yeast cells to grow on a synthetic medium composed solely of known
constituents. This controversy hinged on a discussion as to whether these
media were efficient unless reinforced with something derived from a
living organism. In 1901 Wildier in France published an article in which
he showed that extracts of organic matter when added to synthetic media
had the power to markedly stimulate the growth of yeast organisms. He did
not attempt at the time to identify the nature of this stimulatory
substance, but since it was derived from living organisms, he called it
"Bios." Soon after the discovery of vitamines the bacteriologists began to
discover that they or an analogous factor apparently played a part in the
growth of certain strains of bacteria, especially the meningococcus. In
1919 Roger Williams working in Chicago University was struck with the
bearing of Wildier's work on the vitamine hypothesis and formed the theory
that Wildier's "bios" might be the water-soluble vitamine "B." He
proceeded to test out this theory and demonstrated that extracts of
substances rich in the "B" vitamine had a marked effect on the stimulation
of yeast growth. He developed these experiments and devised a method of
comparing the growth of yeast cells when stimulated by such extracts. The
results were so striking as to appear to justify his view and he then
suggested that his method might be used as a test for the measure of "B"
vitamine in a given source. William's method consisted essentially in
adding the extract of an unknown substance to hanging drops in which were
suspended single yeast cells and observing the rate of growth under the
microscope. Soon after, Miss Freda Bachman reinvestigated the problem with
various types of yeast and found that practically all types of yeast
respond to the stimulation of these "bios" extracts. Her method consisted
in the use of fermentation tubes and the stimulatory effect was measured
by the amount of CO_2 produced in a given time. By this method she
confirmed Williams' view that the "bios" of Wildier was apparently
identical with vitamine "B" and that most yeasts require this vitamine for
their growth. She also suggested that her method might be made the basis
of a test for vitamine content. In 1919 Eddy and Stevenson made extended
experiments with these two methods in the attempt to improve the technique
and make it serve as a quantitative measure. Their experiments served two
purposes, first to bring out certain difficulties in the methods of the
two authors from the quantitative viewpoint and the development of a
technique to correct these difficulties and secondly to add more data
bearing on the specificity of the test. Soon after their publication Funk
became interested and coming to the same conclusions as to specificity
devised a centrifugating method for measuring the yeast growth. Williams
also improved his original method and devised a gravimetric method for the
same purpose. From the viewpoint of methodology we now have methods which
are suitable as quantitive procedures for determining the effect of
extracts of unknown substances on yeast growth and hence if the
stimulatory substance is vitamine "B," a means of determining within a
space of twenty-four hours the approximate content of stimulatory material
in a given source. Since the Funk method is the simplest of these and
illustrates the principles involved it will suffice to describe that.
_Funk method of yeast test with Eddy and Stevenson modification_
1. To a basal diet of 9 cc. of sterile culture medium such as a von Nageli
solution [Footnote: von Nageli's solution consists of the following
ingredients NH_4NO_3, 1 gram; Ca_3(PO_4)_2, 0.005 gram; MgSO_4, 0.25 gram
dextrose 10.0 grams made up to 100 cc. with distilled water. Other culture
media may be used and such combinations will be found in any text on
yeasts. They all permit a certain amount of growth but all are apparently
stimulated by the addition of vitamine extracts.] in a sterile test tube
is added 1 cc. of the sterile, neutral, watery extract of the source of
the vitamine. A pure culture of Fleischman's yeast (Funk prefers brewer's
yeast) is maintained on an agar slant and twenty-four hours before the
test is to be made, a transplant is made to a fresh agar slant. One
standardized platinum loopful of the twenty-four hour yeast growth is then
used to inoculate the contents of the tube, the tube stoppered with cotton
and incubated for from twenty-four to seventy-two hours at a temperature
of 31°C. The seventy-two hour incubation period yields nearly optimum
growth for this purpose.
2. At the end of this time the yeasts are killed by plunging the tube in
water heated to 80°C. and maintained at this temperature for fifteen
minutes. The contents of the tubes are then poured into a Hopkins
centrifuge tube which has a capillary tip graduated in hundredths of a
cubic centimeter. After twenty minutes centrifugating at a speed of about
2400 revolutions per minute the yeasts in the solution have all been
packed into the tip and the volume can then be read accurately to
thousandths of a cubic centimeter (with the aid of a scale and magnifier).
With a control tube containing 9 cc. of the sterile media and 1 cc. of
distilled water in place of the 1 cc. of extract a comparison can be
obtained which is an accurate measure of the stimulatory effect of the
extract. If this stimulus is due purely to vitamine it is obvious that
this procedure would enable us to compare extracts of known weights of and
arrive at comparisons which would be measures of their vitamine content.
In other words the procedure is now in a satisfactory form for testing and
its value depends merely upon our ability to show that the stimulus given
the yeast is due solely to vitamine "B."
The interest of the vitamine student in this test will be easily
understood for it is so simple of manipulation and so rapid in producing
results that it is the nearest approach to a chemical test of satisfactory
nature yet proposed but unfortunately evidence soon began to accumulate to
show that the stimulation produced by extracts of various sources is not a
matter of pure vitamine. If we plot a curve of stimulation for various
dilutions of a given extract we find that the stimulation is not directly
proportional to the concentration of vitamine present but is a composite
of several factors. The chart derived from experiments by Eddy and
Stevenson shows the general nature of this curve. Other experimenters have
reached similar results and some have gone so far as to maintain that the
stimulation is not due to vitamine "B" at all. It is therefore evident
that until this controversy is settled the yeast test cannot be used for
the purpose proposed. Our own experiments at present make us still firm in
our belief that _one_ of the factors and perhaps the most important
factor in the stimulation effect is the vitamine but until we can devise a
basal medium that is comparable to that used in rat feeding experiments,
i.e., one that contains all the elements for optimum growth of yeasts
except vitamine "B" it will be unsafe to draw conclusions from the test as
to vitamine content. It may be possible to so treat our extracts as to
eliminate from them all other stimuli except the vitamine or to destroy
the vitamine in them and thus permit the comparison of an extract with the
vitamine destroyed against one in which it is present and thus arrive at
the result desired. At any rate all we can say at present is that the
yeast test is unreliable as a measure of vitamine content but that if it
can be made quantitative its advantages are so great that it is very much
worth while to continue work upon it until it is certain that it cannot be
made to produce the desired result.
[Illustration: FIG. 7. GROWTH RATE OF YEAST UNDER ALFALFA EXTRACT
STIMULATION
This chart shows the effect of varying concentrations of an alfalfa
extract on the growth rate of the yeast cell. The rate of growth was
determined after the Funk method by centrifuging the cells after seventy-
two hours incubation and measuring the volume in cubic centimeters. The
shape of the curve shows that this method will not give comparative
results unless the extracts tested are dilute enough for the
determinations to fall in the steep part of the curve.]
Another reason for our attention to this test is that if it can be made to
show vitamine effect it provides an excellent medium for investigation of
vitamine "B" reactions, and a method for studying the effect of the
vitamine upon the protoplasm of a single cell.
CHAPTER V
THE SOURCES OF THE VITAMINE
Having now considered the general principles involved in vitamine testing
we may justly ask what information they have yielded us in regard to the
distribution of the vitamines in nature. If we must include vitamines in
our diets it is important to know how to select foods on this basis, hence
a classification of them on the ground of vitamine distribution becomes
essential. The newness of the subject and the limited tests that have been
made as well as the uncertainty residing in the test results make any
classifications presented more or less approximations but we present such
attempts as have been made, with the understanding that these tabulations
are merely guides and not quantitative measurements in the sense that
tables giving calorie values of protein, fat and carbohydrate content are.
The following table (1) has been freely copied from a report of the
British Medical Research Committee to which acknowledgment is hereby
given.
TABLE 1
_Pages 50 and 61 of the British Medical Research Committee's report_
__________________________________________________________________________
| | |
CLASSES OF FOODSTUFFS |VITAMINE "A"|VITAMINE "B"|VITAMINE "C"
___________________________________|____________|____________|____________
| | |
_Fats and oils:_ | | |
Butter . . . . . . . . . . . . | +++ | 0 |
Cream . . . . . . . . . . . . . | ++ | 0 |
Cod-liver oil . . . . . . . . . | +++ | 0 |
Mutton and beef fat or suet . . | ++ | |
Lard . . . . . . . . . . . . . | 0 | |
Olive oil . . . . . . . . . . . | 0 | |
Cotton seed oil . . . . . . . . | 0 | |
Cocoanut oil . . . . . . . . . | 0 | |
Cocoa-butter . . . . . . . . . | 0 | |
Linseed oil . . . . . . . . . . | 0 | |
Fish oil, whale oil, herring | | |
oil, etc. . . . . . . . . . . | ++ | |
Hardened fats (hydrogenated) | | |
of animal or vegetable origin | 0 | |
Margarine from animal fat . . . | In propor- | |
| tion to | |
| animal | |
| fat used | |
Margarine from vegetable fat | | |
or lard . . . . . . . . . . . | 0 | |
Nut butters . . . . . . . . . . | + | |
_Meat, fish, etc.:_ | | |
Lean meat (beef, mutton, etc.) | + | + | +
Liver . . . . . . . . . . . . . | ++ | ++ | +
Kidneys . . . . . . . . . . . . | ++ | + |
Heart . . . . . . . . . . . . . | ++ | + |
Brain . . . . . . . . . . . . . | + | ++ |
Sweetbreads . . . . . . . . . . | + | ++ |
Fish, white . . . . . . . . . . | 0 | Very slight|
| | if any |
Fish fat (salmon, herring, etc.)| ++ | Very slight|
| | if any |
Fish roe . . . . . . . . . . . | + | ++ |
Tinned meats . . . . . . . . . | ? | Very slight| 0
_Milk, cheese, etc.:_ | | |
Milk, cow's whole raw . . . . . | ++ | + | +
Milk, cow's skim . . . . . . . | 0 | + | +
Milk, cow's dried whole . . . . | Less than | + | Less than
| ++ | | +
Milk, cow's boiled whole . . . | ? | + | Less than
| | | +
Milk, cow's condensed sweetened | + | + |
Cheese, whole milk . . . . . . | + | | Less than
| | | +
Cheese, skim milk . . . . . . . | 0 | |
Eggs, fresh . . . . . . . . . . | ++ | +++ | 0?
Eggs, dried . . . . . . . . . . | ++ | +++ | 0?
_Cereals, pulses, etc.:_ | | |
Wheat, maize, rice (whole germ) | + | + | 0
Wheat, maize, rice germ . . . . | ++ | +++ | 0
Wheat, maize, rice bran . . . . | 0 | ++ | 0
White wheat flour, pure corn | | |
flour, polished rice, etc. . | 0 | 0 | 0
Custard powders, egg substi- | | |
tutes prepared from cereal | | |
products . . . . . . . . . . | 0 | 0 | 0
Linseed, millet . . . . . . . . | ++ | ++ | 0
Dried peas, lentils, etc. . . . | | ++ |
Pea-flour, kilned . . . . . . . | | 0 | 0
Soy beans, haricot beans . . . | + | ++ | 0
Germinated pulses or cereals . | + | ++ | ++
_Vegetables and fruits:_ | | |
Cabbage, fresh, raw . . . . . . | ++ | + | +++
Cabbage, fresh, cooked . . . . | | + | +
Cabbage, dried . . . . . . . . | + | + |Very slight
Cabbage, canned . . . . . . . . | | |Very slight
Swedes, raw expressed juice . . | | | +++
Lettuce . . . . . . . . . . . . | ++ | + |
Spinach, dried . . . . . . . . | ++ | + |
Carrots, fresh, raw . . . . . . | + | + | +
Carrots, dried . . . . . . . . |Very slight | | Less than
| | | +
Beetroot, raw, expressed juice | + | + |
Potatoes, raw . . . . . . . . . | | | +
Potatoes, cooked . . . . . . . | | | ++
Beans, fresh scarlet runners raw| | |
Lemon juice, fresh . . . . . . | | | +++
Lemon juice, preserved . . . . | | |
Lime juice, fresh . . . . . . . | | | ++
Lime juice, preserved . . . . . | | |Very slight
Orange juice, fresh . . . . . . | | | +++
Raspberries . . . . . . . . . . | | | ++
Apples . . . . . . . . . . . . | | | +
Bananas . . . . . . . . . . . . | + | + |Very slight
Tomatoes, canned . . . . . . . | | | ++
Nuts . . . . . . . . . . . . . | + | ++ |
_Miscellaneous:_ | | |
Yeast dried . . . . . . . . . . | ? | +++ |
Yeast extract and autolysed . . | ? | +++ | 0
Meat extract . . . . . . . . . | 0 | 0 | 0
Malt extract . . . . . . . . . | | + in some |
| | specimens |
Beer . . . . . . . . . . . . . | | 0 | 0
Honey . . . . . . . . . . . . . | | + |
___________________________________|____________|____________|____________
+++ indicates abundant; ++ relatively large; + present in small amount;
0 absent.
The following table (2) has been compiled from a review of both British
and American data and represents a rather more complete classification
than the British report. The four plus system has also been used to permit
more complete comparisons.
TABLE 2
_________________________________________________________________________
| | |
FOODSTUFF | "A" | "B" | "C"
____________________________________|___________|___________|____________
| | |
_Meats_: | | |
Beef heart . . . . . . . . . . . | + | + | ?
Brains . . . . . . . . . . . . . | ++ | +++ | +?
Codfish . . . . . . . . . . . . | + | + | ?
Cod testes . . . . . . . . . . . | + | |
Fish roe . . . . . . . . . . . . | + | ++ | ?
Herring . . . . . . . . . . . . | ++ | ++ | ?
Horse meat . . . . . . . . . . . | ++ | ++ |
Kidney . . . . . . . . . . . . . | ++ | ++ |
Lean muscle . . . . . . . . . . | 0 | 0 | +?
Liver . . . . . . . . . . . . . | + | + | +?
Pancreas . . . . . . . . . . . . | 0 | +++ |
Pig heart . . . . . . . . . . . | + | + | ?
Placenta . . . . . . . . . . . . | + | |
Thymus (sweetbreads) . . . . . . | 0 | 0 | 0
_Vegetables:_ | | |
Beet root . . . . . . . . . . . | + | + | ++
Beet root juice . . . . . . . . | ? | Little | +++
Cabbage, dried . . . . . . . . . | +++ | +++ | +
Cabbage, fresh . . . . . . . . . | +++ | +++ | ++++
Carrots . . . . . . . . . . . . | +++ | +++ | ++
Cauliflower . . . . . . . . . . | ++ | +++ | ++
Celery . . . . . . . . . . . . . | ? | +++ | ?
Chard . . . . . . . . . . . . . | +++ | ++ | ?
Dasheens . . . . . . . . . . . . | + | ++ | ?
Lettuce . . . . . . . . . . . . | ++ | ++ | ++++
Mangels . . . . . . . . . . . . | ++ | ++ | ?
Onions . . . . . . . . . . . . . | ? | +++ | +++
Parsnips . . . . . . . . . . . . | ++ | +++ |
Peas (fresh) . . . . . . . . . . | + | ++ | +++
Potatoes . . . . . . . . . . . . | 0 | +++ | ++
Potatoes (sweet) . . . . . . . . | +++ | ++ | ?
Rutabaga . . . . . . . . . . . . | | +++ |
Spinach . . . . . . . . . . . . | +++ | +++ | +++
_Cereals:_ | | |
Barley . . . . . . . . . . . . . | + | +++ | ?
Bread (white) . . . . . . . . . | + | +? |
Bread (whole meal) . . . . . . . | + | +++ | ?
Maize (yellow) . . . . . . . . . | + | +++ | ?
Maize (white) . . . . . . . . . | 0 | +++ | ?
Oats . . . . . . . . . . . . . . | + | +++ | 0
Rice polished . . . . . . . . . | 0 | 0 | 0
Rice (whole grain) . . . . . . . | + | +++ | 0
Rye . . . . . . . . . . . . . . | + | +++ | 0
Corn embryo . . . . . . . . . . | | +++ |
Corn (kaffir) . . . . . . . . . | | +++ |
Corn (see maize) . . . . . . . . | | |
Corn pollen . . . . . . . . . . | | ++ |
Malt extract . . . . . . . . . . | 0 | 0 | 0
Wheat bran . . . . . . . . . . . | 0 | + | 0
Wheat embryo . . . . . . . . . . | ++ | +++ | 0
Wheat endosperm . . . . . . . . | 0 | 0 | 0
Wheat kernel . . . . . . . . . . | + | +++ | 0
_Other seeds:_ | | |
Beans, kidney . . . . . . . . . | | +++ |
Beans, navy . . . . . . . . . . | | +++ | 0
Beans, soy . . . . . . . . . . . | + | +++ | 0
Cotton seed . . . . . . . . . . | ++ | +++ |
Flaxseed . . . . . . . . . . . . | ++ | +++ |
Hemp seed . . . . . . . . . . . | ++ | +++ |
Millet seed . . . . . . . . . . | ++ | +++ |
Peanuts . . . . . . . . . . . . | + | ++ |
Peas (dry) . . . . . . . . . . . | +? | ++ | 0
Sun flower seeds . . . . . . . . | + | |
_Fruits:_ | | |
Apples . . . . . . . . . . . . . | | ++ | ++
Bananas . . . . . . . . . . . . | ? | ++ | ++
Grapefruit . . . . . . . . . . . | | +++ | +++
Grape juice . . . . . . . . . . | | + | +
Grapes . . . . . . . . . . . . . | 0 | + | +
Lemons . . . . . . . . . . . . . | | +++ | ++++
Limes . . . . . . . . . . . . . | | ++ | ++
Oranges . . . . . . . . . . . . | | +++ | ++++
Pears . . . . . . . . . . . . . | | ++ | ++
Raisins . . . . . . . . . . . . | | + | +
Tomatoes . . . . . . . . . . . . | ++ | +++ | ++++
_Oils and fats:_ | | |
Almond oil . . . . . . . . . . . | | 0 | 0
Beef fat . . . . . . . . . . . . | + | 0 | 0
Butter . . . . . . . . . . . . . | ++++ | 0 | 0
Cocoanut oil . . . . . . . . . . | 0 | 0 | 0
Cod liver oil . . . . . . . . . | ++++ | 0 | 0
Corn oil . . . . . . . . . . . . | 0 | 0 | 0
Cotton seed oil . . . . . . . . | 0? | 0 | 0
Egg yolk fat . . . . . . . . . . | ++++ | 0 | 0
Fish oils . . . . . . . . . . . | ++ | 0 | 0
Lard . . . . . . . . . . . . . . | 0 | 0 | 0
Oleo, animal . . . . . . . . . . | + | 0 | 0
Oleo, vegetable. . . . . . . . . | 0 | 0 | 0
Olive oil . . . . . . . . . . . | 0 | 0 | 0
Pork fat . . . . . . . . . . . . | 0? | 0 |
Tallow . . . . . . . . . . . . . | 0 | 0 | 0
Vegetable oils . . . . . . . . . | 0? | 0 | 0
_Nuts:_ | | |
Almonds . . . . . . . . . . . . | + | +++ |
Brazil nut . . . . . . . . . . . | | +++ |
Chestnut . . . . . . . . . . . . | | +++ |
Cocoanut . . . . . . . . . . . . | ++ | +++ |
English walnuts . . . . . . . . | | +++ |
Filbert . . . . . . . . . . . . | | +++ |
Hickory . . . . . . . . . . . . | + | + | +
Pine . . . . . . . . . . . . . . | + | + | +
_Dairy products:_ | | |
Butter . . . . . . . . . . . . . | ++++ | 0 | 0
Cheese . . . . . . . . . . . . . | ++ | + | ?
Condensed milk . . . . . . . . . | ++ | + | 0
Cream . . . . . . . . . . . . . | +++ | + | ?
Eggs . . . . . . . . . . . . . . | ++++ | ++ | 0
Milk powder (skim) . . . . . . . | + | +++ | +?
Milk powder (whole) . . . . . . | +++ | +++ | +?
Milk whole . . . . . . . . . . . | +++ | +++ | ++
Whey . . . . . . . . . . . . . . | + | +++ | +
_Miscellaneous:_ | | |
Alfalfa . . . . . . . . . . . . | +++ | +++ | ?
Blood . . . . . . . . . . . . . | Varies with source
Clover . . . . . . . . . . . . . | +++ | ++++ | ?
Honey . . . . . . . . . . . . . | | ++ | 0
Malt extract . . . . . . . . . . | 0 | 0 | 0
Nectar . . . . . . . . . . . . . | 0 | 0 | 0
Timothy . . . . . . . . . . . . | ++ | +++ |
Yeast, brewers . . . . . . . . . | 0 | ++++ | 0
Yeast cakes . . . . . . . . . . | 0 | ++ | 0
Yeast extract . . . . . . . . . | 0 | +++ | 0
____________________________________|___________|___________|____________
CHAPTER VI
THE CHEMICAL AND PHYSIOLOGICAL PROPERTIES OF THE VITAMINE
While the chemists have not yet been able to isolate and identify the
various vitamines they have succeeded in demonstrating many of the
properties of these substances and it is the knowledge of these properties
that has enabled us to produce concentrates and conduct tests. Another
practical consideration involved in this matter of properties lies in the
effect of cooking and commercial methods of food preparation, for not only
must we learn where the vitamine resides but how to prevent injury or
destruction in our utilization of the source.
The properties of the vitamines may therefore be grouped under two heads:
first chemical properties and second physiological properties.
I. CHEMICAL PROPERTIES OF VITAMINE "A"
_a_. This dietary factor's presence in butter fat and egg yolk fat
indicates its solubility in the fat and it would naturally follow that the
fat solvents would suffice to remove it with the fats when food sources
are treated with such a reagent. Experience has shown however that while
ether extraction applied to butter or egg yolk removes the vitamine with
the fat this process fails when it is applied to vegetable sources such as
cotton seed, corn germ, spinach, lettuce, etc. Neither does the cold or
hot press method of oil extraction liberate the vitamine with the oil.
Recent experiments by Osborne and Mendel, to which we have previously
referred, have shown that preliminary treatment of vegetable sources with
alcohol seems to loosen the bond between the source and the vitamine and
that when this binding is once loosened subsequent ether extraction will
take the vitamine out. That the binding is not difficult to break is shown
by the fact that when vegetables are eaten as a source of vitamine the
body is able to separate the complex. It is further evident that the body
does separate this complex and stores it in animal fat from the
experiments with cow feeds and feeding. Milk for example is rich or poor
in vitamine according to the supply of the latter in the food given to the
cow. The only logical conclusion to be drawn from this observation is that
the cow does not synthesize this factor but splits it off from the food
source and then, since it is fat soluble, is able to mobilize it in the
butter fat of the milk or to a more limited extent in the body fat. This
observation as to the dependence of milk content upon food has been
confirmed in the case of nursing mothers and suggests the need of especial
attention to the diet of the mother during the lactating period.
_b_. It has been generally assumed that the "A" vitamine is
comparatively stable to heat. Sherman, MacLeod and Kramer state that "dry
heating at a temperature of 100°C. with free access of air, only very
slowly destroyed fat soluble vitamine." Osborne and Mendel reported that
butter fat treated with steam for two hours and a half did not appear to
have lost its value as a source of this vitamine. Drummond's earlier work
with fish oils and whale oils seemed to confirm this conclusion. Sherman
and his co-workers cited above put it this way: "The results thus far
obtained emphasize the importance of taking full account of the time as
well as the temperature of heating, and of the initial concentration of
the vitamine in the food, as well as of the opportunity for previous
storage of the vitamine by the test animal." More recent work by Steenbock
and his co-workers in America shows that these earlier results are
incorrect in the case of butter fat and that twelve hours exposure of
butter fat to 100°C. may, under certain conditions, destroy the efficiency
of that substance as a source of the vitamine. Drummond and other English
workers have confirmed Steenbock in later experiments. Their work has
shown that the presence or absence of oxygen is a factor, which may
determine the extent of destruction of the vitamine. Heat alone is of very
limited effect but when sources are heated in the presence of oxygen
destruction of the A vitamine may be very rapid. Drummond attributes the
absence of the A vitamine in lard to the oxidation that takes place in the
commercial rendering of this product. We must conclude therefore that
while the vitamine may be destroyed by continuous exposure to a
temperature of 100°C. the effect is largely determined by the nature of
the process and the way the vitamine is held in the source. Cooking of
vegetables therefore will not as a rule result in appreciable destruction
of this factor.
_c_. The process of hydrogenation used in hardening fats appears to
completely destroy the vitamine, hence the many lard substitutes now in
use must in general be considered "A" vitamine-free regardless of the
content of "A" in the fats from which they are derived unless they have
been made by blending instead of hydrogenation.
_d_. Acids and alkalies have apparently little effect on this
particular vitamine.
It may be well to state here however that owing to variability in behavior
with variation in conditions it is dangerous to draw too general
conclusions and until a given source has actually been investigated under
specific cooking conditions one should not rely too strongly on analogies
based on comparative experiments. This statement applies to all vitamines
and presents one of the live subjects of investigation for the cooking
schools and the food factories.
_e_. Little has been learned further about the chemistry of this
substance. [Footnote: Since the above was put in type Steenbock has shown
that the A vitamine resists saponification and that by saponifying fats
which contain the A it may be possible to secure a fraction rich in the
vitamine and free of fat.] Butter fat, nitrogen free and phosphorus free
is shown to carry the vitamine and it is therefore assumed that the
vitamine lacks these elements. It has been claimed that it may be removed
from butter fat by prolonged extraction with water but this has not been
confirmed by more recent experimenters. Steenbock was the first to call
attention to the association of the A vitamine with yellow pigment in
plant and animal sources. Butter, egg yolk, carrots, yellow corn contain
it while white corn and white roots are less rich in this vitamine. This
observation suggested the chemical relation between the vitamine and
carotin. It has however been shown by Palmer and others that carotin is
not vitamine A. This association of the pigment with the vitamine is
therefore apparently a coincidence and this clue has failed as yet to
throw light on the chemical nature of vitamine A.
II. THE CHEMICAL PROPERTIES OF VITAMINE "B"
When Funk first studied this substance he conducted all his evaporations
in vacuo from fear that higher temperatures would prove destructive.
Subsequent investigation however has shown that 100° has very little if
any destructive effect if the vitamine is held in acid or neutral
solution. Temperatures between 100° and 120° maintained in an autoclave at
15 pounds above normal pressure do tend to slowly destroy the factor. The
extent of this destruction also varies with the character of the crude
extract. In general, then, there is little fear of injuring this vitamine
in ordinary cooking temperatures if the use of alkali is avoided.
The effect of alkali depends upon the temperature to a very marked degree.
Osborne has recently reinvestigated this matter and finds that in the
presence of a 0.1N solution of alkali at 20°C. there is very little
destruction but that raising the temperature to 90°C. brings about a
marked destruction. Seidell has shown that if the vitamine is absorbed by
Lloyd's reagent and this reagent be then extracted with dilute alkali the
vitamine passes into the alkaline solution. If the latter is neutralized
quickly it is possible to recover most of the vitamine by this method. The
effect of alkali becomes of practical importance to the housewife because
of certain cooking habits. I refer to the well known practice of adding
soda to the water in which vegetables are cooked to soften the vegetable
and accelerate the cooking. Daniels and Loughlin in this country
investigated this matter and came to the conclusion that this procedure
did not produce enough destruction to be dangerous. Later the matter was
studied by Chick and Hume in England and these investigators brought out a
feature that had perhaps been overlooked in the previous work. Their point
was that in ordinary feeding tests the results merely tell whether there
is enough vitamine present to produce normal growth. Hence if the
substance tested has much vitamine, a large part of it might be destroyed
and this fact not appear in the test because enough might still be left to
induce normal growth. By reducing the amount tested so that it was just
adequate for normal growth and then applying the soda-cooking
experimentation they showed that this method of cookery does do serious
harm to the vitamine. From the practical point of view it is of course
sufficient to show that enough is left after a cooking process to suffice
for normal growth when the substance is taken in the portion sizes
ordinarily eaten. The effect of alkali deserves more attention on the part
of cooks and food preparateurs and we need more data concerning the
minimal dose necessary to protect the human animal.
In neutral and acid solution it is perfectly safe to assume little
destruction of this vitamin through heat and it is now common practice to
boil sources with the extracting reagent and to use the steam bath freely
to concentrate and evaporate these extracts. We have recently investigated
the effect upon cabbage of cooking in a pressure cooker at eight pounds
pressure. The cabbage so cooked, when dried and mixed so as to form 10 per
cent of a basal vitamine free diet, yielded all the "B" vitamine necessary
to produce normal growth in rats.
The very name of this vitamine indicates its ready solubility in water. It
is also soluble in 95 per cent alcohol and either of these extractants may
be used to obtain the vitamine. It is not readily soluble in absolute
alcohol and 95 per cent is not as good an extractant as water. Substances
rich in the vitamine apparently yield the latter more readily if they have
first been subjected to autolysis or if the extracting fluid is acidified.
Funk was the first to show that yeast produced a greater yield if it was
allowed to autolyse before extraction with alcohol. However, Osborne and
Wakeman have produced a method of treating fresh yeast by boiling it with
slightly acidified water which seem as efficient as autolysis in the yield
produced.
The various methods of extraction now in vogue have already been discussed
in Chapter II and need not be repeated here. In general it is apparent
that to obtain concentrates of high potency it is permissible to employ
temperatures of 100°C. if we will maintain an acid or neutral reaction but
that alkali should be avoided wherever possible and when its use is
imperative the temperature must be kept below 20°C. or destruction will
result. In applying this rule to cooking operations the results should be
determined by direct tests rather than by assumptions based on these
generalizations. It should also be noted that the alkalinity of a solution
should be determined on the basis of hydrogen ion concentration and not on
amount of alkali added since many substances have a marked buffer
reaction.
The water-soluble "B" is not only soluble in water but can be dissolved in
other reagents. Thus McCollum has shown that while benzene is of little
value as an extractant of this vitamine, if we will first extract the
vitamine with alcohol or water and deposit this on dextrin by evaporation
it is then possible by shaking the activated dextrin with benzene to cause
the vitamine to pass into solution in benzene. Voegtlin and Meyers have
recently shown that it is soluble in olive oil and in oleic acid and their
data suggest a new means of concentrating the substance which may be of
value in tracing its character.
The "B" vitamine is relatively easily absorbed by finely divided
precipitates. We have already referred to the use of fuller's earth for
this purpose by Seidell. This adsorptive power sometimes manifests itself
in the treatment of plant extracts. A watery extract of alfalfa can be
made to throw down its protein complex by diluting it to 40 per cent with
alcohol. Osborne reports however that this process frequently removes the
vitamine also which appears to be thrown down with the precipitated
material. This adsorptive power therefore often appears as a difficulty in
the handling of the substance as well as a means of extraction. We have
used Osborne's method with alfalfa extracts and find the above result is
not by any means invariable, for in some of our extracts we retained the
greater part of the vitamine. Kaolin and ordinary charcoal are not very
good adsorbents but the latter can be activated to serve this purpose.
The elementary nature of the "B" vitamine remains a mystery. Extracts
which contain it show the presence of nitrogen. Funk's earlier researches
on yeast and rice polishings both yielded crystalline complexes which he
analysed. His data on this subject follow:
_A. The yeast complex_
Crystals melting at 233°C. consisting of:
I. A complex melting at 229°C. and forming needles and prisms nearly
insoluble in water and with the apparent formula of C_24H_19O_2N_5.
II. A complex melting at 222°C. and soluble in water. Formula
C_29H_23O_2N_5.
III. Nicotinic acid melting at 235°C. C_6H_5O_2N.
_B. The rice complex_
Crystals melting at 233°C. consisting of:
I. A complex melting at 233°C. and with a formula of C_26H_20O_9N_4.
II. Nicotinic acid melting at 235°C. C_6H_5O_2N.
Funk held at the time that the possible nature of the compound was:
HN
| \
OC C_16H_18O_6
| /
HN
It was this idea that led him to call it an "amine."
We are unable at present to report any nearer approach to the elementary
analysis and all attempts at purification have shown a tendency to make
the active substance either disappear entirely or else distribute itself
over the several fractions instead of concentrating itself in one. Its
basic nature seems to be well established by its behavior with
phosphotungstic acid and its ready adsorption by carbons activated to take
up basic substances.
III. THE CHEMICAL PROPERTIES OF WATER-SOLUBLE "C"
The properties of this newest member of the family are still less defined.
All are agreed that it is much more sensitive to heat and alkali than the
other two. Temperatures above 50°C. are usually destructive though the
time factor is extremely important as well as the reaction. Hess for
example has found that the temperature used to pasteurize milk continued
for some time, is more destructive to the vitamine than boiling water
temperature continued for only a few minutes. The extent to which orange
juice and tomato juice will resist high temperatures indicates the
protective action of acids to be considerable.
Dr. Delf's experiments at the Lister Institute were especially directed to
the behavior of this vitamine in cabbage. She first determined the minimum
close of raw cabbage required to prevent scurvy in guinea pigs and found
that it was less than 1.5 grams and more than 0.5 gram daily. When the
cabbage was heated in water at 60°C. for an hour, symptoms of severe
scurvy were just prevented by 5 grams of the cooked cabbage fed daily. By
heating at 70°, 80°, 90° and 100° for the same length of time the 5 grams
of cooked material could be made non-effective as a preventive. Her
conclusions are that when cabbage is cooked for one hour at temperatures
ranging from 80° to 100°C. the cabbage leaves lose about 90 per cent of
the antiscorbutic power originally held by the raw equivalent. Sixty
minutes at 60° or twenty minutes at 90° to 100° resulted in about 80 per
cent destruction. Dr. Delf calls attention also to the fact that the
effect of the heat is increased to only a slight degree by rise in
temperature. Assuming that the effect of the rise is orderly, a
temperature coefficient of 1.3 is indicated for each rise of 10°C. This
low result suggests to Delf a contradiction to any theory which imputes to
the vitamine enzyme or protein-like qualities and on the other hand
suggests that the substance is much simpler in constitution. Her results
also confirm Hoist and Fröhlich as showing its great sensitiveness at
temperatures of 100° and below and obviously have a direct bearing upon
cookery methods.
The substance is soluble in water and passes through a parchment membrane
or a porcelain filter. Unlike the "B" it is apparently not adsorbed by
fine precipitates such as fullers' earth or colloidal iron. Harden and
Zilva showed that when a mixture of equal volumes of autolysed yeast and
orange juice is treated with fuller's earth the "B" is removed and the "C"
left unaltered. Eddy and La Mer have treated orange juice with fullers'
earth and then tested the filtered off juice as cure and preventive of
scurvy in guinea pigs. Their results showed that 6-2/3 cc. of the treated
juice was curative, hence the loss due to adsorption must be less than 60
per cent to 70 per cent. Harden and Zilva were among the first to state
that the vitamine is much more stable in acid than in alkali. They have
shown, that even 1/50 N sodium hydrate at room temperature has a rapidly
destructive effect. On the other hand Delf showed that when 0.5 gm. citric
acid is added to the water in which germinated lentils are boiled, the
loss of the antiscorbutic properties is, if anything, greater than when no
addition of acid is made. She therefore concluded that in cooking
vegetables there should be no addition of either acid or alkali to the
cooking water if one wishes to conserve this vitamine. Sherman, La Mer, and
Campbell have been engaged in experiments bearing on this point throughout
the past two years. Some of their results have recently been published and
their observations are worthy of special attention from their bearing on
the character of reaction of the vitamine in general. They first proceeded
to determine the amount of filtered tomato juice just necessary to produce
scurvy in degrees extending from no protection to complete protection and
they also constructed a basal diet which is apparently optimum in
nutrients and all other factors except the "C" vitamine. They found that
at the natural acidity of tomato juice (pH 4.2) boiling for one hour
destroyed practically 50 per cent of the antiscorbutic power and by
boiling for four hours they destroyed 70 per cent, which indicates that
the curve of the destructive process tends to flatten more than that of a
unimolecular reaction. This result was confirmed by heating experiments
conducted at 60°, 80° and 100°. In all cases the temperature coefficients
are low. (Q_10 equals 1.1-1.3) confirming Delf's results. When the natural
acidity of the juice was first neutralized in whole or in part, the juice
then boiled for an hour and immediately cooled and reacidified, it was
found that at less than half neutralization (pH 5.1-4.9) the destructive
effect of an hour's boiling was increased to 58 per cent. When alkali was
added to an initial pH 11 (about N/40 titratable alkali to
phenolphthalein) which fell to 9 during the hour's boiling the destructive
effect was about 65 per cent. When reacidification was omitted and the
neutralized boiled juice stored in a refrigerator for five days before
using the destruction increased 90 to 95 per cent. These particular
observations seem to confirm the view of Harden and Zilva that the
vitamine is especially sensitive to alkali. Hess has recently reported
that oxygen is destructive to this vitamine.
IV. PHYSIOLOGICAL PROPERTIES OF THE "A" VITAMINE
Most authorities are now agreed that both the "A" and "B" types are
essential to growth. Rohmann still holds out against the vitamine
hypothesis. McCollum has recently pointed out that while rats do not have
scurvy it does not at all follow that the absence of the "C" in their diet
is immaterial, but that the contrary is true. Failure to grow, then, may
manifest itself as a result of the absence of either of the first two
types and possibly is affected by the absence of the "C." We have already
seen how this failure may be utilized to measure the vitamine content of a
source. The absence of the "A" type however may also manifest itself in
another way, viz., by the development of an eye disease which McCollum
first designated as xerophthalmia or dry eye and which the British
authorities prefer to designate as keratomalacia. The failure of this
result to always follow the absence of the "A" type in the diet has led
some to question the specificity of this disease. While the infection of
the eye is due to other agents the sum of the evidence supports McCollum
and points to the absence of "A" as the true predisposing cause of the
disease. Bulley, basing her claims on a study of some 500 rats fed on a
synthetic diet, claims that the eye condition is not primarily due to a
dietary deficiency but to an infection resulting from poor hygienic
conditions. In reply to her contentions Emmett has reviewed his own data
and presents them in the following summation:
_________________________________________________________________________
| | | |
RAT | KIND OF VITAMINE | NUMBER CASES | POSITIVE CASES | PER CENT
GROUPS | ABSENT IN THE RATION | REPORTED | OF XEROPH- | POSITIVE
| | | THALMIA |
_______|______________________|______________|________________|__________
| | | |
A | Fat-soluble "A" | 122 | 120 | 98
B | Water-soluble "B" | 103 | 0 | 0
C | None | 216 | 0 | 0
_______|______________________|______________|________________|__________
In these groups special hygienic measures were taken against infection.
Furthermore repeated attempts were made to transmit the eye disease by
using sterile threads, passing them carefully over the edges of the sore
lids and then carefully inoculating the eyes of other rats. These attempts
resulted negatively in all cases where the inoculated rats had plenty of
the "A" vitamine. Treatment of advanced cases of sore eyes with a
saturated solution of boric acid and also with a silver protein solution
failed to relieve the condition while as little as 2 per cent of an
extract containing the "A" vitamine when added to the ration, speedily
resulted in cure and increase of weight. These results combined with
similar data compiled by Osborne and Mendel seem to refute Bulley's
contentions and to justify our acceptance of xeropthalmia as a specific
vitamine deficiency disease.
_Osborne and Mendel data_
Total No. No. with eye
symptoms
Rats on diets deficient in A vitamine . . . . . . . . 136 69
" on diets " " B " . . . . . . . . 225 0
" on diets otherwise deficient . . . . . . . . . 90 0
" on " experimental but probably adequate . 201 0
" on mixed food . . . . . . . . . . . . . . . . 348 0
____ __
Totals . . . . . . . . . . . . . . . . . . . . . 1000 69
On the other hand all workers know that rats often do develop and grow
well for a considerable period of time on a diet free from the "A" and
without manifesting the eye disease. The British authorities explain this
by assuming that animals have the power to lay down a reserve of this
vitamine on which they can draw in emergency. Sherman and his coworkers
confirm this power to store the vitamine. Others have been led to explain
their results as due to contamination of the basal diet. Daniels and
Loughlin recently maintained that the commercial lard used in basal diets
and assumed to be "A" vitamine-free was supplied with sufficient of the
"A" to produce growth and prevent eye disease. Their views have failed of
confirmation by Osborne and Mendel. It is evident therefore that these
occasional lapses from specific response to absence of the "A" vitamine
need further elucidation. It is equally manifest that in the majority of
cases the absence of the "A" will result in both stunted growth and
xeropthalmia. The appearance of the eye disease may be taken however, as a
sure indication of the absence or deficiency in the "A" vitamine.
V. PHYSIOLOGICAL PROPERTIES OF THE "B" VITAMINE
Beri-beri is a disease that is described clinically as a form of severe
peripheral neuritis and may appear in two well marked forms. In one type
there is great wasting, anesthesia of the skin and finally paralysis of
the limbs. In the other, the most marked symptom is excessive edema which
may affect trunk, limbs and extremities. In severe cases the heart is
usually involved and death may occur suddenly from heart failure.
Most observers assume that the antineuritic vitamine discovered by Funk
and the water-soluble "B" are identical. This view is based on the fact
that when sources which yield the water-soluble "B" in rat feeding are
tested for antineuritic power these sources are apparently parallel in
antineuritic power and growth production. Furthermore rats deprived of the
water-soluble "B" develop polyneuroses identical in symptoms with those
shown by rats and pigeons when the latter are placed on a polished rice
diet. The British Medical Board has compiled the following table to
support this view:
_Table compiled from pages 35 and 86, British Medical Research Committee
Report_
_______________________________________________________________________
| |
| | VALUE AS A SOURCE OF
| VALUE AS A SOURCE OF | THE ANTINEURETIC
| WATER-SOLUBLE "B" | FACTOR OR ANTI-BERI-
FOODSTUFF | (SHOWN BY EXPERI- | BERI FACTOR (SHOWN
| MENTS WITH RATS) | BY EXPERIMENTS
| | WITH BIRDS)
_________________________|______________________|_______________________
| |
Rice germ . . . . . . . | +++ | ++++
Wheat germ . . . . . . . | +++ | +++
Yeast . . . . . . . . . | +++ | +++
Egg yolk . . . . . . . . | ++ | +++
Ox liver . . . . . . . . | ++ | +++
Wheat bran . . . . . . . | + | ++
Meat muscle . . . . . . | + | +
Milk . . . . . . . . . . | +++ | Slight
Potatoes . . . . . . . . | + | +
Meat extract . . . . . . | 0 | 0
White bread or flour . . | 0 | 0
Polished rice . . . . . | 0 | 0
_________________________|______________________|_______________________
_________________________________________________________________________
| |
BEHAVIOR | WATER-SOLUBLE "B" | ANTINEURITIC VITAMINE
______________________|________________________|_________________________
| |
Solubility in water . | Very soluble | Very soluble
Solubility in alcohol,| |
dilute . . . . . . | Very soluble | Very soluble
Solubility in absolute| |
alcohol . . . . . . | Insoluble | Insoluble
Solubility in ether, | |
chloroform and | |
benzene . . . . . . | Insoluble | Unusually insoluble
| | but can be extracted
| | with ether from
| | fatty materials such
| | as egg yolk
Stability to heat . . | Stable at 100°C, | Destroyed very slowly
| destroyed rapidly at | at temperatures below
| 120° (in neutral or | 100°C., more rapid at
| acid solution) | temperatures
| | between 110 and 120°C.
Stability to drying . | Stable | Stable
Stability to acids | |
(hot dilute) . . . | Moderately stable | Stable
Stability to acids | |
(cold dilute) . . . | Stable | Stable
Stability to alkalies | |
(hot dilute) . . . | Rapidly destroyed | ?
Stability to alkalies | |
(cold dilute) . . . | Stable |
In dialysis . . . . . | Passes through | Passes through
| parchment membrane | parchment membrane
In adsorption . . . . | Adsorbed from acid | Adsorbed from neutral
| or neutral solution | solutions by fuller's
| by fuller's earth, | earth, colloidal
| charcoal, etc. | ferric hydroxide,
| | animal charcoal, etc.
______________________|________________________|_________________________
Emmett has recently opposed this view and suggests that while the
antineuritic factor and the growth factor are found in the same sources
and have much in common it does not follow that they are identical and
that his experiments tend to show that there are marked differences which
suggest that the "B" type is not a single entity but a group. Mitchell has
summarized very well the controversial phases of this question with an
impartial review of the facts. One of strongest of the opposition
arguments lies in the failure of milk to cure beri-beri except when
administered in large quantities. This objection has been partly allayed
by data bearing on the relation of the milk content to the food of the
cow. Hess, Dutcher, Hart and Steenbock and others have adduced sufficient
evidence to show that the vitamine content of the milk of a cow is largely
determined by the cow's food and as a consequence the milk may be very
poor in vitamine. It is obvious then that the failure of the milk to cure
beri-beri in a given case might be due to this cause and not to lack of
identity of the curative with the growth factor. Osborne and Mendel have
also shown that milk in general must not be classed among the rich sources
of the vitamine, even when the cow's food is rich in vitamine. The
principal facts in the controversy have been presented and at present the
evidence for regarding the vitamines identical seems to be preponderant.
Recently Auguste Lumiere in Paris has put forth the view that polyneuritis
is not merely a vitamine deficiency disease but a nutriment deficiency
disease. He reports that he fed birds on a starvation diet, but with
plenty of vitamine "B". These birds developed polyneuritis and were cured
by adding to the diet plenty of polished rice. The view he wishes us to
take is that all factors must be present and that the absence of the
nutriment is as important as the absence of the vitamine.
In the field of nutrition the absence of the "B" type is particularly
marked by the behavior of the deprived animal. Rats transferred from a
vitamine-free diet to one containing the "B" only, make a much more rapid
recovery toward normal (even in the absence of the "A") than do animals
transferred from the vitamine-free diet to one containing the "A" and not
the "B". This initial jump from addition of the "B" will not continue long
in the absence of the "A", as a general rule. Hess believes that in some
of his infants he was able to show markedly successful growth on the diet
deficient in the "A" but rich in the "B". It is not certain however that
his diets were sufficiently devoid of the "A" factor to be declared "A"
vitamine-free and we know little of the amount of the "A" necessary to
normal infant growth. All results however show that both "A" and "B" are
necessary to growth production and though the term growth vitamine was
applied to the "A" originally the distinction is one that should be
rejected, for both "A" and "B" and possibly "C" are all entitled to this
name.
The manner in which the "B" vitamine acts is still obscure. Voegtlin some
time ago tried to demonstrate that it was identical with secretin and
stimulated pancreatic flow. Recent work at the Johns Hopkins University by
Cowgill and by Aurep and Drummond in England has failed to confirm this.
One of its most marked immediate effects is increase in appetite. Karr in
Mendel's laboratory has shown that dogs which refused their basal diet
would resume eating it if they were allowed to ingest separately a little
dried yeast. Karr studied the metabolism of these dogs as regards nitrogen
partition but the results give little data that is explicatory of the
behavior of the vitamine. In 1915 the author was able to bring about
marked immediate improvement and the ultimate recovery of a number of
infants who were of the marasmic type by merely increasing the "B"
vitamine content of their food. In these cases the vitamine was carried by
Lloyd's reagent and administered mixed with cereal, or the crude extract
was combined with the milk. The pancreas of the sheep was the source used.
In these cases the growth curve changed abruptly from a decline to a sharp
rise and this increase in weight continued and was accompanied by all the
other signs of improved nutrition including increase in appetite. The
change in the growth curve from decline to rise was accomplished without
increasing or changing the basal diet but as the appetite increased the
food had naturally to be increased to keep pace. In these cases the effect
of the vitamine was to enable the child to utilize its normal food and to
increase its appetite for it. This action certainly suggests stimulation
of digestive glands. It also showed that even though the diet may contain
the vitamine as was the case in the milk fed to these children the
addition of the vitamine in concentrated form often gives an upward push
that the food mixture fails to accomplish. Daniels and Byfield have
recently confirmed the effect of increased "B" in infant growth. Cramer
has suggested in a paper published recently in _The American Journal of
Physiology_ that the fatty tissue about the suprarenals may be a
depository of vitamine and that in the absence of vitamine this tissue
loses its supply and that this is the explanation of lessened activity of
that gland in certain metabolic disturbances. This idea tends to support
the idea that vitamines are gland stimulants or hormones and the word food
hormone has been suggested to describe them on that account. A few years
ago Calkins and Eddy tried to determine the effect of the vitamine on the
single cell by use of the paramecium but the results of the experiments
failed to show a vitamine requirement on the part of these animals.
McDougall has recently suggested that the vitamines produce their effect
on yeast cells by increasing hydration. Unfortunately nearly all stimuli
which produce growth are accompanied by hydration effects and it is
difficult to feel that this is a specific vitamine effect although without
denying the possibility. Dutcher has tried to show that vitamines have a
relation to oxidation effects. He observed that the issues of polyneuritic
birds showed a marked reduction in catalase and that this catalase was
restorable by curing the birds with vitamine. The main difficulty lies in
the conflexity of factors that function between cause and effect.
[Illustration: FIG. 8. THE EFFECT OF VITAMINE B ON A MARASMIC INFANT
_1_. On the twentieth day the patient developed a cough. _2_. On
the twenty-first day the cereal was reduced from three times a day to
twice a day. The patient cried during the night. _3_. On the twenty-
second day the stools showed free starch. _4_. On the twenty-third
day an anal abscess was opened. The stools continued to show free starch
until the twenty-fifth day. _5_. On the twenty-fifth day the stools
showed soluble starch but no free starch. _6_. On the twenty-seventh
day the appetite was good and there was no starch. _7_. From the
twenty-eighth to the forty-third day no starch was observed in the stools.
_8_. On the thirty-first day the patient developed a cough. _9_.
From the forty-ninth day to the time of discharge three tablespoonsful of
orange juice were given daily. _10_. On the seventy-third day the
patient developed a bronchitis and mustard paste was applied every four
hours up to the eighty-fourth day.
_V1_ = From the twenty-first day to the forty-third day the patient
received each day 2 grams of Lloyd powder, activated with pancreatic
vitamin. The powder was administered by mixing 1 gram. with each cereal
feeding. The result was 20 ounces gain in twenty-two days, a normal
growth.
_V2_ = After a period of ten days without vitamin, during which the
patient settled down to a level growth curve, the treatment described
under V1 was resumed. This was continued from the fifty-third to the
seventy-sixth day. The result was the resumption of growth but at a slower
rate; 8 ounces were gained in twenty-three days. During the latter part of
the period the patient developed a bronchitis. At the end of this period
the patient was placed on a whole milk formula. From that time to the time
of discharge the patient grew normally.--From the _American Journal of
Diseases of Children,_ 1917, xiv, 189.]
[Illustration: Effects of Vitamines on Growth FIG. 9]
These views are at best speculations. The literature is singularly lacking
in detailed metabolic analyses of excreta of animals during vitamine
stimulation and we know nothing of the possibilities of overdosage, for in
all the work done it has been generally assumed that the presence of an
amount greater than that necessary to produce normal growth is not
material.
The exact manner of the vitamine's action then remains to be determined
and it is obvious that this solution will come much more rapidly if we can
first identify the substance chemically.
VI. THE PHYSIOLOGICAL PROPERTIES OF THE "C" VITAMINE
The steps that led to the acceptance of scurvy as a vitamine deficiency
disease have already been discussed and show how the vitamine acts in such
a disease. Practically all the work done with this vitamine to date has
been concerned either with dosage or with reaction to heat, drying, etc.
The only paper that we have seen that suggests another function than
antiscorbutic power for this vitamine is the one by McCollum and Parsons
in which they suggest that even in animals where scurvy does not exist,
the presence of this factor may be necessary to normal metabolism. The
following table gives some of the data compiled by the British workers as
to the antiscorbutic power of various sources:
_Table compiled from, page 44, British Medical Research Committee
Report_
________________________________________________________________________
| |
| | MINIMUM DAILY
FOODSTUFF | VALUE AGAINST | RATION NECESSARY
| SCURVY | TO PREVENT SCURVY
| | IN GUINEA PIGS
_______________________________|_______________|________________________
| |
_Cereals:_ | |
Whole grains . . . . . . . . | 0 |
Germ . . . . . . . . . . . . | 0 |
Bran . . . . . . . . . . . . | 0 |
Endosperm . . . . . . . . . | 0 |
_Pulses:_ | |
Whole dry . . . . . . . . . | 0 |
Germinated (lentils) . . . . | ++ | 5.0 grams
_Vegetables:_ | |
Cabbage (raw). . . . . . . . | ++++ | 1.0 gram
Cabbage (cooked one-half | |
hour at 100°C) . . . . . . | ++ | 5.0 grams
Runner beans (green pods). . | +++ | 5.0 grams
Carrot (juice) . . . . . . . | + | 20.0 cc.
Beet root (juice). . . . . . | + | More than 20 cc.
Swede (juice) . . . . . . . | +++ | 2.5 cc.
Potatoes (cooked one-half | |
hour at 100°C . . . . . . | + | 20.0 grams
Onions . . . . . . . . . . . | + |
Desiccated vegetables . . . | 0 to + | 60.0 grams expressed
| | as equivalent in
| | fresh cabbage
_Fruits:_ | |
Lemon juice (fresh) . . . . | ++++ | 1.5 cc.
Lemon juice (preserved) . . | ++ | 5.0 cc.
Orange juice (fresh) . . . . | ++++ | 1.5 cc.
Lime juice (fresh) . . . . . | ++ | 10.0 cc.
Lime juice (preserved) . . . | 0 to + |
Grapes . . . . . . . . . . . | Less than + | More than 20.0 grams
Apples . . . . . . . . . . . | Less than + |
Apples dried . . . . . . . . | Less than + |
Tamarind dried . . . . . . . | Less than + |
Mango . . . . . . . . . . . | Less than + |
Kokum . . . . . . . . . . . | Less than + |
_Meat:_ | |
Raw, juice . . . . . . . . . | Less than + | More than 20 cc.
Tinned . . . . . . . . . . . | 0 |
_______________________________|_______________|_______________________
A glance at this table shows the richest sources (see also table on page
59.) To these must be added canned tomato juice which Hess has shown
practically equal to orange juice in efficiency and uses with infants in
the same quantity. This discovery is of great value in instances where the
cost of orange juice is often prohibitive.
La Mer and Campbell have presented some evidence to show that the
antiscorbutic vitamine has a direct effect upon the adrenal glands. In
their scurvy cases they find definite evidence of the enlargement or
hypertrophy of this organ. Whether it affects other organs or not it
remains to be shown.
CHAPTER VII
HOW TO UTILIZE THE VITAMINE IN DIETS
In the preceding chapters it has been the aim to present the findings of
the principal workers in the field. In attempting to summarize the work of
so widely scattered a group as are now engaged in vitamine research it is
impossible to cover completely the many investigations and it is
inevitable that some work will have been overlooked, but the foregoing
covers at least the principal data on the subject. What is the bearing of
all this information on human behavior and what lessons can the layman
draw from it that is of direct application to him? Let us first consider
this question from the dietary viewpoint.
I. INFANT NUTRITION
The limited character of the infant's diet has made the consideration of
vitamine content in his diet much more important than in the case of the
adult with the latter's wide variety of choice. It is evident from the
previous data that a growing infant must not only be provided with a
sufficient supply of calories, nutrients and salts, but must also have a
liberal supply of the three vitamines. Milk has in general been classed as
adequate in all these features, but the vitamine researches have forced us
to reconsider our views in regard to this staple.
The first point to be borne in mind is that the vitamine content of either
cow or human milk is dependent primarily upon the food eaten by the
producer of the milk. In other words milk is merely a mobilization of the
vitamines eaten and if the diet is to yield vitamine-rich milk it must
itself be rich in these factors. Many a cow produces milk low in vitamine
content and the same is true of nursing mothers. There are many "old
wives" prejudices in regard to what food a lactating mother may eat and
unfortunately many of these prejudices are extremely injurious and false.
One of them is the prejudice against green vegetables. Experience has
shown that under ordinary conditions such vegetables are well tolerated by
the mother and from their content of vitamine it is evident that they are
suppliers of these factors. In the case of the cow the fact that cereals
are poor in some of the vitamines and green grasses rich therein, teaches
a lesson that bears directly upon winter feeding of cattle if the milk
supply is to be used for infants. We need a series of diets and cattle
foods for just this purpose of insuring the proper vitamine content in
milk. The preceding tables will enable one to develop such diets fairly
satisfactorily, but more data is urgently needed.
The second point in regard to milk lies in the effect of pasteurization.
This measure is now well nigh universal and in America at least has played
a tremendous part in the reduction of infant mortality, especially during
the summer months. At present, however, we know that this treatment while
removing dangerous germs may also eliminate the antiscorbutic factor. The
sensible attitude then is to recognize this fact and if a clean whole milk
is not available retain the pasteurization and meet the vitamine
deficiency by other agents. Such agents are orange juice and tomato juice
and experience has already shown that these juices can be well tolerated
by infants much earlier than used to be thought possible.
While the pasteurization does not appreciably affect the content of "A" or
"B" vitamines, the variability in content of these vitamines in milk
indicates that it may at times be necessary to supplement them in the
diet. In this connection it must be borne in mind that cereals vary widely
in content and cannot be, as they often are now, considered equivalent in
growth stimulation power. This is a subject that needs special attention
on the part of vitamine experts and dietitians and finally by the food
manufacturers. A good vitamine-rich cereal combination would form an
excellent adjuvant to infant dietaries after they reach the age of
tolerance to such a diet. But even before that time the expressed juice of
various vegetables as well as fruits is found to be well tolerated when
mixed with the milk or given separately, and carrot and spinach juice are
now being used in this connection with good results. These juices like
orange juice contain the B type in abundance and there is no doubt that in
their stimulation to the appetite they play an important part in making
the desirable daily gain.
Fortunately for the layman he has in the scales a good indicator of the
normal progress of his child and so long as growth is normal he can fairly
assume that the diet is adequate but if the scales say otherwise it is
time for him to seek advice and then he is wise who insures that his
medical adviser knows the newer aspects of nutrition. The parent can do
this only by proper selection, but with a little knowledge he can soon
satisfy himself as to whether his pediatrist is the right sort and it is
one of the purposes of this text to bring home to the layman his
responsibility in this matter.
There has grown up in this country a great regard for prepared milk
substitutes in infant feeding and a wide usage of condensed milks,
reinforced milks, diluted milk formulae, etc. All such preparations must
be examined anew in the light of the vitamine discoveries and unless the
given preparation can show a clean bill of health in vitamine content, it
should be either discarded or properly supplemented.
As children grow up, it is fortunate that in their wider choice of
dietaries the danger of vitamine deficiency decreases. But even in
childhood it is unsafe to rely too much on chance. In this country there
are well deserving movements on foot to attract the parents of the
community to the necessity of attention to simple standards of growth
progress, and clinics for this purpose are appearing in increasing numbers
with each year. Such movements are to be most heartily approved. It is
also possible in these measures to not only build better children, but to
make the children themselves intelligent in their rejection of unsuitable
combinations and in that way not only conserve their own health, but
provide an educated body of citizens to pass on the knowledge to future
generations. In a school in New York City I recently had occasion to
discuss the school lunch room and its offerings with the children of the
school in the light of vitamine discoveries. The keenness and intelligence
shown by the children in the discussion that followed has convinced me
that in this matter of vitamines the children themselves can be relied
upon to assist materially in the matter of better food combinations and
intelligent selection.
Finally it must be noted that one of the most common of infant
deficiencies is the failure of the bones to lay down lime. The effect of
this failure is commonly described as rickets. The British workers
consider that this deficiency is a lack of vitamine "A." Their views have
been set forth at greatest length by Mellanby, the principal worker in
this subject. While this view is still debatable and in this country it is
not yet accepted, one fact has come out in the controversy and that is the
remarkable value of cod-liver oil as a preventive of rickets. It may be
that the power of the oil is due to its "A" vitamine content in which it
is known to be rich, or it may be due to a new vitamine, but the fact that
the oil is a preventive in this respect gives the pediatrist another agent
to insure normal growth. The various views on the causes of rickets are
set forth more in detail in Chapter VIII.
II. ADULT DIETS
A study of the dietary habits of various sections of the United States
shows that there is a very general tendency on the part of the majority of
the people to confine their foods to a meat, potato, and cereal diet. The
use of salads is looked upon by many sections as a foreign affectation and
too little attention is paid to the value of eggs, milk and cheese. Enough
has been said already to show that these latter articles have much more
than an esthetic value and one of the missions of the nutrition expert
must be to show the people why dairy products and salads must become
features in the every-day meals of the every-day people. And even if the
salads are still unappreciated, it is necessary that cooked green
vegetables occupy more of a position in the menu than is too often the
case.
There has recently appeared a crusade for the eating of yeast cakes. The
claim made for their use rests on a perfectly firm basis, they are rich in
the "B" vitamine, the proteins of the yeast cake are of good quality and
the cake contains no ingredients poisonous to man. Many people are
reporting beneficial effects from their use. Is there any lesson to be
drawn from this experiment? I feel that the very fact that benefits have
resulted from this yeast feeding is excellent evidence of lack of the
vitamine in the diets of the people affected and a clear argument that the
dietary habits of many people need adjustment to a higher vitamine
content. Whether it is necessary to use yeast cakes or any other
concentrate of vitamine, depends entirely upon whether the ordinary diet
is lacking in these factors and my first advice in the matter would be to
make if possible a selection of the vitamine containing foods and see if
normal conditions did not result before utilizing foods whose taste is not
pleasing or which are taken as medicine. For it is an old experience that
medicines will be taken only so long as the patient is sick and perhaps it
is just as well so. In other words I believe it is possible with
intelligent selection based on such tables as are given in Chapter IV for
people to secure from the butcher and the grocer all their requirements of
these vitamines as a part of their regular palatable diet. To those who
have neglected this selection and find remedy in concentrates, that fact
should lead them to reconstruct their diet rather than persist in
dependence on the medicine to correct faulty diet. In other words the same
arguments apply to the use of medicinal concentrates of vitamines as
applies to the use of laxatives. At times these substances are very
valuable as cures, but it is better by far to so regulate the dietary
habits as to avoid the necessity for their use.
Another phase of this matter that promises to develop in the near future
as a result of the vitamine hypothesis is a reform in food manufacture.
There has been a strong tendency during the past two decades to "purify"
food products. The genesis of this tendency is to be found in a highly
laudable ambition to force the manufacturer to eliminate impurities and
adulterations and provide clean, wholesome, sanitary food. Unfortunately
in attempting to meet this demand on the part of the public, the food
manufacturer has sometimes neglected to seek advice from the nutrition
expert and the latter has failed to appreciate the need of advice. The net
result has been to discover that Nature is often a better chemist than man
and has a much better knowledge of what man needs in his diet than the
chemist. The chemist employed by the manufacturer has, as a result, gone
to such a limit in his development of purification methods as to often
eliminate the essential nutrients and the result has been foods that will
stand analysis for pure nutrients, but which will not stand Nature's
analysis for dietary efficiency. As a secondary result of this tendency we
have acquired habits that in many cases must either be broken or must have
grafted on to them other habits which shall remedy the defective ones.
Take the milling of wheat as an example. Nature put into the wheat grain
most of the elements needed by man and in the early days he was content to
grind up the whole grain and find it palatable. The craze for purity as
expressed by color has gradually replaced this whole meal wheat with a
beautiful white product that is largely pure starch with a few of the
proteins retained. And the principal protein retained lacks one of the
greatest essentials for growth while the vitamines have all been
practically eliminated with the grain germ. Intelligence tells us then
that if, having formed the habit, we will persist in our appetite for
white flour we must see to it that the protein deficiency of the latter
and its lack of vitamines is compensated for by supplementing the diet
with the food-stuffs in which these are rich. We may in other words retain
our bad habits in taste if we will graft on to them the attention to the
eliminated factors and their substitution in other form.
In general then, the adult needs to review his feeding habits and analyze
them in the light of our new knowledge. For this purpose the tables of
Chapter IV supply data useful so far as vitamines are concerned, but it
will be perhaps worth while to repeat here some of this data in more
generalized form.
_a. Sources of the "A" vitamine_
Its most abundant sources are milk, butter, egg yolk fat, and the green
leaves of plants usually classed as salads. Cabbage, lettuce, spinach and
carrots contain this substance in considerable quantity. The germ of
cereals is fairly rich in the factor, but the rest of the grain is
deficient and white flours are therefore poorer than whole meals in this
respect. Cooking temperatures have little effect on this vitamine and
hence little attention need be paid to cooking temperatures as far as this
vitamine is concerned.
_b. Sources of the "B" vitamine_
Its principal sources outside of yeast are the seeds of plants and the
eggs and milk of animals. Meat contains relatively little of this
substance but glandular organs such as the liver and pancreas are fairly
rich in it. In the seeds the distribution is general throughout the whole
body of the seed in the case of beans, peas, etc., but in the cereal
grains it is largely restricted to the embryo portion and hence a high
degree of milling tends to reduce the per cent of this factor in any
highly milled cereal. White flour and polished rice are notable examples
of deficiency of "B" vitamine due to this milling process. Fruits such as
oranges, tomatoes, and lemons are good sources and there is a fair amount
present in the apples and grapes and other common food fruits. Many
vegetables show it in fair abundance, notably potatoes, carrots, and
turnips, but the rule is not general for beets are extremely poor in this
factor. Nuts are also good sources. Eggs, milk and cheese contain it in
fair abundance. Cooking temperatures have little effect on this type if
the temperature does not climb above the boiling point and if the cooking
water is not "alkaline." In the latter case it becomes necessary to
determine the extent of destruction and either eat enough to insure
protection, or reform the method of cookery.
_c. Sources of the "C" vitamine_
Its richest sources are vegetables such as cabbage, swedes, turnips,
lettuce and watercress; fruits such as lemons, oranges, raspberries and
tomatoes. Certain of the vegetables such as potatoes have a substantial
value in this respect, but meat and most prepared milks are low in
antiscorbutic values. The susceptibility of this vitamine to drying, heat
and alkali, make it necessary to scrutinize your cooking methods very
carefully in order not to ruin a good source by a poor preparation of it
for the table.
CHAPTER VIII
AVITAMINOSES OR THE DISEASES THAT RESULT FROM VITAMINE DEFICIENCIES
A survey of the vitamines would be incomplete without a discussion of the
vitamine deficiency diseases in particular, though many of the facts
already cited obviously bear on the treatment and prevention of such
diseases.
The idea of "avitaminoses" or vitamine deficiency as the cause of a
disease of a specific nature was set forth in detail by Funk in his book
_Die Vitamine_. In his discussion of this view he suggests several
types that would, he felt, on examination prove to be due to the absence
of a vitamine in the diet. Of these predicted types beri-beri was the only
one to be established in 1913. Scurvy has now been added to the fold and
rickets or rachitis seems well on the way to acceptance though the
specific vitamine absent in this case is not yet positively identified.
Pellagra still resists the efforts of the vitamine hypothesis to bend it
to that theory and its etiology is still obscure.
I. BERI-BERI
This disease while specifically confined to the oriental in the mind of
the student can be justly considered of much wider distribution for the
mild forms of malnutrition associated with a deficiency in the "B"
vitamine are less acute manifestations of this disease. The disease is not
likely to become marked in well nourished districts in its acute form, but
in famine districts its incidence is always possible. It would be more
than possible were it not for the fact that famine tends to eliminate the
highly milled cereals and throw the people back on to the whole grain,
peas and beans, which are rich in the preventive factor. But when for any
reason diets become limited extra attention is demanded in regard to their
selection and preparation. The main characteristics of this disease have
already been fully covered in what precedes and need not be repeated here.
II. SCURVY
This disease, like beri-beri has already been fully discussed in what
precedes. One of the striking discoveries of this subject has been the
retreat from favor of the time-honored lime juice which is now found to be
much less potent than oranges, lemons, or even canned tomato juice and
which on preservation loses practically all its potency. In the modern
hospital, cases of scurvy rarely appear outside of occasional infant cases
and it might appear that the problem of scurvy prevention is peculiarly
that of the sailor, the explorer and the army rationer. Nevertheless an
insufficient supply of the "C" vitamine may retard growth and well being
in the individual without manifesting itself in its more acute form of
scurvy. In a recent review Hess states: "It is hardly an exaggeration to
state that in the temperate zones the development or non-development of
scurvy depends largely on the potato crop." "This is attributed in part to
the fact that the potato is an excellent antiscorbutic, but to a greater
extent because it is consumed during the winter in amounts that exceed the
combined total of all other vegetables." To the public and to the food
purveyor there is a definite problem in how to best supply the preventive
and how best to concentrate and preserve the sources of this vitamine
without injury to its potency. The following observation is therefore
appended as bearing on this point. In the absence of fruits or other high
potency sources it is possible to develop this factor in cereal grains by
the simple expediency of sprouting. If seeds are soaked in water for
twenty-four hours and then kept moist for from one to three days with the
free access of air, sprouts will develop whose content of the
antiscorbutic vitamine is comparable to that of many fresh vegetables,
even though the dry seeds themselves have little of this factor. In other
words the germination process is a synthesiser of the vitamine. This
observation may be of value where fruits and vegetables are scarce or
expensive. On account of cooking effects, it cannot be too often
reiterated that raw fruits, vegetables and salads, are of more value than
cooked forms of these same sources and that drying processes are extremely
destructive where heat enters into the drying process. Vacuum drying seems
to be much less destructive and it may be possible to develop the drying
of vegetables to a point where retention of this vitamine factor is
practical. At present all dried vegetables should be regarded with
suspicion as a source of vitamine "C." Expressed juices may often be used
where the whole vegetable is scarce or incompatible and this fact is one
to be borne in mind by the worker in famine districts.
III. RACHITIS (RICKETS)
This disease is engaging the attention of many workers on both sides of
the Atlantic at the present time. In England the principal contributor is
Dr. Mellanby, who has accumulated evidence which he believes indicates
that the preventive factor is the A vitamine. This view is not yet
accepted as conclusive by the American workers. McCollum, Howland, Park,
and others at Johns Hopkins University have experimented with various
rickets-producing diets and while the principal deficiency in these diets
seems to be Ca salts and the A vitamine they do not consider that the
disease can as yet be traced to deficiency in any one factor. Hess has
called attention to several new features and the significance of some
older measures. He has shown on the one hand that cod-liver oil is almost
a specific remedy for the disease but that this remedy is not replaceable
by other rich sources of the A vitamine. He has also recently shown that
hygienic measures may have an influence. Schmorl showed that the disease
was seasonal, a high rate maintaining in the winter months and a lower
rate in the summer months. Hess has recently reported beneficial results
from use of the ultra-violet rays which he uses as a substitute for
sunlight. The results seem to confirm Schmorl's view that the sunlight of
the summer months is a preventive factor. He has also suggested that the
specific effect of the cod-liver oil might be due to a new vitamine,
Vitamine D? On the other hand Zilva and Miura in England have recently
shown that crude cod-liver oil is something like two hundred and fifty
times as rich in vitamine A as butter fat, which tends to support the
British view that the A vitamine is the antirachitic factor.
Sherman and Pappenheimer have recently shown that the phosphates exert a
marked preventive effect on rickets and suggest that the utilization of
the calcium by the individual may be determined in part by this factor.
The views in brief are now in an extremely chaotic state and it is
impossible at present to determine whether rickets is a true avitaminose
or a consequence of deficiency in a series of factors. It is however
certain that the disease in its subacute forms is extremely wide-spread
among infants and that its prevention can be most easily secured by the
addition of cod-liver oil to the diet. In this procedure warning is
necessary that the cod-liver oil be as pure a product of oil as possible,
since the market preparations are often almost devoid of the true oil and
hence of the curative agent.
IV. PELLAGRA
This disease has been the subject of exhaustive inquiry and study on this
side of the Atlantic and the findings of the various investigating boards
have added much to the prevention and cure of the scourge, but have failed
as yet to agree on any one etiological factor. The best recent review of
the current findings is to be found in an article by Voegtlin published as
Reprint 597 of the Public Health Reports of the United States Public
Health Service. His conclusions may be quoted in full as representing the
latest summary of evidence now extant:
1. The hypothesis that there is a causal relation between pellagra and a
restricted vegetable diet has been substantiated by direct proof to this
effect and has led to results of considerable practical and scientific
value.
2. The metabolism in pellagra shows certain definite changes from the
normal, which point to decreased gastric secretion and increased
intestinal putrefaction.
3. In the treatment and prevention of pellagra, diet is the essential
factor. The disease can be prevented by an appropriate change in diet
without changing other sanitary conditions.
4. A diet of the composition used by pellagrins prior to their attack by
the disease leads to malnutrition and certain pathological changes in
animals, resembling those found in pellagra. A typical pellagrous
dermatitis has not been observed in animals. Pellagrous symptoms have been
produced in man by the continued consumption of a restricted vegetable
diet.
5. _The nature of the dietary effect has not been discovered_,
although certain observations point to a combined deficiency in some of
the recognized dietary factors as the cause of the pellagrous syndrome.
In elaborating on conclusion 5 Voegtlin states that:
The conception that pellagra is due to a dietary deficiency is, therefore,
not contradicted by the available evidence. This does not imply that the
disease is necessarily due to a deficiency of diet in a specific substance
such as the hypothetical pellagra vitamine of Funk (1913). It is much more
likely that the pellagrous syndrome is caused by a combination of the
deficiencies in some of the well recognized food factors.
V. OTHER AVITAMINOSES
The rôle of the vitamine in the nutrition and growth of organisms other
than the man is becoming a matter of interest in various ways. The
construction of culture media for various strains of bacteria and the
conditions favorable or unfavorable to their growth, are features of study
in which the new hypothesis has demanded attention. It has already been
claimed that vitamines are essential to the growth of the meningococcus,
the influenza bacillus, the typhoid bacillus, the gonococcus, the
pneumococcus Type I, Streptococcus hemolyticus, the diptheria bacillus,
the Bacillus pertussis and certain soil organisms. If these views are
confirmed it becomes evident that the means for prevention of the
development of these forms may lie in the control of the vitamine content
of the materials on which these forms thrive and that in the study of
these types it may be possible to speed up the incubation of strains and
thus hasten diagnostic measures by introducing the necessary vitamines
into the culture media. These observations merely suggest the possible
widening of the scope of the vitamine study in the service of man and give
added reason for our keeping pace with the strides made in this particular
field.
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