Keto-Carotenoids Are the Major Metabolites of Dietary Lutein and Fucoxanthin in Mouse Tissues1,2,3

© 2010 American Society for Nutrition

Keto-Carotenoids Are the Major Metabolites of Dietary Lutein and Fucoxanthin in Mouse Tissues1,2,3


Lina Yonekura4,

Miyuki Kobayashi,

Masaru Terasaki, and

Akihiko Nagao*
+ Author Affiliations

National Food Research Institute, NARO, Tsukuba, Ibaraki 305-8642, Japan



*To whom correspondence should be addressed. E-mail: nagao@affrc.go.jp.





Next Section
Abstract
Fucoxanthin, a xanthophyll present in
brown algae consumed in Eastern Asia, can suppress carcinogenesis and
obesity in rodents.
We investigated the metabolism, tissue
distribution, and depletion of fucoxanthin in ICR mice by comparison
with those of
lutein. The experiments comprised 14-d dietary
supplementation with lutein esters or fucoxanthin, followed by 41- or
28-d,
respectively, depletion periods with
carotenoid-free diets. After lutein ester supplementation,
3′-hydroxy-ε,ε-caroten-3-one
and lutein were the predominant carotenoids in
plasma and tissues, accompanied by ε,ε-carotene-3,3′-dione. The presence
of
these keto-carotenoids in mouse tissues is reported
here for the first time, to our knowledge. Lutein and its metabolites
accumulated most in the liver (7.51 μmol/kg), followed by plasma (2.11 μmol/L), adipose tissues (1.01–1.44 μmol/kg), and kidney (0.87 μmol/kg). The half-life of the depletion (t1/2)
of lutein metabolites varied as follows: plasma (1.16 d) < liver
(2.63 d) < kidney (4.44 d) < < < adipose tissues (>41 d).
Fucoxanthinol and amarouciaxanthin A were the main
metabolites in mice fed fucoxanthin and partitioned more into adipose
tissues
(3.13–3.64 μmol/kg) than into plasma, liver, and kidney (1.29–1.80 μmol/kg). Fucoxanthin metabolites had shorter t1/2
in plasma, liver, and kidneys (0.92–1.23 d) compared with those of
adipose tissues (2.76–4.81 d). The tissue distribution
of lutein and fucoxanthin metabolites was not
associated with their lipophilicity, but depletion seemed to be slower
for more
lipophilic compounds. We concluded that mice
actively convert lutein and fucoxanthin to keto-carotenoids by oxidizing
the
secondary hydroxyl groups and accumulate them in
tissues.



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Introduction
Epidemiologic studies have associated the consumption of carotenoid-rich foods with a reduced risk of cancer and cardiovascular
diseases (1).
Owing to their extended system of conjugated double bonds, the
carotenoids can physically quench singlet oxygen and scavenge
peroxyl radicals, alleviating the oxidative stress
that is associated with the onset and progression of chronic diseases.

Xanthophylls are carotenoids possessing at least 1 oxygenated functional group. Some of the major carotenoids found in the
human diet belong to the xanthophyll class, such as lutein, zeaxanthin, and β-cryptoxanthin. Lutein (Fig. 1A) is the predominant carotenoid in egg yolks and yellow and green leafy vegetables. Lutein and zeaxanthin accumulate in the
macula lutea of the retina and may play a protective role against age-related macular degeneration by filtering blue light, quenching
singlet oxygen and/or acting as chain-breaking antioxidants (2). More polar xanthophylls such as capsanthin, astaxanthin, and fucoxanthin are present in paprika, salmon, and edible algae,
respectively. Fucoxanthin is a marine carotenoid with an allenic bond and a 5,6-epoxide in the molecule (Fig. 1B). This xanthophyll is among the most abundant carotenoids in nature, found in brown algae largely consumed in Asian countries,
such as wakame (Undaria pinnatifida) and hijiki (Hizikia fusiformis). Recent reports have shown that fucoxanthin inhibits proliferation of various cancer cell lines (3–5), suppresses angiogenesis ex vivo (6), and has antiinflammatory (7), antiobesity, and antidiabetic (8, 9)
effects in mice and/or rats. We have found that fucoxanthin is
hydrolyzed to fucoxanthinol in the intestinal tract and oxidized
to amarouciaxanthin A by liver microsomal
dehydrogenase in mice (10), suggesting that xanthophylls are actively metabolized in mammals.


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FIGURE 1
Proposed metabolic transformations of dietary lutein (A) and fucoxanthin (B) in ICR mice.




To assess tissue distribution, metabolic
transformations and rate of depletion of fucoxanthin in mice by
comparison with those
of a typical dietary xanthophyll, lutein, we
conducted 2 experiments consisting of dietary supplementation with
lutein or
fucoxanthin, followed by depletion periods. We
evaluated the rate of depletion of lutein, fucoxanthin, and their
respective
metabolites in plasma, liver, kidney, and adipose
tissues, and identified 2 lutein metabolites whose presence in mice is
reported
here for the first time, to our knowledge.



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Materials and Methods


Carotenoids.
Lutein esters were isolated from a marigold extract suspended in soybean oil (Xangold 15%, a gift from Cognis, Tokyo, Japan;
Supplemental Method 1)
containing a minimum of 15% lutein esters and other minor carotenoids,
which yielded, upon saponification, 7.57% lutein,
0.39% zeaxanthin, and 0.04% cryptoxanthin
(data from the supplier). Fucoxanthin was isolated from dry wakame (Undaria pinnatifida) as previously published (11), with modifications (Supplemental Method 2).

HPLC standards of lutein (12), fucoxanthin (13), fucoxanthinol, and amarouciaxanthin A (10) were prepared as previously reported and lactucaxanthin [(3R,6R,3′R,6′R)-ε,ε-carotene-3,3′-diol; alternative name ‘tunaxanthin F’] was isolated from lettuce (Lactuca sativa; Supplemental Method 3).



Carotenoid-supplemented diets.
Solutions containing purified carotenoids were concentrated under low pressure, below 35°C, and added to soybean oil containing
tert-butylhydroquinone at 200 mg/kg.
The remaining solvents in the carotenoid/oil mixtures were removed by
constantly stirring
in vacuo for several hours until constant
weight was achieved. The diets were prepared under dim yellow light and
were based
on the AIN-93G diet composition (14)
with 2 modifications: the soybean oil was replaced by the
carotenoid-supplemented soybean oil (prepared as described above)
and fiber was replaced by corn starch to
avoid possible interference with carotenoid absorption. The
carotenoid-supplemented
diets were stored at 4°C in vacuum-sealed
packages and consumed within 3 wk. The feeds were analyzed for
carotenoids after
exhaustive extraction with
dichloromethane:methanol 2:1 (v:v). Extracts containing lutein esters
were saponified (as described
below for biological samples) and analyzed
with HPLC setup 1. Fucoxanthin-containing feeds were extracted and
analyzed using
HPLC setup 2 without saponification.



Animals and experimental design.
Five-week-old male ICR mice were
obtained from Charles River Laboratories Japan and housed in individual
wired cages under
a 12 h-light/12-h-dark cycle at 24°C. During
the 5- to 7-d acclimatization period, the mice had free access to tap
water and
a commercial rodent diet (MF; Oriental
Yeast). Preliminary experiments showed that a 14-d feeding period with
lutein- or fucoxanthin-containing
diets was sufficient to load measurable
amounts of carotenoid metabolites into the liver of mice. Thus, the
lutein supplementation/depletion
experiment was comprised of a 14-d carotenoid
supplementation in which the mice (n = 45) consumed ad libitum
a powdered AIN-93G-based diet supplemented with lutein esters, followed
by a 41-d depletion period
when the mice were fed a carotenoid-free
pelleted AIN-93G diet (Oriental Yeast). During the depletion period,
groups of 5
mice were randomly chosen and killed at 0 h, 6
h, 12 h, 1 d, 3 d, 7 d, 14 d, 21 d, and 41 d. The fucoxanthin
supplementation/depletion
experiment followed the same protocol
described above, but the last sampling was done at 28 d. Every 2 d, body
weight and
feed intake were measured and a fresh supply
of feed was offered to each mouse. At each time point, without prior
feed deprivation,
mice were anesthetized with diethyl ether and
blood was collected with heparinized syringes from the caudal vena
cava. Liver,
kidneys, and epididymal, inguinal and
interscapular fat depots were excised, blotted dry, and weighed. All
experiments were
conducted in accordance with the basic
guidelines of the Ministry of Agriculture, Forestry and Fisheries for
laboratory animal
studies.



Source of the human plasma sample.
A plasma sample from a healthy volunteer was obtained in a previous study in our laboratory (15)
and stored at −80°C until analyses. The blood was collected after
overnight fast before the supplementation period (baseline).
None of the participants had taken any
carotenoid-containing supplement during the year before the study began (15).



Preparation of biological samples.
To determine the concentration of lutein and their metabolites, samples of plasma (200 μL) and tissues (60–70 mg) were saponified with 1000 μL
of 4.5% KOH containing 9.5% pyrogallol in 95% ethanol in screw-capped
tubes with argon headspace. After the 30-min reaction
at 60°C, 2 mL of water was added to each tube
and carotenoids were extracted 3 times with 3 mL diethyl ether:hexane
2:1 (v:v).
The supernatants were combined with 60 nmol
of α-tocopherol, dried under reduced pressure, redissolved in methanol,
and analyzed
by reversed-phase HPLC (setup 1). The
recovery of lutein spiked to mouse tissues was higher than 87%. To
evaluate the possible
formation of artifacts and loss of lutein
metabolites during saponification, selected samples of mouse tissues and
human plasma
were also analyzed without saponification (Supplemental Method 4).

The concentration of fucoxanthin and its metabolites was determined in extracts without saponification. Plasma (160 μL)
or tissue samples (50–100 mg) were combined with the internal standard
(neoxanthin) and methanol and hexane were added
to the final ratio of hexane:methanol:water
1:1:0.1 (v:v:v). The samples were vortexed and centrifuged, and the
hexane layer
was discarded and the process repeated 3–4
times, with fresh portions of hexane, to ensure removal of the
lipophilic material.
The methanol:water phase was dried under
reduced pressure, redissolved in methanol:dimethyl sulfoxide:water
70:20:20 (v:v:v),
filtered through a 0.2-μm membrane, and subjected to analysis by reversed-phase HPLC (setup 2). The recovery of fucoxanthin metabolites and neoxanthin
spiked to mouse tissue samples was higher than 90%.



HPLC setups for quantitative analyses.
Quantitative analyses of
carotenoids in feed and biological samples were performed on a Shimadzu
HPLC system (Shimadzu) consisting
of a LC-10AD pump set at 0.2 mL/min, a
SPD-M10A photodiode array detector at a 250- to 550-nm range, and a
CTO-10AS column
oven at 25°C. To achieve satisfactory peak
resolution between lutein and its metabolites, the HPLC conditions were
adapted
from Hudon et al. (16)
and consisted of 2 tandem TSK gel ODS-80Ts columns (2 × 250 mm, Tosoh)
attached to an ODS1 guard column (2 × 10 mm, Tosoh),
run with an acetonitrile:methanol 96:4 (v:v)
mobile phase containing 0.1% (w:v) ammonium acetate (HPLC setup 1).
Fucoxanthin
and its metabolites were analyzed with a
single 2 × 250 mm TSK gel ODS-80Ts column (Tosoh) attached to an ODS1
guard column
(2 × 10 mm, Tosoh) and a mobile phase
consisting of acetonitrile:methanol:water 71.25:14.25:14.5 (v:v:v)
containing 0.1% ammonium
acetate (HPLC setup 2). The peak areas of all
compounds were taken at their absorbance maxima in the mobile phase.
The concentrations
were calculated from the calibration curves
of known standards (lutein, fucoxanthin, and fucoxanthinol). Due to the
unavailability
of standards, lutein metabolites were
estimated from the lutein calibration curve and amarouciaxanthin A was
estimated from
the fucoxanthinol calibration curve.



Identification of carotenoids and their metabolites.
The structural elucidation of
carotenoids in biological samples was based on their UV-vis absorption
spectra in the mobile
phase, coelution with known standards,
chemical derivatization tests for functional groups, iodine-catalyzed
photoisomerization,
and liquid chromatography-MS according to the
amount of samples and availability of standard compounds (Supplemental Method 5).



Calculations and data analyses.
Carotenoid concentration data are presented as mean ± SD or as percentage of total carotenoids. To calculate the half-lives
(t1/2)5 of depletion, the mean carotenoid concentration in plasma and tissues throughout the depletion period was plotted against
time (n = 5 at each time point). As the plots showed that carotenoid depletion followed apparent first-order kinetics, the t1/2 for each carotenoid was calculated by dividing ln 2 by the slope of the ln-transformed concentration vs. time curve. The
t1/2 range was calculated from the highest and lowest slopes within a 95% CI. Half-lives from slopes that did not overlap the
95% CI were considered different.





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Results


Body weight, feed, and carotenoid intake.
The initial body weight of the ICR
mice was 33.2 ± 1.4 g in the lutein experiment and 32.5 ± 1.4 g in the
fucoxanthin experiment.
During the 14-d lutein supplementation, daily
consumption of feed was 5.06 ± 0.38 g, which corresponded to 2.73 ±
0.20 μmol lutein esters/d. For the fucoxanthin supplementation, feed intake was 5.18 ± 0.67 g/d, corresponding to 0.128 ± 0.016
μmol fucoxanthin/d, including 9.5% of a cis
isomer. The lutein ester and fucoxanthin levels in feed were calculated
based on preliminary (L. Yonekura and A. Nagao, unpublished
data) experiments to yield comparable levels
of their metabolites in tissues. We chose to feed the mice lutein esters
instead
of free lutein, because lutein esters are
more easily dissolved in oil and more bioavailable than free lutein (17).



Identification of lutein metabolites.
At the end of the 14-d dietary supplementation with lutein esters, the carotenoid profile of the mouse livers showed measurable
amounts of all-trans-lutein, 13-cis and/or 13’-cis-lutein, and 3 metabolites: ε,ε-carotene-3,3′-dione, 3′-hydroxy-ε,ε-caroten-3-one, and cis-3′-hydroxy-ε,ε-caroten-3-one (Fig. 2).
Common HPLC methods for tissue carotenoid analyses on reversed phase
columns make use of mid-polarity mobile phases to
elute lutein in relatively short retention
times (Rt). Under such conditions, lutein would coelute with its
oxidative metabolites
and those compounds could remain unnoticed.
In the present study, we used 2 tandem 25-cm long ODS columns to attain
suitable
chromatographic resolution between lutein and
its metabolites.


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FIGURE 2
Representative reversed-phase HPLC profiles of carotenoids in saponified (A) and unsaponified (B) liver extracts from mice fed diets containing lutein esters for 14 d and in unsaponified extracts of human plasma sample
(C). Peaks were identified as ε,ε-carotene-3,3′-dione (1), 3′-hydroxy-ε,ε-caroten-3-one (2), cis-3′-hydroxy-ε,ε-caroten-3-one (3), 3-hydroxy-β,ε-caroten-3′-one (3′), all-trans-lutein (4), 13-cis and/or 13'-cis lutein (5). Peaks with the same number in different chromatograms had similar UV-vis and MS spectra, shown in Supplemental
Figure 1.




The high-fat content of mouse liver
and adipose tissue extracts did not allow direct analysis by
reversed-phase chromatography
(HPLC setup 1), because the fat disturbed
reconstitution of carotenoids in the sample solvent for HPLC setup 1 and
a stronger
solvent would cause peak distortion.
Therefore, the plasma and tissue samples were saponified before the
analysis of lutein
and its metabolites. To confirm if the
presumed lutein metabolites were not artifacts formed during
saponification, an extract
from the same sample was also analyzed after
defatting by means of a previous run through reversed-phase HPLC. The
chromatograms
of liver extracts with or without
saponification are shown in Fig. 2A,B. The identity of the major carotenoids was the same in both saponified and unsaponified liver samples, as indicated by the
same Rt (Fig. 2, peaks 1–5) and compatible UV-vis and atmospheric pressure chemical ionization (APCI)-MS spectra (Supplemental Fig. 1).
However, the concentration of ε,ε-carotene-3,3′-dione (peak 1) was much
lower in the saponified sample (as described in
the following section). The identification of
lutein and its metabolites was carried out on pooled extracts of mouse
liver.
Rt, UV-vis, and APCI-MS spectral data were
obtained from the liquid chromatography-MS setup 1 (Supplemental Method
5), as
follows:



3′-Hydroxy-ε,ε-caroten-3-one.
The peak 2 at Rt 23.2 min corresponded to the most abundant carotenoid in mouse liver. (Fig. 2, peak 2) Its UV-vis spectrum with main absorbance at 442 nm and well-defined vibrational bands (Supplemental Fig. 1A2) was consistent with that of authentic lactucaxanthin [(3R,6R,3′R,6′R)-ε,ε-carotene-3,3′-diol], indicating a polyene structure with 9 conjugated double bonds. The APCI-MS (Supplemental Fig. 1B2) showed peaks at mass:charge ratio (m/z) 567 [M+H]+ (relative intensity in percentage, RI, 5.5) and m/z 549 [M+H-18]+ (base peak), and no further loss of water in the MS-MS spectrum of the m/z 549 fragment, indicating the presence of 1 hydroxyl group and presumably 1 keto group in a C40 carotene backbone.

NaBH4-assisted reduction of this compound produced 2 new peaks separated by normal-phase HPLC (Supplemental Fig. 2A,B). One of the reduced compounds coeluted with authentic lactucaxanthin (Supplemental Fig. 2C,D),
which is 1 of the 10 stereoisomers of tunaxanthin
(ε,ε-carotene-3,3′-diol) that includes 6 diastereomers that can be
separated
by normal-phase HPLC (18). Mass spectra of the reduced compounds showed 2 fragment ions corresponding to the loss of water at m/z 551 [M+H-18]+ and 533 [M+H-36]+,
as did those of lactucaxanthin. In addition, the UV-vis spectra of the
reduced compounds were consistent with those of lactucaxanthin.
The 2 peaks produced by NaBH4
reduction were then assigned as tunaxanthin diastereomers, indicating
that the parent compound had a structure similar to
tunaxanthin except for 1 keto group instead
of hydroxyl attached to the ε-end ring. Moreover, the UV-vis spectra of
the parent
compound featured well-defined vibrational
bands, indicating that the keto group was not conjugated to the main
polyene chain.
The parent compound could also be methylated
by methanolic HCl, indicating the presence of a secondary allylic
hydroxyl group.
Therefore, peak 2 was identified as
3′-hydroxy-ε,ε-caroten-3-one (Fig. 1A).



cis-3′-Hydroxy-ε,ε-caroten-3-one.
The peak 3 at Rt 24.1 min had the same response in chemical derivatization tests and APCI-MS (Supplemental Fig. 1B3) as those of peak 2. However, the UV-vis spectrum presented a strong cis peak with ε2/ε1 = 0.585 at 330 nm (Supplemental Fig. 1A3), so the peak 3 was identified as cis-3′-hydroxy-ε,ε-caroten-3-one. After sample handling (solvent evaporation and storage at −20°C), part of the isolated peak
3 spontaneously converted to a compound devoid of the cis peak in the UV-vis spectrum and unchanged APCI-MS, indicating isomerization from cis to all-trans configuration. Because peak 2 had the same Rt as the peak formed spontaneously from peak 3, peak 2 can be regarded as all-trans-3′-hydroxy-ε,ε-caroten-3-one (Fig. 1A).



ε,ε-Carotene-3,3′-dione.
Peak 1 at Rt 21.0 min had UV-vis absorbance maxima and vibrational bands (Supplemental Fig. 1A1) very similar to those of peak 2. The APCI-MS (Supplemental Fig. 1B1) showed a base peak at m/z 565 [M+H]+ and no peaks due to water loss, in agreement with a MS profile of a di-ketocarotenoid. The NaBH4-assisted reduction of peak 1 generated 2 peaks separated by normal-phase HPLC (setup 3; Supplemental Method 5). Their Rt,
UV-vis, and APCI-MS spectra were comparable to those of NaBH4-treated peak 2, indicating formation of tunaxanthin diastereomers from the parent compound. Thus, peak 1 was identified as
ε,ε-carotene-3,3′-dione (Fig. 1A).

All-trans-lutein [(3R,3′R,6′R)-β,ε-carotene-3,3′-diol, Fig. 2, peak 4] at Rt 26.7 min was identified by APCI-MS (Supplemental Fig. 1B4), UV-vis spectral data (Supplemental Fig. 1A4), and coelution with authentic lutein. The absence of carbonyl groups was confirmed by the negative response to NaBH4-assisted reduction.

13-cis-Lutein and 13’-cis-lutein (Fig. 2, peak 5), at Rt 29.9 min, was identified by APCI-MS (Supplemental Fig. 1B5), UV-vis spectral data (Supplemental Fig. 1A5), and coelution with lutein isomers produced by iodine-catalyzed photoisomerization. The blue-shift of 6 nm in absorption
maximum from that of all-trans-lutein (Supplemental Fig. 1A, spectra 4 and 5) and the intensity of the cis peak, ε2/ε1 = 0.427, suggested that C13 or C13’ was the location of isomerization (19). However, 13-cis-lutein and 13’-cis-lutein could not be resolved with the HPLC conditions used in this study.

The peak 3′ of the human plasma chromatogram (Fig. 2C), with Rt very close to that of peak 3 (cis-3′-hydroxy-ε,ε-caroten-3-one) of the mouse liver chromatograms (Fig. 2A,B), was identified as 3-hydroxy-β,ε-caroten-3′-one from the following features: the APCI-MS spectrum showed peaks at m/z 567 [M+H]+ (base peak) and m/z 549 [M+H-18]+ (RI 24.5), which were very similar to previously published MS for that compound (19, 20), whereas m/z 549 [M+H-18]+ was predominant in the fragmentation pattern of cis-3′-hydroxy-ε,ε-caroten-3-one (Supplemental Fig. 1B3); UV-vis absorbance maximum was 5 nm red-shifted relative to that of 3′-hydroxy-ε,ε-caroten-3-one and the spectrum did not
feature a cis peak. ε,ε-Carotene-3,3′-dione and 3′-hydroxy-ε,ε-caroten-3-one were also present in the human plasma extract (Fig. 2C, peaks 1 and 2) but at much smaller amounts relative to all-trans-lutein, the most abundant carotenoid in the human plasma chromatogram.



Identification of fucoxanthin metabolites.
At the end of the fucoxanthin
supplementation period, the mouse plasma had measurable amounts of
fucoxanthinol and amarouciaxanthin
A, consistent with our previous report (10). We also found 3 additional metabolites of fucoxanthin in the present study (Fig. 3; peaks 1, 2, and 5). Peak 5 at Rt 19.8 min had a UV-vis absorbance maximum at 458 nm, unresolved vibrational fine structure,
and a cis peak at 340 nm (ε2/ε1 = 0.590; Supplemental Fig. 3A5). APCI-MS fragmentation was compatible with that of amarouciaxanthin A (Supplemental Fig. 3B5), and the compound coeluted with one of the isomers formed by iodine-catalyzed photoisomerization of amarouciaxanthin A.
Thus, peak 5 was assigned as cis-amarouciaxanthin A. Peak 1 at Rt 7.5 min had absorbance maximum at 470 nm with an unresolved vibrational structure and APCI-MS
base peak at m/z 445. MS-MS of the m/z 445 peak (RI 35.7) showed a fragment ion at m/z 427 (base peak). Peak 2 at Rt 10.5 min had an absorbance maximum at 460 nm with unresolved vibrational structure and APCI-MS
ions at m/z 445 (base peak) and 427 (RI 85.1). The limited amounts of these 2 compounds did not allow a complete identification.


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FIGURE 3
Representative
reversed-phase HPLC profile of carotenoids in the plasma of mice fed
diets containing fucoxanthin for 14 d.
Peaks 1 and 2 could not be fully
identified; other peaks were fucoxanthinol (3), amarouciaxanthin A (4),
and cis-amarouciaxanthin A (5). UV-vis and MS spectra of the identified carotenoids are shown in Supplemental Figure 2.






Distribution of carotenoids in selected mouse tissues and their depletion.
At the end of the dietary supplementation with lutein esters, mouse tissues and plasma had surprisingly high concentrations
of 3′-hydroxy-ε,ε-caroten-3-one and its cis
isomer, which are assumed to be oxidative metabolites of lutein. The
concentrations of those metabolites were even higher
than those of lutein and its isomers in liver
and epididymal, inguinal, and interscapular adipose tissues (Table 1).



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TABLE 1
Concentration of carotenoids in mouse plasma and tissues at the end of the 14-d dietary supplementation with lutein esters1



The losses of lutein and its
metabolites during saponification were evaluated by analyzing the
extracts from saponified and
untreated samples. Both extracts were
defatted by passing through a short ODS column. The ratios of carotenoid
concentration
between saponified and untreated samples
were: 94.0% for the sum of all-trans, 13-cis-, and 13’-cis-lutein, 71.3% for the sum of 3′-hydroxy-ε,ε-caroten-3-one and cis-3′-hydroxy-ε,ε-caroten-3-one,
and 53.9% for ε,ε-carotene-3,3′-dione. The negligible loss of lutein by
saponification was
very consistent with the high recovery of
lutein spiked to tissues, whereas the keto-carotenoids were unstable.
Due to the
large loss of ε,ε-carotene-3,3′-dione by
saponification, we are not reporting the concentrations of this
carotenoid.

Considering the total amount of lutein and its metabolites, we observed the highest concentration in the liver, followed by
plasma, all adipose tissues, and the kidney (Table 1).
In particular, the liver accumulated large amounts of
3′-hydroxy-ε,ε-caroten-3-one and ε,ε-carotene-3,3′-dione.
3′-Hydroxy-ε,ε-caroten-3-one
was also predominant in the adipose tissues (Table 1). On the other hand, in the kidney, all-trans-lutein and 3′-hydroxy-ε,ε-caroten-3-one are present in comparable amounts, while 13-cis- and 13’-cis-lutein are the most abundant carotenoids in plasma. The t1/2
for the total lutein metabolites (sum of the 4 major compounds found in
tissues) varied greatly between tissues, in the following
order: plasma < liver < kidney <
< < adipose tissues (Table 2). The depletion of lutein and its metabolites in epididymal, inguinal, and interscapular fat depots was much slower than
in other tissues, but the t1/2 could not be determined, because the concentrations of lutein metabolites were not significantly reduced even at 41 d of
depletion. Within the same tissue, the compound-specific t1/2 values were similar for all 4 metabolites analyzed (Table 2).



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TABLE 2
t1/2 of carotenoids in mouse plasma and tissues after 14-d dietary supplementation with lutein esters1,2



After the dietary supplementation with fucoxanthin, we observed a higher concentration of total fucoxanthin metabolites in
mouse adipose tissues than in plasma, liver, and kidney (Table 3). Amarouciaxanthin A and its cis
isomer were preferentially accumulated in adipose tissues. Among the
epididymal, inguinal, and interscapular fat tissues,
the distribution of fucoxanthin metabolites
was similar, with 52.8–56.6% amarouciaxanthin A, 22.6–26.5% cis-amarouciaxanthin A, and 18.8–24.1% fucoxanthinol (Table 3). Unlike the profile in adipose tissues, fucoxanthinol was the most abundant metabolite in liver and kidney, followed by
amarouciaxanthin A and cis-amarouciaxanthin A. Amarouciaxanthin A was the most abundant carotenoid in the plasma samples, followed by fucoxanthinol
and cis-amarouciaxanthin A. We did
not detect fucoxanthin in the plasma and tissues analyzed in this study,
consistent with our previous
report (10).



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TABLE 3
Concentration of carotenoids in mouse plasma and tissues at the end of the 14-d dietary supplementation with fucoxanthin1



Overall, t1/2 of fucoxanthin metabolites in plasma and tissues were shorter than those of lutein and its metabolites (Tables 3 and 4). Compared among tissues and plasma, the t1/2 of the total fucoxanthin metabolites were longer in adipose tissues compared with t1/2 in plasma, liver, and kidney (Table 4). In adipose tissues, t1/2 differed significantly according to the site, being the shortest in interscapular fat, followed by inguinal fat and epididymal
fat. Of the individual carotenoids within tissues, the t1/2 of fucoxanthinol was generally shorter than that of amarouciaxanthin A, which in most cases was shorter than that of cis-amarouciaxanthin A (Table 4).



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TABLE 4
t1/2 of carotenoids in mouse plasma and tissues after 14-d dietary supplementation with fucoxanthin1







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Discussion


Metabolism.
The most surprising finding in this study was that keto-carotenoids were the most abundant metabolites in ICR mice fed lutein
esters. 3′-Hydroxy-ε,ε-caroten-3-one (and its cis
isomer) accounted for >50% of the carotenoids in the liver and
adipose tissues. Another keto-carotenoid, ε,ε-carotene-3,3′-dione,
was also present in appreciable amounts in
all samples from lutein-supplemented ICR mice, but quantitative data are
not reported
here due to the loss of this compound during
saponification. Previous reports have indicated that lutein was the
predominant
carotenoid found in BalbC mice after dietary
supplementation with lutein esters (21, 22),
but the use of reversed-phase HPLC with single wavelength detection and
conditions that did not allow chromatographic resolution
of lutein and zeaxanthin may have hindered
the detection of keto-carotenoids. Although there may be differences in
carotenoid
metabolism even among strains of the same
species (23),
we also detected the keto-carotenoids 3′-hydroxy-ε,ε-caroten-3-one and
ε,ε-carotene-3,3′-dione as major carotenoids in
the liver of BalbC mice fed diets containing
lutein (L. Yonekura and A. Nagao, unpublished data). Khachik et al. (19, 24–26)
have also reported the presence of 3-hydroxy-β,ε-carotene-3′-one as
well as 3′-hydroxy-ε,ε-caroten-3-one and ε,ε-carotene-3,3′-dione
in human serum, breast milk, retina, and
liver, and these keto-carotenoids were reported to amount to 34% of the
lutein/zeaxanthin
level in human serum (27). They also suggested that keto-carotenoids were metabolites of dietary lutein, because their concentrations were significantly
raised from the baseline in individuals receiving lutein supplementation (27).
In fact, we also detected a considerable amount of these
keto-carotenoids in human plasma in the present study. Apart from
their presence in human tissues,
3′-hydroxy-ε,ε-caroten-3-one and ε,ε-carotene-3,3′-dione have also been
detected in the feathers
of pin-tailed and golden-winged manakins (Ilicura militaris), where ε,ε-carotene-3,3′-dione was a major carotenoid (16), and in hen egg yolks as a minor carotenoid (28). Our results and the aforementioned reports suggest the existence of a common metabolic pathway for lutein oxidation (Fig. 1A) widely distributed among birds and mammals. Khachik et al. (27) and Matsuno et al. (28)
suggested metabolic pathways for the transformation of dietary lutein,
which encompassed oxidation of lutein to 3-hydroxy-β,ε-caroten-3′-one,
double bond migration yielding
3′-hydroxy-ε,ε-caroten-3-one, and further oxidation to
ε,ε-carotene-3,3′-dione. This may well
be the pathway underlying the formation of
metabolites we found in the present study. The reason why we could not
detect 3-hydroxy-β,ε-caroten-3′-one
could be its faster isomerization to
3′-hydroxy-ε,ε-caroten-3-one in ICR mice compared with in humans.

We previously reported that dietary fucoxanthin is transformed to fucoxanthinol and amarouciaxanthin A (Fig. 1B) in mice (10, 29). In the present study, we observed 2 additional peaks whose APCI-MS featured base peaks at m/z 445, which is also the most abundant fragment in the APCI-MS of amarouciaxanthin A. Further transformation of amarouciaxanthin
A to more polar metabolites may be taking place in ICR mice.

Similarly to the oxidative metabolism we described here for lutein and fucoxanthinol, other authors have reported the oxidation
of 4,4′-dimethoxy-β-carotene at the 4 and 4′ positions to canthaxanthin (30) and at the 3′ position of capsanthin to capsanthone (31)
after ingestion of those carotenoids by humans. Thus, mammals are able
to oxidize secondary hydroxyl groups of various xanthophylls.
In a previous study, we found that a liver
microsomal NAD-dependent dehydrogenase played a role in the oxidation of
fucoxanthinol
to amarouciaxanthin A (10),
but we still do not know whether lutein or capsanthin is oxidized by
the same enzyme. Further studies are needed to determine
the enzymes and mechanisms involved in the
oxidative conversion of xanthophylls. The α,β-unsaturated carbonyl
moiety of keto-carotenoids
derived from lutein have a unique structure,
which has high potential to react with nucleophilic molecules in
biological tissues
(32). Hence, the biological activities of lutein metabolites are worth investigating in terms of the beneficial effects of lutein
on human health.



Tissue distribution.
Surprisingly, lutein and its
metabolites were more concentrated in the liver, whereas the less
lipophilic fucoxanthin metabolites
accumulated mainly in adipose tissues. In the
present study, the total concentration of lutein and its metabolites in
liver
was 3.5-fold that in plasma, whereas in
adipose tissues it ranged from 0.5- to 0.7-fold. In guinea pigs, the
highest lutein
concentration was found in the liver,
followed by kidney and plasma, while there was no lutein in adipose
tissues (33). In quail, the major site of lutein accumulation was also the liver, followed by adipose tissue and serum (34). Previous studies in humans are conflicting, showing a highly variable partition of lutein into adipose tissues, ranging
from 6-fold enrichment to concentrations much lower than those in plasma (35–37). Thus, adipose tissues cannot be assumed to be the major site for lutein accumulation.

Regarding the tissue distribution
of fucoxanthin metabolites, we found that adipose tissues were the main
site of accumulation,
where concentrations were 2.2- to 2.6-fold
relative to plasma. During the preparation of this manuscript, Hashimoto
et al.
(38)
reported that fucoxanthin and its metabolites accumulated mainly in the
adipose tissues, liver, and heart of mice. However,
in their study, liver and adipose tissues had
comparable concentrations of total fucoxanthin metabolites, which may
be due
to the shorter supplementation period (1 wk
compared with 2 wk used in our study). To date, there is no data on the
accumulation
of fucoxanthin metabolites in human adipose
tissue.

A recent report showed differences in carotenoid concentration according to the site of the adipose tissue (39).
We determined the carotenoid concentration in 3 different sites:
epididymal fat, which is the largest visceral fat depot
in mice; inguinal fat, representative of the
subcutaneous white adipose tissue; and interscapular fat, the major
brown fat
depot in mice. However, we did not observe
differences in carotenoid concentrations among those sites.

The distribution profile of individual metabolites differed between tissues. 3′-Hydroxy-ε,ε-caroten-3-one and its cis isomer were more abundant than lutein (all-trans and its cis
isomer) in liver and adipose tissues, whereas the opposite occurred in
plasma and kidney. Lutein metabolites were highly
accumulated in the liver, suggesting the
oxidative conversion of lutein by liver enzymes. Regarding fucoxanthin
metabolites,
in plasma and adipose tissues,
amarouciaxanthin A, including its cis isomer, was more abundant
than fucoxanthinol, whereas the liver and kidneys had comparable
amounts of the 2 metabolites.
Amarouciaxanthin A accumulation in adipose
tissue was remarkable. By contrast, the more lipophilic lutein and its
metabolites
accumulated mainly in the liver. The unique
distribution of carotenoid metabolites in each tissue does not seem to
be related
to the lipophilicity of the metabolites and
tissues and may be associated with other factors, including the
existence of tissue-specific
enzymatic transformation, different metabolic
and transport rates for each compound, and the existence of compound-
and tissue-specific
transport mechanisms. The elucidation of such
mechanisms, however, is beyond the scope of this study.

With regard to the proportion
between carotenoid intake and their accumulation in tissues, we found a
remarkable difference
between lutein esters and fucoxanthin. In
this study, mice ingested 20-fold more lutein esters than fucoxanthin,
and the t1/2 of lutein and its metabolites was longer than
those of fucoxanthin metabolites. Nonetheless, the levels of lutein and
its
metabolites in tissues did not exceed those
of fucoxanthin. These results indicate that fucoxanthin is more readily
absorbed
than lutein esters in mouse intestine.
However, we have previously reported that the bioavailability of dietary
epoxyxanthophylls
such as neoxanthin and fucoxanthin from
spinach and algae is low in humans (15). Thus, the intestinal absorption of epoxyxanthophylls in humans and its difference among species are intriguing issues that
deserve future study.



Depletion rates.
The depletion of lutein and its
metabolites occurred much more slowly than that of fucoxanthin
metabolites. Because lutein
and its oxidative metabolites are more
lipophilic than fucoxanthin metabolites, our results suggest that the
more lipophilic
carotenoids have longer t1/2 in mice, especially in adipose tissues. In addition, we also observed longer t1/2 values for amarouciaxanthin A (all-trans- and cis) compared with those for fucoxanthinol (less lipophilic than amarouciaxanthin A) in adipose tissues. Adipose tissues are
generally regarded as potential sites to assess the long-term intake of lipophilic compounds (40). However, many authors have failed to find any evidence that the carotenoid concentration in adipose tissues reflects the
long-term intake of these phytochemicals (41, 42), which can be partially explained by the highly variable turnover rates of carotenoids found in the present study.

In summary, we identified the
keto-carotenoids 3′-hydroxy-ε,ε-caroten-3-one and
ε,ε-carotene-3,3′-dione as major metabolites
of dietary lutein in ICR mice. Our study
confirms several indications that the oxidation of xanthophylls’
secondary hydroxyl
groups into carbonyl is an active metabolic
pathway in mammals. Tissue accumulation and the distribution of lutein
and fucoxanthin
metabolites were not associated with their
lipophilicity, but the depletion rates seemed to be slower for more
lipophilic
compounds.





Previous SectionNext Section
Acknowledgments
L.Y. designed and conducted research,
analyzed data, and wrote the paper; M.K. conducted carotenoid analyses;
M.T. prepared
carotenoid standards; and A.N. analyzed data and
had primary responsibility for final content. All authors read and
approved
the final manuscript.



Previous SectionNext Section
Footnotes


↵2 Author disclosures: L. Yonekura, M. Kobayashi, M. Terasaki, and A. Nagao, no conflicts of interest.



↵4 Present address: Department of Nutrition, School of Public Health, University of São Paulo, Av. Doutor Arnaldo 715, São Paulo,
SP 01246-904, Brazil; E-mail: linayonekura@usp.br, linayonekura@gmail.com.



↵1 Supported in part by the Research and Development Program for New Bio-industry Initiatives of the Bio-oriented Technology
Research Advancement Institution.



↵3 Supplemental Methods 1–5 and Supplemental are available with the online posting of this paper at jn.nutrition.org.



↵5 Abbreviations used: APCI, atmospheric pressure chemical ionization; m/z, mass:charge ratio Rt, retention time; RI, relative
intensity; t1/2, half-life of depletion.





Manuscript received: May 11, 2010.

Initial review completed: June 2, 2010.

Revision accepted: July 21, 2010.



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