This was disputed by Hopkins and Neville17 who reported that when rats were fed on the experimental ration of Osborne and Mendel, composed of very carefully purified substances they were unable to obtain any appreciable growth, and the animals, in spite of a satisfactory consumption of food, were all dead before the expiration of 40 days. When as little as 2 cc. of milk per day was given in advance of feeding with the Osborne and Mendel diet, steady growth resulted, although distinctly slower than in the earlier experiments of Hopkins.

Osborne and Mendel18 subsequently admitted that the milk-free diet was less satisfactory than they had at first believed. "It is true that in several instances we have succeeded in keeping grown rats in health and in apparent nutritive equilibrium on purely artificial food mixtures over periods far longer than the experience of our predecessors had led us to expect. But the outcome has never been satisfactory in the sense of extending over what may be considered as the larger portion of the life span of an adult animal." Rats fed on purified protein, lard, starch, and protein-free milk grew excellently for sixty to a hundred days, but sooner or later thereafter normal growth stopped. The animals remained at constant weight for a few days, or grew very slowly, and then suddenly declined and died unless a change was made in the diet. They prepared various mixtures of salts designed to imitate as closely as possible those salts present in milk, and found that while under certain conditions there was very considerable growth, nevertheless in most cases this ceased sooner than that induced by the natural protein-free milk.

The manifest superiority of natural milk foods over either "natural" or "artificial protein-free milk," which had been demonstrated by Hopkins, having been thus confirmed by Osborne and Mendel, these investigators arrived at the conclusion that the essential factor must be associated with the cream which had been removed in the preparation of the "protein-free milk." When the lard which they had used as the fat component of the diet was replaced by butter-fat the result.

17 Bioch. J. 7, 96, 1913.

18 J. Biol. Chem. 15, 311, 1913.

Ether extract of egg yolk was found to have the same stimulating effect as butter fat. They conclude: "The fact that a rat of 40 to 50 grams in weight can grow normally during three months or more on such rations (i. e. those containing neither butter-fat nor egg) then cease to grow but maintain its weight and a well-nourished appearance for weeks, and then resume growth on a ration containing certain naturally occurring foodstuffs would lead one to the belief that on these mixtures of purified food substances the animals run out of some organic complex which is indispensable for further growth but without which maintenance in a fairly good nutritive state is possible".

The conception of a food constituent which is present in natural foods in amounts too small for chemical indentification but which is necessary for the preservation of health had been arrived at from quite a different starting point by another group of workers. The disease of beriberi, so common among the poor classes in the East, had been shown by Eijkman in 189721 to follow from a restricted diet of "polished" or highly milled rice, whereas when the food consisted of home-milled rice, in which the germ and pericarp is retained, there is no trouble from the disease. Eijkman found that poultry fed on polished rice developed a polyneuritis in which the symptoms were very similar to those observed in human beriberi, and finally died if the diet was continued, but that a prompt cure could be effected by feeding the rice-polishings. He suggested that some substance was removed in the polishings which was necessary to overcome the injurious effect of a harmful factor in the diet. Fraser and Stanton 22 concluded that the disease was due to a lack of some substance of high physiological importance, the presence of which is essential to the maintenance of health.

Scurvy was another disease which was recognised as due to a deficient diet, and a very considerable amount of literature was in existence dealing with the remarkable preventive and curative power of fresh fruits and vegetables as compared with preserved foods.23

19 Wise. Agric. Exp. Sta. Res. Bull. No. 17, 1911.

20 J. Biol. Chem. 15, 167, 1913.

21 Arch. f. path. Anat. 1488, 523, 149, 197.

To the curative substance which he isolated from the rice-polishings Funk 24 gave the name of "vitamine " in 1912, and at the same time formulated the hypothesis that the failure to promote normal growth in young animals by proteins containing sufficient amino acids to maintain adult animals in nitrogen equilibrium, is due to a deficiency in vitamins. A deficiency in vitamins produces also a predisposition to many other diseases, among which is rickets.

Osborne and Mendel25 verified and extended their earlier researches on the growth-promoting properties of butter and demonstrated very conclusively that the essential factor was concentrated in the fat. They confirm McCollum and Davis' observation as to the efficiency of egg-yolk in promoting growth, and mentioned that all fats are not alike in this respect. While recognizing the importance of Funk's work they were hesitant at that time about accepting his hypothesis. "It is still rather early to generalize on the role of accessory 'vitamins' when the ideal conditions in respect to the familiar fundamental nutrients and inorganic salts adequate for prolonged maintenance are not completely solved. Speculation is quite justifiable in so far as it directs attention to a new phase that needs to be taken into account.11 Funk has expressed the belief that the substance which promotes growth and must be present in order to avert the cessation of growth, which we have described to occur after a certain period of successful growth on our earlier dietaries, is either identical with, or analogous to, the vitamin which plays the role of an antiscorbutic substance. For this we can as yet find no compelling evidence.

Certainly the nitrogen-free butter-fat, so successful in remedying our growth failures, contains no substance chemically related to the nitrogenous products which have lately been credited with this unique physiological efficiency. Furthermore it is well to bear in mind that it is not improbable that the antineuritic and antiscorbutic constituents of foods are not identical with the substances alleged to assist in maintaining body weight".

22 Studies from Inst, for Med. Res. Fed. Malay States, No. 12, The Etiology of beriberi, 1911.

23 For Summary, see Cooper, Brit. Med. J. No. 2727, p. 722, 1913, and Funk, Ergeb. d. Physiol.-13, 126,1913.

24 Brit. Med. J. 1912, ii, 787, J. State Med. 20, 341,1912. Ergeb. d. physiol. 13, 125,1913. Bioch. Bull. 1915, 4, 904.

25J. Biol. Ghem. 16, 423, 1913.