Original Contributions
Comparative study on dynamics of antioxidative action of α-tocopheryl hydroquinone, ubiquinol, and α-tocopherol against lipid peroxidation

https://doi.org/10.1016/S0891-5849(99)00053-2Get rights and content

Abstract

α-Tocopheryl quinone is a metabolite of α-tocopherol (TOH) in vivo. The antioxidant action of its reduced form, α-tocopheryl hydroquinone (TQH2), has received much attention recently. In the present study, the antioxidative activity of TQH2 was studied in various systems in vitro and compared with that of ubiquinol-10 (UQH2) or TOH to obtain the basic information on the dynamics of the antioxidant action of TQH2. First, their hydrogen-donating abilities were investigated in the reaction with galvinoxyl, a stable phenoxyl radical, and TQH2 was found to possess greater second-order rate constant (1.0 × 104 M−1 s−1) than UQH2 (6.0 × 103 M−1 s−1) and TOH (2.4 × 103 M−1 s−1) at 25°C in ethanol. The stoichiometric numbers were obtained as 1.9, 2.0, and 1.0 for TQH2, UQH2, and TOH, respectively, in reducing galvinoxyl. Second, their relative reactivities toward peroxyl radicals were assessed in competition with N,N′-diphenyl-p-phenylenediamine (DPPD) and found to be 6.0 (TQH2), 1.9 (UQH2), and 1.0 (TOH). Third, their antioxidant efficacies were evaluated in the oxidation of methyl linoleate in organic solvents and in aqueous dispersions. The antioxidant potency decreased in the order TOH > UQH2 > TQH2, as assessed by either the extent of the reduction in the rate of oxidation or the duration of inhibition period. The reverse order of their reactivities toward radicals and their antioxidant efficacies was interpreted by the rapid autoxidation of TQH2 and UQH2, carried out by hydroperoxyl radicals. Although neither TQH2 nor UQH2 acted as a potent antioxidant by itself, they acted as potent antioxidants in combination with TOH. TQH2 and UQH2 reduced α-tocopheroxyl radical to spare TOH, whereas TOH suppressed the autoxidation of TQH2 and UQH2. In the micelle oxidation, the antioxidant activities of TQH2, UQH2, and TOH were similar, whereas 2,2,5,7,8-pentamethyl-6-chromanol exerted much more potent efficacy than TQH2, UQH2, or TOH. These results clearly show that the antioxidant potencies against lipid peroxidation are determined not only by their chemical reactivities toward radicals, but also by the fate of an antioxidant-derived radical and the mobility of the antioxidant at the microenvironment.

Introduction

With increasing evidence that shows the involvement of active oxygen species and free radicals in a variety of diseases, cancer, and aging, the beneficial effects of antioxidants have received much attention. The aerobic organisms are protected from such oxidative stress by a variety of antioxidants with different functions. To scavenge active free radicals is one of the important functions of antioxidants, and vitamins E and C are well known as most abundant and potent lipophilic and hydrophilic radical scavenging antioxidants, respectively. TQH2, a reduced form of TQ, which is one of the metabolites of TOH, has been shown to be capable of acting as a radical-scavenging antioxidant.

It has been observed that TQ exists with its reduced form TQH2 in animal tissues including liver, heart, spleen, adipose, and brain [1], [2], [3], [4], [5], [6] and in human tissues [7], [8], [9], [10]. TQ can be reduced to TQH2 in rat liver microsomal, mitochondrial, cytosolic preparations, and isolated hepatocytes [2], [11], [12], and it has been shown that TQ is converted into TQH2 in humans [7]. Moreover, cytochrome P450 reductase and quinone oxidoreductase were found to catalyze TQH2 formation from TQ in cell-free and cellular systems [13]. The role of TQ and TQH2 as antioxidants has received attention. Bindoli reported that TQH2 was a much more potent antioxidant than TQ in inhibiting lipid peroxidation induced by ascorbate/Fe(II) in liposomes and by cumene hydroperoxide in submitochondrial particles [11]. TQ was found to be a more effective antioxidant than TOH in a tissue culture system whenever they were concurrently added or preadded [14]. TQ-pretreated cells were more resistant to lipid peroxidation and cytotoxicity than control cells [15], [16], [17], [18]. The antioxidant activity of TQ in these studies has so far been suggested, presumably because of the production of TQH2, and TQ itself is not considered to be an effective antioxidant [19]. Recently, it has also been reported that TQH2 prevented cumene hydroperoxide-induced lipid peroxidation in rat liver microsomes [13], and it strongly inhibited the oxidation of both surface and core lipids in low-density lipoprotein (LDL) [20].

Ubiquinol (UQH2), a reduced form of coenzyme Q (UQ), has been shown to act as a radical scavenging antioxidant in solution [21], [22], [23], [24], liposomes [24], [25], [26], mitochondria [27], [28], [29], [30], [31], microsomes [21], [28], [29], LDL [23], [32], [33], cell [34], and in whole animal [35], [36].

The antioxidant nature of TQH2, however, has not been studied as extensively as UQH2 and TOH. Moreover, the dynamics and efficacy of ubiquinol as an antioxidant have not been well elucidated either. The present study was carried out, therefore, to investigate and elucidate the inherent activities of TQH2, UQH2, and TOH as radical-scavenging antioxidants and also the interactions between these antioxidants. The basic information is necessary for understanding the antioxidant action in a biologic system.

The reactivity toward radicals and antioxidant activity of TQH2 were assessed and compared to UQH2 and TOH as follows. First, we measured the reactivity and the stoichiometric numbers of the three substances for the reactions with galvinoxyl, a stable phenoxyl radical. Second, their ability to scavenge peroxyl radicals derived from AMVN was studied in various solvents. Third, the oxidation of methyl linoleate induced by AMVN where lipid peroxyl radical is a predominant chain carrier was used to test their ability to inhibit lipid oxidation in organic solution and in aqueous micelle emulsions. Furthermore, parallel experiments were carried out to trace their consumptions in the various reaction systems. Finally, the ability of TQH2 and UQH2 to reduce α-tocopheroxyl radical was investigated in various solvents.

Section snippets

Reagents

The materials, TQ, galvinoxyl, and AMVN, were purchased from Wako Pure Chemical Co. Ltd. (Osaka, Japan). Methyl linoleate was obtained from Sigma Chemical Co. (St. Louis, MO, USA) and purified before use by column chromatography as described previously [37]. DPPD and 2-methyl-6-phenyl-3,7-dihydroimidazo [1,2-a]pyrazin-3-one(CLA), a cyprodina luciferin analog, used as a chemiluminescence probe were obtained from Tokyo Kasei Co. (Tokyo, Japan). TOH, UQ, and 2,2,5,7,8-pentamethyl-6-chromanol (PMC)

Kinetic and stoichiometric studies on the reaction of TQH2, UQH2, and TOH with galvinoxyl

To estimate quantitatively the reactivities toward oxygen radicals, the reactions of TQH2, UQH2, and TOH with galvinoxyl were measured by following a decrease in absorption at 428 nm with a stopped-flow spectrophotometer (Fig. 2A) and the pseudo first-order rate constant k1 was calculated [38]. The results in Fig. 2B show that the pseudo first-order rate constant k1 values obtained for each antioxidant were linearly dependent on their concentrations. The slope of the plots yielded the

Discussion

Our results clearly show that TQH2 possesses higher reactivity toward galvinoxyl and peroxyl radical than does either UQH2 or TOH. The rate constants for the reaction of TQH2, UQH2, and TOH with galvinoxyl obtained in this study at 25°C in ethanol are comparable with those determined by Mukai et al. [50] toward similar, but not identical, hindered phenoxyl radical under similar conditions. The stoichiometric numbers obtained for the reaction with galvinoxyl (Table 1) prove that both TQH2 and UQH

Acknowledgements

We thank Dr. Mareyuki Takahashi’s kind help. This study was supported in part by Grant-in-Aid for Scientific Research and COE Research from the Ministry of Education, Science, Sports and Culture, Japan, and Research for the Future Program by the Japan Society for the Promotion of Science.

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