CYAN EXHIBIT 1012
`
`Title: Transformation of astaxanthin to vitamin A by ocular tissue of the rat in vitro.
`Authors: MASSONET, R.; CONQUY, T.; GRANGAUD, R.
`
`Author Affiliation: Lab. Chim. biol., Fac. Med. Pharm., Algiers.
`Journal: Compte rendu des seances de la Societe de biologie 1961 Vol. 155 pp. 747-750
`
`During the course of previous research, it has been shown that fish are capable of utilizing astaxanthin
`as a precursor of vitamin A. In effect, the administration of this carotenoid to Gambusia holbrooki
`Grd leads, in a deficient animal, to the neoformation of detectable retinol in the intestinal mucosa,
`liver, and eyes (1,2).
`
`(1) R.Grangaud and R. Massonet, C.R.Acad.Sc., 1955, v.241, p. 1087.
`(2) J.P.Moatti, State Doctoral Thesis (Pharmacy), Algiers 1959.
`
`747
`
`

`

`Mammals do not have the same ability as fish with regards to astaxanthin which is not a real
`provitamin for them: administered to the vitamin A deficient white rat, astaxanthin limits its action to
`the ocular apparatus, manifesting properties that we can consider to be selectively antixerophthalmic
`(3,4,5). This topographically-limited activity is, in all aspects, comparable to that which vitamin A
`exercises in the eye and its appendages.
`
`It was therefore logical to try to interpret, on the biochemical level, the yet unknown mechanism of
`the astaxanthin action among rats, to wonder if the latter would be limited to the ocular tissue, as in
`the conversion ability witnessed among fish.
`
`A short note (6) summarizes results of experiments which were conducted in vivo and in vitro and
`which support the verification of that hypothesis.
`
`The objective of the present work is to describe, in detail, the in vitro experimental techniques
`showing the reality of the conversion of astaxanthin into vitamin A by the rat’s ocular tissues.
`
`The concept of these experiments was to incubate, in a complex medium of vitamin A deficient rat
`eyes in the presence or absence of astaxanthin diacetate. The respective concentrations of vitamin A
`of the eyes placed in contact with the carotenoid and the control eyes were then determined.
`
`EXTRACTION OF THE ASTAXANTHIN AND PREPARATION OF DIACETATE. -- In order to avoid the
`
`possible interference of natural provitamin A or pre-formed retinol, the following technique was used
`for the preparation of the astaxanthin diacetate: carotenoid was extracted from the inner wall of
`stomach pockets of two large red penaeid shrimp (Aristeomorphafaliacea and Aristeus antennatas,
`Risso) where there is, in the form of a blue chromoprotein whose physico-chemical characteristics
`closely belong to those of the crustacyanin studied by Wald (7). This chromoprotein is, in effect,
`soluble in water and it is easy to detach it from the prosthetic group. The spectral characteristics of the
`latter (kmax = 492 mu in pyridine) show that it is completely transformed astaxanthin. After
`dissection, the sampled pockets of 5 kg of shrimp were emptied of their contents, agitated with 100 ml
`of distilled water; the limpid solution was diluted with 4 times its volume of acetone; under these
`conditions, the astaxanthin was detached from its protein copula and the color of the solution
`immediately turned orange-red. By an addition of 50 ml of petrol ether and 100 ml of water, the
`pigment was extracted in the light phase which was separated by decantation and dried on anhydrous
`sodium sulfate. The solution was then filtered in a magnesium oxide column (200 mm in length, 20
`mm in diameter). Astaxanthin was adsorbed in the upper part of the column (over a 2 cm length)
`
`(3) R.Grangaud and R. Massonet, C.R.Acad.Sc., 1948, v.227, p.568.
`(4) R.Grangaud and R. Mnssonet, C. R. Acad. Sc., 1950, v.230, p.1319.
`(5) R.Grangaud, Doctoral Thesis in Physical Sciences, Lyon, 1950.
`(6) R.Grangaud, R.Massonet, Th.Conquy, and J.Ridolfo, C. R. Acad. Sc., 1961, v.252, p.1854.
`(7) G.Wald, N.Nathanson, W.P.Jencks and E.Tarr, Biol. Bull., 1948, v.249,
`p.95.
`
`748
`
`

`

`which was washed with 50 ml of hexane diluted with 1 ml of acetone. The purpose of this washing
`was to eliminate the vitamin A carotenes and esters, if they were present. A second washing was
`performed using a mix of 92 ml of hexane and 8 ml of ethanol to eliminate possible traces of retinol
`(8). The column was then sectioned: the pigmented zone was immersed in 20 ml of pyridine which
`immediately induced elution.
`
`The red solution was concentrated under reduced pressure and in an inert atmosphere to a volume of 2
`ml. XL drops of acetic anhydride were added and the entire mix was left for 9 hours at ambient
`temperature. 20 ml of petrol ether and 40 ml of water was then added. The light phase was separated
`by decantation, washed with distilled water then dried on anhydrous sodium sulfate. The solution was
`again chromatographed but this time on aluminum oxide (column of 200 mm in length, 20 mm in
`diameter) which was processed the same as on magnesium oxide by successively passing the hexane-
`acetone mix. The pigment was eluted in pyridine and, finally, treated with 3 ml of hot pyridine. After
`addition of 1 ml of water and 24 hours of rest at 0°C, a crystalline precipitate was obtained which was
`then subjected to two re-crystallizations. The crystallized diacetate was dispersed in Tween 80 at a
`concentration of 1 mg/ml (9).
`
`EXPERIMENTAL PROTOCOLS AND RESULTS. — 12 Wistar rats, weighing exactly 32 grams, were weaned
`and subjected to the previously-described synthetic regime (10). After 40 days, the signs of deficiency
`were being manifested (weight gain termination, beginning of xerophthalmia), the animals were
`decapitated and the eyes were immediately removed: the right eyes of 6 of the subjects and the left
`eyes of the other 5 (Lot 1) were placed in a small colloidion bag which already contained 1 ml of
`fresh blood serum and 1 milli-mole of d-tocopherol dispersed in 1 ml of water. The 12 remaining
`eyes (Lot 11) were placed in a second bag containing the same mix but diluted with 1 ml of
`astaxanthin dispersion. The two bags were immersed in a Krebs-Ringer buffered solution with a pH of
`7 and maintained in an oven at 37°C for 12 hours. At the end of this time, the eyes of each lot were
`saponif1ed in 1 ml of alcoholic potassium hydroxide at 60 pp 100, at 80°C for 15 minutes. The
`unsaponifiable [components] were treated by petrol ether, washed, evaporated using chloroform and,
`in each chloroformic extract, the search for and dosing of vitamin A was performed using the Carr
`and Price reaction, the Meunier and Raoul kinetic technique (11), and measurements were made using
`a photocolorimeter. The following results were obtained: the Lot 1 eyes
`
`(8) S.Y.Thompson, J.Ganguly and S.K.Kon, Brit. J. Nutr., 1949, v.3, p57.
`(9) The technique of preparation is identical to that described by J.G.Bieri (J.Nutrition, 1951, v.44,
`p.2) for obtaining a dispersion of B—carotene.
`(10) R. Massonet, Doctoral Thesis in Natural Sciences, Lyon, 1958.
`(11) P.Meunier and Y.Raoul, Diagnostic chemistry of Vitamin Deficiencies. Masson and Co., Paris
`1942.
`
`749
`
`

`

`(control) contained 0.75 ug of Vitamin A, the Lot 11 eyes (incubation in the presence of astaxanthin)
`contained 1.65 ug.
`
`In parallel to this experiment, in order to verify that the prepared astaxanthin diacetate as it was
`described did not contain carotene or Vitamin A, the following control was again performed: two lots
`of deficient rat intestines were incubated in identical conditions to those of the aforementioned
`
`experiment. 1 mg of B-carotene, dispersed in 1 ml of Tween 80, was added to one of the incubation
`liquids. To the other was added 1 mg of astaxanthin diacetate in the same state of dispersion. Later
`analysis performed on the unsaponifiable [components], using a general technique, revealed the
`presence of Vitamin A, neo-formed in the lot into which the B-carotene had been added; no trace of
`retinol was detected in the lot incubated in the presence of astaxanthin diacetate.
`
`Conclusion. — The conclusion which we gain from these experiments is, therefore, that the neo-
`formed Vitamin in the eye can only be attributed to a transformation of astaxanthin. It is likewise
`concluded that it is the retinal tissue which is the base of the reaction. Additional experiments were
`undertaken to obtain more detailed information.
`
`(Laboratory of Biological Chemistry. Faculty of Medicine and Pharmacy, Algiers).
`
`750
`
`