Reactions of peroxynitrite with γ-tocopherol

Article abstract of DOI:10.1021/tx960200h

The reaction of peroxynitrite with γ-tocopherol (γ-TH) in a methanol/potassium phosphate buffer solution results in the formation of four major products, which were identified as 2,7,8-trimethyl-2-(4,8,12- trimethyldecyl)-5-nitro-6-chromanol (NGT), 2,7,8-trimethyl-2-(4,8,12- trimethyldecyl)-5,6-chromaquinone (tocored), and two diastereomers of 8a- (hydroxy)-γ-tocopherone. NGT was the major product formed in these reactions, and its formation was modestly increased by increasing amounts of Fe³⁺-EDTA. Tocored and NGT also were formed when γ-TH was exposed to 3- morpholinosydnonimine (SIN-1), a compound that decomposes to form peroxynitrite. When γ-TH reacted with the nitrating agent NO₂⁺BF₄^- in acetonitrile or methanol/potassium phosphate buffer, NGT and tocored also were formed, but the major product detected was γ-tocopherol quinone (γ- TQ). This product was not detected in reactions involving peroxynitrite. Oxidation of γ-TH by peroxynitrite involves nitration and electron transfer reactions. Since the product distribution in oxidations with NO₂⁺BF₄^- differed substantially from that in oxidations with peroxynitrite and SIN-1, NO₂⁺ appeared not to be the principal species involved in NGT formation. Nitration of γ-TH may involve either peroxynitrite or some peroxynitrite- derived oxidant other than NO₂⁺. Because of its stability and formation as a novel product of the reaction between γ-TH with peroxynitrite, NGT may be a useful in vivo marker for peroxynitrite interactions with lipid structures that contain γ-TH.

Full text of DOI:10.1021/tx960200h

Chem. Res. Toxicol. 1997, 10, 401-407
401
Rea ction s of P er oxyn itr ite w ith γ-Tocop h er ol
Niel C. Hoglen,*,† Stephen C. Waller,‡ I. Glenn Sipes,† and Daniel C. Liebler†,§
Department of Pharmacology and Toxicology, Center for Toxicology, College of Pharmacy,
The University of Arizona, Tucson, Arizona 85721-0207, and Department of Chemistry,
The University of Arizona, Tucson, Arizona 85721-0041
Received December 9, 1996X
The reaction of peroxynitrite with γ-tocopherol (γ-TH) in a methanol/potassium phosphate
buffer solution results in the formation of four major products, which were identified as 2,7,8-
trimethyl-2-(4,8,12-trimethyldecyl)-5-nitro-6-chromanol (NGT), 2,7,8-trimethyl-2-(4,8,12-tri-
methyldecyl)-5,6-chromaquinone (tocored), and two diastereomers of 8a-(hydroxy)-γ-tocopher-
one. NGT was the major product formed in these reactions, and its formation was modestly
increased by increasing amounts of Fe3+-EDTA. Tocored and NGT also were formed when
γ-TH was exposed to 3-morpholinosydnonimine (SIN-1), a compound that decomposes to form
-
peroxynitrite. When γ-TH reacted with the nitrating agent NO2+BF4 in acetonitrile or
methanol/potassium phosphate buffer, NGT and tocored also were formed, but the major
product detected was γ-tocopherol quinone (γ-TQ). This product was not detected in reactions
involving peroxynitrite. Oxidation of γ-TH by peroxynitrite involves nitration and electron
-
transfer reactions. Since the product distribution in oxidations with NO2+BF4 differed
+
substantially from that in oxidations with peroxynitrite and SIN-1, NO2 appeared not to be
the principal species involved in NGT formation. Nitration of γ-TH may involve either
peroxynitrite or some peroxynitrite-derived oxidant other than NO2+. Because of its stability
and formation as a novel product of the reaction between γ-TH with peroxynitrite, NGT may
be a useful in vivo marker for peroxynitrite interactions with lipid structures that contain
γ-TH.
by the reaction of nitric oxide and superoxide anion (6,
7). The rate constant for formation of peroxynitrite (6.7
× 109 M-1 s-1) exceeds that for the degradation of both
superoxide anion and nitric oxide (8). Cells capable of
generating both superoxide anion and nitric oxide (e.g.,
macrophages, neutrophils) can thus produce peroxyni-
trite, especially when activated (9, 10). Peroxynitrite has
been shown to react with a number of biomolecules
including DNA, proteins, and lipids (11-13) and has been
implicated in a number of disease processes including
atherosclerosis, inflammation, sepsis, and tumor promo-
tion (14-17). In aqueous solutions, peroxynitrite anion
(ONOO-) is rapidly protonated to peroxynitrous acid
(ONOOH, pKa ) 6.8), which has a half-life of about 1 s
at pH 7.4 (7). The decomposition chemistry of peroxyni-
trite/peroxynitrous acid is complex and has recently been
reviewed (18). Peroxynitrite has been postulated to
decompose via two distinct mechanisms. First, peroxy-
nitrite has been postulated to undergo homolytic cleavage
to yield two highly reactive free radicals, namely, NO2
and the hydroxyl radical (•OH) (19). However, Koppenol
et al. suggested that homolytic cleavage is energetically
unfavorable and proposed instead that peroxynitrite may
isomerize to an activated complex with reactivity similar
In tr od u ction
A family of lipid soluble tocopherols and tocotrienols
comprises vitamin E, which is the major chain-breaking
antioxidant within biological membranes. Four major
forms of the tocopherols are R, â, γ, and δ-tocopherol,
which differ only in number and location of methyl
substitutents on the aromatic chromanol ring (1). Of the
tocopherols, R- and γ-tocopherols (R-TH, and γ-TH,1 1,
respectively) are the principal forms found in human and
animal diets and comprise most of the vitamin E content
of tissues (2). R-TH is the most effective antioxidant and
exhibits more bioactivity and reactivity toward reactive
oxygen species than any of the other tocopherols (3, 4).
However, Cooney et al. recently found γ-TH to be a more
effective cellular protectant against nitrogen dioxide
(NO2) and demonstrated that γ-TH suppressed NO2-
induced neoplastic transformation more effectively than
R-TH in vitro (5). Their data suggest that γ-TH 1 may
be more important in protecting organisms from reactive
nitrogen species than R-TH.
One such reactive nitrogen species is peroxynitrite, a
potent oxidizing agent formed both in vivo and in vitro
•
* Corresponding author: telephone, 520-626-6381; fax, 520-626-
2466; e-mail, hoglen@tonic.pharm.arizona.edu.
to OH (20). Increasing evidence for an activated form
of peroxynitrous acid with the capability of undergoing
one- or two-electron oxidations has been shown (18, 21,
22). An alternative reaction pathway is heterolytic
cleavage of peroxynitrite to nitronium (NO2+) and hy-
droxide (OH-) ions, which is catalyzed by coordination
with metal ions (23). This reaction was implicated in the
nitration of tyrosine residues near the active site of Cu/
Zn superoxide dismutase by nitric oxide in the presence
of superoxide (24). This heterolytic pathway contributes
to the metal-catalyzed nitration of low molecular weight
phenols by peroxynitrite (24). 3-Nitrotyrosine, the pre-
† Department of Pharmacology and Toxicology.
‡ Department of Chemistry.
§ To whom reprint requests should be addressed: telephone, 520-
626-4488; fax, 520-626-2466; e-mail, liebler@tonic.pharm.arizona.edu.
X Abstract published in Advance ACS Abstracts, March 15, 1997.
Abbreviations: DTPA, diethylenetriaminepentaacetic acid; Fe3+
-
1
EDTA, ferric-sodium ethylenediaminetetraacetic acid; SIN-1, 3-mor-
pholinosydnonimine; NO2, nitrogen dioxide; NO2+, nitronium cation;
ONOO-, peroxynitrite anion; ONOOH, peroxynitrous acid; R-TH,
R-tocopherol; γ-TH, γ -tocopherol; γ-TQ, γ-tocopherolquinone; γ-T+,
γ-tocopherone cation; 8a-γ-TOH, 8a-(hydroxy)-γ-tocopherone; γ-T•,
γ-tocopheroxyl radical; tocored, 2,7,8-trimethyl-2-(4,8,12-trimethylde-
cyl)-5,6-chromaquinone; NGT, 2,7,8-trimethyl-2-(4,8,12-trimethylde-
cyl)-5-nitro-6-chromanol; 4-HPA, 4-hydroxyphenylacetic acid.
S0893-228x(96)00200-7 CCC: $14.00 © 1997 American Chemical Society

402 Chem. Res. Toxicol., Vol. 10, No. 4, 1997
Hoglen et al.
Ta ble 1. Qu a n tifica tion of P r od u ct F or m a tion fr om
Rea ction of P er oxyn itr ite w ith γ-Tocop h er ola
ratio of
nmol of product formed
peroxynitrite/
γ-TH
additions
NGT
tocored
1:4
1:2
1:1
none
10 µM Fe3+-EDTA
86.0 ( 1.2
89.3 ( 1.1b
3.9 ( 0.2
4.0 ( 0.4
4.0 ( 0.2
3.8 ( 0.3
7.6 ( 0.7
8.7 ( 0.4
8.5 ( 0.6
8.9 ( 0.5b
100 µM Fe3+-EDTA 100.9 ( 1.4b
100 µM DTPA
none
91.1 ( 5.3
171.0 ( 4.0
10 µM Fe3+-EDTA 196.2 ( 2.3b
100 µM Fe3+-EDTA 224.8 ( 6.9b
100 µM DTPA
215.7 ( 5.4b
229.6 ( 10.2 15.4 ( 0.4
none
10 µM Fe3+-EDTA 265.8 ( 12.6b 17.1 ( 0.9
100 µM Fe3+-EDTA 345.5 ( 8.1b 18.5 ( 0.9b
100 µM DTPA
287.0 ( 6.3b 16.7 ( 0.9
a
Peroxynitrite reacted with γ-TH in a methanol/50mM potas-
sium phosphate buffer (3/1, pH 7.6) in the absence or presence of
either Fe3+-EDTA (10-100 µM) or DTPA (100 µM). After 10 min,
reactions were spiked with internal standard, extracted with
hexane, and quantified by HPLC as described in Experimental
Procedures using standard curves generated from authentic NGT
F igu r e 1. Structures of compounds referred to in the text.
dominant product formed from peroxynitrite/protein
interactions, has therefore been used as a marker of in
vivo protein alteration by peroxynitrite (14).
b
and tocored. Indicates significant difference from reactions with
no additions (p < 0.05, ANOVA).
The antioxidant activity of R-TH toward peroxynitrite
has been previously investigated (25, 26). Hogg et al.
demonstrated that successive one-electron oxidations of
R-TH by peroxynitrite in methanol/water led to the
formation of 8a-methoxytocopherone and R-tocopher-
olquinone (26). Because γ-TH possesses an unsubsti-
tuted carbon at C-5 of the chromanol ring ortho to the
phenol (Figure 1), we postulated that nitro substitution
may occur at C-5 upon exposure to peroxynitrite. How-
ever, the ability of peroxynitrite to act as a one-electron
oxidant and as a nitrating agent suggests that other
reactions may occur. This study therefore was under-
taken to investigate the reactions of peroxynitrite with
γ-TH 1, to identify the major products formed, and to
examine the possible mechanisms of formation of the
major products.
derivatives using N,O-bis(trimethylsilyl)trifluoroacetamide
(Pierce, Rockford, IL). Samples were analyzed in the positive
electron impact mode at 70 eV. LC-MS analyses were done
with a Finnigan TSQ 7000 instrument (Finnigan-MAT, San
J ose, CA) equipped with an atmospheric pressure ionization
source. 1H- and 13C-NMR spectroscopy were performed on
either a Varian Gemini 200-MHz NMR or a Varian Unity 300-
1
MHz NMR operating at 200-300 MHz for H-NMR and 75 MHz
for 13C-NMR as described in the Results.
Oxid a tion of γ-TH. Most reactions were carried out in 600
µL of a methanol/50 mM potassium phosphate buffer (3/1 v/v,
pH 7.6) containing 2 mM γ-TH (1.2 µmol). Peroxynitrite in 0.1
N NaOH as a 100× concentrated stock was quickly added to
the buffer containing γ-TH with mixing. pH changes after the
addition of peroxynitrite were minimal in the buffer system
used. After 10 min, samples were spiked with internal standard
(R-tocopheryl acetate, i.s.), extracted three times with hexane
(2 mL), evaporated, and dissolved in methanol prior to HPLC
analysis. Milligram quantities of NGT were prepared by the
reaction of γ-TH with peroxynitrite in acetonitrile/50 mM
potassium phosphate buffer (3/1 v/v, pH 7.6) for 1 h, followed
by extraction with hexane and purification by HPLC. Negative
controls consisted of the addition of degraded peroxynitrite in
0.1 N NaOH. In some reactions, diethylenetriaminepentaacetic
acid (DTPA) or ferric-sodium ethylenediamine tetraacetic acid
(Fe3+-EDTA) were added at concentrations indicated in Table
1.
Exp er im en ta l P r oced u r es
Ch em ica ls. HPLC grade acetonitrile, hexane, and methanol
were obtained from Fisher Scientific (Fairlawn, NJ ). All other
chemicals were purchased from Sigma Chemical Co. (St. Louis,
MO) unless otherwise indicated in the text. Peroxynitrite was
prepared in a quenched-flow reactor with sodium nitrite and
hydrogen peroxide and concentrated by freeze fractionation as
previously described (27). Unreacted hydrogen peroxide was
eliminated by passing the peroxynitrite solution through a
manganese dioxide column prewashed with H2O and 1 N NaOH.
Concentrations of peroxynitrite were determined spectrophoto-
metrically at 302 nm in 1 N NaOH, and peroxynitrite was stored
as stock solutions in 0.1 N NaOH. Authentic standards of γ-TQ
2 and tocored 4 were generated by the reaction of γ-TH with
FeCl3 in ethanol (28).
Reactions with γ-TH and 3-morpholinosydnonimine (SIN-1)
(Alexis Corporation, San Diego, CA) were carried out in methanol/
50 mM potassium phosphate buffer (3/1, v/v, pH 7.6). SIN-1
was freshly prepared in H2O as a 100× concentrated stock and
added to 2 mM solutions of γ-TH. The reaction was carried out
for 4 h in the dark and then extracted and analyzed as above.
Reactions with γ-TH and NO2+BF4- (Aldrich, Milwaukee, WI)
were carried out in a total volume of 1 mL of either acetonitrile
or a methanol/50 mM potassium phosphate buffer (3/1 v/v, pH
7.6) containing 2 mM γ-TH. Reactions in acetonitrile were
An a lytica l Meth od s. Products of reactions were analyzed
by HPLC with a Spectra Physics 8800 solvent delivery system
(Spectra Physics, San J ose, CA), a Spherisorb ODS2 5-µm
reverse-phase column (Alltech Associates, Deerfield, IL), and a
Spectra Physics 100 variable wavelength detector. Compounds
were eluted with a mobile phase consisting of methanol/water
(98/2, v/v) at a flow rate of 1.5 mL min-1 and detected at a
wavelength of 286 nm. UV-vis spectra of reaction products
were obtained using a Hewlett Packard 1040A diode array
detector (DAD) operated by an HP79994A analytical work
station.
-
performed by the addition of freshly prepared NO2+BF4 as a
100-fold concentrated stock solution with vortex mixing and
allowed to react for 1 h at 25 °C in the dark. Methanol was
added to quench the reaction, and the sample was extracted
with hexane and analyzed as described above. For reactions
in methanol/50 mM phosphate buffer (pH 7.6), a 40-fold excess
-
NO2+BF4 was added as a 100× stock, followed by immediate
extraction with hexane to eliminate possible product degrada-
tion by nitric acid.
GC-MS analyses were performed with a Fisons MD800
instrument (Fisons Instruments, Beverly, MA). Samples for
GC-MS analysis were derivatized to trimethylsilyl (TMS)
The reduction of 8a-γ-TOH 5 with ascorbic acid was per-
formed in a sodium formate/ethanol buffer as previously de-

Peroxynitrite Oxidation of γ-Tocopherol
Chem. Res. Toxicol., Vol. 10, No. 4, 1997 403
yielded spectra identical to that for γ-TQ, indicating
rearrangement prior to or during analysis. The UV-vis
spectra are similar to those previously reported for 8a-
(hydroxy)-R-tocopherone (33, 34), which readily arranges
to R-tocopherylquinone. Because the 8a-substituted to-
copherones readily undergo reduction by ascorbic acid to
the corresponding tocopherols (28, 29, 33, 34), we treated
both products with ascorbic acid in sodium formate buffer
(29). HPLC analysis revealed the disappearance of both
products, with a concomitant increase in the formation
of γ-TH. After 1-h incubation, both compounds were
reduced to γ-TH by about 25%, and after 4 h, the
reduction to γ-TH was nearly complete (data not shown).
Because nitration of phenols by peroxynitrite is report-
edly enhanced by ferric chelates (24), we carried out some
reactions in the presence of Fe3+-EDTA. Increasing
concentrations of Fe3+-EDTA increased the amount of
NGT formed when peroxynitrite was added to γ-TH at
three different ratios (Table 1). The addition of 100 µM
Fe3+-EDTA to the reactions increased the formation of
NGT by 15, 24, and 34% at molar ratios of 1:4, 1:2, and
1:1 peroxynitrite/γ-TH, respectively. The addition of 10
µM Fe3+-EDTA also increased the formation of NGT by
13% and 14% at molar ratios of 1:2 and 1:1, respectively.
Furthermore, the presence of 100 µM Fe3+-EDTA mod-
estly but significantly increased the formation of tocored
by 17% when equimolar concentrations of peroxynitrite
and γ-TH were used (Table 1). The effect of Fe3+-EDTA
on formation of the diastereomers of 8a-γ-TOH was
minimal. DTPA was added to some reactions to evaluate
the possible impact of trace metal contamination. DTPA
did not reduce product formation in any of the reactions
studied, which indicates a lack of significant trace metal
contamination. However, DTPA at a concentration of 100
µM significantly increased the amount of NGT formation
in both the 1:2 and 1:1 peroxynitrite/γ-TH reactions as
compared to peroxynitrite with γ-TH alone (Table 1). No
product formation was detected when decomposed per-
oxynitrite was added to γ-TH under similar conditions.
Product yields as a percent of γ-TH oxidized ranged from
approximately 60 to 80%, whereas those for tocored were
approximately 3-5%.
F igu r e 2. HPLC analysis of products formed from peroxynitrite
and γ-TH (1:1 molar ratio) in a methanol/50 mM potassium
phosphate buffer (3/1 v/v, pH 7.6).
scribed (29). Reactions were carried out for up to 4 h in the
dark at 25 °C, extracted as described above, and analyzed by
DAD-HPLC.
Resu lts
Addition of peroxynitrite to γ-TH in methanol/50 mM
potassium phosphate buffer generated a stable, light
yellow-orange color within seconds. Reverse-phase HPLC
analysis separated four major products (Figure 2). The
major product eluting at 15.5 min had an intense yellow
color and was identified as 2,7,8-trimethyl-2-(4,8,12-
trimethyldecyl)-5-nitro-6-chromanol (NGT), 3: UV-vis
λmax (EtOH) at 264 nm (ꢀ ) 2483), 312 nm (ꢀ ) 2464),
and 415 nm (ꢀ ) 904); GC-MS (TMS derivative), m/ z
533 (M•+, 100%), 518 (35), 292 (19), 276 (22), 238 (14),
1
222 (13), and 202 (14); H NMR (300 MHz, CDCl3): δ
(ppm) 0.81-0.85 (12H, m), 1.06-1.54 (21H, m), 1.24 (3H,
s), 2.17 (3H, s), 2.21 (3H, s), 2.99 (2H, t, J ) 6.3 Hz),
10.7 (1H, s). 13C-NMR (75 MHz, CDCl3): δ (ppm) 12.1
(C7′), 13.3 (C8′), 19.7 (C4), 20.9, 21.8, 22.6, 22.7, 23.6,
24.5, 24.8, 28.0, 29.7, 30.9, 32.7, 32.8 (C2′), 37.3 (C3), 37.4,
39.4, 39.6, 75.5 (C2), 113.4 (C4a), 125.3 (C7), 131.9 (C5),
145.2 (C8a), 148.3 (C6). These data are consistent with
assignment as NGT previously characterized by Cooney
et al. from the reaction of γ-TH with NO2 (30). The 13C-
NMR spectrum had not been previously reported.
The reaction of SIN-1 with γ-TH also was investigated.
SIN-1 decomposes with the release of both nitric oxide
and superoxide, which react to form peroxynitrite (35).
This study was done to compare products formed from a
brief exposure of peroxynitrite and a sustained release
of peroxynitrite over time. Reactions with SIN-1 and
γ-TH slowly produced a yellow-orange color over 4 h.
DAD-HPLC analysis confirmed the formation of two
products with identical retention times and UV-vis
spectra for NGT and tocored (Figure 3). The major
product formed in reactions with all molar ratios of SIN-
1/γ-TH was NGT (Table 2), with smaller amounts of
tocored. The ratio of NGT formation to tocored was
greater when γ-TH was exposed briefly to peroxynitrite
than to SIN-1. In the reaction with SIN-1 and γ-TH at
a 5:1 ratio, the yield of NGT was 53% of γ-TH oxidized,
whereas yields of tocored and γ-TQ were both 5%.
The product eluting at 6.8 min was a deep red colored
oil and was identified as 2,7,8-trimethyl-2-(4,8,12-tri-
methyldecyl)-5,6-chromaquinone (tocored), 4: UV-vis
(MeOH/H2O) 282, 465 nm; LC-MS (APCI): m/ z 431 [M
1
+ H]+; MS-MS (dau m/ z 431): 413, 291, 207, 167; H
NMR (200 MHz, CDCl3): δ (ppm) 0.82 (3H, d, J ) 6.2
Hz), 0.84 (9H, d, J ) 6.6 Hz), 0.97-1.55 (2H, m), 1.30
(3H, s), 1.67-1.75 (2H, m), 1.93 (3H, s), 2.00 (3H, s), 2.39
(2H, q, J ) 5.9 Hz). These data are consistent with
previously published data for compound 4 (30-32).
Products eluting at 4.8 and 5.5 min were identified as
diasteromers of 8a-γ-TOH, 5. These compounds were
only moderately stable, the major breakdown product
being γ-TQ. Although the peaks could not be assigned
to specific epimers, virtually identical spectra were
obtained for both compounds: UV λmax (MeOH/H2O) 236,
261 nm. LC-MS (APCI): m/ z 431 [M + H]+; MS-MS
(dau m/ z 431) 403, 207, 167. 1H-HMR analysis of these
+
NO2 has been proposed to be a nitrating species
responsible for phenol nitrations by peroxynitrite (23).
The reaction of γ-TH with NO2+BF4- was thus examined
to determine what products are formed by NO2+. γ-TH
-
reacted with NO2+BF4 in acetonitrile to form a yellow-
red color over 1 h. The reaction yielded three major
products: NGT, tocored, and a yellow product eluting at
5.2 min (Figure 4). This latter product was identified as

404 Chem. Res. Toxicol., Vol. 10, No. 4, 1997
Hoglen et al.
Ta ble 3. Qu a n tifica tion of P r od u ct F or m a tion fr om
Rea ction of NO2+BF 4 w ith γ-THa
-
nmol of product formed
ratio of
NO2+BF4-/γ-TH
NGT
tocored
γ-TH quinone
ACN
1:4
1:1
26.7 ( 2.2
125.3 ( 21.2
5.4 ( 1.1
38.5 ( 6.4
45.8 ( 3.1
243.9 ( 22.5
-
MeOH/KPO4
40:1
16.0 ( 1.9
4.7 ( 0.4
46.5 ( 9.1
a
-
NO2+BF4 was added to a 2 mM solution of γ-TH in either
-
100% ACN or MeOH/KPO4 buffer to a final concentration
indicated. Reactions in ACN were carried out for 1 h, while
reactions in MeOH/KPO4- buffer were extracted immediately after
the addition of reagents. All samples were quenched with MeOH,
followed by the addition of an internal standard (R-TH acetate).
Samples were extracted with hexane, dried, and analyzed by
HPLC as described in Experimental Procedures. Amounts of each
product were calculated based on standard curves for each γ-TH
derivative. Data are expressed as mean ( SD of three separate
reactions.
F igu r e 3. HPLC analysis of products formed from SIN-1 and
γ-TH (1:1 molar ratio) in a methanol/50 mM potassium phos-
phate buffer (3/1 v/v, pH 7.6).
(75 MHz, CDCl3): δ (ppm) 12.0, 12.4, 19.7, 19.8, 21.4,
22.6, 22.7, 24.0, 24.5, 24.8, 26.7, 28.0, 32.8, 37.3, 37.4,
37.6, 39.4, 39.9, 42.4, 72.5, 132.1, 140.6, 141.1, 149.4,
187.6.
In these reactions, the formation of γ-TQ was almost
2-fold greater than NGT (Table 3). In the reaction with
NO2+BF4- and γ-TH in a 1:1 ratio, the yield of NGT was
28% of γ-TH oxidized, whereas those for tocored and γ-TQ
were 9% and 55%, respectively. The reaction was also
performed in methanol/50 mM phosphate buffer (3/1 v/v,
pH 7.6) to determine if the product distribution was
altered in the presence of water. In methanol/50 mM
-
phosphate buffer, a 40-fold molar excess NO2+BF4 was
+
added because of rapid competing hydrolysis of NO2 to
nitric acid. The reaction mixture was immediately
extracted to prevent product decomposition by nitric acid.
As in the acetonitrile system, γ-TQ was the major product
formed, in addition to NGT and tocored (Table 3). The
formation of γ-TQ was over 4-fold greater than NGT
under these reaction conditions.
-
F igu r e 4. HPLC analysis of products formed from NO2+BF4
and γ-TH (1:1 molar ratio) in acetonitrile.
Discu ssion
This study was conducted to determine the major
products formed from the reaction of peroxynitrite with
γ-TH. Because γ-TH contains an unsubstituted carbon
at C-5 (ortho to the phenol), we hypothesized that
nitration at this position by peroxynitrite would occur.
Indeed, we found that NGT was the major product
formed from the reaction of peroxynitrite with γ-TH. This
product was characterized by Cooney et al. in studies on
the reaction of γ-TH with NO2 in hexane (30). In addition
to NGT, tocored and 8a-γ-TOH were identified as prod-
ucts of peroxynitrite oxidation of γ-TH.
Ta ble 2. Qu a n tifica tion of P r od u ct F or m a tion fr om
Rea ction of SIN-1 w ith γ-THa
nmol of product formed
ratio of
SIN-1/γ-TH
NGT
tocored
γ-TQ
ND
trace
45.7 ( 5.8
1:2
1:1
5:1
39.0 ( 6.2
74.2 ( 16.7
484.9 ( 5.6
4.0 ( 0.3
4.4 ( 1.1
41.6 ( 2.9
a
SIN-1 was added to a 1 mM solution of γ-TH in a methanol/
50mM potassium phosphate buffer (3/1 v/v, pH 7.6) to a final
concentration of 1.0-10.0 mM. Samples were mixed and allowed
to react for 4 h. After 4 h, an internal standard (R-TH acetate)
was added, and the reactions were extracted with hexane and
analyzed by HPLC as described in Experimental Procedures.
Amounts of each product were calculated based on standard curves
for each product. Data are expressed as the mean ( SD of 3-5
separate reactions. ND, not detected.
The formation of all the observed products in peroxy-
nitrite oxidations reflects the complex chemistry of per-
oxynitrite as an oxidant. Peroxynitrite and its conjugate
acid, peroxynitrous acid, can act as one-electron oxidants,
whereas an activated form of peroxynitrous acid
([ONOOH]*) can act as a one-electron oxidant or as a
“hydroxyl radical-like” hydroxylating reagent (20, 18).
γ-TQ: UV-vis (MeOH) 255 nm (ꢀ ) 18 035), 330 nm (ꢀ
) 639); GC-MS (TMS derivative) m/ z 504 (M•+, not
detected) 416 (4%), 341 (29), 279 (100), 189 (20), and 151
Alternatively, some metal complexes, such as Fe3+
EDTA can catalyze the heterolysis of peroxynitrous acid
-
1
+
+
(19); H-NMR (200 MHz, CDCl3): δ (ppm), 0.82 (3H, d,
to water and NO2
. Both NO2 and its one-electron
J ) 6.2 Hz), 0.84 (9H, d, J ) 6.6 Hz), 1.20 (3H, s), 1.00-
1.50 (21H, m), 1.54-1.63 (2H, m), 1.98 (3H, s), 2.00 (3H,
s), 2.43-2.51 (2H, m), 6.51 (1H, t, J ) 1.2 Hz). 13C-NMR
reduction product NO2 may act as nitrating agents or
one-electron oxidants. Therefore, in addition to oxida-
tions with peroxynitrite and SIN-1, we also conducted

Peroxynitrite Oxidation of γ-Tocopherol
Chem. Res. Toxicol., Vol. 10, No. 4, 1997 405
-
+
reactions with NO2+BF4 to assess the role of NO2 in
Sch em e 1. Rea ction Mech a n ism of 8a -γ-TOH,
Tocor ed , a n d γ-TQ F or m a tion fr om γ-TH by
P er oxyn itr ite
peroxynitrite-mediated γ-TH oxidation.
γ-TH underwent two types of reactions in oxidations
with peroxynitrite. In the first, nitration at C-5 formed
NGT. In the second, γ-TH was oxidized to 8a-γ-TOH and
tocored, most likely via electron transfer and hydrolysis
reactions (Scheme 1). Electron transfer may be mediated
by any of several peroxynitrite-associated oxidants, in-
cluding peroxynitrite/peroxynitrous acid, [HOONO]*,
NO2+, or NO2. Sequential removal of two electrons from
γ-TH may be followed by hydrolysis at either C5 or C8a
of the resulting γ-T+. Hydrolysis at C-8a yields 8a-γ-
TOH, which can subsequently rearrange to γ-TQ. On the
other hand, hydrolysis at C5 yields a hydroxydienone (6),
which rearranges to the catechol (7). Further two-
electron oxidation forms the more stable o-quinone,
tocored. We propose one-electron oxidation via the
tocopheroxyl radical for three reasons. First, tocopherols
react with a variety of oxidants by initial electron or
hydrogen atom transfer (36, 37). Second, peroxynitrite
oxidizes various substrates by either one- or two-electron
routes (reviewed in ref 18). Finally, Hogg et al. (26)
recently reported that peroxynitrite oxidized R-TH to 8a-
substituted tocopherone products and that the toco-
pheroxyl radical was detected under the reaction condi-
tions. Although these authors concluded that one-
electron oxidation was a minor reaction pathway based
on low levels of the tocopheroxyl radical detected, it
should be noted that tocopheroxyl radicals would not be
expected to accumulate under the reaction conditions
employed in that work or those in our study, as further
oxidation by peroxynitrite or peroxynitrite-derived oxi-
dants (e.g., NO2 or NO2+) should readily occur. Thus,
although it is possible that a concerted two-electron
oxidation formed γ-T+ in our studies, sequential one-
electron oxidations appear to be a more likely reaction
sequence.
cannot evaluate the partitioning between oxidation at C-5
and C-8a. However, comparison of the integrated peak
areas for 8a-γ-TOH in peroxynitrite oxidations with or
without Fe3+-EDTA indicates little variation in yield,
as was also observed for formation of tocored. In reac-
tions with both peroxynitrite and SIN-1, NGT was the
major product formed.
The nitration of phenolic compounds such as tyrosine
has been studied previously (23, 24, 38, 39), but the exact
mechanisms remain unclear and may vary depending on
the substrate (39). Interpretation of the reaction mech-
anism for NGT formation is complicated by the ability
of peroxynitrite to form different oxidants. The formation
of NGT may result, at least in part, from NO2+ generated
The formation of 8a-γ-TOH is analogous to the oxida-
tion of R-TH, which also yields an 8a-hydroxytocopherone
via electron transfer and hydrolysis (28). However,
peroxynitrite oxidation of R-TH in aqueous ethanol (25)
or in acetonitrile (16) yielded R-tocopherolquinone. Al-
though the 8a-hydroxytocopherones of both R-TH and
γ-TH rearrange to the corresponding quinones, the 8a-
γ-TOH appeared to be somewhat more stable than 8a-
R-TOH. The 8a-γ-TOH was stable to HPLC analysis and
approximately 4 h was required to achieve complete
reduction to γ-TH by ascorbate, whereas 8a-R-TOH is
reduced within minutes under the same conditions and
rearranges to R-tocopherolquinone during HPLC analy-
sis. Hogg et al. (16) also reported that 8a-methoxyto-
copherone was formed by R-TH oxidation by peroxynitrite
in methanol, apparently by methanolysis of the tocopher-
one cation. Such 8a-alkoxytocopherones are considerably
more stable than 8a-hydroxy derivatives (37). The ap-
+
by peroxynitrous acid heterolysis. The NO2 could
nitrate the C-5 position of γ-TH by either of two mech-
anisms. The first is an electrophilic aromatic substitu-
tion reaction, in which NO2+ could add to γ-TH to form a
cationic intermediate, which then deprotonates to NGT
(eqs 1 and 2).
γ-TH + NO2+ f [γ-TH-NO2]+
(1)
(2)
[γ-TH-NO2]+ f NGT + H+
+
Alternatively, NO2 could react with γ-TH by initial
electron transfer, followed by recombination of the toco-
pheroxyl and NO2 radical intermediates (eqs 3 and 4).
-
pearance of γ-TQ rather than 8a-γ-TOH in the NO2+BF4
oxidations reflected the ease of rearrangement of 8a-γ-
+
TOH to γ-TQ in the presence of NO2 or nitric acid
generated in the reactions. In the peroxynitrite oxida-
tions, similarly reactive Lewis acids were not present in
high concentrations, and 8a-γ-TOH was comparatively
stable.
γ-TH + NO2+ f γ-T• + NO2 + H+
γ-T• + NO2 f NGT
(3)
(4)
Because of the unavailability of sufficiently stable
standards, we were unable to perform accurate quanti-
fication of 8a-γ-TOH and thus we cannot account com-
pletely for the partitioning of γ-TH between electron
transfer and nitration pathways of oxidation. We also
The electron transfer step (reaction 3) would be consistent
with the high reduction potential of the NO2+ (1.6 V) (20).
It is interesting that nitration of the γ-TH 8a-position
did not occur. This position is frequently the site of

406 Chem. Res. Toxicol., Vol. 10, No. 4, 1997
Hoglen et al.
radical substitution and solvolysis reactions to generate
8a-substituted tocopherones (see above). However, nitra-
tion at C-8a may be disfavored by steric constraints that
limit accomodation of a nitro group. Whereas bulky 8a-
alkyldioxytocopherones may be formed from R-TH (40,
41), bulky 8a-alkoxytocopherones are not (42).
(eq 8), which may yield a nitrocarbonate anion as the
ultimate nitrating species (eq 9; 43-46).
-
ONOO- + CO2 f ONOOCO2
(8)
(9)
ONOOCO2- f O2N-CO3
-
Beckman and co-workers demonstrated that Fe3+
-
EDTA catalyzed the nitration of 4-hydroxyphenylacetic
acid (4-HPA) as well as other phenolic compounds,
apparently by enhancing heterolytic cleavage of peroxy-
nitrous acid to NO2+ (23, 24). We investigated the effect
of Fe3+-EDTA on the formation of NGT in our reaction
system. Fe3+-EDTA modestly increased the nitration
of γ-TH. The increase in γ-TH nitration was maximal
at 100 µM Fe3+-EDTA in reactions with equimolar γ-TH
and peroxynitrite, as compared to reactions with or
without 10 µM addition of Fe3+-EDTA. The fact that
only modest (i.e., 20-30%) increases in NGT yield were
induced by Fe3+-EDTA in our studies may reflect the
ability of NO2+ formed by peroxynitrous acid heterolysis
to act as an electron transfer agent as well as a nitrating
agent. This is consistent with the production of γ-TQ and
Adventitious carbon dioxide/bicarbonate contamination
in our system is likely to be well below the millimolar
concentrations of bicarbonate used to catalyze nitration
in these other studies. Nevertheless, we made no at-
tempt to exclude ambient carbon dioxide from the reac-
tions, and thus these carbon dioxide-derived intermedi-
ates could contribute to the formation of NGT.
In summary, we have demonstrated that the reaction
of γ-TH with peroxynitrite yields several products, in-
cluding NGT. Because γ-TH is associated with biological
lipids, NGT may provide a novel marker for peroxynitrite
interaction with lipid membranes and lipoproteins. NGT
appears to be sufficiently stable to permit extraction from
biological samples and analysis. Although NGT also is
formed by the reaction of NO2 with γ-TH (5), appropriate
experimental controls could exclude this reaction. NGT
thus may be a useful marker for the formation of
peroxynitrite in biological systems.
tocored along with NGT in reactions with the NO2+ donor
-
NO2+BF4
.
+
Our data therefore suggest that than NO2 is not the
principal nitrating species involved in the formation of
NGT. This conclusion is further supported by the
+
observation that, in our studies with NO2 BF4-, the
Ack n ow led gm en t. This research was supported by
USPHS Grants ES 05675 and CA 59585 and by South-
west Environmental Health Sciences Center Grant ES
06694. The authors also acknowledge J eanne A. Burr
and Kathy L. Kaysen for technical assistance and Dr.
Thomas D. McClure of the Southwest Environmental
Health Sciences Center Analytical Core Laboratory for
LC-MS analyses.
combined yield of γ-TQ and tocored exceeded that of NGT
(Table 3). This is in contrast to reactions with peroxy-
nitrite and SIN-1, in which NGT was by far the major
product (Tables 1 and 2). Thus, NO2+ was a less efficient
nitrating agent for γ-TH than was peroxynitrite. In
-
comparing oxidations with NO2+BF4 to those with
peroxynitrite, it is possible that differences in concentra-
+
tions of NO2 generated by these reagents may account
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TX960200H