Protective Effects of a Lutein Ester Prodrug, Lutein Diglutaric Acid, against H2O2-Induced Oxidative Stress in Human Retinal Pigment Epithelial Cells.

Oxidative stress-induced cell damage and death of the retinal pigmented epithelium (RPE), a polarized monolayer that maintains retinal health and homeostasis, lead to the development of age-related macular degeneration (AMD). Several studies show that the naturally occurring antioxidant Lutein (Lut) can protect RPE cells from oxidative stress. However, the poor solubility and low oral bioavailability limit the potential of Lut as a therapeutic agent. In this study, lutein diglutaric acid (Lut-DG), a prodrug of Lut, was synthesized and its ability to protect human ARPE-19 cells from oxidative stress was tested compared to Lut. Both Lut and Lut-DG significantly decreased H2O2-induced reactive oxygen species (ROS) production and protected RPE cells from oxidative stress-induced death. Moreover, the immunoblotting analysis indicated that both drugs exerted their protective effects by modulating phosphorylated MAPKs (p38, ERK1/2 and SAPK/JNK) and downstream molecules Bax, Bcl-2 and Cytochrome c. In addition, the enzymatic antioxidants glutathione peroxidase (GPx) and catalase (CAT) and non-enzymatic antioxidant glutathione (GSH) were enhanced in cells treated with Lut and Lut-DG. In all cases, Lut-DG was more effective than its parent drug against oxidative stress-induced damage to RPE cells. These findings highlight Lut-DG as a more potent compound than Lut with the protective effects against oxidative stress in RPE cells through the modulation of key MAPKs, apoptotic and antioxidant molecular pathways.

Analyzing the Carotenoid Composition of Melilot (Melilotus officinalis (L.) Pall.) Extracts and the Effects of Isolated (All-E)-lutein-5,6-epoxide on Primary Sensory Neurons and Macrophages.

Melilotus officinalis is known to contain several types of secondary metabolites. In contrast, the carotenoid composition of this medicinal plant has not been investigated, although it may also contribute to the biological activities of the drug, such as anti-inflammatory effects. Therefore, this study focuses on the isolation and identification of carotenoids from Meliloti herba and on the effect of isolated (all-E)-lutein 5,6-epoxide on primary sensory neurons and macrophages involved in nociception, as well as neurogenic and non-neurogenic inflammatory processes. The composition of the plant extracts was analyzed by high performance liquid chromatography (HPLC). The main carotenoid was isolated by column liquid chromatography (CLC) and identified by MS and NMR. The effect of water-soluble lutein 5,6-epoxide-RAMEB (randomly methylated-ß-cyclodextrin) was investigated on Ca2+-influx in rat primary sensory neurons induced by the activation of the transient receptor potential ankyrin 1 receptor agonist to mustard-oil and on endotoxin-induced IL-1ß release from isolated mouse peritoneal macrophages. (all-E)-Lutein 5,6-epoxide significantly decreased the percent of responsive primary sensory neurons compared to the vehicle-treated stimulated control. Furthermore, endotoxin-evoked IL-1ß release from macrophages was significantly decreased by 100 µM lutein 5,6-epoxide compared to the vehicle-treated control. The water-soluble form of lutein 5,6-epoxide-RAMEB decreases the activation of primary sensory neurons and macrophages, which opens perspectives for its analgesic and anti-inflammatory applications.

Lutein and Zeaxanthin Isomers in Eye Health and Disease.

Current evidence suggests lutein and its isomers play important roles in ocular development in utero and throughout the life span, in vision performance in young and later adulthood, and in lowering risk for the development of common age-related eye diseases in older age. These xanthophyll (oxygen-containing) carotenoids are found in a wide variety of vegetables and fruits, and they are present in especially high concentrations in leafy green vegetables. Additionally, egg yolks and human milk appear to be bioavailable sources. The prevalence of lutein, zeaxanthin, and meso-zeaxanthin in supplements is increasing. Setting optimal and safe ranges of intake requires additional research, particularly in pregnant and lactating women. Accumulating evidence about variable interindividual response to dietary intake of these carotenoids, based on genetic or metabolic influences, suggests that there may be subgroups that benefit from higher levels of intake and/or alternate strategies to improve lutein and zeaxanthin status.

3'-hydroxy-ε,ε-caroten-3-one inhibits the differentiation of 3T3-L1 cells to adipocytes.

An oxidative metabolite of lutein, 3'-hydroxy-ε,ε-caroten-3-one, inhibited the differentiation of 3T3-L1 cells to adipocytes and the subsequent triacylglycerol production, but lutein did not. The α,ß-unsaturated carbonyl structure of 3'-hydroxy-ε,ε-caroten-3-one was considered to participate in the inhibitory effect, suggesting that this lutein metabolite has the potential to prevent metabolic syndrome.

A 3-hydroxy ß-end group in xanthophylls is preferentially oxidized to a 3-oxo ε-end group in mammals.

We previously found that mice fed lutein accumulated its oxidative metabolites (3'-hydroxy-ε,ε-caroten-3-one and ε,ε-carotene-3,3'-dione) as major carotenoids, suggesting that mammals can convert xanthophylls to keto-carotenoids by the oxidation of hydroxyl groups. Here we elucidated the metabolic activities of mouse liver for several xanthophylls. When lutein was incubated with liver postmitochondrial fraction in the presence of NAD(+), (3'R,6'R)-3'-hydroxy-ß,ε-caroten-3-one and (6RS,3'R,6'R)-3'-hydroxy-ε,ε-caroten-3-one were produced as major oxidation products. The former accumulated only at the early stage and was assumed to be an intermediate, followed by isomerization to the latter. The configuration at the C3' and C6' of the ε-end group in lutein was retained in the two oxidation products. These results indicate that the 3-hydroxy ß-end group in lutein was preferentially oxidized to a 3-oxo ε-end group via a 3-oxo ß-end group. Other xanthophylls such as ß-cryptoxanthin and zeaxanthin, which have a 3-hydroxy ß-end group, were also oxidized in the same manner as lutein. These keto-carotenoids, derived from dietary xanthophylls, were confirmed to be present in plasma of normal human subjects, and ß,ε-caroten-3'-one was significantly increased by the ingestion of ß-cryptoxanthin. Thus, humans as well as mice have oxidative activity to convert the 3-hydroxy ß-end group of xanthophylls to a 3-oxo ε-end group.

Effects of formulation on the bioavailability of lutein and zeaxanthin: a randomized, double-blind, cross-over, comparative, single-dose study in healthy subjects.

PURPOSE: Lutein and zeaxanthin are macular pigments with a protective function in the retina. These xanthophylls must be obtained from the diet or added to foods or supplements via easy-to-use, stable formulations. The technique employed to produce these formulations may affect the bioavailability of the xanthophylls. METHODS: Forty-eight healthy volunteers were randomized into this double-blind, cross-over study investigating the plasma kinetics of lutein provided as two different beadlet formulations. Subjects (n = 48) received a single dose of 20 mg of lutein as either a starch-matrix ("SMB", FloraGLO® Lutein 5 %) or as a cross-linked alginate-matrix beadlet ("AMB", Lyc-O-Lutein 20 %) formulation. Plasma concentrations of lutein and zeaxanthin were measured at 0, 1, 3, 6, 9, 12, 14, 24, 26, 28, 32, 36, 48, 72, 168, and 672 h. RESULTS: The mean plasma AUC(0-72h), AUC(0-672h), and C(max) for total lutein and zeaxanthin and their all-E-isomers were significantly increased (p < 0.001) from pre-dose concentrations in response to SMB and AMB. There was no difference in lutein T max between the two test articles. However, by 14 h post-dose, total plasma lutein increased by 7 % with AMB and by 126 % with SMB. Total lutein AUC(0-72h) and AUC(0-672h) were 1.8-fold and 1.3-fold higher, respectively, for SMB compared to AMB. Both formulations were well tolerated by subjects in this study. CONCLUSION: These findings confirm that the bioavailability of lutein and zeaxanthin critically depends on the formulation used and document a superiority of the starch-based over the alginate-based product in this study.

Effect of nilvadipine on central visual field in retinitis pigmentosa: a 30-month clinical trial.

PURPOSE: To assess the effects of nilvadipine on the progression of central visual field defect in retinitis pigmentosa (RP). DESIGN: Prospective, randomized, nonmasked, single-center trial. METHODS: Patients with RP were randomly divided into a treated group receiving oral nilvadipine at 4 mg/day for ≥30 months and a control group receiving tocopherol nicotinate at 300 mg/day, helenien at 15 mg/day or no medication for the same periods. Progression of RP was evaluated using the 10-2 SITA Fast Program of the Humphrey Visual Field Analyzer, and regression coefficients calculated from the time courses of mean deviation (MD slope) were compared between groups. RESULTS: Nineteen patients in the treated group and 14 patients in the control group completed the follow-up for ≥30 months. The mean (±standard deviation) duration of observation was 48.8 ± 11.8 months (median 48 months, range 30-66 months) for the treated group and 49.2 ± 18.1 months (median 48 months, range 30-90 months) for the control group (p = 0.94). Mean (±standard error of the mean, SEM) regression coefficients of the averaged MD values for the initial 30 months were -0.35 ± 0.17 dB/year in the treated group and -0.75 ± 0.06 dB/year in the control group (p < 0.01). Mean (±SEM) MD slopes for total observational periods were -0.49 ± 0.17 dB/year in the treated group and -0.89 ± 0.16 dB/year in the control group (mean ± SEM, p = 0.042). CONCLUSION: Nilvadipine at 4 mg/day significantly retarded progression of central visual field defects in RP in this small patient series.

Keto-carotenoids are the major metabolites of dietary lutein and fucoxanthin in mouse tissues.

Fucoxanthin, a xanthophyll present in brown algae consumed in Eastern Asia, can suppress carcinogenesis and obesity in rodents. We investigated the metabolism, tissue distribution, and depletion of fucoxanthin in ICR mice by comparison with those of lutein. The experiments comprised 14-d dietary supplementation with lutein esters or fucoxanthin, followed by 41- or 28-d, respectively, depletion periods with carotenoid-free diets. After lutein ester supplementation, 3'-hydroxy-ε,ε-caroten-3-one and lutein were the predominant carotenoids in plasma and tissues, accompanied by ε,ε-carotene-3,3'-dione. The presence of these keto-carotenoids in mouse tissues is reported here for the first time, to our knowledge. Lutein and its metabolites accumulated most in the liver (7.51 µmol/kg), followed by plasma (2.11 µmol/L), adipose tissues (1.01-1.44 µmol/kg), and kidney (0.87 µmol/kg). The half-life of the depletion (t(1/2)) of lutein metabolites varied as follows: plasma (1.16 d) < liver (2.63 d) < kidney (4.44 d) < < < adipose tissues (>41 d). Fucoxanthinol and amarouciaxanthin A were the main metabolites in mice fed fucoxanthin and partitioned more into adipose tissues (3.13-3.64 µmol/kg) than into plasma, liver, and kidney (1.29-1.80 µmol/kg). Fucoxanthin metabolites had shorter t(1/2) in plasma, liver, and kidneys (0.92-1.23 d) compared with those of adipose tissues (2.76-4.81 d). The tissue distribution of lutein and fucoxanthin metabolites was not associated with their lipophilicity, but depletion seemed to be slower for more lipophilic compounds. We concluded that mice actively convert lutein and fucoxanthin to keto-carotenoids by oxidizing the secondary hydroxyl groups and accumulate them in tissues.

Distribution and evolutionary trends of photoprotective isoprenoids (xanthophylls and tocopherols) within the plant kingdom.

The earliest land photosynthesis would have increased the risk of photo-oxidations and the demand of anti-oxidative protection. In this work, we aimed to determine the evolutionary trends in photoprotection across a wide representation of the plant kingdom and to verify whether the non-ubiquitous lutein-epoxide (Lx) cycle is a polyphyletic or an ancient character. Carotenoids and alpha-tocopherol (alpha-toc) were analysed by HPLC in 266 species. Phylogenetic analyses of the presence of photoprotective compounds and zeaxanthin-epoxidase (ZE) sequences were performed. Violaxanthin-cycle pigments (VAZ) and alpha-toc were taxonomically ubiquitous. Ancient groups showed higher contents of VAZ than vascular plants, while alpha-toc showed the opposite pattern. Lutein-epoxide was present in 45% of the species. It showed a remarkable variation across groups but with a clear increasing trend from algae to basal angiosperms. Lutein-epoxide was also related to woody trait and leaf longevity. No correlation between the presence of Lx and recurrent mutations in ZE sequences, including the duplications, was found. Thus, there is an evolutionary trend to increase the content of alpha-toc and to decrease the total amount of VAZ pigments. Absence of Lx in algae discards an ancestral origin. Present results are also inconsistent with a polyphyletic origin of Lx in angiosperms.

Lutein epoxide cycle, light harvesting and photoprotection in species of the tropical tree genus Inga.

Dynamics and possible function of the lutein epoxide (Lx) cycle, that is, the reversible conversion of Lx to lutein (L) in the light-harvesting antennae, were investigated in leaves of tropical tree species. Photosynthetic pigments were quantified in nine Inga species and species from three other genera. In Inga, Lx levels were high in shade leaves (mostly above 20 mmol mol(-1) chlorophyll) and low in sun leaves. In Virola surinamensis, both sun and shade leaves exhibited very high Lx contents (about 60 mmol mol(-1) chlorophyll). In Inga marginata grown under high irradiance, Lx slowly accumulated within several days upon transfer to deep shade. When shade leaves of I. marginata were briefly exposed to the sunlight, both violaxanthin and Lx were quickly de-epoxidized. Subsequently, overnight recovery occurred only for violaxanthin, not for Lx. In such leaves, containing reduced levels of Lx and increased levels of L, chlorophyll fluorescence induction showed significantly slower reduction of the photosystem II electron acceptor, Q(A), and faster formation as well as a higher level of non-photochemical quenching. The results indicate that slow Lx accumulation in Inga leaves may improve light harvesting under limiting light, while quick de-epoxidation of Lx to L in response to excess light may enhance photoprotection.

Short- and long-term modulation of the lutein epoxide and violaxanthin cycles in two species of the Lauraceae: sweet bay laurel (Laurus nobilis L.) and avocado (Persea americana Mill.).

Short- and long-term responses of the violaxanthin (V) and lutein epoxide (Lx) cycles were studied in two species of Lauraceae: sweet bay laurel (Laurus nobilis L.) and avocado (Persea americana L.). The Lx content exceeded the V content in shade leaves of both species. Both Lx and V were de-epoxidised on illumination, but only V was fully restored by epoxidation in low light. Violaxanthin was preferentially de-epoxidised in low light in L. nobilis. This suggests that Lx accumulates with leaf ageing, partly because its conversion to lutein is limited in shade. After exposure to strong light, shade leaves of avocado readjusted the total pools of alpha- and beta-xanthophyll cycles by de novo synthesis of antheraxanthin, zeaxanthin and lutein. This occurred in parallel with a sustained depression of F(v)/F(m). In Persea indica, a closely related but low Lx species, F(v)/F(m) recovered faster after a similar light treatment, suggesting the involvement of the Lx cycle in sustained energy dissipation. Furthermore, the seasonal correlation between non-reversible Lx and V photoconversions and pre-dawn F(v)/F(m) in sun leaves of sweet bay supported the conclusion that the Lx cycle is involved in a slowly reversible downregulation of photosynthesis analogous to the V cycle.

Metabolism of lutein and zeaxanthin in rhesus monkeys: identification of (3R,6'R)- and (3R,6'S)-3'-dehydro-lutein as common metabolites and comparison to humans.

Lutein and zeaxanthin are xanthophylls that can be found highly concentrated in the macula of the retina. They are thought to protect the macula through their role as blue-light filters and because of their antioxidant and singlet oxygen quenching properties. Examination of metabolites unique to lutein and zeaxanthin such as 3'-dehydro-lutein, and of their stereochemistry may provide insight to the mechanism by which they are formed and by which they exert protection. To evaluate the formation of such metabolites, eleven monkeys were raised on a xanthophyll-free diet, and supplemented with pure lutein or pure zeaxanthin (2.2 mg/kg body weight/d). The period of supplementation ranged between 12 and 92 weeks. At study start and throughout the study, serum samples were taken and analyzed for xanthophylls using different HPLC systems. Xanthophyll metabolites were identified using UV/VIS and HR-MS detection. Lutein and zeaxanthin metabolites were found in detectable amounts with 3'-dehydro-lutein being a common metabolite of both. Using chiral-phase HPLC, two diastereomers, (3R,6'R)-3'-dehydro-lutein and (3R,6'S)-3'-dehydro-lutein, were identified and shown to be present in nearly equimolar amounts. A pathway for their formation from either lutein or zeaxanthin is proposed. These findings were comparable to results obtained with human plasma.

All-E lutein and 3'-epilutein in the epidermis of chronic arsenic poisoning.

Identification and quantification of carotenoids in the epidermis of nine patients of chronic arsenic poisoning were done using isocratic reverse phase high performance liquid chromatography (HPLC). The major carotenoids in all the skin biopsies were all-E lutein and 3'-epilutein. Small amount of 2',3'-anhydrolutein, all-E zeaxanthin, and 13-Z zeaxanthin were also present in some of the biopsy samples. Alpha-carotene, beta-carotene, and lycopene were not detected in any sample. The mean (+/- SD) concentration of all-E lutein in the epidermis of healthy volunteers was 1.09 +/- 0.26 microgram/g of wet tissue, whereas it was only 0.29 +/- 0.10 microgram/g in the diffuse dark brown spots of chronic arsenic poisoning. In raindrop-shaped discoloration spots of skin the mean concentration of all-E lutein was 0.86 +/- 0.29 microgram/g of wet tissue. The difference between the concentrations of all-E lutein in the epidermis of healthy volunteers versus patients was for the diffuse dark brown spots statistically significantly (p < 0.05) lower, while this was not significant for the raindrop-shaped discoloration spots. This study suggests that arsenic exposure reduces the number, as well as concentrations of, carotenoids in skin.

Production, separation, and characterization of apo-luteinoids by LC-MS/MS.

It has been postulated that chemical or enzymatic catabolism of carotenoids could produce apo­carotenoids which have biological activity. Our objective was to generate and chemically characterize a series of apo­luteinoids (i.e. products resulting from the catabolism of lutein) which could putatively be found in vivo. Lutein was oxidized using potassium permanganate to produce a series of apo­luteinals/luteinone of subsequently shorter chain lengths, from apo­8'­luteinal to apo­11­luteinal. Sodium borohydride reduced this mixture into the corresponding alcohols (i.e. apo­luteinols). Similarly, Tollens' reagent was employed to oxidize the aldehyde series into carboxylic acids (i.e. apo­luteinoic acids). Mixtures of products were separated via HPLC and characterized in-line using photodiode array (PDA) and tandem mass spectrometry (MS-MS). A global HPLC-PDA-MS/MS method was developed to separate the products, and application of the methods to the symmetric xanthophyll zeaxanthin further confirmed the ε- and ß-ring species. These methods can be employed for the study of lutein oxidation products in plants, foods and biological samples.

Modified saponification and HPLC methods for analyzing carotenoids from the retina of quail: implications for its use as a nonprimate model species.

PURPOSE: To investigate carotenoid content in the retina of Japanese quail (Coturnix japonica), for comparison with carotenoids in human retina, and to assess the effects of different saponification procedures on the recovery of quail retinal carotenoids. METHODS: Extracted retinal carotenoids were saponified with methods adapted from recent studies, then identified and quantified with reverse-phase high-performance liquid chromatography (HPLC). To assess the effects of saponification conditions on carotenoid recovery from quail retina, we varied base concentration and the total time of saponification across a wide range and again used HPLC to compare carotenoid concentrations among conditions. RESULTS: Astaxanthin and galloxanthin were the dominant carotenoids recovered in the quail retina, along with smaller amounts of five other carotenoids (lutein, zeaxanthin, 3'-epilutein, epsilon-carotene, and an unidentified carotenoid). Astaxanthin was sensitive to saponification conditions; recovery was poor with strong bases (0.2 and 0.5 M KOH) and best with weak bases (0.01 and 0.2 M KOH). In contrast, xanthophyll carotenoids (galloxanthin, zeaxanthin, lutein, 3'-epilutein, and the unknown) were best recovered with strong base after 6 hours of saponification at room temperature. The recovery of epsilon-carotene was not affected by saponification conditions. CONCLUSIONS: Separate chemical hydrolysis procedures--using a strong base to recover xanthophylls and a weak base to recover astaxanthin--should be used for maximizing recovery of quail retinal carotenoids. Because the dominant carotenoids in quail retina are absent in human retina, and because of their different packaging (e.g., esterified in oil droplets) and light-absorbance properties compared with xanthophylls in the human eye, use of the quail as a model organism for studying human retinal carotenoids should be approached with caution.

Structure elucidation of deoxylutein II isomers by on-line capillary high performance liquid chromatography-(1)H nuclear magnetic resonance spectroscopy.

Thermal and iodine-catalyzed photochemical (Z/E)-isomerization of deoxylutein II [(3R,6'R)-3-hydroxy-3',4'-didehydro-beta,gamma-carotene, anhydrolutein I] (2), the dehydration product of lutein [(3R,3'R,6'R)-beta,epsilon-carotene-3,3'-diol] (4), yielded multi-component mixtures of (Z)-isomers. By I(2)-catalyzed photoisomerization, (9Z)-2, (9'Z)-2, (13Z)-2, (13'Z)-2 and (15Z)-2 are generated as main products. In addition, this thermodynamic-equilibrium mixture contains traces of (9Z,9'Z)-2 and other (di-Z)-isomers in minor concentrations. Thirteen isomers are chromatographically separated and detected on-line by UV-vis and mass spectrometry. (all-E)-Deoxylutein II (2) and six of its (Z)-configured isomers are separated by capillary HPLC (acetone-d(6)/D(2)O = 85:15) and detected on-line by (1)H NMR spectroscopy in a microprobe. With the microprobe and the active detection volume of 1.5 microl, it is possible to perform structure elucidation with very small amounts available for various (Z)-isomers of deoxylutein II (2) in the isomerization mixture.

Identification of genes coding for functional zeaxanthin epoxidases in the diatom Phaeodactylum tricornutum.

Phaeodactylum tricornutum like other diatoms synthesizes fucoxanthin and diadinoxanthin as major carotenoid end products. The genes involved have recently been assigned for early pathway steps. Beyond ß-carotene, only gene candidates for ß-carotene hydroxylase, zeaxanthin epoxidase and zeaxanthin de-epoxidase have been proposed from the available genome sequence. The two latter enzymes may be involved in the two different xanthophyll cycles which operate in P. tricornutum. The function of three putative zeaxanthin epoxidase genes (zep) was addressed by pathway complementation in the Arabidopsis thaliana Zep mutant npq2. Genes zep2 and zep3 were able to restore zeaxanthin epoxidation and a functional xanthophyll cycle but the corresponding enzymes exhibited different catalytic activities. Zep3 functioned as a zeaxanthin epoxidase whereas Zep2 exhibited a broader substrate specificity additionally converting lutein to lutein-5,6-epoxide. Although zep1 was transcribed and the protein could be identified after import into the chloroplast in A. thaliana, Zep1 was found not to be functional in zeaxanthin epoxidation. The non-photochemical quenching kinetics of wild type A. thaliana was only restored in transformant npq2-zep3.

Isolation and structure elucidation of anhydroluteins from cooked sorrel (Rumex rugosus Campd.).

Anhydrolutein I (= (all-E,3R,6'R)-3',4'-didehydro-beta,gamma-caroten-3-ol; 2) and anhydrolutein II (= (all-E, 3R,6'S)-2',3'-didehydro-beta,epsilon-caroten-3-ol; 3) have been isolated and characterized from the extract of steam-cooked sorrel. The presence of these compounds in cooked vegetable is postulated to be due to acid-catalyzed dehydration of lutein (1; Scheme). The structures of the isolated anhydroluteins were established by UV/VIS, CD, and 1H-NMR spectroscopy, and mass spectrometry.

Identification of lutein and zeaxanthin oxidation products in human and monkey retinas.

PURPOSE: To characterize fully all the major and minor carotenoids and their metabolites in human retina and probe for the presence of the oxidative metabolites of lutein and zeaxanthin. METHODS: Carotenoids of a composite of 58 pairs of human retinas and a monkey retina were elucidated by comparing their high-performance liquid chromatography (HPLC)-ultraviolet/visible absorption spectrophotometry (UV/Vis)-mass spectrometry (MS) profile with those of authentic standards prepared by organic synthesis. RESULTS: In addition to lutein and zeaxanthin, several oxidation products of these compounds were present in the extracts from human retina. A major carotenoid resulting from direct oxidation of lutein was identified as 3-hydroxy-beta, epsilon-caroten-3'-one. Minor carotenoids were identified as: 3'-epilutein, epsilon,epsilon-carotene-3,3'-diol, epsilon,epsilon-carotene-3,3'-dione, 3'-hydroxy-epsilon,epsilon-caroten-3-one, and 2,6-cyclolycopene-1,5-diol. Several of the geometric isomers of lutein and zeaxanthin were also detected at low concentrations. These were as follows: 9-cis-lutein, 9'-cislutein, 13-cis-lutein, 13'-cis-lutein, 9-cis-zeaxanthin, and 13-cis-zeaxanthin. Similar results were also obtained from HPLC analysis of a freshly dissected monkey retina. CONCLUSIONS: Lutein, zeaxanthin, 3'-epilutein, and 3-hydroxy-beta,epsilon-caroten-3'-one in human retina may be interconverted through a series of oxidation-reduction reactions similar to our earlier proposed metabolic transformation of these compounds in humans. The presence of the direct oxidation product of lutein and 3'-epilutein (metabolite of lutein and zeaxanthin) in human retina suggests that lutein and zeaxanthin may act as antioxidants to protect the macula against short-wavelength visible light. The proposed oxidative-reductive pathways for lutein and zeaxanthin in human retina, may therefore play an important role in prevention of age-related macular degeneration and cataracts.

Carotenoid composition in petals of chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura).

Sixteen xanthophylls were isolated from the petals of chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura). Among them, (3S,5S,6R,3'R,6'R)-5,6-dihydro-5,6-dihydroxylutein (1) and five di-Z geometrical isomers of lutein-5,6-epoxide, i.e., 9Z,13'Z (2), 13Z,9'Z (3), 9'Z,13'Z (4), 9Z,13Z (5), and 9Z,9'Z (7), had never before been identified as natural products. All of the carotenoids isolated from chrysanthemum, except for (9Z)-violaxanthin, are beta,epsilon-carotene (alpha-carotene) derivatives. The analyses indicate that carotenoids from the petals of chrysanthemum have a very characteristic composition.