The Vitamine Manual by Walter H. Eddy
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Walter H. Eddy >> The Vitamine Manual
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The reasons for the special precautions just described have arisen from
some recent work of Daniels and Loughlin who claim that commercial lard
contains enough "A" vitamine to permit rats to grow, reproduce and rear
young. The British authorities explain their results as not due to the
presence of the "A" vitamine in the lard but to a reserve store in the
bodies of the animals. They hold that animals may thus store the "A"
vitamine but that apparently they have no storage powers for the "B" that
are comparable to it. Osborne and Mendel repeated the experiments
described by Daniels and Loughlin, using the purification methods just
described, but failed to obtain similar results with either commercial
lard or with the purified fraction. They question the validity of the
British explanation but at the same time reiterate their belief that even
commercial lard contains no "A" vitamine. Whatever the explanation of this
particular phenomenon it is important that the basal diet be of purified
materials and the methods just described supply the procedure necessary to
attain that end.
Before discussing the application of these diets to vitamine testing,
attention is called to other basal diets developed by McCollum. This
worker has paid especial attention to the deficiencies of the cereal
grains and in particular to their salt deficiencies. In his basal diets,
we find, as would be expected, special combinations particularly suited to
the detection of vitamines in such cereals. McCollum has also devised a
method of extracting substances to obtain their "B" vitamine and of
depositing it on dextrin. For that reason he uses dextrin instead of
starch for his carbohydrate and when he wishes to introduce the "B"
vitamine it can be done by his method without having to recalculate the
carbohydrate component. His method consists of first extracting the source
with ether and discarding this extract. Pure ether will not remove the "B"
vitamine. The residue is then reextracted several times with alcohol and
the alcohol extracts combined. If now these alcohol extracts are
evaporated down on a weighed quantity of dextrin the activated dextrin can
be used not only to supply the carbohydrate of the ration but also to
carry the "B" vitamine of a given source that is under investigation.
McCollum's basal diets and salt mixtures are tabulated in the following
chart:
_McCollum's basal diets and salt mixtures_
_______________________________________________________________________
| | |
INGREDIENTS | VITAMINE FREE |"A" ONLY | "B" ONLY
___________________|___________________|_________|_____________________
| | | | | |
Casein . . . . . . |18.0|18.0|18.0|18.0| 18.0 | Same as the vitamine
Dextrin . . . . . |57.3|56.3|76.3|78.3| 71.3 | free diet
Lactose . . . . . |20.6|20.0| | | | with "B" added
Agar . . . . . . . | 2.0| 2.0| 2.0| | 2.0 | as yeasts as
Salt mixture 185 . | 2.7| 3.7| 3.7| 3.7| 3.7 | in the Mendel
Butter fat . . . . | | | | | 5.0 | diets or as
___________________|____|____|____|____|_________| extracts carried
| on the dextrin.
| In the latter
| case a given
| amount of dextrin
Lactose was later discarded when it was shown | carries the
to be usually contaminated with the "B" vitamine.| extract of a
| known weight
| of the source of
| the "B"
_________________________________________________|____________________
Cereal testing combinations
______________________________________________________________________
| | | | | |
Wheat . . . . . . |56.6| | | | 70.0 |
Wheat embryo . . . | |13.3| | | |
Corn . . . . . . . | | |71.3| | |
Oats . . . . . . . | | | |60.0| |
Skim milk powder . | | | | | | 6.0
Dextrin . . . . . |31.5|76.4|18.0|30.3| 20.0 | 81.0
Salt mixture 185 . | | | 3.7| | |
Salt mixture 314 . | | 5.3| | | |
Salt mixture 318 . | 6.9| | | | 5.0 |
Salt mixture 500 . | | | | 4.7| |
Salt mixture ? . . | | | | | | 6.0
Butter fat . . . . | 5.0| 5.0| 5.0| 5.0| 5.0 | 5.0
Agar . . . . . . . | | | 2.0| | | 2.0
___________________|____|____|____|____|_________|____________________
Salt mixtures
__________________________________________________________________________
|
| NUMBER OF MIXTURES
|______________________________________________
| | | | | |
INGREDIENTS | 185 | 314 | 318 | 500 | 211 | ?
___________________________|_______|_______|_______|_______|_______|______
| | | | | |
| grams | grams | grams | grams | grams | grams
| | | | | |
NaCl . . . . . . . . . . . | 0.173 | 1.067 | 1.400 | 0.5148| 0.520 | 15.00
MgSO_4 anhydrous . . . . . | 0.266 | | | | | 1.90
Na_2HPO_4:H_2O . . . . . . | 0.347 | | | | |
K_2HPO_4 . . . . . . . . . | 0.954 | 3.016 | 2.531 | 0.3113| | 34.22
CaH_4(PO_4)_2:H2O . . . . | 0.540 | | | | 0.276 | 0.89
Ca lactate . . . . . . . . | 1.300 | 5.553 | 7.058 | 2.8780| 1.971 | 57.02
Ferrous lactate . . . . . | 0.118 | | | | |
K citrate:H_2O . . . . . . | | 0.203 | 0.710 | 0.5562| 0.799 |
Na citrate anhydrous . . . | | | | | | 3.70
Ferric citrate . . . . . . | | 0.100 | | | | 2.00
Mg citrate . . . . . . . . | | | | | | 7.00
CaCl_2 . . . . . . . . . . | | 0.386 | | 0.2569| |
CaSO_4:2H_2O . . . . . . . | | 0.381 | 0.578 | | |
Fe acetate . . . . . . . . | | | | | 0.100 |
___________________________|_______|_______|_______|_______|_______|______
These diets fall as shown, into two classes. The first group correspond to
those of Osborne and Mendel and are available for general testing of any
unknown. The cereal combinations are so constituted that all deficiencies
of salts are covered and the proportions of the cereal are so selected as
to provide the right proportions of protein, fat and carbohydrate. By
adding enough butter fat to supply the "A" the deficiency in the "B" can
be tested and by adjusting the amounts of "B" on the dextrin the cereal
deficiency in this vitamine can be obtained. It is obvious that by
substituting lard for the butter fat one could use the same mixture
properly supplemented with the "B" to determine the "A" deficiencies of
the wheat.
The most prominent worker in the field of the "A" vitamine measurement in
America is Steenbock. His basal diets are a combination of those already
described.
_Steenbock's basal diets_
per cent
Casein (washed with water containing acetic acid) . . . . . 18.0
Dextrin . . . . . . . . . . . . . . . . . . . . . . . . . . 73.3
Ether extracted wheat embryo as source of vitamine "B" . . . 3.0
Salt mixture (McCollum, no. 185) . . . . . . . . . . . . . . 3.7
Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0
This was his original basal diet but later he modified it by adopting the
McCollum method of carrying his "B" vitamine on the dextrin. This was
usually the alcohol extract of 20 grams of wheat embryo. In the following
diets the presence of this extract is indicated by the letter (x)
following the dextrin.
____________________________________________________________________
| | | | | |
INGREDIENTS | | | | | |
__________________________|______|______|______|______|______|______
| | | | | |
Casein . . . . . . . . . | 18.0 | 18.0 | 16.0 | 18.0 | 16.0 | 12.0
Salt 185. . . . . . . . . | 4.0 | 4.0 | | | |
Salt 32 . . . . . . . . . | | | 4.0 | 4.0 | 2.0 | 2.0
Salt 35 . . . . . . . . . | | | | | 2.5 | 2.5
Dextrin (x) . . . . . . . | 76.0 | 71.0 | 78.0 | 57.0 | |
Butter fat . . . . . . . | | 5.0 | | 5.0 | |
Beets . . . . . . . . . . | | | | 15.0 | |
Potatoes . . . . . . . . | | | | | 79.5 |
Dasheens . . . . . . . . | | | | | | 83.5
Agar . . . . . . . . . . | 2.0 | 2.0 | 2.0 | 1.0 | |
__________________________|______|______|______|______|______|______
_Steenbock's salt mixtures_
McCollum's no. 185; see page 44.
No. 32 consisted of: _grams_
NaCl . . . . . . . . . . . . . . . . . . . . . . . . . 0.202
Anhydrous MgSO_4 . . . . . . . . . . . . . . . . . . . 0.311
K_2HPO_4 . . . . . . . . . . . . . . . . . . . . . . . 1.115
Ca lactate . . . . . . . . . . . . . . . . . . . . . . 0.289
Na_2HPO_4:l2H_2O . . . . . . . . . . . . . . . . . . . 0.526
Ca_2H_2(PO_4)_2:H_2O . . . . . . . . . . . . . . . . . 1.116
Fe citrate . . . . . . . . . . . . . . . . . . . . . . 0.138
No. 35 consisted of:
NaCl . . . . . . . . . . . . . . . . . . . . . . . . . 1.00
CaCO_3 . . . . . . . . . . . . . . . . . . . . . . . . 1.5
The very nature of these basal diets suggests their use. In general
however their utilization for testing purposes is based on the following
principles: Since the basal diet supplies all the requirements of a food
except the vitamine for which one is testing, it is simply necessary to
add the unknown substance as a given percent of the diet and observe the
results. If the amount added is small it is assumed that its addition will
not appreciably effect the optimum concentrations of nutrients, etc., and
for such experiments no allowances are made for the constituents in the
unknown. For example let us assume that we wish to test the value of a
yeast cake as a source of "B" vitamine. We first select a sufficient
member of rats of about thirty days age to insure protection from
individual variations in the animals. The age given is taken as an age
when the rats have been weaned and are capable of development away from
the mother and as furnishing the period of most active growth. These rats
are now placed on one of the basal diets which in this case supplies all
the requirements except the "B" vitamine. In this experiment any of the
diets of Osborne and Mendel or of McCollum will do that have been labelled
"A" _only_. After a week or so on this diet they will have cleared
the system of the influence of previous diets and their weight curves will
be either horizontal or declining. If now we make the diet consist of this
basal diet plus say 5 per cent of yeast cake, the weight curve for the
next few weeks will show whether that amount supplies enough for normal
growth, comparison being made with the normal weight curve for a rat of
that age.
In this method it is assumed that the amount of yeast cake added will not
derange the proportions of protein fat, etc., in the basal diet enough to
affect optimum conditions in these respects. This is a curative type of
experiment. If we wish to develop a preventive experiment the yeast cake
may be incorporated in the diet from the first and the amount necessary to
prevent deviation from the normal curve determined. Both methods are
utilized, the one checking the other. If however the amount of the
substance necessary to supply the vitamine required for normal development
is large such addition would of course disturb the proportions of
nutrients in the normal diet and in that case analysis must be made of the
substance tested to determine its protein, fat, carbohydrate and salt
content and the basal diet corrected from this viewpoint so as to retain
the optimum proportions of these factors. McCollum's cereal testing
combinations are illustrative of such methods applied to cereals. Still
another method is to add a small per cent. of the unknown and then add
just enough of the vitamine tested to make sure that normal growth
results. Such a method gives the results in terms of a known vitamine
carrier. For example, if we add to a basal diet, sufficient in all but the
"A" vitamine (Steenbock's mixture for example), a small per cent of a
substance whose content in "A" is unknown and note that growth fails to
result we can then add butter fat until the amount just produces normal
growth. If now we know just what amount of butter fat suffices for this
purpose when used alone we can calculate the part of the butter which is
replaced by the per cent of unknown used. To put this in terms of figures
will perhaps make the idea clearer. Let us assume that 5 per cent of
butter fat in a given diet is sufficient to supply the "A" necessary for
normal growth. Assume that the addition of 5 grams of the unknown in 100
grams of the butter-free diet fails to produce normal growth but that by
adding 2 per cent of butter fat normal growth is reached. It is obvious
under these conditions that 5 grams of the unknown is equivalent in "A"
vitamine content to 5 minus 2 grams of butter fat, i.e., is equivalent to
3 grams of butter fat or expressed in per cents the substance contains 0.6
or 60 per cent of the "A" found in pure butter fat.
Experience has shown that it is dangerous to draw conclusions from
experiments of too short duration or to base them on too few animals. For
complete data the experiments should be carried through the complete life
cycle of the rat, including the reproductive period. Otherwise it may turn
out that the amount in the unknown while apparently sufficient for normal
growths is incapable of sustaining the drain made in reproduction. It is
this consideration that makes the accumulation of authoritative data on
vitamine contents of foodstuffs so slow and tedious and one of the reasons
why we lack satisfactory tables in this particular at present. Osborne and
Mendel raise another point of methodology and believe that more accurate
results will be obtained if the source of the vitamine is fed separately
than if mixed with the basal diet. It is easily possible that since one of
the effects of lack of vitamine, especially of the "B" type, is poor
appetite, the amount necessary to produce normal growth may be smaller
than would appear from results obtained by mixing it in the basal diet.
When so mixed the animals do not get enough to maintain appetite and
really decline because they do not eat enough rather than because the
amount of vitamine given is inadequate to growth. Details of this kind are
matters however that particularly concern the experimentalist and as our
purpose here is to merely describe the methodology we may perhaps turn now
to other types of testing. Before doing so it is perhaps unnecessary to
suggest that in all experiments it is important that the food intake
consumed be measured. Also that in all such experimentation it is
necessary to run controls on a complete diet rather than to rely too much
on standard figures. For this latter purpose it is merely necessary to add
to the basal diets the "A" as butter fat and the "B" as dried yeast or
otherwise to make them complete. Various special mixtures have been tested
out for this purpose and the data already presented supplies the
information necessary to construct such control diets. Professor Sherman
has given me the following as a control diet on which he has raised rats
at normal growth rate to the fifth generation:
One-third by weight of whole milk powder.
Two-thirds by weight of ground whole wheat.
Add to the mixture an amount of NaCl equal to 2 per cent of the weight
of the wheat.
A control mixture based on Osborne and Mendel's data would have the
following components:
Meat residue 19.6 per cent or casein 18 per cent.
Starch 52.4 per cent or 49 per cent.
Lard 15 per cent or 20 per cent.
Artificial protein-free milk 4 per cent.
Butter fat 9 per cent.
Dried yeast 0.2 to 0.6 gram, daily.
The preceding description has applied especially to testing for the
presence of the "A" or the "B" vitamine. When we come to the methods of
testing for the "C" type it is necessary to change our animal. Rats do not
have scurvy but guinea pigs do. The philosophy of the tests for the
antiscorbutic vitamines then will be identical with that of the
polyneuritic methods with pigeons, viz., preventive and curative tests
with guinea pigs. The "C" vitamine is especially sensitive to heat and
this fact enables us to secure a "C" vitamine-free diet. La Mer, Campbell
and Sherman describe their methods as follows:
First select guinea pigs of about 300 to 350 grams weight. Test these with
the basal diet until you secure pigs that will eat the diet. Those that
will not eat it at first are of no use for testing purposes, for a guinea
pig will starve to death rather than eat food he doesn't like. Having
secured pigs that will eat they should on a suitable basal diet die of
acute scurvy in about twenty-eight days. Their basal diet is as follows:
_per cent_
Skim milk powder heated for two hours at 110 C. in an air
bath to destroy the "C" vitamine that might be present. . 30
Butter fat . . . . . . . . . . . . . . . . . . . . . . . . 10
Ground whole oats . . . . . . . . . . . . . . . . . . . . . 59
NaCl . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
They claim that when fruit juice addenda are given in minimal protective
doses and calculated to unit weight bases, the results are comparable in
precision to those of antitoxin experiments.
Old food should be removed every two days and replaced by new, cups being
cleaned at the same time. Since this is a scurvy-producing diet its use is
obvious. We can let the pig develop scurvy on it and then test the
curative powers of the unknown by adding it to the diet or we can add it
to the diet from the first and determine the dose necessary to prevent
scurvy; or we can determine its effect in terms of a known antiscorbutic
such as orange juice by combining it with measured quantities of the
orange juice.
There are other diets that have been given for this purpose, e.g., Holst
and Frohlich induced scurvy by restricting animals to an exclusive diet of
cereals (oats or rye or barley or corn). Hess and Unger have used hay,
oats and water given ad libitum. All of these and others are subject to
criticism on the basis that they are not necessarily adequate in other
food factors and may therefore not be fair bases for testing the
antiscorbutic powers of the unknown combined with them. Abels has recently
shown that scurvy increases susceptibility to infections and believes that
the scurvy hemorrhages are brought about by the toxic effects of
infection. It is therefore desirable in testing for antiscorbutic power
that the basal diet be itself as complete as possible in all factors
except the absence of "C."
The study of rickets has already progressed to the stage of calculating
rickets-producing diets and the methodology is identical with that for
scurvy but this phase of testing still lacks evidence of an antirachitic
vitamine and in that uncertainty it is hardly worth while to elaborate
these diets here. The British diets are all based on Mellanby's contention
that the "A" vitamine is the antirachitic vitamine. This view is not yet
accepted by American workers.
In concluding this chapter it is sufficient to state that with our present
methodology the accumulation of data for evaluating the vitamine content
of various foods is still far from satisfactory and from the chemist's
viewpoint the methodology is most unsatisfactory as a means of testing
fractional analyses obtained in the search for the nature of the
substance, both because of the time consumed in a single test and from the
difficulty of using the fractions in feeding experiments when these
fractions may themselves be poisonous or otherwise unsuited for mixture in
a diet. It is obvious therefore that interest is keen in any possibility
of devising a test that will be specific, quick and not require
modification of the material tested, because of its unsuitability for
feeding. In 1919 Roger J. Williams proposed a method that seemed to offer
promise in these respects but which is not yet in the form for
quantitative use. It offers promise that entitles it to a special chapter
for discussion and the next chapter presents the present status of the so-
called yeast test for vitamine "B."
Before turning to this test it is well to call attention here to the
importance of the experimental animal. Without the polyneuritic fowls we
might never have cured beri-beri, the guinea pig made the solution of the
scurvy problem possible and if some way of inducing pellagra in an animal
can be devised that scourge may yet be eliminated.
CHAPTER IV
THE YEAST TEST FOR VITAMINE "B"
As far back as the days of Pasteur a controversy arose over the power of
yeast cells to grow on a synthetic medium composed solely of known
constituents. This controversy hinged on a discussion as to whether these
media were efficient unless reinforced with something derived from a
living organism. In 1901 Wildier in France published an article in which
he showed that extracts of organic matter when added to synthetic media
had the power to markedly stimulate the growth of yeast organisms. He did
not attempt at the time to identify the nature of this stimulatory
substance, but since it was derived from living organisms, he called it
"Bios." Soon after the discovery of vitamines the bacteriologists began to
discover that they or an analogous factor apparently played a part in the
growth of certain strains of bacteria, especially the meningococcus. In
1919 Roger Williams working in Chicago University was struck with the
bearing of Wildier's work on the vitamine hypothesis and formed the theory
that Wildier's "bios" might be the water-soluble vitamine "B." He
proceeded to test out this theory and demonstrated that extracts of
substances rich in the "B" vitamine had a marked effect on the stimulation
of yeast growth. He developed these experiments and devised a method of
comparing the growth of yeast cells when stimulated by such extracts. The
results were so striking as to appear to justify his view and he then
suggested that his method might be used as a test for the measure of "B"
vitamine in a given source. William's method consisted essentially in
adding the extract of an unknown substance to hanging drops in which were
suspended single yeast cells and observing the rate of growth under the
microscope. Soon after, Miss Freda Bachman reinvestigated the problem with
various types of yeast and found that practically all types of yeast
respond to the stimulation of these "bios" extracts. Her method consisted
in the use of fermentation tubes and the stimulatory effect was measured
by the amount of CO_2 produced in a given time. By this method she
confirmed Williams' view that the "bios" of Wildier was apparently
identical with vitamine "B" and that most yeasts require this vitamine for
their growth. She also suggested that her method might be made the basis
of a test for vitamine content. In 1919 Eddy and Stevenson made extended
experiments with these two methods in the attempt to improve the technique
and make it serve as a quantitative measure. Their experiments served two
purposes, first to bring out certain difficulties in the methods of the
two authors from the quantitative viewpoint and the development of a
technique to correct these difficulties and secondly to add more data
bearing on the specificity of the test. Soon after their publication Funk
became interested and coming to the same conclusions as to specificity
devised a centrifugating method for measuring the yeast growth. Williams
also improved his original method and devised a gravimetric method for the
same purpose. From the viewpoint of methodology we now have methods which
are suitable as quantitive procedures for determining the effect of
extracts of unknown substances on yeast growth and hence if the
stimulatory substance is vitamine "B," a means of determining within a
space of twenty-four hours the approximate content of stimulatory material
in a given source. Since the Funk method is the simplest of these and
illustrates the principles involved it will suffice to describe that.
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