THE VITAMINE MANUAL

A Presentation of Essential Data

About the

New Food Factors

BY

WALTER H. EDDY

ASSOCIATE PROFESSOR PHYSIOLOGICAL CHEMISTRY

Teachers College, Columbia University



CONTENTS

CHAPTER I

HOW VITAMINES WERE DISCOVERED

CHAPTER II

THE ATTEMPTS TO DETERMINE THE CHEMICAL NATURE OF A VITAMINE

CHAPTER III

THE METHODS USED IN TESTING FOR VITAMINES

CHAPTER IV

THE YEAST TEST FOR VITAMINE B

CHAPTER V

THE SOURCES OF THE VITAMINES

CHAPTER VI

THE CHEMICAL AND PHYSIOLOGICAL PROPERTIES OF THE VITAMINES

CHAPTER VII

HOW TO UTILIZE THE VITAMINES IN DIETS

CHAPTER VIII

AVITAMINOSES OR THE DISEASES THAT RESULT FROM VITAMINE DEFICIENCIES

CHAPTER IX

BIBLIOGRAPHY



PREFACE

The presentation of essential data concerning vitamines to succeeding groups of students has become increasingly difficult with the development of research in this field. The literature itself has assumed a bulk that precludes sending the student to original sources except in those instances when they are themselves to become investigators. The demand on the part of the layman for concise information about the new food factors is increasing and worthy of attention. For all of these reasons it has seemed worth while to collate the existing data and put it in a form which would be available for both student and layman. Such is the purpose of this little book.

It has been called a manual since the arrangement aims to provide the student with working material and suggestions for investigation as well as information. The bibliography, the data in the chapter on vitamine testing, the tables and the subdivision of subject matter have all been arranged to aid the laboratory workers and it is the hope that this plan may make the manual of especial value to the student investigator. The management also separates the details necessary to laboratory investigation from the more purely historical aspects of the subject which we believe will be appreciated by the lay reader as well as the student.

No apologies are made for data which on publication shall be found obsolete. The whole subject is in too active a state of investigation to permit of more than a record of events and their apparent bearing. Whenever there is controversy the aim has been to cite opposing views and indicate their apparent value but with full realization that this value may be profoundly altered by new data.

Since the type of the present manual was set, Drummond of England has suggested that we drop the terminal "e" in Vitamine, since the ending "ine" has a chemical significance which is to date not justified as a termination for the name of the unidentified dietary factors. This suggestion has been generally adopted by research workers and the spelling now in use is Vitamin A, B, or C. It has hardly seemed worth while to derange the entire set up of the present text to make this correction and we have retained the form in use at the time the manuscript was first set up. The suggestion of Drummond, however, is sound and will undoubtedly be generally adopted by the research workers in the subject.

Attempt has been made to cover all the important contributions up to April, 1921. Opportunity has permitted the inclusion of certain data of still later date and undoubtedly other important papers of earlier date will have been overlooked.

It is a pleasure to acknowledge the assistance received in the preparation of the manuscript from Dr. H. C. Sherman, Dr. Mary S. Rose and Dr. Victor La Mer. Their suggestions have been most valuable and greatly appreciated.

WALTER H. EDDY.

Department of Physiological Chemistry, Teachers College, Columbia University, New York City, April, 1921



CHAPTER I

HOW VITAMINES WERE DISCOVERED

In 1911 Casimir Funk coined the name Vitamine to describe the substance which he believed curative of an oriental disease known as beri-beri. This disease is common in Japan, the Philippines and other lands where the diet consists mainly of rice, and while the disease itself was well known its cause and cure had baffled the medical men for many years. Today in magazines, newspapers and street car advertisements people are urged to use this or that food or medicament on the plea of its vitamine content. In less than ten years the study of vitamines has increased to such an extent that it is difficult to find a chemical journal of any month of issue that does not contain one or more articles bearing on the subject. Such a rapid rise to public notice suggests an importance that justifies investigation by the laity as well as the chemist and in the pages that follow has been outlined in simple language the biography of this newest and lustiest of the chemist's children.

Dr. Funk christened one individual but the family has grown since 1911 to three members which for lack of better names are now called vitamines "A," "B," and "C." There are now rumors of another arrival and none dare predict the limits of the family. Had these new substances been limited to their relation to an obscure oriental disease they would have of course commanded the medical attention but it is doubtful whether the general public would have found it worth while to concern themselves. It is because on better acquaintance they have compelled us to reform our ideas on nutrition of both adults and babies and pick out our foods from a new angle, that we accord them the attention they demand and deserve. Granting then, their claim upon our attention, let us review our present knowledge and try to see with just what we are dealing. This will be more easily accomplished if we consider the vitamines first from the historical side and reserve our attention to details of behavior until later.

A limited diet of polished rice and fish is a staple among the peoples of the Orient. When the United States Government took over the Philippine Islands in 1898 it sent there a small group of scientists to establish laboratories and become acquainted with the peculiarities of the people and their troubles. One of the first matters that engaged their attention was the condition of the prisons which were most unsanitary and whose inhabitants were poorly fed and treated. Reforms were put into operation at once and the sanitary measures soon changed these prisons to places not quite so abhorrent to the eye. In trying to improve the diets of the prisoners little change was made in their composition because of the native habits but the reformers saw to it that the rice fed should be clean and white. In spite of these measures the first year saw a remarkable increase in the disease of beri-beri, and the little group of laboratory scientists had at once before them the problem of checking a development that bid fair to become an epidemic. In fact, the logical discoverers of what we now know as the antineuritic vitamine or vitamine "B" should have been this same group of laboratory workers for it was largely due to their work between the years 1900 and 1911 that the ground was prepared for Funk's harvest.

The relation of rice to this disease was more than a suspicion even in 1898. In 1897 a Dutch chemist, Eijkman, had succeeded in producing in fowls a similar set of symptoms by feeding them with polished rice alone. This set of symptoms he called polyneuritis and this term is now commonly used to signify a beri-beri in experimental animals. Eijkman found that two or three weeks feeding sufficed to produce these symptoms and it was he who first showed that the addition of the rice polishings to the diet was sufficient to relieve the symptoms. Eijkman first thought that the cortical material contained something necessary to neutralize the effects of a diet rich in starch. Later however, he changed his view and in 1906 his position was practically the view of today. In that same year (1906) F. Gowland Hopkins in England had come to the conclusion that the growth of laboratory animals demanded something in foods that could not be accounted for among the ordinary nutrients. He gave to these hypothetical substances the name "accessory food factors." To Hopkins and to Eijkman may therefore be justly attributed the credit of calling the world's attention to the unknown substances which Funk was to christen a little later with the name vitamines. Other workers, of course, knew of these experiments of Eijkman and Hopkins and in 1907 two of them, Fraser and Stanton, reported that by extracting rice polishings with alcohol they had secured a product which if added to the diet of a sufferer from beri-beri seemed to produce curative effects. It is obvious that logic would have decreed that some of these workers should be the ones to identify and name the curative material. But history is not bound by the rules of logic and it was so in this case. Another student had been attracted to the problem and was working at the time in Germany where he also became acquainted with Eijkman's results and began the investigation of rice polishings on experimental lines. This student was Casimir Funk and a little later he carried his studies to England where he developed the results that made him the first to announce the discovery of the unknown factor which he christened vitamine. Funk's studies combined a careful chemical fractioning of the extracts of rice polishings with tests for their antineuritic power upon polyneuritic birds, after the manner taught by Eijkman. By carrying out this fractioning and testing he obtained from a large volume of rice polishings a very small amount of a crystalline substance which proved to be curative to a high degree. A little later he demonstrated that this same substance was particularly abundant in brewers' yeast. From these two sources he obtained new extracts and carefully repeated his analytical fractionings. The result was the demonstration that they contained a substance which could be reduced to crystalline form and was therefore worthy of being considered a chemical substance. In 1911, before Fraser and Stanton or any other workers had been able to show to what their curative extracts were due, Funk produced his product, demonstrated its properties and claimed his right to naming the same. At that he barely escaped priority from still another source. The chemists in Japan were naturally interested in this problem and possessed an able worker by the name of Suzuki. Suzuki and his co-workers Odake and Shimamura were engaged in the same fractioning processes with polishings and entirely independently of Funk or other workers they too succeeded in isolating a curative substance and published their discovery the same year as Funk, 1911. Their methods were later shown to be identical up to a certain point. Suzuki called his product "Oryzanin." Funk's elementary analyses had shown the presence of nitrogen in this product and his method of extraction indicated that this nitrogen was present in basic form. For that reason he suggested that his product belonged to a class of substances which chemists call "amines." Since its absence meant death and its presence life what more natural than to call it the Life-amine or Vita-amine. This is the origin of Funk's nomenclature.

Both Funk's original crystals and Suzuki's oryzanin were later shown to be complexes of the curative substances combined with adulterants and we do not yet know just what a vitamine is or whether it is an amine at all but no one since 1911 has been able to get any nearer to the identification than Funk and while he has added much data to his earlier studies he has himself not yet given us the pure vitamine. For that reason it has been suggested by various people that the name vitamine should not be used since it has no sufficient evidence to support it. Hopkins of England had suggested the name "accessory food factors." E. V. McCollum holds that we should call them the "unidentified dietary factors" and added later to this phrase, the terms water-soluble "B" and fat-soluble "A" after the fat soluble form was discovered. Most chemists feel, however, that the purpose of nomenclature is brevity combined with ready recognition of what you are discussing and that it is unnecessary to change the name vitamine until we know exactly what the substances are. The result is that while still a mystery chemically they remain under the name of vitamine and the kinds are distinguished by the McCollum terms "fat-soluble" A, "water-soluble" B, and "C."

We see that beri-beri then was responsible for Funk's adding to our chemical entities a new member but it does not yet appear why this entity concerns our normal nutrition. To get this relation we must turn for a moment to the state of knowledge in 1911 in regard to foods and their evaluation and what was going on in this field of study at the time.

A great advance in measuring food value was the discovery of the isodynamic law. Translated into ordinary language this law states that when a person eats a given amount of a given kind of food, that food may liberate in the body practically the same amount of energy that it would produce if it were burned in oxygen outside of the body. The confirmation of this law permitted us to apply to the measurement of food the same method we had already learned to use in measuring coal. For convenience the physicists devised a heat measure unit for this purpose and naturally called it by a word that means heat, namely, "calorie." Using this unit and applying the isodynamic law it was merely necessary to determine two things; first, how many calories a man produces in any given kind of work, second how many calories a given weight of each kind of food will yield, and then give the man as many calories of food as he needs to meet his requirements when engaged in a given kind of labor. The measurement and tabulation of food values in terms of calories and the investigation of the calorie needs of men and women in various occupations has been one of the great contributions of the past twenty years of nutritional study and to the progress made we owe our power to produce proper rations for every type of worker. Army rations for example are built up of foods that will yield enough calories to supply the needs of a soldier and during the recent war extended studies conducted in training camps all over the United States have shown that when the soldier eats all he wants he will consume on the average about 3600 calories per day. In France the American soldier's ration was big enough to yield him 4200 calories per day if he ate his entire daily allowance.

But calories are not the only necessities. A pound of pure fat will yield all the calories a soldier needs in a day but his language and morals wouldn't stand the strain of such a diet. Neither would his health, for not only does his body demand fuel but also that it be of a special kind. While there are many kinds of foodstuffs, chemical analysis shows that they are mainly combinations of pure compounds of relatively few varieties. The chemists call these proteins, fats, carbohydrates, and salts. Meats, eggs, the curd of milk, etc., are the principal sources of protein. Sugars and starches are grouped together under the name of carbohydrate. By salts is meant mineral matters such as common salt, iron and phosphorus compounds, etc. In selecting foods it was found that the body required that the proportions of these four substances be kept within definite limits or there was trouble. We know now that a man can get along nicely if he eats 50 grams of protein per day and makes up the rest of his calories in carbohydrates and fats, provided that to this is added certain requirements in salts and water.

It is also obvious that the foods given must be digestible and palatable.

We had reached this status some time before 1911. But, a short time before this, there had arisen a controversy as to the relative value of different types of proteins. The animal- vs. vegetable-protein controversy was one of the side shows of this affair. This controversy had led to a careful study of the different kinds of proteins that are found in foodstuffs. Through a brilliant series of chemical investigations for whose description we haven't time or space here, chemists had shown that every protein was built up of a collection of acids which were different in structure and properties, that there were some seventeen of these in all and that any given protein might have present all seventeen or be lacking in one or more and that the proportions present varied for every type of protein. It was then obvious that proteins could not be considered as identities. More than that, it was the necessary task of the food expert to separate all proteins into their acids or building stones and not only show what was present and how much but determine the rle each played in the body. To this task many set their faces and hands.

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

I (from Journ. Biol. Chem., 1913, xv, 167). This chart shows the effect in period III of the addition of an ether extract of egg, 1 gram being given every other day. The diets for periods I-IV were as follows:

Periods . . . . . . . . . . . . . . . I II III IV Salt mixture . . . . . . . . . . . . 6 6 6 6 Casein . . . . . . . . . . . . . . . 18 18 18 18 Lactose . . . . . . . . . . . . . . . 20 0 0 0 Dextrin . . . . . . . . . . . . . . . 0 59 74 74 Starch . . . . . . . . . . . . . . . 31 0 0 0 Agar-agar . . . . . . . . . . . . . . 5 2 2 2 Egg (see above) . . . . . . . . . . . 0 0 * 0 *1 gram extract every other day

II and III (from Journ. Biol. Chem., 1915, xxiii, 231). These charts show the effect (II) of the addition of as little as 2 per cent wheat embryo as sufficient to secure normal growth when it serves as a supply of the B vitamine. Chart III shows that even when the wheat embryo is increased to 30 per cent it is inadequate for growth unless the A is also present. The diets were as follows:

Dextrin . . . . . . . . 69.3 52.8 Salt mixture . . . . . . 3.7 2.6 Butter fat . . . . . . . 5.0 0.0 Agar-agar . . . . . . . 2.0 2.0 Casein . . . . . . . . . 18.0 12.6 Wheat embryo . . . . . . 2.0 30.0]

These results linked up with those of Stepp and Mendel and showed that butter fat and egg yolk fat contained a growth factor which was missing in other fats. McCollum named this the "unidentified dietary factor fat- soluble A."

In the same year F. G. Hopkins in England announced that the addition of 4 per cent of milk to diets consisting of purified nutrients would convert them into growth producers. This was too small an amount to admit of attributing the cause to milk proteins, fats, carbohydrates, or salts. Hopkins therefore suggested the existence of unknown factors in milk of the type to which he had earlier given the name "accessory factors." This work has recently been repeated by Osborne and Mendel who fail to find the high potency in milk ascribed to it by Hopkins but the latter's work, at that time, was accepted without question and became the impetus to important discoveries.

Mendel and Osborne had meanwhile investigated more in detail their milk fractions. They obtained results that confirmed McCollum's findings for butter fat but in addition they showed that by removing all the fat and protein from milk they obtained a residue which played an important part in growth stimulation and that this factor was different from the salts present in the mixture. This specially prepared milk residue they called protein-free milk.

The next few years are a melting pot of investigations. They included some sharp controversies over nomenclature and many apparently contradictory conclusions based on what we now know to be insufficient data. The principal outcome was the identification of the yeast and rice polishing substance with the factor carried by protein-free milk. On the basis of these results Funk put forward the idea that McCollum's butter-fat and egg-yolk factor was merely vitamine which clung to the fats as an adulterant. It was soon shown, however, that butter fat could be obtained that was absolutely free of nitrogen and still be stimulatory to growth. It was therefore clear that whatever the factor present it could not be the Funk vitamine. From out of the smoke of this controversy came an ultimate explanation that was very simple. There were two factors instead of one. McCollum did not discover the presence of the Funk vitamine in his mixtures at first because it was carried by the lactose and he did not know it. Finally, to cut a long story very short, these two factors or vitamines were both found to be essential to growth and in the feeding mixtures that had been used were distributed as follows

Vitamine A Fat-soluble Non-antineuritic Present in butter fat and egg-yolk fat

Vitamine B (Funk's vitamine) Water-soluble Antineuritic Present in protein-free milk, ordinary lactose, yeast and rice polishings

[Illustration: FIG. 2. COMPOSITE CHART OF OSBORNE AND MENDEL PUBLICATIONS

These four charts all show the power of sources of the A vitamine to bring about recovery after failure on diets lacking that vitamine.

I (from Journ. Biol. Chem., 1913-14, xvi, 423). In this group the diet consisted of the following percents: Protein, 18; starch, 26; protein free milk, 28; lard, 28. In the part of the periods marked butter, 18 per cent of butter was substituted for an equal amount of lard.

II (from Jour. Biol. Chem., 1913, xv, 311). Shows recovery on addition of butter fat to a diet containing all the nutrients and artificial protein free milk. These diets contained the following percents: Protein, 18; lactose, 23.8; starch, 26; milk salts, 4.2; total fats, 28.

III (from Journ. Biol. Chem., 1915, xx, 379). These show the effect of various sources of vitamine A such as egg fat, butter fat and oleomargarine. The broken line parts show the failure of laboratory prepared lard to better the commercial lard of the basal diet and the crossed lines the immediate effect when a true source of vitamine A was added. Basal diet: Protein, 18, protein free milk, 28; starch, 24-29; lard, 7-28; other fats, 0-18.

IV (from Journ. Biol. Chem., 1913-14, xvii, 401). This chart shows the failure of almond oil as a source of vitamine A and the prompt recovery when butter fat or cod-liver oil was used. Basal diet: Edestin, 18; starch, 28; protein free milk, 28; lard, 8; almond oil or butter fat or cod-liver oil, 18.]

With these points cleared up each nutrition investigator returned to an analysis of his food mixtures and proceeded to the location in sources of the various factors. The years 1912-1918 are mainly contributory to further knowledge of the properties of these two vitamines, their reactions, source, behavior, etc. In 1912, however, Holst and Frhlich began a study of scurvy that was to culminate later by adding to the list a new member of the family, viz., vitamine "C."

The disease of scurvy and its prevention by use of orange juice potatoes, etc., was a well known phenomenon and to the curative powers of lime juice we owe the name "lime-juicers" as a synonym for the British merchant marine.

Following his discovery of vitamine as the preventative substance to beri- beri, Funk had outlined a theory of "avitaminoses" as the responsible cause of several other types of diseases, including scurvy, rickets, pellagra, and beri-beri. In other words, he suggested that the etiology of these diseases would be found to lie in the lack of the vitamine factors. His views at the time were largely hypothetical since the only one of his avitaminose then demonstrated was beri-beri, but the hypothesis attracted attention and developed a new method of study as it had in matters of normal nutrition.

Between 1907 and 1912 Holst and Frhlich had made exhaustive studies of the causes of scurvy and had reached the conclusion that its cause was due to the absence of some factor, admittedly unknown, but as strongly indicated as in the case of beri-beri. Holst pointed out that a guinea pig restricted to a diet of oats became affected with scurvy. McCollum as well as others were attracted to this problem and in 1918 McCollum stated that scurvy was not due to a lack of a dietary factor but to the absorption from the intestine of the poisonous products resulting from abnormal decomposition of the food and especially of protein food. He studied the guinea pig on an oat diet and drew the conclusion that while it does induce scurvy this result is not due to the absence of any specific factor in the oat diet. He showed that while the oat kernel contains all the chemical elements and complexes necessary for the growth and health of an animal these elements are not in suitable proportions. It lacks certain mineral salts and its content of the "A." vitamine is too low to permit oats alone to give satisfactory growth results. Furthermore its proteins are not of as good quality as those of milk, eggs, and meat. By merely supplementing the oat diet with better protein, salts, and a growth promoting fat, he reported that a guinea pig could be developed normally without further addition and that therefore it was impossible to show that any unknown factor was responsible for the scurvy symptoms. McCollum also reported that the guinea pig could develop scurvy even when his diet was supplemented with fresh milk and since milk was a complete food it followed that the cause of the disease must be sought outside of dietary factors.

Examination of guinea pigs that died of scurvy showed that the cecum was always full of putrefying feces. This observation suggested that the mechanical difficulty these animals have in removing feces from this part of the digestive tract might have something to do with the disease. McCollum and his workers were confirmed in their views by the excellent results that followed the use of a mineral oil as a laxative. Another piece of evidence they gave for their views was that when animals were fed on oats and milk the onset of the scurvy could be delayed by merely adding the cathartic, phenolphthalein, to the mixture. They met the argument of the curative power of orange juice by preparing an artificial juice of citric acid, inorganic salts and cane sugar and showing that this synthetic mixture which held only known substances was capable of protecting animals from scurvy over a long period of time. Without going further into the evidence presented by these workers McCollum was sufficiently convinced of the correctness of his own views to not only state them in his researches but to set them forth at length for public information in his book entitled The Newer Knowledge of Nutrition. In spite of all this evidence his views failed to convince the holders of the vitamine hypothesis. Harden and Zilva and Chick and Hume in England freely criticised his conclusions because whole milk was used in his experiments and no attention paid to the amounts eaten. It was then well known that if enough whole milk is eaten scurvy will not develop. Cohen and Mendel autopsied normal guinea pigs and found that the cecum was nearly always full of feces. On the other hand in autopsies of many pigs dead from scurvy only one-fourth were found to show the impaction of feces claimed by McCollum as cause of the disease. Milk is constipating to guinea pigs. Large amounts of milk should therefore have increased scurvy if the cause stated by McCollum was the real one. On the contrary large amounts of milk prevented scurvy and small doses permitted it to develop. The use of coarse materials as a preventative of constipation failed to prevent scurvy onset. Hess and Unger found that cod-liver oil and liquid petrolatum prevented constipation but failed to prevent scurvy.

The attack on the McCollum view continued from various quarters. Chick and Hume in England examined his grain and milk fed series and showed that those receiving much milk and little grain recovered while those on the reverse diet died. They held that all guinea pigs with scurvy become constipated regardless of the diet. They gave large quantities of dried vegetables well cooked in water, in order to provide bulk, but this did not prevent scurvy and neither did the use of mineral oil. Hess found that in infants with scurvy there is a history of constipation but that while potatoes which are not laxative cure scurvy, malt soups which are laxative permit its development. He found that scurvy in infants is relieved by amounts of orange juice entirely too small to have a marked laxative action and was unable to secure cures with McCollum's artificial orange juice. The most convincing argument was the discovery that orange juice administered intravenously still exerted a curative action which could not in any way be laid to its effect on constipation.

To these attacks McCollum's co-worker, Pitz, suggested a new hypothesis. It was well known that in rats and man the intestinal flora can be changed from a putrefactive form to a non-putrefactive type by feeding milk sugar or lactose. If this were true, as was admitted by all, and the scurvy due to the absorption of putrefactive products, this absorption might still be the causal factor whether constipation was present or absent. To determine this point he fed his guinea pigs on oatmeal to which he added a carbohydrate diet. When the carbohydrate was lactose he was able to cure and prevent scurvy. This evidence was not considered convincing, however, since in his experiments milk was given freely. Furthermore, Cohen and Mendel demonstrated that in their experiments pure lactose neither prevented nor cured scurvy while Harden and Zilva could find no antiscorbutic value in either cane sugar, fructose, or sirup. These authors believed and stated that Pitz's results were entirely attributable to the free use of raw milk.

As this milk factor came increasingly to the attention in the controversy it was natural that students began to rexamine this product more carefully. The vitamine advocates at first believed that its potency as an antiscorbutic was of course due to the vitamines already found present therein, viz., the "A" or the "B." But there began to be difficulties with this view. Hess found that eggs and cod-liver oil, both rich in "A" were of no value as scurvy cures. These experiments eliminated the "A" as the curative factor. Cohen and Mendel used a mixture of yeast and butter in their experiments without success. These experiments threw doubt on the "B" as a curative factor. Studies in heated milk had also shown that the scurvy curing power was destroyed by such procedures as heating and that pasteurized milk was not as good as raw milk. This heating on the other hand did not destroy the antineuritic power of the milk nor its growth- stimulating properties. The combined result of all these studies was to eliminate both the "A" and the "B" as the vitamines with antiscorbutic power without suggesting a better hypothesis than McCollum's.

Gradually, however, it became evident that while scurvy is not prevented by either of these vitamines Funk's hypothesis and Holst and Frhlich's experimental evidence was correct and McCollum's view wrong. The answer lay in the discovery of a third vitamine, water-soluble like "B" but otherwise of entirely different behavior and properties. J. C. Drummond of England finally suggested its inclusion in the family and the name water- soluble "C." As soon as its presence was admitted and its properties roughly determined the way was opened to development of the antiscorbutic vitamine hypothesis and that has now proceeded as rapidly as in the other fields. During the past year many contributions have been made in this field. Sherman, La Mer, and Campbell have recently published results that have taught us much about the measurement of this new member and its manipulation in experimental study of scurvy.

The year 1920, then, has brought us to a recognition of at least three members of the family. Still more recently another deficiency disease has been under investigation and Hess has found in cod-liver oil a remedy for rickets that he cannot believe owes its efficiency to the "A" type. Mellanby of England believes the "A" vitamine is the preventive factor in this disease but Hess's results at least suggest the possibility that the antirachitic vitamine may be separate and distinct from any of those yet named, possibly vitamine "D?" Others are beginning to doubt the identity of the rat growth promoter and the beri-beri curing complexes and feel that the "B" itself may be the name of a group instead of a single entity. All of these features make one feel uncertain to say the least, as to the limits of this vitamine family or of the future possibilities but enough has been given to indicate the historical development to date and we can now turn to more special features of the subject and their bearing on every day affairs.



CHAPTER II

THE ATTEMPTS TO DETERMINE THE CHEMICAL NATURE OF A VITAMINE

The discovery of the existence of an unknown substance is naturally a stimulation to investigation of its nature. In the case of the vitamines we have many researches to this end but extremely meagre results. We are today actually no nearer the goal of identification than we were in 1911 when Funk published his studies on the beri-beri curing type. In brief, we do not know what a vitamine is. Nevertheless, it will be of interest to the student to review the attempts that have been made to isolate these substances for such attempts must furnish the starting point for further studies and their description will help to make clear the nature of the problem involved.

The most extensive investigations have dealt with the first type discovered, namely the vitamine "B" or Funk antineuritic type. In 1911 Cooper and Funk found that the alcoholic extract of rice polishings could be precipitated with phosphotungstic acid and that this procedure permitted them to obtain a fraction that was particularly potent and free from proteins, carbohydrates, and phosphorus. Funk carried this investigation farther and fractioned the phosphotungstic acid precipitate with silver nitrate, following the usual procedure for separating nitrogenous bases. From the silver-nitrate baryta fraction he obtained a crystalline complex melting at 233C. to which he gave the formula C17H20O7N2. This substance was curative for pigeons and the fractioning process was applied by him to yeast and other foodstuffs with similar results. From these results Funk believed the vitamine to belong to a class of substances known as the pyrimidine bases. Later, when working with Drummond, Funk was forced to admit that his crystalline complex was not the pure substance, as analysis showed that it contained large amounts of nicotinic acid. His product might well be considered as nicotinic acid contaminated with vitamines.

Suzuki, Shimamura and Odake also used the phosphotungstic precipitation method and claimed to have prepared the crystalline antineuritic substance which they called oryzanin in the form of a crystalline picrate. Drummond and Funk repeated this work, but were unable to confirm the Japanese results. A group of British chemists (Edie, Evans, Moore, Simpson and Webster) obtained an active fraction from yeast and succeeded in separating this into a crystalline basic member belonging to the pyrimidine group which they called torulin.

None of these three preparations have stood the test of analysis however and their curative properties seem to lie in their greater or less contamination with the actual substance, whatever it is. Numerous modifications of the fundamental method for extracting the substance have been planned and executed. Funk for example has shown that if the phosphotungstic precipitate is treated with acetone it is possible to separate it into an acetone soluble and an acetone-insoluble fraction and that the curative fraction is in the latter. McCollum has reported that while ether, benzene and acetone cannot be used to extract the B vitamine from its source, benzene, (and to a slight extent acetone) will dissolve the vitamine if it is first deposited from an alcohol extract on dextrin. These observations have not yielded any further clew to the nature of the substance.

Recently Osborne and Wakeman have proposed a modification which yields a concentrate of high potency. Their method is to add fresh yeast to slightly acidified boiling water and continue the boiling for about five minutes. This process coagulates the proteins that are present and permits their removal by filtration. The protein-free filtrate appears to contain all of the vitamine originally present in the yeast but attempts to precipitate the vitamine fractionally from the evaporated filtrate by means of increasing concentration of added alcohol has been only partially successful. The method however yields a concentrated extract, and Harris has made use of this process to prepare tablets for medicinal purposes.

Seidell and Williams some time ago devised a procedure which seemed to give promise of good results. Their discovery was that when a filtrate from autolysed yeast is prepared, rich in the vitamine, and is shaken with a specially activated fuller's earth (the preparation produced by Lloyd and known as Lloyd's reagent has this power) in a proportion of 50 grams to the liter of extract the vitamine is absorbed by the earth and when the latter is filtered off it carries the vitamine with it. In their process they shake the mixture for about one-half hour and then remove the earth by filtration. Analysis of the yeast liquor after the extraction shows it to contain practically the same solids as originally present but to have lost practically all its vitamine. The latter is firmly attached to the earth and repeated washing with water fails to remove any appreciable amount of vitamine from it. Furthermore the vitamine-activated fuller's earth retains its active vitamine properties for at least a period of two years. Large amounts of the vitamine can be accumulated in this way and when fed to animals or infants the vitamine is liberated physiologically and produces the usual effects of a vitamine extract. When this discovery was made the discoverers thought that in the fuller's earth they had a means for arriving at the identification of the substance but attempts to recover the vitamine from the earth developed unexpected difficulties. Acids were found to split it off but they also split off aluminium compounds and left an impure mixture little better than the original extract for study. By using a dilute alkali they were able to obtain the substance without aluminium contaminations and by this method they actually obtained some microscopic fibrous needles which were curative. These needles however on recrystallization resulted in the production of a compound contaminated with adenin or rather in adenin contaminated with the curative substance and on standing for some time the adenin crystals gradually lost their curative power. These results led Williams to suggest an interesting hypothesis. By experiments conducted with the hydroxy- pyridines he believed that he had demonstrated a relation between tautomerism or changed space relations in these sort of substances and curative properties. He states his view as follows:

The vitamines contain one or more groups of atoms constituting nuclei in which the curative properties are resident. In a free state these nuclei possess the vitamine activity but under ordinary conditions are spontaneously transformed into isomers which do not possess an antineuritic power. The complementary substances or substituent groups with which these nuclei are more or less firmly combined in nature exert a stabilizing and perhaps otherwise favorable influence on the curative nucleus, but do not themselves possess the vitamine type of physiological potency. Accordingly it is believed that while partial cleavage of the vitamines may result only in a modification of their physiological properties, by certain means disruption may go so far as to effect a complete separation of nucleus and stabilizer, and if it does so will be followed by a loss of curative power due to isomerism. The basis for the assumption that an isomerization constitutes the final and physiologically most significant step in the inactivation of a vitamine is found in the studies of synthetic antineuritic products. This assumption is supported by evidence ... of the existence of such isomerism in the crystalline antineuritic substances obtainable from brewer's yeast.

According to this view the active adenin obtained was not a contamination but an inactive isomer of the active substance. The hydroxy-betaines which Williams prepared in defense of his theory have been repeatedly tested but have in general failed to confirm his view which stands today as an interesting suggestion but without confirmatory evidence. Other attempts by these authors to fraction their alkaline extract of fuller's earth have been unsuccessful. It is of course well known that alkali acts upon the vitamine destructively. On this account the authors of this method operate as rapidly as possible and restore the alkali extract to a neutral or acid medium quickly. The aqueous extract obtained from the earth in this manner has been shown by Seidell to possess only about one-half of the vitamine originally present in the solid but the vitamine in it is shown to be fairly stable. Seidell has not yet determined how long it remains so. Attempts to recover the vitamine from such aqueous solutions have however totally failed to date. To quote Seidell from a recent publication:

By careful evaporation of the solution the products successively obtained show more or less activity by physiological tests but in no case does the resulting material possess the appearance or character which a pure product would be expected to show. Solvents such as benzene, ethylacetate and chloroform fail to effect a separation of active from inactive material. In all fractioning operations the vitamine tends to distribute itself between the fractious rather than to become concentrated in one or the other.

The difficulties encountered by Seidell in this fractioning study have led him to adopt Walsche's idea that vitamines are of the nature of enzymes and hence present all the difficulties of identification and isolation of those substances.

During 1920 Myers and Voegtlin attacked the problem. They have made a discovery that is useful as a separatory process. This that the "B" vitamine is not only soluble in water, but also olive oil and in oleic acid. By shaking an autolysed yeast extract with those solvents in the proportion of 1 cc. of solvent to which 4 cc. of extract the vitamine passes into the oil. When this activated oil is filtered and taken up with eight to ten volumes of ether it in possible to concentrate the ether extract in vacuo and extract from it with 0.1 per cent. HCl an active fraction. Aside from this observation however nothing further has been reported and the possibility of this method of concentration remains yet to be exploited. They did report other methods of fractioning which yielded crystals but failed to produce a pure active substance. Those results add nothing to what has been previously reported except a new method of fractioning and the elimination of the following substances as contributing nothing to vitamine activity (purines, histidine, proteins and albumoses). The crystals they obtained wore contaminated with histamine.

The World War has prevented full knowledge of the work of the German investigators but nothing has appeared that indicates any progress in this field with the exception of a paper by Aberhalden and Schaumann and some work by Hofmeister. The Aberhalden paper yields no new data of any moment and no active substances in pure condition are reported. The reports from Hofmeister are to the effect that he has isolated a very active solution belonging to the pyrimidine series. It yields a crystalline hydrochloride and double salt with gold chloride and has given it the formula C_5H_11NO_2.

The author ban recently been able to obtain a concentrate vitamine from an extract of alfalfa or autolysed yeast with the aid of a carbon specially activated by McKee of Columbia University for the adsorption of basic substance. This adsorbent has been found quite as effective as the fuller's earth and it is possible to recover the vitamine from the carbon with treatment by acid. Glacial acetic and heat are especially favorable for this process. The study of this concentrate has not, however, yet reached a stage where it contributes any real data on the subject but merely provides another method for forming concentrates.

If we were to characterize the present status of the search for the "B" type it might be said to have resolved itself into obtaining concentrates of high potency as the first step in the process and this type of investigation is now going on in many laboratories.

If the data is then meagre in the field of the "B" vitamine it is still more limited in the case of the "A" and the "C." One of the earliest difficulties encountered in the study of the "A" vitamine was the failure of fat solvents to extract the material from its richest vegetable sources. If butter or egg yolk is extracted with ether, the fat obtained is rich in the "A" vitamine. If, however, ether-extraction is applied to green leaves or seeds it removes the oils but these oils contain little or no vitamine. Pressing methods also fail to remove the substance from vegetable sources. For example, if we press or extract cotton seed we obtain the oil but the vitamine is retained in the press cake. McCollum suggested the following explanation for this behavior. His idea is that the "A" vitamine while soluble in fat is so bound up in the vegetable source that extraction methods fail to loosen it. When these vegetables are eaten the vitamine is set free in the process of digestion and being fat-soluble passes into solution in the animal fats. Hence, when these fats contain it in solution, they retain it in the process of extraction while, lacking this separatory process, ether fails to loosen it from the vegetable binding. Recently, however, Osborne and Mendel have presented data in regard to this binding and shown that if for ether we substitute an ether-alcohol mixture the removal of the "A" with the fat is fairly complete even from vegetable sources. They advance the idea that preliminary treatment with alcohol is a process which will materially assist in breaking the attachment of the vitamine and render its removal with the fat solvent effective. Butter-fat rich in the "A" vitamine has been conclusively shown to be free of nitrogen and phosphorus and it is generally assumed that the "A" vitamine is a nitrogen-free and phosphorus free compound. Further than that however we know nothing of its nature.

Concerning the "C" we know only that it is like the "B," water-soluble and we know somewhat of its properties, but nothing of its chemical nature.

One of the greatest difficulties still encountered in the study of chemical fractions is the delay in identification of the active portion. For this purpose we must rely on tests that are far from delicate and time-consuming to a degree. As a result the study of only a few fractions must extend over long periods of time with all the cumulation of difficulties in the way of change in material, etc. that this delay implies. An idea of these difficulties can best be obtained by a review of our present methods for vitamine testing and these methods constitute the subject matter of the next chapter.



CHAPTER III

THE METHODS USED IN TESTING FOR VITAMINES

It will be evident that in the absence of exact tests for a substance which is unknown chemically the problem of detecting its presence must be a matter of indirect evidence. When a chemist is presented with a solution and asked to determine the presence or absence of lead in that solution he knows what he is seeking, what its properties are and how to proceed to not only determine its presence but to measure exactly the amount present. No such possibility is present in a test for vitamines, but this lack of knowledge as to the vitamine structure has not left us helpless. We do know enough of its action to permit us to detect its presence and the technique that has been developed for this purpose is now well standardized and involves no mysteries beyond the comprehension of the layman. In the present chapter is outlined the development of vitamine testing together with a discussion of some of the deficiencies and the problems for the future that these deficiencies suggest.

When Casimir Funk made his original studies of the chemical fractions of an alcohol extract of rice polishings he utilized a discovery of the Dutch chemist Eijkman. We have already referred to this discovery, viz., that by feeding polished rice to fowls or pigeons they could be made to develop a polyneuritis which is identical in symptoms and in response to the curative action of vitamine, to the beri-beri disease. A normal pigeon can be made to eat enough rice normally to develop the disease in about three weeks. The interval can be somewhat shortened by forced feeding. As soon as the symptoms develop the bird is ready to serve as a test for the presence or absence of the antineuritic vitamine. If at this time we have an unknown substance to test it can be administered by pushing down the throat or mixed with the food or an extract can be made and administered intravenously. If the dose is curative, the bird will show the effect by prompt recovery from all the symptoms of the disease in as short a time as six to eight hours. Such a procedure provides a qualitative test which can be made roughly quantitative by varying the dosage until an amount, just necessary to cure the bird in a given time is found and then expressing the vitamine content of the food in terms of this dosage, in such an experiment the value is obviously based on the curative powers of the vitamine source. Another way of applying the test is to determine just how much of the unknown must be added to a diet of polished rice to prevent the onset of polyneuritic symptoms. Such a determination will give the content in terms of preventive dosage. Both methods have been extensively applied and the following tables compiled from the Report of the British Medical Research Committee illustrate both the method and some of its results:

Minimum daily ration that must be added to a diet of polished rice to prevent and to cure polyneuritis in a pigeon of 300 to 400 grams in weight. The weights are given in terms of the natural foodstuff.

AMOUNT NECESSARY FOODSTUFFS AMOUNT NECESSARY FOR DAILY PREVENTION TESTED FOR CURE grams grams 1.5 Wheat germ (raw) 2.5 2.5 Pressed yeast 3.0-6.0[1] 3.0 Egg yolk 60.0[2] 20.0 Beef muscle 140.0[2] 3.0 Dried lentils 20.0[2]

[Footnote 1: Autolysed.] [Footnote 2: Alcohol extract.]

These values illustrate both the method and its value in comparing sources. Unfortunately experience has shown that polyneuritis is amenable to other curative agents to a greater or less extent and it is difficult to be sure whether the curative or preventive dose represents merely the vitamine content of the unknown or is the sum of all the factors present in the curative or preventive material. In comparing the value of different chemical fractions it probably gives a fair enough basis for evaluating their relative power but it is not entirely satisfactory as a quantitive measure of vitamine content.

In America the comparison of vitamine content has been largely based on feeding experiments with the white rat. No other animal has been so well standardized as this one. Dr. Henry Donaldson of the Wistar Institute of Philadelphia has brought together into a book entitled The Rat the accumulated record of that Institution bearing on this animal. This book provides standards for animal comparisons from every view point; weight relation to age, size and age, weight of organs and age, sex and age and weight, etc. This book together with the experience of many workers as they appear in the literature and especially the observations of Osborne and Mendel have made the rat an extremely reliable animal upon which to base comparative data. The omnivorous appetite of the animal, his ready adjustment to confinement, his relatively short life span, all contribute to his selection for experimental feeding tests. Another important reason for his selection is that being a mammal we may reasonably consider that his reactions to foods will be more typical of the human response than would another type, the bird for example. It is perhaps necessary to sound a warning here, however, and point out the danger of too great faith in this comparability of rat and man or in fact of any animal with man. In the case of the rat he has been found useless for the study of "C" vitamine for the simple reason that rats do not have scurvy. In general however his food responses to the vitamines, at least of the "A" and "B" types, have proved, so far as they have been confirmed by infant feeding, to be reasonably comparable.

Provided with the experimental animal the next step was to devise a basal diet which should be complete for growth in every particular except vitamines. Such basal diets have been a process of development. The requirements for such a diet are the following factors:

1. It must be adequate to supply the necessary calories when eaten in amounts normal to the rat's consumption.

2. It must contain the kinds of nutrients that go to make up an adequate diet and in the percents suitable for this purpose.

3. It must contain proteins whose quality is adequate, for growth, i.e., which contain the kinds and amounts of amino acids known to fulfil this function.

4. It must be digestible and palatable.



5. It must be capable of being supplemented by either or both vitamines in response to the particular test it is devised to meet and when both are present in proper amounts it must produce normal growth and serve as a control.



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 vitamines 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" vitamine 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 60C. 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 60C., 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" vitamine it is prepared as follows: Butter is melted in a flask on a water bath at 45C. 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" vitamine 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

(Figures give the per cent of each ingredient in the diet)

INGREDIENTS VITAMINE FREE CONTAINING A ONLY I II III IV V VI VII Purified protein as casein, lactalbumin, edestin, egg albumin, etc. . . . . . . 18.0 18.0 18.0 18.0 18.0 or Meat residue . . . . . 19.6 19.6 Carbohydrates in the form of: Starch . . . . . . . . . . . 29.5 54.0 52.4 29.5 54.0 54.0 52.4 Sucrose . . . . . . . . . . . 15.0 15.0 Fat in the form of: Lard . . . . . . . . . . . 30.0 24.0 24.0 15.0 15.0 15.0 15.0 Butter fat . . . . . . . . . 15.0 9.0 9.0 Egg yolk fat . . . . . . . . 9.0 Cod liver oil . . . . . . . . Salts in the form of: Salt mixture I . . . . . . . 2.5 2.5 or Artificial protein-free milk (Mixt. IV) . . . . . . 4.0 4.0 4.0 4.0 4.0 or Protein-free milk . . . Roughage in the form of: Agar-agar . . . . . . . . . . 5.0 5.0 Total . . . . . . . . . . . . 100.0 100.0 100.0 100.0 100.0 100.0 100.0

INGREDIENTS A ONLY CONTAINING B ONLY VIII IX X XI XII XIII XIV Purified protein as casein, lactalbumin, edestin, egg albumin, etc. . . . . . . 18.0 18.0 18.0 18.0 18.0 18.0 or Meat residue . . . . . 19.6 Carbohydrates in the form of: Starch . . . . . . . . . . . 45.0 45.0 29.5 54.0 52.4 26.0 29.0 Sucrose . . . . . . . . . . . 15.0 Fat in the form of: Lard . . . . . . . . . . . 15.0 27.0 30.0 24.0 24.0 28.0 25.0 Butter fat . . . . . . . . . Egg yolk fat . . . . . . . . Cod liver oil . . . . . . . . 18.0 6.0 Salts in the form of: Salt mixture I . . . . . . . 2.5 or Artificial protein-free milk (Mixt. IV) . . . . . . 4.0 4.0 4.0 4.0 or Protein-free milk . . . 28.0 28.0 Roughage in the form of: Agar-agar . . . . . . . . . . 5.0 Fed Daily "B" vitamine in the form of: 0.2 0.4 0.2 0.04 to gram to gram Dried brewers' yeast 0.6 0.6 gram gram Total . . . . . . . . . . . . 100.0 100.0 100.0 100.0 100.0 100.0 100.0

[Note. Diets I, III and X have been practically discontinued at the present time. Diets II, V and XI are standard. For data on salt mixtures see Osborne, T. B. and Mendel, J. B. The inorganic elements in nutrition, Jour. Biol. Chem. 1918, xxxiv, 131.]

Salt mixture I (after Rohman)

grams Ca3(PO4)2 . . . . . 10.00 K2HPO4 . . . . . . . 37.00 NaCl . . . . . . . . . 20.00 Na citrate . . . . . . 15.00 Mg citrate . . . . . . 8.00 Ca lactate . . . . . . 8.00 Fe citrate . . . . . . 3.00

Total . . . . . . . . 100.00

Artificial protein-free milk

_grams_ CaCO_3 . . . . . . . . 134.8 MgCO_3 . . . . . . . . 24.2 Na_2CO_3 . . . . . . . 34.2 K_2CO_3 . . . . . . . . 141.3 H_3PO_4 . . . . . . . . 103.2 HCl . . . . . . . . . . 53.4 H_2SO_4 . . . . . . . . 9.2 Citric acid: H_2O . . . 111.1 Fe citrate: 1.5H_2O . . 6.34 KI . . . . . . . . . . 0.020 MnSO_4 . . . . . . . . 0.079 NaF . . . . . . . . . . 0.248 K_2Al_2(SO_4)_2 . . . . 0.0245

[N.B.—The ingredients of the artificial protein-free milk are mixed as follows: Making proper allowance for the water in the chemicals the acids are first mixed and the carbonates and citrates added. The traces of KI, MnSO_4, NaF, and K_2Al_2(SO_4)_4 are then added as solutions of known concentration. The mixture is then evaporated to dryness in a current of air at 90 to 100 Centigrade and the residue ground to a fine powder.]

e. When brewers' yeast is used as a source of the "B" vitamine it is first dried over night in an oven at 110C. 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" vitamine to permit rats to grow, reproduce and rear young. The British authorities explain their results as not due to the presence of the "A" vitamine in the lard but to a reserve store in the bodies of the animals. They hold that animals may thus store the "A" vitamine 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" vitamine. 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 vitamine 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 vitamines in such cereals. McCollum has also devised a method of extracting substances to obtain their "B" vitamine 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" vitamine 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" vitamine. 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" vitamine 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

INGREDIENTS VITAMINE FREE "A" ONLY "B" ONLY Casein . . . . . . 18.0 18.0 18.0 18.0 18.0 Same as the vitamine Dextrin . . . . . 57.3 56.3 76.3 78.3 71.3 free diet Lactose . . . . . 20.6 20.0 with "B" added Agar . . . . . . . 2.0 2.0 2.0 2.0 as yeasts as Salt mixture 185 . 2.7 3.7 3.7 3.7 3.7 in the Mendel Butter fat . . . . 5.0 diets or as extracts carried on the dextrin. In the latter case a given amount of dextrin Lactose was later discarded when it was shown carries the to be usually contaminated with the "B" vitamine. extract of a known weight of the source of the "B"

Cereal testing combinations Wheat . . . . . . 56.6 70.0 Wheat embryo . . . 13.3 Corn . . . . . . . 71.3 Oats . . . . . . . 60.0 Skim milk powder . 6.0 Dextrin . . . . . 31.5 76.4 18.0 30.3 20.0 81.0 Salt mixture 185 . 3.7 Salt mixture 314 . 5.3 Salt mixture 318 . 6.9 5.0 Salt mixture 500 . 4.7 Salt mixture ? . . 6.0 Butter fat . . . . 5.0 5.0 5.0 5.0 5.0 5.0 Agar . . . . . . . 2.0 2.0

Salt mixtures NUMBER OF MIXTURES INGREDIENTS 185 314 318 500 211 ? grams grams grams grams grams grams NaCl . . . . . . . . . . . 0.173 1.067 1.400 0.5148 0.520 15.00 MgSO4 anhydrous . . . . . 0.266 1.90 Na2HPO4:H2O . . . . . . 0.347 K2HPO4 . . . . . . . . . 0.954 3.016 2.531 0.3113 34.22 CaH4(PO4)2:H2O . . . . 0.540 0.276 0.89 Ca lactate . . . . . . . . 1.300 5.553 7.058 2.8780 1.971 57.02 Ferrous lactate . . . . . 0.118 K citrate:H2O . . . . . . 0.203 0.710 0.5562 0.799 Na citrate anhydrous . . . 3.70 Ferric citrate . . . . . . 0.100 2.00 Mg citrate . . . . . . . . 7.00 CaCl2 . . . . . . . . . . 0.386 0.2569 CaSO4:2H2O . . . . . . . 0.381 0.578 Fe acetate . . . . . . . . 0.100

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 vitamine 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.

The most prominent worker in the field of the "A" vitamine measurement in America is Steenbock. His basal diets are a combination of those already described.

Steenbock's basal diets per cent Casein (washed with water containing acetic acid) . . . . . 18.0 Dextrin . . . . . . . . . . . . . . . . . . . . . . . . . . 73.3 Ether extracted wheat embryo as source of vitamine "B" . . . 3.0 Salt mixture (McCollum, no. 185) . . . . . . . . . . . . . . 3.7 Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0

This was his original basal diet but later he modified it by adopting the McCollum method of carrying his "B" vitamine on the dextrin. This was usually the alcohol extract of 20 grams of wheat embryo. In the following diets the presence of this extract is indicated by the letter (x) following the dextrin.

INGREDIENTS Casein . . . . . . . . . 18.0 18.0 16.0 18.0 16.0 12.0 Salt 185. . . . . . . . . 4.0 4.0 Salt 32 . . . . . . . . . 4.0 4.0 2.0 2.0 Salt 35 . . . . . . . . . 2.5 2.5 Dextrin (x) . . . . . . . 76.0 71.0 78.0 57.0 Butter fat . . . . . . . 5.0 5.0 Beets . . . . . . . . . . 15.0 Potatoes . . . . . . . . 79.5 Dasheens . . . . . . . . 83.5 Agar . . . . . . . . . . 2.0 2.0 2.0 1.0

Steenbock's salt mixtures

McCollum's no. 185; see page 44. No. 32 consisted of: grams NaCl . . . . . . . . . . . . . . . . . . . . . . . . . 0.202 Anhydrous MgSO4 . . . . . . . . . . . . . . . . . . . 0.311 K2HPO4 . . . . . . . . . . . . . . . . . . . . . . . 1.115 Ca lactate . . . . . . . . . . . . . . . . . . . . . . 0.289 Na2HPO4:l2H2O . . . . . . . . . . . . . . . . . . . 0.526 Ca2H2(PO4)2:H2O . . . . . . . . . . . . . . . . . 1.116 Fe citrate . . . . . . . . . . . . . . . . . . . . . . 0.138 No. 35 consisted of: NaCl . . . . . . . . . . . . . . . . . . . . . . . . . 1.00 CaCO3 . . . . . . . . . . . . . . . . . . . . . . . . 1.5

The very nature of these basal diets suggests their use. In general however their utilization for testing purposes is based on the following principles: Since the basal diet supplies all the requirements of a food except the vitamine for which one is testing, it is simply necessary to add the unknown substance as a given percent of the diet and observe the results. If the amount added is small it is assumed that its addition will not appreciably effect the optimum concentrations of nutrients, etc., and for such experiments no allowances are made for the constituents in the unknown. For example let us assume that we wish to test the value of a yeast cake as a source of "B" vitamine. We first select a sufficient member of rats of about thirty days age to insure protection from individual variations in the animals. The age given is taken as an age when the rats have been weaned and are capable of development away from the mother and as furnishing the period of most active growth. These rats are now placed on one of the basal diets which in this case supplies all the requirements except the "B" vitamine. In this experiment any of the diets of Osborne and Mendel or of McCollum will do that have been labelled "A" only. After a week or so on this diet they will have cleared the system of the influence of previous diets and their weight curves will be either horizontal or declining. If now we make the diet consist of this basal diet plus say 5 per cent of yeast cake, the weight curve for the next few weeks will show whether that amount supplies enough for normal growth, comparison being made with the normal weight curve for a rat of that age.

In this method it is assumed that the amount of yeast cake added will not derange the proportions of protein fat, etc., in the basal diet enough to affect optimum conditions in these respects. This is a curative type of experiment. If we wish to develop a preventive experiment the yeast cake may be incorporated in the diet from the first and the amount necessary to prevent deviation from the normal curve determined. Both methods are utilized, the one checking the other. If however the amount of the substance necessary to supply the vitamine required for normal development is large such addition would of course disturb the proportions of nutrients in the normal diet and in that case analysis must be made of the substance tested to determine its protein, fat, carbohydrate and salt content and the basal diet corrected from this viewpoint so as to retain the optimum proportions of these factors. McCollum's cereal testing combinations are illustrative of such methods applied to cereals. Still another method is to add a small per cent. of the unknown and then add just enough of the vitamine tested to make sure that normal growth results. Such a method gives the results in terms of a known vitamine carrier. For example, if we add to a basal diet, sufficient in all but the "A" vitamine (Steenbock's mixture for example), a small per cent of a substance whose content in "A" is unknown and note that growth fails to result we can then add butter fat until the amount just produces normal growth. If now we know just what amount of butter fat suffices for this purpose when used alone we can calculate the part of the butter which is replaced by the per cent of unknown used. To put this in terms of figures will perhaps make the idea clearer. Let us assume that 5 per cent of butter fat in a given diet is sufficient to supply the "A" necessary for normal growth. Assume that the addition of 5 grams of the unknown in 100 grams of the butter-free diet fails to produce normal growth but that by adding 2 per cent of butter fat normal growth is reached. It is obvious under these conditions that 5 grams of the unknown is equivalent in "A" vitamine content to 5 minus 2 grams of butter fat, i.e., is equivalent to 3 grams of butter fat or expressed in per cents the substance contains 0.6 or 60 per cent of the "A" found in pure butter fat.

Experience has shown that it is dangerous to draw conclusions from experiments of too short duration or to base them on too few animals. For complete data the experiments should be carried through the complete life cycle of the rat, including the reproductive period. Otherwise it may turn out that the amount in the unknown while apparently sufficient for normal growths is incapable of sustaining the drain made in reproduction. It is this consideration that makes the accumulation of authoritative data on vitamine contents of foodstuffs so slow and tedious and one of the reasons why we lack satisfactory tables in this particular at present. Osborne and Mendel raise another point of methodology and believe that more accurate results will be obtained if the source of the vitamine is fed separately than if mixed with the basal diet. It is easily possible that since one of the effects of lack of vitamine, especially of the "B" type, is poor appetite, the amount necessary to produce normal growth may be smaller than would appear from results obtained by mixing it in the basal diet. When so mixed the animals do not get enough to maintain appetite and really decline because they do not eat enough rather than because the amount of vitamine given is inadequate to growth. Details of this kind are matters however that particularly concern the experimentalist and as our purpose here is to merely describe the methodology we may perhaps turn now to other types of testing. Before doing so it is perhaps unnecessary to suggest that in all experiments it is important that the food intake consumed be measured. Also that in all such experimentation it is necessary to run controls on a complete diet rather than to rely too much on standard figures. For this latter purpose it is merely necessary to add to the basal diets the "A" as butter fat and the "B" as dried yeast or otherwise to make them complete. Various special mixtures have been tested out for this purpose and the data already presented supplies the information necessary to construct such control diets. Professor Sherman has given me the following as a control diet on which he has raised rats at normal growth rate to the fifth generation:

One-third by weight of whole milk powder. Two-thirds by weight of ground whole wheat. Add to the mixture an amount of NaCl equal to 2 per cent of the weight of the wheat.

A control mixture based on Osborne and Mendel's data would have the following components:

Meat residue 19.6 per cent or casein 18 per cent. Starch 52.4 per cent or 49 per cent. Lard 15 per cent or 20 per cent. Artificial protein-free milk 4 per cent. Butter fat 9 per cent. Dried yeast 0.2 to 0.6 gram, daily.

The preceding description has applied especially to testing for the presence of the "A" or the "B" vitamine. When we come to the methods of testing for the "C" type it is necessary to change our animal. Rats do not have scurvy but guinea pigs do. The philosophy of the tests for the antiscorbutic vitamines then will be identical with that of the polyneuritic methods with pigeons, viz., preventive and curative tests with guinea pigs. The "C" vitamine is especially sensitive to heat and this fact enables us to secure a "C" vitamine-free diet. La Mer, Campbell and Sherman describe their methods as follows:

First select guinea pigs of about 300 to 350 grams weight. Test these with the basal diet until you secure pigs that will eat the diet. Those that will not eat it at first are of no use for testing purposes, for a guinea pig will starve to death rather than eat food he doesn't like. Having secured pigs that will eat they should on a suitable basal diet die of acute scurvy in about twenty-eight days. Their basal diet is as follows:

per cent Skim milk powder heated for two hours at 110C. in an air bath to destroy the "C" vitamine that might be present. . 30 Butter fat . . . . . . . . . . . . . . . . . . . . . . . . 10 Ground whole oats . . . . . . . . . . . . . . . . . . . . . 59 NaCl . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

They claim that when fruit juice addenda are given in minimal protective doses and calculated to unit weight bases, the results are comparable in precision to those of antitoxin experiments.

Old food should be removed every two days and replaced by new, cups being cleaned at the same time. Since this is a scurvy-producing diet its use is obvious. We can let the pig develop scurvy on it and then test the curative powers of the unknown by adding it to the diet or we can add it to the diet from the first and determine the dose necessary to prevent scurvy; or we can determine its effect in terms of a known antiscorbutic such as orange juice by combining it with measured quantities of the orange juice.

There are other diets that have been given for this purpose, e.g., Holst and Frhlich induced scurvy by restricting animals to an exclusive diet of cereals (oats or rye or barley or corn). Hess and Unger have used hay, oats and water given ad libitum. All of these and others are subject to criticism on the basis that they are not necessarily adequate in other food factors and may therefore not be fair bases for testing the antiscorbutic powers of the unknown combined with them. Abels has recently shown that scurvy increases susceptibility to infections and believes that the scurvy hemorrhages are brought about by the toxic effects of infection. It is therefore desirable in testing for antiscorbutic power that the basal diet be itself as complete as possible in all factors except the absence of "C."

The study of rickets has already progressed to the stage of calculating rickets-producing diets and the methodology is identical with that for scurvy but this phase of testing still lacks evidence of an antirachitic vitamine and in that uncertainty it is hardly worth while to elaborate these diets here. The British diets are all based on Mellanby's contention that the "A" vitamine is the antirachitic vitamine. This view is not yet accepted by American workers.

In concluding this chapter it is sufficient to state that with our present methodology the accumulation of data for evaluating the vitamine content of various foods is still far from satisfactory and from the chemist's viewpoint the methodology is most unsatisfactory as a means of testing fractional analyses obtained in the search for the nature of the substance, both because of the time consumed in a single test and from the difficulty of using the fractions in feeding experiments when these fractions may themselves be poisonous or otherwise unsuited for mixture in a diet. It is obvious therefore that interest is keen in any possibility of devising a test that will be specific, quick and not require modification of the material tested, because of its unsuitability for feeding. In 1919 Roger J. Williams proposed a method that seemed to offer promise in these respects but which is not yet in the form for quantitative use. It offers promise that entitles it to a special chapter for discussion and the next chapter presents the present status of the so- called yeast test for vitamine "B."

Before turning to this test it is well to call attention here to the importance of the experimental animal. Without the polyneuritic fowls we might never have cured beri-beri, the guinea pig made the solution of the scurvy problem possible and if some way of inducing pellagra in an animal can be devised that scourge may yet be eliminated.



CHAPTER IV

THE YEAST TEST FOR VITAMINE "B"

As far back as the days of Pasteur a controversy arose over the power of yeast cells to grow on a synthetic medium composed solely of known constituents. This controversy hinged on a discussion as to whether these media were efficient unless reinforced with something derived from a living organism. In 1901 Wildier in France published an article in which he showed that extracts of organic matter when added to synthetic media had the power to markedly stimulate the growth of yeast organisms. He did not attempt at the time to identify the nature of this stimulatory substance, but since it was derived from living organisms, he called it "Bios." Soon after the discovery of vitamines the bacteriologists began to discover that they or an analogous factor apparently played a part in the growth of certain strains of bacteria, especially the meningococcus. In 1919 Roger Williams working in Chicago University was struck with the bearing of Wildier's work on the vitamine hypothesis and formed the theory that Wildier's "bios" might be the water-soluble vitamine "B." He proceeded to test out this theory and demonstrated that extracts of substances rich in the "B" vitamine had a marked effect on the stimulation of yeast growth. He developed these experiments and devised a method of comparing the growth of yeast cells when stimulated by such extracts. The results were so striking as to appear to justify his view and he then suggested that his method might be used as a test for the measure of "B" vitamine in a given source. William's method consisted essentially in adding the extract of an unknown substance to hanging drops in which were suspended single yeast cells and observing the rate of growth under the microscope. Soon after, Miss Freda Bachman reinvestigated the problem with various types of yeast and found that practically all types of yeast respond to the stimulation of these "bios" extracts. Her method consisted in the use of fermentation tubes and the stimulatory effect was measured by the amount of CO_2 produced in a given time. By this method she confirmed Williams' view that the "bios" of Wildier was apparently identical with vitamine "B" and that most yeasts require this vitamine for their growth. She also suggested that her method might be made the basis of a test for vitamine content. In 1919 Eddy and Stevenson made extended experiments with these two methods in the attempt to improve the technique and make it serve as a quantitative measure. Their experiments served two purposes, first to bring out certain difficulties in the methods of the two authors from the quantitative viewpoint and the development of a technique to correct these difficulties and secondly to add more data bearing on the specificity of the test. Soon after their publication Funk became interested and coming to the same conclusions as to specificity devised a centrifugating method for measuring the yeast growth. Williams also improved his original method and devised a gravimetric method for the same purpose. From the viewpoint of methodology we now have methods which are suitable as quantitive procedures for determining the effect of extracts of unknown substances on yeast growth and hence if the stimulatory substance is vitamine "B," a means of determining within a space of twenty-four hours the approximate content of stimulatory material in a given source. Since the Funk method is the simplest of these and illustrates the principles involved it will suffice to describe that.