Alan Mortensen

Comparative mechanisms and rates of free radical scavenging by carotenoid antioxidants

The comparative mechanisms and relative rates of nitrogen dioxide (NO2 ⋅), thiyl (RS⋅) and sulphonyl (RSO2 ⋅) radical scavenging by the carotenoid antioxidants lycopene, lutein, zeaxanthin, astaxanthin and canthaxanthin have been determined by pulse radiolysis. All the carotenoids under study react with the NO2 ⋅ radical via electron transfer to generate the carotenoid radical cation (Car⋅+). In marked contrast the glutathione and 2‐mercaptoethanol thiyl radicals react via a radical addition process to generate carotenoid‐thiyl radical adducts [RS‐Car]⋅. The RSO2 ⋅ radical undergoes both radical addition, [RSO2‐Car]⋅ and electron abstraction, Car⋅+. Both carotenoid adduct radicals and radical cations decay bimolecularly. Absolute rate constants for radical scavenging were in the order of ∼107–109 M−1 s−1 and follow the sequence HO(CH2)2S⋅>RSO2 ⋅>GS⋅>NO2 ⋅. Although there were some discernible trends in carotenoid reactivity for individual radicals, rate constants varied by no greater than a factor of 2.5. The mechanism and rate of scavenging is strongly dependent on the nature of the oxidising radical species but much less dependent on the carotenoid structure.

Relative stability of carotenoid radical cations and homologue tocopheroxyl radicals. A real time kinetic study of antioxidant hierarchy

Real time detection following laser flash photolysis of transient carotenoid radical cations and tocopheroxyl radicals formed in chloroform and bleaching of the carotenoids has allowed interaction between carotenoids and tocopherols to be studied. It is found that α‐, β‐, and γ‐tocopherol reduce all the carotenoid radical cations investigated whereas the δ‐tocopheroxyl radical can be reduced by lycopene and β‐carotene. Astaxanthin, canthaxanthin, and β‐apo‐8′‐carotenal radical cations are scavenged rapidly by all four tocopherol homologues whereas the other carotenoid radical cations react much more slowly with the tocopherols. The results allow the antioxidant hierarchy to be established: α‐tocopherol>lycopene∼β‐tocopherol∼γ‐tocopherol>β‐carotene>zeaxanthin∼δ‐tocopherol>lutein>echinenone≫canthaxanthin∼β‐apo‐8′‐carotenal>astaxanthin.

Reactivity of β-carotene towards peroxyl radicals studied by laser flash and steady-state photolysis

Peroxyl radicals, as model for peroxyl radicals formed during autoxidation of lipids, have been generated in three solvent systems (cyclohexane, tetrahydrofuran and tert‐butanol/water) by steady‐state and laser flash photolysis, and their reaction with β‐carotene studied. Steady‐state photolysis experiments showed that alkyl, alkoxyl and alkylperoxyl radicals all react with β‐carotene. However, laser flash photolysis experiments indicated that the reaction with peroxyl radicals (second‐order rate constant estimated to be less than 106 M−1 s−1) is slower than with alkyl and alkoxyl radicals, and that β‐carotene is hence a poor direct scavenger of peroxyl radicals. Scavenging of peroxyl radicals by β‐carotene is suggested not to proceed via electron transfer but rather by adduct formation and/or hydrogen abstraction. For different phenoxyl radicals, differences in reactivity towards β‐carotene seem to be correlated with standard reduction potential.

KINETICS OF PHOTOBLEACHING OF BETA -CAROTENE IN CHLOROFORM AND FORMATION OF TRANSIENT CAROTENOID SPECIES ABSORBING IN THE NEAR INFRARED

Upon laser flash photolysis of beta-carotene in chloroform instantaneous bleaching of beta-carotene and concomitant formation of near infrared absorbing species are observed. One species, absorbing with maximum at 920 nm, is formed during the laser pulse (10 ns) and is practically gone in one millisecond, the decay showing a bi-exponential behaviour. The second species, absorbing with maximum at 1000 nm, is formed from the species absorbing at 920 nm by first order kinetics with a rate constant of 4.9.10(4) s-1 at 20 degrees C. This second species decays by second order kinetics and is gone within a few milliseconds. An additional slow bleaching of beta-carotene and formation of the species absorbing at 920 nm is observed. This slow bleaching/formation of transient absorption is probably due to processes involving free radicals generated during the instantaneous bleaching. The species absorbing at 920 nm is suggested to be either (i) a free radical adduct formed from beta-carotene and chloroform or (ii) beta-carotene after abstraction of a hydrogen atom. The species absorbing at 1000 nm is most likely the radical cation. Formation and decay of the near infrared absorbing species and bleaching of beta-carotene are independent of whether oxygen is present or absent in the solutions.

Kinetics of parallel electron transfer from beta-carotene to phenoxyl radical and adduct formation between phenoxyl radical and beta-carotene.

Phenoxyl radicals generated by laser flash photolysis were found to react with beta-carotene with concomitant beta-carotene bleaching in two parallel reactions with similar rates: (i) formation of a beta-carotene adduct with a (pseudo) first order rate constant of 1-1.5 x 10(4) s-1 with absorption maximum around 800 nm, and (ii) formation of a beta-carotene radical cation with a (pseudo) first order rate constant of 2-3 x 10(4) s-1 with absorption maximum around 920 nm. Both beta-carotene radicals decay on a similar time scale and have virtually disappeared after 100 ms, the beta-carotene adduct by a second order process. Oxygen had no effect on beta-carotene bleaching or radical formation and decay. The reduction of phenoxyl radicals by beta-carotene may prove important for an understanding of how beta-carotene acts as an antioxidant.

REAL TIME DETECTION OF REACTIONS BETWEEN RADICALS OF LYCOPENE AND TOCOPHEROL HOMOLOGUES

Laser flash photolysis of lycopene in homogeneous chloroform solution together with tocopherol homologues results in rapid formation of the lycopene radical cation and slower formation of tocopheroxyl radicals. Time-resolved detection by absorption spectroscopy of decay of the lycopene radical cation, of formation of the tocopheroxyl radicals, and of bleaching of lycopene has shown that alpha-tocopherol is able to reduce the lycopene radical cation and thereby partially regenerate lycopene on a ms timescale. In contrast, lycopene is able to reduce the delta-tocopheroxyl radical, whereas an equilibrium exists between the lycopene radical cation and beta- or gamma-tocopherol. The relative stability of these antioxidant radicals is hence: alpha-tocopheroxyl > lycopene radical cation approximately beta-tocopheroxyl approximately gamma-tocopheroxyl > delta-tocopheroxyl.

Free Radical Transients in Photobleaching of Xanthophylls and Carotenes

Carotenoids in chloroform and carbon tetrachloride photobleach upon nanosecond laser flash photolysis in two steps: instantaneously and in a second-order reaction. The rate constant for second-order reaction (first-order in a solvent derived radical and first-order in (excess) carotenoid) is largest for carotenes (9.8.10(8) M-1 for beta-carotene), intermediate for hydroxylated carotenoids, and smallest for carbonyl containing carotenoids (1.0.10(8) M-1 S-1 for astaxanthin) in chloroform at 20 degrees C. Near infrared absorbing transients are formed concomitant with photobleaching in chloroform (not detected in carbon tetrachloride). A species formed instantaneously is tentatively identified as either a carotenoid/solvent adduct or an ion-pair. A second species is formed by decay of instantaneously formed species and is identified as the carotenoid radical action. This species is formed in a first-order reaction with a rate constant of approx. 5.10(4) S-1 and absorbing at longer wavelength than the precursor. The lifetime (second-order decay) of the intermediates appears to be longest for the carotenoids with the longest conjugated system. The results indicate that carotenes are better antioxidants than xanthophylls as the carotenes, at least in the present lipophilic solvents, react faster with free radicals.

Scavenging of benzylperoxyl radicals by carotenoids.

Carotenoids scavenge simple lipid-like alkylperoxyl radicals. However, the rate constant is too low to be determined directly and the mechanism is likewise not known with certainty [Mortensen, A. and Skibsted, L.H. (1998) FEBS Lett . 426 , 392-396]. It is demonstrated that carotenoids react with peroxyl radicals only slightly more reactive than lipidperoxyl radicals neither by electron transfer nor by hydrogen atom donation, but by adduct formation. Benzylperoxyl radicals are scavenged by the carotenoids g -carotene and canthaxanthin with a second-order rate constant of at least 1 u 2 u 10 6 u M m 1 u s m 1 by formation of an adduct which decays in a first-order reaction.

The 1Bu-type singlet state of β-carotene as a precursor of the radical cation found in chloroform solution by sub-picosecond time-resolved absorption spectroscopy

Abstract Generation of the radical cation from 1B u -type states (1B u + and/or 1B u − ) of all- trans -β-carotene in chloroform was demonstrated by sub-picosecond time-resolved absorption spectroscopy. In the visible region, ground-state absorption bleaching completely recovered in n -hexane but not in chloroform. In near-infrared, broad transient absorptions from 1B u + and 1B u − states appeared and decayed within 1 ps in both solvents, while weak transient absorption ascribable to the cation appeared and stayed until 40 ps after excitation in chloroform, but not in n -hexane. In chloroform, direct transformation from the 1B u -type singlet state to the radical cation had a time constant 0.14±0.03 ps.

RE-APPRAISAL OF THE TOCOPHEROXYL RADICAL REACTION WITH BETA -CAROTENE : EVIDENCE FOR OXIDATION OF VITAMIN E BY THE BETA -CAROTENE RADICAL CATION

Photobleached beta-carotene (Car) is regenerated in hexane on a microsecond timescale in the presence of alpha-tocopherol (TOH) but not when alpha-tocopherol is absent, as studied by laser flash photolysis. Beta-carotene radical cations (Car.+) likewise react with (excess) alpha-tocopherol: Car.+ + TOH-->Car + TO. + H+ (second-order rate constant of k = 1.7 +/- 0.1 x 10(7) M(-1) s(-1) in homogeneous di-tert-butylperoxide/benzene at 20 degrees C) rather than alpha-tocopheroxyl radicals (TO.) reacting with beta-carotene. In hexane, hexane radicals formed by pulse radiolysis react considerably faster with beta-carotene (k = 2.1 +/- 0.1 x 10(9) M(-1) s(-1)) than with alpha-tocopherol (k = 4.9 +/- 0.1 x 10(6) M(-1) s(-1)). No evidence was obtained for a slower rate of beta-carotene radical cation formation in beta-carotene/alpha-tocopherol mixtures resulting from alpha-tocopheroxyl radical oxidation of beta-carotene. Steady-state radiolysis experiments confirmed that alpha-tocopherol protects beta-carotene from oxidation by hexane radicals. In both solvent systems, beta-carotene is regenerated from the radical cation by alpha-tocopherol rather than alpha-tocopherol being regenerated by beta-carotene from the alpha-tocopheroxyl radical.