The Endless World of Carotenoids-Structural, Chemical and Biological Aspects of Some Rare Carotenoids.

Carotenoids are a large and diverse group of compounds that have been shown to have a wide range of potential health benefits. While some carotenoids have been extensively studied, many others have not received as much attention. Studying the physicochemical properties of carotenoids using electron paramagnetic resonance (EPR) and density functional theory (DFT) helped us understand their chemical structure and how they interact with other molecules in different environments. Ultimately, this can provide insights into their potential biological activity and how they might be used to promote health. In particular, some rare carotenoids, such as sioxanthin, siphonaxanthin and crocin, that are described here contain more functional groups than the conventional carotenoids, or have similar groups but with some situated outside of the rings, such as sapronaxanthin, myxol, deinoxanthin and sarcinaxanthin. By careful design or self-assembly, these rare carotenoids can form multiple H-bonds and coordination bonds in host molecules. The stability, oxidation potentials and antioxidant activity of the carotenoids can be improved in host molecules, and the photo-oxidation efficiency of the carotenoids can also be controlled. The photostability of the carotenoids can be increased if the carotenoids are embedded in a nonpolar environment when no bonds are formed. In addition, the application of nanosized supramolecular systems for carotenoid delivery can improve the stability and biological activity of rare carotenoids.

Carotenoids: Importance in Daily Life-Insight Gained from EPR and ENDOR.

Carotenoids are indispensable molecules for life. They are present everywhere in plants, algae, bacteria whom they protect against free radicals and oxidative stress. Through the consumption of fruits and vegetables and some carotenoid-containing fish, they are introduced into the human body and, similarly, protect it. There are numerous health benefits associated with the consumption of carotenoids. Carotenoids are antioxidants but at the same time they are prone to oxidation themselves. Electron loss from the carotenoid forms a radical cation. Furthermore, proton loss from a radical cation forms a neutral radical. In this mini-review, we discuss carotenoid radicals studied in our groups by various physicochemical methods, namely the radical cations formed by electron transfer and neutral radicals formed by proton loss from the radical cations. They contain many similar hyperfine couplings due to interactions between the electron spin and numerous protons in the carotenoid. Different EPR and ENDOR methods in combination with DFT calculations have been used to distinguish the two independent carotenoid radical species. DFT predicted larger coupling constants for the neutral radical compared to the radical cation. Previously, INDO calculations miss assigned the large couplings to the radical cation. EPR and ENDOR have aided in elucidating the physisorb, electron and proton transfer processes that occur when carotenoids are adsorbed on solid artificial matrices, and predicted similar reactions in aqueous solution or in plants. After years of study of the physicochemical properties of carotenoid radicals, the different published results start to merge together for a better understanding of carotenoid radical species and their implication in biological systems. Up until 2008, the radical chemistry in artificial systems was elucidated but the correlation between quenching ability and neutral radical formation was an inspiration to look for these radical species in vivo. In addition, the EPR spin-trapping technique has been applied to study inclusion complexes of carotenoids with different delivery systems.

Antioxidant Activity in Supramolecular Carotenoid Complexes Favored by Nonpolar Environment and Disfavored by Hydrogen Bonding.

Carotenoids are well-known antioxidants. They have the ability to quench singlet oxygen and scavenge toxic free radicals preventing or reducing damage to living cells. We have found that carotenoids exhibit scavenging ability towards free radicals that increases nearly exponentially with increasing the carotenoid oxidation potential. With the oxidation potential being an important parameter in predicting antioxidant activity, we focus here on the different factors affecting it. This paper examines how the chain length and donor/acceptor substituents of carotenoids affect their oxidation potentials but, most importantly, presents the recent progress on the effect of polarity of the environment and orientation of the carotenoids on the oxidation potential in supramolecular complexes. The oxidation potential of a carotenoid in a nonpolar environment was found to be higher than in a polar environment. Moreover, in order to increase the photostability of the carotenoids in supramolecular complexes, a nonpolar environment is desired and the formation of hydrogen bonds should be avoided.

Supramolecular Carotenoid Complexes of Enhanced Solubility and Stability-The Way of Bioavailability Improvement.

Carotenoids are natural dyes and antioxidants widely used in food processing and in therapeutic formulations. However, their practical application is restricted by their high sensitivity to external factors such as heat, light, oxygen, metal ions and processing conditions, as well as by extremely low water solubility. Various approaches have been developed to overcome these problems. In particular, it was demonstrated that application of supramolecular complexes of "host-guest" type with water-soluble nanoparticles allows minimizing the abovementioned disadvantages. From this point of view, nanoencapsulation of carotenoids is an effective strategy to improve their stability during storage and food processing. Also, nanoencapsulation enhances bioavailability of carotenoids via modulating their release kinetics from the delivery system, influencing the solubility and absorption. In the present paper, we present the state of the art of carotenoid nanoencapsulation and summarize the data obtained during last five years on preparation, analysis and reactivity of carotenoids encapsulated into various nanoparticles. The possible mechanisms of carotenoids bioavailability enhancement by multifunctional delivery systems are also discussed.

Photo-induced charge separation in hydroxycoumarins on TiO2 and F-TiO2.

The efficiency of photocatalytic charge separation is much higher for 7-hydroxycoumarin (7-CN) and 6,7-dihydroxycoumarin (6,7-CN) adsorbed on the surface modified TiO2 where the surface hydroxyl group was replaced by a fluorine atom (F-TiO2) than on TiO2. EPR measurements find 5- and 12-fold increases in free radical yields for 7-CN and 6,7-CN, respectively. DFT calculations for the coumarins on TiO2 and F-TiO2 were performed to investigate these phenomena. The calculations show that when the coumarins act as the H-bond donors, the driving force for photo-induced electron transfer from the dyes to TiO2 is higher, and the dye's excited state mixes strongly with the TiO2 conduction band states. This is attributed to the shorter distance between the coumarins and the surface of TiO2 when the coumarins act as the H-bond donors. These calculations explain why the efficiency of charge separation of the coumarins is much higher on F-TiO2 than on TiO2.

Photo Protection of Haematococcus pluvialis Algae by Astaxanthin: Unique Properties of Astaxanthin Deduced by EPR, Optical and Electrochemical Studies.

Abstract The antioxidant astaxanthin is known to accumulate in Haematococcus pluvialis algae under unfavorable environmental conditions for normal cell growth. The accumulated astaxanthin functions as a protective agent against oxidative stress damage, and tolerance to excessive reactive oxygen species (ROS) is greater in astaxanthin-rich cells. The detailed mechanisms of protection have remained elusive, however, our Electron Paramagnetic Resonance (EPR), optical and electrochemical studies on carotenoids suggest that astaxanthin's efficiency as a protective agent could be related to its ability to form chelate complexes with metals and to be esterified, its inability to aggregate in the ester form, its high oxidation potential and the ability to form proton loss neutral radicals under high illumination in the presence of metal ions. The neutral radical species formed by deprotonation of the radical cations can be very effective quenchers of the excited states of chlorophyll under high irradiation.

Radicals formed from proton loss of carotenoid radical cations: A special form of carotenoid neutral radical occurring in photoprotection.

In an organized assembly in Arabidopsis thaliana plant, proton loss from the radical cation of zeaxanthin (Zea+) was found to occur under intense illumination, a possible component in photoprotection. A stable neutral radical is formed because of the favorable proton loss at C4(4') position(s) of the terminal ends of Zea+ that extends the unpaired spin density distribution (notation Zea(4) or Zea(4') by symmetry). Proton loss from the radical cation of ß-carotene (ß-car+) to available proton acceptors was also detected in a PSII sample upon irradiation. A controversial optical absorption peak at 750nm predicted by DFT was attributed to ß-carotene neutral radical formed by proton loss at C4(4') position(s) situated on the terminal end(s) (notation ß-car(4) or ß-car(4') by symmetry), also detected in solid copper-containing MCM-41 molecular sieves (Cu-MCM-41) and by hydrogen atom transfer from ß-carotene to hydroxyl radical. Unlike the PSII organized assembly where proton loss occurs from the terminal ends of the radical cation, in the Cu-MCM-41 unorganized assembly proton loss occurs from all positions including the methyl groups on the polyene chain forming neutral radicals with different EPR couplings, different unpaired spin density distribution and different optical absorption peaks. We emphasize here the properties of these neutral radicals for various carotenoids formed under high-light conditions and in different media (solution, solid siliceous materials, and photosynthetic samples).

Hydrogen Bond Formation between the Carotenoid Canthaxanthin and the Silanol Group on MCM-41 Surface.

The formation of one or two hydrogen bonds (H-bonds) between canthaxanthin (CAN), a dye, and the silanol group(s) on the MCM-41 surface has been studied by density functional theory (DFT) calculations and calorimetric experiments. It was found that the formation of the H-bond(s) stabilized the CAN molecule more than its radical cation (CAN(•+)). The charge distribution, bond lengths, and the HOMO and LUMO energies of CAN are also affected. The formation of the H-bond(s) explains the lower photoinduced electron transfer efficiency of CAN imbedded in Cu-MCM-41 versus that for ß-carotene (CAR) imbedded in Cu-MCM-41 where complex formation with Cu(2+) dominates. These calculations show that to achieve high electron transfer efficiency for a dye-sensitized solar cell, H-bonding between the dye and the host should be avoided.

Water soluble biocompatible vesicles based on polysaccharides and oligosaccharides inclusion complexes for carotenoid delivery.

Since carotenoids are highly hydrophobic, air- and light-sensitive hydrocarbon compounds, developing methods for increasing their bioavailability and stability towards irradiation and reactive oxygen species is an important goal. Application of inclusion complexes of "host-guest" type with polysaccharides and oligosaccharides such as arabinogalactan, cyclodextrins and glycyrrhizin minimizes the disadvantages of carotenoids when these compounds are used in food processing (colors and antioxidant capacity) as well as for production of therapeutic formulations. Cyclodextrin complexes which have been used demonstrated enhanced storage stability but suffered from poor solubility. Polysaccharide and oligosaccharide based inclusion complexes play an important role in pharmacology by providing increased solubility and stability of lipophilic drugs. In addition they are used as drug delivery systems to increase absorption rate and bioavailability of the drugs. In this review we summarize the existing data on preparation methods, analysis, and chemical reactivity of carotenoids in inclusion complexes with cyclodextrin, arabinogalactan and glycyrrhizin. It was demonstrated that incorporation of carotenoids into the "host" macromolecule results in significant changes in their physical and chemical properties. In particular, polysaccharide complexes show enhanced photostability of carotenoids in water solutions. A significant decrease in the reactivity towards metal ions and reactive oxygen species in solution was also detected.

Chemistry of carotenoid neutral radicals.

Proton loss from the carotenoid radical cations (Car(+)) to form neutral radicals (#Car) was investigated by numerous electrochemical, EPR, ENDOR and DFT studies described herein. The radical cation and neutral radicals were formed in solution electrochemically and stabilized on solid silica-alumina and MCM-41 matrices. Carotenoid neutral radicals were recently identified in Arabidopsis thaliana plant and photosystem II samples. Deprotonation at the terminal ends of a zeaxanthin radical cation could provide a secondary photoprotection pathway which involves quenching excited state chlorophyll by the long-lived zeaxanthin neutral radicals formed.

DFT and ENDOR Study of Bixin Radical Cations and Neutral Radicals on Silica-Alumina.

Bixin, a carotenoid found in annatto (Bixa orellana), is unique among natural carotenoids by being water-soluble. We stabilized free radicals from bixin on the surface of silica-alumina (Si-Al) and characterized them by pulsed electron-nuclear double resonance (ENDOR). DFT calculations of unpaired electron spin distribution for various bixin radicals predict the EPR hyperfine couplings. Least-square fitting of experimental ENDOR spectra by spectra calculated from DFT hyperfine couplings characterized the radicals trapped on Si-Al. DFT predicts that the trans bixin radical cation is more stable than the cis bixin radical cation by 1.26 kcal/mol. This small energy difference is consistent with the 26% trans and 23% cis radical cations in the ENDOR spectrum. The remainder of the ENDOR spectrum is due to several neutral radicals formed by loss of a H(+) ion from the 9, 9', 13, or 13' methyl group, a common occurrence in all water-insoluble carotenoids previously studied. Although carboxyl groups of bixin strongly affect its solubility relative to other natural carotenoids, they do not alter properties of its free radicals based on DFT calculations and EPR measurements which remain similar to typical water-insoluble carotenoids.

A DFT study of the interaction between olefins and Cu2+ on silica and MCM-41 model surfaces.

The interaction between ethylene and Cu(2+) on a silica model surface was studied by density functional theory (DFT) with nine popular functionals. It is found that B3LYP with BSSE correction is the best method by comparing the calculated results with reported experimental data. This method was also used to study the interactions of Cu(2+) with ß-carotene, 1,3,5,7,9,11,13-tetradecaheptaene and ethylene on a MCM-41 model surface. The relationship between the reorganization energy of an olefin and its conjugation length was studied, and the roles of the electrostatic interaction between the olefin and the Cu(2+) were investigated. It is also found that the different environments of Cu(2+) affect the Cu(2+)-olefin interaction significantly.

Electrochemical study of astaxanthin and astaxanthin n-octanoic monoester and diester: tendency to form radicals.

The carotenoid astaxanthin known for its powerful antioxidant activity was electrochemically investigated along with the synthesized astaxanthin n-octanoic monoester and astaxanthin n-octanoic diester. Cyclic voltammograms (CVs) revealed a two-electron transfer oxidation for all three carotenoids with a difference in the two oxidation potentials (ΔE = E2(0) - E1(0)) slightly increasing from astaxanthin to the monoester to diester. Minimal or no exposure to water prevented the formation of carotenoid neutral radicals from dications and radical cations, causing near absence of the fifth peak in the CVs. This makes the CVs almost reversible and enables a more precise simulation of the redox potentials and the equilibrium constants for the formation of radical cations. The first oxidation potential (E1(0)) of 0.7678, 0.7738, and 0.7753 V versus SCE and the second oxidation potential (E2(0)) of 0.9828, 0.9931, and 0.9966 V versus SCE for astaxanthin, monoester, and diester, respectively, have been standardized to the potential of ferrocene of 0.528 V vs SCE given in a previous study. Reduction potentials (E3(0)) for formation of carotenoid neutral radicals from dications after proton loss from the three studied carotenoids are presented and compared to those of other carotenoids. According to our DFT calculations, the most favorable sites for deprotonation of radical cations and dications are found on the cyclohexene rings. These measurements provide insight into important properties of these carotenoids like radical scavenging of (•)OH, (•)CH3, and (•)OOH by proton abstraction from the carotenoid or the formation of carotenoid neutral radicals from radical cations which can quench photoexcited states. There is no essential difference in first oxidation potentials for the three carotenoids, which suggests a similar scavenging rate of the esters of astaxanthin toward (•)OH, (•)CH3, and (•)OOH radicals when compared to astaxanthin itself. The large equilibrium constants K(com) (102.4, 409.6, and 204.8 for astaxanthin, monoester, and diester) derived from simulation indicate a preference for radical cation formation for both astaxanthin and its esters, while electron transfer to form dications will be unlikely. Proton transfer from the radical cations, which are weak acids, to the neighboring proton acceptors will form neutral radicals, which allows quenching of excited states.

Photochemical and optical properties of water-soluble xanthophyll antioxidants: aggregation vs complexation.

Xanthophyll carotenoids can self-assemble in aqueous solution to form J- and H-type aggregates. This feature significantly changes the photophysical and optical properties of these carotenoids, and has an impact on solar energy conversion and light induced oxidative damage. In this study we have applied EPR and optical absorption spectroscopy to investigate how complexation can affect the aggregation ability of the xanthophyll carotenoids zeaxanthin, lutein, and astaxanthin, their photostability, and antioxidant activity. It was shown that complexation with the polysaccharide arabinogalactan (AG) polymer matrix and the triterpene glycoside glycyrrhizin (GA) dimer reduced the aggregation rate but did not inhibit aggregation completely. Moreover, these complexants form inclusion complexes with both monomer and H-aggregates of carotenoids. H-aggregates of carotenoids exhibit higher photostability in aqueous solutions as compared with monomers, but much lower antioxidant activity. It was found that complexation increases the photostability of both monomers and the aggregates of xanthophyll carotenoids. Also their ability to trap hydroperoxyl radicals increases in the presence of GA as the GA forms a donutlike dimer in which the hydrophobic polyene chain of the xanthophylls and their H-aggregates lies protected within the donut hole, permitting the hydrophilic ends to be exposed to the surroundings.

EPR study of the astaxanthin n-octanoic acid monoester and diester radicals on silica-alumina.

The radical intermediates of the n-octanoic monoester and n-octanoic diester of astaxanthin were detected by pulsed EPR measurements carried out on the UV-produced radicals on silica-alumina artificial matrix and characterized by density functional theory (DFT) calculations. Previous Mims ENDOR for astaxanthin detected the radical cation and neutral radicals formed by proton loss from the C3 (or C3') position and from the methyl groups. Deprotonation of the astaxanthin neutral radical formed at the C3 (or C3') position resulted in a radical anion. DFT calculations for astaxanthin showed that the lowest energy neutral radical forms by proton loss at the C3 (or C3') position of the terminal ring followed by proton loss at the methyl groups of the polyene chain. Contrary to astaxanthin where proton loss can occur at either end of the symmetrical radical, for the diester of astaxanthin, this loss is prevented at the cyclohexene ends and is favored for its methyl groups. The monoester of astaxanthin, however, allows formation of the neutral radical at C3' and prevents its formation at the opposite end where the ester group is attached. At the terminal ring without the ester group attached, migration of proton from hydroxyl group to carbonyl group facilitates resonance stabilization, similarly to already published results for astaxanthin. However, cw EPR shows no evidence of a monoester radical anion formed. This study suggests the different radicals of astaxanthin and its esters that would form in a preferred environment, either hydrophobic or hydrophilic, depending on their structure.

Carotenoid radical formation: dependence on conjugation length.

The relative energy of carotenoid neutral radicals formed by proton loss from the radical cations of linear carotenoids has been examined as a function of conjugation length from n = 15 to 9. For a maximum conjugation length of n = 15 (bisdehydrolycopene, a symmetrical compound), proton loss can occur from any of the 10 methyl groups, with proton loss from the methyl group at position C1 or C1' being the most favorable. In contrast, the most energetically favorable proton loss from the radical cations of lycopene, neurosporene, spheroidene, spheroidenone, spirilloxanthin, and anhydrorhodovibrin occurs from methylene groups that extend from the conjugated system. For example, decreasing the conjugation length to n = 11 (lycopene) by saturation of the double bonds C3-C4 and at C3'-C4' of bisdehydrolycopene favors proton loss at C4 or C4' methylene groups. Saturation at C7'-C8' in the case of neurosporene, spheroidene, and spheroidenone (n = 9, 10, 11) favors the formation of a neutral radical at the C8' methylene group. Saturation of C1-C2 by addition of a methoxy group to a bisdehydrolycopene-like structure with conjugation of n = 12 or 13 (anhydrorhodovibrin, spirilloxanthin) favors proton loss at the C2 methylene group. As a consequence of deprotonation of the radical cation, the unpaired electron spin distribution changes so that larger ß-methyl proton couplings occur for the neutral radicals (13-16 MHz) than for the radical cation (7-10 MHz), providing a means to identify possible carotenoid radicals in biological systems by Mims ENDOR.

Free radical formation in novel carotenoid metal ion complexes of astaxanthin.

The carotenoid astaxanthin forms novel metal ion complexes with Ca(2+), Zn(2+), and Fe(2+). MS and NMR measurements indicate that the two oxygen atoms on the terminal cyclohexene ring of astaxanthin chelate the metal to form 1:1 complexes with Ca(2+) and Zn(2+) at low salt concentrations <0.2 mM. The stability constants of these complexes increased by a factor of 85 upon changing the solvent from acetonitrile to ethanol for Ca(2+) and by a factor of 7 for Zn(2+) as a consequence of acetonitrile being a part of the complex. Optical studies showed that at high concentrations (>0.2 mM) of salt, 2:1 metal/astaxanthin complexes were formed in ethanol. In the presence of Ca(2+) and Zn(2+), salts the lifetime of the radical cation and dication formed electrochemically decreased relative to those formed from the uncomplexed carotenoid. DFT calculations showed that the deprotonation of the radical cation at the carbon C3 position resulted in the lowest energy neutral radical, while proton loss at the C5, C9, or C13 methyl groups was less favorable. Pulsed EPR measurements were carried out on UV-produced radicals of astaxanthin supported on silica-alumina, MCM-41, or Ti-MCM-41. The pulsed EPR measurements detected the radical cation and neutral radicals formed by proton loss at 77 K from the C3, C5, C9, and C13 methyl groups and a radical anion formed by deprotonation of the neutral radical at C3. There was more than an order of magnitude increase in the concentration of radicals on Ti-MCM-41 relative to MCM-41, and the radical cation concentration exceeded that of the neutral radicals.

CIDNP and EPR study of phototransformation of lappaconitine derivatives in solution.

Chemically induced dynamic nuclear polarization (CIDNP) and electron paramagnetic resonance (EPR) techniques have been used to study the paramagnetic species formed during the photolysis of the alkaloid lappaconitine and its synthetic analogues in solution. Lappaconitine is a photosensitive antiarrhythmic and hypertension drug, whose major photoproduct (N-acetyl anthranilic acid) is also a potent photosensitizer. Both these compounds are lipophilic and might bind efficiently to cell membranes thereby causing phototoxic damage. Photolysis of natural lappaconitine (I) as well as its N(20) des-ethyl derivatives (N-Bz (II), N-Me (III), N-H (IV), and N(O)-Et (V)) results in cleavage of the ester bond with the formation of N-acetyl anthranilic acid (VIII) and corresponding enamine. The lappaconitine derivative V shows maximum photostability which correlates with reference data about its low toxicity. It was shown that the primary reaction step is electron transfer from the amino group to the anthranilic fragment of lappaconitine resulting in an intermediate biradical. The final products are formed via fragmentation of the neutral lappaconitine radicals.

Enhancement of the photocatalytic activity of TiO2 nanoparticles by water-soluble complexes of carotenoids.

Photoirradiation of TiO(2) nanoparticles by visible light in the presence of the water-soluble natural polysaccharide arabinogalactan complexes of the hydrocarbon carotenoid ß-carotene leads to enhanced yield of the reactive hydroxyl (OH) radicals. The electron paramagnetic resonance (EPR) spin-trapping technique using α-phenyl-N-tert-butyl nitrone (PBN) as the spin-trap has been applied to detect this intermediate by trapping the methyl and methoxy radicals generated upon reaction of the hydroxyl radical with dimethylsulfoxide (DMSO). The free radicals formed in this system proceed via oxygen reduction and not via the reaction of holes on the TiO(2) surface. As compared with pure carotenoids, carotenoid-arabinogalactan complexes exhibit an enhanced quantum yield of free radicals and stability toward photodegradation. The observed enhancement of the photocatalytic efficiency for carotenoid complexes, as measured by the quantum yield of the desired spin adducts, arises specifically from the decrease in the rate constant for the back electron transfer to the carotenoid radical cation. These results are important for a variety of TiO(2) applications, namely, in photodynamic therapy, and in the design of artificial light-harvesting, photoredox, and catalytic devices.

Measuring Ti(III)-carotenoid radical interspin distances in TiMCM-41 by pulsed EPR relaxation enhancement method.

Interspin distances between the Ti(3+) ions and the carotenoid radicals produced inside TiMCM-41 pores by photoinduced electron transfer from 7'-apo-7'-(4-carboxyphenyl)-beta-carotene (coordinated to Ti(3+)), canthaxanthin (formed as a random distribution of isomers), and beta-ionone (model for a short-chain polyene) to Ti(3+) framework sites were determined using the pulsed EPR relaxation enhancement method. To estimate the electron transfer distances, the temperature dependence of relaxation rates was analyzed in both siliceous and metal-substituted siliceous materials. The phase memory times, T(M), of the carotenoid radicals were determined from the best fits of two-pulse ESEEM curves. The spin-lattice relaxation times, T(1), of the Ti(3+) ion were obtained from the inversion recovery experiment with echo detection on a logarithmic time scale in the temperature range of 10-150 K. The relaxation enhancement for the carotenoid radicals in TiMCM-41 as compared to that in MCM-41 is consistent with an interaction between the radical and the fast relaxing Ti(3+) ion. For canthaxanthin and beta-ionone, a dramatic effect on the carotenoid relaxation rate, 1/T(M), occurs at 125 and 40 K, respectively, whereas for carboxy-beta-carotene 1/T(M) increases monotonically with increasing temperature. The interspin distances for canthaxanthin and beta-ionone were estimated from the 1/T(M) - 1/T(M0) difference, which corresponds to the Ti(3+) contribution at the temperature where the maximum enhancement in the relaxation rate occurs. Determination of the interspin distances is based on calculations of the dipolar interaction, taking into consideration the unpaired spin density distribution along the 20-carbon polyene chain, which makes it possible to obtain a fit over a wider temperature interval. A distribution of the interspin distances between the carotenoid radical and the Ti(3+) ion was obtained with the best fit at approximately 10 A for canthaxanthin and beta-ionone and approximately 9 A for 7'-apo-7'-(4-carboxyphenyl)-beta-carotene with an estimated error of +/-3 A. The interspin distances do not depend on 1/T(M) - 1/T(M0) for carboxy-beta-carotene which shows no prominent peak in the relaxation rate over the temperature range measured.