McCollum and his associates are now applying the above conceptions to the. study of the dietary deficiencies of individual articles of food. In a recent paper * they present their plan of investigation as follows:

"If a single natural food product fails, to nourish an animal adequately, it may be due to: (a) lack of sufficient protein, or to proteins of poor quality; (b) an unsatisfactory mineral content due either to inadequacy of certain elements in amount, or to unsatisfactory proportions among them; (c) an inadequate supply of the fat soluble A; (d) of the water soluble B; (e) or some toxic substance contained therein. One, two, three, four, or all of these factors may operate in inducing nutritive disturbances.

* McCollum, Simmonds, and Pitz. Journal of Biological Chemistry, Vol. 25, pages 105, 132 (May, 1916).

"It should be obvious that a systematic procedure in which we feed the substance under investigation supplemented with (a) pure protein only, (b) salt mixture additions only, (c) butter fat only, (d) extracts known to carry the water soluble B and as little else as is possible, will reveal whether the failure of nutrition involves one factor only, or more than one. If more than one factor is involved, a similar procedure, but with the addition of all possible combinations of pairs of the isolated food ingredients listed above, followed if need be by another series of feeding experiments in which animals are fed the natural foodstuff supplemented with three such uncomplicated additions, in all possible combinations, and if necessary another experiment in which all four additions are made, will give us results which make it possible to consider the components of our rations in an entirely new light. Provided the foodstuffs contain a toxic substance, special procedures will have to be devised for studying its effects.

"Similar studies must also be made by this method of procedure, with pairs of the important foodstuffs (food materials) in varying proportions, the variation of the mixture including sufficient range to reveal the degree to which the deficiencies of the protein mixture of one grain are corrected by the peculiar quantitative relationships among the amino acids yielded by the proteins of the other grain. The same may be said for the factors other than protein. In this way we shall become able to interpret the biological value of the mixtures of natural foodstuffs which make up the rations which are in common use, in which the attempt is now made to make for safety through variety. We have carried our inquiry into the nature of the dietary deficiencies of several natural products far enough to convince us of the practicability of this method of study."

Following this general plan McCollum and his associates have studied the dietary deficiencies of wheat, wheat embryo, rice, maize, oats, and beans. While some of the results thus obtained have already been cited, it may be well to summarize here the chief findings with reference to each of these food materials in succession. In all cases the experiments were chiefly upon rats.

The wheat kernel when fed alone did not induce normal growth in the experimental animals. Addition of either (1) purified casein, (2) butter fat, or (3) a suitable salt mixture such as to make the total ash of the ration resemble milk ash in composition, was found to improve conditions to some degree in each case, but in no case did such a single addition result in normal growth. Neither could fully satisfactory results be secured by the addition to the wheat ration of any two of these three factors mentioned; but when all three were added, the animals showed complete growth and normal reproduction. Hence McCollum concludes that the wheat kernel is deficient as a food (1) in the poor quality of its protein, (2) in that it furnishes an inadequate supply of "fat soluble A," (3) in that it has an unsatisfactory inorganic content. He also believes that when the diet is chiefly made up of the entire wheat kernel, including embryo, the possibility of a mild toxicity, due to a toxic constituent in the embryo, must also be reckoned with.

Wheat embryo when fed alone did not induce growth although it is rich in proteins of high nutritive efficiency and in water soluble B, and not deficient in fat soluble A. It is deficient in its inorganic content; even so simple a modification as the addition of 2 per cent of calcium lactate to the wheat embryo diet may induce noteworthy growth where otherwise no growth takes place. To an important extent, according to these authors, the failure of rats and swine to grow on diets consisting largely of wheat embryo is attributable to a toxic substance contained therein, which appears to be associated with the fat. Extraction of the fat by ether removes in great measure the toxicity of the embryo without necessarily making the food deficient in the fat soluble A. According to the authors the toxicity may be overcome by the simple addition of casein to the diet. That diet may greatly influence susceptibility to toxicity was reported by Hunt in 1910. Hunt found great differences in susceptibility to acetonitrile poisoning, which differences appeared to be due to diet alone.*

Polished rice as a diet for growth was found to be deficient |n four respects: (1) its protein content seemed too low for maximum growth; (2) it contained inorganic elements in insufficient amounts and also not in proper proportions; (3) it was found deficient in fat soluble A; (4) it lacked water soluble B.

Maize when fed alone induced no appreciable growth, nor could a suitable diet be made by mixing the parts of the maize kernel in different proportions. The proteins of the maize kernel contain all the amino acids essential for growth, but it is held that the proportion of certain of them is such that when this is the sole source of protein the growth is never more than about two thirds normal. The maize diet always requires the addition of a suitable salt mixture (or food of suitable ash content). Also the amount of fat soluble A is insufficient in maize to induce growth at the normal rate. Normal growth and reproduction, however, occurred when maize was supplemented by butter fat, purified casein, and a suitable salt mixture.

* "In extreme cases mice after having been fed upon certain diets may recover from forty times the dose of acetonitrile fatal to mice kept upon other diets. It is, moreover, possible to alter the resistance of these animals at will and to overcome the effects of one diet by combining it with another. . . . The experiments with oats and oatmeal and eggs are of especial interest. In the earlier parts of this paper many experiments were quoted showing that a diet of oatmeal or of oats usually leads to a marked resistance of mice to acetonitrile; the experiments quoted in this section which show that the administration of certain iodine compounds with or subsequently to such a diet further increases this resistance, and the experiments previously reported showing that as far as the resistance toward acetonitrile is concerned iodine exerts its action through the thyroid gland, all point to the conclusion that the resistance caused by an oat diet is in part an effect exerted upon the thyroid. This effect is obtained much more markedly and constantly with young, growing mice. From these experiments and considerations it seems very probable that it is possible to influence, in a specific manner, by diet, one of the most important hormones in the body; this is a comparatively new principle in dietetics and one which may prove of much importance" (Hunt, The Effect of a Restricted Diet and of Various Diets upon the Resistance of Animals to Certain Poisons, pages 56, 73).