EPR spin trapping detection of carbon-centered carotenoid and beta-ionone radicals

Free radical intermediates were detected by the electron paramagnetic resonance spin trapping technique upon protonation/deprotonation reactions of carotenoid and beta-ionone radical ions. The hyperfine coupling constants of their spin adducts obtained by spectral simulation indicate that carbon-centered radicals were trapped. The formation of these species was shown to be a result of chemical oxidation of neutral compounds by Fe(3+) or I(2) followed by deprotonation of the corresponding radical cations or addition of nucleophilic agents to them. Bulk electrolysis reduction of beta-ionone and carotenoids also leads to the formation of free radicals via protonation of the radical anions. Two different spin adducts were detected in the reaction of carotenoid polyenes with piperidine in the presence of 2-methyl-2-nitroso-propane (MNP). One is attributable to piperidine radicals (C(5)H(10)N*) trapped by MNP and the other was identified as trapped neutral carotenoid (beta-ionone) radical produced via protonation of the radical anion. Formation of these radical anions was confirmed by ultraviolet-visible spectroscopy. It was found that the ability of carotenoid radical anions/cations to produce neutral radicals via protonation/deprotonation is more pronounced for unsymmetrical carotenoids with terminal electron-withdrawing groups. This effect was confirmed by the radical cation deprotonation energy (H(D)) estimated by semiempirical calculations. The results indicate that the ability of carotenoid radical cations to deprotonate decreases in the sequence: beta-ionone > unsymmetrical carotenoids > symmetrical carotenoids. The minimum H(D) values were obtained for proton abstraction from the C(4) atom and the C(5)-methyl group of the cyclohexene ring. It was assumed that deprotonation reaction occurs preferentially at these positions.     

Reduction of oxidized guanosine by dietary carotenoids: A pulse radiolysis study

Time-resolved pulse radiolysis investigations reported herein show that the carotenoids β-carotene, lycopene, zeaxanthin and astaxanthin (the last two are xanthophylls - oxygen containing carotenoids) are capable of both reducing oxidized guanosine as well as minimizing its formation. The reaction of the carotenoid with the oxidized guanosine produces the radical cation of the carotenoid. This behavior contrasts with the reactions between the amino acids and dietary carotenoids where the carotenoid radical cations oxidized the amino acids (tryptophan, cysteine and tyrosine) at physiological pH.     

The interaction of dietary carotenoids with radical species

Dietary carotenoids react with a wide range of radicals such as CCl3O2*, RSO2*, NO2*, and various arylperoxyl radicals via electron transfer producing the radical cation of the carotenoid. Less strongly oxidizing radicals, such as alkylperoxyl radicals, can lead to hydrogen atom transfer generating the neutral carotene radical. Other processes can also arise such as adduct formation with sulphur-centered radicals. The oxidation potentials have been established, showing that, in Triton X-100 micelles, lycopene is the easiest carotenoid to oxidize to its radical cation and astaxanthin is the most difficult. The interaction of carotenoids and carotenoid radicals with other antioxidants is of importance with respect to anti- and possibly pro-oxidative reactions of carotenoids. In polar environments the vitamin E (alpha-tocopherol) radical cation is deprotonated (TOH*+ --> TO* + H+) and TO* does not react with carotenoids, whereas in nonpolar environments such as hexane, TOH*+ is converted to TOH by hydrocarbon carotenoids. However, the nature of the reaction between the tocopherol and various carotenoids shows a marked variation depending on the specific tocopherol homologue. The radical cations of the carotenoids all react with vitamin C so as to "repair" the carotenoid.     

Excitation energy transfer and carotenoid radical cation formation in light harvesting complexes — A theoretical perspective

Light harvesting complexes have been identified in all chlorophyll-based photosynthetic organisms. Their major function is the absorption of light and its transport to the reaction centers, however, they are also involved in excess energy quenching, the so-called non-photochemical quenching (NPQ). In particular, electron transfer and the resulting formation of carotenoid radical cations have recently been discovered to play an important role during NPQ in green plants. Here, the results of our theoretical investigations of carotenoid radical cation formation in the major light harvesting complex LHC-II of green plants are reported. The carotenoids violaxanthin, zeaxanthin and lutein are considered as potential quenchers. In agreement with experimental results, it is shown that zeaxanthin cannot quench isolated LHC-II complexes. Furthermore, subtle structural differences in the two lutein binding pockets lead to substantial differences in the excited state properties of the two luteins. In addition, the formation mechanism of carotenoid radical cations in light harvesting complexes LH2 and LH1 of purple bacteria is studied. Here, the energetic position of the S₁ state of the involved carotenoids neurosporene, spheroidene, spheroidenone and spirilloxanthin seems to determine the occurrence of radical cations in these LHCs upon photo-excitation. An elaborate pump-deplete-probe experiment is suggested to challenge the proposed mechanism.     

Studies of carotenoid one-electron reduction radicals

The relative reduction potentials of a variety of carotenoids have been established by monitoring the reaction of carotenoid radical anion (CAR1(*-)) with another carotenoid (CAR2) in hexane and benzene. This order is consistent with the reactivities of the carotenoid radical anions with porphyrins and oxygen in hexane. In addition, investigation of the reactions of carotenoids with reducing radicals in aqueous 2% Triton-X 100, such as carbon dioxide radical anion (CO2(*-)), acetone ketyl radical (AC(*-)) and the corresponding neutral radical (ACH(*)), reveals that the reduction potentials for beta-carotene and zeaxanthin lie in the range -1950 to -2100 mV and those for astaxanthin, canthaxanthin and beta-apo-8'-carotenal are more positive than -1450 mV. This illustrates that the presence of a carbonyl group causes the reducing ability to decrease. The radical cations have been previously shown to be strong oxidising agents and we now show that the radical anions are very strong reducing agents.     

Construction and characterization of genetically modified synechocystis sp. PCC 6803 photosystem II core complexes containing carotenoids with shorter pi-conjugation than beta-carotene

Beta-carotene has been identified as an intermediate in a secondary electron transfer pathway that oxidizes Chl(Z) and cytochrome b(559) in Photosystem II (PS II) when normal tyrosine oxidation is blocked. To test the redox function of carotenoids in this pathway, we replaced the zeta-carotene desaturase gene (zds) or both the zds and phytoene desaturase (pds) genes of Synechocystis sp. PCC 6803 with the phytoene desaturase gene (crtI) of Rhodobacter capsulatus, producing carotenoids with shorter conjugated pi-electron systems and higher reduction potentials than beta-carotene. The PS II core complexes of both mutant strains contain approximately the same number of chlorophylls and carotenoids as the wild type but have replaced beta-carotene (11 double bonds), with neurosporene (9 conjugated double bonds) and beta-zeacarotene (9 conjugated double bonds and 1 beta-ionylidene ring). The presence of the ring appears necessary for PS II assembly. Visible and near-infrared spectroscopy were used to examine the light-induced formation of chlorophyll and carotenoid radical cations in the mutant PS II core complexes at temperatures from 20 to 160 K. At 20 K, a carotenoid cation radical is formed having an absorption maximum at 898 nm, an 85 nm blue shift relative to the beta-carotene radical cation peak in the WT, and consistent with the formation of the cation radical of a carotenoid with 9 conjugated double bonds. The ratio of Chl(+)/Car(+) is higher in the mutant core complexes, consistent with the higher reduction potential for Car(+). As the temperature increases, other carotenoids become accessible to oxidation by P(680)(+).     

Macular and serum carotenoid concentrations in patients with malabsorption syndromes

The carotenoids lutein and zeaxanthin are believed to protect the human macula by absorbing blue light and quenching free radicals. Intestinal malabsorption syndromes such as celiac and Crohn’s disease are known to cause deficiencies of lipid-soluble nutrients. We hypothesized that subjects with nutrient malabsorption syndromes will demonstrate lower carotenoid levels in the macula and blood, and that these lower levels may correlate with early-onset maculopathy. Resonance Raman spectrographic (RRS) measurements of macular carotenoid levels were collected from subjects with and without a history of malabsorption syndromes. Carotenoids were extracted from serum and analyzed by high performance liquid chromatography (HPLC). Subjects with malabsorption (n = 22) had 37% lower levels of macular carotenoids on average versus controls (n = 25, P < 0.001). Malabsorption was not associated with decreased serum carotenoid levels. Convincing signs of early maculopathy were not observed. We conclude that intestinal malabsorption results in lower macular carotenoid levels.     

Oxidation of the two beta-carotene molecules in the photosystem II reaction center

We present a spectroscopic characterization of the two nonequivalent beta-carotene molecules in the photosystem II reaction center. Their electronic and vibrational properties exhibit significant differences, reflecting a somewhat different configuration for these two cofactors. Both carotenoid molecules are redox-active and can be oxidized by illumination of the reaction centers in the presence of an electron acceptor. The radical cation species show similar differences in their spectroscopic properties. The results are discussed in terms of the structure and unusual function of these carotenoids. In addition, the attribution of resonance Raman spectra of photosystem II preparations excited in the range 800-900 nm is discussed. Although contributions of chlorophyll cations cannot be formally ruled out, our results demonstrate that these spectra mainly arise from the cation radical species of the two carotenoids present in photosystem II reaction centers.     

Carotenoids from further prasinophytes

The qualitative and quantitative carotenoid composition of seven prasinophytes (eight clones) have been examined by chromatographic (TLC and HPLC) and spectroscopic methods (VIS, CD and mass spectra).

The prasinophytes studied fall into two pigment types: (A) those producing common green algal carotenoids (β,β-carotene, β,ε-carotene, lutein, zeaxanthin and the epoxides violaxanthin and neoxanthin) and (B) prasinophytes synthesising carotenoids peculiar to this algal class (prasinoxanthin, anhydroprasinoxanthin, uriolide, anhydrouriolide, micromonal, anhydromicromonal, micromonol, anhydromicromonol and dihydrolutein), where prasinoxanthin is a major carotenoid.

Mantoniella squamata (clone 2) was grown under both low and high light intensity, revealing differences in carotenoid composition. Lutein together with lesser amounts of zeaxanthin and its epoxides were only detected at high light intensity.

Three previously unidentified carotenoids were identified as prasinoxanthin (xanthophyll K), micromonal and dihydrolutein.     

The reactivity of carotenoid radicals with oxygen

The possibility that carotenoid radicals react with oxygen to form chain-carrying peroxyl radicals has been postulated to account for the reduction in antioxidant effetiveness displayed by some carotenoids at high oxygen concentrations. The primary objective of the work described in this paper was to measure the rate constants for oxygen addition to a series of carotenoid radicals and to examine any influence of carotenoid structural features on these rate constants. Laser flash photolysis has been used to generate long-lived carotenoid radicals (PhS-CAR^√) derived from radical addition reactions with phenylthiyl radicals (PhS^√) in benzene. The PhS-CAR^√ radicals are scavenged by oxygen at rates that display a moderate dependence on the number of conjugated double bonds (n_db) in the carotenoid. The rate constants range from ∼10³ to ∼10⁴ M^- 1 s^- 1 for n_db = 7-11. The data also suggest that the presence of terminal cyclic groups may cause an increase in the rate constant for oxygen addition.     

Free Radical Transients in Photobleaching of Xanthophylls and Carotenes

Carotenoicls in chloroform and carbon tetrachloriclc 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) ccirotenoid) is largest for carotenes (9.8·10⁸ M^-1 s^-1 for β-carotene), intermediate for hydroxylated carotenoids, and smallest for carbonyl containing carotenoids (1.0·10⁸ M^-1 s^-1 for astaxanthin) in chloroform at 20°C. Near infrared, ibsorbing transients are formed concomitant with pliotohleaching in chloroform (not detected in cxbon 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 the instantaneously formed species and is identified as the carotenoid radical cation. This species is formed in a first-order reaction with a rate constant of approx. 5·10⁴ s^-1 and absorbing at longer wavelength than the precursor. The lifetime (second-order decay) of the interniediates appears to be longest for the carotenoids with the longest conjugated system. The results indicate that carotenes are better antioxidants than xantliophylls as the carotenes, at least in the present lipophilic solvents, react faster with free radicals.     

Algal carotenoids 52; secondary carotenoids of algae 3; carotenoids in a natural bloom of Euglena sanguinea

Quantitative carotenoid analysis of a natural bloom of Euglena sanguinea Ehrenberg revealed the presence of β,β-carotene (1% of total carotenoids), monoesters of adonirubin (3%), diesters of (3S, 3′R)-adonixanthin (13%), diesters of (3S, 3′S)-astaxanthin (75%), 19-monoester of (3R, 3′R, 6R)-loroxanthin (1%), (3R, 3′R)-diatoxanthin (6%), diadinoxanthin (1%) and neoxanthin (traces). The carotenoid content amounted to 0.7% of the dry wt. Methods employed included TLC, HPLC, VIS, MS, CD and H NMR (400 and 500 MHz). The high content of ketocarotenoids is characteristic of secondary carotenoids produced under stressed growth conditions. Previously secondary carotenoids were associated with green algae (Chlorophyceae), but have now been encountered in Euglenophyceae.     

Addition of lutein, lycopene, or β-carotene to LDL or serum in vitro: Effects on carotenoid distribution, LDL composition, and LDL oxidation

Carotenoids are dietary antioxidants transported with plasma lipoproteins, primarily low-density lipoprotein (LDL). In this study in vitro methods were used to increase the amounts of specific, individual carotenoids in LDL. By addition of carotenoid to isolated LDL or to serum, followed by (re)isolation of the lipoproteins, samples of LDL were enriched 4- to 150-fold with lutein, 2- to 15-fold with lycopene, or 3- to 25-fold with β-carotene. Enrichment with specific carotenoids was achieved without affecting the electrophoretic mobility of the lipoprotein, its cholesterol to protein ratio, or the levels of other cartenoids or -tocopherol. The distributions among lipoproteins of carotenoid added to serum were similar, but not identical, to the distributions of the endogenous carotenoids. In particular, for added lutein, a greater proportion was found in HDL, and for added β-carotene, more was found in very low-density lipoprotein (VLDL). We then studied the effect of enriching LDL with specific carotenoids on its susceptibility to oxidation by copper ions. Lutein, β-cryptoxanthin, lycopene, and β-carotene, the four major plasma carotenoids, and -tocopherol were destroyed before the formation of lipid peroxidation products. The rates of destruction of the individual carotenoids differed; lycopene was destroyed most rapidly and lutein most slowly. Upon oxidation of β-carotene-enriched LDL, the rates of destruction of β-carotene, lycopene, and lutein were slowed and the lag times before the initiation of lipid peroxidation increased from 19 to 65 min. Neither effect was observed in LDL enriched with lutein or lycopene. Thus, β-carotene was unique among the carotenoids studied in having a small, but significant effect on LDL oxidation in vitro.     

Carotenoid radical cations and dications: EPR, optical, and electrochemical studies

Investigations of the structure and properties of paramagnetic carotenoid radical cations and diamagnetic carotenoid dications using electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) spectroscopy in conjunction with electrochemical, optical, and HPLC measurements, and molecular orbital calculations are described. These methods were applied to determine how the carotenoid radical cations and dications can be formed, their electron-transfer properties and stability in various media, and the mechanism by which carotenoid radical cations can isomerize.     

Carotenoid photooxidation in photosystem II

Carotenoids are known to function as light-harvesting pigments and they play important roles in photoprotection in both plant and bacterial photosynthesis. These functions are also important for carotenoids in photosystem II. In addition, beta-carotene recently has been found to function as a redox intermediate in an alternate pathway of electron transfer within photosystem II. This redox role of a carotenoid in photosystem II is unique among photosynthetic reaction centers and stems from the very highly oxidizing intermediates that form in the process of water oxidation. In this minireview, an overview of the electron-transfer reactions in photosystem II is presented, with an emphasis on those involving carotenoids. The carotenoid composition of photosystem II and the physical methods used to study the structure of the redox-active carotenoid are reviewed. Possible roles of carotenoid cations in photoprotection of photosystem II are discussed.     

The carotenoids of blue-green algae

The carotenoid compositions of Phormidium persicinum, P. luridum, P. faveolarum and Anabaena flos-aquae have been studied, both quantitatively and qualitatively. β-Carotene is the major carotenoid in all species. The xanthophylls comprise zeaxanthin, echinenone, canthaxanthin and the furanoid mutatochrome. Phormidium persicinum lacks glycosidic carotenoids. Myxoxanthophyll (myxol-2′-rhamnoside) and a 4-ketomyxol-2′-methylpentoside (tentatively 4-keto-myxoxanthophyll) are present in the other species. These distribution patterns are compared with those observed in other blue-green algae and some correlations with taxonomy are apparent.     

Carotenoids of Heterosigma akashiwo: A chemosystematic contribution

The presence of C₃₇-norcarotenoids (peridinin and probably pyrrhoxanthin, together 87% of total carotenoids) and the carotenoid pattern in general, including dinoxanthin, diatoxanthin and β,β-carotene, but no fucoxanthin, strongly suggest that H. akashiwo is a dinoflagellate and not a chrysophyte.     

Antioxidant activity of carotenoids: An electron-spin resonance study on β-carotene and lutein interaction with free radicals generated in a chemical system

β-Carotene is thought to be a chain-breaking antioxidant, even though we have no information about the mechanism of its antioxidant activity. Using electron-spin resonance (ESR) spectroscopy coupled to the spin-trapping technique, we have studied the effect of β-carotene and lutein on the radical adducts of the spin-trap PBN (N-t -butyl-α-phenylnitrone) generated by the metal-ion breakdown of different tert -butyl hydroperoxide (t BOOH) concentrations in methylene chloride. The peroxyl radical, along with an oxidation product of PBN (the PBNOx), trapped at room temperature from the breakdown of high concentration of t BOOH (1 M), were quenched by β-carotene or lutein, in competition with the spin-trapping agent. However, carotenoids were not able to quench the alkoxyl and methyl radicals generated in the reaction carried out in the presence of low t BOOH concentration (1 mM). The reaction between carotenoids and the peroxyl radical was also carried out in the absence of the spin trap, at 77 K: Under these different experimental conditions, we did not detect any radical species deriving from carotenoids. In the same system, a further evidence of the peroxyl radical quenching by β-carotene and lutein was obtained. The antioxidant activity of vitamin E was also tested, for comparison with the carotenoids. In the presence of α-tocopherol, peroxyl and alkoxyl radicals were quenched, and the tocopheroxyl radical was detected. Our data provide the first direct evidence that carotenoids quench peroxyl radicals. Under our experimental conditions, we did not detect any carotenoid radical species that could derive from the interaction with the peroxyl radical. The radical-trapping activity of β-carotene and lutein demonstrated in this chemical reaction contributes to our understanding carotenoid antioxidant action in biological systems. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 299–304, 1998     

Carotenoid radical chemistry and antioxidant/pro-oxidant properties

The purpose of this review is to summarise the current state of knowledge of (i) the kinetics and mechanisms of radical reactions with carotenoids, (ii) the properties of carotenoid radicals, and (iii) the antioxidant/pro-oxidant properties of carotenoids.     

What Does Carotenoid-Dependent Coloration Tell? Plasma Carotenoid Level Signals Immunocompetence and Oxidative Stress State in Birds–A Meta-Analysis

Mechanisms maintaining honesty of sexual signals are far from resolved, limiting our understanding of sexual selection and potential important parts of physiology. Carotenoid pigmented visual signals are among the most extensively studied sexual displays, but evidence regarding hypotheses on how carotenoids ensure signal honesty is mixed. Using a phylogenetically controlled meta-analysis of 357 effect sizes across 88 different species of birds, we tested two prominent hypotheses in the field: that carotenoid-dependent coloration signals i) immunocompetence and/or ii) oxidative stress state. Separate meta-analyses were performed for the relationships of trait coloration and circulating carotenoid level with different measures of immunocompetence and oxidative stress state. For immunocompetence we find that carotenoid levels (r = 0.20) and trait color intensity (r = 0.17) are significantly positively related to PHA response. Additionally we find that carotenoids are significantly positively related to antioxidant capacity (r = 0.10), but not significantly related to oxidative damage (r = −0.02). Thus our analyses provide support for both hypotheses, in that at least for some aspects of immunity and oxidative stress state the predicted correlations were found. Furthermore, we tested for differences in effect size between experimental and observational studies; a larger effect in observational studies would indicate that co-variation might not be causal. However, we detected no significant difference, suggesting that the relationships we found are causal. The overall effect sizes we report are modest and we discuss potential factors contributing to this, including differences between species. We suggest complementary mechanisms maintaining honesty rather than the involvement of carotenoids in immune function and oxidative stress and suggest experiments on how to test these.