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."
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 Fröhlich 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.