ARCHIVES
Vol.
OF BIOCHEMISTRY
277, No. 1, February
AND
BIOPHYSICS
15, pp. lOl-108,199O
Physical and Chemical Scavenging Molecular Oxygen by Tocopherols Stephan
Kaiser,’
Paolo
Institut
fiir Physiologische
Received
July
5,1989,
Di Mascio,
Michael
Chemie I, Universitiit
and in revised
form
September
of Singlet
E. Murphy,
Dikseldorf,
Tocopherols (vitamin E) are known to act as biological antioxidants (1). Attention has centered on their function as free radical scavengers, but it has also been demonstrated that tocopherols react with singlet moleci Present address: Colorado State University, Department chemistry and Molecular Biology, Fort Collins, CO 80523. ’ To whom correspondence should be addressed at Institut iologische Chemie I Universitat Dusseldorf, Moorenstrasse Dusseldorf, FRG. Copyright All rights
0 of
$3.00 1990
by Academic Press, Inc. reproduction in any form reserved.
Sies’
27,1989
Singlet molecular oxygen (‘0,) arising from the thermal decomposition of the endoperoxide of 3,3’-(1,4naphthylidene) dipropionate was used to assess the effectiveness of a-, /3-, y-, and &tocopherol in the physical quenching as well as the chemical reaction of ‘Oz. The relative physical quenching efficiencies of the tocopherol homologs were found to decrease in the order of (Y > /3 > y > &tocopherol. The ability of physical quenching depends on a free hydroxyl group in position 6 of the chromane ring. Chemical reactivity of the tocopherol homologs with ‘02 was low, accounting for O.l-1.5% of physical quenching with P-tocopherol showing particularly low reactivity, resulting in the sequence a > y > 6 > @-tocopherol. Tocopheryl quinones were products of all tocopherol homologs, and in addition a quinone epoxide was a major product from y-tocopherol. This quinone epoxide was not cleaved by rat liver microsomal epoxide hydrolase; however, it reacted further with ‘Oz. It is concluded that methylation in position 5 of the chromane ring enhances physical quenching of ‘Oz, whereas chemical reactivity is favored by a methylated position 7. In view of the fact that fi-tocopherol is as effective as a-tocopherol in physical quenching of ‘02 but shows very low chemical reactivity, this tocopherol homolog might be particularly suitable for biological conditions in which an accumulation of oxidation products might weaken the antioxidant defense. 0 1990 Academic Press, Inc.
0003-9861/90
and Helmut
Federal Republic of Germany
of Biofiir Phys5 D-4000
ular oxygen (102)3 (2-8). The generation and possible pathological consequences of singlet oxygen in biological systems via enzymatic reactions have been described (912). ‘02 can be produced by photoexcitation as well as by nonphotochemical processes (chemiexcitation) and has been implicated in the peroxidation of biological lipids (5,13,14). ‘02 was also shown to play a role in the inactivation of cultured human cells by UV-A and near-visible radiation (15). Furthermore, ‘02 has been shown to be capable of inducing DNA damage (16-18) and to be mutagenic (19). Therefore, tocopherols are of biological interest for their ‘02 quenching capability. Scavenging of ‘02 by tocopherols includes physical quenching, in which the excited state of oxygen is deactivated without light emission, and chemical quenching which results in the formation of various oxidation products. Physical quenching by electron energy transfer almost always predominates, the rate depending on solvent polarity. This has led to the suggestion that a charge-transfer intermediate might be involved in the quenching process (2). In addition, it has been pointed out that the balance between physical and chemical quenching is a sensitive function of spin-orbit coupling properties and entropy factors (20). In the quenching of free radicals and also of ‘02, a-tocopherol is more effective than the other homologs in the sequence CY> 0 > y > &tocopherol(21,22). Here we compare the relative physical and chemical quenching ability of the tocopherol homologs toward ‘02 (Table I). This was achieved by using NDPO*, the thermodissociable endoperoxide of 3,3’-(1,4-naphthylidene) dipropionate to generate the singlet oxygen (23, 24), a methodology which offers advantages in comparison to
3 Abbreviations used: IO*, singlet molecular oxygen; UV, ultraviolet; NDP, 3,3’-(1,4-naphthylidene) dipropionate; NDPOP, endoperoxide of 3,3’-(1,4-naphthylidene) dipropionate; BHT, butylated hydroxytoluene; TEA, tetraethylammonium hydroxide; DPPD, diphenyl-p-phenylenediamine; mEPH, microsomal epoxide hydrolase; TQ, tocopheryl quinone; TQO, tocopheryl quinone epoxide; AAPH, 2,2’-azobis(2amidinopropane) HCl. 101
102
KAISER TABLE
Chemical
Structure
I
of the Tocopherol
Homologs
ET
AL.
buffer. As a-tocopherol was noticed to further oxidize to the tocopheryl quinone in the HPLC buffer, the stopping of the oxidation reaction and subsequent sample handling were done butylated hydroxytoluene (BHT) (1 mM). The HPLC system used included a Merck/Hitachi
in the presence Model
of
655A-12
pump and a Nova-Pak Cls 8 mm X 10 cm column (Waters Associates, Milford,
MA).
acetonitrile, adjusted
The
mobile
phase
consisted
of 60%
19.4% water, 0.5% tetraethylammonium
to pH 4.0 with
acetic
acid.
The
flow
2-propanol,
20%
hydroxide TEA,
rate was set at 0.4 ml/
min and a 100~~1 sample injected. An ESA Model 5100A Coulochem Homolog
RI
R2
a-Tocopherol /3-Tocopherol y-Tocopherol F-Tocopherol
CHs CH, H H
CH, H W H
other systems of ‘02 generation. Generation and quenching of ‘02 were directly monitored by the monomol light emission signal arising from the transition of the excited ‘Ap state to the 32, ground state at 1270 nm by the use of a liquid nitrogen-cooled germanium-photodiode detector. The time course and concentration dependence of the loss of tocopherols and the formation of reaction products due to chemical reaction with ‘02 were also examined. MATERIALS
AND
METHODS
electrochemical detector control module was connected to a Model 5021 conditioning cell and a Model 5010 analytical cell, which were installed in that sequence and separated from the HPLC column by an in-line carbon filter. For measurements of tocopherols (26) the redox
potential settings of the conditioning
cell and the first analytical cell
were set at -0.1 V (see Fig. 3). For more sensitive detection of tocopheryl quinones the potentials were set at +0.40 V and -0.70 V, respectively (see Fig. 5). The second cell was set at +0.35 V throughout. Tocopherol oxidation products were identified by HPLC retention times, UV spectra (Shimadzu UV-300), and gas chromatography/ mass spectrometry (Varian 3400 and Finnigan Incas 50). Assuming first-order kinetics, the chemical reaction rate constant (k,) was determined by the relation
km’ r = ln([Ql&Qlo
- [Ql)~‘WA
where [Q],, is the initial concentration of the quencher, [Q] is the quencher concentration at a given time, and [‘O,] is the singlet molecular oxygen concentration calculated as described by Di Mascio and Sies (24). Chemicals. Deuterium oxide (99.8%), butylated hydroxytoluene, and aqueous tetraethylammonium hydroxide (20%) were from Sigma (St. Louis, MO). Diphenyl-p-phenylenediamine (DPPD) was from
NDPO? was synthesized Generation of singlet oxygen by NDPO,. as described (24). NDP02 concentration was determined spectrophotometrically and stock solutions of 0.4 M were kept at -70°C until being used. NDPOz dissociates yielding the 3,3’-(1,4-naphthylidene) dipropionate (NDP) and singlet molecular oxygen (23-25).
NW02 i5mM)
a-Tocopherol iO5mM)
Detection of singlet molecular oxygen monomol emission (1270 nm). Infrared emission of ‘02 was measured using a liquid nitrogencooled germanium-photodiode detector (Model EO-817L, North Coast Scientific Co., Santa Rosa, CA) sensitive in the spectral region of 800 to 1800 nm with a detector area of 0.25 cm2 and a sapphire window. The Ge-diode signal was processed with a lock-in amplifier (Model 5205, EG&G, Brookdeal Electronics Princeton Applied Research, Bracknell, Berks., UK). An oscilloscope (Model 1222A, Hewlett-Packard Co., CO) was simultaneously used with the amplifier, an optical chopper, and the germanium photodiode detector. The chopper (Model OC 400, Photon Technology International, Inc., Princeton, NJ) was used with a frequency of 30 s-l. Measurements were carried out in a cuvette with mirrored walls (35 X 6 X 55 mm). This allowed direct monitoring of both generation and quenching of ‘02 (Fig. 1). The quenching rate constant (12,) was calculated according to SternVolmer plots, using the equation S,/S
= 1 + (k, +
&K’[Ql,
where S and So are the chemiluminescence intensities in the presence and absence of the quencher, k, is the chemical reaction rate constant, kd is the singlet oxygen decay constant in the solvent, lo5 s-l, and [Q] is the concentration of the quencher; as k, 9 k, (compare Figs. 1 and 4A), k, was negligible. Electrochemical detection of tocopherok and tocopherol oxidation products with HPLC. Tocopherols were incubated with NDPOz as described in the individual experiments and subsequently extracted with n-hexane, dried under Nz, and then dissolved in the HPLC
0
FIG. 1.
5 TlmeImin)
10
Effect of a-tocopherol on monomol light emission of singlet oxygen generated by NDPOB. At 37”C, ethanol/chloroform (l/l) was placed in a thermostated glass cuvette of 6 ml (35 X 6 X 55 mm). Twenty microliters of a 0.4 M NDPOz solution, kept at 2”C, was added to the solution under constant stirring with a small magnetic bar. a-Tocopherol dissolved in ethanol was then injected.
TOCOPHEROL
HOMOLOGS
AS
SINGLET
TABLE
OXYGEN
103
QUENCHERS
II
Singlet Oxygen Quenching by Tocopherols and Related Compounds Loss of Ge-diode (%) at 300pM
Compound
signal
k, (lo6
M-Is-l)
a-Tocopherol
49.6 + 0.8
280 450”
P-Tocopherol
49.6 f 1.2
270
y-Tocopherol &Tocopherol a-Tocopherol a-Tocopherol a-Tocopherol a-Tocopherol Trolox DPPD Lycopene B-Carotene Retinoic acid Etretinate Isotretinoin
45.0 f 1.3 37.5 k 0.9 17.2 f 0.3 ndd No loss No loss 67.3 2 2.4 44.6 f 1.1 98.2 + 0.8 96.7 If- 1.4 9.1 + 1.1 7.4 f 0.6 4.0 f 0.9
230 160 -c -
methyl ether ethyl ether acetyl ester succinyl ester
Previous values (Ref.)
kr (lo6 M-l
120-250 (2); 620 (3); 120-670 (5); 31 (8); 42 (22); 260 (30) 23 (22)
3.6
0.23
b
11 (22); 180 (30) 5 (22); 100 (30) nr
2.8 1.7 0.94 0.16 nd nd nd nd nd nd nd
2.6;:30) 0.7 (30) nr nr nr nr nr nr nr nr nr nr nr
Z(2) nr nr
470” 220 nd nd -
Previous values (Ref.)
s-l)
31,Oonf (27) 14,000 (27) nr nr nr
46 (3); 1.9 (8); 6.6 (30)
Note. The loss of the Ge-diode signal is expressed in percentage of the light emitted by ‘02 at a concentration of 300 pM of the quencher. Conditions were as described in the legend to Fig. 1. Values are given as means f SE (n = 3 or 4). The corresponding physical and chemical quenching rate constants were calculated as described under Materials and Methods. a Solvent was D,O/C,H,OH (1:l) for solubility reasons. * Not reported previously. ’ No reaction observed. d Not determined.
Eastman-Kodak (Rochester, NY). All other chemicals were from Merck (Darmstadt, FRG). The endoperoxide of the disodium salt of 3,3’-(1,4-naphthylidene) dipropionate was prepared as described in Refs. (24,25). The product was identified by ‘H NMR and IR spectroscopy. Purified tocopherols and tocopherol derivatives were a gift from Henkel KGaA (Diisseldorf, FRG) and 20 mM stock solutions in ethano1 were prepared and concentrations checked by their UV absorption. Retinoic acid and derivatives were a gift from Hoffmann-LaRoche Co. (Basel, Switzerland). Microsomal epoxide hydrolase (mEPH) from rat liver (specific activity with 13Hlstyrene oxide as substrate, 0.58 rrmol/ min/mg protein) was kindly provided by Professor F. O&h (Department of Toxicology, University of Mainz, FRG).
a-tocopherol at concentrations of 0.3 mM from 49.6 + 0.8% to 65.1+- 0.3% (n = 4). This supports the suggestion that physical quenching of ‘02 by tocopherols involves a charge-transfer mechanism (2, 20). Furthermore, substitution of the hydroxyl group in position 6 of the chromane ring for methyl ether-or an a&y1 or succinyl ester group abolished the ‘02 quenching ability. The observed residual quenching with a-tocopherol methyl
Tocopherol
RESULTS
Physical
Quenching
of ‘0, by Tocopherok
The physical quenching efficiency of the various tocopherols was assessed by their ability to decrease the monomol emission signal. The results in terms of the $ value are shown in Table II and reveal that the physical quenching decreased in the sequence of (Y & p > y > 6tocopherol. The corresponding half-quenching concentrations, i.e., the tocopherol concentration at which 50% of the generated ‘02 was quenched, were 320, 340, 420, and 580 pM, respectively (Fig. 2). Replacing CHC& in the solvent by DzO to create a more polar solvent increased the quenching efficiency of
0
FIG. 2. centration described
I 0.03
0.1 Tocopherol
0.3 ImMl
Dependence of loss of germanium-diode of the different tocopherol homologs. in the legend to Fig. 1.
1.0
signal on the conConditions were as
104
KAISER ~Tocopherol
ET
AL.
copherols (Fig. 4B). Interestingly, /3-tocopherol exhibited very low reactivity. However, at a reaction time of more than 2 h and at a NDPO* concentration of 20 mM all tocopherols had completely reacted (data not shown), in contrast to results reported for the microwave discharge system (22), where the reaction of tocopherols with ‘02 measured in hexadecane as solvent was incomplete leaving about 10% of the tocopherols unreacted. The k, value for Lu-tocopherol (3.6 X lo6 M-* s-l) compares well with the value of 2.1 X lo6 M-’ s-’ obtained in pyridine (2) and 6.6 X lo6 M-l s-l observed in ethanol (30) and reveals that about 1.5% of the total quenching can be accounted for by chemical reaction. For fl-tocoph- erol the chemical reaction accounts for only about 0.1% 0 b 0 b of the total quenching. The NDPOz concentration de1 3 60 m,n pendence of the relative reaction rates suggests other Retention Time components of the reaction exist. FIG. 3. Chromatography and electrochemical detection of tocophReaction products were determined by HPLC with erols after incubation with NDPO*. Tocopherols (300 pM) were incuelectrochemical detection (Fig. 5); the fraction of each bated in an ethanol/D20 (l/l) solution at 37°C in a shaking water peak was collected and UV spectra and gas chromatograbath without (a) and with (b) NDP02. After 60 min the reaction was stopped by adding NaNs (2.5 mM), which quenched the remaining ‘02 phy/mass spectrometry performed. Tocopheryl quinone completely, and placing the samples in liquid Nz. Tocopherols were formation, identified by the retention times reported in determined as described under Materials and Methods. For 01- and j% (26), was detected from all four homologs, whereas meatocopherols, b-tocopherol(300 pM) was added as an internal standard. surable formation of the quinone epoxide was only obSimilarly, for y- and &tocopherols, o-tocopherol was used as an interserved with y-tocopherol (Fig. 5). Tocopheryl quinone nal standard (traces not shown). formation from the tocopherols was low, accounting for lo-15% of the reacted tocopherols, but it increased consistently with time and with NDPOz concentration ether (Table II) is explained by a 1% contamination with (Figs. 6A and 6B). The relative yield of the individual pure a-tocopherol as identified by electrochemical detectocopheryl quinones paralleled the sequence of the tion. Alkylation of the phenolic hydroxyl group should chemical reactivity of the tocopherol homologs, i.e., raise the oxidation potential and thus lower the quench(Y> y > 6 > /3-tocopheryl quinone. ing ability, if a charge-transfer mechanism is involved The y-tocopheryl quinone epoxide was identified by (2). Trolox, the hydrophilic tocopherol analog in which GC-MS (separation temperature 280°C ionizing electhe phytyl side chain has been replaced for a carboxylic tron energy 70 eV, m/e = 448). The formation of the ygroup, showed a quenching efficiency similar to that of tocopheryl quinone epoxide accounted for up to 80% of a-tocopherol, thus excluding participation of the lipothe reacted y-tocopherol; time course and concentration philic side chain in the quenching process (Table II). dependence differed markedly from the tocopheryl quiThe biologically occurring carotenoids lycopene and none (Figs. 6A and 6B). The concentration of this com,&carotene have a ‘02 quenching ability 2 orders of mag- pound rose to a maximum and then decreased with time nitude higher than the tocopherol homologs (Table II; while ‘02 was still being produced. This loss at later time Refs. (3, 27)). In contrast, vitamin A derivatives did not points was enhanced by addition of fresh NDPOz 20 min show quenching of ‘02 (Table II), in line with the obserafter the starting of the initial reaction (Fig. 6A), sugvation that structures with less than seven conjugated gesting that the quinone epoxide may not be the stable double bonds (vitamin A has four) are devoid of a ‘Oi? end product of the y-tocopherol ‘02 oxidation reaction quenching ability (28, 29). On the other hand, the synas assumed so far (22, 30-32). Rather, another product thetic antioxidant DPPD exhibited a quenching capac- or products may be formed from a subsequent reaction of ity similar to that of y-tocopherol (Table II). y-tocopheryl quinone epoxide with ‘02; this additional product(s) is electrochemically inactive. The formation Chemical Reaction of ‘0, with Tocopherols of the y-tocopheryl quinone epoxide and the loss of the y-tocopherol were temperature-dependent. When the As with physical quenching, chemical reactivity reaction was carried out at 4°C for 20 h, with the ‘Oz differed considerably among the various tocopherols. However, the sequence of reactivity toward ‘02 was (Y being released more slowly, less y-tocopherol was lost and relatively more of the quinone epoxide was formed, > y > 6 > P-tocopherol (Figs. 3 and 4). At low concentrawhen compared to the yield obtained at 37°C. tions of NDPOz a-tocopherol showed highest reactivity, The y-tocopheryl quinone epoxide with a concentrawhile at NDPOz concentrations of more than 7 mM there tion of 180 PM as quantified by electrochemical detection was no significant difference between (Y-, y-, and &toP-Tocopheroi
6P
1 L -
-Ii!
TOCOPHEROL
0
AS SINGLET
LO
20 Incubation
FIG. 4. Loss of tocopherol means from four experiments
HOMOLOGS
60
i I
a-Tocopherol
a-TQ
a
b
2
6
L
8
10
NDPO2 (mMI
with singlet oxygen. (A) Time course. For conditions see legend on NDPOz concentration. Reaction time was 30 min.
was incubated with rat liver microsomal epoxide hydrolase for 30 min at 37°C as described (33). While active with the substrate styrene oxide, HPLC analysis of the samples showed no detectable decrease of the y-tocopheryl quinone epoxide compared to controls which were run with inactivated epoxide hydrolase (70°C for 30 min). It is concluded that the epoxide is not a substrate
a
0
105
QUENCHERS
Time (mud
due to chemical reaction (‘I SD). (B) Dependence
a a --A 0
OXYGEN
to Fig. 3. Values
are the
for the microsomal epoxide hydrolase under these conditions and that the disposal of this oxidation product may occur by different means. Apart from the quinones and the quinone epoxide, other products of the reaction of the tocopherol homologs with singlet oxygen appeared on the HPLC. Retention times under the stated HPLC conditions and corre-
y-Tocopherol
I-TQO
I P 1
b-Tocopherol
(I-Tocopherol
I
6
b Y
p-T(
a
b
L, Y-m
J
a
I
a
a
a a-T(
I
a-TQ -d
b
h a
b
-I 60min Retention Time
FIG. 5. Electrochemical detection of tocopherol oxidation products after incubation of tocopherols with Tocopherol oxidation products were determined in samples incubated without (a) and with (b) NDP02 and conditions described under Materials and Methods. Reaction time was 30 min. TQ, tocopheryl quinone; Some minor peaks were due to a slight contamination of OI- by y-tocopherol and of y- and 6- by a-tocopherol,
NDPOP. For conditions see Fig. 3. identified with the electrochemical TQO, tocopheryl quinone epoxide. made visible here at high sensitivity.
106
KAISER
TO.OUd
NW+
ET AL.
ot i.Um~nl
NOP021mMI
FIG. 6. Quinone and quinone epoxide formation from the reaction of tocopherols with singlet oxygen. (A) Time course. Data were obtained as described in the legend to Fig. 5. Open circles, after formation of the y-tocopherol quinone epoxide a second addition of NDPO? (4 mM) was made at 20 min. Values are the means from four experiments (k SD). Conditions were as described in the legend to Fig. 3. (B) Reaction of ytocopherol with NDPOz and corresponding products. The products not identified (right-hand scale) are expressed in percentages of the reacted y-tocopherol.
sponding UV-absorbance maxima for a-tocopherol oxidation products were 15.6 h (X = 258, 264, 271 nm), for P-tocopherol8.1 h (X = 276 nm) and 17.0 h (X = 282 nm), for y-tocopherol 1.1 h (X = 275 nm), 4.8 h (X = 244 nm), 8.0 h (X = 302 nm), and 14.8 h (X = 295 nm) (Fig. 7) and for 6-tocopheroll.5 h (X = 254,272 nm) and 12.2 h (X = 272, 279 nm). For y-tocopherol oxidation products the UV absorption profiles of compounds 4 and 5 (Fig. 7) were similar to those of tocopheryl quinones, whereas the profiles of compounds 6 and 7 were reminiscent of those of tocopherols. The long retention times suggest the formation of dimers or trimers, which have been shown to be formed by the alkaline ferricyanide oxidation of a-tocopherol(34). However, identification by massspectrometry was hampered by low product volatility and perhaps low product stability. It is not known whether any product was the 8a-hydroperoxychromanone identified by Foote et al. (31). When the y-tocopherol was reacted with the thermolabile radical initiator AAPH (50 mM) instead of with NDPOz under otherwise identical conditions, higher amounts of the y-tocopheryl quinone and quinone epoxide were observed as well as compounds with the same retention time as compounds 6 and 7 of the y-tocopherol ‘02 reaction (data not shown). Thus, compounds 4 and 5 are products occurring preferentially in the y-tocopherol ‘02 reaction, not found to an appreciable extent via the radical reaction induced by AAPH.
tivity of the homologs is consistent with results obtained elsewhere using other singlet oxygen-generating systems (22, 30, 35) and is also in line with the relative effectiveness of the homologs in preventing in vitro lipid peroxidation (21) and with their in vivo biological activity (35), as determined by prevention of respiratory decline in rat livers and erythrocyte hemolysis. The quenching ability in C2H50H/CHC13 (l/l) was found to be less than in the more polar solvent of C2H50H/D20
6
!L
6h
Retention
DISCUSSION
Physical Quenching The rate constants of physical quenching of singlet oxygen by the tocopherol homologs were found to be 280, 270, 230, and 160 X lo6 M-l s1 for 01-,/3-, y-, and &tocopherol, respectively (Table II). This sequence of reac-
FIG. 7.
Time
y-Tocopherol oxidation products after incubation of y-tocopherol with NDPOz. Compound 1, y-tocopheryl quinone epoxide; compound 2, y-tocopherol; compound 3, y-tocopherol quinone; compounds 4-7, unidentified products; for spectral characteristics, see text. HPLC conditions are given under Materials and Methods. Incubation conditions were as described in the legend to Fig. 3, except that the reaction was performed at 4°C for 20 h.
TOCOPHEROL
HOMOLOGS
AS SINGLET
(l/l), supporting the possibility that the quenching of ‘02 by tocopherols proceeds through a charge-transfer intermediate (2, 20). Furthermore, esterification or ether formation at the 6-position in the chromane ring of a-tocopherol abolished the quenching ability, underscoring the requirement of a free hydroxyl group at position 6 in the quenching of ‘02 by tocopherols. Trolox showed the same quenching rate as a-tocopherol in CpH50H/D20 (l/l), suggesting that the phytyl chain is not only devoid of antioxidant properties (36) but also does not possess ‘02 quenching ability. Thus, physical quenching of ‘02 appears to depend solely on the chromane ring with a free hydroxyl group in position 6. The quenching ability of a-tocopherol is about lOOfold lessthan that of lycopene or 50-fold less than that of P-carotene, determined under similar conditions (Table II). However, taking into account that plasma ,&carotene levels in humans are only about l/50 of the a-tocopherol levels (37-39), the overall ‘02 quenching capacity of a-tocopherol may be regarded as equivalent to that of P-carotene in plasma; organs with low lycopene or flcarotene but high tocopherol levels might even depend largely on tocopherols for ‘02 quenching. In addition, bilirubin has become of interest in this respect (27, 40). Regarding the increasing epidemiological evidence of an inverse relation between tocopherol plasma levels and certain types of cancer (41), and the observed DNAdamaging effects of singlet oxygen (17-19), the ‘02quenching ability of tocopherols may become of importance in biology. In this respect it is of interest to note that high intracellular levels of tocopherols are not only found in microsomes and mitochondria, but also in the nucleus (42). Further, we recently found that trolox inhibits lop-induced DNA damage in the plasmid pBR 322 (unpublished work). Chemical Reactivity with Singlet Oxygen Following the reaction of 300
tocopherol with 4 52% d-tocopherol, and 72% of P-tocopherol remained detectable (Fig. 4A). Hence, the reactivity of the tocopherol homologs differed as compared to physical quenching of ‘02, with /I-tocopherol showing unexpected low reactivity. This is also in contrast to free-radical reactivity of the tocopherol homologs, shown to occur in the sequence of cy> p > y > d-tocopherol(21). Characterization ofthe oxidation products identified quinones for all four of the tocopherol homologs and a quinone epoxide for y-tocopherol. In previous studies (6, 7,22,31,32) the formation of a quinone epoxide was also shown for CX-,/3-, and y-tocopherol, but not for Stocopherol, via a hydroperoxide intermediate (32). It is possible that a quinone epoxide of (Y-and fl-tocopherol actually forms as an intermediate also under the conditions employed in the present study, but that it is even more susceptible to further reaction than the y-tocopherol quinone epoxide. An expla-
OXYGEN
107
QUENCHERS
nation for this could be the methylated 5-position of (Yand fi-tocopherol, which could destabilize the epoxide between positions 5 and 10. The present data indicate that the y-tocopheryl quinone epoxide seems to react with singlet oxygen again to form a subsequent product (Figs. 6A and 6B). Such further reaction would be in contrast to previous data indicating it to be a stable end product (22, 31, 32). Furthermore, additional reaction products appeared on the HPLC (Fig. 7), reminiscent of tocopheryl quinones or tocopherols by their UV spectra (data not shown), but likely to be dimer and trimer structures as judged by their retention times. However, the products have not yet been isolated and their chemical structures remain to be established; as mentioned above, mass spectrometric data of these compounds were not obtained. When comparing the various quenching and reaction rates of the tocopherol homologs with singlet oxygen it is apparent that a methylated position 5 enhances physical quenching, whereas chemical reactivity is favored by a methyl group in position 7. This model is based on the interesting finding that P-tocopherol has a physical quenching capacity similar to that of a-tocopherol, but shows almost no chemical reactivity. Taking into account the DNA-damaging and mutagenic effects of singlet oxygen (17-19), the high concentrations of tocopherols in the cell nucleus (42), and the potentially hazardous accumulation of tocopherol oxidation products, which may not readily be disposed of enzymatitally, /3-tocopherol might become an important tocopherol homolog in certain biological environments. In this context it is interesting to note that higher plants often contain significant amounts of p-, y-, and &tocopherol homologs (34). As they may be subject to greater exposure to light-generated ‘02 production as compared to animals, the accumulation of the less efficiently quenching tocopherol homologs may be an overall advantage in terms of lower chemical reaction rates.
PM
mM NDPOz, 18% ol-tocopherol, 34% y-tocopherol,
ACKNOWLEDGMENTS We thank Mrs. U. Rabe for technical assistance. We also thank Dr. Matthiessen, Department of Physiologische Chemie II, University of Dusseldorf, for performing mass spectrometry analysis. This work was supported by Deutsche Forschungsgemeinschaft, FRG (Grant Si 255/ 7-21, National Foundation for Cancer Research, USA, and by Fonds der Chemischen Industrie, FRG. M.E.M. was a recipient of a D.A.A.D. fellowship.
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A. L. (1962)
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V&am.
Horm.
20,493-510.
S. R., Doleiden, F. H., Trozzolo, Photo&em. Photobiol. 20,505-509.
A. M., and Lamola,
3. Foote, C. S., Ching, T. Y., and Geller, G. G. (1974) Photochem. Photobiol. 20,511-513. 4. Stevens, B., Small, R. D., and Perez, S. R. (1974) Photochem. Photobiol. 20,515-517. 5. Carlsson, D. J., Suprunchuk, Oil Chem. Sot. 53,656-660.
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Vol.
OF BIOCHEMISTRY
277, No. 1, February
AND
BIOPHYSICS
15, pp. lOl-108,199O
Physical and Chemical Scavenging Molecular Oxygen by Tocopherols Stephan
Kaiser,’
Paolo
Institut
fiir Physiologische
Received
July
5,1989,
Di Mascio,
Michael
Chemie I, Universitiit
and in revised
form
September
of Singlet
E. Murphy,
Dikseldorf,
Tocopherols (vitamin E) are known to act as biological antioxidants (1). Attention has centered on their function as free radical scavengers, but it has also been demonstrated that tocopherols react with singlet moleci Present address: Colorado State University, Department chemistry and Molecular Biology, Fort Collins, CO 80523. ’ To whom correspondence should be addressed at Institut iologische Chemie I Universitat Dusseldorf, Moorenstrasse Dusseldorf, FRG. Copyright All rights
0 of
$3.00 1990
by Academic Press, Inc. reproduction in any form reserved.
Sies’
27,1989
Singlet molecular oxygen (‘0,) arising from the thermal decomposition of the endoperoxide of 3,3’-(1,4naphthylidene) dipropionate was used to assess the effectiveness of a-, /3-, y-, and &tocopherol in the physical quenching as well as the chemical reaction of ‘Oz. The relative physical quenching efficiencies of the tocopherol homologs were found to decrease in the order of (Y > /3 > y > &tocopherol. The ability of physical quenching depends on a free hydroxyl group in position 6 of the chromane ring. Chemical reactivity of the tocopherol homologs with ‘02 was low, accounting for O.l-1.5% of physical quenching with P-tocopherol showing particularly low reactivity, resulting in the sequence a > y > 6 > @-tocopherol. Tocopheryl quinones were products of all tocopherol homologs, and in addition a quinone epoxide was a major product from y-tocopherol. This quinone epoxide was not cleaved by rat liver microsomal epoxide hydrolase; however, it reacted further with ‘Oz. It is concluded that methylation in position 5 of the chromane ring enhances physical quenching of ‘Oz, whereas chemical reactivity is favored by a methylated position 7. In view of the fact that fi-tocopherol is as effective as a-tocopherol in physical quenching of ‘02 but shows very low chemical reactivity, this tocopherol homolog might be particularly suitable for biological conditions in which an accumulation of oxidation products might weaken the antioxidant defense. 0 1990 Academic Press, Inc.
0003-9861/90
and Helmut
Federal Republic of Germany
of Biofiir Phys5 D-4000
ular oxygen (102)3 (2-8). The generation and possible pathological consequences of singlet oxygen in biological systems via enzymatic reactions have been described (912). ‘02 can be produced by photoexcitation as well as by nonphotochemical processes (chemiexcitation) and has been implicated in the peroxidation of biological lipids (5,13,14). ‘02 was also shown to play a role in the inactivation of cultured human cells by UV-A and near-visible radiation (15). Furthermore, ‘02 has been shown to be capable of inducing DNA damage (16-18) and to be mutagenic (19). Therefore, tocopherols are of biological interest for their ‘02 quenching capability. Scavenging of ‘02 by tocopherols includes physical quenching, in which the excited state of oxygen is deactivated without light emission, and chemical quenching which results in the formation of various oxidation products. Physical quenching by electron energy transfer almost always predominates, the rate depending on solvent polarity. This has led to the suggestion that a charge-transfer intermediate might be involved in the quenching process (2). In addition, it has been pointed out that the balance between physical and chemical quenching is a sensitive function of spin-orbit coupling properties and entropy factors (20). In the quenching of free radicals and also of ‘02, a-tocopherol is more effective than the other homologs in the sequence CY> 0 > y > &tocopherol(21,22). Here we compare the relative physical and chemical quenching ability of the tocopherol homologs toward ‘02 (Table I). This was achieved by using NDPO*, the thermodissociable endoperoxide of 3,3’-(1,4-naphthylidene) dipropionate to generate the singlet oxygen (23, 24), a methodology which offers advantages in comparison to
3 Abbreviations used: IO*, singlet molecular oxygen; UV, ultraviolet; NDP, 3,3’-(1,4-naphthylidene) dipropionate; NDPOP, endoperoxide of 3,3’-(1,4-naphthylidene) dipropionate; BHT, butylated hydroxytoluene; TEA, tetraethylammonium hydroxide; DPPD, diphenyl-p-phenylenediamine; mEPH, microsomal epoxide hydrolase; TQ, tocopheryl quinone; TQO, tocopheryl quinone epoxide; AAPH, 2,2’-azobis(2amidinopropane) HCl. 101
102
KAISER TABLE
Chemical
Structure
I
of the Tocopherol
Homologs
ET
AL.
buffer. As a-tocopherol was noticed to further oxidize to the tocopheryl quinone in the HPLC buffer, the stopping of the oxidation reaction and subsequent sample handling were done butylated hydroxytoluene (BHT) (1 mM). The HPLC system used included a Merck/Hitachi
in the presence Model
of
655A-12
pump and a Nova-Pak Cls 8 mm X 10 cm column (Waters Associates, Milford,
MA).
acetonitrile, adjusted
The
mobile
phase
consisted
of 60%
19.4% water, 0.5% tetraethylammonium
to pH 4.0 with
acetic
acid.
The
flow
2-propanol,
20%
hydroxide TEA,
rate was set at 0.4 ml/
min and a 100~~1 sample injected. An ESA Model 5100A Coulochem Homolog
RI
R2
a-Tocopherol /3-Tocopherol y-Tocopherol F-Tocopherol
CHs CH, H H
CH, H W H
other systems of ‘02 generation. Generation and quenching of ‘02 were directly monitored by the monomol light emission signal arising from the transition of the excited ‘Ap state to the 32, ground state at 1270 nm by the use of a liquid nitrogen-cooled germanium-photodiode detector. The time course and concentration dependence of the loss of tocopherols and the formation of reaction products due to chemical reaction with ‘02 were also examined. MATERIALS
AND
METHODS
electrochemical detector control module was connected to a Model 5021 conditioning cell and a Model 5010 analytical cell, which were installed in that sequence and separated from the HPLC column by an in-line carbon filter. For measurements of tocopherols (26) the redox
potential settings of the conditioning
cell and the first analytical cell
were set at -0.1 V (see Fig. 3). For more sensitive detection of tocopheryl quinones the potentials were set at +0.40 V and -0.70 V, respectively (see Fig. 5). The second cell was set at +0.35 V throughout. Tocopherol oxidation products were identified by HPLC retention times, UV spectra (Shimadzu UV-300), and gas chromatography/ mass spectrometry (Varian 3400 and Finnigan Incas 50). Assuming first-order kinetics, the chemical reaction rate constant (k,) was determined by the relation
km’ r = ln([Ql&Qlo
- [Ql)~‘WA
where [Q],, is the initial concentration of the quencher, [Q] is the quencher concentration at a given time, and [‘O,] is the singlet molecular oxygen concentration calculated as described by Di Mascio and Sies (24). Chemicals. Deuterium oxide (99.8%), butylated hydroxytoluene, and aqueous tetraethylammonium hydroxide (20%) were from Sigma (St. Louis, MO). Diphenyl-p-phenylenediamine (DPPD) was from
NDPO? was synthesized Generation of singlet oxygen by NDPO,. as described (24). NDP02 concentration was determined spectrophotometrically and stock solutions of 0.4 M were kept at -70°C until being used. NDPOz dissociates yielding the 3,3’-(1,4-naphthylidene) dipropionate (NDP) and singlet molecular oxygen (23-25).
NW02 i5mM)
a-Tocopherol iO5mM)
Detection of singlet molecular oxygen monomol emission (1270 nm). Infrared emission of ‘02 was measured using a liquid nitrogencooled germanium-photodiode detector (Model EO-817L, North Coast Scientific Co., Santa Rosa, CA) sensitive in the spectral region of 800 to 1800 nm with a detector area of 0.25 cm2 and a sapphire window. The Ge-diode signal was processed with a lock-in amplifier (Model 5205, EG&G, Brookdeal Electronics Princeton Applied Research, Bracknell, Berks., UK). An oscilloscope (Model 1222A, Hewlett-Packard Co., CO) was simultaneously used with the amplifier, an optical chopper, and the germanium photodiode detector. The chopper (Model OC 400, Photon Technology International, Inc., Princeton, NJ) was used with a frequency of 30 s-l. Measurements were carried out in a cuvette with mirrored walls (35 X 6 X 55 mm). This allowed direct monitoring of both generation and quenching of ‘02 (Fig. 1). The quenching rate constant (12,) was calculated according to SternVolmer plots, using the equation S,/S
= 1 + (k, +
&K’[Ql,
where S and So are the chemiluminescence intensities in the presence and absence of the quencher, k, is the chemical reaction rate constant, kd is the singlet oxygen decay constant in the solvent, lo5 s-l, and [Q] is the concentration of the quencher; as k, 9 k, (compare Figs. 1 and 4A), k, was negligible. Electrochemical detection of tocopherok and tocopherol oxidation products with HPLC. Tocopherols were incubated with NDPOz as described in the individual experiments and subsequently extracted with n-hexane, dried under Nz, and then dissolved in the HPLC
0
FIG. 1.
5 TlmeImin)
10
Effect of a-tocopherol on monomol light emission of singlet oxygen generated by NDPOB. At 37”C, ethanol/chloroform (l/l) was placed in a thermostated glass cuvette of 6 ml (35 X 6 X 55 mm). Twenty microliters of a 0.4 M NDPOz solution, kept at 2”C, was added to the solution under constant stirring with a small magnetic bar. a-Tocopherol dissolved in ethanol was then injected.
TOCOPHEROL
HOMOLOGS
AS
SINGLET
TABLE
OXYGEN
103
QUENCHERS
II
Singlet Oxygen Quenching by Tocopherols and Related Compounds Loss of Ge-diode (%) at 300pM
Compound
signal
k, (lo6
M-Is-l)
a-Tocopherol
49.6 + 0.8
280 450”
P-Tocopherol
49.6 f 1.2
270
y-Tocopherol &Tocopherol a-Tocopherol a-Tocopherol a-Tocopherol a-Tocopherol Trolox DPPD Lycopene B-Carotene Retinoic acid Etretinate Isotretinoin
45.0 f 1.3 37.5 k 0.9 17.2 f 0.3 ndd No loss No loss 67.3 2 2.4 44.6 f 1.1 98.2 + 0.8 96.7 If- 1.4 9.1 + 1.1 7.4 f 0.6 4.0 f 0.9
230 160 -c -
methyl ether ethyl ether acetyl ester succinyl ester
Previous values (Ref.)
kr (lo6 M-l
120-250 (2); 620 (3); 120-670 (5); 31 (8); 42 (22); 260 (30) 23 (22)
3.6
0.23
b
11 (22); 180 (30) 5 (22); 100 (30) nr
2.8 1.7 0.94 0.16 nd nd nd nd nd nd nd
2.6;:30) 0.7 (30) nr nr nr nr nr nr nr nr nr nr nr
Z(2) nr nr
470” 220 nd nd -
Previous values (Ref.)
s-l)
31,Oonf (27) 14,000 (27) nr nr nr
46 (3); 1.9 (8); 6.6 (30)
Note. The loss of the Ge-diode signal is expressed in percentage of the light emitted by ‘02 at a concentration of 300 pM of the quencher. Conditions were as described in the legend to Fig. 1. Values are given as means f SE (n = 3 or 4). The corresponding physical and chemical quenching rate constants were calculated as described under Materials and Methods. a Solvent was D,O/C,H,OH (1:l) for solubility reasons. * Not reported previously. ’ No reaction observed. d Not determined.
Eastman-Kodak (Rochester, NY). All other chemicals were from Merck (Darmstadt, FRG). The endoperoxide of the disodium salt of 3,3’-(1,4-naphthylidene) dipropionate was prepared as described in Refs. (24,25). The product was identified by ‘H NMR and IR spectroscopy. Purified tocopherols and tocopherol derivatives were a gift from Henkel KGaA (Diisseldorf, FRG) and 20 mM stock solutions in ethano1 were prepared and concentrations checked by their UV absorption. Retinoic acid and derivatives were a gift from Hoffmann-LaRoche Co. (Basel, Switzerland). Microsomal epoxide hydrolase (mEPH) from rat liver (specific activity with 13Hlstyrene oxide as substrate, 0.58 rrmol/ min/mg protein) was kindly provided by Professor F. O&h (Department of Toxicology, University of Mainz, FRG).
a-tocopherol at concentrations of 0.3 mM from 49.6 + 0.8% to 65.1+- 0.3% (n = 4). This supports the suggestion that physical quenching of ‘02 by tocopherols involves a charge-transfer mechanism (2, 20). Furthermore, substitution of the hydroxyl group in position 6 of the chromane ring for methyl ether-or an a&y1 or succinyl ester group abolished the ‘02 quenching ability. The observed residual quenching with a-tocopherol methyl
Tocopherol
RESULTS
Physical
Quenching
of ‘0, by Tocopherok
The physical quenching efficiency of the various tocopherols was assessed by their ability to decrease the monomol emission signal. The results in terms of the $ value are shown in Table II and reveal that the physical quenching decreased in the sequence of (Y & p > y > 6tocopherol. The corresponding half-quenching concentrations, i.e., the tocopherol concentration at which 50% of the generated ‘02 was quenched, were 320, 340, 420, and 580 pM, respectively (Fig. 2). Replacing CHC& in the solvent by DzO to create a more polar solvent increased the quenching efficiency of
0
FIG. 2. centration described
I 0.03
0.1 Tocopherol
0.3 ImMl
Dependence of loss of germanium-diode of the different tocopherol homologs. in the legend to Fig. 1.
1.0
signal on the conConditions were as
104
KAISER ~Tocopherol
ET
AL.
copherols (Fig. 4B). Interestingly, /3-tocopherol exhibited very low reactivity. However, at a reaction time of more than 2 h and at a NDPO* concentration of 20 mM all tocopherols had completely reacted (data not shown), in contrast to results reported for the microwave discharge system (22), where the reaction of tocopherols with ‘02 measured in hexadecane as solvent was incomplete leaving about 10% of the tocopherols unreacted. The k, value for Lu-tocopherol (3.6 X lo6 M-* s-l) compares well with the value of 2.1 X lo6 M-’ s-’ obtained in pyridine (2) and 6.6 X lo6 M-l s-l observed in ethanol (30) and reveals that about 1.5% of the total quenching can be accounted for by chemical reaction. For fl-tocoph- erol the chemical reaction accounts for only about 0.1% 0 b 0 b of the total quenching. The NDPOz concentration de1 3 60 m,n pendence of the relative reaction rates suggests other Retention Time components of the reaction exist. FIG. 3. Chromatography and electrochemical detection of tocophReaction products were determined by HPLC with erols after incubation with NDPO*. Tocopherols (300 pM) were incuelectrochemical detection (Fig. 5); the fraction of each bated in an ethanol/D20 (l/l) solution at 37°C in a shaking water peak was collected and UV spectra and gas chromatograbath without (a) and with (b) NDP02. After 60 min the reaction was stopped by adding NaNs (2.5 mM), which quenched the remaining ‘02 phy/mass spectrometry performed. Tocopheryl quinone completely, and placing the samples in liquid Nz. Tocopherols were formation, identified by the retention times reported in determined as described under Materials and Methods. For 01- and j% (26), was detected from all four homologs, whereas meatocopherols, b-tocopherol(300 pM) was added as an internal standard. surable formation of the quinone epoxide was only obSimilarly, for y- and &tocopherols, o-tocopherol was used as an interserved with y-tocopherol (Fig. 5). Tocopheryl quinone nal standard (traces not shown). formation from the tocopherols was low, accounting for lo-15% of the reacted tocopherols, but it increased consistently with time and with NDPOz concentration ether (Table II) is explained by a 1% contamination with (Figs. 6A and 6B). The relative yield of the individual pure a-tocopherol as identified by electrochemical detectocopheryl quinones paralleled the sequence of the tion. Alkylation of the phenolic hydroxyl group should chemical reactivity of the tocopherol homologs, i.e., raise the oxidation potential and thus lower the quench(Y> y > 6 > /3-tocopheryl quinone. ing ability, if a charge-transfer mechanism is involved The y-tocopheryl quinone epoxide was identified by (2). Trolox, the hydrophilic tocopherol analog in which GC-MS (separation temperature 280°C ionizing electhe phytyl side chain has been replaced for a carboxylic tron energy 70 eV, m/e = 448). The formation of the ygroup, showed a quenching efficiency similar to that of tocopheryl quinone epoxide accounted for up to 80% of a-tocopherol, thus excluding participation of the lipothe reacted y-tocopherol; time course and concentration philic side chain in the quenching process (Table II). dependence differed markedly from the tocopheryl quiThe biologically occurring carotenoids lycopene and none (Figs. 6A and 6B). The concentration of this com,&carotene have a ‘02 quenching ability 2 orders of mag- pound rose to a maximum and then decreased with time nitude higher than the tocopherol homologs (Table II; while ‘02 was still being produced. This loss at later time Refs. (3, 27)). In contrast, vitamin A derivatives did not points was enhanced by addition of fresh NDPOz 20 min show quenching of ‘02 (Table II), in line with the obserafter the starting of the initial reaction (Fig. 6A), sugvation that structures with less than seven conjugated gesting that the quinone epoxide may not be the stable double bonds (vitamin A has four) are devoid of a ‘Oi? end product of the y-tocopherol ‘02 oxidation reaction quenching ability (28, 29). On the other hand, the synas assumed so far (22, 30-32). Rather, another product thetic antioxidant DPPD exhibited a quenching capac- or products may be formed from a subsequent reaction of ity similar to that of y-tocopherol (Table II). y-tocopheryl quinone epoxide with ‘02; this additional product(s) is electrochemically inactive. The formation Chemical Reaction of ‘0, with Tocopherols of the y-tocopheryl quinone epoxide and the loss of the y-tocopherol were temperature-dependent. When the As with physical quenching, chemical reactivity reaction was carried out at 4°C for 20 h, with the ‘Oz differed considerably among the various tocopherols. However, the sequence of reactivity toward ‘02 was (Y being released more slowly, less y-tocopherol was lost and relatively more of the quinone epoxide was formed, > y > 6 > P-tocopherol (Figs. 3 and 4). At low concentrawhen compared to the yield obtained at 37°C. tions of NDPOz a-tocopherol showed highest reactivity, The y-tocopheryl quinone epoxide with a concentrawhile at NDPOz concentrations of more than 7 mM there tion of 180 PM as quantified by electrochemical detection was no significant difference between (Y-, y-, and &toP-Tocopheroi
6P
1 L -
-Ii!
TOCOPHEROL
0
AS SINGLET
LO
20 Incubation
FIG. 4. Loss of tocopherol means from four experiments
HOMOLOGS
60
i I
a-Tocopherol
a-TQ
a
b
2
6
L
8
10
NDPO2 (mMI
with singlet oxygen. (A) Time course. For conditions see legend on NDPOz concentration. Reaction time was 30 min.
was incubated with rat liver microsomal epoxide hydrolase for 30 min at 37°C as described (33). While active with the substrate styrene oxide, HPLC analysis of the samples showed no detectable decrease of the y-tocopheryl quinone epoxide compared to controls which were run with inactivated epoxide hydrolase (70°C for 30 min). It is concluded that the epoxide is not a substrate
a
0
105
QUENCHERS
Time (mud
due to chemical reaction (‘I SD). (B) Dependence
a a --A 0
OXYGEN
to Fig. 3. Values
are the
for the microsomal epoxide hydrolase under these conditions and that the disposal of this oxidation product may occur by different means. Apart from the quinones and the quinone epoxide, other products of the reaction of the tocopherol homologs with singlet oxygen appeared on the HPLC. Retention times under the stated HPLC conditions and corre-
y-Tocopherol
I-TQO
I P 1
b-Tocopherol
(I-Tocopherol
I
6
b Y
p-T(
a
b
L, Y-m
J
a
I
a
a
a a-T(
I
a-TQ -d
b
h a
b
-I 60min Retention Time
FIG. 5. Electrochemical detection of tocopherol oxidation products after incubation of tocopherols with Tocopherol oxidation products were determined in samples incubated without (a) and with (b) NDP02 and conditions described under Materials and Methods. Reaction time was 30 min. TQ, tocopheryl quinone; Some minor peaks were due to a slight contamination of OI- by y-tocopherol and of y- and 6- by a-tocopherol,
NDPOP. For conditions see Fig. 3. identified with the electrochemical TQO, tocopheryl quinone epoxide. made visible here at high sensitivity.
106
KAISER
TO.OUd
NW+
ET AL.
ot i.Um~nl
NOP021mMI
FIG. 6. Quinone and quinone epoxide formation from the reaction of tocopherols with singlet oxygen. (A) Time course. Data were obtained as described in the legend to Fig. 5. Open circles, after formation of the y-tocopherol quinone epoxide a second addition of NDPO? (4 mM) was made at 20 min. Values are the means from four experiments (k SD). Conditions were as described in the legend to Fig. 3. (B) Reaction of ytocopherol with NDPOz and corresponding products. The products not identified (right-hand scale) are expressed in percentages of the reacted y-tocopherol.
sponding UV-absorbance maxima for a-tocopherol oxidation products were 15.6 h (X = 258, 264, 271 nm), for P-tocopherol8.1 h (X = 276 nm) and 17.0 h (X = 282 nm), for y-tocopherol 1.1 h (X = 275 nm), 4.8 h (X = 244 nm), 8.0 h (X = 302 nm), and 14.8 h (X = 295 nm) (Fig. 7) and for 6-tocopheroll.5 h (X = 254,272 nm) and 12.2 h (X = 272, 279 nm). For y-tocopherol oxidation products the UV absorption profiles of compounds 4 and 5 (Fig. 7) were similar to those of tocopheryl quinones, whereas the profiles of compounds 6 and 7 were reminiscent of those of tocopherols. The long retention times suggest the formation of dimers or trimers, which have been shown to be formed by the alkaline ferricyanide oxidation of a-tocopherol(34). However, identification by massspectrometry was hampered by low product volatility and perhaps low product stability. It is not known whether any product was the 8a-hydroperoxychromanone identified by Foote et al. (31). When the y-tocopherol was reacted with the thermolabile radical initiator AAPH (50 mM) instead of with NDPOz under otherwise identical conditions, higher amounts of the y-tocopheryl quinone and quinone epoxide were observed as well as compounds with the same retention time as compounds 6 and 7 of the y-tocopherol ‘02 reaction (data not shown). Thus, compounds 4 and 5 are products occurring preferentially in the y-tocopherol ‘02 reaction, not found to an appreciable extent via the radical reaction induced by AAPH.
tivity of the homologs is consistent with results obtained elsewhere using other singlet oxygen-generating systems (22, 30, 35) and is also in line with the relative effectiveness of the homologs in preventing in vitro lipid peroxidation (21) and with their in vivo biological activity (35), as determined by prevention of respiratory decline in rat livers and erythrocyte hemolysis. The quenching ability in C2H50H/CHC13 (l/l) was found to be less than in the more polar solvent of C2H50H/D20
6
!L
6h
Retention
DISCUSSION
Physical Quenching The rate constants of physical quenching of singlet oxygen by the tocopherol homologs were found to be 280, 270, 230, and 160 X lo6 M-l s1 for 01-,/3-, y-, and &tocopherol, respectively (Table II). This sequence of reac-
FIG. 7.
Time
y-Tocopherol oxidation products after incubation of y-tocopherol with NDPOz. Compound 1, y-tocopheryl quinone epoxide; compound 2, y-tocopherol; compound 3, y-tocopherol quinone; compounds 4-7, unidentified products; for spectral characteristics, see text. HPLC conditions are given under Materials and Methods. Incubation conditions were as described in the legend to Fig. 3, except that the reaction was performed at 4°C for 20 h.
TOCOPHEROL
HOMOLOGS
AS SINGLET
(l/l), supporting the possibility that the quenching of ‘02 by tocopherols proceeds through a charge-transfer intermediate (2, 20). Furthermore, esterification or ether formation at the 6-position in the chromane ring of a-tocopherol abolished the quenching ability, underscoring the requirement of a free hydroxyl group at position 6 in the quenching of ‘02 by tocopherols. Trolox showed the same quenching rate as a-tocopherol in CpH50H/D20 (l/l), suggesting that the phytyl chain is not only devoid of antioxidant properties (36) but also does not possess ‘02 quenching ability. Thus, physical quenching of ‘02 appears to depend solely on the chromane ring with a free hydroxyl group in position 6. The quenching ability of a-tocopherol is about lOOfold lessthan that of lycopene or 50-fold less than that of P-carotene, determined under similar conditions (Table II). However, taking into account that plasma ,&carotene levels in humans are only about l/50 of the a-tocopherol levels (37-39), the overall ‘02 quenching capacity of a-tocopherol may be regarded as equivalent to that of P-carotene in plasma; organs with low lycopene or flcarotene but high tocopherol levels might even depend largely on tocopherols for ‘02 quenching. In addition, bilirubin has become of interest in this respect (27, 40). Regarding the increasing epidemiological evidence of an inverse relation between tocopherol plasma levels and certain types of cancer (41), and the observed DNAdamaging effects of singlet oxygen (17-19), the ‘02quenching ability of tocopherols may become of importance in biology. In this respect it is of interest to note that high intracellular levels of tocopherols are not only found in microsomes and mitochondria, but also in the nucleus (42). Further, we recently found that trolox inhibits lop-induced DNA damage in the plasmid pBR 322 (unpublished work). Chemical Reactivity with Singlet Oxygen Following the reaction of 300
tocopherol with 4 52% d-tocopherol, and 72% of P-tocopherol remained detectable (Fig. 4A). Hence, the reactivity of the tocopherol homologs differed as compared to physical quenching of ‘02, with /I-tocopherol showing unexpected low reactivity. This is also in contrast to free-radical reactivity of the tocopherol homologs, shown to occur in the sequence of cy> p > y > d-tocopherol(21). Characterization ofthe oxidation products identified quinones for all four of the tocopherol homologs and a quinone epoxide for y-tocopherol. In previous studies (6, 7,22,31,32) the formation of a quinone epoxide was also shown for CX-,/3-, and y-tocopherol, but not for Stocopherol, via a hydroperoxide intermediate (32). It is possible that a quinone epoxide of (Y-and fl-tocopherol actually forms as an intermediate also under the conditions employed in the present study, but that it is even more susceptible to further reaction than the y-tocopherol quinone epoxide. An expla-
OXYGEN
107
QUENCHERS
nation for this could be the methylated 5-position of (Yand fi-tocopherol, which could destabilize the epoxide between positions 5 and 10. The present data indicate that the y-tocopheryl quinone epoxide seems to react with singlet oxygen again to form a subsequent product (Figs. 6A and 6B). Such further reaction would be in contrast to previous data indicating it to be a stable end product (22, 31, 32). Furthermore, additional reaction products appeared on the HPLC (Fig. 7), reminiscent of tocopheryl quinones or tocopherols by their UV spectra (data not shown), but likely to be dimer and trimer structures as judged by their retention times. However, the products have not yet been isolated and their chemical structures remain to be established; as mentioned above, mass spectrometric data of these compounds were not obtained. When comparing the various quenching and reaction rates of the tocopherol homologs with singlet oxygen it is apparent that a methylated position 5 enhances physical quenching, whereas chemical reactivity is favored by a methyl group in position 7. This model is based on the interesting finding that P-tocopherol has a physical quenching capacity similar to that of a-tocopherol, but shows almost no chemical reactivity. Taking into account the DNA-damaging and mutagenic effects of singlet oxygen (17-19), the high concentrations of tocopherols in the cell nucleus (42), and the potentially hazardous accumulation of tocopherol oxidation products, which may not readily be disposed of enzymatitally, /3-tocopherol might become an important tocopherol homolog in certain biological environments. In this context it is interesting to note that higher plants often contain significant amounts of p-, y-, and &tocopherol homologs (34). As they may be subject to greater exposure to light-generated ‘02 production as compared to animals, the accumulation of the less efficiently quenching tocopherol homologs may be an overall advantage in terms of lower chemical reaction rates.
PM
mM NDPOz, 18% ol-tocopherol, 34% y-tocopherol,
ACKNOWLEDGMENTS We thank Mrs. U. Rabe for technical assistance. We also thank Dr. Matthiessen, Department of Physiologische Chemie II, University of Dusseldorf, for performing mass spectrometry analysis. This work was supported by Deutsche Forschungsgemeinschaft, FRG (Grant Si 255/ 7-21, National Foundation for Cancer Research, USA, and by Fonds der Chemischen Industrie, FRG. M.E.M. was a recipient of a D.A.A.D. fellowship.
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