Role of vitamin E as a lipid-soluble peroxyl radical scavenger: in vitro and in vivo evidence

Free Radical Biology and Medicine 66 (2014) 3–12

Contents lists available at ScienceDirect

Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed

Review Article

Role of vitamin E as a lipid-soluble peroxyl radical scavenger: in vitro and in vivo evidence Etsuo Niki n Health Research Institute, National Institute of Advanced Industrial Science & Technology, Ikeda, Osaka 563-8577, Japan

art ic l e i nf o

a b s t r a c t

Available online 2 April 2013

Multiple reactive oxygen/nitrogen species induce oxidative stress. Mammals have evolved with an elaborate defense network against oxidative stress, in which multiple antioxidant compounds and enzymes with different functions exert their respective roles. Radical scavenging is one of the essential roles of antioxidants and vitamin E is the most abundant and important lipophilic radical-scavenging antioxidant in vivo. The kinetic data and physiological molar ratio of vitamin E to substrates show that the peroxyl radicals are the only radicals that vitamin E can scavenge to break chain propagation efficiently and that vitamin E is unable to act as a potent scavenger of hydroxyl, alkoxyl, nitrogen dioxide, and thiyl radicals in vivo. The preventive effect of vitamin E against the oxidation mediated by nonradical oxidants such as hypochlorite, singlet oxygen, ozone, and enzymes may be limited in vivo. The synergistic interaction of vitamin E and vitamin C is effective for enhancing the antioxidant capacity of vitamin E. The in vitro and in vivo evidence of the function of vitamin E as a peroxyl radical-scavenging antioxidant and inhibitor of lipid peroxidation is presented. & 2013 Elsevier Inc. All rights reserved.

Keywords: Antioxidant Free radicals Lipid peroxidation Reactive oxygen species Vitamin E

Contents Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Mechanisms, dynamics, and efficacy of radical scavenging by vitamin E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Rate of scavenging radicals by vitamin E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Rate of scavenging nonradical oxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Fate of vitamin E-derived radicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Distribution and composition of lipids and fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Efficacy of scavenging of radical and nonradical oxidants by vitamin E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 In vitro evidence of scavenging of peroxyl radicals by vitamin E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 In vivo evidence of scavenging of peroxyl radicals by vitamin E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Introduction The aerobic organisms are protected from oxidative stress induced by reactive oxygen/nitrogen species (ROS/RNS)1 by an elaborate defense network in which multiple antioxidants with diverse functions play their roles [1,2]. Some antioxidants are small molecules, whereas others are macromolecules such as proteins and

Abbreviations: AAPH, 2,2′-azobis(2-amidinopropane) dihydrochloride; HETE, hydroxyeicosatetraenoic acid; H(P)ODE, hydro(pero)xyoctadecadienoic acid; PUFA, polyunsaturated fatty acid; RNS, reactive nitrogen species; ROS, reactive oxygen species. n Corresponding author. Fax: +81 3 5313 2555. E-mail addresses: [email protected], [email protected] 0891-5849/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.freeradbiomed.2013.03.022

enzymes. The physiological antioxidant systems have several lines of defense. In the first line, antioxidants prevent the production of ROS/ RNS and other reactive species by, for example, sequestering active metal ions and reducing hydroperoxides and hydrogen peroxide to hydroxides and water, respectively. In the second defense line, antioxidants scavenge, quench, or remove ROS/RNS and other reactive species before they attack biological molecules. In the third defense line, antioxidant compounds and enzymes repair the damage and reconstitute membranes and tissues. Furthermore, low levels of oxidative stress induce an adaptive response, which accelerates the production of antioxidant proteins and enzymes and transfers them to the right site at the right time and in the right amounts [3,4]. Thus, antioxidants act cooperatively and synergistically in the defense network to cope with oxidative stress. Oxidative

4

E. Niki / Free Radical Biology and Medicine 66 (2014) 3–12

stress proceeds by both free radical-mediated mechanisms and nonfree radical mechanisms. Both free radical and nonradical oxidants attack and modify biological molecules, and the disruption of thiol redox circuits induced by, at least in part, ROS/RNS also causes oxidative stress [5]. Radical scavenging is one of the important functions of antioxidants. Several kinds of free radicals are involved in vivo, such as superoxide (O2
−), hydroxyl (HO
), alkoxyl (RO
), peroxyl (RO2
), aryloxyl (ArO
), nitric oxide (
NO), nitrogen dioxide (NO2
), thiyl (RS
), thiyl peroxyl (RSOO
), sulfonyl (RSO2OO
), and carboncentered radicals (R
). Some radicals are quite reactive, whereas others are not. Superoxide and nitric oxide are not reactive enough per se to directly attack biological molecules. Free radicals attack biologically essential molecules such as lipids, proteins, carbohydrates, and DNA by hydrogen atom abstraction, addition to double bond, and electron transfer reactions. Similarly, free radical-scavenging antioxidants (IH) react with free radicals by one of the following three reaction mechanisms. The relative importance of these reactions depends on the radicals, antioxidants, and microenvironment: hydrogen abstraction, X
+IH-XH+I
;

(1)

addition, X
+CQC-X–C–C
;


−

(2)
+

electron transfer, X +IH-X +IH

−




+

-X +I +H .

(3)

Vitamin E, vitamin C, carotenoids, ubiquinol (reduced form of coenzyme Q), uric acid, bilirubin, and thiyl compounds are major radical-scavenging antioxidants in vivo. Notably, vitamin E and vitamin C are the essential lipophilic and hydrophilic radicalscavenging antioxidants, respectively. It was suggested that vitamin E may also function as a signaling mediator independent of antioxidant function, which is a matter of debate [6,7]. This article presents the evidence that vitamin E acts as a peroxyl radicalscavenging antioxidant in vitro and in vivo.

Mechanisms, dynamics, and efficacy of radical scavenging by vitamin E Vitamin E has eight isoforms, α-, β-, γ-, and δ-tocopherol and α-, β-, γ-, and δ-tocotrienol [8]. Tocopherols have a saturated side chain, called a phytyl side chain, at the 2 position of the chromanol ring, whereas tocotrienols have a side chain with three double bonds at positions 3′, 7′, and 11′. α forms have three methyl groups on the chromanol head at positions 5, 7, and 8. There are two methyl groups

Scheme 1. α-Tocopherol action as a radical-scavenging antioxidant against lipid peroxidation. Vitamin E scavenges lipid peroxyl radical (LO2
) before it attacks lipid (LH) to yield lipid hydroperoxide (LOOH) and a lipid radical (L
), which propagates the chain oxidation. The resulting vitamin E radical (E
) may (1) be reduced by ascorbic acid (C) or ubiquinol to regenerate vitamin E, (2) attack LH or LOOH, (3) react with LO2
to yield an adduct, or (4) react with another vitamin E radical to yield a dimer, a nonradical stable product. The rate constants (k) are shown in M−1 s−1; P is phytyl side chain.

on the chromanol head in the β (positions 5 and 8) and γ (positions 7 and 8) forms, whereas the δ forms have one methyl group at position 8. Natural tocopherols synthesized by plants contain only one stereoisomer, RRR, whereas synthetic tocopherols contain equimolar concentrations of all possible stereoisomers at positions 2, 4′, and 8′ (RRR, SRR, RSR, RRS, SSR, SRS, RSS, SSS). The phenolic hydrogen at position 6 is the active site for scavenging radicals and the side chain at the position 2 does not affect the reactivity toward free radicals; that is, tocopherols and the corresponding tocotrienols have the same scavenging capacity for free radicals [9]. The relative reactivity of α, β, γ, and δ forms toward oxygen radicals decreases in the order of α > β ¼ γ > δ [9–13]. Notably, α-tocopherol has the highest bioavailability because of its highest affinity to α-tocopherol transfer protein [14] and low rate of metabolism [15]. Whether the various vitamin E isoforms have specific functions has been the subject of argument, but in this article the focus is on α-tocopherol, which has the highest biological activity. Vitamin E scavenges active free radicals primarily by hydrogen atom transfer reaction to yield a nonradical product and vitamin E radical (Reaction (1)). Under certain conditions, vitamin E may scavenge radicals by a concerted mechanism in which an electron is transferred to yield a vitamin E cation radical, which undergoes rapid deprotonation to give a vitamin E radical (Reaction (3)). When vitamin E scavenges lipid peroxyl radical, lipid hydroperoxide and vitamin E radical are formed. The resulting vitamin E radical may undergo several reactions as shown in Scheme 1 [16]. It may react with another radical to give stable products, attack lipids, or react with a reducing agent such as ascorbate or ubiquinol to regenerate vitamin E. The products of vitamin E with peroxyl radicals have been identified [17,18]. Rate of scavenging radicals by vitamin E The rate of scavenging radicals by vitamin E is determined by the rate constant, kE, and the concentrations of vitamin E and radical (Eq. (4)): rate of radical scavenging ¼ kE[E][radical].

(4)

The relative reactivity of vitamin E toward free radicals has been measured by many groups, but the absolute rate constants, which are essential for understanding the action of vitamin E as a radical-scavenging antioxidant, have been measured in a limited number of studies. The absolute rate constants for scavenging peroxyl radicals by α-tocopherol measured by several groups are summarized in Table 1. The rate constants vary considerably over the range of 3 orders of magnitude depending on the radicals and circumstances. Trichloromethylperoxyl radical is quite reactive and reacts with α-tocopherol at a near diffusion controlled rate, the rate constant being 5.0  108 M−1 s−1 [19]. Lipid peroxyl radicals and related alkyl peroxyl radicals are less reactive and scavenged by α-tocopherol with a rate constant ranging from 6  103 to 7  106 M−1 s−1. One of the important factors that affect the rate constant is the solvent. Notably, the hydrogen bonding between α-tocopherol and solvent such as alcohol reduces the rate constant [20]. Furthermore, the apparent reactivity of vitamin E is reduced in the membranes, lipoproteins, and micelles, probably because of the limited mobility of the molecules [21–24]. The absolute rate constants for the reactions of several free radicals with antioxidants and lipids are compiled in Table 2. The reactivity of methylene groups of lipids, –CH2–, toward free radicals increases in the order of saturated lipids such as stearic acid o mono-olefins such as oleic acid o polyunsaturated fatty acids such as linoleic and arachidonic acids. The reactivity of polyunsaturated fatty acids (PUFAs) increases in proportion to the number of double bonds. The reactivity of oxygen radicals

E. Niki / Free Radical Biology and Medicine 66 (2014) 3–12

5

Table 1 Absolute rate constants for scavenging peroxyl radicals by α-tocopherol reported in the literature. Peroxyl radicala

Temp. (1C)

Cyclohexylperoxyl MeLOO Cumylperoxyl Styrene peroxyl Styrene peroxyl Low-density lipoprotein Cumylperoxyl MeLOO Phenylethylperoxyl LOO LOO PCLOO

Solvent

20 37 25 30 30 37 25 37 25 40 37 37

Rate constant

Ref.

6

6.8  10 3.5  106 3.4  106 3.2  106 2.35  106 5.9  105 5.6  105 5.1  105 1.5  105 6.0  104 3.7  104 5.8  103

Cyclohexane Benzene Benzene Chlorobenzene Chlorobenzene Aqueous suspension tert-Butyl alcohol tert-Butyl alcohol/methanol (3/1) o-Dichlorobenzene SDS micelle SDS micelle Dilinoleoyl PC liposome

[25] [26] [27] [28] [10] [29] [27] [30] [31] [32] [13] [33]

Rate constants are given as M−1 s−1. a

MeLOO, methyllinoleoylperoxyl radical; LOO, linoleoylperoxyl radical; PCLOO, phosphatidylcholine linoleoylperoxyl radical.

Table 2 Absolute rate constants for the reactions of free radicals with lipids and antioxidants. Substrate

Radical Peroxyl o10 o1 62

Stearic acid (18:0) Oleic acid (18:1) Linoleic acid (18:2) Linolenic acid (18:3) Arachidonic acid (20:4) EPA (20:5) DHA (22:6) Cholesterol α-Tocopherol Ascorbic acid β-Carotene Glutathione Reference

197 249 334 11 3.5  106 2.2  106

Alkoxyl

Hydroxyl 6

10 109 9  109 7  109 1010

3.1  108

1010 1010

Rate constants are given as M

[95]

Nitrogen dioxide

Glutathione thiyl

o1 2  105

o 2  106 8  106 1.9  107 3.1  107

3.5  107 1.1  108 2  107 [40,96]

3.6  108 2.2  108

9

2.3  10 3.3  106 8.8  106 1.3  107 2.0  107

a

[94,95] −1

a

−3

1010 [95]

[97,98]

−1

s . DHA, docosahexaenoic acid.

The reactivity of glutathione toward peroxyl radical is less than that of ascorbate, but larger than that of uric acid [99].

increases in the order of peroxyl o alkoxyl o hydroxyl radical, whereas their selectivity decreases in this order. Thus, the reactivity of linoleic acid toward peroxyl radical is 5 orders of magnitude larger than that of stearic acid, and the difference between the two lipids is only about 10-fold toward very reactive yet nonselective hydroxyl radical. Glutathione thiyl radical and nitrogen dioxide react with unsaturated lipids primarily by addition to a double bond rather than a hydrogen atom abstraction reaction [34,35]. A number of thiol compounds play important roles as endogenous antioxidants in vivo. For example, glutathione plays an essential role in the defense network together with glutathione peroxidases [36,37]. At the same time, thiols (RSH) react readily with free radicals to give thiyl radicals, which may undergo several secondary reactions [36]. It has been observed that thiol-derived thiyl radicals initiate and enhance lipid peroxidation [38,39]. Table 2 shows that thiyl radicals react with PUFAs rapidly, the rate constant being as large as 107 M−1 s−1 [38], much larger than that for the reaction of peroxyl radicals with PUFAs. Thiyl radical may react with molecular oxygen to give RSOO
), which rearranges to sulfonyl radical (RSO2
) and reacts with oxygen to give RSO2OO
) [40]. These thiol-derived radicals may react with PUFAs by either hydrogen abstraction or addition to double bond and contribute to lipid peroxidation. Rate of scavenging nonradical oxidants The oxidative stress is mediated not only by free radicals but also by nonradical species. Hydrogen peroxide, singlet oxygen,

ozone, and hypochlorite are important nonradical oxidants. It may be noted that the oxidation by nonradical oxidants is stoichiometric, whereas the free radical-mediated oxidation proceeds by a chain mechanism, that is, one nonradical oxidant oxidizes one molecule of substrate, and one initiating free radical oxidizes many target molecules. Vitamin E is not an efficient quencher of hydrogen peroxide or superoxide. Singlet oxygen is one of the important oxidants produced in vivo [41]. The rate constants for the reactions of singlet oxygen with a variety of compounds have been measured by many groups [42]. α-Tocopherol scavenges singlet oxygen predominantly by physical quenching rather than chemical reaction [43,44] and the reported rate constants range from 1  108 to 7  108 M−1 s−1 [43–48]. The rate decreases in the order α- > β- > γ- > δ-tocopherol [46,49,50]. An ester or ether derivative at the 6 position of the chromanol ring abolished the activity of singlet oxygen scavenging, suggesting that the 6hydroxy group is the active site [46]. Many carotenoids such as lycopene and β-carotene quench singlet oxygen with a rate constant larger than 1010 M−1 s−1 [45], which is about 2 orders of magnitude higher than that for α-tocopherol. Because singlet oxygen reacts with PUFAs with rate constants larger than 105 M−1 s−1 [51] and the ratio of the rate constants kE/kS is about 103, the efficacy of α-tocopherol for inhibiting singlet oxygenmediated oxidation may be limited in vivo (see later text). It may be added that, in contrast to free radical-mediated lipid peroxidation, unsaturated compounds without bis-allylic hydrogen, such as oleic acid, squalene, and cholesterol, may also be important targets of singlet oxygen.

6

E. Niki / Free Radical Biology and Medicine 66 (2014) 3–12

Ozone is a powerful oxidant and induces oxidative damage [52]. Ozone reacts with α-tocopherol and PUFAs at similar rates, the rate constant being about 106 M−1 s−1 [53], and therefore α-tocopherol cannot act as an efficient scavenger of ozone in vivo. Ozone is not itself a radical but produces free radicals in its reactions with olefins and increases lipid peroxidation products [54], which can be suppressed by α-tocopherol. Hypohalous acids such as hypochlorous acid (HOCl), hypobromous acid (HOBr), and hypothiocyanous acid (HOSCN) produced by enzymatic reactions of myeloperoxidase (MPO) with hydrogen peroxide and halides or pseudohalide are also important oxidants and play an important defense role against invading pathogens [55,56]. However, there is considerable evidence that excessive levels of oxidants produced by MPO can result in host damage and MPO has been implicated in the pathogenesis of various inflammatory diseases. Hypochlorite reacts with unsaturated lipids and cholesterol to produce chlorohydrins as a major product [57,58], but it reacts with amines and thiols preferentially, and proteins are the major target [59]. Neither α-tocopherol nor ascorbate was effective against the formation of carbonyl compounds in HOClinduced oxidation of albumin [60] or oxidation of human plasma [59]. Hypochlorous acid reacts with hydrogen peroxide to produce singlet oxygen, but the reaction of hypochlorous acid and lipid hydroperoxides does not produce singlet oxygen [61], although the resulting lipid peroxyl radicals may give singlet oxygen [62]. Furthermore, free radicals are formed from the reaction products of hypochlorous acid and proteins, for example, chloroamines [63], and α-tocopherol may prevent oxidation induced by such free radicals.

Fate of vitamin E-derived radicals The overall capacity of antioxidants is determined not only by the rate of scavenging radicals but also by several other factors as discussed previously [1,64–66]. Among others, the fate of antioxidant-derived radicals formed when the antioxidant scavenges radicals is important in determining the antioxidant efficacy. If the antioxidant-derived radical is reactive enough to continue oxidation, the scavenging of radicals by antioxidant results in simply a chain transfer and this may induce prooxidant actions of the antioxidant under certain circumstances. As discussed above, thiols scavenge radicals rapidly, but the resulting thiyl radicals are reactive and may attack biological molecules and continue oxidation. Hydroquinones such as α-tocopheryl hydroquinone and ubiquinol scavenge radicals faster than α-tocopherol, but the semiquinone radicals derived from hydroquinones are not stable and may react with oxygen to give quinone and hydroperoxyl radical, which may continue oxidation. Therefore, the apparent antioxidant efficacy of α-tocopheryl hydroquinone and ubiquinol may become smaller than that of α-tocopherol despite higher reactivity [67]. As shown in Scheme 1, the α-tocopheroxyl radical may undergo several reactions. It was found that α-tocopherol acted as a prooxidant in the oxidation of low-density lipoprotein (LDL) [68], which may be one of the important initial events in the pathogenesis of atherosclerosis [69]. This pro-oxidant action is ascribed to phase-transfer and chain-transfer reaction by α-tocopheroxyl radical, that is, α-tocopherol present in LDL particles scavenges radicals in aqueous phase and the resulting α-tocopheroxyl radical attacks PUFAs to initiate a chain reaction [70]. This is a special feature of the oxidation of small lipoprotein particles in aqueous dispersions [71]. The α-tocopheroxyl radical is relatively stable but still can abstract a bis-allylic hydrogen atom from PUFAs with a rate constant of 2.7  10−2 M−1 s−1 [72]. It may be noted that this reaction is exothermic, the bond dissociation energy of the

phenolic O–H bond of α-tocopherol and the bis-allylic hydrogen of the PUFA being 78 and 75 kcal/mol, respectively [73]. It is known that ascorbate is an efficient reductant of α-tocopheroxyl radical. It was found by pulse radiolysis [19] and electron paramagnetic resonance [74] studies that ascorbate is capable of reducing α-tocopheroxyl radical rapidly. In fact, α-tocopherol is spared during the oxidation of lipids in the presence of ascorbic acid and α-tocopherol is consumed only after depletion of ascorbic acid [30]. Such a sparing effect of ascorbic acid on α-tocopherol takes place during oxidation not only in homogeneous solution but also in heterogeneous systems such as micelles [76,152], liposomal membranes [77–79], LDL [68,80], plasma [81], platelets [82], whole blood [83], and cultured cells [84]. Barclay et al. [85] found very significant extensions of the efficient inhibition period by a combination of α-tocopherol and ascorbic acid compared to that obtained with α-tocopherol or ascorbic acid alone in the micellar oxidation of methyl linoleate. This interaction is observed with other antioxidants as well: notably, ubiquinol, α-tocopheryl hydroquinone, and polyphenolic compounds reduce α-tocopheroxyl radical and regenerate α-tocopherol [67,86–88]. Glutathione and cysteine also reduce α-tocopheroxyl radical, but uric acid does not [74,89,90]. The results observed in the oxidation of human LDL clearly show that, although uric acid spares α-tocopherol in the oxidation induced by hydrophilic radicals, it cannot spare α-tocopherol in the lipophilic radical-induced oxidation, whereas ascorbic acid can spare α-tocopherol in both cases. Importantly, this suggests that the pecking order of the antioxidants depends on the location of free radicals and antioxidants as well as the reactivity of antioxidants toward free radicals [65,75]. This interaction between α-tocopherol and ascorbic acid is important because it sustains the antioxidant capacity of α-tocopherol by recycling and suppresses pro-oxidant action. Whether such a synergistic interaction between vitamins E and C is important in vivo has been argued, some studies are positive [91,92], whereas others are negative [93]. Distribution and composition of lipids and fatty acids The inhibitory effect against lipid peroxidation depends on the concentration, distribution, and composition of lipid classes and fatty acids as well as the reactivity and concentration of antioxidant. The reactivity of lipids toward peroxyl radical decreases in the order of PUFA > cholesterol > monounsaturated fatty acids > saturated fatty acids (Table 2). The lipid classes and fatty acid composition vary markedly between tissues and possibly between individuals and they depend also on diet. For example, the major PUFA in human plasma, in particular in cholesteryl ester, is linoleate, whereas erythrocytes contain relatively more arachidonate [100,101]. In contrast, arachidonic acid and docosahexaenoic acid (DHA) are the major PUFAs in the brain and retina. Fatty acid composition depends on the brain region. Phosphatidylethanolamine (PE) contains more PUFA than phosphatidylcholine and, interestingly, a considerable amount of adrenic acid (22:4 n–6) was observed in brain PE [102]. The reactivity of bis-allylic hydrogen in free fatty acids and esters is substantially the same. The molar ratio of oxidizable lipids to vitamin E is an important factor that determines antioxidant efficacy of vitamin E. The contents of lipids, fatty acids, and vitamin E in human LDL particles were measured by several groups. For example, the molar ratios of total lipids, fatty acids, and PUFAs to vitamin E were reported as 500/1, 400/1, and 200/1, respectively [103]. LDL acts as a carrier of vitamin E for distribution to various tissues and contains a relatively high proportion of vitamin E. Molar ratios of phospholipid fatty acids to α-tocopherol in rat liver subcellular membrane fractions were on the order of several thousands to 1 [104], whereas the molar ratios of PUFA to α-tocopherol in microsomes

E. Niki / Free Radical Biology and Medicine 66 (2014) 3–12

7

cytotoxic per se and capable of modifying proteins and DNA bases. Therefore, it is important to inhibit lipid peroxidation.

from lung, heart, liver, kidney, testis, and brain of rat were reported as 300, 710, 1820, 1920, 1960, and 2630 to 1, respectively [105]. Thus, the molar ratio of PUFA to vitamin E varies considerably but may be as high as several thousands to 1. Another important issue that determines the antioxidant efficacy of vitamin E is its localization in the membranes. Structural distribution and role of vitamin E in the membranes are not fully understood yet. In the “Singer and Nicholson fluid mosaic model” [106], both lipids and proteins are free to diffuse in the membrane and form a random organization. In the later “lipid domain concept,” it was proposed that lipids as well as proteins were nonhomogeneously distributed and organized in domains in the membranes [107]. More recent studies on the topology and architecture of cellular membranes reveal the heterogeneity of lipid and protein distribution [108–113]. It is assumed that sphingolipids and cholesterol form lipid rafts, which serve as the platform for signaling proteins, whereas PUFAs form nonraft domains. α-Tocopherol is assumed to be enriched in PUFA domains, which facilitate antioxidant function as inhibitors of lipid peroxidation [104,113].

In vitro evidence of scavenging of peroxyl radicals by vitamin E The action of vitamin E as an antioxidant against in vitro lipid peroxidation has been studied extensively [reviewed in 23,65]. The mechanisms and dynamics of lipid peroxidation have also been studied extensively and are now well understood [115,116]. Numerous studies show clearly that vitamin E inhibits lipid peroxidation in the test tube as assessed by oxygen uptake, substrate consumption, and lipid peroxidation products formation. PUFAs are quite vulnerable to oxidation. Methyl linoleate is oxidized spontaneously in the presence of α-tocopherol at room temperature under laboratory light. α-Tocopherol is consumed and methyl linoleate hydroperoxide is accumulated concomitantly, and when α-tocopherol is depleted, a rapid oxidation proceeds. Notably, in the presence of α-tocopherol, cis,trans-hydroperoxides are formed exclusively, whereas after depletion of α-tocopherol, trans, trans-hydroperoxides are formed predominantly [117]. These results suggest that α-tocopherol scavenges peroxyl radicals derived from methyl linoleate rapidly before the peroxyl radical isomerizes from cis,trans form to trans,trans form. An example of the oxidation of plasma induced by free radicals produced at a constant rate is shown in Fig. 1. In the initial stage of the oxidation of fresh plasma, only ascorbic acid was consumed and α-tocopherol was spared efficiently, and then, after complete depletion of ascorbic acid, α-tocopherol began to decrease. The lipid peroxidation was inhibited completely in the presence of ascorbic acid and cholesteryl ester and phosphatidylcholine hydroperoxides and hydroxides began to be accumulated after depletion of ascorbic acid even in the presence of α-tocopherol [118]. Similar results were reported by Frei and his colleagues [81]. When plasma is dialyzed, water-soluble small-molecule antioxidants such as ascorbic acid and uric acid are removed, whereas vitamin E is not. In the oxidation of dialyzed plasma, lipid hydroperoxides are produced without lag phase even in the presence of vitamin E, which as described above acts as a prooxidant [118]. In the oxidation of whole blood induced by free radicals generated in the aqueous phase, the antioxidants decreased in the order of vitamin C > bilirubin > uric acid, plasma vitamin E > erythrocyte thios and vitamin E [83]. Another example is the inhibition by vitamin E of erythrocyte hemolysis induced by free radicals. The role of vitamin E against hemolysis received much attention many years ago [119]. It has been observed that dialuric acid, hydrogen peroxide, xanthine oxidase, peroxynitrate, and cigarette smoke induce hemolysis, which is suppressed by vitamin E. This was applied to assess vitamin E deficiency in humans [120]. The in vitro studies revealed that free radicals induced lipid peroxidation in erythrocyte membranes and hemolysis, which was suppressed by vitamin E and its homologues (Fig. 2) [121]. The extent of hemolysis was directly proportional to the amount of total free radicals produced [122] and oxygen uptake [123]. Interestingly, vitamin E was much more effective at inhibiting hemolysis than Trolox, a water-soluble

Efficacy of scavenging of radical and nonradical oxidants by vitamin E The efficacy of scavenging radicals by vitamin E is determined by the ratio of the rate of radical scavenging by vitamin E to that of the radical attack on the substrate S: kE[X
][IH]/kS[X
][S], where kE and kS are the rate constants for the former and latter reactions, respectively [1]. Thus, the efficacy is determined by the ratio of kE/kL, which is determined inherently by chemical structure and the molar ratio [E]/[S]. The rate constants of kE and kS for α-tocopherol and linoleate with several kinds of free radicals and nonradical oxidants are compiled in Table 3. The rate constants were obtained from Table 2 and the literature. The rate constants for the reaction of HOCl with 3-pentenoic acid and Trolox were reported as 9 and 103 M−1 s−1 [59,114]. The molar ratio of physiological concentrations of vitamin E and substrate may vary and is difficult to estimate. As described above, the molar ratio of PUFA to vitamin E may range from several hundreds to several thousands. For vitamin E to scavenge 90% of radicals or oxidants before they attack substrate, the ratio kE[X
][IH]/kS[X
][S] should be larger than 10, and if we assume the ratio [E]/[S] as 103, kE/kL should be larger than 104. This implies that if the rate constant kS is larger than 106 M−1 s−1 it is difficult for any antioxidant to scavenge , radicals efficiently. This suggests that there is no efficient antioxidant against hydroxyl radical in vivo. It clearly shows that α-tocopherol can scavenge only peroxyl radicals efficiently in vivo. Similarly, nitrogen dioxide and thiyl radicals are also difficult for α-tocopherol to scavenge efficiently. However, it should be noted that, although it may be difficult to inhibit the chain initiation induced by reactive radicals such as hydroxyl and alkoxyl radicals, it is possible for α-tocopherol to break the chain propagation by scavenging lipid peroxyl radicals, which act as chain-carrying radicals independent of the initial attacking species. Lipid peroxidation alters membrane properties, diminishes membrane function, modifies lipoproteins, and produces various products that are

Table 3 Rate constants for the reactions of oxidants with α-tocopherol kE and linoleate kL in M−1 s−1 and their ratio kE/kL. Oxidant

kE kL kE/kL

HO


RO


ROO


NO2


RS


1

O3

HOCl

1010 9  109 1

3.1  108 8.8  106 o 102

3  106 62 5  104

o106 2  105 o 10

o 106 1.9  107 o1

3  108 1.3  105 103

106 106 1

103 10 102

O2

8

E. Niki / Free Radical Biology and Medicine 66 (2014) 3–12

Fig. 1. Oxidation of (A) fresh rat plasma and (B) the same plasma after dialysis in phosphate-buffered saline (1/1 by volume) at 37 1C under air [118]. Oxidation was induced by MeO-AMVN (0.50 mM) and the consumption of α-tocopherol (open circle) and ascorbic acid (solid circle) and the formation of lipid peroxidation products were followed. Solid square, cholesteryl ester (CE) hydroperoxide plus hydroxide; open square, CE hydroperoxide; solid triangle, phosphatidylcholine (PC) hydroperoxide plus hydroxide; open triangle, PC hydroperoxide.

Fig. 2. Transmission electron micrographs of intact erythrocytes (left) and oxidized erythrocytes undergoing hemolysis (right) induced by free radicals generated from AAPH in air [121].

vitamin E homologue, which has the same chemical reactivity toward oxygen radicals but limited access to the radicals within the membrane, suggesting the importance of breaking the chain propagation taking place in the membranes. The preventive effects of vitamin E against oxidative damage were studied by many research groups in cultured cell systems. Various methods have been applied to induce oxidative stress, including free radicals, high oxygen pressure, hydrogen peroxide, lipid oxidation products, modified proteins, toxic metals, glutamate, peroxynitrite, and antioxidant deficiency. Further, various cell types were used. In general, it has been observed that vitamin E suppresses lipid peroxidation and cell damage induced by various stimuli. For example, all eight isoforms of vitamin E suppressed ROS production, lipid peroxidation, and cell death induced by selenium deficiency in Jurkat cells [124] and also by glutamate in immature primary cortical neuron cultures [125]. To assess the protective effects against cytotoxicity, vitamin E is added to the cell culture medium. It should be emphasized that the antioxidant capacity is determined by intracellular concentration, not by the concentration added to the culture medium, and that the rates of incorporation into cultured cells are quite different between tocopherols and tocotrienols [124,125]. It is important to measure the intracellular concentration of antioxidant when assessing the protective capacity accurately. It should be noted that low levels of oxidative stimuli, for example, by lipid peroxidation products, induce the cellular adaptive response to upregulate the defense capacity against subsequent oxidative stress [4,126,127].

In vivo evidence of scavenging of peroxyl radicals by vitamin E The action of vitamin E as a peroxyl radical-scavenging antioxidant in vivo may be assessed by the level and distribution of lipid peroxidation products measured in biological fluids and tissues as a marker. For this purpose, it is important to understand the mechanisms and products of lipid oxidation. Lipids are oxidized in vivo by multiple oxidants and mechanisms to give diverse products [128]. The major lipid oxidation products mediated by free radicals are hydroperoxides, which are reduced in vivo by glutathione peroxidases and selenoproteins to the corresponding hydroxides. Lipid hydroperoxides and hydroxides are produced by lipoxygenases and cytochrome P450 enzymes, respectively, as well as free radical-mediated peroxidation. Furthermore, singlet oxygen and hypochlorite oxidize unsaturated lipids including cholesterol to produce hydroperoxides and chlorohydrins, which may undergo secondary reactions to give epoxides and dihydroxides. Table 4 summarizes the hydroxyeicosatetraenoic acids (HETEs) derived from the oxidation of arachidonic acid by peroxyl radicals, lipoxygenases, cytochrome P450, and singlet oxygen. It is difficult to identify the responsible oxidants from the products, but interestingly trans,trans forms of hydroperoxides and hydroxides are specific products of free radical-mediated peroxidation, because enzymatic oxidation gives only cis,trans forms. trans,trans cholesteryl linoleate hydroperoxides found in healthy human plasma are the evidence for the free radical-mediated lipid peroxidation in vivo [129].

E. Niki / Free Radical Biology and Medicine 66 (2014) 3–12

Table 4 HETE isomers formed in the oxidation of arachidonic acid by various oxidants. Oxidant

Free radical Lipoxygenase Cytochrome P450 Singlet oxygen

9

Table 5 HODE isomers measured in rat liver with and without carbon tetrachloride administration.

Isomer Regio

Stereo

5, 8, 9, 11, 12, 15 5, 12, 15 5, 8, 9, 11, 12, 16, 19, 20 5, 6, 8, 9, 11, 12, 14, 16

cis,trans; trans,trans cis,trans cis,trans cis,trans

Control rat

CCl4 rat

CCl4/control

0.050 0.046 0.004 0.025 0.002 0.021 0.002

0.26 (0.17) 0.23 (0.15) 0.033 (0.016) 0.11 (0.03) 0.015 (0.007) 0.12 (0.08) 0.018 (0.008)

5.2 5.0 8.3 4.4 7.5 5.7 9.0

Enantio R ¼ S R or S R or S

Morrow and Roberts and co-workers [130] found that a series of prostaglandin-like compounds termed isoprostanes were formed by the free radical-mediated oxidation of arachidonic acid independent of cyclooxygenase. An important structural distinction between isoprostanes and cyclooxygenase-derived prostaglandins is that the former contain side chains that are predominantly oriented cis to the prostane ring, whereas the latter possess exclusively trans side chains [130]. Similarly, neuroprostanes are formed in the oxidation of DHA [131]. Isoprostanes and neuroprostanes are accepted as reliable biomarkers for free radical-mediated lipid peroxidation in vivo. Many studies show the association between isoprostane levels and disease progression [132]. Cholesterol is also an important substrate of lipid oxidation and proceeds by multiple mechanisms to give hydroperoxides, hydroxides, ketones, and epoxides as major products [133,134]. Among them, 7β-cholesterol and 7-ketocholesterol are assumed to be formed by free radical oxidation, whereas hydroxylation of side chains is mediated by cytochrome P450. Thus, the effect of vitamin E as a peroxyl radical-scavenging antioxidant in vivo may be assessed from the levels of trans,transhydro(pero)xides of PUFAs, isoprostanes, neuroprostanes, 7β-cholesterol, and 7-ketocholesterol. Biological samples are often measured as free fatty acid and free cholesterol forms after saponification and reduction. Particularly, linoleic acid and its esters are suitable substrates because they are abundant in vivo and oxidized by free radicals by a straightforward mechanism to give four regio- and stereoisomers of hydroperoxyoctadecadienoates (HPODEs): 9- and 13-cis,trans- and 9- and 13-trans,trans-HPODEs. These four isomers of HPODEs and corresponding HODEs can be separately quantified by LC–MS/MS. Vitamin E scavenges lipid peroxyl radicals and thereby decreases the level of H(P)ODE and increases the ratio of cis,trans-HPODE/trans,trans-HPODE. This stereoisomer ratio may be used as a measure of antioxidant efficacy. Clear experimental evidence for scavenging of peroxyl radicals by vitamin E in vivo was observed in rats intoxicated with carbon tetrachloride. Carbon tetrachloride has been applied frequently to induce oxidative damage in the liver and to assess the preventive capacity of antioxidant compounds [135,136]. Carbon tetrachloride is converted by cytochrome P450 to trichloromethyl radical, which reacts with oxygen and then initiates chain oxidation of lipids, resulting eventually in liver damage. The preventive effects of vitamin E have been confirmed in many studies. It was found that administration of carbon tetrachloride to a rat produced a signal ascribed to the cis,trans and trans,trans diene conjugates of microsomal polyunsaturated fatty acids and that the trans,trans isomer was very strongly suppressed by prior administration of vitamin E to the rat [137]. In Table 5 are shown the levels of four stereoisomers of HODE detected in the liver from control and carbon tetrachloride-administered rats [138]. Although, as mentioned above, trichloromethylperoxyl radicals are too reactive to scavenge efficiently, vitamin E can scavenge lipid peroxyl radicals to inhibit lipid peroxidation; that is, vitamin E is unable to inhibit chain initiation, but it can break chain propagation.

Total HODE Total cis,trans-HODE Total trans,trans-HODE 13-cis,trans-HODE 13-trans,trans-HODE 9-cis,trans-HODE 9-trans,trans-HODE

(0.01) (0.01) (0.001) (0.004) (0.001) (0.004) (0.001)

Data are presented as ng/mg tissue [138].

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease that can progress from simple steatosis to nonalcoholic steatohepatitis (NASH) and finally to liver cirrhosis. Recent studies show the involvement of oxidative stress in the pathogenesis of NAFLD and NASH and potential beneficial effects of vitamin E [139]. It was found that intraperitoneal administration of free radical initiator to mice induces fatty liver, which is suppressed by radical-scavenging antioxidants [140,141]. Free radical-generating azo compound induced triacylglycerol (TG) increase and concomitant phospholipid decrease in the liver, whose pattern was quite similar to that induced by high-fat diet [142]. Further, trans,trans-HODEs were increased significantly in the liver in association with increase in TG [142]. Interestingly, it was found that 9- and 13-HODEs and 9- and 13-oxooctadecadienoates were significantly elevated in patients with NASH, compared with patients with steatosis, and that a strong correlation was revealed between these oxidation products and liver histopathology (inflammation, fibrosis, and steatosis) [143]. It was found also that HODEs showed equivalent R and S chiral distribution, suggesting that they are formed by free radicalmediated oxidation, because enzymatic oxidation gives predominantly S-HODE (Table 4). More recent study showed the link between decreased oxidized lipid products and improved histological disease in the setting of a therapeutic trial in NASH [144]. The above results suggest the involvement of free radical-mediated lipid peroxidation in NAFLD and NASH and potential protective effects of vitamin E. Oxidative stress has been implicated also in the pathogenesis of neurodegenerative disorders such as Alzheimer disease and Parkinson disease [145]. Many studies show an association between the level of oxidative stress biomarkers, including lipid peroxidation products, and the progress of neuronal diseases. The effects of vitamin E are still controversial [146]. It has been observed that neurons of Down syndrome patients exhibited a three- to fourfold increase in intracellular ROS and elevated levels of lipid peroxidation [147]. Ts65Dn mice are the most widely used animal model of Down syndrome. It was found that the levels of 8-isoprostaglandin F2α in brain tissue and those of HODE and 7-hydroxycholesterol in the plasma of Ts65Dn mice were higher than those of control mice [148]. α-Tocopherol supplementation decreased the levels of the above lipid peroxidation products and ameliorated the cognitive deficits, with concomitant increase in α-tocopherylquinone, strongly suggesting that α-tocopherol inhibited lipid peroxidation by scavenging lipid peroxyl radicals [148]. It was reported also that the level of F(2)-dihomo-isoprostanes, the peroxidation product of adrenic acid, was increased in Rett syndrome patients [149]. Another interesting example is the effect of the haptoglobin (Hp) genotype on the outcome of vitamin E treatment in diabetic patients [150]. The haptoglobin gene is polymorphic with two common classes of alleles denoted 1 and 2. Haptoglobin binds to hemoglobin and thereby prevents hemoglobin-induced oxidative

10

E. Niki / Free Radical Biology and Medicine 66 (2014) 3–12

tissue damage, but the Hp 2 allelic protein product provides inferior antioxidant protection compared with the Hp 1 allelic product [151]. It was found that vitamin E supplementation reduces cardiovascular events in a subgroup of individuals with both type 2 diabetes mellitus and the Hp 2-2 genotype [152]. It should be noted that we are protected from oxidative stress by an efficient defense network in which vitamin E plays an important role as one of the antioxidants. Vitamin E suppresses the formation of lipid hydroperoxides but produces them when it scavenges lipid peroxyl radicals. The lipid hydroperoxides are detoxified by selenium-containing enzymes and proteins and thus vitamin E and selenium act in collaboration [153].

Concluding remarks Collectively, it may be stated that vitamin E, above all α-tocopherol, exerts antioxidant effects by scavenging lipid peroxyl radicals in in vivo as well as in vitro systems. It should be added that vitamin E is not an efficient scavenger of hydroxyl radical, alkoxyl radical, nitrogen dioxide, thiyl radical, ozone, hypochlorite, and probably singlet oxygen in vivo. It may be noted that the effects of antioxidant supplementation including vitamin E to well-nourished subjects are often small and many large-scale intervention studies have shown disappointing results. This issue has been discussed extensively, but this is outside the scope of this article. Vitamin E should exert beneficial effects on the inhibition of lipid peroxidation and prevention and treatment of various diseases in which free radical-mediated oxidative stress is involved, when given to the right subject at the right time and for the right duration. References [1] Niki, E.; Noguchi, N.; Tsuchihashi, H.; Gotoh, N. Interaction among vitamin C, vitamin E, and β-carotene. Am. J. Clin. Nutr. 62:1322S–1326S; 1995. [2] Halliwell, B., Gutteridge, J. M. C., editors. 4th ed.. Oxford: Clarendon Press; 2007. [3] Niki, E. Do antioxidants impair signaling by reactive oxygen species and lipid oxidation products? FEBS Lett 586:3767–7037; 2012. [4] Higdon, A.; Diers, A. R.; Oh, J. Y.; Landar, A.; Darley-Usmar, V. M. Cell signalling by reactive lipid species: new concepts and molecular mechanisms. Biochem. J. 442:453–464; 2012. [5] Jones, D. P. Radical-free biology of oxidative stress. Am. J. Physiol. Cell Physiol 295:C849–868; 2008. [6] Traber, M. G.; Atkinson, J. Vitamin E, antioxidant and nothing more. Free Radic. Biol. Med. 43:4–15; 2007. [7] Azzi, A. Molecular mechanism of α-tocopherol action. Free Radic. Biol. Med. 43:16–21; 2007. [8] Niki, E.; Traber, M. G. Vitamin E history. Ann. Nutr. Metab. 61:207–212; 2012. [9] Yoshida, Y.; Niki, E.; Noguchi, N. Comparative study on the action of tocopherols and tocotrienols as antioxidant: chemical and physical effects. Chem. Phys. Lipids 123:63–75; 2003. [10] Burton, G. W.; Ingold, K. U. Autoxidation of biological molecules. 1. Antioxidant activity of vitamin E and related chain-breaking phenolic antioxidants in vitro. J. Am. Chem. Soc. 103:6472–6477; 1981. [11] Niki, E.; Tsuchiya, Y.; Yoshikawa, Y.; Yamamoto, Y.; Kamiya, Y. Oxidation of lipids. XIII. Antioxidant activities of α-, β-, γ-, and δ-tocopherols. Bull. Chem. Soc. Jpn. 59:497–501; 1986. [12] Mukai, K.; Watanabe, Y.; Uemoto, Y.; Ishizu, K. Stopped-flow investigation of antioxidant activity of tocopherols. Bull. Chem. Soc. Jpn. 59:3113–3116; 1986. [13] Pryor, W. A.; Strickland, T.; Church, D. F. Comparison of the efficiencies of several natural and synthetic antioxidants in aqueous SDS micelle solutions. J. Am. Chem. Soc. 110:2224–2229; 1988. [14] Hosomi, A.; Arita, M.; Sato, Y.; Kiyose, C.; Ueda, T.; Igarashi, O.; Arai, H.; Inoue, K. Affinity for alpha-tocopherol transfer protein as a determinant of the biological activities of vitamin E analogs. FEBS Lett. 409:105–108; 1997. [15] Brigelius-Flohé, R.; Traber, M. G. Vitamin E: function and metabolism. FASEB J 13:1145–1155; 1999. [16] Niki, E.; Noguchi, N.; Gotoh, N. Dynamics of lipid peroxidation and its inhibition by antioxidants. Biochem. Soc. Trans. 21:313–317; 1993. [17] Liebler, D. C.; Burr, J. A.; Philips, L.; Ham, A. J. Gas chromatography–mass spectrometry analysis of vitamin E and its oxidation products. Anal. Biochem. 236:27–34; 1996. [18] Yamauchi, R. Addition products of alpha-tocopherol with lipid-derived free radicals. Vitam. Horm 76:309–327; 2007.

[19] Packer, J. E.; Slater, T. F.; Willson, R. L. Direct observation of a free radical interaction between vitamin E and vitamin C. Nature 278:737–738; 1979. [20] Valgimigli, L.; Banks, J. T.; Ingold, K. U.; Lusztyk, J. Kinetic solvent effects on hydroxylic hydrogen atom abstractions are independent of the nature of the abstracting radical: two extreme tests using vitamin E and phenol. J. Am. Chem. Soc 117:9966–9971; 1995. [21] Takahashi, M.; Tsuchiya, J.; Niki, E. Scavenging of radicals by vitamin E in the membranes as studied by spin labeling. J. Am. Chem. Soc. 111:6350–6353; 1989. [22] Barclay, L. R. C. Model biomembranes: quantitative studies of peroxidation, antioxidant action, partitioning, and oxidative stress. Can. J. Chem. 71:1–16; 1993. [23] Niki, E.; Noguchi, N. Dynamics of antioxidant action of vitamin E. Acc. Chem. Res. 37:45–51; 2004. [24] Fukuzawa, K.; Ouchi, A.; Shibata, A.; Nagaoka, S.; Mukai, K. Kinetic study of aroxyl radical-scavenging action of vitamin E in membranes of egg yolk phosphatidylcholine vesicles. Chem. Phys. Lipids 164:205–210; 2011. [25] Jovanovic, S. V.; Jankovic, I.; Josimovic, L. Electron-transfer reactions of alkylperoxy radicals. J. Am. Chem. Soc. 114:9018–9021; 1992. [26] Roschek Jr B.; Tallman, K. A.; Rector, C. L.; Gillmore, J. G.; Pratt, D. A.; Punta, C.; Porter, N. A. Peroxyl radical clocks. J. Org. Chem. 71:3527–3532; 2006. [27] Valgimigli, L.; Banks, J. T.; Lusztyk, J.; Ingold, K. U. Solvent effects on the antioxidant activity of vitamin E. J. Org. Chem. 64:3381–3383; 1999. [28] Burton, G. W.; Doba, T.; Gabe, E.; Hughes, L.; Lee, F. L.; Prasad, L.; Ingold, K. U. Autoxidation of biological molecules. 4. Maximizing the antioxidant activity of phenols. J. Am. Chem. Soc. 107:7053–7065; 1985. [29] Culbertson, S. M.; Antunes, F.; Havrilla, C. M.; Milne, G. L.; Porter, N. A. Determination of the alpha-tocopherol inhibition rate constant for peroxidation in low-density lipoprotein. Chem. Res. Toxicol. 15:870–876; 2002. [30] Niki, E.; Saito, T.; Kawakami, A.; Kamiya, Y. Oxidation of lipids. VI. Inhibition of oxidation of methyl linoleate in solution by vitamin E and vitamin C. J. Biol. Chem. 259:4177–4182; 1984. [31] Niki, E.; Tanimura, R.; Kamiya, Y. Oxidation of lipids. II. Rate of inhibition of oxidation by α-tocopherol and hindered phenols measured by chemiluminescence. Bull. Chem. Soc. Jpn. 55:1551–1555; 1982. [32] Castle, L.; Perkins, M. J. Inhibition kinetics of chain-breaking phenolic antioxidants in SDS micelles: evidence that intermicellar diffusion rates may be rate-limiting for hydrophobic inhibitors such as alpha-tocopherol. J. Am. Chem. Soc 108:6381–6382; 1986. [33] Barclay, L. R.; Baskin, K. A.; Dakin, K. A.; Locke, S. J.; Vinqvist, M. R. The antioxidant activities of phenolic antioxidants in free radical peroxidation of phospholipid membranes. Can. J. Chem. 68:2258–2269; 1990. [34] Walling, C.; Helmreich, W. Reactivity and reversibility in the reaction of thiyl radicals with olefins. J. Am. Chem. Soc. 81:1144–1148; 1959. [35] Pryor, W. A.; Lightsey, J. W. Mechanisms of nitrogen dioxide reactions: initiation of lipid peroxidation and the production of nitrous acid. Science 214:435–437; 1981. [36] Winterbourn, C. C.; Hampton, M. B. Thiol chemistry and specificity in redox signaling. Free Radic. Biol. Med. 45:549–561; 2008. [37] Flohé, L.; Toppo, S.; Cozza, G.; Ursini, F. A comparison of thiol peroxidase mechanisms. Antioxid. Redox Signaling 15:763–780; 2011. [38] Schöneich, C.; Dillinger, U.; von Bruchhausen, F.; Asmus, K. D. Oxidation of polyunsaturated fatty acids and lipids through thiyl and sulfonyl radicals: reaction kinetics, and influence of oxygen and structure of thiyl radicals. Arch. Biochem. Biophys 292:456–467; 1992. [39] Kikugawa, K.; Hiramoto, K.; Okamoto, Y.; Hasegawa, Y. Enhancement of nitrogen dioxide-induced lipid peroxidation and DNA strand breaking by cysteine and glutathione. Free Radic. Res 21:399–408; 1994. [40] Everett, S. A.; Dennis, M. F.; Patel, K. B.; Maddix, S.; Kundu, S. C.; Willson, R. L. Scavenging of nitrogen dioxide, thiyl, and sulfonyl free radicals by the nutritional antioxidant beta-carotene. J. Biol. Chem. 271:3988–3994; 1996. [41] Terao, J.; Minami, Y.; Bando, N. Singlet molecular oxygen-quenching activity of carotenoids: relevance to protection of the skin from photoaging. J. Clin. Biochem. Nutr. 48:57–62; 2011. [42] Wilkinson, F.; Brummer, J. G. Rate constants for the decay and reactions of the lowest electronically excited singlet state of molecular oxygen in solution. J. Phys. Chem. Ref. Data 10:809–999; 1981. [43] Fahrenholtz, S. R.; Doleiden, F. H.; Trozzolo, A. M.; Lamola, A. A. On the quenching of singlet oxygen by α-tocopherol. Photochem. Photobiol. 20:505–509; 1974. [44] Foote, C. S.; Ching, T. Y.; Geller, G. G. Chemistry of singlet oxygen. XVIII. Rates of reaction and quenching of α-tocopherol and singlet oxygen. Photochem. Photobiol. 20:511–513; 1974. [45] Di Mascio, P.; Kaiser, S.; Sies, H. Lycopene as the most efficient biological carotenoid singlet oxygen quencher. Arch. Biochem. Biophys. 274:532–538; 1989. [46] Kaiser, S.; Di Mascio, P.; Murphy, M. E.; Sies, H. Physical and chemical scavenging of singlet molecular oxygen by tocopherols. Arch. Biochem. Biophys. 277:101–108; 1990. [47] Kohno, Y.; Egawa, Y.; Itoh, S.; Nagaoka, S.; Takahashi, M.; Mukai, K. Kinetic study of quenching reaction of singlet oxygen and scavenging reaction of free radical by squalene in n-butanol. Biochim. Biophys. Acta 1256:52–56; 1995. [48] Fukuzawa, K.; Inokami, Y.; Tokumura, A.; Terao, J.; Suzuki, A. Rate constants for quenching singlet oxygen and activities for inhibiting lipid peroxidation of carotenoids and alpha-tocopherol in liposomes. Lipids 33:751–756; 1998. [49] Grams, G. W.; Eskins, K. Dye-sensitized photooxidation of tocopherols: correlation between singlet oxygen reactivity and vitamin E activity. Biochemistry 11:606–608; 1972.

E. Niki / Free Radical Biology and Medicine 66 (2014) 3–12

[50] Mukai, K.; Ouchi, A.; Takahashi, S.; Aizawa, K.; Inakuma, T.; Terao, J.; Nagaoka, S. Development of singlet oxygen absorption capacity (SOAC) assay method. 3. Measurements of the SOAC values for phenolic antioxidants. J. Agric. Food Chem. 60:7905–7916; 2012. [51] Doleiden, F. H.; Fahrenholtz, S. R.; Lamola, A. A. H.; Trozzolo, A. M. Reactivity of cholesterol and some fatty acids toward singlet oxygen. Photochem. Photobiol. 20:519–521; 1974. [52] Pryor, W. A. Can vitamin E protect humans against the pathological effects of ozone in smog? Am. J. Clin. Nutr 53:702–722; 1991. [53] Giamalva, D.; Church, D. F.; Pryor, W. A. A comparison of the rates of ozonation of biological antioxidants and oleate and linoleate esters. Biochem. Biophys. Res. Commun. 133:773–779; 1985. [54] Pryor, W. A. Mechanisms of radical formation from reactions of ozone with target molecules in the lung. Free Radic. Biol. Med 17:451–465; 1994. [55] Winterbourn, C. C. Biological reactivity and biomarkers of the neutrophil oxidant, hypochlorous acid. Toxicology 181-182:223–227; 2002. [56] Davies, M. J. Myeloperoxidase-derived oxidation: mechanisms of biological damage and its prevention. J. Clin. Biochem. Nutr. 48:8–19; 2011. [57] van den Berg, J. J.; Winterbourn, C. C.; Kuypers, F. A. Hypochlorous acidmediated modification of cholesterol and phospholipid: analysis of reaction products by gas chromatography–mass spectrometry. J. Lipid Res. 34: 2005–2012; 1993. [58] Spickett, C. M.; Jerlich, A.; Panasenko, O. M.; Arnhold, J.; Pitt, A. R.; Stelmaszyńska, T.; Schaur, R. J. The reactions of hypochlorous acid, the reactive oxygen species produced by myeloperoxidase, with lipids. Acta Biochim. Pol. 47:889–899; 2000. [59] Pattison, D. I.; Hawkins, C. L.; Davies, M. J. What are the plasma targets of the oxidant hypochlorous acid? A kinetic modeling approach Chem. Res. Toxicol. 22:807–817; 2009. [60] Yan, L. J.; Traber, M. G.; Kobuchi, H.; Matsugo, S.; Tritschler, H. J.; Packer, L. Efficacy of hypochlorous acid scavengers in the prevention of protein carbonyl formation. Arch. Biochem. Biophys. 327:330–334; 1996. [61] Noguchi, N.; Nakada, A.; Itoh, Y.; Watanabe, A.; Niki, E. Formation of active oxygen species and lipid peroxidation induced by hypochlorite. Arch. Biochem. Biophys. 397:440–447; 2002. [62] Miyamoto, S.; Martinez, G. R.; Rettori, D.; Augusto, O.; Medeiros, M. H.; Di Mascio, P. Linoleic acid hydroperoxide reacts with hypochlorous acid, generating peroxyl radical intermediates and singlet molecular oxygen. Proc. Natl. Acad. Sci. USA 103:293–298; 2006. [63] Hazell, L. J.; Davies, M. J.; Stocker, R. Secondary radicals derived from chloramines of apolipoprotein B-100 contribute to HOCl-induced lipid peroxidation of low-density lipoproteins. Biochem. J 339:489–495; 1999. [64] Thomas, S. R.; Neuzil, J.; Mohr, D.; Stocker, R. Coantioxidants make alphatocopherol an efficient antioxidant for low-density lipoprotein. Am. J. Clin. Nutr. 62:1357S–1364S; 1995. [65] Prior, R. L.; Wu, X.; Schaich, K. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food Chem. 53:4290–4302; 2005. [66] Niki, E. Antioxidant capacity: which capacity and how to measure it? J. Berry Res 1:169–176; 2011. [67] Shi, H.; Noguchi, N.; Niki, E. Comparative study on dynamics of antioxidative action of α-tocopheryl hydroquinone, ubiquinol, and α-tocopherol against lipid peroxidation. Free Radic. Biol. Med. 27:334–346; 1999. [68] Bowry, V. W.; Ingold, K. U.; Stocker, R. Vitamin E in human low-density lipoprotein: when and how this antioxidant becomes a pro-oxidant. Biochem. J. 288:341–344; 1992. [69] Steinberg, D.; Witztum, J. L. Oxidized low-density lipoprotein and atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 30:2311–2316; 2010. [70] Bowry, V. W.; Stocker, R. Tocopherol-mediated peroxidation: the prooxidant effect of vitamin E on the radical-initiated oxidation of human low density lipoprotein. J. Am. Chem. Soc 115:6029–6044; 1993. [71] Ingold, K. U.; Bowry, V. W.; Stocker, R.; Walling, C. Autoxidation of lipids and antioxidation by alpha-tocopherol and ubiquinol in homogeneous solution and in aqueous dispersions of lipids: unrecognized consequences of lipid particle size as exemplified by oxidation of human low density lipoprotein. Proc. Natl. Acad. Sci. USA 90:45–49; 1993. [72] Watanabe, A.; Noguchi, N.; Fujisawa, A.; Kodama, K.; Tamura, K.; Cynshi, O.; Niki, E. Stability and reactivity of aryloxyl radicals derived from a novel antioxidant BO-653 and related compounds: effects of substituents and side chain in solution and membranes. J. Am. Chem. Soc. 122:5438–5442; 2000. [73] Lucarini, M.; Pedrielli, P.; Pedulli, G. F.; Cabiddu, S.; Fattuoni, C. Bond dissociation energies of O–H bonds in substituted phenols from equilibration studies. J. Org. Chem. 61:9259–9263; 1996. [74] Niki, E.; Tsuchiya, J.; Tanimura, R.; Kamiya, Y. Regeneration of vitamin E from αchromanoxyl radical by glutathione and vitamin C. Chem. Lett. :789–792; 1982. [75] Buettner, G. R. The pecking order of free radicals and antioxidants: lipid peroxidation, α-tocopherol, and ascorbate. Arch. Biochem. Biophys. 300:535–543; 1993. [76] Barclay, L. R.; Locke, S. J.; MacNeil, J. M. Autoxidation in micelles: synergism of vitamin C with lipid-soluble vitamin E and water-soluble Trolox. Can. J. Chem. 63:366–374; 1985. [77] Scarpa, M.; Rigo, A.; Maiorino, M.; Ursini, F.; Gregolin, C. Formation of alphatocopherol radical and recycling of alpha-tocopherol by ascorbate during peroxidation of phosphatidylcholine liposomes: an electron paramagnetic resonance study. Biochim. Biophys. Acta 801:215–219; 1984.

11

[78] Niki, E.; Kawakami, A.; Yamamoto, Y.; Kamiya, Y. Oxidation of lipids. VIII. Synergistic inhibition of oxidation of liposome in aqueous dispersion by vitamin E and vitamin C. Bull. Chem. Soc. Jpn. 58:1971–1975; 1985. [79] Doba, T.; Burton, G. W.; Ingold, K. U. Antioxidant and co-antioxidant activity of vitamin C: the effect of vitamin C, either alone or in the presence of vitamin E or a water-soluble vitamin E analogue, upon the peroxidation of aqueous multilamellar phospholipid liposomes. Biochim. Biophys. Acta 835:298–303; 1985. [80] Sato, K.; Niki, E.; Shimasaki, H. Free radical-mediated chain oxidation of low density lipoprotein and its synergistic inhibition by vitamin E and vitamin C. Arch. Biochem. Biophys 279:402–405; 1990. [81] Frei, B.; Stocker, R.; Ames, B. N. Antioxidant defenses and lipid peroxidation in human blood plasma. Proc. Natl. Acad. Sci. USA 85:9748–9752; 1988. [82] Chan, A. C.; Tran, K.; Raynor, T.; Ganz, P. R.; Chow, C. K. Regeneration of vitamin E in human platelets. J. Biol. Chem. 266:17290–17295; 1991. [83] Niki, E.; Yamamoto, Y.; Takahashi, M.; Yamamoto, K.; Yamamoto, Y.; Komuro, E.; Miki, M.; Yasuda, H.; Mino, M. Free radical-mediated damage of blood and its inhibition by antioxidants. J. Nutr. Sci. Vitaminol. (Tokyo) 34:507–512; 1988. [84] Halpner, A. D.; Handelman, G. J.; Harris, J. M.; Belmont, C. A.; Blumberg, J. B. Protection by vitamin C of loss of vitamin E in cultured rat hepatocytes. Arch. Biochem. Biophys. 359:305–309; 1998. [85] Barclay, L. R.; Locke, S. J.; MacNeil, J. M. The autoxidation of unsaturated lipids in micelles: synergism of inhibitors vitamins C and E. Can. J. Chem. 61:1288–1290; 1983. [86] Mukai, K.; Kikuchi, S.; Urano, S. Stopped-flow kinetic study of the regeneration reaction of tocopheroxyl radical by reduced ubiquinone-10 in solution. Biochim. Biophys. Acta 103:77–82; 1990. [87] Witting, P. K.; Westerlund, C.; Stocker, R. A rapid and simple screening test for potential inhibitors of tocopherol-mediated peroxidation of LDL lipids. J. Lipid Res 37:853–867; 1996. [88] Amorati, R.; Ferroni, F.; Lucarini, M.; Pedulli, G. F.; Valgimigli, L. A quantitative approach to the recycling of alpha-tocopherol by coantioxidants. J. Org. Chem. 67:9295–9303; 2002. [89] Davies, M. J.; Forni, L. G.; Willson, R. L. Vitamin E analogue Trolox C: E.S.R. and pulse-radiolysis studies of free-radical reactions. Biochem. J. 255: 513–522; 1988. [90] Motoyama, T.; Miki, M.; Mino, M.; Takahashi, M.; Niki, E. Synergistic inhibition of oxidation in dispersed phosphatidylcholine liposomes by a combination of vitamin E and cysteine. Arch. Biochem. Biophys. 270:655–661; 1989. [91] Igarashi, O.; Yonekawa, Y.; Fujiyama-Fujihara, Y. Synergistic action of vitamin E and vitamin C in vivo using a new mutant of Wistar-strain rats, ODS, unable to synthesize vitamin C. J. Nutr. Sci. Vitaminol 37:359–369; 1991. [92] Bruno, R. S.; Leonard, S. W.; Atkinson, J.; Montine, T. J.; Ramakrishnan, R.; Bray, T. M.; Traber, M. G. Faster plasma vitamin E disappearance in smokers is normalized by vitamin C supplementation. Free Radic. Biol. Med. 40: 689–697; 2006. [93] Burton, G. W.; Wronska, U.; Stone, L.; Foster, D. O.; Ingold, K. U. Biokinetics of dietary RRR-alpha-tocopherol in the male guinea pig at three dietary levels of vitamin C and two levels of vitamin E: evidence that vitamin C does not spare vitamin E in vivo. Lipids 25:199–210; 1990. [94] Xu, L.; Davis, T. A.; Porter, N. A. Rate constants for peroxidation of polyunsaturated fatty acids and sterols in solution and in liposomes. J. Am. Chem. Soc. 131:13037–13044; 2009. [95] Simic, M. G.; Jovanovic, S. V.; Niki, E. Mechanisms of lipid oxidative processes and their inhibition. ACS Symp. Ser. 500:14–32; 1992. [96] Pryor, W. A.; Houk, K. N.; Foote, C. S.; Fukuto, J. M.; Ignarro, L. J.; Squadrito, G. L.; Davies, K. J. Free radical biology and medicine: it's a gas, man!. Am. J. Physiol. Regul. Integr. Comp. Physiol 291:R491–511; 2006. [97] D'Aquino, M.; Dunster, C.; Willson, R. L. Vitamin A and glutathione-mediated free radical damage: competing reactions with polyunsaturated fatty acids and vitamin C. Biochem. Biophys. Res. Commun. 161:1199–1203; 1989. [98] Schöneich, C.; Asmus, K. D. Reaction of thiyl radicals with alcohols, ethers and polyunsaturated fatty acids: a possible role of thiyl radicals in thiol mutagenesis? Radiat. Environ. Biophys 29:263–271; 1990. [99] Takashima, M.; Shichiri, M.; Hagihara, Y.; Yoshida, Y.; Niki, E. Reactivity toward oxygen radicals and antioxidant action of thiol compounds. Biofactors 38:240–248; 2012. [100] Glaser, C.; Demmelmair, H.; Koletzko, B. High-throughput analysis of fatty acid composition of plasma glycerophospholipids. J. Lipid Res. 51:216–221; 2010. [101] Ichihara, K.; Yoneda, K.; Takahashi, A.; Hoshino, N.; Matsuda, M. Improved method for the fatty acid analysis of blood lipid classes. Lipids 46:297–306; 2011. [102] Söderberg, M.; Edlund, C.; Kristensson, K.; Dallner, G. Fatty acid composition of brain phospholipids in aging and in Alzheimer's disease. Lipids 26:421–425; 1991. [103] Esterbauer, H.; Gebicki, J.; Puhl, H.; Jürgens, G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic. Biol. Med. 13:341–390; 1992. [104] Buttriss, J. L.; Diplock, A. T. The relationship between α-tocopherol and phospholipid fatty acids in rat liver subcellular membrane fraction. Biochim. Biophys. Acta 962:81–90; 1988. [105] Kornbrust, D. J.; Mavis, R. D. Relative susceptibility of microsomes from lung, heart, liver, kidney, brain and testes to lipid peroxidation: correlation with vitamin E content. Lipids 15:315–322; 1980.

12

E. Niki / Free Radical Biology and Medicine 66 (2014) 3–12

[106] Singer, S. J.; Nicolson, G. L. The fluid mosaic model of the structure of cell membranes. Science 175:720–731; 1972. [107] Karnovsky, M. J.; Kleinfeld, A. M.; Hoover, R. L.; Klausner, R. D. The concept of lipid domains in membranes. J. Cell Biol. 94:1–6; 1982. [108] Atkinson, J.; Epand, R. F.; Epand, R. M. Tocopherols and tocotrienols in membranes: a critical review. Free Radic. Biol. Med. 44:739–764; 2008. [109] Wassall, S. R.; Stillwell, W. Polyunsaturated fatty acid–cholesterol interactions: domain formation in membranes. Biochim. Biophys. Acta 1788:24–32; 2009. [110] Quinn, P. J. Lipid–lipid interactions in bilayer membranes: married couples and casual liaisons. Prog. Lipid Res. 51:179–198; 2012. [111] Engelman, D. M. Membranes are more mosaic than fluid. Nature 438:578–580; 2005. [112] Lemaire-Ewing, S.; Desrumaux, C.; Néel, D.; Lagrost, L. Vitamin E transport, membrane incorporation and cell metabolism: is alpha-tocopherol in lipid rafts an oar in the lifeboat? Mol. Nutr. Food Res 54:631–640; 2010. [113] Atkinson, J.; Harroun, T.; Wassall, S. R.; Stillwell, W.; Katsaras, J. The location and behavior of alpha-tocopherol in membranes. Mol. Nutr. Food Res. 54:641–651; 2010. [114] Pattison, D. I.; Hawkins, C. L.; Davies, M. J. Hypochlorous acid-mediated oxidation of lipid components and antioxidants present in low-density lipoproteins: absolute rate constants, product analysis, and computational modeling. Chem. Res. Toxicol. 16:439–449; 2003. [115] Pratt, D. A.; Tallman, K. A.; Porter, N. A. Free radical oxidation of polyunsaturated lipids: new mechanistic insights and the development of peroxyl radical clocks. Acc. Chem. Res. 44:458–467; 2011. [116] Yin, H.; Xu, L.; Porter, N. A. Free radical lipid peroxidation: mechanisms and analysis. Chem. Rev. 111:5944–5972; 2011. [117] Takahashi, M.; Yoshikawa, Y.; Niki, E. Oxidation of lipids. XVII. Crossover effect of tocopherols in the spontaneous oxidation of methyl linoleate. Bull. Chem. Soc. Jpn. 62:1885–1890; 1989. [118] Itoh, N.; Cao, J.; Chen, Z. H.; Yoshida, Y.; Niki, E. Advantages and limitation of BODIPY as a probe for the evaluation of lipid peroxidation and its inhibition by antioxidants in plasma. Bioorg. Med. Chem. Lett. 17:2059–2063; 2007. [119] Gyorgy, P.; Rose, C. S. Tocopherol and hemolysis in vitro and in vivo. Ann. N. Y. Acad. Sci. 52:231–239; 1949. [120] Chow, C. K. Vitamin E and blood. World Rev. Nutr. Diet. 45:133–166; 1985. [121] Niki, E.; Komuro, E.; Takahashi, M.; Urano, S.; Ito, E.; Terao, K. Oxidative hemolysis of erythrocytes and its inhibition by free radical scavengers. J. Biol. Chem. 263:19809–19814; 1988. [122] Miki, M.; Tamai, H.; Mino, M.; Yamamoto, Y.; Niki, E. Free-radical chain oxidation of rat red blood cells by molecular oxygen and its inhibition by α-tocopherol. Arch. Biochem. Biophys. 258:373–380; 1987. [123] Yamamoto, Y.; Niki, E.; Kamiya, Y.; Miki, M.; Tamai, H.; Mino, M. Free radical chain oxidation and hemolysis of erythrocytes by molecular oxygen and their inhibition by vitamin E. J. Nutr. Sci. Vitaminol. (Tokyo) 32:475–479; 1986. [124] Saito, Y.; Yoshida, Y.; Akazawa, T.; Takahashi, K.; Niki, E. Cell death caused by selenium deficiency and protective effect of antioxidants. J. Biol. Chem. 278:39428–39434; 2003. [125] Saito, Y.; Nishio, K.; Akazawa, Y. O.; Yamanaka, K.; Miyama, A.; Yoshida, Y.; Noguchi, N.; Niki, E. Cytoprotective effects of vitamin E homologues against glutamate-induced cell death in immature primary cortical neuron cultures: tocopherols and tocotrienols exert similar effects by antioxidant function. Free Radic. Biol. Med. 49:1542–1549; 2010. [126] Girotti, A. W. Lipid hydroperoxide generation, turnover, and effector action in biological systems. J. Lipid Res. 39:1529–1542; 1998. [127] Chen, Z. H.; Yoshida, Y.; Saito, Y.; Noguchi, N.; Niki, E. Adaptive response induced by lipid peroxidation products in cell cultures. FEBS Lett. 580:479–483; 2006. [128] Niki, E. Lipid peroxidation: physiological levels and dual biological effects. Free Radic. Biol. Med. 47:469–484; 2009. [129] Mashima, R.; Onodera, K.; Yamamoto, Y. Regioisomeric distribution of cholesteryl linoleate hydroperoxides and hydroxides in plasma from healthy humans provides evidence for free radical-mediated lipid peroxidation in vivo. J. Lipid Res 41:109–115; 2000. [130] Morrow, J. D.; Hill, K. E.; Burk, R. F.; Nammour, T. M.; Badr, K. F.; Roberts 2nd L. J. A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. Proc. Natl. Acad. Sci. USA 87:9383–9387; 1990. [131] Roberts 2nd L. J.; Montine, T. J.; Markesbery, W. R.; Tapper, A. R.; Hardy, P.; Chemtob, S.; Dettbarn, W. D.; Morrow, J. D. Formation of isoprostane-like compounds (neuroprostanes) in vivo from docosahexaenoic acid. J. Biol. Chem. 273:13605–13612; 1998.

[132] Davies, S. S.; Roberts 2nd L. J. F2-isoprostanes as an indicator and risk factor for coronary heart disease. Free Radic. Biol. Med. 50:559–566; 2011. [133] Brown, A. J.; Jessup, W. Oxysterols: sources, cellular storage and metabolism, and new insights into their roles in cholesterol homeostasis. Mol. Aspects Med. 30:111–122; 2009. [134] Iuliano, L. Pathways of cholesterol oxidation via non-enzymatic mechanisms. Chem. Phys. Lipids 164:457–468; 2011. [135] Slater, T. F.; Sawyer, B. C. The stimulatory effects of carbon tetrachloride on peroxidative reactions in rat liver fractions in vitro: inhibitory effects of freeradical scavengers and other agents. Biochem. J 123:823–828; 1971. [136] Kadiiska, M. B.; Gladen, B. C.; Baird, D. D.; Dikalova, A. E.; Sohal, R. S.; Hatch, G. E.; Jones, D. P.; Mason, R. P.; Barrett, J. C. Biomarkers of oxidative stress study: are plasma antioxidants markers of CCl4 poisoning? Free Radic. Biol. Med. 28:838–845; 2000. [137] Corongiu, F. P.; Poli, G.; Dianzani, M. U.; Cheeseman, K. H.; Slater, T. F. Lipid peroxidation and molecular damage to polyunsaturated fatty acids in rat liver: recognition of two classes of hydroperoxides formed under conditions in vivo. Chem. Biol. Interact. 59:147–155; 1986. [138] Liu, W.; Yin, H.; Akazawa, Y. O.; Yoshida, Y.; Niki, E.; Porter, N. A. Ex vivo oxidation in tissue and plasma assays of hydroxyoctadecadienoates: Z,E/E,E stereoisomer ratios. Chem. Res. Toxicol. 23:986–995; 2010. [139] Sanyal, A. J.; Chalasani, N.; Kowdley, K. V.; McCullough, A.; Diehl, A. M.; Bass, N. M.; Neuschwander-Tetri, B. A.; Lavine, J. E.; Tonascia, J.; Unalp, A.; Van Natta, M.; Clark, J.; Brunt, E. M.; Kleiner, D. E.; Hoofnagle, J. H.; Robuck, P. R.; Nash, C. R. N. Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. N. Engl. J. Med. 362:1675–1685; 2010. [140] Terao, K.; Niki, E. Damage to biological tissues induced by radical initiator 2,2′-azobis(2-amidinopropane) dihydrochloride and its inhibition by chainbreaking antioxidants. J. Free Radic. Biol. Med 2:193–201; 1986. [141] Shimasaki, H.; Saypil, W. H.; Ueta, N. Free radical induced liver injury. II. Effects of intraperitoneally administered 2,2-azobis(2-amidinopropane) dihydrochloride on the fatty acid profiles of hepatic triacylglycerol and phospholipids. Free Radic. Res. Commun 14:247–252; 1991. [142] Morita, M.; Ishida, N.; Uchiyama, K.; Yamaguchi, K.; Itoh, Y.; Shichiri, M.; Yoshida, Y.; Hagihara, Y.; Naito, Y.; Yoshikawa, T.; Niki, E. Fatty liver induced by free radicals and lipid peroxidation. Free Radic. Res. 46:758–765; 2012. [143] Feldstein, A. E.; Lopez, R.; Tamimi, T. A.; Yerian, L.; Chung, Y. M.; Berk, M.; Zhang, R.; McIntyre, T. M.; Hazen, S. L. Mass spectrometric profiling of oxidized lipid products in human nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. J. Lipid Res. 51:3046–3054; 2010. [144] Zein, C. O.; Lopez, R.; Fu, X.; Kirwan, J. P.; Yerian, L. M.; McCullough, A. J.; Hazen, S. L.; Feldstein, A. E. Pentoxifylline decreases oxidized lipid products in nonalcoholic steatohepatitis: new evidence on the potential therapeutic mechanism. Hepatology 56:1291–1299; 2012. [145] Bonda, D. J.; Wang, X.; Perry, G.; Nunomura, A.; Tabaton, M.; Zhu, X.; Smith, M. A. Oxidative stress in Alzheimer disease: a possibility for prevention. Neuropharmacology 59:290–294; 2010. [146] Lee, H. P.; Zhu, X.; Casadesus, G.; Castellani, R. J.; Nunomura, A.; Smith, M. A.; Lee, H. G.; Perry, G. Antioxidant approaches for the treatment of Alzheimer's disease. Expert Rev. Neurother 10:1201–1208; 2010. [147] Busciglio, J.; Yankner, B. A. Apoptosis and increased generation of reactive oxygen species in Down's syndrome neurons in vitro. Nature 378:776–779; 1995. [148] Shichiri, M.; Yoshida, Y.; Ishida, N.; Hagihara, Y.; Iwahashi, H.; Tamai, H.; Niki, E. α-Tocopherol suppresses lipid peroxidation and behavioral and cognitive impairments in the Ts65Dn mouse model of Down syndrome. Free Radic. Biol. Med. 50:1801–1811; 2011. [149] Durand, T.; De Felice, C.; Signorini, C.; Oger, C.; Bultel-Poncé, V.; Guy, A.; Galano, J. M.; Leoncini, S.; Ciccoli, L.; Pecorelli, A.; Valacchi, G.; Hayek, J. F(2)Dihomo-isoprostanes and brain white matter damage in stage 1 Rett syndrome. Biochimie 95:86–90; 2013. [150] Vardi, M.; Blum, S.; Levy, A. P. Haptoglobin genotype and cardiovascular outcomes in diabetes mellitus—natural history of the disease and the effect of vitamin E treatment: meta-analysis of the medical literature. Eur. J. Intern. Med 23:628–632; 2012. [151] Asleh, R.; Guetta, J.; Kalet-Litman, S.; Miller-Lotan, R.; Levy, A. P. Haptoglobin genotype- and diabetes-dependent differences in iron-mediated oxidative stress in vitro and in vivo. Circ. Res. 96:435–441; 2005. [152] Bisby, R. H.; Parker, A. W. Reaction of ascorbate with the α-tocopheroxyl radical in micellar and bilayer membrane systems. Arch. Biochem. Biophys. 317:170–178; 1995. [153] Ursini, F.; Bindoli, A. The role of selenium peroxidases in the protection against oxidative damage of membranes. Chem. Phys. Lipids 44:255–276; 1987.