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In building up such a diet many experiments have been combined and thanks largely to the efforts of Osborne and Mendel and McCollum in this country, we have a thoroughly standardized procedure even extending to types of cages and care best suited to normal growth and development. For clearer appreciation of the nature of these diets and their preparation we have summarized in the following pages the combinations used by the principal contributors to the subject in this country.

It is at once obvious from the table that the testing value of these basal diets demands the absence of the two vitamins in the protein, carbohydrates and fat fractions. To make sure of this absence various methods have be devised to attain the maximum purity. The authors recommend the following procedure:

a. To purify the casein or other protein used. Boil the protein three successive times (it is assumed that the original is already as pure as it is possible to obtain it by the usual methods of preparation) for an hour each time, with absolute alcohol, using a reflux condenser to prevent loss of alcohol. Filter off the alcohol each time by suction. This process will take off all the adherent fat and hence all the "A" vitamin that might be present. The casein is then dried and ready for use. In certain experiments the authors use meat residues instead of a single protein. This they prepare as follows: Fresh lean round of beef is run through a meat chopper and then ground to a paste in a Nixtamal mill, stirred into twice its weight of water and boiled a few minutes. The solid residue is then strained, using cheese cloth, pressed in the hydraulic press and the cake stirred into a large quantity of boiling water. After repeating this process of washing with hot water the extracted residue is rapidly dried in a current of air at about 60°C. This dried residue may then be further purified with the absolute alcohol treatment as described for casein.

b. To purify the carbohydrate they treat starch in exactly the same way as the casein.

c. To purify the lard. This is melted and poured into absolute alcohol previously heated to 60°C., cooled over night and filtered by suction. This process is repeated three times and the resulting solids dried in a casserole over a steam bath.

d. When butter fat is used to provide a source of "A" vitamin it is prepared as follows: Butter is melted in a flask on a water bath at 45°C. and then centrifugated for an hour at high speed. This results in a separation of the mixture into three layers: (a) Clear fat, containing the "A" vitamin and consisting of 82 to 83 per cent glycerides. This is siphoned off and provides the butter fat named in the diets, (b) An aqueous opalescent layer consisting of water and some of the water-soluble constituents of the milk. This is rejected. (c) A white solid mass consisting of cells, bacteria, calcium phosphate and casein particles. This is also rejected.

Osborne and Mendel's diet

e. When brewers' yeast is used as a source of the "B" vitamin it is first dried over night in an oven at 110°C. and then subjected to the same purification process as the casein and the starch to remove all trace of the "A."

The reasons for the special precautions just described have arisen from some recent work of Daniels and Loughlin who claim that commercial lard contains enough "A" vitamin to permit rats to grow, reproduce and rear young. The British authorities explain their results as not due to the presence of the "A" vitamin in the lard but to a reserve store in the bodies of the animals. They hold that animals may thus store the "A" vitamin but that apparently they have no storage powers for the "B" that are comparable to it. Osborne and Mendel repeated the experiments described by Daniels and Loughlin, using the purification methods just described, but failed to obtain similar results with either commercial lard or with the purified fraction. They question the validity of the British explanation but at the same time reiterate their belief that even commercial lard contains no "A" vitamin. Whatever the explanation of this particular phenomenon it is important that the basal diet be of purified materials and the methods just described supply the procedure necessary to attain that end.

Before discussing the application of these diets to vitamin testing, attention is called to other basal diets developed by McCollum. This worker has paid especial attention to the deficiencies of the cereal grains and in particular to their salt deficiencies. In his basal diets, we find, as would be expected, special combinations particularly suited to the detection of vitamins in such cereals. McCollum has also devised a method of extracting substances to obtain their "B" vitamin and of depositing it on dextrin. For that reason he uses dextrin instead of starch for his carbohydrate and when he wishes to introduce the "B" vitamin it can be done by his method without having to recalculate the carbohydrate component. His method consists of first extracting the source with ether and discarding this extract. Pure ether will not remove the "B" vitamin. The residue is then reextracted several times with alcohol and the alcohol extracts combined. If now these alcohol extracts are evaporated down on a weighed quantity of dextrin the activated dextrin can be used not only to supply the carbohydrate of the ration but also to carry the "B" vitamin of a given source that is under investigation. McCollum's basal diets and salt mixtures are tabulated in the following chart:

McCollum's basal diets and salt mixtures

These diets fall as shown, into two classes. The first group correspond to those of Osborne and Mendel and are available for general testing of any unknown. The cereal combinations are so constituted that all deficiencies of salts are covered and the proportions of the cereal are so selected as to provide the right proportions of protein, fat and carbohydrate. By adding enough butter fat to supply the "A" the deficiency in the "B" can be tested and by adjusting the amounts of "B" on the dextrin the cereal deficiency in this vitamin can be obtained. It is obvious that by substituting lard for the butter fat one could use the same mixture properly supplemented with the "B" to determine the "A" deficiencies of the wheat.

Tags: Vitamins