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Before doing so it is perhaps unnecessary to suggest that in all experiments it is important that the food intake consumed be measured. Also that in all such experimentation it is necessary to run controls on a complete diet rather than to rely too much on standard figures. For this latter purpose it is merely necessary to add to the basal diets the "A" as butter fat and the "B" as dried yeast or otherwise to make them complete. Various special mixtures have been tested out for this purpose and the data already presented supplies the information necessary to construct such control diets. Professor Sherman has given me the following as a control diet on which he has raised rats at normal growth rate to the fifth generation:

One-third by weight of whole milk powder.
Two-thirds by weight of ground whole wheat.
Add to the mixture an amount of NaCl equal to 2 per cent of the weight of the wheat.

A control mixture based on Osborne and Mendel's data would have the following components:

Meat residue 19.6 per cent or casein 18 per cent.
Starch 52.4 per cent or 49 per cent.
Lard 15 per cent or 20 per cent.
Artificial protein-free milk 4 per cent.
Butter fat 9 per cent.
Dried yeast 0.2 to 0.6 gram, daily.

The preceding description has applied especially to testing for the presence of the "A" or the "B" vitamin. When we come to the methods of testing for the "C" type it is necessary to change our animal. Rats do not have scurvy but guinea pigs do. The philosophy of the tests for the antiscorbutic vitamins then will be identical with that of the polyneuritic methods with pigeons, viz., preventive and curative tests with guinea pigs. The "C" vitamin is especially sensitive to heat and this fact enables us to secure a "C" vitamin-free diet. La Mer, Campbell and Sherman describe their methods as follows:

First select guinea pigs of about 300 to 350 grams weight. Test these with the basal diet until you secure pigs that will eat the diet. Those that will not eat it at first are of no use for testing purposes, for a guinea pig will starve to death rather than eat food he doesn't like. Having secured pigs that will eat they should on a suitable basal diet die of acute scurvy in about twenty-eight days.

They claim that when fruit juice addenda are given in minimal protective doses and calculated to unit weight bases, the results are comparable in precision to those of antitoxin experiments.

Old food should be removed every two days and replaced by new, cups being cleaned at the same time. Since this is a scurvy-producing diet its use is obvious. We can let the pig develop scurvy on it and then test the curative powers of the unknown by adding it to the diet or we can add it to the diet from the first and determine the dose necessary to prevent scurvy; or we can determine its effect in terms of a known antiscorbutic such as orange juice by combining it with measured quantities of the orange juice.

There are other diets that have been given for this purpose, e.g., Holst and Fröhlich induced scurvy by restricting animals to an exclusive diet of cereals (oats or rye or barley or corn). Hess and Unger have used hay, oats and water given ad libitum. All of these and others are subject to criticism on the basis that they are not necessarily adequate in other food factors and may therefore not be fair bases for testing the antiscorbutic powers of the unknown combined with them. Abels has recently shown that scurvy increases susceptibility to infections and believes that the scurvy hemorrhages are brought about by the toxic effects of infection. It is therefore desirable in testing for antiscorbutic power that the basal diet be itself as complete as possible in all factors except the absence of "C."

The study of rickets has already progressed to the stage of calculating rickets-producing diets and the methodology is identical with that for scurvy but this phase of testing still lacks evidence of an antirachitic vitamin and in that uncertainty it is hardly worth while to elaborate these diets here. The British diets are all based on Mellanby's contention that the "A" vitamin is the antirachitic vitamin. This view is not yet accepted by American workers.

In concluding this chapter it is sufficient to state that with our present methodology the accumulation of data for evaluating the vitamin content of various foods is still far from satisfactory and from the chemist's viewpoint the methodology is most unsatisfactory as a means of testing fractional analyses obtained in the search for the nature of the substance, both because of the time consumed in a single test and from the difficulty of using the fractions in feeding experiments when these fractions may themselves be poisonous or otherwise unsuited for mixture in a diet. It is obvious therefore that interest is keen in any possibility of devising a test that will be specific, quick and not require modification of the material tested, because of its unsuitability for feeding. In 1919 Roger J. Williams proposed a method that seemed to offer promise in these respects but which is not yet in the form for quantitative use. It offers promise that entitles it to a special chapter for discussion and the next chapter presents the present status of the so- called yeast test for vitamin "B."

Before turning to this test it is well to call attention here to the importance of the experimental animal. Without the polyneuritic fowls we might never have cured beri-beri, the guinea pig made the solution of the scurvy problem possible and if some way of inducing pellagra in an animal can be devised that scourge may yet be eliminated.

Tags: Vitamins