Oxidative stress biomarkers and the role of lipid oxidation in cardiovascular disease

A general problem in studing oxidative stress in biological systems and in the evaluation of the effects of AO in vivo, i.e. in patients, concerns the strategies for reliable measurements of oxidative parameters. Several markers and methods have been used for the assessment of the generation of oxidation products (markers of oxidation) of various biomolecules in vitro, in ex vivo systems and in vivo. The in vitro measurements, although quite effective in the assessment of the antioxidant potential of a given compound in a controlled system, are not greatly predictive of the possible activities in vivo. It should also be added that since different antioxidants act through different mechanisms and different oxidative substrates may yield different types of products, assays should be aimed at measuring various oxidative products using different substrates (Halliwell, 1995).

Ex vivo measurements are often also used in connection with the evaluation of oxidative processes in pathological states, but again in some cases some artefactual modification may occur during the collection of the samples (e.g. cells, plasma preparation). The in vivo assays are made directly on samples collected without any manipulation, e.g. urines, but although they reflect processes occurring in the organism, they do not imitate the site(s) of these events. Measurement of isoprostanes, non-enzymatically produced oxidative metabolites of arachidonic acid, is considered, with the above-mentioned limitations, a valid indicator (biomarker) of lipid peroxidation. Increments of this marker have been observed in conditions in which enhanced lipid peroxidation may be predicted (in people who smoke, or have diabetes or hyper-cholesterolemia) (Pratico et al., 2001).

Concerning specifically the measurements of lipid peroxidation markers, ideal assays should have the following features (Halliwell, 1999):

In vitro (susceptibility of substrates to oxidation under controlled conditions) Substrates/markers:

Substrates = lipids: fats, oils, lipids in membranes and lipoproteins Markers: TBARS, conjugated dienes, lipid peroxides, oxygen uptake, fall of PUFA and vitamin E, isoprostanesSubstrates = Proteins: ±SH groups, amino-acid residues, etc.

Markers: electrophoretic mobility. adduct formation, carbonyl content, etc.

A. Substrates = Nucleic acids: DNA bases, deooxyguanosine

Markers: mass spectrometry (MS) of high performance liquid chromatography

(HPLC) of modified bases, electrophoresis of damaged 5'-GG-3' doublets, `comet

assay’ for DNA bases.

A. Substrates = sugars: ribose and deoxyribose in DNA

Markers: oxidation products

Ex vivo (evaluations on samples, e.g. blood, or cells, obtained from animals/humans without further treatments, except those made in vivo)

Antioxidant/oxidant status, antioxidant capacity, antioxidant levels and activities of AO enzymes, levels of negatively charged LDL (a fraction with different chromatographic behaviour in HPLC systems), antibodies against modified LDL, ex vivo assays of DNA oxidation, ex vivo assays of protein oxidation

In vivo (determinations in biological samples collected non-invasively)

Lipids/lipoprotein oxidation): urinary levels of isoprostanes, hydrocarbons in expired air

DNA damage: urinary levels of modified DNA bases

Quantitation of major products of the peroxidation process.Low coefficients of variation of analyses.No interference by other biomolecules.Methods: Chemically reliable (e.g. mass spectroscopy, MS or high performance liquid chromatography, HPLC) or validated.Possibly not confounded by oxidized lipids ingested with the diet.Assess steady-state levels of peroxidation products and total rates of ongoing lipid peroxidation.Parameters measured should be stable on storage and not produced artefactually.

Measurement of valid biomarkers of oxidative processes should be promoted before conducting studies on the effects of antioxidants in human studies (Mayne, 2003).

A vast literature over the past two decades has been produced, devoted to the possible involvement of oxidative stress and of ROS-derived products in various

pathological states. To some extent the published information is speculative, owing to major conceptual and analytical difficulties in the assessment of oxidative processes in vivo and in the evaluation of their real contribution to pathologies. Uncontrolled free radical production has indeed been advocated as a factor in a number of diseases: atherosclerosis, arthritis, diabetes, pulmonary diseases, cancers, Alzheimer’s disease, lateral amyothrophic sclerosis, neuritis, hepatitis and senile cataracts, but most of the attention has been devoted to the possible involvement of lipid/lipoprotein oxidation in atherogenesis and in cardiovascular disease, CVD (Steinberg, 1997, Berliner and Heinecke, 1996).

As to the issue of oxidative stress and atherosclerotic CVD, certainly rather convincing evidence has been produced in in vitro studies, showing that LDL that have been exposed to oxidative stress (oxLDL) through various mechanisms (exposure to chemicals, to physical factors or to cellular processes) are highly atherogenic. Atherogenesis induced by oxLDL has been shown to activate a sequence of events, involving several types of circulating cells (monocytes, platelets) and cellular components (e.g. smooth muscle cells, SMC) and present within the vessel walls (macrophages).

There are, however, still several issues to be defined. First, LDL are rather etherogenous molecular complexes, with significant individual differences in macro- and micro-components, including a number of lipophilic compounds that are associated to them, and it is difficult to identify and quantify all the products generated after exposure to oxidative stress, which may contribute to atherogenesis. Second, in vitro LDL oxidation is generally carried out in conditions that maximize the oxidative process, e.g. removal or depletion of hydrophilic and amphiphilic antioxidant compounds that are normally present in plasma, exposure to strong pro-oxidant factors that are difficult to compare quantitatively with in vivo free radical generating systems. Therefore the final products, i.e. oxidized LDL, cannot be easily compared with oxLDL possibly

generated in vivo. In vitro studies have also convincingly shown that several types of antioxidants are able to prevent LDL oxidation induced by various agents, but the use of AO, mainly in the form of supplements, in clinical studies has not shown significant protection against CVD. Although some of these issues are considered in detail in other posts, it is worth underlining some the strong and the weak points in the overall relationships between oxidative stress and CVD.

There is evidence that lipoproteins (LP) with some of the general features of oxLP produced in vitro, evaluated with the use of the typical markers of oxidation (see further), are present in atherosclerotic plaques. On the other side, it is not completely clear whether oxLDL are generated within the vessel wall exposed to high oxygen fluxes, from previously accumulated particles, or whether they are deposited in the vessel walls after being produced in the circulation, i.e. whether the presence of oxLDL is a secondary or an associated process, rather than a causative event.

For monocytes, again, the accumulated reactive material could be produced in a secondary process. In addition, the recognition by antibodies has several limitations: poor characterization of the oxLDL used as antigens for the preparation of the antibody, and eventual (epitope) differences between the artificially produced oxLDL and those generated in vivo. In addition there may be some lack of specificity and poor quantitative responses in the reaction.

Some of the previously mentioned limitations may apply to the presence of autoantibodies against oxLDL in sera of atherosclerotic patients. There is also some evidence that antioxidant consumption may slow the progression of the disease. This, however, is a rather controversial aspect. In essence, the difficulties in the evaluation of the outcome of the studies concern the form and doses of administration of the AO and in the selection of the people to be treated.

In addition to the role of oxidized LDL in the atherogenetic process, a number of studies have been devoted to assess the involvement of oxidative stress in several CV conditions and functions, as discussed in the following reviews: endothelial functions (Cai and Harrison, 2000; Lum and Roebuck, 2001; Matsuoka, 2001; Terada, 2002), neutrophil activation (Kaminski et al., 2002), macrophage involvement (Jessup et al., 2002), smooth muscle cell function (Bomzon and Ljubuncic, 2001), vascular ageing (Yu and Chung, 2001), congestive heart failure (Mak and Newton, 2001), arterial hypertension (Zalba et al., 2001) and diabetes (Bayraktutan, 2002). However, as already discussed, most of the evidence is derived from in vitro models, animal studies or ex vivo situations, i.e. in somewhat artefactual conditions where some of the processes may be amplified. It is therefore rather problematic to assess and quantify the actual role and relevance of oxidative stress in CVD.

Based on all the direct and indirect evidence in support of the hypothesis that free radical-mediated processes and specific products arising from them may play a role in CVD, great interest has been devoted to the possible protective effects of AO in the diet, or as pure compounds, on biomarkers and on clinical endpoints in population studies.

A vast number of studies have been carried out since 1990 on various aspects of the issue of AO protection: they range from epidemiological investigations to controlled trials and have involved a great number of participants. In reality, early observations on the relationships between dietary antioxidant vitamins and disease date back to the 1930s (Seventh-Day Adventists) and the 1950s (Mormons) (reported by Enstrom et al., 1992), and the whole area has been recently reviewed systematically (Asplund, 2002). This review is based on the following inclusion criteria: human studies only, published after 1989, reporting only original data, obtained in case-control, cohort or randomized controlled trials; related to AO vitamins only; mainly reporting on morbidity and mortality of clinically meaningful manifestations of ischaemic heart disease or stroke. The following contexts have been considered: primary prevention of various endpoints (ischaemic heart disease, stroke or combined cardiovascular events), the effects on intermediary endpoints (e.g. blood lipids and blood pressure), studies on secondary prevention in patients with manifest CV disease.

The main conclusions are: in observational studies (case-control or cohort design) people with high intake of AO vitamins by regular diet or as food supplements generally have a lower risk of myocardial infarction and stroke than low consumers. In randomized controlled trials, however, AO vitamins as food supplements have no beneficial effects in the primary prevention of myocardial infarction and stroke, with some report also of adverse events. In addition, in contrast with the initial favourable reports on AO in the secondary prevention of CVD, recent reports apparently failed to show beneficial effects. Some of the negative findings on the effects of AO vitamins, however, may be attributed to pitfalls in the design of the experiments: inadequate characterization of subjects under investigation in terms of ongoing oxidative stress, inappropriate formulations and dosages, especially in comparison with the situation in natural sources: single compounds rather than mixtures, concentrations too high (possibly pro-oxidant) or too low (ineffective), administered as a bolus (capsules or tablets) rather than in the context of foods (better absorption, protection vs. oxidation of dietary components, balance between various ingredients with maintenance of natural structural and functional relationships).

In summary, some relationship exists between intakes/plasma levels of some risk factor for vitamin C (reduction of cholesterol and blood pressure with high intakes/levels), for vitamin E (reduced platelet adhesiveness with high intakes) and for multivitamin supplementation (reduced platelet aggregation), but correlations are generally weak and the area has not been investigated in detail. For case-control studies there is some support for low plasma concentrations of beta-carotene, and possibly of vitamin E, being linked to increased risk of myocardial infarction. The same does not apply to vitamin C. Altogether, owing to rapid changes in plasma AO vitamins during CV events, the data must be interpreted with caution. Concerning cohort studies, people with high intakes of AO vitamins (regular food or food supplements) have a modest reduction of risk for CV events. Plasma levels of carotene and vitamin C are stronger predictors of future CV events than dietary intakes.

Primary prevention in healthy subjects: 1 out of 8 studies has shown protective effects with beta-carotene vs. retinol on a limited number (1203) of subjects. 1 study with beta-carotene show enhanced risk of lung cancer in smokersSecondary prevention of CVD in patients with manifestations of the diseaseOut of 14 studiesIn 5, reduction of CV eventsIn 9, no effectIn 1 increase of CV events (beta-carotene).

The effects of dietary supplements of AO in the primary and secondary preventions of CVD in randomized controlled trials are summarized above. The general conclusions from these studies are as follows:

People affected by ischaemic heart disease and stroke, and populations with high occurrence of CVD often have low intakes/plasma levels of AO vitamins (causal or unfavourable lifestyle factors?).In case-control or cohort studies, people with high intakes of AO vitamins (food or supplements) have a low risk of myocardial infarction and stroke.In randomized controlled trials, AO vitamins as supplements have no beneficial effect on risk for MI or stroke (not recommendable for prevention).Some support from observational studies that low intakes of fresh fruits/ vegetables may confer a high risk for CVD.

Diets, however, especially those rich in fruits and vegetables, contain several factors or mechanisms other than AO or AO other than vitamins, exerting protective effects on various systems (Halliwell, 1999). The issue of the effects of bioactive compounds in foods and their role in the prevention of CV disease is therefore quite complex, since a large number of potentially health beneficial substances have been described (Kris-Etherton et al., 2002).

Flavonoids in particular have been investigated in relation to possible health benefits (Ross and Kasum, 2002), owing to their potential antioxidant and free-radical scavenging activities observed in vitro. Human feeding studies have shown that their absorption and bioavailability are higher than originally believed, but their overall function in vivo has yet to be clarified, whether antioxidant, anti-inflammatory, enzyme inhibitor, enzyme inducer, inhibitor of cell division, or some other function (Rice-Evans, 2001). Epidemiological studies exploring the role of flavonoids in human health have been inconclusive: some studies support a protective effect of their consumption on CVD and cancer, other studies demonstrate no effect and a few studies suggest potential harm (Ross and Kasum, 2002). Additional selected classes of bioactive compounds with antioxidant and other types of potentially healthful activities are the large groups of phenolics that are present in edible fluids — obtained from fruits of plants exposed to stressful conditions, such as grapes and olives ­ which, since the beginning of recorded history, have been part of the diet of populations living in certain areas, such as the Mediterranean basin, i.e. wine and olive oil. A vast literature is available on the properties of these compounds (German and Walzem, 2000; Visioli et al., 2002), although the impact of their consumption on health through the diet has not yet been fully assessed.

Compounds           Examples                        Sources

Flavonoids

Flavones             Apigenin, luteolin             Parsley, thyme, celery

Flavonols            Quercetin, myricetin         Onions, broccoli, apples, cherries,

berries, tea

Flavanones         Naringenin, hesperedin     Cirtus foods, prunes

Catechins            Epicatechin, gallocatechin Tea, apples, cocoa

Anthocyanidins    Pelargonin, malvadin        Cherries, grapes

Isoflavones         Genistein, daidzein           Soya beans, legumes

Phytoestrogens

Lignans,                    Enterolatone, coumestrolk         Flaxseed oil, clover
coumestran

Resveratrol                                               Grapes, red wine, peanuts

Lycopene                                                 Tomatoes, tomato products

Organosulphur              Allicin, diallyl sulphide                 Garlic, onion, leek
compounds

Isothiocyanates            Phenethyl benzyl,                       Cruciferous vegetables
sulphoranes

Monoterpenes        d-Limonene, perillic acid   Essential oils of citrus fruit, rice

bran oil, cherries, mint

Plant sterols          Sitostanol, stigmasterol    Tall oil, soybean oil, rice bran oil

Olive oil                Hydroxytyrosol, oleuropein Olives, virgin olive oil

Oxidative Stress and Cardiovascular Disease

Interest in the role of uncontrolled oxidative processes in response to oxidative stress in the onset and progression of disease, and as contributing factors in organ and system dysfunctions related to ageing, is underlined by the volume of research work and reviews devoted to this area since 1990. These studies cover diverse fields, from the chemistry of oxidative reactions of susceptible substrates in a test-tube, to the occurrence and detection of oxidative processes in living systems, and the relevance of these events in pathophysiology. Although these processes are considered factors in altering or modulating biological functions, it is difficult to reach reliable quantitative estimates of their contribution to multifactorial events such as diseases. This review discusses the links between oxidative stress, with special relevance to lipid oxidation, and cardiovascular functions and disease.

Oxygen is a key factor in energy metabolism in the animal kingdom since it participates in the major energy-releasing reactions, such as substrate utilization and formation of high-energy compounds. Oxygen is also directly involved in oxidative reactions catalysed by several enzymes, such as the oxygenases, resulting in the formation of a large number of bioactive products from lipids (e.g. the eicosanoids), or the hydroxylating enzymes involved in the formation of neurotransmitters from amino acids (e.g. the catecholamines), or in the metabolism of xenobiotics (e.g. hydroxylations or other types of oxygen-dependent reactions).

Under aerobic conditions, in addition to the participation of oxygen to redox reactions, highly reactive unstable and short-lived chemical entities, the so-called reactive oxygen species (ROS) are produced. ROS can be considered as the outcome of oxidative stress, a condition that, in addition to resulting from endogenous processes, can also be induced by a wide range of environmental factors, including UV exposure, pathogen invasion (hypersensitive reactions) and tissue reperfusion after oxygen deprivation. The term ROS, generally referring to the superoxide anion radical (O2 ), H2O2, and the hydroxylradical (OH’—), includes also hypochlorous acid (HOCl), chloramines, singlet oxygen and peroxyradicals. ROS are highly reactive and interact with major biomolecules: they bind to proteins, break DNA strands, react with vital cellular components, and alter structural lipids in biomembranes by attacking double bonds of polyunsaturated fatty acids (PUFA) in membrane phospholipids.

The oxidative modification of proteins and lipids is commonly defined as protein oxidation and lipid peroxidation, respectively. Free iron (Fe3+) and, in general, bivalent metal ions accelerate the decomposition of lipid hydroperoxydes (LOOH—) into compounds such as alkoxyl and peroxyl radicals, 4-hydroxynonenal (4-HNE) and malonylaldehyde (MDA). These compounds, in addition to being end products of peroxidative decomposition of polyenoic fatty acids in the lipid peroxidation process, are also reactive compounds that in turn can continue, amplify and extend the process beyond the initial oxidative event by oxidizing cellular thiol groups.

Lipid peroxidation can spread throughout the cell, and disrupt lipid membranes, even at sites not immediately associated with those where ROS have originated. Lipid peroxidation may be considered a major alteration in ROS-induced cellular derangements for various reasons. Interactions between ROS and cellular structures mainly take place at interfaces between water and cells. Cell membranes, the most obvious site for these interactions, are largely composed of structural lipids. The route oxygen takes from the atmosphere to animal cells and tissues involves a sequence of highly expanded cellular membranes and of membrane-bound particles: red blood cells (highly enriched in oxygen), other types of circulating cells, lipoproteins of different size and composition and endothelial cells.

The area of the surface of red blood cell membranes is in the order of about 0.5 m2/mL blood and that the global surface of the lipoprotein particles is over 1 m2/mL blood, an enormous value that suggests a high surface of exposure to lipid peroxidation. Of particular relevance in this respect is the role of erythrocytes, loaded with `quanta’ of oxygen, and endowed with highly expanded membranes (the peculiar shape of these cells results in a very high surface/volume ratio) enriched in PUFA in structural lipids. Erythrocytes in the arteries continually `bombard’ endothelial cells with `quanta’ of oxygen, and create a persisting highly oxygenated `background’ condition for the major components of vessel walls. In addition, the endothelium interacts with subpopulations of polymorpho-nuclear neutrophils (PMN) and leuckocytes that, upon activation, release ROS.

Oxidative Stress and Cardiovascular Disease

This post will focus on the potential roles of fat-soluble nutrients and fat-soluble antioxidants in preventing cardiovascular disease (CVD). Two fat-soluble vitamins will be discussed in detail, i.e. vitamin E and vitamin D. Vitamin E (tocopherols and tocotrienols) is generally considered an antioxidant nutrient, although it may have important functions unrelated to its antioxidant functions (as discussed below). Antioxidant nutrients function by preventing damage to biological systems caused by reactive oxygen species (ROS) and/or reactive nitrogen oxide species (RNOS). Vitamin D (calciferols) is not a true vitamin since it is not required in our diet, can be produced in skin tissue, and is generally not present in plants. Vitamin D is, perhaps, best described as a steroid hormone precursor. Although vitamin D may function as a membrane antioxidant under in vitro conditions (Wiseman, 1993), its primary biological role is to maintain plasma calcium and phosphorus homeostasis.

The additional fat-soluble antioxidant nutrient reviewed here will be coenzyme Q10 (ubiquinone or CoQ10), which has strong antioxidant properties. Vitamin E and CoQ10 can protect lipid±protein complexes, such as biological membranes and lipoproteins, from lipid peroxidation. During lipid peroxidation, highly reactive lipid hydroperoxides, peroxyl radicals and reactive aldehydes, such as malondialdehyde (MDA) and 4-hydroxynonenol (4-HNE), are generated. The peroxyl radicals support chain reactions that can rapidly damage oils, biological membranes or lipoproteins containing polyunsaturated fatty acids (PUFA).

The literature reviewed below strongly suggests that oxidative stress plays a key role in the etiology of cardiovascular disease. Oxidative stress is a physiological condition in which pro-oxidant factors outweigh antioxidant defences. Accordingly, the role of oxidative stress in promoting cardiovascular disease and the roles of fat-soluble antioxidant nutrients in potentially protecting from this disease process will be discussed in some detail. Oxidative stress is likely to occur during inflammatory processes, during exercise and from cigarette smoking. The evidence presented below also suggests that vitamin D plays an important and significant role in preventing cardiovascular disease but it is very unlikely that this effect is related to its potential role as an antioxidant.

Owing to the enormous worldwide impact of cardiovascular disease it must be emphasized that even very modest reductions in risk factors, brought about by the appropriate design and use of functional foods, can have very important health related and economic significance. Statistics from the American Heart Association (see http://www.americanheart .org /statistics/03cardio.html) indicate the enormous impact of CVD. Over 61 million Americans have one or more types of CVD. CVD causes more mortality each year than the next seven leading causes of death combined and the estimated cost of cardiovascular diseases and stroke in the United States in 2003 was $352 billion. In developed countries, childhood obesity has reached epidemic proportions and this will certainly translate into a dramatic increase in type 2 diabetes which is charac­terized by elevated levels of triglycerides, LDL-C (low-density lipoprotein­ cholesterol) and decreased levels of HDL-C (high-density lipoprotein­ cholesterol), i.e. a shift towards a highly atherogenic lipid profile. Moreover, the World Health Organization (see http://www.who .int/ncd/cvd ) makes a very convincing argument that CVD impact is not just limited to Westernized countries but will reach epidemic proportions in developing countries as well because of demographic and lifestyle changes. It has been estimated that by the year 2020, CVD will be the number one cause of deaths in the world.

Both lipid-soluble and water-soluble antioxidants present in blood may be important in preventing cardiovascular disease owing to their ability to prevent the oxidation of lipid—protein complexes called lipoproteins. Lipoproteins are extremely important in cardiovascular disease since we know with certainty that high levels of LDL-C cause atherosclerosis, which is the underlying cause of most cardiovascular disease. In contrast, high levels of HDL-C are a negative risk factor for CVD. Atherosclerosis is the gradual build-up of `plaque’ in the arterial wall. LDL-C is the major source of the lipids occurring in these plaques.

There is now considerable evidence that LDL lipids (primarily cholesteryl esters) make their way into plaques by cells in the arterial wall called macrophages. These macrophages take up so much LDL that they become `foamy’ in appearance and are, therefore, called `foam cells.’ This is the very first step (called fatty streak formation) in atherosclerosis and this process begins in childhood. It is surprising, however, that LDL incubated with macrophages does not transform into foam cells. After LDL is oxidized (oxLDL) it will, however, cause macrophages to transform into foam cells. Macrophages have receptors for native LDL but the expression of these receptors is down-regulated by the accumulation of intracellular cholesterol. Unlike native LDL, chemically modified forms of LDL can be taken up by scavenger receptors whose expression is not down-regulated by the accumulation of intracellular cholesterol.

LDL is the primary plasma carrier for both vitamin E and CoQ10, both of which act as antioxidants in LDL by inhibiting lipid peroxidation of lipids containing polyunsaturated fatty acid moieties. Work by Jessup et al. (1990) indicates that most of the endogenous vitamin E in LDL must be oxidized before it is converted into a `high uptake’ form of oxLDL capable of transforming macrophages into foam cells. Since antioxidants, such as vitamin E, prevent the oxidation of LDL (Jessup et al., 1990) it is logical to suggest that antioxidants could prevent foam cell formation and thereby retard the process of atherosclerosis. This suggestion is called the `oxidative modification hypothesis.’ Although most in vitro experiments support this view, not all evidence is supportive (Asmis and Jelk, 2000).

Whether or not oxLDL formation occurs in vivo and what the mechanism (s) might be for this oxidation are still open issues (Chisolm and Steinberg, 2000). Despite intensive efforts, there is little evidence for the existence of oxLDL in fresh human plasma. This has led to the hypothesis that LDL could be oxidized in the subendothelial space of arteries rather than in plasma. It is significant, therefore, that LDL isolated from human aortic atherosclerotic intima has extremely high levels of 3-nitrotyrosine (Leeuwenburgh et al., 1997). Although the origin of this 3-nitrotyrosine is not clear, it is probably due to the reaction of peroxynitrite (ONOO) with tyrosine residues in apoB100 (the primary protein component of LDL). The addition of ONOO to LDL or bovine serum albumin in vitro certainly gives rise to 3-nitrotyrosine. Furthermore, LDL-treated ONOO undergoes lipid peroxidation accompanied by the oxidation of alpha­tocopherol to alpha-tocopheryl quinone and is converted to a form recognized by macrophage scavenger receptors (Graham et al., 1993; Hogg et al., 1993).

If oxLDL were the source of lipids in atherosclerotic plaques one might expect that these lipids would have a very low content of vitamin E. Paradoxically, Suarna et al. (1995) have found that human atherosclerotic plaques contain relatively large amounts of alpha-tocopherol and ascorbate (a water-soluble antioxidant). Foam cells are not likely, therefore, to be formed by the uptake of large amounts of oxLDL with very low levels of endogenous tocopherol. Nevertheless, Suarna et al. (1995) also found that plaque contains large amounts of oxidized lipids and a significant level of alpha-tocopheryl quinone, an oxidation product of alpha-tocopherol. These data support the view that oxidative stress is an important factor in atherosclerosis but indicate that oxidized lipids in atherosclerotic plaques may not be derived from oxLDL.

Risk factors for coronary heart disease (CHD): the role of oxidative stress

Endothelial dysfunction and intimal±media thickness are considered the early steps in atherosclerosis. Rassel Ross has modified atherosclerosis patho­genetical theories because numerous pathophysiological observations in humans and animals have led to the formulation of the response-to-injury hypothesis of atherosclerosis. Each characteristic lesion of atherosclerosis represents a different stage in a chronic inflammatory process in the artery. The lesions of atherosclerosis represent a series of highly specific cellular and molecular responses that can be described as an inflammatory disease. Possible causes of endothelial dysfunction leading to atherosclerosis include hypercholesterol­aemia, hypertension, diabetes mellitus, cigarette smoking, elevated plasma homocysteine concentrations, infectious microrganisms and ageing. Framingham’s studies have shown how each factor and combination of these factors are associated with atherosclerotic diseases. All these factors can be associated with oxidative stress.10±15 The beneficial effect of alpha-tocopherol and ascorbic acid is mediated by their antioxidant actions in preventing atherosclerosis. On the other hand, the effect of alpha-tocopherol could also be mediated by its antiplatelet and anti-coagulant actions, which would prevent the thrombotic consequences of atherosclerosis.

Cigarette smoking, hypertension, diabetes mellitus, genetic alterations, elevated plasma homocysteine concentrations, infectious microorganisms, such as herpes viruses or Chlamidia pneumoniae, have proinflammatory actions, increasing the formation of hydrogen peroxide and free radicals such as superoxide anion and hydroxyl radicals in plasma. These substances reduce the formation of nitric oxide (NO) by endothelium. Nitric oxide is a free radical with an unpaired electron in its highest orbital. This is why it behaves as a potential antioxidant agent by virtue of its ability to reduce other molecules. In vitro experiments support this concept inasmuch as NO is able to inhibit lipid peroxidation. However, NO is rapidly inactivated by the peroxide anion (O2?) to form peroxynitrite (NOO which is a potent oxidant. Therefore, in the presence of O2?, NO behaves as a potent pro-oxidant. This is the mechanism that accounts for the low-density lipoprotein (LDL) oxidation that occurs when NO and O2? are simultaneously present in the medium. As NO and O2? are simultaneously released by cells, such as endothelial cells, the balance between these two radicals is crucial in understanding the net effect of NO on lipid peroxidation. Thus an excess of NO will favour lipid peroxidation inhibition, while an excess of O2? or equimolar concentrations of NO and O2? will induce lipid peroxidation. Modulation of this balance may have important clinical implications, particularly in the atherosclerotic process, in which oxidative stress seems to play a pivotal role in the onset and progression of vascular lesions.

Several studies have strongly suggested that enhanced oxidative stress may represent an important trigger for atherogenesis elicited by angiotensin II (Ag II). Free radical formation mediates some of the effects of hypertension. Angiotensin II concentrations are often elevated in patients with hypertension and it is a potent vasoconstrictor. It also increases smooth-muscle hypertrophy and lipoxygenase activity, which, in turn, can increase inflammation and the oxidation of LDL.

Grienling et al. examined the effect of Ag II on superoxide anion (O2?) production by smooth muscle cells and demonstrated that 4 to 6 hour exposure of these cells to Ag II elicited enhanced production of O2?. This effect was mediated by NADH and NADPH oxidase activation probably via intracellular mobilization of fatty acids such as arachidonic acid. Experimental studies in animals demonstrated that Ag II infusion enhanced simultaneously blood pressure and vascular production of O2?; this last effect was dependent upon NADH/NADPH oxidase, further suggesting the role of this pathway in Ag II-mediated O2? production. These findings have important pathophysiological implications owing to the effect of O2? on vascular motility.

The oxidative stress may have a role in hypertensive patients, in whom a reduced vasodilating response to acetylcholine has been demonstrated. Thus, in patients with hypertension, the administration of the antioxidant vitamin C has been able to restore acetylcholine-induced vasorelaxation, suggesting a role for oxygen free radicals in inducing vascular dysfunction in patients with hypertension. Cigarette smoke contains large amounts of free radicals which may degrade nitric oxide release from the endothelium and also produce highly reactive intermediates resulting in endothelial injury. Antioxidants such as vitamin E can also reduce free-radical formation by modified LDL.

Blood analysis of lipid peroxides or measurement of urinary excretion of isoprostanes has provided evidence that oxidative stress is enhanced in patients with diabetes. The impact of these data in the context of atherosclerosis progression is still unclear, but there is some evidence supporting a role for oxidative stress in contributing to deteriorating vascular disease. For instance, an important finding is the demonstration that endothelium-dependent vasodilation is reduced in patients with diabetes and that vitamin C is able to prevent it, so indicating a role for oxygen free radicals in reducing vasodilatory property of endothelium. Oxidative stress could also contribute to worse metabolic disturbance by interfering with glycaemic control. Thus it has been demonstrated that, in diabetes, oxidative stress impairs insulin activity and antioxidants prevent it. That hyperglycaemia is a risk for enhanced oxidative stress has been further corroborated by a study in patients with type II diabetes, in whom an increased urinary excretion of PGF2m-III, which derives from arachidonic acid and interaction with oxygen free radicals, has been demonstrated. It is of note that a significant reduction of urinary PGF2cx-III was observed when patients underwent a strict glycaemic control, further reinforcing the relationship between hyperglycaemia and oxidative stress.

Hyperglycaemia may enhance oxidative stress and in turn induce vascular damage via several pathways, including the formation of the advanced glycated end products that are proatherogenic and prothrombotic. Furthermore, glucose may alter the balance between free radicals such as O2? and NO in endothelial cells; thus NO exerts its vasodilatory and antioxidant effect unless it is converted to ONOO by interaction with O?. This deleterious effect occurs in endothelial cells exposed to glucose, which, in fact, favours the formation of O2? and in turn promotes oxidation.

Hyper­glycaemia was shown to enhance endothelial O2? generation via activation of cyclooxygenase pathway which is known to generate ROS with a mechanism involving NAD(P)H oxidase. The potential role of this enzyme in inducing oxidative stress has been recently demonstrated by Guzik et al. who studied the expression of NAD(P)H oxidase in the vessel wall of people with and without diabetes.27 They found that, compared with controls, vascular expression of NAD(P)H oxidase submits, p22 phox and p47 phox, were overexpressed in those with diabetes.

There is experimental and clinical evidence indicating that hyper­cholesterolaemia is associated with enhanced oxidative stress. Oxygen free radicals, such as O2', and F2-isoprostanes, have been found elevated in the artery of hypercholesterolaemic animals and in the urine of patients with high serum cholesterol respectively. The relevance of these findings in the context of the pathophysiology of atherosclerosis is unclear, even if there is some evidence that in this setting oxidative stress may have a role in reducing the vasodilation of endothelium. Conversely, these is no evidence yet that the increase of these markers actually represents a marker of progression of atherosclerotic disease.

Two hypotheses can be suggested to explain why hypercholesterolaemia enhances oxidative stress. Cholesterol has been recently shown to activate the metabolism of the arachidonic acid pathway, which in turn seems to be associated with NAD(P)H oxidase activation. This hypothesis has been recently underscored by our group showing that platelet incubation with cholesterol enhanced O2~ production and that inhibition of PLA2 or NADPH oxidase enzymes significantly reduced O2 formation.

The cascade of cholesterol biosynthesis may represent another pathway leading to enhanced oxidative stress. Intracellular metabolism of mevalonate leads, in fact, to the formation of protein isoprenylation, which has a key role in the production of proinflammatory and pro-oxidant cytokines such as tumor necrosis factor alpha. Accordingly, treatment of hypercholesterolaemic patients with an inhibitor of HMG-CoA-reductase was associated with reduced monocytes formation of TNF, suggesting a relationship between cholesterol and intracellular formation of pro-oxidant cytokines.

The association between hypercholesterolaemia and oxidative stress has been further corroborated by an interventional study with statin in people with hypercholesterolaemia in whom simvastatin reduced the urinary excretion of PGF2cx-III, probably by lowering serum cholesterol. However, the existence of a mechanism independent of cholesterol lowering was not investigated. The relationship between hypertriglyceridaemia and oxidative stress has not been fully investigated. We found only one report aimed at analysing whether people with hypertriglyceridaemia had enhanced oxidative stress. Pronai et al. measured scavenging property and O2~ formation by peripheral monocytes of hypertriglyceridaemic patients with and without diabetes. They found a significant positive correlation between O2~ generation and plasma triglycerides and a significant negative correlation between superoxide scavenging property and plasma triglycerides.