CYAN EXHIBIT 1010
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`Title: Transformation of astaxanthin to vitamin A in the albino rat: neoformation in
`vivo and in vitro.
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`Authors: GRANGAUD, René; MASSONET, Renée; CONQUY, Thérese; RIDOLFO,
`J.
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`Author Affiliation: Lab. Chim. biol., Fac. Med. Pharm., Algiers.
`Journal: Comptes Rendus de l'Academie des Sciences 1961 Vol. 252 pp. 1854-1856
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`PHYSIOLOGY — Transformation of Astaxanthin into Vitamin A in the Albino Rat: Neo-formation
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`in vivo and in vitro. Note (*) by Mr. Rene Grangaud, Ms. Renee Massonet, Madams Therese
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`Conquy and Jacqueline Ridolfo, presented by Mr. Robert Courrier.
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`In deficient rats, the per os administration of astaxanthin diacetate causes neoformation
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`of vitamin A which is detectable in the eye. In vitro experimentation can be used to specify
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`that this organ is the seat of the enzymatic transformation which converts astaxanthin to
`retinol.
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`In vitamin A deficient albino rats, astaxanthin (3,3'-dihydroxy-4,4‘-diketo-[3-carotene) manifests a
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`decidedly antixerophthalmic activity. In fact, the daily administration of 10 to 15 ug of pigment heals
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`ocular lesions rapidly without resulting in weight increase which is only observed with much greater
`dosages (1) t° (4). In fish, however, astaxanthin is an authentic provitamin A because, in Gambusia
`holbrooki, it leads to a neoformation of retinol which is particularly detectable in the intestinal
`mucous <3)’ <6)’ (7). Confrontation of these facts suggests that the rat might have preserved the restricted
`ability, in the eye, of converting astaxanthin into vitamin A. This is, at least, the hypothesis which was
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`subjected to experimental examination.
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`In a first experiment, some astaxanthin diacetate was administered to vitamin A deficient rats which
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`were then sacrificed at the same times as control rats and vitamin A was administered, in parallel, in
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`the eyes of each category of subjects. In a second experiment, the eyes of deficient animals were
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`divided into 2 lots: the first lot was incubated in the presence of astaxanthin diacetate, the second was
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`treated in the same manner but without any addition of pigment; the respective concentrations of
`retinol were then determined.
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`ASTAXANTHIN DIACETATE PREPARATION — The inner walls of the stomach pockets ofAr1'ste0m0rpha
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`foliacea, Risso and Aristeus antennatus, Risso (jumbo red penaeid shrimp) contain a blue
`chromoprotein whose prosthetic group is astaxanthin (4). After dissection, the pockets were emptied
`of their contents and agitated with distilled water in which chromoprotein was dissolved. The solution
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`was then diluted with four times its volume of acetone: the astaxanthin was detached from its protein
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`copula and the solution turned an orange-red. With the addition of petrol ether and water, the pigment
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`passed into the light phase which was separated and then dried on anhydrous sodium sulfate. The
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`solution was filtered in a magnesium oxide column which was then washed with petrol ether, then
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`with hexane diluted with 2% volume acetone (elimination of vitamin A carotenes and
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`esters) and, finally, with a mix of 92 ml of hexane and 8 ml of ethanol (elimination of the retinol). The
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`column was then sectioned, the pigmented zone was immersed in pyridine which, by elution, turned
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`red. The concentrated solution under reduced pressure and under nitrogen was diluted with acetic
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`anhydride (XX drops/ml). After 9 hours at ambient temperature, the diacetate was then treated by
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`petrol ether. The solution was washed, dried and chromatographed on aluminum oxide. The column
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`was processed similar to the magnesium oxide. The pyridinic solution which was obtained was diluted
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`with petrol ether and water. The light phase was separated, dried and concentrated. The residue was
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`treated with hot pyridine, diluted by a third volume of water and left for 24 hours at 0°C. The
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`crystalline precipitate was purified by two re-crystallizations.
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`PROTOCOL AND RESULTS. — a. In vivo experiment — 18 Wistar rats, weighing 32 g, were weaned and
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`subjected to the base regime which was free of all vitamin A factors. After 40 days, the signs of
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`deficiency were manifest, the animals were divided into 3 lots which received the following daily
`doses:
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`Lot A: traces; lot B: 1 ug; lot C: 2.1 ug.
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`b. In vitro experiments. — 12 deficient rats, under the same conditions as the preceding, were
`decapitated on the 40th day. 1 ml of blood serum and 1 mmole of or-tocopherol dispersed in 1 ml of
`water was placed into two small colloidion bags. The right eyes of 6 subjects and the left eyes of the
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`other 6 subjects were placed into one of the bags (Lot 1).; The 12 remaining eyes and 1 mg of
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`astaxanthin diacetate in dispersion in 1 ml of Tween 80 were placed into the other bag (Lot 11). The
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`bags were immersed into a buffered Krebs-Ringer solution and kept in an oven at 37°C for 12 hours.
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`The dosage of vitamin A produced in the unsaponifiable [tissues] of all the eyes of each lot provided
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`the following values:
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`Lot 1: 0.75 ug; Lot 11: 1.65ug.
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`Discussion. — A spectrophotometric study of the pigment solution before chromatography did not
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`reveal any vitamin A nor carotene. If traces of these substances had, however, escaped examination,
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`they would have been eliminated by the washings of the columns performed for that purpose in
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`accordance with (10). Re-crystallization of the astaxanthin diacetate constitutes a final purification.
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`The following control excludes the interference of impurities: under the same conditions as that in the
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`in Vitro experiment, two lots of deficient rat intestinal fragments were incubated, 1 lot in the presence
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`of [3-carotene and the other in the presence of astaxanthin diacetate. Neoformation of vitamin A was
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`only detected in the first lot.
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`Thus, in the experimental conditions described, in vivo as well as for in vitro, neoformed and detected
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`Vitamin A in the eye can only be due to astaxanthin transformation. Additional experiments will try to
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`determine whether, since it is likely, it is the retina which performs the enzymatic conversion.
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`Conclusion. — The recorded results Verify the working hypothesis which dictated these experiments:
`in Vitamin A deficient albino rats, the administration of astaxanthin diacetate causes neoformation of
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`retinol in the eye. The in vitro experimentation led to the same results and also localized the reaction
`seat to the ocular tissue.
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`(*) Meeting of 13 March 1961.
`(1) R.Grangaud and R. Massonet, C.R.Acad.Sc., 1948, V.227, p.568.
`(2) R.Grangaud and R. Massonet, C. R. Acad. Sc., 1950, V.230, p.1319.
`(3) R.Grangaud, Doctoral Thesis in Physical Sciences, Lyon, 1950.
`(4) Mlle R. Massonet, Doctoral Thesis in Natural Sciences, Lyon, 1958.
`(5) R.Grangaud and R. Massonet, C.R.Acad.Sc., V241, 1955, p. 1087.
`(6) R.Grangaud, P.Vignais, Mlle R. Massonet, and J.P.Moatti, C.R.Acad.Sc., V243, 1956, p.1170.
`(7) J .P.Moatti, State Doctoral Thesis (Pharmacy), Algiers, 1959.
`(8) 2% dot-tocopherol is added to a solution of deVitaminized Vegetable oil.
`(9) J.M.Lewis, O.Bodansky, K.G.Falk and G. McGuire, .1. Nutrition, v23, 1941, p.351.
`(10) S.Y.Thompson, J.Ganouly and S.K.KON, Brit. J. Nutr.,V.3, no.1, 1949, p. 57.
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`(Laboratory of Biochemistry, Faculty of Medicine and Pharmacy of Algiers.)
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