(Received for publication, August 14, 1995; and in revised form, November 10, 1995)
From the
Mechanisms of free radical scavenging by the nutritional
antioxidant -carotene have been investigated by pulse radiolysis.
Free radicals, which can initiate the chain of lipid peroxidation,
including nitrogen dioxide (NO
), thiyl
(RS
), and sulfonyl (RSO
)
radicals, are rapidly scavenged by
-carotene. Absolute rate
constant k[NO
+
-carotene] = (1.1 ± 0.1)
10
M
s
and for the
glutathione thiyl radical k[GS
+
-carotene] = (2.2 ± 0.1)
10
M
s
have been
determined. The mechanisms however are mutually exclusive, the former
involving electron transfer to generate the radical-cation
[
-carotene]
and the
latter by radical-addition to generate an adduct-radical
[RS
-carotene]
. Rate
constants for thiyl radical-addition reactions vary from 10
to 10
M
s
and correlate with the lipophilicity of the thiyl radical under
study. Sulfonyl radicals undergo both electron abstraction,
[
-carotene]
and
radical-addition,
[RSO
-carotene]
in an approximate 3:1 ratio. The
-carotene radical-cation
and adduct-radicals are highly resonance stabilized and undergo slow
bimolecular decay to non-radical products. These carotenoid-derived
radicals react differently with oxygen, a factor which is expected to
influence the antioxidant activity of
-carotene within tissues of
varying oxygen tension in vivo.
-Carotene (provitamin A) is a major lipid soluble
antioxidant nutrient present in a range of fresh fruit and vegetable
produce(1) . Epidemiological evidence that correlates
-carotene dietary supplementation with a reduced risk of
contracting cancer and heart disease (2) has provided the
impetus for ongoing human intervention
trials(3, 4, 5, 6) , particularly on
smokers(7, 8) . The antioxidant properties of
-carotene have been implicated in the molecular basis for disease
prevention(9, 10) , primarily because of the putative
role of oxidative stress in disease initiation and
progression(11, 12, 13, 14) . In
vitro studies on cellular membrane damage (15, 16) and low density lipoprotein (LDL) (
)oxidation (17) indicate that
-carotene may
modulate free radical processes in vivo by behaving as a
chain-breaking antioxidant in lipid peroxidation(9, 18) Paradoxically, other studies have shown that
-carotene offers little protection from metal-catalyzed LDL
oxidation (19, 20) and can kill tumor cells via
prooxidative pathways(21) .
-Carotene in combination with
-tocopherol has recently been found to reduce radiation effects in
normal tissues, suggesting a potential application as a radioprotector
in clinical cancer radiotherapy(22) .
Although the role of
-carotene as a singlet oxygen quencher has been thoroughly
characterized(23, 24, 25, 26, 27) ,
surprisingly fewer mechanistic studies on radical-scavenging properties
of the carotenoid have been pursued. Pulse radiolysis has shown that
halogenated peroxyl radicals (e.g. CCl
OO
) are rapidly scavenged by
-carotene (28, 29, 30) and that the
reactivity toward the superoxide radical-anion
(O
) is much less significant than for
the related carotenoid lycopene(31) .
Nitric oxide, a major
component of cigarette smoke (500-1000 ppm), may contribute
to the development of oral/lung cancers and heart disease in smokers by
generating more damaging nitrogen oxides, including nitrogen dioxide
(NO
) radicals (32) . In vitro studies have demonstrated that NO
radicals can initiate the autoxidation of polyunsaturated fatty
acids (33) and that cigarette smoke can induce LDL
oxidation(34) . Paradoxically, although
-carotene does not
appear to prevent the oxidation of LDL by cigarette smoke(34) ,
it can protect lymphocytes from NO
radical-induced membrane damage(16) .
A number of
thiols, including the endogenous thiol antioxidant glutathione (GSH),
have been shown to enhance nitrogen dioxide-induced lipid peroxidation
in model systems by generating thiyl (RS) and
thiyl-derived radical species(35) . Thiyl radicals are
synonymous with the classical repair reactions of thiols in vivo(36, 37) and are also generated by
peroxidase-catalyzed oxidation of thiols (38, 39) .
Although the fate of RS
radicals within cells remains
a matter for debate(40) , there is increasing evidence to
suggest that they are capable of initiating the chain of lipid
peroxidation at least in model
systems(41, 42, 43) . Thiyl radicals undergo
conjugation with molecular oxygen to generate the thiyl peroxyl
(RSOO
) radical (44) which can rearrange to
thiyl-sulfonyl (RSO
) radical(45) ,
another potent initiator of lipid peroxidation(46) . A
thiol-specific antioxidant enzyme considered widely distributed in
mammalian tissues which catalyzes the removal of thiyl radicals has
provided evidence for thiyl radical-induced damaging
reactions(47) . Moreover, human (THP-1) macrophages oxidize LDL
by a thiol-dependent mechanism, which is believed to involve thiyl and
thiyl-derived radical species.
Following a preliminary
communication(48) , we now report a detailed pulse radiolysis
study on the oxidation of -carotene by
NO
, RS
, and
RSO
radicals and characterization of the
resultant
-carotene-derived radical species.
The nitrogen dioxide radical
(NO) was generated by radiolysis of
N
-saturated tert-butyl alcohol/water mixtures
(50%, v/v) containing 0.1 mM NO
in 1 mM phosphate buffer at pH 5. Under these conditions
the radiolysis of tert-butyl alcohol/water mixtures generates
the primary radical species (52) in .
In and the hydrated electron
(e) and solvated electron
(e
) reduce nitrate to nitrate radical
anions (NO
) which rapidly
protonate at pH 5, then dissociate (<1 µs) into the
NO
radicals(53) .
Both hydroxy (OH) and hydrogen
(H
) radicals are scavenged by tert-butyl
alcohol to generate a radical that is unreactive toward
-carotene
and does not absorb above 300
nm.
Thiyl radicals were generated by radiolysis of
NO-saturated tert-butyl alcohol/water mixtures
(50%, v/v) containing 10 mM thiol and 1 mM phosphate
buffer at pH 5. In this case hydrated electron is converted to
OH radicals that becomes the principal product of
water radiolysis in .
Thiyl radicals (RS) are then rapidly formed
(<1 µs) after the pulse by the repair of the carbon-centered
radicals () generated in and by the
thiol(54, 55, 56) .
Thiyl-sulfonyl radicals were generated on radiolysis of
N-saturated tert-butyl alcohol/water mixtures
(50%, v/v) containing 10 mM C
H
SO
Cl and 1 mM phosphate buffer at pH 5. In this case RSO
radicals are generated by dissociative electron capture (57) in , while both
OH and
H
radicals are once again scavenged by the alcohol in .
The free radical yields are represented by their respective G-values, where G(NO)
= 0.9, G(RS
) = 5.5 and G(RSO
) = 0.9 were
determined by monitoring the oxidation of ABTS
(2,2`-azinobis(3-ethylbenzothiazoline-6-sulfonate)) to the stable
radical-cation ABTS
(
max
= 417 nm,
= 34,700 dm
mol
s
)(53, 58) .
Prior to
experimentation a N-saturated stock solution of
-carotene in toluene was prepared and kept in the dark. Solutions
were prepared immediately before experimentation using purified water
from a Milli-Q system (Millipore). Solutions required for pulse
radiolysis experiments contained no more than 20 µmol
dm
-carotene to allow adequate light
transmission in the 300-500 nm region, where
-carotene
exhibits a strong ground-state absorption(59) . Before
irradiation solutions were bubbled with nitrous oxide (N
O,
oxygen content < 10 ppm) or with nitrogen (N
, oxygen
content < 10 ppm) from the British Oxygen Co., United Kingdom. All
experiments were performed at ambient room temperature (20 ± 2
°C).
Bleaching of the -carotene ground-state absorption in
the 300-500 nm has proven a useful method of probing the
mechanism of free radical and enzymatic destruction of
carotenoid(9, 10, 60) . To assess the
possibility of
-carotene-derived products interfering at these
wavelengths. Steady-state radiolysis experiments were performed in a
field of a 2000 Ci of
Co
-source at a dose rate of 5
Gy min
(total dose
25 Gy equals < 30%
conversion of
-carotene to products) as determined by Fricke
dosimetry(61) . EDTA 10 mmol dm
was included
to prevent metal-catalyzed degradation of either
-carotene or
-carotene-derived products (60) , otherwise the
experimental conditions were as for pulse radiolysis. Products formed
on irradiation of
-carotene were separated by high performance
liquid chromatography using a Hypersil 5ODS 125
4.6-mm column.
Samples were separated isocratically with a
methanol/acetonitrile/chloroform (25:60:15%, v/v) eluent (62) and components detected spectrophotometrically at 451 and
300 nm by a Waters 486 variable wavelength detector. Although
NO
, RS
,
RSO
radicals depleted
-carotene in a
dose-dependent manner no products of the reactions were detected above
420 nm which would interfere with the pulse radiolysis kinetic
measurements.
Figure 1:
Oxidation of -carotene by nitrogen
dioxide. Absorption spectra showing the formation and decay of the
-carotene radical-cation as measured by pulse radiolysis of
N
O-saturated tert-butyl alcohol/water mixtures
(50%, v/v) containing nitrate (0.1 M) and
-carotene (10
µM) at pH 5. The spectra were built from measurements of
absorbance at discrete wavelengths 10 ms (solid circles) and
0.1 s (open circles) after pulses of approximately 1 Gy. Insets: optical traces showing exponential bleaching of the
-carotene ground-state absorption at 450 nm and the corresponding
exponential growth of the
-carotene radical-cation absorption at
910 nm.
The absorption of the solution measured 10 ms after the electron
pulse showed a decrease in the wavelength range 300-600 nm (where
-carotene absorbs) accompanied by an increase in the range
600-1000 nm. These spectral changes are ascribed to the reaction of
NO
radicals with
-carotene causing a
bleaching of the parent chromophore and the formation of a new
absorbing species with
910 ± 5 nm. The
extinction coefficient of the radical species was calculable from the
radical yield of G
= 8.5 ±
0.2
10
M
cm
. Under the experimental conditions
employed, the radiation chemical yield of nitrogen dioxide radicals was G(NO
) = 0.9 giving an
extinction coefficient for the
-carotene radical species of
= 9.4 ± 0.2
10
M
cm
. By
comparison with an analogous transient generated by flash photolysis (25) and one-electron oxidation of
-carotene by the
halogenated peroxyl radical
CCl
OO
(28, 29) , the
transient species was identified as the
-carotene radical-cation
[carotene]
generated by
electron abstraction from
-carotene by NO
radicals via . The extinction coefficient of the
[carotene]
radical-cation
compares well with that measured in aqueous Triton X-100 (
1
10
M
cm
)(63) .
By analogy to the reversible addition reactions of nitrogen
dioxide with the double bonds of alkenes to form nitro alkyl radicals (64) , the nitrogen dioxide -carotene adduct-radical
[NO
-carotene]
was considered a possible precursor to radical-cation
formation(16) . Indeed, a characteristic shoulder at
650-800 nm in the
[
-carotene]
radical-cation absorption spectrum was observed and thought to be
a separate radical species quite possibly a
[NO
-carotene]
radical. However, the reaction kinetics between 600 and 1000 nm
were identical, and it was therefore concluded that the entire
absorption in this region was due to the
[carotene]
radical-cation
alone.
Both the decline in the -carotene ground-state
absorption at 451 nm (left inset, Fig. 1) and the
increase in the [
-carotene]
radical-cation absorption at 910 nm (right inset, Fig. 1) were exponential and first-order in
-carotene
concentration. The rate constant for the reaction obtained from the
slope of the linear plot of the first-order rate constant of the
build-up of absorption at 910 nm versus the concentration of
-carotene was identical to the corresponding plot for the decline
in
-carotene absorption at 451 nm (not shown here), both giving k
= (1.1 ± 0.1)
10
M
s
. This provided
further confirmation that no other radical species are generated under
these experimental conditions and that NO
radicals react with
-carotene exclusively by electron
abstraction to form the carotenoid radical-cation. If intermediate
[NO
-carotene]
radicals are formed they must be highly unstable, rapidly
eliminating NO
to generate the
radical-cation in <10 µs.
After 0.1 s the
[-carotene]
radical-cation absorption has decayed almost to zero (see in Fig. 1). The absorption at 910 nm decays predominantly by
second-order kinetics with a half-life which decreased with increased
radiation dose (0.2-2 Gy) as shown in Fig. 2(i.e. with increasing initial concentration of radicals). This is
attributed principally to the bimolecular decay of the radical-cation
according to .
Figure 2:
Decay kinetics of the -carotene
radical-cation. Transient absorption at 910 nm observed on reaction of
NO
radical with
-carotene showing
decay of the
-carotene radical-cation (experimental conditions as
per Fig. 1). Inset: dependence of the reciprocal of the
first half-life of the
-carotene radical-cation on the dose per
pulse (approximately 0.2-2 Gy).
The reciprocal of the first half-life of the
[-carotene]
radical-cation varied linearly with the initial radical
concentration and from the slope of the fitted straight line the rate
constant 2k
= 4.1
10
M
s
was
determined. An intercept of
< 6 s
indicated that a small fraction of the
[
-carotene]
radical-cation decays by a first-order process, quite possibly
solvation or base-catalyzed formation of the carotene hydroxy
adduct-radical, and ,
respectively.
However, due to the constraints of pH on
NO radical (see and ) and
-carotene concentration within this
experimental system, this particular aspect of the mechanism could not
be pursued. The decay of the radical-cation absorption after 0.1 s
paralleled a partial restitution of the
-carotene ground-state
absorption in the 300-600 nm region. These spectral changes were
consistent with the partial regeneration of
-carotene as a
consequence of .
Figure 3:
Carotene scavenging of the
-mercaptoethanol thiyl radical. Absorption spectra obtained on the
pulse radiolysis of N
-saturated tert-butanol/water
mixtures (50%, v/v) containing
-mercaptoethanol (10 mM)
and
-carotene (10 µM) at pH 5. Bleaching of the
-carotene ground-state absorption was observed 50 µs (solid circles) and 0.2 s (open circles) after
approximately 1-Gy pulses. Insets: optical traces at 450 nm
obtained in the absence and presence of
-mercaptoethanol. In the
presence of thiol a characteristic biphasic exponetial decline in the
-carotene ground-state absorption is
observed.
The decline in absorption at 451 nm is biphasic
with a fast step complete in 50 µs and a much slower step
complete some 80 ms later. The fast step was exponential and
first-order in
-carotene concentration, while the slower phase was
second-order, independent of
-carotene concentration, but
dependent on the radiation dose per pulse.
The observations have
been attributed to a radical-addition process in which RS radicals are scavenged by
-carotene to generate a resonance
stabilized adduct-radical
[RS
-carotene]
via . Two isobestic points are located near 380 and 520 nm,
respectively, thus indicating that the adduct-radical absorbs in the
same region of the spectrum as that of the
-carotene ground-state
absorption.
The rate constant, k = 2.5 ±
0.1
10
M
s
for the
-mercaptothiyl radical plus
-carotene was
obtained from the slope of the linear plot of the first-order rate
constant for the fast step versus
-carotene
concentration. At such low doses per pulse (
1 Gy), the slower
decline in absorption at 451 nm could have been easily mistaken for
first-order processes, including hydrogen abstraction from
-carotene (), radical-addition at alternative sites
along the polyconjugated backbone of
-carotene or as a consequence
of a reverse repair reaction, e.g.in which
-carotene could perturb a possible equilibrium reaction () by irreversibly scavenging thiyl radicals via . In the latter case the observed biphasic bleaching of
the
-carotene chromophore could reflect the fast formation of the
[RS
-carotene]
radical-adduct via and followed by
a slower formation of the
[RS
-carotene]
radical-adduct reflecting the contribution from the reverse
repair reaction ().
However, the radical species decayed slowly by pure second-order
kinetics with a half-life which decreased with increased radiation dose
(0.2-2 Gy), suggesting the
[RS-carotene]
radicals undergo bimolecular decay to products via . In Fig. 4the plot of the reciprocal of the
first half-life of the radical species varied linearly with the initial
radical concentration and from the slope of the fitted straight line
the rate constant 2k
= (2.1 ± 0.1)
10
M
s
was determined.
Figure 4:
Decay kinetics of the -carotene-thiyl
adduct-radical. Transient absorption at 450 nm observed on the reaction
of HO(CH
)
S
radical with
-carotene at pH 5 initiated by a pulse of approximately 1.8 Gy. Inset: dependence of the reciprocal of the first half-life of
the [RS
-carotene]
adduct-radical on the dose/pulse.
No intercept is observed in Fig. 4, ruling out any
contribution from the aforementioned first-order processes. The
O radical-anion has been shown to
undergo addition-elimination reactions with
-carotene(31) . An analogous reversible reaction can be
envisaged for the reaction of thiyl radicals with
-carotene, .
However, in this system the elimination of the thiyl radical
from the [RS-carotene]
radical-adduct is unable to compete with , which
effectively pulls the equilibrium position () toward
[RS
-carotene]
radical-adduct formation.
No evidence was obtained for thiol
repair of the [RS-carotene]
adduct-radical, since hydrogen atom transfer via would have regenerated another RS
radical-capable of initiating a chain reaction and further
bleaching of the
-carotene via .
Indeed no changes in the second-order decay at 450 nm were
observed when [RSH] 5-30 mM, which would
suggest that the resultant carbon-centered adduct-radical is relatively
unreactive, presumably due to resonance stabilization through
delocalization of the radical electron spin density along the
polyconjugated backbone of
-carotene.
Under the same
experimental conditions the glutathione thiyl radical
(GS) gave the same biphasic bleaching of the
-carotene ground-state absorption and exhibited similar spectral
changes previously observed for the
-mercaptothiyl radical (see Fig. 5, left and right insets). Again no
evidence was obtained for carotenoid radical-cation formation by the
GS
radical, which clearly favors the radical-addition
pathway. In this case, however, the rate constant for GS
radical-addition to
-carotene, k
= (2.2 ± 0.1)
10
M
s
is almost an
order of magnitude slower than that previously measured for the
-mercaptoethanol thiyl radical. Rate constants, k
for a variety of thiyl radical reactions with
-carotene
(together with other rate constants measured during this study) are
displayed in Table 1. Values for k
vary by
almost 2 orders of magnitude, depending on the degree of hydrophilicity
of the thiyl radical under study. The lowest rate constant, k
= (4.2 ± 0.3)
10
mM
s
, was measured
for the thiyl radical of WR-1065, which contains a doubly protonated
polyamino side chain(65) . A similar trend has been observed
for rates of thiyl radical abstraction of biallylic hydrogen atoms from
PUFAs(42) .
Figure 5:
-Carotene scavenging of the
glutathione thiyl radical. Absorption spectra obtained on the pulse
radiolysis of N
-saturated tert-butyl alcohol/water
mixtures (50%, v/v) containing GSH (10 mM) and
-carotene
(10 µM) at pH 5. bleaching of the
-carotene
ground-state absorption was observed 50 µs (solid circles)
and 0.1 s (open circles) after approximately 1-Gy pulses. Insets: optical traces at 450 nm showing exponential fast then
slower bleaching of
-carotene as observed for other
thiols.
Since thiyl radicals are also capable of
abstracting a hydrogen atom from the activated C-H bonds of
alcohols and ethers(42) , it was necessary to rule out the
possibility of hydrogen abstraction from -carotene (). Provided the experimental conditions remained
constant the rate constants for the bimolecular decay of
[
-carotene]
radicals following were expected to be independent of the nature of the
attacking thiyl radical. However, further evidence supporting the
assignment of the observed thiyl radical-mediated biphasic bleaching of
the
-carotene ground-state absorption to formation and decay of
the [RS
-carotene]
adduct-radical via and was derived
from comparing second-order rate constants, 2k
for thiyl radicals of
-mercaptoethanol and WR-1065. Addition
of the WR-1065 thiyl radical to
-carotene effectively lowered the
rate of bimolecular decay of the adduct-radicals due to mutual
electrostatic repulsion of the polyamino side chains(65) , i.e. where R =
H
N(CH
)
H
N(CH
)
-.
The measured rate constant 2k
= (5.2
± 0.1)
10
M
s
was slower than the glutathione thiyl
radical-adduct (1.3 ± 0.1)
10
M
s
and almost an
order of magnitude slower than that of the more hydrophobic
-mercaptoethanol thiyl radical-adduct (2.1 ± 0.1)
10
M
s
.
Thiyl radical-addition to
-carotene is kinetically favored
relative to hydrogen abstraction from the polyconjugated C-H
bonds.
Figure 6:
Carotene scavenging of sulfonyl radicals.
Absorption spectra showing the bleaching of the -carotene
ground-state absorption measured by pulse radiolysis (200 µs and
0.2 s after approximately 1.5-Gy pulse, respectively) of
N
-saturated tert-butyl alcohol/water mixtures
(50%, v/v) containing ethanesulfonyl chloride (10 mM) and
-carotene (10 µM) at pH 5. Insets: optical
traces showing a biphasic decline in the
-carotene ground-state
absorption at 450 nm and the exponential growth of the
-carotene
radical-cation absorption at 910 nm.
The radiation chemical yield of the
[-carotene]
radical-cation at 910 nm was determined as G
= 6.1 ± 0.2
10
M
cm
. Taking
9.4 ± 200
10
M
cm
for the
[
-carotene]
radical-cation gave a yield of G[
-carotene]
= 0.64, i.e. 71% of the original radical yield G(C
H
SO
)
= 0.9. Formation of the
[C
H
SO
-carotene]
radical-adduct therefore accounts for the remaining 29% of the
C
H
SO
radical
yield.
The fast decline in absorption at 451 nm was exponential and
first-order in -carotene concentration and reflects the
contributions from both and . The overall
rate constant for C
H
SO
radical attack on
-carotene was obtained from the slope of
the plot of observed first-order rate constant (k
) versus
-carotene concentration
and was found to be k
/k
= 5.5 ± 0.1
10
M
s
. From the
relative yields of
[
-carotene]
radical-cation to the
[C
H
SO
-carotene]
radical-adduct (i.e. 71 to 29%, respectively), the
first-order rate constants k
and k
were determined. The observed rate constants
for electron transfer (k
) and radical-addition (k
) are given by k
=
(0.71
k
) and k
= [(1 - 0.71)
k
], respectively. Fig. 7shows the
linear plots of k
and k
versus [
-carotene] the slopes of which
correspond to the rate constants k
= (3.9
± 0.1)
10
M
s
and k
= (1.6
± 0.1)
10
M
s
. The rate constant for obtained from the slope of the plot of the first-order
build up of the [
-carotene]
radical-cation absorption at 910 nm versus [
-carotene] gave k
=
(3.4 ± 0.1)
10
M
s
, in excellent agreement with that obtained
from the corresponding kinetics at 450 nm.
Figure 7:
Kinetics associated with -carotene
scavenging of the sulfonyl radical. Transient absorption at 450 nm
observed on reaction of
C
H
SO
radical
with
-carotene at pH 5 initiated by a pulse of 1.5 Gy. Inset: plots of the observed and calculated first-order rates: k
at 450 nm (solid circles), k
at 910 nm (open squares), k
at 450 nm (solid triangles) and k
at 450 nm (solid squares) versus
-carotene concentration.
Free radical oxidants, which are recognized as potential
initiators of the chain of lipid peroxidation, including
NO, RS
, and
RSO
radicals, were rapidly scavenged by
the lipid-soluble antioxidant
-carotene. Although
NO
and RS
radicals are
both moderately strong one-electron oxidants with similar redox
potentials (e.g.
E
(NO
/NO
)
= + 1.04 V) (66) and E
(HO(CH
)
S
,
H
/HO(CH
)
SH) = +
1.3 V)(67) , respectively), they interact with
-carotene
via distinct mechanisms. Nitrogen dioxide reacts exclusively by
electron transfer to generate the
[
-carotene]
radical-cation which decays via a bimolecular charge transfer
process. In marked contrast all the RS
radicals under
study undergo rapid addition reactions to generate
[RS
-carotene]
adduct-radicals. Thiyl radicals are particularly electrophilic,
and it is therefore not surprising that they add to centers of
relatively high electron density such as the polyconjugated
-system of
-carotene. This electrophilicity may be enhanced
by the ability of sulfur to use d orbitals to accommodate
negative charge. Thiyl-sulfonyl radicals can abstract an electron to
generate the polyene radical-cation (71%) or add to the polyconjugated
double bonds to generate
[RSO
-carotene]
adduct-radicals (29%).
Hydrogen abstraction by nitrogen
dioxide, thiyl, and thiyl-sulfonyl radicals from PUFAs generate
pentadienyl radicals(41, 42, 68) , which act
as radical precursors in the chain of lipid peroxidation(43) .
Although deviations from homogeneous reaction kinetics are expected
within cells, rate constants for radical-scavenging by -carotene
compare favorably with the corresponding rates of hydrogen abstraction
from PUFAs. For example, the rate constant for the oxidation of
-carotene by GS
radical, k
= (2.2 ± 0.1)
10
M
s
is almost 1
order of magnitude faster than with linoleic, linolenic, and
arachidonic acids, k = 0.8-3.1
10
M
s
(42) .
However, more lipophilic thiyl radicals, for example the
HO(CH
)
S
radical, the
differential is greater, k
= (2.5 ±
0.1)
10
M
s
as opposed to 3.1-6.8
10
M
s
for the same
range of PUFAs(42) .
The antioxidant properties of
-carotene will of course not only reflect rates of free radical
scavenging, but also the reactivity of the resultant
-carotene-derived radicals. Carotenoid radical-cations and
adduct-radicals are highly resonance stabilized and must therefore be
relatively unreactive compared with the attacking free radical
species(10) . Within lipid-rich environments, such as
biomembranes or LDL carotene, the bimolecular decay of radical-adducts
and radical-cations will generate non-radical products probably
incapable of exerting prooxidative effects. Carotenoid radical-adducts
are also likely to contribute to the antioxidant properties of
-carotene by scavenging PUFA radicals, thereby terminating the
chain of lipid peroxidation as exemplified by and .
The antioxidant efficiency of -carotene against peroxyl
radical-initiated lipid peroxidation in homogeneous solutions is
diminished with increasing partial pressure of oxygen (pO
) (18) Corroborating observations in
liposomes (69, 70) and in LDL (19) suggest
that carotenoid-peroxyl radicals may prove important in vivo particularly in tissues of high pO
such as
the lung. Based on the classical mechanisms of peroxyl radical
formation, the conjugation of molecular oxygen with
-carotene
adduct-radicals to generate a carotenoid-peroxyl radical () represents a most feasible decay pathway following
RS
radical oxidation of
-carotene. Due to
resonance stabilization within the carotenoid adduct-radical, the
reaction with oxygen may be represented as a reversible equlibrium .
High pO is likely to drive to the right, thereby promoting the autoxidation of
-carotene or PUFAs in a similar manner to other carotenoid-peroxyl
radicals(9, 18) . Theoretically, any radical species
likely to generate carotenoid adduct-radicals when scavenged by
-carotene, including thiyl-sulfonyl radicals, may initiate
prooxidative pathways at high pO
. No evidence was
obtained for the reaction of carotenoid radical-cations with molecular
oxygen. In the case of nitrogen dioxide,
-carotene is likely to
behave as a chain-breaking antioxidant, since the carotenoid
radical-cations may undergo bimolecular decay by charge transfer to
generate non-radical products, which are unlikely to exhibit
prooxidative effects. However, GSH has been shown to enhance nitrogen
dioxide-induced lipid peroxidation and DNA strand breaks in model
systems(35) , possibly via the formation of thiyl and
thiyl-derived radical species according to .
represents a stoichiometric conversion of
nitrogen dioxide to the glutathione thiyl
radical(53, 71) , which in the context of free radical
scavenging by -carotene may produce a corresponding shift from
carotenoid radical-cation to adduct-radical formation. represent the formation of
glutathione thiylperoxyl (GSOO
), sulfonyl
(GSO
), sulfonylperoxyl
(GSO
OO
), and sulfinyl
(GSO
) radicals, moderately strong oxidants which may
exert damaging effects in
vivo(44, 45, 46, 72, 73) .
The interplay of free radical reactions in oxidative stress has been
largely overlooked in studies of the antioxidant effects of
-carotene(9, 10) , a factor that may contribute
to some of the contradictory results obtained from in vitro models.