The Chemical Nature of a Vitamin : Part 1

The Vitamine Manual
by Walter H. Eddy

(Page 2 of 8)

The discovery of the existence of an unknown substance is naturally a stimulation to investigation of its nature. In the case of the vitamins we have many researches to this end but extremely meagre results. We are today actually no nearer the goal of identification than we were in 1911 when Funk published his studies on the beri-beri curing type. In brief, we do not know what a vitamin is. Nevertheless, it will be of interest to the student to review the attempts that have been made to isolate these substances for such attempts must furnish the starting point for further studies and their description will help to make clear the nature of the problem involved.

The most extensive investigations have dealt with the first type discovered, namely the vitamin "B" or Funk antineuritic type. In 1911 Cooper and Funk found that the alcoholic extract of rice polishings could be precipitated with phosphotungstic acid and that this procedure permitted them to obtain a fraction that was particularly potent and free from proteins, carbohydrates, and phosphorus. Funk carried this investigation farther and fractioned the phosphotungstic acid precipitate with silver nitrate, following the usual procedure for separating nitrogenous bases. From the silver-nitrate baryta fraction he obtained a crystalline complex melting at 233°C. to which he gave the formula C 17H 20O 7N 2. This substance was curative for pigeons and the fractioning process was applied by him to yeast and other foodstuffs with similar results. From these results Funk believed the vitamin to belong to a class of substances known as the pyrimidine bases. Later, when working with Drummond, Funk was forced to admit that his crystalline complex was not the pure substance, as analysis showed that it contained large amounts of nicotinic acid. His product might well be considered as nicotinic acid contaminated with vitamins.

Suzuki, Shimamura and Odake also used the phosphotungstic precipitation method and claimed to have prepared the crystalline antineuritic substance which they called oryzanin in the form of a crystalline picrate. Drummond and Funk repeated this work, but were unable to confirm the Japanese results. A group of British chemists (Edie, Evans, Moore, Simpson and Webster) obtained an active fraction from yeast and succeeded in separating this into a crystalline basic member belonging to the pyrimidine group which they called torulin.

None of these three preparations have stood the test of analysis however and their curative properties seem to lie in their greater or less contamination with the actual substance, whatever it is. Numerous modifications of the fundamental method for extracting the substance have been planned and executed. Funk for example has shown that if the phosphotungstic precipitate is treated with acetone it is possible to separate it into an acetone soluble and an acetone-insoluble fraction and that the curative fraction is in the latter. McCollum has reported that while ether, benzene and acetone cannot be used to extract the B vitamin from its source, benzene, (and to a slight extent acetone) will dissolve the vitamin if it is first deposited from an alcohol extract on dextrin. These observations have not yielded any further clew to the nature of the substance.

Recently Osborne and Wakeman have proposed a modification which yields a concentrate of high potency. Their method is to add fresh yeast to slightly acidified boiling water and continue the boiling for about five minutes. This process coagulates the proteins that are present and permits their removal by filtration. The protein-free filtrate appears to contain all of the vitamin originally present in the yeast but attempts to precipitate the vitamin fractionally from the evaporated filtrate by means of increasing concentration of added alcohol has been only partially successful. The method however yields a concentrated extract, and Harris has made use of this process to prepare tablets for medicinal purposes.

Seidell and Williams some time ago devised a procedure which seemed to give promise of good results. Their discovery was that when a filtrate from autolysed yeast is prepared, rich in the vitamin, and is shaken with a specially activated fuller's earth (the preparation produced by Lloyd and known as Lloyd's reagent has this power) in a proportion of 50 grams to the liter of extract the vitamin is absorbed by the earth and when the latter is filtered off it carries the vitamin with it. In their process they shake the mixture for about one-half hour and then remove the earth by filtration. Analysis of the yeast liquor after the extraction shows it to contain practically the same solids as originally present but to have lost practically all its vitamin. The latter is firmly attached to the earth and repeated washing with water fails to remove any appreciable amount of vitamin from it. Furthermore the vitamin-activated fuller's earth retains its active vitamin properties for at least a period of two years.

Large amounts of the vitamin can be accumulated in this way and when fed to animals or infants the vitamin is liberated physiologically and produces the usual effects of a vitamin extract. When this discovery was made the discoverers thought that in the fuller's earth they had a means for arriving at the identification of the substance but attempts to recover the vitamin from the earth developed unexpected difficulties. Acids were found to split it off but they also split off aluminium compounds and left an impure mixture little better than the original extract for study. By using a dilute alkali they were able to obtain the substance without aluminium contaminations and by this method they actually obtained some microscopic fibrous needles which were curative. These needles however on recrystallization resulted in the production of a compound contaminated with adenin or rather in adenin contaminated with the curative substance and on standing for some time the adenin crystals gradually lost their curative power. These results led Williams to suggest an interesting hypothesis. By experiments conducted with the hydroxy- pyridines he believed that he had demonstrated a relation between tautomerism or changed space relations in these sort of substances and curative properties. He states his view as follows:

The vitamins contain one or more groups of atoms constituting nuclei in which the curative properties are resident. In a free state these nuclei possess the vitamin activity but under ordinary conditions are spontaneously transformed into isomers which do not possess an antineuritic power. The complementary substances or substituent groups with which these nuclei are more or less firmly combined in nature exert a stabilizing and perhaps otherwise favorable influence on the curative nucleus, but do not themselves possess the vitamin type of physiological potency.

Accordingly it is believed that while partial cleavage of the vitamins may result only in a modification of their physiological properties, by certain means disruption may go so far as to effect a complete separation of nucleus and stabilizer, and if it does so will be followed by a loss of curative power due to isomerism. The basis for the assumption that an isomerization constitutes the final and physiologically most significant step in the inactivation of a vitamin is found in the studies of synthetic antineuritic products. This assumption is supported by evidence ... of the existence of such isomerism in the crystalline antineuritic substances obtainable from brewer's yeast.