Aroxyl-Radical-Scavenging Rate Increases ... - ACS Publications

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Aroxyl-Radical-Scavenging Rate Increases Remarkably under the Coexistence of α‑Tocopherol and Ubiquinol-10 (or Vitamin C): Finding of Synergistic Effect on the Reaction Rate Kazuo Mukai,* Aya Ouchi, Saori Nakaya, and Shin-ichi Nagaoka Department of Chemistry, Faculty of Science, Ehime University, Matsuyama 790-8577, Japan ABSTRACT: Measurements of aroxyl radical (ArO•)-scavenging rate constants (ksAOH) of antioxidants (AOHs) (αtocopherol (α-TocH), ubiquinol-10 (UQ10H2), and sodium ascorbate (Na+AsH−)) were performed in 2-propanol/water (2-PrOH/H2O, 5/1, v/v) solution using stopped-flow spectrophotometry. ksAOH values were measured not only for each AOH but also for the mixtures of two AOHs ((i) α-TocH and UQ10H2 and (ii) α-TocH and Na+AsH−). A notable synergistic effect that the ksAOH values increase 1.6, 2.5, and 6.8 times for α-TocH, UQ10H2, and Na+AsH−, respectively, was observed for the solutions including two kinds of AOHs. Furthermore, measurements of the regeneration rates of αtocopheroxyl radical (α-Toc•) to α-TocH by UQ10H2 and Na+AsH− were performed in 2-PrOH/H2O using double-mixing stopped-flow spectrophotometry. Second-order rate constants (kr) obtained for UQ10H2 and Na+AsH− were 2.01 × 105 and 1.19 × 106 M−1 s−1, respectively. In fact, UV−vis absorption of α-Toc• (λmax = 428 nm), which had been produced by reaction of αTocH with ArO•, disappeared under the existence of UQ10H2 or Na+AsH− due to the above fast regeneration reaction. The result indicates that the prooxidant effect of α-Toc• is suppressed by the coexistence of UQ10H2 or Na+AsH−. As α-TocH, UQ10H2, and ascorbate monoanion (AsH−) coexist in relatively high concentrations in plasma, blood, and various tissues, the above synergistic effect, that is, the increase of the free-radical-scavenging rate and suppression of the prooxidant reaction, may function in biological systems.

kr

1. INTRODUCTION It is well known that vitamin E (α-tocopherol, α-TocH) is one of the most important lipophilic antioxidants (AOHs) in foods and biological systems.1−3 α-TocH functions as an efficient inhibitor of lipid peroxidation in plasma, LDL, and biomembranes.1,2,4,5 The antioxidant action of α-TocH has been ascribed to the scavenging reaction of lipid peroxyl radical (LOO•), producing the corresponding α-tocopheroxyl (αToc•) radical (reaction 1).1,6 On the other hand, if α-TocH exists in biomembranes and edible oils, α-Toc• radicals may react with unsaturated lipids (LHs) (reaction 2). Reaction 2 is known as a prooxidant reaction, which induces degradation of unsaturated lipids.7−10 k inh

LOO• + α ‐TocH ⎯→ ⎯ LOOH + α ‐Toc•

α ‐Toc• + UQ 10H 2 → α ‐TocH + UQ 10H•

where UQ10H• denotes a ubisemiquinone radical. Therefore, kinetic studies were performed for reactions 3 and 4 in organic solvents using chemiluminescense,18 stopped-flow spectrophotometry,19−21 and O2 consumption method.22 Results indicated that both reactions are important for the antioxidant actions of UQ10H2. On the other hand, vitamin C (Vit C) (ascorbate monoanion, AsH−) is well known as a representative watersoluble AOH. Hydrophilic Vit C (AsH−) also enhances the antioxidant activity of α-TocH by regenerating α-Toc• to αTocH (reaction 5)23−26 kr

α ‐Toc• + AsH− → α ‐TocH + As−•

(1)

kp

α ‐Toc• + LH → α ‐TocH + L•

k inh

LOO + UQ 10H 2 ⎯→ ⎯ LOOH + UQ 10H

•

© 2013 American Chemical Society

(5)

where As−• is ascorbate free radical. Furthermore, it was also reported that AsH− functions as an LOO• scavenger in water phase.1,27

(2)

Ubiquinol-10 (reduced ubiquinone-10, UQ10H2) is also well known as a representative lipophilic antioxidant.11,12 It has been suggested that UQ10H2 functions as an AOH (i) by scavenging LOO• (reaction 3) and (ii) by regenerating α-Toc• to α-TocH (reaction 4)1,12−17 •

(4)

k inh

LOO• + AsH− ⎯→ ⎯ LOOH + As−•

(6)

Received: April 1, 2013 Revised: June 17, 2013 Published: June 18, 2013

(3) 8378

dx.doi.org/10.1021/jp403239q | J. Phys. Chem. B 2013, 117, 8378−8391

The Journal of Physical Chemistry B

Article

It is well known that mixtures of (i) α-TocH and UQ10H2 and (ii) α-TocH and Vit C may function synergistically as AOHs in various tissues.1,27 The synergistic effect of α-TocH and UQ10H2 (or Vit C) is considered to be due to the fast regeneration reaction from α-Toc• to α-TocH by UQ10H2 (or Vit C).19−21,23−26 In previous works, we measured the reaction rates (ks) of many kinds of natural AOHs (such as TocH derivatives including α-, β-, γ-, δ-TocHs,28−32 biological hydroquinones including UQ10H2,32 Vit C (or sodium ascorbate),33 and many polyphenols including catechins and flavone derivatives33,34) with 2,6-di-tert-butyl-4-(4-methoxyphenyl)phenoxyl (aroxyl, ArO•) (see Figure 1) in ethanol, 2-propanol/water (2-PrOH/

2. EXPERIMENTAL METHODS 2.1. Materials. α-Tocopherol was supplied from Eisai Co. Ltd. Sodium ascorbate (Na+AsH−) is commercially available (Wako Chemicals, Japan). Ubiquinone-10 (UQ10) was kindly supplied by Kaneka Co. Ltd. Ubiquinol-10 (UQ10H2) was prepared by reduction of UQ10 with sodium hydrosulfite in nhexane under a nitrogen atmosphere.38 It was recrystallized from ethanol/petroleum ether solution. Mp: 46−47 °C. UV spectrum (ethanol): λmax = 289 nm (ε = 4340 M−1 cm−1) and λmax = 290 nm (ε = 4010 M−1 cm−1), see ref 38)). The UQ10H2 prepared was kept under vacuum in a refrigerator at −20 °C. The purity of UQ10H2 was checked spectrophotometrically before use using the above molar extinction coefficient (ε) in ethanol. Experiments were always performed using pure UQ10H2. ArO• radical was prepared according to the method of Rieker et al.40 2.2. Measurements. Measurement of the second-order rate constant (ks) for reaction of ArO• with AOH (reaction 7) was performed with a Unisoku single-mixing stopped-flow spectrophotometer (Model RSP-1000) by mixing equal volumes of 2-PrOH/H2O (5/1, v/v) solutions of ArO• and AOH under nitrogen atmosphere.32,33 The kr value (reactions 4 and 5) was measured with a Unisoku double-mixing stopped-flow spectrophotometer (model RSP-1000-03F).21,26 By the first mixing of equal volumes of 2-PrOH/H2O solutions of α-TocH (cell A) and ArO• (cell B), α-Toc• radical was prepared (reaction 7), and after 2 s the second mixing of equal volumes of α-Toc• solution and UQ10H2 (or Na+AsH−) solution (cell C) (reactions 4 and 5) was performed using a double-mixing unit of the RSP-1000. The rate constant (kr) was determined by analyzing the decay curve of α-Toc• as described later. The time between mixing two solutions and recording the first data point (that is, dead time) was 10−20 ms. The reaction was monitored with either single-wavelength detection (Figure 2b) or a photodiode array detector (Figure 2a) attached to the stopped-flow spectrophotometer. All measurements were performed at 25.0 ± 0.5 °C. Experimental errors in the rate constants (ks and kr) were estimated to be about 5% in 2-PrOH/H2O solution. Ethanol had been generally used for measurements of the ks and kr values. However, as Na+AsH− is insoluble in ethanol, 2-PrOH/ H2O (5/1, v/v) solution was used in the present work.

Figure 1. Molecular structures of α-tocopherol (α-TocH), αtocopheroxyl radical (α-Toc•), ubiquinol-10 (UQ10H2), sodium ascorbate (Na+AsH−), and aroxyl radical (ArO•).

H2O) (5/1, v/v), and micellar solutions (reaction 7) using stopped-flow spectrophotometry. ArO• can be regarded as a model for active oxygen radicals (LOO• and others) in biological systems, as described in previous works.2,28−32 ks

ArO• + AOH → ArOH + AO•

(7)

As described above, α-TocH, UQ10H2, and Vit C are the most important natural antioxidants in biological systems. These AOHs coexist in relatively high concentrations in human (and rat) plasma and various tissues.35−39 Furthermore, it is well known that various AOHs coexist in many foods.3 However, a detailed kinetic study has not been performed for mixtures of these AOHs, as far as we know. Therefore, in the present work, first, measurements of the second-order rate constant (ks) were performed for reaction of ArO• radical with α-TocH, UQ10H2, and Na+AsH− in 2-PrOH/H2O (5/1, v/v) solution at 25 °C using stopped-flow spectrophotometry. Second, measurements of the ks value were performed for the solutions including two kinds of AOHs ((i) α-TocH and UQ10H2 and (ii) α-TocH and Na+AsH−) in order to investigate the synergistic effect of AOHs on the ArO•-scavenging rate. Third, measurements of the rate constant (kr) for reactions of α-Toc• radical with UQ10H2 and Na+AsH− (reactions 4 and 5) were performed in 2-PrOH/H2O solution using double-mixing stopped-flow spectrophotometry.

3. RESULTS 3.1. Measurements of the Aroxyl Radical-Scavenging Rates (ks (alone)) for α-Tocopherol, Ubiquinol-10, and Sodium Ascorbate in 2-Propanol/Water (5/1, v/v) Solution. Measurement of the rate constant (ksα‑TocH) for reaction of ArO• radical with α-TocH was performed in 2PrOH/H2O solution (reaction 7). By reacting α-TocH with ArO• radical, the absorbances at 375 and 578 nm of the ArO• decrease and the absorbance at 428 nm of α-Toc• radical increases, as shown in Figure 2a. The scavenging rate of ArO• was measured by following the decrease in absorbance at 375 or 578 nm of the ArO• radical, as shown in Figure 2b.29,32 The pseudo-first-order rate constants (kobsd) at 375 or 578 nm were linearly dependent on the concentration of α-TocH [α-TocH], and thus, the rate equation is expressed as −d[ArO• ]/dt = kobsd[ArO• ] = ks α ‐ TocH[α ‐ TocH][ArO• ] (8) 8379



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Figure 2. (a) Change in electronic absorption spectra of ArO• and α-Toc• radicals during reaction of ArO• with α-TocH in 2-propanol/water (5/1, v/v) solution at 25.0 °C. Initial concentration is [ArO•] = 7.78 × 10−5 M and [α-TocH] = 4.11 × 10−4 M. Spectra were recorded at 100 ms intervals. Arrow indicates a decrease (ArO•) and an increase (α-Toc•) in absorbance with time. Time dependences of the absorbance of (b) ArO• radical (at 375 nm) and (d) α-Toc• radical (at 428 nm) in solutions including six and five different concentrations of α-TocH at 25.0 °C, respectively. (c) Pseudo-first-order rate constant (kobsd) versus [α-TocH] plot.

the ε value of α-Toc• radical at λmax = 428 nm using the relation (absorbance (of α-Toc• at tmax) = ε × [ArO•]t = 0), as reported in a previous work.42 The ε value of α-Toc• radical obtained was 4470 M−1 cm−1 in 2-PrOH/H2O solution. Similar measurements were performed for reaction of UQ10H2 with ArO• in 2-PrOH/H2O solution. By reacting UQ10H2 with ArO• radical, the absorbance at 375 nm of ArO• decreases rapidly, as shown in Figure 3a. kobsd values at 375 nm were linearly dependent on [UQ10H2], and the rate constant (ksUQ10H2 (alone)) was obtained by plotting kobsd against [UQ10H2], as shown in Figure 3b. As shown in Figure 3a, we could not observe the absorption spectrum of UQ10H• radical because of its instability.43 Similarly, by reacting Na+AsH− with ArO•, the absorbance at 375 nm of ArO• decreases rapidly, as shown in Figure 4a. The rate constant (ksNa+AsH‑ (alone)) was also obtained by plotting kobsd against [Na+AsH−] (see Figure 4b). Na+As−• radical is stable, but the absorption of Na+As−• radical is not seen in the spectrum because a weak broad absorption of Na+As−• at 370 nm overlaps with the strong absorption of ArO•.26,44

where ksα‑TocH is the second-order rate constant for oxidation of α-TocH by ArO• radical. The rate constant (ksα‑TocH) was obtained by plotting kobsd against [α-TocH], as shown in Figure 2c. The ksα‑TocH (alone) value obtained is 8.87 × 103 M−1 s−1 (see Table 1), where “ksα‑TocH (alone)” means the ArO•-radical scavenging rate constants obtained in solution including only one component of antioxidant. As described above, by reacting α-TocH with ArO• in 2PrOH/H2O solution, α-Toc• is produced rapidly. α-Toc• is unstable at 25.0 °C; its absorption peak decreases gradually after passing though the maximum and disappears by a bimolecular reaction (reaction 9) (see Figure 2d).6,41,42 2k d

α ‐Toc• + α ‐Toc• ⎯→ ⎯ nonradical products(NRP)

(9)

As shown in Figure 2d, the maximum absorbance (that is, the concentration) of α-Toc• observed at tmax increases with increasing [α-TocH] and approaches a constant value, because at high [α-TocH] α-Toc• appears rapidly and the decay of αToc• is very small and negligible. Therefore, we can estimate 8380



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Table 1. Second-Order Rate Constants (ksAOH (alone)) for Reaction 7 of Antioxidants (AOHs) (α-Tocopherol, Ubiquinol-10, and Sodium Ascorbate) with ArO• Radical, Relative Rate Constants (ksAOH (alone)/ksα‑TocH (alone)), and Second-Order Rate Constants (krAOH) for Reactions 4 and 5 of Ubiquinol-10 and Sodium Ascorbate with α-Toc• Radical in 2-Propanol/Water (5:1, v/v) Solution at 25.0 °C antioxidant

ksAOH (alone)/M−1 s−1 a

av ksAOH (alone)/M−1 s−1

α-TocH

8.87 × 103

avb 8.65 × 103

1.00

av 8.63 × 103

0.998

2.01 × 105

av 1.02 × 104

1.18

1.19 × 106

UQ10H2

Na+AsH−

a

8.26 8.58 8.89 9.05 8.46 8.39 9.60 1.01 1.10

× × × × × × × × ×

ksAOH (alone)/ ksα‑TocH (alone)

krAOH/M−1 s−1 a

3

10 103 103 103 103 103 103 104 104

Experimental errors are ±5%. bav denotes the average value.

Measurements of ksAOH (alone) values were repeated three or four times for every α-TocH, UQ10H2, and Na+AsH− to obtain reliable ksAOH (alone) values. ksAOH (alone) value and average ksAOH (alone) values obtained are summarized in Table 1. ksAOH (alone) values of α-TocH, UQ10H2, and Na+AsH− are similar to one another. ksAOH (alone) values of UQ10H2 and Na+AsH− are 0.998 and 1.18 times as large as that of α-TocH, respectively. 3.2. Measurements of the Regeneration Rates (kr) of α-Tocopherol by Ubiquinol-10 and Sodium Ascorbate in 2-Propanol/Water. It is well known that mixtures of (i) αTocH and UQ10H2 and (ii) α-TocH and Vit C may function synergistically as antioxidants in various tissues.1,5,12 Synergitic effects of α-TocH and UQ10H2 (or Vit C) are considered to be due to the fast regeneration reaction from α-Toc• to α-TocH by UQ10H2 (or Vit C) in biological systems.19,23 In the present work, sodium ascorbate (Na+AsH−) was used for the measurement because Vit C (AsH2) takes ascorbate monoanion (AsH−) structure at pH 7.4.24 As described in Experimental Methods, measurement of the kr value for reaction of α-Toc• radical with UQ10H2 (reaction 4) was performed in 2-PrOH/H2O solution using a double-mixing stopped-flow spectrophotometer.21 α-Toc• radical was prepared by the first mixing of equal volumes of α-TocH (cell A) and ArO• (cell B) solutions (reaction 7), and after 2 s the second mixing of equal volumes of α-Toc• solution and UQ10H2 solution (cell C) (reaction 4) was made. Typical concentrations in cells A and B are 2.16 × 10−3 and 2.01 × 10−4 M, respectively. Decay curves of the absorbance of α-Toc• at 428 nm in 2-PrOH/H2O are shown in Figure 5a, indicating that the decay rates increase with increasing [UQ10H2]. The pseudo-first-order rate constants (kobsd) observed at 428 nm were linearly dependent on [UQ10H2], and thus, the rate equation is expressed as follows

Figure 3. (a) Change in electronic absorption spectrum of ArO• radical during reaction of ArO• with UQ10H2 in 2-propanol/water at 25.0 °C. Initial concentration is [ArO•] = 7.73 × 10−5 M and [UQ10H2] = 3.36 × 10−4 M. Absorption of UQ10H• was not observed. (b) Plots of kobsd versus [UQ10H2] for reactions of ArO• radical with (i) UQ10H2 only (○) and (ii) mixture of α-TocH and UQ10H2 (●). (c) Plots of kobsd versus [UQ10H2] for reactions of ArO• radical with (i) α-TocH only (○) and (ii) mixture of α-TocH and UQ10H2 (measurements 1 (▲), 2 (■), and 3 (●), see Table 2B and text). Dotted lines show the plots for which any synergistic effect is absent between α-TocH and UQ10H2.

−d[α ‐Toc•]/dt = kobsd[α ‐Toc•] = k r[UQ 10H 2][α ‐Toc•] (10)

The kr value was obtained by plotting kobsd against [UQ10H2], as shown in Figure 5b. Similar measurements were performed for reaction of α-Toc• with Na+AsH− in 2-PrOH/H2O solution. The kobsd versus 8381



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Figure 5. (a) Time dependences of the absorbance of α-Toc• radical at 428 nm in 2-propanol/water including six different concentrations of UQ10H2 at 25.0 °C. (b) kobsd versus [UQ10H2 (or Na+AsH−)] plot.

[Na+AsH−] plot is also shown in Figure 5b. kr values obtained for UQ10H2 and Na+AsH− are 2.01 × 105 and 1.19 × 106 M−1 s−1, respectively, as listed in Table 1. The rate constant (kr) of Na+AsH− is 5.92 times larger than that of UQ10H2. kr values obtained are very fast and about 1−2 orders of magnitude larger than those for ksα‑TocH (alone) (av 8.65 × 103 M−1 s−1), ksUQ10H2 (alone) (av 8.63 × 103 M−1 s−1), and ksNa+AsH‑ (alone) (av 1.02 × 104 M−1 s−1). Furthermore, kr values are 2−3 orders of magnitude larger than the rate constant (2kd = 1.30 × 103 M−1 s−1 in ethanol) for the self-bimolecular reaction of αToc•.42 3.3. Measurements of the Aroxyl Radical-Scavenging Rates (ks) for Mixtures of α-Tocopherol and Ubiquinol10 in 2-Propanol/Water. a. Measurement of the ksα‑TocH (+UQ10H2) Value by Keeping Ubiquinol-10 at a Constant Concentration and Varying Concentration of α-Tocopherol. Measurements of the ksα‑TocH (+UQ10H2) and ksUQ10H2 (+αTocH) values were performed for the solution including αTocH and UQ10H2. “ksα‑TocH (+UQ10H2) and ksUQ10H2 (+αTocH)” indicate the rate constants obtained in the solution

Figure 4. (a) Change in electronic absorption spectrum of ArO• radical during reaction of ArO• with Na+AsH− in 2-propanol/water at 25.0 °C. [ArO•] = 7.32 × 10−5 M and [Na+AsH−] = 4.00 × 10−4 M. Absorption of Na+As−• was not observed. (b) Plots of kobsd versus [Na+AsH−] for reactions of ArO• radical with (i) Na+AsH− only (○) and (ii) mixture of α-TocH and Na+AsH− (■). (c) Plots of kobsd versus [Na+AsH−] for reactions of ArO• radical with (i) Na+AsH− only (○) and (ii) mixture of α-TocH and Na+AsH− (measurements 1 (▲), 2 (■), and 3 (●), see Table 3B and text). 8382



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If α-TocH and UQ10H2 coexist in solution, reactions 11 and 12 will occur competitively in solution because the ksα‑TocH (alone) and ksUQ10H2 (alone) values are very similar to each other, as listed in Table 1.

including two components of AOHs. First, measurement of ksα‑TocH (+UQ10H2) was performed by keeping [UQ10H2] constant (9.80 × 10−5 M) and varying [α-TocH] (0−3.27 × 10−4 M). By mixing the solution of ArO• radical with the solution including α-TocH and UQ10H2, absorption of the ArO• at 375 and 578 nm decreases rapidly, as shown in Figure 6a. Absorption of α-Toc• at 428 nm was not observed at low [α-TocH] (0−8.16 × 10−5 M), but weak absorption appeared at high [α-TocH] ((1.63−3.27) × 10−4 M)) (data not shown). By analyzing the decay curves of ArO• at 375 nm, kobsd values were determined. Figure 6b shows the kobsd versus [α-TocH] plot.

ArO• + α ‐ TocH + UQ 10H 2 ks α‐ TocH

⎯⎯⎯⎯⎯⎯⎯→ ArOH + α ‐ Toc• + UQ 10H 2

(11)

ArO• + α ‐ TocH + UQ 10H 2 ks UQ 10H2

⎯⎯⎯⎯⎯⎯⎯→ ArOH + α ‐ TocH + UQ 10H•

(12)

In such a case, we can expect that the kobsd value depends on eq 13 if the interaction between α-TocH and UQ10H2 is negligible. kobsd = ksα‐ TocH(alone)[α ‐ TocH] + ksUQ 10H2(alone) [UQ 10H 2]

(13)

Next, we will assume that a synergistic effect is present. By substituting ksα‑TocH (alone) and ksUQ10H2 (alone) values and the value of [UQ10H2] (= 9.80 × 10−5 M) used for measurement 1 into eq 13, kobsd was plotted against [α-TocH] (see a dotted line in Figure 6b). Further, the result of the kobsd versus [αTocH] plot (open circle) obtained for the solution including only α-TocH (see eq 8) is also shown in Figure 6b. As described above, measurement 1 was performed by keeping UQ10H2 at a constant concentration and varying [α-TocH]. Consequently, the ksα‑TocH (+UQ10H2) value was determined from the gradient of the kobsd versus [α-TocH] plot (closed square) in Figure 6b using eq 14. kobsd = ksα‐ TocH(+UQ 10H 2)[α ‐ TocH] + ksUQ 10H2(alone) [UQ 10H 2]

(14)

As expected from the gradient in Figure 6b, the ksα‑TocH (+UQ10H2) value (1.22 × 104 M−1 s−1) is 1.41 times larger than ksα‑TocH (alone) (8.65 × 103 M−1 s−1) obtained for the solution including only α-TocH. A notable effect due to the coexistence of α-TocH and UQ10H2 in solution was observed for the rate constant (ksα‑TocH (+UQ10H2)). In order to investigate the effect of the concentration of UQ10H2 on the reaction rate (ksα‑TocH (+UQ10H2)), a similar measurement was performed for the mixture including higher constant [UQ10H2] (see measurement 2 in Table 2A). As shown in Figure 6b, the ksα‑TocH (+UQ10H2) value (1.41 × 104 M−1 s−1) obtained is larger than that (1.22 × 104 M−1 s−1) for the lower [UQ10H2]. b. Measurement of the ksUQ10H2 (+α-TocH) Value by Keeping α-Tocopherol at a Constant Concentration and Varying Concentration of Ubiquinol-10. As a notable effect of the coexistence of α-TocH and UQ10H2 was observed for the rate constants (ksα‑TocH (+UQ10H2)), similar measurements were performed for solutions including α-TocH and UQ10H2 by keeping [α-TocH] constant and varying [UQ10H2] (see measurements 1, 2, and 3 in Table 2B). As shown in Figure 7a−d, absorption of α-Toc• at 428 nm decreased with increasing concentration of UQ10H2, suggesting that the αToc• produced is regenerated quickly to α-TocH by reaction with UQ10H2 (reaction 4) because the regeneration rate constant (kr) (2.01 × 105 M−1 s−1) is very fast compared with the ksα‑TocH (+UQ10H2) value.

Figure 6. (a) Change in electronic absorption spectrum of ArO• radical during reaction of ArO• with a mixture of α-TocH and UQ10H2 in 2-propanol/water at 25.0 °C. [ArO•] = 8.15 × 10−5, [α-TocH] = 1.63 × 10−5, and [UQ10H2] = 9.80 × 10−5 M. Absorption of α-Toc• was not observed due to reaction with UQ10H2. (b) Plots of kobsd versus [α-TocH] for reactions of ArO• radical with (i) α-TocH only (○) and (ii) mixture of α-TocH and UQ10H2 (measurements 1 (■) and 2 (●), see Table 2A and text). 8383



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Table 2. Second-Order Rate Constants (ksα‑TocH (+UQ10H2) and ksUQ10H2 (+α-TocH)) Obtained for Mixtures of Two Kinds of Antioxidants (α-TocH and UQ10H2) and Ratios (ksα‑TocH (+UQ10H2)/ksα‑TocH (alone) and ksUQ10H2 (+α-TocH)/ksUQ10H2 (alone)) (A) measurements 1 and 2 were performed by keeping [UQ10H2] constant and varying [α-TocH] [UQ10H2]/M measurement 1 measurement 2

−4

ksα‑TocH (+UQ10H2)/ksα‑TocH (alone)

9.80 × 10 (lower) (0−3.27) × 10 1.22 × 10 1.96 × 10−4 (higher) (0−3.06) × 10−4 1.41 × 104 (B) measurements 1−3 were performed by keeping [α-TocH] constant and varying [UQ10H2] [α-TocH]/M

a

ksα‑TocH (+UQ10H2)/M−1 s−1 a

[α-TocH]/M

−5

measurement 1

6.68 × 10−5 (lower)

measurement 2

1.25 × 10−4 (intermediate)

measurement 3

2.17 × 10−4 (higher)

4

[UQ10H2]/M (0−0.124) (0.206−0.825) (0.825−1.65) (0−0.391) (0.586−1.56) (0−0.660) (0.660−1.32)

× × × × × × ×

10−4 10−4 10−4 10−4 10−4 10−4 10−4

1.41 1.63

ksUQ10H2 (+α-TocH)/M−1 s−1 a

ksUQ10H2 (+α-TocH)/ksUQ10H2 (alone)

× × × × × × ×

2.68 1.34 1.10 2.33 1.32 2.47 1.61

2.31 1.16 9.47 2.01 1.14 2.13 1.39

104 104 103 104 104 104 104

Experimental errors are ±5%.

Figure 7. Change in electronic absorption spectra of ArO• and α-Toc• radicals during reaction of ArO• with a mixture of α-TocH and UQ10H2 in 2propanol/water at 25.0 °C. [ArO•] = 6.22 × 10−5 and [α-TocH] = 1.25 × 10−4 M. Spectra were recorded at 200 ms intervals. Absorption of α-Toc• decreased with increasing concentrations of UQ10H2. [UQ10H2] = (a) 0, (b) 7.82 × 10−6, (c) 1.95 × 10−5, and (d) 9.77 × 10−5 M.

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Table 3. Second-Order Rate Constants (ksα‑TocH (+Na+AsH−) and ksNa+AsH‑ (+α-TocH)) Obtained for Mixtures of Two Kinds of Antioxidants (α-TocH and Na+AsH−) and Ratios (ksα‑TocH (+Na+AsH−)/ksα‑TocH (alone) and ksNa+AsH‑ (+α-TocH)/ks Na+AsH‑ (alone)) (A) measurements 1 and 2 were performed by keeping [Na+AsH−] constant and varying [α-TocH] [Na+AsH−]/M

−4

ksα‑TocH (+Na+AsH−)/ksα‑TocH (alone)

1.17 × 10 (lower) (0−3.00) × 10 1.23 × 10 2.21 × 10−4 (higher) (0−3.28) × 10−4 1.02 × 104 (B) measurements 1−3 were performed by keeping [α-TocH] constant and varying [Na+AsH−]

measurement 1 measurement 2

[α-TocH]/M

a

ksα‑TocH (+Na+AsH−)/M−1 s−1 a

[α-TocH]/M

−4

−5

measurement 1

5.56 × 10

measurement 2

1.34 × 10−4 (intermediate)

measurement 3

2.22 × 10−4 (higher)

(lower)

4

[Na+AsH−]/M (0−0.209) (0.419−1.47) (0−0.225) (0.450−1.80) (0−0.200) (0.200−0.790) (0.999−1.60)

× × × × × × ×

ksNa+AsH‑ (+α-TocH)/M−1 s−1a −4

10 10−4 10−4 10−4 10−4 10−4 10−4

2.70 9.62 5.59 8.61 6.95 1.25 4.84

× × × × × × ×

4

10 103 104 103 104 104 103

1.42 1.18

ksNa+AsH‑ (+α-TocH)/ks

Na+AsH‑

(alone)

2.64 0.943 5.48 0.844 6.81 1.23 0.474

Experimental errors are ±5% ksα‑TocH.

as large as the ksα‑TocH (alone) value (av 8.65 × 103 M−1 s−1). However, the former value obtained for a lower concentration of Na+AsH− is larger than the latter one obtained for a high concentration of Na+AsH−. b. Measurements of the ksNa+AsH‑ (+α-TocH) Value by Keeping α-Tocopherol at a Constant Concentration and Varying Concentration of Sodium Ascorbate. The ksNa+AsH‑ (+α-TocH) value was measured by keeping [α-TocH] constant (1.34 × 10−4 M) and varying [Na+AsH−] (0−1.80 × 10−4 M) (see measurement 2 in Table 3B). Figure 4b shows the kobsd versus [Na+AsH−] plot. The plot shows two different gradients at low and high concentrations of Na+AsH−. From the gradients, two different values of ksNa+AsH‑ (+α-TocH) (= 5.59 × 104 and 8.61 × 103 M−1 s−1) were obtained. These values are 5.48 and 0.844 times as large as ksNa+AsH‑ (alone) (1.02 × 104 M−1 s−1), respectively. At the low-concentration region of Na+AsH−, a very large synergistic effect was observed. On the other hand, at the high-concentration region, the rate constant (ksNa+AsH‑ (+α-TocH)) was smaller than ksNa+AsH‑ (alone), although the kobsd value (that is, the total aroxyl radical scavenging rate (−d[ArO•]/dt)) was still larger than that expected from eq 13 (that is, reaction of Na+AsH− independent of that of α-TocH) (dotted line in Figure 4b). Furthermore, two different constant concentrations of αTocH ([α-TocH] = 5.56 × 10−5 and 2.22 × 10−4 M) were used in measurements 1 and 3, respectively. Figure 4c shows the kobsd versus [Na+AsH−] plot, indicating that the plot consists of two or three different gradients. The ksNa+AsH‑ (+α-TocH) values obtained are listed in Table 3B. As observed for measurements 1 and 3, ksNa+AsH‑ (+α-TocH) values obtained at low concentration of Na+AsH− are larger than the corresponding ones at high concentration. 3.5. UV−Vis Absorption Spectra of the Mixtures of αTocopherol and Ubiquinol-10 (or Sodium Ascorbate) in 2-Propanaol/Water. Measurements of the UV−vis absorption spectra of α-TocH, UQ10H2, and Na+AsH− were performed in 2-PrOH/H2O. Similarly, UV−vis absorption spectra were measured for solutions including (i) α-TocH and UQ10H2 and (ii) α-TocH and Na+AsH−. Spectra obtained were explained by the simple sum of the spectrum of each antioxidant included (data are not shown), indicating that the interaction between α-TocH and UQ10H2 (or Na+AsH−) is negligible in the mixture. The result suggests that the increase

As shown in Figure 3b, the kobsd versus [UQ10H2] plot (closed circle) for measurement 3 consists of two lines having different gradients. Analysis of the rate constant (ksUQ10H2 (+αTocH)) was performed tentatively using eq 15 similar to eq 14. kobsd = ks α‐ TocH(alone)[α ‐TocH] + ks UQ 10H2( +α ‐TocH) [UQ 10H 2]

(15)

The ksUQ10H2 (+α-TocH) values obtained at low and high [UQ10H2] (0−0.660 × 10−4 and 0.660−1.32 × 10−4 M) are 2.13 × 104 and 1.39 × 104 M−1 s−1, respectively (see Table 2B). The ksUQ10H2 (+α-TocH) values of the former and the latter are 2.47 and 1.61 times larger than the ksUQ10H2 (alone) value, respectively. Similar measurements were performed for solutions including various [α-TocH] and [UQ10H2] (see measurements 1 and 2 in Table 2B). As shown in Figure 3c and listed in Table 2B, two and three different ksUQ10H2 (+α-TocH) values appeared along eq 15 for solutions including intermediate and lower constant [α-TocH] (closed quare and closed triangle), respectively. The larger ksUQ10H2 (+α-TocH) values were obtained at the low concentration region of UQ10H2. Furthermore, ksUQ10H2 (+α-TocH) values obtained were larger than the ksUQ10H2 (alone) value in every concentration of αTocH and UQ10H2. 3.4. Measurements of the Aroxyl Radical-Scavenging Rates (ks) for Mixtures of α-Tocopherol and Sodium Ascorbate in 2-Propanol/Water. a. Measurements of the ksα‑TocH (+Na+AsH−) Value by Keeping Sodium Ascorbate at a Constant Concentration and Varying Concentration of αTocopherol. Similar measurements (see measurements 1 and 2 in Table 3A) were performed for the solution including αTocH and Na+AsH− by keeping [Na+AsH−] constant and varying [α-TocH]. Absorption of α-Toc• at 428 nm was not observed at all concentrations of α-TocH ((0−3.00) × 10−4 M) used (see Figure 8a), indicating that a rapid regeneration reaction (reaction 5) proceeded in solution. The kobsd versus [α-TocH] plot is shown in Figure 8b. As performed for the mixture of α-TocH and UQ 10 H 2 (see eq 14), k s α‑TocH (+Na + AsH − ) values were determined. The k s α ‑ T o c H (+Na+AsH−) values (1.23 × 104 M−1 s−1) (measurement 1) and (1.02 × 104 M−1 s−1) (measurement 2) obtained under the coexistence of Na+AsH− are 1.42 and 1.18 times, respectively, 8385



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On the other hand, if UQ10H2 coexists in the above solution, absorption of α-Toc• radical at 428 nm decreases largely with increasing [UQ10H2] and disappears at higher [UQ10H2], as shown in Figure 7. The time dependence of [α-Toc•] observed at λmax (= 428 nm) is shown in Figure 9a, where [α-Toc•] was

Figure 8. (a) Change in electronic absorption spectrum of ArO• radical during reaction of ArO• with a mixture of α-TocH and Na+AsH− in 2-propanol/water at 25.0 °C. [ArO•] = 8.02 × 10−5, [αTocH] = 3.00 × 10−4, and [Na+AsH−] = 1.17 × 10−4 M. Absorption of α-Toc• was not observed due to reaction with Na+AsH−. (b) Plots of kobsd versus [α-TocH] for reaction of ArO• radical with (i) α-TocH only (○) and (ii) mixture of α-TocH and Na+AsH− (measurements 1 (■) and 2 (●), see Table 3A and text).

Figure 9. (a) Time dependences of the concentration of α-Toc• radical [α-Toc•] (at 428 nm) in 2-propanol/water including nine different concentrations of UQ10H2 at 25.0 °C. (b) [α-Toc•] at 2 s in part a versus [UQ10H2] plot.

of the rate constants (ksAOH) observed for mixtures of α-TocH and UQ10H2 (or Na+AsH−) may not be explained by such an interaction. 3.6. Decrease in UV−Vis Absorption of α-Tocopheroxyl Radical under the Coexistence of α-Tocopherol and Ubiquinol-10 (or Sodium Ascorbate). By reacting ArO• radical with α-TocH, absorption of α-Toc• radical appears rapidly and decreases gradually, as shown in Figure 2d. The concentration of α-Toc• radical ([α-Toc•]) produced by reaction with ArO• ([ArO•] = 7.78 × 10−5 M) is similar to that of ArO• if a high concentration of α-TocH was used for the reaction, as described in a previous section.42

calculated from the absorbance of α-Toc• using the relation (absorbance = εcl, ε = 4470 M−1 cm−1).42 Figure 9b shows the [α-Toc•] (at 2 s in Figure 9a) versus [UQ10H2] plot. Absorption of α-Toc• disappears at [UQ10H2] ≈ 6.0 × 10−5 M. The result indicates that one molecule of UQ10H2 may quickly regenerate one molecule of α-Toc• to one molecule of α-TocH because the initial [α-Toc•] is 7.78 × 10−5 M as mentioned above. Similar measurements were performed for solutions including α-TocH and Na+AsH−. As shown in Figure 10a, 8386



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pherol and Ubiquinol-10 (or Sodium Ascorbate). The free-radical-scavenging AOHs function not only individually but also synergistically with other AOHs. The most well-known interaction is the one between α-TocH and Vit C.1,27 Hydrophilic Vit C present in the aqueous phase efficiently reduces α-Toc• radical located within the membranes and lipoproteins to regenerate α-TocH and inhibit initiation of a chain reaction induced by α-Toc• (that is, the prooxidant effect of α-TocH). Similarly, lipophilic UQ10H2 regenerates α-TocH during lipid peroxidation in solution, liposomal membranes, low-density protein, and mitochondrial membranes.11−17 Measurements of the inhibition rate constant (kinh) for the scavenging of peroxyl (LOO•) radical were performed by Niki et al.27 for α-TocH and Vit C in tert-butyl alcohol/methanol (3/1, v/v) solution at 37 °C. Measurement was repeated several times for each reaction. The values obtained were kinh = av 5.1 × 105 (3.89 × 105 to 7.12 × 105) M−1 s−1 and av 0.75 × 105 (0.45 × 105 to 1.08 × 105) M−1 s−1 for α-TocH and Vit C, respectively. The kinh value obtained for α-TocH was several times larger than that for Vit C because Vit C in tert-butyl alcohol/methanol (3/1, v/v) solution will not take the monoanion form (AsH−) at pH = 7.4 in aqueous solution but takes the reduced form (AsH2). The kinh value for the monoanion form (AsH−) is larger than that for the reduced form (AsH2).24 Furthermore, the measurement was performed for the solution including α-TocH and Vit C.27 kinh values measured under the coexistence of α-TocH and Vit C were av 4.0 × 105 (3.21 × 105 to 4.64 × 105) M−1 s−1. The kinh value obtained for the mixture of α-TocH and Vit C was similar to that for α-TocH only. However, the details are not clear because the kinh values reported include large experimental errors, as described above. In the present work, measurements of ArO• radicalscavenging rate constants (ksAOH) of AOHs (α-TocH, UQ10H2, and Na+AsH−) were performed in 2-PrOH/H2O solution. ksAOH values were measured not only for each AOH but also for mixtures of two kinds of AOHs ((i) α-TocH and UQ10H2 and (ii) α-TocH and Na+AsH−). As described in the Results section, a notable synergistic effect was observed for the ksAOH values. The rate constants (ksα‑TocH (+UQ10H2), ksα‑TocH (+Na+AsH−), ksUQ10H2 (+α-TocH), and ksNa+AsH‑ (+α-TocH) values) obtained showed different values depending on the concentrations of two AOHs included in solution. For example, ksα‑TocH (+UQ10H2) values ((1.22 and 1.41) × 104 M−1 s−1) obtained in solution including α-TocH and UQ10H2 were 1.41 and 1.63 times larger than the ksα‑TocH (alone) value (av 8.65 × 103 M−1 s−1) in solution including only α-TocH (see Tables 1 and 2). Similarly, ksUQ10H2 (+α-TocH) values ((9.47−23.1) × 103 M−1 s−1) were 1.10−2.68 times larger than the ksUQ10H2 (alone) value (av 8.63 × 103 M−1 s−1) in solution including only UQ10H2. Furthermore, the ksα‑TocH (+Na+AsH−) value ((1.02 and 1.23) × 104 M−1 s−1) obtained in solution including α-TocH and Na+AsH− was 1.18 and 1.42 times larger than the ksα‑TocH (alone) value (av 8.65 × 103 M−1 s−1) obtained in solution including only α-TocH (see Tables 1 and 3). The effect of the coexistence of α-TocH and Na+AsH− was more notable for ksNa+AsH‑ (+α-TocH). The ratios of the rate constants (ksNa+AsH‑ (+α-TocH)/ksNa+AsH‑ (alone)) varied notably 6.81, 1.23, and 0.474, depending on [Na+AsH−] (see measurement 3 in Table 3B). In particular, at the low-concentration region of Na+AsH− ((0−0.200) × 10−4 M), the ksNa+AsH‑ (+α-TocH) value (6.95 × 104 M−1 s−1) obtained in solution including α-TocH and

Figure 10. (a) Time dependences of the concentration of α-Toc• radical [α-Toc•] (at 428 nm) in 2-propanol/water including nine different concentrations of Na+AsH− at 25.0 °C. (b) [α-Toc•] at 2 s in part a versus [Na+AsH−] plot.

[α-Toc•] decreases with increasing [Na+AsH−]. The [α-Toc•] (at 2 s in Figure 10a) versus [Na+AsH−] plot is shown in Figure 10b. The result indicates that one molecule of Na+AsH− may regenerate more than one molecule of α-Toc• to α-TocH. Not only the H atom of the OH group at 2 position of Na+AsH− but also the H atom of the −CH− group at the 4 position may contribute to the regeneration reaction of α-Toc• radical because the −CH− hydrogen atom will be activated by the πelectron systems (CCCHOCO) and may show high reactivity with α-Toc• radical.10,45 However, the details are not clear at present.

4. DISCUSSION 4.1. Finding of Synergistic Effect on the Free-RadicalScavenging Rates under the Coexistence of α-Toco8387



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Na+AsH− was 6.81 times larger than the ksNa+AsH‑ (alone) value (av 1.02 × 104 M−1 s−1). In human plasma, the concentrations of α-TocH, Vit C, and UQ10H2 included are as follows; [α-TocH] = av 22 μM (12.0− 36.0 μM), [Vit C] = 34 μM, [UQ10H2] = av 0.71 μM (0.36− 1.10 μM).35−39 Similar (or several times higher) concentrations of AOHs were used for measurements in the cases of the coexistence of α-TocH and Vit C, as listed in Table 3. Consequently, we may expect a similar synergistic effect on the free-radical-scavenging rate functions in human plasma. α-TocH and ubiquinone (UQ) are present in all cellular membranes, blood plasma, and plasma lipoproteins.11,15,37,38 Although the relative distribution of UQ in tissues varies by species, the highest concentrations are found in heart, muscle, liver, kidney, and brain.11,37,38 Lass et al.37 reported the amounts of α-TocH and UQ homologues (UQ9 and UQ10) in (i) serum, (ii) homogenates of the heart, hindlimb skeletal muscle, liver, kidney, and brain, (iii) mitochondria of these tissues, and (iv) brain synaptosomes of mice, determined by HPLC. The molar amount of α-TocH in the serum of mice was ∼17 times higher than the total amount of UQ9 and UQ10. Similarly, the molar amount of α-TocH in the plasma of human was also ∼31 times higher than the total amount of UQ10. In contrast to the serum, the amount of α-TocH in the homogenates of all tissues and their mitochondria in the mice was ∼3−90 times lower than the total amount of UQ9 and UQ10. The results obtained by the present kinetic study suggest that α-TocH and UQ10H2 function synergistically in many biological systems. It is important to know the concentrations of reduced and oxidized UQ in the tissues, because oxidized UQ9 and UQ10 do not show free-radical-scavenging activity.18−20,22 In the case of human liver, pancreas, and intestine, the UQ isolated was completely reduced and the major part of UQ in other tissues was also to a large extent in the reduced form.46 Only in the brain and lung, most of UQ, about 80%, was in the oxidized state. Tang et al.47 reported using HPLC analysis that the percentage of UQ10H2 in total UQ10 was ∼96% in healthy human plasma. Consequently, we may expect a similar synergistic effect on the free-radical-scavenging rate (ks) functions in various human tissues. 4.2. Mechanism for the Increase of the Aroxyl RadicalScavenging Rate (ks) under the Coexistence of αTocopherol and Ubiquinol-10 (or Ascorbate). In previous works, measurements of the scavenging rate (ks) of ArO• by many phenolic AOHs including vitamin E homologue (α-, β-, γ-, and δ-TocH) were performed in ethanol solution (reaction 7).28−32 The logarithms of the rate constants (log ks) were found to correlate well with their peak oxidation potentials (EP); the AOHs which have smaller EP values show higher scavenging rates.28−31 From detailed analysis of the temperature dependence of ks values of the TocHs, the activation energy (Eact) for the reaction was determined.29 The Eact values obtained were also found to correlate well with the EP values. A similar result was obtained for biological hydroquinones including UQ10H2. These facts suggest that the transition states in the above ArO•-scavenging reactions by TocH and UQ10H2 have the property of electron-transfer intermediates [ArO:−---α-TocH•+] and [ArO:−---UQ10H2•+], respectively (see Scheme 1 (eqs 16 and 17)),28−31 as also reported for reaction of LOO• with vitamin E homologue.48,49 We can expect that Na+AsH− will also take a similar electron-transfer intermediate [ArO:− ---Na+AsH•] at the transition state

Scheme 1. One-Component System

(Scheme 1 (eq 18)). As described above, both ArO• radicalscavenging rate constants (ksα‑TocH (+Na+AsH−) and ksNa+AsH‑ (+α-TocH)) increased notably due to the coexistence of Na+AsH− and α-TocH in 2-PrOH/H2O solution, respectively. In such a case, Scheme 2 (eqs 19 and 20) is regarded as the mechanism of the reactions. Scheme 2. Two-Component Systems

As described in the Results section, measurement of the UV−vis absorption spectra was performed under the coexistence of two AOHs. Spectra obtained were simply explained by the sum of the spectrum of each AOH, suggesting that the interaction between AOHs is negligible. The results suggest that the total energy levels of the reactants (ArO• + αTocH + Na+AsH−) in reaction 19 (or 20) and (ArO• + αTocH + UQ10H2) in reaction 11 (or 12) do not vary under the coexistence of two AOHs in solution (see Figure 11). On the other hand, the reaction rate constants (ksAOH) showed a notable increase under the coexistence of two AOHs in solution.

Figure 11. Potential curves of the ArO• radical-scavenging reactions of α-TocH in the presence and absence of Na+AsH−. Figure shows that the transition state for reaction of ArO• with α-TocH has the property of an electron-transfer intermediate [ArO:−---α-TocH•+] (see Scheme 1). 8388



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Recently, kinetic studies of the ArO• radical-scavenging reaction (reaction 7) of α-TocH have been performed in the presence of several alkali and alkaline earth metal salts (such as LiI, NaClO4, and Mg(ClO4)2) in methanol and acetonitrile solutions using stopped-flow spectrophotometry.50,51 The ks values increased linearly with increasing the concentration of metal salts. As given in Scheme 1 (reaction 16), the hydrogen transfer reaction of α-TocH proceeds via an electron transfer intermediate from α-TocH to ArO• radical. Both coordination of metal cations to the one-electron-reduced anions of ArO• (ArO:−) and of counteranions to the one-electron-oxidized cations of α-TocH (α-TocH+•) may stabilize the intermediate, resulting in acceleration of electron transfer, that is, an increase of the rate constant (ksα‑TocH) (see Figure 9 in ref 50). Since Na+AsH− salt consists of Na+ cation and AsH− anion moieties, the effect of the coexistence of Na+AsH− and α-TocH on the reation rate (ksα‑TocH (+Na+AsH−)) will be similarly explained in the following way. As Scheme 2 and Figure 11 indicate, if the transition state of reaction 7 by α-TocH has the property of an electron-transfer intermediate [ArO:−---α-TocH•+], a Na+ cation of Na+AsH− will interact with ArO:− anion by Coulomb interaction (e2/ 4πεor) and form a complex with an ArO:− anion [ArO:−--Na+].50,51 Similarly, the anion moiety (AsH−) of Na+AsH− will also interact with α-TocH•+ cation and form a complex with a α-TocH•+ cation [α-TocH•+---AsH−]. As a result, the energy level of the electron-transfer intermediate [ArO:−---α-TocH•+] will be stabilized (that is, the activation energy (Eact) will decrease), and thus, the ksα‑TocH (two) value increases. As described above, both the ArO:− anion and α-TocH•+ cation are surrounded by many cations and anions of Na+AsH− at the transition state, respectively. The kobsd value increased with increasing concentration of Na+AsH−, as shown in Figure 4b and 4c. If both the ArO:− and α-TocH+ form only (1:1) complex with cation (Na+) and anion (AsH−), respectively, at the transition state, such an increase in the kobsd value would not be observed. The [ArO:−---α-TocH•+] complex is considered to be stabilized by the Coulomb interation with many cations and anions existing in the 2-PrOH/H2O solution. As shown in Figure 4b and 4c, the rate constants (kobsd) increase with increasing concentrations of the Na+AsH−. At the high-concentration region of Na+AsH−, the effect of Na+AsH− is suppressed, the rapid increase of kobsd value stops, and the smaller ksNa+AsH‑ (+α-TocH) values are obtained, as listed in Table 3B. At a low concentration of ascorbate (AsH−), ascorbate anion may occupy the position near the [α-TocH•+] cation, showing the strong Coulomb interaction with [αTocH•+]. At a high concentration of ascorbate, the average distance between ascorbate and [α-TocH•+] will increase, resulting in the decrease of the rate constant (ksNa+AsH‑). However, the detailed mechanism for the remarkable change of the kobsd value (that is, ksNa+AsH‑ (+α-TocH) value) is not clear at present. As described above, both the ksα‑TocH (+UQ10H2) and the UQ10H2 (+α-TocH) values increased notably due to the ks coexistence of two AOHs (α-TocH and UQ10H2) in 2-PrOH/ H2O solution. As the pKa1 value of UQ10H2 is 11.4, UQ10H2 will not take a monoanion form (UQ 10 H − ) but an undissociated form (UQ10H2) in 2-PrOH/H2O.32 Consequently, we cannot expect strong interaction between an electron-transfer intermediate [ArO:−---α-TocH•+] and an UQ10H2 at the transition state. Similarly, we cannot explain the increase of the ksUQ10H2 (+α-TocH) value because the pKa

value of α-TocH is 13.1.32 The mechanism for the notable increase of ksα‑TocH (+UQ10H2) and ksUQ10H2 (+α-TocH) values due to the coexistence of α-TocH and UQ10H2 is not clear at present. 4.3. UV−Vis Absorption of α-Tocopheroxyl Radical Disappears under the Coexistence of α-Tocopherol and Ubiquinol-10 (or Sodium Ascorbate): Suppression of Prooxidant Effect of α-Tocopherol. As described in the Introduction, α-Toc• is an important key radical which appears in the process of the antioxidant and prooxidant actions of αTocH (see reactions 1, 2, 4, and 5). In the present work, a detailed kinetic study was performed for the solution including two kinds of AOHs, and it was found that the rate constants (ksα‑TocH (+UQ10H2), ksα‑TocH (+Na+AsH−), ksUQ10H2 (+αTocH), and ksNa+AsH‑ (+α-TocH)) increase remarkably under the coexistence of two AOHs. Furthermore, as described in the Results, formation of α-Toc• radical was suppressed remarkably under the coexistence of α-TocH and UQ10H2 (or Na+AsH−). As shown in Figures 5−10, we could directly ascertain that αToc• radical produced by reaction with ArO• radical may immediately disappear through the regeneration reaction with UQ10H2 and Na+AsH− by observing the decrease of the UV− vis absorption of α-Toc• radical. In fact, the regeneration rate constants (kr) obtained for UQ10H2 and Na+AsH− were very fast, as listed in Table 1. An example for such a direct observation of the disappearance of αToc• radical under the coexistence of α-TocH and UQ10H2 (or Na+AsH−) has not been reported, to our knowledge. As described above, in similar concentrations α-TocH and Vit C coexist in human plasma.35,36,39 In mitochondria membrane systems (heart, kidney, lung, etc.), α-TocH and UQ10H2 coexist in high concentrations.37 The result of the present work clearly indicates that the prooxidant effect of αTocH, which is usually observed in aprotic edible oils including a high concentration of α-TocH,9 may be suppressed by the coexistence of α-TocH with UQ10H2 (or Vit C) in biological systems.

5. SUMMARY A kinetic study of the ArO• radical-scavenging activity of αTocH, UQ10H2, and Na+AsH− has been performed for the solution, including not only each AOH but also the mixtures of two AOHs ((i) α-TocH and UQ10H2 and (ii) α-TocH and Na+AsH−). The ArO• radical-scavenging rate (ksAOH) for each AOH increased remarkably under the coexistence of two AOHs, depending on the concentrations of two AOHs included in solution. The mechanism for the increase of ksAOH values was discussed based on the results obtained. UV− vis absorption of α-Toc• (λmax = 428 nm), which had been produced by reaction of α-TocH with ArO•, disappeared under the existence of UQ10H2 or Na+AsH− due to the fast regeneration reaction between α-TocH and UQ10H2 (or Na+AsH−). The result indicates that the prooxidant effect of α-Toc• is suppressed by the coexistence of UQ10H2 or Na+AsH−. We can expect a similar synergistic effect between α-TocH and the other antioxidants (such as catechins, flavone derivatives, caffeic acids, etc.), which coexist in biological systems. Measurements of the reaction rates (ksAOH) under the coexistence of α-TocH and the other AOHs are now in progress in our laboratory. 8389



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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partly supported by a Grant-in-Aid for Challenging Exploratory Research (No. 24658123) from the Japan Society for the Promotion of Science.

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