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
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