The Vitamine Manual by Walter H. Eddy

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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 Frohlich
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 Frohlich 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 reexamine 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 Frohlich'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.

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