The Methods Used In Testing For Vitamins : Part 1

The Vitamine Manual
by Walter H. Eddy

(Page 3 of 10)

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 vitamins, but this lack of knowledge as to the vitamin 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 vitamin 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 vitamin, 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 vitamin. 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 vitamin content of the food in terms of this dosage, in such an experiment the value is obviously based on the curative powers of the vitamin 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.

These values illustrate both the method and its value in comparing sources. Unfortunately experience has shown that polyneuritis is amenable to other curative agents to a greater or less extent and it is difficult to be sure whether the curative or preventive dose represents merely the vitamin content of the unknown or is the sum of all the factors present in the curative or preventive material. In comparing the value of different chemical fractions it probably gives a fair enough basis for evaluating their relative power but it is not entirely satisfactory as a quantitive measure of vitamin content.

In America the comparison of vitamin content has been largely based on feeding experiments with the white rat. No other animal has been so well standardized as this one. Dr. Henry Donaldson of the Wistar Institute of Philadelphia has brought together into a book entitled The Rat the accumulated record of that Institution bearing on this animal. This book provides standards for animal comparisons from every view point; weight relation to age, size and age, weight of organs and age, sex and age and weight, etc. This book together with the experience of many workers as they appear in the literature and especially the observations of Osborne and Mendel have made the rat an extremely reliable animal upon which to base comparative data.

The omnivorous appetite of the animal, his ready adjustment to confinement, his relatively short life span, all contribute to his selection for experimental feeding tests. Another important reason for his selection is that being a mammal we may reasonably consider that his reactions to foods will be more typical of the human response than would another type, the bird for example. It is perhaps necessary to sound a warning here, however, and point out the danger of too great faith in this comparability of rat and man or in fact of any animal with man. In the case of the rat he has been found useless for the study of "C" vitamin for the simple reason that rats do not have scurvy. In general however his food responses to the vitamins, at least of the "A" and "B" types, have proved, so far as they have been confirmed by infant feeding, to be reasonably comparable.

Provided with the experimental animal the next step was to devise a basal diet which should be complete for growth in every particular except vitamins. Such basal diets have been a process of development. The requirements for such a diet are the following factors:

1. It must be adequate to supply the necessary calories when eaten in amounts normal to the rat's consumption.

2. It must contain the kinds of nutrients that go to make up an adequate diet and in the percents suitable for this purpose.

3. It must contain proteins whose quality is adequate, for growth, i.e., which contain the kinds and amounts of amino acids known to fulfil this function.

4. It must be digestible and palatable.

5. It must be capable of being supplemented by either or both vitamins in response to the particular test it is devised to meet and when both are present in proper amounts it must produce normal growth and serve as a control.