747

Kinetic Study of the Prooxidant Effect of Tocopherol. Hydrogen Abstraction from Lipid Hydroperoxides by Tocopheroxyls in Solution Kazuo Mukai a,*, Kouhei Sawada a, Yasuhiro K o h n o a and Junji Terao b aDepartment of Chemistry, Faculty of Science, Ehime University, Matsuyama 790 and bNational Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305, Japan

A kinetic study of the prooxidant effect of vitamin E (tocopherol, TocH) has been carried out. The rates of hydrogen abstraction (k_ I) from methyl Unoleate hydroperoxide (ML-OOH) by a-tocopheroxyl (a-Toc') (1) and eight types of alkyl substituted Toc" radicals (2-9) in benzene solution have been determined spectrophotometrically. The results show that the rate constants decrease as the total electron-donating capacity of the alkyl substituents on the aromatic ring of Toc" increases. The k-1 value (5.0 X 10-1M-is -l) obtained for a-Toc" (1) was found to be about seven orders of magnitude lower than the k I value (3.2 X 106M-is -1) for the reaction of a-TocH with peroxyl radical, which is well known as the usual radical-scavenging reaction of a-TocH. The above reaction rates (k_ l) obtained were compared with those (ks) of methyl linoleate with Toc- (1-9) in benzene solution. The rates (k_l) were found to be about six times larger than those (ks) of the corresponding Toc'. The results suggest that both reactions may relate to the prooxidant effect of a-TocH at high concentrations in foods and oils. The effect of the phytyl side chain on the reaction rate of Toc" in micellar dispersions has also been studied. We have measured the rate constant, k_ 1, for the reaction of phosphatidylchoHne hydroperoxide with a Toc" radical in benzene, tert-butanol and in Triton X-100 micellar dispersions, and compared the observed k-i values with the corresponding values for ML-OOH. Lipids 28, 747-752 (1993).

oxidant effect of a-TocH leads to an increase of the level of hydroperoxides (LOOH) with conjugated diene structure. Loury e t al. (3) and Terao and Matsushita (9) have proposed t h a t Toc- radicals participate in this prooxidant effect through Reactions [3] and [4]: k3 Toc" + LH --) TocH + L" [3] k- 1

Toc" + LOOH --* TocH + LOO~

[4]

where Reaction [3] is the chain transfer reaction and Reaction [4] is the reverse of Reaction [1]. A more complex reaction scheme, including a great number of elementary reaction steps, has been proposed by Rousseau-Richard e t al. (10,11). However, so far the kinetics of Reactions [3] and [4] related to the prooxidant effect of vitamin E have not been studied. The reactions are considered important to the understanding of the prooxidant properties of vitamin E. We have recently reported the second-order rate constant, k 3, for the reaction of L H esters (ethyl linoleate, ethyl linolenate, ethyl arachidonate and all cis4,7,10,13,16,19-docosahexaenoic acid ethyl ester) with Toc" (5,7-diisopropyltocopheroxyl 5) (12,13). The rate constant, k 3, obtained was 1.82 to 9.05 • 10-2M-is -1 in benzene at 25~ The rate of k 3 is about 7 to 8 orders of magnitude lower t h a n k 1 for Reaction [1] of a-TocH with LOO" (1). Furthermore, we recently succeeded in measuring the rate c o n s t a n t (k-l) for the reaction of 5,7-diisopropylThe role of vitamin E (a-,/~, )- and &tocopherols) as an im- tocopheroxyl radical 5 with n-, sec- and t e r t - b u t y l hydroportant biological antioxidant has been well recognized peroxides used as lipid hydroperoxide model systems (14). in recent years. The antioxidant properties of tocopherols The observed rate, k-l, was 1.34 to 3.65 X 10-1M-is -' in (TocH) have been ascribed to hydrogen abstraction from benzene solution at 25~ The values of k-1 are only the OH group in TocH by a peroxyl radical (LOO'). The about one order of magnitude higher t h a n those of k3. In the present work, we have determined the secondhydrogen abstraction produces a tocopheroxyl radical {Tom), which combines with another LOO" (Reactions [1] order rate constant, k-l, for the reactions of methyl linoleate hydroperoxide (ML-OOH) with nine types of and [2]) (Refs. 1,2). alkyl substituted Toc" radicals 1-9 in benzene (see Fig. 1). kl LO0 9 + TocH -,- LOOH + Toe[1] The rate, k_~, was compared with k, for Reaction [1] and with k3 for Reaction [3]. The effect of alkyl substituents on the aromatic ring of Toc" has also been studied. It is LOO" + Toc" ~ LOO-Toc(nonradical product) [2] of interest to know how the rate of the reaction of ML-OOH with Toc- is different from t h a t of the phosphoIn recent years, several investigators demonstrated that lipid hydroperoxide We have thus measured the rate cona-TocH at high concentrations acts as a prooxidant dur- stant, k_l, for the reaction of phosphatidylcholine ing the autoxidation of polyunsaturated f a t t y acids (LH) hydroperoxide (PC-OOH) with a Toc" radical in benzene, in aqueous medium and in bulk phase (3-9). This pro- tert- butanol and in micellar dispersions, and compared the observed k-1 values with the corresponding values *To whom correspondence should be addressed. for ML-OOH. The effect of the phytyl side chain on the Abbreviations: LH, polyunsaturated fatty acid(s) and/or polyun- reaction rate of Toc" in micellar dispersion also has been saturated fatty acid ester(s); LOOH, lipid hydroperoxide; LOO', peroxyl radical; ML-OOH,methyl linoleate hydroperoxide; PC, egg studied. In Figure 1 we give the structures of the molephosphatidylcholine; PC-OOH, phosphatidylcholine hydroperoxide; cules studied in this work. The study was intended to p r o PhO ~ 2,6-di-tert-butyl-4-(4"-methoxyphenyl)phenoxyl;TLC, thin- vide a basis for the interpretation of similar reactions in layer chromatography;TocH, tocopherol;Toc', tocopheroxylradical more complex biological systems. Copyright 9 1993 by the American Oil Chemists' Society

LIPIDS, Vol. 28, no. 8 (1993)

748

K. MUKAI ET AL. The preparation of TocH 2p-gp was reported previously (19-21). The Toc" radicals 3, 5, 6, 7, 8 and 9 are fairlystable, and were prepared by PbO2 oxidation of the corresponding TocH in benzene solution under a nitrogen atmospher~ i 2 3 However, in the cases of TocH lp, 2p and 4p, the Toc" radicals 1, 2 and 4 produced were not very stable, and absorption spectra decreased rapidly with time Therefore, Toc. 1, 2 and 4 were prepared by reaction between the stable P h O o radical 10 and the corresponding TocH in benzene at 25~ under a nitrogen atmosphere and were 4 5 6 reacted immediately with M L - O O H solution. Micellar dispersions of Triton X-100 (5.0 wt%) containing Toc" 5 were prepared as follows {22). 5,7-Diisopropyltocopherol 5p {15-20 rag, 32-42 pmol) was dissolved in 5-mL of diethyl ether, and the solution was poured into 7 B g a small flask. The diethyl ether was removed on a rotary evaporator to obtain a thin film on the flask wall. Twenty m L of aqueous Triton X-100 {5.0wt%, in 0.1 M phosphate buffer, p H 7) was added, and the flask was shaken vigorously in a Vortex mixer for 1 rain. PhO'-containing solutions of ~ t o n X-100 (5.0 wt%) were prepared similarly and reacted with the above TocH-cont~inlng micellar dispersions. The PhO" radical is very stable in the absence Methyl llnoleate hydroperoxide (IL-001I) Phenoxlfl radicaL (Ph0.} of 5,7-diisopropyltocopherol and shows absorption peaks at Am~ = 377 and 577 nm in aqueous Triton X-100 dispersions (5.0 wt%). Upon mixing the micellar disperF'V!fe,,, e0. sion of TocH (0.66 mM) with the micellar dispersion of the PhO" radical (0.60 mM) (1:1, vol]vol) at 25.0~ the absorption spectrum characteristic of the PhO" radical immediately changed to that of TOc" 5. The new absorption oI~(cH=)= maxima in the visible region (;tm~ = 397 and 417 rim) a l e due to Toe" 5 (23). Because Toc- 5 is stable at 25.0~ the Phosphtldylchoiiae hydroperexlde (~-OOF[) absorption intensity decreases only gradually with time Measurements. The kinetic data were obtained on a FIG. 1. Molecular structures of tocopheroxyl radicals 1-9, PhO ~ Shimadzu UV-2100S {Kyot~ Japan) spectrophotometer ML-OOH (methyl linoleate hydroperuxide) and PC-OOH (phosphatidylcholine hydroperoxide). by mixing equal volumes of solutions of Toc" and ML-OOH {or PC-OOH). The oxidation reactions were studied under pseudo-first-order conditions, and the ol~ MATERIALS AND METHODS served rate constants (kob~d)were calculated in the usual Sample preparatior~ Methyl linoleate (linoleic acid methyl way, using standard least-squares analysis. All measure ester, LH) (>99%) was obtained from Sigma Chemical Ca ments were performed at 25.0~ because Toc" 1, 2 and (St. Louis, M e ) and used without further purification. 4 used are not stable at 37~ Hydroperoxide levels in methyl linoleate were estimated by measuring absorption at 234 nm 05) and were found to be less than 0.3%. Egg phosphatidylcholine (PC) (>95%) RESULTS AND DISCUSSION was kindly supplied by Nippon Oil Fats Ca {Amagasaki, Reaction between M L - O O H and Toc" 1-9 in benzene Japan). The average molecular weight of the PC was 5,7-Diisopropyltocopberoxyl 5 is comparatively stable in assumed to be 774 (13). The amount of hydroperoxides in the absence of ML-OOH and shows absorption peaks at = 417 nm and 397 nm in benzene (Fig. 2). When addthe sample was found to be negligible as judged by thinlayer chromatography (TLC). a-T0cH was supplied by Eisai ing a benzene solution of excess ML-OOH to a benzene Ca (Tokyo, Japan). Triton X-100 was purchased from solution of Toc', the absorption spectrum of the Toc" Nacalai Tesque Inc {Kyot~ Japan) and was used as re~ gradually disappeared. Figure 2 shows an example of the ceived. The 2,6-di-tert-butyl-4-(4'-methoxyphenyl)phenoxyl results of the interaction between 5,7
=--o-po-0,,-?,

LIPIDS, Vol. 28, no. 8 (1993)

749 REACTION B E T W E E N TOCOPHEROXYL AND L I P I D H Y D R O P E R O X I D E

(x I0 -3)

0.8

.

.

.

""17 ~4

.

nm

'

'

15

'

1////

0.6

t

10

~

2 . 0.4

0.2

0.0 340

380

420

460

500

0~

1

0

Wavelength(nm)

2

[LOOHJ (M)

FIG. 2. Changes in the absorption spectrum of 5,7-diisopropyltocopheroxyl radical 5 in the course of the reaction of Toc ~ 5 with ML-OOH in benzene at 25.0~ ~Ibc']t= 0 = ca. 0.15 mM and [ML'OOH]t = o = 21.5 raM. The spectra were recorded at 100-s intervals. Arrow indicates decrease (4) of absorbance with t i m e . Abbreviation as in Figure 1. TOC', tocopheroxyl radical.

3

(x 10-2)

FIG. 3. Dependence of t h e pseudo-first~rder rate constants, kobsa, of Toc" radicals 1, 2, 3, 5, 6 and 7 on the concentration of ML-OOH in benzene at 25.0~ Abbreviations as in Figures 1 and 2.

tives (Reaction [1]) (27,28, and references therein). The most reliable kl value was reported by Burton et aL (27), using the inhibited autoxidation of styrene method. For example the kz value obtained for the reaction of a-TocH with poly(peroxystyryl)peroxyl radical was 3.20 • [6] kob,d = k o + k s [LOOH] 10eM-ls -1 in chlorobenzene at 30~ Therefor~ the k_l where k o is the rate constant for the natural decay of value (5.0 • 10-1M-is -1) obtained for wToc" I in the presToc- in benzene solution, and ks is the apparent second- ent study is about seven orders of magnitude lower than order rate constant for the reaction of Toc. with added the above-mentioned kl value Effect of alkyl substituents in Toc radicals. In the presMI~OOH. These rate parameters are obtained by plotting kob,d against [ML-OOH]. Similar measurements were ent work, we have determined the second-order rate conmade for the reactions of Toc- 1-9 with ML-OOH. As stants, k-l, for the reaction of ML-OOH with Toc" 1-9 in shown in Figure 3, the first-order decay constant, kob~d, benzene As listed in Table 1 for the Toc- radicals 4, 5, 6 and 7, having two alkyl substituents at the ortho posiis proportional to the concentration of ML-OOH. As reported by Mahoney and DaRooge (24,25), the tions to the OH group, the k_l values obtained in benzene are 2.5 X 10-1M-is -1 for radical 4, 1.33 X results are consistent with Reactions [7] and [2]: the pseudo-first-order rate constant, bleaching is given by Equation 6:

kobsd,

for Woc"

k-1 Toc" + LOOH -~ TocH + LOO" kl

[7]

k2 LOO" + Toc" ~ LOO-Toc

[2]

TABLE 1

where k2[Woc" ] >> k,[TocH]; k 2 is the second-order rate constant for the reaction of Toc- with LOO'. A combination product of Toc- and LOO', that is, LOO-Toc, was isolated by Yamauchi et aL (26). Under these conditions, the rate of disappearance of Toc- is given by the expression (24,25):

Secondg)rder (k_ z and k3) Rate Constants for t h e Reaction of Toc" 1 - 9 with Methyl Linoleate Hydroperoxide and Methyl Linoleate, R e s p e c t i v e l y , in Benzene at 25.0~ and Relative Rate Constants

(k-l/k3) k_

(M-is -i )

1 2

5.0 X 10 - 1

3 - d [ T o c ' ] / d t - - {ko + 2k_I[LOOH]} [Toc']

[8]

The values of k_ 1 calculated from kob,d are listed in Table 1. The experimental errors in the k-1 value for Toc" radicals 3, 5, 6, 7, 8 and 9 were less than +7%. The natural decays of Toc" 1, 2 and 4 are much faster, and therefore we could only estimate the approximate rate constants. Several investigators have measured the rate constant, kz, for the reaction between LOO" and vitamin E deriva-

1a

Toc"

4

5 6 9

1.3 X 10 -1 2.11 X 10 -2 2.5 X 10 - I 1.33 X 10 - I 9.14 X 10 -2 7.77 X 10 -3 1.31 X 10 -1 8.41 X 10 -2

k3a

(M-Zs - I ) --

2.3 3.27 5.0 1.86 1.61 1.10 1.83 2.01

k-l/k3 --

X X X X X

10 - 2 10-3L 10-2.~ 10-2.0 10-2.0 X 10 -30 X 10 -2 X 10 -2

5.7 6.45 5.0 7.15 5.68 7.06 7.16 4.18

aEx~erimental errors in k-1 and k 3 values were less than 7% for T o c ; 3 and 5-9 and 20% for Toc" 1, 2 and 4. Toc', tocopheroxyl radical. bThese values were previously reported (Ref. 13).

LIPIDS, Vol. 28, no. 8 (1993)

750

K. MUKAI ET AL. 10-1M-is -1 for 5, 9.14 • 10-2M-is -1 for 6 and 7.77 • 10-3M-is -1 for 7. The values of k_ 1 decrease in the order of 4 > 5 > 6 > 7, as the size of the two ortho-alkyl groups in the Toc" radical increases. The Toc- 4 is 32-times as reactive as the Toc. 7. The results clearly indicate t h a t the decrease in k_~ is due to steric factors. Inductive effects on the reaction rate of these Toc" radicals with ML-OOH would not be significant, because methyl, e t h y l isopropyl and tert-butyl groups have electron-donating properties t h a t are not too different. Further, the rate constants of Toc" radicals 1, 2 and 3, which have two alkyl substituents at the ortho positions and a methyl substituent at C-8 decrease in the order 1 > 2 > 3. This result also suggests that the effect of steric hindrance on the reaction rate is considerable B o t h 5,7-diisopropyl-8-methyltocopheroxyl 3 and 5,7diisopropyltocopheroxyl 5 have two isopropyl substituents at the ortho positions and, in addition, 3 has a methyl substituent at the meta position. When comparing the rate constant, k_~, observed for Toc. 5 with t h a t for Toc" 3, compound 5 is 6.3 times more reactive than 3. Similarly, 5,7-diethyltocopheroxyl 4 is 1.9 times as reactive as the 5,7-diethyl-8-methyltocopheroxyl 2. The results indicate t h a t substitution on the aromatic ring by an electrondonating methyl group (at C-8) results in a decrease in the second-order rate constant, k_l. In other words, in the reaction between the Toc" and ML-OOH, the Toc 9 acts as an electron acceptor, and thus the rate constants will decrease as the total electron-donating capacity of the alkyl substituents on the aromatic ring of the Toc" increases (29). Effect of alkyl side chain of TOe" radical on its reaction rate in benzene and in miceUar dispersion. In order to clarify the effect of the phytyl side chain of the Toc. radical on the reaction rat~ we have measured the rate constants, k_l, for the reaction of ML-OOH with 5,7-diisopropyltocopheroxyl 5 and 5,7-diisopropyltocopheroxyl model 8 in benzene and in micellar dispersion. As shown in Table 2, the Toc" 5 and Toc- 8 without phytyl side chain in 2-position reacted with the ML-OOH at a similar rate in benzene. On the other hand, in 5 wt% Triton X-100 micellar dispersion, the k_~ value (6.93 X 1 0 - 2 M - s -1) of Toc. 5 is 0.61 t i m e s t h a t (1.14 X 10-1M-is -1) of Toc" 8. The result shows t h a t the p h y t y l

side chain of vitamin E has little effect on reactivity in homogeneous solution, whereas it has considerable effect on reactivity in micellar dispersion (1,2,30-33). The siguificance of the above changes in the rate constants, k-l, is not clear at present, b u t it may relate to the biological activity of TocH. For instance, although b o t h a-TocH models and a-TocH have high in vitro antioxidant activit y in solution, the former has no in vivo vitamin E activity (34,35). Reaction between PC-OOH and Toe" 5. It is of interest to examine whether or not the rate constant of hydrogen abstraction of PC-OOH is the same as t h a t of M I r O O H , which is one of the hydroperoxide derivatives (LOOH) of the f a t t y acid moieties contained in the PC. The rate constants for hydrogen abstraction from PCO O H and M L - O O H by Toc- 5 (ko~d) have been measured in benzene tert-butanol and 5 wt% Triton X-100. The second-order rate constants, k-l, are obtained by plotting kob.d vS. [LOOH]. The k_ I values obtained are given in Table 3. The observed k-1 value of P C - O O H was only 8 % of that of M L - O O H in the nonpolar benzene solvent, whereas the k_1 value of P C - O O H was 1.9 times larger than that of M I ~ O O H in the more polar tert-butanol solvent. Furthermor~ in micellar dispersion, PC-OOH reacted at a rate t h a t was only about 30% of t h a t observed for ML-OOH. There is, as yet, no unambiguous explanation for this apparent contradiction, b u t some possible explanations are offered in the following. Firstly, the formation of reverse rnicelles in benzene solution m u s t be considered, as PC molecules are known to aggregate into reverse micelles in nonpolar solvents (36,37). The solubility of Toc- 5, which has a nonpolar p h y t y l side-chain, is high in benzene b u t low in PC liposom~ Thus, formation of reverse micelles might prevent Toc" 5 from reacting with the hydroperoxide group of PC-OOH, causing a smaller rate constant, k_~, for PCOOH than for ML-OOH. This would be in agreement with the results obtained for the hydrogen abstraction reaction from PC and from u n s a t u r a t e d f a t t y acids (LH) by Toc. 5 in benzene (13). Therefore, we measured the rate cons t a n t , k - l , i n polar tert-butanol solvent in which PC-OOH does not form reverse miceUes. As the two f a t t y acid moieties are quite close in PC-OOH, Toc- 5 may not

TABLE 2 Pseud~First-Order (kot~d) and Second-Order (k_ t) Rate Constants for the Reaction of Toe" 5 and 8 with M L - O O H in Benzene or 5 wt% Triton X-100 (pH = 7.0, 0.1 M phosphate buffer) at 25.0~

Solution

[ML-OOH] (raM}

Toc" 5 kobsd (s- i}

k_ 1a (M- 1s - ]}

[ML-OOH] (raM)

Toe" 8 kobsd (s- 1} 1.42 X 1 0 - 3 2.79 4.86

Benzene

3.12 6.24 9.37 12.49

8.81 • 10 -4 17.3 25.5 33.9

1.33 • 10 -1

4.68 9.37 17.64

Micellar dispersion

3.96 7.46 11.22

5.29 • 1 0 - 4 9.82 15.3

6.93 X 1 0 - 2

4.02 7.37 11.08 14.69

9.6 • 1 0 - 4 16.9 24.5 34.3

k_l a

(M-ls-1) 1.31 • 10-1

1.14 X 10-1

aFor each Toc', the experimental error in k_ 1 value was less than 7%. Toe', tocopheroxyl radical; ML-OOH, methyl linoleate hydroperoxide. LIP!DS, Vol. 28, no, 8 (!993)

751

REACTION BETWEEN TOCOPHEROXYL AND LIPID HYDROPEROXIDE TABLE 3

Pseudo-First-Order (kobsd)and Second-Order (k_ 1) Rate Constants for the Reaction of Toc" 5 with M L - O O H and P C - O O H in Benzene, t e r t - b u t a n o l or 5 wt% Triton X-100 (pH = 7.0, 0.1 M phosphate buffer) at 25.0~

Solution

[ML-OOH] (raM)

ML-OOH kobsd {s-i)

k_ i~ (M-is -1 )

3.12 8.81 X 10-4 1.33 6.24 17.3 9.37 25.5 12.49 33.9 tert-Butanol 3.92 8.56 X 10-5 2.49 7.47 10.5 11.51 12.5 15.00 14.1 Micellar dispersion 3.96 5.29 • 10-4 6.93 7.46 9.82 11.22 15.3 =Experimental error in k_ z values was less than 7%. Abbreviations Benzene

be able to easily approach the active sit~ t h a t is, the -OOH group of PC-OOH. In such a case, the k_ t value of PCO O H will be smaller t h a n t h a t of ML-OOH. However, the observed k_l value (4.68 X 10-3M-is -1) of PC-OOH was 1.9 t i m e s l a r g e r t h a n t h a t (2.49 X 10-3M-ls-~) of M L - O O H in tert-butanol. The reason for this is not clear at present. In Triton X-100, PC-OOH reacted with Toc- 5 at a rate t h a t was only about 30% of t h a t observed with ML-OOH. As the accessibility of the M I ~ O O H appears to be greater t h a n t h a t of PC-OOH in micellar dispersion, the reactivity of M L - O O H was higher t h a n t h a t of PC-OOH. The cause of the prooxidant effect of TocH. We recently reported the second-order rate constants, k 3, for the reactions of the Toc. radicals 4-7 with methyl linoleate (LH) in benzene using a spectrophotometric monitoring system (Reaction [3]) (12,13). We have also measured the rate constants, k 3, for the reactions of Toc- 1, 2, 3, 8 and 9 with L H in benzene at 25.0~ As described in a previous section, wToc" 1 is unstable, and we could not determine the rate constant, k3. The rate constants, k3, obtained are s u m m a r i z e d in Table 1, together with those reported for Toc" 4, 5, 6 and 7. The results indicate t h a t the effect of s u b s t i t u t i o n on the reaction rates, k3, observed for Toc" 2-9 is very similar to that on k_ i. The values of k_ i were plotted against k 3. A s shown in Figure 4, the k_ i values were found to correlate linearly with the k 3 values (correlation coefficient= 0.96). The ratio of k-i to k3 was estimated to be about 6 • 1 from the values given in Table i (Eq. 9). k_l=(6---+l) X k 3

[9]

I t is well known t h a t tocopherols (vitamin E) are present in b i o m e m b r a n e s and in oils, where they function as antioxidants. The antioxidant properties of TocH have been ascribed to the initial oxidation by the LOO ~ radical of the phenolic hydroxyl group, producing a Toc, radical (Reaction [1]) (1,2). On the other hand, several investigators have d e m o n s t r a t e d t h a t a-TocH at high concentrations acts as a prooxidant during the autoxidation of L H in aqueous m e d i u m and in bulk p h a s e (3-11). This proox-

[PC-OOH] (raM)

PC--OOH kobsd (s-i)

2.68 5.09 7.66 9.97 2.92 5.52 7.71 10.35 3.05 5.99 7.98

9.61 X 10-s 14.4 20.9 25.2 6.37 X 10-s 7.91 10.1 13.3 2.60 X 10-4 3.65 4.61

• 10-1

)< 10-3

X 10-2

k_l a

(M-ls-1) 1.09 X 10-2

4.68 X 10 -3

2.02 X 10-2

as in Figure 1.

idant effect of a-TocH leads to an increase of the level of L O O H with a conjugated diene structure~ L o u r y et aL (3) and Terao and M a t s u s h i t a (9) have proposed t h a t Tocradicals participate in this prooxidant effect through Reactions [3] and [4]. In the present work, we have measured the rate constants, k-i, for the reaction of M I ~ O O H with Toc" 1-9. The k_~ values were found to be a b o u t six times larger t h a n those (k3) of the corresponding Toc" radicals for Reaction [3]. The results suggest t h a t b o t h Reactions [3] and [4] m a y relate to the p r o o x i d a n t effect of a-TocH at high concentrations. Therefor~ if L H coexists with L O O H in edible oils or in membranes, the rate of disappearance of Toc" will be represented b y E q u a t i o n 10: -d[Toc']/dt = ka[LH][Toc'] + k-l[LOOH][Toc*]

[10]

(x 10-1) 3

,

'

'

'

'

0 4

2 I

P 1

6

0

9

0

9

i

I

i

I

I

1

2

3

4

5

k3

(M-is -1)

6

(• 10-2)

FIG. 4. Plot of k - l vs. k 3 for tocopheroxyl radicals 2-9.

LIPIDS, Vol. 28, no. 8 (1993)

752

K. MUKAI E T AL. I n t h e initial s t a g e of lipid degradation, the concentration of L O O H will be m u c h lower t h a n t h a t of L H , a n d t h u s t h e second t e r m in E q u a t i o n 10 is negligible Consequently, t h e p r o o x i d a n t effect of a-TocH in edible oils a n d fats will be induced b y the hydrogen abstraction Reaction [3] between Toc- a n d L H . On t h e o t h e r hand, if the autoxidation proceeds, t h e level of L O O H increases. W h e n t h e c o n c e n t r a t i o n of L O O H a p p r o a c h e s a p p r o x i m a t e l y 17% of t h a t of L H , the radical d e c a y Reactions [3] a n d [4] will a p p r o a c h similar rates. These facts s u g g e s t t h a t n o t only the chain transfer Reaction [3] is due to Toc., b u t also t h a t Reaction [4] between Toc" a n d L O O H p a r t i c i p a t e s in t h e p r o o x i d a n t effect of a-TocH.

ACKNOWLEDGMENTS We are very grateful to Dr. Shiro Urano for his advice on synthesizing TocH 2p and 3p. We are also grateful to Prof. Shin-ichi Nagaoka for valuable discussions. We wish to thank Himnobu Hosose, Seiji Kikuchi, Hitoshi Morimoto and Aya Kuranaka for the preparation of TocH 2p-gp. We also wish to thank Yuji Okanchi for the measure~ ment of reaction rates, k3, of Toc~ 3, 8 and 9. We express our gratitude to the Eisai Ca Ltd. and Nippon Oil Fats Ca Ltd. for the generous gifts of a~TocH and egg PC, respectively.

REFERENCES 1. Burton, G.W., and Ingold, K.U. (1981) J. Am. Chem. Soa 103, 6472-6477. 2. Niki, E., Kawakami, A., Saito, M., Yamamoto, Y., Tsuchiya, J., and Kamiya, Y. (1985) J. BioL Chem. 260, 2191-2196. 3. Loury, M., Bloch, C, and Francois, R. (1966)Rev. F~. Corps Gras 13, 747-752. 4. Cillard, J., Cillard, P., Corrnier, M., and Girre, L. (1980) J. Am. Oil Chem. Soc 57, 252-255. 5. Cillard, J., Cillard, P., and Cormier, M. (1980) J. Am. Oil Chem. Soa 57, 255-261. 6. Peers, K.E., Coxon, D.T., and Chan, H.W.-S~ (1981) J. Sci. Food Agria 32, 898-904. 7. Peers, K.E., and Coxon, D.T. (1983) Chem. Phys. Lipids 32, 49-56. 8. Koskas, J.P., Cillard, J., and Cillard, P. (1984) J. Am. Oil Chem. Soa 61, 1466-1469. 9. Terao, J., and Matsushita, S. (1986) Lipids 21, 255-260. 10. Roussean-Richard, C, Richard, C, and Martin, R. (1988)3:. Ch/m. Phys. 85, 167-173. 11. Rousseau-Richard, C., Richard, C., and Martin, R. (1988)J. Ch/m. Phys. 8~ 175-184. 12. Mukai, K., and Okanchi, Y. (1989) Lipids 24, 936-939.

LIPIDS, Vol. 28, no. 8 (1993)

13. Nagaoka, &, OkauchL Y., Uran(~ S., Nagashima, U., and Mukai, K. (1990)J. Am. Chem. Soc 112, 8921-8924. 14. Mukai, K., Kohno, Y., and Ishizu, K. (1988) Biochem. Biophys. Res. Commun. 155, 1046-1050. 15. Kaneda, T., and Ueta, N. (1983) Kasanka Shishitsu Jikhen Hc~ Ishiyaku Syuppan, Toky~ 36-39. 16. Rieker, A., and Scheffler, K. (1965) Liebigs Ann. Chem. 689, 78-92. 17. Terao, J., and Matsushita, S. (1977) J. Am. Oil Chem. Soa 54, 234-238. 18. Teraa J., Asano, I., and Matsushita, S. (1985) Lipids 20, 312-317. 19. Mukai, K., Kageyama, Y., Ishida, T., and Fukuda, K. (1989) J. Org. Chem. 54, 552-556. 20. Mukai, K., Okabe, K., and Hosose, H. (1989) J. Org. Chem. 54, 557-560. 21. Mukai, K., Kikuchi, S., and Urano, S. (1990) Biochim. Biophys. Acta 1035, 77-82. 22. Mukai, K., Nishimura, M., and Kikuchi, S. (1991) J. Biot Chem. 266, 274-278. 23. Mukai, K., Watanabe, Y., and Ishizn, K. (1986)Bull Chem. Soa Jpn. 59, 2899-2900. 24. Mahoney, L.A., and DaRooge, M.A. (1970)J. Am. Chem. Soa 92, 4063-4067. 25. DaRooge, M.A., and Mahoney, L.R. (1967)J. Org. Chem. 32, 1-6. 26. Yamanchi, R., Matsui, T., Kato, K., and Ueno, Y. (1990) Lipids 25, 152-158. 27. Burton, G.W., Doba, T., Gabe, E.J., Hughes, L., Lee, EL., Prasad, L., and Ingold, K.U. (1985)J. Am. Chem. Soc. 107, 7053-7065. 28. Niki, E. (1987) Chem. Phys. Lipids 44, 227-253. 29. Nagaoka, S., Kuranaka, A., Tsuboi, H., Nagashima, U., and Mukai, K. (1992)J. Phys. Chem. 96, 2754-2761. 30. Fukuzawa, K., Chida, H., Tokumura, A., and Tsukatani, H. (1981) Arch. Biochem. Biophys. 206, 173-180. 31. Mnk~i; I"L,Yokoyama, ~, Fukuda, K., and Uemota Y. (1987) Bull. Chem. Soc JprL 60, 2163-2167. 32. Pryor, W.A., Strickland, T., and Church, D.E (1988)J. Am. Chem. Soa 110, 2224-2229. 33. Liu, Z.L., Han, Z.X., Chert, E, and Liu, Y.C. (1990) Chem. Phys. Lipids 56~ 73-80. 34. Skinner, W.A., Parkhurst, R.M., SchoUer, J., Alaupovic, P., Crider, QE., and Schwartz, K. (1967) J. Meal Chem. 10, 657-660. 35. Skinner, W.~, and Parkhurst, R.M. (1970) Lipids 5, 184-189. 36. Barclay, LR.C., MacNeil, J.M., VanKessel, J., Forrest, B.J., Porter, N.A., Nehman, L.S~, Smith, K.J., and EUingyon, Jr., J.C. (1984) J. Am. Chem. Soa 106, 6740-6747. 37. Lehman, L.S., and Porter, N.A. (1984) in Oxygen Radicals Chem. BioL, Proc InL Conf., 3rd (Bors, W., Saran, M., and Tait, D., eds.) pI~ 281-284g, Waiter de Gruyter and Ca, Berli~ [Received July I, 1992, and in revised form April 22, 1993; Revision accepted May 8, 1993]