Evaluation of vitamin A and carotenoid data in food composition tables.

Vitamin A and carotenoid data in food composition tables have been generated with the use of the Association of Official Analytical Chemists procedures. These procedures fractionate complex mixtures of retinoids and carotenoids into only three fractions, e.g., retinoids, carotenes, and xanthophylls. Food tables lack uniformity in the presentation of vitamin A and carotenoid data. For example, the revisions of Agriculture Handbook, No. 8 (Washington, D.C.: U.S. Govt Print Off, 1963), report both international units and retinol equivalents of vitamin A in foods. Food tables published in the United Kingdom (Paul AA, Southgate DA. McCance and Widdowson's: The composition of foods. Amsterdam: Elsevier/North-Holland, 1978) report preformed vitamin A as retinol and carotenoids as carotene on a weight basis (micrograms/100 g food). The vitamin A and carotenoid data currently available in food composition tables allow only the intake of total vitamin A activity to be estimated. Information about the intake of specific species of retinoids or carotenoids is not available and could not be calculated from existing food tables. Limited applications of new separation techniques and chemical instrumentation have demonstrated the potential for generating detailed analytic information in regard to the retinoid and carotenoid contents of foods.

Carotenoid analyses of selected raw and cooked foods associated with a lower risk for cancer.

We examined the carotenoid content of selected foods consistently found to be associated with a lower risk for various epithelial cancers in epidemiologic studies. Both raw and cooked samples of green, leafy vegetables and yellow or orange vegetables were quantitatively examined by high-performance liquid chromatography for individual carotenoid content. The results indicated that fresh, green, leafy vegetables were moderately high in beta carotene (0.5-14.6 mg/100 g) and very high in oxygenated carotenoids or xanthophylls, primarily lutein and its stereoisomers (2.3-63.0 mg/100g) [corrected]. The fresh, yellow or orange vegetables examined were very high in beta carotene (16.0-120.5 mg/100 g) [corrected] but had no detectable nonhydrocarbon carotenoids. Cooking differentially reduced the lutein content compared with the beta carotene content in green, leafy vegetables. These analyses suggest that consumption of carotenoids in addition to beta carotene may be associated with a lower risk for cancer.

Non-invasive raman spectroscopic detection of carotenoids in human skin.

Carotenoids are thought to play a significant part in the skin's anti-oxidant defense system, and may help prevent malignancy. Inability to measure skin carotenoid content readily has, however, made it difficult to establish the relationship between carotenoid concentration and the occurrence of cutaneous malignancy. We have measured in vivo carotenoid concentration using a noninvasive optical method, Raman spectroscopy. To validate our instrumentation, abdominoplasty skin was evaluated by both Raman spectroscopy and high-performance liquid chromatography determination for carotenoid content. Evaluation of the Raman signal in specific carotenoid solutions was also performed. Precision of Raman measurements within skin sites, within subjects, and between subjects was measured. Sensitivity of the method was evaluated as a function of anatomical region and the distribution of carotenoids within the stratum corneum. Lastly, we evaluated the Raman signal in actinic keratosis and basal cell carcinoma lesions and perilesional skin and compared this with region-matched sites in healthy subjects. Our results indicate that the Raman scattering method reflects the presence of carotenoids in human skin and is highly reproducible. Evaluation of five anatomical regions demonstrated significant differences in carotenoid concentration by body region with the highest carotenoid concentration noted in the palm. Comparison of carotenoid concentrations in basal cell carcinomas, actinic keratosis, and their perilesional skin demonstrate a significantly lower carotenoid concentration than in region-matched skin of healthy subjects. These results represent the first evidence that carotenoid concentration in the skin correlate with the presence or absence of skin cancer and precancerous lesions.

Plasma carotenoid response to chronic intake of selected foods and beta-carotene supplements in men.

We determined serial changes in four major plasma carotenoid fractions (alpha-carotene, beta-carotene, lutein/zeaxanthin, and lycopene) in 30 men consuming defined daily doses of carotenoids from foods (broccoli, carrots, or tomato juice) or from purified beta-carotene in capsules (12 or 30 mg) for 6 wk while fed a controlled diet. Compared with baseline, beta-carotene increased in the 30- and 12-mg-capsule and carrot groups whereas alpha-carotene increased in the carrot group and lutein increased in the broccoli group. Lower lutein concentrations in recipients of beta-carotene capsules suggested an interaction between these two carotenoids. Lycopene declined in all groups except the tomato-juice group. Total carotenoid concentration changes only reflected the large increases in beta-carotene concentrations and not the smaller changes observed in other individual carotenoids. Overall, purified beta-carotene produced a greater plasma response than did similar quantities of carotenoids from foods sources. However, some foods increased plasma concentrations of certain carotenoids.

Chronic ingestion of lycopene-rich tomato juice or lycopene supplements significantly increases plasma concentrations of lycopene and related tomato carotenoids in humans.

The bioavailability of lycopene from tomato juice and 2 dietary supplements, each containing 70-75 mg lycopene, was studied in 15 healthy volunteers in a randomized, crossover design. Subjects ingested lycopene-rich tomato juice, tomato oleoresin, lycopene beadlets, and a placebo for 4 wk each while consuming self-selected diets. Treatment periods were separated by 6-wk washout periods. Plasma lycopene concentrations, assessed at baseline and weekly throughout the treatment periods, were significantly higher during tomato juice, oleoresin, and lycopene beadlet ingestion than during placebo ingestion. Mean (+/-SEM) increases in plasma lycopene at week 4 of tomato juice, oleoresin, and lycopene beadlet ingestion were not significantly different: 0.24 +/- 0.07, 0.23 +/- 0.05, and 0.24 +/- 0.06 micromol/L, respectively. Plasma concentrations of phytofluene and phytoene, which were present in small amounts in tomato juice, oleoresin, and lycopene beadlets, increased significantly with ingestion of these 3 products. Beta-carotene, zeta-carotene, and 2,6-cyclolycopene-1,5-diol (a metabolite of lycopene)--also present in tomato juice and supplements--were significantly increased with consumption of the tomato juice and lycopene beadlets, but not with oleoresin consumption. A marked increase in plasma concentrations of an unknown compound was observed; it was detected in trace amounts in tomato juice, oleoresin, and lycopene beadlets, and had a maximum absorbance at 448 nm and a molecular weight of 556. Concentrations of plasma lycopene and other carotenoids with potential for enhancing human health can be increased by ingestion of realistic amounts of tomato juice. Lycopene appears to be equally bioavailable from tomato juice and the supplements used in this study.

Phase II randomized clinical trial of lycopene supplementation before radical prostatectomy.

An inverse association has been observed between dietary intake of lycopene and the risk of prostate cancer. We investigated the effects of lycopene supplementation in patients with prostate cancer. Twenty-six men with newly diagnosed, clinically localized (14 T(1) and 12 T(2)) prostate cancer were randomly assigned to receive 15 mg of lycopene (n = 15) twice daily or no supplementation (n = 11) for 3 weeks before radical prostatectomy. Biomarkers of differentiation and apoptosis were assessed by Western blot analysis on benign and malignant parts of the prostate gland. Prostatectomy specimens were entirely embedded, step-sectioned, and evaluated for pathological stage, Gleason score, volume of cancer, and extent of high-grade prostatic intraepithelial neoplasia. Plasma levels of lycopene, insulin-like growth factor-1 (IGF-1), IGF binding protein-3, and prostate-specific antigen were measured at baseline and after 3 weeks of supplementation or observation. Eleven (73%) subjects in the intervention group and two (18%) subjects in the control group had no involvement of surgical margins and/or extra-prostatic tissues with cancer (P = 0.02). Twelve (84%) subjects in the lycopene group and five (45%) subjects in the control group had tumors <4 ml in size (P = 0.22). Diffuse involvement of the prostate by high-grade prostatic intraepithelial neoplasia was present in 10 (67%) subjects in the intervention group and in 11 (100%) subjects in the control group (P = 0.05). Plasma prostate-specific antigen levels decreased by 18% in the intervention group, whereas they increased by 14% in the control group (P = 0.25). Expression of connexin 43 in cancerous prostate tissue was 0.63 +/- 0.19 absorbance in the lycopene group compared with 0.25 +/- 0.08 in the control group (P = 0.13). Expression of bcl-2 and bax did not differ significantly between the two study groups. IGF-1 levels decreased in both groups (P = 0.0002 and P = 0.0003, respectively). The results suggest that lycopene supplementation may decrease the growth of prostate cancer. However, no firm conclusions can be drawn at this time because of the small sample size.

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.

Inhibitory effects of natural carotenoids, alpha-carotene, beta-carotene, lycopene and lutein, on colonic aberrant crypt foci formation in rats.

Inhibitory effect of four carotenoids prevalent in human blood and tissues against the formation of colonic aberrant crypt foci was examined in Sprague-Dawley rats. They received three intrarectal doses of N-methylnitrosourea in weak 1, and a daily gavage of de-escalated doses of carotenoids during weeks 2 and 5. Lycopene, lutein, alpha-carotene and palm carotenes (a mixture of alpha-carotene, beta-carotene and lycopene) inhibited the development of aberrant crypt foci quantitated at week 6, but beta-carotene did not. The results suggested that lycopene and lutein in small doses may potentially prevent colon carcinogenesis.

Isolation and structural elucidation of (13Z,13'Z,3R,3'R,6'R)-lutein from marigold flowers, kale, and human plasma.

(13Z,13'Z,3R,3'R,6'R)-Lutein has been isolated and purified from extracts of marigold flowers, fresh raw kale (Brassica oleracea var. Acephala), and human plasma and fully characterized by (1)H and (13)C NMR, UV/vis, and MS. While the concentration of (13Z, 13'Z)-lutein in kale and human plasma compared to (all-E,3R,3'R, 6'R)-lutein was found to be quite low, this compound was readily isolated by fractional crystallization of lutein from marigold extracts. Thus, the mother liquors from two consecutive crystallizations of lutein from a saponified extract of marigold flowers were enriched in (13Z,13'Z)-lutein (8.7% of total carotenoids) and employed for the isolation of this compound by HPLC. The identity of the di-Z-lutein in kale and human plasma has been established by comparison of the HPLC-UV/vis-MS profiles of the purified compounds with those of a fully characterized sample, isolated from marigolds. 3-Hydroxy-beta,epsilon-caroten-3'-one and 3'-epilutein have also been identified in extracts from marigolds.

Cancer prevention by natural carotenoids.

Various natural carotenoids were proven to have anticarcinogenic activity. Epidemiological investigations have shown that cancer risk is inversely related to the consumption of green and yellow vegetables and fruits. Since beta-carotene is present in abundance in these vegetables and fruits, it has been investigated extensively as possible cancer preventive agent. However, various carotenoids which co-exist with beta-carotene in vegetables and fruits also have anti-carcinogenic activity. And some of them, such as alpha-carotene, showed higher potency than beta-carotene to suppress experimental carcinogenesis. Thus, we have carried out more extensive studies on cancer preventive activities of natural carotenoids in foods; i.e., lutein, lycopene, zeaxanthin and beta-cryptoxanthin. Analysis of the action mechanism of these natural carotenoids is now in progress, and some interesting results have already obtained; for example, beta-cryptoxanthin was suggested to stimulate the expression of RB gene, an anti-oncogene, and p73 gene, which is known as one of the p53-related genes. Based on these results, multi-carotenoids (mixture of natural carotenoids) seems to be of interest to evaluate its usefulness for practice in human cancer prevention.

Separation of carotenol fatty acid esters by high-performance liquid chromatography.

Employing isocratic and gradient-elution high-performance liquid chromatography (HPLC) a number of straight-chain fatty acid esters (decanoate, laurate, myristate, palmitate) of violaxanthin, auroxanthin, lutein, zeaxanthin, isozeaxanthin, and beta-cryptoxanthin, prepared by partial synthesis, have been separated on a C18 reversed-phase column. Several chromatographic conditions were developed that separated a mixture of di-fatty acid esters (dimyristate, myristate palmitate mixed ester, dipalmitate) of violaxanthin, auroxanthin, lutein, and zeaxanthin in a single chromatographic run. Hydroxycarotenoids such as lutein, zeaxanthin, and isozeaxanthin that are not easily separated by HPLC on C18 reversed-phase columns, can be readily separated after derivatization with fatty acids and chromatography of their esters. Chromatographic conditions for optimum separation of carotenoids from various classes are discussed.

Decapreno-beta-carotene as an internal standard for the quantification of the hydrocarbon carotenoids by high-performance liquid chromatography.

The application of decapreno-beta-carotene as an internal standard in quantification of the hydrocarbon carotenoids extracted from raw carrots has been thoroughly examined. Decapreno-beta-carotene is a C50 beta-carotene that has most of the requirements of an internal standard and it can be commercially synthesized in high purity. An isocratic high-performance liquid chromatographic (HPLC) system has been developed that separated all-trans-alpha-carotene, all-trans-beta-carotene, and its 15,15'-cis-isomer from this internal standard. Quantitative determination of the hydrocarbon carotenoids in carrots by HPLC using the internal standard technique gave values for alpha- and beta-carotene similar to those obtained from alpha- and beta-carotene standards alone.

Lutein, lycopene, and their oxidative metabolites in chemoprevention of cancer.

Numerous epidemiological studies have demonstrated that consuming large quantities of fruits and vegetables reduces the risk for several types of human cancers. Carotenoids are abundant in fruits and vegetables and have been extensively studied as cancer preventive agents. A proposed mechanism of action for the protective effect of carotenoids against cancer is based on their antioxidant capability. Recently, we have isolated and characterized 14 new carotenoids, including seven metabolites from the extracts of human serum/plasma. This brings the total number of identified blood carotenoids to 21. Lutein and lycopene, abundant in most fruits and vegetables as well as human serum, have been shown to possess strong antioxidant capability. Among the metabolites of lutein, four results from oxidation and two from non-enzymatic dehydration. The metabolite of lycopene has been identified as 5,6-dihydroxy-5,6-dihydrolycopene, which apparently results from oxidation of lycopene to an intermediate, lycopene epoxide. This intermediate may undergo metabolic reduction to form the lycopene metabolite. Although in vivo oxidation of lutein to its metabolites has been demonstrated based on data obtained from two human studies, in vivo oxidation of lycopene to its metabolite has not yet been established. Recent preliminary studies involving healthy subjects ingesting purified lutein and zeaxanthin (a dietary dihydroxycarotenoid isomeric to lutein) are presented. We propose a possible antioxidant mechanism of action for lutein and lycopene that leads to formation of the oxidation products of these promising chemopreventive agents.

Effect of dietary lycopene on N-methylnitrosourea-induced mammary tumorigenesis.

Epidemiological studies suggest protective effects of lycopene-rich foods on several types of cancer, including prostate and gastrointestinal tract. Moreover, an inverse association between serum lycopene concentrations and several types of cancer has been reported. However, few studies have focused on breast cancer, and they have shown little association between lycopene consumption and cancer risk. In this report, we used the N-methylnitrosourea (NMU)-induced rat mammary tumor model to compare the effects of pure lycopene with a lycopene-rich tomato carotenoid oleoresin (TCO) on NMU-induced mammary tumorigenesis. Rats were fed diets supplemented with 250 and 500 ppm crystalline lycopene or TCO beginning seven days before initiation with NMU (55 days of age) to termination (18 wk after NMU). Neither pure lycopene nor lycopene in the form of a mixed carotenoid oleoresin exerted an inhibitory effect on tumor incidence, latency, multiplicity, volume, or total tumors per group compared with unsupplemented controls. Weight gains in all groups were similar. Assay of serum lycopene concentrations in lycopene-supplemented groups indicated that median levels of 7,12,60, and 87 ng/ml were attained in blood of groups supplemented with 250 and 500 ppm lycopene and 250 and 500 ppm TCO, respectively. The results of this animal study are consistent with epidemiological reports indicating that lycopene does not protect against breast cancer.

Separation and identification of carotenoids and their oxidation products in the extracts of human plasma.

Eighteen carotenoids as well as vitamin A and two forms of vitamin E (gamma- and alpha-tocopherol) have been separated from extracts of human plasma by high-performance liquid chromatography (HPLC) on reversed-phase and sillca-based nitrile-bonded columns. In the order of chromatographic elution on a C18 reversed-phase column, the carotenoids were identified as (3R,3'R,6'R)-beta, epsilon-carotene-3,3'-diol [(3R,3'R,6'R)-lutein], (3R,3'R)-beta, beta-carotene-3,3'-diol [(3R,3'R)-zeaxanthin], 5,6-dihydroxy-5,6-dihydro-psi,psi-carotene, 3-hydroxy-2',3'-didehydro-beta,epsilon-caroten-3-ol, 3-hydroxy-beta-carotene,psi,psi-carotene, 7,8-dihydro-psi,psi-carotene, beta,psi-carotene, 7,8,7',8'-tetrahydro-psi,psi-carotene, beta,epsilon-carotene, beta,beta-carotene, 7,8,11,12,7',8'-hexahydro-psi,psi-carotene, and 7,8,11,12,7',8'-11',12'-octahydro-psi,psi-carotene. The polar carotenoids, which eluted in the vicinity of lutein and were unresolved on the C18 column, have been separated on a nitrile-bonded column employing isocratic HPLC conditions. In the order of elution, the carotenoids were epsilon,epsilon-carotene-3,3'-dione, 3'-hydroxy-epsilon,epsilon-caroten-3-one, 5,6-dihydroxy-5,6-dihydro-psi,psi-carotene, 3-hydroxy-beta,epsilon-caroten-3'-one, (all-E,3R,3'R,6'R)-lutein, (all-E,3R,3'R)-zeaxanthin, and (all-E,3R,3'S,6'R)-beta,epsilon-carotene-3,3'-diol (3'-epilutein) followed by several geometrical isomers of lutein and zeaxanthin.

Lycopene uptake and tissue disposition in male and female rats.

Epidemiologic and clinical studies suggest that tomato consumption may reduce the risk of cancer. Lycopene, a hydrocarbon carotenoid, is the major carotenoid in tomatoes and, as a potent singlet oxygen quencher, has been considered by some to be the biologically active agent responsible for the reduction of cancer risk associated with tomato consumption. However, little is known concerning lycopene absorption or biological activity in rodent models of cancer. Therefore, the present study was designed to provide information regarding the uptake and tissue disposition of lycopene and related carotenoid after feeding a diet containing a carotenoid mixture extracted from tomatoes (Betatene). Betatene was added to the diet at 2.3, 0.9, 0.45, 0.23, 0.09 and 0 (mM/kg diet) and fed to male and female Fischer-344 rats for a period of 10 weeks. Using reverse phase HPLC methods, it was found that approximately 55% of administered lycopene was excreted in the feces. In both males and females, lycopene concentrations were highest in the liver (120-42 microg/g wet wt.); physiologically significant levels were detected in prostate (97-47 ng/g), lung (227-134 ng/g), mammary gland (309-174 ng/g) and serum (285-160 ng/ml). Tissue concentrations were related to dose with the exception of serum, and differences between males and females were minimal. Other carotenoids present in Betatene (i.e., phytoene, phytofluene, z-carotene and beta-carotene) were also absorbed and stored in the liver. These results indicate that lycopene, when incorporated into the semipurified AIN-76A diet, is absorbed in both male and female rats in a dose-related manner and can be detected at nanogram levels in a variety of target organs.

Isolation, structural elucidation, and partial synthesis of lutein dehydration products in extracts from human plasma.

All-E-(3R,6'R)-3-hydroxy-3',4'-didehydro-beta,gamma-carotene (anhydrolutein I) and all-E-(3R,6'R)-3-hydroxy-2',3'-didehydro-beta,epsilon-carotene (2',3'-anhydrolutein II) have been isolated and characterized from extracts of human plasma using semipreparative high-performance liquid chromatography (HPLC) on a C18 reversed-phase column. The identification of anhydroluteins was accomplished by comparison of the UV-Vis absorption and mass spectral data as well as HPLC-UV-Vis-mass spectrometry (MS) spiking experiments using fully characterized synthetic compounds. Partial synthesis of anhydroluteins from the reaction of lutein with 2% H2SO4 in acetone, in addition to anhydrolutein I (54%) and 2',3'-anhydrolutein II (19%), also gave (3'R)-3'-hydroxy-3,4-dehydro-beta-carotene (3',4'-anhydrolutein III, 19%). While anhydrolutein I has been shown to be usually accompanied by minute quantities of 2',3'-anhydrolutein II (ca. 7-10%) in human plasma, 3',4'-anhydrolutein III has not been detected. The presence of anhydrolutein I and II in human plasma is postulated to be due to acid catalyzed dehydration of the dietary lutein as it passes through the stomach. These anhydroluteins have also been prepared by conversion of lutein diacetate to the corresponding anhydrolutein acetates followed by alkaline hydrolysis. However, under identical acidic conditions, loss of acetic acid from lutein diacetate proceeded at a much slower rate than dehydration of lutein. The structures of the synthetic anhydroluteins, including their absolute configuration at C(3) and C(6') have been unambiguously established by 1H NMR and in part by 13C NMR, and circular dichroism.