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Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, IA 50011
3To whom correspondence should be addressed. E-mail: abarua{at}iastate.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • violaxanthin • lutein • lutein 5,6-epoxide • lutein esters • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The chemical structures of lutein, zeaxanthin and some of their epoxy analogs that occur widely in nature are shown in Figure 1
. Epidemiologic studies show that an increased intake of fruits and vegetables is associated with decreased risk of cancer (2
). Whether the observed association between consumption of fruits and vegetables and the incidence of cancer and other chronic diseases is due to ß-carotene or to some other carotenoids and/or phytochemicals is still not clear (3
). In two recent studies from this laboratory, ß-carotene 5,6-epoxide and ß-carotene 5,8-epoxide were both found to be much more active than ß-carotene in inducing the differentiation of NB4 cells (4
) and to be absorbed well by humans (5
).
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). However, no information is available about the absorption of xanthophyll epoxides by humans. To study whether xanthophyll epoxides are absorbed by humans, lutein 5,6-epoxide and zeaxanthin 5,6,5'6'-diepoxide were chemically prepared, and the plasma response of these two orally administered carotenoids in humans was examined. |
SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Anhydrous diethyl ether, acetone, acetonitrile, hexane, methanol, dichloromethane, ethyl acetate and NaOH were purchased from Fisher Scientific (Fair Lawn, NJ). Whenever available, HPLC-grade solvents were used for HPLC. 3-Chloroperoxybenzoic acid was purchased from Aldrich (Milwaukee, WI). Neutral alumina (Brockman activity I; Woelm Pharma, GmbH, Eschwege, Germany) was deactivated with water (5 mL/100 g). Lutein (standard) was a gift from Kemin Industries (Des Moines, IA). The sources for other carotenoids were reported earlier (7
).
Preparation of lutein 5,6-epoxide.
The preparation of lutein 5,6-epoxide has been described in detail (8
). In brief, lutein (75 mg) was dissolved in diethyl ether (20 mL) and stirred with 3-chloroperoxybenzoic acid (75 mg). As judged by TLC analysis, most lutein had been converted to the epoxide in 
2 h. The solution was first washed with aqueous NaOH solution (50 g/L) and then with water. The ether phase was dried over anhydrous sodium sulfate and then was evaporated to dryness. The residue was dissolved in diethyl ether (2 mL) and subjected to column chromatography on neutral alumina (2.5 x 25 cm). The column was developed first with hexane and then with increasing volumes of diethyl ether in hexane (5–30%). Fractions of 5 mL were collected. Lutein eluted first with 10–15% ether, followed closely by 5,6-epoxylutein (20–30% ether). Based on an extinction coefficient (E1%, 1 cm) = 2800 in ethanol (9
), the yield of lutein 5,6-epoxide was 56 mg. The identity of lutein 5,6-epoxide (
max 472, 443, 420 nm) was confirmed by treatment with a trace of dilute HCl, which converted the 5,6-epoxide quantitatively to the 5,8-furanoid compound (max 448, 420, 390 nm) (9
). Lutein [retention time (tR) = 13.1 min; max 475, 448, 422 nm in the HPLC solvent mixture] separated very well from lutein 5,6-epoxide (tR = 8.6 min; max 472, 443, 420 nm in HPLC solvent mixture) and from lutein 5,8-epoxide (tR = 8.8 min; max 448, 420, 395 nm in HPLC solvent mixture).
Preparation of zeaxanthin 5,6,5',6'-diepoxide (violaxanthin).
The preparation of violaxanthin from zeaxanthin by stirring with 3-chloroperoxybenzoic acid has been described (8
). As in the case of 5,6-epoxylutein, the reaction was stopped, as judged from TLC, when most of the zeaxanthin and initially formed antheraxanthin were converted to violaxanthin. Violaxanthin was purified by crystallization. Pure violaxanthin showed max 470, 438, 405 nm in methanol (9
). With a trace of HCl, violaxanthin was converted to auroxanthin, max 425, 400, 375 nm. Violaxanthin (tR = 6.7 min) separated very well from antheraxanthin (tR = 10.2 min) and zeaxanthin (tR = 13.9 min). Violaxanthin thus prepared coeluted with violaxanthin isolated from mustard green leaves.
HPLC procedure.
The simultaneous analysis of xanthophyll epoxides, xanthophylls, xanthophyll esters and hydrocarbon carotenes, along with retinol and tocopherols, has been described (8
). In brief, Waters Associates (Milford, MA) components were used with a photodiode array detector (Model 991 or 996). A Microsorb MV 3-µm C-18 column (0.36 x 10 cm) (Rainin, Woburn, MA) was preceded by a guard column packed with C-18 material. A combination of isocratic and gradient programs was primarily used. An isocratic elution with acetonitrile/water [97.5:2.5 (v/v) containing ammonium acetate (10 mmol/L), solvent 1] was run for 5 min at a flow rate of 0.6 mL/min. A linear gradient from solvent 1 to solvent 2 [acetonitrile/dichloromethane/water, 70:30:1 (v/v/v) containing ammonium acetate (10 mmol/L)] and an increase in flow rate from 0.6 to 1.2 mL were applied from 5 to 25 min. Then solvent 2 was on hold from 25 to 65 min at a flow rate of 1.2 mL/min. Thus, the total run time for one analysis was 65 min. If xanthophyll esters were absent, the total run time was 30 min. At the end of the run, the gradient was reversed to original conditions in 5 min, and the column was allowed to equilibrate with solvent 1 for 10 min. Chromatograms (520–280 nm range) were stored in the software memory. Chromatograms were plotted at 445, 420 and 400 nm for carotenoids, at 325 nm for retinoids and at 285 nm for the tocopherols.
Human subjects.
The study was carried out on three healthy human adults, two men and one woman, ages 50–60 y. All three were nonvegetarians, and their daily diet included fruits and vegetables. The vegetables consisted of tomatoes (cooked and raw), greens such as lettuce, spinach (raw and cooked) and mustard (cooked). The fruits consisted of mangoes, bananas, apples, peaches and plums. Any carotenoid-containing food was excluded from the diet during the experiment.
Preparation of the oral doses and collection of blood.
The doses of lutein 5,6-epoxide and zeaxanthin 5,6,5'6'-diepoxide were prepared in the same way as the ß-carotene 5,6-epoxide dose (5
), namely, crystalline lutein 5,6-epoxide (50 mg, 85.5 µmol), or, crystalline zeaxanthin 5,6,5'6'-diepoxide (50 mg, 83 µmol) was ground with 5 mL of corn oil in a small mortar until a clear solution was obtained. The concentration of the carotenoids in the dose was determined spectrophotometrically by use of E (1%, 1 cm) value of 2800 at 443 nm for lutein 5,6-epoxide, and 2550 at 438 nm for zeaxanthin 5,6,5',6'-diepoxide (9
). The oily solution of lutein 5,6-epoxide (1 mL = 17.1 µmol) or zeaxanthin 5,6-epoxide (1 mL = 16.6 µmol) was poured quantitatively by means of a positive displacement pipette onto a piece of thick bread, and the bread was eaten by the volunteer at breakfast. The dose size of violaxanthin, or lutein 5,6-epoxide was 10–100 times the amount of either carotenoid present in one 100-g serving of boiled green beans (6
), fresh spinach or fresh red bell peppers (10
). The breakfast included tea or coffee and toast with butter.
Blood was collected at the Student Health Center of the University by a certified phlebotomist before and at 3, 6 and 9 h after the dose of lutein 5,6-epoxide or zeaxanthin 5,6,5'6'-diepoxide. These time points were selected because it was found in a previous study that the concentration of orally administered ß-carotene 5,6-epoxide peaked at 6 h after the dose (5
). The blood was centrifuged at 1200 x g for 20 min, and the plasma was stored at -20°C until analysis within 2 d. The procedures used were in full accord with the guidelines for the use of human subjects devised by the NIH and with the guidelines set by the University’s Institutional Review Board. The subjects signed an informed consent form.
Extraction of carotenoids, retinoids and tocopherols from plasma.
Extraction of carotenoids, retinoids and tocopherols was carried out by a slight modification of the procedure described previously (11
), namely, plasma (0.5 mL) was mixed with ethanol (1 mL) containing retinyl acetate (internal standard) and BHT (100 mg/L), ethyl acetate (1 mL) and hexane (1 mL). The mixture was vortexed (30 s), and then centrifuged (1 min at 500 x g). The supernatant solution was separated and kept cold. The pellet was vortexed and centrifuged as before with hexane (1 mL), twice. Water (0.5 mL) was added to the pooled supernatant solution, which was vortexed and centrifuged as before. The hexane phase was carefully pipetted into a glass tube and evaporated to dryness under a slow stream of argon. The residue was dissolved in a mixture of acetonitrile/dichloromethane (7:3, v/v) (100 µL). An aliquot of 50 µL was injected onto the HPLC injector by means of a Hamilton syringe.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Analysis by HPLC of plasma samples after an oral dose of 16.6 µmol of zeaxanthin 5,6,5',6'-diepoxide (violaxanthin) to human adults showed that the chromatogram of plasma extract before the dose (0 h) was identical with the chromatograms of plasma extracts 3, 6 and 9 h after the dose. No additional peak due to violaxanthin or any of its metabolites including mono- or diesters appeared, nor did the area of any existing peak increase significantly in any of the plasma extracts. For comparison, a chromatogram of the plasma carotenoids of one nonsupplemented subject is shown in Figure 2A
, along with the chromatogram of the plasma carotenoids (9 h postdosing) of a subject who received an oral dose of violaxanthin (Fig. 2C
). The elution of spiked violaxanthin (peak 1) and lutein 5,6-epoxide (peak 2) and of spiked lutein monoesters (peaks 10–12) and lutein diesters (peaks 13–16) in marigold flower extract (8
) in the 0-h plasma extract of the same subject, is shown in Figure 2B
. The small peaks 3 and 4 found in the region of lutein epoxide at zero time as well as later were metabolites of lutein and are normally present in human plasma (12
). Furthermore, when extracts of plasma taken at 3, 6 and 9 h were treated with HCl and rechromatographed, no shift in max from 438 to 420 (for one 5,6-epoxy group to 5,8-epoxy group), or 400 nm (for two 5,6-epoxy groups to two 5,8-epoxy groups), characteristic of the isomerization of the 5,6,5'6'-diepoxide to either 5,6,5'8'-diepoxide, or, 5,8,5'8'-diepoxide, was noted in any of the peaks.
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Analysis of plasma extracts by HPLC after an oral dose of 17.1 µmol of lutein 5,6-epoxide to human adults, showed that the chromatogram of plasma extract before the dose (0 h) of lutein 5,6-epoxide was identical to the chromatograms of extracts of plasma obtained 3, 6 and 9 h after the dose. There was no indication of the appearance of lutein 5,6-epoxide, lutein 5,8-epoxide, or any of their metabolites, or the enhancement of any existing carotenoid peaks. Furthermore, when the extracts of plasma were treated with HCl and rechromatographed, no shift in max from 443 to 420 nm, characteristic of the isomerization of the 5,6- to 5,8-epoxide, was noted in any of the peaks.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,10
), which are consumed by most people in the United States. Besides these vegetables, cooked mustard greens (leaves) represent a rich source of xanthophyll epoxides (13
) and are popular among Americans of Asian origin. It has been shown that the xanthophyll epoxides, neoxanthin and violaxanthin, constitute 30% of the total carotenoids in mustard green leaves (13
). However, monoepoxy-hydrocarbon carotenes, such as ß-carotene 5,6-epoxide, which are also common constituents of fruits such as mangoes and some vegetables, enhance the differentiation of NB4 cells much better than ß-carotene (4
). In addition, both 5,6- and 5,8-monoepoxy-ß-carotenes are efficiently absorbed by humans, and the absorption of ß-carotene 5,6-epoxide is as high as 60% of the dose (5
). Although it was thought that HCl present in the stomach would convert any 5,6-epoxy-carotenoid to the corresponding 5,8-epoxy-carotenoid, no appreciable conversion was observed during intestinal absorption of ß-carotene 5,6-monoepoxide by humans (5
). The identification of ß-carotene 5,6-epoxide as an in vivo product of ß-carotene in the stomach of rats (14
) and after incubation with intestinal homogenates (15
) was reported. In contrast to the finding that ß-carotene 5,6- and 5,8-epoxides were efficiently absorbed by humans, it was found during the present study that lutein 5,6-epoxide and zeaxanthin 5,6,5',6'-diepoxide were not detectable in the plasma after oral administration. The HPLC method was capable of analysis of esters of epoxyxanthophylls, which were also not detected.
It has been reported that only 4 of 11 human subjects showed plasma response after an oral dose of ß-carotene (16
). The present study was carried out with only 3 subjects. Although it is very unlikely that all three subjects were nonresponders, a larger study with more participants is warranted to confirm the present findings. It is to be noted that one of the subjects in the present study also participated in the ß-carotene 5,6-epoxide absorption study that was carried out in this laboratory and was a very good responder to ß-carotene 5,6-epoxide (5
).
In the present study, under the conditions of extraction and analysis, not a trace of violaxanthin or taraxanthin was detected in plasma within 9 h after the dose. It has been reported that the concentration of ß-carotene after an oral dose of the compound peaked at 24 h, although absorption of carotene was noted at earlier time points also (17
). In view of this finding, future studies on the absorption of xanthophyll epoxides should be aimed at a longer duration of study.
In very careful studies, no occurrence of these epoxy-xanthophylls has been reported as components of human plasma or breast milk, although their nonepoxylated precursors lutein and zeaxanthin, occur in human plasma and milk (12
,18
,19
). The only known difference between these monoepoxy-carotenes and epoxy-xanthophylls is that although monoepoxycarotenes contain at least one unsubstituted ß-ionone ring and can act as provitamin A, the epoxyxanthophylls do not contain an unsubstituted ß-ionone ring and cannot act as provitamin A (20
). Whether the nonprovitamin A carotenoid epoxides including xanthophyll epoxides are unable to enter absorptive cells of the intestinal mucosa is unclear. The mechanism of this effect clearly deserves further attention.
The HPLC method used in the present study could resolve xanthophyll epoxides, xanthophylls, xanthophyll esters and other carotenoids that occur in plasma, along with retinol and tocopherols (8
). It was found that the plasma of all three human adults showed traces of carotenoids (Fig. 2C
, peaks 10, 11) that eluted in the region in which lutein monoesters in marigold petals eluted (Fig. 2B
) (8
). The concentration of these esters was very low, but visible spectra of these peaks, obtained with a photodiode array detector, were identical with that of lutein or its esters. Moreover, saponification of the plasma extract resulted in the disappearance of these peaks, whereas the lutein peak was slightly enhanced. Therefore, it is very likely that the plasma of these adults contained traces of lutein monoesters. It is to be noted that these adults consumed several servings of carotenoid-rich fruits and vegetables daily before, but not during the study. Unlike marigold flowers which are rich in lutein diesters (8
), none of the fruits or vegetables consumed by these volunteers contained substantial amounts of lutein esters. It is not known whether the observed plasma lutein esters resulted from the absorption of the lutein esters from their diet or from dietary lutein that underwent esterification. In view of the report that small amounts of lutein esters could be detected in human plasma after supplements with lutein (21
) and also in human skin (22
), further study is warranted to confirm the identity of these minor peaks that eluted after ß-carotene as lutein monoesters.
In conclusion, neither violaxanthin nor taraxanthin could be detected in human plasma after oral administration of these carotenoids in oil. The observed lack of plasma response of these carotenoids in the present study may be due to several factors, i.e., only a single dose of each carotenoid was administered, all three human subjects were nonresponders and the absorption study was not carried out for a sufficiently long time for carotenoids to appear in the blood. Therefore, future studies should be designed to include repeated doses of the xanthophyll epoxides, a larger number of human subjects and a longer follow-up time.
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FOOTNOTES |
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2 Supported by the U.S. Department of Agriculture NRICGP 00–37200-4290. Journal Paper no. J-19480 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA. Project no. 3335, and supported by Hatch Act and State of Iowa Funds.
4 Deceased (September 22, 2000).
Manuscript received July 20, 2001. Revision accepted September 6, 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Goodwin, T. W. (1980) 2nd ed. The Biochemistry of the Carotenoids 1:96-203 Chapman and Hall London, UK .
2. Block, G., Patterson, B. & Subar, A. (1992) Fruit, vegetables, and cancer prevention: a review of the epidemiological evidence. Nutr. Cancer 18:1-29.[Medline]
3. Olson, J. A. (1999) Shils, M. E. Olson, J. A. Shike, M. Ross, C. A. eds. Carotenoids. In: Modern Nutrition in Health and Disease 9th ed. 1999:520-541 Williams & Wilkins Baltimore, MD .
4. Duitsman, P. K., Barua, A. B., Becker, B. & Olson, J. A. (1999) Effects of epoxycarotenoids, ß-carotene and retinoic acid on the differentiation and viability of the leukemic cell line NB4 in vitro. Int. J. Vitam. Nutr. Res. 69:303-308.[Medline]
5. Barua, A. B. (1999) Intestinal absorption of epoxy-ß-carotenes by humans. Biochem. J. 339:359-362.
6. Khachik, F., Beecher, G., Goli, M. B. & Lusby, W. R. (1991) Separation, identification, and quantification of carotenoids in fruits, vegetables, and human plasma by high-performance liquid chromatography. Pure Appl. Chem. 63:71-80.
7. Barua, A. B. & Furr, H. C. (1992) Extraction and analysis by high-performance liquid chromatography of carotenoids in human serum. Methods Enzymol 213:273-281.[Medline]
8. Barua, A. B. (2001) Improved normal-phase and reversed-phase gradient high-performance liquid chromatographic procedures for the analysis of retinoids and carotenoids in human serum, plant and animal tissues. J. Chromatogr. (in press). .
9. De Ritter, E. & Purcell, A. E. (1981) Carotenoid analytical methods. Bauernfeind, J. C. eds. Carotenoids as Colorants and Vitamin A Precursors: Technological and Nutritional Applications 1981:815-923 Academic Press New York, NY. .
10. Klaui, H. & Bauernfield, J. C. (1981) Carotenoids as food colors. Bauernfeind, J. C. eds. Carotenoids as Colorants and Vitamin A Precursors: Technological and Nutritional Applications 1981:47-317 Academic Press New York, NY. .
11. Barua, A. B. & Olson, J. A. (1998) Reversed-phase gradient high-performance liquid chromatographic procedure for analysis of very polar to non-polar retinoids, carotenoids and tocopherols in animal and plant tissues. J. Chromatogr. B. 707:69-79.
12. Khachik, F., Spangler, C. J., Smith, J. C., Jr, Canfield, L. M., Steck, A. & Pfander, H. (1997) Identification, quantification, and relative concentrations of carotenoids and their metabolites in human milk and serum. Anal. Chem. 69:1873-1881.[Medline]
13. Barua, A. B. & Olson, J. A. (2001) Epoxyxanthophylls, unlike monoepoxycarotenes, are not absorbed by humans. FASEB J 15:A296(abs.).
14.
Barua, A. B. & Olson, J. A. (2000) ß-Carotene is converted primarily to retinoids in rats in vivo. J. Nutr. 130:1996-2001.
15. Handelman, G., van Kujik, F. J., Chatterjea, A. & Krinsky, N. I. (1991) Characterization of products formed during the oxidation of ß-carotene. Free Radic. Biol. Med. 10:427-437.[Medline]
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Johnson, E. J. & Russell, R. M. (1992) Distribution of orally administered ß-carotene among lipoproteins in healthy men. Am. J. Clin. Nutr. 56:128-135.
17. Bowen, P. E., Mobarhan, S. & Smith, J. C., Jr (1993) Carotenoid absorption in humans. Methods Enzymol 214:3-17.[Medline]
18.
Kostic, D., White, W. S. & Olson, J. A. (1995) Intestinal absorption, serum clearance, and interactions between lutein and ß-carotene when administered to human adults in separate or combined oral doses. Am. J. Clin. Nutr. 62:604-610.
19. Parker, R. S. (1993) Analysis of carotenoids in human plasma and tissues. Methods Enzymol 214:86-93.[Medline]
20. Karrer, P. & Jucker, E. (1950) In: Carotenoids 1950:13-15 Elsevier London, UK. .
21. Granado, F., Olmedilla, B., Gil-Martinez, E. & Blanco, I. (1998) Lutein ester in serum after lutein supplementation in human subjects. Br. J. Nutr. 80:445-449.[Medline]
22. Wingerath, T., Sies, H. & Stahl, W. (1998) Xanthophyll esters in human skin. Arch. Biochem. Biophys. 355:271-274.[Medline]
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