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diff --git a/.gitattributes b/.gitattributes new file mode 100644 index 0000000..6833f05 --- /dev/null +++ b/.gitattributes @@ -0,0 +1,3 @@ +* text=auto +*.txt text +*.md text diff --git a/7983-8.txt b/7983-8.txt new file mode 100644 index 0000000..a1487c3 --- /dev/null +++ b/7983-8.txt @@ -0,0 +1,5403 @@ +The Project Gutenberg EBook of The Vitamine Manual, by Walter H. Eddy + +Copyright laws are changing all over the world. Be sure to check the +copyright laws for your country before downloading or redistributing +this or any other Project Gutenberg eBook. + +This header should be the first thing seen when viewing this Project +Gutenberg file. Please do not remove it. Do not change or edit the +header without written permission. + +Please read the "legal small print," and other information about the +eBook and Project Gutenberg at the bottom of this file. Included is +important information about your specific rights and restrictions in +how the file may be used. You can also find out about how to make a +donation to Project Gutenberg, and how to get involved. + + +**Welcome To The World of Free Plain Vanilla Electronic Texts** + +**eBooks Readable By Both Humans and By Computers, Since 1971** + +*****These eBooks Were Prepared By Thousands of Volunteers!***** + + +Title: The Vitamine Manual + +Author: Walter H. Eddy + +Release Date: April, 2005 [EBook #7983] +[Yes, we are more than one year ahead of schedule] +[This file was first posted on June 8, 2003] + +Edition: 10 + +Language: English + +Character set encoding: ISO-Latin-1 + +*** START OF THE PROJECT GUTENBERG EBOOK THE VITAMINE MANUAL *** + + + + +Produced by Juliet Sutherland, Richard Prairie, Charles Franks +and the Online Distributed Proofreading Team. + + + + +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 rôle 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 Fröhlich +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 Fröhlich 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 reëxamine 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 Fröhlich'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 233°C. to which he gave the formula +C_17H_20O_7N_2. 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. + +[Illustration: FIG. 3. TWO TYPES OF EXPERIMENT CAGES DEVISED BY OSBORNE +AND MENDEL + +These are manufactured by the Herpich Co. of New Haven, Conn.] + + 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. + +[Illustration: FIG. 4. A METABOLISM CAGE DEVISED FOR USE IN THE AUTHOR'S +LABORATORY + +The cages being bottomless are readily cleaned. They are set on circles of +wire mesh over galvanized iron funnels permitting urine and feces to pass +through. A second screen over the collecting cup and of fine mesh +separates the feces from urine and also collects scattered food.] + +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. + +[Illustration: FIG. 5. ILLUSTRATING THE USE OF THE CHATILLON SCALE FOR +RAPID WEIGHING OF ANIMALS + +The dial is so made that it can be set to counterbalance the weight of the +cage and the weights read directly. This is also used for weighing food.] + +[Illustration: FIG. 6. SAMPLE LABORATORY RECORD] + +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 60°C. 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 60°C., 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 +45°C. 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_ +Ca_3(PO_4)_2 . . . . . 10.00 +K_2HPO_4 . . . . . . . 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 110°C. 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 +MgSO_4 anhydrous . . . . . | 0.266 | | | | | 1.90 +Na_2HPO_4:H_2O . . . . . . | 0.347 | | | | | +K_2HPO_4 . . . . . . . . . | 0.954 | 3.016 | 2.531 | 0.3113| | 34.22 +CaH_4(PO_4)_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:H_2O . . . . . . | | 0.203 | 0.710 | 0.5562| 0.799 | +Na citrate anhydrous . . . | | | | | | 3.70 +Ferric citrate . . . . . . | | 0.100 | | | | 2.00 +Mg citrate . . . . . . . . | | | | | | 7.00 +CaCl_2 . . . . . . . . . . | | 0.386 | | 0.2569| | +CaSO_4:2H_2O . . . . . . . | | 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 MgSO_4 . . . . . . . . . . . . . . . . . . . 0.311 + K_2HPO_4 . . . . . . . . . . . . . . . . . . . . . . . 1.115 + Ca lactate . . . . . . . . . . . . . . . . . . . . . . 0.289 + Na_2HPO_4:l2H_2O . . . . . . . . . . . . . . . . . . . 0.526 + Ca_2H_2(PO_4)_2:H_2O . . . . . . . . . . . . . . . . . 1.116 + Fe citrate . . . . . . . . . . . . . . . . . . . . . . 0.138 +No. 35 consisted of: + NaCl . . . . . . . . . . . . . . . . . . . . . . . . . 1.00 + CaCO_3 . . . . . . . . . . . . . . . . . . . . . . . . 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 110°C. 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 Fröhlich 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. + +_Funk method of yeast test with Eddy and Stevenson modification_ + +1. To a basal diet of 9 cc. of sterile culture medium such as a von Nageli +solution [Footnote: von Nageli's solution consists of the following +ingredients NH_4NO_3, 1 gram; Ca_3(PO_4)_2, 0.005 gram; MgSO_4, 0.25 gram +dextrose 10.0 grams made up to 100 cc. with distilled water. Other culture +media may be used and such combinations will be found in any text on +yeasts. They all permit a certain amount of growth but all are apparently +stimulated by the addition of vitamine extracts.] in a sterile test tube +is added 1 cc. of the sterile, neutral, watery extract of the source of +the vitamine. A pure culture of Fleischman's yeast (Funk prefers brewer's +yeast) is maintained on an agar slant and twenty-four hours before the +test is to be made, a transplant is made to a fresh agar slant. One +standardized platinum loopful of the twenty-four hour yeast growth is then +used to inoculate the contents of the tube, the tube stoppered with cotton +and incubated for from twenty-four to seventy-two hours at a temperature +of 31°C. The seventy-two hour incubation period yields nearly optimum +growth for this purpose. + +2. At the end of this time the yeasts are killed by plunging the tube in +water heated to 80°C. and maintained at this temperature for fifteen +minutes. The contents of the tubes are then poured into a Hopkins +centrifuge tube which has a capillary tip graduated in hundredths of a +cubic centimeter. After twenty minutes centrifugating at a speed of about +2400 revolutions per minute the yeasts in the solution have all been +packed into the tip and the volume can then be read accurately to +thousandths of a cubic centimeter (with the aid of a scale and magnifier). +With a control tube containing 9 cc. of the sterile media and 1 cc. of +distilled water in place of the 1 cc. of extract a comparison can be +obtained which is an accurate measure of the stimulatory effect of the +extract. If this stimulus is due purely to vitamine it is obvious that +this procedure would enable us to compare extracts of known weights of and +arrive at comparisons which would be measures of their vitamine content. +In other words the procedure is now in a satisfactory form for testing and +its value depends merely upon our ability to show that the stimulus given +the yeast is due solely to vitamine "B." + +The interest of the vitamine student in this test will be easily +understood for it is so simple of manipulation and so rapid in producing +results that it is the nearest approach to a chemical test of satisfactory +nature yet proposed but unfortunately evidence soon began to accumulate to +show that the stimulation produced by extracts of various sources is not a +matter of pure vitamine. If we plot a curve of stimulation for various +dilutions of a given extract we find that the stimulation is not directly +proportional to the concentration of vitamine present but is a composite +of several factors. The chart derived from experiments by Eddy and +Stevenson shows the general nature of this curve. Other experimenters have +reached similar results and some have gone so far as to maintain that the +stimulation is not due to vitamine "B" at all. It is therefore evident +that until this controversy is settled the yeast test cannot be used for +the purpose proposed. Our own experiments at present make us still firm in +our belief that _one_ of the factors and perhaps the most important +factor in the stimulation effect is the vitamine but until we can devise a +basal medium that is comparable to that used in rat feeding experiments, +i.e., one that contains all the elements for optimum growth of yeasts +except vitamine "B" it will be unsafe to draw conclusions from the test as +to vitamine content. It may be possible to so treat our extracts as to +eliminate from them all other stimuli except the vitamine or to destroy +the vitamine in them and thus permit the comparison of an extract with the +vitamine destroyed against one in which it is present and thus arrive at +the result desired. At any rate all we can say at present is that the +yeast test is unreliable as a measure of vitamine content but that if it +can be made quantitative its advantages are so great that it is very much +worth while to continue work upon it until it is certain that it cannot be +made to produce the desired result. + +[Illustration: FIG. 7. GROWTH RATE OF YEAST UNDER ALFALFA EXTRACT +STIMULATION + +This chart shows the effect of varying concentrations of an alfalfa +extract on the growth rate of the yeast cell. The rate of growth was +determined after the Funk method by centrifuging the cells after seventy- +two hours incubation and measuring the volume in cubic centimeters. The +shape of the curve shows that this method will not give comparative +results unless the extracts tested are dilute enough for the +determinations to fall in the steep part of the curve.] + +Another reason for our attention to this test is that if it can be made to +show vitamine effect it provides an excellent medium for investigation of +vitamine "B" reactions, and a method for studying the effect of the +vitamine upon the protoplasm of a single cell. + + + +CHAPTER V + + +THE SOURCES OF THE VITAMINE + +Having now considered the general principles involved in vitamine testing +we may justly ask what information they have yielded us in regard to the +distribution of the vitamines in nature. If we must include vitamines in +our diets it is important to know how to select foods on this basis, hence +a classification of them on the ground of vitamine distribution becomes +essential. The newness of the subject and the limited tests that have been +made as well as the uncertainty residing in the test results make any +classifications presented more or less approximations but we present such +attempts as have been made, with the understanding that these tabulations +are merely guides and not quantitative measurements in the sense that +tables giving calorie values of protein, fat and carbohydrate content are. +The following table (1) has been freely copied from a report of the +British Medical Research Committee to which acknowledgment is hereby +given. + + +TABLE 1 + +_Pages 50 and 61 of the British Medical Research Committee's report_ +__________________________________________________________________________ + | | | +CLASSES OF FOODSTUFFS |VITAMINE "A"|VITAMINE "B"|VITAMINE "C" +___________________________________|____________|____________|____________ + | | | +_Fats and oils:_ | | | + Butter . . . . . . . . . . . . | +++ | 0 | + Cream . . . . . . . . . . . . . | ++ | 0 | + Cod-liver oil . . . . . . . . . | +++ | 0 | + Mutton and beef fat or suet . . | ++ | | + Lard . . . . . . . . . . . . . | 0 | | + Olive oil . . . . . . . . . . . | 0 | | + Cotton seed oil . . . . . . . . | 0 | | + Cocoanut oil . . . . . . . . . | 0 | | + Cocoa-butter . . . . . . . . . | 0 | | + Linseed oil . . . . . . . . . . | 0 | | + Fish oil, whale oil, herring | | | + oil, etc. . . . . . . . . . . | ++ | | + Hardened fats (hydrogenated) | | | + of animal or vegetable origin | 0 | | + Margarine from animal fat . . . | In propor- | | + | tion to | | + | animal | | + | fat used | | + Margarine from vegetable fat | | | + or lard . . . . . . . . . . . | 0 | | + Nut butters . . . . . . . . . . | + | | +_Meat, fish, etc.:_ | | | + Lean meat (beef, mutton, etc.) | + | + | + + Liver . . . . . . . . . . . . . | ++ | ++ | + + Kidneys . . . . . . . . . . . . | ++ | + | + Heart . . . . . . . . . . . . . | ++ | + | + Brain . . . . . . . . . . . . . | + | ++ | + Sweetbreads . . . . . . . . . . | + | ++ | + Fish, white . . . . . . . . . . | 0 | Very slight| + | | if any | + Fish fat (salmon, herring, etc.)| ++ | Very slight| + | | if any | + Fish roe . . . . . . . . . . . | + | ++ | + Tinned meats . . . . . . . . . | ? | Very slight| 0 +_Milk, cheese, etc.:_ | | | + Milk, cow's whole raw . . . . . | ++ | + | + + Milk, cow's skim . . . . . . . | 0 | + | + + Milk, cow's dried whole . . . . | Less than | + | Less than + | ++ | | + + Milk, cow's boiled whole . . . | ? | + | Less than + | | | + + Milk, cow's condensed sweetened | + | + | + Cheese, whole milk . . . . . . | + | | Less than + | | | + + Cheese, skim milk . . . . . . . | 0 | | + Eggs, fresh . . . . . . . . . . | ++ | +++ | 0? + Eggs, dried . . . . . . . . . . | ++ | +++ | 0? +_Cereals, pulses, etc.:_ | | | + Wheat, maize, rice (whole germ) | + | + | 0 + Wheat, maize, rice germ . . . . | ++ | +++ | 0 + Wheat, maize, rice bran . . . . | 0 | ++ | 0 + White wheat flour, pure corn | | | + flour, polished rice, etc. . | 0 | 0 | 0 + Custard powders, egg substi- | | | + tutes prepared from cereal | | | + products . . . . . . . . . . | 0 | 0 | 0 + Linseed, millet . . . . . . . . | ++ | ++ | 0 + Dried peas, lentils, etc. . . . | | ++ | + Pea-flour, kilned . . . . . . . | | 0 | 0 + Soy beans, haricot beans . . . | + | ++ | 0 + Germinated pulses or cereals . | + | ++ | ++ +_Vegetables and fruits:_ | | | + Cabbage, fresh, raw . . . . . . | ++ | + | +++ + Cabbage, fresh, cooked . . . . | | + | + + Cabbage, dried . . . . . . . . | + | + |Very slight + Cabbage, canned . . . . . . . . | | |Very slight + Swedes, raw expressed juice . . | | | +++ + Lettuce . . . . . . . . . . . . | ++ | + | + Spinach, dried . . . . . . . . | ++ | + | + Carrots, fresh, raw . . . . . . | + | + | + + Carrots, dried . . . . . . . . |Very slight | | Less than + | | | + + Beetroot, raw, expressed juice | + | + | + Potatoes, raw . . . . . . . . . | | | + + Potatoes, cooked . . . . . . . | | | ++ + Beans, fresh scarlet runners raw| | | + Lemon juice, fresh . . . . . . | | | +++ + Lemon juice, preserved . . . . | | | + Lime juice, fresh . . . . . . . | | | ++ + Lime juice, preserved . . . . . | | |Very slight + Orange juice, fresh . . . . . . | | | +++ + Raspberries . . . . . . . . . . | | | ++ + Apples . . . . . . . . . . . . | | | + + Bananas . . . . . . . . . . . . | + | + |Very slight + Tomatoes, canned . . . . . . . | | | ++ + Nuts . . . . . . . . . . . . . | + | ++ | +_Miscellaneous:_ | | | + Yeast dried . . . . . . . . . . | ? | +++ | + Yeast extract and autolysed . . | ? | +++ | 0 + Meat extract . . . . . . . . . | 0 | 0 | 0 + Malt extract . . . . . . . . . | | + in some | + | | specimens | + Beer . . . . . . . . . . . . . | | 0 | 0 + Honey . . . . . . . . . . . . . | | + | +___________________________________|____________|____________|____________ + ++++ indicates abundant; ++ relatively large; + present in small amount; +0 absent. + +The following table (2) has been compiled from a review of both British +and American data and represents a rather more complete classification +than the British report. The four plus system has also been used to permit +more complete comparisons. + +TABLE 2 + +_________________________________________________________________________ + | | | + FOODSTUFF | "A" | "B" | "C" +____________________________________|___________|___________|____________ + | | | +_Meats_: | | | + Beef heart . . . . . . . . . . . | + | + | ? + Brains . . . . . . . . . . . . . | ++ | +++ | +? + Codfish . . . . . . . . . . . . | + | + | ? + Cod testes . . . . . . . . . . . | + | | + Fish roe . . . . . . . . . . . . | + | ++ | ? + Herring . . . . . . . . . . . . | ++ | ++ | ? + Horse meat . . . . . . . . . . . | ++ | ++ | + Kidney . . . . . . . . . . . . . | ++ | ++ | + Lean muscle . . . . . . . . . . | 0 | 0 | +? + Liver . . . . . . . . . . . . . | + | + | +? + Pancreas . . . . . . . . . . . . | 0 | +++ | + Pig heart . . . . . . . . . . . | + | + | ? + Placenta . . . . . . . . . . . . | + | | + Thymus (sweetbreads) . . . . . . | 0 | 0 | 0 +_Vegetables:_ | | | + Beet root . . . . . . . . . . . | + | + | ++ + Beet root juice . . . . . . . . | ? | Little | +++ + Cabbage, dried . . . . . . . . . | +++ | +++ | + + Cabbage, fresh . . . . . . . . . | +++ | +++ | ++++ + Carrots . . . . . . . . . . . . | +++ | +++ | ++ + Cauliflower . . . . . . . . . . | ++ | +++ | ++ + Celery . . . . . . . . . . . . . | ? | +++ | ? + Chard . . . . . . . . . . . . . | +++ | ++ | ? + Dasheens . . . . . . . . . . . . | + | ++ | ? + Lettuce . . . . . . . . . . . . | ++ | ++ | ++++ + Mangels . . . . . . . . . . . . | ++ | ++ | ? + Onions . . . . . . . . . . . . . | ? | +++ | +++ + Parsnips . . . . . . . . . . . . | ++ | +++ | + Peas (fresh) . . . . . . . . . . | + | ++ | +++ + Potatoes . . . . . . . . . . . . | 0 | +++ | ++ + Potatoes (sweet) . . . . . . . . | +++ | ++ | ? + Rutabaga . . . . . . . . . . . . | | +++ | + Spinach . . . . . . . . . . . . | +++ | +++ | +++ +_Cereals:_ | | | + Barley . . . . . . . . . . . . . | + | +++ | ? + Bread (white) . . . . . . . . . | + | +? | + Bread (whole meal) . . . . . . . | + | +++ | ? + Maize (yellow) . . . . . . . . . | + | +++ | ? + Maize (white) . . . . . . . . . | 0 | +++ | ? + Oats . . . . . . . . . . . . . . | + | +++ | 0 + Rice polished . . . . . . . . . | 0 | 0 | 0 + Rice (whole grain) . . . . . . . | + | +++ | 0 + Rye . . . . . . . . . . . . . . | + | +++ | 0 + Corn embryo . . . . . . . . . . | | +++ | + Corn (kaffir) . . . . . . . . . | | +++ | + Corn (see maize) . . . . . . . . | | | + Corn pollen . . . . . . . . . . | | ++ | + Malt extract . . . . . . . . . . | 0 | 0 | 0 + Wheat bran . . . . . . . . . . . | 0 | + | 0 + Wheat embryo . . . . . . . . . . | ++ | +++ | 0 + Wheat endosperm . . . . . . . . | 0 | 0 | 0 + Wheat kernel . . . . . . . . . . | + | +++ | 0 +_Other seeds:_ | | | + Beans, kidney . . . . . . . . . | | +++ | + Beans, navy . . . . . . . . . . | | +++ | 0 + Beans, soy . . . . . . . . . . . | + | +++ | 0 + Cotton seed . . . . . . . . . . | ++ | +++ | + Flaxseed . . . . . . . . . . . . | ++ | +++ | + Hemp seed . . . . . . . . . . . | ++ | +++ | + Millet seed . . . . . . . . . . | ++ | +++ | + Peanuts . . . . . . . . . . . . | + | ++ | + Peas (dry) . . . . . . . . . . . | +? | ++ | 0 + Sun flower seeds . . . . . . . . | + | | +_Fruits:_ | | | + Apples . . . . . . . . . . . . . | | ++ | ++ + Bananas . . . . . . . . . . . . | ? | ++ | ++ + Grapefruit . . . . . . . . . . . | | +++ | +++ + Grape juice . . . . . . . . . . | | + | + + Grapes . . . . . . . . . . . . . | 0 | + | + + Lemons . . . . . . . . . . . . . | | +++ | ++++ + Limes . . . . . . . . . . . . . | | ++ | ++ + Oranges . . . . . . . . . . . . | | +++ | ++++ + Pears . . . . . . . . . . . . . | | ++ | ++ + Raisins . . . . . . . . . . . . | | + | + + Tomatoes . . . . . . . . . . . . | ++ | +++ | ++++ +_Oils and fats:_ | | | + Almond oil . . . . . . . . . . . | | 0 | 0 + Beef fat . . . . . . . . . . . . | + | 0 | 0 + Butter . . . . . . . . . . . . . | ++++ | 0 | 0 + Cocoanut oil . . . . . . . . . . | 0 | 0 | 0 + Cod liver oil . . . . . . . . . | ++++ | 0 | 0 + Corn oil . . . . . . . . . . . . | 0 | 0 | 0 + Cotton seed oil . . . . . . . . | 0? | 0 | 0 + Egg yolk fat . . . . . . . . . . | ++++ | 0 | 0 + Fish oils . . . . . . . . . . . | ++ | 0 | 0 + Lard . . . . . . . . . . . . . . | 0 | 0 | 0 + Oleo, animal . . . . . . . . . . | + | 0 | 0 + Oleo, vegetable. . . . . . . . . | 0 | 0 | 0 + Olive oil . . . . . . . . . . . | 0 | 0 | 0 + Pork fat . . . . . . . . . . . . | 0? | 0 | + Tallow . . . . . . . . . . . . . | 0 | 0 | 0 + Vegetable oils . . . . . . . . . | 0? | 0 | 0 +_Nuts:_ | | | + Almonds . . . . . . . . . . . . | + | +++ | + Brazil nut . . . . . . . . . . . | | +++ | + Chestnut . . . . . . . . . . . . | | +++ | + Cocoanut . . . . . . . . . . . . | ++ | +++ | + English walnuts . . . . . . . . | | +++ | + Filbert . . . . . . . . . . . . | | +++ | + Hickory . . . . . . . . . . . . | + | + | + + Pine . . . . . . . . . . . . . . | + | + | + +_Dairy products:_ | | | + Butter . . . . . . . . . . . . . | ++++ | 0 | 0 + Cheese . . . . . . . . . . . . . | ++ | + | ? + Condensed milk . . . . . . . . . | ++ | + | 0 + Cream . . . . . . . . . . . . . | +++ | + | ? + Eggs . . . . . . . . . . . . . . | ++++ | ++ | 0 + Milk powder (skim) . . . . . . . | + | +++ | +? + Milk powder (whole) . . . . . . | +++ | +++ | +? + Milk whole . . . . . . . . . . . | +++ | +++ | ++ + Whey . . . . . . . . . . . . . . | + | +++ | + +_Miscellaneous:_ | | | + Alfalfa . . . . . . . . . . . . | +++ | +++ | ? + Blood . . . . . . . . . . . . . | Varies with source + Clover . . . . . . . . . . . . . | +++ | ++++ | ? + Honey . . . . . . . . . . . . . | | ++ | 0 + Malt extract . . . . . . . . . . | 0 | 0 | 0 + Nectar . . . . . . . . . . . . . | 0 | 0 | 0 + Timothy . . . . . . . . . . . . | ++ | +++ | + Yeast, brewers . . . . . . . . . | 0 | ++++ | 0 + Yeast cakes . . . . . . . . . . | 0 | ++ | 0 + Yeast extract . . . . . . . . . | 0 | +++ | 0 +____________________________________|___________|___________|____________ + + + +CHAPTER VI + + +THE CHEMICAL AND PHYSIOLOGICAL PROPERTIES OF THE VITAMINE + +While the chemists have not yet been able to isolate and identify the +various vitamines they have succeeded in demonstrating many of the +properties of these substances and it is the knowledge of these properties +that has enabled us to produce concentrates and conduct tests. Another +practical consideration involved in this matter of properties lies in the +effect of cooking and commercial methods of food preparation, for not only +must we learn where the vitamine resides but how to prevent injury or +destruction in our utilization of the source. + +The properties of the vitamines may therefore be grouped under two heads: +first chemical properties and second physiological properties. + +I. CHEMICAL PROPERTIES OF VITAMINE "A" + +_a_. This dietary factor's presence in butter fat and egg yolk fat +indicates its solubility in the fat and it would naturally follow that the +fat solvents would suffice to remove it with the fats when food sources +are treated with such a reagent. Experience has shown however that while +ether extraction applied to butter or egg yolk removes the vitamine with +the fat this process fails when it is applied to vegetable sources such as +cotton seed, corn germ, spinach, lettuce, etc. Neither does the cold or +hot press method of oil extraction liberate the vitamine with the oil. +Recent experiments by Osborne and Mendel, to which we have previously +referred, have shown that preliminary treatment of vegetable sources with +alcohol seems to loosen the bond between the source and the vitamine and +that when this binding is once loosened subsequent ether extraction will +take the vitamine out. That the binding is not difficult to break is shown +by the fact that when vegetables are eaten as a source of vitamine the +body is able to separate the complex. It is further evident that the body +does separate this complex and stores it in animal fat from the +experiments with cow feeds and feeding. Milk for example is rich or poor +in vitamine according to the supply of the latter in the food given to the +cow. The only logical conclusion to be drawn from this observation is that +the cow does not synthesize this factor but splits it off from the food +source and then, since it is fat soluble, is able to mobilize it in the +butter fat of the milk or to a more limited extent in the body fat. This +observation as to the dependence of milk content upon food has been +confirmed in the case of nursing mothers and suggests the need of especial +attention to the diet of the mother during the lactating period. + +_b_. It has been generally assumed that the "A" vitamine is +comparatively stable to heat. Sherman, MacLeod and Kramer state that "dry +heating at a temperature of 100°C. with free access of air, only very +slowly destroyed fat soluble vitamine." Osborne and Mendel reported that +butter fat treated with steam for two hours and a half did not appear to +have lost its value as a source of this vitamine. Drummond's earlier work +with fish oils and whale oils seemed to confirm this conclusion. Sherman +and his co-workers cited above put it this way: "The results thus far +obtained emphasize the importance of taking full account of the time as +well as the temperature of heating, and of the initial concentration of +the vitamine in the food, as well as of the opportunity for previous +storage of the vitamine by the test animal." More recent work by Steenbock +and his co-workers in America shows that these earlier results are +incorrect in the case of butter fat and that twelve hours exposure of +butter fat to 100°C. may, under certain conditions, destroy the efficiency +of that substance as a source of the vitamine. Drummond and other English +workers have confirmed Steenbock in later experiments. Their work has +shown that the presence or absence of oxygen is a factor, which may +determine the extent of destruction of the vitamine. Heat alone is of very +limited effect but when sources are heated in the presence of oxygen +destruction of the A vitamine may be very rapid. Drummond attributes the +absence of the A vitamine in lard to the oxidation that takes place in the +commercial rendering of this product. We must conclude therefore that +while the vitamine may be destroyed by continuous exposure to a +temperature of 100°C. the effect is largely determined by the nature of +the process and the way the vitamine is held in the source. Cooking of +vegetables therefore will not as a rule result in appreciable destruction +of this factor. + +_c_. The process of hydrogenation used in hardening fats appears to +completely destroy the vitamine, hence the many lard substitutes now in +use must in general be considered "A" vitamine-free regardless of the +content of "A" in the fats from which they are derived unless they have +been made by blending instead of hydrogenation. + +_d_. Acids and alkalies have apparently little effect on this +particular vitamine. + +It may be well to state here however that owing to variability in behavior +with variation in conditions it is dangerous to draw too general +conclusions and until a given source has actually been investigated under +specific cooking conditions one should not rely too strongly on analogies +based on comparative experiments. This statement applies to all vitamines +and presents one of the live subjects of investigation for the cooking +schools and the food factories. + +_e_. Little has been learned further about the chemistry of this +substance. [Footnote: Since the above was put in type Steenbock has shown +that the A vitamine resists saponification and that by saponifying fats +which contain the A it may be possible to secure a fraction rich in the +vitamine and free of fat.] Butter fat, nitrogen free and phosphorus free +is shown to carry the vitamine and it is therefore assumed that the +vitamine lacks these elements. It has been claimed that it may be removed +from butter fat by prolonged extraction with water but this has not been +confirmed by more recent experimenters. Steenbock was the first to call +attention to the association of the A vitamine with yellow pigment in +plant and animal sources. Butter, egg yolk, carrots, yellow corn contain +it while white corn and white roots are less rich in this vitamine. This +observation suggested the chemical relation between the vitamine and +carotin. It has however been shown by Palmer and others that carotin is +not vitamine A. This association of the pigment with the vitamine is +therefore apparently a coincidence and this clue has failed as yet to +throw light on the chemical nature of vitamine A. + +II. THE CHEMICAL PROPERTIES OF VITAMINE "B" + +When Funk first studied this substance he conducted all his evaporations +in vacuo from fear that higher temperatures would prove destructive. +Subsequent investigation however has shown that 100° has very little if +any destructive effect if the vitamine is held in acid or neutral +solution. Temperatures between 100° and 120° maintained in an autoclave at +15 pounds above normal pressure do tend to slowly destroy the factor. The +extent of this destruction also varies with the character of the crude +extract. In general, then, there is little fear of injuring this vitamine +in ordinary cooking temperatures if the use of alkali is avoided. + +The effect of alkali depends upon the temperature to a very marked degree. +Osborne has recently reinvestigated this matter and finds that in the +presence of a 0.1N solution of alkali at 20°C. there is very little +destruction but that raising the temperature to 90°C. brings about a +marked destruction. Seidell has shown that if the vitamine is absorbed by +Lloyd's reagent and this reagent be then extracted with dilute alkali the +vitamine passes into the alkaline solution. If the latter is neutralized +quickly it is possible to recover most of the vitamine by this method. The +effect of alkali becomes of practical importance to the housewife because +of certain cooking habits. I refer to the well known practice of adding +soda to the water in which vegetables are cooked to soften the vegetable +and accelerate the cooking. Daniels and Loughlin in this country +investigated this matter and came to the conclusion that this procedure +did not produce enough destruction to be dangerous. Later the matter was +studied by Chick and Hume in England and these investigators brought out a +feature that had perhaps been overlooked in the previous work. Their point +was that in ordinary feeding tests the results merely tell whether there +is enough vitamine present to produce normal growth. Hence if the +substance tested has much vitamine, a large part of it might be destroyed +and this fact not appear in the test because enough might still be left to +induce normal growth. By reducing the amount tested so that it was just +adequate for normal growth and then applying the soda-cooking +experimentation they showed that this method of cookery does do serious +harm to the vitamine. From the practical point of view it is of course +sufficient to show that enough is left after a cooking process to suffice +for normal growth when the substance is taken in the portion sizes +ordinarily eaten. The effect of alkali deserves more attention on the part +of cooks and food preparateurs and we need more data concerning the +minimal dose necessary to protect the human animal. + +In neutral and acid solution it is perfectly safe to assume little +destruction of this vitamin through heat and it is now common practice to +boil sources with the extracting reagent and to use the steam bath freely +to concentrate and evaporate these extracts. We have recently investigated +the effect upon cabbage of cooking in a pressure cooker at eight pounds +pressure. The cabbage so cooked, when dried and mixed so as to form 10 per +cent of a basal vitamine free diet, yielded all the "B" vitamine necessary +to produce normal growth in rats. + +The very name of this vitamine indicates its ready solubility in water. It +is also soluble in 95 per cent alcohol and either of these extractants may +be used to obtain the vitamine. It is not readily soluble in absolute +alcohol and 95 per cent is not as good an extractant as water. Substances +rich in the vitamine apparently yield the latter more readily if they have +first been subjected to autolysis or if the extracting fluid is acidified. +Funk was the first to show that yeast produced a greater yield if it was +allowed to autolyse before extraction with alcohol. However, Osborne and +Wakeman have produced a method of treating fresh yeast by boiling it with +slightly acidified water which seem as efficient as autolysis in the yield +produced. + +The various methods of extraction now in vogue have already been discussed +in Chapter II and need not be repeated here. In general it is apparent +that to obtain concentrates of high potency it is permissible to employ +temperatures of 100°C. if we will maintain an acid or neutral reaction but +that alkali should be avoided wherever possible and when its use is +imperative the temperature must be kept below 20°C. or destruction will +result. In applying this rule to cooking operations the results should be +determined by direct tests rather than by assumptions based on these +generalizations. It should also be noted that the alkalinity of a solution +should be determined on the basis of hydrogen ion concentration and not on +amount of alkali added since many substances have a marked buffer +reaction. + +The water-soluble "B" is not only soluble in water but can be dissolved in +other reagents. Thus McCollum has shown that while benzene is of little +value as an extractant of this vitamine, if we will first extract the +vitamine with alcohol or water and deposit this on dextrin by evaporation +it is then possible by shaking the activated dextrin with benzene to cause +the vitamine to pass into solution in benzene. Voegtlin and Meyers have +recently shown that it is soluble in olive oil and in oleic acid and their +data suggest a new means of concentrating the substance which may be of +value in tracing its character. + +The "B" vitamine is relatively easily absorbed by finely divided +precipitates. We have already referred to the use of fuller's earth for +this purpose by Seidell. This adsorptive power sometimes manifests itself +in the treatment of plant extracts. A watery extract of alfalfa can be +made to throw down its protein complex by diluting it to 40 per cent with +alcohol. Osborne reports however that this process frequently removes the +vitamine also which appears to be thrown down with the precipitated +material. This adsorptive power therefore often appears as a difficulty in +the handling of the substance as well as a means of extraction. We have +used Osborne's method with alfalfa extracts and find the above result is +not by any means invariable, for in some of our extracts we retained the +greater part of the vitamine. Kaolin and ordinary charcoal are not very +good adsorbents but the latter can be activated to serve this purpose. + +The elementary nature of the "B" vitamine remains a mystery. Extracts +which contain it show the presence of nitrogen. Funk's earlier researches +on yeast and rice polishings both yielded crystalline complexes which he +analysed. His data on this subject follow: + +_A. The yeast complex_ + +Crystals melting at 233°C. consisting of: + +I. A complex melting at 229°C. and forming needles and prisms nearly +insoluble in water and with the apparent formula of C_24H_19O_2N_5. + +II. A complex melting at 222°C. and soluble in water. Formula +C_29H_23O_2N_5. + +III. Nicotinic acid melting at 235°C. C_6H_5O_2N. + +_B. The rice complex_ + +Crystals melting at 233°C. consisting of: + +I. A complex melting at 233°C. and with a formula of C_26H_20O_9N_4. + +II. Nicotinic acid melting at 235°C. C_6H_5O_2N. + +Funk held at the time that the possible nature of the compound was: + + HN + | \ + OC C_16H_18O_6 + | / + HN + +It was this idea that led him to call it an "amine." + +We are unable at present to report any nearer approach to the elementary +analysis and all attempts at purification have shown a tendency to make +the active substance either disappear entirely or else distribute itself +over the several fractions instead of concentrating itself in one. Its +basic nature seems to be well established by its behavior with +phosphotungstic acid and its ready adsorption by carbons activated to take +up basic substances. + +III. THE CHEMICAL PROPERTIES OF WATER-SOLUBLE "C" + +The properties of this newest member of the family are still less defined. +All are agreed that it is much more sensitive to heat and alkali than the +other two. Temperatures above 50°C. are usually destructive though the +time factor is extremely important as well as the reaction. Hess for +example has found that the temperature used to pasteurize milk continued +for some time, is more destructive to the vitamine than boiling water +temperature continued for only a few minutes. The extent to which orange +juice and tomato juice will resist high temperatures indicates the +protective action of acids to be considerable. + +Dr. Delf's experiments at the Lister Institute were especially directed to +the behavior of this vitamine in cabbage. She first determined the minimum +close of raw cabbage required to prevent scurvy in guinea pigs and found +that it was less than 1.5 grams and more than 0.5 gram daily. When the +cabbage was heated in water at 60°C. for an hour, symptoms of severe +scurvy were just prevented by 5 grams of the cooked cabbage fed daily. By +heating at 70°, 80°, 90° and 100° for the same length of time the 5 grams +of cooked material could be made non-effective as a preventive. Her +conclusions are that when cabbage is cooked for one hour at temperatures +ranging from 80° to 100°C. the cabbage leaves lose about 90 per cent of +the antiscorbutic power originally held by the raw equivalent. Sixty +minutes at 60° or twenty minutes at 90° to 100° resulted in about 80 per +cent destruction. Dr. Delf calls attention also to the fact that the +effect of the heat is increased to only a slight degree by rise in +temperature. Assuming that the effect of the rise is orderly, a +temperature coefficient of 1.3 is indicated for each rise of 10°C. This +low result suggests to Delf a contradiction to any theory which imputes to +the vitamine enzyme or protein-like qualities and on the other hand +suggests that the substance is much simpler in constitution. Her results +also confirm Hoist and Fröhlich as showing its great sensitiveness at +temperatures of 100° and below and obviously have a direct bearing upon +cookery methods. + +The substance is soluble in water and passes through a parchment membrane +or a porcelain filter. Unlike the "B" it is apparently not adsorbed by +fine precipitates such as fullers' earth or colloidal iron. Harden and +Zilva showed that when a mixture of equal volumes of autolysed yeast and +orange juice is treated with fuller's earth the "B" is removed and the "C" +left unaltered. Eddy and La Mer have treated orange juice with fullers' +earth and then tested the filtered off juice as cure and preventive of +scurvy in guinea pigs. Their results showed that 6-2/3 cc. of the treated +juice was curative, hence the loss due to adsorption must be less than 60 +per cent to 70 per cent. Harden and Zilva were among the first to state +that the vitamine is much more stable in acid than in alkali. They have +shown, that even 1/50 N sodium hydrate at room temperature has a rapidly +destructive effect. On the other hand Delf showed that when 0.5 gm. citric +acid is added to the water in which germinated lentils are boiled, the +loss of the antiscorbutic properties is, if anything, greater than when no +addition of acid is made. She therefore concluded that in cooking +vegetables there should be no addition of either acid or alkali to the +cooking water if one wishes to conserve this vitamine. Sherman, La Mer, and +Campbell have been engaged in experiments bearing on this point throughout +the past two years. Some of their results have recently been published and +their observations are worthy of special attention from their bearing on +the character of reaction of the vitamine in general. They first proceeded +to determine the amount of filtered tomato juice just necessary to produce +scurvy in degrees extending from no protection to complete protection and +they also constructed a basal diet which is apparently optimum in +nutrients and all other factors except the "C" vitamine. They found that +at the natural acidity of tomato juice (pH 4.2) boiling for one hour +destroyed practically 50 per cent of the antiscorbutic power and by +boiling for four hours they destroyed 70 per cent, which indicates that +the curve of the destructive process tends to flatten more than that of a +unimolecular reaction. This result was confirmed by heating experiments +conducted at 60°, 80° and 100°. In all cases the temperature coefficients +are low. (Q_10 equals 1.1-1.3) confirming Delf's results. When the natural +acidity of the juice was first neutralized in whole or in part, the juice +then boiled for an hour and immediately cooled and reacidified, it was +found that at less than half neutralization (pH 5.1-4.9) the destructive +effect of an hour's boiling was increased to 58 per cent. When alkali was +added to an initial pH 11 (about N/40 titratable alkali to +phenolphthalein) which fell to 9 during the hour's boiling the destructive +effect was about 65 per cent. When reacidification was omitted and the +neutralized boiled juice stored in a refrigerator for five days before +using the destruction increased 90 to 95 per cent. These particular +observations seem to confirm the view of Harden and Zilva that the +vitamine is especially sensitive to alkali. Hess has recently reported +that oxygen is destructive to this vitamine. + +IV. PHYSIOLOGICAL PROPERTIES OF THE "A" VITAMINE + +Most authorities are now agreed that both the "A" and "B" types are +essential to growth. Rohmann still holds out against the vitamine +hypothesis. McCollum has recently pointed out that while rats do not have +scurvy it does not at all follow that the absence of the "C" in their diet +is immaterial, but that the contrary is true. Failure to grow, then, may +manifest itself as a result of the absence of either of the first two +types and possibly is affected by the absence of the "C." We have already +seen how this failure may be utilized to measure the vitamine content of a +source. The absence of the "A" type however may also manifest itself in +another way, viz., by the development of an eye disease which McCollum +first designated as xerophthalmia or dry eye and which the British +authorities prefer to designate as keratomalacia. The failure of this +result to always follow the absence of the "A" type in the diet has led +some to question the specificity of this disease. While the infection of +the eye is due to other agents the sum of the evidence supports McCollum +and points to the absence of "A" as the true predisposing cause of the +disease. Bulley, basing her claims on a study of some 500 rats fed on a +synthetic diet, claims that the eye condition is not primarily due to a +dietary deficiency but to an infection resulting from poor hygienic +conditions. In reply to her contentions Emmett has reviewed his own data +and presents them in the following summation: + +_________________________________________________________________________ + | | | | + RAT | KIND OF VITAMINE | NUMBER CASES | POSITIVE CASES | PER CENT +GROUPS | ABSENT IN THE RATION | REPORTED | OF XEROPH- | POSITIVE + | | | THALMIA | +_______|______________________|______________|________________|__________ + | | | | + A | Fat-soluble "A" | 122 | 120 | 98 + B | Water-soluble "B" | 103 | 0 | 0 + C | None | 216 | 0 | 0 +_______|______________________|______________|________________|__________ + +In these groups special hygienic measures were taken against infection. +Furthermore repeated attempts were made to transmit the eye disease by +using sterile threads, passing them carefully over the edges of the sore +lids and then carefully inoculating the eyes of other rats. These attempts +resulted negatively in all cases where the inoculated rats had plenty of +the "A" vitamine. Treatment of advanced cases of sore eyes with a +saturated solution of boric acid and also with a silver protein solution +failed to relieve the condition while as little as 2 per cent of an +extract containing the "A" vitamine when added to the ration, speedily +resulted in cure and increase of weight. These results combined with +similar data compiled by Osborne and Mendel seem to refute Bulley's +contentions and to justify our acceptance of xeropthalmia as a specific +vitamine deficiency disease. + +_Osborne and Mendel data_ + + Total No. No. with eye + symptoms + +Rats on diets deficient in A vitamine . . . . . . . . 136 69 + " on diets " " B " . . . . . . . . 225 0 + " on diets otherwise deficient . . . . . . . . . 90 0 + " on " experimental but probably adequate . 201 0 + " on mixed food . . . . . . . . . . . . . . . . 348 0 + ____ __ + + Totals . . . . . . . . . . . . . . . . . . . . . 1000 69 + +On the other hand all workers know that rats often do develop and grow +well for a considerable period of time on a diet free from the "A" and +without manifesting the eye disease. The British authorities explain this +by assuming that animals have the power to lay down a reserve of this +vitamine on which they can draw in emergency. Sherman and his coworkers +confirm this power to store the vitamine. Others have been led to explain +their results as due to contamination of the basal diet. Daniels and +Loughlin recently maintained that the commercial lard used in basal diets +and assumed to be "A" vitamine-free was supplied with sufficient of the +"A" to produce growth and prevent eye disease. Their views have failed of +confirmation by Osborne and Mendel. It is evident therefore that these +occasional lapses from specific response to absence of the "A" vitamine +need further elucidation. It is equally manifest that in the majority of +cases the absence of the "A" will result in both stunted growth and +xeropthalmia. The appearance of the eye disease may be taken however, as a +sure indication of the absence or deficiency in the "A" vitamine. + +V. PHYSIOLOGICAL PROPERTIES OF THE "B" VITAMINE + +Beri-beri is a disease that is described clinically as a form of severe +peripheral neuritis and may appear in two well marked forms. In one type +there is great wasting, anesthesia of the skin and finally paralysis of +the limbs. In the other, the most marked symptom is excessive edema which +may affect trunk, limbs and extremities. In severe cases the heart is +usually involved and death may occur suddenly from heart failure. + +Most observers assume that the antineuritic vitamine discovered by Funk +and the water-soluble "B" are identical. This view is based on the fact +that when sources which yield the water-soluble "B" in rat feeding are +tested for antineuritic power these sources are apparently parallel in +antineuritic power and growth production. Furthermore rats deprived of the +water-soluble "B" develop polyneuroses identical in symptoms with those +shown by rats and pigeons when the latter are placed on a polished rice +diet. The British Medical Board has compiled the following table to +support this view: + +_Table compiled from pages 35 and 86, British Medical Research Committee +Report_ + +_______________________________________________________________________ + | | + | | VALUE AS A SOURCE OF + | VALUE AS A SOURCE OF | THE ANTINEURETIC + | WATER-SOLUBLE "B" | FACTOR OR ANTI-BERI- + FOODSTUFF | (SHOWN BY EXPERI- | BERI FACTOR (SHOWN + | MENTS WITH RATS) | BY EXPERIMENTS + | | WITH BIRDS) +_________________________|______________________|_______________________ + | | +Rice germ . . . . . . . | +++ | ++++ +Wheat germ . . . . . . . | +++ | +++ +Yeast . . . . . . . . . | +++ | +++ +Egg yolk . . . . . . . . | ++ | +++ +Ox liver . . . . . . . . | ++ | +++ +Wheat bran . . . . . . . | + | ++ +Meat muscle . . . . . . | + | + +Milk . . . . . . . . . . | +++ | Slight +Potatoes . . . . . . . . | + | + +Meat extract . . . . . . | 0 | 0 +White bread or flour . . | 0 | 0 +Polished rice . . . . . | 0 | 0 +_________________________|______________________|_______________________ +_________________________________________________________________________ + | | + BEHAVIOR | WATER-SOLUBLE "B" | ANTINEURITIC VITAMINE +______________________|________________________|_________________________ + | | +Solubility in water . | Very soluble | Very soluble +Solubility in alcohol,| | + dilute . . . . . . | Very soluble | Very soluble +Solubility in absolute| | + alcohol . . . . . . | Insoluble | Insoluble +Solubility in ether, | | + chloroform and | | + benzene . . . . . . | Insoluble | Unusually insoluble + | | but can be extracted + | | with ether from + | | fatty materials such + | | as egg yolk +Stability to heat . . | Stable at 100°C, | Destroyed very slowly + | destroyed rapidly at | at temperatures below + | 120° (in neutral or | 100°C., more rapid at + | acid solution) | temperatures + | | between 110 and 120°C. +Stability to drying . | Stable | Stable +Stability to acids | | + (hot dilute) . . . | Moderately stable | Stable +Stability to acids | | + (cold dilute) . . . | Stable | Stable +Stability to alkalies | | + (hot dilute) . . . | Rapidly destroyed | ? +Stability to alkalies | | + (cold dilute) . . . | Stable | +In dialysis . . . . . | Passes through | Passes through + | parchment membrane | parchment membrane +In adsorption . . . . | Adsorbed from acid | Adsorbed from neutral + | or neutral solution | solutions by fuller's + | by fuller's earth, | earth, colloidal + | charcoal, etc. | ferric hydroxide, + | | animal charcoal, etc. +______________________|________________________|_________________________ + +Emmett has recently opposed this view and suggests that while the +antineuritic factor and the growth factor are found in the same sources +and have much in common it does not follow that they are identical and +that his experiments tend to show that there are marked differences which +suggest that the "B" type is not a single entity but a group. Mitchell has +summarized very well the controversial phases of this question with an +impartial review of the facts. One of strongest of the opposition +arguments lies in the failure of milk to cure beri-beri except when +administered in large quantities. This objection has been partly allayed +by data bearing on the relation of the milk content to the food of the +cow. Hess, Dutcher, Hart and Steenbock and others have adduced sufficient +evidence to show that the vitamine content of the milk of a cow is largely +determined by the cow's food and as a consequence the milk may be very +poor in vitamine. It is obvious then that the failure of the milk to cure +beri-beri in a given case might be due to this cause and not to lack of +identity of the curative with the growth factor. Osborne and Mendel have +also shown that milk in general must not be classed among the rich sources +of the vitamine, even when the cow's food is rich in vitamine. The +principal facts in the controversy have been presented and at present the +evidence for regarding the vitamines identical seems to be preponderant. + +Recently Auguste Lumiere in Paris has put forth the view that polyneuritis +is not merely a vitamine deficiency disease but a nutriment deficiency +disease. He reports that he fed birds on a starvation diet, but with +plenty of vitamine "B". These birds developed polyneuritis and were cured +by adding to the diet plenty of polished rice. The view he wishes us to +take is that all factors must be present and that the absence of the +nutriment is as important as the absence of the vitamine. + +In the field of nutrition the absence of the "B" type is particularly +marked by the behavior of the deprived animal. Rats transferred from a +vitamine-free diet to one containing the "B" only, make a much more rapid +recovery toward normal (even in the absence of the "A") than do animals +transferred from the vitamine-free diet to one containing the "A" and not +the "B". This initial jump from addition of the "B" will not continue long +in the absence of the "A", as a general rule. Hess believes that in some +of his infants he was able to show markedly successful growth on the diet +deficient in the "A" but rich in the "B". It is not certain however that +his diets were sufficiently devoid of the "A" factor to be declared "A" +vitamine-free and we know little of the amount of the "A" necessary to +normal infant growth. All results however show that both "A" and "B" are +necessary to growth production and though the term growth vitamine was +applied to the "A" originally the distinction is one that should be +rejected, for both "A" and "B" and possibly "C" are all entitled to this +name. + +The manner in which the "B" vitamine acts is still obscure. Voegtlin some +time ago tried to demonstrate that it was identical with secretin and +stimulated pancreatic flow. Recent work at the Johns Hopkins University by +Cowgill and by Aurep and Drummond in England has failed to confirm this. +One of its most marked immediate effects is increase in appetite. Karr in +Mendel's laboratory has shown that dogs which refused their basal diet +would resume eating it if they were allowed to ingest separately a little +dried yeast. Karr studied the metabolism of these dogs as regards nitrogen +partition but the results give little data that is explicatory of the +behavior of the vitamine. In 1915 the author was able to bring about +marked immediate improvement and the ultimate recovery of a number of +infants who were of the marasmic type by merely increasing the "B" +vitamine content of their food. In these cases the vitamine was carried by +Lloyd's reagent and administered mixed with cereal, or the crude extract +was combined with the milk. The pancreas of the sheep was the source used. +In these cases the growth curve changed abruptly from a decline to a sharp +rise and this increase in weight continued and was accompanied by all the +other signs of improved nutrition including increase in appetite. The +change in the growth curve from decline to rise was accomplished without +increasing or changing the basal diet but as the appetite increased the +food had naturally to be increased to keep pace. In these cases the effect +of the vitamine was to enable the child to utilize its normal food and to +increase its appetite for it. This action certainly suggests stimulation +of digestive glands. It also showed that even though the diet may contain +the vitamine as was the case in the milk fed to these children the +addition of the vitamine in concentrated form often gives an upward push +that the food mixture fails to accomplish. Daniels and Byfield have +recently confirmed the effect of increased "B" in infant growth. Cramer +has suggested in a paper published recently in _The American Journal of +Physiology_ that the fatty tissue about the suprarenals may be a +depository of vitamine and that in the absence of vitamine this tissue +loses its supply and that this is the explanation of lessened activity of +that gland in certain metabolic disturbances. This idea tends to support +the idea that vitamines are gland stimulants or hormones and the word food +hormone has been suggested to describe them on that account. A few years +ago Calkins and Eddy tried to determine the effect of the vitamine on the +single cell by use of the paramecium but the results of the experiments +failed to show a vitamine requirement on the part of these animals. +McDougall has recently suggested that the vitamines produce their effect +on yeast cells by increasing hydration. Unfortunately nearly all stimuli +which produce growth are accompanied by hydration effects and it is +difficult to feel that this is a specific vitamine effect although without +denying the possibility. Dutcher has tried to show that vitamines have a +relation to oxidation effects. He observed that the issues of polyneuritic +birds showed a marked reduction in catalase and that this catalase was +restorable by curing the birds with vitamine. The main difficulty lies in +the conflexity of factors that function between cause and effect. + +[Illustration: FIG. 8. THE EFFECT OF VITAMINE B ON A MARASMIC INFANT + +_1_. On the twentieth day the patient developed a cough. _2_. On +the twenty-first day the cereal was reduced from three times a day to +twice a day. The patient cried during the night. _3_. On the twenty- +second day the stools showed free starch. _4_. On the twenty-third +day an anal abscess was opened. The stools continued to show free starch +until the twenty-fifth day. _5_. On the twenty-fifth day the stools +showed soluble starch but no free starch. _6_. On the twenty-seventh +day the appetite was good and there was no starch. _7_. From the +twenty-eighth to the forty-third day no starch was observed in the stools. +_8_. On the thirty-first day the patient developed a cough. _9_. +From the forty-ninth day to the time of discharge three tablespoonsful of +orange juice were given daily. _10_. On the seventy-third day the +patient developed a bronchitis and mustard paste was applied every four +hours up to the eighty-fourth day. + +_V1_ = From the twenty-first day to the forty-third day the patient +received each day 2 grams of Lloyd powder, activated with pancreatic +vitamin. The powder was administered by mixing 1 gram. with each cereal +feeding. The result was 20 ounces gain in twenty-two days, a normal +growth. + +_V2_ = After a period of ten days without vitamin, during which the +patient settled down to a level growth curve, the treatment described +under V1 was resumed. This was continued from the fifty-third to the +seventy-sixth day. The result was the resumption of growth but at a slower +rate; 8 ounces were gained in twenty-three days. During the latter part of +the period the patient developed a bronchitis. At the end of this period +the patient was placed on a whole milk formula. From that time to the time +of discharge the patient grew normally.--From the _American Journal of +Diseases of Children,_ 1917, xiv, 189.] + +[Illustration: Effects of Vitamines on Growth FIG. 9] + +These views are at best speculations. The literature is singularly lacking +in detailed metabolic analyses of excreta of animals during vitamine +stimulation and we know nothing of the possibilities of overdosage, for in +all the work done it has been generally assumed that the presence of an +amount greater than that necessary to produce normal growth is not +material. + +The exact manner of the vitamine's action then remains to be determined +and it is obvious that this solution will come much more rapidly if we can +first identify the substance chemically. + +VI. THE PHYSIOLOGICAL PROPERTIES OF THE "C" VITAMINE + +The steps that led to the acceptance of scurvy as a vitamine deficiency +disease have already been discussed and show how the vitamine acts in such +a disease. Practically all the work done with this vitamine to date has +been concerned either with dosage or with reaction to heat, drying, etc. +The only paper that we have seen that suggests another function than +antiscorbutic power for this vitamine is the one by McCollum and Parsons +in which they suggest that even in animals where scurvy does not exist, +the presence of this factor may be necessary to normal metabolism. The +following table gives some of the data compiled by the British workers as +to the antiscorbutic power of various sources: + +_Table compiled from, page 44, British Medical Research Committee +Report_ + +________________________________________________________________________ + | | + | | MINIMUM DAILY + FOODSTUFF | VALUE AGAINST | RATION NECESSARY + | SCURVY | TO PREVENT SCURVY + | | IN GUINEA PIGS +_______________________________|_______________|________________________ + | | +_Cereals:_ | | + Whole grains . . . . . . . . | 0 | + Germ . . . . . . . . . . . . | 0 | + Bran . . . . . . . . . . . . | 0 | + Endosperm . . . . . . . . . | 0 | +_Pulses:_ | | + Whole dry . . . . . . . . . | 0 | + Germinated (lentils) . . . . | ++ | 5.0 grams +_Vegetables:_ | | + Cabbage (raw). . . . . . . . | ++++ | 1.0 gram + Cabbage (cooked one-half | | + hour at 100°C) . . . . . . | ++ | 5.0 grams + Runner beans (green pods). . | +++ | 5.0 grams + Carrot (juice) . . . . . . . | + | 20.0 cc. + Beet root (juice). . . . . . | + | More than 20 cc. + Swede (juice) . . . . . . . | +++ | 2.5 cc. + Potatoes (cooked one-half | | + hour at 100°C . . . . . . | + | 20.0 grams + Onions . . . . . . . . . . . | + | + Desiccated vegetables . . . | 0 to + | 60.0 grams expressed + | | as equivalent in + | | fresh cabbage +_Fruits:_ | | + Lemon juice (fresh) . . . . | ++++ | 1.5 cc. + Lemon juice (preserved) . . | ++ | 5.0 cc. + Orange juice (fresh) . . . . | ++++ | 1.5 cc. + Lime juice (fresh) . . . . . | ++ | 10.0 cc. + Lime juice (preserved) . . . | 0 to + | + Grapes . . . . . . . . . . . | Less than + | More than 20.0 grams + Apples . . . . . . . . . . . | Less than + | + Apples dried . . . . . . . . | Less than + | + Tamarind dried . . . . . . . | Less than + | + Mango . . . . . . . . . . . | Less than + | + Kokum . . . . . . . . . . . | Less than + | +_Meat:_ | | + Raw, juice . . . . . . . . . | Less than + | More than 20 cc. + Tinned . . . . . . . . . . . | 0 | +_______________________________|_______________|_______________________ + +A glance at this table shows the richest sources (see also table on page +59.) To these must be added canned tomato juice which Hess has shown +practically equal to orange juice in efficiency and uses with infants in +the same quantity. This discovery is of great value in instances where the +cost of orange juice is often prohibitive. + +La Mer and Campbell have presented some evidence to show that the +antiscorbutic vitamine has a direct effect upon the adrenal glands. In +their scurvy cases they find definite evidence of the enlargement or +hypertrophy of this organ. Whether it affects other organs or not it +remains to be shown. + +CHAPTER VII + + + +HOW TO UTILIZE THE VITAMINE IN DIETS + + +In the preceding chapters it has been the aim to present the findings of +the principal workers in the field. In attempting to summarize the work of +so widely scattered a group as are now engaged in vitamine research it is +impossible to cover completely the many investigations and it is +inevitable that some work will have been overlooked, but the foregoing +covers at least the principal data on the subject. What is the bearing of +all this information on human behavior and what lessons can the layman +draw from it that is of direct application to him? Let us first consider +this question from the dietary viewpoint. + +I. INFANT NUTRITION + +The limited character of the infant's diet has made the consideration of +vitamine content in his diet much more important than in the case of the +adult with the latter's wide variety of choice. It is evident from the +previous data that a growing infant must not only be provided with a +sufficient supply of calories, nutrients and salts, but must also have a +liberal supply of the three vitamines. Milk has in general been classed as +adequate in all these features, but the vitamine researches have forced us +to reconsider our views in regard to this staple. + +The first point to be borne in mind is that the vitamine content of either +cow or human milk is dependent primarily upon the food eaten by the +producer of the milk. In other words milk is merely a mobilization of the +vitamines eaten and if the diet is to yield vitamine-rich milk it must +itself be rich in these factors. Many a cow produces milk low in vitamine +content and the same is true of nursing mothers. There are many "old +wives" prejudices in regard to what food a lactating mother may eat and +unfortunately many of these prejudices are extremely injurious and false. +One of them is the prejudice against green vegetables. Experience has +shown that under ordinary conditions such vegetables are well tolerated by +the mother and from their content of vitamine it is evident that they are +suppliers of these factors. In the case of the cow the fact that cereals +are poor in some of the vitamines and green grasses rich therein, teaches +a lesson that bears directly upon winter feeding of cattle if the milk +supply is to be used for infants. We need a series of diets and cattle +foods for just this purpose of insuring the proper vitamine content in +milk. The preceding tables will enable one to develop such diets fairly +satisfactorily, but more data is urgently needed. + +The second point in regard to milk lies in the effect of pasteurization. +This measure is now well nigh universal and in America at least has played +a tremendous part in the reduction of infant mortality, especially during +the summer months. At present, however, we know that this treatment while +removing dangerous germs may also eliminate the antiscorbutic factor. The +sensible attitude then is to recognize this fact and if a clean whole milk +is not available retain the pasteurization and meet the vitamine +deficiency by other agents. Such agents are orange juice and tomato juice +and experience has already shown that these juices can be well tolerated +by infants much earlier than used to be thought possible. + +While the pasteurization does not appreciably affect the content of "A" or +"B" vitamines, the variability in content of these vitamines in milk +indicates that it may at times be necessary to supplement them in the +diet. In this connection it must be borne in mind that cereals vary widely +in content and cannot be, as they often are now, considered equivalent in +growth stimulation power. This is a subject that needs special attention +on the part of vitamine experts and dietitians and finally by the food +manufacturers. A good vitamine-rich cereal combination would form an +excellent adjuvant to infant dietaries after they reach the age of +tolerance to such a diet. But even before that time the expressed juice of +various vegetables as well as fruits is found to be well tolerated when +mixed with the milk or given separately, and carrot and spinach juice are +now being used in this connection with good results. These juices like +orange juice contain the B type in abundance and there is no doubt that in +their stimulation to the appetite they play an important part in making +the desirable daily gain. + +Fortunately for the layman he has in the scales a good indicator of the +normal progress of his child and so long as growth is normal he can fairly +assume that the diet is adequate but if the scales say otherwise it is +time for him to seek advice and then he is wise who insures that his +medical adviser knows the newer aspects of nutrition. The parent can do +this only by proper selection, but with a little knowledge he can soon +satisfy himself as to whether his pediatrist is the right sort and it is +one of the purposes of this text to bring home to the layman his +responsibility in this matter. + +There has grown up in this country a great regard for prepared milk +substitutes in infant feeding and a wide usage of condensed milks, +reinforced milks, diluted milk formulae, etc. All such preparations must +be examined anew in the light of the vitamine discoveries and unless the +given preparation can show a clean bill of health in vitamine content, it +should be either discarded or properly supplemented. + +As children grow up, it is fortunate that in their wider choice of +dietaries the danger of vitamine deficiency decreases. But even in +childhood it is unsafe to rely too much on chance. In this country there +are well deserving movements on foot to attract the parents of the +community to the necessity of attention to simple standards of growth +progress, and clinics for this purpose are appearing in increasing numbers +with each year. Such movements are to be most heartily approved. It is +also possible in these measures to not only build better children, but to +make the children themselves intelligent in their rejection of unsuitable +combinations and in that way not only conserve their own health, but +provide an educated body of citizens to pass on the knowledge to future +generations. In a school in New York City I recently had occasion to +discuss the school lunch room and its offerings with the children of the +school in the light of vitamine discoveries. The keenness and intelligence +shown by the children in the discussion that followed has convinced me +that in this matter of vitamines the children themselves can be relied +upon to assist materially in the matter of better food combinations and +intelligent selection. + +Finally it must be noted that one of the most common of infant +deficiencies is the failure of the bones to lay down lime. The effect of +this failure is commonly described as rickets. The British workers +consider that this deficiency is a lack of vitamine "A." Their views have +been set forth at greatest length by Mellanby, the principal worker in +this subject. While this view is still debatable and in this country it is +not yet accepted, one fact has come out in the controversy and that is the +remarkable value of cod-liver oil as a preventive of rickets. It may be +that the power of the oil is due to its "A" vitamine content in which it +is known to be rich, or it may be due to a new vitamine, but the fact that +the oil is a preventive in this respect gives the pediatrist another agent +to insure normal growth. The various views on the causes of rickets are +set forth more in detail in Chapter VIII. + +II. ADULT DIETS + +A study of the dietary habits of various sections of the United States +shows that there is a very general tendency on the part of the majority of +the people to confine their foods to a meat, potato, and cereal diet. The +use of salads is looked upon by many sections as a foreign affectation and +too little attention is paid to the value of eggs, milk and cheese. Enough +has been said already to show that these latter articles have much more +than an esthetic value and one of the missions of the nutrition expert +must be to show the people why dairy products and salads must become +features in the every-day meals of the every-day people. And even if the +salads are still unappreciated, it is necessary that cooked green +vegetables occupy more of a position in the menu than is too often the +case. + +There has recently appeared a crusade for the eating of yeast cakes. The +claim made for their use rests on a perfectly firm basis, they are rich in +the "B" vitamine, the proteins of the yeast cake are of good quality and +the cake contains no ingredients poisonous to man. Many people are +reporting beneficial effects from their use. Is there any lesson to be +drawn from this experiment? I feel that the very fact that benefits have +resulted from this yeast feeding is excellent evidence of lack of the +vitamine in the diets of the people affected and a clear argument that the +dietary habits of many people need adjustment to a higher vitamine +content. Whether it is necessary to use yeast cakes or any other +concentrate of vitamine, depends entirely upon whether the ordinary diet +is lacking in these factors and my first advice in the matter would be to +make if possible a selection of the vitamine containing foods and see if +normal conditions did not result before utilizing foods whose taste is not +pleasing or which are taken as medicine. For it is an old experience that +medicines will be taken only so long as the patient is sick and perhaps it +is just as well so. In other words I believe it is possible with +intelligent selection based on such tables as are given in Chapter IV for +people to secure from the butcher and the grocer all their requirements of +these vitamines as a part of their regular palatable diet. To those who +have neglected this selection and find remedy in concentrates, that fact +should lead them to reconstruct their diet rather than persist in +dependence on the medicine to correct faulty diet. In other words the same +arguments apply to the use of medicinal concentrates of vitamines as +applies to the use of laxatives. At times these substances are very +valuable as cures, but it is better by far to so regulate the dietary +habits as to avoid the necessity for their use. + +Another phase of this matter that promises to develop in the near future +as a result of the vitamine hypothesis is a reform in food manufacture. +There has been a strong tendency during the past two decades to "purify" +food products. The genesis of this tendency is to be found in a highly +laudable ambition to force the manufacturer to eliminate impurities and +adulterations and provide clean, wholesome, sanitary food. Unfortunately +in attempting to meet this demand on the part of the public, the food +manufacturer has sometimes neglected to seek advice from the nutrition +expert and the latter has failed to appreciate the need of advice. The net +result has been to discover that Nature is often a better chemist than man +and has a much better knowledge of what man needs in his diet than the +chemist. The chemist employed by the manufacturer has, as a result, gone +to such a limit in his development of purification methods as to often +eliminate the essential nutrients and the result has been foods that will +stand analysis for pure nutrients, but which will not stand Nature's +analysis for dietary efficiency. As a secondary result of this tendency we +have acquired habits that in many cases must either be broken or must have +grafted on to them other habits which shall remedy the defective ones. +Take the milling of wheat as an example. Nature put into the wheat grain +most of the elements needed by man and in the early days he was content to +grind up the whole grain and find it palatable. The craze for purity as +expressed by color has gradually replaced this whole meal wheat with a +beautiful white product that is largely pure starch with a few of the +proteins retained. And the principal protein retained lacks one of the +greatest essentials for growth while the vitamines have all been +practically eliminated with the grain germ. Intelligence tells us then +that if, having formed the habit, we will persist in our appetite for +white flour we must see to it that the protein deficiency of the latter +and its lack of vitamines is compensated for by supplementing the diet +with the food-stuffs in which these are rich. We may in other words retain +our bad habits in taste if we will graft on to them the attention to the +eliminated factors and their substitution in other form. + +In general then, the adult needs to review his feeding habits and analyze +them in the light of our new knowledge. For this purpose the tables of +Chapter IV supply data useful so far as vitamines are concerned, but it +will be perhaps worth while to repeat here some of this data in more +generalized form. + +_a. Sources of the "A" vitamine_ + +Its most abundant sources are milk, butter, egg yolk fat, and the green +leaves of plants usually classed as salads. Cabbage, lettuce, spinach and +carrots contain this substance in considerable quantity. The germ of +cereals is fairly rich in the factor, but the rest of the grain is +deficient and white flours are therefore poorer than whole meals in this +respect. Cooking temperatures have little effect on this vitamine and +hence little attention need be paid to cooking temperatures as far as this +vitamine is concerned. + +_b. Sources of the "B" vitamine_ + +Its principal sources outside of yeast are the seeds of plants and the +eggs and milk of animals. Meat contains relatively little of this +substance but glandular organs such as the liver and pancreas are fairly +rich in it. In the seeds the distribution is general throughout the whole +body of the seed in the case of beans, peas, etc., but in the cereal +grains it is largely restricted to the embryo portion and hence a high +degree of milling tends to reduce the per cent of this factor in any +highly milled cereal. White flour and polished rice are notable examples +of deficiency of "B" vitamine due to this milling process. Fruits such as +oranges, tomatoes, and lemons are good sources and there is a fair amount +present in the apples and grapes and other common food fruits. Many +vegetables show it in fair abundance, notably potatoes, carrots, and +turnips, but the rule is not general for beets are extremely poor in this +factor. Nuts are also good sources. Eggs, milk and cheese contain it in +fair abundance. Cooking temperatures have little effect on this type if +the temperature does not climb above the boiling point and if the cooking +water is not "alkaline." In the latter case it becomes necessary to +determine the extent of destruction and either eat enough to insure +protection, or reform the method of cookery. + +_c. Sources of the "C" vitamine_ + +Its richest sources are vegetables such as cabbage, swedes, turnips, +lettuce and watercress; fruits such as lemons, oranges, raspberries and +tomatoes. Certain of the vegetables such as potatoes have a substantial +value in this respect, but meat and most prepared milks are low in +antiscorbutic values. The susceptibility of this vitamine to drying, heat +and alkali, make it necessary to scrutinize your cooking methods very +carefully in order not to ruin a good source by a poor preparation of it +for the table. + + + +CHAPTER VIII + + +AVITAMINOSES OR THE DISEASES THAT RESULT FROM VITAMINE DEFICIENCIES + +A survey of the vitamines would be incomplete without a discussion of the +vitamine deficiency diseases in particular, though many of the facts +already cited obviously bear on the treatment and prevention of such +diseases. + +The idea of "avitaminoses" or vitamine deficiency as the cause of a +disease of a specific nature was set forth in detail by Funk in his book +_Die Vitamine_. In his discussion of this view he suggests several +types that would, he felt, on examination prove to be due to the absence +of a vitamine in the diet. Of these predicted types beri-beri was the only +one to be established in 1913. Scurvy has now been added to the fold and +rickets or rachitis seems well on the way to acceptance though the +specific vitamine absent in this case is not yet positively identified. +Pellagra still resists the efforts of the vitamine hypothesis to bend it +to that theory and its etiology is still obscure. + +I. BERI-BERI + +This disease while specifically confined to the oriental in the mind of +the student can be justly considered of much wider distribution for the +mild forms of malnutrition associated with a deficiency in the "B" +vitamine are less acute manifestations of this disease. The disease is not +likely to become marked in well nourished districts in its acute form, but +in famine districts its incidence is always possible. It would be more +than possible were it not for the fact that famine tends to eliminate the +highly milled cereals and throw the people back on to the whole grain, +peas and beans, which are rich in the preventive factor. But when for any +reason diets become limited extra attention is demanded in regard to their +selection and preparation. The main characteristics of this disease have +already been fully covered in what precedes and need not be repeated here. + +II. SCURVY + +This disease, like beri-beri has already been fully discussed in what +precedes. One of the striking discoveries of this subject has been the +retreat from favor of the time-honored lime juice which is now found to be +much less potent than oranges, lemons, or even canned tomato juice and +which on preservation loses practically all its potency. In the modern +hospital, cases of scurvy rarely appear outside of occasional infant cases +and it might appear that the problem of scurvy prevention is peculiarly +that of the sailor, the explorer and the army rationer. Nevertheless an +insufficient supply of the "C" vitamine may retard growth and well being +in the individual without manifesting itself in its more acute form of +scurvy. In a recent review Hess states: "It is hardly an exaggeration to +state that in the temperate zones the development or non-development of +scurvy depends largely on the potato crop." "This is attributed in part to +the fact that the potato is an excellent antiscorbutic, but to a greater +extent because it is consumed during the winter in amounts that exceed the +combined total of all other vegetables." To the public and to the food +purveyor there is a definite problem in how to best supply the preventive +and how best to concentrate and preserve the sources of this vitamine +without injury to its potency. The following observation is therefore +appended as bearing on this point. In the absence of fruits or other high +potency sources it is possible to develop this factor in cereal grains by +the simple expediency of sprouting. If seeds are soaked in water for +twenty-four hours and then kept moist for from one to three days with the +free access of air, sprouts will develop whose content of the +antiscorbutic vitamine is comparable to that of many fresh vegetables, +even though the dry seeds themselves have little of this factor. In other +words the germination process is a synthesiser of the vitamine. This +observation may be of value where fruits and vegetables are scarce or +expensive. On account of cooking effects, it cannot be too often +reiterated that raw fruits, vegetables and salads, are of more value than +cooked forms of these same sources and that drying processes are extremely +destructive where heat enters into the drying process. Vacuum drying seems +to be much less destructive and it may be possible to develop the drying +of vegetables to a point where retention of this vitamine factor is +practical. At present all dried vegetables should be regarded with +suspicion as a source of vitamine "C." Expressed juices may often be used +where the whole vegetable is scarce or incompatible and this fact is one +to be borne in mind by the worker in famine districts. + +III. RACHITIS (RICKETS) + +This disease is engaging the attention of many workers on both sides of +the Atlantic at the present time. In England the principal contributor is +Dr. Mellanby, who has accumulated evidence which he believes indicates +that the preventive factor is the A vitamine. This view is not yet +accepted as conclusive by the American workers. McCollum, Howland, Park, +and others at Johns Hopkins University have experimented with various +rickets-producing diets and while the principal deficiency in these diets +seems to be Ca salts and the A vitamine they do not consider that the +disease can as yet be traced to deficiency in any one factor. Hess has +called attention to several new features and the significance of some +older measures. He has shown on the one hand that cod-liver oil is almost +a specific remedy for the disease but that this remedy is not replaceable +by other rich sources of the A vitamine. He has also recently shown that +hygienic measures may have an influence. Schmorl showed that the disease +was seasonal, a high rate maintaining in the winter months and a lower +rate in the summer months. Hess has recently reported beneficial results +from use of the ultra-violet rays which he uses as a substitute for +sunlight. The results seem to confirm Schmorl's view that the sunlight of +the summer months is a preventive factor. He has also suggested that the +specific effect of the cod-liver oil might be due to a new vitamine, +Vitamine D? On the other hand Zilva and Miura in England have recently +shown that crude cod-liver oil is something like two hundred and fifty +times as rich in vitamine A as butter fat, which tends to support the +British view that the A vitamine is the antirachitic factor. + +Sherman and Pappenheimer have recently shown that the phosphates exert a +marked preventive effect on rickets and suggest that the utilization of +the calcium by the individual may be determined in part by this factor. + +The views in brief are now in an extremely chaotic state and it is +impossible at present to determine whether rickets is a true avitaminose +or a consequence of deficiency in a series of factors. It is however +certain that the disease in its subacute forms is extremely wide-spread +among infants and that its prevention can be most easily secured by the +addition of cod-liver oil to the diet. In this procedure warning is +necessary that the cod-liver oil be as pure a product of oil as possible, +since the market preparations are often almost devoid of the true oil and +hence of the curative agent. + +IV. PELLAGRA + +This disease has been the subject of exhaustive inquiry and study on this +side of the Atlantic and the findings of the various investigating boards +have added much to the prevention and cure of the scourge, but have failed +as yet to agree on any one etiological factor. The best recent review of +the current findings is to be found in an article by Voegtlin published as +Reprint 597 of the Public Health Reports of the United States Public +Health Service. His conclusions may be quoted in full as representing the +latest summary of evidence now extant: + +1. The hypothesis that there is a causal relation between pellagra and a +restricted vegetable diet has been substantiated by direct proof to this +effect and has led to results of considerable practical and scientific +value. + +2. The metabolism in pellagra shows certain definite changes from the +normal, which point to decreased gastric secretion and increased +intestinal putrefaction. + +3. In the treatment and prevention of pellagra, diet is the essential +factor. The disease can be prevented by an appropriate change in diet +without changing other sanitary conditions. + +4. A diet of the composition used by pellagrins prior to their attack by +the disease leads to malnutrition and certain pathological changes in +animals, resembling those found in pellagra. A typical pellagrous +dermatitis has not been observed in animals. Pellagrous symptoms have been +produced in man by the continued consumption of a restricted vegetable +diet. + +5. _The nature of the dietary effect has not been discovered_, +although certain observations point to a combined deficiency in some of +the recognized dietary factors as the cause of the pellagrous syndrome. + +In elaborating on conclusion 5 Voegtlin states that: + +The conception that pellagra is due to a dietary deficiency is, therefore, +not contradicted by the available evidence. This does not imply that the +disease is necessarily due to a deficiency of diet in a specific substance +such as the hypothetical pellagra vitamine of Funk (1913). It is much more +likely that the pellagrous syndrome is caused by a combination of the +deficiencies in some of the well recognized food factors. + +V. OTHER AVITAMINOSES + +The rôle of the vitamine in the nutrition and growth of organisms other +than the man is becoming a matter of interest in various ways. The +construction of culture media for various strains of bacteria and the +conditions favorable or unfavorable to their growth, are features of study +in which the new hypothesis has demanded attention. It has already been +claimed that vitamines are essential to the growth of the meningococcus, +the influenza bacillus, the typhoid bacillus, the gonococcus, the +pneumococcus Type I, Streptococcus hemolyticus, the diptheria bacillus, +the Bacillus pertussis and certain soil organisms. If these views are +confirmed it becomes evident that the means for prevention of the +development of these forms may lie in the control of the vitamine content +of the materials on which these forms thrive and that in the study of +these types it may be possible to speed up the incubation of strains and +thus hasten diagnostic measures by introducing the necessary vitamines +into the culture media. These observations merely suggest the possible +widening of the scope of the vitamine study in the service of man and give +added reason for our keeping pace with the strides made in this particular +field. + + + +BIBLIOGRAPHY + + +ABDERHALDEN, E., AND LAMPE, A. E.: Z. ges. exp. Med., (1913), i, 296. + +ABDERHALDEN, E., AND SCHATJMANN: Beitrag zur Kenntnis von organischer +Nahrungsstoffen mit spezifischer Wirkung I. Pflüger's Archiv. f. d. ges. +Physiol., (1918), clxxii, 1. + +ABDERHALDEN, E.: Weitere Beitrage zur Kenntnis von organischen +Nahrungsstoffen mit spezifischer Wirkung II. Pfülger's Archiv. f. d. ges. +Physiol., (1920), clxxviii, 133. + +ABDERHALDEN, E.: Weitere Beitrage zur Kenntnis von organischen +Nahrungsstoffen mit spezifischer Wirkung III. Pflüger's Archiv. f. d. ges. +Physiol., (1920), clxxviii, 260. + +ABELS, H.: Wiener klinische Wochenschr., (1920), xxxiii, 899. + +ABEL, J. 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R., AND SALEEBY: Experimental treatment of human beri-beri +with constituents of rice polishings. Ph. J. Sci., 1915, x, B, 99. + +WILLIAMS, R. R., AND CROWELL: The thymus gland in beri-beri. Ph. J. Sci., +1915, x, B, 121. + +WILLIAMS, R. R., AND JOHNSTON: Miscellaneous notes and comments on beri- +beri. Ph. J. of Sci., 1915, x, B, 337. + +WILLIAMS, R. R.: The chemical nature of the vitamines. I. Antineuritic +properties of the antineuritic substances. J. Biol. Chem., 1916, xxv, 437. + +WILLIAMS, R. R., AND SEIDELL, A.: The chemical nature of the vitamines. +II. Isomerism in natural antineuritic substances. J. Biol. Chem., 1916, +xxvi, 431. + +WILLIAMS, R. R., AND SEIDELL, A.: The chemical nature of the vitamines. +III. The structure of the curative modifications of the hydroxypyridines. +J. Biol. Chem., 1917, xxix, 495. + +WILTSHIRE, H. W.: Value of germinated beans in the treatment of scurvy. +The Lancet, London, 1918, ii, 811. + +WRIGHT, A. E.: Scurvy and acidosis. Army Med. Dept. Report, 1895-96, +xxxvii, 394. + +WOOD, E. J.: Pellagra. Edinburgh Med. J., xxv, 363 (1920). + +WOOD, E. J.: The diagnosis of pellagra. Arch. Diagnosis, N. Y., 1917, +April. + +ZILVA, S. S.: The extraction of the fat-soluble factor of cabbage and +carrot by solvents. Biochem. J., 1920, xiv, 494. + +ZILVA, S. S.: The action of ozone on the fat-soluble factor in fats. +Biochem. J., 1920, xiv, 740. + +ZILVA, S. S., AND STILL, G. F.: Orbital hemorrhage with protopsis in +scurvy. Lancet, London, 1920, i, 1008. + +ZILVA, S. S., AND WELLS, F. M.: Changes in the teeth of guinea pigs on a +scorbutic diet. Proc. Roy. Soc., ex, B, 505 (1919). + +ZILVA, S. S.: The action of the ultra-violet rays on the accessory food +factors. Biochem. J., 1919, xiii, 164. + +ZILVA, S. S.: The action of deficient nutrition on the problem of +agglutinins, complement and amboceptor. Biochem. J., 1919, xiii, 172. + +ZUNTZ, E.: Les facteurs accessories de la croissance et de l'equilibre. +Scalpel, 1920, June 19, No. 25. + + + + + + +End of the Project Gutenberg EBook of The Vitamine Manual, by Walter H. Eddy + +*** END OF THE PROJECT GUTENBERG EBOOK THE VITAMINE MANUAL *** + +This file should be named 7983-8.txt or 7983-8.zip + +Produced by Juliet Sutherland, Richard Prairie, Charles Franks +and the Online Distributed Proofreading Team. + +Project Gutenberg eBooks are often created from several printed +editions, all of which are confirmed as Public Domain in the US +unless a copyright notice is included. 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