1. IntroductionNutrition and other lifestyle factors have been shown to have an important impact on the incidence and outcomes of most of the common non-communicable diseases that have been associated with aging, such as neurodegenerative and cardiovascular diseases, type 2 diabetes and cancer [1]. Aging is a biological process of progressive decline in physiological functions with advancing chronological age, leading to an increased vulnerability to disease and, subsequently, death [2]. The characteristic functional changes that precede these diseases, such as physical impairment and cognitive decline, are driven by multiple biomolecular mechanisms, including the accumulation of cellular damage and epigenetic alterations, which collectively result in altered functioning at the cellular, tissue and organism levels [3,4]. These characteristic mechanisms have collectively been described as the “hallmarks of ageing” [5] and might comprise effective targets for preventive and curative treatments of multiple age-related disease conditions. Age-related diseases, such as neurodegenerative and cardiovascular diseases, type 2 diabetes and cancer, are affected by the hallmarks of aging [2]. Besides well-known pharmacological therapies such as statins, management of body weight and physical exercise have been shown to be preventive (lifestyle) strategies [6,7]. However, effective regulation of the age-associated cellular damage described through the hallmarks has not been accomplished yet.One of the processes contributing to age- and adverse lifestyle related disease is mitochondrial dysfunction, of which oxidative damage may be an important cause and consequence [8]. The process of oxidative phosphorylation in the mitochondria produces reactive oxygen species (ROS). ROS encompass a group of molecules, either free radical or non-radical species, derived from molecular oxygen (O2) formed during reduction-oxidation (redox) reactions or by electronic excitation [9]. Free radicals have an unpaired electron, making them less stable and thus more reactive with various organic substrates than non-radical species. Non-radical species can, however, easily lead to free radical reactions in living organisms in the presence of transitions metals such as iron or copper [10]. Sources of ROS include endogenous sources (e.g., mitochondria, peroxisomes and NADPH oxidases) and exogenous sources (e.g., ultraviolet light, pollutants and ionizing radiation). These ROS can cause damage to macromolecules and mitochondria when the balance between ROS compounds and antioxidant defense mechanisms is disrupted. In turn, mitochondrial dysfunction will promote further free radical and non-radical ROS generation [9,11], for example, via the decreased expression of crucial proteins for electron transport due to damaged mitochondrial DNA (mtDNA). Oxidative stress refers to an “imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage” [11]. Importantly, redox signaling by ROS compounds is required for normal cellular functioning and host defense mechanisms. When ROS generation is deficient or excessive, this may lead to a broad range of phenotypic changes including altered gene expression, cellular senescence and inhibited growth [9].To prevent cellular damage and maintain ROS homeostasis, a complex system of different antioxidants exists. For example, antioxidant enzymes are involved in the neutralization of ROS in the mitochondria, including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX). Non-enzymatic antioxidants comprise dietary vitamins such as vitamin C and vitamin E (α-tocopherol), which intercept free radical chain reactions. Alteration in acting antioxidant levels could result in a disruption of ROS production and removal, leading to disruption of ROS signaling or in oxidative-stress induced damage. Antioxidants have therefore been hypothesized to play an important role in the development of multiple diseases. In line with this hypothesis, a promising antioxidant in observational studies is α-tocopherol [12]. However, although many prospective cohort studies have observed associations between higher α-tocopherol levels and a lower risk of overall and chronic disease mortality [13,14,15], randomized clinical trials comparing α-tocopherol supplementation with placebo have failed to demonstrate any beneficial clinical effect of higher α-tocopherol levels on the onset and development of disease, particularly cardiovascular diseases [16,17,18,19].

To date, it remains difficult to make causal inferences about oxidative stress and the use of antioxidant supplementation in nutrition, and the implications in human health and disease. In the present review, we focus on the paradox of the therapeutic role of (dietary) antioxidants in disease with regard to the rapidly evolving field of nutrition and medical sciences, integrating important recent studies that used novel research techniques such as Mendelian randomization. Accordingly, we first provide a brief overview of the chemical processes resulting in oxidative damage and the role of (anti)oxidants, focusing on the non-enzymatic antioxidant α-tocopherol. We then summarize the pertinent evidence on antioxidant supplementation in both the general and disease population. The final part of the review addresses the controversy between the circulating levels and capacity of antioxidants and discusses directions for future research.

4. Discussion and Concluding Remarks 4.1. Antioxidant Supplements: Is There Really Any Benefit?To date, there is an ongoing controversy about the use of antioxidant supplements for the prevention and treatment of multiple diseases. There is ample molecular evidence: an imbalance in ROS production and elimination can lead to oxidative damage, which triggers a cascade of the hallmarks of ageing and may contribute to the onset and development of numerous diseases [5,20,37,99]. Rationally, research has subsequently focused on enhancing the system that can effectively eliminate ROS: the complex network of antioxidants. Although it may seem only reasonable that increasing antioxidant levels to eradicate excessive ROS molecules should alleviate the burden caused by the overproduction of various ROS compounds, randomized clinical trials and MR studies to date have failed to provide evidence supporting this rationale [17,18,83,84,85,86]. A large discrepancy exists between the molecular indication and clinical outcomes for antioxidant supplementation. Therefore, the question is whether antioxidant supplementation truly provides considerable benefits on health status. Notably, the intake of antioxidant supplements as a therapy for low antioxidant status, due to, e.g., antioxidant deficiency diseases, may improve the patients’ health status and quality of life. However, this category of exceptionally low antioxidants levels only covers a small part of the dynamic and transient range of ROS. The greater part of the range of ROS, where defense mechanisms are sufficient for efficient ROS elimination, can be identified in the general population. These individuals with adequate antioxidant levels at baseline may only increase the circulating levels of antioxidants through the intake of supplements, but not the actual antioxidant capacity to eliminate part of the produced ROS. In other words, the network of antioxidant compounds may not become more effective by augmenting the pool of individual antioxidants with supplements in the general population (Figure 3).The balance between ROS and antioxidants can also tilt toward excessive antioxidant levels (Figure 3, left panel). Through increased endogenous production, enhanced daily food intake or a combination of the two, antioxidant levels could theoretically exceed its healthy boundaries and cause adverse effects. Although little is known about the possible detrimental effects of antioxidant supplementation, non-enzymatic antioxidants, including vitamin C and α-tocopherol, have been shown to have pro-oxidant effects at high concentrations, leading to ROS generation and contributing to a state of oxidative stress [100,101]. It has also been shown that α-tocopherol may interact with other vitamins to enhance or interfere with their function [102]. Accordingly, α-tocopherol can interfere with the blood clotting capacity of vitamin K [102], resulting in reduced blood clot formation. Although this aspect may be beneficial in certain patients, including in women with recurrent abortion due to impaired uterine blood flow [103], it may also increase the risk of bleedings in healthy individuals. However, it is important to consider that these adverse clinical effects of α-tocopherol antioxidant use could also be observed due to chance or possible flaws in the study design and/or selection of the study population. Taken together, these results indicate that antioxidant supplementation, particularly α-tocopherol, should be used with caution for adverse effects. It is therefore important to determine whether an individual genuinely requires antioxidant supplementation before intake.To this end, it is essential to measure oxidative damage markers and ROS turnover in the human body. However, measuring these endpoints forms a challenge in research. No single parameter has been recommended as a gold standard for measuring redox status in clinical studies thus far [104]. A major limitation is the identification of reliable biomarkers [105]. Some biomarkers have been identified in experimental and population-based epidemiological studies. Examples of current biomarkers for lipid peroxidation include plasma malondialdehyde, 4-hydroxynonenal and isoprostanes; for nucleic acid oxidation, examples include 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoG) for DNA and RNA, respectively [106]; and protein carbonyl can be used as a biomarker of protein oxidation [107]. Despite the potency of measuring these biomarkers of oxidative stress, the measured oxidative damage is often the result of a complex, interacting mechanism of numerous endogenous and exogenous antioxidants. Furthermore, these biomarkers cannot reflect the complete oxidative damage that has been brought to the body since they are mostly exclusive to certain macromolecule damage [22]. In addition to biomarkers, measuring ROS as a representation of oxidative stress has its limitations [104]. Some ROS molecules are highly reactive (particularly hydroxyl radicals) and therefore have a relatively short half-life, which makes their measurement in biological systems a complex task. Since accurate measurements of pro- and antioxidant levels are crucial to make inferences about the use of antioxidant supplementation, it is important to define an integrative yet clinically applicable approach to determine an individual’s redox status. 4.2. Final Remarks and Conclusions

Regarding the key role of oxidative damage in ageing and the onset and development of several diseases, research on decreasing oxidative damage with antioxidants has emerged in the last few decades. However, since clinical trials to date have not supported the use of antioxidant supplementation in oxidative stress-related diseases, a paradox exists: does supplementation of antioxidants delay aging and/or treat oxidative stress-related diseases?

In summary, there are three critical points to consider when examining the use of antioxidant supplementation. First, identifying reliable biomarkers for antioxidant capacity and levels of oxidative species that reflect the overall redox status in vivo, as well as transient redox status in specific tissues or cells, is crucial for further research. To date, there is still little consensus about the gold standard for measurements of oxidative stress in vivo. An optimal biomarker should be easily accessible, simple to detect accurately in human tissue and/or body fluid and reasonably stable. Second, the difference between antioxidant activity and capacity should be recognized in further research. Supplementation of antioxidants may increase their circulating levels and bioactivity, but this does not imply that the capacity of antioxidants is enhanced. Furthermore, several mechanisms may contribute to the difference between antioxidant activity and capacity, including its metabolism. Third, regarding the physiological importance of ROS signaling, it is necessary to develop strategies in redox studies that selectively address disease-associated mechanisms without disrupting the signaling pathways of ROS compounds. Future research should therefore focus on exploring novel markers for measuring oxidative stress and antioxidant status in vivo. Reliable yet simple measurements can facilitate in-depth studies examining the effects of antioxidant supplementation in aging and the development and progression of oxidative stress-related diseases, as well as in the general population, providing crucial knowledge that is indispensable to make inferences about the use of antioxidant supplements by healthy and diseased individuals.