The author ban recently been able to obtain a concentrate vitamine 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 vitamine 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" vitamine 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" vitamine 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" vitamine. If, however, ether-extraction is applied to green leaves or seeds it removes the oils but these oils contain little or no vitamine. 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 vitamine is retained in the press cake. McCollum suggested the following explanation for this behavior. His idea is that the "A" vitamine 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 vitamine 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 vitamine and render its removal with the fat solvent effective. Butter-fat rich in the "A" vitamine has been conclusively shown to be free of nitrogen and phosphorus and it is generally assumed that the "A" vitamine 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 vitamine testing and these methods constitute the subject matter of the next chapter.
CHAPTER III
THE METHODS USED IN TESTING FOR VITAMINES
It will be evident that in the absence of exact tests for a substance which is unknown chemically the problem of detecting its presence must be a matter of indirect evidence. When a chemist is presented with a solution and asked to determine the presence or absence of lead in that solution he knows what he is seeking, what its properties are and how to proceed to not only determine its presence but to measure exactly the amount present. No such possibility is present in a test for vitamines, but this lack of knowledge as to the vitamine structure has not left us helpless. We do know enough of its action to permit us to detect its presence and the technique that has been developed for this purpose is now well standardized and involves no mysteries beyond the comprehension of the layman. In the present chapter is outlined the development of vitamine testing together with a discussion of some of the deficiencies and the problems for the future that these deficiencies suggest.
When Casimir Funk made his original studies of the chemical fractions of an alcohol extract of rice polishings he utilized a discovery of the Dutch chemist Eijkman. We have already referred to this discovery, viz., that by feeding polished rice to fowls or pigeons they could be made to develop a polyneuritis which is identical in symptoms and in response to the curative action of vitamine, to the beri-beri disease. A normal pigeon can be made to eat enough rice normally to develop the disease in about three weeks. The interval can be somewhat shortened by forced feeding. As soon as the symptoms develop the bird is ready to serve as a test for the presence or absence of the antineuritic vitamine. If at this time we have an unknown substance to test it can be administered by pushing down the throat or mixed with the food or an extract can be made and administered intravenously. If the dose is curative, the bird will show the effect by prompt recovery from all the symptoms of the disease in as short a time as six to eight hours. Such a procedure provides a qualitative test which can be made roughly quantitative by varying the dosage until an amount, just necessary to cure the bird in a given time is found and then expressing the vitamine content of the food in terms of this dosage, in such an experiment the value is obviously based on the curative powers of the vitamine source. Another way of applying the test is to determine just how much of the unknown must be added to a diet of polished rice to prevent the onset of polyneuritic symptoms. Such a determination will give the content in terms of preventive dosage. Both methods have been extensively applied and the following tables compiled from the Report of the British Medical Research Committee illustrate both the method and some of its results:
Minimum daily ration that must be added to a diet of polished rice to prevent and to cure polyneuritis in a pigeon of 300 to 400 grams in weight. The weights are given in terms of the natural foodstuff.