The Vitamine Manual by Walter H. Eddy
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_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.
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