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JFS C: Food Chemistry and Toxicology

Singlet Oxygen Oxidation Ratesof ∝-, γ -, and δ-TocopherolsH.J. KIM, M.Y. LEE, AND D.B. MIN

ABSTRACT: The reaction rate constants of 5 × 10−4 M, 10 × 10−4 M, and 20 × 10−4 M α-, γ-, and δ-tocopherols withsinglet oxygen in methylene chloride containing 1 × 10−5 M chlorophyll under light at 25 ◦C for 60 min were studied.The oxidation of tocopherols determined by a spectrophotometric method showed that the losses of 20 × 10−4 M α-,γ-, and δ-tocopherols after 60 min under light were 21%, 16%, and 9%, respectively. The degradation of α-, γ-, andδ-tocopherols was undetectable in the absence of chlorophyll under light or in the presence of chlorophyll in dark.The losses of tocopherols under light were mainly due to singlet oxygen oxidation. The degradation rates of 20 ×10−4 M α-, γ-, and δ-tocopherols were 6.6 ×10−6 M/min, 5.0 × 10−6 M/min, and 2.9 × 10−6 M/min, respectively. Thereaction rates between α-, γ-, or δ-tocopherol and singlet oxygen were 4.1 ×106/M s, 3.3 × 106/M s, and 1.4 × 106/M s,respectively. The singlet oxygen oxidation rate of δ-tocopherol was significantly lower than α- or γ-tocopherol atα = 0.05. As the electron density in the chromanol ring of tocopherol increased, the singlet oxygen oxidation wasincreased.

Keywords: chlorophyll, photosensitized oxidation, reaction rate, singlet oxygen, tocopherols

Introduction

Soybean oil is susceptible to oxidation due to the presence of alarge amount of linoleic and linolenic acids. Lipid oxidation is

responsible for the deterioration of flavor stability and the decreaseof nutritional quality of foods. Lipid oxidation is due to a combina-tion of triplet oxygen and singlet oxygen oxidation (Frankel 1985;Min and Boff 2002). Singlet oxygen can directly react with doublebonds without the formation of free radicals. Singlet oxygen oxida-tion is very rapid in lipids due to the low activation energy requiredfor the chemical reaction. The rate of singlet oxygen with linoleicacid is about 1500 times greater than that of ordinary atmospherictriplet oxygen (Min and Boff 2002; Choe and Min 2005). Soybeanoil reacts with singlet oxygen at 1.4 × 105/M s (Lee and Min 1990).Soybean oil contains 1 to 1.5 ppm chlorophylls (Brekke 1980) andchlorophylls have been reported as good photosensitizers for singletoxygen formation (Endo and others 1984; Jung and others 1991).

Tocopherols are the best known antioxidants in nature to pro-tect lipids from oxidation (Burton and Ingold 1981; Min and Boff2002). Commercial soybean oil contains about 1000 to 1500 ppmtocopherols (Carpenter 1979; Jung and others 1989) and the compo-sitions of α-, β-, γ -, and δ-tocopherols in soybean oil were 4:1:66:30(Jung and others 1989). Tocopherols have been reported to minimizefood oxidation by scavenging free radicals (Sherwin 1978; Christo-pher and Ho 1985; Jung and Min 1990) and quenching singlet oxy-gen by charge transfer mechanism (Foote and others 1974; Foote1979). Tocopherols react with singlet oxygen to produce diepox-ide intermediates by 1,4-cycloaddition of singlet oxygen to toco-pherols (Yamauchi and Matsushita 1977) and form further oxidationproducts such as tocoquinone and tocopherone. The oxidized toco-pherols act as prooxidants in oils (Jung and Min 1992) and lose thevitamin E activities. It is important to protect tocopherols in foodsfrom triplet and singlet oxygen oxidation. However, the chemical

MS 20060341 Submitted 6/15/2006, Accepted 8/2/2006. Dept. of Food Scienceand Technology, The Ohio State Univ., 2015 Fyffe Rd., Columbus, OH 43210,U.S.A. Direct inquiries to author Min (E-mail: min.2@osu.edu).

reaction rates of photosensitized singlet oxygen oxidation of toco-pherols have not been reported. The objective of this study was todetermine the reaction rates of α-, γ -, and δ-tocopherols with singletoxygen.

Materials and Methods

MaterialsAlpha-, γ -, and δ-tocopherols from Hoffman-La Roche Inc. (Nut-

ley, N.J., U.S.A.), American Chemical Society certified methylenechloride from Fisher Scientific (Cincinnati, Ohio, U.S.A.), chloro-phyll b from Sigma Chemical Co. (St. Louis, Mo., U.S.A.), and spec-trophotometric grade ethanol from Aldrich Co. (Milwaukee, Wis.,U.S.A.) were used.

Sample preparationMethylene chloride solutions containing 5 × 10−4 M, 10 × 10−4 M,

and 20 × 10−4 M α-, γ -, and δ-tocopherols, and 1 × 10−5 M chloro-phyll b solution were prepared. Five milliliters of the solution wastransferred into a 10-mL serum bottle and the sample bottle wassealed air-tight with a TeflonTM coated rubber septum (Supelco,Bellefonte, Pa., U.S.A.) and an aluminum cap. The samples wereprepared in duplicate.

Light storage for singlet oxygen oxidationThe light storage box consists of a glass chamber and a wooden

box (Lee and Min 1988). The light sources were 4 Sylvania 15 watt flu-orescent lamps (Danver, Mass., U.S.A.) and the light intensity was3000 lux. Samples containing 5 × 10−4 M, 10 × 10−4 M, and 20 ×10−4 M α-, γ -, and δ-tocopherols, and 1 × 10−5 M chlorophyll bwere stored in the light box at 25 ◦C. A sample that was wrappedwith aluminum foil to protect the sample from light and a samplecontaining no chlorophyll were also prepared as control samplesand stored in the same light box with tocopherol samples. The sam-ples were placed on the wire net that was 10 cm above the bottomof a light lamp for 60 min at 25 ◦C. The tocopherol contents wereanalyzed every 10 min during the storage of 60 min.

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Singlet oxygen oxidation rates . . .

Evaluation of singlet oxygen oxidation of tocopherolsThe singlet oxygen oxidation of tocopherol was determined

by the spectrometric Emmerie–Engel method (Wong and others1988) using a Milton Roy Spectronic 1001 spectrophotometer (Univ.Park, Pa., U.S.A.). The method was calibrated by standard of pureα-tocopherol. The sample bottles, during the sample preparationand analysis, were protected from light by wrapping the samplebottle with aluminum foil. The samples were stored in the light boximmediately after sample preparation.

Statistical analysisTocopherol contents were the mean of duplicate samples. Tukey’s

range test (SAS, Cary, N.C., U.S.A., 1998) was used to ascertain thequalitative effects of tocopherols on their reactions rates with singletoxygen at α = 0.05.

Results and Discussion

Chlorophyll-photosensitized oxidationof tocopherols

The photosensitized oxidation of 5 × 10−4 M, 10 × 10−4 M, and20 × 10−4 M ∝-, γ -, and δ-tocopherols in methylene chloride con-taining 1 × 10−5 M chlorophyll during storage for 60 min at 25 ◦Cis shown in Table 1. The coefficient of variation for the determi-nations of 5 × 10−4 M tocopherols by spectrophotometric methodwas 2.9%. As the concentrations of tocopherols increased from 5 ×10−4 M to 10 × 10−4 M and 20 × 10−4 M, the coefficients of varia-tions decreased. The 2.9% coefficient of variation was thought tobe a good reproducibility of determination. Alpha-, γ -, and δ-tocopherol contents of 5 × 10−4 M, 10 × 10−4 M, and 20 × 10−4 Msignificantly decreased atα =0.05 as the storage time increased from0 to 60 min. The contents of 20 × 10−4 M α-, γ -, and δ-tocopherolsafter 60 min of storage under light were 15.8 × 10−4 M, 16.8 × 10−4

M, and 18.2 × 10−4 M, respectively (Table 1). That is, the α-, γ -, andδ-tocopherols after the storage of 60 min lost 21%, 16%, and 9%,respectively. Similarly, the 5 × 10−4 M or 10 × 10−4 M α-, γ -, and δ-tocopherols after the storage of 60 min lost approximately 22%, 17%,and 9%, respectively. The losses of tocopherols in the absence ofchlorophyll under light or in the presence of chlorophyll in the darkfor 60 min at 25 ◦C were not detectable (data not shown). Since toco-pherols were not degraded in the absence of chlorophyll under lightor in the presence of chlorophyll in the dark, the degradation of to-copherols in methylene chloride containing chlorophyll under lightstorage was mainly due to the chlorophyll-photosensitized singletoxygen oxidation. It is well known that chlorophylls are good pho-

Table 1 --- Photosensitized oxidation of α-, γ-, and δ-tocopherols in methylene chloride containing 1 × 10−5 Mchlorophyll at 25 ◦C for 60 min

Tocopherol content (× 104 M)∗

Reaction time (min)Tocopherol(× 104 M) 0 10 20 30 40 50 60 Mean∗∗

20 (α-T) 20 18.9 18.6 17.8 17.3 16.6 15.8 17.9a

(γ -T) 20 19.2 18.9 18.5 18.1 17.4 16.8 18.4b

(δ-T) 20 19.4 19.0 18.8 18.5 18.3 18.2 18.9c

10 (α-T) 10 9.5 9.2 8.7 8.3 8.2 7.9 8.8a

(γ -T) 10 9.5 9.3 8.9 8.5 8.3 8.2 9.0a

(δ-T) 10 9.7 9.5 9.4 9.3 9.1 9.1 9.4b

5 (α-T) 5 4.7 4.4 4.2 4.1 4.1 3.8 4.3a

(γ -T) 5 4.8 4.6 4.5 4.3 4.1 4.0 4.5b

(δ-T) 5 4.9 4.8 4.7 4.5 4.5 4.5 4.7c

∗Tocopherol content is the mean value of duplicate samples.∗∗Mean of tocopherol contents after 0, 10, 20, 30, 40, 50, and 60 min under lightand means with the different superscripts in a same column with the sameamounts of tocopherols are significantly different at α = 0.05.

tosensitizers for the formation of singlet oxygen under light (Rawlsand Van Santen 1970; Foote 1979; Endo and others 1984; Lee andMin 1988).

The linear regression equations were prepared from Table 1 forthe reactions between 20 × 10−4 M α-, γ -, or δ-tocopherol and sin-glet oxygen produced by chlorophyll photosensitization during thestorage of 60 min. The linear regression equations of δ-, γ -, andδ-tocopherols were Y = 19.8 – 0.066X (R2 = 0.99), Y = 19.9 – 0.050X(R2 = 0.98), and Y = 19.8 – 0.029X (R2 = 0.94), respectively, where Y isthe tocopherol content in the sample (×10−4 M) and X is the storagein minutes. The 20×10−4 Mα-,γ -, and δ-tocopherols were degradedwith the rate of 6.6 × 10−6, 5.0 × 10−6, and 2.9 × 10−6 M/min, re-spectively. The reaction rate of 20×10−4 Mα-tocopherol with singletoxygen was faster than γ - or δ-tocopherol at α = 0.05 (Table 1).

Electrophilic singlet oxygen can directly react with electron-richdouble bonds to fill its highest degenerate vacant molecular orbital(Min and Boff 2002). The singlet oxygen reaction with double bondsincreases as the electron density increases. The reaction rate of sin-glet oxygen and an electron-rich aromatic phenol ring was high at106/M s to 107/M s (Martire and others 1993). Singlet oxygen can re-act with double bonds on the chromanol ring of tocopherol. α-, γ -,and δ-tocopherols have 3, 2, and 1 methyl groups in their chromanolrings, respectively, as shown in Figure 1. Kearns (1971) reported thatthe reaction rate of singlet oxygen with olefins increased as the num-ber of methyl groups on the olefinic double bond increased. Thissuggested that the more the number of methyl groups on the dou-ble bonds of molecule, the greater electron-rich the double bondsof the molecule become. It could be assumed that the electron den-sity in the chromanol ring of tocopherol increases as the number ofmethyl groups increases from 1 to 2 or 3 (Figure 1). As the number ofmethyl groups on the chromanol ring of tocopherol increases, thesinglet oxygen oxidation with tocopherol will increase. Our resultsshowed that the α-, γ -, and δ-tocopherols were degraded with therate of 6.6 ×10−6 M/min, 5.0 × 10−6 M/min, and 2.9 × 10−6 M/min,respectively. α-Tocopherol having 3 methyl groups on chromanolring was more reactive with singlet oxygen and degraded faster thanγ - or δ-tocopherol as was expected.

The results of Table 1 showed that the degradation rates of to-copherols by singlet oxygen oxidation increased in the order ofα-tocopherol > γ -tocopherol > δ-tocopherol as the number ofmethyl groups increased in the chromanol ring of tocopherol. Thisconfirmed that the reaction rate between tocopherol and singletoxygen increased as the electron density in the chromanol ring oftocopherol increased.

Singlet oxygen oxidation rates of α-, γ-,and δ-tocopherol

The schematic diagram for the formation of singlet oxygen bysensitizer and the singlet oxygen oxidation is shown in Figure 2.When a sensitizer (Sen) such as chlorophyll absorbs light energy, itbecomes an excited singlet chlorophyll (1Sen∗) and then becomesan excited triplet chlorophyll (3Sen∗) by an intersystem crossing(ISC) mechanism. The energy of excited triplet chlorophyll is trans-ferred to ordinary triplet-state oxygen (3O2) to produce singlet oxy-gen (1O2) by triplet–triplet annihilation. Singlet oxygen undergoes4 major available pathways (Figure 2). Singlet oxygen reacts withsubstrates (A) to form oxidized products (AO2), or singlet oxygenis quenched physically or chemically by quenchers or is decayedwithout radiation.

The steady-state kinetic equation for singlet oxygen formation isaccordingly given as (Foote 1979):

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Singlet oxygen oxidation rates . . .

Trivial Name Chemical Name R1 R2 R3

α-Tocopherol 5,7,8-Trimethyltocopherol CH3 CH3 CH3

γ-Tocopherol 7,8-Dimethyltocopherol H CH3 CH3

δ-Tocopherol 8-Methyltocopherol H H CH3

R3

R2

CH3HO

R1

O

CH3CH3CH3

CH3

23

44a5

67 8

8a1’ 3’ 5’ 7’ 9’ 11’

2’ 4’ 6’ 8’ 10’ 12’

Figure 1 --- Structure of α-, γ-, orδ-tocopherol

{− d[O2]

dt

}−1

={

d[AO2]dt

}−1

= K −1(

1 + (kq + kox−Q)[Q] + kd

kr× [A]−1

)

where K denotes the rate of singlet oxygen formation; AO2, oxidizedproducts; kr , reaction rate constant of substrate with singlet oxygen;A, substrate; kq, reaction rate constant of physical singlet oxygenquenching; kox−Q, reaction rate constant of chemical singlet oxygenquenching; Q, quencher; and kd, decay rate of singlet oxygen.

Tocopherols quench singlet oxygen by a combination of physi-cal and chemical mechanisms as shown in Figure 2. Singlet oxygenis quenched by the charge transfer of tocopherols in the physicalquenching mechanism. Tocopherols are not degraded in the phys-ical quenching mechanism. Tocopherols quench singlet oxygen byreacting with singlet oxygen to form oxidized tocopherols such as to-copherol quinone and tocopherol quinone epoxide in the chemicalquenching mechanism (Kamel-Eldin and Appelqvist 1996). Toco-pherols are destroyed after the chemical quenching mechanism. Thechemical quenching could be determined by measuring the loss oftocopherols. Tocopherols were substrate and chemical quencher inthis study.

The equation for the singlet oxygen oxidation without a quenchercan be simplified to the following (Foote 1979; Jung and others 1995;Lee and others 1997):

{d[AO2]

dt

}−1

= K −1(

1 + kd

kr× [A]−1

)

where { d[AO2]dt } is the rate of oxidized tocopherol formation and

[A] is the concentration of tocopherol. When plotting [the degra-dation rate of tocopherol]−1 against [the concentration of toco-pherol]−1, the ratio of slope to intercept (S/I) is kd/kr . If the decayrate of singlet oxygen (kd) is known, it can determine the reactionrate constant, kr , between tocopherol and singlet oxygen (Foote1979). The decay rate of singlet oxygen (kd) in methylene chlo-ride has been reported to be 1.1 × 104/s (Salokhiddinov and others1981).

The reciprocal plot of the degradation rate of tocopherol (1/V,min/M) against the initial tocopherol concentrations (1/To, M−1)of 5 × 10−4 M, 10 × 10−4 M, and 20 × 10−4 M in methylene chlo-ride containing chlorophyll stored under light at 25 ◦C for 60 min isshown in Figure 3. The 3 regression line equations containing 5 ×10−4 M, 10 × 10−4 M, and 20 × 10−4 M of α-, γ -, and δ-tocopherolare Y = 194.79X + 5.705 × 104 (R2 = 0.99), Y = 246.55X + 7.685 × 104

(R2 = 0.99), and Y = 556.64X + 8.585 × 104 (R2 = 0.99), respectively,where X and Y are the reciprocal of initial tocopherol concentrationand degradation rate of tocopherol. Our previous study found thatα-tocopherol quenched only singlet oxygen and not an excitedtriplet sensitizer (Jung and others 1991). The y-intercept in a linear

Sen 1Sen* 3Sen*1O2 AO2

hv Kisc O2 A

ko kr

1Sen Sen

Q kq

Q kox-Q

kd

3O2 QO23O2

Figure 2 --- Schematic diagram for the formation of singletoxygen by chlorophyll and the singlet oxygen oxidationof tocopherol: Sen = chlorophyll; A = tocopherol; AO2 =oxidized tocopherol; Q = quencher

0

20

40

60

80

100

120

140

0 0.05 0.1 0.15 0.2

1/To (x 104 M-1)

1/V

( x 1

04 M-1

min

)

α-tocopherol

γ-tocopherol

δ-tocopherol

Figure 3 --- Linear regressions of the reciprocal plot of thedegradation rate of tocopherols against the concentra-tion of tocopherols

regression line is equal to [the rate of singlet oxygen formation]−1

(K−1). It suggested that the y-intercepts of α-, γ -, and δ-tocopherolsare constant. The slopes of 3 regression lines (194.79, 246.55, 556.64)were significantly different at α = 0.05. The y-intercepts (5.705× 104, 7.685 × 104, 8.585 × 104) were not significantly different

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Singlet oxygen oxidation rates . . .

at α = 0.05. The y-intercepts should be the average of 3 inter-cepts because the intercepts were statistically the same. There-fore, the regression line equations of the plot 1/V against 1/To forα-, γ -, and δ-tocopherol are Y = 194.79X + 7.325 × 104, Y = 246.55X +7.325 × 104, and Y = 556.64X + 7.325 × 104, respectively.

The linear regression equation for α-tocopherol was Y =194.79X + 7.325 × 104. The (S/I) of this regression line is equal tokd/kr , which is 26.59 × 10−4 M. When considering kd in methylenechloride is 1.1 × 104/s, the reaction rate (kr) between α-tocopheroland singlet oxygen is (1.1 × 104/s)/(26.59 × 10−4 M) = 4.1 × 106/M s.The reaction rates between γ - and δ-tocopherol and singlet oxygenwere 3.3×106/M s and 1.4×106/M s, respectively. Tocopherols actedas substrate and chemical quencher in this study. The chemicalquenching was determined by measuring the loss of tocopherols bysinglet oxygen oxidation. The reaction rate (kr) of tocopherols withsinglet oxygen is the same as the singlet oxygen chemical quenchingrate (kox−Q) of tocopherols.

Neely and others (1988) reported the physical quenching rates(kq) of singlet oxygen byα-,γ -, and δ-tocopherols were 4.2×107/M s,1.1 × 107/M s, and 0.5 × 107/M s, respectively. The ratios of thephysical quenching rate to the chemical quenching of α-, γ -, andδ-tocopherol for singlet oxygen are 10.2:3.3:3.6, respectively. Thephysical quenching of tocopherol is more prevalent in singlet oxygenoxidation than in the chemical quenching. The important mecha-nism for singlet oxygen quenching of tocopherols is the physicalquenching mechanism.

Conclusion

The losses of α-, γ -, and δ-tocopherol in methylene chloride con-taining chlorophyll under light were studied. Alpha-, γ -, and

δ-tocopherol contents significantly decreased at α = 0.05 as thestorage time under light increased from 0 to 10, 20, 30, 40, 50, or60 min. The degradation of tocopherols under light was mainly dueto the singlet oxygen oxidation. The reaction rates between α-, γ -, orδ-tocopherol and singlet oxygen were 4.1 × 106/M s, 3.3 × 106/M s,and 1.4 × 106/M s, respectively. The singlet oxygen oxidation rateof α-tocopherol was higher than γ - or δ-tocopherol due to higherelectron density in the chromanol ring of α-tocopherol. Tocopherolquenches singlet oxygen by a combination of physical and chem-ical mechanisms. The physical quenching rates for α-, γ -, andδ-tocopherols were 10.2, 3.3, and 3.6 times the chemical quench-ing rates.

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