Number 237
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`CYAN EXHIBIT 1004
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`TH ES ES
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`PRESENTED at the
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`UNIVERSITY OF LYON
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`FACUULTY OF SCIENCES
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`TO OBTAIN
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`DOCTORATE OF NATURAL SCIENCES
`
`BY
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`Renée MASSONET
`
`Assistant at the Algiers’ School of Medicine
`
`FIRST THESIS:
`
`RESEARCH ON ASTAXANTH|N’S BIOCHEMISTRY
`
`SECOND THESIS:
`
`PROPOSALS GIVEN BY THE SCHOOL
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`Presented orally on April 22, 1958 before the Examination Jury
`
`....President
`Mr. CORDIER...
`MENTZER ............ ..Examiner
`CHOPIN ............. ..Examiner
`DESSAUX ............. ..Examiner
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`LYON
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`1958
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`RESEARCH ON ASTAXANTH|N’S BIOCHEMISTRY
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`

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`INTRODUCTION
`
`biological science can often find, by the consideration of the majority, the rigorous nature that it
`seems at first to lose as a result of individual fluctuation
`
`André DOGNON
`
`The studies on vitamin A deficiency have for a long time determined that among the signs of
`
`deficiency, the most constant and most apparent expressions were the arrested weight growth and
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`the damage in the eye and its related organs; the administration of vitamin A or a substance having
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`vitamin A activity results in the deficient animal in the recovery of weight followed by the receding
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`and curing of the eye injuries. This double nature should make vitamin A be referred to as anti-
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`xerophthalmic, growth-promoting vitamin.
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`When the structure of vitamin A (or vitamin A or retinol) was elucidated in the light of Karrer’s work,
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`it appeared that the vitamin activity was very closely related to the chemical composition, and it is a
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`typical notion today that all factors which may prevent or cure the deficiency are chemically related
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`to vitamin A. We can even add that they only have vitamin activity to the extent that the body is
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`capable of converting them into Vitamin A: all substances having in common with retinol the
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`property to restore growth and heal xerophthalmia in the vitamin A deficient animal, have in fact in
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`their molecular structure the axerophthyl group:
`
`CH3
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`CH3
`
`fH3
`§"3
`— cu = CH — c = cu — cu = cu — c = cH_- CH =
`
`‘
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`that is, a potential vitamin A molecule (*).
`
`Revealing in the Risso Aristaeomorpha foliacea hepatopancreas oils a substance that - at least at
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`certain doses - shows in the vitamin A-deficient white rat a selectively anti-xerophthalmic action, as
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`well as the demonstration that the substance involved is astaxanthin (83), an oxygenated-nucleus
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`carotenoid considered as lacking vitamin activity, should for sure give rise to significant biochemical
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`problems.
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`(*) Vitamin A of fresh water fish has, however, a double additional bond at 3-4.
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`8
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`Their study has been the objective of research work carried out for over ten years in the biological
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`chemistry laboratory ofthe Algiers’s School of Medicine and Pharmacy. I had the pleasure ofjoining
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`that research from the beginning and this work integrates into them as a whole. As a matter of fact,
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`there is no doubt it was ofgreat importance to gather in a paperthe information, that until now was
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`quite fragmented, on the materials examined, the biological test methods, the results that establish
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`the dissociation between the anti-xerophthalmic activity and the action on growth.
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`On the other hand, although it
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`is very true that the action on weight growth is nil
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`in the deficient
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`white rat for daily doses from 5 to 10 pg astaxanthin, this is no longer the same when the amount
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`administered is three or four times as big. Then, a normal growth is obtained without the least
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`addition to the vitamin or pro-vitamin A diet. Therefore,
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`it
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`is possible to study a true vitamin A
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`deficiency in the adult animal and to explore in particularthe troubles ofthe reproduction functions.
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`It was also interesting to specify the localization of the pigment in the body of the treated rat, to
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`conduct the anatomical and pathological study of the animals in the experiment and to investigate,
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`particularly in the liver, whetherthe administration of astaxanthin causes the build-up and storage of
`vitamin A. Our results show that this is not at all the reason that leads to attribute to astaxanthin a
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`specific activity.
`
`In addition, astaxanthin, the main shellfish pigment, is plentiful in fish food. In the second part of our
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`work we have studied the effects of pigment administration to a small fresh water Cyprinodontidae,
`Gambusia holbrooki Grd, We could see the neo-formation of vitamin A from astaxanthin, that
`
`appears then as a pro-vitamin A for Gambusia holbrooki.
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`PART ONE
`
`CHAPTER I
`
`MATERIALS AND METHODS
`
`10
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`

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`FIG. 2 - Aristeus antennatus - RISSO
`
`a) Carapace ofthe female
`b) Rostrum ofthe male
`0) Maxilla I
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`11
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`Fig. 1 - Aristaeomorpha foliacea — RISSO
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`a) Cephalothorax of the female
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`b) Rostrum of the male
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`0) Maxilla I
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`12
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`MATERIAL UNDER STUDY
`
`The shellfish species that provided the pigments examined belong to the Decapoda order. They
`are:
`
`- three Penaeidae:
`
`Aristaeomorpha foliacea, Risso
`
`Aristeus antennatus, Risso
`
`Parapenaus Iongirostris, H. Lucas;
`
`- one Pandalidae:
`
`Plesionika edwardsii, Brandt;
`
`- one Scyllaride:
`
`Scyllarus Iatus, Latr.
`
`As a comparison, we made some determinations on Squilla Mantis, Latr, that is a stomatopod, and
`we thought we should report the literature data relating to the vitamin A content of various
`Euphausiacea species.
`
`A - GEOGRAPHIC DISTRIBUTION
`
`ECOLOGICAL DATA (1)
`
`- Aristaeomorpha foliacea, Risso.
`
`Apart from the Algerian coasts, this shrimp is found in some places of the Mediterranean Sea
`(Sardinia, Sicily) and in the Atlantic Ocean it is found along the Moroccan coasts. It seems not to
`migrate beyond 400 meters. The trawlers that bring large amounts of Aristeus antennatus from
`bottoms of 300, 350 and 400 meters, fish very small amounts of Aristaeomorpha foliacea.
`
`- Aristeus antennatus, Risso
`
`This animal seems quite localized in the Mediterranean Sea, the temperate and subtropical regions
`of the Atlantic Ocean from Portugal to Cape Verde islands. This is a meso-abyssal species. The
`trawlers fishing in the Algerian coasts catch plenty of this fish between 300 and 400 meters and
`only at these depths.
`
`- Parapenaus Iongirostris, H. Lucas
`
`Very common in the Mediterranean Sea, this species is captured off Algiers and Oran, between 200
`and 300 meters. It is plentiful in Morocco between 200 and 400 meters (Gruvel) (96).
`It can also be
`found from 70 to 80 meters.
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`(1) These short reminders were essentially borrowed from Argilas (2) and Dieuzeide (43).
`
`13
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`Fig. 3 - Parapenaeus Iongirostris - H. LUCAS
`
`a) Carapace ofthe female
`b) Rostrum of the male
`0) Maxilla I
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`34567009
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`14
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`

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`- Plesionika edwardsii, Brandt;
`
`This species is caught with Aristeus antennatus and Aristaeomorpha foliacea in bottoms between
`200 and 500 meters.
`
`- Scyllarus Iatus, Latr.
`
`This species found in the Mediterranean Sea is also found in the Atlantic Ocean; it lives in the
`vicinity of bottoms between 4 and 10 meters.
`
`- Squillidae (Squilla mantis, Latr.) lives on sandy but mainly silty bottoms in the vicinity of the littoral
`and hardly descends beyond 100 meters. It is a carnivore that lives a fossorial life but hunts other
`shellfish during the night.
`
`B - MORPHOLOGY
`
`The first comprehensive work on the morphology of Penaeidae was conducted by Bouvier (10).
`Argilas (2), directed by Boutan, specially studied the species of the Algerian coasts. Dieuzeide (43)
`wrote a monograph on these same species. The data in connection with Pandalidae were borrowed
`from his research on the fauna of bottoms suitable fortrawling in the Castiglione bay (44).
`
`- Aristaeomorpha foliacea is a large-sized shrimp that reaches 20 centimeters in length. It is easy to
`recognize through the features ofthe rostrum that has 9 dorsal teeth in the female and 6 in the male
`(Figure 1). Aristaeomorpha foliacea is dark red with purplish reflections. Unequal pigmentation is
`stronger in the branchial region and the marginal region of the lower portion of the carapace. The
`red pigment is localized in the dermis chromatophores the pseudopods of which extend to the outer
`layer of the carapace or epicuticle. Under the carapace there is an epidermal layer topping the
`dermis (*); a conjunctival membrane separates the dermis from the central cavity. The stomach
`pouch (formed by the union of a cardiac pouch and a pyloric pouch) and the intestine are totally
`enveloped by a strongly pigmented conjunctival sheath. This red pigment
`is also found in the
`branchiae and appendices (antennules, antennae, maxillipeds, maxillas).
`
`the
`If the red connective tissue that envelops the stomach pouch is removed by scratching,
`stomach pouch can be seen as a blue membrane, verging on mauve. The genital glands that are
`initially whitish gradually become colored following the development of the eggs; grayish in the first
`stage, the eggs change to violet upon maturation.
`
`- Aristeus antennatus, Risso: it is generally smaller than Aristaeomorpha in size; however, some
`specimens that reach 20 centimeters can be found. The rostrum has three teeth on the dorsal edge
`in both sexes; this clearly differentiates Aristeus antennatus from Aristaeomorpha foliacea (Figure
`2).
`
`
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`(*) The whole is improperly referred to as hypodermis.
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`15
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`28
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`Aristeus antennatus is washed red in color, with purplish stains in the cephalothorax carapace and
`the abdomen.
`In the Aristeus antennatus the eggs pass from cyclamen pink to clear mauve upon
`maturation.
`
`- Parapenaeus Iongirostris, H. Lucas: this species is clearly smallerthan the two preceding species.
`Its size is 8 to 10 centimeters in average. Its rostrum has 8 teeth and shows slightly marked sexual
`differences (Figure 3).
`It is a pale pink shrimp. Like in the two other Penaeidae, a red-pigmented
`conjunctival sheath is found that envelops the gastric pouch. The genital glands and their content
`are blue-greenish in colorthe intensity of which increases with the development ofthe eggs.
`
`- Plesionika edwardsii, Brandt (former Pandalus narval H. Milne-Edwards): This is a small-sized,
`pale-pink colored Pandalidae; it
`is recognized by the length of its rostrum and the teeth thereof
`(Figure 4).
`
`
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`FIG. 4 - PLESIONIKA EDWARDSII, Brandt
`
`Unlike the Penaeidae, Pandalidae carry their eggs; they are in the form of a quite characteristic
`brilliant blue granular mass. The females are ovigerous from January to May.
`
`Scyllarus latus, Latr. is a Decapoda species that reaches 30 to 40 centimeters in length.
`-
`is brown red in color. The female carries its eggs.
`-
`Squilla mantis, Latr. is a Stomatopod sized from 12 to 15 centimeters. The abdomen is very
`large in proportion to the cephalothorax; the carapace, relatively short, covers only the first four
`thoracic segments. The rostrum is comprised of a small articulated plate. The thoracic appendices
`are quite particular; the second pair of pereiopods is represented by a large-sized biting claw. The
`orangey red pigmentation is very unequally distributed: certain items are strongly pigmented while
`other items are colorless. The thoracic segments are also more intensely colored than the
`abdominal segments. The pigment distribution at the gastric pouch is very different from the
`Penaeidae: the red conjunctival mass is only present at the pouch base. The eggs, plentiful, are
`retained by the female in the first abdominal appendices.
`
`It
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`16
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`All these species have plenty of pigments. The study below will show the close relationship
`between those pigments.
`
`C - BIOLOGICAL STUDY
`
`The main carotenoid pigment of shellfish was successively described under the names of
`crustaceo-rubin (117), zooerythrin (165), vitellorubin (150),
`tetronerythrin (148), haematochrome
`(104) and finally astacene (or astacin) (131). However, Kuhn and Sorensen (132) show that most of
`the extraction processes cause an alteration and that astacene is actually a product of the oxidation
`ofthe natural pigment that has been named astaxanthin.
`
`The chemical composition of astaxanthin determined by Karrer’s works (120) shows that it is the
`3,3’-dihydroxy-4,4’-diketo-[3-carotene. Tischer (225) was the first to establish the spectral properties
`(broadband spectrum with a single maximum located at X = 492 nm in pyridine) (*).
`
`is found in a considerable number of animal and
`Astaxanthin, which is wide spread in nature,
`vegetable species (133), (121), (161), (67). Among shellfish, the pigment exists both in a free,
`esterified form and as differently colored chromoproteins such as the egg ovoverdin (217) and the
`crustacyanin of the lobster carapace (237).
`In all the species we have studied, we have identified
`astaxanthin and evidenced the presence of the three forms. Before reporting their physical and
`chemical properties (cf. p. 27),
`it
`is appropriate to discuss another important matter: the possible
`presence of carotenes and vitamin A in shellfish.
`
`1. VITAMIN A AND CAROTENE IN SHELLFISH
`
`a) VITAMIN A
`
`History
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`The issue ofthe possible presence of vitamin A in shellfish deserves in fact to be examined with the
`greatest care, because it can allow addressing the general problem of the origin of this vitamin
`factor in the animal kingdom.
`
`Lederer (136), Euler, Hellstrom and Klussmann (53) did not find any vitamin A in Copepods; Kon
`and Thompson (127) (128), Fisher, Kon and Thompson (59) obtained the same negative result on
`three Copepod species, as well as on two Amphipod species and one Cladocera species.
`
`Wald (234) was the first to show the presence of vitamin A in the lobster eyes. Fisher, Kon and
`Thompson (60) found vitamin A in fourteen Decapoda species.
`
`(*) The three—maximum spectrum (476, 493 and 513 mu in pyridine) described by Kuhn, Sténe and Sorensen
`(133) is, according to Wald (235), certainly marred by mistakes.
`
`17
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`

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`The Euphausiacea, an order close to Decapoda, was thoroughly studied by Kon (124), Fischer et
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`al. (58) (60).
`
`In fact, the Euphausiacea are plentiful
`
`in Krill that is eaten by whales. Both in the
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`fished species and those taken from the stomach, the vitamin A contents proved considerably
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`higher than in all the other shellfish examined. The Table below (Table I) reproduced according to
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`Kon, emphasizes the fact that, from the standpoint of their vitamin A content, the Euphausiacea are
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`in a quite dominating position.
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`Insofar as the localization is determined precisely, almost all the vitamin is found in the eye
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`(Fischer Kon and Thompson (58) (60), Batham, Fischer, Henry, Kon and Thompson (5),
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`Fischer, Kon, Plack and Thompson (61)). Tables II, III, IV are illustrative in this regard.
`
`TABLE I
`
`(According to Kon (126))
`
`Vitamin A content of marine shellfish
`
`Species or groups
`
`"9
`per g
`
`Locations
`
`Common
`name
`
`Euphausiacea
`
`Amphipod
`Euphausiacea
`Amphipod
`Shrimps
`Hermit crabs
`
`Shrimps
`lsopods
`Copepods
`Crabs
`Lobsters
`
`Mysidacea
`Branchiopods
`
`A.M.
`
`A A A A P P P P A
`
`)>§fi>§§
`
`78
`
`51.3
`16.5
`14.1
`3.9
`3.9
`3.3
`3.3
`
`2.8
`2.8
`2 0
`
`0 3
`
`P: Pacific Ocean
`
`M: Mediterranean Ocean
`
`18
`
`Meganyctiphanes norvegica
`Thysanoessa raschii
`Thysanoessa inermis
`Thysanoessa gregaria
`Stylocheiron elongatum
`Stylocheiron maximum
`Euphausia pacifica
`Nematoscelis difficilis
`
`Thysanoessa spinifera
`Onisimus plautus
`Euphausia superba
`Syrrhoe crenulata
`Caridea (17 species)
`Anomura (7 species)
`Penaeidae
`
`lsopoda (3 species)
`Copepod (3 species)
`Brachyuran (9 species)
`Astacura (2 species)
`Mysidacea (5 species)
`Branchiopoda
`
`An: Antarctica
`
`A: Atlantic Ocean
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`TABLE II
`
`(According to BATHAM et al. (5))
`
`Vitamin A distribution in Euphausiacea and Decapoda organs
`
`Average weight
`of a specimen
`
`Vitamin A contained
`in a specimen
`
`S
`
`'
`
`Wh I
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`\Alh I
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`Eupagurus benhardus
`
`Meganyctiphanes norvegica
`Meganyctiphanes norvegica
`Meganyctiphanes norvegica
`Meganyctiphanes norvegica
`Thysanoessa raschii
`Carcinus maenas
`
`TABLE III
`
`(According to KON (126))
`
`Vitamin A distribution in Euphausiacea and Decapoda organs
`
`Species and organs
`
`per organ
`pg
`
`er
`grgaa
`Hg
`
`Total amount of
`_vitamin A
`In the eyes
`
`Thysanoessa raschii
`Body less the eyes
`Eyes
`
`Meganyctiphanes norvegica
`Body less the eyes
`Eyes
`
`Crangon allmani
`Body less the eyes
`Eyes
`
`Eupagurus bernhardus
`Body less the eyes
`Eyes
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`Nephrops norvegicus
`Body less the eyes
`Eyes
`
`Cancer pagurus
`Body less the eyes
`Eyes
`
`Maja squinado
`Body less the eyes
`Eyes
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`19
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`TABLE IV
`
`(According to KON (126))
`
`Vitamin A distribution in Meganyctiphanes norvegica (Euphausiacea) organs
`
`Lipids
`per 100
`
`Average
`weight
`in mg
`
`22
`
`Vitamin A
`
`Vitamin A
`
`in g.
`per organ
`
`in pg
`Per 9
`
`Organs
`
`Hepatopancreas
`Stomach
`
`Eyes (pair)
`Rest of body
`
`Total
`
`The examination of these results shows that, apart from the Euphausiacea characterized by
`exceptional contents, vitamin A exist in the other crustaceans only as traces (0.15 pg per gram in
`Penaeidae, cf. Table I) or is completely absent. When it is present, it is substantially concentrated in
`the eye; vitamin A ofthe body is almost totally contained in this organ.
`
`Experimental section
`
`Materials and methods
`
`Vitamin A assessment and determination are conducted using the Carr-Price reaction (19) with
`antimony trichloride in a chloroform solution. The absorption is measured using the Beckman
`spectrophotometer by monitoring decolorization as a function of time using the Meunier and Raoul
`(167) method (*). For series measurements, a photo colorimeter with photocell was also used;
`experimental controls allowed verifying that the sensitiveness and fidelity of the device allowed
`determinations of sufficient accuracy.
`
`The method was designed for assessing vitamin A in the Aristaeomorpha foliacea hepatopancreas.
`The operating method that will be described is valid for all the other organs and tissues. If variants
`should be introduced, they will be quoted with regard to every particular case. However, it is worthy
`of note now that each extract was controlled using UV spectrophotometry.
`
`(*) The measurement was made at X = 620 nm ( E —————————————— = 5070)
`1 cm
`
`1 %
`
`20
`
`

`
`
`
`LDOO\lO\U'|-bu)l\JI4
`
`29
`
`30
`
`ARISTAEOMORPHA FOLIACEA
`
`Hepatopancreas
`
`Saponification: 10 g hepatopancreas were saponified using a 60 per 100 (1 mL per gram tissue)
`aqueous solution of potash according to the Lewis and Bodansky (140) method. Saponification
`continued in a water bath for 20 minutes under a nitrogen atmosphere. The content ofthe flask was
`fast cooled and taken up with an alcohol-water (1
`: 2) mixture and extracted three times with
`petroleum ether. The -petroleum ether solutions gathered, washed with distilled water, then with
`aqueous potash at 3 per 100 and again with distilled water (50, 50 and 50 mL distilled water), were
`dehydrated by contact with anhydrous sodium sulfate. The -petroleum ether solution was finally
`evaporated under partial vacuum under nitrogen atmosphere. The residue was taken up by 1 mL of
`rectified chloroform and kept on calcium chloride.
`
`Carr-Price reaction: the chloroform solution is added 0.5 mL acetic anhydride, then 5 mL Carr and
`Price reagent. A color is developed, the absorption of which is measured at the 15th second, then
`every 30 seconds until 2 minutes. The kinetic curve is built and graphic extrapolation to zero time is
`performed.
`
`Results: it can be seen right away that neither the color nor the kinetics are typical of vitamin A
`(168). As a matter of fact, a greenish color is obtained that turns pure green increasing its intensity,
`and the behavior ofthe curve alone shows that it cannot be vitamin A (Fig. 5 - curve I).
`
`Experimental control (77). It consists in making the determination on the same oil to which
`known amounts of vitamin A were added.
`
`30 pg vitamin A is added to 10 g hepatopancreas and the mixture is saponified in the
`above-described conditions. A clearly blue color is initially developed by the Carr-Price
`reaction, the downward kinetic curve is characteristic. The interpretation of this curve leads
`to the following results: at time 120 sec. the curve is no longer downward and shows a
`plateau (Fig. 5. - curve ll).
`
`Photo calorimeter
`di'w':3i'r3n3
`
`50
`
`#6‘
`
`50
`
`26'
`
`I9
`
`
`
`Time in seconds
`
`
`#5
`
`50 45 60 Id:
`
`9.2: mi
`
`21
`
`

`
`Ifthe plateau height is deducted (corresponding to base absorption), the absorption value, so
`corrected, corresponds only to vitamin A; thus, the value found is that of the amount introduced,
`apart from any experimental errors. This control confirms the results recorded above; vitamin A is
`absent from hepatopancreas.
`
`Hypodermis
`
`Vitamin A was searched in a 10 g test sample according to the above-described method.
`
`No trace of vitamin A was detected.
`
`Pyloric pouch
`
`The peristomal connective tissue enveloping the pouch was removed by scraping and emptied of
`any content.
`
`The pouch and the content were treated separately. Work is conducted on 5 g.
`negative for the two samples.
`
`the result
`
`is
`
`Peristomal connective tissue
`
`10 g of connective tissue were treated. A negative result was obtained.
`
`Flesh
`
`20 g were taken. The search was conducted on a sample collected from the cephalothorax and a
`sample from the abdomen. The results were negative.
`
`Carapace
`
`The search was conducted on 30 g. carapace finely ground. A negative result was obtained.
`
`Intestine
`
`A 5 g test sample was used, the Carr - Price reaction was positive, the vitamin A content is 6.0 to
`6.6 pg.
`
`However, an important remark must be made: the intestine content could not be emptied before
`saponification. Therefore it was not possible to determine whether the vitamin found belonged to
`the intestinal tissue or came from the content.
`
`Eggs
`
`5 g of eggs were treated: the Carr - Price reaction was weakly positive, 1.2 to 1.5 pg per gram of
`fissue.
`
`Eyes
`
`The above-described method was also used. For verification purposes, we removed the pigment by
`chromatography and then dosing was made in the usual conditions.
`
`A 2g test sample was used: the Carr - Price reaction was clearly positive; the concentration of
`vitamin A is higher in the species fished during summer; for winter, the concentrations range from
`1.8 to 2.5 pg per gram while for summerthey range from 6 to 9.5 pg per gram (cf. Table V).
`
`0O\lO\U'|-l>UUl\Jl4
`
`9
`10
`
`11
`12
`
`13
`
`14
`
`15
`
`16
`17
`
`18
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`19
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`20
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`21
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`23
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`25
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`26
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`29
`
`30
`31
`
`32
`33
`34
`
`35
`
`36
`
`22
`
`

`
`ARISTEUS ANTENNATUS
`
`Vitamin A examined by the same method could be detected neither in the hepatopancreas nor in
`the hypodermis, the pyloric pouch (pouch and content treated separately), flesh and carapaces.
`
`It was found in the intestine (not emptied) at 6 to 7.5 pg per gram: in the eggs, 1.2 to 1.3 pg, in the
`eyes, 2.4 to 4.8 pg (winter Aristeus), 6 to 9.6 pg per gram (summer Aristeus).
`
`PARAPENAEUS LONGIROSTRIS
`
`The results are negative for the hypodermis, the pyloric pouch, flesh, carapaces and eggs. Traces
`are present in the hepatopancreas; the values found in the intestine are 3 pg per gram (winter
`fishing), 4.5 pg (summer fishing); in the eyes, 7.5 pg per gram (winter fishing), 11.5 pg per gram
`(summer fishing)
`
`PLESIONIKA EDWARDSII
`
`The Carr — Price reaction in Plesionika edwardsii was positive only for egg extracts: however, only
`traces were found.
`
`SCYLLARUS LATUS
`
`Vitamin A was only searched in the eggs; 0.18 pg per gram were found.
`
`All results of these determinations are shown in Table VI.
`
`The examination of this Table shows that the vitamin is present only in the intestines, the eggs and
`the eyes. The values found in the eggs are very low.
`In the intestines,
`it was not possible to
`measure separately the vitamin in the content and in the organ, due to the small diameter of the
`latter. Therefore, one cannot presume whether the vitamin is present in the mucosa or in the
`digestive waste. However, it is worthy of note that it is absent from the content of the pyloric pouch;
`accordingly, one can think that it will not be present either in the intestine content. Nevertheless, a
`decisive conclusion could not be made from this argument alone.
`
`It is interesting to report the results per gram of eye lipids. The content of lipids (extracted with the
`alcohol-ether mixture) is 1.2 to 1.5%, resulting in concentrations of 150 to 750 pg per gram of lipid
`in the eye tissue.
`
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`b) CAROTENES
`
`History
`
`Euler, Hellstrom and Klussman (54) show that among three Calanus finmarchicus pigments
`isolated, one is
`from carotene. Lederer
`(136)
`found that
`this pigment
`is actually present
`simultaneously with astacine (astaxanthin). He found 0.4 mg per 120 mg of astacine in 500 grams
`ofCopepods.
`
`Vlfith regard to Decapoda, carotene traces were reported by Kuhn and Lederer (131), by Kuhn,
`Lederer and Deutsch (130)
`(Maja squinado) by Goodwin and Srisukh (127),
`(128) (Pandalus
`bonnieri, Crangon vulgaris).
`
`For Euphausiacea, Wagner (232) had thought he could assert the presence of high amounts of
`carotene. This issue was important because the Euphausiacea constitute most of the Krill eaten by
`the whales, and it is known that large vitamin A reserves are found in whales. The Wagner’s results
`could then lead to accept that carotene should be considered as the main source of supply of
`vitamin A for whales. However,
`the conclusions made by Wagner are disputable due to the
`identification procedures used. Kon (125) took up this study and, using chromatographic analysis
`and spectrophotometry, stated the almost total absence of carotenes in the Euphausiacea from the
`Arctic and Antarctic oceans, both in the material collected from whale stomach and in directly fished
`animals.
`
`Experimental section
`
`Methods
`
`Identification of carotenes is essentially based on their behavior vis-a-vis the adsorbents and on
`their spectral features. The extraction method that leads to the dissolution of the pigments in
`petroleum ether is described above (cf. page 33).
`
`The direct spectrophotometric examination of the -petroleum ether solution of organs or tissues
`does not reveal the 3-maximum system typical of carotenes (78). However, one can dread that the
`abundance of astaxanthin that absorbs over a neighboring range of wavelength avoids detecting
`the pigment if it
`is present only in small amounts. This is the reason why a chromatographic
`separation is essential. In fact, as shown by Lederer (136), carotenes are less strongly retained on
`an alumina column that the oxygenated carotenoid pigments of the astacine (or astaxanthin) type.
`
`a -petroleum ether solution containing both
`As a matter of fact, we have verified that when
`astaxanthin (free or esterified) and carotenes is chromatographed on alumina, the carotenes slowly
`descend the column to finally appear in the filtrate, while astaxanthin is adsorbed in the upper
`portion ofthe chromatogram.
`
`Not the least trace of carotenes was found in the extracts examined (Aristaeomorpha foliacea,
`Aristeus antennatus).
`
`25
`
`

`
`2 - ASTAXANTHIN IN SHELLFISH
`
`As we have just shown, in the species studied, vitamin A is essentially localized in the eyes, while
`the other organs or tissues do not contain it, apart from mature ovaries where traces have been
`detected. Vlfith regard to carotene, it is totally absent. On the contrary, astaxanthin is found in large
`amounts, mainly in the Aristaeomorpha foliacea that is the most strongly pigmented species we
`have examined. It is also plentiful in Aristeus antennatus and, in these two Penaeidae, the pigment
`is mainly localized in the hypodermis and the peristomal connective tissue.
`
`a) FREE, ESTERIFIED ASTAXANTHIN
`
`Little information is available on the coexistence of the equilibrium relationships which could exist
`among the various forms in the different tissues or organs. In fact, for small shellfish, the extraction
`is made on the animal as a whole (Calanus finmarchicus, Lederer (136), Holopedium gibberum,
`Daphnia magna, Gammarus pulex), Sorensen (215).
`
`their size generally allows studying the various tissues or organs
`Vlfith regard to Decapoda,
`separately: Kuhn and Lederer (131) show that the pigment extracted from the hypodermis of
`Homarus vulgaris and Nephrops norvegicus is in the esterified form, while in the carapace, the free
`and esterified forms coexist. According to Goodwin and Srisukh (69), taking up this study on the
`same species, the hypodermis pigment is esterified astaxanthin. However, according to them, the
`free form would be only present in the carapace. Therefore, these data are in part conflicting.
`
`EXPERIMENTAL CONTRIBUTION
`
`Materials and methods
`
`the carapaces, hypodermis, peristomal connective tissue,
`a) dissection - For every species,
`hepatopancreas, genital glands are removed separately. The tissues or organs are immediately
`covered with anhydrous sodium sulfate that dehydrates them and also allows protecting the mass
`from oxidation.
`
`b) extraction - The tissue coated with sodium sulfate is finely ground and extracted with acetone.
`Extractions are repeated several times. To the acetone solutions gathered in a decantation glass
`are added distilled water and petroleum ether. The proportions of water and ether depend upon the
`amount of tissue water and the lipids present (136). For hypodermis and peristomal connective
`tissue, the most favorable proportions are as follows: at 1,000 mL acetone solution, 300 mL water
`and 200 mL petroleum ether are added. For the acetone solution of hepatopancreas or genital
`glands with a higher lipid load (10 to 11% on average), passing to the -petroleum ether phase is
`favored by the addition of water and petroleum ether in equal proportions (300 : 300). The
`extraction of
`
`00\lO\U'|-l>UUl\JI4
`
`11
`12
`13
`
`14
`15
`16
`17
`18
`19
`
`20
`
`21
`
`22
`
`23
`24
`25
`26
`
`27
`28
`29
`30
`31
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`
`36
`
`26
`
`

`
`OLD0O\lO\U'|-l>UUl\JI4
`
`carapaces needs the addition of hydrochloric acid to the acetone. After maceration, the acetone
`solution is treated as stated above. After stirring and rest, the -petroleum ether phase is separated,
`washed several times with distilled water and dehydrated by contact on anhydrous sodium sulfate.
`
`This -petroleum ether solution contains both free astaxanthin and its esters.
`
`DISTRIBUTION BETWEEN SOLVENTS
`
`According to their solubility in petroleum ether and in 90% methanol, the following is separated:
`
`- on the one hand,
`(epiphase pigments);
`
`the pigments that are more soluble in petroleum ether than in methanol
`
`- on the other hand, the pigments that are more soluble in methanol than in petroleum ether
`(hypophase pigments).
`
`The astaxanthin esters are epiphase esters, free astaxanthin is hypophase in nature.
`
`The -petroleum ether solution of the pigments is stirred with 90% methanol. Two phases are
`separated. After rest, the alcohol phase is extracted and the petroleum ether is extracted again by
`stirring one more time with methanol. These extractions are repeated until the alcohol phase is no
`longer noticeably colored.
`
`The methanol solutions gathered are stirred with petroleum ether after addition of water. Finally, two
`-petroleum ether solutions are obtained:
`
`-
`-
`
`The first one is an ester solution;
`The second one is free astaxanthin.
`
`Measurement of the optical densities allows easily determining the relative concentrations of both
`solutions.
`
`c) Results
`
`ARISATEOMORPHA FOLIACEA
`
`In the peristomal connective tissue in win