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"