Does Pharmacodynamic Interaction of Nonenzymatic Antioxidants Modify Response to Antioxidant Therapy in the Process of Atherosclerosis?

Abstract

A discrepancy exists between clinical trials and epidemiological studies on the effect of antioxidants on cardiovascular disease. This discrepancy could be attributed to the lack of knowledge on the effect of interaction of exogenous antioxidant supplementation with one another or on the effect of interaction of exogenously administered antioxidant vitamins with endogenous ones. This study attempts a systematic review of available data on possible synergistic, additive, or antagonistic action of nonenzymatic antioxidants in atherosclerosis. Electronic databases were searched with the available search terms up to and including February 2010. Eligibility criteria were full publications, clinical trials, epidemiological studies, or in vitro or in vivo studies that investigated the effect of pharmacodynamic interaction of 2 or more antioxidants in the process of atherosclerosis and /or the mechanism of interaction. Eligible clinical trials should have at least 4 arms, 1 arm for the study of each antioxidant alone, 1 for the effect of both antioxidants, and 1 arm for the effect of placebo. In vitro data as well as the limited number of identified randomized clinical trials suggested that coadministration of antioxidants results in synergistic or additive interaction in the process of atherosclerosis. No study demonstrated antagonistic interaction between antioxidants.

Keywords

antioxidants atherosclerosis interaction synergistic effect additive effect antagonistic effect

Introduction

Atherosclerosis is a progressive disease characterized by the accumulation of cholesterol deposits in the macrophages of large and medium size arteries. This deposition leads to the proliferation of certain cell types within the arterial wall which gradually impinge on the vessel lumen and impede blood flow.^1
–3 Oxidative stress is defined as a disturbance in the generation of reactive oxygen species (ROS) leading to enhanced ROS level with subsequent oxidative damage to cellular constituents, that is, DNA, proteins, lipids, and sugars.³ Thus, oxidative stress, in particular oxidative modification of low-density lipoprotein (LDL) cholesterol, initiated by a free radical-driven lipid peroxidation process, is considered to play a key role in initiation and progression of atherosclerosis, although a direct causative role of LDL oxidation for atherosclerosis has not been established.⁴ However, currently atherosclerosis is regarded an inflammatory process in which oxidative stress is seen as a proinflammatory and atherogenic factor.²

Animal models of atherosclerosis support the notion that ROS released from nicotinamide adenine dinucleotide phosphate oxidases, xanthine oxidases, and lipooxygenases and enhanced ROS production from dysfunctional mitochondrial respiratory chain indeed have a causative role in atherosclerosis. Impairment of vascular function and enhanced atherogenesis have been observed in animal models that have deficiencies in antioxidant enzymes. Investigation in humans supports the oxidative stress hypothesis of atherosclerosis. Experimental evidence suggests that pharmacological modulation of oxidative stress response is feasible^5
–7 and might alter the natural history of a number of diseases. Thus, antioxidants could inhibit the initiation, progression, and development of atherosclerosis. Proposed mechanisms include inhibition of LDL oxidation, inhibition of leukocyte adhesion to the endothelium, and vascular endothelial dysfunction.⁸ Experimental studies in animals have shown that antioxidant vitamins can slow the progression of atherosclerosis. Epidemiological studies show an inverse relationship between antioxidant vitamin consumption and cardiovascular disease. The National Health and Nutrition Examination Survey epidemiological follow-up study of 11 348 participants, aged 25 to 74 years, reported that individuals who received a high dose of vitamin C (>50 mg/d) had lower overall total mortality rate after 10 years, and in particular lower mortality from cardiovascular disease.⁹ The Health Professionals Follow-up Study reported that participants whose vitamin C intake exceeded 50 mg/d tended to have a lower rate of death from all cardiovascular diseases.¹⁰ In a prospective cohort study of 54 251 women and 42 148 men who were followed for 8 years and were free of cardiovascular disease, cancer, and diabetes, the authors found that the consumption of fruits and vegetables, particularly green leafy vegetables and vitamin C-rich fruits and vegetables, appeared to have a protective effect against coronary heart disease (ie, nonfatal acute myocardial infarction [AMI] or fatal coronary heart disease).¹¹ In contrast, clinical trials have been giving a more confused picture than expected, with results ranging from a significant protective action to the absence of any effect. Thus, a double-blind, randomized, placebo-controlled, cardiovascular and cancer prevention trial of a combination of antioxidants (120 mg vitamin C, 30 mg vitamin E, 6 mg β-carotene, 100 μg selenium, and 20 mg zinc) showed no beneficial effects of long-term daily low-dose supplementation of antioxidant vitamins and minerals on carotid atherosclerosis and arterial stiffness.¹² The Physicians Health Study, a randomized, prospective, double-blind, placebo-controlled study of 14 641 male physicians, which investigated the effect of oral supplementation of 400 IU of vitamin E every other day and 500 mg of vitamin C daily, did not show a significant difference between the treated and control groups in respect to the risk of major cardiovascular events.¹³

The discrepancy between the clinical trials and epidemiological studies has been attributed to inclusion of patients without biochemical evidence of oxidative stress, short duration of trials, use of suboptimal doses of antioxidant vitamins, lack of inclusion of markers of oxidative stress and markers of vascular response, inappropriate administration of vitamins relative to meal ingestion, poor patient compliance, and lack of monitoring vitamin levels. Another cause for the discrepancy between epidemiological and clinical studies could be that the effect of interaction of exogenous antioxidant supplementation with one another or the effect of interaction of exogenously administered antioxidant vitamins with endogenous ones has not been fully investigated. Indeed, the suppression of γ-tocopherol by α-tocopherol and the use of vitamin E supplementation without the concurrent use of vitamin C have been proposed as possible causes for the discrepancy between epidemiological studies of fruits and vegetables and clinical studies of supplementation with 1 or 2 antioxidants. Experimental data suggest that antioxidants interact in different biological systems with resultant synergistic, additive, or antagonistic action. The present study is a systematic review of the available data on possible synergistic, additive, or antagonistic action of nonenzymatic antioxidants in atherosclerosis.

Methods

Pubmed, Scopus and Google Scholar were searched with the search terms “antioxidant vitamins,” “vitamin C,” “vitamin E,” “vitamin A,” “tocopherol,” “alpha lipoic acid,” “carotenoids,” “lycopene,” “glutathione,” “flavonoids,” “carotenoids,” “phenolics,” “atherosclerosis,” “lipid peroxidation,” “LDL oxidation,” “vascular disease,” “interaction,” “synergism,” “synergistic action,” “synergistic effect,” “additive action,” additive effect,” and “antagonism” up to and including February 2010. Eligibility criteria were full publications, clinical trials, epidemiological studies, or laboratory in vitro or in vivo studies that investigated the effect of pharmacodynamic interaction of 2 or more antioxidants in the process of atherosclerosis and /or the mechanism of interaction. Atherosclerosis was defined either with pathological–biochemical criteria (ie, LDL oxidation, lipid peroxidation, and arterial wall cell proliferation) or in clinical terms (ie, fatal myocardial infarction). Additive interaction was defined as the combined effect of 2 antioxidants being the sum of each antioxidant administered separately. Synergistic interaction was defined as the combined effect of 2 antioxidants being larger than the sum of each antioxidant administered separately. Both endogenous and exogenous nonenzymatic antioxidants were investigated. Antioxidants considered included antioxidant vitamins, α-lipoic acid, carotenoids, flavonoids, and nonflavonoid phenolics. There was no language restriction. The reference list of all the identified trials was checked for more relevant articles.

Eligible clinical trials should have at least 4 arms, 1 arm for the study of each antioxidant alone, 1 for the effect of both antioxidants, and 1 arm for the effect of placebo. Data extracted included the antioxidants administered, the doses of antioxidants, duration of treatment, efficacy of single antioxidant, efficacy of the combination of antioxidants, and safety issues of antioxidants.

Results

The electronic search identified 397 articles. The titles were reviewed and the abstracts of 297 articles were selected for further review. Articles studying the effect of a combination of antioxidant vitamins without investigating the effect of each antioxidant vitamin alone were excluded as they could provide no data on interaction of antioxidants. Yet, articles investigating the effect of antioxidants not specified in the Methods section or the effect of antioxidant interaction in other pathological processes were also excluded.

In Vitro Studies of LDL Oxidation

A synergistic interaction has been shown among ferulic acid, α-tocopherol, β-carotene, and ascorbic acid in inhibiting lipid peroxidation in rat liver microsomal membranes and reactive oxygen production in NIH 3T3 fibroblasts induced by oxidants.¹⁴

The effect of antioxidants on LDL oxidation in the presence of copper oxidation system in vitro has been extensively studied. However, a few articles investigate the effect of interaction of antioxidants on LDL oxidation (Table 1).^{15

–26} These studies have shown synergistic action of phenolics toward LDL oxidation. Synergistic effects have also been shown for carotenoids as well as for mixtures of flavonoids with carotenoids and for mixtures of carotenoids with vitamin E. It has been shown that although individual components may have prooxidant action when administered alone, their combination exerts antioxidant action. Negre-Salvayre et al¹⁵ demonstrated the additive effect between α-tocopherol and ascorbic acid or rutin and synergistic effect between ascorbic acid and rutin on peroxidation processes investigated in xanthine–xanthine oxidase systems, linolenic acid ufasomes, and human erythrocyte membranes.¹⁵

Table 1.

In Vitro Studies of the Effect of Interaction of Nonenzymatic Antioxidants in the Process of Atherosclerosis

Author	Antioxidants	Interaction
Negre-Salvayre et al¹⁵	Ascorbic acid, α-tocopherol, and rutin	Synergistic action among the 3 of them
Dobreanu and Mody¹⁶	Vitamins C, E, and A	Synergistic action among hydrophilic and lipophilic vitamins
Fuhrman et al¹⁷	Lycopene, vitamin E, flavonoids (glabridin derived from licorice), phenolics (rosmarinic acid or carnosic acid derived from rosemary), garlic acid (mixture of antioxidants), and β-carotene	Synergism between lycopene and vitamin E, lycopene and glabridin, lycopene and rosmarinic acid, lycopene and garlic acid, lycopene, and β-carotene
Hwang et al¹⁸	Soy and alfafa phytoestrogen extracts (rich in flavonoids) and acerola cherry extract (rich in ascorbic acid)	Synergistic effect
Ivanov et al¹⁹	Red wine extracts and ascorbic acid	Additive effect
Shafiee et al²⁰	Polyphenol or tocopherol rich extracts	Synergistic action dependent on the oxidation system
Milde et al²¹	Flavonoids (rutin) with hydrophilic antioxidant (ascorbic acid) and lipophilic antioxidant (γ-terpinene)	Synergistic action between rutin and ascorbate as well as between rutin and γ-terpinene
Briante et al²²	Nonflavonoid phenols from Olea europea L	Antioxidant or prooxidant effect of individual nonflavonoids compounds dependent on their concentration. When administered in mixture, synergistic effects, that enhance their antioxidant capacities, thus avoiding prooxidant action
Chen et al²³	Oat phenolics (avenanthramides and simple phenolic acids) and vitamin C	Synergistic action
Dalby-Brown et al²⁴	Constituents of Echinacea purpurea (alkamides, caffeic acid derivatives, and polysaccharide fractions)	Synergistic action
Cirico et al²⁵	Four phenolic compounds: catechin, hesperidin, ferrulic acid, and quercetin	Two of the phenolics had prooxidant effect while the mixture of 4 had possibly synergistic action
Milde et al²⁶	Phenolics (rutin) and carotenoids (lycopene and lutein)	Synergistic action

In Vitro Studies of Human Cells

Ulrich-Merzenich et al²⁷ investigated the effect of vitamin C and vitamin E interaction on human vascular endothelial and smooth muscle cell DNA synthesis and proliferation. Coadministration of vitamin C and vitamin E potentiated the effect of each vitamin alone on human umbilical endothelial cell growth.²⁷ Negre-Salvayer et al²⁸ showed synergistic effect of α-tocopherol, ascorbic acid, and rutin in protecting cultured endothelial cells incubated in the presence of oxidized LDL either through reduction of LDL oxidation or through increasing their resistance against oxidized LDL. This synergistic effect permitted maximal antioxidant effect to be achieved with lower concentrations of antioxidants, thus avoiding higher cytotoxic concentrations.²⁸

Zhang and Omay²⁹ demonstrated that β-carotene, α-tocopherol, and ascorbic acid produced concentration-dependent antioxidant effect on human lung cells under hypoxic conditions. Coadministration of the 3 compounds exhibited greater protection than any individual compound.²⁹

Similarly, Schmidt et al³⁰ showed additive effect between the hydrophilic antioxidants rutin and ascorbic acid and the lipophilic antioxidant ascorbic acid in protecting cultured endothelial cells against the cytotoxic effect of oxidized LDL. Rutin and ascorbic acid when administered alone had a biphasic protective effect at low doses and cytotoxic effect at high doses, but the biphasic effect was abolished upon combination with α-tocopherol.³⁰ On the contrary, Sabharwal et al,³¹ showed no interaction between α-lipoic acid and ascorbate in protecting EA.hy296 endothelial cells against intracellular and extracellular oxidative stress.³¹

Randomized Controlled Trials

A few randomized controlled trials investigating the effect of antioxidant interaction on atherosclerosis were identified (Tables 2 and 3).^32

–35 Although available data were quite limited, a synergistic or additive interaction of the limited investigated antioxidants was suggested for the combination of vitamin C with vitamin E. In the Antioxidant Supplementation in Atherosclerosis Prevention (ASAP) study, the authors investigated the efficacy of vitamins C and E supplementation on the progression of carotid atherosclerosis, hypothesizing synergism between vitamins.³³ Participants were randomized to either vitamin C (250 mg/d), vitamin E (91 mg/d), vitamin C, vitamin E, or placebo. Combined supplementation with both vitamin E and slow-release vitamin C delayed the progression of common carotid atherosclerosis in men, although the respective treatment effect was nonsignificant in groups that received only vitamin E or vitamin C, although there were trends toward protection.³³ These data were replicated in the 6-year evaluation of ASAP study.³⁵

Table 2.

Randomized Controlled Trials Investigating the Effect of Antioxidant Interactions on Atherosclerosis-Descriptive Data

Author	Study Design	Antioxidants Tested and Doses	Number of Patients	Study Duration
Rapola et al³²	RCT	Vitamin E (50 mg/d), β-carotene (20 mg/d), both agents, or placebo	1862 men enrolled in the α-tocopherol cancer prevention study	5 years
Salonen et al³³	RCT 2 × 2 factorial design	Twice daily either vitamin E 91 mg/d 136 IU, vitamin C slow release 250 mg/d, both agents, or placebo	520 smoking and nonsmoking men and menopausal women 45-69 years, all hypercholesterolemic	3 years
Huang et al³⁴	RCT 2 × 2 factorial design	Vitamin C (500 mg ascorbate/d), vitamin E (400 IU RRR α-tocopherylacetate/d), both agents, and placebo	148 smokers	2 months
Salonen et al³³	RCT 2 × 2 factorial design	Twice daily either vitamin E 91 mg/d 136 IU, vitamin C slow release 250 mg/d, both agents, or placebo	520 smoking and nonsmoking men and menopausal women 45-69 years, all hypercholesterolemic	6 years

Table 3.

Randomized Controlled Trials Investigating the Effect of Antioxidant Interactions on Atherosclerosis-Outcome Data

Author	Endpoint	Outcome	Adverse Events
Rapola et al³²	The first major coronary heart event either nonfatal myocardial infarction or fatal myocardial infarction	Incidence of fatal myocardial infarctions decreased among recipients of vitamin E and increased among recipients of beta carotene. Interaction between vitamin E and β-carotene was found in the nonfatal myocardial infarction group but not in the fatal coronary heart disease group.	None reported apart from myocardial infarctions
Salonen et al³³	Progression of atherosclerosis	Combination of vitamins was able to retard progression of carotid artery atherosclerotic disease in smoking men. The effect was not statistically significant for either vitamin alone although a trend toward protection was evidenced.	6 deaths, 1 in the placebo group due to cardiac arrhythmia, 3 deaths in the vitamin E group, 1 of which is accidental, 1 due to alcohol intoxication and 1 sudden coronary death, 1 death in the vitamin C group due to subarachnoid hemorrhage and 1 in the vitamin combination group due to complications of carotid endarterectomy. 13 severe adverse events and 15 other adverse events.
Huang et al³⁴	Lipid peroxidation	Supplementation with each vitamin alone reduced in vivo lipid peroxidation, while supplementation with both vitamins conferred no extra reduction	Not reported
Salonen et al³⁵	Progression of atherosclerosis	3-year findings were replicated	22 deaths, 21 severe adverse events, and 6 other adverse events

Another placebo controlled clinical trial investigated the effect of vitamin C (500 mg/d) and vitamin E (400 IU) either alone or in combination on in vivo lipid peroxidation.³⁴ The study demonstrated that supplementation with vitamin C or vitamin E alone reduced lipid peroxidation, while the combination of vitamin C with vitamin E conferred no benefit beyond that of either vitamin alone.

The Alpha Tocopherol Beta Carotene Cancer Prevention study evaluated antioxidant supplementation in 1862 male smokers with a previous MI.³² Participants were randomized to α-tocopherol (50 mg/d), β-carotene (20 mg/d), both, or placebo. The median follow-up was 5 years. Neither α-tocopherol nor β-carotene supplements decreased the proportion of major coronary events. Moreover, the risk of fatal coronary heart disease increased in the groups that received either β-carotene or the combination of α-tocopherol and β-carotene and there was a nonsignificant trend of increased deaths in the α-tocopherol group.³²

Mechanisms of Antioxidant Interaction in Atherosclerosis

A quite limited number of studies focus on the investigation of the mechanisms of antioxidant interaction in the case of atherosclerosis. It has been suggested that vitamin E that is transported by LDL plays a critical role in protecting against LDL oxidation. Thus, it has been shown that vitamin C may enhance the effects of vitamin E against LDL oxidation by reducing tocopheryl radicals. Kinetic analysis and studies of vitamin E regeneration in protein-denaturated systems have shown that ascorbate regenerates vitamin E by nonenzymatic mechanisms, whereas glutathione regenerates vitamin E enzymatically.^36,37

In addition, antioxidants may exert their antiatherogenic actions entirely or in part through redox-independent mechanisms. A series of pleiotropic effects has been suggested for α-tocopherol. Thus α-tocopherol inhibits protein kinase C, smooth muscle cell proliferation, platelet aggregation, cell adhesion, enhances nitric oxide bioavailability, and increases protein phosphatase A21 activity. Hence, evidence suggests that α-tocopherol counteracts inflammation and improves endothelium-dependent vasodilatory function. All the above-mentioned pleiotropic effects of vitamin E are active, only if α-tocopherol is in its reduced form. Thus, vitamin C or other cooxidants contribute to the anti-atherosclerotic effect of vitamin E.^38
–40

Discussion

Atherosclerosis is a multifactorial process and oxidative/nitrosative stress, imbalance of vasoconstrictor–vasodilator production, platelet aggregation, and modification of LDLs are all implicated in its pathophysiology. Evidence suggests that antioxidants could not only modify almost all the pathophysiological parameters of atherosclerosis but also affect the progression and complications of atherosclerosis. Antioxidants comprise a great number of endogenous and exogenous substances that modulate diverse cellular and molecular processes. They have different mechanisms of action suggesting that pharmacodynamic interactions among different antioxidants could be expected with beneficial effects in multifactorial processes such as atherosclerosis. However, although combinations of antioxidants are labeled and often prescribed, the evidence on pharmacodynamic interactions of antioxidants remains quite limited. In the case of atherosclerosis, most in vitro studies focus on the interaction of antioxidants against LDL oxidation, while there is paucity of data on the interaction of antioxidants in modulation of other parameters of initiation of atherosclerosis. Only a few possible combinations of antioxidants have been investigated. Available experimental data suggest synergistic or additive interaction of antioxidants in atherosclerosis. Mechanisms of synergistic interaction include different localization of antioxidants, diversity of defense mechanisms provided by antioxidant vitamins and inhibition of prooxidant action of antioxidant-derived radicals. No study demonstrated the antagonistic interaction between antioxidants in the process of atherosclerosis, although it has been suggested in studies of cancer prevention.⁴¹ Synergistic action of antioxidants has been observed in cancer chemoprevention, especially when antioxidants are combined in low doses or when antioxidants with different mechanism of actions are combined.⁴² On the contrary, combinations of high doses of antioxidants have antagonistic or additive effect, possible because of saturation of effect or of cytotoxicity.^41,43

Safety is a major issue in drug development today. Existing data have raised concerns on the safety profile of antioxidant therapy of cardiovascular disease. Large randomized trials with β-carotene supplementation have shown an increase in cardiovascular mortality.^32,44 The Beta-Carotene and Retinol Efficacy trial was interrupted because of an increase in the incidence of lung cancer and total mortality among 17 000 participants who received a supplement of a daily combination of β-carotene (30 mg) and retinol (25 000 IU).⁴⁴

Further research is needed on the interaction of antioxidants. Synergism of antioxidants might lead to the design of novel agents with clinical implications in cardiovascular disease and other processes whose pathophysiology includes oxidative stress. In conclusion, in vitro data as well as the limited number of identified randomized clinical trials suggested that coadministration of antioxidants results in synergistic or additive interaction in the process of atherosclerosis. No study demonstrated antagonistic interaction between antioxidants.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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