From the results there has accrued much progress in the evaluation of proteins but an unexpected development was the part played by these investigations in the story of the vitamines.
About 1909-1910 Professors Osborne and Mendel under a grant from the Carnegie Institution began a detailed investigation into the value of purified proteins from various sources. In their experiments they used the white rat as the experimental animal and proceeded to feed these animals a mixture consisting of a single purified protein supplemented with the proper proportions of fat carbohydrate, and mineral salts. Since the food furnished was composed of pure nutrients and always in excess of the appetite of the rat the necessary number of calories was also present. These researches were published as a bulletin (No. 156) by the Carnegie Institution in 1911, the same year that Funk announced his Vitamine discoveries. It was timely in this respect for one of Osborne and Mendel's discoveries was that no matter how efficient the mixture in all the requirements then known to the nutrition expert, the rats failed to grow unless there was added to the diet a factor which they found in milk. In searching for this factor they made a still further discovery for on fractioning the milk they soon learned that the unknown factor was distributed in two different parts of the milk, namely in the butter fat and in the protein free and fat-free whey. The absence of either milk fraction was sufficient to prevent growth. The 1911 publication merely described these results without attempting to explain the nature of the growth producing factors but the vitamine hypothesis of Funk naturally suggested to these authors that their two unknown factors might be similar in nature to his beri-beri curative factor and their announcement may be justly considered a point of junction of nutrition theories with the vitamine hypothesis.
The peculiarity of butter fat as a growth stimulus had been considered from another angle by a German worker, Stepp. In 1909 this student of nutrition had tried to estimate the importance of various types of fats in the same way that was later done with proteins, to determine whether, like proteins, the quality of the fats varied in nutritive efficiency. His experiments were also conducted with white rats and the main outlines of his methods and observations were as follows: Rats fed on a bread and milk diet grew normally. If now the bread and milk mixture was extracted with alcohol-ether the residue was found to be inadequate for growth or maintenance. Stepp assumed that this failure could naturally be ascribed to the removal of the fat by the alcohol-ether mixture. To determine the efficiency of different kinds of fats he then proceeded to substitute in combination with the alcohol-ether extracted diet amounts of purified fats corresponding to what was removed by the alcohol-ether. The results were totally unexpected for none of the purified fats substituted were adequate to secure growth! When, however, he evaporated off his alcohol- ether from the extract of the bread and milk and returned that residue to the diet, growth was resumed as before. The conclusion was obvious, viz., that alcohol-ether takes out of a mixture of bread and milk some factor that is necessary to growth and that factor is not fat but something removed by the extraction with the fat. These results led Stepp to suspect the existence of an unidentified factor but he was unable to identify it as a lipoid. He makes the following statement which is now significant: "It is not impossible that the unknown substance indispensable to life goes into solution in the fats and that the latter thereby become what may be termed carriers for these substances." These studies were published between the years 1909 and 1912 and were therefore concurrent with those of Funk and Osborne and Mendel.
But there was still another set of studies that led up to this vitamine work. In 1907 E. V. McCollum began the study of nutrition problems at the Wisconsin Experiment Station. At the time he was especially interested in two papers that had been published just previous to his entrance into the problem. One of these papers by Henriques and Hansen told how the authors had attempted to nourish animals whose growth was already complete on a mixture consisting of purified gliadin (the principal protein from the quantity viewpoint in wheat), carbohydrates, fats, and mineral salts. In spite of the fact that the nitrogen of this mixture was sufficient to supply the body needs, as proved by analysis of the excreta, the animals steadily declined in weight from the time they were confined to this diet. The authors had assumed that the gliadin was deficient in a substance necessary to growth (lysine) but since their studies were begun only after the animals had reached maximum growth they expected that the growth factor would not be necessary. Why had their animals declined in weight?
The second paper that interested McCollum was by Wilcock and Hopkins. These authors carried out experiments similar to those of the paper just cited but using corn protein (zein) in place of gliadin. This protein had already been shown to be deficient in a chemical constituent known as tryptophan. Animals fed on the zein mixture died in a few days but the inexplicable thing was that when the missing tryptophan was added to the diet the animals lived a little longer but finally declined and died. Why?
McCollum wished to answer this "Why?" These experimenters had complied with every known law of nutrition and yet their mixtures failed to nourish the animals. What was lacking? Earlier work at the Station by Professor Babcock suggested an interesting line of attack and in collaboration with Professors Hart and Humphries, McCollum began a series of studies that have become classic contributions to the vitamine hypothesis and brought this worker into the field as one of the most important contributors to the subject. His initial experiments may be briefly summarized as follows: Young heifer calves weighing 350 pounds at the start and as nearly alike in size and vigor as could be obtained were selected as experimental animals. These were divided into groups and fed with rations so made up as to be alike in so far as chemical analysis could determine, but differing in that the sources of the ration were divided between three plants. One group was supplied with a ration obtained entirely from the wheat plant. A second group derived their ration solely from the corn plant. A third from the oat plant and a fourth or control group from a mixture of oat, wheat and corn. By chemical analysis each group received enough of its particular plant to produce exactly the same amount of protein, fat and carbohydrate and all were allowed to eat freely of salt. All groups ate practically the same amount of feed, and digestion tests showed that there was no difference in the digestibility of the different rations. Exercise was provided by allowing them the run of a yard free of all vegetation. It was a year or more before any distinct change appeared in the different groups. At that time the cornfed animals were in fine condition. On the contrary, the wheat-fed group were rough coated, gaunt in appearance and small of girth. The oat-fed group were better off than the wheat-fed but not in so good shape as the corn-fed. In reproduction the corn-fed animals carried their young well. They were carried for the full term and the young after birth were well formed and vigorous. The wheat-fed mothers gave birth to young from three to five weeks before the end of the normal term. The young were either born dead or died within a few hours after birth. All were much under weight. The oat-fed mothers produced their young about two weeks before the normal period. Of four young, so born, one was born dead, two so weak that they died within a day or two and the fourth was only saved by special measures. The young of the oat-fed mothers were of nearly the same size, however, as those of the corn-fed mothers. After the first reproduction period, the mothers were kept on this diet another year and the following year repeated the same process with identical results. During the first milk-producing period the average production per day was 24.03 pounds per day for the corn-fed, 19.38 pounds for the oat-fed, and 8.04 pounds for the wheat-fed. During the second period it was 28.0, 30.1, and 16.1 pounds per day respectively during the first thirty days.
Every chemical means was now employed to determine the causes of these differences and without success. McCollum then decided to attempt to solve the problem by selecting small animals (the rat was used) and experiment with mixtures consisting of purified proteins from different sources, combined with fats, carbohydrates and mineral salts until a clue was obtained to the nature of the deficiencies. His early results in this direction confirmed the results of other investigators, animals lived no longer on these diets than when allowed to fast. What was missing? Up to 1911 the main result of these experiments had been to call attention to the peculiar deficiencies of cereals and especially in mineral salts, but without unlocking the mystery.
These collateral investigations show how in all parts of this country and on the other side of the ocean events were marching toward the same goal. The year 1911 then is a significant epoch, for from this time the various independent efforts began to link up and the next few years carried us far toward the goal.
In 1912 McCollum was working with a mixture consisting of 18 per cent. purified protein in the form of milk curd or casein, 20 per cent. lactose or milk sugar, 5 per cent. of a fat and a salt mixture made up to imitate the salt content of milk. The remainder of that mixture was starch. With this mixture McCollum found that growth could be produced if the fat were butter fat but not if it were olive oil, lard, or vegetable oils of various sorts. Carrying out the lead here suggested he tried egg yolk fats. They proved as effective as butter fat.
[Illustration: FIG. 1. COMPOSITE CHART OF MCCOLLUM AND DAVIS PUBLICATIONS