The Vitamine Manual Part 1

The Vitamine Manual.

by Walter H. Eddy.

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 a.s.sumed 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 ma.n.u.script 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 a.s.sistance received in the preparation of the ma.n.u.script 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 j.a.pan, 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 advertis.e.m.e.nts 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 l.u.s.tiest 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 a.n.a.lytical 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 j.a.pan 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 a.n.a.lyses 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 cla.s.s 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 ent.i.ties a new member but it does not yet appear why this ent.i.ty 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 a.n.a.lysis 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 princ.i.p.al 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 s.p.a.ce 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 ident.i.ties. 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 role 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...o...b..rne and Mendel under a grant from the Carnegie Inst.i.tution 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 appet.i.te of the rat the necessary number of calories was also present.

These researches were published as a bulletin (No. 156) by the Carnegie Inst.i.tution 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 b.u.t.ter 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 b.u.t.ter 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 a.s.sumed 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 subst.i.tute 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 subst.i.tuted 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 princ.i.p.al protein from the quant.i.ty 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 a.n.a.lysis of the excreta, the animals steadily declined in weight from the time they were confined to this diet.

The authors had a.s.sumed 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 Wilc.o.c.k 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 const.i.tuent 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 Babc.o.c.k suggested an interesting line of attack and in collaboration with Professors Hart and Humphries, McCollum began a series of studies that have become cla.s.sic 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 a.n.a.lysis 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 a.n.a.lysis 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 b.u.t.ter 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 b.u.t.ter fat.

[Ill.u.s.tration: 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 b.u.t.ter 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 b.u.t.ter 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 b.u.t.ter 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 princ.i.p.al 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 b.u.t.ter-fat and egg-yolk factor was merely vitamine which clung to the fats as an adulterant. It was soon shown, however, that b.u.t.ter fat could be obtained that was absolutely free of nitrogen and still be stimulatory to growth.