UVB Induced Photooxidation of Vitamin E - ACS Publications

UVB Induced Photooxidation of Vitamin E. Kimberly A. Kramer and Daniel C. Liebler*. Department of Pharmacology and Toxicology, College of Pharmacy, Th...
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Chem. Res. Toxicol. 1997, 10, 219-224

219

UVB Induced Photooxidation of Vitamin E Kimberly A. Kramer and Daniel C. Liebler* Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona 85721-0207 Received September 16, 1996X

The photochemistry of R-tocopherol (R-TH, vitamin E) may contribute to its inhibition of UVB (290-320 nm) photocarcinogenesis. Photochemical reactions of R-TH were studied by monitoring the fate of R-TH in UVB irradiated liposomes and solution. Soy phosphatidylcholine (SPC) and dioleoylphosphatidylcholine (DOPC) liposomes were supplemented with R-TH (1.0 mol % R-TH/phospholipid) and irradiated with UVB at a dose rate of 6.0 J m-2 s-1 for up to 90 min. R-TH was rapidly depleted in UVB irradiated liposomes. Oxidative damage, assessed by monitoring lipid peroxidation, was suppressed in SPC liposomes until R-TH was depleted to 20% of initial levels. R-TH also was rapidly depleted by UVB irradiation in acetonitrile/ H2O (4:1 v/v) solution. In SPC liposomes, products previously identified as marker products for peroxyl radical scavenging by R-TH were observed, including R-tocopherol quinone, 5,6epoxy-R-tocopherol quinone, and 2,3-epoxy-R-tocopherol quinone. These products also were formed in DOPC liposomes, which are resistant to lipid peroxyl radical formation. In addition, an R-tocopherol dihydroxy dimer and several 8a-(hydroperoxy)epoxytocopherones were identified by HPLC and HPLC-MS. The dimer appears to result from recombination of photoinduced tocopheroxyl radicals. Products associated with peroxyl radical scavenging (quinones, epoxyquinones, 8a-(hydroperoxy)epoxytocopherones) and with UVB dependent production of tocopheroxyl radicals (dihydroxy dimer) also were found when R-TH was oxidized by UVB in acetonitrile. Because the acetonitrile contained no autoxidizable substrate, formation of peroxyl radical derived products may occur via intermediate tocopherone peroxyl radicals. These results indicate that R-TH photooxidation proceeds via competing reactions of UVB induced tocopheroxyl radicals.

Introduction

R-T• + ROO• f epoxytocopherones

R-TH1

(3)

Topically applied (1, Figure 1) recently has been shown to prevent UVB induced carcinogenesis and immunosuppression in mice (1). R-TH is thought to inhibit UVB induced photocarcinogensis in part by scavenging UVB induced oxidants and preventing oxidative damage (2-4). Its potential for use as a topical chemopreventative agent has led us to further investigate the photochemistry of vitamin E. R-TH prevents oxidative damage primarily by scavenging peroxyl radicals and preventing the propagation of lipid peroxidation through the formation of the resonance-stabilized tocopheroxyl radical (2, R-T• ) (5, 6):

The photochemistry of R-TH has not been well characterized; however. R-T• is formed upon UVB irradiation of R-TH, which has an absorbance maximum at 292 nm (9). Photoexcitation leads to photolysis and release of R-T• (eq 6), which could either undergo further reactions with other free radicals or undergo reductive recycling back to R-TH (5, 9).

R-TH + ROO• f R-T• + ROOH

R-TH 98 R-T• + H•

H2O

8a-substituted tocopherones 98 3 H2O

epoxytocopherones 98 4, 5



(1)

Reaction of R-T• with a second peroxyl radical leads to the formation of 8a-substituted tocopherones, epoxytocopherones, and their respective hydrolysis products quinone 3, and epoxyquinones 4/5 (eqs 2-5). These products can serve as markers of the antioxidant reactions of R-TH (7, 8).

R-T• + ROO• f 8a-substituted tocopherones (2)

(4) (5)

(6)

Here we describe the UVB induced photooxidation of R-TH in lipid bilayers and in homogeneous solution. Products previously associated with peroxyl radical scavenging are the principal products of these reactions. The peroxyl radicals scavenged may arise from autoxidizable lipids or from R-T• itself. The results indicate that competing reactions of R-T• dictate the fate of R-TH in UVB photooxidations.

Experimental Procedures * To whom correspondence should be addressed. Phone: (520) 6264504; FAX: (520) 626-2466; e-mail: [email protected]. X Abstract published in Advance ACS Abstracts, January 1, 1997. 1 Abbreviations: R-TH, R-tocopherol; R-TAc, R-tocopherol acetate; R-T•, R-tocopheroxyl radical; APCI, atmospheric pressure chemical ionization; BHT, butylated hydroxytoluene; BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; DOPC, dioleoylphosphatidylcholine; SDS, sodium dodecyl sulfate; SPC, soy phosphatidylcholine; TMCS, trimethylchlorosilane.

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Materials. R-TH ((R,R,R)-R-tocopherol) was a gift from Henkel Fine Chemicals (La Grange, IL). R-Tocopheryl acetate, R-TAc (6, Figure 1) (all-rac-R-tocopherol acetate), was purchased from Sigma (St. Louis, MO). Soy phosphatidylcholine, (SPC) and dioleylphosphatidylcholine (DOPC) in chloroform were purchased from Avanti Polar Lipids (Alabaster, AL). Westinghouse FS-20 UVB lamps were purchased from National Biologi-

© 1997 American Chemical Society

220 Chem. Res. Toxicol., Vol. 10, No. 2, 1997

Kramer and Liebler

Figure 1. Structures referred to in the text. cal Corp. (Twinsburg, OH), and a UVX digital radiometer with a UVX-31 sensor was purchased from Ultraviolet Products, Inc. (San Gabriel, CA). Approximately 80% of the lamp output was in the UVB (290-320 nm), whereas the remainder was in the UVA (320-400 nm). Other reagents were obtained commercially from standard sources and used without purification. Preparation of Liposomes. Liposomes containing R-TH or R-TAc were prepared by a modification of the ethanol injection method (10). Briefly, R-TH or R-TAc (5-500 nmol) and SPC or DOPC (5 µmol) were dissolved in 100 µL of ethanol and injected into 10 mL of 50 mM Tris-HCl (pH 7.4) over a period of 2 min with vortex mixing. Incubations with [14C]-R-TH (1.00 mCi mmol-1) also were done in liposomes at the concentrations indicated above. Depletion of r-TH. Liposomal suspensions containing R-TH (5 or 50 nmol of R-TH/5 µmol of SPC) were irradiated in a 5.0 cm glass Petri dish with UVB at a dose rate of 6.0 J m-2 s-1 for 90 min (1). The solution depth was approximately 6.0 mm. UVB dose was determined by measuring UVB flux with a UVX radiometer. Aliquots (1.0 mL) were taken in triplicate from the liposomal suspension at 20 min intervals, and 500 fmol of R-THd6 (an internal standard), 20 nmol of butylated hydroxytoluene (BHT), 10 µmol of sodium dodecyl sulfate (SDS), and 2.0 mL of ethanol were added to each aliquot. The samples then were extracted with hexane, and the hexane extract was evaporated under N2. Samples then were converted to O-TMS derivatives with 90 µL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 10 µL of trimethychlorosilane (TMCS) in 100 µL of dimethylformamide at 25 °C for 2 h, and R-TH content of samples was evaluated by GC-MS (11). Measurement of Lipid Peroxidation in UVB Irradiated Liposomes. Liposomes containing 5 µmol of SPC with or without 5 or 50 nmol of R-TH or R-TAc were prepared by ethanol injection. Liposome suspensions were irradiated for 90 min under a UVB lamp at a dose rate of 6.0 J m-2 s-1. Aliquots (0.5 mL) were taken in triplicate at 30 min intervals and extracted twice with 2 mL of chloroform/methanol (2:1 v/v). A 0.5 mL aliquot of the chloroform extract was then evaporated to dryness under N2. This sample was resuspended in hexane/2-propanol (3:2 v/v) and analyzed for conjugated diene content by measuring UV absorbance at 232 nm (12). Generation of r-TH Products in UVB Irradiated Incubations. Liposome suspensions containing 5 µmol of SPC and 0.5 µmol of R-TH in 10 mL of 50 mM Tris-HCl (pH 7.4) or solutions containing 200 nmol of R-TH in 4 mL of acetonitrile/

water (4:1 v/v) were irradiated at 6.0 J m-2 s-1 for 90 min. Liposomes were irradiated as above, in Petri dishes, whereas acetonitrile/water solutions were irradiated in a UV-transparent quartz Thumberg cell (NSG Precision Cells, Farmingdale, NY). Incubations with [14C]-R-TH were done in the same manner. R-TH and products were isolated from irradiated liposomes by hexane extraction as outlined above. R-TH and products were isolated from UVB irradiated solution by evaporation of acetonitrile/H2O under N2. HPLC Analysis of Oxidation Products of r-TH in UVB Irradiated Liposomes and Solution. R-TH and its oxidation products from UVB irradiated incubations were initially separated by reverse-phase HPLC on a Spherisorb ODS-2 5 µm, 4.6 × 250 mm column with a gradient elution. The initial mobile phase was MeOH/H2O/acetic acid (85:14:1 v/v) programmed to 100% MeOH over 50 min, followed by 20 min elution with MeOH/ethyl acetate (1:1 v/v) at a flow rate of 1.5 mL min-1. HPLC of R-TH and its products allowed comparison of UV spectra and retention times of sample peaks with those of authentic standards of known oxidation products of R-TH. An early eluting fraction of polar products was collected for a normal-phase HPLC separation on a Spherisorb CN, 5 µm, 4.6 × 250 mm column with a mobile phase of hexane/isopropyl alcohol (95:5 v/v), at a flow rate of 1.5 mL min-1. Radiolabeled products from incubations with [14C]-R-TH were analyzed by reverse-phase HPLC as described above. Fractions of the column effluent were collected at 0.4 min intervals and were assayed for radioactivity by liquid scintillation counting. A radiochromatogram was constructed for comparison of product elution times with those of authentic standards. GC-MS and LC-MS Analyses of Products. Products of UVB irradiated R-TH were analyzed by GC-MS with a Fisons MD 800 mass spectrometer coupled to a Carlo Erba 5000 series GC (Fisons Instruments, Beverly, MA) and were separated on a 30 m DB5ms column (J&W Scientific, Folsom, CA). Products were converted to trimethylsilyl derivatives prior to analysis by treatment with BSTFA/TMCS in dimethylformamide as described above. Full-scan electron impact mass spectra and GC retention time were used to confirm product identity by comparison with those of authentic standards. Products also were analyzed by APCI-HPLC-MS with a Finnigan TSQ 7000 (Finnigan MAT, San Jose, CA) instrument in the Southwest Environmental Health Sciences Center Analytical Core laboratory at The University of Arizona. Products were analyzed by reverse- and normal-phase HPLC as described above.

Photooxidation of Vitamin E

Figure 2. (A) Depletion of R-TH (0.1 mol %) by UVB. (B) Depletion of R-TH and R-TAc (1.0 mol %) by UVB. SPC (b) and DOPC (2) liposomes containing R-TH and SPC liposomes containing R-TAc (9) were irradiated with UVB for 90 min. Depletion was monitored by GC-MS.

Chem. Res. Toxicol., Vol. 10, No. 2, 1997 221

Figure 3. Suppression of lipid peroxidation by R-TH. (A) Liposomes containing 0.1 mol % R-TH or R-TAc. (B) Liposomes containing 1.0 mol % R-TH or R-TAc. Liposomes containing SPC alone (4), or with R-TH (b) or R-TAc (9), were irradiated with UVB for 90 min. Lipid peroxidation was measured by monitoring conjugated diene formation at 232 nm.

Results Oxidation of r-TH in UVB Irradiated Liposomes. UVB irradiation of liposomes containing 0.1 mol % R-TH resulted in rapid depletion of R-TH (Figure 2A). Approximately 90% of the R-TH was depleted over the first 30 min, and depletion was greater than 90% at 90 min. In liposomes containing 1.0 mol % R-TH, approximately 70% of the R-TH was depleted at 30 min and depletion was greater than 90% at 90 min (Figure 2B). In contrast, UVB had no effect on levels of R-TAc (Figure 2B). To assess the role of phospholipid peroxidation, R-TH depletion was also monitored in DOPC liposomes, which contain only singly unsaturated oleate chains and thus are virtually resistant to lipid peroxidation. The time course of R-TH depletion in DOPC liposomes was similar to that seen in SPC liposomes (Figures 2A,B), which indicates that peroxidation of liposomal lipid was not required for R-TH depletion. UVB irradiation of SPC liposomes caused measurable lipid peroxidation by 30 min (Figure 3A,B). Lipid peroxidation continued to increase over the remaining 60 min of irradiation, as measured by increases in the levels of conjugated dienes. Liposome supplementation with 0.1 mol % R-TH or R-TAc did not decrease lipid peroxidation (Figure 3A). However, in SPC liposomes supplemented with 1.0 mol % R-TH, lipid peroxidation was completely suppressed for 60 min of irradiation, followed by a modest increase in lipid peroxidation from 60 to 90 min (Figure 3B). Approximately 85% of the R-TH was depleted before lipid peroxidation commenced. Liposome supplementation with 1.0 mol % R-TAc decreased lipid peroxidation by approximately half. This may have been due to UVB absorbance by R-TAc, since R-TAc is antioxidant-inactive, and HPLC and GC-MS analyses indicated that it was not consumed (Figure 2B). Analysis of Photooxidation Products of [14C]-rTH in Liposomes. UVB irradiation of liposomes containing [14C]-R-TH yielded three groups of products, which were observed upon reverse-phase HPLC analysis

Figure 4. Radiochromatograms of R-TH products. HPLC separations of UVB irradiated incubations containing [14C]-RTH. Top panel: Product distribution in DOPC liposomes. Bottom panel: Product distribution in SPC liposomes. For product group identification, see the Results.

(Figure 4). Product group A, which eluted between 14 and 22 min, was a poorly resolved mixture of polar products and accounted for 34% of R-TH consumed. Product group B eluted between 24 and 35 min and contained three peaks. These products had HPLC retention times and UV spectra identical to those of quinone 3 and epoxyquinones 4/5. The identities of products 3, 4, and 5 from each system were confirmed by GC-MS of TMS derivatives. Products 4 and 5 accounted for 17% while product 3 accounted for 6% of the product associated radiolabel.

222 Chem. Res. Toxicol., Vol. 10, No. 2, 1997

Product group C contained one major peak, which eluted at 55-60 min. This product had an HPLC retention time and UV spectrum identical to those of dihydroxy dimer 7 (13, 14) and accounted for 43% of the product radioactivity. The identity of 7 was confirmed by UV-vis spectrophotometry, with spectral maxima identical to those of an authentic standard (UVmax at 290 and 340 nm), and by LC-MS, with retention time and APCI mass spectrum identical to those of an authentic standard. Dimer 7 was collected and subjected to flowinjection MS/MS analysis of the [M + H]+ ion at m/z 858. The resulting product ion spectrum contained major fragment ions at m/z 442 and 165, and the entire product ion spectrum was identical to that observed with an authentic standard. The polar products in group A were further resolved by normal-phase HPLC. These peaks displayed UV maxima at approximately 245-255 nm, which is characteristic of UV spectra of epoxytocopherones 8 and 9 (15, 16). Also, the HPLC retention of these products relative to epoxyquinones 4 and 5 was similar to that observed previously (17). These products appeared to be somewhat unstable under the conditions of the reverse-phase HPLC analysis. Much of the radiolabel present in product group A collected from reverse-phase HPLC subsequently eluted on normal-phase HPLC as epoxyquinones 4 and 5. The remainder of the radiolabel eluted as a series of 6-8 peaks. These peaks could represent stereoisomers of epoxytocopherones 8 and 9, as each has four possible diastereomers. Reverse-phase APCI-HPLC-MS was employed to further investigate the identity of the putative epoxytocopherones 8 and 9. The APCI spectra of several peaks eluting in the region of product group A displayed weak ions at m/z 479, which corresponds to [M + H]+ for epoxytocopherones 8 and 9. More intense fragment ions at m/z 463 and 445 corresponded to losses of oxygen and hydrogen peroxide, respectively. When products in this fraction were collected and subjected to silylation and GCMS analysis, only TMS derivatives of epoxyquinones 4 and 5 were observed. This is consistent with the facile hydrolysis of epoxytocopherones 8 and 9 to epoxyquinones 4 and 5 during workup (15-17). Products 3, 4, and 5 are typically associated with peroxyl radical scavenging reactions of R-TH (3, 4). In order to determine whether formation of these products was due to lipid peroxyl radical scavenging, the oxidation of [14C]-R-TH was investigated in DOPC liposomes. As discussed above, these liposomes are resistant to lipid peroxidation and [14C]-R-TH oxidation by lipid peroxyl radicals is unlikely in this system. The product profile was similar to that seen in SPC liposomes, with the exception that dimer 7, rather than product group A (putative epoxytocopherones 8 and 9), was the major product. Epoxyquinones 4 and 5 accounted for 22% of products, quinone 3 for 6%, dimer 7 for 53%, and epoxytocopherones 8 and 9 for 19% of products. Analysis of Photooxidation Products of [14C]-rTH in Homogeneous Solution. The R-TH product profile from SPC and DOPC liposomes suggested that R-TH photooxidation was largely independent of membrane lipid peroxidation, which could occur in the SPC liposomes, but not in the DOPC liposomes. In order to determine whether a complete absence of oxidizable lipid affected R-TH photooxidation, we examined the oxidation of [14C]-R-TH in acetonitrile solution. In this system, there is no source of lipid peroxyl radicals, and R-TH

Kramer and Liebler Table 1. Product Distributions from UVB Irradiated SPC and DOPC Liposomes and ACN/H2O Solutiona % of total products product

DOPC

SPC

ACN/H2O

3 4 and 5 7 8 and 9

6 22 53 19

6 17 43 34

3 19 12 66

a Product percentages were determined from radioactive counts in UVB irradiated incubations containing [14C]-R-TH.

oxidation thus should result from tocopherol photochemical reactions rather than lipid peroxidation. HPLC analysis of products formed by UVB photooxidation in acetonitrile/water indicated that products typically associated with peroxyl radical scavenging were formed. Quinone 3 accounted for 3% and epoxyquinones 4 and 5 for 19% of the products, respectively. In this system, however, polar fraction A, which contained the putative epoxytocopherones, was the major product fraction, and accounted for 66% of the products, whereas dimer 7 accounted for 12% of the products. Product distributions in liposomes and homogeneous solution are outlined in Table 1.

Discussion The effectiveness of R-TH as a chemopreventative agent in mice is thought to be due in part to its ability to act as an antioxidant (2-4). Radical scavenging by R-TH could prevent membrane lipid peroxidation and protein and DNA oxidation caused by UVB radiation. Although the antioxidant chemistry of R-TH is well understood (5, 7), the photochemistry of vitamin E exposed to UVB light has not been extensively investigated. We have shown that UVB irradiation causes a rapid consumption of R-TH and the formation of a number of oxidation products. Although it is clear that R-TH is acting as an antioxidant in our liposome system, much of the R-TH oxidation in liposomes and solution seems to be independent of lipid peroxyl radical scavenging. It was no surprise that R-TH (1.0 mol %) was able to prevent UVB induced lipid peroxidation in our liposome system. However, the ability of R-TAc to reduce lipid peroxidation was somewhat surprising. This effect of R-TAc presumably resides in its abilty to absorb UVB, since it cannot act directly as an antioxidant and did not undergo ester hydrolysis under any of the reaction conditions employed. Since R-TH and R-TAc have very similar UV absorption spectra (R-TH λmax 292, R-TAc λmax 285), R-TH may act partially by absorbing UVB in this system as well. In contrast to R-TAc, R-TH undergoes extensive photooxidation. In liposomes containing 0.1 mol % R-TH, lipid peroxidation was not prevented, whereas in liposomes containing 1.0 mol % R-TH, lipid peroxidation was prevented until 85% of the R-TH was depleted. This indicates that approximately 0.2 mol % was the minimum R-TH level needed to prevent UVB induced peroxidation in this system. R-TH concentrations in biological membranes range from 0.01 to 0.2 mol % (18, 19), and R-TH in this concentration range can prevent lipid peroxidation induced by diverse oxidants (5) depending on the nature and intensity of the oxidant stress and the presence of other antioxidants (20). The products identified in UVB irradiated liposomes and solution included quinone 3, epoxyquinones 4 and

Photooxidation of Vitamin E

5, dimer 7, and epoxytocopherones 8 and 9. Quinone 3 and epoxyquinones 4 and 5 have previously been established as marker products for the antioxidant reactions of R-TH (5, 7). R-TH can scavenge a peroxyl radical, to form R-T•, which reacts with another peroxyl radical to form intermediate tocopherones and epoxytocopherones. These are hydrolyzed to 3 and 4/5, respectively. The formation of these products in a peroxyl radical generating system results in the scavenging of two peroxyl radicals and the turnover of R-TH (21). Epoxytocopherones 8 and 9 have also been identified previously as products of peroxyl radical scavenging in aqueous/organic mixtures, but were found to be rapidly hydrolyzed to 4 and 5 (17). Dimer 7, however, seems to be uniquely formed in photochemical oxidations, as it does not occur as a major product in peroxyl radical generating systems (15-17, 21, 22). The end products associated with peroxyl radical scavenging reactions of R-TH (3, 4, 5, 8, and 9) accounted for 57% of products in SPC liposomes. This is somewhat lower than in azo compound initiated autoxidations in liposomes (23), in which these products accounted for essentially all the R-TH consumed. These same products collectively accounted for 88% of the product total in UVB irradiated solution and for 47% in UVB irradiated DOPC liposomes. In these systems, there is no peroxidizable substrate, and thus these products may be formed by the UVB driven autoxidation of R-TH. The remainder of the oxidized R-TH is accounted for by dimer 7, which results from UVB induced dimerization of R-T•. The combination of two R-T• to form the dihydroxy dimer may be favored by high concentrations of R-T• achieved by photoexcitation in the absence of high concentrations of other radicals (e.g., lipid peroxyl radicals). This may explain why the highest levels of dimer 7 are seen in the nonperoxidizable DOPC liposomes and in homogeneous solution. Another structurally distinct dimer, the spirodimer 10 (13), is not formed in UVB irradiated liposomes and solution. The spirodimer has been observed previously in R-TH oxidations in low polarity solvents and is formed via a quinone methide intermediate which results from hydrogen abstraction from R-T• (21). Dimer 7 may be a useful marker for R-TH photooxidation. The products formed in UVB irradiated liposomes and solution result from competing reactions of R-T•. As mentioned above, two R-T• can dimerize and form dimer 7. Alternatively, R-T• can undergo further oxidation to 8a-hydroxytocopherone 11 and epoxytocopherones 8 and 9, which hydrolyze to quinone 3 and epoxyquinones 4 and 5, respectively. Although the mechanism of autoxidation is not clear, an 8a-peroxyl radical derived by oxygen addition to R-T• may be involved (16). Previous work by Stocker and colleagues (24) indicates that R-T• may act as a prooxidant in low density lipoprotein in the absence of other antioxidants that reduce R-T• to R-TH. Although one might hypothesize that UVB could induce oxidations via R-T•, the overall effect of R-TH incorporation was to inhibit lipid peroxidation in our system. This probably reflects the presence of R-TH in excess over R-T• in the steady state, in contrast to the LDL particle, which may contain few R-TH molecules (24). Scavenging of free radicals may be a principal mechanism by which R-TH prevents UVB induced carcinogenic damage in the skin. However, photochemical reactions such as those described here also may contribute to R-TH turnover in UVB exposure. Indeed, we have recently

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shown that topically applied R-TH prevents UVB induction of mutagenic thymine dimers in mice.2 This action reflects a sunscreen effect due to R-TH UVB absorbance. This UVB absorbance may initiate R-TH degradation as described here. Our results indicate that distinguishing R-TH turnover due to peroxyl radical scavenging versus photochemically induced R-TH autoxidation may be difficult. However, the formation of dimer 7 may be a useful marker for the occurrence of photochemically induced R-TH oxidation. Further studies to examine the fate of topically applied R-TH in UVB irradiated skin will provide further insight into the mechanisms of prevention of photocarcinogenesis by R-TH.

Acknowledgment. This work supported by NIH Grant CA59585, NIEHS Center Grant ES06694, and The Flinn Foundation.

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224 Chem. Res. Toxicol., Vol. 10, No. 2, 1997 (19) Sevanian, A., Hacker, A. D., and Elsayed, N. (1982) Influence of vitamin E and nitrogen dioxide on lipid peroxidation in rat lung and liver microsomes. Lipids 17, 269-277. (20) Liebler, D. C., Kling, D. S., and Reed, D. J. (1986) Antioxidant protection of phospholipid bilayers by R-tocopherol. Control of R-tocopherol status and lipid peroxidation by ascorbic acid and glutathione. J. Biol. Chem. 261, 12114-12119. (21) Liebler, D. C., and Burr, J. A. (1995) Antioxidant stoichiometry and the oxidative fate of vitamin E in peroxyl radical scavenging reactions. Lipids 30, 789-793. (22) Liebler, D. C., Burr, J. A., Matsumoto, S., and Matsuo, M. (1993) Reactions of the vitamin E model compound 2,3,5,7,8-penta-

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