The Vitamine Manual by Walter H. Eddy
W >>
Walter H. Eddy >> The Vitamine Manual
Pages:
1 |
2 | 3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11
According to this view the active adenin obtained was not a contamination
but an inactive isomer of the active substance. The hydroxy-betaines which
Williams prepared in defense of his theory have been repeatedly tested but
have in general failed to confirm his view which stands today as an
interesting suggestion but without confirmatory evidence. Other attempts
by these authors to fraction their alkaline extract of fuller's earth have
been unsuccessful. It is of course well known that alkali acts upon the
vitamine destructively. On this account the authors of this method operate
as rapidly as possible and restore the alkali extract to a neutral or acid
medium quickly. The aqueous extract obtained from the earth in this manner
has been shown by Seidell to possess only about one-half of the vitamine
originally present in the solid but the vitamine in it is shown to be
fairly stable. Seidell has not yet determined how long it remains so.
Attempts to recover the vitamine from such aqueous solutions have however
totally failed to date. To quote Seidell from a recent publication:
By careful evaporation of the solution the products successively obtained
show more or less activity by physiological tests but in no case does the
resulting material possess the appearance or character which a pure
product would be expected to show. Solvents such as benzene, ethylacetate
and chloroform fail to effect a separation of active from inactive
material. In all fractioning operations the vitamine tends to distribute
itself between the fractious rather than to become concentrated in one or
the other.
The difficulties encountered by Seidell in this fractioning study have led
him to adopt Walsche's idea that vitamines are of the nature of enzymes
and hence present all the difficulties of identification and isolation of
those substances.
During 1920 Myers and Voegtlin attacked the problem. They have made a
discovery that is useful as a separatory process. This that the "B"
vitamine is not only soluble in water, but also olive oil and in oleic
acid. By shaking an autolysed yeast extract with those solvents in the
proportion of 1 cc. of solvent to which 4 cc. of extract the vitamine
passes into the oil. When this activated oil is filtered and taken up with
eight to ten volumes of ether it in possible to concentrate the ether
extract in vacuo and extract from it with 0.1 per cent. HCl an active
fraction. Aside from this observation however nothing further has been
reported and the possibility of this method of concentration remains yet
to be exploited. They did report other methods of fractioning which
yielded crystals but failed to produce a pure active substance. Those
results add nothing to what has been previously reported except a new
method of fractioning and the elimination of the following substances as
contributing nothing to vitamine activity (purines, histidine, proteins
and albumoses). The crystals they obtained wore contaminated with
histamine.
The World War has prevented full knowledge of the work of the German
investigators but nothing has appeared that indicates any progress in this
field with the exception of a paper by Aberhalden and Schaumann and some
work by Hofmeister. The Aberhalden paper yields no new data of any moment
and no active substances in pure condition are reported. The reports from
Hofmeister are to the effect that he has isolated a very active solution
belonging to the pyrimidine series. It yields a crystalline hydrochloride
and double salt with gold chloride and has given it the formula
C_5H_11NO_2.
The author ban recently been able to obtain a concentrate vitamine from an
extract of alfalfa or autolysed yeast with the aid of a carbon specially
activated by McKee of Columbia University for the adsorption of basic
substance. This adsorbent has been found quite as effective as the
fuller's earth and it is possible to recover the vitamine from the carbon
with treatment by acid. Glacial acetic and heat are especially favorable
for this process. The study of this concentrate has not, however, yet
reached a stage where it contributes any real data on the subject but
merely provides another method for forming concentrates.
If we were to characterize the present status of the search for the "B"
type it might be said to have resolved itself into obtaining concentrates
of high potency as the first step in the process and this type of
investigation is now going on in many laboratories.
If the data is then meagre in the field of the "B" vitamine it is still
more limited in the case of the "A" and the "C." One of the earliest
difficulties encountered in the study of the "A" vitamine was the failure
of fat solvents to extract the material from its richest vegetable
sources. If butter or egg yolk is extracted with ether, the fat obtained
is rich in the "A" vitamine. If, however, ether-extraction is applied to
green leaves or seeds it removes the oils but these oils contain little or
no vitamine. Pressing methods also fail to remove the substance from
vegetable sources. For example, if we press or extract cotton seed we
obtain the oil but the vitamine is retained in the press cake. McCollum
suggested the following explanation for this behavior. His idea is that
the "A" vitamine while soluble in fat is so bound up in the vegetable
source that extraction methods fail to loosen it. When these vegetables
are eaten the vitamine is set free in the process of digestion and being
fat-soluble passes into solution in the animal fats. Hence, when these
fats contain it in solution, they retain it in the process of extraction
while, lacking this separatory process, ether fails to loosen it from the
vegetable binding. Recently, however, Osborne and Mendel have presented
data in regard to this binding and shown that if for ether we substitute
an ether-alcohol mixture the removal of the "A" with the fat is fairly
complete even from vegetable sources. They advance the idea that
preliminary treatment with alcohol is a process which will materially
assist in breaking the attachment of the vitamine and render its removal
with the fat solvent effective. Butter-fat rich in the "A" vitamine has
been conclusively shown to be free of nitrogen and phosphorus and it is
generally assumed that the "A" vitamine is a nitrogen-free and phosphorus
free compound. Further than that however we know nothing of its nature.
Concerning the "C" we know only that it is like the "B," water-soluble and
we know somewhat of its properties, but nothing of its chemical nature.
One of the greatest difficulties still encountered in the study of
chemical fractions is the delay in identification of the active portion.
For this purpose we must rely on tests that are far from delicate and
time-consuming to a degree. As a result the study of only a few fractions
must extend over long periods of time with all the cumulation of
difficulties in the way of change in material, etc. that this delay
implies. An idea of these difficulties can best be obtained by a review of
our present methods for vitamine testing and these methods constitute the
subject matter of the next chapter.
CHAPTER III
THE METHODS USED IN TESTING FOR VITAMINES
It will be evident that in the absence of exact tests for a substance
which is unknown chemically the problem of detecting its presence must be
a matter of indirect evidence. When a chemist is presented with a solution
and asked to determine the presence or absence of lead in that solution he
knows what he is seeking, what its properties are and how to proceed to
not only determine its presence but to measure exactly the amount present.
No such possibility is present in a test for vitamines, but this lack of
knowledge as to the vitamine structure has not left us helpless. We do
know enough of its action to permit us to detect its presence and the
technique that has been developed for this purpose is now well
standardized and involves no mysteries beyond the comprehension of the
layman. In the present chapter is outlined the development of vitamine
testing together with a discussion of some of the deficiencies and the
problems for the future that these deficiencies suggest.
When Casimir Funk made his original studies of the chemical fractions of
an alcohol extract of rice polishings he utilized a discovery of the Dutch
chemist Eijkman. We have already referred to this discovery, viz., that by
feeding polished rice to fowls or pigeons they could be made to develop a
polyneuritis which is identical in symptoms and in response to the
curative action of vitamine, to the beri-beri disease. A normal pigeon can
be made to eat enough rice normally to develop the disease in about three
weeks. The interval can be somewhat shortened by forced feeding. As soon
as the symptoms develop the bird is ready to serve as a test for the
presence or absence of the antineuritic vitamine. If at this time we have
an unknown substance to test it can be administered by pushing down the
throat or mixed with the food or an extract can be made and administered
intravenously. If the dose is curative, the bird will show the effect by
prompt recovery from all the symptoms of the disease in as short a time as
six to eight hours. Such a procedure provides a qualitative test which can
be made roughly quantitative by varying the dosage until an amount, just
necessary to cure the bird in a given time is found and then expressing
the vitamine content of the food in terms of this dosage, in such an
experiment the value is obviously based on the curative powers of the
vitamine source. Another way of applying the test is to determine just how
much of the unknown must be added to a diet of polished rice to prevent
the onset of polyneuritic symptoms. Such a determination will give the
content in terms of preventive dosage. Both methods have been extensively
applied and the following tables compiled from the Report of the British
Medical Research Committee illustrate both the method and some of its
results:
_Minimum daily ration that must be added to a diet of polished rice to
prevent and to cure polyneuritis in a pigeon of 300 to 400 grams in
weight. The weights are given in terms of the natural foodstuff._
____________________________________________________________
AMOUNT NECESSARY | FOODSTUFFS | AMOUNT NECESSARY
FOR DAILY PREVENTION | TESTED | FOR CURE
______________________|__________________|__________________
| |
_grams_ | | _grams_
1.5 | Wheat germ (raw) | 2.5
2.5 | Pressed yeast | 3.0-6.0[1]
3.0 | Egg yolk | 60.0[2]
20.0 | Beef muscle | 140.0[2]
3.0 | Dried lentils | 20.0[2]
______________________|__________________|__________________
[Footnote 1: Autolysed.]
[Footnote 2: Alcohol extract.]
These values illustrate both the method and its value in comparing
sources. Unfortunately experience has shown that polyneuritis is amenable
to other curative agents to a greater or less extent and it is difficult
to be sure whether the curative or preventive dose represents merely the
vitamine content of the unknown or is the sum of all the factors present
in the curative or preventive material. In comparing the value of
different chemical fractions it probably gives a fair enough basis for
evaluating their relative power but it is not entirely satisfactory as a
quantitive measure of vitamine content.
In America the comparison of vitamine content has been largely based on
feeding experiments with the white rat. No other animal has been so well
standardized as this one. Dr. Henry Donaldson of the Wistar Institute of
Philadelphia has brought together into a book entitled _The Rat_ the
accumulated record of that Institution bearing on this animal. This book
provides standards for animal comparisons from every view point; weight
relation to age, size and age, weight of organs and age, sex and age and
weight, etc. This book together with the experience of many workers as
they appear in the literature and especially the observations of Osborne
and Mendel have made the rat an extremely reliable animal upon which to
base comparative data. The omnivorous appetite of the animal, his ready
adjustment to confinement, his relatively short life span, all contribute
to his selection for experimental feeding tests. Another important reason
for his selection is that being a mammal we may reasonably consider that
his reactions to foods will be more typical of the human response than
would another type, the bird for example. It is perhaps necessary to sound
a warning here, however, and point out the danger of too great faith in
this comparability of rat and man or in fact of any animal with man. In
the case of the rat he has been found useless for the study of "C"
vitamine for the simple reason that rats do not have scurvy. In general
however his food responses to the vitamines, at least of the "A" and "B"
types, have proved, so far as they have been confirmed by infant feeding,
to be reasonably comparable.
Provided with the experimental animal the next step was to devise a basal
diet which should be complete for growth in every particular except
vitamines. Such basal diets have been a process of development. The
requirements for such a diet are the following factors:
1. It must be adequate to supply the necessary calories when eaten in
amounts normal to the rat's consumption.
2. It must contain the kinds of nutrients that go to make up an adequate
diet and in the percents suitable for this purpose.
3. It must contain proteins whose quality is adequate, for growth, i.e.,
which contain the kinds and amounts of amino acids known to fulfil this
function.
4. It must be digestible and palatable.
[Illustration: FIG. 3. TWO TYPES OF EXPERIMENT CAGES DEVISED BY OSBORNE
AND MENDEL
These are manufactured by the Herpich Co. of New Haven, Conn.]
5. It must be capable of being supplemented by either or both vitamines
in response to the particular test it is devised to meet and when both are
present in proper amounts it must produce normal growth and serve as a
control.
[Illustration: FIG. 4. A METABOLISM CAGE DEVISED FOR USE IN THE AUTHOR'S
LABORATORY
The cages being bottomless are readily cleaned. They are set on circles of
wire mesh over galvanized iron funnels permitting urine and feces to pass
through. A second screen over the collecting cup and of fine mesh
separates the feces from urine and also collects scattered food.]
In building up such a diet many experiments have been combined and thanks
largely to the efforts of Osborne and Mendel and McCollum in this country,
we have a thoroughly standardized procedure even extending to types of
cages and care best suited to normal growth and development. For clearer
appreciation of the nature of these diets and their preparation we have
summarized in the following pages the combinations used by the principal
contributors to the subject in this country.
[Illustration: FIG. 5. ILLUSTRATING THE USE OF THE CHATILLON SCALE FOR
RAPID WEIGHING OF ANIMALS
The dial is so made that it can be set to counterbalance the weight of the
cage and the weights read directly. This is also used for weighing food.]
[Illustration: FIG. 6. SAMPLE LABORATORY RECORD]
It is at once obvious from the table that the testing value of these basal
diets demands the absence of the two vitamines in the protein,
carbohydrates and fat fractions. To make sure of this absence various
methods have be devised to attain the maximum purity. The authors
recommend the following procedure:
_a_. To purify the casein or other protein used. Boil the protein
three successive times (it is assumed that the original is already as pure
as it is possible to obtain it by the usual methods of preparation) for an
hour each time, with absolute alcohol, using a reflux condenser to prevent
loss of alcohol. Filter off the alcohol each time by suction. This process
will take off all the adherent fat and hence all the "A" vitamine that
might be present. The casein is then dried and ready for use. In certain
experiments the authors use meat residues instead of a single protein.
This they prepare as follows: Fresh lean round of beef is run through a
meat chopper and then ground to a paste in a Nixtamal mill, stirred into
twice its weight of water and boiled a few minutes. The solid residue is
then strained, using cheese cloth, pressed in the hydraulic press and the
cake stirred into a large quantity of boiling water. After repeating this
process of washing with hot water the extracted residue is rapidly dried
in a current of air at about 60 C. This dried residue may then be further
purified with the absolute alcohol treatment as described for casein.
_b_. To purify the carbohydrate they treat starch in exactly the same
way as the casein.
_c_. To purify the lard. This is melted and poured into absolute
alcohol previously heated to 60 C., cooled over night and filtered by
suction. This process is repeated three times and the resulting solids
dried in a casserole over a steam bath.
_d_. When butter fat is used to provide a source of "A" vitamine it
is prepared as follows: Butter is melted in a flask on a water bath at
45 C. and then centrifugated for an hour at high speed. This results in a
separation of the mixture into three layers: (a) Clear fat, containing the
"A" vitamine and consisting of 82 to 83 per cent glycerides. This is
siphoned off and provides the butter fat named in the diets, (b) An
aqueous opalescent layer consisting of water and some of the water-soluble
constituents of the milk. This is rejected. (c) A white solid mass
consisting of cells, bacteria, calcium phosphate and casein particles.
This is also rejected.
_Osborne and Mendel's diet_
(Figures give the per cent of each ingredient in the diet)
_________________________________________________________________________
| | |
INGREDIENTS | VITAMINE FREE | CONTAINING A ONLY |
_______________________________|_________________|_______________________|
| | | | | | | |
| I | II | III | IV | V | VI | VII |
Purified protein as casein, | | | | | | | |
lactalbumin, edestin, egg | | | | | | | |
albumin, etc. . . . . . . | 18.0|18.0 | | 18.0| 18.0| 18.0| |
or Meat residue . . . . . | | | 19.6| | | |19.6 |
| | | | | | | |
Carbohydrates in the form of: | | | | | | | |
Starch . . . . . . . . . . . | 29.5| 54.0| 52.4| 29.5| 54.0| 54.0| 52.4|
Sucrose . . . . . . . . . . . | 15.0| | | 15.0| | | |
| | | | | | | |
Fat in the form of: | | | | | | | |
Lard . . . . . . . . . . . | 30.0| 24.0| 24.0| 15.0| 15.0| 15.0| 15.0|
Butter fat . . . . . . . . . | | | | 15.0| 9.0| | 9.0|
Egg yolk fat . . . . . . . . | | | | | | 9.0| |
Cod liver oil . . . . . . . . | | | | | | | |
| | | | | | | |
Salts in the form of: | | | | | | | |
Salt mixture I . . . . . . . | 2.5| | | 2.5| | | |
or Artificial protein-free | | | | | | | |
milk (Mixt. IV) . . . . . . | | 4.0| 4.0| | 4.0| 4.0| 4.0|
or Protein-free milk . . . | | | | | | | |
| | | | | | | |
Roughage in the form of: | | | | | | | |
Agar-agar . . . . . . . . . . | 5.0| | | 5.0| | | |
_______________________________|_____|_____|_____|_____|_____|_____|_____|
| | | | | | | |
Total . . . . . . . . . . . . |100.0|100.0|100.0|100.0|100.0|100.0|100.0|
_______________________________|_____|_____|_____|_____|_____|_____|_____|
_________________________________________________________________________
| |
INGREDIENTS | A ONLY | CONTAINING B ONLY
_______________________________|___________|_____________________________
| | | | | | |
| VIII| IX | X | XI | XII | XIII| XIV
Purified protein as casein, | | | | | | |
lactalbumin, edestin, egg | | | | | | |
albumin, etc. . . . . . . | 18.0|18.0 | 18.0| 18.0| | 18.0| 18.0
or Meat residue . . . . . | | | | | 19.6| |
| | | | | | |
Carbohydrates in the form of: | | | | | | |
Starch . . . . . . . . . . . | 45.0| 45.0| 29.5| 54.0| 52.4| 26.0| 29.0
Sucrose . . . . . . . . . . . | | | 15.0| | | |
| | | | | | |
Fat in the form of: | | | | | | |
Lard . . . . . . . . . . . | 15.0| 27.0| 30.0| 24.0| 24.0| 28.0| 25.0
Butter fat . . . . . . . . . | | | | | | |
Egg yolk fat . . . . . . . . | | | | | | |
Cod liver oil . . . . . . . . | 18.0| 6.0| | | | |
| | | | | | |
Salts in the form of: | | | | | | |
Salt mixture I . . . . . . . | | | 2.5| | | |
or Artificial protein-free | | | | | | |
milk (Mixt. IV) . . . . . . | 4.0| 4.0| | 4.0| 4.0| |
or Protein-free milk . . . | | | | | | 28.0| 28.0
| | | | | | |
Roughage in the form of: | | | | | | |
Agar-agar . . . . . . . . . . | | | 5.0| | | |
_______________________________|_____|_____|_____|_____|_____|_____|_____
| | |
| | | Fed Daily
| | |_____________________________
"B" vitamine in the form of: | | | | | | |
| | | 0.2 | 0.4 | 0.2 | 0.04|
| | | to | gram| to | gram|
Dried brewers' yeast | | | 0.6 | | 0.6 | |
| | | gram| | gram| |
_______________________________|_____|_____|_____|_____|_____|_____|_____
| | | | | | |
Total . . . . . . . . . . . . |100.0|100.0|100.0|100.0|100.0|100.0|100.0
_______________________________|_____|_____|_____|_____|_____|_____|_____
[_Note_. Diets I, III and X have been practically discontinued at the
present time. Diets II, V and XI are standard. For data on salt mixtures
see Osborne, T. B. and Mendel, J. B. The inorganic elements in nutrition,
Jour. Biol. Chem. 1918, xxxiv, 131.]
_Salt mixture I (after Rohman)_
_grams_
Ca_3(PO_4)_2 . . . . . 10.00
K_2HPO_4 . . . . . . . 37.00
NaCl . . . . . . . . . 20.00
Na citrate . . . . . . 15.00
Mg citrate . . . . . . 8.00
Ca lactate . . . . . . 8.00
Fe citrate . . . . . . 3.00
______
Total . . . . . . . . 100.00
_Artificial protein-free milk_
_grams_
CaCO_3 . . . . . . . . 134.8
MgCO_3 . . . . . . . . 24.2
Na_2CO_3 . . . . . . . 34.2
K_2CO_3 . . . . . . . . 141.3
H_3PO_4 . . . . . . . . 103.2
HCl . . . . . . . . . . 53.4
H_2SO_4 . . . . . . . . 9.2
Citric acid: H_2O . . . 111.1
Fe citrate: 1.5H_2O . . 6.34
KI . . . . . . . . . . 0.020
MnSO_4 . . . . . . . . 0.079
NaF . . . . . . . . . . 0.248
K_2Al_2(SO_4)_2 . . . . 0.0245
[N.B.--The ingredients of the artificial protein-free milk are mixed as
follows: Making proper allowance for the water in the chemicals the acids
are first mixed and the carbonates and citrates added. The traces of KI,
MnSO_4, NaF, and K_2Al_2(SO_4)_4 are then added as solutions of known
concentration. The mixture is then evaporated to dryness in a current of
air at 90 to 100 Centigrade and the residue ground to a fine powder.]
_e_. When brewers' yeast is used as a source of the "B" vitamine it
is first dried over night in an oven at 110 C. and then subjected to the
same purification process as the casein and the starch to remove all
trace of the "A."
Pages:
1 |
2 | 3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11