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

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


Preparation of postmitochondrial fraction from mouse liver
Male ICR mice (6 weeks old; Clea Japan, 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., Tokyo, Japan) and tap water . After 7 days of feeding, the mice were anesthetized with isofl urane and euthanized by exsanguination. The livers were excised and rinsed with ice-cold saline. The livers were homogenized in a Potter-Elvehjem homogenizer with four volumes of ice-cold 50 mM HEPES-KOH buffer (pH 7.4) containing 0.154 M KCl, 1.0 mM EDTA, 1.0 mM EGTA, and 0.1 mM DTT. The postmitochondrial supernatant obtained by centrifugation at 10,000 g for 10 min was subjected to fractionation with a Sephadex G25 column. The protein fraction of the eluate was used as an enzyme source unless otherwise specifi ed, and is here referred to as postmitochondrial fraction. All procedures involving mice were approved by the Animal Care Committee of the National Food Research Institute (approval # H23-035) and were conducted in accordance with the guidelines of the National Food and Agriculture Research Organization for laboratory animal studies.

Oxidation of xanthophylls by liver postmitochondrial fraction
The oxidation of several xanthophylls by postmitochondrial fraction of mouse liver was evaluated in the following conditions. The standard reaction mixture contained 20 M xanthophyll, 2.4 mM NAD + , 0.1 M glycine-KOH buffer (pH 9.5), 0.5 mM DTT, 1 mM EDTA, 1 mM EGTA, 0.2% Tween 20, and postmitochondrial fraction (0.2-0.5 mg protein) in a total volume of 0.1 ml. Xanthophylls were solubilized in Tween 20 micelle before being added to the reaction mixture, as described ( 28 ). The reaction mixture was incubated at 37°C for 60 min, unless otherwise specifi ed. The reaction was terminated by adding 0.5 ml ethanol containing 40 nmol/ml ␣ -tocopherol and 35 mol/ml acetic acid. Subsequently, 0.4 ml deionized water, 0.5 ml ethyl acetate, and 0.5 ml n-hexane were added and the mixture was vortexed after each addition. The upper phase was collected and the lower phase was mixed with the same volumes of ethyl acetate and n -hexane as noted above. The combined upper phase was evaporated to dryness in vacuo. The extract was subjected to an HPLC analysis after being dissolved in 400 l of the eluting solvent.

Isomerization of 3 ′ -hydroxy-␤ , -caroten-3-one
The isomerization of 3 ′ -hydroxy-␤ , -caroten-3-one to 3 ′ -hydroxy-, -caroten-3-one was evaluated in the following conditions. 12 M 3 ′ -Hydroxy-␤ , -caroten-3-one were incubated at 37°C for 30 min in the reaction mixture containing 0.1 M buffer, 5 mM DTT, 1 mM EDTA, 1 mM EGTA, and 0.2% Tween 20, either in the presence or in the absence of postmitochondrial fraction (0.32 mg protein) in a total volume of 0.1 ml. The pH of the reaction mixture was adjusted with the following buffer: MES-KOH buffer for pH 6.0, HEPES-KOH buffer for pH 7.0 and 7.4, TRI-CINE-KOH buffer for pH 8.0 and 8.5, and glycine-KOH buffer for pH 9.5. After the reaction was terminated, the extract was prepared and subjected to an HPLC analysis as described above.

Preparation of oxidation products of xanthophylls
The following preparative reactions were conducted to obtain suffi cient amounts of the oxidation products to determine the chemical structure by NMR and circular dichroism (CD) spectroscopy. The reaction mixture contained 100 M xanthophyll, 2.4 mM NAD + , 0.1 M glycine-KOH buffer (pH 9.5), 5 mM DTT, 1 mM EDTA, 1 mM EGTA, 0.5% Tween 20, and the postmitochondrial cleavage enzyme to apocarotenoid and a small molecule, as a recombinant murine ␤ -carotene 9 ′ ,10 ′ -oxygenase in Escherichia coli was demonstrated to produce the specifi c cleavage products from lutein and zeaxanthin ( 12 ).
In fi sh and birds, diverse xanthophylls are synthesized from dietary carotenoids, and a metabolic conversion that retains a polyene backbone occurs in their tissues. For example, goldfi nches oxidized hydroxyl groups of C3 and C3 ′ in lutein to 3-oxo carotenoids and cardinals oxidized C4 and C4 ′ of ␤ -carotene and zeaxanthin to canthaxanthin and astaxanthin ( 19 ). Several 3-oxo carotenoids were detected in hen egg yolk ( 20 ). Fish have the same oxidative activity as birds and the reductive metabolism of astaxanthin to tunaxanthins was also reported in yellowtail ( 21 ). In human tissues, several keto-carotenoids derived from lutein and zeaxanthin were detected and assumed to be formed by a reaction with reactive oxygen species or by their enzymatic conversions ( 22 ).
The metabolic conversions have been elucidated mainly by identifying carotenoids that were not present in feed and by tracing labeled carotenoids ( 23 ). However, no enzyme reaction for the metabolic conversion of xanthophylls besides the cleavage reactions had been revealed until we found the NAD + -dependent oxidation of fucoxanthinol to amarouciaxanthin A by mouse liver microsomes ( 24 ). These two xanthophylls were the metabolites accumulated in the tissues of mice fed fucoxanthin, which is a major xanthophyll of edible brown algae. This conversion comprised the oxidation of a hydroxyl group at C3 of fucoxanthinol and the subsequent opening of 5,6-epoxide. We also found that mice fed lutein accumulated two ketocarotenoids as major carotenoids in the tissues ( 25 ), indicating that one of the two hydroxyl groups in lutein is oxidized to a carbonyl group in either a ␤ -end group or -end group, and that the ␤ -end group is isomerized to the -end group by double bond migration.
In the present study, we found that mouse liver has a metabolic activity to oxidize the 3-hydroxy ␤ -end group in xanthophylls to a 3-oxo -end group via an intermediate of a 3-oxo ␤ -end group, and that the same oxidative conversion occurs in humans.

Analysis of keto-carotenoids in human plasma
Four healthy volunteers (mean age of 43.3 years) were recruited from the National Food Research Institute (Tsukuba, Japan). None of the participants had consumed any supplement containing carotenoids during the 3 months before the study was started or during the study period. The human study was approved by the Ethics Committee of the National Food Research Institute and all of the participants signed the informed consent form.
The participants ingested canned mandarin orange juice (190 g) containing 1.9 mg of ␤ -cryptoxanthin at lunch or dinner each day for 13 consecutive days. Blood was collected by venipuncture into vacutainer tubes with sodium EDTA as an anticoagulant from overnight-fasted participants in the morning on the fi rst day and on the day after the fi nal day. The blood was centrifuged at 1,000 g for 15 min at 4°C and the plasma was collected and stored at Ϫ 80°C.
Xanthophylls in 0.4 ml of the plasma were extracted by the modifi ed procedures described ( 30 ), using lutein monomethyl ether as an internal standard. The plasma extract was dissolved in 200 l of n -hexane:dichloromethane:methanol (95:5:0.15, v/v/v) and an aliquot was subjected to HPLC analysis on the cyanopropyl column as described above, except that the gradient elution was performed at a fl ow rate of 0.15 ml/min, using solvent A, n -hexane:dichloromethane:methanol: N , N -diisopropylethylamine (95:5:0.15:0.1, by volume) and solvent B, n -hexane:dic hloromethane:methanol: N , N -diisopropylethylamine (75:25:0.15:0.1, by volume) as follows: 0-25 min at 100% solvent A; 25-50 min, a linear gradient to 100% solvent B; and 50-80 min at 100% solvent B. The recovery of each analyte spiked to human plasma in this procedure was more than 96%, and the limit of quantifi cation was 1.2 nmol/l plasma.

Effect of keto-carotenoids on nitric oxide production by RAW 264 cells
RAW 264 mouse macrophage cells (RIKEN BioResource Center, Tsukuba, Japan) were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 4 mmol/l L -glutamine, 40,000 units/l penicillin, and 40 mg/l streptomycin at 37°C in a humidifi ed atmosphere of 95% air and 5% CO 2 . The cells were maintained by passing every 2 days. The cells were seeded at a density of 3 × 10 4 cells per well in a 96-well plate and incubated for 4 h. Thereafter, the cells were cultured with the medium (0.1 ml) in which xanthophylls and keto-carotenoids were solubilized by the solvent evaporation method, as described ( 31 ). After 24 h cultivation, lipopolysaccharide (LPS) from E. coli (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the medium (fi nal concentration of 40 ng/ml ). After incubation for an additional 24 h, the cell supernatants were collected and subjected to an analysis of nitrite by using Griess reagent (Promega, Tokyo, Japan) as a measure of nitric oxide (NO) formation. The cellular protein supernatant before fractionation with a Sephadex G25 column (120-300 mg protein) in a total volume of 60 ml.
The reaction mixture was placed in a conical fl ask with a screw cap and was incubated at 37°C under anaerobic conditions by displacing the air with argon gas to eliminate the oxidative degradation of xanthophylls during incubation. The reaction times for the preparations of 3 ′ -hydroxy-␤ , -caroten-3-one, 3 ′ -hydroxy-,caroten-3-one, and , -carotene-3,3 ′ -dione from lutein were 1.5, 9.5, and 24 h, respectively. The reaction times for the preparations of ␤ , ␤ -caroten-3-one and ␤ , -caroten-3 ′ -one from ␤ -cryptoxanthin were 20 min and 9.5 h, respectively. The reaction times for the preparations of 3-hydroxy-␤ , -caroten-3 ′ -one and , -carotene-3,3 ′ -dione from zeaxanthin were 7.5 and 32 h, respectively. The reactions were terminated by adding 60 ml ethanol containing 20 nmol/ml ␣ -tocopherol. Subsequently, 60 ml ethyl acetate and 60 ml n -hexane were added and vigorously mixed. The upper phase was collected and the lower phase was mixed with the same volumes of ethyl acetate and n -hexane as above. The combined upper phase was evaporated to dryness and subjected to the purifi cation of oxidation products as described in the supplementary Methods.

Liver samples of mice fed lutein
The liver samples of mice fed lutein esters were obtained in our previous study ( 25 ) and stored at Ϫ 80°C until the isolation of the metabolites of lutein. The liver samples were homogenized as described in the section above and mixed well with an equal volume of the following solvents: ethanol, ethyl acetate, and n -hexane. The upper phase was collected and the lower phase was extracted with the same volume of ethyl acetate and n -hexane, as noted above. The combined upper phase was subjected to fractionation on an open column of silicic acid. The fractions rich in keto-carotenoids were purifi ed by semi-preparative HPLC on a cyanopropyl column of Inertsil CN-3 (10 × 250 mm; GL Sciences, Tokyo, Japan) attached to an Inertsil CN-3 guard column (7.6 × 30 mm) with the following mobile phase at a fl ow rate of 5.0 ml/min: n -hexane:dichloromethane:methanol (75:25:0.15, by volume). The eluates were further purifi ed by reverse-phase HPLC on a Wakopak Navi C30-5 column (100 × 250 mm; Wako Pure Industries, Osaka, Japan), with methanol containing 0.05% ammonium acetate as a mobile phase.

HPLC analyses of keto-carotenoids
We analyzed the extract of the reaction mixture with xanthophylls and liver postmitochondrial fraction by conducting normalphase HPLC with an HP-1100 system (Agilent Technologies, Palo Alto, CA) equipped with a photodiode array detector and a column oven at 25°C. A cyanopropyl column of Inertsil CN-3 (2.1 × 250 mm; GL Sciences) attached to an Inertsil CN-3 guard column (1.5 × 10 mm) was used with the following mobile phase at a fl ow rate of 0.2 ml/min: n -hexane:dichloromethane:methanol: N , Ndiisopropylethylamine (75:25:0.15:0.1, by volume) for lutein, zeaxanthin, and lactucaxanthin and their oxidation products, and n -hexane:dichloromethane:methanol: N , N -diisopropylethylamine (95:5:0.15:0.1, by volume) for ␤ -cryptoxanthin and its oxidation products.
The preparation of 3 ′ -hydroxy-, -caroten-3-one isolated from livers of mice fed lutein was also separated to two peaks on a chiral column ( Fig. 3B ). They had the same retention times as those prepared by the oxidation of lutein with postmitochondrial fraction. The forward peak and the cytotoxic effects (lactate dehydrogenase leakage) of cell treatments were evaluated as described ( 32 ).
We used Western blotting to examine the effect of ketocarotenoids on the protein expression of inducible NO synthase (iNOS). The RAW 264 cells were seeded at a density of 23 × 10 4 cells per well in a 12-well plate containing 1 ml of medium per well, and treated with ␤ , -caroten-3 ′ -one and then with LPS, as described above. The cells washed with Hanks' balanced salt solution were recovered with a lysis buffer (50 mM Tris-HCl, 1 mM EDTA, and 1 mM phenylmethanesulfonyl fl uoride). The recovered cells were homogenized on ice by sonication and then centrifuged (10,000 g , 15 min). The supernatants were collected and their protein concentrations were determined by the BCA method. Equal amounts of protein from each sample were subjected to SDS-PAGE and then electroblotted on a polyvinylidene difl uoride membrane . Rabbit iNOS antibody (1:1,000, #2982; Cell Signaling Technology Japan, Tokyo, Japan) and ␤ -actin antibody (1:1,000, #4967; Cell Signaling Technology Japan) were used as primary antibodies. The secondary immunoreaction and chromogenic reaction were carried out using a WesternBreeze chromogenic Western blot immunodetection kit (Life Technologies Japan, Tokyo, Japan).

Spectroscopic analysis
High-resolution mass spectra were recorded on a Fouriertransform ion cyclotron resonance mass spectrometer with an interface of ESI (Apex II 70e; Bruker Daltonics, Billerica, MA) and on a Kingdon trap-type mass spectrometer with an interface of atmospheric pressure chemical ionization (Orbitrap Veros Pro ETD; Thermo Fisher Scientifi c, Waltham, MA). The 1 H NMR (800 MHz) and 13 C NMR (201 MHz) spectra were recorded on a Bruker AVANCE 800 instrument at 4°C in CDCl 3 with tetramethylsilane as an internal standard. The CD spectra of the xanthophylls and oxidation products were measured with a J-820 CD system (Jasco, Tokyo, Japan) at 20°C in ethanol.

Statistical analysis
Values are expressed as means and SDs. The data were analyzed by one-way ANOVA, followed by the Tukey-Kramer test and the paired Student's t-test. P values <0.05 were considered signifi cant.

Formation of 3 ′ -hydroxy-, -caroten-3-one from lutein
After the incubation of lutein with postmitochondrial fraction of mouse liver in the presence of coenzyme NAD + for 60 min, the extract of the reaction mixture showed an unknown peak (peak 2, 8.5 min) before that of lutein (peak 3, 14.1 min) in the elution profi le of a normal-phase HPLC on a cyanopropyl column ( Fig. 1A ). Peak 2 was not detected in the reaction mixtures containing no NAD + ( Fig. 1B ) and the postmitochondrial fraction treated with protease ( Fig. 1C ). The peak 2 formation was optimum at pH 9.5, and the reaction dependent on NADP + was only 6.6% of that on NAD + . These results indicate that peak 2 was formed from lutein by a NAD + -dependent dehydrogenase reaction. Its UV-VIS spectrum showed the maximum absorption at 443 nm with well-defi ned vibrational bands ( Fig. 1H ), suggesting the loss of one double bond in the conjugated polyene structure of lutein. The retention time and UV-VIS spectrum of peak 2 were identical to those of 3 ′ -hydroxy-, -caroten-3-one that was prepared by the oxidation of lactucaxanthin with nickel peroxide ( Fig. 1E, J ). with those reported for lutein, indicating that the confi guration of -end group would be 3 ′ R ,6 ′ R or 3 ′ S ,6 ′ S.

Formation of , -carotene-3,3 ′ -dione from lutein
An unknown peak (peak 1) in addition to 3 ′ -hydroxy-,caroten-3-one was detected in a HPLC profi le of the reaction C3 was assigned to be R , as in the case of ␤ -cryptoxanthin and zeaxanthin. The 3-hydroxy-␤ , -caroten-3 ′ -one formed from zeaxanthin was a mixture of the two diastereomers of (3 R ,6 ′ R ) and (3 R ,6 ′ S ). Indeed, they were separated to two peaks which were eluted at close retention times with the equal peak height in the HPLC profi le on a chiral column.
, -Carotene-3,3 ′ -dione was detected in the HPLC profi le of the reaction mixture of zeaxanthin and liver postmitochondrial fraction after incubation for >18 h (supplementary Fig. 3B). It was separated into three possible steric isomers on a chiral column as in case of the , -carotene-3,3 ′ -dione isolated from egg yolk ( Fig. 3G, I ).

The oxidation of ␤ -cryptoxanthin
In a normal phase HPLC profi le, the extract from the reaction mixture of ␤ -cryptoxanthin with postmitochondrial fraction of mouse liver gave two peaks before ␤ -cryptoxanthin was eluted ( Fig. 5B ). The UV-VIS spectrum of peak 3 showed a maximum absorption at 450 nm ( Fig. 5C ), consistent with that of ␤ -cryptoxanthin (peak 5). The level of peak 3 was increased to the maximum at the early stage of the reaction and then declined in a way similar to 3 ′ -hydroxy-␤ ,caroten-3-one in the reaction mixture of lutein. We identifi ed peak 3 as ␤ , ␤ -caroten-3-one, based on high-resolution MS m / z 551.4245 [M+H] + (calculated for C 40 H 55 O: 551.4247) and 1 H and 13 C NMR data (supplementary Table 1). The

Effect of keto-carotenoids on NO production by RAW 264 cells
We found that the treatment of murine macrophage RAW 264 cells with lutein and ␤ -cryptoxanthin did not suppress the NO production induced by LPS, whereas keto-carotenoids such as 3 ′ -hydroxy-, -caroten-3-one, ,carotene-3,3 ′ -dione, and ␤ , -caroten-3 ′ -one repressed the peak 4 showed a UV-VIS spectrum with a maximum absorption at 447 nm ( Fig. 5C ). We identifi ed peak 4 as ␤ ,caroten-3 ′ -one, based on high-resolution MS m / z 551.4246 [M+H] + (calculated for C 40 H 55 O: 551.4247) and 1 H and 13 C NMR data (supplementary Table 1). The isolated ␤ ,caroten-3 ′ -one showed no CD signal, and it was separated to two peaks which were eluted at the close retention times with the equal peak height in the HPLC profi le on a chiral column. The isolated ␤ , -caroten-3 ′ -one was thus a racemic mixture of 6 ′ R and 6 ′ S isomers.

Substrate specifi city
The oxidative activity of the liver postmitochondrial fraction against several xanthophylls under the standard condition is shown in Table 1 . ␤ -Cryptoxanthin, lutein, zeaxanthin, and 3-hydroxy-␤ , -caroten-3 ′ -one, which have a hydroxyl group in their ␤ -end group, were converted to the corresponding keto-carotenoids with 3-oxo -end groups. In contrast, only a small amount of keto-carotenoids with 3-oxo -end groups were formed from lactucaxanthin and 3 ′ -hydroxy-, -caroten-3-one, of which hydroxyl groups bind only to -end groups. Oxidation of the 3 ′ -hydroxy -end group in lutein can produce 3-hydroxy-␤ , -caroten-3 ′ -one, whereas its formation was not confi rmed on an HPLC profi le due to overlapping with other peaks.
The residual amount of the xanthophylls in the minus NAD + control was >66% of the initial level after a 60 min incubation. In a complete incubation, the xanthophylls with a ␤ -end group markedly decreased, while the other xanthophylls without ␤ -end group were kept >83% of the initial level. The amount of keto-carotenoids formed from in their tissues ( 25 ). In the present study, several oxidation products were successfully prepared from xanthophylls using the reaction system with liver postmitochondrial fraction. From their structures, we elucidated the oxidative metabolism of xanthophylls by liver enzymes in mammals. The most important study points in the conversion of lutein to 3 ′ -hydroxy-, -caroten-3-one are the determination of which hydroxyl group is oxidized in lutein and how double bond migration occurs. From the confi guration of the oxidation products of lutein, we could specify a hydroxyl group oxidized by liver enzyme. Lutein was oxidized to two diastereomers of (6 R ,3 ′ R ,6 ′ R )-and (6 S ,3 ′ R ,6 ′ R NO production in a dose-dependent manner ( Fig. 7 ). In particular, ␤ , -caroten-3 ′ -one signifi cantly repressed NO production at a low concentration (5 M) and reduced the protein level of iNOS. Cytotoxic effects and growth inhibition were not observed in the cells treated with the xanthophylls and their oxidation products.

DISCUSSION
We previously found that mice fed lutein accumulated 3 ′ -hydroxy-, -caroten-3-one and , -carotene-3,3 ′ -dione of postmitochondrial fraction of mouse liver ( Fig. 8 ). The two diastereomers of 3 ′ -hydroxy-, -caroten-3-one were also found in the liver of mice fed lutein, indicating that the oxidative reaction of lutein found in vitro certainly worked in vivo.
We previously found that fucoxanthinol was oxidized to amarouciaxanthin A NAD + -dependently by microsomes of mouse liver ( 24 ). This oxidative conversion followed two reactions: the dehydrogenation of the 5,6-epoxy-3hydroxy-5,6-dihydro-␤ -end group of fucoxanthinol and the isomerization to an -end group by cleavage of the epoxide. The oxidations of lutein and fucoxanthinol differed in isomerization, which was caused by double bond migration and by the cleavage of epoxide, respectively. The intermediates produced by dehydrogenation would have unstable conformations, which led to isomerization to an -end group by double-bond migration or the cleavage of epoxide. Thus, these two reactions would be mediated by the same microsomal dehydrogenase.
Lutein has 3-hydroxy ␤ -end and 3 ′ -hydroxy -end groups. The former was preferentially oxidized, but it is uncertain whether the latter was oxidized to produce 3-hydroxy-␤ ,caroten-3 ′ -one. However, a small amount of 3 ′ -hydroxy-,caroten-3-one and lactucaxanthin, in which hydroxyl groups bind only to -end groups, were oxidized to the corresponding keto-carotenoids NAD + -dependently by postmitochondrial fraction, although the oxidative activities were much lower than that for lutein.
3 ′ )-hydroxy-, -caroten-3-one by postmitochondrial fraction. These confi gurations indicated that the (3 ′ R ,6 ′ R )-3 ′hydroxy -end group of lutein was retained intact in the oxidation product and that the ␤ -end group of lutein was converted to (6 R )-and (6 S )-3-oxo -end groups by both dehydrogenation and the migration of a double bond.
Based on the metabolic activity of postmitochondrial fraction of mouse liver observed in the present study, we speculate in the plasma of humans and rhesus monkeys fed zeaxanthin ( 36,37 ). In particular, the presence of equal amounts of two diastereomers (3 R ,6 ′ R and 3 R ,6 ′ S ) of 3-hydroxy-␤ ,caroten-3 ′ -one in the plasma of rhesus monkeys was quite consistent with the oxidation of a ␤ -end group proposed in the present study.
In contrast to mouse plasma, the relative levels of ketocarotenoids to parental xanthophylls were apparently low in human plasma. The ratio of 3 ′ -hydroxy-, -caroten-3-one to lutein in the plasma of mice fed lutein was 0.57 ( 25 ), whereas that of human subjects before the intake of mandarin orange juice in the present study was 0.14. This apparent difference in the ratio between the two species suggests that mice have higher oxidative activity of 3-hydroxy ␤ -end groups to 3-oxo -end groups compared with humans.
In human plasma, the level of 3-hydroxy-␤ , -caroten-3 ′one was higher than that of 3 ′ -hydroxy-, -caroten-3-one. The former is produced from zeaxanthin, and the latter is produced from lutein, according to the oxidation pathway of the 3-hydroxy ␤ -end group to the 3-oxo -end group. However, the level of 3-hydroxy-␤ , -caroten-3 ′ -one is assumed to be lower than that of 3 ′ -hydroxy-, -caroten-3one, considering that the lutein levels of the human diet and plasma are much greater than those of zeaxanthin. This discrepancy suggests that a signifi cant portion of human plasma and that its concentration increased signifi cantly after the intake of mandarin orange juice rich in ␤ -cryptoxanthin. Therefore, the presence of ␤ , -caroten-3 ′ -one in human plasma certainly indicates that the oxidative conversion of a 3-hydroxy ␤ -end group to a 3-oxo -end group occurs in human tissues. The oxidation of a ␤ -end group in humans is also supported by the reported increase of 3-hydroxy-␤ , -caroten-3 ′ -one   9. Proposed oxidation pathway of xanthophylls in mammals. The solid arrows indicate the oxidation conversion of a 3-hydroxy ␤ -end group to a 3-oxo -end group. The dashed arrows indicate the oxidative conversion of a 3-hydroxy -end group to a 3-oxo -end group. The latter reaction was not characterized in the present study. ( 17 ). The postprandial response of lutein in chylomicrons showed large interindividual differences. The responses were related to single nucleotide polymorphisms in 15 genes known to participate in the intestinal absorption of lutein and chylomicron metabolism ( 41 ). It is most likely that genetic variants of an unknown dehydrogenase responsible for the oxidation of lutein to 3 ′ -hydroxy-, -caroten-3-one affect the long-term accumulations of lutein and zeaxanthin in human plasma, macula lutea, and other tissues. The characterization of the responsible enzyme and its gene would help clarify the causes of the interindividual variation in xanthophyll accumulation.
The oxidative metabolism of dietary xanthophylls has the potential to affect not only their bioavailability but also their biological activity. All of the oxidative metabolites of xanthophylls, as described above, have a characteristic structure of ␣ , ␤ -unsaturated carbonyl, which is highly reactive to nucleophiles to form a Michel-type adduct. These carbonyl compounds have diverse biological activities by reacting with sulfhydryl groups of target proteins. Various phytochemicals and medicines with an ␣ , ␤ -unsaturated carbonyl structure were reported to have biological activity such as anti-tumor, anti-viral, and anti-infl ammatory activities (42)(43)(44). In the present study, three oxidative metabolites of xanthophylls were found to suppress the NO production by mouse macrophage RAW 264 cells stimulated with LPS. The suppression would be caused by reducing the expression of inducible nitric acid synthase by treatment of the oxidative metabolites. This biological activity of the oxidative metabolites was consistent with that of terpenes with an ␣ , ␤ -unsaturated carbonyl group, which suppressed NO production by inhibiting iNOS expression through the Narft-2 pathway. Thus, we suggest that the metabolites of xanthophylls reduce the formation of reactive oxygen species by macrophages and ameliorate oxidative damage by infl ammation.
In conclusion, we found that the 3-hydroxy ␤ -end group of xanthophylls was oxidized to a 3-oxo -end group via an unstable intermediate with a 3-oxo ␤ -end group by a NAD +dependent dehydrogenase of mouse liver. The increase of ␤ , -caroten-3 ′ -one in human plasma by the ingestion of ␤ -cryptoxanthin indicated that the oxidative conversion found in mouse liver also occurred in humans.
This assumption is supported by the previous report that the plasma level of 3-hydroxy-␤ , -caroten-3 ′ -one was increased in humans and rhesus monkeys fed lutein ( 37,38 ). However, the oxidative activity of the ␤ -end group in zeaxanthin is suggested to be higher than that of the -end group in lutein, because the formation rate constant of 3-hydroxy-␤ , -caroten-3 ′ -one from zeaxanthin was found to be higher than that from lutein in Cohn and colleagues' kinetic study of lutein and zeaxanthin orally administered to human subjects ( 36,38 ). Thus, these results indicate that the oxidation of both the 3-hydroxy ␤ -end group and the 3-hydroxy -end group occur in human tissues as in mouse, as mentioned above.
Regarding the bioavailability of xanthophylls, the oxidative metabolism of xanthophylls, as described above, would naturally decrease the level of intact xanthophylls in tissues. The 3-oxo -end group in the oxidation products has a hydrogen atom at C6, which is unstable to oxidation particularly under alkaline condition ( 39 ), and its ␣ , ␤ -unsaturated carbonyl moiety is highly reactive with nucleophilic molecules of biological tissues ( 40 ). Therefore, the oxidation products might be readily decreased in the biological tissues by oxidative degradation and reaction with nucleophiles, suggesting that the oxidative metabolism might decrease the level of total xanthophylls in tissues.
Moreover, the intermediate (3-oxo ␤ -end group) formed by the dehydrogenation of a 3-hydroxy ␤ -end group is extremely unstable due to steric hindrance of the ␤ -end group. A 3-oxo-␤ -end group cannot form a stable half-chair conformation because of the presence of a carbonyl group at C3 and a double bond between C4 and C5. Moreover, methylene protons at C2 and C4 in a 3-oxo-␤ -end group are easily eliminated in basic medium because of the presence of a carbonyl group at neighbor position of C3. Therefore, xanthophylls with a 3-oxo-␤ -end group are very labile.
In fact, the isomerization of the intermediate (3 ′ -hydroxy-␤ , -caroten-3-one) to a 3-oxo -end group in the incubation mixtures was not stoichiometric. Only a small portion of the intermediate was isomerized, and a large part disappeared (probably due to degradation to smaller molecules). The residual amounts of xanthophylls with a 3-hydoxy ␤end group after incubation with liver postmitochondrial fraction were remarkably lower than those of xanthophylls with a hydroxyl group only in the -end group. The amounts of the products with a 3-oxo -end group were far less than those of the consumed substrates. These results indicate that the oxidation of the 3-hydroxy ␤ -end group in xanthophylls might cause the oxidative degradation of xanthophylls, accompanied by a little conversion to anend group. Therefore, the oxidation of 3-hydroxy ␤ -end group in xanthophylls might participate in the elimination of xanthophylls from the body.
The genetic variants related to the intestinal absorption, distribution, and metabolism of carotenoids have been found to be associated with the carotenoid level in human plasma