Pure &Appl. Chem., Vol. 63, No. 1, pp. 89-100, 1991.
`HmmdmGmmBmmm
`©1991|UPAC
`
`206922209l00009
`
`CYAN EXHIBIT 1031
`
`Recent progress on carotenoid metabolism in
`animals
`
`Katharina Schiedt‘
`
`, Stefan Bischof‘
`
`and Ernst Glinzb
`
`" Department of Vitamin and Nutrition Research, 5’ Central Research Units
`F. Hoffmann—La Roche Ltd, CH—4002 Basel, Switzerland
`
`Abstract - The influence of dietary astaxanthin, canthaxanthin and zeaxanthin
`on the carotenoid content and composition of the oil droplets in chicken retina
`was investigated. From a "racemic" astaxanthin mixture,
`the (3§,3'§)-isomer
`was deposited almost selectively in the retina. Both oxidative and reductive
`metabolic pathways were followed by all three carotenoids. Astaxanthin,
`the
`main carotenoid in avian oil droplets, was obviously formed from both dietary
`zeaxanthin and canthaxanthin.
`— Egg yolk pigmentation was studied in relation to carotenoid structure.
`Depositon rates and metabolites of some C30 and C40 carotenoids have been
`determined. Beta- and w—apo-carotenoids with a terminal methyl group in the
`y—position were gradually shortened by a type of 6~oxidation.
`— Various yellow metabolites of astaxanthin have been identified in the prawn
`Penaeus vannamei and their absolute configurations determined. The 4,4’-oxo
`groups of astaxanthin were reduced stereospecifically, resulting in (4§,4’§)—
`tetrahydroxypirardixanthin. The presence of the novel, naturally occurring
`isoastaxanthin [(6§,6'§)~4,4’-dihydroxy-e,s-carotene-3,3’-dione] offered an
`explanation for a racemization of astaxanthin in 3133, which was proved in
`Penaeus japonicus after administration of optically active [3H]—label1ed
`astaxanthin.
`
`INTRODUCHON
`
`three years since the last Symposium, numerous publications have appeared
`In the past
`which document
`the importance of carotenoids in the industrial farming of animals. By
`far the greatest number deal with the biological effects of carotenoids in cattle,
`poultry and aquaculture and are aimed at improving production and quality of the
`product. A relatively small number of papers deal with metabolic transformations of
`carotenoids and with their function on a molecular basis. Since,
`in this meeting,
`some
`of the experts in this field reported on their own investigations or reviewed results
`obtained on animal carotenoid metabolism, I shall present in this article some studies
`carried out
`in our own laboratories. These concern:
`- deposition of dietary carotenoids and their metabolites in the chicken retina,
`- influence of structure on the rate of deposition of carotenoids and metabolites
`in egg yolk,
`- metabolites of astaxanthin in the prawn Penaeus.
`
`CAFIOTENOIDS IN CHICKEN RETINA
`
`three years ago, we discussed the occurrence of some tissue-specific carot-
`In Boston,
`enoids in the avian retina and their possible formation in the chicken embryo (ref. 1).
`Those studies had emanated from a co-operation with Dr. Brian Davies, University of
`Wales, Aberystwyth, UK. Ve had seen that distinct, stereospecific carotenoids were
`responsible for the colour of the red, yellow and greenish oil droplets located in the
`photoreceptor cells,
`the cones. Avian oil droplets were assumed to improve acuity in
`colour vision and were classified by Goldsmith 55 El. by microphotometric measure-
`ments and by analytical procedures (ref. 2).
`In 1984, Bethan Davies showed that labelled zeaxanthin was mobilized from the egg yolk
`to the retina of the chick embryo (ref. 3).
`
`89
`
`

`
`90
`
`K. SCHIEDT, S. BISCHOF AND E. GLINZ
`
`Deposition and transformation of dietary carotenoids in the chicken retina
`Dr. Harald Weiser of the Roche Biochemical Animal Section carried out growth tests by
`feeding (3§.3'§)—zeaxanthin, "racemic" astaxanthin (i.e. a mixture of the
`stereoisomers RR:RS:SS - 1:2:1) and canthaxanthin to one-day-old chicks (Strain
`Lohmann) (ref._z):_The control diet contained a minimal amount of 450 IU vitamin A and
`was virtually free of carotenoids. The experimental groups were fed astaxanthin,
`canthaxanthin or zeaxanthin in dosages of 36-144 mg/kg feed.
`
`For supporting growth, canthaxanthin and zeaxanthin could replace vitamin A partly or
`entirely. Astaxanthin was no growth factor and the animals that were fed astaxanthin
`without vitamin A supplementation became weak and moribund and were sacrificed after 21
`days. The chicks of the canthaxanthin and zeaxanthin groups were sacrificed after 39
`days,
`the retinas removed and the retinal carotenoids analysed.
`
`Control diet
`
`Q 8 I -Carotene
`
`zeaxanthin
`
`ado» I rub In
`E Canthaxan th l 11
`
`E Epl-/luteln
`Gallexunthln
`
`Fig.
`
`1 Carotenoid composition in the retina of chickens fed canthaxanthin,
`astaxanthin, zeaxanthin or control diet.
`
`Figure 1 presents the relative abundance of carotenoids in the retinas of the four
`groups that were fed 36 mg carotenoid/kg feed. It is evident
`that dietary astaxanthin
`and zeaxanthin did not alter the range of carotenoids present but only changed the
`ratio of the basic retinal carotenoids, by increasing the amounts of astaxanthin,
`galloxanthin, zeaxanthin, lutein of various chiralities and of s,s—carotene. In the
`canthaxanthin-fed group, not only were small amounts of this dietary carotenoid found,
`but also adonirubin and 6,5-carotene, which are not usually encountered in the chicken
`retina (Figs. 2 and 3).
`
`Based on quantitative analyses of the main retinal carotenoids, namely astaxanthin,
`zeaxanthin and galloxanthin including stereoisomers,
`the following metabolic processes
`may be assumed: all three dietary carotenoids were deposited in the retina; astaxanthin
`was partly metabolized to zeaxanthin and galloxanthin, zeaxanthin increased the amount
`of astaxanthin and galloxanthin. After administration of astaxanthin or zeaxanthin, a
`slight increase of 6,8-carotene was also observed. Canthaxanthin obviously follows
`an oxidative pathway to astaxanthin as well as a reductive one to B,B—carotene. This is
`interesting from an evolutionary point of view; apparently,
`the capability of some
`crustaceans to transform canthaxanthin into astaxanthin (ref. 5) is preserved in the
`chicken retina,
`though this modification is lost in many fishes such as salmonids
`(ref. 6).
`
`

`
`Recent progress on carotenoid metabolism in animals
`
`91
`
`ug/Retina
`10
`
`
`
`.81
`
`Astoxanthln
`
`
`
`Adonlrubln
`Canthaxanthln
`
`§.B-Carotene
`- H-4-oxo-pt -carotene ‘I
`Lutcln/Epl-lutnln
`Galloxnnthln
`
`Zoaxanthln
`
`i.8'Corotnne
`
`2. 18
`
`1.0
`
`0,1
`
`0.01
`
`control diet
`
`36 ppm Canthax.
`
`Fig. 2 Carotenoids in chicken retina (ug/retina)
`after canthaxanthin feeding (36 mg/kg feed) for 39 days
`
` dietary carotenoids
`
`°’‘
`
`_
`
`\ P
`
`\
`
`HO
`
`(3R,3'R)-
`Ioaxanthin
`
`0
`
`on
`
`\
`
`P \
`
`HO
`
`O
`ss:ns:an = 25:50:25
`nttnxanthin
`
`O
`
`\
`
`P~\
`
`O
`
`canthnxanthin
`
`0
`
`O
`
`» mag:
`
`3,3-carotene
`
`1
`
`53 [go]
`
`1:)
`
`03
`=
`
`\~
`
`.51“ 10;:
`
`no
`
`‘OH
`.‘
`
`47 [10] *)
`
`¢
`
`J
`
`98
`
`1
`
`[90] SS *)
`
`[10] RS *)
`
`l
`\
`ii‘ \
`
`s
`
`Oadonirubin
`
`H0
`
` °H2°H
`
`gnllosnnthin
`
`30
`
`I5
`
`I E
`
`~§\‘
`
`(53, 5 I 5)
`-E, S-carotene
`
`H0
`
`~\
`
`lutoin
`
`P '
`
`\ \ \ \ \ \ \
`
`Fig. 3 Deposition and transformation of dietary carotenoids in chicken retina
`
`*) without brackets: Percent of optical isomers in control animals
`in brackets [ 1: Percent of optical isomers after carotenoid feeding
`
`

`
`92
`
`K. SCHIEDT, S. BISCHOF AND E. GLINZ
`
`Selective absorption of (3S,3’S)-astaxanthin from a racemic mixture and non-
`racemization of (3R,3’R)-zeaxanthin
`
`These studies may be open to criticism because they were carried out without radio-
`labelled compounds. However, by means of sophisticated analytical methods, and by
`isolation of the respective all-trans-isomers and subsequent derivatization (refs. 8,
`9, 10) it was possible to determine the quantitative stereoisomeric composition.
`Table 1 clearly demonstrates that (3§,3'§)-astaxanthin was deposited preferentially,
`followed by (33,3’§;meso)-astaxanthin. The (3§,3'§)-isomer from the dietary mixture was
`not deposited at all. Regarding zeaxanthin, we had, at the last Symposium, discussed
`the unexpected finding that zeaxanthin in chicken retina was a mixture of the (5,5)-
`and the meso-isomers. This was intriguing, as zeaxanthin had been considered a pre-
`cursor of astaxanthin which is optically pure (3§,3'§) (refs. 1, 10). From our experi-
`ments, it is now evident
`that
`the ingested (3§,3'§)-zeaxanthin was deposited as such;
`no racemization could be observed. It may therefore be concluded that
`the meso-isomer
`is a secondary metabolite of (3§,3'§)—zeaxanthin formed by a redox—system perhaps via
`3'-dehydrolutein and lutein (ref. 1, 11). To determine why such an obviously species-
`specific equilibrium of xanthophyll stereoisomers is maintained in the eye and to
`establish the possible function of this equilibrium in vision requires further
`research.
`
`Table 1 Quantity and configuration of astaxanthin and zeaxanthin in the chicken retina
`after administration of the respective carotenoid
`
`Experimental
`
`groups
`
`A s
`
`t
`
`a
`
`x
`
`a
`
`n
`
`t
`
`ng/retina
`
`Rac- Astax.
`
`*)
`
`Control
`
`6250
`
`4640
`
`Increase
`
`in
`
`astaxanthin
`
`1610
`
`(3R..3'R.)-Zeax-
`
`*)
`
`Control
`Increase
`
`in
`
`astaxanthin
`
`1260
`
`(%)
`
`(10)
`
`(2)
`
`(33)
`
`625
`
`93
`
`532
`
`ng
`
`(%)
`
`5625
`
`4547
`
`1078
`
`(90)
`
`(98)
`
`(67)
`
`72
`
`(2)
`
`3528
`
`(98)
`
`(93)
`2293
`(2)
`42
`(98)
`1235
`(2)
`30
`nRetina
`
`ng/retina
`
`ng
`
`(%)
`
`RR
`
`ng
`
`(%)
`
`(3R_’3-R_),zeax_
`
`tr)
`
`")
`
`36 mg/kg feed
`
`500
`
`40°
`
`50
`
`(10)
`
`450
`
`(90)
`
`397
`
`<99)
`
`CAROTENOIDS AS POTENTIAL EGG YOLK P|GME_NTERS
`
`The consumer expects high quality eggs not only direct from the farm but also in in-
`dustrial poultry products. one mark of perceived egg quality is the yolk colour which
`depends almost entirely on the carotenoid content and composition in the layers’ feed.
`The appealing appearance of an egg yolk depends not only on the colour hue but also on
`its saturation and the dominant wavelength that are also responsible for its luminos-
`ity.
`
`Influence of carotenoid structure on absorption and deposition
`
`The deposition rate in egg yolk has been tested for some acyclic, cyclic and apo-
`carotenoids of various structures synthesized by Drs. K. Bernhard and U. Hengartner
`(ref. 12).
`
`

`
`Recent progress on carotenoid metabolism in animals
`
`93
`
`Rather than merely a comparison of pigmenting efficacies by statistical means, some
`biochemical, analytical studies regarding deposition rate and metabolism have been
`undertaken. Deposition rate is defined as the amount deposited in egg yolk as a per-
`centage of the quantity ingested. When a compound was converted into metabolites,
`the
`amounts of the original compound plus metabolites were summed and considered as total
`deposition. The aim was to obtain some basic knowledge about how structure influences
`absorption, deposition and metabolism.
`The following compounds were added as beadlets to the basic feed (10 mg/kg) of laying
`hens (Shaver Starcross 288):
`torularhodin aldehyde §, B-apo-8'—caroten-
`torularhodin 2,
`torularhodin ethyl ester 1,
`oic acid ethyl ester 5, B-apo-8’~carotenal §, B-apo-2’-carotenal Z, 6'-apo-lycopenoic
`acid ethyl ester lg, 6'-apo-lycopenal 12, and 8'-apo-lycopenal lg. The pigmentation
`trial was carried out by Dr; J. Broz of our 'Animal Nutrition’ department. Eggs were
`collected during days 16-20, when the yolk colour had reached plateau values; 20-30
`yolks were pooled per group and the administered compound as well as the metabolites
`were analysed. Numerous chromatographic separations (column, TLC, HPLC) on adsorption
`and reversed phases were involved as well as chemical reactions and derivatizations
`such as esterification of the carboxylic acids with diazomethane and finally
`characterization by MS (Mr. W. Meister) and 1H-NMR (Dr. G. Englert).
`
`The results obtained from a first group,
`agents are compiled in Fig. 4.
`
`fed C40 and C30 carotenoids as pigmenting
`
`Carotenoid supplements 1n teed —-*— Compounds deposited in egg yolk
`
`Dep.
`rate
`
`.
`
`6 ,‘ cmat cmEt
`
`15-
`
`16'
`
`(496)
`
`J.
`
`15'
`
`3-
`
`15:
`
`9 ‘I CW", ¢WH
`
`(499)
`
`Z
`
`16'
`
`
`
`2
`
`15'
`
`10% Ejfgiyayk/§/fifiw«Y§¢fiAw«wmm
`
`Ej:séy§«L4§4*ay§r§nfiAs4wcam
`
`(506)
`
`3
`
`8|
`
`(499)
`
`2
`
`8|
`
`49 %
`
`\ \ \ \ \ \ \ \ m“mt
`
`__,_
`
`\ \ \ \ \ \ \ x ”“mt
`
`(443)
`
`A
`
`(443)
`
`A
`
`_ w° ¢WH
`
`av
`
`1.}
`
`av
`
`1)
`
`(452)
`
`5
`
`(443)
`
`.6.
`
`in brackets:
`
`l,.x, nm (n-hexane)
`
`1) Wildfeuez, 1969 (ref. 13).
`
`Fig. 4
`
`C40 and C30 carotenoids l—§ fed to laying hens and compounds deposited
`in egg yolk
`
`the C40 hydrocarbons 5,6-carotene and lycopene are virtually not
`It is known that
`deposited at all in egg yolk. By the introduction of oxygen functions into the
`molecule, however,
`the absorption rate can be improved markedly.
`
`- From the comparison of the red C40 carotenoid l and the yellow C30 apo-carotenoid 4
`(Fig. 4) it is evident
`that
`the length of the molecule significantly influences the
`deposition rate in egg yolk. However,
`the type of the functional group, aldehyde,
`carboxylic acid or ester, does not change the order of magnitude of the deposition
`rate.
`
`- The ethyl esters 1 and 4 were deposited as such. Only minor amounts of 3-6% of the
`total deposited carotenoid had been hydrolyzed to the corresponding acid. The
`carboxylic acid 2 was deposited unchanged. The aldehydes Q and §, however, were
`oxidized to the respective carboxylic acids.
`
`

`
`
`
`2'
`
`(I59)
`
`3,
`
`(465)
`
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`
`2-
`
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`
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`
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`(47!)
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`(459)
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`(443)
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`**>
`//////////
`
`*>
`
`re)
`
`*‘k)
`
`*)
`
`94
`
`K. SCHIEDT, S. BISCHOF AND E. GLINZ
`
`2'
`
`,.
`
`0:: id .
`
`(493)
`
`‘‘°’’
`
`11
`
` CEO /\r§,C003
`
`\\\\\\\
`
`(454)
`
`Artefact
`
`1.9
`
`(424)
`
`L5
`
`R = not identified
`
`*)
`
`identified
`
`in brackets: 7\,,,,,,
`
`run (n—hexane)
`
`33 mm’-hY1 93"-933 bY V13: M3
`M) vrs, MS, 1n-mm
`
`Fig. 5 B—Apo-2'-carotenal 2 (C37) fed to hens and metabolites detected in egg yolk
`Deposition rate in total: 9%,
`in boxes: relative Z of metabolites (not identified: 33 Z)
`
`— After feeding the C3, aldehyde 1 (Fig. 5), again none of the original material was
`found in egg yolk but only carboxylic acids. It was evident
`that
`these acids were by
`no means homogeneous. Another, comparatively apolar, fraction was found which at
`first was assumed to be a reduction product, namely a 5-apo-carotenol with an un-
`decaene structure according to its chromophore, but
`this fraction was found to con-
`sist of two compounds, an ester and a y-lactone. Obviously,
`the latter must be con-
`sidered as an artefact arising by spontaneous lactone ring closure of the carboxylic
`group with the y—C of the 3',4’—dihydrogenated acid and concomitant retro-rearrange-
`ment of the polyene system. The question of why the chicken stabilizes the majority
`of this labile compound by esterification deserves further consideration. In con-
`trast, xanthophyll or retinyl esters are hydrolyzed and deposited in egg yolk in
`their free form. Details of isolation and identification will not be presented but it
`should be mentioned that
`the carboxylic acids were esterified with diazomethane,
`the
`methyl esters separated by column chromatography on Mgo according to the number of
`double bonds in the polyene system,
`the all-trans isomers of the single esters
`isolated by HPLC and finally characterized by MS and in some cases also by 1H—NMR.
`
`It should be noted that, after administration of the originally carmine red 2'—apo—
`carotenal, a number of carotenoids with a much shorter conjugated polyene system
`ranging in colour from orange to bright yellow were found in egg yolk. As seen in
`Fig. 4,
`the C40 and C30 carotenoids l, l, l, 5, § were not degraded in chain length in
`egg yolk. What is the structural difference between 3-apo-2’-carotenal Z and the
`homologues torularhodin aldehyde l and B—apo—8’—carotenal § that
`they should be
`metabolized so differently? Certainly, it seems not
`to be the length of the molecule
`but
`the fact
`that compound 1 possesses a y—methyl group, whereas the others have an
`a—methyl, which obviously causes a steric hindrance to enzymic attack.
`
`three acyclic model compounds lg, ll, l§ and their metabolites
`For further comparison,
`are depicted in Fig. 6. The relative proportions of the metabolites are marked in the
`corresponding boxes. Characterization was again carried out by VIS, MS and 1H—NHR.
`
`the apo-lycopenals ll and lg were oxidized to the corresponding carboxylic
`— Again,
`acids, but while 8’-apo-lycopenal l§ with its apmethyl substituent maintained its
`chain length, compound ll with the methyl group in the Y-position was readily de-
`graded to acids of shorter chain length.
`
`

`
`Recent progress on carotenoid metabolism in animals
`
`95
`
`Carotenoids administered to laying hens and their metabolites in egg yolk
`
`Deposition
`
`21%
`
`.3 .
`
`/
`
`
`
`.'
`
`."
`
`._.
`.
`.
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`(435)
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` mH
`(416)
`1 25.
`
`turther degradation
`to acids of shorter
`chain length
`
`in brackets: L“, M (n-hexane)
`
`Fig. 6 6'-Apo-lycopenoic acid ethyl ester lg, 6'—apo-lycopenal 11
`and 8’-apo-lycopenal lg fed to laying hens and identification of metabolites
`in egg yolk
`
`— Another metabolic peculiarity of these acyclic carotenoids was hydrogenation in the
`5,6-position.
`- The 6'-apo-lycopenoic acid ethyl ester lg showed a much higher deposition rate (21 2)
`than the apo—1ycopena1s 11 and lfi (4% and 6%). In this case, however,
`the ethyl ester
`lg was only partly deposited unchanged. In part, epoxidation occurred in the 1,2—pos—
`ition, partly the ester was hydrolyzed and hydrogenated in the 5,6-position. Finally,
`once hydrolyzed,
`the 5,6-dihydrogenated 6'-carboxylic acid g; was also shortened by
`two carbons resulting in 5,6-dihydro-8'-apo-lycopenoic acid gg.
`
`Comparative metabolic considerations
`
`It has been shown that some carotenoids tested as egg yolk pigmenters were metabolized,
`while others were not.
`
`- The ethyl esters l and Q of the C40 carotenoid torularhodin and of the C30 5—apo-
`carotenoic acid were both deposited unchanged in egg yolk. 6’—Apo—1ycopenoic acid
`ethyl ester lg with the methyl group in the y—position was hydrolyzed and sub-
`sequently underwent shortening by B—oxidation. Obviously,
`the hydrolase can attack
`carotenoids without a terminal methyl group more readily.
`those
`- The apo-carotenals were oxidized and deposited as carboxylic acids; again,
`carboxylic acids g, Q, g; that arose from an aldehyde with a terminal methyl group
`were not degraded further, whereas those originating from aldehydes 1, ll with a
`methyl group in the v—position were gradually oxidized by two and three carbons,
`respectively.
`g
`- Hydrogenation of the 5,6—double bond occurred in the acyclic apo-carotenoids 16, ll,
`and ;§. B—Apo—2’—carotenoic acid ll was hydrogenated in the 3',4'-position. The loss
`of up to four double bonds gives a chromophore which exhibits quite different pig-
`menting characteristics.
`
`

`
`96
`
`K.SCHEDT,S.mSCHOFANDE.GUNZ
`
`An explanation for this structure-specific behaviour may be offered by comparing the
`enzymes involved in the fl—oxidation of fatty acids (ref. 14). When the enzymic cascade
`of fatty acid oxidation is compared with that of carotenoic acids,
`two things become
`apparent; First,
`the oxidation itself is an intramitochondrial process and, secondly,
`for the passage of the fatty acyl moiety through the mitochondrial membrane,
`two
`enzymic steps are required, namely activation to acyl-CoA and then transfer as a
`carnityl—acy1—complex.
`these may be the limiting steps. Thus if activation to
`In the case of carotenoic acids,
`the acyl—CoA is not possible because of steric hindrance by the terminal methyl group,
`then passage through the inner mitochondrial membrane would not be possible. In turn,
`if carotenoic acids with their methyl group in the 7-position can form the activated
`acyl-CoA,
`then the acyl group may enter the mitochondria by means of the carnitine-acyl
`transferase, and only then would the cascade of chain-shortening be possible.
`In our studies on egg yolk pigmentation, neither the sites of these conversions,
`presumably intestinal mucosa or liver, nor the question of whether further degradation
`to retinoic acid occurs, were investigated. Retinal and retinol were not measured.
`However,
`these studies may be an example of an eccentric cleavage of B—apo—carotenoids
`in non—mammals. It may be asked whether the chain shortening as shown above is due to
`an unspecific dioxygenase or to enzymes that are also involved in the fi—oxidation of
`fatty acids. Possibly, glucuronides are formed and then excreted. The presence of the
`C3, y-lactone lg, originally formed from the 3',4'-dihydrogenated acid 2, may show some
`analogy to vitamin A metabolism where similar shorter lactone metabolites have been
`found in bile and urine of rats and humans (ref. 15).
`
`METABOLITES OF ASTAXANTHIN IN THE CRUSTACEAN PENAEUS
`
`Tetrahydroxypirardixanthin (isolated from P. vannamei) (5,6,5,’6’-tetrahydro-B,
`B-carotene-3,4,3’,4’-tetrol)
`
`the previous Symposium, we reported the occurrence of yellow carotenoid esters
`At
`(20-30%) besides the main astaxanthin esters in some wild species of Penaeidae
`(ref. 1). Those yellow carotenoids were not identical with lutein or zeaxanthin, but
`a major part corresponded to 3,4,3’,4'—tetro1s with 5,6-dihydro-B-rings. The same
`5,6-saturated tetrols have previously been reported by Matsuno gl al.
`in the spindle
`shell Fusinus perplexus (ref. 16),
`in three species of Fusinus and also in the prawn
`Penaeus japonicus by Katagiri El al. (refs. 17, 18).
`In none of those papers was the
`absolute configuration described. We considered the assignment of the absolute config-
`uration of these tetrols with 8 centres of asymmetry something of a challenge, partic-
`ularly that for the one isolated from Penaeus, where the compound is assumed to be the
`reduction product of astaxanthin. Since astaxanthin in these higher crustaceans of the
`order Decapoda is an almost "racemic" mixture of all three configurational isomers,
`different stereoisomers of tetrahydroxypirardixanthin could also be expected. We re-
`isolated the yellow fraction from the carapace of cultured Penaeus vannamei
`that had
`been fed racemic astaxanthin and compared the spectroscopic data (13-NMR, CD) with
`those of samples obtained from the wild species. The esters were again saponified and,
`for reasons of higher stability of the product, acetylated in the presence of the
`catalyst 4—dimethy1—aminopyridine (DMAP) (ref. 19). Dr. G. Englert assigned the
`relative configurations of these tetraacetates by ‘H-NMR spectroscopy and Dr. K. Noack
`determined CD spectra with minute amounts of the isolated material. However, only after
`the total synthesis of the optically active (3§,4§,5§,6§,3'§,4'§,5’§,6’§)—tetraacetoxy—
`pirardixanthin by our colleague Dr. U. Hengartner (ref. 20), could the spectra be
`assigned unequivocally. The metabolites depicted in Fig. 7 were of various absolute
`configurations and occurred in various combinations. All were characterized by MS,
`1H-NMR and CD.
`
`in the
`The 4—hydroxy group exhibits the same configuration in both types of end-group,
`saturated cyclohexane rings A (3§,4§,5§,6§) and B (3§,4§,5§,6§) of tetrahydroxypirardi-
`xanthins lg, lg and in the B—end-groups D (3§,4§) and E (3§,4§) of crustaxanthins ll
`and ll. Obviously,
`the reduction of astaxanthin was again stereospecific, but opposite
`to that found in Atlantic salmon (refs. 1, 21).
`
`In Boston, we reported the occurrence of the novel natural 4-hydroxy-e-caroten-3-one
`end-group 9 (ref. 1), but only recently succeeded in isolating also the symmetrical
`isoastaxanthin lg.
`In 1981, a possible racemization of astaxanthin lg vivo was suggest-
`ed by Buchecker (ref. 22) and Zell gl al. proposed a similar reaction mechanism for the
`synthesis of isoastaxanthin via the enediol (ref. 23). The biological product isolated
`from E. vannamei was optically active and exhibited the (6§,6'§)—configuration accord-
`ing to CD. Also the pirardixanthin derivatives showed uniformly the same configuration
`at C-6,0-6’.
`
`

`
`Recent progress on carotenoid metabolism in animals
`
`97
`
`
`
`
`crustaxanthin 32
`
`D - P - E
`
`33
`
`R - gcetyl
`
`
`
`Isoastaxanthin
`C-P-A
`
`c-2-3
`
`C-P-D
`
`
`
`3.1
`
`Fig. 7 Metabolites of astaxanthin in.Penaeus vannamei:
`_tetrahydroxypirardixanthin,
`isoastaxanthin, crustaxanthin and related compounds
`
`Since in these higher crustaceans all three astaxanthin isomers were found, it was a
`challenge to investigate a possible racemization in vivo. Unfortunately,
`the number of
`animals was very small but, for the first time, it became possible to maintain the
`species of E.
`japonicus in our Basel laboratories and finally,
`the feeding trial was
`carried out successfully by Mr. P. Home with some animals that survived quite well.
`
`Experimental conditions and analytical procedures
`
`[15,15'-3H2]-(3§,3'§)-Astaxanthin with a specific radioactivity of 198 uCi/mg or
`439 600 dpm/ug dissolved in cod liver oil was applied onto pellets of a commercial feed
`(Gold coin). The astaxanthin supplement
`in the feed amounted to 200 ppm. The average
`live weight of the prawns (N = 5) was 10 grams at
`the beginning and 12.3 grams at
`the
`end of a 21-day feeding period.
`
`0
`
`03
`
`HO
`
`O
`
`(35, 3 ' S)
`
`\.P \ ~
`in feed
`
`
`racemization
`
`O
`
`on
`
`\
`
`P‘\
`
`30
`
`O
`
`—_>
`
`HO
`
`O
`
`0
`
`_,.oa
`
`\
`
`R\
`
`-:> W
`HO
`
`O
`
`O
`
`_,.oa
`
`\
`
`‘R\
`
`(3S,3'S)
`
`(3s,3'R; mesa)
`
`(3R,3'R)
`
`Fig. 8
`
`I_n vivo racemization of optically active [15,15'—3H,]—(3§,3'§_)—astaxanthin
`in Penaeus japonicus
`
`

`
`98
`
`K. SCHIEDT, S. BISCHOF AND E. GLINZ
`
`Table 2
`
`Isolation and analysis of astaxanthin, and
`mono- and diacetylenic asterinic acid from the carapace of Penaeus jagonicus
`
`SEPARATION AND ANALYS I S
`
`1. Chrom. on sio,
`
`spec. act.
`dpm/#9
`
`
`pg (VIS)
`
`dpm.10‘
`
`Esters (carot. equiv.)
`Free "astax."
`
`1 343
`223
`
`18.83
`8.18
`
`14 020
`36 680
`
`total
`
`1 566
`
`27
`
`2. Prep. TLC on S10,
`"Astaxanthin"
`
`other carotenoids
`
`160
`
`39
`
`7.29
`
`0.22
`
`45 560
`
`5 640
`
`3. HPLC,
`
`isolation of
`
`all-trans isomers
`
`7,8,7',8’-tetra-
`dehydro-astax.
`
`7,8—didehydro-
`astaxanthin
`
`area %
`
`pg (VIS)
`
`dpm.10‘
`
`dpm/ug
`
`28
`
`19
`
`20
`
`15
`
`0.06
`
`0.11
`
`2 140
`
`5 790
`
`94 420
`4.06
`43
`53
`Astaxanthin
`
`
`
`
`4. HPLC of dicanphanates
`
`
`
`
`
` Astaxanthin
`dpm
`(3§,3’§)
`13
`0.28
`21 000
`
`75 000
`
`
`
`(3§,3’§;g§§g)
`43
`1.10
`97 400
`88 000
`
`
`113 000 (3§,3’§) 44 1.39 157 000
`
`
`
`7,8,7',8'-tetra-
`
`
`
`
`
`
`
`dehgdro-astax.
`
`(3§,3’§)
`
`(3§,3’§;9g§g)
`
`(3§,3'§)
`
`7,8-didehgdro-
`astaxanthin
`
`(3§,3’§)
`
`(3§,3'§;gg§g)
`
`25
`
`46
`
`29
`
`15
`
`37
`
`2
`
`4.3
`
`2.6
`
`1 000
`
`200
`
`120
`
`500
`
`50
`
`50
`
`0.64
`
`1.65
`
`940
`
`1 680
`
`1 470
`
`1 020
`
`1 18a
`2 480
`2.1
`4a
`(3§,3'§)
`
`
`

`
`Recent progress on carotenoid metabolism in animals
`
`99
`
`Carotenoids were extracted with acetone from the carapaces, exclusively. The four steps
`of separation,
`isolation and analysis as well as the distribution of the radioactivity
`in the various fractions are summarized in Table 2. Chiral analysis of astaxanthin and
`asterinic acids was performed via the dicamphanate derivatives (ref. 8). It has only
`been possible so far to investigate in detail the free astaxanthin fraction, although a
`much larger portion of radioactivity was found in the esters. The free astaxanthin
`fraction showed a considerable specific radioactivity, which increased continuously
`during the purification procedure. Finally, a specific radioactivity of approximately
`100 000 dpm/ug astaxanthin was attained, corresponding to a dilution of 1:4 with endo-
`genous astaxanthin (Table 2, section 4).
`
`The following conclusions may be drawn:
`— The ratio of astaxanthin stereoisomers found was similar to that reported previously
`in P. vannamei (ref. 1) and in P.
`japonicus (ref. 18).
`— The fact
`that an almost equal specific radioactivity was observed in all three
`stereoisomers, although only the (§,§)—isomer had been fed, allows the conclusion
`that racemization of the astaxanthin end-group took place during or after absorption.
`— Moreover, it may be noted that a considerable portion of 7,8,7',8'-tetradehydro- and
`7,8-didehydro-astaxanthin (28% and 192) was present
`in the "astaxanthin fraction".
`From the fact
`that
`these acetylenic compounds are also "racemic" mixtures,
`two con-
`clusions may be drawn: acetylenic compounds have not previously been considered as
`metabolites of astaxanthin, but are assumed to be transformation products of the
`algal carotenoids diatoxanthin [(3§,3’§)—7,8-didehydro-B,B-carotene—3,3'-diol] and
`alloxanthin [(3R,3'§)—7,8,7',8’-tetradehydro—B,B-carotene-3,3’-dio1] in many aquatic
`animals. According to the very low specific activity found in the two acetylenic
`compounds in these prawns, also here 7,8—didehydro- and 7,8,7',8’-tetradehydro-
`astaxanthin may be excluded as metabolites of astaxanthin. However, it is surprising
`that
`the astaxanthin end—groups in these two compounds are no longer optically pure,
`but exhibit
`the same "racemic" mixture as seen in astaxanthin. This fact again
`supports the finding of a racemization of the astaxanthin end-group ig vivo,
`in
`contrast
`to the optically pure asterinic acid found in members of the Euphausiaceae
`together with all three stereoisomers of astaxanthin (ref. 24).
`
`that contain
`We plan to work up also the labelled ester fractions (Table 2, section 1)
`also the the yellow metabolites depicted in Fig. 7. It may then be possible to unravel
`metabolic pathways of astaxanthin in P.
`japonicus and to find out whether the yellow
`compounds are involved in the racemization of astaxanthin. If so, it would be another
`objective of research to find out whether those reactions occur at the level of free or
`esterified carotenoids or of carotenoproteins. — With these prospects,
`the specialists
`in the cultivation of crustaceans should be encouraged to pursue further the bio-
`chemistry of these fascinating animals.
`
`Acknowledgements
`
`We are greatly indebted to the following co—workers at Hoffmann—La Roche, Basel,
`their valuable contributions to these studies: Dr. J. Broz, Dr. J. Gabaudan,
`(NMR),
`Mr. P. Horne, Mr. A. Kormann, Dr. H. Weiser (animal experiments); Dr. G. Englert
`Mr. V. Meister and Dr. V. Vetter (MS), Dr. K. Noack (CD), Dr. M. Vecchi
`(HPLC), Dr. K.
`Bernhard, Dr. U. Hengartner, Mr. K. Holzhauser (chemical syntheses), Dr. T. Latscha
`(marketing aspects and provision of samples), Dr. H. Mayer for his expert advice and
`continuous support, Ms. H. Vicki and Ms. U. Hartmann for the preparation of the
`manuscript.
`
`for
`
`REFERENCES
`
`in Carotenoids: Chemistry and Biology (N.I. Krinsky, M.M. Matthews-Roth
`1. K. Schiedt,
`and R.F. Taylor, eds.), pp. 247-268, Plenum Press, New York (1990)
`2. T.H. Goldsmith, J.S. Collins and S. Licht, Vision Res. 24, 1661-1667 (1984)
`3. B.V. Davies, A. Akers and B.H. Davies, Abstracts 7th Int. Symp. on Carotenoids,
`Munich (1984), p. 14.
`4. H. Veiser, F. Hoffmann—La Roche Ltd, Basel, unpublished (1988).
`5. Y. Tanaka, Mem. Fac. Fish., Kagoshima University, 21, 335-422 (1978).
`6. K. Schiedt, F.J. Leuenberger, M. Vecchi and E. Glinz, Pure Appl. Chem. §Z, 685-692
`(1985).
`7. A. Riittimann, K. Schiedt and M. Vecchi, J. High Res. Chrom. Chrom. Commun.
`612-616 (1983).
`
`_6_,
`
`

`
`100
`
`K. SCHIEDT, S. BISCHOF AND E. GLINZ
`
`8. M. Vecchi and R.K. Mfiller, J. High Res. Chrom. Chrom. Commun. 2, 195-196 (1979).
`9. K. Schiedt, F.J. Leuenberger and M. Vecchi, Helv. Chim. Acta Q4, 449-457 (1981).
`10. B.W. Davies, Ph.D.
`thesis, University of Vales, 1986.
`11. K. schiedt and F.J. Leuenberger, Abstracts 6th Int. Sxmg. on Carotenoids,
`Liverpool (1981).
`12. K. Bernhard and U. Hengartner, F. Hoffmann—La Roche Ltd, Basel, unpublished.
`13. J. Wildfeuer, Z. Lebensm. Unters. Forsch. 140-144 (1969).
`14. L. Stryer, Biochemie, p. 298, Vieweg & Sohn, Braunschveig/Viesbaden (1985).
`15. G. Wolf, Phxsiol. Rev. 64, 873-937 (1984).
`16. T. Matsuno, K. Katagiri, T. Maoka and T. Kamari, Comg. Biochem. Phzsiol. §1§,
`905-908 (1985).
`17. K. Katagiri, T. Maoka and T. Matsuno, Comp. Biochem. Phxsiol. 843, 473-476 (1986).
`18. K. Katagiri, Y. Koshino, T. Maoka and T. Matsuno, Comg. Biochem. Phzsiol. §Z§,
`161-163 (1987).
`19. G. H6f1e, V. Steglich and H. Vorbrfiggen, An ew. Chemie 29, 602-615 (1978).
`20. U. Hengartner, Abstracts 9th Int. Sxmg. on Carotenoias, Kyoto (1990), p.22.
`21. K. Schiedt, H. Mayer, M. Vecchi, E. Glinz and T. Storebakken, Helv. Chim. Acta, 71,
`881-886 (1988).
`(G. Britton and
`22. R. Buchecker,
`in Carotenoid Chemistry and Biochemistry,
`T.V. Goodwin, eds.) pp. 175-193, Pergamon Press, Oxford (1982).
`23. R. Ze11, E. Vidmer, T. Lukéc, H.G.V. Leuenberger, P. Schfinholzer and E.A. Broger,
`Abstracts 6th Int.SXmg.on Carotenoids, Liverpool (1981).
`24. K. Schiedt, Dr.
`techn.
`thesis, University of Trondheim-NTH, 1987.