Transformation of naturally occurring (3R,3'R,6'R)-lutein and its fatty acid esters to (3 R)-ß-cryptoxanthin and (3R,6'R)-α-cryptoxanthin.

The objective of this study was to develop straightforward processes that could be applied to the large-scale production of ß-cryptoxanthin in an attempt to facilitate investigation of its biological activity. An oleoresin obtained from crude extracts of marigold flowers (Tagetes erecta) with approximately 24% total lutein fatty acid ester content was directly used as starting material for partial synthesis of (3 R)-ß-cryptoxanthin under mild reaction conditions at ambient temperature. Therefore, acid-catalyzed deoxygenation of lutein esters from marigold oleoresin followed by hydrogenation in the presence of catalytic amount of platinum (Pt) supported on alumina (5%) at ambient temperature gave a mixture of (3 R)-ß-cryptoxanthin fatty acid esters (major) and (3 R,6'R)-α-cryptoxanthin fatty acid esters (minor). Saponification and Z-to-E isomerization of the product followed by crystallization gave a mixture of (3 R)-ß-cryptoxanthin as the major product. Similarly, acid-catalyzed hydrogenation of unesterified (3 R,3'R,6'R)-lutein with Pt/alumina in ethyl acetate gave a mixture of (3 R,6'R)-α-cryptoxanthin acetate (minor) in a one-pot reaction. Alkaline hydrolysis and Z-to-E isomerization of the mixture followed by crystallization provided (3 R)-ß-cryptoxanthin.

Partial synthesis of serum carotenoids and their metabolites.

Human serum and tissues contain in excess of 12 dietary carotenoids and several metabolites that originate from consumption of fruits and vegetables. Among these are hydroxycarotenoids: (3R,3'R,6'R)-lutein (1), (3R,3'R)-zeaxanthin (2), (3R,6'R)-α-cryptoxanthin (3), and (3R)-ß-cryptoxanthin (4). In addition, several dehydration products of 1 have also been identified in human serum, these are: (3R,6'R)-3-hydroxy-3',4'-didehydro-ß,γ-carotene (5), (3R,6'R)-3-hydroxy-2',3'-didehydro-ß,ε-carotene (6), and (3R)-3-hydroxy-3',4'-didehydro-ß,ß-carotene (7). Several metabolites of 1 and/or 2, namely, (3R,3'S,6'R)-lutein (3'-epilutein, 8) and (3R,3'S;meso)-zeaxanthin (9) have also been characterized in human serum and ocular tissues. Semi-synthetic processes have been developed that separately transform commercially available 1 into 4 via 7 as well as 1 into 8. While 8 is converted into 2 by base-catalyzed isomerization, 7 is transformed into 2 and its (3R,3'S;meso)-stereoisomer (9) by regioselective hydroboration.

Total synthesis of (3R,3'R,6'R)-lutein and its stereoisomers.

(3R,3'R,6'R)-lutein (1) is a major dietary carotenoid that is abundant in most fruits and vegetables commonly consumed in the U.S. and that accumulates in the human plasma, major organs, and ocular tissues. Numerous epidemiological and experimental studies have shown that 1 has important biological activities and may play an important role in the prevention of age-related macular degeneration (AMD). While the total synthesis of 1 has been previously reported in a poor overall yield, the total synthesis of the other seven stereoisomers of lutein has not yet been accomplished. We have developed a relatively straightforward methodology for the total synthesis of 1 and three of its stereoisomers, (3R,3'S,6'S)-lutein (2), (3R,3'S,6'R)-lutein or 3'-epilutein (3), and (3R,3'R,6'S)-lutein (4) by C(15) + C(10) + C(15) Wittig coupling reactions. Employing this methodology, the other four stereoisomers of lutein that are enantiomeric to the aforementioned lutein isomers can be similarly prepared. One of the important features of this strategy is its application to the total synthesis of (13)C-labeled luteins and their metabolites with appropriate stereochemistry for metabolic studies in animals and humans. This synthesis also provides access to the C(15)-precursors of optically active carotenoids with a 3-hydroxy-epsilon end group that are otherwise difficult to synthesize.

Dietary tomato powder supplementation in the prevention of leiomyoma of the oviduct in the Japanese quail.

Spontaneous leiomyomas of the oviduct are common tumors of the Japanese Quail (Coturnix coturnix japonica), which makes it a good animal model for screening potential agents for testing in the prevention and treatment of human myoma uteri. We have previously reported a decreased incidence of leiomyomas in the oviduct of Japanese quail with lycopene supplementation. Although the major carotenoid in tomatoes is lycopene, tomatoes also contain other compounds, which may contribute to their health benefit. Therefore, in this study, we investigated the effects of tomato powder supplementation on the development of leiomyomas in the oviduct of Japanese quail. We also measured serum levels of malondialdehyde (MDA), carotenoids, and vitamins C, E, and A. A total of 150 quails (3 mo old) were assigned to 3 treatment groups consisting of 5 replicates of 10 birds in each group. Birds were fed either a basal diet (control group) or the basal diet supplemented with 25 g (Treatment I) or 50 g (Treatment II) of tomato powder (0.8 mg lycopene per g of tomato powder) per kg of diet. The animals were sacrificed after 365 days, and the tumors were identified. Tomato powder supplementation significantly decreased the number of leiomyomas as compared to control birds (P < 0.01). The tumors in tomato powder fed birds were smaller than those found in control birds (P < 0.01). Serum lycopene, lutein, zeaxantin, and vitamins C, E, and A increased (P = 0.01), whereas MDA concentrations decreased (P = 0.01) with tomato powder supplementation. No measurable lycopene could be detected in the serum of control birds, whereas a dose-dependent increase was observed in the serum of birds supplemented with tomato powder. The results indicate that dietary supplementation with tomato powder reduces the incidence and size of spontaneously occurring leiomyoma of the oviduct in the Japanese quail. Clinical trials should be conducted to investigate the efficacy of tomato powder supplementation in the prevention and treatment of uterine leiomyoma in humans.

Partial synthesis of (3R,6'R)-alpha-cryptoxanthin and (3R)-beta-cryptoxanthin from (3R,3'R,6'R)-lutein.

(3R,3'R,6'R)-Lutein (1), (3R,3'R)-zeaxanthin (2), (3R,6'R)-alpha-cryptoxanthin (3), and (3R)-beta-cryptoxanthin (4) are among dietary hydroxycarotenoids that have been identified in human serum, milk, and ocular tissues. While 1 containing 6% of 2 is commercially available, industrial production of optically active 3 and 4 has not yet been accomplished. Several processes have been developed that transform 1 into 3, 4, and minor quantities of (3R,5'RS,6'R)-3',4'-didehydro-5',6'-dihydro-beta,beta-caroten-3-ol (5) (a regioisomer of 3). In one process, lutein (1) was cleanly deoxygenated to 3 in the presence of trifluoroacetic acid (TFA) and Me3N.BH3 in CH2Cl2 at ambient temperature in nearly 90% yield. Reaction of lutein (1) with a Lewis acid (AlCl3, ZnBr2, ZnI2) and a hydride donor (Me3N.BH3, Na[BH3(OCOCF3)], NaCNBH3) in solvents such as CH2Cl2, THF, and TBME produced similar results. In a two-step process, high-temperature acid-catalyzed dehydration of 1 (propanol/water/acid, 90 degrees C) gave a mixture of anhydroluteins 6, 7, and 8 in 86% yield. In the second step, these dehydration products underwent ionic hydrogenation with TFA/Me3N.BH3 in CH2Cl2 to afford a mixture of 3 and 4 in nearly 80% yield that contained only 1% of 5.

Dose-ranging study of lutein supplementation in persons aged 60 years or older.

PURPOSE: To examine the dose-response relationship between oral lutein supplementation and serum lutein concentrations in persons aged 60 years and older, with or without age-related macular degeneration (AMD). METHODS: Forty-five participants with no AMD, large drusen, or advanced AMD, were randomized to receive one of three doses (2.5, 5, or 10 mg) of lutein for 6 months and to be observed for 6 additional months after the cessation of lutein supplementation. RESULTS: The mean age of the participants (33 women) was 71 years (range: 60-91). The serum lutein concentrations of each dose group were similar before supplementation, increased at 1 month, and peaked by 3 months. Median serum concentrations of the 2.5-, 5-, and 10-mg groups from baseline to month 6 increased from 18.7 to 35.1 microg/dL (2-fold increase), from 17.8 to 59.2 microg/dL (2.9-fold increase), and from 15.1 to 66.8 microg/dL (4-fold increase), respectively (all P < 0.001). The increases in lutein serum concentrations did not vary with AMD disease severity (P = 0.98). No toxicity was observed with any dose of lutein. No significant changes were detected in visual acuity or visual field tests. CONCLUSIONS: Increasing doses of lutein supplements significantly increased the serum levels of lutein and zeaxanthin, and doses up to 10 mg were safely administered. A long-term large clinical trial is necessary to investigate the safety and efficacy of lutein in reducing the risk of the development of advanced AMD.

The effect of lutein and zeaxanthin supplementation on metabolites of these carotenoids in the serum of persons aged 60 or older.

PURPOSE: To investigate the effect of lutein supplementation at doses of 2.5, 5.0, and 10 mg/d for 6 months on distribution of these carotenoids and their metabolites in the serum of elderly human subjects, with and without age-related macular degeneration. To determine whether supplementation with lutein can interact with the serum levels of other dietary carotenoids, retinol, and alpha-tocopherol. METHODS: Forty-five subjects received daily supplements of lutein (containing 5% zeaxanthin) for 6 months and were followed up for another 6 months after supplementation. Blood was collected at various intervals and lutein, zeaxanthin, and their metabolites in the sera were quantified by normal-phase high-performance liquid chromatography (HPLC)-UV/visible detection. Other dietary carotenoids, retinol, and alpha-tocopherol were identified and quantified on a C18 reversed phase HPLC column. RESULTS: After 6 months of supplementation with 10 mg of lutein, the increases in the mean serum levels from baseline were: 210 to 1000 nM/L (P < 0.0001) for lutein and 56 to 95 nM/L (P < 0.0001) for zeaxanthin. Similarly, the mean concentrations (nM/L) of carotenoid metabolites increased from 49 to 98 (P < 0.0001) for 3-hydroxy-beta,epsilon-caroten-3'-one (3'-oxolutein); 31 to 80 (P < 0.0001) for 3'-hydroxy-epsilon,epsilon-caroten-3-one; and 19 to 25 (P < 0.0001) for epsilon,epsilon-carotene-3,3'-dione. The serum levels of these carotenoids gradually decline within 6 months after supplementation. CONCLUSIONS: The increase in the serum levels of lutein/zeaxanthin correlates with increases in the serum levels of their metabolites that have previously been identified in the ocular tissues. Elderly human subjects with and without AMD can safely take supplements of lutein up to 10 mg/d for 6 months with no apparent toxicity or side effects.

Chronic ingestion of (3R,3'R,6'R)-lutein and (3R,3'R)-zeaxanthin in the female rhesus macaque.

PURPOSE: To investigate how supplementation of the monkey's diet with high doses of lutein (L), zeaxanthin (Z), or a combination of the two affects the plasma levels and ocular tissue deposition of these carotenoids and their metabolites over time and to determine whether these high doses can cause ocular toxicity. METHODS: Eighteen female rhesus monkeys were divided into groups of control (n = 3 control), L-treated (n = 5, 9.34 mg lutein/kg and 0.66 mg zeaxanthin/kg), Z-treated (n = 5, 10 mg zeaxanthin/kg), and L/Z-treated (n = 5, lutein and zeaxanthin, each 0.5 mg/kg). After 12 months of daily supplementation, one control animal, two L-treated animals, two Z-treated animals, and all the L/Z-treated animals were killed. The rest of the monkeys were killed after an additional six months without supplementation. Plasma and ocular tissue carotenoid analyses, fundus photography, and retina histopathology were performed on the animals. RESULTS: Supplementation of monkeys with L and/or Z increased the mean plasma and ocular tissue concentrations of these carotenoids and their metabolites. The mean levels of L and Z in the retinas of the L- and Z-treated animals after 1 year increased significantly over baseline. High dose supplementation of monkeys with L or Z did not cause ocular toxicity and had no effect on biomarkers associated with kidney toxicity. CONCLUSIONS: The mean levels of L and Z in plasma and ocular tissues of the rhesus monkeys increase with supplementation and in most cases correlate with the levels of their metabolites. Supplementation of monkeys with L or Z at high doses, or their combination does not cause ocular toxicity.

Lycopene supplementation prevents the development of spontaneous smooth muscle tumors of the oviduct in Japanese quail.

Leiomyomas (fibroids) are benign tumors of the uterus affecting millions of women. Spontaneous leiomyomas of the oviduct are common tumors of the Japanese quail (Coturnix coturnix japonica), which makes it a good animal model for screening potential agents for testing in the prevention and treatment of human myoma uteri. Because dietary intake of lycopene has been associated with a reduced risk of a variety of human cancers, we investigated the effects of lycopene supplementation on the development of leiomyomas in the oviduct of Japanese quail. We also measured serum levels of oxidative stress markers [malondialdehyde (MDA) and homocysteine], lycopene, vitamins C, E, and A, and tissue biomarkers Bcl-2 and Bax expression. One hundred twenty quails (6 mo old) were assigned to 3 treatment groups consisting of 4 replicates of 10 birds in each group. Birds were fed either a basal diet (group C) or the basal diet supplemented with 100 mg (group L1) or 200 mg (group L2) of lycopene per kilogram of diet. The animals were sacrificed after 285 days and the tumors were identified. Lycopene supplementation decreased the number of leiomyomas compared with control subjects (P=0.056). The tumors in lycopene-fed birds were smaller than those found in control birds (P=0.01). There were no significant differences in the expression of tissue Bcl-2 and Bax among the study groups. Serum vitamins C, E, and A increased (P=0.01), whereas MDA and homocysteine concentrations decreased (P=0.01) with lycopene supplementation. No measurable lycopene could be detected in the serum of control birds, whereas a dose-dependent increase was observed in the serum of lycopene-supplemented birds. The results indicate that dietary supplementation with lycopene reduces the incidence and size of spontaneously occurring leiomyoma of the oviduct in the Japanese quail. Clinical trials should be conducted to investigate the efficacy of lycopene supplementation in the prevention and treatment of uterine leiomyoma in humans.

Lutein interacts with ascorbic acid more frequently than with alpha-tocopherol to alter biomarkers of oxidative stress in female zucker obese rats.

The influence of dietary lutein, with and without moderate amounts of vitamin C (VC) or vitamin E (VE), on biomarkers of oxidative stress was examined in rats. Nine groups of immature Zucker obese (fa/fa) and lean female rats (8/group) consumed ad libitum for 8 wk the AIN-93G diet (Control) to which was added either dl-alpha-tocopherol acetate (VE) at 0.60 mg/kg or ascorbic acid (VC) at 0.75 mg/kg diet. Each of these diets contained lutein oil (FloraGlo) at 0.5 (Lut0.5) or 1.0 (Lut1.0) mg/kg diet. Weight gain, food efficiency and relative liver weight were higher in obese than in lean rats. Although liver malondialdehyde (MDA) concentrations were significantly higher in obese than in lean rats, levels were significantly lower in obese rats fed VE, VE-Lut and VC-Lut0.5 compared with other obese groups. The accumulation of alpha-tocopherol in liver was 6- and 3-times greater in the VE and VE-Lut1.0 groups, respectively, compared with the obese and lean control groups. Lutein reduced the activity of superoxide dismutase (SOD) in obese rats, independent of VC or VE, and raised the activity of glutathione peroxidase to higher levels in lean rats when combined with VC. Plasma insulin levels were dramatically higher in obese compared with lean rats, but significantly lower in obese rats fed VC-Lut0.5, VE-Lut1.0 and Lut1.0 compared with the Control group. These results suggest that lutein independently reduces the activity of SOD and alters more biomarkers of oxidative stress when combined with vitamin C than with vitamin E, and that vitamin E reduces liver lipid peroxidation in obese rats when the accumulation of liver alpha-tocopherol is very high.

Distribution of lutein, zeaxanthin, and related geometrical isomers in fruit, vegetables, wheat, and pasta products.

Quantitative data with regard to dietary (3R,3'R,6'R)-lutein, (3R,3'R)-zeaxanthin, and their (E/Z)-geometrical isomers are scarce, and in most cases, only the combined concentrations of these two carotenoids in foods are reported. Lutein and zeaxanthin accumulate in the human macula and have been implicated in the prevention of age-related macular degeneration (AMD). The qualitative and quantitative distributions of lutein, zeaxanthin, and their (E/Z)-isomers in the extracts from some of the most commonly consumed fruits, vegetables, and pasta products were determined by HPLC employing a silica-based nitrile-bonded column. Green vegetables had the highest concentration of lutein (L) and zeaxanthin (Z), and the ratios of these carotenoids (L/Z) were in the range 12-63. The yellow-orange fruits and vegetables, with the exception of squash (butternut variety), had much lower levels of lutein in comparison to greens but contained a higher concentration of zeaxanthin. The ratio of lutein to zeaxanthin (L/Z) in two North American bread varieties of wheat (Pioneer, Catoctin) was 11 and 7.6, respectively, while in a green-harvested wheat (Freekeh) imported from Australia, the ratio was 2.5. Between the two pasta products examined, lasagne and egg noodles, the latter had a much higher concentration of lutein and zeaxanthin. The levels of the (E/Z)-geometrical isomers of lutein and zeaxanthin in these foods were also determined.

An efficient conversion of (3R,3'R,6'R)-lutein to (3R,3'S,6'R)-lutein (3'-epilutein) and (3R,3'R)-zeaxanthin.

Two dietary carotenoids, (3R,3'R,6'R)-lutein (1) and (3R,3'R)-zeaxanthin (2), and their metabolite (3R,3'S,6'R)-lutein (3'-epilutein) (3) accumulate in human serum, milk, and ocular tissues. There is increasing evidence that compounds 1 and 2 play an important role in the prevention of age-related macular degeneration. Therefore, the availability of these carotenoids for metabolic studies and clinical trials is essential. Compound 1 is isolated from extracts of marigold flowers (Tagete erecta) and is commercially available, whereas 2 is only accessible by a lengthy total synthesis, and a viable method for synthesis of 3 has not yet been developed. This report describes an efficient conversion of technical grade 1 to 2 via 3. Acid-catalyzed epimerization of 1 yields an equimolar mixture of diastereomers 1 and 3. The mixture was separated by enzyme-mediated acylation with lipase AK from Pseudomonas fluorescens that preferentially esterified 3 and after alkaline hydrolysis yielded this carotenoid in 90% diastereomeric excess (de). Compound 3 was also separated from 1 in 56-88% de by solvent extraction and low-temperature crystallization, Soxhlet extraction, or supercritical fluid extraction. Base-catalyzed isomerization of 3 gave 2 in excellent yield, providing a convenient alternative to the total synthesis of this important dietary carotenoid.

Chemistry, distribution, and metabolism of tomato carotenoids and their impact on human health.

Recent epidemiological studies have suggested that the consumption of tomatoes and tomato-based food products reduce the risk of prostate cancer in humans. This protective effect has been attributed to carotenoids, which are one of the major classes of phytochemicals in this fruit. The most abundant carotenoid in tomato is lycopene, followed by phytoene, phytofluene, zeta-carotene, gamma-carotene, beta-carotene, neurosporene, and lutein. The distribution of lycopene and related carotenoids in tomatoes and tomato-based food products has been determined by extraction and high-performance liquid chromatography-UV/Visible photodiode array detection. Detailed qualitative and quantitative analysis of human serum, milk, and organs, particularly prostate, have revealed the presence of all the aforementioned carotenoids in biologically significant concentrations. Two oxidative metabolites of lycopene, 2,6-cyclolycopene-1,5-diols A and B, which are only present in tomatoes in extremely low concentrations, have been isolated and identified in human serum, milk, organs (liver, lung, breast, liver, prostate, colon) and skin. Carotenoids may also play an important role in the prevention of age-related macular degeneration, cataracts, and other blinding disorders. Among 25 dietary carotenoids and nine metabolites routinely found in human serum, mainly (3R,3'R,6'R)-lutein, (3R,3'R)-zeaxanthin, lycopene, and their metabolites were detected in ocular tissues. In this review we identified and quantified the complete spectrum of carotenoids from pooled human retinal pigment epithelium, ciliary body, iris, lens, and in the uveal tract and in other tissues of the human eye to gain a better insight into the metabolic pathways of ocular carotenoids. Although (3R,3'R,6'R)-lutein, (3R,3'R)-zeaxanthin, and their metabolites constitute the major carotenoids in human ocular tissues, lycopene and a wide range of dietary carotenoids have been detected in high concentrations in ciliary body and retinal pigment epithelium. The possible role of lycopene and other dietary carotenoids in the prevention of age-related macular degeneration and other eye diseases is discussed.

Transformations of selected carotenoids in plasma, liver, and ocular tissues of humans and in nonprimate animal models.

PURPOSE: To determine the stereochemistry of carotenoids in human ocular tissues in comparison with plasma and liver and to elucidate the possible transformations of dietary (3R,3'R,6'R)-lutein and (3R,3'R)-zeaxanthin in the eye. Similarly, to characterize the carotenoid profiles in the eye tissues, plasma, and liver of quails and frogs to determine whether these can serve as appropriate nonprimate animal models for metabolic studies. METHODS: Configurational isomers of carotenoids and their nondietary by-products from pooled human plasma, liver, retinal pigment epithelium (RPE-choroid), ciliary body, iris, and lens were characterized and quantified by high-performance liquid chromatography (HPLC) on a chiral column. Carotenoids and their nondietary by-products in pooled extracts from quail and frog plasma, liver, retina, RPE-choroid, iris, and lens were similarly characterized and quantified. RESULTS: (3R,3'R,6'R)-lutein, (3R,3'R)-zeaxanthin, (3R,3'S; meso)-zeaxanthin, (3R,3'S,6'R)-lutein (3'-epilutein), 3-hydroxy-beta, epsilon -carotene-3'-one, and 5Z- and all-E-lycopene were detected in nearly all human ocular tissues examined. (3R,3'S; meso)-zeaxanthin was not detected in the human plasma and liver but was present in human macula, retina, and RPE-choroid. (3S,3'S)-zeaxanthin was detected in human macula in minute quantities. The carotenoid profiles in quail and frog ocular tissues were somewhat similar to those in humans, with the exception that lycopene was absent. Frog retina, plasma, and liver revealed the presence of (3S,3'S)-zeaxanthin. CONCLUSIONS: The most likely transformations of carotenoids in the human eye involve a series of oxidation-reduction and double-bond isomerization reactions. Quail and frog appear to possess the appropriate enzymes for conversion of dietary (3R,3'R,6'R)-lutein and (3R,3'R)-zeaxanthin to the same nondietary by-products observed in humans and thus may serve as excellent nonprimate animal models for metabolic studies.