Mechanisms of free-radical scavenging by the nutritional antioxidant beta-carotene.

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 [8lo]. 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 ) . l.0Xld