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The Medaka, Oryzias latipes, a cyprinodont fish, is widely distributed in Japan and a material used for experiments of genetics, embryology and physiology. A number of cultivated color varieties have been known. The chromatophores responsible for the body color are melanophores, xanthophores, leucophores and iridophores. The main kinds of color varieties are as follows: blackish brown (wild, BR), orange red (bR), blue (Br) and white (br). These color phenotypes result from the action of genes. The genes B, b and R, r concern the development of melanophore and xanthophore respectively. In the study on the relations among pteridines, fats and carotenoids of Oryzias xanthophore, the presence of carotenoid and acarotenoid xanthophores was suggested by one of the authors, i.e., the carotenoid containing xanthophores found in the wild and orange red color varieties (R strain) and the carotenoid lacking xanthophores found in the blue and white ones (r strain). The mechanism of carotenoid deposition in the xanthophore remains to be solved 1), 2).
On the other hand, fish contains various sorts of carotenoids, the dominant of which is peculiar to the species concerned. Dominant carotenoids for the fish are astaxanthin which is common to red fishes, lutein common to freshwater fishes, tunaxanthin common to Scombrina, Carangina and Percina fishes and a few other carotenoids common to some groups of fishes though their chemical properties are not fully elucidated yet 3)-7). In addition to the dominant the fish usually contains other various carotenoids in smaller amounts, the proportion of which often differs between samples possibly due to their physiological and/or dietal conditions. Whether this stands for Oryzias remains to be opened. On the componental carotenoids of Oryzias, Goodrich 8) was probably the first person to report. He found lutein in its adult skin, but his data seems to require more detailed confirmation. This paper concerns with the analysis of the carotenoids in the adult skin of orange red color variety.
For the better chromatographical separation of the sample carotenoids, an apparatus illustrated in Fig. l was devised. The column consists of two tubes of different sizes, the outer one of which is about twice as large as the inner, and the adsorbent is filled in the gap between the two. The sample carotenoids loaded on the column are developed into daughnut shaped bands which descend almost horizontally without being contaminated by the neighboring components. As the results of chromatographic analysis on the two different samples were similar, most of the data described in this paper were of the sample collected in April 1966.
Experiment 1: 2342 mg of unsaponifiable matter was dissolved in petroleum ether and loaded on alumina column and developed with mixtures of petroleum ether and ethyl ether of different proportions. The developed color zones were carefully observed and collected as separately as possible, thus 30 fractions containing carotenoids were obtained as shown in Table l.
The fractions were immediately examined of their absorption spectra using Hitachi EPS II spectrophotometer and the results are shown in the Table and Fig. 2. The fractions which did not contain carotenoid were omitted from the Table. As the crystallization of the sample carotenoids were not successful, their identification was made basing on their chromatographic and spectroscopic properties. It is known for a carotenoid that in addition to its absorption maxima measured in various solvents, the whole figure of the spectra is peculiar to the kind of carotenoid concerned. The absorption spectra in Fig. 2 were all figured in the same size so that the comparison one after another was more feasible.
As seen in the Fig. 2 the Fr. 2 overlapped the authentic beta-carotene. Fr. 5 was also nearly identical with the former except a slight inflexion around 400 m micron probably due to a cis-configuration in the structure. Frs. 8, 9 and 10 had single maximum around 451-456 m micron and spectroscopically they closely resembled each other. Frs. 8 and 10 indicated, however, a small but prominient second maximum around 300 m micron which Fr. 9 lacked. These three were deduced to be a group of stereoisomers of a kind of carotenoid although not identified yet. The authors tentatively named them as Carotenoid M-I inclusive of the possible isomers. Fr. 11-1 may be a remainder of the preceding carotenoid containing much of non-carotenoid substances. Frs. 11-2 and 12-1 resembled closely but were not identified. Fr. 12-2 resembled the above two except that it had conspicuous second maxima around the shorter wavelengths probably due to its isomerized structure or contamination by other substances. These three were collectively named as Carotenoid M-II. It may be of interest that Fr. 13-1, though it seemed to contain much of non-carotenoid impurity, showed an entirely different spectra from either preceding or succeeding carotenoid groups. This phenomenon was observed on the two sample materials of Oryzias used in this experiment. Frs. 13-2 and 14-1 more or less resembled Carotenoid M-I though their maxima shifted to shorter wavelengths by 2-7 m micron than the latter. They were not identified and named as Carotenoid M-III. Fr. 13-2 seemed to contain much impurities which may be the cause that the two spectra were not in entire congruence, or rather Fr. 13-2 may be an isomer for Fr. 14-1. Frs. 14-2, 14-3 and 14-4 resembled the preceder Fr. 14-1 but the former three lacked the inflexion around 400 m micron which was conspicuous with the latter. These three may be a group of stereoisomers of a carotenoid and were named inclusively as Carotenoid M-IV. Fr. 15-1 may be a remainder of the preceder mixed with non-carotenoid substances. Fr. 15-2 was unlike the former; its two maxima coincided with that of the successive fraction Fr. 16-1, but the figures of spectra of the two were far apart. Fr. 16-1 was one of the main carotenoid in the sample amounting 42.54 per cent of the total. From its spectroscopic and chromatographic properties it was noted to be almost identical with tunaxanthin which was common to marine fish but has not been known in the freshwater fish. The dotted line in the Figure was of crystals obtained from fins of yellowfin tuna, whose m.p. was 167oC, and E(1%,1cm) 438 m micron=2200 (in petroleum ether). In this experiment, however, Fr. 16-1 failed to crystallize and more detailed work for identification has to be carried on in the future. In this paper, however, the authours will call this fraction as tunaxanthin. Fr. 16-2 was also a dominant carotenoid in the sample, being 34.06 per cent of the total, but was not identified. The absorption spectrum took an intermediate figure between tunaxanthin and lutein, though it cannot be the latter. This carotenoid was not crystallized and has to be studied again in the future. Fr. 16-3 amounted to 5.81 per cent of the total and and resembled Fr. 16-2. Though these two may not be the same, they were named as Carotenoid M-V. Frs. 17-1, 17-2 and 17-3 resembled each other and the nearest of known carotenoid to them was lutein judging from their figures and the chromatographic behavior etc. Along with the spectra of these three, the spectra of lutein crystallized from the leaf of Ginkgo biloba is shown in the Fig. 2. They were, however, not exactly the same. They are probably stereoisomers of lutein, but in this paper the author will call them collectively as lutein. Frs. 17-4 and 18 may be a group of isomers though not identified, and were named as Carotenoid M-VI. Frs. 19, 20, 21 and 22 showed broad main maxima around 446-458 m micron with second maxima around 468-474 m micron locating much closer to the first one than ever noted on other carotenoids. They were not identified and collectively named as Carotenoid M-VII. Probably they contain keto group in the molecule though has to be examined in the future. Fr. 22 was the last carotenoid to remain on the column, and neither astacene which was frequently encountered in a saponified sample of fish skin nor astaxanthin was detected.
Color reaction for epoxide group in the molecule was tested on all these 30 fractions with conc. HCl in ethyl ether, but none of them was found positive.
It is possible that some of these various carotenoids separated in this experiment were not present in the original sample fish but artifacts which newly appeared in the course of analytical process. This possibility, however, seemed to be less as will be discussed later.
Experimental 2: To elucidate the proportions of both free and esterified carotenoids in the sample, the whole oil was chromatographed on alumina and separated into ten fractions. Each fraction was saponified and rechromatographed on alumina and fractionated according to the color zones. Table 2 shows the results on whole oil. More than 64 per cent of loaded oil by weight passed through the column in the first four fractions suggesting that these mostly consisted of glycerides. Fr. 5 was found to contain much sterols and its unsaponifiable matter amounted to 62.5 per cent, which was interpreted to consist of esterified sterols almost exclusively. More than 95 per cent of the total carotenoids passed through the column in the first five fractions. They were suggested to be esterified xanthophylls and carotenes. Frs. 6, 7 and 8 contained smaller amounts of carotenoids, possibly free xanthophylls. Frs. 9 and 10 contained no carotenoids.
Table 3 shows results of rechromatography on unsaponifiable matter of each fraction obtained above. The positions of color zones sketched in the Table correspond to the kind of developers used. From the spectroscopic and chromatographic coincidence with those carotenoids separated in Experiment I, identification was made as shown in the Table. On this occasion a check was made to confirm if any spectral change might occur during the analytical procedure of the sample. After the absorption spectra were measured, the fractions were mixed again by each group and the spectra of these mixture were compared respectively with the original non-saponified fractions. As far as the visible range of wavelength is concerned the coincidence of the two spectra was perfect on each group tested, and the recovery of carotenoids after the saponification and rechromatography processes was found ranging 92-84 per cent of the original.
From the findings in this series of experiment, most of Oryzias carotenoids were concluded to occur originally in the esterified form. Lutein and the Carotenoid M-VI, however, occur in both free and esterified forms.
Table 4 shows carotenoid proportion in the two different samples. In both cases tunaxanthin and Carotenoid M-V were dominant and lutein came next. There was a slight difference between the two samples collected in different seasons due to some unknown cause. As far as beta-carotene is concerned, however, according to the authors' unpublished data on Ayu, Plecoglossus altivelis a freshwater Salmoniana fish contained more beta-carotene in the skin in the fall than in summer probably due to their physiological and dietal differences by the seasons. This phenomenon was in accordance with that of Oryzias in this experiment.
It was a matter for regret that none of the carotenoids separated in this experiment was crystallized and fully identified. Further experiments remain to be continued. So long as the present data is concerned, however, Oryzias as a freshwater fish possesses a unique feature. The reasons are follows: this fish contains lutein in rather small amounts while other freshwater fish such as Carassius, Cyprinus, Misgurnus, Parasilurus, Plecoglossus etc. contain lutein as a dominant carotenoid. On the other hand, Oryzias contains dominantly tunaxanthin which is commonly found in ocean fish but has never been known in the freshwater fish. Another fact was noted, that is, when the Carr-Price reaction was tested on the unsaponifiable matter chromatographed, the absorption spectra of one fraction showed a single peak at 620 m micron typical to the vitamin A1. Another fraction neighbouring the vitamin A1 containing fraction showed a peak at 664 m micron due to the presence of vitamin A1 aldehyde. None of other fractions, however, showed a peak around 693 m micron typical to the vitamin A2. This vitamin is common to freshwater fish. From the above facts, it seems that Oryzias belongs rather to marine fish type. In fact, Oryzias can survive in highly saline water for a long time contrary to the common freshwater fish.
According to the authors' experience, carotenoid composition of Oryzias is the most complex. It contains more than ten different kinds of carotenoids while most of marine and freshwater fish contain usually a few or at most several kinds of carotenoids. A possibility exists that some of the carotenoids separated in this experiment may be artifacts instead of being original. However, no anhydrovitamin A was detected in the sample. This vitamin has been known to be a common artifact appearing in the process of analysis in the oil carelessly treated. In view of this, it can be said that our analysis has been done successfully and most of fractionated carotenoids were original. Then, a question remains as to the diversity of the sorts of carotenoid in Oryzias. Many of them might be intermediates in the biosynthetic processes from one carotenoid to the other. Katayama et al. separated some intermediates in the goldfish and suggested the final conversion of dietal lutein into astaxanthin. The authors' data may be the case. In the case of Oryzias, what kind of carotenoid is the starting or final one? According to Mimoto and the authors' data, roe of tuna and mackerel contain only lutein but no tunaxanthin while the latter is found in the skin dominantly or almost solely. Mackerel skin at young stage contains both lutein and tunaxanthin in various proportions. The dietal origin of tunaxanthin remains open. Pitardixanthin and epsiron-carotene are spectroscopi-cally similar to tunaxanthin but their wide occurrence in the fish diet seems doubtful. Is it unnatural to assume that Oryzias converts the dietal lutein into tunaxanthin and many other carotenoids in question are the intermediates of the two? Further experiments remain to be done on this line.
The skin carotenoid of orange red color variety of Oryzias was analyzed chromatographycally and spectroscopically. The carotenoids consisted of ten different members: beta-carotene, tunaxanthin, lutein and seven unidentified carotenoids. Tunaxanthin occupied about 40 per cent of the total, and an unidentified Carotenoid M-V similar to tunaxanthin amounted to about the same. Lutein was found to be about 4 per cent of the total. From these findings Oryzias seems to be a fish of marine type rather than a freshwater fish so far as the carotenoid components are concerned. Most of the xanthophylls was found to occur in esterified form except that lutein and Carotenoid M-VI were found in both free and esterified forms.
2) K. TAKEUCHI: Embryologia, 5, 170~177 (1960).
3) S. HIRAO, J. YAMADA and R. KIKUCHI: Bull. Tokai. Reg. Fish. Res. Lab., 16, 53~58 (1957).
4) G.F. CROZIER and D. W. WILKIE: Comp. Biochem. Physiol., 18, 801~804 (1966).
5) R. ESTABLIER: Invest. Pesquera, 30, 497~500 (1966).
6) S. HIRAO: This Bull., 33, 866~871, 908 (1967).
7) N. TSUKUDA and K. AMANO: ibid., 32, 334~345 (1966).
8) H.B. GOODRICH, G.A. HILL and M.S. ARRICK: Genetics, 26, 573~586 (1941).
9) S. HIRANO, R. KIKUCHI and H. TAGUCHI: This Bull., 29, 371~381 (1963).
10) T. KATAYAMA and C.O. CHICHESTER: Oral report at the Annual Meeting of Jap. Soc. Sci. Fish., April, 1968, Tokyo.
11) K. MIMOTO and S. HIGASA: Report of Nankai Reg. Fish. Res. Lab.,8, 93~103(1958).