Carotenoids enhance vitamin E antioxidant efficiency

Full text of DOI:10.1021/ja962512c

J. Am. Chem. Soc. 1997, 119, 621-622
621
Carotenoids Enhance Vitamin E Antioxidant
Efficiency
Fritz Bo¨hm,† Ruth Edge,‡ Edward J. Land,§
David J. McGarvey,‡ and T. George Truscott‡,*
Department of Dermatology (Charite´)
Humboldt UniVersity, 10117 Berlin, Germany
Department of Chemistry
Keele UniVersity, ST5 5BG, UK
CRC Department of Biophysical Chemistry
Paterson Institute for Cancer Research
Christie Hospital NHS Trust, Manchester, M20 9BX, UK
ReceiVed July 22, 1996
The principal role of vitamin E (of which R-tocopherol is
the major component) is that of a free radical scavenging
antioxidant, and there is evidence that a high intake of
carotenoids (particularly â-carotene) and R-tocopherol may
prevent cancer1 and other diseases. However, some trials
indicate that â-carotene can increase the incidence of lung cancer
amongst heavy smokers.2 In addition, there is some evidence
that other carotenoids may be more efficient antioxidants than
â-carotene.3,4
We report here results of pulse radiolysis and laser flash
photolysis studies of the reactions of the R-tocopheroxyl radical
with a range of carotenoids and of the interaction of ascorbic
acid with the carotenoid radical cations. The results of this work
are used to support a proposed molecular mechanism for the
antioxidant protection of human lymphoid cells from the NO2
radical, to be reported elsewhere.
Carotenoid and R-tocopherol radicals were generated in
hexane by pulse radiolysis5 using a 9-12 MeV Vickers linear
accelerator with pulses of 20 ns duration and a dose of 0.3 nC.
Solutions were nitrous oxide saturated (to capture the electron)
and were irradiated in quartz flow-through cells with internal
volumes of either 0.7 or 3 cm3 and a monitoring optical path
length of 2.5 cm. With a solution of R-tocopherol alone (10
mM) the solvent radicals react with the R-tocopherol producing
a species with an absorption maximum at 420 nm assigned to
the R-tocopheroxyl radical.6 In methanol, carotenoid radical
cations were generated using laser flash photolysis via electron
transfer quenching ([CAR] ) 10 µM) of the 1-nitronaphthalene
triplet state, as described previously.7
Figure 1. (A, top) Representative kinetics profile of the formation of
77DH•+ following pulse radiolysis of 10 µM 77DH in the absence and
presence of 100 µM R-tocopherol. (B, bottom) Representative kinetics
profile of the decay of â-CAR•+ following pulse radiolysis of 100 µM
â-CAR in the absence and presence of 100 µM ascorbic acid in aqueous
Triton X 100, pH 7.
•
near infrared (800-1100 nm).8,9 The growths of the radical
cations of all the carotenoids studied were observed at their
respective absorption maxima, and the rate constants for these
growths were much lower than in the absence of R-tocopherol
where the CAR•+ is produced directly from the solvent radical
cation. Typical traces are shown in Figure 1A for 7,7′-dihydro-
â-carotene (77DH). These observations show that electron
transfer occurs from carotenoids (CAR) to the R-tocopheroxyl
radical (TO•).
H+
For both carotenoid alone and carotenoid/R-tocopherol mix-
tures (100 µM R-tocopherol and 10 µM carotenoid) in hexane,
the carotenoid radical cation formation was monitored in the
TO• + CAR 8 TOH + CAR•+
(1)
It has been proposed that R-tocopherol protects â-carotene.10
However, except for astaxanthin, see below, our results show
that the reverse of reaction 1 does not occur. Therefore
R-tocopherol is unlikely to protect â-carotene by a repair
mechanism.
The second-order rate constants ((10%) for reaction 1 were
calculated from the pseudo-first-order rate constants of the
formation of the carotenoid radical cations in the presence of
R-tocopherol11 using k ) kformation/[CAR]. All of the second-
order rate constants of reaction 1 are close to diffusion controlled
* To whom correspondence should be addressed.
† Department of Dermatology (Charite´).
‡ Keele University.
§ Christie Hospital NHS Trust.
(1) Blot, W. J.; Li, J.-Y.; Taylor, P. R.; Guo, W.; Dawsey, S.; Wang,
G.-Q.; Yang, C. S.; Zheng, S.-F.; Gail, M.; Li, G.-Y.; Yu, Y.; Liu, B.-Q.;
Tangrea, J.; Sun, Y.-H.; Liu, F.; Fraumeni, J. F., Jr.; Zhang Y.-H.; Li, B.
J. Nat. Cancer Inst. 1993, 85, 1483-1492.
(2) (a) The R-tocopherol, â-carotene cancer prevention study group: New
Engl. J. Med. 1994, 330, 1029-1035. (b) Peterson, K. Science 1996, 271,
441. (c) Omenn, G. S.; Goodman, G. E.; Thornquist, M. D.; Balmes, J.;
Cullen, M. R.; Glass, A.; Keogh, J. P.; Meyskens, F. L., Jr.; Valanis, B.;
Williams, J. H., Jr.; Barnhart, S.; Hammar, S. New Engl. J. Med. 1996,
334, 1150-1155.
(8) The weakly absorbing R-tocopheroxyl radical could not be monitored
in the mixture due to strong carotenoid ground state absorption in that region
(e.g. â-carotene has ꢀ420 ) 90 000 M-1 cm-1).
(3) (a) Tinkler, J. H.; Bo¨hm, F.; Schalch, W.; Truscott, T. G. J.
Photochem. Photobiol. B: Biology 1994, 26, 283-285. (b) Rousseau, E.
J.; Davison, A. J.; Dunn, B. Free Rad. Biol. Med. 1992, 13, 407-433.
(4) Bo¨hm, F.; Tinkler, J. H.; Truscott,T. G. Nature Med. 1995, 1, 98-
99.
(5) Butler, J.; Hodgson, B. W.; Hoey, B. M.; Land, E. J.; Lea, J. S.;
Lindley, E. J.; Rushton, F. A. P.; Swallow, A. J. Radiat. Phys. Chem. 1989,
34, 633-646.
(6) (a) Packer, J. E.; Slater, T. F.; Willson, R. L. Nature 1979, 278, 737-
738. (b) Bisby, R. H.; Parker, A. W. FEBS Lett. 1991, 290, 205-208.
(7) Tinkler, J. H.; Tavender, S. M.; Parker, A. W.; McGarvey, D. J.;
Mulroy, L.; Truscott, T. G. J. Am. Chem. Soc. 1996, 118, 1756-1761.
(9) Dawe, E. A.; Land, E. J. J. Chem. Soc., Faraday Trans. 1975, 71,
2162-2169.
(10) Palozza, P.; Krinsky, N. I. Arch. Biochem. Biophys. 1992, 297, 184-
187.
(11) The lifetime of EO• in the absence of â-carotene is long compared
to the lifetime in the presence of the carotenoid. Thus, the rate equation
kformation ) kdecay + k[CAR] can be simplified to kformation ) k[CAR] (where
kformation is the rate constant for the formation of CAR•+ and kdecay is the
rate constant for the decay of EO• other than by electron transfer from the
carotenoid).
S0002-7863(96)02512-7 CCC: $14.00 © 1997 American Chemical Society

622 J. Am. Chem. Soc., Vol. 119, No. 3, 1997
Communications to the Editor
(21 × 109 M-1 s-1 in hexane). The values in hexane, obtained
at the λmax of each carotenoid radical cation,12 are (109 M-1
s-1) 5.3, 8.8, 10, 13, 14, 18, and 26 for lutein, canthaxanthin,
â-carotene, septapreno-â-carotene, lycopene, 7,7′-dihydro-â-
carotene, and zeaxanthin, respectively.
Scheme 1
Of course, the rate constants reported here are not measured
in a biological environment, but the high values obtained do
indicate the direction of electron transfer, from carotene to TO•,
and, therefore, the repair of the R-tocopheroxyl radical by the
carotenoids. Doubling the â-carotene concentration did not
affect the amount of radical cation formed, but the rate of
formation doubled indicating complete scavenging. Hence, all
the carotenoids studied are capable of regenerating R-tocopherol
from its radical. However, in a recent experiment, we have
found that astaxanthin does not recycle R-tocopherol, but it may
be recycled by R-tocopherol, and this will be the subject of
further work.
Our investigations of the reactivity of ascorbic acid (AscH2)
with carotenoid radical cations, using pulse radiolysis and laser
flash photolysis, have shown an enhanced CAR•+ decay rate in
the presence of ascorbic acid (see Figure 1B), consistent with
the following reactions in methanol (2) and water, Triton X
100 pH 7 (3):
may reflect differing cellular locations of â-carotene and
R-tocopherol. It is generally accepted that â-carotene is more
lipophilic than R-tocopherol14 and will be more likely to be in
the interior of a membrane. However, the â-carotene radical
(CAR•+) is a charged species, while the R-tocopherol radical
(TO•) is uncharged. Thus, the CAR•+ may reorientate so that
the charge is near the polar interface of the cell membrane and
hence be more accessible to aqueous phase antioxidants such
as ubiquitous ascorbic acid. Indeed, the disscussion of Pryor
et al.15 also suggests the role of carotenoids as antioxidants
depends on their environment. We are currently using time-
resolved resonance Raman spectroscopy of carotenoid radical
ions to investigate such effects.7
Our findings are in agreement with Palozza and Krinsky10
who showed that â-carotene and R-tocopherol can act syner-
gistically in the membrane system, rat liver microsomes;
however, we differ in that our findings show that R-tocopherol
protects â-carotene and not vice versa. Willson16 proposed that
R-tocopherol and â-carotene have similar electron donor abili-
ties, while Tappel17 suggests â-carotene is the weaker antioxi-
dant. Our results show clearly that most carotenoids (including
â-carotene) are better electron donors than R-tocopherol.
Furthermore, this scheme allows us to suggest that the low levels
of antioxidants, such as ascorbic acid, in smokers compared to
nonsmokers, may be related to the apparent failure of â-carotene
to offer any benefit to this group, as reported recently.2
In conclusion, we show that carotenoids could substantially
enhance vitamin E/C protection, and our results suggest a
molecular mechanism (Scheme 1) for this effect.
CAR•+ + AscH2 f CAR + AscH• + H+
CAR•+ + AscH- f CAR + Asc•- + H+
(2)
(3)
These reactions, for â-carotene, have second-order rate constants
((10%) of 9.4 × 106 M-1 s-1 in aqueous 2% Triton X 100
detergent micelles, pH 7, and 0.35 × 109 M-1 s-1 in methanol.
The corresponding rate constants in methanol for the other
carotenoids studied are (109 M-1 s-1) 1.3, 1.1, 1.4, 0.91, and
7.7, for lutein, canthaxanthin, septapreno-â-carotene, 7,7′-
dihydro-â-carotene, and zeaxanthin, respectively. These were
calculated from a plot of concentration of ascorbic acid (0-
100 µM) against the pseudo-first-order rate constants for the
decay of CAR•+. Unfortunately, due to solubility problems we
could not obtain a value for lycopene.
A synergistic effect we have observed in cell protection by
â-carotene and vitamins E and C13 may be related to the fact
that â-carotene is not only quenching oxy-radicals but is also
repairing the R-tocopheroxyl radical (b in Scheme 1), which is
produced when R-tocopherol scavenges an oxy-radical (a in
Scheme 1). Such a synergistic mechanism requires that the
CAR•+ is reconverted to CAR (c in Scheme 1). Of course, in
hexane the â-carotene and R-tocopherol simply compete in
scavenging any radicals present. However, a synergistic effect
Acknowledgment. Experiments were performed at the Paterson
Institute for Cancer Research Free Radical Research Facility, The
Christie Hospital NHS Trust, Manchester. The facility is supported
by the European Commission TMR programme/access to large scale
facilities. We thank AICR, CRC (UK), and the Nuffield Foundation
for financial support and Dr. K. Bernhard of Hoffmann-La Roche for
the carotenoids, and T.G.T. and F.B. thank the British Council and
DAAD (Germany) for support.
JA962512C
(14) Niki, E.; Noguchi, N.; Tsuchihashi, H.; Gotoh, N. Am. J. Clin. Nutr.
1995, 62, 1322S-1326S.
(15) Pryor, W. A.; Cornicelli, J. A.; Devall, L. J.; Tait, B.; Trivedi, B.
K.; Witiak, D. T.; Wu, M. D J. Org. Chem. 1993, 58, 3521-3532.
(16) Willson, R. L. In Biology of Vitamin E; Porter, R., Whelan, J., Eds.;
Ciba Foundation Symposium, 101, Pitman Press: London, 1993; pp 19-
44.
(12) Bensasson, R. V.; Land, E. J.; Truscott, T. G. Flash Photolysis and
Pulse Radiolysis; Pergamon Press: Oxford, 1983.
(13) Bo¨hm, F.; Edge, R.; Land, E. J.; McGarvey, D. J.; Truscott, T. G.
To be published.
(17) Tappel, A. L. Inform. 1995, 6, 778-783.