The investigations, the object of which was to find the cause of the failure of an animal to grow when restricted to wheat as its sole source of nutriment, were carried out in 1913, soon after the publication by Funk of his first work on polyneuritis. In the same year Hopkins called attention to the remarkable effects produced by the addition of small amounts of milk to diets composed of purified food-stuffs. The vitamin hypothesis had just been formulated by Funk (19). McCollum and Davis were, therefore, aware of the relation of a diet of polished rice to experimental beri-beri. They believed, in the light of their experiences with the diet of purified protein, milk sugar, fats and inorganic salts, that such a mixture was capable of inducing growth when certain fats were supplied, but not when others were substituted. The further fact that wheat could be supplemented by purified protein, a growth-promoting fat, and a suitable salt mixture, that is, with food-stuffs of known character, seemed to indicate that there was but a single unidentified substance necessary in the diet. They decided to apply to polished rice the same procedure which had shown so clearly the nature of the dietary deficiencies of wheat. Rice, they reasoned, could be nothing less than a mixture of proteins, starch, fat, and of inorganic salts, similar to that contained in wheat, but in different proportions. It should, therefore, be supplemented with a suitable salt mixture, a purified protein, and a growth-promoting fat, so as to induce growth and maintain animals for a long time in a state of health. This seemed to be a necessary corollary, since they had secured growth and well-being in animals fed strictly upon a mixture of purified protein (casein), starch, milk sugar, butter fat and a mixture of inorganic salts of suitable composition (20).

It was a great surprise to McCollum and Davis to find that polished rice, even when supplemented with the purified protein, casein, butter fat and a salt mixture properly constituted, failed utterly to induce any growth in young rats (21). Not only did they fail to grow, but in the course of a few weeks they developed in some cases a state of paralysis which was suggestive of polyneuritis. Here was an apparent contradiction. The polished rice could be nothing less than a mixture of protein, carbohydrate, fat and salts. The only difference between this and the mixture of supposedly purified food-stuffs with which they had achieved success was in the 20 per cent of milk sugar which the synthetic diet contained. They, therefore, decided to repeat the experiments with the latter mixture, with the milk sugar replaced by starch. It was found that this change in the composition of the food mixture made the difference between success and failure. No growth could be secured when the milk sugar was omitted. Later experiments showed that if milk sugar sufficiently purified by repeated crystallization was added to the purified food mixture it was no longer effective in inducing growth whereas the water from which the sugar had been crystallized would, when evaporated upon the food mixture, render it capable of inducing growth. This made it evident that there is indeed a second dietary essential, of which an animal needs but a very small amount, but which is absolutely necessary for both growth in the young and for the maintenance of health in the adult.