The Biological Analysis of Food Stuff

THE BIOLOGICAL ANALYSIS OF FOOD-STUFF

FOOD-STUFF

Our knowledge of nutrition has progressed hand
in hand with the development of the science of
Chemistry. Chemical science gave us the clue to an
understanding of the nature of the food-stuffs and
the changes which take place in digestion, as well
as an appreciation of some of the secrets of the
metabolic processes which take place within the
tissues of the body. Chemistry will continue, as
time goes on, to aid in extending our knowledge of
the finer processes of physiology. Nevertheless,
it has been possible for a time to advance very rapidly
in the study of nutrition, from the technical as well
as from the practical standpoint, by a systematic
feeding of simplified diets to animals. The results
were interpreted on the observations as to the ability,
or failure, of the animals to develop normally, as
the diets were modified. Progress has resulted in
the past, and will continue in the future to come
from the judicious division of labor between the

1

2 THE NEWER KNOWLEDGE OF NUTRITION

study of food problems by chemical methods, and
by animal experimentation. In this brief exposition
of the present situation respecting our knowledge of
foods and nutrition, it is desirable that the reader
should appreciate the viewpoint of the investigator,
and should understand the line of reasoning by which
the successive steps in the progress of the last few
years have been attained. A brief historical account
of the steps by which research in this field have been
developed will serve this purpose, and at the same
time, will illustrate the mental processes of a student
engaged in the task of bringing order into a field of
scientific inquiry where before there was no clear
understanding.

A plant structure, or an animal body is an exceed-
ingly complex mixture of chemical substances, many
of which are themselves individually as complicated
in their structure as the most complex machine.
The first step in the direction of reaching an under-
standing of the chemistry of the living mass, must
involve the separation and study of the structural
units of which the tissues are composed. This was,
indeed, the field of activity of many organic and
physiological chemists during the nineteenth century.
The fats and the simpler substances into which they
can be converted as in soap making; the starches and
the simpler sugars, and the manner in which they
are related chemically; the proteins, bodies having
the properties of egg white, the casein of milk, hair,
etc., yet very closely related in their chemical nature,

BIOLOGICAL ANALYSIS OF FOOD-STUFF 3

since they can all be resolved into the same digestion
products in the animal body, or in the chemical
laboratory, have all been carefully studied and with
marked success. These and a long list of a thousand
or more relatively simple chemical substances have
been discovered, and isolated in a state of purity from
plant and animal tissues. They have been studied
to determine their special properties, composition
and the tests by means of which they may be recog-
nized and identified.

Through a century of patient labor by many able
men, an understanding of the number and character
of simple structural units into which the tissues of
animal or plant can be separated, became realized.
Furthermore, certain of these simple bodies could be
recognized as intermediate products on their way
toward being built up into the most Jiighly organized
units of the living tissues; others were shown to be
degradation products resulting from the physiological
activity of the living tissues of the plant or animal.*
Through these studies it became established that the
body of an animal or the tissues of a plant consists
essentially of: proteins, which are peculiar in that
they contain about sixteen per cent of the element
nitrogen, and are complex in structure; starch-like
substances and sugars, into which the starches can
be easily converted, and fats and a number of closely
related, and, in many respects, similar substances
known collectively as lipoids. With these there are
always associated in the living tissues more or less

4 THE NEWER KNOWLEDGE OF NUTRITION

water and a number of mineral salts. Numerous
special varieties of each of these types of substances
became known, and their less obvious characteristics
were described. Certain substances were found to
be special products, found only at certain times and
in certain special localities, and these became re-
garded in their true light, as of subordinate interest.
Examples of such are the alkaloids, quinine, strich-
nine, etc., the cellulose which serves as skeletal tissue
for the plant but is not necessary for the animal, and
in the same category belong the waste products of
the life processes of the animal body, most of which
are not found in plant substances. Living tissues,
although always associated with numerous sub-
stances, the exact importance of which could not
be determined, were found to consist essentially of
the proteins, fats, sugars, mineral salts and water.
These came to be regarded even as early as 1840, as
the essential and never failing constituents of plant
tissues and were regarded as the essential constituents
of an adequate diet for an animal.

The processes of the digestion of food have excited
the wonderment and have occupied the patient
attention of some of the most earnest students of
physiology and biochemistry. The chemistry of the
fats, and the starches and sugars being simpler, or
rather less complex than that of the proteins, came
to be earlier understood in their essential features.
It was not until toward the close of the nineteenth
century that the nature and extent of protein diges-

BIOLOGICAL ANALYSIS OF FOOD-STUFF 5

tion became clearly appreciated. Soon after 1900
the researches of Fischer revealed the great variation
in the composition of proteins from different sources. 1
This discovery introduced into nutrition studies the
idea of quality in addition to quantity which had
heretofore seemed satisfactory to students of nutri-
tion. Most proteins were found to be resolved into
eighteen simple digestion products called amino-
acids, and it was found that the proportions in which
these were present in the protein molecule varied
greatly in the proteins from different sources. All
or nearly all of these digestion products appear to
be indispensable constituents of an adequate diet.
All natural foods contain several proteins as the
extensive and valuable studies of Osborne have
shown, 2 and although there are individual proteins
which are entirely lacking in one or more of the essen-
tial digestion products of proteins, every natural food
appears to contain more or less of each of them.
The proteins of any single food-stuff may be regarded
as biologically complete, but their biological values
differ greatly, depending upon the yield of the
several amino-acids which can be obtained from
them.

Food Analysis. — Since proteins, carbohydrates,
such as starches and sugars, fats and mineral salts
came to be regarded as the essential constituents of
the normal diet, it early became the principal activity
of the investigator of nutrition problems to analyze
foods of every sort by chemical methods in order to

6 THE NEWER KNOWLEDGE OF NUTRITION

determine their content of what were supposed to
be the only essential food complexes. Pronounced
differences were observed in the composition of the
many substances which serve as food for man and
animals. Meats, milk, eggs, and a few seeds such
as the pea and bean are very rich in protein, the
cereal grains contain less of this food substance,
whereas the tubers and vegetables, especially in the
fresh condition, contain but very little. Equally
great variations are observable in the water content
of foods, and in their yields of fats and carbohydrates.
One of the great epochs in the development of the
science of nutrition, is that in which Atwater and
his associates examined and tabulated in classified
form the chemical composition of an extensive list
of human foods. 3 Following this, similar data were
accumulated in the Agricultural Experiment Sta-
tions concerning substances used for animal foods.
Up to about 1900 the idea that there was any varia-
tion in the quality of the proteins from different
sources did not become generally appreciated.

In the light of the revelations in the field of nutri-
tion during the last few years, it seems remarkable
that close students of animal nutrition accepted for
so long, without proof, the belief that the results of a
chemical analysis revealed the dietary values of
food-stuffs.

Disease and Diet. — Restricted diets of monotonous
character have produced, for centuries, diseases in
man in several parts of the world. The only one

BIOLOGICAL ANALYSIS OF FOOD-STUFF 7

of these which was at all general in the Western
hemisphere was scurvy, a disease which caused much
suffering among sailors in the days of the long sailing
voyages. It was well understood that the disease
was the sequel to the consumption of a faulty diet,
composed usually of biscuit and salt meats, and that
prompt recovery resulted from the consumption of
liberal amounts of fresh vegetables and fruits.
Decades passed without any systematic attempt to
determine the cause of the peculiar value of this
class of foods.

Pellagra was a scourge among the poorest of peas-
ants in parts of Europe for centuries, and its etiology
has been referred by many to the poor quality of the
simple and monotonous diet. This disease was not
observed in America until after 1900. Since then
it has been steadily increasing in the Southern States.

Beri-beri is a disease common among the poorest
classes of the Orient, who limit their food supply
principally to polished rice and fish. It is remarkable
that not until the year 1897 was the first fertile
suggestion made by Eijkman, 4 as to the nature of
the dietary fault which was responsible for the
development of this disease.

Man has been sufficiently industrious in most
parts of the world to secure for himself a varied
diet, derived from the cereal grains and legumes,
fruits, roots and tubers, meats and certain leaves,
which he foupd edible. Beginning with the dawn
of the era of his most rapid advance toward achieve-

8 THE NEWER KNOWLEDGE OF NUTRITION

ment, he has in many parts of the world been the
possessor and protector of flocks and herds, which
provided him with clothing, and a constant supply
of both meat and milk. The importance of this last
item in his food supply we have just now come to
really appreciate. It is in order that it may be fully
appreciated how great are the differences in the
nutritive value of foods of such a composition as to
appear alike from the results of chemical analysis
that the present account of the investigations of
recent years was prepared.

In the year of 1907 the author began the study of
nutrition problems at the Wisconsin Experiment
Station. An inspection of the literature which re-
lated to nutrition at that time disclosed the fact that
the diet was supposed to consist essentially of pro-
tein, carbohydrates and fats, and a suitable amount
of several mineral salts. There were in the literature
two papers which were highly suggestive that a new
era was about to dawn in this field of research.
Henriques and Hansen, 5 believing that gliadin, one
of the proteins of wheat, was free from the amino-
acid lysine, had made up a diet of purified gliadin,
carbohydrate, fats and mineral salts, and had at-
tempted to nourish on this food mixture, animals
whose growth was complete. It was reported that
rats had been kept in a state of nitrogen equilibrium,
and even retention of nitrogen (protein) was reported
during an experimental period covering nearly a
month. In most of their trials the animals failed

BIOLOGICAL ANALYSIS OF FOOD-STUFF 9

steadily from the time they were confined to food
of this character.

Willcock and Hopkins 6 had conducted experiments
with similar food mixtures, composed of carefully
purified food-stuffs in which all the constituents
were known. When the protein of the diet consisted
solely of zein, from maize, the mice lived but a few
days. When to this food the amino-acid trypto-
phane, which is not obtained on the digestion of
zein, was added to the diet, the animals lived dis-
tinctly longer than without this addition. All ex-
perimental work with such diets indicated that they
were unable to support well-being in a young animal
during growth over a prolonged period. It was an
interest in these results, and a desire to know why
such food mixtures, which complied with all the re-
quirements of the chemist and the dietitian, failed
to properly nourish an animal that led to the decision
that the study of nutrition offered a promising field
of activity.

At the Wisconsin Experiment Station there was in
progress at that time an experiment which greatly
strengthened the author's conviction that the most
profitable point of attack for the study of the prob-
lems of nutrition, lay in the study of greatly sim-
plified diets so made up that every component should
be known. It seemed that, employing such diets,
and by the systematic addition of one or more purified
substances known to be found in natural foods, or in
animal tissues, it should be possible to arrive at the

10 THE NEWER KNOWLEDGE OF NUTRITION

solution of the problem of just what chemical com-
plexes are necessary in the diet of the higher ani-
mals.

The above experiment was based upon earlier
work by Professor S. M. Babcock, and was suggested
by him, and carried out at first by Professors Hart
and Humphrey, and later with the cooperation of
Mr. Steenbock and the author. 7 In this experiment
the object was to determine whether rations, so made
up as to be alike, in so far as could be determined by
chemical analysis, but derived each from a single
plant, would prove to be of the same value for growth
and the maintenance of vigor in cattle.

The ration employed for one group of animals was
derived solely from the wheat plant, and consisted
of wheat, wheat gluten and wheat straw; for a second
group the ration consisted entirely of corn plant
products, and included the corn kernel, corn gluten,
a by-product of the corn starch industry, and the
leaves and stalks of the corn plant (corn stover);
the third group derived their ration solely from the
oat plant, being fed entirely upon rolled oats and
oat straw. There was a fourth group which it was
supposed would serve as controls, which was fed a
ration having the same chemical composition, but
derived from about equal parts of wheat, corn and
oat products.

The animals employed were young heifer calves
weighing about 350 pounds, and were as nearly
comparable in size and vigor as could be secured.

BIOLOGICAL ANALYSIS OF FOOD-STUFF 11

They were restricted absolutely to the experimental
diets, and were well cared for. They were given all
the salt (NaCl) they cared to eat, and were allowed
to exercise in an open lot free from vegetation. Their
behavior during growth, and in performing the func-
tions of reproduction were extremely interesting.
All groups ate practically the same amount of feed,
and digestion tests showed that there was no dif-
ference in the digestibility of the three rations.

It was not until the animals had been confined to
their experimental rations for a year or more that
distinct differentiation in their appearance was easily
observable. The corn fed group were sleek and fine
and were evidently in an excellent state of nutrition.
In marked contrast stood the wheat fed group.
These animals were rough coated and gaunt in
appearance and small of girth as compared with
those fed the corn plant ration. The weights of
the two groups did not differ in a significant degree.
The groups fed the oat plant ration and the mixture
of the three plants, leaf and seed, stood intermediate
between the two lots just described. The assumption
that the animals receiving the mixture of products
would do better than any of the others, and thus
serve as the standard group for controls was not
realized. The corn fed animals were at all times in
a better state of nutrition than were those receiving
the greater variety of food materials.

The reproduction records of these animals are of
special interest. The corn fed heifers invariably

12 THE NEWER KNOWLEDGE OF NUTRITION

carried their young the full term, and the young
showed remarkable vigor. All were normal in size
and were able to stand and suck within an hour
after birth as is the rule with vigorous calves. All
lived and developed in a normal manner. The young
of the wheat fed mothers were the reverse in all
respects. All were born three to four weeks too
soon, and all were small and weighed on an average
forty-six pounds, whereas the young of the corn fed
animals weighed 73 to 75 pounds each. This weight
is normal for new-born calves. The young were
either dead when born or died within a few hours.
The young of the mothers which had been grown on
the oat plant were almost as large as those from the
corn fed mothers, the average weight being 71
pounds. All of them produced their calves about
two weeks too soon. One of the four was born dead,
two were very weak and died within a day or two
after birth, the fourth was weak, but with care it
was kept alive. The young of the cows fed the
mixture of the three plants were weak in most cases,
and one was born dead and one lived but six days.
The mothers were kept on their experimental rations,
and the following year they repeated in all essential
details the reproduction records observed in the first
gestation period.

Records were kept of the milk production during
the first thirty days of the first lactation period. The
average production per day by each individual in
the corn-fed lot was 24.03 pounds; for the wheat- fed

BIOLOGICAL ANALYSIS OF FOOD-STUFF 13

animals 8.04 pounds, and for the oat-fed animals
19.38 pounds. Those fed the mixture of the three
plants produced 19.82 pounds of milk per cow per
day during the first thirty days. In the second lacta-
tion period the figures for milk production were
28.0; 16.1; 30.1; 21.3 pounds, respectively, per day
during the first thirty days.

Through autopsy and analysis of tissues of the
young, and analysis of the feces and urines of the
animals in the several groups, an elaborate attempt
was made to solve the problem of the cause of the
marked differentiation of the animals fed these
restricted diets. Interesting data were secured
which showed marked differences in the character
of the fat in the milk of cows from the different lots,
and the observation was made that the urines of the
wheat fed animals were invariably distinctly acid
in reaction, whereas those from the other lots were
alkaline or neutral to litmus indicator. It was not
possible by any means known to physiological chem-
istry, to obtain a clue to the cause of the pronounced
differences in the physiological well-being of the
different lots of cows. This experiment confirmed
the author's conviction that the only way in which
the problems of nutrition could ever be solved, would
be to solve the problem of the successful feeding of
the most simplified diets possible. If this were
accomplished it would be possible to proceed from
the simple to the complex diets employed in prac-
tical nutrition, ascertaining the nature of the dietary

14 THE NEWER KNOWLEDGE OF NUTRITION

faults in each of the natural foods, singly, the seed
alone, and the leaf alone before attempting to inter-
pret the cause of malnutrition in animals fed the
more complex mixtures.

Such an undertaking as that just described, viz.,
the solution of the problem of why animals do not
thrive on a diet of purified protein, starch, sugars,
fats and inorganic salts which contained all the
elements known to be left, as ash, on the incineration
of an animal body, necessitated the employment of
small laboratory animals. This was true for several
reasons: First, because it is difficult and laborious
to prepare isolated and purified food substances in
sufficient amounts for the conduct of feeding experi-
ments; second, it is both necessary and desirable to
shorten the length of the experiments as much as
possible, consistent with obtaining data regarding
growth and reproduction, in order that data may
accumulate sufficiently fast to make progress reason-
ably rapid. The domestic rat seemed to be the most
suitable animal, and accordingly it was selected.
The rat has a gestation period of but 21 days, and
the young are ready to wean at the age of 25 days.
The female usually produces her first Utter of young
at the age of about 120 days, and will as a rule have
five Utters by the time she reaches the age of four-
teen months, which age marks the end of her fertiUty.
The span of Ufe of a rat which is well nourished is
about 36 months. When such an animal is employed,
it is possible to accompUsh within a relatively short

BIOLOGICAL ANALYSIS OF FOOD-STUFF 15

time, the accumulation of data regarding growth
and reproduction which it would take years to secure
with domestic animals of large size, long period of
gestation and long span of life.

A sufficient number of comparable experiments
have now been conducted with several species of
animals to make it appear certain that the chemical
requirements of one species are the same as that of
another among all the higher animals. The require-
ments with respect to the physical properties of the
food vary greatly. The ruminants must have bulky
food with the right consistency, whereas the omnivora
(man, pig, rat, etc.), cannot, because of the nature
of their digestive tracts, consume enough of such
foods as leaves and coarse vegetables, to meet their
energy requirements.

The early efforts to nourish young rats on diets
composed of purified proteins, carbohydrates, fats
and mineral salts, confirmed the results of the earlier
investigators. The animals lived no longer on such
food mixtures, than when allowed to fast. The
rations employed were of such a character that the
most thorough chemical analysis could reveal no
reason why they should not adequately nourish an
animal. It seemed obvious that there was some-
thing lacking from such mixtures which is indis-
pensable for the nutrition of an animal, and a system-
atic effort was made during the years that followed
to discover the cause of failure of animals to develop
on diets of purified and isolated food-stuffs. It was

. 16 THE NEWER KNOWLEDGE OF NUTRITION

not until 1912 that light began to be shed upon the
problem.

The diet which was most in use at that time con-
sisted of purified casein to the extent of about 18
per cent, lactose 20 per cent (supposed to be pure),
about 5 per cent of some fat, together with a salt
mixture which was made up in imitation of the min-
eral content of milk, and the remainder of starch to
make 100 per cent. 8 This food mixture was sup-
posed to be composed of materials sufficiently pure
to comply with the requirements of such work;
that is, they were supposed to contain too little of
any impurities which would in any way influence
the results. With this diet the interesting observa-
tion was made that growth could be secured when the
fat in the food mixture was butter fat , whereas no growth
could be secured when the butter fat was replaced by
lard, olive oil or other vegetable oils. Egg yolk fats
were next tried and were found to induce growth in
the same manner as butter fat. It was definitely
established that, contrary to the past beliefs, the
fats are not all of the same dietary value. Certain
fats contain some substance which is not dispensable
from the diet, whereas other fats do not contain the
dietary essential in question.

The portion of the food mixture other than fat,
appeared to contain only substances of known com-
position, i. e n protein, carbohydrate and inorganic
salts, and for a time it appeared that the unknown
substance in butter fat was the only element of

BIOLOGICAL ANALYSIS OF FOOD-STUFF 17

mystery in the diet. The lactose or milk sugar was
carefully examined as to its purity and was judged
to be sufficiently pure to be satisfactory for such
experimental work, since it was practically free from
nitrogen. The tentative conclusion was reached
that the essential factors in an adequate diet included
one substance or a group of substances which had
not been appreciated jn. the past, and that these,
if there should be more than one, were associated
with certain fats but not with all.

This observation was in harmony with the pub-
lished work of Stepp 9 which had appeared in 1909.
Stepp observed that grown mice were satisfactorily
nourished by a bread which was made with milk,
but that early failure and death followed when the
animals were fed the same bread which had been
previously extracted with alcohol. When the sub-
stances extracted from the bread by alcohol were
replaced, the bread was again rendered efficient for
the maintenance of life and health. He demonstrated
in other experiments that the bread could be ex-
tracted with ether or with chloroform without re-
moving the substance which was soluble in alcohol,
and without which the animals steadily failed.
Stepp considered the unknown substance or sub-
stances with which he was dealing in his feeding work,
as belonging to the not well defined group of sub-
stances generally called lipoids. This group includes
fats and related substances more complex in char-
acter some of which contain the elements, phos-

18 THE NEWER KNOWLEDGE OF NUTRITION

phorous and nitrogen. Stepp was not able to secure
with any known lipoid, the effects which resulted
from the administration of the alcohol-soluble por-
tion of his milk bread.

A new viewpoint was suggested by F. G. Hopkins
of Cambridge, England, in 1912. 10 He had as early
as 1906 conducted experiments in the feeding of mix-
tures of purified protein, carbohydrate, fats and
mineral salts and was aware of the fact that neither
maintenance of body weight, nor growth could be
secured with such diets. He then tried the addition
of such amounts of milk as would furnish 4 per cent
of the total dry matter of the food mixture and ob-
served that growth could proceed when such milk
additions were made. Hopkins suggested the exist-
ence of certain unidentified food substances which
were supplied by the milk and to these he gave the
name " accessory' J articles of the diet.

Attention has been called to the fact that Eijk-
man, a student of the disease, beri-beri, made the
discovery in 1897 that pigeons fed solely upon pol-
ished rice, develop usually within three or four weeks,
a state of paralysis which is called polyneuritis, and
is analogous to beri-beri in man. He found that
when the birds were given the entire rice kernel, or
unpolished rice the disease did not develop. It was
found, f urthermore,that the administration of rice pol-
ishings to pigeons suffering from polyneuritis, caused
prompt relief of their symptoms. Eijkman's ob-
servations attracted little attention until 1911, when

BIOLOGICAL ANALYSIS OF FOOD-STUFF 19

Funk took up the study of beri-beri, and made an
elaborate attempt to isolate and study the " curative "
substance in rice polishings. 11 Fraser and Stanton
had, however, in 1907, employed alcoholic extracts
of rice polishings for the relief of experimental
polyneuritis. 12 In the work of these investigators
the erroneous assumption seems to have prevailed
that the process of polishing consists essentially of
the removal of the outer covering, or bran layer of
the rice kernel. As a matter of fact the rice germ
is in a very exposed position, and is easily rubbed
off during the process of polishing. As was later
shown by McCollum and Davis, for the wheat
kernel, the germ is a very different thing from the
seed from the dietary standpoint. 13 The reason for
this will be made clear later.

The studies of Eijkman, Hopkins, Fraser and
Stanton and Funk, referred to above, clearly sug-
gested that there was required in the diet something
other than protein, carbohydrate, fats and inorganic
salts. When McCollum and Davis succeeded in
securing growth in young rats fed upon a mixture of
"purified" food-stuffs, when the mixture contained
butter fat, but no growth when vegetable fats or
the body fats of animals were substituted, it appeared
to them that the only element of mystery in the diet
was that associated with certain fats. This could
not at first be harmonized with the observation of
Funk, namely, that butter fat had no favorable
influence on pigeons which were suffering from ex-

20 THE NEWER KNOWLEDGE OF NUTRITION

perimental beri-beri. 14 His studies seemed to in-
dicate that there is necessary in the normal diet at
least one other substance, the absence of which
brought on the attack of polyneuritis. Later experi-
ments by McCollum and Davis cleared up the prob-
lem, but not without a considerable amount of
experimenting and delay.

McCollum and Davis arrived at the conclusion
that aside from the long recognized constituents of
the normal diet, there is some unknown substance in
butter fat which must likewise be furnished in the
food, and began a systematic investigation of the
problem of why a young animal cannot grow when
restricted to a single grain such as wheat, maize
(corn), oats, peas, beans, etc. They had tried many
times to limit young rats to whole wheat, or other
grain as their sole food, and had found that they
not only failed to grow, but would not live many
weeks. Chemical analysis shows the cereal grains
to contain all the essential food substances, for which
we know how to analyze, viz : proteins, starch, sugar,
fats and all the mineral salts which occur in the body
of an animal.

It was reasoned that, since all the dietary essen-
tials, except possibly the one which is not present in
vegetable fats, are certainly present in the wheat
kernel, the faults in the latter must depend upon a
lack of the unknown substance contained in butter
fat, or on the quality of some one or more of the well
recognized constituents of the diet. It seemed

BIOLOGICAL ANALYSIS OF FOOD-STUFF 21

possible to discover by means of a systematic series
of feeding experiments in which the quality of the
seed should be improved with respect to one dietary
.factor at a time, which factor was interfering with
growth. Accordingly they fed wheat in the following
combinations, and with the results noted:

(1) Wheat alone no growth, short life.

(2) Wheat plus purified protein no growth, short life.

(3) Wheat plus a salt mixture which gave it a mineral content

similar to that of milk very little growth.

(4) Wheat plus a growth promoting fat (butter-fat)

no growth.

From these results it seemed apparent that either
their working hypothesis regarding the factors which
are necessary in an adequate diet, must be wrong,
or there must be more than a single dietary factor
of poor quality, and jointly responsible for the poor
nutrition of the animals. In order to test this theory
they carried out another series of feeding experiments,
in which wheat was fed, supplemented with two
purified food additions.

(5) Wheat plus protein, plus the salt mixture Good

growth for a time. Few
or no young. Short life.

(6) Wheat plus protein, plus a growth-promoting fat (butter-

fat) No growth. Short life.

(7) Wheat plus the salt mixture, plus the growth-promoting fat,

(butter-fat) Fair growth for a time. Few

or no young. Short life.

22 THE NEWER KNOWLEDGE OF NUTRITION

The behavior of the animals fed wheat with two
purified food additions was highly suggestive that
there are three dietary factors of poor quality in
the wheat kernel. This was demonstrated to be
true by a feeding trial in which wheat was fed with
three purified food additions :

(8) Wheat plus protein, plus the salt mixture, plus a growth-
promoting fat (butter-fat) .... Good growth, normal

number of young, good
success in rearing young ;
life approximately the
normal span.

McCollum and Davis were, in 1912, more than
ever convinced that the only element of mystery in
the normal diet was the unidentified substance in
butter fat, for with the improvement of three dietary
factors wheat became a satisfactory food for the
nutrition of an animal during growth and for the
support of all the functions of reproduction and
rearing of young.

This series of experiments brought to light two
new viewpoints in animal nutrition, one of which was,
that the inorganic content of the wheat kernel, although
it furnishes all the necessary elements, does not contain
enough of certain of these to meet the requirements of a
young animal during the growing period. It is true
that some years earlier Henry, 15 had called at-
tention to the deficiency of the corn kernel in ash
constituents, and had in some of his experiments

BIOLOGICAL ANALYSIS OF FOOD-STUFF 23

added wood ashes to the diet, with noticeable im-
provement in the well-being of the animals. The
fact that seeds such as wheat fail to supply enough
of any of the essential inorganic elements was not
genexally appreciated and was given but little at-
tention in works on nutrition. Later, work by Mc-
Collum and Simmonds, demonstrated that the de-
ficiency in mineral elements in wheat and other
seeds is limited to three elements, caJcim, sodium
and chlorine.

A second new viewpoint brought out by these ex-
periments was the fact that the wheat kernel is indeed
too poor in its content of the unidentified substance
which butter fat contains, to satisfactorily nourish an
animal over a long period of time.

It has already been mentioned that the studies of
Kossel, Fischer and of Osborne, had made it clear
that there should exist very pronounced differences
in the value of the proteins from different sources.
The proteins were prepared in a state of relative
purity and were digested in the laboratory by means
of acids, and were analyzed by the methods of
Fischer and of Kossel. Certain of the eighteen di-
gestion products, the amino-acids, were determined
quantitatively so far as the methods would permit.
Although the methods were never perfected so as to
give results which were approximately quantitative,
except in the case of less than a third of the amino-
acids which were known to be formed in the di-
gestion of proteins, it was shown in the case of these

24 THE NEWER KNOWLEDGE OF NUTRITION

few that there were very great variations in the pro-
portions among them in the mixture obtained from
proteins from different sources. Thus the proteins
of the muscle tissues of several species of animals
were shown to yield between 12 and 14 per cent of
glutamic acid, one of the digestion products obtained
from practically all proteins. The same amino-acid
is present in the two principal proteins of the wheat
kernel to the extent of about 40 per cent of the
total protein. These two proteins together make
about 85 per cent of tbe total protein of the wheat
kernel. Other equally great differences were shown
to exist in the composition of proteins of our common
food-stuffs and those of the tissue proteins which
are formed during growth.

A good illustration of the problems which the
animal meets in its protein nutrition, may be had by
comparing the digestion products of the protein
molecule to the letters of the alphabet. The pro-
teins of the food and of the tissues are made up
of the same letters arranged in different orders and
present in different proportions. In growth the
animal takes as food, proteins which are very unlike
those of its tissues, digests these into the simple com-
plexes, the amino-acids, and then, after absorbing
these, puts together the fragments in new order,
and in new proportions to form the tissue proteins.

If the muscle tissue of an animal be likened to a
block of printer's type so arranged as to print the
rhyme beginning "Jack Spratt, who could eat no

BIOLOGICAL ANALYSIS OF FOOD-STUFF 25

fat, and his wife could eat no lean," the proteins of
which the muscle consists are represented by the
individual words, and the digestion products of the
proteins by the letters of which the words consist.
Now if the animal could take in its food proteins
which correspond to a block of type which would
print the jingle beginning: " Peter Piper picked a
peck of pickled peppers," it is easy to understand
that when the proteins of the food are resolved to
their constituent letters, and an effort made to form
the body proteins of the new and different type
from the letters supplied by the food, the trans-
formation cannot be made. In setting up the first
line, "Jack Spratt could eat no fat and his wife
could eat no lean," we need four of the letter t, but
the food proteins contain but one. The first line of
the Jack Spratt rhyme, which represents the muscle
proteins, requires but one letter p, whereas the food
proteins represented by the Peter Piper rhyme
yield nine in the first fine. The first line of the
Jack Spratt rhyme contains the letters j and n,
whereas the Peter Piper rhyme contains none, so
that even with the entire stanza:

Peter Piper picked a peck of pickled peppers
If Peter Piper picked a peck of pickled peppers,
Where's the peck of pickled peppers,
That Peter Piper picked?

it is not possible to reproduce even the first line of
the Jack Spratt rhyme, and in order that growth

26 THE NEWER KNOWLEDGE OF NUTRITION

might become possible, it would be necessary to
take proteins of another character, which would
supply the missing letters.

Such a comparison between food proteins and
tissue proteins gives a good illustration of the kind
of problem which the animal meets in its protein
nutrition. The most conspicuous protein of the corn
kernel (zein) is wholly lacking in three of the amino-
acids or digestion products which are obtainable
from most tissue proteins. In accord with what we
should expect on theoretical grounds, this protein
is, when taken as the sole source of amino-acids, not
capable of supporting growth, or of maintaining an
animal in body weight* This illustration shows how
we may have excellent, good or poor food proteins
for the formation of body proteins in growth.

The investigations described above, the object of
which was to find the cause of the failure of an
animal to grow when restricted to wheat as its sole
source of nutriment, were carried out in 1912, the
year following the publication of the first work by
Funk on polyneuritis. In the same year Hop-
kins called attention to the remarkable effects pro-
duced by the addition of small amounts of milk to
diets composed of purified food-stuffs. The " vita-
mine" hypothesis had just been formulated by
Funk. 16 McCollumand Davis were, therefore, aware
of the relation of a diet of polished rice to experi-
mental beri-beri. They believed, in the light of
their experiences with the diet of purified protein,

BIOLOGICAL ANALYSIS OF FOOD-STUFF 27

carbohydrate, fats and inorganic salts, which, they
observed, was capable of inducing growth when cer-
tain fats were supplied, but not when others were
substituted, and the further fact that wheat could
be supplemented by purified protein, a growth-
promoting fat, and a suitable salt mixture, i. e. with
food-stuffs of known character, that there was but
a single unidentified substance necessary in the diet.
They decided to next apply to polished rice the
same procedure which had shown so clearly the
nature of the dietary deficiencies of wheat. Rice,
they reasoned, could be nothing less than a mixture
of proteins, starch, traces of fat, and a mixture of
inorganic salts, similar to that contained in wheat,
but smaller in amount. It should, therefore, be
supplemented with a suitable salt mixture, a purified
protein, and a growth-promoting fat, so as to in- '
duce growth and maintain animals for a long time
in a state of health. This seemed to be a necessary
conclusion, since they had secured growth and well-
being in animals fed strictly upon a mixture of puri-
fied protein (casein), starch, milk-sugar, butter fat
and a mixture of inorganic salts of suitable com-
position.

It was a great surprise to McCollum and Davis
to find that polished rice, even when supplemented
with the purified protein, casein, butter fat and a
salt mixture properly constituted, failed utterly to
induce any growth in young rats. 17 Not only did
they fail to grow, but in the course of a few weeks

28 THE NEWER KNOWLEDGE OF NUTRITION

they developed in some cases a state of paralysis
which was suggestive of polyneuritis. Here was an
apparent contradiction. The polished rice could be
nothing less than a mixture of protein, carbohydrate
and salts. The only difference between this and the
mixture of supposedly purified food-stuffs with which
they had achieved success was in the 20 per cent of
milk sugar which the latter contained. It was,
therefore, decided to repeat the experiments with
the latter mixture, with the milk sugar replaced by
starch. It was found that this change in the com-
position of the food mixture made the difference be-
tween success and failure. No growth could be se-
cured when the milk sugar was omitted. Later
experiments showed that if milk sugar was suf-
ficiently purified by repeated crystallization it was
no longer effective in inducing growth when added
to the purified food mixture, whereas the water from
which the sugar had been crystallized would, when
evaporated upon the food mixture, render it capable
of inducing growth. This made it evident that there
is indeed a second dietary essential, of which an
animal needs but a very small amount, but which
is absolutely necessary for both growth in the young
and the maintenance of health in the adult.

Further experiments were then conducted to find
whether this unidentified substance which was being
added accidentally as an impurity in the milk sugar,
was the same as the substance which Fraser and
Sta;;ton and Funk were dealing with in their studies

BIOLOGICAL ANALYSIS OF FOOD-STUFF 29

of beri-beri. It was found that pigeons which had
developed beri-beri as the result of being fed ex-
clusively upon polished rice, could be temporarily
"cured" with any preparation which would, when
added to the diet of purified food-stuffs, containing
a growth-promoting fat, cause animals to grow.

Following the method introduced by Fraser and
Stanton, McCollum and Davis, 18 next employed
alcoholic extracts of various natural foods, adding
the alcohol soluble matter to the standard mixture
of purified protein (casein), starch (dextrinized) ,
salts and butter fat, and soon became convinced
that the substance which relieves the condition of
polyneuritis in pigeons was always present in the
preparations which render the purified food mixture
capable of promoting growth. They finally adopted
an alcoholic extract of wheat germ as a source of
this dietary factor in their investigations. Funk
and his co-workers had previously shown that the
curative substance is present in many natural
foods. 16 Repeated trials showed that the inclusion
of the alcoholic extract of wheat germ or of other
food, was not sufficient to induce growth unless
the butter fat was likewise added to the purified food
mixture. Both the growth-promoting fat and the
trace of unidentified substance in the alcoholic extract
of wheat germ are necessary for the promotion of
growth or the preservation of health.

It has been pointed out that Funk, in his examip* 1
tion of the various natural foods for the purpo

30 THE NEWER KNOWLEDGE OF NUTRITION

determining the distribution of the antineuritic sub-
stance (substance which relieves polyneuritis) found
butter fat ineffective. This was later confirmed by
McCollum and Kennedy. 19

Through the "vitamine" hypothesis, Funk at-
tempted to account for the diseases beri-beri, scurvy,
pellagra and rickets, as being each due to the lack
of a specific chemical substance, a " vitamine," in
the diet. 16 This was a very logical conclusion from
the data available to Funk. Scurvy, it had long
been known is relieved in a very spectacular manner
by the inclusion of fresh vegetables or orange juice
in the diet, and there was no doubt that the disease
developed as the result of a diet of poor quality. On
first consideration it seemed very reasonable to
assume that there is an " antiscorbutic vitamine"
in certain fruits and vegetables.

Pellagra has long been suspected of being due to
faulty diet, although the exact manner in which the
diet is unsatisfactoiy remained obscure. It was
generally appreciated by clinicians that a change to
a highly nutritious diet in which milk and eggs were
conspicuous was the best prophylactic measure for
the treatment of the disease, and that without diet-
ary measures, all remedies fail. It was not surpris-
ing that Funk should have regarded pellagra as one
of the " deficiency" diseases, due to lack of a " vita-
mine" in the diet. As will be shown later (Chapter V)
there has since been secured much experimental evi-
dence in support of the view that scurvy and pellagra

BIOLOGICAL ANALYSIS OF FOOD-STUFF 31

do not arise from deficiency in the diet of specific
chemical substances in the sense in which Funk
suggested. This seems to be true also of rickets.
In view of the considerations just mentioned rel-
ative to the cause of scurvy and pellagra, and the
convincing evidence that beri-beri is actually caused
by specific starvation for a substance, "vitamine,"
as Funk suggested, McCollum and Davis formulated
in the following way, their working hypothesis as to
what constitutes an adequate diet. The diet must
contain, in addition to the long recognized dietary
factors, viz: protein, a source of energy in the form
of proteins, carbohydrates and fats ; a suitable supply
of certain inorganic salts, two as yet unidentified
substances or groups of substances. 18 One of these
is associated with certain fats, and is especially
abundant in butter fat, egg yolk fats and the fats
of the glandular organs such as the liver and kidney,
but is not found in any fats or oils of vegetable origin.
The second substance or group of substances of
chemically unidentified nature, is never associated
with fats or oils of either animal or vegetable origin.
It is widely distributed in natural foods, and can be
isolated in a concentrated, but not in a pure form,
from natural food-stuffs by extraction of the latter
with either water or alcohol. This water or alcoholic
extract always contains the substance which cures
polyneuritis. At the time it seemed possible that
it also contained several other "vitamines," pro-
tective against the other diseases mentioned. The

32 THE NEWER KNOWLEDGE OF NUTRITION

former substance or group of substances, which is
associated with certain fats is not " curative" for
any of the list of diseases which Funk designated as
"vitamine" deficiency diseases. Indeed, butter fat,
which is the food containing one of the indispensable
substances in greatest abundance, was stated by
Funk to contain no "vitamine." 14

Nomenclature of the Unidentified Dietary Essen-
tials. — The ending amine has a definite and specific
meaning in organic chemistry, and applies only to
substances containing the element nitrogen. Since
butter fat, which is very rich in one of the dietary
essentials in question is practically, if not entirely,
free from nitrogen, it seems almost certain that the
physiologically indispensable substance which it con-
tains is free from nitrogen, and could not with pro-
priety be designated by any name ending in amine.
For this reason, and because it is possible to divide
the unidentified constituents of the normal diet into
two classes on the basis of their solubility, McCollum
and Kennedy 19 proposed the terms fat-soluble A
and water-soluble B to designate them. The former
prevents the development of a pathological condition
of the eyes, 20 the latter prevents the development of
beri-beri. As will be shown later, there is much
evidence for and none against the view that what we
designate by each of these terms is in reality but a
single physiologically indispensable substance and
not a group of substances. This necessitates the
further assumption that certain of the diseases of

BIOLOGICAL ANALYSIS OF FOOD-STUFF 33

dietary origin, which Funk held to be due to "vi-
tamine" starvation, are in reality due to other causes.
This view will be supported by further evidence later.
Indeed it is not possible to longer regard scurvy as
a "vitamine" deficiency disease.

The " vitamine" hypothesis of Funk was extremely
attractive and seemed to account for the etiology
of several diseases in a most satisfactory way. It
seemed to rest upon sound observations, but in
reality it rested only upon suggestive chemical ev-
idence. It failed to stand the test of a systematic
investigation of all the more important natural
food-stuffs, by the biological method which was
described in its essential features in illustrating the
nature of the dietary deficiencies of the wheat kernel.

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