THE SEPARATION OF A SUBSTANCE FROM OILS WHICH INHIBITS THE DESTRUCTION OF VITAMIN A

BY FERROUS SULFATE.

BY HOWARD W. ESTILL AND E. V. McCOLLUM.

(From the Department of Chemical Hygiene, School of Hygiene and Public Health, the Johns Hopkins University, Baltimore.)

(Received for publication, July 26, 1927.)

In a previous paper (1) from this laboratory it was reported that the ophthalmia induced in rats by feeding ferrous sulfate in the food mixture can be prevented or cured by the subsequent addition of wheat germ oil to the ferrous sulfate-containing ration. In the case of a fat which contains vitamin A, it has been shown that ferrous sulfate causes the destruction of the vitamin, and this takes place rapidly with butter fat but more slowly with cod liver oil (2). This destruction, or possibly, inactivation, is inhibited by the inclusion of a small amount of wheat germ oil in the diet.

In investigating other possible avenues of attack than those used heretofore in the isolation of fat-soluble vitamins, we have separated a most interesting substance from cod liver oil in the form of its lithium chloride derivative; this substance, or active fraction, is very effective in inhibiting the destruction of vitamin A by ferrous sulfate.

Zwikker (3) reported that when lithium chloride is dispersed in a fat by the addition to the latter of a solution of the anhydrous salt in pyridine the cholesterol is precipitated practically quantita- tively in the form of a crystalline compound consisting of 1 mol each of cholesterol and lithium chloride. It was recommended that the system be diluted with petroleum ether to facilitate filtering. We have not found the reagent so effective for the quantitative removal of cholesterol from a fat or oil as Zwikker believed it to be because of the slight solubility of the lithium chloride compound in the oil-pyridine system. The compound is easily prepared from anhydrous lithium chloride and cholesterol dissolved in dry pyridine.

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158 Vitamin Isolation

It is well known that the more common alcohols form double compounds with a number of salts. The best known of these are the complexes formed from an alcohol with LiC1, MgCl,, CaC12, BaO, Al&& SnCl,, SbC&, GuSOh, Mg(NO&, and PtCL. The lithium chloride derivative of the substance described in this paper is typical of several derivatives which can be produced from the higher alcohols and sterols accompanying fats and oils by employ- ing certain of the salts listed above under appropriate conditions in non-aqueous systems; the usefulness of this type of reaction in the isolation of the fat-soluble vitamins is under investigation in this laboratory.

Since the studies of Takahashi (4) on vitamin A, and those of Steenbock (5)) Hess (6)) and Hess and Windaus (7) on the develop- ment of antirachitic properties in cholesterol or ergosterol by irradiation with ultra-violet light appear to indicate that both vitamins A and D are related to the sterols (which contain hy- droxyl groups), it was thought possible that one or more of the fat-soluble vitamins could be separated from the fats in which they are dissolved by forming derivatives with those salts which combine with alcohols in non-aqueous systems. We report at this time the separation from cod liver oil of a lithium chloride complex which induces in rats a recovery from the “salt ophthal- mia” due to feeding ferrous sulfate in a manner comparable to the inclusion of wheat germ oil in the ration. The method of preparing the substance is as follows: To 200 gm. of cod liver oil diluted with 216 cc. of petroleum ether were added quickly and with constant rotating 3 gm. of anhydrous lithium chloride dis- solved in 38 cc. of anhydrous pyridine. Because pyridine is a rather strong base, and since its effect on the fat-soluble vitamins is entirely unknown, it seemed best to exclude air from the reaction mixture by means of carbon dioxide; this also prevented any oxidation of vitamin A by contact with air. The latter was ex- cluded by saturating the cod liver oil-petroleum ether mixture with carbon dioxide before adding the lithium chloride-pyridine reagent. At the time of mixing, the temperature of the diluted cod liver oil was 11’ and that of the lithium chloride solution, 20”. As soon as the solutions were mixed, a white precipitate formed in rather large amount; most of this was doubtless lithium chloride. ‘The mixture was rotated vigorously for $ hour in a stoppered

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H. W. Estill and E. V. McCollum 159

1 liter Erlenmeyer flask; during this time carbon dioxide from a cylinder was introduced frequently and any ether lost was replaced. Toward the end of the reaction period one noticed that that part of the precipitate which had settled out most readily had assumed a very light pink color; the supernatant liquid was very turbid. After centrifuging for about 30 minutes at a moderate speed, the virtually clear solution was decanted as much as possible, and the precipitate (some of it pasty, some granular) transferred quickly to a No. 50 Whatman filter, and filtered as dry as possible under diminished pressure; the very deliquescent precipitate was pro- tected by means of pure dry carbon dioxide. Petroleum ether was used to wash the precipitate free from cod liver oil and pyri- dine; the precipitate was then removed to a 100 cc. centrifuge tube (filled with dry C03) and 50 cc. of petroleum ether added at once. After stirring the precipitate thoroughly, one noticed that some of it in the form of small pellets had risen to the surface of the liquid. Below this part of the precipitate the solution was very turbid, while a rather coarsely granular light pink colored precipitate was noted at the bottom of the tube. That portion of the precipitate which floated on the surface of the petroleum ether consisted entirely of imperfectly spherical granular pellets (diameter about 1 mm.) which resembled particles of sago. It was impossible to obtain a clear idea of the physical nature of the individual pellets of the precipitate of low specific gravity, since their ‘appearance under the microscope varied greatly with the method of illumination; but one gained the impression that essen- tially they consisted of thin shells enclosing irregularly formed masses. As the precipitate aggregated at the surface of the liquid it had a faint pink cast; this precipitate will be referred to as “the precipitate of low specific gravity. ” There was nothing particularly characteristic about that portion of the initial precipi- tate which rendered the solution turbid; it was designated “the finely divided precipitate. ” The coarsely granular portion of the initial precipitate was definitely cystalline; it was called “the coarse precipitate. ”

It is believed that a fairly clean cut separation of the precipitate of low specific gravity was effected; certainly none of the coarse precipitate separated with it, and the small amount of the fine precipitate carried with it was easily washed out with petroleum

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160 Vitamin Isolation

ether. Much less satisfactory was the separation of the fine and coarse precipitates; but it was thought that it was sufficiently exact to enable one to decide which precipitate, if either, had carried down one or more of the fat-soluble vitamins.

The precipitate of low specific gravity was removed with a small flexible spatula to a small glass evaporating dish (or “shell”) con- taining petroleum ether in contact with a gentle stream of pure dry carbon dioxide. After removing as much of this precipitate as possible, the precipitate in the centrifuge tube was again stirred up gently but thoroughly, and more of the precipitate of low specific gravity removed to the glass shell. This operation was repeated until no more of the precipitate floated on the surface of the petroleum ether.

The exposure of a small amount of this precipitate to the air showed that it was extremely hygroscopic. It was therefore care- fully protected against moisture with the aid of petroleum ether and dry carbon dioxide. It was necessary to use carbon dioxide, for some of the precipitate rose to the surface of the petroleum ether. The precipitate was then washed with petroleum ether to remove the remaining cod liver oil and pyridine, and the latter was tested for by shaking a given portion of the filtrate with water and adding an excess of a saturated solution of mercuric chloride to the aqueous layer (in the presence of pyridine a white crystalline precipitate is formed). The precipitate must have been free from cod liver oil for from 500 to 1000 cc. of petroleum ether were necessary to remove the pyridine.

In order to remove lithium chloride, 4 cc. of water were added to the precipitate, which dissolved immediately. Above the aqueous layer was a narrow, very light yellow colored oily layer. The solution was treated at once with 10 cc. of ethyl ether, then trans- ferred to a separatory funnel, and the glass shell rinsed with 5 cc. of ether. The aqueous layer was washed with three 10 cc. por- tions of ethyl ether, and the washings added to the main portion. The colorless ethereal solution was evaporated on dextrin in a current of air and tested for the relief of salt ophthalmia.

In order to investigate the active substance or fraction, the ether was removed from the ether extract of the hydrolyzed precipitate of low specific gravity with the aid of dry carbon dioxide, and at a temperature not to exceed 40’. A small amount of a pale

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H. W. Estill and E. V. McCollcm

yellow pungent oil remained. This concentrate gave an entirely negative Liebermann-Burchard reaction, sulfuric acid reaction, and Drummond AsC13 reaction (7) for vitamin A. Chemical studies, including other color reactions, are now in progress.

The ether extracts of the three hydrolyzed precipitates, that is, the precipitate of low specific gravity, the fine precipitate, and the coarse precipitate, were combined and tested for vitamin A; other ether extracts similarly prepared from the three precipitates were combined and tested for vitamin D. In each case the animal tests were negative. The treatment of both the fine and coarse precipitates was substantially the same as described above for the precipitate of low specific gravity. It may be mentioned that ether extracts of the separately hydrolyzed fine and coarse precipi- tates were found to be entirely negative when tested individually for vitamins A and D and for the relief of salt ophthalmia.

From the manner in which the lithium chloride complex sepa- rates from the accompanying material it would seem probable that the preparation consists largely or wholly of an individual organic substance. We have employed this lithium chloride-free material (lithium chloride being toxic to rats) in the type of experiment described in a previous paper (1). In most instances the entire yield of the organic portion of the precipitate of low specific gravity obtained from 200 gm. of cod liver oil was thoroughly mixed with 40 gm. of dextrin; this dextrin was then used to make up to 200 gm. of ration.

An attempt to remove the same substance from wheat germ oil resulted in the separation of the active principle with lithium chloride; but in contrast to the case of cod liver oil, a clean cut physical separation from accompanying substances was not effected. We are pursuing further the perfection of a method of separation of the components of the mixture obtained from wheat germ oil.

From the known property of lithium chloride to form compounds with alcohols, it is of interest that a very small amount of a sub- stance with the properties described can be separated by combin- ing it with lithium chloride from fats containing vitamin E (wheat germ oil and cod liver oil). Careful tests of the material compos- ing the organic portion of the lithium chloride complex for vitamins A and D were entirely negative. We have experienced much

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Vitamin Isolation

difficulty in securing female rats of proven sterility by using the original diet of Evans (8) for inducing resorptions in rats. For this reason we have not yet been able to make conclusive tests on the material described for vitamin E with female rats, but such results as have been obtained and certain observations on male rats which have been kept on diets which induce sterility in males seem to indicate that the substance which we have separated by the method described may be vitamin E. Further studies of this interesting material are being conducted.

CONCLUSIONS.

1. The destruction of vitamin A by ferrous sulfate is inhibited by a substance separated from certain oils by means of lithium chloride dissolved in pyridine; this substance may be vitamin E.

2. The alcohol-salt addition product type of reaction promises to be useful in vitamin isolation.

BIBLIOGRAPHY.

1. Simmonds, N., Becker, J. E., and McCollum, E. V., J. Am. Med. Assn., 1927, lxxxviii, 1047.

2. McCollum, E. V., Simmonds, N., and Becker, J. E., Proc. Sot. Exp. Biol. and Med., 1927, xxiv, 952.

3. Zwikker, J. J. L., Pharm. Weekbl., 1917, liv, 101. 4. Takahashi, K., Nakamiya, Z., Kawakami, K., and Kitasato, P., Scien-

tific papers of the Institute of Physical and Chemical Research of Japan, Komagome, Hongo, and Tokyo, 1925, iii, 81.

5. Steenbock, H., and Black, A., J. Biol. Chem., 1925, lxiv, 263. 6. Hess, A. F., Weinstock, M., and Sherman, E., J. Biol. Chem., 1925, lxvi,

145; 1926, Ixvii, 413; 1926, lxx, 123. 7. Hess, 9. F., and Windaus, A., PTOC. Sot. Exp. Biol. and Med., 1927, xxiv,

461. 8. Evans, H. M., and Bishop, K. S., J. Metabol. Research, 1923, iii, 233.

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Howard W. Estill and E. V. McCollumFERROUS SULFATE

DESTRUCTION OF VITAMIN A BYFROM OILS WHICH INHIBITS THE

THE SEPARATION OF A SUBSTANCE

1927, 75:157-162.J. Biol. Chem. 

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