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Fact Sheet 4

Scientific Basis for Olive Oil, monounsaturated fatty acids, antioxidants and LDL oxidation

Author
Prof. Dr. med Gerd Assmann
Dr. troph. Ursel Wahrburg
The Institute of Arteriosclerosis Research,
University of Münster, Germany

1. Introduction

A high concentration of plasma low-density lipoprotein (LDL) cholesterol is a dominant risk factor for the development of atherosclerosis. However, the precise mechanisms by which LDL causes atherosclerosis, i.e. the steps between infiltration of LDL into the arterial wall and the formation of an atherosclerotic lesion, are not well understood. There is increasing evidence that LDL must be modified in some way before it can become pathogenic, and recent data from biochemical, animal and epidemiological studies strongly support the hypothesis that oxidative modification of LDL plays a crucial and causative role in the pathogenesis of atherosclerosis. The susceptibility of LDL to become oxidised is determined by a variety of endogenous and exogenous factors. Among the latter, nutritional factors are of outstanding importance, in particular the types of dietary fatty acids and antioxidant vitamins. The present paper outlines the oxidation hypothesis of atherosclerosis and the role nutritional factors could play in modulating this process.

2. The role of LDL oxidation in atherogenesis

2.1 LDL oxidation

LDL is a spherical particle consisting of a large protein, termed apolipoprotein B (apo B), which is embedded in an outer mono-layer of phospholipids and free cholesterol molecules. This monolayer surrounds a central core of cholesterylesters and triglycerides. One such LDL-particle contains about 3600 fatty acids, roughly half of them being polyunsaturated fatty acids (PUFA). Additionally, LDL contains several antioxidants, the most important being -tocopherol.

Oxidation of LDL is a lipid peroxidation chain reaction, initiated by so-called free radicals. Radicals are molecules with an unpaired free electron, making them highly reactive. In particular, oxygen radicals (eg. hydroxyl-, hydroperoxyl-, superoxidradical), which are produced in the cells as by-products of oxidative metabolic processes, are of importance. The chain reaction begins when a reactive free radical removes a hydrogen atom from a polyunsaturated fatty acid molecule in the LDL particle. PUFAs are highly susceptible to lipid peroxidation, because the susceptibility of a fatty acid to oxidation increases with its number of double bonds. Lipid peroxyl radicals are formed which in turn can initiate oxidation in neighbouring fatty acids. This process leads to a breakdown of PUFA, yielding a variety of reactive aldehydes, ketones, and other products, some of which form covalent bonds with LDL apo B).

It is well known that oxidation of LDL can be initiated in vitro by incubating isolated LDL particles with cells (macrophages, lymphocytes, smooth muscle cells, and endothelial cells), metal ions (copper or iron), enzymes, oxygen radicals, or UV-light (10,19,40). However, little is known about the mechanisms by which LDL becomes oxidised in vivo. There is evidence that LDL in the plasma is protected against oxidation, because the plasma contains a lot of water-soluble antioxidative substances, such as ascorbic acid, ureic acid, or bilirubin. Thus, it is likely that the majority of oxidative modification of LDL occurs in the artery wall and not intravascularly. In the arterial wall LDL is largely isolated from the many antioxidants present in the plasma. Furthermore, the LDL particles are exposed to a variety of free radical species and oxidative enzymes produced by artery wall cells.

If LDL is exposed to pro-oxidative conditions, it becomes depleted of its antioxidants, with -tocopherol being the first to be lost. The oxidation of the LDL PUFA to lipid hydroperoxides starts when most of the antioxidant defence has been lost. The rapid decomposition of PUFA leads to a lot of further modifications in the LDL-particle, eg. oxidation of cholesterol, modifications of apo B and release of several bioactive substances.

Nilsson et al., Herz 1992;263

2.2 LDL oxidation and atherosclerosis

Early atherosclerotic lesions are characterised by the presence of fatty streaks, which are composed of so-called foam cells, derived from smooth muscle cells and monocyte-macrophages. In a first step monocytes invade from the bloodstream into the subendothelial space and become resident macrophages. They then take up lipids and lipoproteins, predominantly cholesterylesters, infiltrated and deposited in those regions. However, the precise mechanisms of this lipid uptake and accumulation is not known. It is known that the LDL-receptor is down-regulated when the intracellular cholesterol content increases, so the uptake of cholesterol via the classic LDL-receptor pathway cannot result in a pathological cholesterol accumulation. On the other hand, oxidatively modified LDL (ox-LDL) is no longer recognised by the LDL-receptor, but can be taken up by the so-called scavenger receptor on macrophages which is not regulated by intracellular cholesterol. Thus, the scavenger receptor mediated uptake of ox-LDL could lead to a substantial cholesterylester accumulation in macrophages.

The study of ox-LDL has shown that oxidation of LDL changes it in many ways that make it more atherogenic than native LDL. These effects are summarised in Table 1.

Table 1: Atherogenic properties of ox-LDL.

3. Olive oil and LDL oxidation

There is no doubt that nutrition is of great importance in LDL oxidation. In particular, the amount and type of fat in the diet as well as the content of antioxidative components affect the susceptibility of LDL and cells to oxidative damage. There are several potential ways by which dietary fatty acids may influence the oxidation of LDL. First of all, the amount and composition of dietary fat affects the amount of LDL particles present in the artery wall. Replacement of dietary saturated fatty acids with MUFA or PUFA lowers total and LDL cholesterol levels . This reduction in LDL levels would likely decrease the amount of LDL entering the artery wall and theoretically would directly reduce the amount of LDL available for oxidation. Dietary fatty acids may also directly influence LDL susceptibility to oxidation by changing its fatty acid composition. Furthermore, dietary fatty acids may change the fatty acid composition of the artery wall cells, thus altering their pro-oxidant activity and their response to oxidative stress.

Due to its high MUFA content olive oil seems to have protective properties with regard to LDL oxidation. Additionally, olive oil may further provide some protection by supplying LDL with potent antioxidants, such as Vitamin E and polyphenolic compounds. These protective effects of olive oil are detailed below.

3.1 Effects of dietary fatty acids on LDL oxidation

Several investigators have compared the influence of dietary MUFA and PUFA on LDL oxidation. First, it could be shown in rabbits, that oleate-rich LDL particles were remarkably resistant to oxidative modification (32). In subsequent studies small groups of human subjects were fed diets differing in their MUFA and PUFA content.

Reaven et al. provided their study participants with high-fat liquid formula diets which had a MUFA content of about 80% of total fatty acids or a PUFA content of about 60%. After these diets extremely enriched with MUFA (derived from high-oleic sunflower oil) or PUFA (derived from sunflower oil) the fatty acid composition of isolated LDL particles reflected the fatty acid composition of the diet, and the fatty acid distribution was similar in the different lipid fractions of the LDL-particle. The linoleic acid (C18:2) content of LDL was strongly related to the rate and the extent of oxidation, whereas the amount of oleic acid (C18:1) in the LDL-particles was inversely correlated to the extent of oxidation.

Some other studies were conducted with solid food diets. For instance, Bonanome et al. compared a grapeseed oil-enriched diet (45 % fat; 5 % MUFA, 30 % PUFA) to a diet enriched in olive oil (45% fat; 30% MUFA, 5% PUFA) in 12 healthy subjects. Again, the rate of LDL oxidation during the PUFA diet was increased compared to the MUFA diet. In the other studies it could be confirmed that the linoleic acid content of LDL strongly correlated with either the rate or the extent of oxidation.

Although these results were unambiguous, several questions remain open. On the basis of the studies conducted so far, it is not clear whether only the decrease in easily oxidisable linoleic acid in LDL is responsible for the decrease in lipid peroxidation after a MUFA-rich diet, or whether this decrease is due to direct antioxidant properties of oleic acid? Or, are both mechanisms together involved in reducing the susceptibility of LDL to oxidation? Do PUFA enhance or do MUFA decrease LDL oxidation?

Until now, there are only two studies dealing with these questions: Aviram and Eias compared the effects of an olive oil supplement (50g/day) to the baseline diet (30% fat, 50% carbohydrates). It could be demonstrated, that the LDL obtained after one and two weeks of the olive oil-rich diet showed a reduced susceptibility to oxidation as well as reduced cellular uptake by macrophages. Thus, it can be suggested that MUFA supplementation can cause an absolute decrease in LDL susceptibility to oxidation. Berry et al confirmed these results in their study in which they compared a MUFA-rich diet (17% of energy, total fat: 33% of energy) with a carbohydrate-rich diet (65% of energy; MUFA: 7%), keeping the PUFA content constant in both diets. The MUFA-diet led to a significant reduction in the susceptibility of LDL to oxidative stress. These data support the concept, that oleic acid enriched diets may reduce LDL oxidation both through intrinsic antioxidant properties of the MUFA, as well as by reducing the content of linoleic acid in LDL.

3.2 Effects of dietary fatty acids on cellular pro-oxidant activity and cellular susceptibility to oxidative stress

Dietary fatty acids can also have effects on cellular pro-oxidant activity. Several investigators have demonstrated that dietary supplementation with different types of fatty acids leads to changes in the fatty acid composition of the monocyte membrane composition and that this influences the production of oxygen radicals, particularly superoxide anions, in monocytes and macrophages. The production of superoxide anions by these cells undoubtedly contributes to LDL oxidation. In a comparison of the effects of dietary supplementation with MUFA, n-3-, or n-6-PUFA on superoxide anion generation a decrease in superoxide production was only observed after n-3 fatty acid supplementation, while the monocytes from the MUFA or n-6-PUFA supplemented groups showed no change or had increased superoxide anion levels. The mechanism by which n-3 fatty acids may reduce the oxygen radicals is not known, and other studies could not confirm these effects. Further investigation is needed to exactly evaluate the role of the different fatty acids on cellular pro-oxidant activity.

Furthermore, dietary fatty acids can influence the susceptibility of cells to oxidative stress, probably also by changing cell membrane fatty acid composition. Cells enriched with MUFA have been shown to be less susceptible to oxidative damage, whereas n-6 PUFA increased the susceptibility to oxidative damage. Oxidative damage of cells of the artery wall can contribute to the progression of atherosclerotic lesions. For instance, oxidation-induced injury to endothelial cells may increase the likelihood of plaque rupture and clot formation.

3.3 Antioxidative constituents of olive oil and LDL oxidation

3.3.1 Vitamin E (-Tocopherol)

Oxidative injury is assumed to play a crucial role in the development of several chronic diseases, eg. coronary heart disease (CHD) and cancer, and the possibility that dietary antioxidants may protect against LDL oxidation and oxidative injury has received growing attention in the past few years.

Since the 1980s several epidemiologic studies have been carried out to evaluate the relationship between the intake of antioxidants, with main emphasis on vitamin E, and cardiovascular disease. It could be observed, that high-dose vitamin E supplements (>100 IU/d = 67 mg -tocopherol/d) over at least two years significantly lowered the CHD risk (risk reduction 31-65%) (reviewed in Jha et al 95). On the other hand, short-term supplementations as well as low-dose supplementations (< 100 IU/d) had no significant effects on CHD.

By contrast, the majority of randomised intervention trials completed so far showed no significant reduction in cardiovascular disease with vitamin E supplementation (for review see. However, it should be mentioned that these trials had several limitations: They were not specifically designed to assess cardiovascular disease, did not provide data on nonfatal cardiovascular events, the treatment duration was insufficient and they used suboptimal vitamin E doses. It can be expected that the contrast between epidemiological and interventional findings could be resolved after completion of the ongoing large-scale and long-term randomised intervention trials designed specifically to evaluate the effects of antioxidants on cardiovascular disease.

Until now, only the CHAOS (Cambridge Heart Antioxidant Study) has been completed. In this double-blind, placebo-controlled study 2000 patients with established coronary atherosclerosis received either a vitamin E supplementation ( 400 or 800 IU daily) or a placebo for about one year. The -tocopherol treatment led to a substantial reduction of non-fatal myocardial infarction.

However, intervention trials cannot provide sufficient evidence for a causal relationship between the intake of antioxidants and LDL oxidation and atherogenesis. In addition, there are several unresolved questions with respect to antioxidant intervention studies. It can be speculated that a study duration of only a few years may be inadequate, because - based on the hypothesis that the anti-atherogenic effects of antioxidants are mediated by the inhibition of LDL oxidation and that LDL oxidation is one of the earliest steps in atherogenesis - there might be no demonstrable effect on clinical events for some time. It is not known how long it takes for a new fatty streak to become a clinically relevant lesion, and thus it may be sensible to investigate the effects of antioxidant supplementation over a longer period, possibly over twenty or more years.

In addition to epidemiological observations and intervention studies the effects of antioxidants on oxidative modifications of LDL have been investigated directly with controlled experimental studies. As already pointed out (see pt. 2.1), -tocopherol is the most important antioxidant in LDL particles, and the main research interest has focused on the relationships between vitamin E and LDL oxidation.

Several studies with healthy subjects whose diet was supplemented with vitamin E were conducted in order to investigate how increased vitamin E intake would affect the oxidation resistance of LDL. In a study by Esterbauer et al. healthy volunteers were given -tocopherol doses from 150 to 1200 IU for three weeks. The plasma and LDL antioxidant status and the oxidation resistance of LDL (lag time and oxidation rate to copper-induced LDL oxidation) were measured before, during and after supplementation. It could be demonstrated that the -tocopherol supplementation led to an increase in the amount of -tocopherol in the plasma and in the LDL particles. During the supplementation period the isolated LDL showed in vitro a higher oxidation resistance compared to the initial value determined before the study. The degree of oxidation resistance correlated closely with the dosage of vitamin E. One week after the supplements were stopped the oxidation resistance had returned to the initial basal values. Similar results were obtained in comparable studies.

Furthermore, it could be observed that the oxidation resistance of LDL was also increased in non-vitamin E supplemented men at high vitamin E plasma levels as compared to subjects with a lower plasma content, suggesting that even an amount of -tocopherol that can be taken up only with vitamin E rich foods would result in a plasma vitamin E level which decreases the susceptibility of LDL to oxidation.

3.3.2 Phenolic compounds

In addition to its vitamin E content olive oil contains a variety of further minor components that are responsible for its unique flavour and taste. This is related to the fact that olive oil is the only vegetable oil obtained from whole fruits rather than from seeds, which allows it to retain all the organoleptic properties of olives.

Among these minor constituents (adding up to 2-3% of unrefined oil), phenolic compounds are of outstanding importance. The amount of phenolic compounds – in a range from 50 to 800 mg/kg oil - depends on factors such as climate, cultivation, stage of maturation. Furthermore, unrefined olive oil has a much higher content of phenolic compounds than refined oils.

Phenolic compounds in foods include simple phenols and phenolic acids, eg. hydroxycinnamic acid derivatives, and flavonoids. These phenolic classes contain numerous compounds that are widespread in plant foods. Phenolic compounds influence the quality, palatability, and stability of foods by acting as flavourants, colourants, and antioxidants. The presence of conjugated ring structures and hydroxyl groups allows phenolics to actively scavenge and detoxicate free radicals, and they can sequester metal ions through liganding. They have been shown to inhibit lipid oxidation in biological systems. Additionally, phenolic compounds are able to inhibit the activities of the pro-oxidant enzymes lipoxygenase and cyclooxygenase. In numerous animal models phenolic compounds exhibit further pharmacological effects, such as anticarcinogenic, antiinflammatory, and antihaemorrhagic effects (inhibit lipid oxidation in biological systems.

Until now, not all of the numerous phenolic compounds in olive oil have been chemically identified. Some of the major compounds with potent antioxidative properties are dihydroxyphenylethanol (DHPE), hydroxytyrosol, caffeic acid and oleuropein. The concentration of DHPE in olive oil is considered to be a marker of its quality and has been shown to be a good index of its stability. Various phenolic components have been tested for their ability to prevent the accumulation of peroxides in the oil, but only little is known about their potential biological activities.

In a recent study isolated LDL particles obtained from healthy subjects were incubated with different olive oil polyphenols, leading to an inhibition of LDL oxidation, measured as effects on various parameters of lipid oxidation, such as formation of lipid peroxides and conjugated dienes. An animal experiment in rats which were fed with different MUFA-rich oils with standardised vitamin E contents demonstrated that the LDL particles from the olive oil-fed rats were more resistant to oxidative modification than those of the triolein fed-rats, suggesting that the phenolic compounds present in the olive oil may be responsible for the increased resistance of LDL to oxidation. These results could be confirmed with comparable experiments in rabbits.

The findings available so far are encouraging and indicate that phenolic compounds present in olive oil, particularly in unrefined oils, may contribute to the prevention of processes, such as lipoprotein oxidation, that are considered to be relevant in promoting atherogenesis. However, this evidence is only based on in vitro and animal studies, and much more research work, especially controlled human dietary studies, has to be done to evaluate the possible beneficial effects of phenolic compounds in olive oil in vivo.

As described above, phenolic compounds are not only found in olive oil, but are widespread in vegetable foods. In particular, flavonoids as a large group of potent polyphenolic antioxidants are naturally present in vegetables, fruits, and in beverages such as tea and wine. Some of the major food flavonoids are quercetin, kaempferol, myricetin, and luteolin. In the Seven Countries Study as well as in the Zutphen Elderly Study the average intake of flavonoids was inversely and independently correlated with mortality from CHD. It can be assumed that the flavonoids may be partially responsible for the positive effects of fruit and vegetable consumption on CHD. Nevertheless, more supportive epidemiological data and more experimental studies on the mechanisms involved are needed before firm conclusions on the protective effects of flavonoids on CHD risk can be drawn.

4. Summary and conclusions

There is extensive evidence that oxidative modifications of LDL play a crucial and causative role in the pathogenesis of atherosclerosis. Oxidation of LDL begins with peroxidation of the PUFA in the LDL particle. Thus, LDL fatty acid composition undoubtedly contributes to the process of LDL oxidation. The fatty acid composition of LDL is influenced by dietary fatty acids, and, as a consequence, the amount and type of fat in the diet also affects the susceptibility of LDL to oxidative damage. Diets rich in MUFA render LDL more resistant to oxidative modifications compared with diets rich in linoleic acid, due to an enrichment of LDL particles with oleic acid instead of linoleic acid. In addition, the fatty acid composition of cell membranes is diet-dependent, and MUFA-rich diets also lead to a higher MUFA content of cell membranes, and therefore to a higher cellular resistance to oxidative damage.

A second important dietary factor which provides protection against oxidative stress are antioxidants mainly derived from plant foods, such as vitamin E, -carotene, vitamin C, flavonoids and other phenolic compounds. Recent studies indicate that not only -tocopherol, but also several phenolic compounds in olive oil may inhibit also LDL oxidation, leading to a reduced risk of atherosclerosis. Although these results are promising, many questions remain unanswered and more research work is needed to investigate the exact mechanisms of actions of the phenolic compounds and to confirm the protective effects in vivo.

Up to now, attention to the benefits of the Mediterranean diet has been focused on its favourable effects on hyperlipidaemia and other established cardiovascular risk factors, due to its low content of SAFA and its high content of MUFA as well as of complex carbohydrates and dietary fibre. Currently evidence is provided that additional components typically present in abundance in the Mediterranean diet, namely antioxidants derived from vegetables, fruit and beverages, but also from olive oil, might contribute to the protection against CHD and, probably, cancer and other diseases. Furthermore, the high intake of MUFA in the Mediterranean diet, due to the olive oil consumption may combine the advantages of lowering cholesterol levels and decreasing LDL and cell susceptibility to oxidation.