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According to this view the active adenin obtained was not a contamination but an inactive isomer of the active substance. The hydroxy-betaines which Williams prepared in defense of his theory have been repeatedly tested but have in general failed to confirm his view which stands today as an interesting suggestion but without confirmatory evidence. Other attempts by these authors to fraction their alkaline extract of fuller's earth have been unsuccessful. It is of course well known that alkali acts upon the vitamin destructively. On this account the authors of this method operate as rapidly as possible and restore the alkali extract to a neutral or acid medium quickly. The aqueous extract obtained from the earth in this manner has been shown by Seidell to possess only about one-half of the vitamin originally present in the solid but the vitamin in it is shown to be fairly stable. Seidell has not yet determined how long it remains so. Attempts to recover the vitamin from such aqueous solutions have however totally failed to date. To quote Seidell from a recent publication:

By careful evaporation of the solution the products successively obtained show more or less activity by physiological tests but in no case does the resulting material possess the appearance or character which a pure product would be expected to show. Solvents such as benzene, ethylacetate and chloroform fail to effect a separation of active from inactive material. In all fractioning operations the vitamin tends to distribute itself between the fractious rather than to become concentrated in one or the other.

The difficulties encountered by Seidell in this fractioning study have led him to adopt Walsche's idea that vitamins are of the nature of enzymes and hence present all the difficulties of identification and isolation of those substances.

During 1920 Myers and Voegtlin attacked the problem. They have made a discovery that is useful as a separatory process. This that the "B" vitamin is not only soluble in water, but also olive oil and in oleic acid. By shaking an autolysed yeast extract with those solvents in the proportion of 1 cc. of solvent to which 4 cc. of extract the vitamin passes into the oil. When this activated oil is filtered and taken up with eight to ten volumes of ether it in possible to concentrate the ether extract in vacuo and extract from it with 0.1 per cent. HCl an active fraction. Aside from this observation however nothing further has been reported and the possibility of this method of concentration remains yet to be exploited. They did report other methods of fractioning which yielded crystals but failed to produce a pure active substance. Those results add nothing to what has been previously reported except a new method of fractioning and the elimination of the following substances as contributing nothing to vitamin activity (purines, histidine, proteins and albumoses). The crystals they obtained wore contaminated with histamine.

The World War has prevented full knowledge of the work of the German investigators but nothing has appeared that indicates any progress in this field with the exception of a paper by Aberhalden and Schaumann and some work by Hofmeister. The Aberhalden paper yields no new data of any moment and no active substances in pure condition are reported. The reports from Hofmeister are to the effect that he has isolated a very active solution belonging to the pyrimidine series. It yields a crystalline hydrochloride and double salt with gold chloride and has given it the formula C 5H 11NO 2.

The author ban recently been able to obtain a concentrate vitamin from an extract of alfalfa or autolysed yeast with the aid of a carbon specially activated by McKee of Columbia University for the adsorption of basic substance. This adsorbent has been found quite as effective as the fuller's earth and it is possible to recover the vitamin from the carbon with treatment by acid. Glacial acetic and heat are especially favorable for this process. The study of this concentrate has not, however, yet reached a stage where it contributes any real data on the subject but merely provides another method for forming concentrates.

If we were to characterize the present status of the search for the "B" type it might be said to have resolved itself into obtaining concentrates of high potency as the first step in the process and this type of investigation is now going on in many laboratories.

If the data is then meagre in the field of the "B" vitamin it is still more limited in the case of the "A" and the "C." One of the earliest difficulties encountered in the study of the "A" vitamin was the failure of fat solvents to extract the material from its richest vegetable sources. If butter or egg yolk is extracted with ether, the fat obtained is rich in the "A" vitamin. If, however, ether-extraction is applied to green leaves or seeds it removes the oils but these oils contain little or no vitamin. Pressing methods also fail to remove the substance from vegetable sources. For example, if we press or extract cotton seed we obtain the oil but the vitamin is retained in the press cake. McCollum suggested the following explanation for this behavior. His idea is that the "A" vitamin while soluble in fat is so bound up in the vegetable source that extraction methods fail to loosen it.

When these vegetables are eaten the vitamin is set free in the process of digestion and being fat-soluble passes into solution in the animal fats. Hence, when these fats contain it in solution, they retain it in the process of extraction while, lacking this separatory process, ether fails to loosen it from the vegetable binding. Recently, however, Osborne and Mendel have presented data in regard to this binding and shown that if for ether we substitute an ether-alcohol mixture the removal of the "A" with the fat is fairly complete even from vegetable sources. They advance the idea that preliminary treatment with alcohol is a process which will materially assist in breaking the attachment of the vitamin and render its removal with the fat solvent effective. Butter-fat rich in the "A" vitamin has been conclusively shown to be free of nitrogen and phosphorus and it is generally assumed that the "A" vitamin is a nitrogen-free and phosphorus free compound. Further than that however we know nothing of its nature.

Concerning the "C" we know only that it is like the "B," water-soluble and we know somewhat of its properties, but nothing of its chemical nature.

One of the greatest difficulties still encountered in the study of chemical fractions is the delay in identification of the active portion. For this purpose we must rely on tests that are far from delicate and time-consuming to a degree. As a result the study of only a few fractions must extend over long periods of time with all the cumulation of difficulties in the way of change in material, etc. that this delay implies. An idea of these difficulties can best be obtained by a review of our present methods for vitamin testing and these methods constitute the subject matter of the next chapter.

Tags: Vitamins