Antioxidant Micronutrients in the Prevention of Age-related Diseases
The role and functions of antioxidant micronutrients such as ascorbate (vitamin C), a-tocopherol (vitamin E) and carotenoids that are provided through the diet in aging and in the prevention of age-related diseases are discussed in the present work. In general, a healthy lifestyle involving regular exercise and avoidance of tobacco or alcohol abuse are the key to the prevention of several age-related diseases including cardiovascular diseases, dementia and cancer. A balanced and regular nutrition with at least five portions of fruit and vegetables per day is a critical constituent of such a healthy lifestyle.
The increase in average life expectancy is resulting in an increasing prevalence of major invalidating illnesses, such as dementia, cardiovascular disease (CVD) and cancer., We need to understand the mechanisms underlying the aging process so that we can help delay it. This is an absolute priority.
The so-called free radical theory of aging might constitute a common link between all the aging theories formulated so far. Reactive species like those of oxygen (reactive oxygen species, ROS), nitrogen (reactive nitrogen species, RNS) and chlorine (reactive chlorine species, RCS) are produced during normal metabolism: a certain amount of ROS/RNS/RCS production is, in fact, necessary for proper health: for example, it helps the body's immune system to kill microorganisms. ROS are mainly produced in mitochondria, and are formed in much larger amounts during activity than during rest. ROS, RNS and RCS are oxidants - i.e., molecules or atoms which can oxidize a substrate and are reduced in this reaction. They are able to damage several key cellular components like membrane lipids, nucleic acids, carbohydrates and proteins, thereby severely disturbing major cellular and organic physiologic functions. This type of damage occurs when the host defenses against oxidants (for this reason called antioxidants) are quantitatively and/or qualitatively unable to counteract the production and effects of oxidants themselves. This state is referred to as oxidative stress, and is known to be associated with a number of disease states in humans. To date, compelling evidence supports the important role played by oxidative and nitrative stresses in the aging process as well as in the pathophysiology of age-related illnesses such as CVD and AD.
The antioxidant defense system provides protection against oxidative reactions and is organized at the levels of prevention, interception and repair. Prevention comprises of strategies that avoid the generation of ROS/RNS/RCS; e.g. diminished light exposure to lower photooxidative reactions or caloric restriction to decrease side reactions in the sequence of the respiratory chain. Proteins, tightly binding metal ions which otherwise catalyze prooxidant reactions, are also involved in prevention.
For interception, a network of antioxidant enzymes, i.e. endogenous- and exogenous molecules, is available to scavenge ROS/RNS/RCS once they are generated. Superoxide dismutase, catalase, glutathione and glutathione-dependent enzymes as well as other sulfur- or selenium-containing proteins and low molecular weight compounds are synthesized by the organism for defense. Small molecular weight compounds with antioxidant properties such as ascorbate (vitamin C), a-tocopherol (vitamin E) and carotenoids, instead, are provided to the organism through the diet - particularly by fruits and vegetables - and are therefore called antioxidant micronutrients. They are an essential component of the antioxidant defense network; an inadequate supply with the diet has been epidemiologically correlated with an increased risk for degenerative diseases. Repair, finally, is the domain of enzymes, which recognize oxidatively damaged molecules and initiate repair, degradation or removal. The interplay of all processes and compounds in the network provides optimal protection of the organism. During aging, and in oxidative stress-related disease states, antioxidant micronutrients are consumed and their levels may fall below normal ranges. Furthermore, age-related biological and often socioeconomic changes induce critical risk factors for malnutrition; surveys indicate that the elderly are particularly at risk for marginal deficiency of vitamins and trace elements.,, The biological explanations for malnutrition in the elderly include mechanisms related to decreased energy intake, sensory changes, changes of the gastrointestinal tract (oral health problems, digestive changes), the body composition, physical activity, neuropsychological function, as well as to components able to modify the bioavailability of the ingested micronutrients in the aging organism - for instance, use of multiple medications and drug-nutrient interactions.,
Although vitamin deficiency is rarely encountered in developed countries, a suboptimal vitamin status puts aged individuals at high risk for diseases like CVD, dementia, and osteoporosis., The assessment of circulating antioxidants can be used, together with other information, to evaluate conditions of oxidative stress in humans. Rather than absolute antioxidant concentrations, however, it is the antioxidant pattern observed in a specific condition that may be both used to uncover the importance of one compound over another and to determine a particular nutritional need of the subject studied. In the Nun Study, a strong negative association was found between dependence in self-care in 77- to 98-year-old women and plasma levels of lycopene, but not of other carotenoids or vitamin E.
Interestingly, even usual aging (without disease but at risk) and successful aging (low risk and high function) show a specific antioxidant pattern. For example in the former, there exists an inverse correlation between lycopene plasma concentrations and age, with lowest levels among subjects older than 80 years, as was shown in an elderly subpopulation of the Framingham Heart Study. As far as successful aging is concerned, healthy Italian centenarians have significantly lower levels and activities of antioxidant non-enzymatic and enzymatic compounds compared to younger controls, with the exception of plasma levels of vitamins A and E which are significantly higher in centenarians than in younger subjects.,, This observation has been recently confirmed in a larger group of healthy centenarian subjects, and is particularly interesting in light of the role played by these vitamins in protecting against immune and cognitive disturbances - from which our centenarians were in fact completely free.
Neurodegenerative diseases including Alzheimer's Disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS) share in common both aging as a major risk factor and oxidative stress as an important pathophysiological mechanism., Interestingly, an involvement of oxidative stress has been suggested in accelerated aging processes such as progeria and Werner syndromes, which are also associated with neurodegenerative processes. Collectively, there is enough evidence to suggest that oxidative damage plays an important role in brain dysfunction seen in dementias, especially in AD. The potential implication of antioxidant patterns also displays in this disease: while we found high peripheral levels of biomarkers of oxidative damage to DNA, lipids and proteins (Polidori et al, not yet published data) paralleling a dramatic decrease in levels and activities of several enzymatic and non-enzymatic antioxidants, only some antioxidants appear to be specifically associated to a particular biomolecular damage. This is, for instance, the case of lutein, lycopene, a-and b-carotene, whose plasma levels have been found to be inversely related to the lymphocyte DNA content of an oxidized base, 8-hydroxy-2-deoxyguanosine, in AD patients. This observation might be of relevance in AD prevention or in slowing disease progression, since lycopene has been shown to exert protective effects against oxidative DNA damage and since supplementation with xanthophylls and lycopene was able to decrease the amount of biomarkers of DNA and lipid oxidation in humans. The critical question is whether oxidative stress is a consequence rather than a causative step in the development of neurodegenerative processes. It can be, at least, partially answered with the observation that subjects with mild cognitive impairment (MCI), a condition frequently associated with AD, have increased plasma, urine and CSF levels of 8,12-iso-iPF2a-VI - a sensitive marker of in vivo lipid peroxidation; compared to healthy subjects. These results, together with the report of increased levels of cerebral biomarkers of oxidative damage in the early AD stage compared to the most advanced one as well as the observation of broad and dramatic antioxidant depletion noted in MCI and AD subjects support the hypothesis that oxidative damage occurs as one of the earliest pathophysiological events in AD. An early stage of AD, MCI, precedes AD by several years. A large number of studies found an association between high dietary antioxidant intake and with a decreased risk for AD.,, However, intervention trials demonstrated no major benefit with antioxidant supplementation in the treatment of AD. It is possible that when the clinical symptoms of AD appear a large proportion of neuronal cells might already be destroyed and therefore the intervention with antioxidants could be too late.
Several lines of evidence support a role for oxidative stress in atherogenesis. Epidemiological studies suggest that low levels of antioxidants are associated with increased risk for CVD, while high vitamin E plasma levels have been shown to be associated with the absence of atherosclerosis in octogenarians.
After brain injury by ischemic or hemorrhagic stroke or trauma, the production of ROS may increase, sometimes drastically, leading to tissue damage via several different cellular molecular pathways, including complex cascades of metabolic events that involve glutamate and its transporter proteins, NMDA receptors, nitric oxide synthases, etc. We showed that plasma levels of vitamin C - but not of vitamin E, ubiquinol-10 and uric acid - were inversely correlated with the size of the brain lesion in patients with brain hemorrhage or head trauma. In addition, plasma vitamin C levels were lower in jugular compared with peripheral blood samples in a small subset of patients. Independent of dietary intake, lipid profile, smoking habit and vitamin supplementation only plasma levels of vitamin C and of lutein, but not of other antioxidants, were shown to be associated with functional outcome in patients with neurological impairment due to ischemic stroke of recent onset. In these individuals, lipid peroxidation was shown to be significantly higher compared to controls., In classic experiments, it was shown that under conditions of oxidative stress, until all vitamin C is consumed there is neither a significant loss of other antioxidants nor an increase in lipid peroxidation in human plasma. The protection against free radicals exhibited by vitamin C was confirmed even in the presence of bleomycin-detectable free iron, an effect that might be of relevance in brain injury when iron is released by damaged cells. Two recent intervention studies showed that short-term and long-term vitamin C supplementation increases the resistance of plasma to lipid peroxidation both in healthy humans and in stroke patients (Polidori et al., not yet published data).
Antioxidant micronutrients have been postulated to exert protective effects against coronary heart disease (CHD). A meta-analysis has indicated that the relative risk reduction of ischemic heart disease in high consumers of fruit and vegetables may be in the order of 15%. Vitamin E is thought to prevent atherosclerotic disease not only by its antioxidant effects, but also through its inhibitory effects upon smooth muscle proliferation and platelet adesion. In supplementation studies in humans, a-tocopherol decreases lipid peroxidation and platelet aggregation and adhesion and is anti-inflammatory in nature. Of 11 studies evaluating the effects of various combinations of antioxidants including a-tocopherol, ascorbic acid and b-carotene on cardiovascular events, 4 showed a benefit with regards to the primary endpoint. Randomized, controlled trials of specific antioxidant supplements, however, have failed to demonstrate a consistent or significant effect of any single vitamin or combinations of vitamins on incidence of or on death from CVD.
We studied 30 elderly patients with CHF due, in half of the cases, to ischemic heart disease. The sample population, once again, was rigorously controlled for several potential oxidative stress-related confounding variables, including comorbidity, drug therapy, antioxidant vitamin supplementation, dietary intake, smoking habit, lipid profile, and degree of physical activity. Independently of the ischemic origin of the failure, the left ventricular ejection fraction (the ratio of the volume of blood the heart empties during systole to the volume of blood in the heart at the end of the diastole, expressed as a percentage of the total diastolic blood volume in the heart and normally ranging between 60% and 80%) significantly and directly correlated with plasma levels of vitamin A, vitamin E, lutein and lycopene - but not with other carotenoids. This observation is substantiated by the synergistic action and quenching activity of lutein and lycopene and by their potential ability to neutralize free radicals implicated as a cause of endothelial dysfunction in heart disease., In our study a simultaneous inverse correlation was found between ejection fraction and malondialdehyde, a marker of lipid peroxidation. Recently, an inverse correlation between ejection fraction and plasma levels of a more sensitive and specific biomarker of lipid peroxidation (8,12-isoprostane F2a-VI, one of the most abundant F2 -isoprostanes produced in vivo in humans) was also found in another group of patients with moderate and moderately severe CHF of ischemic origin (Polidori et al., not yet published data). It is, therefore, interesting to note that the relationship between the antioxidant pattern observed in CHF and the heart functionality might be, once again, mediated by and intimately linked to the pathophysiological step of lipid peroxidation.
It is often difficult to correlate a dietary deficiency with clinical symptoms that may arise, because many vitamins and minerals have multiple roles in metabolism. Vitamin metabolites, for instance, show various kinds of activities, both antioxidative and non-antioxidative.,,,
Antioxidant vitamins and micronutrients are able to exert non-antioxidant biological activities in addition to their free radical-scavenging capacity. Vitamin C, due to its participation in hydroxylation reactions and involvement in collagen synthesis, might play an important role in the prevention of pressure sores and ulcers in the elderly; vitamin E might exert regulatory effects on cell proliferation and shows a beneficial effect in improving glucose transport and the insulin sensitivity; lycopene is a very efficient antioxidant and is superior to b-carotene in scavenging singlet oxygen but its anti-cancer effects may not be or only in part be, related to its antioxidant activity. In several in vitro studies it was shown that the inhibition of cell proliferation by lycopene and b-carotene is associated with a delay in cell cycle progression.,
The fact that there is a multitude of oxidants as well as of antioxidants with overlapping reactivities, renders a biochemically rigorous assessment of the implication of oxidative stress difficult. Moreover, helpful as epidemiological studies can be, sometimes there is a confounding of associations with cause-effect relationships, leading to erroneous conclusions. For example while studying the implications of oxidative stress in CVD, controlling for risk factors like diabetes or smoking, that are themselves associated with free radical-induced damage constitutes a fundamental step for a subsequent clear interpretation of data. The results of cross-sectional studies (whether ecological or based on comparisons between individuals) might be, therefore, difficult to interpret, because there is usually an abundance of possible confounding factors, such as diet, lifestyle and physical activity. Inter-individual variations should also be taken into account: similar mean intakes of vitamin E, for instance, do not relate to similar mean plasma a-tocopherol concentrations while the same mean plasma concentrations of a-tocopherol may be achieved by different mean vitamin E intakes in humans. Geographic issues might be extremely relevant when conducting transnational studies, as the concentrations of antioxidant-rich foods vary considerably from country to country: plasma concentrations of g-tocopherol the most abundant form of vitamin E in the US diet, for instance, have been shown to be 2.15 ± 0.85 µM in a group of healthy German women and 6.4 ± 3.2 µM in healthy American women participating in the Framingham Heart Study., For antioxidant measurements, the issues of sampling, sample preparation, storage conditions and operator competence are of great importance. Even when attempts are made to carefully and rigorously control as many variables as possible, a statistically significant correlation between two parameters (such as a biological and a clinical marker of disease) does not always allow to infer a cause-effect relationship, as shown in the storks vs. babies correlation.
As far as randomized controlled trials are concerned, it is not redundant to mention once again that there are several explanations to the conflicting results obtained. These have been clearly and succintly summarized by Asplund and include, among others, the lack of specificity of the design to test a particular antioxidant, the lack of attention for the above mentioned confounding factors, the short duration of treatment and inappropriate choice of “time window” (to achieve efficacy, the antioxidant must be given during the time available between the damaging event and irreversible cell loss), the synthetic vs. natural source of antioxidant used (for instance, hepatocytes selectively enrich nascent very low-density lipoproteins with the natural 2R-stereoisomers of a-tocopherol, thereby loading the tissue only with this form of vitamin E14), and the absence of evaluation of markers of oxidative stress as intermediate end-points. With respect to the latter point, it is indeed essential to measure biomarkers of lipid peroxidation when conducting studies on CVD or of DNA oxidation when studying cancer and in nutritional studies in general, to prove that antioxidant vitamins are materially able to decrease disease-related oxidative damage. For neurodegeneration and stroke, agents that cross the blood-brain-barrier, penetrate the brain and are safe for long term consumption are needed.
In conclusion, a large body of scientific evidence doubtlessly indicates that an association exists between inadequate antioxidant status and increased risk for or poor outcome of several age-related diseases, including AD, stroke, CHF, CVD, osteoporosis, cancer, osteoarthritis, degenerative diseases of the eye,, and peripheral arterial disease. Antioxidant micronutrients do also show beneficial effects in the prevention of several of these diseases. This effect, however, is much stronger and consistent for antioxidant-rich fruits and vegetables than for single vitamin supplements. In addition, fruits and vegetables contain compounds such as sulforaphane - that induces GSH transferase thereby helping detoxifying many kinds of carcinogens - and phytochemicals such as genistein, whose intake is associated with a lower risk for several types of cancer. Increased consumption of fruits and vegetables can enhance the plasma antioxidant capacity in humans and is associated with a lower risk for CVD and cancer. Nutritional interventions effectively increase plasma levels of carotenoids, vitamins and antioxidants and decrease plasma levels of homocysteine in humans.,, They also constiute feasible strategy in the population in comparison to other strategies such as drug treatment or caloric restriction. While the National Cancer Institute and the National Research Council recommend 5-9 servings of fruit and vegetables per day be part of daily diet - with the most beneficial effect shown for 9 servings per day, 80% of American children and adolescents and 68%of adults did not meet this intake. It is the utmost hope that this situation, applicable to most industrialized countries, will promptly improve through the help of large health campaigne such as the “5-a-day” in the US (www.5aday.com) and the “5-am-Tag” in Germany (www.5amtag.de). In general, a healthy lifestyle involving, among others, regular exercise and avoidance of tobacco or alcohol abuse is key for the prevention of several age-related diseases of our time. A balanced and regular diet with at least five portions of fruits and vegetables per day is a critical constituent of such healthly lifestyle.
The author is grateful to Prof. Helmut Sies and Prof. Wilhelm Stahl (Institute of Biochemistry and Molecular Biology I, Heinrich-Heine University Düsseldorf) for their continued support, to Prof. Patrizia Mecocci (Institute of Gerontology and Geriatrics, University Hospital of Perugia, Italy) for the collaboration on nutritional studies in age-related diseases, and to the EU Marie-Curie Fellowship # QKL6-CT-1999-51332 for sponsoring in part the studies described here.