Chem. Res. Toxicol. 1991,4, 89-93
89
Peroxyl Radical Trapping and Autoxidation Reactions of a-Tocopherol in Lipid Bilayers Daniel C. Liebler,* Kathryn L. Kaysen, and Jeanne A. Burr Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721 Received July 26,1990
A phospholipid liposome system was employed t o model peroxyl radical trapping reactions of a-tocopherol (1) in biological membranes. Peroxyl radicals generated by thermolysis of 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) a t 37 "C oxidized 1 t o sa-[(2,4-dimethyl-1nitrilopent-2-yl)dioxy]tocopherone (3a), 8a-(hydroperoxy)tocopherone (3b), a-tocopherol quinone (4), 4a,5-epoxy-8a-hydroperoxytocopherone (6), 2,3-epoxy-a-tocopherol quinone (7), and 5,6epoxy-a-tocopherol quinone (8). T h e products were purified by high-performance liquid chromatography and characterized by UV-vis spectroscopy, mass spectrometry, and cochromatography with authentic standards. Products accumulated in approximately constant proportion as 1 was consumed. Tocopherones 3a/3b decomposed in bhe bilayer primarily by hydrolyzing to produce 4. Tocopherone decomposition also produced small amounts of epoxides 6-8, apparently by unimolecular tocopherone decomposition rather than by peroxyl radical dependent oxidation, since neither AMVN nor 1 affected the rate of 3a loss or the distribution of products. Epoxides 6-8 appear t o be formed primarily by autoxidation reactions that compete with the peroxyl radical trapping reactions that form tocopherone 3a. Epoxide products may thus serve as biochemical markers for irreversible oxidation of 1 by peroxyl radicals in membranes.
Introduction Vitamin E (a-tocopherol, 1;see Chart I) protects living tissues against oxidative injury by trapping reactive free radicals generated by chemical intoxication, by radiation, or by endogenous processes of oxygen metabolism (I). Exceptional reactivity toward peroxyl radicals allows 1 to effectively suppress membrane lipid peroxidation at concentrations typically as low as one molecule of 1 per 1000 phospholipids (2-4). Compound 1 reacts with peroxyl radicals to produce a hydroperoxide and the relatively stable tocopheroxyl radical (2). Sustained antioxidant protection has been postulated to depend on reductive regeneration of 1 from 2 by ascorbic acid (5, 6) or by a glutathione-dependent enzyme (7,8). Alternatively, 2 may trap a second peroxyl radical to form other products. In homogeneous solution, reactions of peroxyl radicals with radical intermediate 2 yield multiple products. Peroxyl radicals generated by thermolysis of AMVN' oxidize 1 to 8a-(alkyldioxy)tocopherone 3a as the principal product, which may either undergo reduction to 1 (9) or hydrolyze in the presence of water to a-tocopherol quinone (4) ( 1 0 , I I ) . A small fraction of intermediate 2 reacts with oxygen and a hydrogen donor to form the 8a-hydroperoxytocopherone 3b (10,12), which also hydrolyzes to 4. In a competing reaction, 2 undergoes an apparent peroxyl radical dependent epoxidation, followed by reaction with oxygen and a hydrogen donor to form epoxy-8a-hydroperoxytocopherones 5 and 6 (10). In the presence of water, these readily hydrolyze t o epoxyquinones 7 and 8, respectively. The reactions of 2 that form 3b and epoxytocopherones 516 do not trap radicals and therefore do not produce an antioxidant effect. The extent to which epAbbreviations: AMVN, azobis(2,4-dimethylvaleronitrile);DADHPLC, high-performanceliquid chromatographywith diode-array spectrophotometric detection; Tris-HC1, tris(hydroxymethy1)aminomethane hydrochloride. The term 'mol %", which is used to describe reactant concentrations in the lipid bilayer, is defined as (moles of reactant/moles of phospholipid) X 100.
Chart I. Structures of Compounds Referred to in the Text
16H33
2
1
1BH33
OH
1~3~33
5
4
0
m
1
6 .+" H 3
3
OOH
6 OH
7
OH
8
oxidation of 2 competes with radical trapping presumably dictates how far the reaction stoichiometry of 1 deviates from the theoretical ratio of 2 peroxyl radicals trapped for every 1 consumed (2). Experimentally determined values
0893-22~~/91/2104-0089$02.50/0 0 1991 American Chemical Society
90 Chem. Res. Toxicol., Vol. 4, No. 1, 1991
for this ratio vary between 1.4 and 2.0, depending on the test system used (13-15), which suggests that competition between radical-trapping and autoxidation reactions of 2 varies with reaction environment or with the structure of the attacking peroxyl radical. Indeed, oxidation of 1 by tert-butylperoxyl radicals in excess tert-butyl hydroperoxide yields epoxytocopherone 6 without producing an 8a-(alkyldioxy)tocopherone product (12). To determine whether the reaction chemistry of 1 observed in homogeneous solution occurs in phospholipid bilayers, we studied the reactions of 1 with peroxyl radicals generated from AMVN in a liposome system. This experimental system provides a reaction environment similar to biological membranes, but without other redox-active components that could obscure some reactions of 1 and 2. Here we report that 1 reacted with peroxyl radicals to produce 8a-(alky1dioxy)tocopherone 3a, epoxytocopherone 6, 8a-hydroperoxytocopherone 3b, and the hydrolysis products 4,7, and 8. Compounds 3a and 3b decomposed slowly to 4, epoxytocopherone 6, and epoxyquinones 7 and 8. These results indicate that both radical trapping and autoxidation reactions occur when 1 reacts with peroxyl radicals in a phospholipid bilayer.
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Liebler et al.
2oo
150 100
t
50
5 i o
200
-3 a-
100 0 0
20
40
60
time, min
Figure 1. HPLC analysis of products formed from [14C]-1and AMVN-derived peroxyl radicals in phosphatidylcholineliposomes during incubation at 37 O C for 2 h (top) and 6 h (bottom).
a Hewlett-Packard 1040A diode-array HPLC detector operated with an HP79994A Analytical Workstation and Hewlett-Packard operating software. Unlabeled 1was quantitated by revene-phase HPLC with coulometric detection as described previously (9). Mass spectrometry wm done with a Finnegan MAT-90 instrument in the College of Pharmacy Mass Spectrometry Facility. Samples Experimental Procedures were introduced by direct probe insertion and analyzed in the Chemicals. [5-14C-methyl]-d-a-Tocopherol ([“CI-1) (1.0 mCi electron impact mode at an ionizing voltage of 70 eV. mmol-’) was synthesized from d-y-tocopherol and purified as described previously (10, 16). [5-14C-methyl]-8a-[(2,4-DiResults methyl-l-nitrilopent-2-yl)dioxy] tocopherone ( [14C]-3a)was preOxidation of 1 and [14C]-1. Incubation of 0.1 mol % pared by oxidation of [“Cl-l with AMVN and purified by HPLC (10). [5-14C-methyl]-8a-Hydroperoxytocopherone ( [14C]-3b)was 1 with 20 mol ’70 AMVN in liposomes caused complete prepared by photochemical oxidation of [14C]-1(17)and purified oxidation of 1 within 10 h. Oxidation required AMVN, by HPLC as described (10). A mixture of tocopherol dimers for as only 11% of 1 was oxidized in a parallel incubation use as HPLC markers was synthesized by alkaline ferricyanide without AMVN. In liposomes containing [14C]-l and oxidation of 1 (18). L-a-Phosphatidylcholine (from soybean,type AMVN at these concentrations, two groups of radiolabeled III-S) was purchased from Sigma. AMVN from Polysciences products were detected in samples taken at 2 and 6 h (Warrington, PA) was used as supplied. Unlabeled 1 (99+% (Figure 1). The first product group eluted between 10 and d-a-tocopherol)was a gift from Henckel Corp., Fine Chemicals 20 min and contained four peaks. These were identified Division, LaGrange, IL. as epoxyquinones 7 and 8, epoxytocopherone 6, and aLiposome Preparation and Incubations. Liposomes were tocopherol quinone 4. Product identities were established prepared by the ethanol injection method (19). Phosphatidylcholine (5 pmol),AMVN (1pmol), and 1 (1nmol) were dissolved by coelution of radiolabeled products with authentic in 0.1 mL of ethanol and injected into 10 mL of rapidly stirred standards. The identities of compounds 4,7, and 8 were 50 mM Tris-HC1and 50 mM NaC1, pH 7.0. Liposomes containing confirmed by DAD-HPLC analysis and mass spectrometry 3a and 3b were prepared in the same manner, except that the of products isolated from incubations with unlabeled 1. phosphatidylcholine was first treated with dilute ammonia/ The UV and mass spectra thus obtained matched those methanol to remove traces of HCl present from lipid storage in of authentic standards and are summarized as follows. CHC13solution (9). Failure to remove the acid causes tocopherone Compound 4 displayed UV,, a t 262 and 267 nm (in hydrolysis during liposome preparation. Incubations were done HPLC mobile phase) and mass spectral signals at m / z 446 at 37 “C under air in an orbital shaking incubator. Large-scale (M+, 17.7%), 430 (25.0), 221 (100.0), 178 (69.2), and 165 incubations to generate products for mass spectral analysis contained 1, AMVN, and phosphatidylcholine in the same ratios at (43.3). Compound 7 displayed a UV,, at 273 nm (in 10-fold higher concentrations. HPLC mobile phase) and mass spectral signals at m / z 444 Product Analyses. Incubation mixtures were extracted twice (M+- 18, 2.3%), 430 (14.2), 419 (4.6), 402 (4.9), 239 (loo), with 2 volumes of hexane/2-propanol(3:2v/v), and the extracts 237 (42.9),and 195 (76.2). Compound 8 displayed a UV, were evaporated to dryness in vacuo. Extraction of radiolabel at 275 nm (in HPLC mobile phase) and mass spectral from incubations with [14C]-1was at least 95%. Phospholipids signals at m / z 444 (M+ - 18, 2.5%), 430 (16.5), 419 (26.81, and their oxidation products, which coelute with products of 1, 402 (15.8),237 (100.0), 194 (44.8), and 167 (78.0). UV and were removed by solid-phaseextraction (20) prior to analysis by mass spectral characterization of the putative epoxytocoDAD-HPLC. Lipid extracts were dissolved in toluene/ethyl pherone 6 was not attempted. However, we observed that acetate (3:2 v/v) and applied to a 3-mL Extract-Clean diol solthis peak had a retention time identical with that of 6 id-phase extraction column (Alltech Associates, Deerfield, IL) equilibrated with the same solvent. Products of 1 were eluted isolated from homogeneous solution oxidations of 1 by with an additional portion of toluene/ethyl acetate. Phospholipids AMVN. Moreover, this peak was eliminated from the then were eluted with methanol. Oxidation products of 1 and chromatogram by treatment of the product extract with [14C]-1were analyzed by reverse-phase HPLC on a Spherisorb dilute HC1, which causes 6 to hydrolyze to epoxyquinone ODS-2, 5-pm, 4.6 X 250 mm column eluted with methanol/l N 8 (10). sodium acetate, pH 4.25 (93:7v/v), at a flow rate of 1.5mL mi&. The second product group consisted of three broader Tocopherol dimers were eluted with a 1:l (v/v) combination of peaks eluting between 35 and 46 min (Figure 1). These the above mobile phase with ethyl acetate at the same flow rate. products were tentatively identified as diastereomeric toRadiolabeled products were detected by collecting 0.4-min fraccopherones 3a by coelution with a mixture of four unlations, which were assayed for 14Cby liquid scintillation counting. Products from incubations with unlabled 1 were detected with beled tocopherone 3 s diastereomers synthesized by A M ”
Chem. Res. Toxicol., Vol.4, No.1, 1991 91
Oxidation of a-Tocopherol
Table I. Product Distributions from Oxidation of [14C]-1by AMVN-Derived Peroxyl Radicals in Lipid Bilayers and in Homogeneous Solution experimental system liposomesb CH3CN/H20C(6:4 v/v) CHSCN'
reaction time, min 2400 60 60
recovery of 1, % 29 40 20
yield, % 3a 11 32 51
3b 6
4 7
1 3
2 ndd
516
718
% epoxides4
23 6 22
20 13
64
nd
29
35
'Percent of total products present as 5 / 6 and 7/8. bThese data are from the experiment described in Figure 3. cThese data are taken from ref 10. dnd, not detected. 8 O00
loo a, 75
[y--
25 u
-
UlOO
430
1209
:75 .I I
1751
j
I 30
0 0
100 200 300 400 500 600
m/z Figure 2. Electron impact mass spectra of synthetic 8a-(alkyldioxy)tocopherone 3a (top) and compound 3a isolated from phosphatidylcholine liposomes incubated with 1 and AMVN (bottom). oxidation of 1 in homogeneous solution (IO). In this HPLC system, two of the diastereomers are incompletely resolved. To confirm the identity of these products, product peaks from incubations with unlabeled 1 were collected and analyzed as the diastereomeric mixture by UV-vis spectroscopy and mass spectrometry. As expected for 8a(alkyldioxy)tocopherones,the products exhibited a UVin ethanol a t 234 nm (10,11,15). The absorbance maximum rapidly shifted to 265 nm upon addition of 5% (v/v) 1 N HCl, indicating rapid hydrolysis to 4 (9). The product mixture exhibited an electron impact mass spectrum identical with that of authentic 3a (Figure 2). A minor radiolabeled peak that eluted at 22-23 min in Figure 1was identified as [14C]-3bon the basis of its coelution with an authentic standard of unlabeled 3b and its disappearance from the chromatogram upon treatment of the lipid extract with dilute HCl, which causes 3b to hydrolyze to 4. To better characterize the relationships between oxidation products, the distribution of products was determined by HPLC radiochromatography a t various times during [14C]-1 oxidation. The disappearance of [14C]-1 and the appearance of products are depicted in Figure 3. All products accumulated together as [14C]-1 were consumed; there was no apparent lag in the formation of the epoxide containing products 5-8. Relative product proportions remained approximately constant while unoxidized [14C]-1 remained in the system. After complete [14C]-1oxidation, levels of 3b and 6 rapidly declined. Table I compares the relative product yields during oxidation of [14C]-1 by AMVN-derived peroxyl radicals in lipid bilayers and in homogeneous solution (10). Although the same products are formed in both systems, the ratio of epoxide products (5-8) to non-epoxide products (3 and 4) varies between model systems. In solution, this ratio is about 0.4-0.6, whereas in the liposome system, epoxides predominate and the ratio is about 2.4. Stability and Fate of Tocopherones 3a and 3b. Since tocopherone 3a can hydrolyze and rearrange to more polar
0
2
4
6
8
1012
time, h r
Figure 3. Time course of depletion of [14C]-1 (top) and product appearance (bottom) in phosphatidylcholine liposomes containing 0.1 mol % [14C]-1 and 20 mol % A W N incubated at 37 "C.The svmbols used are I (e),3a (n),3b (m), 4 (v),5 / 6 (v),and 7 / 8
500 0
-I
20 40 time, min
60
Figure 4. HPLC analysis of products formed during incubation of [*%]-3a(0.1 mol %) with AMVN (20 mol %) and without (top) or with (bottom) 0.1 mol % unlabeled 1 for 6 h at 37 OC. products in homogeneous solution (lo), we examined its fate in the lipid bilayer. Liposomes containing 0.1 mol % [14C]-3aand 20 mol % AMVN were incubated a t 37 "C. After 6 h, radiolabeled products were extracted and analyzed by HPLC (Figure 4, top). Approximately 70% of the radiolabel eluted as unchanged 3a. The remaining radioactivity eluted primarily as compound 4 (31%) and as compounds 6-8 (approximately 1.5%). Compound 4 arises from simple hydrolysis of 3a, but formation of compounds 6-8 involves further oxidation and hydrolysis (10). To determine whether these secondary reactions were due to further oxidation of 3a by peroxyl radicals, the experiment was repeated with liposomes containing 0.1 mol % unlabeled 1, in addition to [14C]-3aand AMVN (Figure 4, bottom). Unlabeled 1 was added to trap peroxyl radicals
Liebler et al.
92 Chem. Res. Toxicol., Vol. 4, No. 1, 1991
.-
0 0
2
4
6
time, h r
Figure 5. Decomposition of [14C]-3a( 0 ) to [‘*C]-4(m) and [“C]-7/8 (A) during incubation with AMVN and unlabeled 1. Data are from integration of radiolabeled peaks corresponding to 3a, 4, and 7/8 in HPLC analyses similar to that depicted in Figure 4. that might otherwise react with [14C]-3a. HPLC analysis of the incubation mixture after 6 h indicates that 1 did not alter the distribution of [14C]-3adecomposition products. Since [14C]-3adecomposed to epoxyquinones 7 and 8 in addition to quinone 4, the origin of the epoxides was examined further. Epoxide formation from 3a could result either from peroxyl radical dependent epoxidation of 4, from peroxyl radical dependent oxidation of 3a, or from rearrangement and hydrolysis of 3a by a mechanism not involving peroxyl radicals. The latter two pathways would simultaneously form quinone 4 and epoxyquinones 7/ 8, whereas the former could produce a delay or lag in epoxyquinone formation as the precursor quinone 4 first accumulates and then is further oxidized. Moreover, suppressing peroxyl radical reactions either by including 1 or by omitting AMVN would decrease epoxide product yield in the former two pathways. The decomposition of 3a in the bilayer under the reaction conditions was therefore studied. Figure 5 depicts the time course of formation of [ 14C]-4and [14C]-7/8 in liposomes containing 0.1 mol 5% [14C]-3a, 0.1 mol 5% unlabeled 1, and 20 mol % AMVN. Both [14C]-4and [14C]-7/Saccumulated together in a ratio of approximately 25:l. A similar result was obtained when [14C]-3awas incubated in liposomes without 1 or AMVN (not shown). It therefore appears that the principal fate of 3a is hydrolysis to 4, although a small fraction decomposes to 718 by a separate pathway. Tocopherone 3b, which was present in small amounts among products of [14C]-1 (Figure l),also may yield 4 by hydrolysis and epoxytocopherone 6 by undergoing further oxidation. The fate of [14C]-3bwas therefore examined to determine whether it could yield such secondary products in liposomes. Decomposition of [14C]-3bin liposomes containing 0.1 mol ‘70 unlabeled 1 and 20 mol % AMVN was complete within 18h and yielded [14C]-4and [l4C]-7/8 in approximately a 3:l ratio (Figure 5). Moreover, the ratio of products was identical in the absence of 1 and/or AMVN (not shown). This observation suggests that 3b is not directly oxidized to epoxyquinones 718 by peroxyl radicals. The results collectively indicate that tocopherones 3a/3b decompose primarily by hydrolyzing to 4 but also decompose to epoxides 6-8 by reactions not initiated by peroxyl radicals.
Discussion Peroxyl radicals derived from AMVN react with 1 in lipid bilayers to yield products identical with those ob-
served previously in homogeneous solution (10). The products observed here include tocopherones 3a and 3b, their common hydrolysis product 4, epoxytocopherone 6, and epoxyquinones 7 and 8. One interesting aspect of these results is the apparent absence of tocopherol dimers or trimers as oxidation products. Product mixtures from liposome oxidation of [14C]-1were analyzed on an HPLC system capable of eluting and resolving tocopherol dimers, but no radiolabel eluted in these fractions. Two major oxidative pathways for 1 in liposomes can be proposed from the data presented here and from the results of studies in homogeneous solution (10). The first forms 8a-(alkyldioxy)tocopherones3a via initial hydrogen abstraction from 1 followed by peroxyl radical addition to 2. Each molecule of 1 consumed thus traps 2 peroxyl radicals. This mechanism accounts for the formation of 3a and its hydrolysis product 4 and is consistent with the behavior of 1 as a chain-breaking antioxidant (13). The second product-forming pathway yields epoxytocopherones 5 and 6 by peroxyl radical dependent epoxidation of 2, followed by oxygen addition to the 8a-position and hydrogen abstraction:
-
+ ROO’ epoxy-2 + RO’ epoxy-2 + O2 epoxy-2-00’ epoxy-2-00’ + R’H 5/6 + R” 2
-
-
(1) (2) (3)
This pathway, which results in no net radical trapping, can consume 1 by autoxidation if 1 acts as the H’ donor in reaction 3. Reactions 1-3 would consume 1 without producing an antioxidant effect, unless reaction 1proceeded much more slowly than competing lipid peroxyl radical propagation. Although not observed directly in these experiments, epoxytocopherone 5 is postulated as the precursor to epoxyquinone 7 (10). Epoxytocopherones 5 and 6 yield the corresponding epoxyquinones 7 and 8 by hydrolysis. The appearance of 3b among the products may also indicate that autoxidation of 2 occurs in this system. Matsuo et al. (12) proposed that, in a tert-butylperoxyl radical generating system, 3b arose via oxygen addition to 2 and hydrogen abstraction. Alternatively, 3b could be formed by singlet oxygen oxidation of 1 (17). Singlet oxygen in our system could arise via the Russell reaction between two peroxyl radicals (21). Decomposition of 3b in singlet oxygen oxidations also yields epoxyquinones 718. An alternative route to epoxytocopherones 516 and epoxyquinones 718 in this system is by decomposition of 8a-(alky1dioxy)tocopherone 3a and 8a-hydroperoxytocopherone 3b (10). However, our data argue that virtually all of the epoxide formation occurs directly from 2 as described above rather than from tocopherone decomposition. First, epoxides comprise only about 4% of the products formed by tocopherone 3a decomposition (Figure 5). Consequently, for tocopherone 3a decomposition to be the sole source of epoxides during 1 oxidation, 3a levels would need to be at least 25-fold higher than epoxide levels. However, in this system, epoxide levels were actually 2.5-fold higher than tocopherone 3a levels (Table I). Second, decomposition of tocopherones 3a/3b produces quinone 4 in considerable excess over epoxides 5-8 (see Figures 4-6). Epoxide products formed during [14C]-1 oxidation, on the other hand, are formed in excess over quinone 4. This pattern is opposite that expected were epoxide products formed by tocopherone decomposition. Although tocopherones are apparently not obligatory precursors to epoxide products formed during 1 oxidation, the mechanism of tocopherone decomposition is never-
Oxidation of a-Tocopherol 1500
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E 1000 500
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