QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Asai, A. Articles by Nagao, A. Search for Related Content PubMed PubMed Citation Articles by Asai, A. Articles by Nagao, A.

BIOTRANSFORMATION OF FUCOXANTHINOL INTO AMAROUCIAXANTHIN A IN MICE AND HEPG2 CELLS: FORMATION AND CYTOTOXICITY OF FUCOXANTHIN METABOLITES

National Food Research Institute, Tsukuba, Japan

(Received June 16, 2003; Accepted October 1, 2003)

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Fucoxanthin, a major carotenoid in edible brown algae, potentially inhibits the proliferation of human prostate cancer cells via apoptosis induction. However, it has been postulated that dietary fucoxanthin is hydrolyzed into fucoxanthinol in the gastrointestinal tract before absorption in the intestine. In the present study, we investigated the further biotransformation of orally administered fucoxanthin and estimated the cytotoxicity of fucoxanthin metabolites on PC-3 human prostate cancer cells. After the oral administration of fucoxanthin in mice, two metabolites, fucoxanthinol and an unknown metabolite, were found in the plasma and liver. The unknown metabolite was isolated from the incubation mixture of fucoxanthinol and mouse liver preparation (10,000gsupernatant of homogenates), and a series of instrumental analyses identified it as amarouciaxanthin A [(3S,5R,6'S)-3,5,6'-trihydroxy-6,7-didehydro-5,6,7',8'-tetrahydro-ß,-carotene-3',8'-dione]. The conversion of fucoxanthinol into amarouciaxanthin A was predominantly shown in liver microsomes. This dehydrogenation/isomerization of the 5,6-epoxy-3-hydroxy-5,6-dihydro-ßend group of fucoxanthinol into the 6'-hydroxy-3'-oxo-end group of amarouciaxanthin A required NAD(P)⁺ as a cofactor, and the optimal pH for the conversion was 9.5 to 10.0. Fucoxanthinol supplemented to culture medium via HepG2 cells was also converted into amarouciaxanthin A. The 50% inhibitory concentrations on the proliferation of PC-3 human prostate cancer cells were 3.0, 2.0, and 4.6 µM for fucoxanthin, fucoxanthinol, and amarouciaxanthin A, respectively. To our knowledge, this is the first report on the enzymatic dehydrogenation of a 3-hydroxyl end group of xanthophylls in mammals.

Although the conversions of ß-carotene and other provitamin A carotenoids into retinol are well known, little information is available on the metabolism of nonprovitamin A carotenoid. In particular, the mammalian metabolism of dietary epoxy-xanthophylls, including fucoxanthin, remains unknown despite their abundance in foodstuffs and the potential health benefits. Recently, we demonstrated in mice that orally administered fucoxanthin is incorporated into blood circulation as fucoxanthinol, a deacetylated metabolite (Sugawara et al., 2002). In the present study, we investigated further biotransformation of fucoxanthinol in mice and HepG2 cells and estimated the antiproliferative effect of fucoxanthin metabolites on PC-3 human prostate cancer cells.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials. Cholesterol esterase from porcine pancreas, lysophosphatidylcholine (1-palmitoyl-sn-glycero-3-phosphocholine), mono-olein, sodium taurocholate and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)¹ were purchased from Sigma-Aldrich (St. Louis, MO). Boiled and dried wakame was purchased from a local market in Tsukuba, Japan. Other chemicals and solvents were of reagent grade.

Apparatus. Visible absorption (VIS) spectra were measured on a U-3310 spectrophotometer (Hitachi, Ltd., Tokyo, Japan). High-resolution mass spectra were recorded on a Fourier transform ion cyclotron resonance mass spectrometer (APEX II 70e; Nihon Bruker Daltonics, Tsukuba, Japan) with an interface of electrospray ionization. ¹H NMR spectra were recorded on a Bruker AVANCE 800 instrument at 4°C in CDCl₃ (99.96%; Isotech, Miamisburg, OH). Chemical shifts () were related to the signals of tetramethylsilane, and only relevant coupling constant values (J) were given. Circular dichroism (CD) spectrum was measured with a J-820 CD System (Jasco, Tokyo, Japan). Ellipticity () was expressed as actual values in millidegrees observed with a cell thickness of 0.1 cm.

Analytical HPLC was carried out with an HP-1100 System (Agilent Technologies, Palo Alto, CA) equipped with a photodiode array detector. A TSK-gel ODS 80Ts column, 2 x 250 mm (Tosoh Corporation, Tokyo, Japan), attached to an ODS guard column (2 x 10 mm; Tosoh) was used with an appropriate mobile phase for each analysis. The eluate was monitored with the photodiode array detector (range 250–550 nm), and carotenoids were quantified from their peak area at 450 nm by use of calibration curves of authentic standards.

LC/MS analysis was performed with the HPLC system connected to an LCQ mass spectrometer (Thermo Finnigan, San Jose, CA) equipped with an interface of atmospheric pressure chemical ionization (APCI). HPLC was carried out on a TSK-gel ODS 80Ts column, 2 x 250 mm (Tosoh), with acetonitrile/methanol/water (75:15:10, v/v) containing 0.1% (w/v) ammonium acetate as a mobile phase at a flow rate of 0.2 ml/min. APCI was performed in the positive mode with the HPLC eluate flowing into the ion source with the capillary set at 240°C, the vaporizer at 500°C, the source voltage at 8 kV, the source current at 5 µA, and the nitrogen sheath gas flow and auxiliary gas flow set at 80 and 10 (arbitrary units), respectively.

Preparation of Fucoxanthin and Fucoxanthinol. All procedures for carotenoid preparation and extraction were carried out under dim yellow light to minimize degradation and isomerization by light irradiation. Fucoxanthin was isolated from wakame as previously reported (Haugan et al., 1992). Fucoxanthinol was prepared from fucoxanthin by enzymatic hydrolysis. Approximately 5 µmol of fucoxanthin and 100 mg of taurocholate were together dissolved in a small volume of dichloromethane/methanol (2:1), and the solvent was dried up under a stream of argon gas in a centrifuge tube (50-ml volume). Then, 10 units of cholesterol esterase in 10 ml of potassium phosphate buffer (0.1 M, pH 7.0) were added to the residue and dissolved with a vortex mixer. After incubation at 37°C for 2 h, 4 volumes of dichloromethane/methanol (2:1, v/v) were added to the reaction mixture, and fucoxanthinol was extracted into the dichloromethane phase. The extracted fucoxanthinol was then purified by preparative HPLC on a TSK-gel ODS 80Ts column, 10 x 250 mm (Tosoh), with acetonitrile/methanol/water (67.5:13.5:19, v/v) containing 0.1% (w/v) ammonium acetate as a mobile phase. The purified fucoxanthinol was confirmed by VIS, high-resolution MS, LC/MS, and NMR spectra.

Fucoxanthinol: VIS _max (acetone) 447.5 nm; high-resolution MS m/z 639.4020 [M+Na]⁺ (calc. C₄₀H₅₆O₅Na: 639.4020); LC/MS t_R 7.1 min, m/z (% rel. int.) 617 [M+H]⁺ (16), 599 [M+H-H₂O]⁺ (100), 581 [M+H-2H₂O]⁺ (65), 563 [M+H-3H₂O]⁺ (5); ¹H NMR (800 MHz, CDCl₃) = 0.96 s (3H, Me-17), 1.04 s (3H, Me-16), 1.07 s (3H, Me-17'), 1.23 s (3H, Me-18), 1.34 s (3H, Me-18'), 1.35 s (3H, Me-16'), 1.42 m (1H, H-2), 1.50 m (1H, H-2), 1.79 m (1H, H-4), 1.81 s (3H, Me-19'), 1.94 s (3H, Me-19), 1.96 m (1H, H-2'), 2.00 s (6H, Me-20, 20'), 2.27 m (1H, H-4'), 2.33 m (1H, H-4), 2.60 d (J_gem = 18 Hz, 1H, H-7), 3.82 br (1H, H-3), 4.33 br (1H, H-3'), 6.04 s (1H, H-8'), 6.13 d (J_10',11' = 11 Hz, 1H, H-10'), 6.27 d (J_14',15' = 12 Hz, 1H, H-14'), 6.36 d (J_12',11' = 15 Hz, 1H, H-12'), 6.42 d (J_14,15 = 12 Hz, 1H, H-14), 6.58 dd (J_11,10 = 11 Hz, J_11,12 = 15 Hz, 1H, H-11), 6.61 dd (J_11',10' = 11 Hz, J_11',12' = 15 Hz, 1H, H-11'), 6.65 dd (J_15,14 = 12 Hz, J_15,15' = 14 Hz, 1H, H-15), 6.68 d (J_12,11 = 15 Hz, 1H, H-12), 6.76 dd (J_15',14' = 12 Hz, J_15',15 = 14 Hz, 1H, H-15'), 7.16 d (J_10,11 = 11 Hz, 1H, H-10).

The purities of fucoxanthin and fucoxanthinol were >99%, based on the peak area of all components absorbed at each specific wavelength in HPLC analysis. The extinction coefficient of each carotenoid was used for quantification (Haugan et al., 1992; Strand et al., 1998). The carotenoids were dissolved in acetone and stored at -80°C.

Animal Studies. All procedures involving animals were conducted in accordance with the Guidelines for Experimental Animals of the National Food Research Institute, Japan. Male ICR mice (7 weeks old; Clea Japan, Inc., Tokyo, Japan) were housed at 25°C with a 12-h light/dark cycle and acclimated with free access to a standard rodent chow (MF; Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water. After 7 days of feeding, mice were deprived of food for 15 h before the administration trials described below.

For the oral administration study, mixed micelles containing fucoxanthin were prepared as described previously (Sugawara et al., 2002) with a slight modification. In brief, appropriate volumes of the stock solutions were transferred to a glass tube, and the solvent was dried up under a stream of argon gas. The residue was then dissolved in distilled water with a vortex mixer. The final concentrations were 12 mM sodium taurocholate, 2.5 mM mono-olein, 7.5 mM oleic acid, 1.25 mM lysophosphatidylcholine, and 0.2 mM fucoxanthin. The micellar solution (40 nmol of fucoxanthin in 0.2 ml) was administered to each mouse by direct stomach intubation. At 0 (untreated), 1, 2, 3, 6, or 9 h after the administration, each mouse was anesthetized with diethyl ether, and a blood sample was collected from the caudal vena cava by a heparinized syringe. Plasma was prepared from each blood sample by centrifugation of blood at 1000g for 15 min at 4°C. Immediately after each blood sample was taken, the liver was excised and rinsed with ice-cold saline.

For the intravenous injection study, an emulsion containing fucoxanthinol was prepared as follows. Appropriate amounts of fucoxanthinol were dissolved with soybean oil (20 mg) and soybean phosphatidylcholine (5 mg) in a small amount of dichloromethane in a glass tube, and the solvent was dried under a stream of argon gas. After the residue was suspended in 0.5 ml of 0.15 M NaCl with a vortex mixer, the suspension was sonicated three times for 2 min each under ice-cold conditions, and then filtrated with a sterilized 0.2-µm filter. The concentration of fucoxanthinol in the filtrated emulsion was confirmed by HPLC. The emulsion (1.5 nmol of fucoxanthinol in 50 µl) was injected into the tail vein of each mouse. Two hours after the injection, plasma and liver samples were prepared as described above.

Metabolites of fucoxanthin were extracted as follows. Plasma (0.1 ml) diluted with 0.3 ml of phosphate-buffered saline (PBS) or 0.4 ml of liver homogenates (10% in PBS, w/v) was mixed with 1.5 ml of dichloromethane/methanol (1:2, v/v) and vortexed for 1 min. Then, dichloromethane (1.9 ml) and distilled water (1.9 ml) were added to the mixture and further vortexed for 1 min. After the centrifugation at 1000g for 10 min, the dichloromethane phase was transferred and evaporated to dryness in vacuo. The residue was dissolved in a mixture of dimethyl sulfoxide (DMSO)/methanol (1:1, v/v), and an aliquot was subjected to HPLC analysis.

Analytical HPLC was carried out with a mobile phase of acetonitrile/methanol/water (75:15:10, v/v) containing 0.1% (w/v) ammonium acetate at a flow rate of 0.3 ml/min (system 1). The quantity of the unknown metabolite, identified as amarouciaxanthin A in this report, was estimated from the peak area at 450 nm by use of the calibration curve of authentic fucoxanthinol and expressed as fucoxanthinol equivalence. The molar extinction coefficient of amarouciaxanthin A could not be determined because an insufficient amount was obtained. It was necessary to wash out the nonpolar compounds on the column with methanol/ethyl acetate (70:30, v/v) for 15 min at a flow rate of 0.3 ml/min and then equilibrate the column with the mobile phase described above for 10 min before a subsequent injection.

Isolation and Identification of Amarouciaxanthin A. The unknown metabolite shown in mouse plasma and liver was synthesized by incubating fucoxanthinol with liver homogenates. Livers of male ICR mice (8 weeks old) were excised immediately after cervical dislocation and rinsed with ice-cold saline. The livers were then homogenized in a Potter-Elvehjem homogenizer with 4 volumes of ice-cold 0.154 M KCl containing 50 mM Hepes-KOH, pH 7.4, 1.0 mM EDTA, and 0.1 mM dithiothreitol, and the homogenates were centrifuged at 10,000g for 10 min at 4°C. The 10,000g supernatant (7 ml) was then incubated at 37°C for 3 h with 50 µM fucoxanthinol, 2.4 mM NAD⁺, and 0.2% Tween 20 (final concentrations) in 0.1 M glycine-KOH buffer, pH 10.0, in a total volume of 20 ml. After the incubation, the reaction mixture was mixed with 30 ml of dichloromethane/methanol (2:1, v/v), and the metabolite was extracted into the dichloromethane phase. The extract was then dried up under an argon gas stream, and the residue was applied to an alumina column. Neutral alumina (ICN Biomedicals Inc., Costa Mesa, CA) was deactivated to activity III before use. The metabolite was washed with increasing amounts of diethyl ether in hexane (2:8–5:5, v/v). It was then eluted with diethyl ether and further purified by preparative HPLC on a TSK-gel ODS 80Ts column, 10 x 250 mm, with acetonitrile/methanol/water (50:25:25, v/v) containing 0.1% (w/v) ammonium acetate as a mobile phase. The purified metabolite was identified as amarouciaxanthin A by VIS, MS, NMR, and CD spectral analyses.

Characterization of the Hepatic Conversion of Fucoxanthinol into Amarouciaxanthin A. Subcellular fractions were also prepared from the livers of male ICR mice (8 weeks old, 15 h of food deprivation). The livers were homogenized in a Potter-Elvehjem homogenizer with 4 volumes of ice-cold 0.25 M sucrose containing 10 mM Tris-HCl, pH 7.4, and 0.1 mM EDTA (sucrose buffer). Nuclei and debris were removed by centrifugation at 600g for 10 min at 4°C. The postnuclear supernatant was then centrifuged at 9000g for 10 min at 4°C, and the mitochondrial precipitates were washed by resuspension in the sucrose buffer and centrifuged at 9000g for 10 min at 4°C. The postmitochondrial supernatant was further centrifuged at 105,000g for 60 min at 4°C, and the precipitates of microsomes were washed by resuspension in the sucrose buffer and centrifuged at 105,000g for 60 min at 4°C. The postmicrosomal supernatant was centrifuged again at 105,000g for 90 min at 4°C, and the resulting supernatant was used as the cytosolic fraction. Prepared subcellular fractions were suspended in the sucrose buffer and were stored at -80°C. Protein concentrations were determined using a DC Protein Assay kit (Bio-Rad, Hercules, CA), and glucose 6-phosphatase activity was measured as a marker enzyme of microsomes (Swanson, 1955).

A standard incubation mixture unless otherwise stated contains 10 µM fucoxanthinol, 2.4 mM NAD⁺, 0.1 M glycine-KOH buffer (pH 10.0), 0.2% Tween 20, and the subcellular fraction (0.5 mg of protein) in a total volume of 0.2 ml. The mixture was incubated at 37°C for 60 min. The incubation was terminated by adding 0.8 ml of dichloromethane/methanol (2:1, v/v). The mixture was then vortexed for 1 min. After the centrifugation at 1000g for 10 min at 4°C, 0.4 ml of the dichloromethane phase was transferred and evaporated to dryness in vacuo. The residue was dissolved in a mixture of acetonitrile/methanol/water (50:25:25, v/v), an aliquot of which was subjected to HPLC analysis in a mobile phase of acetonitrile/methanol/water (50:25:25, v/v) containing 0.1% (w/v) ammonium acetate at a flow rate of 0.2 ml/min (system 2). The quantity of amarouciaxanthin A was estimated as a fucoxanthinol equivalence as described above.

Conversion of Fucoxanthinol into Amarouciaxanthin A by HepG2 Cells. HepG2 human hepatoma cells (RIKEN BioResource Center, Tsukuba, Japan) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 4 mM L-glutamine, 40,000 U/l penicillin, and 40 mg/l streptomycin at 37°C in a humidified atmosphere of 5% CO₂ in air. Cells that reached 90% confluence in 60-mm dishes were cultured in fresh medium (4 ml/dish) supplemented with 1.0 µM fucoxanthinol and 0.4% DMSO (vehicle). After 24 h in culture, the media were transferred and filtrated with a 0.2-µm filter to remove cell debris, and the cell layers were washed twice with ice-cold PBS (4 ml/dish). Amarouciaxanthin A was extracted from the medium by using 4 volumes of dichloromethane/methanol (2:1, v/v) and from the cell layers twice by using a mixture of hexane/2-propanol (3:2, v/v) (Brown et al., 1980). The quantity of amarouciaxanthin A was estimated by analytical HPLC (system 2).

Antiproliferative Activity on PC-3 Cells. PC-3 human prostate cancer cells (American Type Culture Collection, Manassas, VA) were cultured, and MTT assay was performed as previously reported (Kotake-Nara et al., 2001) with slight modifications. In brief, PC-3 cells were seeded at a density of 3 x 10³ cells per well containing 100 µl of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in 96-well plates. After 24 h in culture, the medium was replaced with fresh medium supplemented with fucoxanthin, fucoxanthinol, or amarouciaxanthin A. The carotenoids, dissolved in DMSO, were added to the culture medium at final concentrations of 0.625 to 20 µM. The final concentration of the vehicle (DMSO) was 0.4%, and the control culture received the vehicle alone. After 72 h in culture, cell viability was evaluated by MTT assay (Mosmann, 1983) with a modified extraction buffer (Hansen et al., 1989). The IC₅₀ on PC-3 proliferation was calculated from the regression equation of a four-parameter sigmoid in SigmaPlot ver. 6.0 statistical software (SPSS Inc., Chicago, IL).

To determine the cellular uptake of carotenoids, the PC-3 cells that had reached up to 90% confluence in 24-well plates were cultured in fresh medium (0.5 ml/dish) supplemented with 1.0 µM carotenoids and 0.4% DMSO. After 4 h in culture, the carotenoids were extracted from cells as described above and were then identified by the analytical HPLC (system 1).

	Results

Top Abstract Materials and Methods Results Discussion References

 
Fucoxanthin Metabolites in Mice. After the oral administration of fucoxanthin, two metabolite peaks at 4.7 and 6.3 min of retention time (t_R) appeared in the chromatograms of plasma (Fig. 1A) as well as in the chromatogram of liver (Fig. 1B) extracts. The metabolite at t_R 4.7 min was identified as fucoxanthinol from the retention time, VIS spectrum (Fig. 1C), and characteristic ions at m/z 617 [M+H]⁺, 599 [M+H-H₂O]⁺, 581 [M+H-2H₂O]⁺, and 563 [M+H-3H₂O]⁺ in LC/MS. The UV-visible spectrum of the metabolite at t_R 6.3 min was different from that of fucoxanthinol (Fig. 1C). The metabolite (t_R 6.3 min) was identified as amarouciaxanthin A, as described below. Fucoxanthinol and amarouciaxanthin A in plasma and liver reached almost maximum concentrations within 2 h after the single administration of fucoxanthin and remained at detectable levels for 9 h (Fig. 2). In its nonmetabolized, intact form, fucoxanthin was not detected from the plasma or the liver at any time in the 9-h period after oral administration.

View larger version (26K): [in this window] [in a new window]   FIG. 1. Representative HPLC chromatograms of fucoxanthin metabolites in mouse plasma (A) and liver (B) and VIS spectra of the metabolites in plasma (C). The HPLC condition (system 1) was described under Materials and Methods. Chromatograms of plasma (A) and liver (B) extracts of mice before (dashed lines) and 2 h after (solid lines) an oral administration of fucoxanthin (40 nmol/mouse). C, VIS spectra of peaks at tR 4.7 min (fucoxanthinol, solid line) and tR 6.3 min (amarouciaxanthin A, dashed line) in panel A.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Time course changes of fucoxanthin metabolites in mouse plasma (A) and liver (B) after a single oral administration of fucoxanthin. Mice were administered fucoxanthin (40 nmol/mouse) solubilized in mixed micelles. Values are means ± S.D. of five mice. , fucoxanthinol; , amarouciaxanthin A.

After the i.v. injection of fucoxanthinol, amarouciaxanthin A appeared in the plasma and liver of mice. Two hours after the i.v. injection (1.5 nmol of fucoxanthinol/mouse), the concentrations of fucoxanthinol and amarouciaxanthin A were 29.1 and 17.2 pmol/ml in plasma and 108.9 and 18.2 pmol/g in liver, respectively (means of two mice).

Identification of Amarouciaxanthin A. After the incubation of fucoxanthinol with the mouse liver preparation (10,000g supernatant), a metabolite was generated from fucoxanthinol (Fig. 3). From the retention times (6.3 and 60 min for systems 1 and 2, respectively) and VIS spectrum (_max 468 nm) in analytical HPLC, we decided that the metabolite was identical to that found in the fucoxanthin-administered mice. From the spectral data, we identified the metabolite as amarouciaxanthin A, which has been isolated from marine animals, a tunicate (Amaroucium pliciferum) (Matsuno et al., 1985a), and a bivalve (Paphia euglypta) (Matsuno et al., 1985b).

View larger version (19K): [in this window] [in a new window]   FIG. 3. HPLC chromatogram (A) and UV-visible spectrum (B) of amarouciaxanthin A produced by the incubation of fucoxanthinol with mouse liver homogenates. The incubation and HPLC (system 2) conditions are described under Materials and Methods. A, chromatogram obtained from the incubation mixture after 3-h incubation. B, spectra of fucoxanthin (solid line) and amarouciaxanthin A (dashed line) shown in panel A.

Amarouciaxanthin A [(3S,5R,6'S)-3,5,6'-trihydroxy-6,7-didehydro-5,6,7',8'-tetrahydro-ß,-carotene-3',8'-dione]: VIS _max (acetone) 465 nm, (ethanol) 467.5 nm; CD (nm) () [ethanol, A_467.5 = 0.945 (l = 0.1 cm)] 215 (1.35), 225 (0), 246 (-6.99), 263 (0), 271 (2.89); high-resolution MS m/z 637.3875 [M+Na]⁺ (calc. C₄₀H₅₄O₅Na: 637.3864); LC/MS, shown in Fig. 4; ¹H NMR (800 MHz, CDCl₃) = 1.05 s (3H, Me-17'), 1.07 s (3H, Me-17), 1.08 s (3H, Me-16'), 1.34 s (3H, Me-18), 1.34 s (3H, Me-16), 1.34 m (1H, H-2), 1.42 m (1H, H-4), 1.82 s (3H, Me-19), 1.91 s (3H, Me-18'), 1.95 m (1H, H-2), 1.96 s (3H, Me-19'), 1.99 s (3H, Me-20'), 2.01 s (3H, Me-20), 2.28 m (1H, H-4), 2.34 d (J_gem = 18 Hz, 1H, H-2'), 2.49 d (J_gem = 18 Hz, 1H, H-2'), 2.94 d (J_gem = 15 Hz, 1H, H-7'), 3.05 d (J_gem = 15 Hz, 1H, H-7'), 5.85 s (1H, H-4'), 4.33 br (1H, H-3), 5.95 s (1H, OH-6', disappeared by additional D₂O), 6.04 s (1H, H-8), 6.13 d (J_10,11 = 11 Hz, 1H, H-10), 6.27 d (J_14,15 = 12 Hz, 1H, H-14), 6.36 d (J_12,11 = 15 Hz, 1H, H-12), 6.46 d (J_14',15' = 12 Hz, 1H, H-14'), 6.57 dd (J_11',10' = 11 Hz, J_11',12' = 15 Hz, 1H, H-11'), 6.62 dd (1H, H-11), 6.64 dd (1H, H-15'), 6.72 d (J_12',11' = 15 Hz, 1H, H-12'), 6.76 dd (1H, H-15), and 7.11 d (J_10',11' = 11 Hz, 1H, H-10'). The proton assignment in the ¹H NMR spectrum was confirmed by double quantum-filtered correlation spectroscopy, heteronuclear single quantum correlation, heteronuclear multiple-bond correlation spectroscopy, and nuclear Overhauser effect spectroscopy spectra. The hydrogen/deuterium exchange performed in high-resolution MS with CH₃OD/D₂O (1:1, v/v) as solvent gave the shift of molecular ion (+ 3 u), m/z 640.4089 [M_D+Na]⁺ (calc. for C₄₀H₅₁D₃O₅Na: 640.4057); i.e., three exchangeable hydrogen atoms exist in the structure (three hydroxyl groups in amarouciaxanthin A). The reduction by sodium tetrahydroborate (Eugster, 1995) gave a hypsochromic shift with increased spectral fine structure in the VIS spectrum, _max (HPLC solvent) 400, 424, and 450 nm [% III/II (Britton, 1995) = 95], and the shift of molecular-related ions (+ 4 u) in LC/MS, t_R 6.0 min, m/z (% rel. int.) 619 [M+H]⁺ (10), 601 [M+H-H₂O]⁺ (83), 583 [M+H-2H₂O]⁺ (100), and 565 [M+H-3H₂O]⁺ (32); i.e., two carbonyl groups exist in the structure, and at least one carbonyl group is conjugated with the main polyene chain (Britton, 1995).

View larger version (22K): [in this window] [in a new window]   FIG. 4. APCI-MS spectrum of purified amarouciaxanthin A in LC/MS analysis. The proposed fragment pattern in the MS spectrum is indicated. Inset, total ion chromatogram (tR 9.3, amarouciaxanthin A).

Characterization of the Hepatic Conversion Activity. Figure 5A shows the subcellular distribution of the activity by which fucoxanthinol is converted into amarouciaxanthin A in mouse liver. The activity was recovered predominantly from the microsomes (74% of total activity). The mitochondrial proportion (26%) of conversion activity was probably due to microsomal contamination in the mitochondrial fraction, since that fraction contained 33% of the activity of glucose 6-phosphatase, a marker enzyme of microsomes. NAD⁺ was the preferred cofactor for this microsomal dehydrogenation/isomerization of 5,6-epoxy-3-hydroxy-5,6-dihydro-ß end group of fucoxanthinol into 6'-hydroxy-3'-oxo- end group of amarouciaxanthin A, whereas NADP⁺ was less preferred (Fig. 5B). The optimal pH for the conversion was 9.5 to 10.0 (Fig. 5C).

View larger version (18K): [in this window] [in a new window]   FIG. 5. Characterization of hepatic conversion of fucoxanthinol into amarouciaxanthin A in mice. A, subcellular distribution of the conversion activity (solid bars) and glucose 6-phosphatase (open bars). Values are means ± S.D. of three mice. B, effect of cofactors on the microsomal conversion activity. Microsomes were incubated without (none) or with 2.4 mM NAD(P)+. Other conditions were described under Materials and Methods. Values are means ± S.D. of three mice. C, pH dependence of the microsomal conversion activity. The incubation mixture was buffered by Hepes (pH 7.0), N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine (pH 8.0), or glycine (pH 9.0–11.0). Other conditions were described under Materials and Methods. Duplicate incubations were performed on each pH point.

Conversion of Fucoxanthinol into Amarouciaxanthin A by HepG2 Cells. When HepG2 cells were cultured in fucoxanthinol-supplemented medium, amarouciaxanthin A was produced in the cells and in the cultured medium. After 24 h in culture with fucoxanthinol (4 nmol/dish), the amounts of amarouciaxanthin A in the cells and in the cultured medium were 68 ± 1 and 31 ± 5 pmol/dish, respectively (means ± S.D. of three dishes). On the other hand, no amarouciaxanthin A was produced by incubating fucoxanthinol for 24 h with the medium only, regardless of whether the medium was fresh or conditioned with HepG2 cells of 90% confluence for 24 h before the incubation. No morphological changes were observed in HepG2 cells cultured with fucoxanthinol (1.0 µM) for 24 h compared with the cells cultured with the vehicle alone.

Antiproliferative Effect on PC-3 Cells. Figure 6A shows the dose-dependent effects of fucoxanthin, fucoxanthinol, and amarouciaxanthin A on the viability of PC-3 human prostate cancer cells. The IC₅₀ values on the proliferation of PC-3 cells were 3.0, 2.0, and 4.6 µM for fucoxanthin, fucoxanthinol, and amarouciaxanthin A, respectively.

View larger version (24K): [in this window] [in a new window]   FIG. 6. Antiproliferative effect (A) and cellular uptake (B) of fucoxanthin and its metabolites on PC-3 human prostate cancer cells. A, PC-3 cells were cultured with fucoxanthin- (), fucoxanthinol- (), or amarouciaxanthin A- () supplemented medium for 72 h. Cell viability was estimated by MTT assay and is expressed relative to that of the control cells treated with vehicle alone. The viability corresponding to that of the cells before the carotenoid treatment (initial cells) is indicated as a solid horizontal line. The intersection of the dotted line and the line plotted for each carotenoid indicates the IC50 value on cell proliferation during the 72-h culture. Values are means ± S.D. of eight wells. Replicate experiments demonstrated a similar trend. B, PC-3 cells were cultured with fucoxanthin-, fucoxanthinol-, or amarouciaxanthin A-supplemented medium (500 pmol/well) for 4 h. The solid bar indicates the combined value of fucoxanthin (dotted bar) and fucoxanthinol (hatched bar) in the cells cultured with fucoxanthin-supplemented medium. Values are means ± S.D. of three wells.

The cellular uptakes of fucoxanthin, fucoxanthinol, and amarouciaxanthin A after 4 h in culture are shown in Fig. 6B. No morphological changes were seen in PC-3 cells cultured for 4 h with the carotenoids at the concentration of 1.0 µM. The cellular uptake of fucoxanthin was estimated as a combined value of fucoxanthin and fucoxanthinol, since fucoxanthin was hydrolyzed into fucoxanthinol in PC-3 cells (Fig. 6B). No amarouciaxanthin A was generated in PC-3 cells cultured with fucoxanthin- or fucoxanthinol-supplemented medium.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Several reports have noted that fucoxanthin, the major carotenoid in edible brown algae, has potential health benefits. However, little is known about the absorption and metabolism of dietary fucoxanthin in mammals. As reported previously (Sugawara et al., 2002), fucoxanthinol and an unknown metabolite, identified in the present study as amarouciaxanthin A, were confirmed in mouse plasma after the oral administration of fucoxanthin. Both metabolites were also found in the liver. Since these metabolites were detected in plasma for 9 h (Fig. 2), they and/or their further metabolites may act as functional molecules in vivo.

In the present study, no unchanged fucoxanthin was found in the plasma or liver of the fucoxanthin-administered mice. Two hours after the oral administration, unchanged fucoxanthin was not detected in the contents of the small intestine, whereas fucoxanthinol was present predominantly (data not shown). Orally administered fucoxanthin therefore seems to be rapidly hydrolyzed to fucoxanthinol in the gastrointestinal tract, although it could be in part hydrolyzed after absorption within 2 h after the administration. The enzymes that hydrolyze fucoxanthin in the gastrointestinal tract might be lipase, cholesterol esterase, carboxylesterase, or others. Fucoxanthin was actually deacetylated into fucoxanthinol in vitro by cholesterol esterase in the present study and by lipase in our previous study (Sugawara et al., 2002). It has been suggested that fatty acid esters of carotenoids are hydrolyzed in the small intestine in humans, because no esters have been detected in chylomicrons or serum (Khachik et al., 1992; Wingerath et al., 1995). Strand et al. (1998) reported that fucoxanthinol but not fucoxanthin was transferred to the egg yolks of laying hens fed a diet containing 15% brown alga meal. This hydrolysis of fucoxanthin is consistent with the general phenomenon that animals absorb carotenoids as a nonesterified form.

Amarouciaxanthin A was shown in plasma and liver of mice also after the i.v. injection of fucoxanthinol. This indicates that amarouciaxanthin A was generated from fucoxanthinol in vivo. In the present study, fucoxanthinol was actually converted into amarouciaxanthin A by the liver homogenate preparation. Although the conversion did not occur by a similar homogenate preparation of small intestinal mucosa (data not shown), the possibility of extrahepatic conversion of fucoxanthin into amarouciaxanthin A cannot be excluded. Also, in HepG2 cell culture, amarouciaxanthin A was produced from fucoxanthinol. This suggests that fucoxanthinol is metabolized to amarouciaxanthin A also in human liver, since HepG2 is a highly differentiated cell line used in numerous studies on liver functions as a model of human hepatocytes. No amarouciaxanthin A was produced from fucoxanthinol by incubation with the medium only, even when the medium was conditioned with HepG2 cells. Therefore, amarouciaxanthin A in cultured medium should be secreted from HepG2 cells but should not be converted from fucoxanthinol in the medium by the secreted materials. From these findings, we propose the metabolic pathway of dietary fucoxanthin shown in Fig. 7. Until now, amarouciaxanthin A has been found only in two marine invertebrates, a tunicate (A. pliciferum) (Matsuno et al., 1985a) and a bivalve (P. euglypta) (Matsuno et al., 1985b). From the structural similarity, Matsuno et al. (1985a) hypothesized that amarouciaxanthin A would be one of the metabolites of fucoxanthin in A. pliciferum. However, there has been no experimental evidence for the conversion of fucoxanthin into amarouciaxanthin A. In particular, the metabolism of dietary fucoxanthin in mammals has been quite unknown until the present study.

View larger version (21K): [in this window] [in a new window]   FIG. 7. Proposed metabolic pathway of dietary fucoxanthin in mammals.

From the structures of fucoxanthinol and amarouciaxanthin A, we presumed that dehydrogenase participates in the conversion (dehydrogenation/isomerization) of fucoxanthinol into amarouciaxanthin A. As shown in Fig. 5, the conversion was observed to occur predominantly in the microsomes when microsomal contamination into the mitochondrial fraction was taken into account. The cofactor and pH dependences indicate that NAD(P)⁺-dependent dehydrogenase participates in the conversion. In mammals, a number of NAD(P)⁺-dependent dehydrogenases are involved in steroid and retinoid metabolisms and belong to short-chain dehydrogenase/reductase (SDR) (Jörnvall et al., 1995) and aldo-keto reductase (Jez et al., 1997) superfamilies. Interestingly, in the plant kingdom, an SDR family enzyme plays an important role in the biosynthesis of abscisic acid, a plant hormone (Cheng et al., 2002; Seo and Koshiba, 2002). Cheng et al. (2002) reported that the conversion of xanthoxin, a cleaved compound of 9'-cis neoxanthin, into abscisic aldehyde is catalyzed by Arabidopsis SDR (SDR1). This conversion of the 5,6-epoxy-3-hydroxy-5,6-dihydro-ß end group in xanthoxin into the 6-hydroxy-3-oxo- end group in abscisic aldehyde is identical to the conversion of fucoxanthinol into amarouciaxanthin A confirmed in the present study. Thus, the dehydrogenase responsible for the conversion of fucoxanthinol into amarouciaxanthin A might belong to the mammalian SDR superfamily. To our knowledge, this is the first report in mammals on the enzymatic dehydrogenation of a 3-hydroxyl end group of xanthophylls. Khachik et al. (1997, 2002) reported the presence of (3R,6'R)-3-hydroxy-ß,-carotene-3'-one (3'-oxolutein) in human blood plasma and ocular tissues and presumed that 3'-oxolutein is converted from dietary lutein in vivo. Similar to the conversion medium found in the present study, the oxidation of lutein to 3'-oxolutein might be enzymatically mediated.

5,6-Epoxy-xanthophylls such as fucoxanthin, neoxanthin, and violaxanthin are widely distributed in nature and represent a major portion of dietary carotenoids in a number of vegetables, fruits, and edible algae. Neoxanthin and violaxanthin are major carotenoids in leafy green vegetables. Recently, we demonstrated that fucoxanthin and neoxanthin remarkably reduce the viability of prostate cancer cells via apoptosis induction (Kotake-Nara et al., 2001). Apoptosis induction by fucoxanthin has also been observed in HL-60 human leukemia cells (Hosokawa et al., 1999). In the present study, two metabolites of fucoxanthin, fucoxanthinol and amarouciaxanthin A, also reduced the viability of PC-3 cells (Fig. 6A). Since 50% of incorporated fucoxanthin was hydrolyzed to fucoxanthinol in PC-3 cells within the 4-h culture (Fig. 6B), the antiproliferative effect of fucoxanthin may be attributable to the action of fucoxanthinol or its metabolites. Since epoxides are generally supposed to be chemically reactive, the 5,6-epoxide in fucoxanthin was considered to play important roles in cytotoxicity. However, amarouciaxanthin A, a nonepoxy metabolite, also reduced the viability of PC-3 cells (Fig. 6A). This suggests that the 5,6-epoxide is not essential for the cytotoxicity of epoxy-xanthophylls, although the IC₅₀ value of amarouciaxanthin A is higher than those of fucoxanthin and fucoxanthinol. One or more other mechanisms not based on the cytotoxicity of epoxide should be involved in the antiproliferative effect of epoxy-xanthophylls on PC-3 cells.

Despite their abundance in foodstuffs and potential health benefits, little is known about the absorption and metabolism of dietary epoxyxanthophylls. In the present study, we demonstrated that orally administered fucoxanthin is hydrolyzed into fucoxanthinol and subsequently converted into amarouciaxanthin A in mice. Therefore, fucoxanthinol and amarouciaxanthin A, rather than fucoxanthin itself, should be considered in mechanistic studies of the biological actions of dietary fucoxanthin. To clarify the nutritional and therapeutic effects of epoxy-xanthophylls, including fucoxanthin, further studies should be required on the tissue distributions and the biological actions of these metabolites.

	Footnotes

 
Supported in part by Special Coordination Funds of the Ministry of Education, Culture, Sports, Science and Technology, the Japan Government (to A.N.) and by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (to A.A).

¹ Abbreviations used are: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; VIS, visible absorption; MS, mass spectrometry; CD, circular dichroism; HPLC, high-performance liquid chromatography; LC/MS, liquid chromatography/mass spectrometry; APCI, atmospheric pressure chemical ionization; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide;t_R, retention time; SDR, short-chain dehydrogenase/reductase.

Address correspondence to: Dr. Akihiko Nagao, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. E-mail: nagao{at}nfri.affrc.go.jp

	References

Top Abstract Materials and Methods Results Discussion References

 


Britton G (1995) UV/Visible spectroscopy, in Carotenoids: Spectroscopy, vol. 1B (Britton G, Liaaen-Jensen S, and Pfander H eds) pp 13-62, Birkhäuser Verlag, Basel, Switzerland.

Brown MS, Ho YK, and Goldstein JL (1980) The cholesteryl ester cycle in macrophage foam cells. J Biol Chem 255: 9344-9352.[Free Full Text]

Cheng WH, Endo A, Zhou L, Penney J, Chen HC, Arroyo A, Leon P, Nambara E, Asami T, Seo M, et al. (2002) A unique short-chain dehydrogenase/reductase in Arabidopsis glucose signaling and abscisic acid biosynthesis and functions. Plant Cell 14: 2723-2743.[Abstract/Free Full Text]

Eugster CH (1995) Chemical derivatization: microscale tests for the presence of common functional groups in carotenoids, in Carotenoids: Isolation and Analysis, vol. 1A (Britton G, Liaaen-Jensen S, and Pfander H eds) pp 71-80, Birkhäuser Verlag, Basel, Switzerland.

Funahashi H, Imai T, Mase T, Sekita M, Yokoi K, Hayashi H, Shibata A, Hayashi T, Nishikawa M, Suda N, et al. (2001) Seaweed prevents breast cancer? Jpn J Cancer Res 92: 483-487.[Medline]

Funahashi H, Imai T, Tanaka Y, Tsukamura K, Hayakawa Y, Kikumori T, Mase T, Itoh T, Nishikawa M, Hayashi H, et al. (1999) Wakame seaweed suppresses the proliferation of 7,12-dimethylbenz(a)anthracene-induced mammary tumor in rats. Jpn J Cancer Res 90: 922-927.[CrossRef][Medline]

Greenlee RT, Murray T, Bolden S, and Wingo PA (2000) Cancer statistics, 2000. CA-Cancer J Clin 50: 7-33.[Abstract]

Hansen MB, Nielsen SE, and Berg K (1989) Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Methods 119: 203-210.[CrossRef][Medline]

Haugan JA, Aakermann T, and Liaaen-Jensen S (1992) Isolation of fucoxanthin and peridinin. Methods Enzymol 213: 231-245.

Hosokawa M, Wanezaki S, Miyauchi K, Kurihara H, Kohno H, Kawabata J, Odashima S, and Takahashi K (1999) Apoptosis-inducing effect of fucoxanthin on human leukemia cell line HL-60. Food Sci Technol Res 5: 243-246.

Jez JM, Bennett MJ, Schlegel BP, Lewis M, and Penning TM (1997) Comparative anatomy of the aldo-keto reductase superfamily. Biochem J 326: 625-636.

Jörnvall H, Persson B, Krook M, Atrian S, Gonzàlez-Duarte R, Jeffery J, and Ghosh D (1995) Short-chain dehydrogenases/reductases (SDR). Biochemistry 34: 6003-6013.[CrossRef][Medline]

Khachik F, Beecher GR, Goli MB, Lusby WR, and Smith JC Jr (1992) Separation and identification of carotenoids and their oxidation products in the extracts of human plasma. Anal Chem 64: 2111-2122.[Medline]

Khachik F, de Moura FF, Zhao DA, Aebischer CP, and Bernstein PS (2002) Transformations of selected carotenoids in plasma, liver and ocular tissues of humans and in nonprimate animal models. Investig Ophthalmol Vis Sci 43: 3383-3392.[Abstract/Free Full Text]

Khachik F, Spangler CJ, and Smith JC Jr (1997) Identification, quantification and relative concentrations of carotenoids and their metabolites in human milk and serum. Anal Chem 69: 1873-1881.[Medline]

Kim JM, Araki S, Kim DJ, Park CB, Takakusa N, Baba-Toriyama H, Ota T, Nir Z, Khachik F, Shimidzu N, et al. (1998) Chemopreventive effects of carotenoids and curcumins on mouse colon carcinogenesis after 1,2-dimethylhydrazine initiation. Carcinogenesis 19: 81-85.[Abstract/Free Full Text]

Kotake-Nara E, Kushiro M, Zhang H, Sugawara T, Miyashita K, and Nagao A (2001) Carotenoids affect proliferation of human prostate cancer cells. J Nutr 131: 3303-3306.[Abstract/Free Full Text]

Matsuno T, Ookubo M, and Komori T (1985a) Carotenoids of tunicates, III. The structural elucidation of two new marine carotenoids, amarouciaxanthin A and B. J Nat Prod 48: 606-613.[Medline]

Matsuno T, Ookubo M, and Komori T (1985b) Isolation and identification of amarouciaxanthin A from the bivalve Paphia euglypta (Sudaregai in Japanese). Nippon Suisan Gakkaishi–Bull Jpn Soc Sci Fisheries 51: 1909.

Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55-63.[CrossRef][Medline]

Okuizumi J, Nishino H, Murakoshi M, Iwashima A, Tanaka Y, Yamane T, Fujita Y, and Takahashi T (1990) Inhibitory effects of fucoxanthin, a natural carotenoid, on N-myc expression and cell cycle progression in human malignant tumor cells. Cancer Lett 55: 75-81.[CrossRef][Medline]

Okuizumi J, Takahashi T, Yamane T, Kitao Y, Inagake M, Ohya K, Nishino H, and Tanaka Y (1993) Inhibitory effects of fucoxanthin, a natural carotenoid, on N-ethyl-N'-nitro-N-nitrosoguanidine-induced mouse duodenal carcinogenesis. Cancer Lett 68: 159-168.[CrossRef][Medline]

Seo M and Koshiba T (2002) Complex regulation of ABA biosynthesis in plants. Trends Plant Sci 7: 41-48.[CrossRef][Medline]

Strand A, Herstad O, and Liaaen-Jensen S (1998) Fucoxanthin metabolites in egg yolks of laying hens. Comp Biochem Physiol A 119: 963-974.[CrossRef]

Sugawara T, Baskaran V, Tsuzuki W, and Nagao A (2002) Brown algae fucoxanthin is hydrolyzed to fucoxanthinol during absorption by Caco-2 human intestinal cells and mice. J Nutr 132: 946-951.[Abstract/Free Full Text]

Swanson MA (1955) Glucose-6-phosphatase from liver. Methods Enzymol 2: 541-543.[CrossRef]

Willis MS and Wians FH Jr (2003) The role of nutrition in preventing prostate cancer: a review of the proposed mechanism of action of various dietary substances. Clin Chim Acta 330: 57-83.[CrossRef][Medline]

Wingerath T, Stahl W, and Sies H (1995) ß-cryptoxanthin selectively increases in human chylomicrons upon ingestion of tangerine concentrate rich in ß-cryptoxanthin esters. Arch Biochem Biophys 324: 385-390.[CrossRef][Medline]

Yamamoto I and Maruyama H (1985) Effect of dietary seaweed preparations on 1,2-dimethylhydrazine-induced intestinal carcinogenesis in rats. Cancer Lett 26: 241-251.[CrossRef][Medline]

Yamamoto I, Maruyama H, and Moriguchi M (1987) The effect of dietary seaweeds on 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis in rats. Cancer Lett 35: 109-118.[CrossRef][Medline]

This article has been cited by other articles:

				A. Asai, M. Terasaki, and A. Nagao An Epoxide-Furanoid Rearrangement of Spinach Neoxanthin Occurs in the Gastrointestinal Tract of Mice and In Vitro: Formation and Cytostatic Activity of Neochrome Stereoisomers J. Nutr., September 1, 2004; 134(9): 2237 - 2243. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Asai, A. Articles by Nagao, A. Search for Related Content PubMed PubMed Citation Articles by Asai, A. Articles by Nagao, A.