Pure :51 Appi. Chem. Vol. 63. No. 1. pp. 31-88, 1991.
Printed in Great Britain.
2(]6922209l0O008
@ 1991 IUPAC
Xanthophylls as precursors of retinoids
Takao Matsuno
Department of Natural Products Research, Kyoto Pharmaceutical
University, Misasagi, Yamashina-ku, Kyoto 607, Japan
Abstract — Xanthophylls such as canthaxanthin, astaxanthin,
zeaxanthin, lutein and tunaxanthin that are widely distributed
in Nature have been found to be precursors of retinoids not
only in freshwater fish and marine fish (yellowtail) but also
in mammals (rat).
Reductive metabolic pathways from 3—dehydroretinol to retinol
and from 3—dehydroretinal to retinal have been newly discovered
by feeding experiments with rat and yellowtail, respectively.
INTHODUCHON
In many animals, the most important metabolic products of carotenoids are
the retinoids, and the metabolic reactions of carotenoids in animals are
essentially oxidative. However, pathways of reductive metabolism have
recently been discovered, and this has opened up the possibility that
xanthophylls could be precursors of retinoids (ref. 1).
A review on xanthophylls as retinoid precursors will be presented, based upon
the original literature and on the results of our feeding experiments.
The retinoids discussed so far in these studies are retinol (1), retinal (2),
3—dehydroretino1 (3) and 3-dehydroretinal (4) (Fig. l).
Retinal {1}
Retinal (2)
E:jtf§,ia/Q;/i§,»CH20H fi:j:o§/Lp/e%,i§V/CH0
3-Dehydroretinol (3)
Fig.
3~De1-iydroretinal 14'.)
1. Retinoids.
only about 60 of the 600 known carotenoids (ref. 2) have been reported to be
precursors of retinol, the main ones being a—carotene, Y-carotene, B-cryptoxanthin, echinenone, B-apo-12'-carotenal and, of course,B-carotene (Fig. 2}.
From the structural point of view, retinol precursors must have at least one
unsubstituted B—ring attached to a conjugated polyene structure that is
intact from C-7 to C-15 (Fig. 2].
fiiek-v¥«~Map3Q
Q_CCIrCtenE»
B-Cryptoxcnthm
Echrnenone
,'3~Corotene
f_ccmtenE
Fig.
6
,6-Apo—‘|2‘—coro‘te-not
2. Typical retinol precursors.
81
CYAN EXHIBIT
EXHIBIT 1070
1070
CYAN
32
T. MATSUNO
The familiar reactions of carotenoid metabolism in animals are essentially
oxidative. Figure 3 shows the typical oxidative metabolic pathways that have
been demonstrated in goldfish Carassius auratus (refs. 3 & 4) and fancy red
carp Cyprinus cargio (ref. 5) and Figure 4 shows typical oxidative metabolic
pathways In crustaceans (refs. 6 - 10).
OH
OH
.-‘
H
Lutein A
‘J
H0 |'.3R.3'R) zeaxanrhm
\
‘
.-‘
HO
Lutan B
_.oOH
H0 .\ \
&
(3S.4R.3'R.5'RJ-;3..S-
.‘_ ‘ ’§JE2;0H
'I3S.3'R}-g
0
Adonixanlhin
:3S'4R'3'S‘6'R lrfi‘ '5'
“\OH
0”
Carotene‘3. 4. 3'-trmi
so * 0H
\ ‘A:Q;oH
-- 3
HO
"
()FritschieI}axanthln H0 0 a.—]joradexan{hjn
Fig. 3. Oxidative metabolic pathways of
carotenoids in goldfish and fancy red carp.
Carotene
d
O
1
CI) Echnrnone 0 HOW
O
I.’ .. _l
HO
., .
Lulein A
1
‘N H0 0 Fr-itschiel axanth
0
O Canthaxanlhm
¥ \'|JJ‘7Q
oxan 1!‘!
0
{3S.TS)-Asmxamhm
OH .
_ _ _ _Ȥ:[:T (35. 3'R}-Adonixanthin
.
th'
'5 1 OH
H0
{3R. 3'R }- Zeaxanlhin
t in
OH
6
H0 Carotene-3.4.3" trial
_
H
‘°
H0
H0
---- OH H0
H0 (3S.4R. 3‘R]—,6‘ ;- HO
Carotene-3.4.3'*triol
“‘
‘
l
m
V"?
0
Q' Papilioerythrinone
_ ._ _ _/mftxd
O
Adoniruhin
[3S.3'S'J-Astaxanthin
C.’
{‘
AjiQ/0H
Hojipfifh-‘_ ”
o{3RS.TRS)-Asmxamhm
Fig. 4. oxidative metabolic pathways of
carotenoids in crustaceans.
However, pathways of reductive metabolism have recently been discovered.
Figure 5 shows the reductive metabolic pathways of ketocarotenoids in rainbow
trout Salmo gairdneri (refs. ll — 12 ). These reductive metabolic reactions
involve the stepwise removal of the keto-groups at C-4 and C-4‘.
Figure 6 shows the reductive metabolic pathways of astaxanthin to tunaxanthin
in yellowtail Seriola guingueradiata (refs.13 & 14}, flying fish Prognichthys
agco and dolphin Coryghaena higgurus (ref. 15), respectively. These metabolic
pathways also involve the elimination of keto groups from C-4 and C-4' and
also the reduction of keto-groups at C-3 and C-3‘ to secondary alcohols.
Grangaud et al. (ref. 16) first demonstrated that astaxanthin is converted
into retinol in the liver of retinol—depleted freshwater fish in 1962.
Gross and Budowski (ref. 17) in 1966 found evidence of the conversion of
astaxanthin, canthaxanthin and isozeaxanthin, via B—carotene, into retinol
Xanthoph}/H's as precursors of retinoids
Q
HO 0 Astaxinthin
Q
0
H
;:r-uI:’°
Red‘-lctl-V9 meta-130115
pathways of carote-
OH
gr»
zjcmib
H0 Ofidonirubin
OCanthait&nthin
Adonixanthin
noi s in rain ow rou
H0
83
Asteroidenone
*
*
Z""““h‘“
Cryploxanthin
0
Echinenone
*
£3‘CaI'DtEI1e
,.0,&*“*°iJE2’°” $
Antheraxanthin
r_
i
_
Hob?--_"§:g
OH
H5 -3 3S. 4R5. 3’R . -,6. ,5-Carolena*3. 4. 3'-trio}
“O
.3R.3'R‘.-Zeaxanthin
i
HOIEIVN-&:‘Qg
0
'2 3R. 6'5 L‘-3-I-iydroxy-.8. 2-carolen-3'-one
‘
Fig.
6.
Reduotive metabolic pathways of
9
‘f2’0
Ob’\*~=~"""*-~',
carotenoids in yellowtail, flying
fish and dolphin.
.68. ES '5. e-carotene-3. 3'-dione
\
HO -:3s.es.e‘s.-—3-Hyd..m--
'.3R.6S.6‘ST'-3-Hydro!!!‘
£‘£_cam[en_3-_oM_,
,\
{
5.:-carolen-3 -one
\¢~’°-H
‘V
H0 /3%».
Tunaxanthan A
. \
/
H0 Aflx
, "’
Tunaxanlnm B
on
\
on
H0 ._.-\«¢-x
, "
Tunaxanthan C
and 3—dehydroretinol in freshwater fish, guppies and platies. Barua and
Goswami (ref. 18] reported in 1977 that lutein is the precursor of
3—dehydroretinol in some freshwater fish. Recently, Schiedt et al. showed
quite clearly that labelled retinol and 3-dehydroretinol are formed in
retinol-depleted rainbow trout from any of a number of labelled carotenoids,
particularly astaxanthin, zeaxanthin and canthaxanthin (refs. 11 5 19}.
B. W. Davies and B. H. Davies have reported the formation of retinol and
3—dehydroretinol from radio-labelled xanthophylls, i.e., canthaxanthin,
zeaxanthin and lutein, in goldfish (ref. 20). All these investigations have
been Carried out with freshwater fish.
our feeding trials were conducted to demonstrate the possibility of the
bioconversion of some xanthophylls such as astaxanthin, zeaxanthin, cantha—
xanthin, lutein and tunaxanthin into retinoids in retinoid—depleted
freshwater fishes, marine fish and mammals.
84
T.MAT5UNO
TILAPIA
Figure 7 shows the contents of retinol and 3-dehydroretinol in liver of
tilapia Tilapia nilotica after feeding trials with a retinoid-depleted diet
supplemented with xanthophylls such as astaxanthin, oanthaxanthin, zeaxanthin, lutein and tunaxanthin (ref. 21}. The retinoid content increased
in all these xanthophyll-fed groups, compared to that of the control group.
In the groups administered B—carotene and canthaxanthin, retinol
predominated over 3—dehydroretino1, whilst in the groups given xanthophylls,
such as astaxanthin, zeaxanthin, lutein and tunaxanthin, 3—dehydroretinol
predominated over retinol.
From these results, we can conclude that these xanthophylls, astaxanthin,
zeaxanthin, lutein and tunaxanthin, were probably directly bioconverted into
3-dehydroretinol without being first transformed into retinol (Fig. 8).
Recently, the bioconversion of astaxanthin into 3—dehydroretinol in mature
rainbow trout has been reported by Guillou et al. (ref. 22).
Mg/Ind.
253 *
E
.1
203‘
n
5
zoo -
Re-..noI
1
1
O
Afi
_
1 3-Defiydroret
no!
0. C=|'\I"\ax=‘.'-rm
m .
H: (j,
I
'i -
__
:
10:.‘ ?.
-uh
935
"_
79:."
5”
"C /Ej\/\\_._/ujg
'5!-{Zor:t:=r.s
I I
_,
CH
-'-\5tcI:Ita”1T.hIn
I
KJK
92?"
‘I’ ,
'
ZE€xCH“‘.i'1 n
I _
5”2°” _. CH20H
Q9’!-I’-O1
Cor~.+:'o| E-Ccro.Con1ho.Asto.
Zeo
Lutem
T.Jno
** Significant difference from control
r_p-10.01)
Fig.
7.
Contents of retinoids in liver
of tilapia after the feeding
experiments.
3-D-9n\,-drorehnol
xfx./CH
H0
Zi:‘*~‘r><
'-u‘.€-’
Fig.
'35-‘
H3
Tu~cxon1mr~
8. Xanthophylls as retinoid
precursors in tilapia.
BLACKBASS
Figure 9 shows the results of the feeding experiments with black bass
Mioropterus salmoides (ref. 23}. In all the xanthophyll-fed groups, i.e.
with oanthaxanthin, astaxanthin, zeaxanthin and lutein, the retinoid concentration markedly increased, compared with that of the control group, and the
retinoid fractions comprised a large amount of 3—dehydroretino1 and only a
trace of retinol except for the canthaxanthin group.
From these results, it is assumed that these xanthophylls were directly bioconverted into 3—dehydroretinol, whereas canthaxanthin was reductively bioconverted into retinol and the retinol thus formed was oxidised to 3-dehydroretinol.
with respect to its liver retinoids, the black bass is a typical 3-dehydroretinol-type freshwater fish.
AYU
Figure 10 shows the results of feeding experiments with ayu Plecoglossus
altivelis (ref. 24}. In all test groups that were fed the xanthophylls and
those that were fed B—carotene and B—cryptoxanthin which are known as retinol
precursors, the concentration of retinoids increased compared with that
of the control group.
with respect to its liver retinoids, the ayu is a retinol—type freshwater
fish.
Xsnrhophyfls as precursors of retinoids
85
gig/Ind.
30 _
- 3—Deh~_.rdrore+.inol
25.2”
E
D Rennm
20 P 1
+—Ret?nol
E
K!)
_ 3-Dehydroretanol
20"
*-—3—Dehydrore‘.ino!
I
H
[II
Control
C:'\IhO.
Asto.
Zea.
Luiein
it significant di;ferenCe from control
r1_=<oTo1)
Fig.
9.
Contents of retinoids in
liver of black bass after
the feeding experiments.
Control fi'Cc1ro_ Ccmthc. 5-Cryp, Astc.
Zed.
Lutem
** significant difference from control
‘P‘°'°l’
Fig.
10.
Contents of retinoids in
liver of ayu after the
feeding experiments.
YELLOWTAIL
We have studied the bioconversion of administered astaxanthin in the eggs of
a marine fish, namely yellowtail Seriola guingueradiata during embryonic
development (ref. 25). Figure ll shows the composition of the feed used in
this experiment and procedures for sampling eggs at different stages
during embryonic development. The yellowtail was reared first on fresh
mackerel, followed by moist pellets, and finally with moistpellets containing
antarctic krill oil, and were then injected with gonadotropic hormone to
promote ovulation. Eggs were removed after two days; both imature eggs
(Sample No. 1} and ovulated eggs (Sample No. 2) were collected. After fertilir
zation, fertilized eggs were collected {sample No. 3), and then further eggs
collected one day after fertilization (Sample No. 4) and two days after
fertilization [the eyed stage) (Sample No. 5). Finally larval fish were
collected (Sample No. 6). The six samples (Sample No. l - 6) were taken from
the same one individual female yellowtail.
Figure 12 shows the dynamic aspects of the bioconversicn of administered
astaxanthin in yellowtail eggs during embryonic development. Adonixanthin,
B-carotene-3,4,3‘-triol and zeaxanthin found in immature eggs of yellowtail
are considered to be reductive metabolic products of astaxanthin derived from
the administered antarctic krill oil. Both the carotenoid and retincid
contents of the eggs markedly increased with time and finally reached a
maximum from the stage of immature eggs (Sample No. 1) to the stage of
ovulated eggs {Sample No. 2). Both carotenoid and retincid contents
decreased progressively during the embryonic development. It was observed
that the content of 3-dehydroretinal (4) which is derived from zeaxanthin,
decreased, whilst the retinal (2) content increased from Sample No. 2 to
No. 5. From the experimental results we have proposed a possible reductive
pathway for the metabolism of 3—dehydroretina1 (4) to retinal {2} (Fig. 13),
because no transport of material occurs through the egg membrane in such a
closed system as fish eggs during embryonic development. From the results,
which show a dramatic decrease in both carotenoid and retincid contents
from ovulated eggs (sample No. 2) to fertilized eggs (Sample No. 3), it may
be concluded that both carotenoids and retinoids play an important role
during fertilization.
86
T.MATSUNO
sf ——Recree with living mackerel for 3? Guys
moist pellet for 116 days
, moist pellet Nlth antarctic krill oil {Eng/fish} for 1a days
' "Injection of Qonodotrenlc hormone
'
'__
/='=*r.-.—
Fertilization
11.
{1,7;,,ng;
Mo. 2 {Jvuloted eggs
{95,3g}
No. 3 Fertiltzed eggs
£33.19}
-3I
No. it
oI
No. 5 Eggs
two days after fertllizotmn (23.09)
I yea stage:
5 ‘
Fig.
sample
No. l [mature eggs
Eggs one only after fertlllzatlen
(58.09)
No. 5 Larval nsn
(59.09:
Feed of yellowtail in the feeding experiment and procedures for
sampling of eggs at different stages during embryonic development.
mfiflfls eggs
3.5 I‘
3.0
3—Dehyuroretln:l
-'
Carotencids F-‘I '3
Otnerturutenoius
Tuncxontnin 2.5
Luteln
Adenixnnthln
Astuxontnln
Sfimfllfl
NO. 1
Fig.
SUHDIE
N0. 2
Sflflflle
NO. 3
sanole
N0.H
501919
N0. 5
SGMDIE
ND. 6
12. Dynamic aspects of the bioconversion of administered astaxanthin
in yellowtail eggs during embryonic development.
CH0
CH0
j
3-Dehydroretinol
Fig.
Retinal
13. Reduction of 3—dehydroretinal to retinal in yellowtail during
embryonic development.
RAT
Figure 14 shows the retinol content in the liver of weanling male rats after
feeding trials (ref. 26}. There is a significant difference between most
xanthophyll-fed groups and the control group, except that there is little
significant difference between the tunaxanthin and lutein groups and the
control group. A large amount of unchanged 3-dehydroretinol and a comparatively small amount of retinol were found in the liver of the rats that had
been administered 3-dehydroretinol. This indicated that some of the orally
administered 3-dehydroretinol was reductively metabolized to retinol.
Xanrhophyfls as precursors of rat.-'no."ds
8?
‘I2?
IBOL
CHZOH
3—Dehydroretino1
3-De'r~ydrore*;mo!—.
I
CHZOH
r'-ie‘t:no|—Retinal
Fig.
Control &ro. Ocm.
Ast.
Zea.
15. Reduction of 3—dehydroretino1
in rat liver.
Tuna Lut. Dehydro
reunol
* Values with different superscripts are
significantly different (p -(0.05) .
Fig. 14. Contents of retinoids in liver
of rat after the feeding experiments.
0
H
Astemnthin
O3lJ,.:Q°“
I1
0
Adonixcmthin
1
HO
0.
jjf\—-/%j£2/
H0
B—Coro'rene-3,4,3‘-triol
.,i3e'«<QO”
Tunexclnthin
zeqxanfhin
I
E:fi:eJ§«ee4e/CH0
3—3ehydroreti no.1
3—Dem'droretinoI
/‘L/
Retinoi
Fig.
16. Xanthophylls as retinoid precursors.
C H0
88
T.MATSUNO
To our knowledge, this is the first finding of the reductive bioconversion
of 3—dehydroretino1 into retinol (Fig. 15).
A large amount of retinol was found in the group fed 8-carotene, compared
with that in any xanthophyll—fed group. Each of the xanthophylls was less
efficiently bioconverted into retinol than was B—carotene. After feeding
rats with canthaxanthin, we have observed the presence of echinenone in the
rat liver extract, and have concluded that canthaxanthin was reductively
metabolized via echinenone to B—carotene and then finally to retinol.
In the case of the astaxanthin-fed group, a reasonable explanation for the
formation of retinol is that astaxanthin is bioconverted reductively into
zeaxanthin, and the zeaxanthin thus formed is metabolized to retinol via
3-dehydroretinol by a reductive metabolic reaction. A similar explanation
can be proposed for the formation of retinol in the groups fed tunaxanthin
and lutein.
In conclusion, xanthophylls such as canthaxanthin, astaxanthin, zeaxanthin,
lutein and tunaxanthin, which are widely distributed in Nature have been
found to be precursors of retinoids not only in freshwater fish and marine
fish (yellowtail) but also in a mammal (rat) as shown in Fig. 16.
Acknowledgements
I am most grateful to the coworkers responsible for
most of the experimental work outlined in this article:- Dr. M. Katsuyama,
Miss M. Tsushima and Mr. E. Yamashita, of the Kyoto Pharmaceutical University
and Dr. S. Arai, of the National Research Institute of Aquaculture, for the
feeding experiments with tilapia, yellowtail, ayu, black bass and rat,
and Dr. T. Maoka, of the Kyoto Pharmaceutical University, for numerous
HPLC analyses of carotenoids and retinoids.
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