shown that plant sterols and stanols derived from wood pulp and vegetable oils lower total and LDL cholesterol by inhibiting cholesterol absorption from the intestine in humans. As plant stanols are virtually unabsorbable, they are more ideal

hypocholesterolaemic agents than plant sterols (Nguyen, 1999).

39

Sitosterol

Stigmasterol

Campesterol

Figure 1.6

Chemical structure of phytosterols

40

Esterification of plant stanols has allowed their incorporation into various food products such as margarine without having an adverse effect on the taste or texture of such foods. It has been shown that plant stanol esters at a level of 2-3 g per day can reduce LDL cholesterol by 10-15%. However, it has been shown that plant sterols may also reduce the level of carotenoids although these reductions are well within the variation observed in dietary intake of carotenoids between winter and summer and are therefore not biologically significant (Weststrate and Meijer, 1998). Recent studies have shown that dietary advice to consume an additional daily serving of a high-carotenoid fruit or vegetable when consuming spreads containing esterified plant sterols or stanols will maintain plasma carotenoid concentrations and at the same time significantly lower LDL cholesterol concentrations (Noakes et al., 2002).

1.4.3 1.4.3.1

Sulphur-containing compounds Sulfides

Naturally occurring sulphur-containing compounds (the allium family) are found in large quantities in bulbous plants such as garlic, onions, and leeks. Such vegetables contain a range of S compounds which are derivatives of L-cysteine sulphoxides (CSO) and a potential source of protective compounds in the diet against degenerative disease (Rhodes, 1996).

Figure 1.7

Chemical structure of allicin

41

The 1-propenyl derivative is particularly associated with onions and the allyl derivative with garlic, the compounds being odourless in undamaged plant tissues. Only when the garlic is crushed or the onion sliced, does the enzyme allinase gain access to its substrate and the flavour becomes obvious. In garlic, dimerisation of the allyl sulfenic acid leads to the formation of allicin which is well known for its antibacterial properties (Coultate, 2001). There has been a long history of the

medicinal properties of garlic and allicin although results in clinical trials with human subjects have been variable. Studies have shown that consumption of garlic may have benefits with regard to cholesterol, blood pressure, platelet aggregration and coagulation time (Lawson et al., 1992).

1.4.3.2

Glucosinolates

The glucosinolates (Figure 1.8) are a large group of sulfur-containing compounds that occur principally in the family Cruciferae, including the brassicas (cabbage, Brussels sprouts, broccoli) and many plants renowned for their pungency when raw, namely, horse-radish, black and white mustards, and radish. compounds have been described thus far (Rhodes, 1996). Over 100 such

S D-glucose RC N OSO3-

Figure 1.8

Structure of glucosinolates

Total glucosinolates are normally present at levels of around 2 g/kg fresh weight with the highest amounts being found in the most pungent species or varieties (Coultate, 2001). The glucosinolates are not themselves pungent but when the plant tissue is

42

disrupted by chewing, chopping in the kitchen or suffers insect or other damage in the field, the glucosinolates are hydrolysed by the enzyme myrosinase to form isothiocyantes or nitriles. The volatile isothiocyanates are the most likely products to the formed (collectively referred to as 'mustard oils') and it is these that have the pungent taste. With regard to health benefits of consumption of fruits and

vegetables, it is recognised that at least some of the beneficial effects especially in relation to cancers of the colon, rectum and thyroid, can be related to the consumption of glucosinolates in cruciferous vegetables. Various experiments

carried out with laboratory animals and tissue cultures of human cells have shown that allylisothyiocyanate, from sinigrin, and sulforophane, from glucoraphanin at realistic concentrations have been shown to kill cancerous cells (Zhang and Talalay, 1994). It appears that sulforophane causes cell to increase production of glutathione transfereases, a group of enzymes involved in the neutralisation of carcinogens.

1.4.4

Phenolic phytochemicals

This group of phytochemicals includes a broad range of compounds that are widespread in commonly consumed fruits, vegetables and beverages derived from plants, such as tea. The term 'phenolic' encompasses a variety of plant compounds containing an aromatic ring with one or more hydroxyl groups. They are partly responsible for the colour, taste and smell of many foods and are influenced by factors such as growing conditions, cultivar, ripeness, processing, and storage (Cassidy and Dalais, 2003). Research has highlighted the potential role of these phytochemicals as important contributing factors to the antioxidant activity of the diet and hence protective effects for human health (Rice-Evans and Millar, 1996;

43

Rice-Evans et al., 1997). The flavonoids form the most important group of phenolic phytochemicals found in foods and beverages.

1.5

Flavonoids

To date more than 4000 chemically unique flavonoids have been identified in plant material being found in the upper epidermal layer of leaves, flowers, stems, roots, seeds and fruits. They are derived biosynthetically from phenylalanine, the general structure being two benzene groups connected by a three-carbon bridge (C6-C3-C6 structure) with phenolic OH groups (Figure 1.9). Three moles of malonyl-coenzyme A (CoA) from glucose metabolism condense to form ring A, the reaction being catalysed by chalcone synthetase. Rings B and C also come from glucose

metabolism, but via the shikimate pathway through phenylalanine, which is converted to cinnamic acid and then to coumaric acid. Subsequently, coumaric acid CoA and three malonyl CoAs are condensed in a single enzymatic step to form naringenin chalcone. The C-ring closes and becomes hydrated to form 3-

hydroxyflavonoids (e.g. catechins), 3,4-dio flavonoids (e.g. quercetin), and procyanidins (Merken and Beecher, 2000). In plant foods, the flavonoids are present mainly as glycosides in which phenolic hydrogen or hydrogens are substituted to the sugar moiety (Terao, 1999). Glucose is the most commonly encountered sugar with galactose, rhamnose, xylose and arabinose not uncommon and mannose, fructose, glucuronic and galacturonic acids being rare (Robards and Antolovich, 1997).

44

Figure 1.9

Basic structure of flavonoids

From a dietary point of view the most important classes of flavonoids are the flavones and flavonols, the catechins and related tannins based on the flavan-3-ol structure and the anthocyanins, these being the pigments responsible for the red and blue colours of many fruits and vegetables (Rhodes, 1996). Furthermore, the

isoflavones are related to these compounds and have activity as phytoestrogens, although this group of compounds are largely limited to the pea family whereas the flavonoids are found in nearly all fruits and vegetables.

1.5.1

Flavonols, flavones and flavanols

The three major sub-classes of flavonoids are the flavonols, flavones and flavanols. Common flavonols include quercetin, kamepferol, myricetin, and isohamnetin (Figure 1.10). Glycosylation occurs preferentially at the 3-hydroyl group.

45

Quercetin

Kaempferol

Myricetin

Figure 1.10

Chemical structure of flavonols

The two most common flavones of dietary importance are luteolin and apigenin (Figure 1.11). They are structurally different to the flavonols in that they lack a hydroxyl group on the 3-position of the C ring. Other flavones include nobiletin and sinensetin found in sweet orange peel and taneretin found in tangerine oil (Robards et al., 1997).

46

Apigenin

Luteolin

Figure 1.11

Chemical structure of flavones

Flavanols (or flavan-3-ols) differ from the flavonols and flavones as they lack an oxygen molecule at the 4 position of the C ring. The principal flavanols found in the plant materials include catechin, epicatechin (Figure 1.11) and gallocatechin.

Figure 1.12

Chemical structure of epicatechin

47

The flavanols are also called tea flavonoids are these compounds are only rich in tea leaves where catechins may constitute up to 25% of dry leaf weight. One of the most concentrated dietary sources of catechins is green tea (Camellia sinensis) where epicatechin, epigallocatechin, and their gallate esters are commonly found. During the preparation of black tea, oxidative polymerization of flavanols occurs with theaflavin, theaflavingallates, thearubigins, and epitheaflavic acid being formed (Figure 1.12).

(a)
(a)
Compound Thearubigin Thearubigin-3-gallate Thearubigin-3'-gallate Thearubigin-3,3'digallate R H galloyl H galloyl R1 H H galloyl galloyl

(b)
(b)
Compound Theaflavin Theaflavin-3-gallate Theaflavin-3'-gallate Theaflavin-3,3'digallate R H galloyl H galloyl R1 H H galloyl galloyl

(c)
(c)
Compound Epitheaflavic acid Epitheaflavic gallate R1 H gallate

Figure 1.13

Chemical structures of thearubigin (a), theaflavin (b), epitheaflavic (c) and related compounds

The flavanols are also important constituents of fruits in oligomeric or polymeric forms as proanthocyanidins or condensed tannins (Figure 1.14). They are widely distributed in foods such as apples, grapes, strawberries, plums, grape seeds, and barley.

48

Dimer Procyanidin B3 (R3' = H) Prodelphinidin B3 (R3' = OH) Figure 1.14

Trimer

Chemical structure of proanthocyanidins

1.5.2.

Flavanones

Flavanones occur in small amounts compared with other flavonoids but are the predominant flavonoid in citrus fruits (Robards et al., 1997). The most commonly found flavanones in citrus include naringenin (Figure 1.15), naringin, hesperidin, and hesperetin. In the case of sweet oranges, mandarins, lemons and citrons, the

predominant flavanone is hesperidin while the major flavanone in grapefruit is naringin.

Figure 1.15

Chemical structure of naringenin

49

1.5.3.

Anthocyanins

The anthocyanins are natural plant pigments occurring in nature as glycosides with the aglycones being commonly known as anthocyanidins. It is these compounds that give the pink, red, mauve, violet and blue colours of flowers, fruit, and vegetables. They are particularly rich in berries, and the red grape.

Anthocyanidins (R3 = R5 = H)

Anthocyanin (R3, R5 = glycosides)

Compound Delphinidin (De) Cyanidin (Cy) Pelargonidin (Pe) Peonin (Pn) Malvidin (M) Petunidin (Pt)

R3' OH OH H OCH3 OCH3 OCH3

R5' OH H H H OCH3 OH

Compound De-3-glucosides De-3,5-diglucosides

R3 glucose glucose

R5 H glucose

Figure 1.16

Chemical structures of anthocyanins and related glycosides

In total, six anthocyanidins (Figure 1.16) occur in nature although the diversity of the patterns of glycosylation means there are innumerable different anthocyanins. Cyanidin is the most common followed by delphinidin, peonin, pelargonidin, 50

petunidin and malvidin. Of the six anthocyanidins only pelargonidin is not found in grapes. It is also interesting that there is much more variety in the patterns of glycosylation and acetylation than in most plants (Coultate, 2001). With regard to vegetables, cyanidin is the anthocyanidin of red cabbage, while pelargonidin and delphinidin occur in radishes and aubergines, respectively.

1.5.4

Hydroxycinnamates

Closely related to the flavonoids are some derivatives of cinnamic acid, such as caffeic, -coumaric, and ferrulic acids (Figure 1.17) also known as

hydroxycinnamates. Such acids may acylate with the sugar moieties of the glycoside and are effective antioxidants. Caffeic acid, ferulic acid and -coumaric acid are found in fruits and vegetables such as asparagus, cabbage, olives, tomatoes and white grapes while chlorogenic acid is present in apple, cherry, pear, tomato, and peach (Rice-Evans et al., 1997).

Compound Caffeic acid p-Coumaric Ferulic acid

R1 OH H OCH3

Figure 1.17

Chemical structure of cinnamic acid and derivatives

51

1.5.5

Isoflavones (Phyoestrogens)

Isoflavonids are a class of flavonoids also known as phytoestrogens due to the fact that their structure (Figure 1.18) is similar to that to the mammalian hormone oestrogen. They are found principally in legumes with soy being the main dietary source, although they can be derived from other legumes. isoflavones are present as glycosides. In soya beans, the

Following digestion these conjugated

isoflavones are hydrolysed by glycosidases and bioactive aglycones are formed. The major dietary aglycones are daidzein, glycetin and genistein, their glycosides being glycitin, daidzin and genistin. The isoflavone content of soybean varieties ranges from 1.2 - 3.8 mg/g seed and depends on the variety, growing conditions, and planting season. Phytoestrogens produce both agonisitic and antagonistic activity as estrogen receptrors depending upon the level of circulating endogenouse estrogen and the type of estrogen receptor (Alpro, 2003).

Daidzein

Genistein

Figure 1.18

Chemical structure of isoflavones

52

1.6

Flavonoids and Health

Interest in plant-based diets is increasing because, as noted previously, this type of diet may offer protection against some of the chronic diseases common in Western countries.

1.6.1

The Mediterranean diet

The well known term "the Mediterranean Diet" was first popularised in the 1970s when studies showed that Mediterranean countries have diets associated with low incidences of cardiovascular disease with later studies showing that these countries also enjoy a low incidence of cancers of the colon and breast. There is now little doubt that the Mediterranean countries enjoy a low risk of many of the diet-related diseases of affluence (Hill, 1995). The Mediterranean diet is characterised by

consumption of fruit, vegetables, olive oil and dietary fibre and low intakes of meat and saturated fats. It not only produces favourable effects on blood lipids but also protects against oxidative stress, the latter being thought to represent one of the mechanisms leading to chronic diseases such as atherosclerosis and cancer.

Many studies suggest that a link exists between fruit and vegetables or the amounts of plasma antioxidant vitamins and risk of death from cancer or coronary heart disease (Ghiselli et al., 1997). The preference for fresh fruit and vegetables, typical of Mediterranean populations, results in a higher consumption of raw foods, involving a reduced production of cooking-related antioxidants, with a consequent decreased waste of nutritional and endogenous antioxidants. It is thought that the health benefits of the Mediterranean diet are mainly due to its high antioxidant

53

potential due to the abundance of flavonoids and other phytochemicals which may be additionally or complimentarily involved in the reported protective effects of this diet (Peluso and Vineis, 2000).

1.6.2.

The French Paradox

The low incidence of coronary heart disease (CHD) in France, especially in southern France, despite having a diet high in fat and a high incidence of smoking has been termed the "French Paradox". The incidence of CHD is also low in countries such as Italy, Greece and Spain and so maybe the phenomenon should be known as the Mediterranean Paradox. Mortality from ischaemic heart disease in France is about a quarter of that in Britain although the risk factors are similar. Undercertification of ischaemic heart disease in France could account for about 20% of the difference. It has been postulated, however, that the high consumption of red wine by the French could act as a protective factor, particularly as it has been shown that red wine could inhibit the oxidation of low density lipoprotein (LDL) (Kondo et al., 1993).

It was postulated by Frankel et al. (1993) that it is the phenolic substances extracted from red wine that could potentially inhibit LDL oxidation, the active antioxidants being flavonoids. The flavonoid in the greatest amount is quercetin, which

comprises 60% of all the flavonoids in wine. Other flavonoids in red wine such as caffeic acid have greater antioxidant potential but are present in small amounts. The flavonoids resveratrol and epicatechin are also found in red wines but not in white wines. As red wine is fermented with the grape skin, red wine contains procyanidins (tannins) the absorption of which is facilitated by the 5-11% alcohol. Frankel et al.

54

(1993) showed that quercetin and phenolic substances isolated from red wine effectively impaired copper ion-catalyzed oxidation of LDL, while -tocopherol exhibited only 60% of the potency of wine phenolics or quercetin.

According to Renaud and deLorgeril (1992) moderate alcohol intake does not prevent CHD through an effect on atherosclerosis, but rather through a haemostatic mechanism. Data has shown that blood platelet aggregation, which is related to CHD, is inhibited significantly by alcohol at levels of intake associated with reduced risk of CHD. This inhibition of platelet activity by wine (alcohol) may be one explanation for protection from CHD in France, since studies showed that the tendency of platelets to aggregate is considerably less lower in subjects from the south of France than in Scotland. Most flavonoids decrease eicosanoid synthesis. Red wine has been shown to decrease concentration (inhibits synthesis) of thromboxane B2. Overall the decrease in eicosanoid metabolism decreases thrombaxane and increases prostacyclin. Most flavonoids increase the formation of endothelium-dependent relaxation factor maybe by increasing the formation of nitric oxide (NO) (Fitzpatrick et al., 1993). This effect of red wine, if it occurs in vivo, could help to prevent vasospasm of coronary arteries or to inhibit platelet aggregation, as NO inhibits platelet aggregation. The vasorelaxation effect of red wine seems to be mediated by an increase in cyclic GMP resulting in vascular smooth muscle relaxation.

Other possibilities to explain the French Paradox are those eluded to earlier when referring to the Mediterranean diet such as the high intake of monounsaturated fatty acids mainly in the form of olive oil, which also contains antioxidants. The higher

55

temperatures of the Mediterranean countries compared to Northern Europe may be another possible contributory factor (Leake, 1995).

It is there thought that the cardiovascular benefits of red wine are a result of its alcohol and flavonoid content. Alcohol alone in doses of 20 to 30 g per day has been shown to decrease CHD by 40%. Red wine also contains a large amount of folic acid, other vitamins and potassium and magnesium, which may also have beneficial cardiovascular effects.

The health benefits of phytochemicals such as the flavonoids from fruits and vegetables are thus becoming more evident and have increasing importance in nutritional science.

1.6.3

Flavonoid intake and cardiovascular disease

Epidemiological studies have demonstrated that dietary flavonoid intake is inversely associated with mortality from coronary heart disease (CHD) and incidence of stroke (Hertog et al., 1993, Knekt et al., 1996).

Studies were undertaken by Hertog et al. (1995) to determine whether flavonoid intake could explain differences in mortality rates from chronic diseases between populations. The workers determined the mean intake of flavonoids at baseline (1960) in the 16 participant cohorts of the Seven Countries Study by chemical analysis of equivalent food composites and related it to 25 year mortality from CHD, cancer at various sites, and all causes. It was found that the mean flavonoid intake of the cohorts varied from 2.6 mg per day in West Finland to 68.2 mg per day in

56

Ushibuka, Japan, with quercetin representing 100% (e.g. West Finland) to 39% (Zutphen, the Netherlands) of total flavonoid intake. Hertog and co-workers (1995) showed that flavonoid and quercetin intake was highly correlated (r = 0.92) and the relationhips between quercetin intake and disease mortality similar to those observed for total flavonoid intake. Results showed that the average flavonoid intake was inversely related to CHD mortality, with variance in flavonoid intake explaining about 25% of the total variance in CHD mortality across the 16 cohorts. Using multivariate analysis, it was shown that intake of saturated fat (73%, p = 0.001), flavonoid intake (8%, p = 0.01), and percentage of smokers per cohort (9%, p = 0.03) explained together, independent of alcohol and antioxidant vitamins, 90% of the variance in CHD rates. Adjustment for intake of vitamin E, ascorbic acid, carotene, or alcohol did not increase the total explained variance.

In the same studies by Hertog et al. (1995) the average flavonoid intake was not associated with lung or colorectal cancer mortality. There was a strong and positive correlation of flavonoid intake with stomach cancer mortality. However, using

multivariate analysis, after additional adjustment for intake of ascorbic acid, the association was no longer statistically significant. Average flavonoid intake was not related to all-cause cancer mortality, and adjustment for fat intake and percentage of smokers did not change the association.

Hertog et al. (1995) concluded that average flavonoid intake may contribute to differences in mortality from CHD across populations, but not to differences in cancer mortality. The results obtained for this cross-cultural comparison by Hertog and co-workers (1995) were in agreement to those of the prospective cohort Zutphen

57

Elderly Study (Hertog et al., 1993, 1994) that involved a cohort of 806 elderly men (aged 65 - 84 years) in the Netherlands at baseline in 1985. In that particular study, flavonoid intake was inversely associated with CHD mortality, but not with mortality from cancer at all sites. It was hypothesised by Hertog et al. (1995) that diets rich in flavonoids protect LDL against oxidation by free radicals. The relative incidence of heart disease among men from the Zutphen study who had the highest intake of flavonoids was only on third of those who had the lowest intake of flavonoids. The result was the same even when adjustments were made for age, body fat, smoking, cholesterol, blood pressure, physical activity, coffee consumption, and the intake of calories, vitamin C, vitamin E, -carotene, and dietary fibre. Their findings also supported the suggestion noted previously that flavonoids present in red wine could be partly responsible for low CHD mortality in red wine drinkers.

A Finnish cohort study undertaken to study the effect of flavonoid intake and coronary mortality (Knekt et al., 1996) used a large sample size of 2748 men and 2385 women. The study was based on data collected at the Finnish mobile clinic health examination survey from 1967-72 and followed up until 1992, and took into account 30 communities from different parts of Finland. In line with the Zutphen Elderly Study, the Finnish study indicated an inverse association between intake of flavonoids and coronary mortality. It was found that there was an inverse

relationship between flavonoid intake and total mortality both in men and women when the major cardiovascular risk factors of smoking, serum cholesterol concentration, blood pressure, and body mass index were adjusted for. It was found that the intake of the antioxidant vitamins C and E and carotenoids were low in Finland during the baseline study thereby suggesting the possibility that even smaller

58

amounts of dietary flavonoids are important in circumstances where the intake of other dietary antioxidant agents is low. For women, coronary mortality was

significantly inversely associated with flavonoid intake, but for men the inverse association was not significant. Further adjustment for intake of energy, fatty acids, fibre, and antioxidant vitamins weakened the associations in women. The results are in line with previous findings of Knekt et al. (1994) which showed antioxidants are of particular benefit to women. It was therefore concluded by Knekt et al. (1996) that people with very low intakes of flavonoids have risks of coronary disease.

A study carried out by Rimm et al. (1996) investigated the association between intake of flavonols and flavones and CHD in 34,789 men enrolled in the US Health Professionals Follow-up Study and followed prospectively for six years. Results of the study showed that no individual flavonoids were associated with an appreciable reduction in risk from CHD, and controlling for intake of dietary fibre, saturated fat, or cholesterol did not alter the results. The data did not therefore support a strong inverse association between flavonoid intake and total CHD, but did not exclude the possibility that flavonoids have a protective effect in men with established CHD.

Hertog et al. (1997a) also investigated whether flavonol intake predicted a lower rate of ischemic heart disease (IHD) in 1900 Welsh men aged between 45 and 59 years, who were followed up for 14 years (The Caerphilly Study). In this case flavonol intake, mainly from tea to which milk was normally added, was not related to IHD incidence but was weakly positively related to IHD and cancer mortality and strongly related to total mortality. Hertog and co-workers (1997a) concluded that the intake of antioxidant flavonols, the major food source of which is black tea, is not inversely

59

associated with IHD risk in the UK. These results contrasted strongly with the findings of the Zuthphen Elderly Study (Hertog et al., 1993). There was a possibility that the flavonols from tea to which milk is added are not absorbed, experimental evidence suggesting that adding milk to tea abolishes the plasma antioxidant-raising capacity of tea.

Using data from the Zutphen study, Arts et al. (2001) evaluated the association between catechin intake and the incidence of and mortality from IHD and stroke. They calculated the mean catechin intake in 1985 to be approximately 72 mg, mainly from black tea, apples, and chocolate. It was found that catechin intake was

inversely associated with IHD disease mortality which is in contrast to the findings of Hertog et al. (1997a) noted previously for the Caerphilly Study. However, Arts et al. (2001) found no association between intake and stroke incidence or mortality.

Work carried out by Arai et al. (2000) calculated the intake of flavonols, flavones and isoflavones by 115 Japanese women aged between 29 and 78 years. The total mean intakes of flavonoids (sum of flavonols and flavones) and isoflavones were 16.7 and 47.2 mg per day, respectively. It was found that the total intake of

isoflavones exceeded that of other dietary antioxidants, such as flavonoids, carotenoids and vitamin E, and was approximately half that of the vitamin C intake. After adjustment for age, body mass index and total energy intake, it was demonstrated that the total intake of flavonoids was inversely correlated with the plasma total cholesterol concentration (TC) and plasma LDL cholesterol (LDL-C). Quercetin, as a single component, was also inversely correlated with both TC and LDL-C. The results of the work carried out by Arai et al. (2000) suggested that a

60

high consumption of both flavonoids and isoflavones by Japanese women may contribute to their low incidence of CHD compared with women in other countries.

As part of the Women's Health Study, Sesso et al. (2003) examined whether flavonoids are associated with incident cardiovascular disease (CVD) in a large cohort of female health professionals (38,445) in the US. They evaluated the

association between individual selected flavonols and flavones, including quercetin, kaempferol, myricetin, apigenin, and luteolin, plus specific food sources of flavonoids and the risk of CVD. The data obtained from the large cohort of middleaged and older US women indicated that higher flavonoid intake was not associated with a reduced risk of CVD after adjustment for lifestyle and dietary factors. No individual flavonol or flavone was associated with CVD.

Knekt et al. (2002) studied the association between flavonoid intake and risk of several chronic diseases in Finland. The total dietary intakes of 10,054 men and women were determined during the year preceding the baseline examination. The flavonoid intakes were estimated based mainly on the flavonoid concentrations in Finnish foods and the incident cases of the diseases considered were identified from different national public health registers. Overall, it was found that people with a higher total flavonoid intake tended to have a lower total mortality. Results from the study suggested the presence of an inverse association between flavonoid intake and subsequent occurrence of IHD, cerebrovascular disease, lung and prostate cancer, type 2 diabetes, and asthma. The potential benefits of the flavonoids were mainly attributed to quercetin, known to be the most potent antioxidant, but also in some cases to kaempferol, myricetin, hesperitin, and naringenin. The lower risk found for

61

IHD mortality was found to be mainly in those people with the higher intakes of apples and onions, and accordingly to the flavonols quercetin and kaempferol. However, the authors did note that although their findings were independent of the intake of antioxidant vitamins, the potential importance of other biologically active compounds in fruit and vegetables could not be ruled out. They did, however, conclude that the risk of some chronic diseases may be lower at higher dietary flavonoid intakes.

Recent work published by Mennen et al. (2004) evaluated the relationship between consumption of foods rich in flavonoids and estimated cardiovascular risk in a large French population. A cross-sectional analysis was carried out on 1286 women and 1005 men from the SU.VI.MAX (SUpplementation en VItamines et Minraux AntioXidants) Study, an 8 year trial evaluating the effect of antioxidant vitamin (C, E and -carotene) and mineral (selenium and zinc) supplementation on the incidence of major chronic diseases (cancers of all sites and ischemic heart disease) in France. In women, flavonoid-rich food consumption was inversely related to systolic blood pressure. No such relationship was obtained for men. There was little difference between genders with respect to intake of flavonoid-rich foods, except for the consumption of wine, which was higher in men and the consumption of tea, which was higher in women. Overall, it was concluded that a diet rich in flavonoid-rich foods, such as apples, tea, chocolate, onions, citrus fruit, and red fruit, may be useful in the prevention of CVD in women.

The body of evidence available suggests that dietary flavonoids are beneficial to health as they have a wide range of different biological activities, including

62

antibacterial, antithrombotic, vasodilatory, antiinflammatory, and anticarcinogenic effects mediated by different mechanisms (Knekt et al., 2002; Middleton et al., 2000; Nijveldt et al., 2001). However, it should be noted that the results obtained with regard to an association between flavonoids and CHD are mixed. In vitro studies have indicated that there are considerable differences in the antioxidative potential of different flavonoid sub-groups, depending on their chemical structure (Rice-Evan et al., 1997). As knowledge on the bioavailability, absorption and metabolism of the polyphenols in human is limited, it is likely that different groups of flavonoids have different effects on human health.

1.6.4

Mode of action of flavonoids in relation to coronary heart disease

Flavonoids can function as: metal chelators and reducing agents; scavengers of ROS (reactive oxygen species); chain-breaking antioxidants; quenchers of the formation of singlet oxygen; and protectors of ascorbic acid, or conversely, ascorbic acid can protect flavonoids against oxidative degradiation. (Middleton et al., 2000).

In many of the cases reported it is not certain whether flavonoids inhibit the formation of ROS or scavenge them. Nonetheless, flavonoids have been shown to be react with the hydroxyl radical and, therefore, can act as highly important chainbreaking antioxidants. ROS that can be scavenged or whose formation can be

prevented by flavonoids have been presented previously in Table 1.3.

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Table 1.3 Reactive oxygen species that can be scavenged or whose formation can be inhibited by flavonoids O2- (Superoxide ion) One-electron reduction product of O2. Produced by phagocytes, formed in autooxidation reactions (flavoproteins, redox cycling), and generated by oxidaseses (heme proteins). Protonated form of O2- Two-electron reduction product of O2 formed from O2- (HO2- ) by dismutation or directly from O2-. Reactivity of O2- and H2O2 is amplified in the presence of heme proteins. Three-electron reduction product of O2 generated by Fenton reaction, transition metal (iron, copper)catalyzed Haber-Weiss reaction; also formed by decomposition of peroxynitrite produced by the reaction of O2- with NO. (nitric oxide radical). Example: Lipid radical (LO.). Example: Lipid peroxy radical (LOO.) produced from organic hydroperoxide (e.g. lipid hydroperoxide, LOOH), ROOH by hydrogen abstraction. Singlet oxygen.

HO2- (Perhydroxy radical) H2O2 (Hydrogen peroxide)

OH (Hydroxyl radical)

RO (Alkoxyl radical) ROO (Peroxyl radical)

O2

(from Middleton et al., 2000)

1.6.4.1

Inhibition of lipid peroxidation

It has been reported that several flavonoids can lipid peroxidation by scavenging OH. The chain breaking antioxidant action of the flavonoids (F) can be represented as follows (Middleton et al., 2000): LOO + FLOH LOOH + FLO where FLOH represents the flavonoid.

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Termination of lipid radical (L), lipid peroxyl radical (LOO) and alkoxyl radical (LO) by phenolic antioxidants is as follows:

LOO / L / LO + AOH LOOH / LH / LOH + AO

where AOH represents phenolics (e.g. -tocopherols, flavonoids) and AO represents the phenoxyl radical.

There has also been a proposal that flavonoids react with lipid peroxyl radicals (LOO) leading to termination of radical chain reactions (Takahama, 1983).

Differences in the modes of action of the flavonoids as to whether they act as free radical scavengers, chain-breaking antioxidants or act by terminating radical chain reactions indicate that different constituents are important for different biological activities as noted previously.

The characteristics of flavonoid structure for most effective radical-scavenging activity are presented in Table 1.4 [taken from Middleton et al. (2000)]. It appears that quercetin is an extremely efficient radical scavenger, with myricetin being even more active due to the presence of the third (pyrogallol) OH group on the B ring. Kaempferol has been shown to be a very good scavenger despite the fact that is has only one OH group on the B ring (4' -OH) possibly because of the combination of the other characteristics (C2-C3 double bond, 3-OH group, and 4-oxo group on ring C). Although catechin has the catechol group on ring B and the 3-OH group on ring C, it

65

is a weak scavenger as it lacks the C2-C3 double bond and the 4-oxo group on ring C (Rice-Evans et al., 1997).

Table 1.4

Characteristics of flavonoid structure for most effective radicalscavenging activity

The catechol (O-dihydroxy) group in the B ring confers great scavenging ability, with exceptions such as those described by Ratty and Das (1983), who thought it did not contribute towards lipid peroxidation in rat brain mitochondria. A pyrogallol (trihydroxy) group in B ring of a catechol, as in myricetin, produces even higher activity. The C2-C3 double bond of the C ring appears to increase scavenger activity because it confers stability to the phenoxy radicals produced. The 4-oxo (keto double bond at position 4 of the C ring), especially in association with the C2-C3 double bond, increases scavenger activity by delocalizing electrons from the B ring. The 3-OH group on the C ring generates an extremely active scavenger; in fact, the combination of C2-C3 double bond and 4-oxo group appears to be the best combination on top of the catechol group. The 5-OH and 7-OH groups may also add scavenging.

(from Middleton et al., 2000)

Thus, there appears to be a hierachy of flavonoid and isoflavonoid antioxidant activities that is dependent on structure and defines the relative abilities of the compounds to scavenge free radicals (Rice-Evans et al., 1997). The antioxidant activities relative to Trolox, the water-soluble vitamin E analogue, are consistent with their structure (Rice-Evan et al., 1997). The half-peak reduction (Ep/2) has also been ascribed as a suitable parameter for representing the scavenging activity of the flavonoids (Van Acker et al., 1996). A flavonoid with a low value for half-peak reduction potential (i.e. <0.2) is a good scavenger. The half-peak reduction 66

potentials can be compared with the total antioxidant activity, measured as the Trolox Equivalent Antioxidant Capacity (TEAC). From Table 1.5 (Rice-Evans et al., 1997) it can be seen that there is broad agreement between the TEAC value (pH 7.4) and the Ep/2 value (same pH) in that flavonoids with efficient scavenging properties have a TEAC value of 1.9 mM and Ep/2 values of 0.2 mV, the exception being kaempferol.

Table 1.5. Hierarchy of flavonoid antioxidant activities and the relationship with reduction potentials Flavonoid Quercetin Rutin Catechin Luteolin Taxifolin Apigenin Naringenin Hesperetin Kaempferol
a

Antioxidant activitya (mM) 4.7 2.4 2.4 2.1 1.9 1.5 1.5 1.4 1.3

Half-peak reduction potentialb (mV) 0.03 0.18 0.16 0.18 0.15 >1 0.6 0.4 0.12

Measured as the TEAC (Trolox equivalent antioxidant activity)- the concentration of Trolox with the equivalent antioxidant activity of 1mM concentration of the experimental substance. b Designated Ep/2. An Ep/2 of <0.2 indicates a chemical that is readily oxidizes and therefore an efficient free radical scavenger.

(from Rice-Evans et al., 1997) 1.6.4.2 Decrease in LDL oxidation by flavonoids

As oxidation of LDL is implicated in the pathogenesis of atherosclerosis, the enhancement of the resistance of LDL to oxidation is one of the models used by researchers to investigate the efficacy of dietary phytochemicals as antioxidants

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against radicals generated in the lipophilic phase (Rice-Evans et al., 1997). DeWhalley et al. (1990) reported that flavonoids inhibited cell-free oxidation of LDL oxidation mediated by CuSO4. They appeared to act by protecting LDL against oxidation caused by macrophages, as they inhibited the generation of lipid hydroperoxides and protected -tocopherol from being consumed by oxidation in the LDL. Consequently, the flavonoids protected -tocopherol, and possibly other endogenous antioxidants, in LDL from oxidation, maintained their levels for longer periods of time, and delayed the onset of lipid peroxidation.

Thus, there is now good experimental evidence that flavonoids are potent inhibitors of LDL oxidation, although the mechanism by which they do so are not certain. The following possibilities, however, have been put forward (Middleton et al., 2000):

Firstly, the flavonoids may reduce the generation or release of free radicals in the macrophages or may protect the -tocopherol in LDL from oxidation by being oxidized by free radicals themselves.

Secondly, the flavonoids could regenerate active -tocopherol by donating a hydrogen atom to the -tocopheryl radical. The latter radical is formed when it transfers its own OH hydrogen atom to a lipid peroxyl radical to terminate the chain reaction of lipid peroxidation as noted previously.

Thirdly, the flavonoids may sequest metal ions, such as iron and copper, thereby diminishing the endangered free radicals in the medium.

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Investigations by Negre-Salvayre et al. (1991) have shown that the cytotoxicity of oxidized LDL could be prevented by flavonoids (quercetin, rutin and catechin) either by inhibiting the lipid peroxidation of LDL (induced by UV radiation) or by blocking at the cellular level the cytotoxicity of previously oxidized LDL. Later work carried out by Negre-Salvayre et al. (1995) showed that LDL mildly oxidized by copper ions or UV radiation exhibited a cytotoxic effect on cultured endothelial cells that could be inhibited by rutin, ascorbic acid and -tocopherol. It appeared that these

antioxidants acted to inhibit oxidation of LDL and increase the resistance of the cells to the cytotoxic effects of oxidized LDL, the mixture of the three compounds having a 'supra-additive' effect.

It has also been suggested that flavonoids may be protective again CHD by influencing several other processes such as (i) an increase in HDL levels, (ii) a reduction of cardiac mast cell mediator release; or by (iii) decreasing cardiovascular inflammation (Middleton et al., 2000).

1.6.5

Bioavailability of flavonoids

Demonstration of the fact that flavonoids are bioavailable after they are consumed from fruits, vegetables and beverages is essential in order to support the belief that the flavonoids derived from these sources are of benefit to health. Indirect evidence of their absorption through the gut barrier is the increase in the antioxidant capacity of the plasma after the consumption of flavonoid-rich foods (Hollman et al., 1997). More direct evidence on the bioavailability of a few flavonoids has been obtained by measuring their concentrations in plasma and urine after the ingestion of either pure compounds or of foodstuffs with a known content of the compound of interest

69

(Scalbert and Williamson, 2000). The chemical structure of the flavonoids will determine their rate and extent of absorption through the small intestine and the nature of the metabolites circulating in the plasma.

Limited information is available on the metabolism of flavonoids in humans. Ring scission occurs under the influence of intestinal bacteria, which also accounts for the subsequent demethylation and dehydroxylation of the resulting phenolic acids (cinnamic acid derivatives and simple phenols). Intestinal microorganisms also

possess glycosidases capable of cleaving sugar residues from flavonoid residues. The flavonoids can also undergo oxidation and reduction reactions, as well as methylation, glucuronidation, and sulfation in animal species (Middleton et al., 2000). The metabolism of quercetin is proposed to be via the splitting of the

flavonoid molecule to form hydroxyaromatic acids with a two-carbon side chain a OH group is on the 3-carbon of the pyrone ring. When a OH group is lacking in this position, as in hesperidin, hyroxyaromatic acids with 3-carbon side chains are formed (Rice-Evans et al., 1996).

Gugler et al. (1975) studied the metabolism of quercetin in six subjects (four males and two females) aged between 21 and 32 years. After oral administration of a single dose of 4 g, no measurable concentrations of quercetin or its derivatives were detected in plasma or urine. However, it was found that approximately 53% of the oral dose were recovered unchanged in the faeces, and it was calculated that only 1% of the original 4 g dose of quercetin or approximately 40 mg was absorbed through the gastrointestinal tract.

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Work by Hollman et al. (1997) investigated the relative bioavailabiltiy of quercetin from onions and apples. The participants in this study (five women and four men, mean age 24.8 years) followed a quercetin-free diet during three experimental periods each of five days, these periods being separated by nine days without treatment or without a prescribed diet. On day four of each experimental period, the subjects were provided with one out of three different quercetin-containing supplements in random order, namely fried onions, apples and rutinoside (a glycoside of quercetin). These supplements were given at breakfast with blood samples being collected periodically over the next 36 hours and urine continuously for 24 hours. Results showed that the quercetin was found in the circulation after consumption of the major dietary sources of quercetin. However, it was found that the bioavailability and absorption kinetics varied markedly between sources, a major difference being the type of glycosides. Most rapidly absorbed was the quercetin from onions, which contains only glucosides. Bioavailability from apples, which contain a variety of glycosides, and pure quercetin-3-rutinoside, the major species in tea, was 30% relative to onions. Peak levels were achieved less than 0.7 hours after ingestion of the onions, 2.5 hours for apples and 9 hours after ingestion of the rutinoside. The half-life of elimination was 28 hours for onions and was found to be 23 hours for apples. The workers concluded that the experimental results pointed to a predominant role of the sugar moiety in the bioavailability and absoprtion of dietary quercetin in the human body. They could not rule out, however, that

differences between apples and onions in cell wall structures, location of glyosides in cells, or their binding to cell constituents also effect the liberation of quercetin from these foods in the digestive tract.

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Olthof et al. (2000) tested whether the position of the glucose moiety affected the bioavailability of quercetin glucosides in humans by comparing quercetin-3glucoside with quercetin-4'-glucoside, the former being produced by splitting of the rhamnose molecule from the quercetin-3-rutinoside. The quercetin-3-glucoside

differs only from the highly bioavailable quercetin-4'-glucoside in the position of the glucose moiety on the quercetin aglycone. The workers fed five healthy men and four healthy women (19 - 57 years) with a single dose of each compound and followed the plasma quercetin glucosides. The bioavailability was found to be the same for both quercetin glucosides and it was thus concluded that irrespective of the position of the glucose moiety, the quercetin glucosides are rapidly absorbed in humans.

Table 1.6, taken from the review by Scalbert and Williamson (2000) provides a good summary of the bioavailability in humans of polyphenols consumed alone or in foods. As noted by these authors, bioavailability is particularly low for quercetin and rutin (0.3-1.4%), but reaches higher values for catechins in green tea, isoflavones in soy, flavanones in citrus fruits or anthocyanidins in red wine (3-26%). Interindividual variations have also been observed, e.g. 5-57% of the naringin consumed with grapefruit juice is found in urine according to the individual (Fuhr and Kummert, 1995).

According to the work reviewed by Scalbert and Williamson (2000), the concentration of intact parent polyphenols in the plasma are often low (Table 1.6), the maximum rarely exceeding 1 M after the consumption of 10-100 mg of a single phenolic compound. It has also been found that they do not account on their

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own for the increase in antioxidant capacity in the plasma which is probably higher due to the presence of metabolites formed in the body's tissues or by the colonic microflora. These metabolites are still for the main unknown and not accounted for. Work carried out by Donovan et al. (1999) demonstrated that catechin was found almost exclusively as metabolites after the consumption of red wine being present both as a sulfate conjugate and a conjugate containing both glucuronide and sulfate residues. They concluded that if flavonoids are protective nutrients, the active forms are likely to be metabolites, which are far more abundant than the forms that exist in foods.

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Table 1.6.

Bioavailability in humans of polyphenols consumed alone or in foods1

Quantity of polyphenol ingested mg 1000 68 98 202 144 139 6.4 88 82 33 32 105 Maximum concentration in plasma M 0.74 0.30 0.30 3.2 1.34 0.8 0.5 ND 3.6 ND 6.2 Unno et al. 1996 Maiani et al. 1997 Nakagawa et al. 1997 Donovan et al. 1999 Balant et al. 1979 Xu et al. 1994 Cassidy et al. 1994 Gooderham et al. 1996 8.7 26 8.8 6.8 24.4 4.9 3.0 1.06.7 Karr et al. 1997 Fuhr and Kummert 1995 Ameer et al. 1996 Excretion in urine Reference % 27 Jacobson et al. 1983 1.39 Hollman et al. 1997 0.44 Hollman et al. 1997 0.35 Hollman et al. 1997 Hollman et al. 1999 Aziz et al. 1998 Young et al. 1999 Lee et al. 1995

Polyphenol Caffeic acid Quercetin Quercetin Quercetin-4-Orhamnoglucoside Quercetin-4-Oglucoside Quercetin Quercetin Epigallocatechin gallate Epigallocatechin Epicatechin gallate Epicatechin Epigallocatechin gallate Epigallocatechin gallate Epigallocatechin gallate Catechin Catechin Genistein Daidzein Genistein Daidzein Genistein Daidzein Genistein Daidzein Naringin Naringin Hesperidin Naringin Hesperidin Anthocyanins
1

Source

Onion Apple Pure compound Pure compound Onion Mixed black currant and apple juice, 1000 ml/d for 7 d Green tea infusion, 1.2 g

0.33 0.67 ND 0.27 0.130.31 5.0

Green tea infusion, 5 g Green tea infusion, 6 g Green tea extract Red wine, 120 ml Pure compound Soy milk Soy proteins, 60 g/d for 1 mo Soy proteins, 60 g/d for 28 d

525 34 500 19 25 20 25 80 36 23 13 43 689 89 500 500 218

4.4 0.072 2.0 0.74 0.79

0.45 19.8 5.3 9.2 2.5

0.50 0.91

Soy proteins, 20 g/d for 9 d Grapefruit juice, 120 ml Grapefruit and orange juice, 1250 ml each Pure compounds Red wine, 300 ml

<4

Ameer et al. 1996 Lapidot et al. 1998

Polyphenols, principally in the form of conjugated metabolites, as sulfate esters or glucuronides, in plasma and urine, were hydrolyzed by acid or enzymes before chromatrographic or colorimetric analysis. ND, not detected.

(from Scalbert and Williamson, 2000)

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1.7

Previous studies

As noted previously evidence from epidemiological and in vitro studies suggest dietary flavonoids may be able to protect against CHD. A collaborative study between the Department of Food Science and the Department of Clinical Biochemistry (Queens University-Belfast) Dietary Flavonoids and their Possible Role as Antioxidants in Preventing Atherosclerosis (McAnlis, 1998); was investigated both the in vitro and in vivo effects of the main dietary flavonoids. The work of McAnlis (1998) found that the flavonoids quercetin, myricetin, kaempferol and apigenin were found at high concentrations in onions, lettuce, tomatoes, cabbage greens and black tea. A series of in vivo studies investigated the ability of these dietary flavonoids to protect LDL against oxidation. All the flavonoids, with the exception of apigenin, inhibited the oxidation of LDL in vitro in a dose dependent manner. The results from these series of initial studies suggested that the

consumption of flavonoid rich foods may be able to protect LDL against oxidation, thereby slowing the progress of atherosclerosis.

The in vivo effects of the main dietary flavonoids were consequently investigated by McAnlis and co-workers (McAnlis et al., 1997; McAnlis, 1998; McAnlis et al., 1999). The studies carried out indicated that flavonoids can be absorbed from

dietary sources thereby increasing the total antioxidant capacity of the plasma but not reducing the susceptibility of LDL to oxidation. Further investigation revealed that the flavonoids become bound to plasma proteins thus they were not present in the LDL particle to protect against lipid oxidation. It was therefore concluded that the

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ingestion of flavonoid-rich foods does not directly protect the LDL against oxidation but may protect against CHD by another mechanism.

1.8

Objectives of the present investigations

The studies carried out in the present investigations followed on from those of McAnlis and co-workers as summarised in Section 1.7. The first main objective of the present studies was to determine flavonoid content in a range of fruits and vegetables selected for analysis based on those listed in a food frequency questionnaire currently being used as part of the EUREYE Study, the aim of which is to determine the effect of diet on eye disease in the over-sixties age group in Europe. The concentration of flavonoids in the raw fresh fruit and vegetables was to be analysed and subsequently a range of fruits and vegetables subjected to the cooking processes of baking, boiling and frying.

The second objective of the work was to use the values obtained from analysis of the raw fruits and vegetables to estimate the flavonoid intake, both profile and content, of the Northern Ireland cohort of the EUREYE Study participants.

The third main objective of the work was to carry out a series of in vitro studies to study the ability of the selected flavonoids to protect VLDL (very low density lipoprotein), LDL (low density lipoprotein) and HDL (high density lipoprotein) against oxidation, a process of significant importance in the pathogenesis of atherosclerosis.

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