230s BiochemicalSociety Transactions (1995) 23
MECHANISMS O F FREE-RADICAL SCAVENGING BY
THE NUTRITIONAL ANTIOXIDANT p-CAROTENE
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STEVEN A. EVERETT*, SUBHAS. C. KUNDU,
SUSAN MADDIX and ROBIN L. WILLSON
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*Cancer Research Campaign Gray Laboratory, PO Box 100, Mount
Vernon Hospital, Northwood, Middx. HA6 2JR, UK.
Department of Biology and Biochemistry, Brunel University,
Middx. UB8 3PH, UK.
p-Carotene (pro-vitamin A) is a lipid-soluble antioxidant
capable of quenching singlet oxygen and scavenging free-radical
species [ 1-31, Epidemiological studies have established a
correlation between p-carotene dietary supplementation with a
reduced risk of cancer and heart disease thus providing a basis for
clinical investigation [4, 51. The antioxidant properties of pcarotene, perhaps in combination with its immunomodulating
properties are believed to play an important role in preventing
disease initiation and progression [6]. Despite on-going clinical
studies utilizing p-carotene in disease prevention few mechanistic
studies on the radical scavenging properties of this antioxidant
have been pursued.
A few investigations have however focused on the interaction
of reactive oxygen species (e.g. ROO', '0, and O;-) with pcarotene in either aqueous organic solvents or micellar-membrane
models utilizing the techniques of flash photolysis 171 and pulse
radiolysis [8- lo]. Halogenated peroxyl radicals (e.g. CC1,OO')
rapidly oxidise the polyconjugated diene backbone ( k = 5 X lo9
mol-' dm3 s-') predominantly by electron abstraction to generate a
p-carotene radical-cation [p-carotene]" which exhibits a
characteristic absorption at 910 nm [7, lo]. In an attempt to
diversify knowledge of radical interactions with 0-carotene a study
has been made of a range of other biologically relevant radical species
including nitrogen dioxide (NO,'), thiyl (RS') and thiyl sulphonyl
(RSO,') radicals utilizing the pulse radiolysis technique combining
optical and conductivity detection.
Cigarette smoke contains nitrogen oxides in abundance
including NO,' radicals which deplete lipid-phase antioxidants
particularly p-carotene. The [p-carotene]' radical-cation has been
generated by pulse radiolysis of solutions in which nitrate ions are
used to scavenge the hydrated electron [ 1 I]. Hydrolysis of the
intermediate NO?- generate NO,' radicals which reacts with pcarotene by electron transfer to generate the characteristic [pcarotene]* radical-cation spectrum A,,
= 910 nm. The rate of
formation of the absorption at 910 nm and corresponding rate of
bleaching of the p-carotene ground state absorption at 450 nm was
exponential and first-order in [p-carotene] gave an identical rate
constant k = I x 10' mol-' dm's-' indicating that NO,' radicals
reacts exclusively by electron abstraction.
Despite being slightly stronger oxidants than NO,' radicals
(E"(NO,'/NO,-) = 1 V), RS' radicals (E"(RS', H+/RSH) = 1.3 V)
[ 121 do not react with 0-carotene by electron abstraction. Pulse
radiolysis of an N,O-saturated ten-butanollwater (60:40%) mixtures
containing 2-mercaptoethanol generate RS' radicals immediately
after the pulse. In the presence of barotene no absorption above 600
nm and no change in conductivity were observed indicating a
complete absence of the [ p-carotene]" radical-cation. However,
optical traces recorded at 450 nm showed a fast then slow biphasic
bleaching of the p-carotene absorption (figure 1). The first fast step
was first-order in [p-carotene] but the second slower step was
second-order and dependent on the dose per pulse. The rate
constant for the first step was determined as k = 2.5 x lo9 mol-'
dm's.' indicating a rapid scavenging of RS'radicals by p-carotene
by radical-addition. The resultant thiyl-p-carotene adduct-radical
[RS...p-carotene]' is fully formed SO ps after the pulse then decays
to products 80 ms later (figure I).
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450
500
550
600
650
700
750
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Figure 1. Optical absorption spectra obtained on pulse radiolysis
of N,O-saturated terr-butanollwater (60:40%) solution containing
0-mercaptoethanol (10 mmol dm3) and p-carotene (10 pmol drn-,)
recorded at SO ps and 80 ms after a dose of = 2 Gy. Insert:
Observed bleaching of the p-carotene ground-state absorption at
4.50 nm.
No evidence was obtained for thiol repair of the [RS...P- carotene]'
adduct-radical as hydrogen transfer would have regenerated another
RS' radical to initiate a chain reaction and further bleaching of pcarotene. Indeed no changes in the second-order decay at 450 nm were
observed when [RSH] 5 - 20 mmoldm3 indicating that the carboncentred adduct-radical is relatively unreactive presumably due to
resonance stabilization.
Conjugation of RS' radicals with molecular oxygen generates
RSO,' radical which are relatively good oxidant and potential
innitiator of lipid peroxidation. Pulse radiolysis of N,-saturated
tert-butanollwater mixtures containing C,H,SO,CI generate the
C,H,SO,'radicals [ 131 which react with p-carotene by both addition
and electron abstraction as indicated by a composite absorption
spectrum containing both the radical-cation and an adduct-radical.
Clearly, p-carotene scavenges radical species by at least two
independent pathways, electron transfer and radical-addition.
-
This work is supported by the Cancer Research Campaign and the
Association for International Cancw Research
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