Vitamins in human arteriosclerosis with emphasis on vitamin C and vitamin E

Clinica Chimica Acta 339 (2004) 11 – 25 www.elsevier.com/locate/clinchim

Review

Vitamins in human arteriosclerosis with emphasis on vitamin C $ and vitamin E Ntei Abudu a, James J. Miller a, Mohammed Attaelmannan a, Stanley S. Levinson a,b,* a

Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, KY 40292, USA b Laboratory Service, VAMC, 800 Zorn Avenue, Louisville, KY 40206-1466, USA Received 9 June 2003; received in revised form 25 September 2003; accepted 29 September 2003

Abstract Introduction: This review focuses on the process of arteriosclerosis arising from oxidative stress on lipoproteins and the general failure of randomized human trials using vitamins to retard this process. Review: As well as clinical trials, the paper reviews the mechanisms by which a variety of oxidants act. Antioxidants are discussed, emphasizing interactions of vitamins C and E with transition metals that can lead to prooxidation. There is a focus on interactions between supplemental or coantioxidants that counterbalance prooxidant effects of one another. Conclusions: It is concluded that normal cellular supplementation mechanisms are poorly accessible in the arteriosclerotic plaque leading to a prooxidant environment in which the haphazard introduction of vitamins could potentially be hazardous. Continued investigations into basic and clinical redox interactions of the kind discussed in this review using new measuring techniques may lead to approaches whereby antioxidants can be introduced into tissue in controlled ways for reducing arteriosclerosis. D 2003 Elsevier B.V. All rights reserved. Keywords: Co-antioxidants; Vitamin C; Vitamin E; Transition metals

1. Focus of this article Oxidation of lipoproteins appears essential for the natural process of arteriosclerosis to proceed. Thus, many assumed that vitamin antioxidants would retard arteriosclerosis [1 – 3]. Yet, recent randomized human $

For a fuller discussion of the attributes of vitamin C, the interested reader is referred to Padayatty SJ, Katz A, Wang Y, Eck P, Kwon O, Lee JH, Chen S, Corpe C, Dutta A, Dutta SK, Levine M. Vitamin C as an antioxidant: evaluation of its role in disease prevention. J Am Coll Nutr 22 (2003) 18 – 35. * Corresponding author. Laboratory Service, VAMC, 800 Zorn Avenue, Louisville, KY 40206-1466, USA. Tel.: +1-502-8953401x5565/5572; fax: +1-502-894-6265. E-mail address: [email protected] (S.S. Levinson). 0009-8981/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2003.09.018

clinical trials indicate that vitamin antioxidants failed to retard arteriosclerosis and may have caused adverse effects [4– 6]. In some circumstances, vitamin E and vitamin C act as prooxidants. This prooxidant effect has been widely proposed for explaining the failure of vitamins in clinical trials [4 – 6]. Under normal conditions, physiological antioxidants, so called supplemental or co-antioxidants, can interact to negate prooxidant effects [7]. This review contends that failure of coantioxidant interactions within the arteriosclerotic plaque may explain why vitamins have not retarded arteriosclerosis in the trials. The review is organized in the following way: (2) A brief review of the process of arteriosclerosis and the oxidative modification of

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low-density lipoprotein (LDL) hypothesis, and a discussion of the types of oxidation products found in humans; (3) an outline of human randomized clinical trials examining the relationships between antioxidant vitamin treatments and arteriosclerosis; (4) a discussion of the mechanisms of oxidation, antioxidation and co-antioxidant interactions; and (5) discussion and conclusions pointing out how, in an arteriosclerotic plaque, these mechanisms may cause an overall prooxidant environment, whereby, in the absence of co-antioxidant interactions nutrient antioxidants may facilitate rather than inhibit arteriosclerosis. Emphasis will be placed on mechanisms of vitamins E and C, especially in interactions with transition metals.

2. The process of arteriosclerosis and the oxidative modification of LDL hypothesis Substantial evidence links arteriosclerosis with endothelial impairment, inflammation and thrombosis [8,9], and arteriosclerosis appears to be a systemic process [10 – 12]. Hyperlipidemia, and especially oxidized (ox) LDL, appears instrumental in inducing this process leading to plaque formation [1,3,9,13 – 16]. Microcirculatory dysfunction allows entry of lipoproteins into the arterial wall leading to the release of inflammatory mediators which promotes further binding of LDL to the vessel endothelium and a continuing cycle of inflammation [8,9]. Elevated LDL cholesterol correlates with elevated levels of lipid oxidation products, lipid lowering in animals and humans leads to decreased oxidative products and improved endothelial function [17 –19]. Peroxidation of unsaturated fatty acids gives rise to reactive aldehydes and ketones that may complex positively charged amino acid residues of apo B found in lipoproteins [20,21]. Oxidation also otherwise modifies and fragments apo B [10,20]. Uncomplexed lysine residues are required for normal controlled LDL uptake by the LDL receptor, while oxLDL containing modified apo B is taken up in an unregulated way by macrophage scavenger receptors. As a result, macrophages form lipid laden foam cells, an early event in fatty streak formation and an integral part of the necrotic core within a maturing plaque [22,23]. OxLDL not adducted to apo B may also be recognized by scavenger receptors [24].

Other proatherogenic effects of oxLDL and oxphospholipids include the ability to attract monocytes, to inhibit the motility of macrophages, to prevent the release of vasodilatory nitric oxide (NO) from endothelial cells and to promote abnormal proliferation of vascular smooth muscle cells [9,13,14,25]. These adverse effects of oxLDL on coronary artery vasomotion and coagulation pathways may play a role in the latter stages of arteriosclerosis leading to acute ischemic syndromes [9,13,14]. Humans with arteriosclerosis show elevations in oxidized lipid products including malondialdehyde (MDA), F2-isoprostanes, 4-hydroxynonenals (HNE), hydroxyoctadecadienoic acid derivatives (HODEs) [21,26,27], and both oxLDL and MDA-LDL [28 – 30]. OxLDL found in serum may originate from the arteriosclerotic wall [30,31]. OxLDL may be a powerful marker for assessing risk of coronary artery disease (CAD) [32]. Besides, elevated levels of antibodies against MDA-LDL have been found associated with arteriosclerosis [33]. These oxidation products may be a manifestation of lipoprotein oxidation consistent with the LDL oxidation hypothesis of arteriosclerosis. Hence, it would seem that dietary antioxidants should retard arteriosclerosis in animals and humans [1].

3. Antioxidant studies in animals and humans 3.1. Animal studies Animal studies have been well reviewed by Neuzil et al. [4] and are discussed only briefly here. Studies with rabbits show that antioxidants such as probucol, butylated hydroxytoluene and diphenyl-phenylenediamine can inhibit arteriosclerosis [34]. In mouse models, vitamin E has been effective alone and in combination with other antioxidants in retarding arteriosclerosis [3], but studies with rabbits have been inconsistent. The main isomer of vitamin E found in lipoproteins is a-tocopherol (a-TOH). Rabbits supplemented with 1% a-TOH for 36 months showed decreased aortic lesion development and decreased plasma lipid concentrations in one study and decreased aortic thickness in another [4]. Yet, in another study, various amounts of a-TOH up to 1% did not inhibit arteriosclerosis nor lower plasma cholesterol

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levels [4]. Similarly, studies with WHHL rabbits and hamsters were contradictory [4]. 3.2. Human studies 3.2.1. Vitamins as antioxidants and protectors of endothelial tissue Ingestion of a-TOH, in amounts greater than 400 IU/day, substantially reduced oxidative susceptibility in lipoproteins of humans ex vivo [21,35]. Vitamin C (ascorbate) has been shown to be superior to other water-soluble antioxidants in protecting lipoproteins in plasma from oxidative damage in vitro [36]. Vitamin E inhibits monocyte production of inflammatory mediators, inhibits monocyte adhesion to human endothelial cells in vitro [37] and preserves endothelial function in vivo [38,39]. Likewise, vitamin C improves endothelial vasodilation [40], restores coronary microcirculation in smokers [41], protects human vascular smooth muscle cells in culture [42] and retards a variety of pro-inflammatory mechanisms in vitro and in human forearm models, and animal models [43]. Thus, vitamins appear to enhance both antioxidant capacity of lipoproteins and preservation of endothelial tissue (a surrogate marker that predicts cardiovascular events) [44] and should protect from arteriosclerosis. Yet, as discussed below, the vast majority of large interventional studies with vitamins have shown no reduction in coronary events or even an adverse effect. For additional insight, see the recent reviews by Neuzil et al. [4] and by Kritharides and Stocker [45]. 3.2.2. Vitamin E preparations Vitamin E includes four tocopherols and four tocotrienols (each, a, h, y, g) [46]. Supplements usually contain only a-TOH, either unesterfied or as esters of acetate, succinate or nicotinate, but this variation does not substantially effect plasma levels since free and esterfied a-TOH have similar bioavailability [46]. More important is whether or not the preparation is natural d-RRR or synthetic all-racemic (DL), which consists of eight stereoisomers, since after ingestion plasma concentrations of synthetic a-TOH reach levels of only about 50% of natural a-TOH [46].

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3.2.3. Results of randomized studies In a primary prevention study (of a-TOH and hcarotene), 29,134 male smokers age 50 – 69 were assigned to 55 IU of synthetic vitamin E or vitamin E and 20 mg h-carotene for a median follow up of 6.1 years [47]. As compared to placebo, there was a significant increase in cancer among those taking h-carotene. In a follow-up study of 1862 men, with a previous myocardial infarction, there was no difference in coronary events in any group. But significantly more men died from fatal coronary heart disease in the h-carotene group [48]. There was also an increased relative risk of cardiac death in the vitamin E group that did not reach significance. The authors recommended against the use of supplemental vitamins in smokers. One criticism of the study was that the vitamin E concentration was below that required to effect oxidative susceptibility of lipoproteins. The randomized secondary prevention Cambridge Heart Antioxidant Study used higher doses of vitamin E [49]. Here, 1035 of 2002 patients were fed 400 or 800 IU/day of natural vitamin E for 510 days. Although a reduction in nonfatal myocardial infarction was highly significant in the treated group, which was encouraging, the study was not robust since the treated group also showed a nonsignificant increase in cardiovascular death [4]. Contrary results were found in the GISSI study of 11,324 [50] and the HOPE study of 9541 men and women [51]. In these studies, 300 mg/day of synthetic (equivalent to about 450 IU of a-TOH) and 400 IU/day of natural vitamin E did not significantly reduce cardiac events. In a subset of the HOPE study, an angiotensin converting enzyme inhibitor retarded arteriosclerosis but vitamin E did not [52]. The secondary prevention Heart Protection Study randomized 20,536 adults to receive 600 mg (about 660 IU) of synthetic vitamin E, 250 mg vitamin C and 20 mg h-carotene per day or placebo. There was no significant difference in cardiac outcomes over a 5-year period [53]. Recently, a randomized, secondary prevention study of hormonal replacement therapy and vitamin supplementation in women showed that 800 IU/day of vitamin E and 1000 mg vitamin C for more than two years did not reduce coronary disease. It actually caused a positive trend towards more death, nonfatal MI and stroke [54].

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In a small randomized study, 6 months of probucol treatment significantly reduced the risk of restenosis in 80 patients, while treatment with a cocktail of hcarotene, 700 IU/day of a-DL-TOH and 500 mg of vitamin C did not [55]. In a randomized study of 652 elderly healthy people, it was shown that 200 mg of a-TOH acetate (about 220 IU) per day did not show a favorable effect on the incidence of respiratory infections, but rather showed an adverse effect on illness severity [56]. Moreover, up to 2000 IU/day vitamin E treatment for up to 8 weeks caused no reduction of 4-HNE, or F2-isoprostane concentrations [6,19]. Increased intima-media thickness (IMT) in the common carotid artery is a strong surrogate predictor for future cardiac events [57]. The ASAP study of aD-TOH (136 IU/day) and vitamin C (250 mg) on 520 individuals showed a decrease in IMT progression over 3 years in each of the vitamin E and vitamin C groups and a significant decrease in a combination group of men but not women [58]. The finding that men were protected but not women is puzzling. Another randomized study, of 353 men and women without CAD, but with elevated LDLC, treated with 400 IU of a-DL-TOH/day showed no significant change in either sex over a 3-year period. Besides, both sexes showed a nonsignificant trend towards increased IMT progression [59]. Recently, 146 patients with CAD were randomized to receive either placebo, lipid lowering statin drugs, lipid lowering drugs plus the antioxidants (800 IU/day a-DL-TOH, 1000 mg/day vitamin C, 25 mg h-carotene and selenium), or antioxidants alone. Although progression of disease measured by angiography nonsignificantly decreased in those treated with antioxidant as compared to placebo, those treated with antioxidants and statins showed greater progression of stenosis and significantly more cardiovascular events than those treated with statins alone [60]. This diminished effect has been attributed to a blunting of the high-density lipoprotein cholesterol (HDLC) subfraction 2 response to the statin therapy [61]. Yet, this remains no more than a hypothesis, since it is unclear that HDL2C is more antiatherogenic than other subfractions of HDL [62]. Still the conclusion that the vitamin cocktail caused adverse effects in patients on statin therapy seems reasonable.

4. Mechanisms of oxidation and antioxidation 4.1. Oxidants 4.1.1. Oxidation of lipids in lipoproteins Oxidation of fatty acids in lipoproteins may be mediated by reactive species such as radicals, transition metals, other electrophiles and by enzymes [63]. Once initiated, oxidation of fatty acids in triglycerides and cholesterol esters may proceed by a chain reaction. Within the lipoprotein, the chain can be terminated by a lipid soluble antioxidant that donates an electron. Cholesterol itself is not as susceptible to oxidation but products such as 7-ketocholesterol have been detected [64]. 4.1.2. Reactive oxygen (ROS) and nitrogen species (RNS) Normally, cells produce superoxide radical via NADPH oxidase as a defense against microorganisms and injured cells may release radicals [65]. Oxidation of lipoproteins within the arterial wall may be mediated by leukocytes, endothelial cells or transition metals [65]. Radicals produced by leukocytes seem especially important because of the instrumental role played by the monocyte/macrophage system in arteriosclerosis. These cells enzymatically generate the ROS superoxide anion radical from oxygen that in turn can give rise to the hydroxyl radical, the most powerful ROS found in biological systems. Fig. 1 illustrates mechanisms that generate ROS. Oxygen itself is a radical [63], because oxygen contains two unpaired electrons, each with the same spin direction. Due to this spin restriction, it reacts sluggishly. Although a very weak radical, it can be induced to react with macromolecules via transition to superoxide or by enzymes or transition metals. Neutrophils and monocytes secrete the enzyme myeloperoxidase that can catalyze the production of the potent oxidant hypochlorous acid from hydrogen peroxide and chloride ion and tyrosyl radical from tyrosine [20,65]. Endothelial cells produce the weak radical NO [66,67]. Although, normally, NO levels are well regulated and NO is considered anti-atherogenic [67], it is induced by cytokines during inflammation from phagocytic and endothelial cells which can lead to sustained production [66,68]. Under this situation,

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Fig. 1. Production of reactive species. ROS can be produced from the sluggish radical oxygen in the mitochondria and endoplasmic reticulum, by various enzymatic reactions and from oxyhemoglobin. RNS can be produced by reaction of superoxide anion radical with the weak radical nitric oxide. Normally nontoxic hydrogen peroxide can give rise to the powerful hydroxyl radical in the presence of transition metals. Oxygen can also be induced to react with biomolecules by transition metals. See the text for details. SOD, superoxide dismutase.

as illustrated in Fig. 1, NO reacts with superoxide anion to produce the powerful reactive nitrogen species (RNS) peroxynitrite. It can directly promote oxidation of lipoproteins [65] or give rise to other species that can promote oxidation [69 –72]. Myeloperoxidase from leukocytes can also generate RNS [20,73,74]. 4.1.3. Transition metals Iron and copper are powerful catalysts of oxidation. As illustrated in Fig. 1, in the reduced form, these metals can reduce hydrogen peroxide to hydroxyl radical-the Fenton-type reaction. As described by Haber, Weiss and Willsta¨tter, in the oxidized form, they can react with superoxide anion to revert to the reduced form [75]. Thus, in a biological system, when iron or copper is present, there is a potential for the continuous production of hydroxyl radicals. Analysis of tissue from arteriosclerotic tissue by isotopic dilution mass spectrometry suggests that metal catalyzed radical production may not be physiologically important in oxidizing LDL proteins in early arteriosclerosis [20]. Nevertheless, there are other ways in which metals can facilitate oxidation. Both iron and copper can directly catalyze the peroxidation of lipids in lipoproteins [65,76]. Transition metals can exist in several spin states. As such, they can arrest the spin restriction of oxygen [77] and can react with oxygen to produce potent metal-containing oxidants [78]. Furthermore, they may allow simultaneous binding or bridging of a biomolecule and oxygen [76,77]. Lipid peroxides in the presence

of oxygen can reduce Cu2 + to Cu+, creating a peroxyl radical. Cu+ can be oxidized to Cu2 + by lipid peroxides to produce an alkoxyl radical, thereby propagating a chain of autoxidation [79]. Similarly, iron can facilitate lipid peroxidation [76]. Protein-bound transition metals catalyze lipoprotein oxidation, either directly or after release from the protein. Iron is bound as Fe3 + in the storage form, which is normally not toxic, but reducing agents may convert bound iron to Fe2 + causing its release, whereby it becomes reactive [76,77,80]. Free cellular iron may reside in a labile chelatable pool [80]. Increases in this pool may facilitate oxidation [81,82]. One of the seven coppers in the acute phase protein ceruloplasmin can catalyze the oxidation of lipoproteins in vitro as readily as free copper, and hence ceruloplasmin is a potentially important physiological prooxidant, although evidence of this in animals and humans has not yet been reported [83 – 85]. 4.1.4. Amino acid and protein damage Various oxidizing agents can cause carbonyl formation with peptide bond cleavage, cross-linking or modification of side chains, especially lysine, arginine and proline [86,87]. Reactive aldehydes, many of which are strong electrophiles, can form Michael adducts with cystine, histidine and lysine [87,88]. Tyrosine residues can be modified by ROS, RNS and hypochlorous acid to o,oV-tyrosine, dihydroxy-phenylalanine, and nitro- and chloro-forms [20,87].

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4.2. Antioxidants Antioxidants may be characterized into various groups such as: (1) enzymes that eliminate ROS or ROS precursors such as superoxide dismutase (SOD) and catalase; (2) thiol oxidative/reduction systems that are also enzyme catalyzed; (3) endogenously synthesized substances such as low molecular weight uric acid, bilirubin and high molecular weight proteins; (4) vitamins and other dietary antioxidants. 4.2.1. Enzymes The three forms of SOD are oxidoreductases that contain iron, copper or manganese at the active site. These act as an antioxidant because they can catalyze the simultaneous oxidation and reduction of two molecules of the superoxide anion radical to hydrogen peroxide that normally is nonreactive. But hydrogen peroxide can be reduced by transition metals to produce the hydroxyl radical. Hydrogen peroxide is readily dissipated by glutathione peroxidase and catalase. 4.2.2. Thiol oxidation/reduction systems Glutathione is considered to be the major thiodisulfide redox buffer in biological systems [89]. As illustrated in Fig. 2, it can cycle between the oxidized disulfide form (GSSH) and reduced glutatione (GSH). These reactions are facilitated by glutathione peroxidase and by glutathione reductase, respectively. Other important thio systems include the protein thioredoxin that can be reduced by thioredoxin reductase [90], and lipophilic a-lipoic acid that can be reduced by lipoamide dehydrogenase from its intra-molecular disulfide form to dihydrolipoic acid [91]. 4.2.3. Endogenously synthesized nonthiol substances Low molecular weight uric acid and bilirubin bound to albumin appear to be important aqueous antioxidants that can scavenge ROS [7,92,93]. Albumin alone is also an antioxidant [94]. Bilirubin appears to protect against both ROS and RNS [95]. Another acute phase protein—fibrinogen—has been shown to be an efficient scavenger of ROS and RNS [96,97]. It also protects lipoproteins [98]. Weak radicals such as NO detoxify more powerful radicals [99].

4.2.4. Vitamins a-TOH is by far the most predominant vitamin E isomer in lipoproteins because of a marked discrimination by the liver in secreting it [100]. Other antioxidants contained in lipoproteins include h-carotene, g-tocopherol and coenzyme Q. h-carotene is a precursor of vitamin A. The carotenoids include both a- and h-carotene, lycopene, cryptoxanthin and lutein. These are more effective in savaging radicals generated from within the lipid environment than vitamin E [101]. These vitamin A type antioxidants act as a radical trap, with the peroxyl radical covalently binding to the polyene chain of the carotenoid. In this way, breaking the chain reaction of lipid peroxidation [102]. The phenolic ring of the chromanol nucleus of tocopherols acts as an electron donating reducing agent. Although the phenolic end of tocopherols exhibits polar properties, the molecule is insoluble in aqueous solution because of the long aromatic isoprenoid side chain. These are arranged with the more polar chromanol rings toward the surface of the lipoprotein particle and the aromatic chain towards the core. Each LDL molecule contains five to nine vitamin E molecules [103]. Although vitamin E acts as a free radical scavenger that can react with oxygen, superoxide anion radical and hydroxyl radical [104], due to its lipid solubility, it is mainly a chain breaking antioxidant within the lipoprotein. Because of its very polar chromanol nucleus, it is a better antioxidant than vitamin A at savaging radicals generated from an aqueous environment [101]. It reacts poorly with RNS and does not appear to protect lipoproteins from hypochlorous acid or tyrosyl radical, most likely because these are targeting apo B [68]. Vitamin C in plasma proved superior to other water soluble antioxidants at protecting lipids from ROS [105]. Once vitamin C is depleted, uric acid, albumin bound bilirubin and protein thiols only partially reduce lipid peroxidation [105]. Vitamin C can scavenge hypochlorous acid and tyrosyl radical, thereby, protecting apo B. It also protects lipids from oxidation by RNS [68]. 4.3. Vitamins C and E and other antioxidants as prooxidants and co-antioxidants 4.3.1. Antioxidants as prooxidants As a chain breaking antioxidant within the lipoprotein, vitamin E reacts with a lipid peroxyl radical in

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Fig. 2. Supplemental co-antioxidant recycling of vitamin E and vitamin C. Under normal conditions and in blood, potentially toxic vitamin E and vitamin C radicals can be depleted by cellular recycling mechanisms, as illustrated. Species within the arterial wall spaces may not have adequate access to these mechanisms. As a result, prooxidative conditions may be encouraged. As illustrated, under these conditions, the introduction of additional vitamin E or vitamin C may lead to an increase in radical production due to TMP and transition metal catalysis. See the text for details. GSSG and GSH, oxidized and reduced glutathione, respectively; Asc, ascorbic acid; LOOH, lipid peroxide; LOOb, lipid peroxyl radical.

a single electron reaction resulting in a vitamin E radical (illustrated in Fig. 2). This breaks the chain reaction of lipid peroxidation. If the vitamin E radical reacts with a second peroxyl radical or with another vitamin E radical, the reaction comes to termination [104]. Alternatively, studies in vitro, ex vivo and with liposomes indicate vitamin E, may act as a prooxidant that facilitates rather than terminates lipid peroxidation [4,7,106]. This process that has been called tocopherol-mediated peroxidation (TMP) occurs as follows: (1) vitamin E reacts with an aqueous radical from outside the lipoprotein. (2) The range of movement of the vitamin E molecule within the lipoprotein is limited. As such, if another aqueous radical appears, the vitamin E may react with it, or the vitamin E radical must react with another lipid soluble antioxidant within the lipoprotein. Otherwise, the vitamin E radical must react with an unsaturated fatty acid. (3) In the latter case, a lipid peroxyl radical will be formed that can initiate a chain reaction of lipid peroxidation until it is terminated by another vitamin E or other antioxidant. When a lipid peroxyl radical is formed, vitamin E has transferred the radical reaction from outside to inside the lipoprotein [7,106]. This prooxidant effect occurs more readily under conditions of low radical flux when relatively few aqueous radicals are colliding with the lipoprotein [7,106]. It also occurs in the presence of transition

metals. Especially, when the metals are in low concentrations relative to the concentration of vitamin [107], and when the concentration of vitamin E in the lipoprotein is very low or the initial level of hydroperoxides in the lipid is high [104]. These prooxidant effects appear to be general properties of the chromanol nucleus since the water soluble vitamin E analog trolox and other phenolic nutrients act similarly [108,109]. Vitamin C at low concentrations can also undergo a one electron reaction to form an ascorbyl radical (Fig. 2). Experiments with cell-free systems, isolated membrane systems and under culture conditions indicate that, in the presence of transition metals, vitamin C can readily switch from an antioxidant to a prooxidant [77,105]. The concentration at which this occurs will depend on the concentrations of metal and vitamin C in the solution. Because vitamin C is an antioxidant, at high concentrations, there will be little radical in the solution, but at low concentrations the rate of antioxidant reactions will be slowed, thus, the amount of radical will be increased relatively causing more oxidative damage to biomolecules. Likewise, high concentrations of transition metals will increase prooxidation by vitamin C [77]. Other small water-soluble antioxidants behave similarly in vitro. Thus, uric acid and even glutathione can promote oxidation in the presence of transition metals

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[110,111]. Sulfur species may also induce oxidative stress by reacting as reactive species and by consuming glutathione and other thiol antioxidants [112]. Even vitamin A can exhibit prooxidant properties since additions to the polyene chain lead to liberation of radical cleavage products [101]. 4.3.2. Co-antioxidants In cell-free systems, cell culture, isolated membrane systems and liposomes, several antioxidants, which include h-carotene, ubiquinol-10 (reduced form of coenzyme Q), 3-hydroxyanthranilic acid, albuminbound bilirubin and ascorbic acid, can supplement vitamin E in a way that reduces TMP [4,7,106,113]. It appears that the latter four antioxidants can recycle vitamin E radical back to vitamin E [4,7]. For this purpose, vitamin C appears to be most important [114,115]. It preserves vitamin E in LDL during oxidative stress [116] and, as shown in Fig. 2, protects lipoproteins by coupling the glutathione redox system to vitamin E [117]. It can actually produce a synergistic effect where the combined effect is greater than the sum of the individual antioxidants [114,118]. This is true for synthetic trolox as well as natural vitamin E

[119,120]. The synergistic effect between vitamin C and vitamin E is illustrated in Fig. 3, where the lag phase difference between the copper control and vitamin E and vitamin C alone equals 39 min each, while that for the vitamins together equals 133 min. This amounts to an 80% increase over the sum of each alone. Recycling of vitamin E radical by vitamin C gives rise to the ascorbyl radical (semidehydroascorbic acid) that disproportionates to ascorbic acid and dehydroascorbate (DHA) [113,122]. As illustrated in Fig. 2, much DHA is reduced back to ascorbic acid by recycling with glutathione and NADPH within cells [4,123]. Leukocytes accumulate external DHA and recycle it back to ascorbic acid rapidly [124]. Neither glutathione nor NADP can directly recycle vitamin E but can act only through vitamin C [119,125,126]. aLipoic acid and possibly thioredoxin can also couple with DHA to regenerate vitamin C [90,91]. Although the degree to which these recycling systems actually function in vivo remains unclear, it appears that under normal cellular conditions, a system exists whereby TMP or prooxidation by other nutrients can be retarded and peroxidation caused by such effects reduced.

Fig. 3. Supplemental antioxidant interactions. Oxidation of 0.03 g/l LDL was catalyzed by 5 Amol/l copper in vitro and antioxidants added as previously described [98]. LDL, density between 1.020 and 1.063, was isolated by sequential ultracentrifugation in the presence of 100 mmol/l EDTA and frozen at 70 jC in aliquots. EDTA was removed by dialysis prior to the experiment and oxidation was followed by conjugated diene formation at 234 nm [98]. Lag phase time was determined as the average slope of the propagation phase from each set of duplicates [121]—indicated by the point at which the dotted lines cross the x-axis. The water soluble vitamin E analog trolox was used as a substitute for natural vitamin [98]. Its chromanol nucleus and oxidation properties are very similar to a-tocopherol.

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Recycling is not a necessary prerequisite for antioxidants to act as co-antioxidants. The requirement is only that a prooxidant effect be reduced.

5. Discussion and conclusions Much experimental data suggests that oxidative stress contributes to arteriosclerosis and it is well established that vitamin E and vitamin C are important antioxidants that should protect tissue [3,14]. Still, as indicated above, most data linking oxidative mechanisms to arteriosclerosis has been obtained from in vitro or animal models and direct evidence for the oxidative modification hypothesis in humans is sparse [1,3]. It was widely thought that randomized human trials with ingested antioxidants in humans would be direct evidence supporting this hypothesis in humans [1]. Yet, to date in vivo studies using vitamins failed to show a reduction in heart disease with antioxidant therapy [127]. Although most studies did not measure the levels of plasma vitamin E or other antioxidants with treatment, the overall consistency of the results strongly support the conclusions that vitamin E alone or as an ingredient in vitamin cocktails failed to retard arteriosclerosis. The failure of randomized antioxidant trials to retard arteriosclerosis is not sufficient for rejecting a hypothesis that is supported by so much indirect evidence including epidemiological studies in humans and animal models [3]. Rather, there are a number of reasons why vitamin antioxidants might not be effective inhibitors of arteriosclerosis in humans [3]. One reason for the failure to achieve a positive effect may be due to the prooxidant effects of the therapeutic agents themselves. As illustrated in Fig. 2, data from in vitro studies indicate vitamin E and vitamin C may produce toxic radicals. Besides, supplementation with 500 mg/day vitamin C has been shown to have prooxidant effects in humans in vivo [128], although serious criticisms have been raised regarding artifactual ex vivo oxidation and other deficiencies in this study [129]. Also, after muscle injury in the human arm that caused the release of free iron, ingestion of an antioxidant cocktail of vitamin C and N-acetyl-cysteine caused an increase in serum lipid hydroperoxides and prostaglandins indicating a prooxidant effect [130]. Recent evidence suggest arteriosclerosis is a systemic process occurring at multiple sites [10 – 12].

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This may be due to a systemic insult such as hypertension, glucose intolerance, or hypercholesterolemia that damages many sites or due to a generalized facilitation of the oxidative or inflammatory state of individuals. Endothelial dysfunction occurs in coronary and peripheral vascular beds [131,132]. Damage allows the entry of serum proteins and small watersoluble antioxidants as well as lipoproteins into the intima wall. The definition of this intimal microenvironment is not entirely clear [68], but it is known that the oxLDL concentration is about 70X higher in plaques than in plasma and is associated with plaque instability [133]. Superoxide is generated more in unstable angina as compared to more stable plaques [134,135]. Plaques contain cholesterol and fatty acid oxidation products including 7-ketocholesterol, MDA, HODEs and F2-isoprostanes, and nitrotyrosine [136 – 138]. Even transplant associated arteriosclerosis seems dependent on this pathogenesis since oxLDL antibodies appear elevated in this condition [139], and lipid lowering treatment reduces graft rejection [140]. Plasma antioxidants may retard peroxidation initially, but ultimately reactive species from leukocytes, metals in proteins such as ceruloplasmin, and metals from other sources should predominate in some sites. Seeding peroxides, either produced by 15-lipoxygenase or reactive species, are found in plaque LDL [34,79]. Myeloperoxidase is found associated with macrophages in lesions [20]. Redox active iron and copper are found in advanced atherosclerotic plaques but not normal tissue [20,34,79], and copper levels are associated with enhanced arteriosclerosis and elevations in LDL auto-antibodies. Vitamin E and vitamin C are found in the plaque milieu and vitamin C is accumulated by leukocytes [4,34,124,136]. Normal cellular recycling capabilities and co-antioxidant interactions such as those discussed above and illustrated in Fig. 2 [122,123,141] may be greatly reduced. Thus, glutathione peroxidase and reductase activity were markedly lower in plaques as compared to normal artery tissue [142]. The compartmentalization of phagocytes with lipoproteins and transition metals are apt to produce conditions of low radical flux that facilitate TMP and transition metal catalyzed peroxidation. Moreover, inducible NO is apt to increase, leading to RNS. Although it is unlikely that vitamin C acts as a prooxidant in plasma [105,143], the sequestering of vitamin C along with transitional

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metals could produce toxic combinations [77]. Since the interior of advanced human arteriosclerotic lesions is a highly prooxidant environment [144] containing enzymatic and nonenzymatic oxidation products [135,137], the haphazard introduction of additional amounts of vitamins or other phenolic nutrients for therapeutic purposes could potentially be hazardous. Some have suggested that statins, angiotensin converting enzyme inhibitors [145] or other substances that may have antioxidant effects by blocking the production of radicals, rather than acting as antioxidants per se, should be the way future research is directed [5]. We agree that these areas of research are important. But, at the same time, we believe that continued research into the interactions between antioxidants and prooxidants that occurs in living system may provide sufficient understanding so that beneficial antioxidant cocktails can be developed. It is likely that mechanisms can be manipulated towards recycling or otherwise supplementing antioxidants for benefit. Thus, it appears that cellular vitamin C and vitamin E within a limited range of concentrations can compensate for depletions in glutathione [146]. The recently completed 6 year ASAP study suggests that vitamin C may be a better supplement when given in time release form [147]. Some therapeutic lipophilic phenoxy antioxidants such as probucol may retard arteriosclerosis [107]. Other antioxidants such as a-lipoic acid are readily absorbed from the diet and could conceivably supplement vitamins synergistically [88]. Antiinflammatory effects of a-TOH are largely due to natural vitamin E, hence, synthetic all-rac vitamin E may not produce full benefit [148]. Elegant advances in identifying and quantifying specific oxidation products by mass spectrometry (MS) should provide information on basic and clinical redox interactions of the kind discussed in this review that may lead to techniques whereby antioxidants can be introduced into tissue in a controlled way for reducing arteriosclerosis [20]. Besides, the lipid oxidation products HNE, MDA, F2-isoprostanes and HODEs, MS can measure protein oxidation products from metals, hypochlorous acid, RNS and ROS [20]. Thus, it was shown that nitrotyrosine levels but not Creactive protein was significantly decreased with statin therapy implicating peroxynitrite or myeloperoxidase [149]. This type of information will be useful in

identifying interventions that specifically effect relevant physiological pathways to be tested in clinical trials [20].

Acknowledgements This work was supported by the Department of Veterans Affairs, Louisville, KY. We thank the Reviewers for thoughtful and helpful suggestions that improved this article.

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