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a HIST, Institute of Food Science and Technology, N-7004 Trondheim, Norway, b AKVAFORSK, Institute of Aquaculture Research AS, N-6600 Sunndalsøra, Norway and c Norwegian University of Science and Technology (NTNU), Faculty of Chemistry and Biology, Organic Chemistry Laboratories, N-7034 Trondheim, Norway
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • Astaxanthin E/Z isomers • astaxanthin R/S isomers • bioavailability • rainbow trout • Oncorhynchus mykiss
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Animals (Zechmeister 1937
), including fish (Goodwin 1951
), have a
different ability to absorb and deposit dietary carotenoids. Salmonids
are quite unique among fish in their ability to deposit substantial
amounts of dietary carotenoids in their muscle tissues. Astaxanthin
(3,3'-dihydroxy-ß,ß-carotene-4,4'-dione, Fig. 1
.Structures 1–3) is the major carotenoid of wild salmonids (Khare et al. 1973
) and the most common carotenoid used for commercial salmonid
pigmentation (Storebakken and No 1992
, Torrissen et al. 1989
).
Salmonids preferentially absorb and deposit more polar carotenoids,
particularly astaxanthin rather than canthaxanthin
(ß,ß-carotene-4,4'-dione) and zeaxanthin
(ß,ß-carotene-3,3'-diol), or carotenes including ß,ß-carotene
(Foss et al. 1984
, Guillou et al. 1992
, Schiedt et al. 1985
).
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, Parker 1996
). (3S,3'S)-Astaxanthin
(1) contains nine conjugated double bonds in the central
chain (Fig. 2
) and may theoretically form 271 Z-isomeric forms and 1
all-E-form (Zechmeister 1944
). However, in nature the
highest amounts are expected to be found of the sterically unhindered
(Pauling 1939
) all-E, 9Z, 13Z and
15Z isomers (Fig. 2)
.
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;
Sweeney and Marsh 1973
), chicken liver (Ben-Amotz et al. 1989
), ferret
liver and adrenal tissues and serum (White et al. 1993
) and human serum
(Gaziano et al. 1995
, Jensen et al. 1987
, Stahl et al. 1992
) generally
is attributed to a discrimination in absorption (Parker 1996
). Little
is known about the bioavailability of the different astaxanthin
(1bcd, 2bcd, 3bcd) Z
isomers. The results of Schiedt et al. (1981, 1989)
indicated that the
different E/Z isomers were utilized equally well
for fillet pigmentation and that no metabolic E/Z
isomerization took place in wild Atlantic salmon and Oncorhynchusspecies. In contrast, Arai et al. (1987)
reported that astaxanthin
Z isomers (1bcd, 2bcd,
3bcd) only comprised 3–8% of total astaxanthin deposited
in the flesh of coho salmon fed diets supplied with Antarctic krill,
indicating a selective absorption or deposition of
all-E-astaxanthin (1a, 2a,
3a). The Antarctic krill oil is expected to contain high
amounts of Z isomers due to the relatively high temperature
(
125°C) used during processing (see Arai et al. 1987
)
The most important commercial source of astaxanthin (Carophyll Pink,
Hoffmann-La Roche, Basel, Switzerland) contains ~25% Z
isomers of the total astaxanthin. Astaxanthin is by far the most costly
feed ingredient on a weight basis, and because normally only 15% of
the ingested astaxanthin may be recovered from the muscle, depending on
dose and species (Storebakken and No 1992
, Torrissen et al. 1989
),
knowledge about the bioavailability and metabolic fate of the different
geometrical stereoisomers is important.
However, only one previous report has addressed the utilization of
E/Z isomers of astaxanthin in fish (Bjerkeng et al. 1997
). Our present objective was to investigate the tissue
distribution of astaxanthin E/Z and
R/S isomers in rainbow trout fed primarily
all-E-astaxanthin (1a, 2a,
3a) as the carotenoid source in comparison with trout fed a
mixture of all-E-astaxanthin (1a, 2a,
3a), 9Z- (1b, 2b,
3b), 13Z- (1c, 2c,
3c) and 15Z- astaxanthin (1d,
2d, 3d) (Fig. 2)
. Astaxanthin isomers with
different chirality at C-3, 3' are here referred to as
R/S (optical) isomers, consisting of the two
enantiomers (3S,3'S) (1) and
(3R,3'R) (3) and the meso
form (3R,3'S) (2). Isomers with
different configuration (a,b,c,d) of in-chain double bonds are referred
to as E/Z (geometrical) isomers. The optical
isomers are separated as dicamphanates (Vecchi and Müller 1979
)
and the geometrical isomers by direct HPLC resolution on a
H3PO4-modified silica gel column (Vecchi et al. 1987
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Analytical standards of all-E-astaxanthin (cf. structures 1, 2, 3; Fig. 1 ; ratio ca. 1:2:1) were gifts from Hoffmann-La Roche Inc. (Basel, Switzerland). All high-performance liquid chromatography (HPLC) grade solvents for chromatography and solvents for extraction (pro analysis quality) were purchased from Merck (Darmstadt, Germany). Dry pyridine and (-)-camphanoyl chloride were obtained from Fluka Chemie (Buchs, Switzerland). CDCl3 for recording of 1H-NMR spectra was purchased from Merck.
Animals and diets.
Two cold-pelleted diets were fed to duplicate groups of 16-mo-old
rainbow trout (Oncorhynchus mykiss), obtained from AKVAGEN
(Sunndalsøra, Norway) for 69 d. Initially, each fiberglass tank
contained 25 fish with a mean weight of 0.4 kg. The tanks were supplied
with freshwater (flow rate ca. 18 /min, 8–9°C), and the oxygen
concentration of the outlet water was not <220 µmol/L. The fish
were maintained indoors in 1 m2 tanks (ca. 600 L) covered
with a lid to minimize disturbance. Each tank was supported with a
separate light source supporting continuous illumination. Continuous
illumination results in increased growth rate in salmonids (Kråkenes et al. 1991
). The fish were fed slightly in excess, using electrically
driven disc feeders (Akvaprodukter, Sunndalsøra, Norway). No
mortalities occurred during the experiment, and the average final
weight was –1.0 kg for both treatments. Care and treatment of the
rainbow trout followed recommended guidelines for laboratory animals
(The Council of Europe 1986
).
The two experimental diets were produced by coating the basal diet with either predominantly all-E-astaxanthin (allE-AX4; 36.9 mg/kg, 97% all-E- (1a, 2a, 3a), 0.4% 9Z- (1b, 2b, 3b), 1.5% 13Z-astaxanthin (1c, 2c, 3c) and 1.1% other isomers, respectively) or an I2-catalyzed quasi-equilibrium mixture of all-E and Z isomers of astaxanthin [allE/Z-AX; 35.4 mg/kg, 64% all-E- (1a, 2a, 3a), 18.7% 9Z- (1b, 2b, 3b), 12.3% 13Z-astaxanthin (1c, 2c, 3c), and 2.0% other isomers, respectively] solubilized in dietary oil (Table 1 ). Diets were prepared in batches of 5 kg when required.
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has been described previously (Bjerkeng et al. 1997
). Respective
proximate composition of the allE-AX and
allE/Z-AX diets were (g/kg): dry matter 896 and
905, crude protein 497 and 499, fat 192 and 195, ash 104 and 104, and
gross energy 23.1 and 22.4 MJ/kg. Growth, feed utilization, and
astaxanthin digestibilities of the all-E- (1a,
2a, 3a), 9Z- (1b,
2b, 3b), 13Z- (1c,
2c, 3c) and 15Z-
astaxanthin (1d, 2d, 3d) in rainbow
trout fed the two diets was reported by Bjerkeng et al. (1997)
. Sampling and tissue sample preparation.
Before the feeding experiment started, 10 fish were weighed and fillet samples collected for estimation of the initial astaxanthin concentration. After 69 d, five fish per tank were collected for analyses of total carotenoid, all-E- (1a, 2a, 3a), 9Z- (1b, 2b, 3b) and 13Z-astaxanthin (1c, 2c, 3c) concentration and distribution of (3S,3'S) (1abcd), (3R,3'S)- (2abcd) and (3R,3'R)-astaxanthin (3abcd) optical isomers (Fig. 2) . The fish were anesthetized by transfer to water containing metacaine (60 mg/L, NMD, Trondheim Norway). All fish samples were analyzed individually, except for feces samples, which were pooled per tank. Blood (10–15 mL) was drawn from vena caudalis by punctuation slightly below the sideline between the anal fin and the adipose dorsal fin using Li-heparinized vacuum sampling tubes (Vacutainer, Venoject, Terumo, Belgium). The blood samples were stored on ice (maximum 30 min) before being centrifuged (10 min, 1700 x g). The blood plasma was collected and stored at -80°C until analyzed. After blood sampling, the fish were killed by cutting a gill arch and cut open, and the viscera were excised. The viscera was dissected, and the liver, intestinal tissues (consisting of intestinal tract, pyloric ceca and intestinal fat) and anterior and posterior kidney tissues were extracted and packed separately. The intestinal tissues were rinsed in isotonic saltwater. The carcass was filleted, and the muscle of one fillet and skin were packed separately into plastic bags. The samples were stored at -20°C before being analyzed. Feces for determination of astaxanthin stereoisomers (1abcd, 2abcd, 3abcd) were collected by stripping after 60 d and frozen immediately (-80°C). The reason for different sample storage temperatures was primarily logistic, and samples were anticipated not to have been negatively affected by temperature before being analyzed within the 3 months of frozen storage. All analyses were performed in duplicate.
Quantification of total carotenoids and astaxanthin geometrical stereoisomers.
Total carotenoids, including all-E- (1a,
2a, 3a), 9Z- (1b,
2b, 3b) and 13Z-astaxanthin
(1c, 2c, 3c), in all except the skin
samples were quantified by HPLC using a
H3PO4-modified silica gel column (Vecchi et al. 1987
), with all-E-astaxanthin (1, 2,
3; Hoffmann-La Roche) as an external standard, as described
by Bjerkeng et al. (1997)
, Figure 3.
The HPLC
instrument used was a Hewlett Packard liquid chromatograph (Hewlett Packard,
Palo Alto, CA) connected to a Hewlett Packard photo diode array UV-VIS
detector. The flow was either 1.0 or 1.5 ml/min and the pressure ~22
or 38 bar, respectively. The mobile phase was renewed daily. Detection
wavelength was set at 470 nm. All chromatograms were reintegrated for
baseline adjustment. The retention times (RT) of
all-E- (1a, 2a, 3a),
9Z- (1b, 2b, 3b) and
13Z-astaxanthin (1c, 2c,
3c) were ~9.7, 10.5 and 11.3 or 5.9, 6.4 and 6.9 min for
flow 1.0 and 1.5 mL/min, respectively.
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) and 1350 and 1750 for 13Z-
(1b, 2b, 3b) and
9Z-astaxanthin (1c, 2c,
3c), respectively. The E1cm,1%-values for
13Z- (1b, 2b, 3b) and
9Z-astaxanthin (1c, 2c,
3c), were estimated based on the respective HPLC response
factors relative to all-E-astaxanthin (1a,
2a, 3a) reported by Schüep and Schierle (1995)
. Quantification of the total amount of carotenoids in skin
extracts was performed spectrophotometrically (U-2000 UV-VIS recording
spectrophotometer, Hitachi Instruments, San Jose, CA), and the relative
amount of individual carotenoids determined by HPLC as described above.
Compensation for background absorption was performed as described by
Bjerkeng (1992)
. Parallel samples were analyzed repeatedly if the
difference between them were >10%. The detection limit of carotenoids
was <0.05 mg/kg biological sample. Quantification of optical isomers of astaxanthin.
The astaxanthin fraction was purified by thin-layer chromatography
(TLC) on silica gel plates (0.5 mm; Kieselgel 60 G, product no. 7731;
Merck) and converted into the corresponding diesters of (-)-camphanic
acid by reacting a dry sample of astaxanthin with (-)-camphanoyl
chloride in dry pyridine (1 mL) at 0–4°C, according to Bjerkeng et al. (1990)
. Astaxanthin mono- and diesters from the skin extracts were
saponified according to Bjerkeng et al. (1990)
, using methanolic KOH
(0.18 mol/L) in dichloromethane after removal of O2 by
careful flushing with N2 to prevent formation of
autoxidation products. The relative amount of the optical isomers of
astaxanthin were determined quantitatively by HPLC of the dicamphanates
according to Vecchi and Müller (1979)
, Figure 4
.Synthetic astaxanthin from Carophyll Pink (Hoffmann-La Roche)
consisting of the (3S,3'S)- (1abcd),
(3R,3'S)- (2abcd) and
(3R,3'R)-astaxanthin (3abcd) optical
isomers in a 1:2:1 ratio (here determined to 25.1:49.6:25.3), was used
as a reference.
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Liver extracts from fish fed the allE-AX was purified by precipitating polar lipids in a ultrafreezer (-80°C) followed by centrifugation (Sorvall RC-5B Plus superspeed centrifuge) at ca. 8000 x g (-15°C, 15 min). The supernatant was dissolved in hexane to yield a 3% solution and partitioned with an equal amount of dimethylsulfoxide (DMSO). The carotenoids were reextracted from the DMSO with diethyl ether after dilution with a saturated aqueous NaCl-solution and the solvent evaporated. The crude carotenoids were chromatographed repeatedly by TLC on silica gel, and the samples were purified by semipreparative HPLC using the conditions described above. Proton nuclear magnetic resonance spectra (1H-NMR) of geometrical isomers of astaxanthin from liver were recorded in CDCl3 solution with a Bruker FT-NMR instrument. No satisfactory spectra were obtained due to lipid impurities. The individual HPLC peaks (geometrical isomers) from liver corresponded by RT values and VIS data to the authentic geometrical isomers characterized below.
Authentic geometrical isomers of astaxanthin (1,
2, 3)5
were prepared from Carophyll Pink after I2-catalyzed
stereomutation (Bjerkeng et al. 1997
). The isomers were separated by
semipreparative HPLC, and individual isomers (peaks) were characterized
by VIS spectra and one-dimensional 1H-NMR and
two-dimensional 1H 1H COSY spectra
(CDCl3, 400 MHz, Bruker FT-NMR instrument). Assignments
were made according to reported data for geometrical isomers of
astaxanthin (1) diacetate (Englert and Vecchi 1980
) and for
the all-E C14 and 9Z C14
end groups (Englert 1995
). The assignments made were compatible with
two-dimensional 1H 1H COSY spectra recorded
here for the individual geometrical isomers.
Statistical analyses.
Mean results of five individual samples per replicate were analyzed
statistically by one-way ANOVA using the SAS computer software
(SAS/STAT Version 6, SAS Institute, Cary, NC
). Comparisons between
samples were performed by Tukey tests, when significance was indicated
by the general linear model. Significant differences was indicated when
P < 0.05. Results are given as means and pooled
SEM for two replicates for each dietary treatment.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A small amount of
Z isomers of astaxanthin [0.4% 9Z
(1b, 2b, 3b), 1.5%
13Z-astaxanthin (1c, 2c,
3c) and 1.1% other Z isomers, of total
astaxanthin, respectively] was present in the allE-AX diet,
whereas the Z isomers of astaxanthin accounted for a total
of 34% in allE/Z-AX diet [18.7% 9Z
(1b, 2b, 3b), 12.3%
13Z-astaxanthin (1c, 2c,
3c), and 2.0% other Z isomers, of total
astaxanthin, respectively], Table 1
. The total carotenoid
concentration in the allE-AX diet was not significantly
higher (1.5 mg/kg) than in the allE/Z-AX diet.
Estimates were based on the average values for all batches prepared for
the experiment (Table 1)
. The total plasma carotenoid and all-E-astaxanthin (1a, 2a, 3a) concentration was higher in the trout fed the allE-AX diet than in trout fed the allE/Z-AX diet (P < 0.05, Table 2 ). The hepatic concentration of total carotenoids and all-E-astaxanthin (1a, 2a, 3a) were significantly higher (P < 0.05), and 13Z-astaxanthin (1c, 2c, 3c) significantly lower (P < 0.05) when the trout was fed the allE-AX diet compared with the allE/Z-AX diet (Table 2) . Accumulation of 13Z-astaxanthin (1c, 2c, 3c) relative to the all-E and 9Z isomer (1b, 2b, 3b) was pronounced in the liver of both treatments compared with the other samples (P < 0.05). 13Z-Astaxanthin (1c, 2c, 3c) comprised ca. 39% of total hepatic astaxanthin of trout fed the allE-AX diet and ca. 49% of total astaxanthin in the other group, whereas 9Z-astaxanthin (1b, 2b, 3b) comprised only ca. 3% of total astaxanthin in both groups.
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No significant differences in anterior and posterior kidney tissues of total carotenoid concentration (1.5–1.6 and 3.2–3.5 mg/kg, respectively), or individual carotenoid concentration were observed between treatments. The relative astaxanthin E/Z-isomer distribution in anterior and posterior kidney tissues was similar and ranged from 89.4 to 91.7% for all-E-astaxanthin (1a, 2a, 3a), 2.9 to 3.8% for 9Z-astaxanthin (1b, 2b, 3b) and 6.0 to 7.4% for 13Z-astaxanthin (1c, 2c, 3c). Dietary treatment did not influence the total carotenoid concentration of the skin (12.1–12.5 mg/kg). Neither was the relative amount of all-E-astaxanthin (1a, 2a, 3a), 9Z-astaxanthin (1b, 2b, 3b) nor 13Z-astaxanthin (1c, 2c, 3c) affected (89.1–89.5%, 2.3–2.5% and 8.2–8.4%, respectively).
The dietary concentration of astaxanthin E/Z isomers (1abcd, 2abcd, 3abcd) did not influence the distribution of the (3S,3'S)- (1abcd), (3R,3'S)- (2abcd) and (3R,3'R)-astaxanthin (3abcd) optical isomers significantly in the two treatments (Table 3 ).The astaxanthin optical isomer distributions between treatments were observed for muscle, liver, intestinal tissues and blood, were similar to the astaxanthin optical isomer distribution in the diet. The ratio between (3S,3'S)-astaxanthin (1) and (3R,3'R)-astaxanthin (3) in the skin was ~2:1, and 3:1 in the posterior kidney of both treatment groups.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The present results provide data on tissue distribution of the
E/Z and R/S isomers of
astaxanthin in rainbow trout fed diets with different
E/Z-isomer composition. Most studies on
carotenoid E/Z-isomer bioavailability have
focused on ß,ß-carotene in mammals (Krinsky et al. 1990
, Parker 1996
). At present only limited information is available on other
carotenoids and species. Due to the lack of pertinent literature on
carotenoid E/Z-isomer metabolism in fish, the
results are discussed with reference to available reports on carotenoid
E/Z-isomer metabolism in other species including
humans.
Quantification of astaxanthin E/Z isomers (Fig. 2)
in the present experiment was based on individual extinction
coefficients. Extinction coefficients of purified isomers are
determined spectrophotometrically based on gravimetric quantification,
cf. Schüep and Schierle (1995)
.
In vivo and in vitro E/Z isomerization.
The relative instability of carotenoids and their possible in vivo and
in vitro isomerization should be recognized when utilization of
E/Z isomers of carotenoids is evaluated. Proper
care should be taken to avoid isomerization during sample work-up and
analysis (Schiedt and Liaaen-Jensen 1995
). Usually the carotenoid
all-E isomers are most prevalent but
E/Z isomerization may occur readily for instance
in the presence of acids, on contact with active surfaces, in light,
and in elevated temperatures (Zechmeister 1960
). This is illustrated by
the reduction in astaxanthin Z-isomer (1bcd,
2bcd, 3bcd) concentration during preparation of
the experimental diets. The isomer mixture of astaxanthin
(1abcd, 2abcd, 3abcd) added to the
diets contained 50% all-E-astaxanthin (1a,
2a, 3a) of total astaxanthin, whereas the
relative concentration in the allE/Z-AX diet was
64% (Bjerkeng et al. 1997
). In vivo E/Z
isomerization of carotenoids during intestinal passage was recognized
ca. 50 years ago (Kemmerer and Fraps 1945
), and it results in a near
absence of 9Z-ß,ß-carotene in postprandial plasma in
humans orally administered
13C-9Z-ß,ß-carotene (You et al. 1996
).
The 10% higher fecal than dietary relative astaxanthin Z-isomer (1bc, 2bc, 3bc) concentration might be explained by Z isomerization of all-E-astaxanthin (1a, 2a, 3a) during gastrointestinal residence. However, the high fecal level, low circulating and intestinal tissue levels of total astaxanthin (1abcd, 2abcd, 3abcd) of 144, 70 and 66%, respectively, in trout fed the allE/Z-AX diet compared with trout fed the allE-AX diet strongly indicated that absorption of astaxanthin Z isomers (1bc, 2bc, 3bc) was less efficient than absorption of all-E-astaxanthin (1a, 2a, 3a). A less efficient absorption of astaxanthin Z-isomers (1bc, 2bc, 3bc) than all-E-astaxanthin (1a, 2a, 3a) would lead to a gradual enrichment of the former isomers in the feces. The rate of intestinal isomerization cannot be quantified based on the present results.
Absorption of astaxanthin all-E- (1a, 2a, 3a) and Z isomers (1bcd, 2bcd, 3bcd).
E/Z-isomerization leads to considerable spatial rearrangement of carotenoids (Fig. 2) . Factors influencing the intestinal absorption rate such as solubility characteristics, diffusion rates and lipid bilayer incorporation and penetration rates, may be different between isomers.
The significantly higher fecal carotenoid concentration (ca. 50%) of trout fed the allE/Z-AX diet was due to a higher concentration of astaxanthin Z isomers (1bcd, 2bcd, 3bcd). Together with a similar fecal all-E-astaxanthin (1a, 2a, 3a) concentration for both treatments, it indicates that intestinal absorption of 9Z- (1b, 2b, 3b) and 13Z-astaxanthin (1c, 2c, 3c) is lower than for all-E-astaxanthin (1a, 2a, 3a). However, quantification of precise amounts of the E/Z isomers absorbed by the intestine is not possible.
Intestinal wall permeability is sensitive to small differences in
chemical structures (Pithavala et al. 1995
). The 9Z and
13Z isomers of lycopene have higher absorption rate than the
all-E isomer in humans (Stahl and Sies 1992
). Similarly,
ferrets fed diets containing canthaxanthin primarily of the
all-E-form have higher serum concentrations of
Z-canthaxanthins than of the all-E isomer (Tang et al. 1993, 1995
, Yu et al. 1995
). The opposite is reported for
ß,ß-carotene E/Z isomers in humans (Gaziano et al. 1995
, Jensen et al. 1987
, Johnson et al. 1997
, Stahl et al. 1992
, You et al. 1996
) and for astaxanthin E/Z
isomers in the present experiment. Structural differences cannot
explain the observed differences in absorption of astaxanthin
(1, 2, 3) and canthaxanthin in ferrets
and rainbow trout. Thus factors other than molecular weight dependant
diffusion rates seem to govern the discrimination between carotenoid
E/Z isomers. Equal uptake rates for
all-E- and 9Z-ß,ß-carotene in rat brush
border membrane vesicles (Moore et al. 1996
) agree with this view. A
discriminatory mechanism for intracellular translocation of different
astaxanthin E/Z isomers (1abcd,
2abcd, 3abcd) from the site of uptake until
incorporation into chylomicrons thus appears to be in operation.
Different metabolic transformation rates between the
E/Z isomers may contribute to the observed
differences between carotenoids. However, these hypotheses need to be
confirmed experimentally.
Blood transport and tissue accumulation.
The blood clearance rate of carotenoids is slow and limited by the low
elimination rate of the blood lipoprotein fractions (Guillou et al. 1992
, Kübler 1989
). After absorption, in vivo isomerization of
carotenoids is likely to be relatively slow because it is possible to
distinguish between E/Z-isomer composition
patterns in different tissues (Tang et al. 1995
). A
relatively high selectivity between E/Z isomers
was exhibited during absorption and deposition in the present
experiment. Regardless of dietary E/Z-isomer
composition, muscle Z-astaxanthin (1bcd,
2bcd, 3bcd) comprised 5% of total astaxanthin
similar to that of juvenile coho salmon fed diets containing
astaxanthin from Antarctic krill (Arai et al. 1987
). However, it is
considerably less than reported for wild and farmed Atlantic salmon and
Oncorhynchus species by Schiedt et al. (1981, 1989)
(up to
ca. 30% Z isomers; 1bcd, 2bcd,
3bcd). The results of the latter authors indicate that
utilization of astaxanthin Z isomers (1bc,
2bc, 3bc) equals the all-E-isomer
(1a, 2a, 3a).
E/Z isomerization may have occurred during their
work-up and purification procedures that involved active surfaces
(MgSO4 and silica gel) and derivatization into astaxanthin
(1, 2, 3) diacetates. The degree of
isomerization occurring in the present experiment was low as evidenced
by low Z-astaxanthin (1bc, 2bc,
3bc) levels.
A selective mechanism led to an accumulation of astaxanthin
Z isomers, notably 13Z-astaxanthin
(1c, 2c, 3c) in the liver, whereas
predominantly all-E-astaxanthin (1a,
2a, 3a) seemed to be incorporated into the
transport proteins and delivered to the muscle cells. In salmonid
blood, carotenoids are transported in plasma lipoproteins, mainly HDL
(Ando 1986
), and as recently shown as a plasma protein (Aas et al. 1998
). The relatively low concentrations of astaxanthin Z
isomers (1bc, 2bc, 3bc) in the skin
and kidney tissues confirmed this view and supported the theory
E/Z isomerization in these tissues is negligible.
The pronounced accumulation of 13Z-astaxanthin
(1c, 2c, 3c) in the liver may indicate
that the all-E (1a, 2a, 3a)
and 9Z isomers (1b, 2b, 3b)
are preferred substrates for metabolic transformation or incorporation
into the blood transport vehicles by the liver. In addition to
bioavailability differences between different carotenoid E/Z
isomers, species differences occur. According to Stahl et al. (1992)
9Z- and 13Z-ß,ß-carotene accumulate in human
liver compared with all-E-ß,ß-carotene. Ben-Amotz et al. (1989)
reported 9Z-/all-E-ß,ß-carotene ratios
of ca. 0.5 and 2.9 in rat and chicken liver, respectively. The hepatic
ratios of 9Z- (1b, 2b, 3b)
to all-E-astaxanthin (1a, 2a,
3a) in rainbow trout was considerably lower, whereas the
13Z (1c, 2c, 3c) to
all-E-astaxanthin (1a, 2a,
3a) ratio was close to 1. The low levels of astaxanthin
(1, 2, 3) in the liver show that it is
not stored in this organ.
Optical isomer distribution.
Astaxanthin (1, 2, 3) absorption was
not R/S selective or dependant on the relative
dietary E/Z-isomer concentration as shown by
similar intestinal tissue, plasma and dietary astaxanthin optical
isomer (1, 2, 3) ratios in both
groups. The muscle (3S,3'S)- (1)
(3R,3'S)- (2) and
(3R,3'R)-astaxanthin (3) optical
isomer distribution resembled that of the diet, although the amount of
(3S,3'S)-astaxanthin (1) compared to
(3R,3'R)-astaxanthin (3) was slightly
higher than reported earlier (Bjerkeng et al. 1990, 1992
, Foss et al. 1984
, Schiedt et al. 1985
). The higher ratio between
(3S,3'S)-astaxanthin (1) and
(3R,3'R)-astaxanthin (3) in the skin
indicates some selectivity in the tissue metabolism of the optical
isomers of astaxanthin (1, 2, 3).
Excretion of astaxanthin (1, 2, 3)
metabolites into the bile (Hardy et al. 1990
) and esterification of
astaxanthin (1, 2, 3) in the skin thus
may proceed by selective mechanisms.
Geometrical E/Z and optical R/S isomerism has a
profound effect on astaxanthin absorption (Bjerkeng et al. 1997
) and
plasma appearance and on tissue deposition. This may be related to the
large impact the former isomerism has on the steric arrangement of the
molecule, whereas the latter may be related to enzyme
stereospecificity. No clear picture of the factors governing the
bioavailability of E/Z carotenoids in different species is
presently available. Even though the all-E isomer of
astaxanthin (1a, 2a, 3a) seems to be
better absorbed and deposited than its Z isomers
(1bcd, 2bcd, 3bcd) in rainbow trout,
this may not be true for the all-E isomer of other
carotenoids. The lack of detailed knowledge merits further studies on
the mechanisms of absorption, transport, and metabolic transformation
of different astaxanthin isomers (1abcd, 2abcd,
3abcd). Experiments should be carried out using purified
isotope-labelled stereoisomers to develop a reliable compartmentalized
model for the metabolic transformation and turnover. Details on the
tissue specific metabolic transformation of carotenoids in salmonids
awaits further elucidation.
|
FOOTNOTES |
|---|
1 The work at the Institute of Aquaculture
Research AS (AKVAFORSK) was partly funded by a grant from BioMar AS,
Myre, Norway and BioMar AS, Brande, Denmark.
3 Abbreviations used: allE-AX, diets
supplemented with predominantly all-E-astaxanthin
(1a, 2a, 3a);
allE/Z-AX, diets supplemented with a isomerized
mixture of all-E- (1a, 2a,
3a), 9Z- (1b, 2b,
3b), 13Z- (1c, 2c,
3c) and 15Z-astaxanthin (1d,
2d, 3d).
4 All-E-Astaxanthin (1a,
2a, 3a). HPLC RT = 10.7
(flow 1.0 mL/min); VIS 
max nm (HPLC solvent) 472; 400
MHz 1H-NMR (CDCl3), (
values) 1.21
(s, ~6H, Me-17 and Me-17'), 1.32 (s, ~6H,
Me-16 and Me-16'), 1.81 (t, ~2H, H-2 ß,
J1 = 13.1 Hz,
J2 = 13.4 Hz), 1.94 (s,
~6H, Me-18 and Me-18'), 1.99 (s, ~6H, Me-20 and Me-20'),
2.00 (s, ~6H, Me-19 and Me-19'), 2.16 (dd,
~2H, H-2 
, Jvic = 5.6 Hz,
Jgem = 12.7 Hz), 6.21 (d,
~2H, H-7 and H-7, J = 16.0 Hz), 6.28–6.31
(m, ~4H, H-10, H-10', H-14 and H-14'), 6.43 (d,
~2H, H-8 and H-8, J = 16.1 Hz), 6.45 (d,
~2H, H-12 and H-12', J = 14.9 Hz), 6.6–6.7
(m, ~4H, H-11, H-11', H-15 and H-15').
9Z-Astaxanthin (1b, 2b,
3b). HPLC RT = 11.7 (flow 1.0
mL/min); VIS max nm (HPLC solvent) 370, 468,
DB/DII (Ke et al. 1970
) = 23%; 400 MHz
1H-NMR (CDCl3), ( values) 1.21
(s, ~6H, Me-17 and Me-17'), 1.32 (s, ~3H,
Me-16'), 1.34 (s, ~3H, Me-16), 1.94 (s, ~3H,
Me-18'), 1.95 (s, ~3H, Me-20), 1.97 (s, ~3H,
Me-18), 1.98 (s, ~3H, Me-20'), 1.99 (s, ~3H,
Me-19'), 2.01 (s, ~3H, Me-19), 6.19 (d, ~1H,
H-7', J = 16.0 Hz), 6.22 (d, ~1H, H-7,
J = 16.0 Hz), 6.24 (d, ~1H, H-10,
J ~ 16.0 Hz), 6.29 (dd, ~3H, H-14, H-14'
and H-10', J1 ~J2 ~12.0 Hz), 6.36 (d, ~1H,
H-12, J = 14.7 Hz), 6.42 (d, ~1H, H-8',
J = 16.0 Hz), 6.44 (d, ~1H, H-12',
J = 15.3 Hz), 6.64–6.67 (m, ~3H, H-11,
H-15 and H-15'), 6.71 (d, ~1H, H-11, J =
15.0 Hz), 6.96 (d, ~1H, H-8, J = 15.9 Hz).
13Z-Astaxanthin (1c, 2c,
3c). HPLC RT = 12.5 (flow 1.0
mL/min); VIS max nm (HPLC solvent) 370, 465,
DB/DII (Ke et al. 1970
) = 49%; 400 MHz
1H-NMR (CDCl3), ( values) 1.21
(s, ~6H, Me-17 and Me-17'), 1.32 (s, ~6H,
Me-16 and Me-16'), 1.81 (dd, ~2H, H-2 ß,
J1 =13.1 Hz,
J2 =13.4 Hz), 1.95 (s,
~6H, Me-18 and Me-18'), 1.99 (s, ~3H, Me-20'), 2.00
s and 2.01 s (~9H, Me-19, Me-19' and Me-20),
6.18 (d, ~1H, H-14, J = 12.0 Hz), 6.20
(d, ~1H, H-7', J = 16.0 Hz), 6.22
(d, ~1H, H-7, J = 15.9 Hz), 6.26–6.35
(m, ~3H, H-10, H-10' and H-14'), 6.43 (d,
~2H, H-8 and H-8', J = 16.1 Hz), 6.44 (d,
~1H, H-12', J = 16.0 Hz), 6.56–6.69 (m,
~3H, H-11, H-11' and H-15'), 6.97 (d, ~1H, H-12,
J = 15.8 Hz).
Manuscript received March 27, 1998. Initial review completed July 20, 1998. Revision accepted November 9, 1998.
|
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