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.     

Molecular cloning and sequence analysis of the crtB gene of Thermus thermophilus HB27, an extreme thermophile producing carotenoid pigments.

We have cloned and sequenced a 1.5-kb chromosomal fragment of Thermus thermophilus which promoted the overproduction of carotenoids in T. thermophilus. An open reading frame (ORF-A) coding for a polypeptide with 289 amino acids was responsible for carotenoid overproduction. The putative ORF-A protein showed significant homology with the amino acid sequences of crtB gene products (phytoene syntheses) of other microorganisms. The clone containing the ORF-A on a multicopy plasmid produced about three times as much carotenoid as that produced by the host strain, suggesting that the crtB gene product is a rate-limiting enzyme for carotenoid biosynthesis in T. thermophilus.     

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.     

Carotenoid synthesis in a suspension culture of carrot cells

Biosynthesis of carotenoid in cultured carrot cells was studiedin relation to cell growth and acetate metabolism. Of the twostrains tested, one (GD-1) predominantly produces ß-caroteneand the other (GD-2) lycopene. In both strains, carotenoid wasproduced in parallel with cell growth. Incorporations of acetate-¹⁴Cinto carotenoids, organic acids and amino acids were acceleratedby increasing the concentration of 2,4-D in the medium. (Received November 17, 1970; )     

Identification of carotenoid pigments and their fatty acid esters in an avian integument combining HPLC-DAD and LC-MS analyses

Yellow-orange-red ornaments present in the integuments (feathers, bare parts) of birds are often produced by carotenoid pigments and may serve to signal the quality of the bearer. Although carotenoid esterification in tissues is a common phenomenon, most of the work on avian carotenoids has been focused on the identification of free forms or have been done after sample saponification. Here we determined free and esterified carotenoid composition in a bird species with red ornaments: the red-legged partridge (Alectoris rufa). Carotenoids from leg integument were extracted and processed by TLC to separate three major carotenoid groups (free form, mono- and diesters with fatty acids), whereas saponified extracts gave only free forms of carotenoids. TLC fractions were then analyzed by HPLC-DAD with C18 phase column for a preliminary identification of carotenoid groups. The final characterization of free carotenoids and its esters with fatty acids was performed with direct extracts analyzed by LC-MS and LC-MS/MS with a C30 phase, always with a system coupled to DAD. The main carotenoid (λ(max) 478 nm and [M+H](+) at m/z 597.2) was identified as astaxanthin by comparison with standards. A second carotenoid (λ(max) between 440 and 480 nm and [M+H](+) at m/z 581.3) was not identified among any of the commercially available carotenoid standards, although it could correspond to pectenolone according to its fragmentation pattern. Both the unidentified carotenoid and astaxanthin formed monoesters with fatty acids, but only astaxanthin was in its diesterified form. Monoesters were mainly formed with palmitic, stearic, oleic and linoleic acids. Complementary analyses of fatty acid composition in partridge integument by GC-MS revealed high amounts of these and other fatty acids, such as myristic, arachidic and docosanoic acids. The combination of HPLC-DAD and LC-MS/MS spectra was especially useful to identify the carotenoids present in the esterified forms and the probable masses of the fatty acids included in them, respectively.     

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.     

One-electron reduction potentials of dietary carotenoid radical cations in aqueous micellar environments

The one-electron reduction potentials of the radical cations of five dietary carotenoids (β-carotene, canthaxanthin, zeaxanthin, astaxanthin and lycopene) in aqueous micellar environments have been obtained from a pulse radiolysis study of electron transfer between the carotenoids and tryptophan radical cations as a function of pH, and lie in the range of 980–1060 mV. These values are consistent with our observation that the carotenoid radical cations oxidise tyrosine and cysteine. The decays of the carotenoid radical cations in the absence of added reactants suggest a distribution of exponential lifetimes. The radicals persist for up to about 1 s, depending on the medium.     

Development and Quality Evaluation of Hypoallergenic Bakery Products Suitable for Patients with Wheat-associated Allergy

The influence of growth temperature (21–41°C) and light intensity on compositions of four carotenoids, and of fatty acids from carotenoid glucoside ester (carotenoid K-G-FA) and from the cellular lipids in Rhodococcus rhodochrous RNMS1 were quantitatively investigated. Lowering the temperature increased the total carotenoid content and the proportion of carotenoid K-G-FA. It increased the proportion of unsaturated fatty acids in both carotenoid K-G-FA and the cellular lipids, decreased that of saturated ones, and slightly decreased that of branched-chain ones. This bacterium adapted itself to low temperature by desaturating its fatty acids. The light intensity affected neither the content and the composition of carotenoids nor the composition of the fatty acids in carotenoid K-G-FA and the cellular lipids. This bacterium was a scotochromogenic strain.     

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.     

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)(+).     

Isolation,characterization, and cDNA sequence of a carotenoid binding protein from the silk gland of Bombyx mori larvae

A carotenoid binding protein (CBP) has been isolated from the silk glands of Bombyx mori larvae. The protein has an apparent molecular mass of 33 kDa and binds carotenoids in a 1:1 molar ratio. Lutein accounts for 90% of the bound carotenoids, whereas alpha-carotene and beta-carotene are minor components. Immunological analysis demonstrated the presence of CBP only in the yellow-colored tissues of the silk gland, midgut, testis, and ovary. Several phenotypes of B. mori mutants linked to carotenoid transport have been utilized to characterize CBP. The Y (yellow hemolymph) gene controls uptake of carotenoids from the midgut lumen into the midgut epithelium, and larvae with the +(Y) gene lack this property. Immunoblotting analysis confirmed the presence of CBP in mutants with the dominant Y gene only. Immunohistochemistry verified the localization of CBP in the villi of the midgut epithelium, indicating that CBP might be involved in absorption of carotenoids. A cDNA clone for CBP encoding a protein of 297 amino acids has been isolated from the B. mori silk gland cDNA library. The deduced amino acid sequence revealed that CBP is a novel member of the steroidogenic acute regulatory (StAR) protein family with its unique structural feature of a StAR-related lipid transfer domain, known to aid in lipid transfer and recognition. Lutein-binding capacity of the recombinant CBP (rCBP) determined by incubating rCBP with lutein followed by immunoprecipitation using anti-CBP IgG conjugated to protein A-Sepharose, demonstrated the formation of a lutein-rCBP complex. Sequence analyses coupled with binding specificity suggest that CBP is a new member of the StAR protein family that binds carotenoids rather than cholesterol.     

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.     

An experimental test of the dose-dependent effect of carotenoids and immune activation on sexual signals and antioxidant activity

Carotenoid-based sexual traits are thought to be reliable indicators of male quality because they might be scarce and therefore might indicate the ability of males to gather high-quality food and because they are involved in important physiological functions (as immune enhancers and antioxidants). We performed an experiment where male and female zebra finches (Taeniopygia guttata) were provided with increasing carotenoid doses in the drinking water during 4 weeks (bill color of this species is a carotenoid-based sexual signal). Simultaneously, birds were split into two groups: one receiving weekly injections of Escherichia coli lipopolysaccharide in order to activate the immune system, the other being injected with the same volume of phosphate buffered saline. We assessed how carotenoid availability and immune activation affected the amount of circulating plasma carotenoids, the beak color, and the antioxidant defenses (assessed as the resistance of red blood cells to a controlled free radical attack). Carotenoid availability affected the amount of circulating carotenoids and beak color; both variables reached a plateau at the highest carotenoid doses. Immune activation diverted carotenoids from plasma, and this in turn affected the expression of the sexual trait. Finally, we found a positive correlation between the change in circulating carotenoids and antioxidant defenses. These results support the idea that carotenoids have important physiological properties that ensure the honesty of carotenoid-based sexual traits.     

Interplay of oxygen, vitamin E, and carotenoids in radical reactions following oxidation of Trp and Tyr residues in native HDL3 apolipoproteins. Comparison with LDL. A time-resolved spectroscopic analysis

It has been recently shown that the inhibition of apolipoprotein A-I (apoAI) reverse cholesterol transport activity during oxidation of HDL by myeloperoxidase may involve myeloperoxidase electron transfer pathways other than those leading to tyrosine chlorination. To better understand how such mechanisms might be initiated, the role of semioxidized Tyr and Trp residues in loss of apoAI and apolipoprotein A-II (apoAII) integrity has been assessed using selective Trp and Tyr one-electron oxidation by *Br2(-) radical-anions in HDL3 as well as in unbound apoAI and apoAII. Behavior of these radicals in apolipoprotein B of LDL has also been assessed. Formation of semioxidized Tyr in HDL3 is followed by partial repair during several milliseconds via reaction with endogenous alpha-tocopherol to form the alpha-tocopheroxyl radical. Subsequently, 2% of alpha-tocopheroxyl radical is repaired by HDL3 carotenoids. With LDL, a faster repair of semioxidized Tyr by alpha-tocopherol is observed, but carotenoid repair of alpha-tocopheroxyl radical is not. Only a small fraction of HDL3 particles contains alpha-tocopherol and carotenoids, which explains limited repair of semioxidized Tyr by alpha-tocopherol. All LDL particles normally contain multiple alpha-tocopherol and carotenoid molecules, and the lack of repair of alpha-tocopheroxyl radical by carotenoids probably results from hindered mobility of carotenoids in the lipid core. Western blots of gamma-irradiated HDL3 comparable to those reported for apoAI myeloperoxidase oxidation show that the incomplete repair of semioxidized Tyr and Trp induces apoAI and apoAII permanent damage including formation of a heterodimer of one apoAI with a monomeric apoAII at about 36 kDa.     

Identification and Subcellular Distribution of Carotenoids in the Aerobic Photosynthetic Bacterium, Pseudomonas radiora Strain MD-1

The carotenoid compositions in the aerobic methylotrophic photosyntheticbacterium, Pseudomonas radiora (Methylobacterium radiotolerans)strain MD-1, were investigated. Carotenoids bound to the BChl-proteincomplex were identified as spirilloxanthin, anhydrorhodovibrinand rhodovibrin, which have been generally found in purple photosyntheticbacteria. The ratio of BChl a to carotenoid in the BChl-proteincomplex was about 2 to 1. The transfer of excitation energyfrom carotenoids to BChl in the BChl-protein complex was observedalthough its efficiency was as low as that in the purple bacterialcomplexes which have spirilloxanthin as the major carotenoidcomponent. By contrast, carotenoic acids were found as majorforms of carotenoids in the cells, and most of them were notbound to the BChl-protein complex. These carotenoic acids werealso found as the major carotenoids in other methylotrophs thathave been characterized as aerobic photosynthetic bacteria. (Received November 28, 1994; Accepted April 21, 1995)     

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.     

Enhancing the carotenoid content of <Emphasis Type="Italic">Brassica napus</Emphasis> seeds by downregulating lycopene epsilon cyclase

The accumulation of carotenoids in higher plants is regulated by the environment, tissue type and developmental stage. In Brassica napus leaves, beta-carotene and lutein were the main carotenoids present while petals primarily accumulated lutein and violaxanthin. Carotenoid accumulation in seeds was developmentally regulated with the highest levels detected at 35-40 days post anthesis. The carotenoid biosynthesis pathway branches after the formation of lycopene. One branch forms carotenoids with two beta rings such as beta-carotene, zeaxanthin and violaxanthin, while the other introduces both beta- and epsilon-rings in lycopene to form alpha-carotene and lutein. By reducing the expression of lycopene epsilon-cyclase (epsilon-CYC) using RNAi, we investigated altering carotenoid accumulation in seeds of B. napus. Transgenic seeds expressing this construct had increased levels of beta-carotene, zeaxanthin, violaxanthin and, unexpectedly, lutein. The higher total carotenoid content resulting from reduction of epsilon-CYC expression in seeds suggests that this gene is a rate-limiting step in the carotenoid biosynthesis pathway. epsilon-CYC activity and carotenoid production may also be related to fatty acid biosynthesis in seeds as transgenic seeds showed an overall decrease in total fatty acid content and minor changes in the proportions of various fatty acids.     

Variation in the composition of fatty acids of zeaxanthin and astaxanthin monoesters in the ovary and hepatopancreas of Penaeus schmitti during ovogenesis

The carotenoid esters of the shrimp, Penaeus schmitti, were investigated by thin layer chromatography and absorption spectrophotometry. Astaxanthin monoester and zeaxanthin monoester were identified in hepatopancreas and ovaries during the sexual development. The nature of fatty acids derived from these natural esters has been determined quantitatively by gas chromatography of their methyl esters. The variations of linkage between fatty acids and carotenoids during ovogenesis are measured. The role of zeaxanthin monoester in carotenoids transfer from hepatopancreas to ovaries during this sexual development, and relations between lipid and carotenoid metabolism are discussed.     

Carotenoids and fatty acids in red yeasts Sporobolomyces roseus and Rhodotorula glutinis

Rhodotorula glutinis and Sporobolomyces roseus, grown under different aeration regimes, showed differential responses in their carotenoid content. At higher aeration, the concentration of total carotenoids increased relative to biomass and total fatty acids in R. glutinis, but the composition of carotenoids (torulene > beta-carotene > gamma-carotene > torularhodin) remained unaltered. In contrast, S. roseus responded to enhanced aeration by a shift from the predominant beta-carotene to torulene and torularhodin, indicating a biosynthetic switch at the gamma-carotene branch point of carotenoid biosynthesis. The overall levels of total carotenoids in highly aerated flasks were 0.55 mol-percent and 0.50 mol-percent relative to total fatty acids in R. glutinis and S. roseus (respectively), and 206 and 412 microg g(-1) dry weight (respectively).     

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.