Part 2 (1/2)
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 polis.h.i.+ngs 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 pus.h.i.+ng 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 quant.i.tative 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 ill.u.s.trate 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 ill.u.s.trate 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 quant.i.tive 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 Inst.i.tute of Philadelphia has brought together into a book ent.i.tled _The Rat_ the acc.u.mulated record of that Inst.i.tution 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, s.e.x 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 appet.i.te 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.
[Ill.u.s.tration: 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.
[Ill.u.s.tration: 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 pa.s.s 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 princ.i.p.al contributors to the subject in this country.
[Ill.u.s.tration: FIG. 5. ILl.u.s.tRATING 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.]
[Ill.u.s.tration: 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 a.s.sumed 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 quant.i.ty of boiling water. After repeating this process of was.h.i.+ng with hot water the extracted residue is rapidly dried in a current of air at about 60C. 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 60C., cooled over night and filtered by suction. This process is repeated three times and the resulting solids dried in a ca.s.serole over a steam bath.
_d_. When b.u.t.ter fat is used to provide a source of ”A” vitamine it is prepared as follows: b.u.t.ter is melted in a flask on a water bath at 45C. 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 b.u.t.ter fat named in the diets, (b) An aqueous opalescent layer consisting of water and some of the water-soluble const.i.tuents of the milk. This is rejected. (c) A white solid ma.s.s 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,
lactalb.u.min, edestin, egg
alb.u.min, 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
b.u.t.ter 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,