Relative one-electron reduction potentials of carotenoid radical cations and the interactions of carotenoids with the vitamin E radical cation

Article abstract of DOI:10.1021/ja974191q

Pulse radiolysis studies have been used to determine the electron- transfer rate constants between various pairs of carotenoids, one of which is present as the radical cation. These dietary carotenoids include those of importance to vision, namely zeaxanthin and lutein. These results have suggested the order of relative ease of electron transfer between six carotenoids. Additional experiments, involving electron transfer between astaxanthin (ASTA), β-apo-8'-carotenal (APO), and vitamin E (TOH), lead to the following order in terms of relative ease of electron transfer for the seven carotenoid radical cations studied: astaxanthin > β-apo-8'-carotenal > canthaxanthin > lutein > zeaxanthin > β-carotene > lycopene, such that lycopene is the strongest reducing agent (the most easily oxidized) and astaxanthin is the weakest, and the radical cations of the visual carotenoids, lutein (LUT) and zeaxanthin (ZEA), are reduced by lycopene (LYC) but not by β-carotene (β-CAR). Work on 7,7'-dihydro-β-carotene (77DH) and vitamin E allows us to better understand the interaction of the vitamin E radicals with carotenoids.

Full text of DOI:10.1021/ja974191q

J. Am. Chem. Soc. 1998, 120, 4087-4090
4087
Relative One-Electron Reduction Potentials of Carotenoid Radical
Cations and the Interactions of Carotenoids with the Vitamin E
Radical Cation
†
‡
†
†
Ruth Edge, Edward J. Land, David McGarvey, Louise Mulroy, and
T. George Truscott*,†
Contribution from the Chemistry Department, Keele UniVersity, Staffordshire ST5 5BG, U.K., and
CRC Department of Drug DeVelopment and Imaging, Paterson Institute for Cancer Research,
Christie Hospital NHS Trust, Manchester M20 9BX, U.K.
ReceiVed December 10, 1997
Abstract: Pulse radiolysis studies have been used to determine the electron-transfer rate constants between
various pairs of carotenoids, one of which is present as the radical cation. These dietary carotenoids include
those of importance to vision, namely zeaxanthin and lutein. These results have suggested the order of relative
ease of electron transfer between six carotenoids. Additional experiments, involving electron transfer between
astaxanthin (ASTA), â-apo-8′-carotenal (APO), and vitamin E (TOH), lead to the following order in terms of
relative ease of electron transfer for the seven carotenoid radical cations studied: astaxanthin > â-apo-8′-
carotenal > canthaxanthin > lutein > zeaxanthin > â-carotene > lycopene, such that lycopene is the strongest
reducing agent (the most easily oxidized) and astaxanthin is the weakest, and the radical cations of the visual
carotenoids, lutein (LUT) and zeaxanthin (ZEA), are reduced by lycopene (LYC) but not by â-carotene (â-
CAR). Work on 7,7′-dihydro-â-carotene (77DH) and vitamin E allows us to better understand the interaction
of the vitamin E radicals with carotenoids.
Introduction
or via other processes, such as, an addition reaction to a carbon-
carbon double bond
The role of carotenoids as antioxidants and their possible
switch to prooxidant behavior is of much current interest.
One aspect of this is the possibility that singlet oxygen
quenching by carotenoids (CAR) is partly responsible for their
antioxidant behavior. It has been claimed that in some
environments lycopene (LYC) is a more efficient singlet oxygen
1
-3
•
•
RO + CAR f [RO - - -CAR]
(2)
(3)
2
2
and hydrogen-atom transfer
4,5
•
2
•
quencher than â-carotene (â-CAR).
Nevertheless, we have
RO + CAR f ROOH + CAR(-H)
5
shown that singlet oxygen is quenched only about 1.3 times
faster by lycopene compared to â-carotene in both benzene and
toluene and this small difference is unlikely to be of any
significance in either chemical or biological systems. However,
the major role of carotenoids with respect to antioxidant (and
prooxidant) behavior may well involve radical scavenging
processes including electron transfer and consequent free radical
production. For example, an oxygen-centered radical such as
The electron-transfer reaction 1 will clearly be favored where
the R groups are electron withdrawing (e.g., CCl3O2 ). Clearly,
•
the ease of oxidation of the carotenoids will be an important
factor governing the relative rate constants of these radical
scavenging processes. In the present paper, we investigate the
rates of electron transfer between pairs of carotenoids, one of
which is present as the radical cation, to establish the order of
the ease of electron transfer of a series of carotenoid radical
cations:
•
6
a peroxyl radical (RO2 ) may react either via electron transfer
to produce a carotenoid radical cation
•
2
-
•+
RO + CAR f RO + CAR
(1)
CAR1•+ + CAR2 f CAR1 + CAR2•+
2
(4)
7
*
To whom correspondence should be addressed.
Keele University.
Christie Hospital NHS Trust.
We have recently reported the formation of carotenoid radical
cations following quenching of a vitamin E radical in hydro-
carbon solvents. In the present paper, we also use pulse
radiolysis to further investigate the vitamin E radical reaction
with 7,7′-dihydro-â-carotene (77DH) and show that the vitamin
†
‡
(
(
(
1) Burton, G. W.; Ingold, K. U. Science 1984, 224, 569-573.
2) Palozza, P.; Krinsky, N. I. Methods Enzymol. 1992, 213, 403-420.
3) Truscott, T. G. J. Photochem. Photobiol., B: Biol. 1996, 35, 233-
2
35.
•+
E radical quenched is, in fact, the radical cation TOH . The
structures of the carotenoids studied, including several xantho-
phylls (XAN), are given in Figure 1.
(4) Di Mascio, P.; Kaiser, S.; Sies, H. Arch. Biochem. Biophys. 1989,
2
74, 532-538.
(5) Conn, P. F.; Schalch, W.; Truscott, T. G. J. Photochem. Photobiol.,
B: Biol. 1991, 11, 41-47.
6) Hill, T. J.; Land, E. J.; McGarvey, D. J.; Schalch, W.; Tinkler, J. H.;
Truscott, T. G. J. Am. Chem. Soc. 1995, 117, 8322-8326.
(
(7) B o¨ hm, F.; Edge, R.; Land, E. J.; McGarvey, D. J.; Truscott, T. G. J.
Am. Chem. Soc. 1997, 119, 621-622.
S0002-7863(97)04191-7 CCC: $15.00 © 1998 American Chemical Society
Published on Web 04/09/1998

4088 J. Am. Chem. Soc., Vol. 120, No. 17, 1998
Edge et al.
Figure 2. Spectrum of â-carotene radical cation in argon-flushed
benzene. The spectrum is taken at a long enough time for the radical
anion to have completely decayed.
Table 1. Wavelengths of the Carotenoid Radical Cation
Absorption Band Maxima (λMax) in Benzene and Hexane
carotenoid
λ
max in benzene/nm
λ
max in hexane/nm
b
astaxanthin
920a
940
890
a
a
â-apo-8'-carotenal
canthaxanthin
lutein
zeaxanthin
â-carotene
lycopene
880
940a
950a
960
c
b
973
1040
1040
1070
1000a
b
a
d
1020
1050a
d
a
This work. b Reference 6. c Reference 10. d Reference 11.
Results and Discussion
Relative One-Electron Reduction Potentials of Carotenoid
Radical Cations. The radical cations of a range of carotenoids
6
,10,11
have previously been characterized in hexane,
dichlo-
1
2
romethane, and more recently by Skibsted and co-workers in
13
chloroform. In the present work benzene is used as the solvent
because it affords a higher solubility of carotenoids than hexane,
especially those containing oxygen (the xanthophylls). Also,
the radiation chemistry of benzene is well understood, and
Figure 2 shows the absorption spectrum of the â-carotene radical
cation in this solvent. For comparison, the corresponding
wavelengths (nm) of the radical cation absorption band maxima
in benzene and hexane are given in Table 1. The solvent
polarizabilities for benzene and hexane (based on refractive
indices of 1.501 and 1.375, respectively) and the observed
solvent shifts for carotenoid radical cations fit the trends noted
1
4
by Andersson et al. for carotenoid ground states.
Figure 3 shows the changes in spectra with time on pulse
-
4
-5
radiolysis of 1 × 10 M ASTA in the presence of 1 × 10
M
Figure 1. Structures of carotenoids and xanthophylls.
•+
LYC. This illustrates positive charge transfer from ASTA to
•+
LYC, i.e. electron transfer from LYC to ASTA . Similar data
have allowed the electron-transfer second-order rate constants
to be determined for 10 such pairs as given in Table 2. In
general, these pulse radiolysis kinetic studies show that lycopene
(LYC) efficiently quenches (reduces) the radical cation of all
the oxy-containing carotenoids studied
Methods
Experiments were carried out using a 9-12-MeV Vickers linear
8
,9
accelerator as previously described, using 50-200-ns pulses. Solu-
tions were studied using quartz flow-through cells with an optical path
3
length of 2.5 cm and an internal volume of 3 cm . The vitamin E was
supplied by Sigma and purified, via spinning plate chromatography,
to >99.5% pure (by HPLC) before use. The carotenoids were a gift
from Hoffmann-La Roche and were used as supplied, after confirming
their purity by HPLC (>99.5%). The lutein sample, however, contained
about 4% zeaxanthin. The benzene and hexane were super purity
solvents from Romil.
XAN•+ + LYC f XAN + LYC•+
(5)
whereas â-carotene only reduces the radical cation of astaxanthin
ASTA), canthaxanthin (CAN), and â-apo-8′-carotenal (APO),
(
(
11) Dawe, E. A.; Land, E. J. J. Chem. Soc., Faraday Trans. 1 1971,
(
8) Keene, J. P. J. Sci. Instrum. 1964, 41, 493-496.
71, 2162-2169.
(9) Butler, J.; Hodgson, B. W.; Hoey, B. M.; Land, E. J.; Lea, J. S.;
(12) Khaled, M.; Hadjipetrou, A.; Kispert, L. D. J. Phys. Chem. 1991,
95, 2438-2442.
Lindley, E. J.; Rushton, F. A. P.; Swallow, A. J. Radiat. Phys. Chem. 1989,
4, 633-646.
10) Lafferty, J.; Roach, A. C.; Sinclair, R. S.; Truscott, T. G.; Land, E.
J. J. Chem. Soc., Faraday Trans. 1 1977, 73, 416-429.
3
(13) Mortensen, A.; Skibsted, L. H. Free Rad. Res. 1996,25, 355-368.
(14) Andersson, P. O.; Gillbro, T.; Ferguson, L.; Cogdell, R. J.
Photochem. Photobiol. 1991, 54, 353-360.
(

Reduction Potentials and Interactions of Carotenoids
J. Am. Chem. Soc., Vol. 120, No. 17, 1998 4089
-
2
hF ei gx ua nr ee .4. Transient spectra of 1 × 10 M vitamin E in N
2
O-flushed
-
4
Figure 3. Spectra obtained on pulsing 1 × 10 M ASTA with 1 ×
M LYC in argon-flushed benzene.
-
5
1
0
•
+
Table 2. Bimolecular Rate Constants for Electron Transfer
between Carotenoid Pairs (CAR1 + CAR2 f CAR1 + CAR2 )
electron transfer to CAN . Indeed, we find that the introduction
of the oxygen heteroatom into a carotenoid (to give a XAN)
leads to a radical cation which is a stronger oxidizing species
and this is especially so if the oxygen is present as part of a
carbonyl group. Consistent with this, we find that ZEA reduces
the radical cations of ASTA and APO (see Table 2).
•
+
•+
rate constant ((10%)/10 M s-1
9
-1
carotenoid
radical cation
lycopene
â-carotene
zeaxanthin
•
+
ASTA
9
11
8
7
5
8
6
5
<1
<1
5
8
•
+
APO
•
+
CAN
<1
s
<1
Based on these studies of pairs of carotenoids, we propose
that the carotenoid radical cations can be placed in the following
relative order in terms of reduction potential:
•
+
ZEA
LUT
•
+
(see Table 2). However, for several pairs, spectral overlap
precluded the observation of the electron-transfer process.
It is noteworthy that the radical cations arising from the two
carotenoids in the human macular [lutein (LUT) and zeaxanthin
(
ZEA)] are both repaired efficiently by lycopene but not by
It should be noted that this is just an order of reduction potentials
and in some cases the difference may be small, leading to only
very slow reactions (e.g., ZEA/â-CAR pair).
â-carotene. The retina is the only organ in the body which is
continually exposed to high levels of focused radiation and is
in a highly oxygenated environment. This combination of light
and oxygen, together with the presence of photosensitizers, gives
a potential for oxy free radical and singlet oxygen generation.
Lutein and zeaxanthin both contain terminal hydroxyl groups
Reactions of Carotenoids with the Vitamin E Radical
•
+
7
Cation (TOH ). In a previous paper, we reported that seven
carotenoids underwent analogous electron-transfer reactions
•
+
leading to CAR formation from the vitamin E radical
(see Figure 1) which may allow one or both of them to span
•
•+
(
interpreted as TO but shown below to be TOH ). Also, we
the outer segment membrane. Hence, they may play a
particularly efficient role in antioxidant processes by being more
accessible to species, such as vitamin C in the extracellular
environment, so as to regenerate the carotenoid from its radical
cation. While it is well established that the retina does not
contain high concentrations of hydrocarbon carotenoids such
suggested that this reaction did not occur with astaxanthin. We
now confirm this result for astaxanthin and have also shown
•
+
that TOH accelerates the decay of ASTA so that the reverse
•
+
•+
process ASTA + TOH f ASTA + TOH is suggested.
These studies were performed in hexane rather than in benzene
as solvent due to the more prominent triplet absorptions in
benzene which interfere at the wavelengths of interest.
Figure 4 shows the transient absorption spectrum at various
15
as â-carotene and lycopene, nevertheless Mares-Perlman et al.
have shown a correlation between age-related macular degen-
eration (in which the yellow spot of the macular, which contains
the xanthophylls, may be photodamaged) and low levels of
serum lycopene. Also, there is other evidence that dietary
factors, involving xanthophylls and other carotenoids, may well
be related to age-related macular degeneration.16,17
It is also established that canthaxanthin can accumulate in
the retina after high-dose ingestion. While the presence of
canthaxanthin does not appear to have any significant clinical
consequence, we find that both lycopene and â-carotene undergo
-
2
times following pulse radiolysis of 1 × 10 M vitamin E in
N2O-flushed hexane. As can be seen, there are two peaks (420
and 460 nm maxima) but the kinetics associated with these
absorption maxima are clearly quite different. The species with
λmax ) 420 nm does not decay on the time scale of our
experiments. However, the species at 460 nm decays with two
lifetimes of about 250 ns and 6 µs. The shorter of these is not
of interest in the present work. On the basis of previous
1
8-21
work,
we interpret the species absorbing at 460 nm as the
•+
(
15) Mares-Perlman, J. A.; Brady, W. E.; Klein, R.; Klein, B. E. K.;
Bowen, P.; Stacewicz-Sapuntzakis, M.; Palta, M. Arch. Opthalmol. 1995,
13, 1518-1523.
16) Hammond, B. R., Jr.; Johnson, E. J.; Russell, R. M.; Krinsky, N.
radical cation (TOH ) and that at 420 nm as the neutral radical
1
(18) Depaw, M. C.; Craw, M. T.; MacCormick, K.; Wan, J. K. S. J.
Photochem. Photobiol., B: Biol. 1987, 1, 229-239.
(19) Parker, A. W.; Bisby, R. H. J. Chem. Soc., Faraday Trans. 1993,
89, 2873-2878.
(20) Nagaoka, S.; Kuranaka, A.; Tsuboi, H.; Nagashima, U.; Mukai, K.
J. Phys. Chem. 1992, 96, 2754-2761.
(21) Nagaoka, S.; Mukai, K.; Itoh, T.; Katsumata, S. J. Phys. Chem.
1992, 96, 8184-8187.
(
I.; Yeum, K.-J.; Edwards, R. B.; Snodderly, D. M. InVest. Opthalmol. Vis.
Sci. 1997, 38, 1795-1801.
(17) Seddon, J. M.; Ajani, U. A.; Sperduto, R. D.; Hiller, R.; Blair, N.;
Burton, T. C.; Farber, M. D.; Gragoudas, E. S.; Haller, J.; Miller, D. T.;
Yannuzzi, L. A.; Willett, W. JAMA, J. Am. Med. Assoc. 1994, 272, 1413-
1
420.

4090 J. Am. Chem. Soc., Vol. 120, No. 17, 1998
Edge et al.
increased with a second-order rate constant of ∼10 M-1 s .
1
0
-1
(
The ground-state absorption spectrum of this carotenoid allows
•
+
us to monitor the TOH at 460 nm, whereas this is not so for
the other carotenoids.) Figure 5 compares the kinetics of the
decay of the species at 460 nm with and without 77DH and
shows the formation of the 77DH radical cation at 830 nm in
the presence of 1 × 10 M vitamin E. Figure 5c shows that
the rate of formation of the 77DH radical cation occurs at the
same rate as the enhanced decay of the slow component at 460
nm. We interpret this as the electron-transfer process:
-
2
TOH•+ + CAR f TOH + CAR•+
(6)
While we can monitor TOH•+ (at 460 nm), unfortunately,
spectral overlap with the strongly absorbing carotenoid ground
state prevents us from monitoring the effects of the carotenoid
•
on TO (at 420 nm) by optical methods. However, the ESR
2
2
•
work of Ingold and co-workers has clearly shown that TO is
unaffected by â-carotene.
We have also studied the interaction of APO with TOH•
and found that this xanthophyll behaves like all the other
carotenoids studied, except ASTA. This allows us to distinguish
between ASTA and APO in the above scheme, such that
ASTA has the higher reduction potential. Also, it is interesting
to note that the lifetime of TOH in nonpolar environments is
+
•+
•
+
19
markedly longer than had been previously suggested. It is
2
3
•+
also noteworthy that ASTA , a carotenoid radical cation
containing both carbonyl and hydroxyl groups, has a reduction
•
+
potential that is higher than E(TOH /TOH).
In conclusion, while there is no direct evidence for the
production of carotenoid radical cations in vivo as a result of
radical scavenging, the relative ease of oxidation of the
carotenoids is worthy of future study and may well be a
significant parameter in their dietary antioxidant role.
•
+
Figure 5. (A) Decay trace of TOH at 460 nm with and without 77DH
-
5
•+
(
1 × 10 M) and formation of 77DH at 830 nm in N
2
O-flushed
hexane. For the traces at 460 nm, the residual absorption (A
∞
) 0.0021
and 0.00124 for the traces with and without, respectively) has been
subtracted out. (B) Plot of ln(∆A - ∆A ) for the decay of TOH with
∞
Acknowledgment. We thank AICR, CRC (U.K.), and the
Wellcome Trust for financial support, Dr. K Bernhard of
Hoffmann-La Roche for the gift of the carotenoids, and Drs.
K. U. Ingold and N. Krinsky for useful discussions.
•
+
and without 77DH.
•
TO ). Our results are consistent with the decay of the TOH•+
(
being, in part, due to deprotonation with a corresponding growth
in the species absorbing at 420 nm.
On adding 77DH (10µM) the decay rate of the slow
component of the species absorbing at 460 nm is markedly
JA974191Q
(22) Valgimigli, L.; Lucarini, M.: Pedulli, G. F.; Ingold, K. U. J. Am.
Chem. Soc. 1997, 119, 8095-8096.
(23) Miki, W. Pure Appl. Chem. 1991, 63, 141-146.