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Biochemistry 1989, 28, 9772-9171
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Redox Cycles of Vitamin E: Hydrolysis and Ascorbic Acid Dependent Reduction of 8a-(Alkyldioxy )tocopherones+ Daniel C . Liebler,* Kathryn L. Kaysen, and Todd A. Kennedy Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721 Received June 19, 1989
E; T H ) by peroxyl radicals yields 8a-(alkyldioxy)tocopherones,which either may hydrolyze to a-tocopheryl quinone (TQ) or may be reduced by ascorbic acid to regenerate TH. T o define the chemistry of this putative two-electron TH redox cycle,
ABSTRACT: Oxidation of the biological antioxidant a-tocopherol (vitamin
we studied the hydrolysis and reduction of 8a- [ (2,4-dimethyl- 1-nitrilopent-2-yl)dioxy]tocopherone (1) in acetonitrile/buffer mixtures and in phospholipid liposomes. T Q formation in acetonitrile/buffer mixtures, which was monitored spectrophotometrically, declined with increasing p H and could not be detected above pH 4. The rate of TQ formation from 1 first increased with time and then decreased in a first-order terminal phase. Rearrangement of 8a-hydroxy-a-tocopherone( 2 ) to T Q displayed first-order kinetics identical with the terminal phase for TQ formation from 1. Both rate constants increased with decreasing pH. Hydrolysis of 1 in acetonitrile/H2180 yielded [ 180]TQ. These observations suggest that 1 loses the 8a-(alkyldioxy) moiety to produce the tocopherone cation (T’), which hydrolyzes to 2, the TQ-forming intermediate. Incubation of either 1 or 2 with ascorbic acid in acetonitrile/buffer yielded TH. Reduction of both 1 and 2 decreased with increasing pH. In phosphatidylcholine liposomes at p H 7, approximately 10% of the T+ generated from 1 was reduced to TH by 5 m M ascorbic acid. T h e results collectively demonstrate that T+ is the ascorbic acid reducible intermediate in a two-electron TH redox cycle, a process that probably would require biocatalysis to proceed in biological membranes. %e lipophilic antioxidant vitamin TH’ (vitamin E) is the principal nonenzymatic defense against cellular membrane damage by reactive free radicals (Machlin & Bendich, 1987). This work was supported in part by USPHS Grant CA47943, by American Cancer Society Institutional Research Grant IN 1 10, and by a Basic Research Support Grant awarded to D.C.L. by the College of Pharmacy, University of Arizona.
T H interrupts radical chains by reacting with peroxyl radicals to form the relatively stable tocopheroxyl radical (Burton & I Abbreviations: AMVN, azobis(2,4-dimethylvaleronitrile); HPLC, high-performance liquid chromatography; T H , d-a-tocopherol; TQ, da-tocopheryl quinone; Tris-HC1, tris(hydroxymethy1)aminomethane hydrochloride. The term “ascorbic acid” refers to both the un-ionized and anionic forms of the molecule.
0006-2960/89/0428-9772$0 1.50/0 0 1989 American Chemical Society
(A1kyldioxy)tocopherone Hydrolysis and Reduction
A 1 FIGURE
2
1: Structures of 1 and 2 (R- = phytyl, -C,6H33).
Ingold, 1986). Tappel et al. (1961) proposed that the tocopheroxyl radical could be reduced by the water-soluble antioxidant ascorbic acid to regenerate TH. This reaction would make the reducing power of much larger water-soluble cellular antioxidant pools available for the reduction of peroxyl radicals in membranes. The chemical feasibility of a one-electron T H redox cycle was confirmed when Packer et al. (1979) directly observed tocopheroxyl radical reduction by ascorbic acid in homogeneous solution. T H and ascorbic acid also display a synergistic antioxidant effect in phosphatidylcholine liposome suspensions (Leung et al., 1981; Doba et al., 1985; Niki et al., 1985; Liebler et al., 1986). This finding suggests that electron transfer from the aqueous phase into the phospholipid bilayer can complete a one-electron T H redox cycle in biological membranes. Tocopheroxyl radicals that do not undergo reduction may react with a second peroxyl radical to produce nonradical products. Among these are 8a-(alkyldioxy)tocopherones, which are adducts formed between peroxyl radicals and the tocopheroxyl radical 8a-position. 8a-(Alkyldioxy)tocopherones account for approximately half of the T H consumed by peroxyl radicals in homogeneous solution (Winterle et al., 1984). T H oxidation in other systems yields analogous 8a-alkoxytocopherones (Durckheimer & Cohen, 1964; Goodhue & Risley, 1965), which decompose to TQ in aqueous acid but are easily reduced to T H by aqueous ascorbic acid. McCay (1985) recently suggested that their facile reduction to T H could permit tocopherones to complete a two-electron T H redox cycle in cellular membranes. To investigate this hypothesis further, we synthesized the model 8a-(alkyldioxy)tocopherone 8a[(2,4-dimethyl- 1-nitrilopent-2-yl)dioxy] tocopherone (1, Figure 1) and studied its hydrolysis and reduction in acetonitrile/ buffer mixtures and in phosphatidylcholine liposomes. We found that hydrolysis of 8a-(alkyldioxy)tocopherones is an acid-catalyzed reaction that yields the tocopherone cation (T+), which hydrolyzes to 8a-hydroxy-a-tocopherone(2, Figure l), the TQ-forming intermediate. Ascorbic acid reduces neither 1 nor 2 directly but reacts instead with T+. The slow rates of these reactions at neutral pH suggest that biochemical catalysis would be required to complete a two-electron T H redox cycle in biological membranes. EXPERIMENTAL PROCEDURES Chemicals and Instrumentation. Ascorbic acid and L-aphosphatidylcholine (from soybean, type 111-S) were purchased from Sigma. AMVN from Polysciences, Warrington, PA, was used as supplied. T H (99+% d-a-tocopherol) was from Henkel Corp., Fine Chemicals Division, LaGrange, IL. Acetonitrile (Omni-Solv) from EM Science was used without further purification. Mass spectral analyses were done with a Finnegan MAT-90 instrument equipped with a Micro PDP-I 1/73 computer ( U S . Design, Palo Alto, CA) and a Finnegan ICIS data system. Samples were introduced by
Biochemistry, Vol. 28, No. 25, 1989 9773 direct probe insertion. For chemical ionization analysis, ammonia was used as the reagent gas. HPLC analyses were done with a Spectra Physics 8800 solvent delivery system and either a Kratos SF757 variable-wavelength UV-vis detector or an ESA Coulochem electrochemical detector with standard ESA guard and analytical cells. Samples were injected with a Spectra Physics 8775 autosampler. Synthesis of 1 and 2. T H (10 mg, 0.023 mmol) and AMVN (43.3 mg, 0.175 mmol) were dissolved in 5 mL of oxygen-saturated acetonitrile, and the mixture was heated in a screw-capped test tube at 50 OC for 3 h. UV spectroscopy of the reaction mixture indicated that the T H had been completely consumed. 1 was separated from other components of the product mixture by reverse-phase HPLC on a Hamilton PRP-1 IO-pm, 4.6 X 250 mm analytical column, which was eluted with methanol/2-propanol (75:25 v/v) at a flow rate of 2 mL min-I. Products were detected by UV absorbance at 240 nm. Removal of the solvent in vacuo afforded 1 as a clear oil in approximately 50% yield. The UV spectrum (acetonitrile) indicated the presence of the tocopherone chromophore with an absorbance maximum at 240 nm (e = 13 700 M-' cm-I). An exact mass measurement for 1 was obtained by chemical ionization analysis with tetrakis(trimethylsily1)adenosine (MH+ = 556.263) as the mass reference standard. The measured mass for 1 (MH') was 572.469 f 0.004 (calculated MH' = 572.479). Analysis in the electron impact mode produced fragments consistent with the proposed structure: m/z 571 [M'], 1.4%; 461 [M' - -C(CN)(CH,)CH,CH(CH,),], 2.5%; 445 [M' - -OC(CN)(CH,)CH$H(CH,)J, 44.2%; 430 [M+ - -OOC(CN)(CH,)CH,CH(CH3)J, 100%; 417 [445 - CO], 68.9%; 401, 15.8%. Analysis of this material by reverse-phase HPLC on a Spherisorb ODs-2 5-pm, 4.6 X 250 mm analytical column, which was eluted with methanol/l N sodium acetate, pH 4.25 (93:7 v/v), at 1.5 mL min-', indicated that the product was a mixture of four diastereomers due to chiral centers at C-8a and C-2' of the AMVN-derived alkyldioxy group. The mixture was used for all the experiments described here. 2 was synthesized by the N-bromosuccinimide oxidation of T H as described previously (Durckheimer & Cohen, 1964). This procedure consistently yielded a mixture of 2 (7040%) and TQ (20-30%). Attempts to purify the mixture by reverse-phase HPLC as described above yielded only TQ, apparently because 2 rearranged to TQ on the column. Quantification of 2 in freshly prepared solutions by UV absorbance at 240 nm was subject to up to 25% error due to some absorbance from TQ (UV,,, = 266 nm). The significance of this limitation is discussed under Results. Incubation Conditions and Product Analyses. Formation of TQ was monitored spectrophotometrically with a Hewlett-Packard 8452A diode array UV-vis instrument operated with a Zenith 248 microcomputer and Hewlett-Packard 85930A MS-DOS UV-vis operating software. 1 or 2 was dissolved in 0.6 mL of acetonitrile added to 0.4 mL of 50 mM sodium formate buffer in a 1-mL quartz cuvette. The absorbance at 266 nm versus that at a reference wavelength of 350 nm then was monitored with time. All experiments were done at 25 "C. For studies of ascorbic acid dependent reduction, 20 nmol of 1 or 2 was diluted in 0.6 mL of acetonitrile, and then 0.4 mL of buffer containing ascorbic acid was added to begin the incubation. Samples (0.05 mL) of the mixture were quickly transferred to silanized tubes, which contained 0.5 mL of water, 0.5 mL of ethanol, 2 mL of hexane, and 0.5 nmol of y-tocopherol and y-tocopheryl quinone internal standards. After mixing, the organic layer was removed to
9774 Biochemistry, Vol, 28, No. 25, 1989 -3.5
---- --h
e
Liebler et al.
-35,
I
B I
\
2
c1
2 6
31
2 8
5 '3 2
4
6
firre, m i l :T e , vir Rates of TQ formation from 1 (solid symbols) and 2 (open symbols) versus time in acetonitrile/50 mM sodium formate, pH 3.6 (6:4 v/v). Data points used for linear regression analysis of the terminal phase are represented by solid triangles. (B) Residual plot of differences between extrapolated terminal-phase line and measured rates between 1 and 5 min in (A). See text for discussion.
FIGURE 2: (A)
another silanized tube, the solvent evaporated under a stream of nitrogen, and the residue dissolved in 1 mL of methanol for HPLC analysis. Despite the use of y-tocopherol as an internal standard, analyses after a single extraction underestimated the TH concentration by about 15%; an additional extraction with hexane was necessary to approach 100% recovery. In experiments where the time course of TH generation by ascorbic acid from 1 or 2 was measured, only single extractions were possible and the TH levels were underestimated by about 15% (vide infra). TH and TQ were separated by reverse-phase H P L C on Spherisorb ODS-2 as described above. TH was detected by oxidation a t a potential of +0.3 V and TQ by reduction a t -0.5 V. For studies of the incorporation of l 8 0 into TQ, 100 nmol of 1 was dissolved in 0.75 mL of acetonitrile. To this was added 0.5 mL of HzO or H2I8Oacidified to pH 2.9 with 3.6 pL of acetyl chloride. After 15 min, the TQ product was extracted with hexane and the extract was analyzed by chemical ionization mass spectrometry as described above. Liposome Incubations. A stock solution of soybean phosphatidylcholine (0.5 rmol) in chloroform was evaporated to dryness under a nitrogen stream, and then 0.1 mL of methanol and 0.01 mL of methanol/concentrated NH40H (9:l v/v) were added to dissolve the residue. The mixture was immediately evaporated again to dryness. This ammonia treatment was used to remove traces of HC1 from the phospholipid that arise from its storage in chloroform. Failure to remove the acid caused partial hydrolysis of 1 to TQ during liposome preparation. A solution of 1 (1 5 nmol in 0.01 7 mL of hexane) was added to the lipid residue, and the hexane was evaporated under nitrogen. The lipid mixture was then dissolved in 0.1 mL of ethanol, and unilamellar liposomes were prepared in 10 mL of 50 m M Tris-HC1 and 50 m M NaC1, pH 7.0, by the ethanol injection method (Kremer et al., 1977). Liposome suspensions were incubated at 37 "C under an air atmosphere. Aliquots of the liposome suspension (0.5 mL) were removed for analysis of TH as described above.
RESULTS Kinetics of TQ Formation from 1. Incubation of 1 in acetonitrile/buffer mixtures yielded TQ, which was monitored by its absorbance at 266 nm. The overall rate of TQ formation decreased with increasing pH. Above pH 4, no increase in absorbance due to TQ could be detected. The reaction at pH 3.6 was then studied in detail. The rate of TQ formation initially increased and then declined in an apparent first-order terminal phase with a rate constant k,, = 0.036 mi& (Figure 2A). We hypothesized that this kinetic profile reflected the
32
3a
26
38
..-,
8
Dependence on pH of the terminal-phase rate constant for TQ formation from 1 (k,em) (solid circles) and the first-order rate constant for rearrangement of 2 to TQ ( k , ) (open circles). FIGURE3:
formation of TQ via three sequential reactions, which are the first-order dissociation of 1 to T+ (eq I ) , the fast pseudofirst-order hydrolysis of T+ to 2 (eq 2), and the first-order rearrangement of 2 to TQ (eq 3). The apparent first-order
kl
1
T+
T+
+ H 2 0 2 2 + H+
(1)
(2)
k3
2-TQ (3) rate constant for hydrolysis of 1 to 2, kok, was estimated with the method of residuals, in which the terminal-phase decline in TQ formation from 1 was extrapolated to t = 0. A semilog plot of the difference between the measured and extrapolated rates at early time points between 1 and 5 min was linear and yielded an estimate of kobs= 0.296 min-I (Figure 2B). Although this rate constant describes the two-step conversion of 1 to 2, the linear residual plot indicates that one step is rate-limiting. Since eq 1 and 2 describe an SN1 hydrolysis reaction, in which k l
